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Modulation of tumor angiogenesis through the use of antisense oligodeoxynucleotodes targeted to VEGF and BFGF.

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Title:
Modulation of tumor angiogenesis through the use of antisense oligodeoxynucleotodes targeted to VEGF and BFGF.
Creator:
Shi, Wenyin, 1974-
Publication Date:
Language:
English
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vii, 171 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Angiogenesis ( jstor )
Cancer ( jstor )
Cell growth ( jstor )
Endothelial cells ( jstor )
Fibroblast growth factors ( jstor )
Human growth ( jstor )
Liposomes ( jstor )
Receptors ( jstor )
Renal cell carcinoma ( jstor )
Tumors ( jstor )
Angiogenesis Inducing Agents -- genetics ( mesh )
Angiogenesis Inducing Agents -- physiology ( mesh )
Carcinoma, Renal Cell -- genetics ( mesh )
Carcinoma, Renal Cell -- therapy ( mesh )
Department of Pharmacology and Therapeutics thesis Ph.D ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Pharmacology and Therapeutics -- UF ( mesh )
Endothelial Growth Factors -- genetics ( mesh )
Endothelial Growth Factors -- physiology ( mesh )
Fibroblast Growth Factors -- genetics ( mesh )
Fibroblast Growth Factors -- physiology ( mesh )
Oligodeoxyribonucleotides, Antisense -- genetics ( mesh )
Oligodeoxyribonucleotides, Antisense -- therapeutic use ( mesh )
Research ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 2002.
Bibliography:
Bibliography: leaves 135-170.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Wenyin Shi.

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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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50681577 ( OCLC )
ANV9844 ( NOTIS )

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MODULATION OF TUMOR ANGIOGENESIS THROUGH THE USE OF
ANTISENSE OLIGODEOXYNUCLEOTIDES TARGETED TO VEGF AND BFGF












By

WENYIN SHI













A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


2002





























Dedicated to
my parents, Xuehui and Ying
my wife, Weiwen
and
my daughter, Julia.















ACKNOWLEDGMENTS


I would like to express my sincere gratitude to Dr. Dietmar W. Siemann for

providing me the very precious opportunity to work in a wonderful laboratory, and for his

immeasurable support and encouragement.

I would also like to extend my gratitude to the members of my supervisory

committee, Dr. Ian Phillips, Dr. Steven Sugrue, and Dr. Edwin Meyer, for their valuable

advice and continuous encouragement in the completion of my studies. I also would like

to thank Dr. Clare Yuan Zhang for her kind help and assistance in establishing the

studies. I also would like to express my gratitude to Dr. Jeffrey Hughes, Dr. Fuxing Tang

for assistance in liposome preparation and to Neal Benson for his help with FACS

analysis.

In addition, I would like to express my appreciation to the past and present

members of Dr. Siemann's laboratory, including Dr. Lingyun Li, Sharon Lepler, Chris

Pampo, Dr. Gustavo Cabrera, Dr. Kenneth Warrington, Jr., Howard Salmon, Heather

Newlin, Emma Mercer, and Destry Taylor, for their help and providing a pleasant

working environment. I also greatly appreciate all the faculty, staff and student groups at

the Department of Pharmacology and Therapeutics for the countless help they have

rendered and the stimulating intellectual atmosphere they provided.















TABLE OF CONTENTS
page

ACKNOW LEDGM ENTS............................................................ ..........................iii

A B ST RA C T ............................................................................ ..................................... vi

CHAPTERS

1 IN TRO D U CTIO N .............................. ..................................... .......................... 1

Tum or A ngiogenesis.................................................................... ............................. 1
Antiangiogenesis Targets in the Treatment of Cancer................................................ 4
Antisense Oligodeoxynucleotides Technology............................................... 16
Renal Cell Carcinom a ............................ .................... .............. .......... 19
Significance .......................................................................... ...................................22

2 CELLULAR DELIVERY OF ANTISENSE OLIGODEOXYNUCLEOTIDES......... 28

Introduction ............................................................. 28
M materials and M ethods..................................... ................................................. 30
R results ........................................................................ ..................... 34
D discussion ............................................................................ .................................. 36

3 AS-ODNS DESIGN AND IN VITRO ASSESSMENT............................................. 49

Introduction .................. .................... ................................................ 49
M materials and M ethods................................... ....................................................... 52
Results ................................................. ........................ ...................... 57
Discussion ........................................ ...................... ........................ 60

4 ANTI-ANGIOGENIC EFFICACY STUDIES......................... ......... ............. 79

Introduction ........................................ ....................... ....................... 79
Materials and Methods.................. .......................................................... 81
Results ................................................. ....................... ...................... 85
Discussion ........................................... ........................ ...................... 87









5 EFFICACY OF AS-ODNS TREATMENT IN CAKI-I XENOGRAFTS ................. 97

Introduction ............................ .... ..................................................................... 97
M materials and M ethods......................................... ............................................. 99
Results ............................................... ....................... ....................... 102
Discussion ........................................ ...................... ........................ 104

6 COMBINATION STUDIES................................................. .. 114

Introduction ................................... ........................................................................ 114
M material and M ethods............................................................ .......................... 116
Results ............................................. ....................... ....................... 118
Discussion ................................................................ ......................... 120

7 SUMMARY AND PERSPECTIVE............................. ................ ......... 130

R EFER EN C ES........................................................ .................................................. 135

BIOGRAPHICAL SKETCH ......................................................................... 171
































v















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

MODULATION OF TUMOR ANGIOGENESIS THROUGH THE USE OF
ANTISENSE OLIGODEOXYNUCLEOTIDES TARGETED TO VEGF AND BFGF
By

Wenyin Shi

August 2002

Chair: Dietmar W. Siemann
Department: Pharmacology and Therapeutics

Angiogenesis is critical for the growth and metastatic spread of solid tumors, It is

tightly controlled by specific regulatory factors. Vascular endothelial growth factor

(VEGF) and basic fibroblast growth factor (bFGF) have been implicated as the key

factors in tumor angiogenesis. The present studies were undertaken to evaluate the effects

of blocking VEGF/bFGF production by antisense phosphorothioate

oligodeoxynucleotides (AS-ODNs) on the angiogenic activity and growth of a preclinical

model of renal cell carcinoma (Caki-1).

Efficient deliveries of AS-ODNs were achieved using cationic liposome

(DOTAP:DOPE) based delivery systems both in vitro and in vivo.

AS-ODNs sequences against VEGF and bFGF have been designed and their

efficacies were tested in vitro. Effective AS-ODNs against VEGF (V515) and bFGF

(B460) were identified. Treatment of Caki-l cells with V515 or B460 led to a reduction

in VEGF or bFGF expression levels sufficient to impair the proliferation and migration









potential of co-cultured endothelial cells. The observed effects were AS-ODNs sequence

specific, dose dependent and were achieved at a low, non-toxic dose. The treatment of

Caki-1 cells with V515 or B460 was also sufficient to impairthe Caki-l tumor cell

induced angiogenesis in vivo. When V515 or B460 treated Caki-l cells were injected into

nude mice and evaluated for their angiogenic potential, the number of vessels initiated

were significantly reduced.

To test antitumor efficacy of VEGF/bFGF AS-ODNs treatment, V515 and B460

were administrated to Caki-l xenograft tumor bearing mice. The results showed that

systemic administration of VEGF/bFGF AS-ODNs significantly inhibited the growth of

Caki-1 tumors. More importantly, a better response was observed when these two AS-

ODNs treatments were combined. A combination of VEGF/bFGF AS-ODNs treatment

with VEGF/bFGF receptor inhibitor or single dose local radiation also showed enhanced

tumor responses when compared to single treatment alone.

These results indicate that AS-ODNs against pro-angiogenic factors VEGF and

bFGF may have great utilities in the treatment of renal cell carcinoma either alone or in

combination with other anti-cancer therapies.













CHAPTER 1
INTRODUCTION

Cancer is a group of diseases characterized by uncontrolled growth and spread of

abnormal cells. If the spread is not controlled, it can result in death. In spite of ever

increasing efforts to understand its process and improve treatment, its incidence in the

population is rising. In the US, about 1 in 2 men and 1 in 3 women will develop cancer

in their lifetime (American cancer society, 2002). As a cause of mortality overall in the

Western World, cancer is second only to cardiovascular disease. Cancer can be treated by

surgical removal or destroyed with toxic chemicals or radiation. However, these

approaches all have drawbacks. Surgery will work for many primary tumors, but

metastases are difficult to identify let alone remove at an early stage. Radiation and

chemotherapy are generally toxic to normal cells as well. If even a few cancerous cells

remain, they can proliferate to produce a resurgence of the disease; moreover, unlike the

normal cells, cancer cells are genetically dynamic and may evolve resistance to the

chemicals used against them. New therapeutic approaches providing more tumor-specific

targeting still need to be exploited. One promising new development of cancer therapy is

the anti-angiogenic strategies, following the recognition that tumor growth and metastasis

depend on establishment of new blood vasculature (Folkman, 1971; Folkman, 1972b).

Tumor Angiogenesis

Angiogenesis is the formation of new blood vessels out of pre-existing capillaries.

It is a sequence of events that is of key importance in a broad array of physiologic and

pathologic processes (Folkman and Shing, 1992). While it plays a key role in








development, in adults, it is a rare event under normal physiological circumstances,

occurring almost exclusively in the female reproductive system. Under normal conditions

such as wound healing, the angiogenic process switches on and then off at the appropriate

times indicating tight regulation of stimulatory and inhibitory factors (Hanahan and

Folkman, 1996). However, angiogenesis can be activated with a variety of pathological

conditions and occur in a less controlled manner (O'Reilly, 1997). These including

cardiovascular diseases (atherosclerosis), rheumatoid arthritis, diabetic retinopathy,

psoriasis, etc (Folkman, 2001). In addition, angiogenesis is critical in the growth and

metastatic dissemination of cancer (Folkman, 1995; Folkman, 1992; Folkman, 1972a;

Folkman and Shing, 1992).

It has been observed for more than one hundred years that tumors appear to be

more vascular than normal tissues. It was not until in the early 1970s that Drs Folkman

and Denekamp put forward the idea that tumors are highly vascularized and thereby

vulnerable at the level of their blood supply. This is the initial recognition of

angiogenesis being a therapeutically interesting process in the area of oncology. Folkman

proposed the hypothesis that angiogenesis was a requirement for the growth and

metastatic spread of solid tumors (Figure 1-1) (Folkman, 1971; Folkman, 1972a). He

further hypothesized that solid tumors could only grow to a size of-1-2 mm in diameter

without developing new blood supply, and if the development of vascular supply could

be prevented, tumor growth could be limited to a small size (Folkman, 1971; Folkman,

1972a). This hypothesis implied that by destroying the newly developing vessels of the

tumor, all the tumor cells supported by these vessels could also be killed (Denekamp J,

1972).









The process of angiogenesis consists of multiple, sequential, and interdependent

steps (Figure 1-2). It begins with local degradation of the basement membrane

surrounding capillaries, which is followed by the invasion of the surrounding stroma by

underlying endothelial cells in the direction of the angiogenic stimulus. Endothelial cell

migration is accompanied by the proliferation of endothelial cells and their organization

into three-dimensional structures that join with other similar structures to form a network

of new blood vessels (Figure 1-2) (Auerbach and Auerbach, 1994).

The process of angiogenesis is mediated by the balance between pro-angiogenic

and anti-angiogenic factors. Angiogenesis is rapidly initiated in response to hypoxic or

ischemic conditions. In all types of angiogenesis, under either physiologic or pathologic

conditions, endothelial cell activation seems to be the first process to take place. In

tumors, angiogenesis begins by mutual stimulation between tumor cells and endothelial

cells by paracrine mechanisms (Gasparini, 1999). Cytokines from various sources

including tumor and stromal cells are released in response to hypoxia or ischemia It is

suggested that vascular endothelial growth factor (VEGF) is a major player in

angiogenesis initiation (Ziche et al., 1997). Besides affecting vasodilation and vascular

permeability, VEGF can induce the expression ofproteases and receptors important in

cellular invasion and tissue remodeling and is able to prevent endothelial cell apoptosis

(Ferrara and Keyt, 1997; Gupta et al., 1999). Following the releasing of pro-angiogeneic

factors, endothelial cells can release proteolytic enzymes (matrix metalloproteinases,

MMPs) to degrade the extracellular matrix for migration, proliferation, and endothelial

penetration into new areas of the body (Stetler-Stevenson, 1999).









Endothelial cell proliferation and migration is stimulated by pro-angiogenic

growth factors, like VEGF and bFGF. VEGF and bFGF are direct acting pro-angiogenic

growth factors. BFGF exists in both low molecular weight form and high molecular

weight forms due to alternative translation (Florkiewicz et al., 1991). It is suggested that

during angiogenesis, low molecular weight bFGF binding to endothelial cell surface FGF

receptors leads to increased motility, proliferation and proteinase activity, whereas the

high molecular weight forms may act on endothelial cell proliferation after nuclear

translocation (Gleizes et al., 1995; Klein et al., 1997). VEGF, besides its effect on

angiogenesis initiation, also affects endothelial cell proliferation through high affinity

receptors (KDR/flk-1 and Fit-1) expressed on endothelial cells (Ferrara, 1999; Veikkola

and Alitalo, 1999). Finally, the neovasculature become mature and stable through the

interaction of endothelial cells with extracellular matrix and mesenchymal cells. After

endothelial cell proliferation and migration and maturation and formation of endothelial

tube structures, surrounding vessel layers composed of mural cells need to be recruited.

Endothelial cells may accomplish this via the synthesis and secretion of platelet-derived

growth factor (PDGF), a mitogen and chemo-attractant for a variety of mesenchymal

cells (George, 2001; LaRochelle et al., 2002). Subsequent differentiation of the mural

precursor cells into pericytes and smooth muscle cells is believed to be cell-cell contact

dependent process (Griffioen and Molema, 2000).

Anti-angiogenic Targets in the Treatment of Cancer

Understanding angiogenesis and its unique characteristics in tumor growth and

metastasis has provided insights to a variety of ways to interrupt the process. During the

past decade, research on anti-angiogenic agents has exploded and with ever increasing

interest in its potential (Kerbel, 2000). We now have a much clearer understanding of









tumor angiogenesis, including key regulatory factors, differences between normal and

tumor vasculature, along with endogenous inhibitors and methods to study and quantify

angiogenesis (Kerbel, 2000).

The complex process of tumor angiogenesis of tumor provides multiple potential

targets for anti-angiogenic strategies. The formation of new blood vessels involves

basement membrane degradation, endothelial cell migration, endothelial proliferation and

tube formation. Anti-angiogenic strategies under evaluation target at least one of the

several stages (Figure 1-2). These strategies vary from regulation of angiogenic factor

expression in tumors, to endogenous inhibitors of angiogenesis. Currently, there are over

80 clinical trials employing such strategies underway (http://cancertrials.nci.nih.gov/)

reflecting the high pace of development. Based on the biological activities, these

strategies can be categorized into several broad classes. The first class consists ofMMP

inhibitors, compounds that block the degradation of the basement membrane. The second

class of agents includes those designed to inhibit endothelial cell function, such as TNP-

470, thalidomide, endostatin, etc. The third class of agents specifically targets angiogenic

growth factors. It includes trysine kinase inhibitors of VEGF/bFGF, antibodies or AS-

ODNs against pro-angiogenic growth factors or their receptors. The last class of agents

target survival factors ofneovascular blood supply, such as intergrin antagonists, or anti-

VEGF therapy (Reinmuth et al., 2001; Fidler et al., 2000).

I will now review some of the important angiogenic factors that have potential as

therapeutic targets in anti-angiogenic therapies.









VEGF-A and Its Receptors

VEGF-A and its receptor system is among the most substantial mediators of

angiogenesis. Considerable evidence has accumulated indicating that VEGF is an

angiogenic cytokine of central importance. Its angiogenic activities have been

demonstrated in numerous experimental models (Takeshita et al., 1994; Wilting et al.,

1992; Wilting et al., 1993; Potgens et al., 1995; Kondo et al., 1995). The central role of

VEGF in tumor angiogenesis has also been suggested. Over-expression of VEGF has

been reported to occur in the vast majority of clinically important cancers (Zhu and

Witte, 1999; Hemmerlein et al., 2001; Ferrara and Keyt, 1997). High serum and urine

levels of VEGF have been associated with poor survival and treatment outcome to

patients of different cancers (Hemmerlein et al., 2001; Maeda et al., 1996; Gasparini et

al., 1997; Edgren et al., 1999; Sliutz et al., 1995). Tumor associated endothelial cells

frequently demonstrate increased expression of VEGF receptors (Zhu and Witte, 1999).

Moreover, high VEGF expression is found to associate with increased microvessel

density and increased metastatsis in cancers (Zhu and Witte, 1999; Tsuji et al., 2002;

Fontanini et al., 2002; Ng et al., 2001).

Disruption of VEGF signal transduction provides a potential effective target for

anti-angiogenic approaches. Different strategies have been designed and evaluated.

Specific VEGF antibodies are a way of stopping the angiogenic effects of this growth

factor. Systemic administration of monoclonal VEGF antibody into tumor bearing nude

mice significantly suppressed tumor growth in several tumor models (Kim et al., 1993;

Borgstrom et al., 1998). A humanized antibody, rhuMAb-VEGF also has been developed

and is undergoing Phase II clinical trials (Lin et al., 1999; Presta et al., 1997).









Alternatively, AS-ODNs or antisense RNA were also been used to directly disrupt VEGF

protein expression and lead to inhibition of tumor growth in different tumor systems

(Smyth et al., 1997; Ellis et al., 1996; Nguyen et al., 1998). Another approach is the

coupling of a toxin to VEGF itself When active parts of diphtheria toxin (DT390) are

linked to VEGF165 or VEGFI21, the chimeric molecule exerts highly selective toxic

effects on endothelial cells. It disrupts neovascularisation in the chicken chorioallantoic

membrane assay and slows down the growth of tumors in preclinical tumor models

(Arora et al., 1999).

Blocking the interaction of VEGF with it receptor and receptor signal

transduction pathway provides another option for anti-angiogenic treatment. VEGF

receptors (flt-1, flk-l and fit-4) are almost exclusively expressed on endothelial cells,

with VEGFR-2 (flk-1) believed to play a central role in VEGF signal transduction

(Ortega et al., 1999; Bematchez et al., 1999). Antibodies against VEGF receptors also

showed efficacy in inhibiting tumor growth in preclinical tumor models (Brekken et al.,

2000; Klement et al., 2000; Witte et al., 1998). Targeting VEGF receptor expression

through the use of antisense also has been shown to be effective at inhibiting tumor

angiogenesis and growth (Kamiyama et al., 2002; Marchand et al., 2002). Purified

soluble VEGFR-1 binds VEGF with high affinity and blocks VEGF induced endothelial

cell proliferation (Kendall et al., 1996). Over-expression of VEGF soluble receptor

through gene therapy significantly inhibits tumor growth and metastasis and leads to

higher survival rates (Goldman et al., 1998; Hasumi et al., 2002; Shiose et al., 2000;

Takayama et al., 2000). Recently, small molecular compounds that can inhibit VEGF

receptor tyrosine kinase activities have been developed and initial preclinical studies









showed promising anti-angiogenic and antitumor effects (Solorzano et al., 2001; Ning et

al., 2002; Hess et al., 2001). To interfere in the binding of VEGF with its receptors, novel

peptides have been developed (Fairbrother et al., 1998). This approach provides another

potentially effective means to disrupt the VEGF signal transduction pathway and a

provide treatment for cancer.

FGFs and Their Receptors

Fibroblast growth factor was originally identified as an activity in pituitary

extracts that stimulates the proliferation of Balb/c 3T3 cells (Armelin, 1973;

Gospodarowicz, 1974; Gospodarowicz, 1975). Currently, FGFs consist of a family of

over 20 structurally related proteins (Basilico and Moscatelli, 1992;Omitz and Itoh,

2001). They bind and activate high-affinity tyrosine kinases, FGFR 1-4 (Lee et al., 1989;

Dionne et al., 1990; Ruta et al., 1989; Reid et al., 1990). Among all the FGFs, FGF-2 also

called basic fibroblast growth factor (bFGF) is one most extensively investigated and

important in angiogenesis.

BFGF was originally purified from bovine pituitary gland (Esch et al., 1985). It

acts in a paracrine and autocrine manner and is released by tumor cells, macrophages or

the extracellular matrix. BFGF can stimulate endothelial proliferation and also is

chemotactic for endothelial cell migration (Gospodarowicz et al., 1987; Moscatelli et al.,

1986; Moscatelli et al., 1986). It can also up-regulate other important pro-angiogenic

factors like VEGF or plasminogen activator (Seghezzi et al., 1998; Montesano et al.,

1986). Blocking bFGF expression and function can be achieved by using vaccine,

antisense against bFGF or its receptors (Plum et al., 2000; Maret et al., 1995; Wang and









Becker, 1997; Redekop and Naus, 1995; Ensoli et al., 1994; Murphy et al., 1992; Becker

et al., 1989).

Blocking the intrinsic tyrosine kinase activity of FGF receptors is a promising

new approach in anti-angiogenic strategies targeting the bFGF signal transduction

pathway. Some experimental compounds have been found to specifically block signaling

of FGFR- I and inhibit angiogenesis (Mohammadi et al., 1998; Perollet et al., 1998).

The Tie-angiopoietin System

The tie-receptor family consists of two know endothelial tyrosine kinases: TIE

and TIE2/Tek. They are identified in vascular endothelium and hematopoietic cells

(Dumont et al., 1992; Iwama et al., 1993; Partanen et al., 1992; Schnurch and Risau,

1993). Mice lacking TIE 1 or TIE 2 are lethal (Puri et al., 1995). Ties may represent the

earliest endothelial cell lineage marker and may regulate the endothelial cell proliferation,

differentiation, and proper patterning during vasculogenesis.

The first Tie-2 ligand, Aniopoietin-1 (Ang-1) was identified from human

neuroepithelioma and mouse myoblast cell lines (Davis et al., 1996). Ang-1 is a novel

endothelial regulatory factor that has been found to promote angiogenic remodeling by

vascular supporting as well as vessel maturation and stabilization. Another related ligand,

Ang-2 also has been found. However, binding of Ang-2 to Tie-2 did not induce

phosphorylation of Tie-2 in endothelial cells. Moreover, it seems to block Ang-l activity

and suggesting Ang-2 may be antagonize the activation of Tie-2 (Maisonpierre et al.,

1997). These findings together underline the feasibility to use the Tie-angiopoietin

system to control angiogenesis (Lin et al., 1998). But before these molecules can be used

for therapy, their effects on adult human vasculature and their interaction with other









angiogenic molecules during physiological and pathological angiogenesis need to be

further investigated.

Angiogenin

Angiogenin is a 14 kD single chain basic protein found in human adenocarcinoma

cells. It is a potent inducer of angiogenesis in vivo, which functions in the picomolar

range (Vallee and Riordan, 1997; Fett et al., 1985). It stimulates the proliferation of

endothelial cells and promotes the adhesion of endothelial and tumor cells (Soncin et al.,

1994; Hu et al., 1997). Blocking angiogenin with a monoclonal antibody can impair

subcutaneous tumor growth of a colon adenocarcinoma in a dose dependent manner

(Olson et al., 1994). In 40-50% of the cases, growth of human breast carcinoma

xenografts in athymic mice could be completely inhibited by a humanized version of the

monoclonal antibody (Piccoli et al., 1998). In addition to its efficacy of inhibit tumor

growth, it may also inhibited the establishment and metastatic growth of tumor cells

(Olson et al., 2002). Besides the use of antibodies, other strategies to abolish angiogenin-

induced angiogenesis include the use of DNA aptamers or antisense (Olson et al., 2001;

Nobile et al., 1998). Anti-angiogenic therapy using angiogenin as a target may become an

important tool because angiogenin mediates angiogenesis by mechanisms distinct from

VEGF and bFGF (Lixin et al., 2001; Moroianu and Riordan, 1994)

Endogenous Inhibitors (Endostatin, Angiostatin)

Angiostatin and endostatin are two endogenous peptides that have been found to

have potent anti-angiogenic effect (O'Reilly et al., 1997; O'Reilly et al., 1994b; O'Reilly

et al., 1994a). Angiostatin is a prolytic fragment of plasminogen and endostatin is a 20

kD fragment of collagen XVIII. These factors make endothelial cells resistant to









angiogenic stimuli and induce "dormancy" of metastases. Administration of the

recombinant protein, or expressing angiostatin or endostatin by means of gene therapy,

illicits potent anti-tumor effects in the preclinical studies (O'Reilly et al., 1997; O'Reilly

et al., 1994b; O'Reilly et al., 1994a; Bertolini et al., 2000; Jin et al., 2001; Yamanaka et

al., 2001; Feldman et al., 2001; Sacco et al., 2001; Szary and Szala, 2001; Wen et al.,

2001; Shi et al., 2002). The anti-tumor activities of angiostatin and endostatin are

currently undergoing clinical evaluation.

In addition to angiostatin and endostatin, there are other endogenous angiogenic

inhibitors including restin, vasostatin, etc (Pike et al., 1998; Ramchandran et al., 1999).

Also, human prolactin, growth hormone, placental lactogen and growth hormone variant

are angiogenic factors, whereas their 16 kD N-terminal fragments are anti-angiogenic

(Struman et al., 1999).

Integrins

Integrins are heterodimeric transmembrane proteins consisting of a and p

subunits with large ectodomains and short cytoplasmic tails. They control cell motility,

differentiation and proliferation via interactions with extracellular matrix molecules.

Integrins avP3 and avPi are up-regulated on proliferating endothelial cells in angiogenic

blood vessels during wound healing as well as in tumor vasculature (Brooks et al., 1994;

Friedlander et al., 1996). The avyf integrin, an adhesion receptor for extracellular matrix

components with an exposed RGD sequence, is an attractive target for anti-angiogenic

therapy. This integrin is almost exclusively present on the cell surface of activated

endothelial cells, but absent on quiescent endothelium or other cell types (Eliceir and

Cheresh, 1999). Antibodies against avP3 were found to inhibit adhesion-dependent signal









transduction by angiogenic factors, leading to apoptosis of activated endothelial cells.

Consequently, these compounds could block endothelial tube formation and angiogenesis

in tumors (Brooks et al., 1994; Brooks et al., 1995). Currently, integrin antagonists are

being evaluated in phase I and phase II clinical trials (Brower, 1999).

Matrix Metalloproteinases (MMPs) and Tissue Inhibitor of Metalloproteinases

(TIMPs)

To form new blood vessels, endothelial cells of existing blood vessels must

degrade the underlying basement membrane and invade into the stroma of the

neighboring tissue (Mignatti and Rifkin, 1993; Mignatti and Rifkin, 1996). These

processes of endothelial cell invasion and migration require the cooperative activity of

the plasminogen activator and the MMPs.

The MMPs are a family of structurally related zinc-dependent endopeptideases

collectively capable of degrading extracellular matrix (ECM). MMPs play an important

role in the degradation of ECM, both in physiological conditions, such as morphogenesis

and tissue repair and in pathologic conditions, such as tumor invasion and metastasis. The

activity of MMPs is controlled at different levels (Liekens et al., 2001) First, the

expression of MMPs is up-regulated by angiogenic growth factors (Giuliani et al., 1999;

Bond et al., 1998; Wang and Keiser, 1998). Secondly, MMPs need to be activated

proteolytically (Murphy et al., 1999). Lastly, the MMPs activities are also regulated by

their inhibitors TIMPs (Blavier et al., 1999; Henriet et al., 1999). However, a large body

of evidence suggests that this regulation is lost during tumor growth and metastasis

(Rasmussen and McCann, 1997). An imbalance between MMPs and their TIMPs is

responsible for the invasive phenotype of breast, colon and lung tumors and a low









survival rate in urothelial cancers (Kossakowska et al., 1996; Gohji et al., 1996b; Gohji et

al., 1996a).

Inhibition of MMPs activities thus has been extensively studied as an approach to

inhibit growth and invasion of neoplastic cells. Important anigiogenesis inhibitors in

clinical trials based on MMP blocking are Metastat, Neovastat, BMS-2752291,

Mariamstat, AG3340, Bay 12-9556 and CGS 27023A (Vihinen and Kahari, 2002).

MMPs inhibitors currently in clinical trials are synthetic peptides or non-peptidic

molecules, chemically modified tetracyclines, bisphosphonates or natural MMP inhibitors

(neovastat). Further trials using MMPs in combination with classical chemotherapy are

also underway.

Plasminogen Activator (uPA) and its Inhibitors (PAl-1)

Proteases of the fibrinolytic cascade also contribute to the regulation of

angiogenesis. Expression of urokinase-type plasminogen activator (uPA) by malignant

cells results in an aggressive phenotype with increased tumor angiogenesis and metastatic

invasion. PAl-I, the natural uPA inhibitor, is paradoxically also up-regulated in human

tumor samples (Landau et al., 1994). Clinically, expression of both, uPA and PAI-I

correlate with a poor prognosis of several cancers (Rosenquist et al., 1993; Heiss et al.,

2002; Osmak et al., 2001). Taken together, inhibition of uPA rather than PAI-1 activity

might be a possible therapeutic target to treat cancer and other angiogenesis dependent

diseases.

Thrombospondin (TSP)

Extracellular matrix molecules play an important role in maintaining tissue

integrity and endothelial cell viability. However, thrombospondin, one such extracellular









matrix molecules, first identified in 1979 from platelets is also a very powerful inhibitor

of endothelial cell adhesion, migration, motility and proliferation and angiogenesis in

vivo (Lawler et al., 1978; Taraboletti et al., 1990; Good et al., 1990). TSP-1 is a member

of a family of structurally related proteins encoded by different genes, which includes 4

recent identified members, TSP 2-5 (Bomstein and Sage, 1994). TSP-I inhibits

endothelial cell proliferation, migration and can induce endothelial apoptosis (Vogel et

al., 1993; Tolsma et al., 1993). In addition, tumor cells transfected with TSPI developed

smaller tumors than the parental cell lines (Volpert et al., 1998; Streit et al., 1999). In

clinical studies, expression of TSP1 has been inversely correlated with malignant

progression of breast cancer, melanoma, and lung carcinomas (Zabrenetzky et al., 1994).

These data indicate that TSP-I can be utilized to inhibit tumor growth by an anti-

angiogenic mechanism.

Platelet Factor 4 (PF-4)

Platelet factor 4 belongs to the CXC cytokine superfamily (Strieter et al., 1995). It

is a 7.8 kD protein of 70-amino acid in length that shares homologies with p-

thromboglobulin and interleukin-8 (Deuel et al., 1977). It has been known for a while that

PF-4 inhibits angiogenesis (Maione et al., 1990). First, PF-4 inhibits endothelial cell

proliferation, migration and angiogenesis in vivo (Gupta and Singh, 1994; Maione et al.,

1990). Second, PF-4 is targeted to endothelial cells that undergo angiogenesis in vivo

(Hansell et al., 1995), Moreover, it has been shown the tumor angiogenesis could be

inhibited by PF-4 (Sharpe et al., 1990; Kolber et al., 1995). In addition, human glioma

cells infected with a secretable PF-4 cDNA grew slowly in vivo and were hypovascular









(Tanaka et al., 1997). Finally, data exist suggesting the PF-4 may counteract angiogenic

factor activity at the sties of platelet activation (Watson et al., 1994).

Administration of recombinant PF-4 protein or delivery of the PF-4 gene showed

efficacy against tumor growth in preclinical models (Maione et al., 1990; Tanaka et al.,

1997; Kolber et al., 1995; Maione et al., 1991). These findings suggest that it may have

great potential as an effective anti-angiogenic factor in the treatment of cancer. Currently,

recombinant PF-4 is being evaluated in clinical trials (Belman et al., 1996).

Interleukins

Interleukins have been known for a long time for their immunomodulatory

activities but their role in angiogenesis is just becoming a hot topic in cancer research.

Some of the interleukins have anti-angiogenic properties (interleukin-10, -12, -18) while

others seems to be pro-angiogenic (interleukin-1, -6, -8, -15), some may even have both

effects (interleukin-4). The mechanisms by which interleukins achieve their effects on

endothelial cells are quite different and not fully understood (El Awad et al., 2000; Huang

et al., 1996; Voest et al., 1995). Interleukins may be useful tools to treat angiogenesis-

related diseases including cancer. However, better understanding of their specific

functions, as well as their interactions is needed.

Anti-angiogenic Factors Summary

Among all these angiogenic regulatory factors, the most important pro-angiogenic

growth factors in cancer are VEGF and bFGF Both factors are found to stimulate

endothelial cell proliferation and migration (Leung et al., 1989; Plate et al., 1992;

Schweigerer et al., 1987). Indeed, the expression of VEGF has been related to

fundamental features of tumors, such as growth rate (Kim et al., 1993; Nagao and









Nishikawa, 1989), microvessel density (Toi et al., 1994; Straume and Akslen, 2002) and

vascular architecture (Drake and Little, 1999; Faridi et al., 2002) as well as the

development of tumor metastasis (Weidner et al., 1991; Faridi et al., 2002). A correlation

between VEGF and/or bFGF expression and survival has been noted in some cancer

patients (Gasparini et al., 1997; Yiangou et al., 1997; Dietz et al., 2000). Given the

importance of VEGF and bFGF in the angiogenic process of cancer, these two growth

factors were chosen as the targets for the presents investigations.

Antisense Oligodeoxynucleotides Technology

In order to inhibit the expression or function of specific gene products, such as

VEGF and bFGF, a strategy with promise is the use of antisense oligodeoxynucleotides

(AS-ODNs).

In 1977, it is first described that gene expression can be modified with exogenous

nucleic acids by using single strand DNA to inhibit translation of a complementary RNA

in a cell-free system (Paterson et al., 1977). Soon after, Zamecnik and Stephenson also

demonstrated that AS-ODNs targeted to 3' end of virus could inhibit viral replication in

vitro (Zamecnik and Stephenson, 1978). These initial findings showed that AS-ODNs

could inhibit gene expression in a sequence specific manner. One of the first studies

showing in vivo activities of AS-ODNs was published in 1991 (Whitesell et al., 1991).

Since then, particularly with the introduction of efficient methods for DNA sequencing

and ODNs synthesis, various targets have been analyzed in vitro and in animals with

encouraging results (Jansen et al., 1998; Tamm et al., 2001; Pawlak et al., 2000; Golden

et al., 2002; Braasch and Corey, 2002; Corey, 2002).









The essential steps in drug design are the identification of an appropriate target

responsible for a certain disease and the development of a drug with specific recognition

of and affinity to that target. For most conventional drugs the mechanism of fairly broad.

In contrast, the basis of the use of AS-ODNs is that the introduction of ODNs

complementary to target mRNA sequences into the cytoplasm can result in decreased

expression of the gene being targeted (Dias N and Stein, 2002). Since AS-ODNs inhibit

gene expression in a sequence dependent way, selective alteration of specific gene

expression is possible. The AS-ONDs approaches generally used are mainly to inhibit

oncogene expression, to induce apoptosis, to overcome multidrug resistance, or to inhibit

pro-angiogenic growth factors (Pawlak et al., 2000).

In 1998, the first antisense drug (fomivirsen) was approved by the US Food and

Drugs Administration (FDA) for the treatment of cytomegalovirus-induced retinitis in

AIDS patients (de Smet et al., 1999). Although fomivirsen is administrated locally, the

approval shows the feasibility of AS-ODNs as drugs for the treatment of human diseases.

With continuous development and understanding of tumor biology, inappropriate

expression of certain genes was found to be basic to the pathophysiology of cancer.

Consequently the use AS-ODNs as therapeutic strategies in the treatment of cancer has

attracted much attention and intensive investigation. The currently more than 8 ongoing

clinical trials illustrate the growing interest in AS-ODNs in the treatment of cancer

(Koller et al., 2000; Tamm et al., 2001). Besides these approaches, AS-ONDs may also

have a role in overcoming multidrug resistance in cancer. For example, the use of anti-

mdrl AS-ODNs has been shown to lead to reduction of the gene product gp170









expression, restore chemotherapy drug sensitivity, and even lead to eventual prolongation

of survival (Cucco and Calabretta, 1996; Kuss et al., 2002; Pan et al., 2001).

Oncogene over-expression is one of the most common molecular events that may

lead to cancer development. AS-ODNs are specific tools to inhibit expression of certain

oncogenes and so can be used as potential drugs to reverse the harmful effects of

dysregulated gene expression. AS-ODNs against ras. myc, myb, bcr-abl, as well as viral

oncogenes, such as E6, E7 or HBx have been evaluated (Gray et al., 1993; Szczylik et al.,

1996; Venturelli et al., 1990; Leonetti et al., 1996; Citro et al., 1994; Szczylik et al.,

1991; Beer-Romero et al., 1997; Lappalainen et al., 1996). Methods to regulate the

mechanisms of cell death and survival in order to shift the balance toward apoptosis are

of great interest in the treatment of cancer. Studies have focused on targeting the vital

anti-apoptotic genes, such as bcl-2, p53 and CRIPTO and MDM-2 proteins using AS-

ODNs (Ziegler et al., 1997; Campbell et al., 1998; Normanno et al., 1999; Chen et al.,

1998).

The application of AS-ODNs to target the pro-angiogenic growth factors is a new

and promising strategy in cancer management. Studies with AS-ODNs against VEGF

showed such treatment can significantly impair tumor angiogenesis and lead to tumor

growth inhibition in VEGF dependent tumors (Masood et al., 2001; Masood et al., 1997).

AS-ODNs directed at inhibiting the expression of bFGF also showed both anti-

angiogenic and anti-tumor efficacy (Wang and Becker, 1997). The strategy of using AS-

ODNs against both VEGF and bFGF as therapeutic intervention of cancer was explored

in detail in studies described in this dissertation.









To date proof of clinical efficacy of AS-ODNs in oncology is very limited (de

Smet et al., 1999). However, data providing proof of principle exist. Future development

of AS-ODNs holds considerable promise in the treatment of cancer. Further development

of new various new targets, assessment of combination treatments with several PS-

ODNs, and investigations focused on strategies targeting tumor mechanisms should

improve their therapeutic activities.

Renal Cell Carcinoma

Renal cell carcinoma (RCC) is the sixth leading cause of cancer death in the

United States, accounting for 3% of adult malignancies. There were an estimated 30,800

RCC cases diagnosed in 2001, with approximately 12,100 deaths in the United States

(Jemal et al., 2002). The incidence of RCC deaths in the United States has been steadily

increasing during the past 25 years, possibly partly because of increased sensitivity and

greater use of various imaging modalities (Chow et al., 1999; Homma et al., 1995; Jayson

and Sanders, 1998).

Although the etiology of RCC is unknown, several risk factors, including obesity,

smoking, hypertension, diuretic use, consumption of fired meat, asbestos exposure,

petroleum exposure, and frequent analgesic use have been consistently implicated (Dhote

et al., 2000). Renal transplantation, with its associated immunosuppression, acquired

cystic kidney disease also increase the risks of developing RCC (Hoshida et al., 1999).

RCCs are clinically, histologically, and genetically a very heterogeneous group of

tumors. Clear cell RCC is the most common type of RCC, accounting for over 70%/ of the

cases. It is a highly vascularised neoplasm demonstrating clear evidence of abundant

angiogenesis and abnormal blood vessel development (Yoshino et al., 2000).









Clinically, RCC patients can present with a multiplicity of manifestations ranging

from the classic presenting triad of hematuria, pain and palpable renal mass to more

obscure symptoms, such as those of paraneoplastic syndromes. Unfortunately, the classic

triad usually indicates patients with far advanced disease, and it is seen in less than 10%

of patients at presentation (Gibbons et al., 1976). More commonly, renal tumors are

discovered incidentally during the course of various diagnostic studies.

The treatment of choice for RCC is surgical removal. Radical nephrectomy is

accomplished by early ligation of the renal artery, renal vein, and en bloc removal of the

kidney with the surrounding Gerota's fascia (Robson et al., 1969). Despite the

remarkable improvements and response rates of single and combination chemotherapy in

some solid tumors, RCC remains a chemoresistant tumor. The most common agents,

vinblastine and floxiuridine, have response rates of 7%/ and 16%, respectively (Yagoda et

al., 1995). In a review of 72 agents, evaluated in 3500 patients, between 1983 and 1992,

an overall objective response rate of only 5.6% was found, mostly of short duration

(Yagoda et al., 1995). Hormone therapy has been found to be equally ineffective

(deKemion and Lindner, 1982). Consequently, there currently is no role for

chemotherapy or hormone therapy in the treatment of RCC. Unlike chemotherapy,

radiotherapy has been shown to provide some benefits to patients with RCC (Rost and

Brosig, 1977). Preoperative radiation can reduce the risk of tumor dissemination at the

time of nephrectomy; reduce primary tumor size; increase resectability and reduce tumor

vascularity. Postoperative radiation therapy has the theoretical benefit of providing local

control of tumor in patients with positive surgical margins, incompletely rejected primary

tumors, or lymph node involvement (Riches, 1966; Mantyla et al., 1977). Still, currently,









radiation therapy in RCC is generally reserved for palliation, most often for symptomatic

bony metastases (Halperin and Harisiadis, 1983).

Taken together, five-year survival rates after radical nephrectomy for stage 1 RCC

is approximately 94%, and stage II 79%. Patients with renal vein or inferior vena caval

involvement have a survival rate of 25-50%, and patients with regional lymph node

involvement or expracapsular extension have a survival rate of 12-25%. Five year

survival rate for patients with stage IV disease is less than 5%.

The unsatisfactory management of RCC with conventional anticancer therapies

warrants novel approaches to augment the tumor response and treatment outcome.

Histopathological studies of RCC reveal it to be a highly vascularised neoplasm

demonstrating clear evidence of abundant angiogenesis and abnormal blood vessel

development (Figure 1-3) (Yoshino et al., 2000). Therefore it may provide an excellent

target for anti-angiogenic therapeutic approaches. Basic fibroblast growth factor (bFGF)

and vascular endothelial growth factor (VEGF) are of particular interest. Both factors

have been shown to be expressed in renal cell carcinoma tissues and renal cell carcinoma

cell lines (Mydlo et al., 1993; Gospodarowicz et al., 1986; Mydlo et al., 1988; Sato et al.,

1999; McLaughlin and Lipworth, 2000; Ferrara and Keyt, 1997). Serum levels of VEGF

and bFGF often are elevated in RCC patients (Nguyen et al., 1994b; Fujimoto et al.,

1991; Wechsel et al., 1999; Tomisawa et al., 1999; Paradis et al., 2000) and renal cell

carcinoma VEGF and bFGF mRNA levels have been reported to be much higher than

those found in surrounding normal tissues (Tricarco et al., 1999; Thelen et al., 1999;

Tsuchiya et al., 2001; Eguchi et al., 1992). In addition, elevated serum/urine bFGF levels

have been shown to associated with malignant progression and poor treatment outcome









(Song et al., 2001; Jacobsen et al., 2000; Rasmuson et al., 2001; Edgren et al., 1999;

Dosquet et al., 1997; Fujimoto et al., 1995; Plunkett and Hailey, 1990; Miyake et al.,

1996; Nguyen et al., 1994a; Huang et al., 1996). Taken together, these findings suggest

that VEGF and bFGF are key factors involved in the angiogenic process of RCC. For

these reasons, RCC was the tumor of choice for the current investigation of anti-

angiogenic therapeutic approaches with the use of VEGF and bFGF AS-ODNs. An RCC

cell line (Caki-1) was used. This cell line is a human clear cell renal cell carcinoma

originally derived from a 49 year-old Caucasian male patient (Fogh, 1978). It grows in

vitro as an anchored cell culture and also forms tumor in athymic nude mice. The

histology of the Caki-l xenograft displays many of the features of clinical samples of

RCC (Figure 1-5).

Significance

Angiogenesis is unique process that contributes to a variety of pathologic

processes and is especially critical to tumor growth and metastasis. It therefore has been

proposed and utilized as both a prognostic indicator as well as a possible target for

therapeutic intervention in certain malignant states. In the present studies, the feasibility

of inhibiting tumor angiogenesis by utilizing AS-ODNs directed against VEGF and bFGF

were investigated. A human renal cell carcinoma cell line grown in vitro or as solid tumor

xenografts in nude mice was used as the tumor model. Through the investigations, a

viable means of intervening with the angiogenic process and in situ growth of renal

carcinoma cells was developed. It is further believed that this approach will not be

confined to RCC, but will be applicable to other neoplasms and may even provide a basis

for selective intervention in other diseases characterized by angiogenesis.













Anti-angiogenic

Pro-angiogenic


Figure 1-1. The angiogenic process. Tumor cells or host cells secrete pro-anglogenic
growth factors, which then bind to specific receptors on endothelial cells. This ligand-
receptor interaction leads to endothelial cell proliferation, migration, invasion and.
eventually, capillary tube formation (Fidler et al, 2000)

















Lo3 ritem M g

4 EC ativloum






WMigr d

7 ECM rmodong



Mp Liallrmage
8 rb fniat




acliar Stabizaion


Figure 1-2. Cascade of events in tumor anglogenesis. (Source: The Angiogenesis
Foundation, www.anglo.org)











pro-angiogenic
I I- to

Iv- t- an I ,






I .






anti-angiogenic



Figure 1-3. Major regulators of angiogenesis and their receptors. On the upper part of the
cell pro-angiogenesis regulators are shown, on the lower part are inhibitory molecules.
(Hagedom and Bikfalvi, 2000)



































Figure 1-4. Resin cast of RCC tumor microvascular network. (Gerwins et al., 2000)









































Figure 1-5. H&E staining histology section ofCaki-1 xenograft tumor.














CHAPTER 2
CELLULAR DELIVERY OF ANTISENSE OLIGONUCLEOTIDES

Introduction

Many of the limitations of current cytotoxic therapies of cancer result from a lack

of specificity of the anti-cancer agents. The advances in molecular biology over the past

The application of two decades have made possible the concept of genetically based,

targeted treatment. Antisense oligonucleotides (AS-ODNs) is one approach to

specifically inhibit gene expression (Stein and Cheng, 1993; Wagner, 1994). These

molecules, usually 18-20 bases in length, can undergo Watson-Crick hybridization to

target mRNAs, ultimately resulting in decreased expression of the gene products. AS-

ODNs technology has attracted great interest and shown great promise as agents to

inhibit the expression of specific genes that regulate physiological functions or mediate

various diseases (Harrison, 1993; Wagner, 1994; Wagner, 1995; Pan etal., 2002; Mani et

al., 2002; Morris et al., 2002).

However, one of the biggest challenges in the application of AS-ODNs as

therapeutic agents is the development of ways to maximize their cellular uptake (Wagner,

1995; Crooke, 1993). AS-ODNs are negatively charged molecules that behave as

polyanions. In general this property leads to poor cellular uptake and intracellular

distribution. Furthermore, the commonly used phosphorothioate modified AS-ODNs

have quite high affinity for proteins, especially heparin-binding proteins (Fennewald and

Rando, 1995). In addition, some new cell surface ODNs-binding proteins have also been

identified recently (Beltinger et al., 1995; Hawley and Gibson, 1996). Even after









internalization into the cell through endocytosis (Yakubov et al., 1989; Crooke et al.,

1995), naked AS-ODNs are localized to endosomes or lysosomes, that are topologically

still "outside" of the cell (Tonkinson et al., 1994). ODNs may then either be released

from the cell via exocytosis or may be partially digested (Tonkinson et al., 1994). Still,

exists a growing literatures of antisense effects after naked AS-ODNs delivery (Anfossi

et al., 1989; Gewirtz and Calabretta, 1988). Nontheless, it should be recognized that these

effects were achieved at high AS-ODNs concentration and may be the result of AS-

ODNs release into the cytoplasm through spontaneous endosomal/lysosomal rupture.

Many techniques have thus been then used to enhance AS-ODNs uptake, the most

widely used being based on the application of cationic lipids. Cellular uptake as well as

the activity in cell cultures can be improved greatly by cationic liposomes (Bennett et al.,

1992; Lappalainen et al., 1994; Zelphati and Szoka, Jr., 1996b; Zelphati and Szoka, Jr.,

1996a). Highly polar, water soluble molecules including AS-ODNs can be entrapped in

the internal aqueous space of the liposme, while the lipids form into bilayers. Cationic

liposomes spontaneously bind the negatively charged AS-ODNs and protect them against

degradation. The macromolecular complexes have a positive charge at the surface, this

results in a high affinity for most cell membranes, which are negatively charged under

physiological conditions. Following the attachment to the membrane, the complexes are

taken up via endocytosis. To help facilitate the release from endosomes and lysosomes, a

helper lipid such as 1,2-dioleoyl-3-sn-phosphatidylethanolamine (DOPE) is often used in

the liposome preparation. This inverted-cone-shaped lipids thought to facilitate cytosolic

release through the fusion and disruption of endosomal membranes (Farhood et al.,

1992). The flip-flop of anionic phospholipids in the endosome membrane, lead to









neutralization of the cationic lipid charge, displacement of the bound oligonucleotides,

and release form the endosome (Figure 2-1) (Koltover et al., 1998; Lebedeva et al.,

2000). Studies have demonstrated that ODNs can readily dissociate from the liposome

complexes and are in bioavailable form within the cells (Tari, 2000; Abe et al.,

1998).Cationic liposomes facilitated delivery of AS-ODNs has proven to be effective in

many different cell lines and to be of general utility (Bennett et al., 1992).

However, studies in mammalian cell lines have demonstrated that AS-ODNs

efficacy varies with different cationic lipids and lipid complexes, cationic lipid/DNA

ratio, and cell type (Flanagan and Wagner, 1997; Lappalainen et al., 1997). This suggests

that as a general rule, the best liposome composition and optimal liposome/ODNs ratio

needs to be established for each cell lines to achieve best results.

In the present studies, fluorescein isothioicyanate (FITC) labeled ODNs were

used to study the cellular uptake of ODNs using a cationic liposome delivery system

(Noonberg et al., 1992). Although the biological activity of AS-ODNs against the

molecular target is highly sequence-dependent, this is typically not the case for

pharmacokinetics and toxicology. Indeed, the pharmacokinetics and toxicology of AS-

ODNs of widely differing sequences directed against vastly disparate gene products have

proven surprising similar (Srinivasan and Iversen, 1995). Thus, the same ODNs sequence

was used for the present cellular uptake and toxicity studies.

Materials and Methods
Cell Culture

The clear cell RCC cell lines Caki-l, Caki-2 and A498 were gifts from Dr. Susan

Knox (Stanford University). These cells were grown in Dulbecco's modified minimum

essential medium (DMEM, Invitrogen, Grand Island, NY) supplemented with 10% fetal









bovine serum (FBS, Invitrogen, Grand Island, NY), 1% penicillin-streptomycin

(Invitrogen, Grand Island, NY) and 1% 200 mmol/L L-glutamine (Invitrogen, Grand

Island, NY).

FITC Labeled Phosphorothioate Oligodeoxynucleotides

The 20-mer ODNs, sequence: 5'- CAC CCT GCT CAC CGC ATG GC -3' (20-

mers) were customer synthesized by Geno Mechanix (Alachua, FL). The entire backbone

was phosphorothioate modified and FITC was labeled at the 5' end of the ODNs. The

ODNs were suspended in sterile and endotoxin-free water at a concentration of 1 mM,

aliquoted and stored at -20C.

Liposome Preparations

Cationic liposomes of different lipid compositions were obtained from Dr. Jeffrey

Hughes' lab (University of Florida, Gainesville, FL). DOTAP:DOPE is composed of

cationic lipid 1,2-dioleoyloxy-3-(trimethylammonium) propane (DOTAP) and a helper

lipid 1,2-dioleoyl-3-sn-phosphatidylethanolamine (DOPE) at a molar ration of 1:1.

DS3DOPC is composed of 1,2-Dioleoyl-sn-Glycero-3-Phophoserine-N-Citraconyl and

dioleoyl-phosphatidylcholine (DOPC) at a molar ratio of 1:1. DOGSDSO is composed of

l',2'-dioleoyl-sn-glycero-3'-succinyl-2-hydroxyethyl disulfide omithine conjugate (Tang

and Hughes, 1998). CHDTAEA is composed of cholesterol hemidithiodiglycolyl

tris(aminoethyl)amine (Tang and Hughes, 1999). PEG-PE is composed of DOTAP,

DOPE and polyethylene glycol distearoyphospatidylethanolamine (PEG-PE) at a molar

ration of 25:25:3 (Meyer et al., 1998). All the lipids were obtained from Avanti Polar-

Lipids (Alabaster, Al).









Briefly, the lipid mixture was evaporated to dryness in a round-bottomed flask

using a rotary evaporator at room temperature. The resulting lipid film was dried by

nitrogen for an additional 10 min to evaporate any residual chloroform. The lipid film

was re-suspended in sterile water to a final concentration of 1 mg/ml based on the weight

of cationic lipid. The resultant mixtures were shaken in a water bath at 35C for 30 min.

The suspensions then were sonicated using a Sonic Dismembrator (Fisher Scientific,

Pittsburgh, PA) for 1 min at room temperature to form homogenized liposomes. The

particle-size distribution of liposomes was measured using a NICOMP 380 ZLS

instrument (Santa Barbara, CA). The average particle diameter was 144.0 77.0 nm.

Liposomes were stored at 40C and used within 3 months.

Antisense Treatment

Caki-1, Caki-2, A498 cells were set at 1 x 105 in 60 mm dishes and allowed to

attach overnight. For comparison of delivery efficiency by different liposomes, FITC

labeled ODNs were mixed with different liposomes in serum free medium or 10% FBS

medium and incubated at room temperature for 30 min. For other studies, only

DOTAP:DOPE liposome was used. The medium of cells was then changed with that

containing ODNs-liposome complex at a ODNs concentration of 1 gM/ml and incubated

at 370C for 3 hours. Equal amounts of 20% FBS medium were added to dishes and

continued to incubate for a total of 24 hr, except for the time course studies, in which,

cells were incubated for different lengths of time.

Fluorescence Microscope

After FITC labeled ODNs treatment, the medium containing ODNs was removed

and cells were washed 4 times with PBS. The cells were then fixed in 1% p-









formaldehyde for 1 hr. Fluorescent microscope pictures were then taken using a Zeiss

Axioplan 2 Florescence Microscope (Zeiss, Thomwood, NY) made available by the

Optical Microscopy Facility at Brain Institute, University of Florida.

Flow Cytometry Analysis

After FITC labeled ODNs treatment, the medium containing AS-ODNs was

removed and cells were washed 4 times with PBS. The cells were then collected by

trypsin digestion. After fixation in 1% p-formaldehyde for I hr, cells were re-suspended

in PBS at the concentration of I x 106 cells/ml and kept in the dark. Green fluorescent

intensities of the cells derived from FITC were then analyzed by FACS on a Becton

Dickinson flow cytometer made available through the University Cole Facility for Flow

Cytometry at the University of Florida.

Toxicity Studies

Caki-1 cells were set in 96-well dishes at 1 x 104 cells per well and allowed to

attach overnight. The culture medium was then changed to 100 pl serum free or 10%

FBS medium containing various doses of DOTAP:DOPE. The cells were incubated for

24 hr at 37C. The viable cells after treatment were measured using a

CellTiter96AQueous Assay System (Promege, Madison, WI). Briefly, 100 ld of

phenylmethasulfazone (PMS) solution was added to 2 ml 3-(4,5-dimethylthiazol-2-yl)-5-

(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tet razolium (MTS) solution and mixed

by gentle swirl. 20 pl of the combined MTS/PMS solution was added into each well and

incubated at 37C for 2 hr. After incubation, 25 il of 10% SDS was added into each well

to stop the reaction. The absorbance was then measured at 490 nm using a microplate

reader.









Results

Uptake of FITC labeled ODNs measured by flow cytometry and fluorescence

microscopy, allowed the assessment of cellular delivery of ODNs. Delivery of naked

ODNs resulted in poor internalization into the cells, either in serum free or 10% FBS

conditions. Only a small portion of cells treated showed moderate increases in

fluorescence intensity derived from FITC (Figure 2-2). However, the use of cationic

liposomes (DOTAP:DOPE) was found to significantly improve the up-take efficiency of

ODNs in Caki-1 cells, in both serum free and 10% FBS conditions (Figure 2-2).

In order to optimize the delivery system for ODNs, other cationic liposome

compositions were evaluated for their delivery efficiency of ODNs in Caki-1 cells. The

cells were treated with FITC labeled ODNs delivered by different cationic liposomes

(DOTAP:DOPE, DS3DOPC, DOGSDSO, CHDTAEA and PEG-PE) for 24 hr at a AS-

ODNs concentration of 1 uM/ml. Both serum free and 10% FBS conditions were studied

and compared. Significant enhancements of ODNs cellular up-take were observed in all

liposome treatment groups, with DOTAP:DOPE and DS3DOPC being most efficient and

with minimal serum resistance (Figure 2-3). Since DOTAP:DOPE is of simple

composition and easy to prepare, it was chosen as the delivery vehicle for the rest of

studies.

Studies to optimize the delivery efficiency of ODNs by DOTAP:DOPE were then

carried out. One major factor that determines the delivery efficiency is the liposome to

ODNs charge ratio. In order to determine the optimal charge ratio in Caki-1 cells, the

cells were treated for 24 hr with I iM/ml FITC labeled ODNs prepared with different

amount of DOTAP:DOPE to achieve various charge ratios (1, 1.25, 1.5, 1.75, 2, 2.5). The









fluorescent intensities of Caki-l cells after the treatment were then compared (Figure 2-

4). Increases in delivery efficiency and as well as resistance to serum were observed as

the charge ratio increased. A plateau in these effects occurred at a charge ratio of about 2.

This charge ratio was used in all subsequent studies evaluating the delivery of AS-ODNs

in Caki-1 cells.

The delivery efficiency ofODNs by DOTAP:DOPE as the function of time was

then evaluated. Caki-l cells were treated with 1 uM/ml FITC labeled ODNs delivered by

DOTAP:DOPE at the pre-determined optimal charge ratio of 2. The fluorescent intensity

of Caki-l cells after different lengths of treatment were determined by FACS (Figure 2-

5). Very fast up-take of ODNs by Caki-l cells were observed when delivered by

DOTAP:DOPE liposomes. The up-take of ODNs by Caki-1 cells reached a plateau

within 24 hr of incubation (Figure 2-5).

With the optimized delivery system based on cationic liposomes (DOTAP:DOPE)

very efficient cellular up-take of ODNs was achieved in Caki-1 cells. Significant up-take

of FITC labeled ODNs was observed in Caki-I cells after 24 hr treatment of 1 pM ODNs

delivered by DOTAP:DOPE at a charge ratio of 2. ODNs were delivered into -100% of

the cells with uniform cellular distribution and enhanced nuclear concentration (Figure 2-

6). This result was achieved in both exponential phase and plateau phase Caki-1 cells

(Figure 2-7).

This optimized delivery system was then further tested for ODNs delivery

efficiency in two other RCC cell lines. The results showed that similar highly efficient

cellular up-take of ODNs also could be achieved in Caki-2 and A498 cells (Figure 2-8)









ODNs as fragments of DNA sequences have very low toxicity (Rubenstein et al.,

1997; Agrawal et al., 1997). The toxicity of the cationic liposome delivery vehicle was

also examined in both serum free and 10% FBS conditions. No significant toxicity of

DOTAP:DOPE in Caki-I cells was observed with doses up to 150 mg/ml in 10% serum

and 100 mg/ml in serum free conditions. At doses higher than 100 mg/ml some

cytotoxicity was observed using DOTAP:DOPE in serum medium. The DOTAP:DOPE

dose used in the delivery studies was only 10 mg/ml, which resulted in no toxicity in

Caki-l cells either in serum free or 10% FBS conditions.

Discussion

AS-ODNs can block the expression of specific target genes involved in the

development of human diseases. Therapeutic applications of antisense techniques are

currently under investigation in many different fields. In order for AS-ODNs to down-

regulate gene expression, it must penetrate into the target cells. Phospholipid bilayers

represent a strong barrier to the movement of ions. Studies on the diffusion of ODNs

through model membranes have led to the general conclusion that it is of little

importance. Meanwhile, internalization of ODNs into cells has been clearly

demonstrated, implying the existence of other mechanisms other than passive diffusion

(Garcia-Chaumont et al., 2000). To date, the precise mechanisms involved in ODNs

penetration are still not totally clear. Though it has been found that up-take of AS-ODNs

can occur through receptor mediated active transport (Wu-Pong et al., 1994), which

depends on temperature (Loke et al., 1989; Yakubov et al., 1989), the structure and the

concentration of ODNs (Vlassov et al., 1994), and the cell lines. At the present time, it is

generally believed that adsorptive endocytosis and fluid phase endocytosis are the major

mechanisms of ODNs interalization (Dias N and Stein, 2002; Garcia-Chaumont et al.,









2000). At relatively low ODNs concentrations, it is likely that intemalization occurs via

interaction with a memberane-bound receptors (Loke et al., 1989; Yakubov et al., 1989;

de Diesbach et al., 2000). While at higher ODNs concentrations, these receptors are

saturated, and the endocytotic process assumes larger importance. Numerous reports have

demonstrated that naked ODNs are internalized poorly by cells (Gray et al., 1997; Stein

et al., 1993)(Figure 2-2). More importantly, naked ODNs tend to localize in

endosomes/lysosomes, where they are unavailable for antisense purposes. As has been

demonstrated in numerous experiments, the sine qua non of antisense activity appears to

be nuclear localization (Dias N and Stein, 2002).

To improve cellular uptake and ODNs spatial and temporal activity, delivery

vehicles were developed. Among them, cationic liposomes are most commonly used.

Cellular up-take of ODNs, as well as their activity in cell cultures, can be improved by

cationic liposomes (Bennett et al., 1992; Lappalainen et al., 1994; Zelphati and Szoka, Jr.,

1996b, Zelphati and Szoka, Jr., 1996a). Cationic liposomes are safe, simple and easy to

produce on a large scale (Nabel et al., 1993; Nabel et al., 1994a; Nabel et al., 1994b).

They have been approved by FDA for clinical use. However, when tested for their ability

to promote delivery of FITC-ODNs in mammalian cell lines, their efficacy varied

significantly (Lewis et al., 1996). Important variables include cationic lipsome/ODNs

ratio, composition of lipid and cell type tested (Flanagan and Wagner, 1997; Lappalainen

et al., 1997; Vellon et al., 2002). This suggests that even though cationic liposomes have

been proven to be effective and to be of general utility, a best reagent and optimal

liposome/ODNs ratio could and should be empirically established for each cell type.









In the present studies, delivery efficiencies of FITC tagged ODNs by different

liposome compositions were first evaluated. All cationic liposome compositions

significantly enhanced cellular up-take of ODNs. However, the incorporation of PEG-PE

significantly inhibited the delivery efficiency of cationic liposomes (DOTAP:DOPE).

PEG-PE, when incorporated into conventional liposomes, can provide a steric barrier at

the surface of lipsomes that inhibits opsonization, and therefore can extend the residence

time of liposomes in the blood (Webb et al., 1998). However, even though the use of

PEG-PE in liposomes have minimal effect on the binding and subsequent endocytosis of

lipid/DNA complexes, it did severely inhibit the endosomal release of AS-ODNs into the

cytoplasm (Figure 2-3) (Song et al., 2002).

Given the efficiency of delivery, minimum serum resistance and simplicity of

liposome composition and preparation, DOTAP:DOPE was chosen as the delivery

vehicle for AS-ODNs in Caki-1 cells (Figure 2-3). A major problem associated with

cationic liposomes is low transfection efficiency due to inactivation of cationic liposomes

by serum (Feigner et al., 1987). Much effort has been devoted to resolving this problem.

The charge ratio of liposome to DNA has been proven previously to be critical for high

efficiency oflipofection and serum resistance (Yang and Huang, 1997; Yang and Huang,

1998). The optimal transfection efficiency and minimal serum inactivation was achieved

at a DOPTA:DOPE/ODNs charge ratio of about 2 (Figure 2-4). Using this charge ratio,

cellular up-take of ODNs by Caki-l cells reaches a plateau within 24 hr (Figure 2-5).

ODNs in Caki-1 cells were evenly distributed in the cytoplasum with enhanced nuclear

concentration (Figure 2-6). This distribution is believed to best facilitate antisense

function of AS-ODNs (Wagner, 1994; Hogrefe, 1999). With the optimized delivery







39

system, efficient cellular up-take of ODNs were achieved in both plateau and exponential

phases Caki-l cells (Figure 2-7) as well as two other RCC cell lines (Caki-2, A498)

(Figure 2-8).

Cytotoxic evaluation of DOTAP:DOPE confirmed that cationic liposomes are

safe unless very high doses are used (Porteous et al., 1997; Gao and Huang, 1995). The

dose used in the present studies (10 mg/ml) was far below the doses that resulted in

cytotoxic effects in Caki-1 cells (>100 mg/ml)(Figure 2-9).

In conclusion, cationic liposomes (DOTAP:DOPE) enhanced the cellular up-take

of ODNs in RCC cell lines. The simplicity of preparation, efficiency of up-take and

safety features have rendered the cationic liposome (DOTAP:DOPE) an attractive vehicle

for AS-ODNs therapy.










.._a_.



m......Ic o


& 1A' arM aby lrar ar
wrrt, ,4
u hinofs ha h a
..ene or--- --Lm "
It".' *v.aa h, p U ,


Figure 2-1. Proposed mechanisms of internalization of cationic liposome into cells and
release ofODNs in cytoplasm. Modified from Lebedeva (Lebedeva et al., 2000).







41



Control Naked PS-ODNS with IOTAP:DOPE







Serum Free"


:IT






10% FBS

Figure 2-2. Flow cytometry histogram of Caki-1 cell fluorescece intensity after
treatments of 1 LiM FITC labeled ODNs either with or without DOTAP:DOPE liposome.












i0000 -
100 Serum Free
SD10% FBS




S
Q 100-
o








Con, DOTMIOPE DS3DOPC DOOSOSO CHDTAMA PEPE

Liposomes


Figure 2-3. Efficiency of cellular up-take of FITC labeled ODNs delivered by different
liposmes in Caki-I cells. Caki-1 cells were treated with 1 pM FITC labeled ODNs in
serum free or 10% FBS conditions for 24 hr. Each bar represents the mean + S.E. of 3
independent experiments.













10000
S Serum Free
0 10% FBS


1000-
C


S100,



o
10
LL 1



0.5 1 1.25 1.5 1.75 2 2.5

Charge ratio


Figure 2-4. Effect of liposome to ODNs charge ratio on the delivery efficiency of ODNs
in Caki-l cells. Caki-l cells were treated for 24 hours with I pM FITC labeled ODNs
delivered by different amounts ofDOTAP:DOPE liposomes at the charge ratio indicated.
Each bar represents the mean S.E. of 3 independent experiments.












1000




I W
100



L io,
0 Serum free
0 E10% FBS



0 24 48 72
Time (hr)


Figure 2-5. Time course of up-take of ODNs delivered by DOTAP:DOPE in Caki-1 cells.
Caki-1 cells were treated with 1 p.M FITC labeled ODNs delivered by DOTAP:DOPE
liposome at a charge ratio of 2.0. Results are the mean S.E. of 3 independent
experiments.













































Figure 2-6. Fluorescent microscopic pictures of Caki-I cells after FITC labeled ODNs
treatment. Caki-1 cells were treated with FITC labeled ODNs for 24 hr at a dose of 1 pM.
A) low magnification, 5x; B) high magnification, 20x


































Figure 2-7. Fluorescent microscopic pictures showing cellular uptake of FITC labeled
ODNs in Caki-l cells. Caki-1 cells were treated with FITC labeled ODNs at a dose of I
pM for 24 hr. A) bright field picture of confluent Caki-1 cells; B) fluorescent field
picture of confluent Caki-I cells: C) bright field picture of exponential phase Caki-1
cells; D) fluorescent field picture of exponential phase Caki-1 cells.











A498 Caki-2









Control









1 iM FITC-AS-ODNs

Figure 2-8. Delivery efficiency of FITC labeled ODNs by DOTAP:DOPE liposome in
other RCC cell lines (A498, Caki-2).The cells were treated with FITC labeled ODNs at a
dose of 1 p|M for 24 hr.













120-


100-


so-

so


s 40-

> 20
S. 10%FBS
2 D Serum Free




0 50 100 150

DOTAP:DOPE concentration (mglml)


Figure 2-9. Caki-1 cell viability after 24 hr treatment with different concentrations of
DOTAP:DOPE liposomes in serum free or 10% FBS conditions.














CHAPTER 3
AS-ODNS DESIGN AND IN VITRO ASSESSMENT

Introduction

Angiogenesis, a complex multi-step process involving the formation of new blood

vessels from pre-existing ones, is tightly regulated by both positive and negative

regulatory factors (Risau, 1997). These regulators, which include pro-angiogenic factors

such as basic fibroblast growth factor (bFGF) (Montesano et al., 1986), angiogenin (Gho

and Chae, 1997) and vascular endothelial growth factor (VEGF) (Leung et al., 1989;

Saleh et al., 1996; Asano et al., 1995; Borgstrom et al., 1996; Cheng et al., 1996), as well

as angiostatic peptides such as endostatin (O'Reilly et al., 1997; Perletti et al., 2000),

angiostatin (O'Reilly et al., 1994a; O'Reilly et al., 1994b; O'reilly et al., 1994) and

thrombospondin (Folkman and Shing, 1992; Folkman, 1995) are potential targets for

anti-angiogenic therapy of solid tumors (Smith et al., 1999; Bicknell and Harris, 1992;

Denekamp, 1999; Bicknell R and Harris A.L., 1992; Folkman, 1971; Denekamp J.,

1999). Among all these factors, VEGF and bFGF are believed to be most important

regulators in tumor angiogenesis (Risau, 1997;Siemeister et al., 1998).

VEGF is an endothelial cell specific mitogen, secreted as a 45 kDahomo dimer

protein. There are five human isoforms derived from alternative splicing (VEGF 121,

145, 165, 189, 206) as illustrated in Figure 3-1 (Tischer et al., 1991; Houck et al., 1991;

Poltorak et al., 1997). VEGFi21 and VEGFi65 are the only soluble isoforms and also the

most abundant, with VEGF165 being the major isoform and most powerful stimulator of

endothelial cell proliferation (Houck et al., 1992; Soker et al., 1997). VEGFi65 is









commonly expressed in a wide variety of human and animal tumors (Hanahan and

Folkman, 1996) and has been shown to induce angiogenesis both in vitro and in vivo

(Leung et al., 1989; Plate et al., 1992a; Plate et al., 1992b). It is currently believed that

this diffusible molecule is probably a key mediator of tumor angiogenesis (Ferrara,

1999a; Ferrara, 1999b). Indeed, the expression of VEGF has been related to fundamental

features of tumors, such as growth rate (Kim et al., 1993), microvessel density (Toi et al.,

1994) and vascular architecture (Drake and Little, 1999) as well as the development of

tumor metastasis (Weidner et al., 1991). A correlation between VEGF expression and

survival has been noted in some cancer patients (Gasparini et al., 1997; Maeda et al.,

1996).

Basic fibroblast growth factor is a prototype of a large family of 13 structurally

related, heparin-binding growth factors. It affects the growth, differentiation, migration

and survival of a wide variety of cell types (Bikfalvi et al., 1997). BFGF was originally

purified from the bovine pituitary gland as a 146-amino acid protein with a molecular

weight of 15 kD (Gospodarowicz, 1975). It was later found to represent a proteolytic

product of the primary 18 kD form (Bikfalvi et al., 1997). The amino acid sequence of 18

kD bFGF is highly conserved among species with 89-95% identity among human, bovine

and rat (Abraham et al., 1986a; Abraham et al., 1986b). This low level of divergence

suggests that there may be functional importance for all regions of bFGF. Larger forms of

bFGF have also been identified resulting from alternative CUG translation starting sites

(Florkiewicz et al., 1991a; Florkiewicz et al., 1991b). The use of different in-frame CUG

codons upstream of the conventional AUG start codon allows translation of several bFGF

isoforms with different molecular weight (Figure 3-6) (Okada-Ban et al., 2000). In









addition to the 18 kD isoform, alternative translation of 22, 22.5, 24 and 34 kD isoforms

are also possible (Araud et al., 1999). The main structural feature of the four high

molecular weight forms of bFGF is the presence of nuclear localization sequence which

directs the growth factors to the nucleus, whereas the 18 kD bFGF isoform initiated from

AUG start codon is essentially cytosolic.

BFGF is a multifunctional growth factor which has various effects in a large panel

of cells and tissues. It plays key role in development, remodeling and disease states in

almost every organ system (Bikfalvi et al., 1997). One of best characterized activities of

bFGF is its ability to regulate the growth and function of vascular cells such as

endothelial cell and smooth muscle cells. BFGF also regulates the expression of several

molecules thought to mediate critical steps during angiogenesis. These include interstitial

collagenase, urokinase type plasminogen activator (uPA), plasminogen activator inhibitor

(PAI-1), uPA receptor, and pi integrins (Montesano et al., 1992; Mignatti and Rifkin,

1993; Klein et al., 1993).. It is a potent angiogenic factor involved in tumor angiogenesis

and metastasis (Basilico and Moscatelli, 1992). Up-regulation of bFGF and its receptors

have been found in tumor tissues compare to normal tissues (Smith et al., 1999;

Dellacono et al., 1997; Arbeit et al., 1996). Clinically, associations between serum/urine

bFGF and cancer outcome have been shown in several tumor systems, including RCC

(Wechsel et al., 2000; Edgren et al., 1999; Nanus et al., 1993; Fujimoto et al., 1991),

breast cancer (Yiangou et al., 1997), head and neck cancer (Dietz et al., 2000), cervical

cancer (Sliutz et al., 1995), liver cancer (Poon et al., 2001), pancreas cancer (Ohta et al.,

1995), thyroid cancer (Sasaki et al., 2001) and glioma, neuroblastoma (Bredel et al.,

1997; Komuro et al., 2001).









In light of their important roles in tumor angiogenesis, VEGF and bFGF may be

attractive targets for anti-angiogenic therapeutic interventions applied to the treatment of

cancer. Attempts to abrogate the angiogenic activity of VEGF and bFGF have focused on

inactivating VEGF/bFGF through the use of antibodies against VEGF/bFGF or their

receptors (Mordenti et al., 1999; Kim et al., 1993; Aonuma et al., 1999; Lu et al., 2002;

Brekken et al., 2000) and VEGF soluble receptors (Lin et al., 1998), inhibiting

VEGF/bFGF receptor tyrosine kinases (Hennequin et al., 1999; Laird et al., 2002;

Solorzano et al., 2001; Ning et al., 2002) or suppressing VEGF and bFGF messages

(Smyth et al., 1997; Ellis et al., 1996a; Ellis et al,, 1996b; Nguyen et al., 1998a; Nguyen

et al., 1998b; Inoue et al., 2000). The latter relied on antisense oligonucleotides (AS-

ODNs) or antisense RNA (Eguchi et al., 1991; Mercola and Cohen, 1995a; Mercola and

Cohen, 1995b) to modulate gene expression by disrupting RNA expression. AS-ODNs

technology provides an approach for inhibiting gene expression with target specificity as

a particular advantage (Stein and Cheng, 1993; Engelhard, 1998a; Engelhard, 1998b).

AS-ODNs are also easy to produce in large quantities which make them potentially more

practical than antisense RNA vector delivery approaches.

In the present studies, AS-ODNs against VEGF and bFGF were designed and

their efficacy tested in vitro in the model of human RCC (Caki-1).

Materials and Methods

Cell Culture

The clear cell RCC cell lines Caki-l, Caki-2 and A498 were gifts from Dr. Susan

Knox (Stanford University, CA). Caki-1 cells were grown in Dulbecco's modified

minimum essential medium (DMEM, Invitrogen, Grand Island, NY) supplemented with









10% fetal bovine serum (FBS, Invitrogen, Grand Island, NY), 1% penicillin-streptomycin

(Invitrogen, Grand Island, NY) and 1% 200 mmol/L L-glutamine (Invitrogen, Grand

Island, NY).

Antisense Phosphorothioate Oligodeoxynucleotides (AS-ODNs)

Antisense and control ODNs (20-mers) were custom synthesized by Geno

Mechanix (Alachua, FL). AS-ODNs V515 was complementary to 5' UTRjust up-stream

of the translation start site (AUG codon) of VEGF mRNA: 5' CTC ACC CGT CCA

TGA GCC CG 3'. Scramble sequence: 5'- CAC CCT GCT CAC CGC ATG GC 3';

sense sequence: 5' CGG GCT CAT GGA CGG GTG AG 3' and an inverted sequence:

5'-GCC CGA GTA CCT GCC CAC TC 3', were used as controls ODNs. AS-ODNs

B460 was complementary to the translation start site (AUG codon) of bFGF mRNA: 5'

TCC CGG CTG CCA TGG TCC CT 3', AS-ODNs B471 was complimentary to the

coding region of bFGF mRNA: 5' CGT GGT GAT GCT CCC GGC TG 3'; AS-ODNs

B931 was complimentary to the 3' UTR: 5' GAT GTG GCC ATT AAA ATC AG 3'.

Scramble sequence: 5' GCC TGG ACC CTG GCT CTC TC 3'; sense sequence: 5' AGG

GAT GGC TGC CGG GA 3' and an inverted sequence: 5' TCC CTG GTA CCG TCG

GCC CT 3' were used as controls. All AS-ODNs were suspended in sterile and endotoxin

free water at a concentration of I mM, aliquoted and stored at -200C.

DOTAP:DOPE Liposome Preparation

Cationic liposomes were prepared using the method described by Tang (Tang and

Hughes, 1999). Briefly, cationic lipid 1,2-dioleoyloxy-3-(trimethylammonium) propane

(DOTAP) was dissolved in chloroform and mixed with a helper lipid 1,2-dioleoyl-3-sn-

phosphatidylethanolamine (DOPE) (Avanti Polar-Lipids, Alabaster, Al) at a molar ratio









of 1:1. The mixture was evaporated to dryness in a round-bottomed flask using a rotary

evaporator at room temperature. The resulting lipid film was dried by nitrogen for an

additional 10 min to evaporate any residual chloroform. The lipid film was re-suspended

in sterile water to a final concentration of I mg/ml based on the weight of cationic lipid.

The resultant mixtures were shaken in a water bath at 35C for 30 min. The suspensions

then were sonicated using a Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA) for 1

min at room temperature to form homogenized liposomes. The particle-size distribution

of liposomes was measured using a NICOMP 380 ZLS instrument (Santa Barbara, CA).

The average particle diameter was 144.0 + 77.0 nm. Liposomes were stored at 4C and

used within 3 months.

VEGF Enzyme Immunoassay

Caki-l cells (1 x 105) were set in 60 mm dishes and allowed to attach overnight.

The medium then was removed and replaced with AS-ODNs in serum free medium with

liposome (DOTAP:DOPE) and incubated for 5 hr. Fresh medium containing 10% FBS

then was added. After 24 hr of incubation, or at different time points for the time course

studies, the VEGF concentration was determined in the medium using a human VEGF

ELISA kit (R &D Systems, Minneapolis, MN).

Enzyme Immunoassay of bFGF

Caki-l cells (I x 105) were set in 60 mm dishes and allowed to attach overnight.

The medium then was removed and replaced with AS-ODNs in serum free medium with

liposome (DOTAP:DOPE) and incubate for 5 hr. Fresh medium containing 10% FBS

then was added. Caki-1 cells were collected 72 hr later, washed and suspended 1 x 106

cells in 1 ml PBS containing protease inhibitors (100 pg/ml Phenylmethanesulphonyl









fluoride, 20 [pg/ml leupeptin, 3 lpg/ml aprotinin). The suspension was subjected to 3

freeze-thawing cycles, ultrasonication for 5 s (100 W) on ice, and was centrifuged at

14,000 g for 10 min. The supernatant containing the intracellular bFGF was used for the

bFGF concentration determination (human bFGF immunoassay kit, R & D Systems,

Minneapolis, MN).

VEGF and bFGF Relative Quantitative RT-PCR

Caki-I cells were set at 3 x 105 in 100 mm dishes and allowed to attach overnight.

The cells were then treated with 1 JiM VEGF antisense (V515), bFGF antisense (B460)

or control ODNs as described. 24 hr later the cells were collected and the total RNA was

isolated using RNeasy Mini Kit (Qiagen, Valencia, CA) and RNA concentrations were

determined by UV spectrophotometry. A 2 pg total RNA sample was used to reverse

synthesize cDNA using Superscript II reverse transcriptase (Invtrogen, Grand Island,

NY). A 2.5 pl aliquot of the reverse transcriptase reaction product then was used for the

PCR reaction. VEGF PCR reactions were carried out with a VEGF gene specific relative

RT-PCR Kit (Ambion, Austin, TX). BFGF PCR reactions were carried out using a

forward primer: 5'GCA GCC GGG AGC ATC ACC A 3' and reverse primer: 5' GCC

CAG TTC GTT TCG GTG CCC A 3' (Campbell et al., 1999). The PCR reactions were

run 22 cycles (denature 940C 30 s, anneal 600C 60 s, extension 720C 60 s) in aDNA

Engine 200 (MJ research, Waltham, MA). PCR products then were run in 2% agrose gel

and stained by ethidium bromide. The gels were visualized and analyzed (Gel Doc 2000

gel documentation system, Bio-Rad, Hercules, CA). All PCR preparations were carried

out in a laminar flow hood using aerosol resistant plugged pipette tips.









FGF Receptors 1-4 RT-PCR

Total RNA of exponential phase Caki-l cell was isolated using RNeasy Mini Kit

(Qiagen, Valencia, CA) and RNA concentrations were determined by UV

spectrophotometry. A 2.5 [l aliquot of the reverse transcriptase reaction product then was

used for the PCR reaction. Primers for human FGFR 1-4 designed by Tartaglia etc were

used (Tartaglia et al., 2001). The PCR reactions were run for 30 cycles (denature 94C 30

s, anneal 60"C 60 s, extension 720C 60s) in a DNA Engine 200 (MJ research, Waltham,

MA). The specificity of the cDNA amplifications were then verified by endonuclease

restriction analyses (Tartaglia et al., 2001). All PCR preparations were carried out in a

laminar flow hood using aerosol resistant plugged pipette tips. Negative controls without

template DNA were included in each assay. 18S primer set (Ambion, Austin, TX) was

used as positive control.

Cell Cycle Assays

Caki-l cells were plated in 60 mm dishes at 2 x 105 cells per dish and allowed to

attach overnight. The cells were then treated with I pM B460 or control ODNs mixed

with DOTAP:DOPE as described above. 48 hr later, the cells were trypsinized, counted

and fixed in 50% ethanol overnight. Before analyzed by FACS, the cells were treated

with 1 mg/ml RNase (in PBS) for 30 min. The samples were then washed with PBS twice

and resuspended in 25 mg/ml propidium iodine (PI) in PBS at a concentration of 1 x 106

cells/ml. The cells were stained with PI in darkness for 15 min and were analyzed by

FACS for cell cycle distribution on a Beckman Dickinson flow cytometer made available

through the University of Florida Core Facility for Flow Cytometry.









Apoptosis Assays

Caki-1 cells were set in 2-well chamber slides and treated with 1 gM B460 or

control ODNs as described earlier. 48 hr later, the cells were washed and fixed in 4%

para-formaldehyde solution for Fluorometric TdT-mediated dUTP Nick-End labeling

(TUNEL) assay. Briefly, the cells were permeabilized in 0.2% Triton X-100 solution for

5 min. DNA strand breaks were then labeled with fluorescein-12-dUTP in TdT

incubation buffer at 370C for 1 hr. The samples were then counterstained with 1 pg/ml

PI in PBS, which binds to the A-T rich regions of DNA. Localized green fluorescence of

apoptotic cells (fluorescein-12-dUTP) in a red background (PI) was detected by

fluorescence microscopy. The percentage of apoptotic cells was obtained by dividing the

number of cells with green fluorescence by the total number of cells counted A

minimum of 300 cells were counted for each condition.

Results

VEGF AS-ODNs Design and Assessment

Since VEGF has multiple isoforms resulting from alternative splicing (Figure 3-

1), AS-ODNs design was targeted at the common region of all isoforms, regions around

the AUG start codon. After screening several different designs, AS-ODNs V515 which is

complimentary to the 5'UTR region just up-stream of the AUG start codon of the VEGF

gene, was found to be most effective. The results showed that after 24 hr treatment with 1

pM VEGF AS-ODNs (V515) delivered by cationic liposome (DOTAP:DOPE), the

medium VEGF levels were significantly reduced from a normal of 850 pg/ml/106 cells to

250 pg/ml/106 cells (p<0.05, student's I-test) (Figure 3-2). This antisense effect was

sequence, and target region specific. Treating Caki-1 cells with liposome vehicles









(DOTAP: DOPE) or control scramble ODNs did not affect VEGF levels. Similarly,

treatment with sense or inverted sequence ODNs failed to reduce VEGF expression.

Continued exposure of Caki- cells to the VEGF AS-ODNs (V515) resulted in a

constant repression of VEGF in the culture medium (Figure 3-3). However, if the culture

medium containing V515 was replaced with fresh medium 24 hr later, the VEGF levels

in the Caki-l cell medium gradually recovered, and reached about -80% of that found in

the untreated Caki-l cell medium in about 7 days (Figure 3-3).

This repression of VEGF expression by V515 was also dose dependent (Figure 3-

4). For example, a 24 hr treatment with 0.5 giM, reduced the medium VEGF level to 56%

of control (p<0.05, student's M-test) whereas a 1 gM dose down-regulated the VEGF level

to 22% of control (p<0.05, student's t-test).

VEGF mRNA levels in different AS-ODNs treatment groups also were

determined (Figure 3-5). The results indicated a marked inhibition of VEGF mRNA after

treatment with V515 which was absent in cells treated with scramble control ODNs. This

result indicated that RNase H plays an important role in the function of V515.

BFGF AS-ODNs Design and Assessment

Alternative translation utilizing CUG start codons other than the AUG start codon

leads to different isoforms bFGF gene products (Figure 3-6). In order to target all the

bFGF isoforms using the same AS-ODNs sequence, the AS-ODNs were designed to

target the common regions of all isoforms, especially around the AUG start codon.

Effective AS-ODNs sequences against bFGF were identified and Caki-l cells treated

with them showed bFGF levels significantly lower than those normally observed (720

pg/ml/106 cells) (Figure 3-7). This effect was sequence and target region specific. The









AS-ODNs complimentary to the start codon (AUG) region (B460) was found to be the

most effective. For example, the cellular bFGF level of B460 treated Caki-1 cells was

found to be about 41% of that found in control or untreated cells (p<0.05, student's t-

test). By comparison, the AS-ODNs complimentary to the 3' UTR (B931) or coding

region (B471) were less effective at down regulating bFGF expression (57% and 65% of

control values respectively, p<0.05, student's t-test). Since B460 had the most prominent

inhibitory effect, it was used in all subsequent studies. Treating Caki-1 cells with control

scramble ODNs or liposome vehicles did not affect bFGF levels in Caki-1 cells.

Similarly, treatment with sense or inverted sequence ODNs failed to reduce bFGF

expression. This inhibitory effect was also found to be AS-ODNs dose dependent (Figure

3-8). While a low dose of 0.5 pM B460 reduced the cellular bFGF level to about 80% of

control, a high dose of 5 [.M B460 led to a reduction by 65%.

BFGF mRNA levels in different PS-ODNs treatment groups also were determined

(Figure 3-9). The results indicated a marked inhibition of bFGF mRNA after treatment

with B460 which was absent in cells treated with scramble ODNs. Again, this suggests a

role for RNase H in the efficacies of bFGF AS-ODNs B460.

Because FGF can have mitogenic effects in renal cells (Gospodarowicz et al.,

1986; Issandou and Darbon, 1991), the influence of antisense and control ODNs

treatment on Caki-1 cell growth was investigated. Control ODNs or liposome vehicles

showed no effect on Caki-1 cell growth (Figure 3-10). In contrast, Caki-1 cell growth was

significantly inhibited by AS-ODNs targeted against different regions of bFGF mRNA.

B460 was found to be the most effective while AS-ODNs targeting the 3' UTR (B931) or

coding region (B471) showed less cell growth inhibition. When comparing the data of









Figures 3-7 and 3-10, it also is apparent that the extent of Caki-1 cell growth inhibition

by different AS-ODNs was closely related to their potency in down regulating bFGF

expression.

In order to gain a better understanding of the underlying mechanisms of the

observed growth inhibitory effect, FGF receptor expression was determined in Caki-1

cells (Figure 3-11). It was found that 3 out of 4 FGF receptors involved in the bFGF

signal transduction pathway were expressed by Caki-1 cells, indicating that bFGF may

play an autocrine role in Caki-l cells. Additional studies indicated that B460 treatment

had small but significant effects on Caki-l apoptosis and cell cycle distribution (Figures

3-12 and 3-13). However, clonogenicity studies showed no significant difference between

B460 treated and control cells indicating that B460 treatment had no direct cell killing

effect on Caki-I cells (data not shown). These findings suggest that blocking the bFGF

signal transduction pathway may affect Caki-I cell growth through cell cycle inhibition

and induction of apoptotic cell death.

Discussion

Although dramatic advances have been made in the treatment of cancer, the

development of efficacious anticancer agents still lags behind the rapid strides in our

understanding of cancer biology, especially with the advent of molecular biology. The

continued progress in our knowledge of the biology of neoplasm and in the identification,

cloning and sequencing of genes critical to tumor cell function permits the exploitation of

this information to develop specific agents that may directly modulate the function of

these genes or their protein products. One methodology that takes direct advantage of









molecular sequencing data involves the use of antisense oligonucleotides (Ho and

Parkinson, 1997).

The antisense-mediated gene inhibition was first introduced in 1978 by

Stephenson and Zamecnik (Zamecnik and Stephenson, 1978; Stephenson and Zamecnik,

1978). The underlying concept is relatively straightforward: the use of a sequence,

complementary by virtue of Watson-Crick basepair hybridization, to a specific mRNA

can inhibit its expression and then induce a blockade in the transfer of genetic

information from DNA to protein.

The selection of an appropriate target sequence is the first step in the process of

AS-ODNs drug development. As a matter of fact, the hybridization between AS-ODNs

and the target mRNA, which has a particular three dimensional shape resulting from

secondary and tertiary structures, depends on the accessibility of the target sequence.

Only limited stretches of mRNA sequences are actually available for heteroduplex

formation with AS-ODNs. Still there is no sure way to determine a prior which AS-

ODNs sequence would work best (Cohen, 1989;Woolf et al., 1992; Brysch and

Schlingensiepen, 1994). The region surrounding the start codon (AUG) is probably the

most popular target, followed by 5'UTR, coding regions or splicing sites. In the present

studies, AS-ODNs sequence design and selection focused mainly on the start codon

region of VEGF and bFGF genes. This region is also common to all isoforms of these

two growth factors (Figure 3-1 and 3-6).

The most commonly used AS-ODNs are 18-20 bases in length. According to

statistical calculations, a particular sequence of 13 bases in RNA and of 17 bases in DNA

should be found only once in the entire human genome, thus representing unique









elements within the cell (Helene and Toulme, 1990). It also has been found that the

activities of AS-ODNs increased with AS-ODNs length, but the increased

thermodynamic stability of hybridization observed with AS-ODNs binding to non-target

mRNA sequences that may be similar, but not identical to the target sequence, results in

reduced specificity (Monia et al., 1992). Thus, an AS-ODNs length of 20-mer is usually

considered optimal and therefore was used in the present studies for all the designed AS-

ODNs. To ensure the specificity of the target sequence, all designed AS-ODNs sequences

were checked for global sequence comparison using the Basic Local Alignment Search

Tool (BLAST) from the National Center for Biotechnology Information (NCBI,

http.//www.ncbi.nlm.nih.gov/BLAST/). Only sequences specific for the VEGF/bFGF

gene and having at least 4 miss-match bases with other genes were used. In addition,

polyguaosine (GGGG), which is known to exert non-antisense effects was avoided

(Benimetskaya et al., 1997). Lastly, all the AS-ODNs designed were examined for

secondary structures such as hairpins, self-dimers, and cross-dimers using Netprimer

(PREMIER Biosoft International, Palo Alto, CA).

Since cells contain a variety of exo- and endonucleases that can degrade ODNs,

nucleotide modifications have been made to make the AS-ODNs more resistant to

nuclease digestion than the native ODNs that have phosphodiester linkages in their

backbones. The most widely explored analogues have been the phosphorothioates, in

which one of the nonbridging oxygen atoms in each intemucleoside phosphate linkage is

replaced by a sulfur atom. This modification is easily adapted to automated synthesis and

confers metabolic stability because its resistance to degradation by DNase (Stein et al.,

1988). In addition, these analogues retain water solubility and permit RNase H mediated









hydrolysis of the target mRNA strand. This modification has been successfully used in a

variety of investigations (Galderisi et al., 1999; Ho and Parkinson, 1997; Crooke, 1998).

Based on these favorable properties and extensive information available for its

application, phosphorothioate modification was used in the current investigations of

VEGF/bFGF AS-ODNs.

AS-ODNs may exert biological activity through a variety of mechanisms.

Although some of the mechanisms of inhibition have been characterized, rigorous proof

for others is still frequently lacking. Two classes of AS-ODNs can be discerned: (a) the

RNase-H dependent ODNs, which induce the degradation of mRNA; and (b) the steric-

blocker ODNs, which physically prevent or inhibit the progress of splicing or

translational machinery (Crooke, 1992; Dias N and Stein, 2002).

The most commonly implicated antisense mechanisms relate to RNase H

mediated hydrolysis of the target mRNA. This is also the case for most of the antisense

drugs investigated in the clinic. RNase H is a ubiquitous enzyme that hydrolyzes the

RNA strand of the RNA/DNA hybrid. Thus the binding of AS-ODNs to its target mRNA

may induce digestion of the message. AS-ODNs assisted RNase H dependent reduction

of target RNA expression can be quite efficient, reaching 80-95% down-regulation of

protein and mRNA expression (Dias N and Stein, 2002). Furthermore, in contrast to the

steric-blocker ODNs, RNase H dependent ODNs can inhibit protein expression when a

much wider region of the mRNA is targeted. Thus, unlike most steric-blocker ODNs that

are efficient only when targeted to certain 5' UTR or AUG start codon regions, RNase H

dependent AS-ODNs can exert effects when targeted to widely separated areas in the

coding region as well (Dean and McKay, 1994; Larrouy et al., 1992). In the present









studies, mRNA levels of VEGF and bFGF were significantly down-regulated after the

AS-ODNs treatment (Figures 3-5 and 3-9). These findings suggest that current AS-ODNs

function mainly through the RNase H mechanism.

Successful design and evaluation of AS-ODNs relies on their efficient delivery of

into the cytoplasm, where they can exert their antisense effect. A cationic liposome

(DOATP:DOPE) based delivery system was used to deliver AS-ODNs in the present

studies. The evaluation and optimization of this delivery system has been discussed in

detail in Chapter 2.

Following the aforementioned guidelines in AS-ODNs design and selection,

effective AS-ODNs against VEGF and bFGF have been identified. Treatment of Caki-1

cell with the AS-ODNs led to significant repression of VEGF and bFGF expression

levels (Figures 3-2 and 3-7). These effects were ODNs sequence specific, dose dependent

and could be achieved at low, non-toxic doses. These results indicate that AS-ODNs can

be used to efficiently modulate the specific target gene expressions.













VEGF mRNA


VEF 121 mm 121 3 I 4

VEOF 145 1 2 3 4

VEGF 165 Im 1 2 3 1 4 //

VEGF 189 1 2 3 4

VEGF206 1 M 2 3 I4 IS //
V515





Figure 3-1. VEGF mRNA structure showing all isoforms derived from alternative

splicing.














100

0



0
I.
LJ 20
> 0


Untreaed DOTAP Scrarble Snse Invrted V515

Treatment




Figure 3-2. VEGF levels in culture medium of Caki-1 cells treated with different AS-
ODNs. Caki-1 cells were untreated, treated with vehicle (DOTAP) only, treated with I
gM control ODNs or VEGF AS-DONs (V515) for 24 hr. The 100% VEGF expression
level of the untreated group corresponds to -800 pg/ml/106 cells. Each bar represents the
mean S.E. of 3 independent experiments. The star indicates a statistically significant
difference from the untreated group (p<0.05, student's t-test).













120


S100





ua
S so,


s*


U 20.
> 20,


O Continued V515 treatment
* 24 Ih V515 treatment


0-*
0 1 2 3 4 5 6 7 8

Time (day)



Figure 3-3. VEGF levels in the culture medium of Caki-1 cells at different times after
V515 treatment. Media containing V515 were either unchanged (1) or replaced with
fresh medium after 24 hr (0). Each datum point represents the mean S.E. of 3
independent experiments.













120-





8 80-

a0-
So

40
240-
u. *
20- *
^0---- --------------a


0 1 2 3 4 5

Antisense PS-ODNs dose (pM)




Figure 3-4. VEGF levels in culture medium of Caki-1 cells after treatment with different
doses of V515. The 0 dose represents the Caki-1 cells treated with 5 giM control scramble
ODNs. Each datum point represents the mean S.E. of 3 independent experiments. Stars
indicate statistical significance compared to the control ODNs treated (p<0.05, student's
t-test).











A Untreated Scramble V515

18W

VZi/F


B 200-

S180-



1 120

so

S60-
E 40
IL
0 20
> 0-


Untreated
Untreated


Scramble V515


Treatment


Figure 3-5. Message RNA levels in Caki-l cells either untreated, treated with a 1 JM
dose of scramble ODNs or VEGF AS-ODNs (V515). A) Representative relative RT-PCR
results; B) Relative VEGF mRNA levels of Caki-l cells after the treatment. The star
indicates a statistical significance compared to untreated control (p<0.05, student's t-test).







70





bFGF mRNA
CUG CUG CUGCUG AUG STOP
86 319 346 361 486 951
s' I I I I I I
5'

bFGF proteins
S18 kD
I 22 kD
I I 22.5 kD
I I I 24 kD
SI 34kD
SNuclear localization signal




Figure 3-6. Message RNA structure and protein products of bFGF gene. Modified from
Okada-Ban (Okada-Ban et al., 2000).

















= 000
600



400
2soo *
L. 300
U-
200

100

0
Untreated DOTAP Scramble Sense Intted B460 B471 B31

Treatment





Figure 3-7. Cellular bFGF levels of Caki-l cells after AS-ODNs treatment. Caki-1 cells
were either untreated, treated with liposome alone, I IM control ODNs or bFGF AS-
ODNs for 24 hr. Each bar represents the mean S.E. of 3 independent experiments. Stars
indicate statistically significant difference from the untreated control group (p<0.05,
student's t-test).
















100




60



2 \

0 20 -
0-


0 1 2 3 4 5

B460 dose (pM)



Figure 3-8. Cellular bFGF levels in culture medium of Caki-1 cells after treatment with
different doses of B460. The 0 dose Caki-1 cells treated with 5 ltM control scramble
ODNs. Each datum point represents the mean S.E. of 3 independent experiments. Stars
indicate statistically significant differences compared to the control ODNs treated group
(p<0.05, student's t-test).










A Untreated DOTAP


18s
bFGF


amur a" me i- h man A* 1-


o Iso


Sloo *


z
E
0
U.
0
Untreated DOTAP Scramble B460
Treatment

Figure 3-9. Message RNA levels in Caki-l cells either untreated, treated with a 1 gM
dose of scramble ODNs or bFGF AS-ODNs (B460). A) Representative relative RT-PCR
results; B) Relative bFGF mRNA levels of Caki-1 cells after the treatment. The star
indicates a statistically significant difference compared to the untreated control group
(p<0.05, student's t-test).


Scramble B460















T 100-

s 80-
08 *


U 4-


S20-

0
Untreated DOTAP Scramble Sense Inverted 480 B471 BM31

Treatment




Figure 3-10. Effect of AS-ODNs treatment on Caki-1 cell growth. Caki-I cells were
either untreated, treated with liposome alone, 1 gM control ODNs or bFGF AS-ODNs for
4 days and the number of cells determined. Each bar represents the mean S.E. of 3
independent experiments. Stars indicate statistically significant differences compared to
untreated control group (p<0.05, student's t-test).
































Figure 3-11. FGF receptor expression in Caki-l cells. RT-PCR results of FGF receptors
1-4 and control 18s expression in Caki-l cells.















































Figure 3-12. Apoptotic cell death in Caki-1 cells either untreated or treated with bFGF
AS-ODNs. A) Representative picture of untreated Caki-1 cells stained with DeadEndTM
Fluorometric TUNEL System: B) representative picture of Caki-1 cells after treatment
with B460 stained with DeadEndTM Fluorometric TUNEL System.







77




10-
9
a 8
S 7
6.* -6



S 3-
< 4


0 2
C I
0
Untreated DOTAP Scramble B460

Treatment



Figure 3-13. Apoptotic cell death in Caki-1 cells after AS-ODNs treatment. Caki-l cells
were either untreated, treated with liposome alone, treated with I iM scramble control
ODNs or bFGF AS-ODNs. Each bar represents the mean S.E. of 3 independent
experiments. The star indicated a statistically significant difference from the untreated
control group (p<0.05, student's 1-test).














Untreated
C Control
60 [O B460
0


4 45


C 30

1 -




GO-G1 S G2-M

Cell cycle distribution



Figure 3-14. Effect of bFGF AS-ODNs treatment on the cell cycle distribution of Caki-l
cells. Each bar represents the mean S.E. of 3 independent experiments. Stars indicate
statistical significant differences compared to the untreated control group (p<0.05,
student's -test).














CHAPTER 4
ANTI-ANGIOGENIC EFFICACY STUDIES

Introduction

The greater understanding of the process of tumor angiogenesis, coupled with the

notion that tumors require a blood supply to grow and metastasize, has fueled the

research for strategies that block or disrupt the angiogenic process. Moreover, because

normal vascular endothelial cells turn over so slowly, conventional wisdom suggests that

an anti-angiogenic approach to cancer therapy should offer improved efficacy and

reduced toxicity, with much less potential for drug resistance.

Angiogenesis is a complex process with multiple, sequential and interdependent

steps (Fidler, 1999). This complexity creates many potential targets for inhibition. Key

characteristic of the immature vasculature of tumors have allowed the development of

several categories of anti-angiogenic agents (Kerbel, 2000). Preclinical studies have

identified agents that (i) inhibit endothelial cell activation (ii) inhibit endothelial

proliferation/migration (iii) inhibit basement membrane degradation and (iv) inhibit

integrin receptor activation.

Angiogenesis can be qualitatively and quantitatively measured in a large variety

of in vitro and in vivo model systems. As mentioned before the angiogenic cascade can be

dissected into different sequential steps so that can be studied separately in vitro.

Research has mainly focused on the proliferation and migration of endothelial cells as

key elements for angiogenic potential in vitro. For this research, different endothelial cell

sources can be utilized. For human tumor research most laboratories make use of









HUVECs. Although readily available, a major advantage, the major drawback of these

cells is their macrovascular origin, which makes them less suitable for studies on

angiogenesis, a microvascular process. In recent years, microvascular endothelial cells

derived from different organs have been established and become commonly available.

Protocols for isolating purified tumor endothelial cells also have been developed (St

Croix et al., 2000).

Assays to study proliferation of endothelial cells are based on cell counting, radio-

labeled thymidine incorporation, or on colorimetric assays for measurement of

mitochondrial activity. Detection of cell death also is used to determine cell growth

effects. To measure endothelial cell migration, Boyden chambers are primarily used.

Though an easier system based on wounding of a confluent monolayer of endothelial

cells and measuring wound width or invading cells as a function of time is also available,

it maybe physiologically less relevant to tumor angiogenesis. In the present studies, we

established a co-culture system based on modified boyden chambers to evaluate

endothelial cell proliferation and migration potential (Figure 4-1 and 4-4). In order to

mimic the in vivo interaction of tumor and endothelial cells, co-cultured tumor cells were

the primary source ofpro-angiogenic growth factors for the endothelial cells.

The advantage of the in vitro assays is clearly the control that can be exerted over

selected parameters. However the angiogenic cascade consists of multiple steps in their

entirety, in vivo investigations are needed. The most frequently used in vivo assay

systems are the chicken chorioallantoic membrane assay (Nguyen et al., 1994a), the

comeal pocket assay (Conrad et al., 1994), transparent chamber preparations such as the

dorsal skin-fold chamber (Algire G.H., 1943; Lichtenbeld et al., 1998), the cheek pouch









window (Shubik et al., 1976) and polymer matrix implants (Mahadevan et al., 1989;

Plunkett and Hailey, 1990). A simpler system using intradermal implantation of tumors

cells is also available to study the tumor angiogenic process in vivo (Sidky and Auerbach,

1976). This assay has been validated for evaluating tumor-induced angiogenesis in vivo

using a variety of different tumor models and treatment interventions (Lindner and

Borden, 1997; McMillan et al., 1999; Danielsen and Rofstad, 1998) and was applied in

the present studies.

Through the utilization of both the co-culture system in vitro and the intradermal

angiogenesis assay in vivo, objective and reasonable assessments of the anti-angiogenic

efficacy of treatment interventions can be achieved. In the present studies, the anti-

angiogenic efficacy of VEGF and bFGF AS-ODNs treatments were evaluated using these

in vitro and in vivo models.

Materials and Methods

Cell Culture

The clear cell RCC cell line Caki-1 was a gift from Dr. Susan Knox (Stanford

University, CA). Caki-1 cells were grown in Dulbecco's modified minimum essential

medium (DMEM, Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine

serum (FBS, Invitrogen, Grand Island, NY), 1% penicillin-streptomycin (Invitrogen,

Grand Island, NY) and 1% 200 mmol/L L-glutamine (Invitrogen, Grand Island, NY).

The mouse heart endothelial cell line (MHE) was a gift from Dr. Robert Auerbach

(University of Wisconsin, WI). MHE cells were grown in DMEM supplemented with

10% heat inactivated FBS, 1% penicillin-streptomycin and 1% 200 mmol/L L-glutamine.

Human microvascular endothelial cell from the lung (HMVEC-L) cells were obtained









from Clonetics (San Diego, CA). HMVEC-L cells were grown in EBM-2-MV (Clonetics,

San Diego, CA) supplemented with 5% FBS.

Antisense Phosphorothioate Oligodeoxynucleotides (AS-ODNs)

Antisense and control ODNs (20-mers) were custom synthesized by Geno

Mechanix (Alachua, FL). The entire backbone of all ODNs was phosphorothioate

modified. AS-ODNs V515 was complementary to 5' UTR just up-stream of the

translation start site (AUG codon) of VEGF mRNA: 5' CTC ACC CGT CCA TGA

GCC CG 3'. Scramble sequence: 5'- CAC CCT GCT CAC CGC ATG GC 3'; sense

sequence: 5' CGG GCT CAT GGA CGG GTG AG 3' and an inverted sequence: 5'-

GCC CGA GTA CCT GCC CAC TC 3', were used as ODNs controls. AS-ODNs B460

was complementary to the translation start site (AUG codon) of bFGF mRNA: 5' TCC

CGG CTG CCA TGG TCC CT 3'. Scramble sequence: 5' GCC TGG ACC CTG GCT

CTC TC 3'; sense sequence: 5' AGG GAT GGC TGC CGG GA 3' and an inverted

sequence: 5' TCC CTG GTA CCG TCG GCC CT 3' were used as controls. All ODNs

were suspended in sterile and endotoxin free water at a concentration of I mM, aliquoted

and stored at -200C.

DOTAP:DOPE Liposome Preparation

Cationic liposomes were prepared using the method described by Tang (Tang and

Hughes, 1999). Briefly, cationic lipid 1,2-dioleoyloxy-3-(trimethylammonium) propane

(DOTAP) was dissolved in chloroform and mixed with a helper lipid 1,2-dioleoyl-3-sn-

phosphatidylethanolamine (DOPE) (Avanti Polar-Lipids, Alabaster, Al) at a molar ratio

of 1:1. The mixture was evaporated to dryness in a round-bottomed flask using a rotary

evaporator at room temperature. The resulting lipid film was dried by nitrogen for an









additional 10 min to evaporate any residual chloroform. The lipid film was re-suspended

in sterile water to a final concentration of 1 mg/ml based on the weight of cationic lipid.

The resultant mixtures were shaken in a water bath at 35C for 30 min. The suspensions

then were sonicated using a Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA) for 1

min at room temperature to form homogenized liposomes. The particle-size distribution

of liposomes was measured using a NICOMP 380 ZLS instrument (Santa Barbara, CA).

The average particle diameter was 144.0 77.0 nm. Liposomes were stored at 4C and

used within 3 months.

Co-culture Assay

Transwell (Coming, Coming, NY) 6-well dishes with a membrane pore size of

0.4 uM were used. Caki-1 cells were seeded at 5 x 104 in the transwell inserts and MHE

or HMVEC-L cells were plated at 5 x 104 per well in the 6-well dishes and allowed to

attach overnight. The Caki-l cell medium then was replaced with serum free medium

containing 1 pM V515 or B460 AS-ODNs or control ODNs delivered with liposome

(DOTAP:DOPE). After a 5 hr of treatment, medium containing 10% heat inactivated

FBS was added to yield a final FBS concentration of 2.5%. The transwells containing

treated Caki-l cells were assembled with 6-well dished containing MHE and HMVEC-L

cells and incubated at 370C for 72 hr at which time the numbers of MHE or HMVEC-L

cells were determined by haemocytometer count (Figure 4-1).

Migration Assay

Caki-1 cells were set at 1 x 105 per well in 24-well dishes and allowed to attach

overnight. The Caki-1 cells then were treated with 1 .M V515 or B460 AS-ODNs or

control ODNs for 24 hr. HTS FluoroBlok inserts (Becton Dickinson, Franklin Lakes, NJ)









with a pore size of 8.0 urm were assembled into the 24-well dish with the Caki-l cells.

MHE or HMVEC-L cells were grown in T-150 flasks to about 80% confluence. The

endothelial cells were stained in medium containing 10 p.g/ml Di-l (Molecular Probes,

Eugene, OR) for 24 hr, washed 4 times with PBS, collected and added into the

FluoroBlok inserts (5 x 104 MHE or HMVEC-L) and incubated for another 24 hr. The

number of migrated endothelial cells then was determined by direct measurement of the

fluorescence in the bottom well using a CytoFluor 4000 plate reader (Perceptive

BioSystems, St. Paul, MN). (Figure 4-4)

Intradermal Angiogenesis Assay

Caki-1 cells were treated with AS-DONs for 5 hr in vitro as described before. The

cells were then collected and inoculated intradermally (5 x 104) in a volume of 10 .1 at 4

sites on the ventral surface of nude mice. One drop of 0.4% trypan blue was added to the

cell suspension before injection, which making it lightly colored, simplified subsequent

location of the sites of injection. Three days later the mice were killed, the skin was

carefully separated from the underlying muscle and the number of vessels counted using

a dissecting microscope (Sidky and Auerbach, 1976). Scoring of all of the reaction areas

was carried out at the same magnification (5x) and only vessels readily detected at this

magnification were counted. The sites of injection, recognized by local swelling and blue

staining, were exposed by carefully removing fat or other tissue covering the area. All

vessels that touched the edge of the tumor inoculates were counted. All the animals in the

experiments were pre-coded and vessel counts in each animal were scored twice. The

resultant data points for each treatment group were pooled for statistical analysis

(Wilcoxon rank sum test).









Results

Anti-angiogenic efficacy of VEGF/bFGF AS-ODNs treatments was first

evaluated in vitro. The transwell co-culture system was used to examine the effect of AS-

ODNs treatment of Caki-1 cells on the proliferation of co-cultured endothelial cells

(Figure 4-1). This setting allowed the constant exchange of growth factors without direct

tumor-endothelial cell-cell interaction and mimicked the paracrine interaction between

tumor and endothelial cells. Since a human RCC tumor cell line and xenograft in nude

mice were used in these studies, both human (HMVEC-L) and mouse (MHE) endothelial

cells were studied. Caki-1 tumour cells were grown in transwells with 0.4 pmr membrane

pores. The effects of pretreating Caki-1 tumor cells with VEGF or bFGF AS-ODNs on

endothelial cell proliferation then were determined (Figure 4-2 and 4-3). The results

showed that compared to untreated Caki-l cells, Caki-l cells pre-treated with V515 or

B460 significantly inhibited both HMVECV-L and MHE cell proliferation. Once again,

treating Caki-1 cells with a variety of control PS-ODNs had no effect on HMVEC-L or

MHE cell growth.

To test whether a reduction in VEGF or bFGF expression by tumor cells could

affect endothelial cell migration, HMVEC-L or MHE cells were stained with 10 gg/ml

Di-I for 24 hours and added into Fluoroblok inserts placed into 24 well dishes containing

Caki-l tumour cells treated with V515. The number of pre-labelled endothelial cells

which migrated through the 8 mu pore size membranes in a 24 hr period were quantified

by determining the fluorescence intensity in the bottom well (Figure 4-4). The results

showed (Figure 4-5) that 24 hr after co-culturing the two cell populations -45%

(p<0.05, student's t-test) and 37% (p<0.05, student's t-test) fewer MHE or HMVEC-L









cells respectively migrated through the membrane in the presence of V515 treated Caki-1

cells compared to untreated or scramble control AS-ODNs treated Caki-1 cells.

Similarly, -37% (p<0.05, student's t-test) and -33% (p<0.05, student's t-test) fewer

MHE or HMVEC-L cells respectively migrated through the membrane in the presence of

B460 treated Caki-I cells compared to untreated or scramble treated control AS-ODNs

(Figure 4-6).

Although, the in vitro studies indicated that treating Caki-l tumor cells with

VEGF or bFGF mRNA targeted AS-ODNs down-regulated VEGF/bFGF protein

production sufficiently to affect the proliferation and migration of endothelial cells, it was

important to demonstrate that such treatments also could affect Caki-1 cell induction of

angiogenesis in vivo. To examine this possibility Caki-1 cells that had been treated with

V515, B460 or control ODNs were injected intradermally and the number of vessels

induced were counted 3 days later (Figure 4-7). While untreated Caki-l cells and control

ODNs treated Caki-l cells had very similar angiogenic potency in vivo (both groups

induced -44-46 new vessels in the assay period), the angiogenic potential of Caki-I cells

that had been pre-treated with VEGF AS-ODNs (V515) was found to be significantly

impaired; only -25.5 (p<0.05, Wilcoxon rank sum test) new blood vessels were observed.

Similarly, bFGF AS-ODNs (B460) treated tumor cells also induced less vessels, -27 new

blood vessels (p<0.05, Wilcoxon rank sum test). More importantly, the most significant

inhibition of formation of new blood vessels was observed when the Caki-1 cells were

treated with both V515 and B460, only -20 new vessels developed (p<0.05, Wilcoxon

rank sum test).









Discussion

Anti-angiogenesis treatment strategies represent a new approach to cancer

management. Given that solid tumors cannot progress effectively without the generation

of new blood vessels, various tacks have been taken to interfere tumor angiogenesis. One

possible target which has received considerable attention is the pro-angiogenic factor

VEGF. VEGF can induce endothelial cell proliferation and migration in vitro (Soker et

al., 1997; Hanahan and Folkman, 1996a; Hanahan and Folkman, 1996b) and

angiogenesis in vivo (Leung et al., 1989; Plate et al., 1992a; Plate et al., 1992b). Its

expression level has been associated with a variety of tumors and correlated to treatment

outcome (Gasparini et al., 1997; Maeda et al., 1996a; Maeda et al., 1996b). Basic

fibroblast growth factor (bFGF) is another important pro-angiogenic factor (Yoshida et

al., 1996; Hoying and Williams, 1996). It also has been found to associated with different

tumors and correlated to treatment outcome, especially RCC (Nguyen et al., 1994b;

Nanus et al., 1993; Miyake et al., 1996). Antisense oligodeoxynucleotide technology

provides an approach for inhibiting gene expression with target specificity as a particular

advantage (Stein and Cheng, 1993; Engelhard, 1998a; Engelhard, 1998b). Effective AS-

ODNs against VEGF and bFGF have been identified and tested in vitro (Chapter 3).

To evaluate the VEGF/bFGF suppression through the use of AS-ODNs on Caki-1

tumor angiogenesis, both in vitro and in vivo efficacies were studied. The co-culture

system, which allows constant exchange of growth factors between tumor and endothelial

cells was used to study the treatment on endothelial proliferation. The tumor cells also

served as primary source of pro-angiogenic growth factors for endothelial cells. This

provides a setting that closely mimics the in vivo situation. Significantly impaired









proliferation potential of both human and mouse microvascular endothelial cells were

observed after VEGF/bFGF AS-ODNs treatment (Figure 4-2 and 4-3). In order to

evaluate the AS-ODNs treatment on endothelial cell migration potential, a very similar

co-culture system (FluoroBlok system) utilizing 24 well dishes and larger pore sized

membranes (8 pM) was used. The results showed that suppression of VEGF/bFGF

expression by AS-ODNs was sufficient to inhibit the migration of endothelial cells in

response to pro-angiogenic growth factors produced by tumor cells (Figure 4-5 and 4-6).

VEGF/bFGF AS-ODNs treatment of Caki-1 cells led to inhibition of endothelial

cell proliferation and migration in both human and mouse microvascular endothelial cells

(Figure 4-2, 4-3, 4-5 and 4-6) suggesting that such treatments should also exert their

effects in a mouse model. In deed, the anti-angiogenic efficacy of VEGF/bFGF AS-

ODNs was readily demonstrated in vivo using the intradermal angiogenesis assay (Figure

4-8).

These results not only support the role of VEGF and bFGF as important pro-

angiogenic growth factors in Caki-1 cell induced angiogenesis, but also clearly suggest

that inhibition of cancer cell VEGF or bFGF expression may ultimately impact tumor

growth. The use of AS-ODNs against VEGF/bFGF was sufficient to illicit anti-

angiogenic effects both in vitro and in vivo.

Taken together, these findings suggest that AS-ODNs targeted to VEGF and

bFGF are effective in inhibiting Caki-l tumor cell induced angiogenesis. They further

implying that such a treatment strategy may have utility in the treatment of RCC.







89












I Endothellal cells


Figure 4-1 Transwell co-culture system for evaluating VEGF/bFGF AS-ODNs treatment
of Caki-1 cells on endothelial cell proliferation.







90




4.0x10 -
0 Untreated
:S0_ DOTAP
3.5x10 0 Cortrol
O V515
3.0x10s

S2.5x10

U 2.Ox0O.

O 1.5x10



5.OxtO


MHE HMVEC-L
Cell lines



Figure 4-2. Treatment of Caki-l cells with VEGF AS-ODNs on the growth of co-
cultured endothelial cell. Caki-1 cells were either untreated, treated with liposome alone,
treated with 1 tM scramble control ODNs or VEGF AS-ODNs. Each bar represents the
mean S.E. of 3 independent experiments. Stars indicate statistical significance
compared to untreated control group (p<0.05, student's t-test).












4.0x105
T Urtreated
3.5x10s DOTAP
Control
3.ox0 B46

S2.5x10l

2.sxlo"

O 1.5x10s

1.0x10.

5.0x10


MHE HMVEC-L

Cell lines



Figure 4-3. Treatment of Caki-1 cells with bFGF AS-ODNs on the growth of co-cultured
endothelial cell. Caki- cells were either untreated, treated with liposome alone, treated
with 1 vM scramble control ODNs or bFGF AS-ODNs. Each bar represents the mean
S.E. of 3 independent experiments. Stars indicate statistical significance compared to
untreated control group (p<0.05, student's 1-test).








92





Endothellal cells

Sabed with D-I- k




Fluorescence
opaque
membrane

Tumor cells










Figure 4-4. Transwell co-culture system for evaluating VEGF/bFGF AS-ODNs treatment
of Caki-1 cells on the migration potential of endothelial cells.












900
00 Untreated
800. DOTAP
SScramble
S700 V515




0 400-

300

20000
100


MHE HWMEC-L

Cell line


Figure 4-5. Treatment of Caki-l cells with VEGF AS-ODNs on the migration potential of
co-cultured endothelial cells. Caki-1 cells were either untreated, treated with liposome
alone, treated with pLM scramble control ODNs or VEGF AS-ODNs. Each bar
represents the mean S.E. of 3 independent experiments. Stars indicate statistical
significance compared to untreated control group (p<0.05, student's t-test).




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MODULATION OF TUMOR ANGIOGENESIS THROUGH THE USE OF ANTISENSE OLIGODEOXYNUCLEO I IDES T ARGE I ED TO VEGF AND BFGF By WENYINSIIl A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PIIlLOSOPHY UNIVERSITY OF FLORIDA 2002

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Dedicated to my parents, Xitehui and Ying my wtfe, Weiwen and m y daughter Julia

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ACKNOWLEDGMENTS I would like to expres s my s incere gratitude to Dr Dietmar W Siemann for providing me the very preciou s opportunity to work in a wonderful laboratory and for hi s immeasurable support and encouragement. I would also like to extend my gratitude to the member s of my s upervisory committee, Dr Ian Phillips Dr Steven Sugrue and Dr Edwin Meyer for their valuable advice and continuous encouragement in the completion of m y s tudie s I al so would like to thank Dr Clare Yuan Zhang for her kind help and assistance in establishing the s tudies I also would like to expre ss m y gratitude to Dr Jeffrey Hugh es Dr. Fuxing Tang for assistance in liposome preparation and to Neal Ben so n for his help with FACS analysi s. In addition I would like to express my appreciation to the pa s t and pre se nt members of Dr Siemann s laboratory including Dr. Lingyun Li Sharon Lepler Chris Pampo Dr Gustavo Cabrera Dr Kenneth Warrington Jr ., Howard Salmon H ea ther Newlin, Emma Mercer and Destry Taylor for their h e lp and providing a pleasant working environment I also greatly appreciate all the faculty s taff and s tudent groups at the Department of Pharmacology and Therapeutic s for the countle ss help they have rendered and the stimulating intellectual atmosphere they provided 111

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TABLE OF CONTENTS page ACKNOWLE DGME NTS ................. .. .............. .. ...... .. ........... .. .................. ...... .. .. ...... ....... .... .... .. .. .... ........................ .. ........ .. .............................. .. .. .. 111 ABSTRACT ............. .. .. .. .. .. ............ .. .. .. .. ........................ .. .... .. .... .... .................... . .. ... .. . ... ....... .... .. .. V l CHA PTERS 1 WTR ODU CTI ON .. .... ... ....... .. .. . .. .. .. .. .. .. .. .. . .. ......... ..... . .. .. ... .. .... .. . .... ............. . 1 Tumor Angiogenesis .. . .. ....... .. .. . .......... . ....... .. ... .... . ........ . .. .. . .. 1 Antiangiogenesis Targets in the Treatment of Cancer .. .. . ..... ... . . .. .. .. ..... .. .... .. . ... .... 4 Antisense Oligodeoxynucleotides Technology .. .. .. . .. . ...... ........ .... . .. . .... .. ........... . 16 Renal Cell Carcinoma 1 9 Significance ..... ...... .. 22 2 CELLULA R DELIVERY OF ANTISENSE OLIGODEOXYNUCLEOTIDES 28 Introduction . Materials and Methods Re s ults Discussion 3 AS-ODNS DESIGN AND IN VITRO ASSESSMENT Introduction . Materials and Method s Re sults .. .. . Discussion 28 30 34 36 49 49 52 57 60 4 ANTI -ANGIOG EN IC EFFICACY STUDIES .... . ..... ... .. .. ...... . ............. ... ...... . .. .. .. 79 Introduction Materials and Methods Re sults .. . . ... ... . ..... ...... ......... . . ...... .. .. .. . .. .... .... .... .. .. ... . .. ........ ......... ... Discussion lV 79 81 85 87

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5 EFFICACY OF AS-ODNS TREATMENT IN CAKI -1 XENOGRAFTS 97 Introduction .. 97 99 Materials and Methods Re s ults ...... Discu ss ion 6 COMBINATION STUDIES Introduction Material and Methods. Result s ..... Di s cussion 102 104 114 114 116 118 120 7 S _... .Y AND PERSPECTNE . ..... .. ...... . .. .. ................. . .......... ... .. ....... . ....... 130 REFERENCES ..... .. . .. ... .. ....... ... .... ...... .. .. . .. .. .. .... .. .. .. ..... .. . .. .. . . .. .. .. .... .... .... .. .. 1 3 5 BIOGRAPHICAL SKETCH 171 V

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philo s ophy MODULATION OF TUMOR ANGIOGENESIS THROUGH THE USE OF ANTISENSE OLIGODEOXYNUCLEO l'IDES TARGETED TO VEGF AND BFGF By Wenyin Shi August 2002 Chair : Dietmar W Siemann Department : Pharmacology and Therapeutic s Angiogenesis is critical for the growth and metastatic spread of solid tumors It is tightly controlled by specific regulatory factors Vascular e11dotheJiaI growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been implicated as the key factors in tumor angiogenesis. The present studies were undertaken to evaluate the effects of blocking VEGF/bFGF production by antisense phosphorothioate oligodeoxynucleotides (AS-ODNs) on the angiogenic activity and growth of a preclinical model of renal cell carcinoma (Caki-1 ) Efficient deliveries of AS-ODNs were achieved using cationic liposome (DOTAP : DOPE) based delivery systems both in vitro and in vivo AS-ODNs sequences against VEGF and bFGF have been designed and their efficacies were tested in vitro Effective AS-ODNs against VEGF (V515) and bFGF (B460) were identified Treatment of Caki-1 cells with V515 or B460 led to a reduction in VEGF or bFGF expression levels sufficient to impair the proliferation and migration Vl

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potential of co-cultured endothelial cells The observed effects were AS-ODNs sequence specific, dose dependent and were achieved at a low non-toxic dose The treatment of Caki-1 cells with V515 or B460 was also sufficient to impair the Caki-1 tumor cell induced angiogenesis in vivo When VS 15 or B460 treated Caki-1 cells were injected into nude mice and evaluated for their angiogenic potential the nt1mber of vessels initiated were significantly reduced To test antitumor efficacy ofVEGF/bFGF AS-ODNs treatment V515 and B460 were administrated to Caki-1 xenograft tumor bearin g mice The re s ult s showed that systemic administration of VEGF/bFGF AS-ODNs significantly inhibited the growth of Caki-1 tumors More importantly a better response was observed when these two AS ODNs treatments were combined A combination ofVEGF/bFGF AS-ODNs treatment with VEGF/bFGF receptor inhibitor or single dose local radiation al s o showed enhanced tumor responses when compared to single treatment alone These results indicate that AS-ODNs against pro-angiogenic factors VEGF and bFGF may have great utilities in the treatment of renal cell carcinoma either alone or in combination with other anti-cancer therapies Vil

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CHAPTER 1 INTRODUCTION Cancer is a group of diseases characterized by uncontrolled growth and spread of abno1r1al cells. If the spread is not controlled, it can result in death. In spite of ever increasing efforts to understand its process and improve treatment its incidence in the population i s rising. In the US about 1 in 2 men and 1 in 3 women will develop cancer in their lifetime (American cancer society 2002). As a cause of mortality overall in the W estem World, cancer is second only to cardiovascular disease. Cancer can be treated b y surgical removal or destroyed with toxic chemicals or radiation However these approaches all have drawbacks. Surgery will work for many primary tumors but metastases are difficult to identify let alone remove at an early stage Radiation and chem otherapy are generally toxic to normal cells as well If even a few cancerous cells remain they can proliferate to produce a resurgence of the disease ; moreover unlike the normal cells cancer cells are genetically dynamic and may evolve resistance to the chemicals used against them New therapeutic approaches providing more tumor-specific targeting still need to be exploited. One promising new development of cancer therapy is the anti-angiogenic strategies following the recognition that tumor growth and metasta s i s depend on establishment of new blood vasculature (Folkman 1971; Folkman 1972b). Tumor Angiogenesis Angiogenesis is the fortnation of new blood vessel s out of pre-exi s ting capillarie s It is a sequence of events that is of key importance in a broad array of physiologic and pathologic processes (Folkman and Shing 1992). While it plays a key role in I

PAGE 9

2 development in adults, it is a rare event under normal physiological circumstances occurring almost exclusively in the female reproductive system Under normal conditions such as wound healing, the angiogenic process switche s on and then off at the appropriate times indicating tight regulation of stimulatory and inhibitory factor s (Hanahan and Folkman 1996). However angiogenesis can be activated with a variety of pathological conditions and occur in a less controlled manner (O'Reilly 1997) These including cardiovascular diseases (atherosclerosis) rheumatoid arthritis diabetic retinopathy psoriasis etc (Folkman 2001) In addition angiogenesi s is critical in the growth and metastatic dissemination of cancer (Folkman 1995 ; Folkman 1992 ; Folkman 1972a; Folk1nan and Shing 1992) It has been observed for more than one hundred year s that tumor s appear to be more vascular than normal tissues It was not until in the early 1970s that Drs Folkman and Denekamp put forward the idea that tumors are highly vascularized and thereby vulnerable at the level of their blood supply This i s the initial recognition of angiogenesis being a therapeutically interesting proce ss in the area of oncology. Folkman proposed the hypothesis that angiogene s is wa s a requirement for the growth and metastatic spread of solid tumors (Figure 1-1) (Folkman 1971 ; Folkman 1972a). He further hypothesized that solid tumors could only grow to a size of ,.., 1-2 mm in diameter without developing new blood supply, and if the development of va s cular s upply could be prevented tumor growth could be limited to a small s ize (Folkman 1971 ; Folkman 1972a) This hypothesis implied that by destroying the newly developing vessels of the tumor all the tumor cells supported by these vessel s could also be killed (Denekamp J 1972).

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3 The process of angiogenesis consists of multiple sequential and interdependent steps (Figure 1-2) It begins with local degradation of the basement membrane surrounding capillaries which is followed by the invasion of the surrounding stroma by underlying endothelial cells in the direction of the angiogenic s timulus Endothelial cell migration is accompanied by the proliferation of endothelial cells and their orgru1ization into three-dimensional structures that join with other similar structures to form a network of new blood vessels (Figure 1-2) (Auerbach and Auerbach 1994) The process of angiogenesis is mediated by the balance between pro-angiogenic and anti-angiogenic factors Angiogenesis is rapidly initiated in response to hypoxic or ischemic conditions In all types of angiogenesis under either physiologic or pathologic conditions endothelial cell activation seems to be the first process to take place In tumors angiogenesis begins by mutual stimulation between tumor cell s and endothelial cells by paracrine mechanisms (Gasparini 1999) Cytokine s from various sources including tumor and stromal cells are released in response to hypoxia or ischemia It is suggested that vascular endothelial growth factor (VEGF) i s a major player in angiogenesis initiation (Ziche et al ., 1997) Besides affecting vasodilation and vascular permeability VEGF can induce the expression of proteases and receptors important in cellular invasion and tissue remodeling and is able to prevent endothelial cell apoptosis (Ferrara and Keyt, 1997; Gupta et al ., 1999). Following the releasing of pro-angiogeneic factors, endothelial cells can release proteolytic enzymes (matrix metalloproteinases MMPs) to degrade the extracellular matrix for migration, proliferation and endothelial penetration into new areas of the body (Stetler-Stevenson 1999)

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4 Endothelial cell proliferation and migration is stimulated by pro-angiogenic growth factors, like VEGF and bFGF VEGF and bFGF are direct acting pro-angiogenic growth factors. BFGF exists in both low molecular weight forrn and l1igh molecular weight forrns due to alternative translation {Florkiewicz et al ., 1991 ) It is suggested that during angiogenesis, low molecular weight bFGF binding to endothelial cell surface FGF receptors leads to increased motility proliferation and proteinase activity whereas the high molecular weight for rns may act on endothelial cell proliferation after nuclear translocation (Gleizes et al ., l 995~ Klein et al ., 1997) VEGF, besides its effect on angiogenesis initiation, also affects endothelial cell proliferation through high affinity receptors (KDR/flk-1 and Flt-I) expressed on endothelial cells (Ferrara, 1999 ; Veikkola and Alitalo 1999) Finally the neovasculature become mature and sta ble through the interaction of endothelial cells with extracellular matrix and mesenchymal cells After endothelial cell proliferation and migration and maturation and formation of endothelial tube structures surrounding vessel layers composed of mural cells need to be recruited Endothelial cells may accompl is h this via the synthesis and sec retion of platelet-derived g rowth factor (PDGF), a mitogen and chemo-attractant for a variety of 1nesenchymal cells (George, 2001 ; LaRochelle et al ., 2002) Subsequent differentiation of the mural precursor cells into pericytes and smooth muscle cells is believed to be cell-cell contact dependent process (Griffioen and Molema 2000) Anti-angiogenic Ta1gets in the T1eatment of Cancer Understanding angiogenesis and it s unique characteristic s in tumor growth and metastasis has provided in s i g hts to a variety of ways to interrupt the process During the past decade research on anti-angiogenic agents has exploded and with ever increasing interest in its potential (Kerbel 2000) We now ha ve a much clearer understandin g of

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5 tumor angiogenesis, including key regulatory factors differences between normal and tumor vasculature, along with endogenous inhibitors and methods to study and quantify angiogenesis (Kerbel 2000) The complex process of tumor angiogenesis of tumor provides multiple potential targets for anti-angiogenic strategies The formation of new blood vessels involves basement membrane degradation, endothelial cell migration endothelial proliferation and tube forn1ation Anti-angiogenic strategies under evaluation target at least one of the several stages (Figure 1-2). These strategies vary from regulation of angiogenic factor expression in tumors, to endogenous inhibitors of angiogenesis Currently there are over 80 clinical trials employing such strategies underway (bttp : // cancertrial s. nci.nih govD reflecting the high pace of development Based on the biological activities these strategies can be categorized into several broad classes The first class consists of MMP inhibitors, compounds that block the degradation of the basement membrane The second class of agents includes those designed to inhibit endothelial cell function such as TNP 470, thalidomide endostatin "' etc The third class of agents specifically targets angiogenic growth factors It includes trysine kinase inhibitors of VEGF/bFGF, antibodies or AS ODNs against pro-angiogenic growth factors or their receptors The last class of agents target survival factors of neovascular blood supply such as intergrin antagonists or anti VEGF therapy (Reinmuth et al. 2001 Fidler et al ., 2000). I will now review some of the important angiogenic factors that have potential as therapeutic targets in anti-angiogenic therapies

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6 VEGF-A and Its Recepto1s VEGF-A and its receptor system is among the most substantial mediators of angiogenesis Considerable evidence has accumulated indicating that VEGF is an angiogenic cytokine of central importance Its angiogenic activities have been demon s trated in numerous experimental models (Takeshita et al 1994 ; Wilting et al ., 1992 ; Wilting et al ., 1993 ; Potgens et al ., 1995 ; Kondo et al ., 1995) The central role of VEGF in tumor angiogenesis has also been suggested Over-expression of VEGF has been reported to occur in the vast majority of clinically important cancers (Zhu and Witte 1999; Hemmerlein et al ., 2001 ; Ferrara and Keyt 1997) Hi g h serum and urine levels of VEGF have been associated with poor survival and treatment outcome to patients of different cancers (Hemmerlein et al 2001 ; Maeda et al ., 1996 ; Gasparini et al ., 1997 ; Edgren et al 1999 ; Sliutz et al ., 1995) Tumor as s ociated endothelial cells frequently demonstrate increased expression of VEGF receptor s (Zhu and Witte, 19 9 9) Moreover high VEGF expression is found to associate with increased microvessel density and increased metastatsis in cancers (Zhu and Witte 1999 ; Tsuji et al ., 2002 ; Fontanini et al ., 2002 ; Ng et al ., 2001) Disruption of VEGF signal transduction provides a potential effective tar g et for anti-angiogenic approaches Different strategies have been de s igned and evaluated Specific VEGF antibodies are a wa y of stopping the angio g enic effects of this growth factor Systemic administration of monoclonal VEGF antibody into tumor bearing nude mice significantly suppressed tumor growth in several tumor model s (Kim et al ., 1993 ; Borg s trom et al ., 1998) A humanized antibody rhuMAb-VEGF al s o has been developed and is undergoing Phase II clinical trials (Lin et al ., 1999 ; Presta et al ., 1997)

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7 Alternatively, AS-ODNs or antisense RNA were also been used to directly disrupt VEGF protein expression and lead to inhibition of twnor growth in different tumor systems (Smyth et al 1997 ; Ellis et al 1996 ; Nguyen et al ., 1998). Another approach is the coupling of a toxin to VEGF itself When active parts of diphtheria toxin (DT390) are linked to VEGF16 5 or VEGF1 2 1, the chimeric molecule exerts highly selective toxic effects on endothelial cells It disrupts neovascularisation in the chicken chorioallantoic membrane assay and slows down the growth of tumors in preclinical tumor models (Arora e t al ., 1999) Blocking the interaction of YEGF with it receptor and receptor signal transduction pathway provides another option for anti-angiogenic treatment. VEGF receptors (flt-I flk-1 and flt-4) are almost exclusively expressed on endothelial cells with VEGFR-2 (flk-1) believed to play a central role in VEGF signal transduction (Ortega et al 1999 ; Bernatchez et al. 1999) Antibodies against VEGF receptors also showed efficacy in inhibiting tumor growth in preclinical tumor models (Brekken et al ., 2000 ; Klement et al ., 2000 ; Witte et al 1998) Targeting VEGF receptor expression through the use of antisense also has been shown to be effective at inhibiting tumor angiogenesis and growth (Kamiyama et al ., 2002 ; Marchand et al 2002) Purified soluble VEGFR-1 binds VEGF with high affinity and blocks VEGF induced endothelial cell proliferation (Kendall et al ., 1996) Over-expression of VEGF solub le receptor through gene therapy significant l y inhibits tumor growth and metastasis and leads to higher survival rates (Goldman et al ., 1998 ; Hasumi et al ., 2002 ; Shiose et al ., 2000; Takayama et al ., 2000). Recently smal l molecular compounds that can inhibit VEGF receptor tyrosine kinase activities have been developed and initial preclinical studies

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8 showed promising anti-angiogenic and antitumor effects (Solorzano et al ., 2001 ; Ning et al 2002 ; Hess et al ., 2001) To interfere in the binding of VEGF with its receptors novel peptides have been developed (Fairbrother et al ., 1998) This approach provides another potentially effective means to disrupt the VEGF signal tran s duction pathway and a provide treatment for cancer FGFs and Their Receptors Fibroblast growth factor was originally identified as an activity in pituitary extracts that stimulates the proliferation of Balb / c 3T3 cells (Armelin 1973 ; Gospodarowicz 1974 ; Gospodarowicz 1975) Currently FGF s consist of a family of over 20 structurally related proteins (Basilico and Moscatelli l 992 ; 0rnitz and Itoh 2001 ). They bind and activate high-affinity tyrosine kinase s, FGFR 1-4 (Lee et al. 1989 ; Dionne et al ., 1990 ; Ruta et al ., 1989 ; Reid et al ., 1990) Among all the FGFs FGF-2 also called basic fibroblast growth factor (bFGF) is one most extensively investigated and im portant in angiogenesis. BFGF was originally purified from bovine pituitary gland (Esch et al ., 1985) It acts in a paracrine and autocrine manner and i s released by tumor cell s, macrophage s or the extracellular matrix BFGF can stimulate endothelial proliferation and also is chemotactic for endothelial cell migration (Gospodarowicz et al ., 1987 ; Moscatelli et al ., 1986 ; Moscatelli et al 1986) It can also up-regulate other important pro-angiogenic factors like VEGF or plasminogen activator (Seghezzi et al. 1998 ; Montesano et al ., 1986) Blocking bFGF expression and function can be achieved by using vaccine anti s ense against bFGF or its receptors (Plum et al ., 2000 ; Maret et al ., 1995 ; Wang and

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9 Becker 1997 ; Redekop and Naus 1995 ; Ensoli et al 1 99 4 ~ Murphy et al ., 1992 ; Becker et al ., 1989) Blocking the intrinsic tyrosine kinase activity of FGF receptors is a promising new approach in anti-angio ge nic strategies tar get in g the bFGF s ignal transduction pathway Some experimental compounds ha ve be e n found to s pecifically block signa lin g of FGFR-1 and inhibit angio ge ne s i s (Mohammadi et al ., 1998 ; Perollet et al ., 1 998) The Tie-angiopoietin System The tie-receptor family consists of two know endothelial tyrosine kinase s : 1 1El and 1 1E2 / Tek They are identified in vascular endothelium and hematopoiet jc cells (Dumont et al 1992 ~ Iwama et al 1 993 ; Partanen et al ., 1992 ; Schnurch and Ri sa u 1993) Mice lacking TIE 1 or IIE 2 are lethal (Puri et al ., 1 995) Ties may r e pre se nt the earliest endothelial cell lineag e marker and ma y re g ulate the endothelial cell proliferation differentiation, and proper patterning during vasculogenesis The fir s t Tie-2 ligand Aniopoietin-1 (Ang-1) was identified from human neuroepithelioma and mou se myoblast cell line s (Davis e t al ., 1996) Ang-1 i s a no vel endothelial regulatory factor that ha s been found to promote angiogenic remodelin g by vasc ular s upporting as well as vesse l maturation and sta bilization Another related li gan d An g -2 al so has been found Howe ve r bindin g of An g -2 to Tie-2 did not induce pho s phorylation of Tie-2 in endothelial cells Moreover it seems to block Ang-I activity and s u ggesting Ang-2 may be antagonize the activation of Tie-2 (Maiso npi er re et al ., 1 997) The se finding s together underline th e feasibility to use the Tie-angiopoietin syste m to control angio ge ne s i s (Lin et al ., 1 998). But before these molecul es can b e u se d for therap y their effects on adult human vasc ulature and their interaction with other

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10 angiogenic molecules during physiological and pathological angiogenesis need to be further investigated Angiogenin Angiogenin is a 14 kD single chain basic protein found in human adenocarcinoma cells It is a potent inducer of angiogenesis in vivo which functions in the picomolar range (Vallee and Riordan 1997 ; Fett et al ., 1985) It stimulates the proliferation of endothelial cells and promotes the adhesion of endothelial and tumor cells (Soncin et al ., 1994 ; Hu et al ., 1997) Blocking angiogenin with a m onoclonal antibody can impair subcutaneous tumor growth of a colon adenocarcinoma in a dose dependent manner (Olson et al ., 1994) In 40-50% of the cases growth of human breast carcinoma xenografts in athymic mice could be completely inhibited by a humanized version of the monoclonal antibody (Piccoli et al ., 1998) In addition to its efficacy of inhibit tumor growth it may also inhibited the establishment and metastatic growth of tumor cells (Olson et al ., 2002). Besides the use of antibodies, other strategies to abolish angiogenin induced angiogenesis include the use of DNA aptamers or antisense (Olson et al. 2001 ; Nobile et al ., 1998) Anti-angiogenic therapy using angiogenin as a target may become an important tool because angiogenin mediates angiogenesis by mechanisms distinct from VEGF and bFGF (Lixin et al. 2001 ; Moroianu and Riordan 1994) Endogenous Inhibito1s (Endostatin, Angiostatin) Angiostatin and endostatin are two endogenous peptides that ha v e been found to have potent anti-angiogenic effect (O'Reilly et al ., 1997 ; O'Reilly et al ., 1994b ~ O'Reilly et al ., 1994a) Angiostatin is a prolytic fragment of plasminogen and endostatin is a 20 kD fragment of collagen XVIII These factors make endothelial cell s re s istant to

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1 1 angiogenic stimuli and induce ''dormancy'' of metastase s Admini s tration of the recombinant protein or expre ss ing angio s tatin or endostatin by means of ge ne therapy illicit s potent anti-tumor effects in the preclinical s tudie s (O'Reilly et al ., 1 997 ; O Reilly et al ., 1994b ; O'Reilly et al 1 99 4a ; Bertolini et al 2000 ; Jin et al ., 2001 ; Yamanaka et al ., 200 1 ; Feldman et al 2001 ; Sacco et al 2001; Szary and Szala, 2001 ; Wen et al ., 2001 ; Shi et al 2002 ) The anti-tumor activitie s of an g io s tatin and endostatin ar e currently undergoing clinical evaluation In addition to angio s tatin and endostatin there are other endogenous angiogenic inhibitors includin g re s tin vasostatin etc (Pike et al 1998 ; Ramchandran et al 1999) Al so, human prolactin growth hormone placental lacto ge n and g rowth hormone variant ar e angiogenic factor s wherea s their 1 6 kD N-terminal fragments are anti-angio ge nic (Struman et al ., 1999) Integrins Inte g rins are beterodim e ric tran s membrane prot e in s con s i s tin g of a and f3 s ubunit s with lar g e ectodomains and s hort cytoplasmic tail s They control cell motility differentiation and proliferation via interaction s with extracellular matrix molecule s Integrins a v f3 3 and a v f3 s are up-regulated on proliferatin g endothelial cells in an g io ge nic blood vessels during wound healin g as well as in tumor vasculature (Brook s et al ., 1 99 4 ; Friedlander et al., 1996) The a v {33 integrin an adhe s ion receptor for extracellular matrix components with an exposed RGD se qu e nce i s an attractive tar ge t for anti-angio ge nic therapy Thi s integrin i s almo s t exclusively pre se nt on the c e ll s urface of activated endothelial cells but absent on quie s cent endothelium or other cell type s (Eliceiri and Cheresh, 1 999). Antibodie s a g ain s t a v f3 3 were found to inhibit adhe s ion-dependent sign al

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12 tran s duction by angiogenic factors leading to apoptosis of activated e ndothelial celJs Co n se quentl y the se compounds could block e ndoth el ial tube for1nation and an gioge n esis in tumor s (Brook s et al ., 1 99 4 ; Brook s et al ., 1 995) C urr e ntl y inte gri n ant ago ni s t s are b e in g eva luated in phase I and phase II clinical trial s (Brower 1 999) Matrix Metalloproteinases (MMPs) and Tissue Inhibito1 of Metalloproteinases (TIMPs) To form new blood vessels e ndothelial cells of existing blood vessels must degrade the underlying basement m e mbrane and in va de into the s troma of th e n eig hborin g ti ss ue (Mignatti and Rifkin 1 993 ; Mignatti and Rifkin 1 996) Th ese proce sses of endothelial ceU invasion and mi g ration require the cooperative activity o f the plasminogen activator and the MMP s The !vflv1P s ar e a famil y of st ructurall y r e lat e d zinc -dep e nd e nt en dop e ptid eases co ll ec ti ve ly capable of d egra din g extracellular matrix (ECM) MMPs pla y an important rol e in the de g radation of ECM both in ph ysio l ogic al condition s, s uch as morpho ge n esis and ti ss ue repair and in path o lo g ic conditions s u c h as turnor invasion and m etas t as i s Th e activity of MrvtP s is controlled at different lev e l s (Liekens et al ., 2001 ) First the expre s s ion of MMP s i s up-regulated b y an giogenic g rowth factors (Giuliani et al 1 999 ; Bond e t al ., 1 998 ; Wan g and K e i se r 1 99 8) S eco ndl y :M:MPs need to be activated proteol y tically (Murphy et al. 1 999) Lastly, the M1v1P s activi ti es are al so r eg ulated b y their inhibitor s TIMP s (Blavier et al ., 1 999 ; H e nri et et a l ., 1 999) However a lar ge bod y of evidence s ugge s t s that thi s re g ulation i s lo s t durin g tumor gro wth and 1n etas tasi s (R as mus se n and McCann 1 997) An imbalanc e b e tw ee n l\.11v1Ps and th e ir TIMPs i s responsible for the in va s i ve ph enotype of breast colon and lun g tumors and a low

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13 survival rate in urothelial cancers (Kossakowska et al ., l 996 ~ Gohji et al ., 1996b ; Gohji et al 1996a) Inhibition of MMPs activities thus has been extensively studied as an approach to inhibit growth and invasion of neoplastic cells Important anigiogenesis inhibitors in clinical trials based on l\1M.P blockin g are Metastat Neovastat BMS-2752291 Mariamstat, AG3340, Ba y 12-9556 and CGS 27023A (Vihinen and Kahari, 2002) MMPs inhibitors currently in clinical trials are synthetic peptides or non-peptidic molecules chemically modified tetracyclines bispho s phonates or natural MMP inhibitors (neovastat) Further trials using MMPs in combination with classical chemotherapy are also underway Plasminogen Activator ( uP A) and its Inhibitors (PAI-1) Proteases of the fibrinolytic cascade also contribute to the regulation of angiogenesis Expression of urokinase-type plasminogen activator (uP A) by malignant cells results in an aggressive phenotype with increa se d tumor angiogenesis and metastatic invasion PAI-1 the natural uPA inhibitor is paradoxically also up-r egu lated i n human tumor samp le s (Landau et al ., 1 994) Clinically, expression of both uPA and PAI-1 correlate with a poor prognosis of seve ral cancers (Rosenquist et al ., 1993 ; Heiss et al ., 2002 ; Osmak etal ., 2001) Taken together inhibition ofuPA rather than PAI-I activity might be a possible therapeutic target to treat cancer and other angiogenesis dependent diseases Thrombospondin (TSP) Extracellular matrix molecules play an important rol e in maintaining tissue integrity and endothelial cell viability However, thrombospondin, one s uch extracellular

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14 matrix molecules, first identified in 1979 from platelets is also a very powerful inhibitor of endothelial cell adhesion, migration motility and proliferation and angiogenesis in , ;vo (Lawler et al ., 1978 ; Taraboletti et al ., 1990 ; Good et al ., 1990) TSP-I is a member of a family of structurally related proteins encoded by different genes which include s 4 recent identified members TSP 2-5 (Bornstein and Sage 1994) TSP-I inhibits endothelial cell proliferation migration and can indt1ce endothe lial apoptosis (Vogel et al ., 1993; Talsma et al ., 1 993) In addition tumor cells transfected with TSPI developed smaller tumors than the parental cell line s (Volpert et al ., 1998 ; Streit et al. 1999) In clinical st udi es, expression of TSPI has been inversely correlated with malignant progression of breast cancer, m e lanoma, and lun g carcinomas (Zabrenetzky et al ., 1994) These data inclicate that TSPI can be util ized to inhibit tumor g rowth by an anti angiogenic mechanism Platelet Factor 4 (PF-4) Platelet factor 4 belongs to the CXC cytokine s uperfamil y (Strieter et al 1995) It is a 7. 8 kD protein of 70-amino acid in length that sha re s homologies with thromboglobulin and interleukin-8 (Deuel et al., 1977) It has been known for a while that PF-4 inhibits angiogenesis (Maione et al ., 1 990) First, PF-4 inhibits endothe lial cell proliferation, migration and angjogenesis in vivo (Gupta and Singh 1994 ; Maione et al ., 1990) Second, PF-4 is targeted to endothelial cells that undergo angiogenesis in vivo (Hansel l et al ., 1 995) Moreover it has been shown the tumor angiogenesis could be inhibited by PF-4 (Sharpe et al ., 1990 ; Kolber et al ., 1995) In addition human g li oma cells infected with a secretable PF-4 cDNA grew s lowl y in vivo and were h ypovascular

PAGE 22

15 (Tanaka et al ., 1997) Finally data exist s u gges tin g the PF-4 ma y counteract an g io ge nic factor activity at the s ties of platelet activation (Watson et al ., 1 99 4 ) Administration of recombinant PF-4 protein or d el i very of th e PF-4 ge ne s howed efficacy against tumor growth in preclinical model s (Maione e t al l 990~ Tanaka et al ., 1 997 ; Kolber et al ., 1995 ; Maione et al 1 99 1 ) These findin gs s u gges t that it may ha ve great pot en tial as an effective anti-an gioge nic factor in th e treatment of cancer C urr e ntl y, recombinant PF 4 is bein g evaluated in clinical trial s (Belman e t al ., 1996) Interleukins Interl e ukin s have been known for a lon g time for their immunomodulatory activities but their role in an g io g en es is is ju s t becomin g a hot topic in canc e r re searc h Some of the interleukin s ha ve anti-an gi o ge nic properties ( interl e ukin-I 0 -12 -18 ) while others see m s to be pro angio ge nic (interleukinI 6 -8 1 5) so me may even ha ve both effec t s ( interleukin-4) The mechani s ms by which interleukins achjeve their effects on en dothelial cells are quite differ e nt and not full y und e r s tood (E l Awad et al 2000 ; Huan g e t al ., 1 996 ; Yoe st e t al 1 995) Int e rl e ukit1 s ma y be u sefu l tool s to tr ea t an giogenes i s r e lated di seases includin g canc e r Howe ve r better under s tandin g of their s pecific function s as well as their interaction s i s n eeded Anti-angiogenic Factors Summary Among all the s e angio ge nic regulatory factors th e mo st important pro-an gioge nic growt h factor s in cancer are VEGF and bFGF Both factors are found to s t i mulat e endothelial cell proliferation and mi g ration (Leung e t al ., 1 989; Plate et al ., 1 992 ; Schweigerer e t al 1987) Indeed the expression of VEGF has b ee n r el at ed to fundamental feature s of tumor s s uch as growth rate ( Kim et al ., 1 993 ; Nagao and

PAGE 23

16 Nishikawa 1989) microvessel density (Toi et al 1994 ; Straume and Akslen 2002) and vascular architecture (Drake and Little 1999; Faridi et aJ 2002) as well as the development of tumor metastasis (Weidner et al 19 9 1 ; Faridi et al ., 2002) A correlation between VEGF and/or bFGF expression and s urvival has been noted in s ome cancer patients (Gasparini et al ., 1997 ; Yiangou et al ., 1997 ; Dietz et al ., 2000) Given the importance of VEGF and bFGF in the angio ge nic proce ss of cancer the s e two g rowth factor s were chosen as the targets for the present s investigation s Antisense Oligodeoxynucleotides Technology In order to inhibit the expression or function of s pecific ge n e product s s uch as VEGF and bFGF, a strategy with promi s e i s the use of anti s ense oli go deox-ynucleotide s (AS-ODNs) In 1977 it is first de s cribed that ge ne expression can be modified with exogenous nucleic acids by using s ingle s trand DNA to inhibit tran s lation of a complementary RNA in a cell-free system (Paterson et al ., 1977) Soon after Zamecnik and Stephenson also demon st rated that AS-ODN s tar get ed to 3 end of virus could inhibit viral replication in l itr o (Zamecnik and Stephen so n 1978) The s e initial finding s showed that AS-ODN s could inhibit gene expression in a sequence s pecific manner One of th e first s tud ies s howing in vivo activities of AS-ODN s wa s published in 1 99 1 (White se ll et al. 19 9 1 ) Since then particularly with the introduction of efficient methods for DNA se quencin g and ODN s sy nthesis variou s targets have been analy ze d in vitfo and in animal s with encouraging results (Jansen et aJ 1998 ~ Tamm et al 2001 ; Pawlak et al 2000; Golden et al 2002 ; Braasch and Corey 2002 ; Corey 2002)

PAGE 24

17 The essential steps in dru g design are the identification of an appropriate target respon s ible for a certain disease and the de velo pment of a drug with s pecific recognition of and affinity to that target For mo s t conventional drug s the mechani s m of fairly broad In contrast the basi s of the use of AS-ODN s is that the introduction of ODN s complementary to target rnRNA se quence s into the cytoplasm can result in decreased expression of the g ene being targeted (Dias N and Stein 2002) Since AS-ODNs inhibit ge ne expression in a sequence dependent way se lective alteration of spec ific ge ne expression i s pos s ible The AS-OND s approaches ge nerally used are mainly to inhibit oncogene expression to induce apoptosis to overcome multidru g re s i s tanc e or to inhibit pro-an g io ge nic growth factors (Pawlak et al 2000) In 1998 the fir st anti se n se dru g (fomivirsen) was approved by the US Food and Drug s Ad.ministration (FDA) for the tr eatme nt of cy tome galovirusinduced retiniti s in AIDS patient s (de Smet et al ., 1999) AJthou g h fomivirsen i s admini s trated locally the approval shows the feasibility of AS-ODNs as dru gs for the tre a tment of human disea ses With continuous development and under s tandin g of tumor biology inappropriate expression of certain genes was found to be basic to the pathophysiology of cancer Consequently the use AS-ODN s as th e rapeutic s trategie s in the treatment of cancer ha s attracted much attention and inten sive inv estiga tion The currently mor e than 8 ongoing clinical trial s illu str ate the growing intere s t in AS-ODNs in the treatment of cancer (Koller et al 2000 ~ Tamm et al ., 2001 ) Be s ides the se approaches AS-OND s ma y also hav e a rol e in overcoming multidrug re sist ance in cancer For examp l e the u se of anti md,l AS-ODNs has been s hown to lead to reduction of the gene product gpl 70

PAGE 25

18 expression restore chemotherapy dru g sens itivity 1 and even lead to eventual prolongation of s urvival (Cucco and Calabretta, 1996 ; Ku ss e t al ., 2002 ; Pan et al 2001) Onco ge n e over-expression is one of th e mo s t common m o l ec ular eve nt s that ma y l ea d to cancer d eve lopment AS-ODNs are s p ec ifi c t oo l s to inhibit ex pr essio n of certain oncogenes and s o can be used as pot e ntial dru gs to r eve r se th e harmful effects of d ys re g ulated ge ne expres s ion AS-ODNs against ra s m yc 1t1 yb, b c -abl as well as viral oncogenes s uch as E6 E7 or HBx hav e been evaluated (Gray et al 1 993 ; S zczyl ik et al ., 1 996 ; Venturelli et al ., 1 990 ; Leonetti e t al ., 1 996 ; C itro e t al ., 1994; Szczylik et al ., 1 991 ; B eer -Rom e ro et al ., 1 997 ; Lappalainen et al ., 1996) Methods to r eg ul a t e the mechani s m s of cell death and survival in orde r to sh ift th e balance toward apoptosis are of g r eat int eres t in the treatment of cancer Studies ha ve focused on tar ge tin g the vi tal anti-apoptotic genes s uch as b c l-2 p 53 and CRIPTO and MDM-2 prot e in s u sing AS ODN s (Ziegler et al ., 1 99 7 ; Campbell et al 1 99 8 ; Normanno e t al 1 999 ~ Chen et al ., 1 998) The application of AS ODN s to tar ge t the pro-an g io ge nic g rowth factors i s a n ew and promi si ng strategy in cancer management Studi es with AS-ODNs against VEGF s howed s uch treatment c an s ignificantl y impair twnor angiogenesis and lead t o tumor growt h inhibition in VEGF dep e nd e nt ttunor s (Masood et al ., 2001 ; Masood et al 1 997) AS-ODNs directed at inhibitin g the exp re ss ion of bFGF al so showe d both anti an gioge nic and anti-tumor effic ac y ( Wang and Beck e r l 997) The s trate gy of u sing AS OD Ns against both VEGF and bFGF as th e rapeutic int erve ntion of cancer was explored in detail in s tudie s described in thi s di sse rtation

PAGE 26

19 To date proof of clinical efficacy of AS -OD Ns in oncology is very limited (de Smet et al ., 1999) However data providing proof of principle exist Future development of AS-ODNs holds considerable pronuse in the treatment of cancer Further de ve lopment of new various new targets assessment of combination treatment s with several PS ODN s, and investigations focused on s trategie s targetin g tumor mechat1i s m s s hould improve their therapeutic activitie s Renal Cell Ca,cinoma Renal cell carcinoma (RCC) i s the sixth leadin g cause of cancer death in the U nited States accounting for 3o/o of adult mali g nancie s There were an estimated 30 800 RCC cases diagnosed in 2001 with approximately 12 100 death s in the United States (Jemal et al 2002) The incidence of RCC death s in th e U nited States has been s teadil y increasi11g during the past 25 ye ar s, po ss ibly partly becau se of increased se n s itivity and greater use of various imaging modalitie s (Chow et al 1 999 ; Homma et al ., 1995 ; Jay s on and Sanders, 1998) Although the etiology of RCC i s unknown seve ral ri s k factors includin g obesity, smoking, hypertension diuretic use consumption of fir e d meat asbestos exposure, petroleum exposure and frequent anal ges ic u se ha ve been consistently implicated (Dhote et al ., 2000) Renal transplantation with it s associated i1nmunosuppression, acquired cystic kidney disease a l so increase the ri s k s of d eve lopin g RCC (Hoshida et al ., 1 999) R CCs are clinically, histolo g icall y and genetically a very hetero ge neou s g roup of tumor s Clear cell RCC i s the mo s t common type ofRCC ac countin g for over 70% of the cases It i s a highly vascularised neopl as m demon s tratin g clear evi dence of abundant angiogenesis and abnormal blood vesse l dev e lopment (Yo s hino e t al 2000)

PAGE 27

20 Clinically RCC patient s can present with a multiplicity of manife s tations rangin g from the classic presentin g triad of hematuria, pain and palpable renal mass to more obscure symptoms such as tho se of paraneoplastic syn drome s Unfortunately the classic triad u s ually incticates patients with far advanced disease and it is see n in le ss than 10% of patient s at pre se ntation (Gibbons et al ., 1 976). More commonly renal tumor s are discovered incidentally during the course of various diagno stic s tudie s. The treatment of choice for RCC i s s ur g ical removal Radical nephrectomy is accomplished by early ligation of the renal artery renal vein and e n bloc removal of the kidney with the s urroundin g Gerota 's fascia (Robson et al ., 1969) De s pite the remarkable improvement s and re s pon se rates of sing le and combination chemotherapy in some s olid tumor s, RCC remain s a chemoresistant tumor The most common agents vinblastine and floxiuridine have response rate s of 7 % and l 6 o/ o re spec tively (Yagoda et al ., 1995) In a review of 72 agents evaluated in 3 500 patients between 1983 and 19 92, an overall objective re s ponse rate of only 5 6% was found mostly of short duration (Yagoda et al ., 1995) Hon11one therapy has been found to be equally ineffective ( deKernion and Lindner 1982) Consequently there currently is no role for chemotherapy or hormone therapy in the treattnent of RCC Unlike chemotherapy radiotherapy has been shown to provide so me benefit s to patient s with RCC (Rost and Bro sig, 1977) Preoperati ve radiation can reduce the risk of tumor dis s emination at the ti me of nephrectomy reduce primary tumor size~ increase re secta bili ty and reduce tumor vasc ularity Postoperative radiation therapy has the theoretical b e nefit of providing local contro l of tumor in patients with po si tive s urgical mar gi n s incompletely r esec ted primary tumor s, or lymph node involvement (Riches 19 66; Mantyla et al ., 1977) Still, currently

PAGE 28

21 radiation therapy in RCC is generally reserved for palliation most often for symptomatic bony metastases (Halperin and Harisiadis 1983 ) Taken together five-year survival rate s after radical nephrectomy for stage I RCC is approximately 94%, and stage II 79 o/ o Patients with renal vein or inferior vena caval involvement have a survival rate of 25-50%, and patient s with regional lymph node involvement or expracapsular extension have a survival rate of 12-25 %. Five year survival rate for patients with stage IV disease is less than 5% The unsatisfactory management of RCC with conventional anticancer therapie s warrants novel approaches to augment the tumor respon se and treatment outcome Histopathological studies of RCC reveal it to be a highly vascularised neoplasm demonstrating clear evidence of abundant angiogenesis and abnor1nal blood vessel development (Figure 1-3) (Yoshino et al ., 2000) Therefore it may provide an excellent target for anti-angiogenic therapeutic approaches Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are of particular interest Both factors have been shown to be expressed in renal cell carcinoma tissues and renal cell carcinoma cell lines (Mydlo et al ., 1993 ; Gospodarowicz et al ., 1986 ; Mydlo et al ., 1988 ; Sato et al ., 19 99; McLaughlin and Lipwortl1 2000 ; Ferrara and Keyt 1997) Serum levels of YEGF and bFGF often are elevated in RCC patients (Nguyen et al 1994b ; Fujimoto et al ., 1991 ; Wechsel et al. 1999 ; Tomisawa et al ., 1999 ; Paradis et al ., 2000) and renal cell carcinoma VEGF and bFGF mRNA levels have been reported to be much higher than those found in surrounding normal tissues (Tricarico et al ., 1999 ; Thelen et al., 1999 ; Tsuchiya et al ., 2001 ; Eguchi et al ., 1992) In addition, elevated se rum/urine bFGF levels have been shown to associated with malignant progres s ion and poor treatment outcome

PAGE 29

22 ( Son g et aJ ., 2001 ; Jacob se n et al. 2000 ; Rasmu so n et al 2001 ; Edgren et aJ 1 999 ; Do s quet et al ., 1 997 ; Fujimoto et al 1 995 ; Plunkett and Hailey 1 990 ; Miyake e t al 1 996 ; Nguye n et aJ 1 994 a ; Huan g et al ., 1 996) Taken to ge th e r the se fm din gs suggest that VEGF and bFGF are key factors in vo lved in th e angiogenic process of R CC For th ese rea so n s RC C was the tumor of choice for the current investigation of anti angiogenic therapeutic approaches with the u se of VEGF and bFGF AS-ODNs An R CC cell line (Caki-1) was u se d This cell line i s a human clear cell r en al cell carcinoma originally derived from a 49 year-old Ca ucasian male patient (Fogh, 1978) It g rows in vit r o as an anchored cell culture and aJ so forms tumor in athymic nude mice The hi s tolo gy of the Caki-1 xeno g raft di s pla ys many of the fea ture s of clinical samples of R CC (Figure 1-5) Significance Angiogenesis i s unique proce ss that contributes t o a variety o f patholo gic proce sses and i s especially critical to tumor g rowth and metastasis It ther efore has been propo se d and utili ze d as both a progno s tic indicator as well as a possible target for therap e utic intervention in certain mali g nant s tat es In the present s tudi es the feasibility of inhibiting tumor an g iogene s i s by utilizin g AS-ODNs directed agai n s t VEGF and bFGF were investigated A human renal cell carcinoma ce ll lin e grown in v itr 'O or as sol id tumor xenog raft s in nud e mic e was u se d as the tum or mod e l Through the investigations a v iable mean s o f int erve nin g with the an giogenic proce ss and in si tu growt h of renal carcinoma cells was developed It i s further b e lie ve d that thi s approach will not be confmed to RCC but will be applicable to other neoplasm s an d ma y even provide a basi s for se l e cti ve intervention in other diseases characterized b y angiogenesis

PAGE 30

Anti-angiogenic Pro-angiogenic Invasio 2 3 t>e e f> ee e f>e f> e e ltroliferation Fi g ur e 1-1 Th e an g io ge ni c pro cess Turnor ee l l s or ho s t ce ll s se cr e t e pro -a n gi o ge n ic g rowt l1 fa c tor s wh ic h th e n b ind to s p ec ifi c r ece ptor s on e n do th e I i a l ce ll s Thi s l i gand r e c e ptor int e raction l e ad s to e ndoth e l ia l e e l I pro I if e r a tion mi g ration in v a s ion a11d eve ntu a ll y capilla ry tltb e form a t i on ( Fidl e r e t al ., 2000)

PAGE 31

24 3 EC receptor binding -bltnlc:ellular ......... ... 4 ec ~vatlon BM radatlon t; EC prolJfuatlo 6 Dtrectlonal Migration 7 l!CM remodeling ~formation 9 Loop formation A,,V dlffvu:ntiatloa Fi g ur e 12 Cascade ot eve nt s i n tL 1 mor a n g i oge n es i s (So ur ce : Th e An g iogenesi s Foundation www a n g io or g)

PAGE 32

wo,, wo, .. VEOF-C/.O J -1 HtV tat 25 pro-ang io gen i c lnterleuklft ,1~-1$ 11.. R' wo,11-, IL R 's lnt orl11 uk 1 n 10 12. 11 FOF-1+2 1 FOfJI 1-4 antl-ang io geni c Arigiogattln ECM VN, r: 1 I Oateop0tttln t_! DGF ~ __ 170 kDa llecaptor ECM, VN Figure 1-3 Major regulators of an g io ge ne sis and their receptor s On the upper part of the cell pro-angiogenesis regulators are s hown on the lower part are inhibitory molecule s. (Hagedorn and Bikfalvi 2000)

PAGE 33

26 Figure 1-4 Resin cast ofRCC tumor microvascular network (Gerwins et al. 2000)

PAGE 34

27 Figure L 5 H&E sta ining hi sto l ogy sec ti on of Caki-1 xenograft ttimor

PAGE 35

CHAPTER2 CELLULAR DELIVERY OF ANTISENSE OLIGONUCLEOTIDES Many of the limitations of current cytotoxic therapies of cancer result from a lack of specificity of the anti-cancer agents Tl1e advances in molecular biology over the past The application of two decades have made possible the concept of genetically based targeted treatment Antisense oligonucleotides (AS-ODNs) is one approach to specifically inhibit gene expression (Stein and Cheng 1993 ; Wagner, 1994). These molecules usual l y 18-20 bases in length, can undergo Watson-Crick hybridization to target mRNAs ultimately resulting in decreased expression of the gene products AS ODNs technology has attracted great interest and shown great promise as agents to inhibit the expression of specific genes that regulate physiological functions or mediate various diseases (Harrison 1993 ; Wagner 1994 ; Wagner 1995 ; Pan et al ., 2002 ; Mani et al ., 2002 ; Morris et al ., 2002) However, one of the biggest challenges in the application of AS ODNs as therapeutic agents is the development of ways to maximize their cellular uptake (Wagner, 1995 ; Crooke, 1993) AS-ODNs are negatively charged molecules that behave as polyanions. In general this property leads to poor cellular uptake and intracellular distribution Furthermore, the commonly used phosphorothioate modified AS-ODNs have quite high affinity for proteins especially heparin binding proteins (Fennewald and Rando 1995) In addition some new cell surface ODNs-binding proteins have also been identified recently (Beltinger et al. 1995 ; Hawley and Gibson 1996) Even after 28

PAGE 36

29 internalization into the cell through endocytosis (Yakubov et al 1989 ~ Crooke et al. 1995), naked AS-ODNs are localized to endosomes or lysosomes that are topologically still 'outside ' of the cell (Tonkinson et al. 1994) ODNs may then either be released from the cell , ia exocytosis or may be partially digested (Tonkinson et al ., 1994 ) Still exists a growing literatures of antisense effects after naked AS-ODNs delivery (Anfossi et al., 1989 ; Gewirtz and Calabretta 1988) Nontheless, it should be recognized that these effects were achieved at high AS ODNs concentration and may be the result of AS ODNs release into the cytoplasm through spontaneous endosomal/lysosomal rupture. Many techniques have thus been then used to enhance AS-ODNs uptake the most widely used being based on the application of cationic lipids Cellular uptake as well as the activity in cell cultures can be improved greatly by cationic liposomes (Bennett et al ., 1992 ; Lappalainen et al ., 1994 ; Zelphati and Szoka, Jr ., 1996b ; Zelphati and Szoka, Jr ., 1996a) Highly polar water soluble molecules including AS-ODNs can be entrapped in the internal aqueous space of the liposme while the lipids fonn into bilayers. Cationic liposomes spontaneously bind the negatively charged AS-ODNs and protect them against degradation The macromolecular complexes have a positive charge at the surface this results in a high affinity for most cell membranes which are negatively charged under physiological conditions Following the attachment to the membrane the complexes are taken up via endocytosis. To help facilitate the release from endosomes and lysosomes, a helper lipid such as 1 2-dioleoyl-3-sn-phosphatidylethanolamine (DOPE) is often used in the liposome preparation This inverted-cone-shaped lipids thought to facilitate cytosolic release through the fusion and disruption of endosomal membranes (Farhood et al ., 1992) The flip-flop of anionic phospholipids in the endosome membrane lead to

PAGE 37

30 neutralization of the cationic lipid charge displacement of the bound oligonucleotides and release forn1 the endosome (Figure 2-1) (Koltover et al ., 1998 ; Lebedeva et al ., 2000). Studies have demonstrated that ODNs can readily dissociate from the liposome complexes and are in bioavailable form within the cells (Tari 2000 ; Abe et al ., 1998).Cationic liposomes facilitated delivery of AS-ODNs has proven to be effective in many different cell lines and to be of general utility (Bennett et al ., 1992). However studies in mammalian cell lines have demonstrated that AS-ODNs efficacy varies with different cationic lipids and lipid complexes, cationic lipid/DNA ratio and cell type (Flanagan and Wagner, 1997 ; Lappalainen et al ., 1997). This suggests that as a general rule, the best liposome composition and optimal liposome / ODNs ratio needs to be established for each cell lines to achieve best results In the present studies fluorescein isothioicyanate (FITC) labeled ODNs were used to study the cellular uptake of ODNs using a cationic liposome delivery system (Noonberg et al ., 1992) Although the biological activity of AS-ODNs against the molecular target is highly sequence-dependent this is typically not the case for pharmacokinetics and toxicology Indeed the pharmacokinetics and toxicology of AS ODNs of widely differing sequences directed against vastly disparate gene products have proven surprising similar (Srinivasan and Iversen, 1995) Thus, the same ODNs sequence was used for the present cellular uptake and toxicity studies Materials and Methods Cell Culture The clear cell RCC cell lines Caki-1 Caki-2 and A498 were gifts from Dr Susan Knox (Stanford University). These cells were grown in Dulbecco's modified minimum essential medium (DMEM, Invitrogen Grand Island, NY) supplemented with I 0% fetal

PAGE 38

31 bovine s erum (FBS Invitrogen Grand Island NY) I% penicillin-streptomycin (Invitrogen Grand Island NY) and I % 200 mmol/L L g lutamine (Invit r ogen Grand Island NY) FITC Labeled Phosphorothioate Oligodeoxynucleotide s The 20-mer ODNs sequence : 5 CAC CCI GCT CAC CGC ATG GC 3 (20mer s ) were cu s tom er synthesized by Geno Mechanix (Alachua, FL) The entire backbone wa s phosphorothioate modified and FI'I'C was l abeled at the 5 end of the ODNs The ODNs were suspended in sterile and endotoxin free water at a concentration of I mM aliquoted and stored at -20 C Liposome Prepa1ations Cationic l iposomes of different lipid compositions were obtained from Dr Jeffrey Hughes lab (University of Florida, Gaine s vi ll e FL) DOT AP : DOPE is compo s ed of cationic lipid l 2 -di o l eoy l oxy 3-(trimethylammonium) propane (DOT AP) and a helper lipid 1 2 -di oleoyl-3-sn-phosphatidylethano lamin e (DOPE) at a molar ration of I : 1 DS3DOPC is composed of 1 2-Dioleoyl-sn-Glycero-3-Phopho s erine-N-Citraconyl and dioleoyl-pho s phatidylcholine (DOPC) at a molar ratio of I : 1 DOGSDSO i s compo s ed of l' 2 -di oleoyl sn glycero-3'-succinyl-2-hydroxyethy l di s ulfide ornithine conju g ate (Tang and Hughe s, 1998) CHDTAEA is composed of cholesteryl hemidithiodiglycolyl tri s (aminoethyl)amine (Tang and Hughes 1999) PEG-PE i s composed of DOT AP DOPE and polyethylene glycol distearoyphospatidylethanolamine (PEG-PE) at a molar ration of25 : 25 : 3 (Meyer et al 1998) All the lipid s were obtained from Avanti Polar Lipids (Alabaster Al)

PAGE 39

32 Briefly the lipid mixture was evaporated to dryne ss in a ro1md-bottomed flask u s in g a rotary evaporator at room temperature The resulting lipid film was dri e d by nitrogen for an additional IO min to evapora te any r esi dual chlorofor1r1 The lipid film was re-suspended in s terile water to a final concentration of I mg/ml b ase d o n the weight of cationic lipid The re s ultant mixture s were s haken in a water bath at 35 C for 30 min The s u s pen sio n s then wer e so nicated u s in g a Sonic Di s membr ato r (Fisher Scientific Pitt s burgh PA) for I min at room temperature to forrn homo ge ni zed lipo somes The particle-size distribution of lipo so me s was me as ured u sing a NICOMP 380 ZLS in s trum e nt (Santa Barbara, CA) The a ve ra ge particle diameter was 144 .0 + 77.0 nm Liposomes were s tored at 4 C and u sed within 3 month s Antiseose Treatment Caki -1 Caki -2 A4 98 ce ll s were se t at I x I 0 5 in 60 mm dishes and allowed to attach overnight For comparison of delivery efficiency b y di ffe rent lipo s ome s FITC lab ele d OD Ns were mixed with diff e r e nt l i po so me s in se rum free medium or I 0% FBS medium and incubated at room t e mperature for 30 m in For o th er s tudie s only DOTAP : DOP E lipo so me was used Th e m e dium of cells was th e n changed with that containing ODN s -lipo s ome complex a t a OD Ns concentration of 1 M/ml and incubated at 37 C for 3 hour s Equal amount s of 20% FBS medium were added to di s he s and co ntinu ed to incubate for a total of 24 hr except for the time course studies, in which cells were incubated for different l e ngth s of ti me Fluorescence Microscope After FIT C labeled OD Ns treatment the mediun1 co nt ai nin g OD Ns was r e m oved and cells were washed 4 times with PBS The cells were th e n fixed in I % p

PAGE 40

33 formaldehyde for 1 hr Fluorescent microscope pictures were then taken using a Zeiss Axioplan 2 Florescence Microscope (Zeiss Thomwood NY) made available by the OpticaJ Microscopy Facility at Brain Institute University of Florida Flow Cytometry Analysis After FITC 1abeled ODNs treatment the medium containing AS-ODNs was removed and cells were washed 4 times with PBS. The cells were then collected by trypsin digestion After fixation in 1 o/o p formaldehyde for l hr cells were re-suspended in PBS at the concentration of l x I 0 6 cells / ml and kept in the dark Green fluorescent intensities of the cells derived from FITC were then analyzed by F ACS on a Becton Dickinson flow cytometer made available through the University Cole Facility for Flow Cytometry at the University of Florida. Toxicity Studies Caki-1 ceJls were set in 96-welJ dishes at 1 x I 0 4 cells per well and allowed to attach overnight The culture medium was then changed to 100 I serum free or 10% FBS medium containing various doses ofDOTAP:DOPE The cells were incubated for 24 hr at 3 7 C The viable cells after treatment were measured using a CellTiter96AQueous Assay System (Promege Madison WI) Briefly, I 00 l of phenylmethasulfaz.one (PMS) solution was added to 2 mJ 3-( 4 5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2 ( 4-sulfophenyl)-2H tet razolium (MTS) solution and mixed by gentle swirl 20 I of the combined MTS / PMS solution was added into each well and incubated at 3 7 C for 2 hr After incubation, 25 l of I 0% SDS was added into each well to stop the reaction The absorbance was then measured at 490 nm using a microplate reader

PAGE 41

34 Results Uptake of FITC labeled ODNs measured by flow cytometry and fluorescence micro sc opy allowed the assessment of cellular delivery ofODNs Delivery of naked ODNs resuJted in poor internalization into the cells either in serum free or 10 % FBS conditions OnJy a small portion of cells treated s howed moderate increase s in fluorescence intensity derived from FITC (Figure 2-2) However the u se of cationic lipo so mes (DOT AP : DOPE) was found to s ignificantly improve the up-take efficiency of ODNs in Ca kil cells, in both serum free and I 0% FBS conditions (Figure 2-2) In order to optimize the delivery system for ODN s, other cationic liposome compositions were evaluated for their delivery efficiency of ODNs in Caki-1 cells The cells were treated with FITC labeled ODN s delivered by different cationic lipo so mes (DOT AP : DOPE DS3DOPC DOGSDSO CHDT AEA and PEG-PE) for 24 hr at a AS ODNs concentration of 1 Mimi Both serum free and 10 % FBS conditions were studied and compared Significant enhancements of ODNs cellular up-take were observed in all liposome treatment groups with DOT AP : DOPE and DS3DOPC being most efficient and with minimal se rum resistance (Figure 2-3) Since DOT AP : DOPE is of s imple composition and easy to prepare it was chosen as the delivery vehicle for the re s t of stt1dies Studies to optimize the delivery efficiency of ODN s by DOT AP : DOPE were then carried out One major factor that dete1 r1une s the delivery efficiency i s the lipo so me to ODN s charge ratio In order to determine the optimal charge ratio in Caki-1 cells the cel l s we r e treated for 24 hr with I M/ml FITC labeled ODNs prepared with different amount ofDOTAP : DOPE to achieve various charge ratio s (1 1 25, 1 5, 1 75, 2, 2 5) The

PAGE 42

35 fluorescent intensities of Caki-1 eel ls after the treatment were then compared (Figure 24 ) Increases in delivery efficiency and as well as resistance to serum were observed as the charge ratio increased A plateau in these effects occurred at a charge ratio of about 2 This charge ratio was used in all subsequent studies evaluating the delivery of AS-ODNs in Caki-1 cells The delivery efficiency of ODNs by DOT AP : DOPE as the function of time was then evaluated Calci-1 cells were treated with I Mimi FITC labeled ODNs delivered by DOT AP : DOPE at the pre-determined optimal charge ratio of 2 The fluorescent intensity of Caki-1 cells after different lengths of treatment were determined by FACS (Figure 25) Very fast up-take of ODNs by Caki-1 cell s were observed when delivered by DOT AP : DOPE liposomes The up-take of ODN s by Caki-1 cells reached a plateau within 24 hr of incubation (Figure 2-5) With the optimized delivery system based on cationic liposomes (DOTAP : DOPE) very efficient cellular up-take of ODNs was achieved in Caki-1 cells Significant up-take of FITC labeled ODNs was observed in Caki-1 cells after 24 hr treatment of I M ODN s delivered by DOT AP : DOPE at a charge ratio of 2 ODN s were delivered into ~ I 00 % of the cells with uniform cellular distribution and enhanced nuclear concentration (Figure 26). This result was achieved in both exponential phase and plateau phase Caki-1 cells (Figure 27) This optimized delivery system was then further te s ted for ODN s delivery efficiency in two other RCC cell lines The re s ult s s howed that s imilar highly efficient cellular up-take of ODNs also could be achieved in Caki-2 and A498 cells (Figure 2-8)

PAGE 43

36 ODN s as fragments of DNA se quences have very low toxicity (Rubenstein et al l 997 ~ Agrawal et al ., 1997) The toxicity of the cationic lipo so me deli very vehicle was also examined in both serum free and 10 % FBS conditions No significant toxicity of DOT AP : DOPE in Caki-1 cells was observed with do ses up to 150 m g/ ml in 1 0% se rum and 100 m g/ ml in se rum free conditions At do ses higher than 100 m g/ ml so me cytotoxicity was observed u s ing DOT AP : DOPE in se rum medium The DOT AP : DOPE do se u se d in the delivery s tudie s was only 10 m g/ ml which re s ulted in no to xicity in Caki-1 cells either in serum free or I 0/4 FBS conditions Discussion AS-ODNs can block the expression of s p ec ific tar ge t ge n es involved in the development of human disease s. Therapeutic applications of anti se n se techniques are currently under investigation in many different field s. In order for AS ODNs to down regulate gene expression it mu s t penetrate into the tar ge t cells Phospholipid bila ye r s repre se nt a s trong barrier to the mo ve ment of ions Studie s on the diffusion of ODN s throu g h model membrane s have led to tl1 e ge neral conclu s ion that it is of little importance Meanwhile internali za tion of ODN s into cells has been clearly demon s trated implying the existence of other mechanism s other than pa ss ive diffusion (Ga rcia-Chaumont et al ., 2000) To date th e preci se mechanisms involved in OD Ns penetration are still not totall y clear Thou g h it has been found that up-tak e of AS OD Ns can occur through receptor mediated active tran s port (Wu-Pong et al 1994), which depend s on temperature (Loke et al 1989 ; Yakubov et al 1 9 8 9), the s tructure and the concentration of ODNs (Vlas s ov et al ., 19 9 4) and the cell line s At the pre se nt time it is gene rally b e lieved that adsorpti ve e ndocyto s i s and fluid phase e ndoc y to sis are the major mechani s ms of ODN s internalization (Dias N and Stein 2002 ; Garcia-Chaumont et al .,

PAGE 44

37 2000) At relatively low ODNs concentrations it is likely that internalization occurs via interaction with a memberane-bound receptors (Loke et al ., 1989 ; Yakubov et al ., 198 9; de Diesbach et al ., 2000) While at higher ODNs concentrations these receptors are saturated, and the endocytotic process assumes larger importance Numerous reports have demonstrated that naked ODNs are internalized poorly by cells (Gray et al ., 1997 ; Stein et al ., 1993 )(Figure 2-2) More importantly naked ODN s tend to localize in endosomes/lysosomes where they are unavailable for antisense purposes As has been demon s trated in numerous experiments the s ine qi,a non of antisense activity appears to be nuclear localization (Dias N and Stein 2002) To improve cellular uptake and ODNs spatial and temporal activity delivery vehicles were developed Among them cationic lipo s omes are most commonly used Cellular up-take of ODNs as well as their activity in cell ct1ltures can be improved by cationic liposomes (Bennett et al ., 1992 ; Lappalainen et al ., 1994 ; Zelphati and Szoka, Jr ., 1996b ; Zelphati and Szoka Jr ., 1996a) Cationic lipo s ome s are s afe simple and easy to produce on a large scale (Nabel et al ., 1993 ; Nabel et al ., 1 9 94a ; Nabel et al ., 1994b ) They have been approved by FDA for clinical use However when tested for their ability to promote delivery of FITC-ODNs in mammalian cell line s, their efficacy varied significantly (Lewis et al ., 1996) Important variables include cationic lipsome / ODNs ratio composition of lipid and cell type tested (Flanagan and Wagner 1 99 7 ; Lappalainen et al 1997 ; Yellen et al ., 2002) Thi s s ugge s ts that even though cationic liposomes have been proven to be effective and to be of general utility a best reagent and optimal liposome / ODNs ratio could and should be empirically established for each cell type

PAGE 45

38 In the present studies deli very efficiencies of FITC ta gge d OD Ns b y different lipo so me compo s ition s were first evaluated All cationic liposome compositions s ignifi can tly enhanced ce llul ar up-tak e of ODN s How ever, th e incorporation of PEG-PE s ignificantly inhibited the d e li very efficiency of cationic lipo so m es (DOT AP : DOPE ) PEG-P E, when incorporated int o conventional lipo so m es can provide a s teric barrier at th e surface of lip so me s that inhibits op so nization, and ther efore can extend the re s id e nc e time of lipo s ome s in the blood (Webb e t al ., 19 9 8) Howe ve r even though the use of PEG-PE in lipo so me s have minimal effect on the bindin g and s ub seq u e nt endocytosis of lipid/DNA complexes it did seve rely inhibit the endosomal rele ase of AS-ODNs into the cytoplasm (Figure 2-3) (Song et al ., 2002) Given the efficiency of delivery minimum se rum re sis tance and si mplicity of liposom e composition and preparation DOT AP : DOP E was chosen as the d e li very ve hicl e for AS-ODN s in Caki-1 cells (Figure 2 3) A major problem associa ted with cationic lipo s ome s i s low tran sfec tion efficiency du e to inactivation of cationic lipo so me s by se rum (Feigner et al ., 1 9 87) Much effort has been devoted to re so lvin g thi s problem The charge ratio of liposome to D NA h as been proven pr evio u s l y to be critical for hi g h efficiency of lipof e ction and se rum re s i s tance (Yang and Huan g 1 997; Yan g and Huan g, 1 998) The optimal tran s fection efficie nc y and minimal se rum inacti va tion was achieved at a DOPT A : DOPE / ODN s charg e ratio of about 2 (Figure 2 4) Using thi s charge ratio cellular up-take of OD Ns b y Caki-1 cells r eac hes a plateau within 24 hr (F i g ur e 2-5) ODN s i n Ca ki-1 c e ll s were eve nly di s tributed in th e cytoplasum with enhanced nucl ear concentration (Figure 2 6). Thi s di s tribution i s beli eve d to b es t facilitate anti se n se function of AS-ODN s (Wagner 1 99 4 ; Ho g refe 1 999). With th e optimized delivery

PAGE 46

39 system, efficient cellular up-take of ODN s were achieved in both plateau and exponential phases Caki-1 cell s (Figure 27) as well as two other RCC cell lines (Caki-2 A498) (Figure 2-8) Cytotoxic evaluation of DOTAP : DOPE confirmed that cationic lipo so mes are safe unless very high doses are used (Porteous et al ., 1 997 ; Gao and Huang 1995) The dose used in the present studies (IO m g/ ml) was far below the doses that resulted in cytotoxic effects in Caki-1 cells( > 100 m g/ ml)(Figure 2-9) In conclusion cationic liposome s (DOTAP : DOPE) enhanced the cellular up-take of ODNs in RCC cell line s The s implicity of preparation efficiency of up-take and safety features have rendered the cationic lipo so me (DOT AP : DOPE) an attractive vehic le for AS-ODNs therapy

PAGE 47

N,w,ellular I l ate r ac tl on n, ... ,uepheUplcla ss wllll cura-1 ...... 40 I \ ......... of M-ODN Figure 21 Proposed mechanisms of inten1alization of cationic l iposome in to cel l s and r elease of ODN s in c y toplasm M odified from Lebedeva (Lebedeva et al ., 2000)

PAGE 48

41 control Naked P S OONS with DOTAP:DO P E I l J a 1: ,i 1,1 i: \I 11 1 3i M ~ I <: 0 o o o' ~ -., o' I J .0 10 1 ~ ~ serum F ree lll'C g .. ! : 0 0: '-' I ~ YI M l !~ li i 3a II ? 1 1 0 0 ~l lOJ 1 0 ,o o 1 0 3 o oo ,o "' \, 1-II C 10% FBS Figure 2 2 F low cy t o m etry histog ram of Caki -1 ce l l fl u o r escece i nt e n sity after tr ea tm e nt s o f 1 M FIT C l a b e l e d OD Ns e ith e r wi th o r wit h o ut DOT AP : DOP E lipo s om e

PAGE 49

Cl) C s C CD u C CD u Cl) e 0 ::s LL 10000 100 42 Serum Free C 10%FBS Control DOTAP:DOPE 0S3DOPC DOGSO S O CHDTAEA PEG-PE Liposomes Figure 2-3 Efficiency of ce llul ar up-take of FITC labeled ODNs de l ivered by different liposmes in Caki-1 cells Caki-1 cells were treated with I M FI'l C labeled ODN s in serum free or l 0% FBS condition s for 24 hr Each bar repre s ent s the mean + S E of 3 independent experiments

PAGE 50

10000 1000 fl) C: s C: G) u 100 C: G) (.) fl) 0 :, 10 u. Serum Free D 10%FBS 1 ..1,,,,-....,,j 0 5 1 43 1.25 1.5 1.75 2 2 5 Charge ratio Figure 2-4 Effect of liposome to OD Ns charge ratio on th e delivery efficiency of OD Ns in Caki -1 cells Caki-1 cells were tre a ted for 24 hour s with 1 M FITC lab e l e d OD Ns deli ve red by different amount s of DOT AP : DOP E lipo so m es at th e charge ratio indicated Eac h bar represents the mean S.E of 3 independent experi ment s

PAGE 51

1000 ..., "' C: ,! 100 C: Cl) (.) C: Cl) (.) "' e 10 0 :l LL 44 1 ,,,,,,,,,,,, I 0 24 Time (hr) I I I 48 Serum free C 10%FBS I I I I I 72 Figure 2 5. Time co ur se of up-tak e of ODNs delivered by DOTAP : DOPE in Caki 1 cells Caki -1 ce ll s were treated with 1 M FITC lab e led OD Ns delivered by DOT AP:DOPE liposome at a charge rati o of 2 0 Results are th e mean S E of3 independent experiments

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45 Figure 2-6 Fluorescent rnicro sc opic pictur es of Cakil cells after FITC labeled ODNs treatmer1t Caki-1 cells were treated with FITC lab e l ed ODNs for 24 hr at a dose of I A) low magnification 5x ; B) high magnification 20x

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4 6 Figure 2-7 Fluor esce nt micro scopic pi c tur es s ho w in g ce llular Ltptak e of FIT C lab e l e d ODNs in Cak i-1 cells Cak i-1 ce ll s we r e tr ea t e d with FITC la beled ODN s at a do se of 1 M for 2 4 hr A ) bri g ht fi e ld pi c tur e of co nflu e nt Caki1 cells ~ B ) flt1ore sce nt fi e ld pi c tur e of c onflu e nt Cak i-1 ce ll s ~ C) bright field pi c tur e of exponential pha se Caki -1 ce ll s ~ D ) tlttor esce nt field pi c tur e of ex pon e ntial ph ase Cak i-1 cells

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47 A498 Cak l -2 nl r l I 1 l 1 1 M FITC AS-ODNs Figure 2-8 Delivery efficiency of FITC labeled ODNs by DOTAP : DOPE lipo so me in other RCC cell lines (A498 Caki 2) The cel l s were treated with FITC labeled ODNs at a do se of 1 M for 24 hr

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120 100 G) C, ca ., C G) (.) .. G) c.. ,,, G) (.) G) J:2 ca > 80 60 40 20 0 0 10 % FBS Serum Free 50 48 100 DOTAP:DOPE concentration (mg/ml) 150 Figure 2-9 Caki-1 cell viability after 24 hr treatment with different concentration s of DOT AP : DOPE liposome s in serum free or I 0 % PBS conclition s

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CHAPTER3 AS-ODNS DESIGN AND IN VITRO ASSESS.MENT Angiogenesi s, a complex multis tep proce ss involvin g the formation of new blood vessels from pre-existing ones is tightly regulated by both positive and negative regulatory factors (Risau, 1997) These regulator s, which include pro-angiogenic factors such as basic fibroblast growth factor (bFGF) (Monte s ano et al. 1986) angiogenin (Gho and Chae 1997) and vascular endothelial growth factor (VEGF) (Leung et al ., 1989 ; Saleh et al ., 1996 ; Asano et al ., 1995 ; Borgstrom et al 199 6; Cheng et al ., 1996) as well as angiostatic peptides such as endostatin (O'Reilly et al ., 1 997; Perletti et al ., 2000), angiostatin (O'Reilly et al ., 1994a ; O'Reilly et al ., 1994b ; O'reilly et al ., 1994) and thrombospondin (Folkman and Shing 1992 ; Folkman 1995) are potential targets for anri-angiogenic therapy of solid tumors (Smith et al 1999 ; Bicknell and Harris, 1992 ; Denekamp 1999 ; Bicknell Rand Harri s A L 1992 ; Folkman 1971 ; Denekamp J 1999) Among all these factors VEGF and bFGF are believed to be most important regulators in tumor angiogenesis (Risau, l 997 ; Siemeister et al ., 1998) VEGF is an endothelial cell specific mitogen secreted as a 45 kDa homo dimer protein There are five human isoforms derived from alternative splicing (VEGF 121 145 165 189 206) as illustrated in Figure 3-1 (Tischer et al ., 1991 ; Houck et al ., 1991 ; Poltorak et al ., 1997) VEGf 1 2 1 and VEGF1 65 are the only soluble isoforms and also the most abundant with VEGF1 6s being the major isoform and most powerful stimulator of endothelial cell proliferation (Houck et al ., 1992 ; Soker et al ., 1997) VEGF 1 6 5 is 49

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50 commonly expressed in a wide variety of human and animal tumors (Hanahan and Folkman 1996) and has been shown to induce angiogenesis both in vitro and in vivo (Leung et al 1989 ; Plate et al ., 1992a ; Plate et al 1992b) It is currently believed that this diffusible molecule is probably a key mediator of tumor angiogenesis (Ferrara, 1999a ; Ferrara 1999b ) Indeed the expression of VEGF has been related to fundamental features of tumors such as growth rate (Kim et al ., 1993 ) mi crovessel density (Toi et al ., 1994) and vascular architecture (Drake and Little 1999) as well as the development of tumor metastasis (Weidner et al 1991 ) A correlation between VEGF expression and survival has been noted in some cancer patient s (Ga s pariru et al ., l 997 ~ Maeda et al ., 1996) Basic fibroblast growth factor is a prototype of a large family of 13 structurally related heparin-binding growth factors It affects the growth differentiation migration and survival of a wide variety of cell types (Bikfalvi et al ., 1997) BFGF was originally purified from the bovine pituitary gland as a 146-amino acid protein with a molecular weight of 15 kD (Gospodarowicz, 1975) It was later found to represent a proteolytic product of the primary 18 kD form (Bikfalvi et al ., 1997) The amino acid sequence of 18 kD bFGF is highly conserved among species with 89-95% identity among human bovine and rat (Abraham et al 1986a ; Abraham et al ., 1986b) This low level of divergence suggests that there may be functional importance for all regions of bFGF Larger forms of bFGF have also been identified resulting from alternative CUG translation starting sites (Florkiewicz et al. 1991a ; Florkiewicz et al ., 1991 b ) The use of different in-frame CUG codons upstream of the conventional AUG start codon allows translation of several bFGF isoforms with different molecular weight (Figure 3-6) (Okada -Ban et al 2000) In

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51 addition to the 18 kD isoform, alternative translation of 22 22 5 24 and 34 kD isoforms are also possible (Arnaud et al ., 1999) The main structural feature of the four high molecular weight forms of bFGF is the presence of nuclear localization sequence which directs the growth factors to the nucleus whereas the 18 kD bFGF isofor1n initiated from AUG start codon is essentially cytosolic BFGF is a multifunctional growth factor which has various effects in a large panel of cells and tissues It plays key role in development remodeling and disease states in almost every organ system (Bikfalvi et al 1997) One of best characterized activities of bFGF is its ability to regulate the growth and function of vascular cells such as endothelial cell and smooth muscle cells BFGF also regulates the expression of several molecules thought to mediate critical steps during angiogene s is These include interstitial collagenase urokinase type plasminogen activator (uPA) plasminogen activator inhibitor (PAI-1) uPA receptor and Pl integrins (Montesano et al ., 1992 ; Mignatti and Rifkin, 1993 Klein et al ., 1993) .. It is a potent angiogenic factor involved in tumor angiogenesis and metastasis (Basilico and Moscatelli 1992) Up-regulation of bFGF and its receptors have been fotn1d in tumor tissues compare to normal tissues (Smith et al 1999 ~ Dellacono et al ., 1997 ; Arbeit et al. 1996) Clinically associations between serum/urine bFGF and cancer outcome have been shown in several tumor systems including RCC (Wechsel et al 2000 ; Edgren et al ., 1999 ; Nanus et al ., l 993 ~ Fujimoto et al 1991 ) breast cancer (Yiangou et al ., 1997) head and neck cancer (Dietz et al ., 2000) cervical cancer (Sliutz et al ., 1995) liver cancer (Poon et al ., 2001 ) pancreas cancer (Ohta et al ., 1995) thyroid cancer (Sasaki et al ., 2001) and glioma, neuroblastoma (Bredel et al ., l 997 ~ Komuro et al ., 200 I)

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52 In light of their important roles in tumor angiogenesis VEGF and bFGF may be attractive targets for anti-angiogenic therapeutic interventions applied to the treatment of cancer Attempts to abrogate the angiogenic activity of VEGF and bFGF have focused on inactivating VEGF/bFGF through the use of antibodies against VEGF/bFGF or their receptors (Mordenti et al ., l 999 ~ Kim et al 1993 ; Aonuma et al ., 199 9 ; Lu et al 2002 ~ Brekken et al ., 2000) and VEGF soluble receptor s (Lin et al ., 19 9 8) inhibiting VEGF/bFGF receptor tyrosine kinases (Hennequin et al ., 1 99 9 ; Laird et al ., 2002 ; Solorzano et al 2001 ; Ning et al ., 2002) or suppre s sin g VEGF and bFGF messages (Smyth et al 1997 ; Ellis et al 1996a ; Elli s et al 1996b ; Nguyen et al ., 1998a ; Nguyen et al 1998b ; Inoue et al ., 2000) The latter relied on antisense oligonucleotides (AS ODN s ) or anti sense RNA (Eguchi et al ., 19 9 1 ; Mercola and Cohen 1995a ; Mercola and Cohen, 1995b) to modulate gene expression by disrupting RNA expression AS-ODNs technology provides an approach for inhibiting gene expre ss ion with tar g et specificity as a particular advantage (Stein and Cheng 1993 ; Engelhard, 1998a ; Engelhard 1998b ) AS-ODNs are also easy to prodt1ce in large quantities which make them potentially more practical than antisense RNA vector delivery approaches In the present studies AS-ODNs against VEGF and bFGF were designed and their efficacy tested in vitro in the model of human RCC (Caki-1) Materials and Methods Cell Culture The clear cell RCC cell line s Caki-1 Caki-2 and A4 9 8 were gifts from Dr Susan Knox (Stanford University, CA) Caki-1 cells were grown in Dulbecco's modified minimum essential medi urn (DMEM, Invitrogen Grand Island NY) supplemented with

PAGE 60

53 10 % fetal bovine serum (FBS Invitrogen Grand Island, NY) 1 % penicillin-streptomycin (Invitrogen, Grand Island NY) and 1 % 200 mmol/L L-glutamine (Invitrogen, Grand I s land NY) Antisense Phospho1othioate Oligodeoxynucleotides (AS-ODNs) Antisense and control ODNs (20-mers) were custom sy nthesized by Geno Mechanix (Alachua, FL) AS-ODNs V515 was complementary to 5 UTRjust up-stream of the translation start site (AUG codon) ofVEGF mRNA : 5 CTC ACC CGT CCA TGA GCC CG3' Scramble sequence : 5' CAC CCT GCT CAC CGC ATG GC 3' ; se n se sequence : 5' CGG GCT CAT GGA CGG GTG AG 3' and an inverted sequence : 5'-GCC CGA GTA CCT GCC CAC TC 3 were used as controls ODNs AS-ODNs B460 was complementary to the translation s tart site (AUG codon) of bFGF mRNA: 5' TCC CGG CTG CCA TGG TCC CT 3' AS-ODNs B471 was complimentary to the coding region ofbFGF mRNA : 5 CGT GGT GAT GCT CCC GGC TG 3 '; AS-ODNs B931 was complimentary to the 3' UTR : 5' GAT GTG GCC ATT AAA ATC AG 3 '. Scramble sequence : 5' GCC TGG ACC CTG GCT CTC TC 3' ; sense sequence : 5' AGG GAT GGC TGC CGG GA 3' and an inverted sequ ence : 5' TCC CTG GTA CCG TCG GCC CT 3' were u se d as controls All AS-ODN s were suspended in ste rile and endotoxin free water at a concentration of 1 mM aliquoted and stored at -20 C DOT AP:DOPE Liposome Prepa1ation Cationic liposome s were prepared u s ing the method described by Tang (Tang and Hughes, 1999) Briefly cationic lipid l 2-dioleoyloxy-3-(trimethylammonium) propane (DOTAP) was dissolved in chlorofor1n and mixed with a helper lipid 1 2-dioleoyl-3-sn phosphatidyletbanolamine (DOPE) (Avanti Polar-Lipid s, Alabaster Al) at a molar ratio

PAGE 61

54 of 1 : 1 The mixture was evaporated to dryn ess in a round-b o ttomed flask using a rotary evaporator at room temperature The r es ultin g lipid film was dried b y nitro ge n for an additional IO min to evaporate any r esi dual chlorofor1r1 Th e lipid film was re-suspended in s terile water to a final concentration of 1 m g/ nu b ase d on the weight of cationic lipid Th e r es ultant mixture s wer e s haken in a water bath at 35 C for 30 min T he s u s pen sio n s then were s onicated u s in g a Sonic Di s membrator (F i s her Scientific Pitt s burgh PA) for l min at room temperature to fortn homo genize d l i po so me s The particl e size di s tribution of liposomes was measured u s in g a NICOMP 380 ZLS instrument (S anta Bar bara CA) The average particle diameter was 144 0 77 0 nm Liposomes were s tored at 4 C and u se d within 3 month s VEG F Enzyme Immunoassay Caki-1 cells (I x 1 0 5 ) were se t in 60 mm di s he s and allowed to attach overnight The medium then was remo ve d and replac e d with AS-ODN s in se rum f ree medium with lipo so me (DOT AP : DOPE) and incubated for 5 hr Fre s h m e dium containing I 0% FBS th e n was added After 24 hr of incubation or at different time point s for the time course s tudie s the VEGF concentration was d e termined in th e medium u s in g a human VEGF ELISA kit (R &D Systems Minneapoli s MN) Enzyme Immunoassay of bFG F Ca ki-1 cells (1 x 1 0 5 ) were se t in 60 mm di s h es and allowed to attach overnight The medium then was remo ve d and r ep laced with AS-OD Ns in ser um fr ee m e dium with lipo so me (DOT AP : DOPE) and incubate for 5 hr Fresh m edi um co ntainin g 10 % FBS then was added Caki-1 cells were collected 72 hr later, washed and suspe nded 1 x 1 0 6 cells in 1 ml PBS containin g prot ease inhibitors ( 1 00 g/ml Phen y lmethane s uJphon yl

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55 fluoride 20 / ml leupeptin 3 g/ rnl aproti11in) The suspension was subjected to 3 freeze-thawing cycles ultrasonication for 5 s (100 W) on ice and was centrifuged at 14 000 g for 10 min The supernatant containing the intracellular bFGF was used for the bFGF concentration determination (human bFGF immunoassay kit, R & D Systems, Minneapolis, MN) VEGF and bFGF Relative Quantitative RT-PCR Caki-1 cells were set at 3 x 10 5 in 100 mm dishes and allowed to attach overnight The cells were then treated with l M VEGF antisense (VS 15) bFGF antisense (B460) or control ODNs as described. 24 hr later the cells were collected and the total RNA was isolated using RNeasy Mini Kit (Qiagen Valencia, CA) and RNA concentrations were determined by UV spectrophotometry A 2 g total RNA sa mple was u se d to reverse synthesize cDNA using Superscript II reverse tran scri ptase (Invtrogen, Grand Island, NY) A 2 5 l aliquot of the reverse transcriptase reaction product then was used for the PCR reaction VEGF PCR reaction s were carried out with a VEGF gene specific relative RT-PCR Kit (Ambion Austin, TX). BFGF PCR reactions were carried out using a forward primer: S GCA GCC GGG AGC ATC ACC A 3' and rever se primer : 5 GCC CAG ITC GIT TCG GTG CCC A 3 (Campbell et al ., 1999). The PCR reactions were run 22 cycles (denature 94 C 30 s, anneal 60 C 60 s extension 72 C 60 s) in a DNA Engine 200 (MJ research Waltham MA) PCR products then were run in 2% agrose gel and stai ned by ethidiurn bromid e. The gels were visualized and analyzed (Gel Doc 2000 ge l documentation system Bio-Rad Hercule s, CA) All PCR preparations were carried out in a laminar flow hood using aerosol resistant plug ge d pipette tips.

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56 FGF Receptors 1-4 RT-PCR Total RNA of exponential phase Caki-1 cell was isolated using RNeasy Mini Kit (Qiagen Valencia, CA) and RNA concentrations were determined by UV spectrophotometry. A 2 5 I aliquot of the reverse transcriptase reaction product then was used for the PCR reaction Primers for human FGFR 1-4 designed by Tartaglia etc were used (Tartaglia et al. 2001 ) The PCR reactions were run for 30 cycles (denature 94 C 30 s anneal 60 C 60 s extension 72 C 60s) in a DNA Engine 200 (MJ research Waltham, MA) The specificity of the cDNA amplifications were then verified by endonuclease re s triction analyses (Tartaglia et al ., 2001 ) All PCR preparations were carried out in a laminar flow hood using aerosol resistant plugged pipette tips Negative controls without template DNA were included in each assay 18S primer se t (Ambion, Austin TX) wa s used as positive control Cell Cycle Assays Caki-1 cells were plated in 60 mm dishes at 2 x I 0 5 cells per dish and allowed to attach overnight. The cells were then treated with I M B460 or control ODNs mixed with DOT AP:DOPE as described above 48 hr later the cells were trypsinized counted and fixed in 50% ethanol overnight Before analyzed by FACS the cells were treated with 1 mg/ml RNase (in PBS) for 30 min The samples were then washed with PBS twice and re s uspended in 25 mg/ml propidium iodine (PI) in PBS at a concentration of 1 x I 0 6 cells / ml The cells were stained with PI in darkne ss for 15 min and were analyzed by FACS for cell cycle distribution on a Beckman Dickin s on flow cytometer made available through the University of Florida Core Facility for Flow Cytometry

PAGE 64

57 Apoptosis Assays Caki-1 cells were set in 2-well chamber slides and treated with 1 M B460 or control ODNs as described earlier 48 hr later the cells were washed and fixed in 4% para-forrnaldehyde solution for Fluorometric TdT-mediated dUTP Nick-End labeling (TUNEL) assay Briefly, the cells were permeabilized in 0 2% Triton X-100 solution for 5 min DNA strand breaks were then labeled with fluorescein-12-dUTP in TdT incubation buffer at 3? C for 1 hr The samples were then counterstained with l g/ml PI in PBS, which binds to the A-T rich regions of DNA Localized green fluorescence of apoptotic cells (fluorescein 12-dUTP) in a red background (PI) was detected by fluorescence microscopy The percentage of apoptotic cells was obtained by dividing the number of cells with green fluorescence by the total number of cells counted A minimum of 300 cells were counted for each condition Results VEGF AS-ODNs Design and Assessment Since VEGF has multiple isofor 111s resulting from alternative splicing (Figure 31 ) AS-ODNs design was targeted at the common region of all isofor1ns regions around the AUG start codon After screening several different designs, AS-ODNs VS 15 which is complimentary to the 5'UTR region just up-stream of the AUG start codon of the VEGF gene was found to be most effective The results showed that after 24 hr treatment with l M VEGF AS-ODNs {V515) delivered by cationic liposome (DOT AP : DOPE) the medium VEGF levels were significantly reduced from a norn1al of 850 pg/ml/10 6 cells to 250 pg / ml / 10 6 cells (p < 0 05 student s t-test) (Figure 3-2) This antisense effect was sequence and target region specific Treating Caki-1 cells with liposome vehicles

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58 ( DOT AP : DOPE) or control scramb l e ODNs did not affect VEGF levels Similarly treatment with sense or inverted sequence ODNs failed to reduce VEGF expression Co ntinued exposure of Caki-1 cells to the VEGF AS-ODNs (VS 15) resulted in a constant repression of VEGF in the culture medium (Figure 3-3) However if the culture medium containing VS 15 was replaced with fresh medium 24 hr later the VEGF levels in the Caki-1 ce ll medium gradually recovered, and reached about ~ 80% of that found in the untreated Caki-1 cell medium in about 7 day s (Figure 3 3) This repression of VEGF expression by VS 15 was also dose dependent (Figure 3 4) For example a 24 hr treatment with 0 5 M, reduced the medium VEGF level to 56o/o of control (p < 0 05, student's t-test) whereas a 1 M dose down-regulated the VEGF level to 22% of control (p < 0 05 student s /-test) VEGF mRNA lev els in different AS-ODNs treatment groups also were determined (Figure 3-5) The results indicated a marked inhibition of VEGF mRNA after treatment with VS 15 whic}1 was absent in cells treated with scramble control ODNs This result indicated that RNase H plays an important role in the function of VS 15 BFGF AS-ODNs Design and Assessment Alternative translation utilizing CUG start codons other than the AUG start codon leads to different i sofor1ns bFGF gene products (Figure 3-6) In order to target all the bFGF isoforms using the same AS-ODNs sequence, the AS-ODNs were designed to target the common regions of all isoforms, especially around the AUG start codon Effective AS-ODNs sequences against bFGF were identified and Caki-1 cells treated with them showed bFGF l evels significantly lo wer than those normally observed (720 p g/ ml / 10 6 cells) (Figure 37) Thi s effect wa s sequence and target region specific The

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59 AS-ODNs complimentary to the start codon (AUG) region (B460) was found to be the most effective For example the cellular bFGF level of B460 treated Caki-1 cells was found to be about 41% of that found in control or untreated cells (p < 0 05 student's t test) By comparison, the AS-ODNs complimentary to the 3' UTR {B93 l) or coding region (B471) were less effective at down regulating bFGF expression (57% and 65% of control values respectively p < 0 05 student s t-test) Since B460 had the most prominent inhibitory effect it was used in all subsequent studies Treating Caki 1 cells with control scramble ODNs or liposome vehic le s did not affect bFGF levels in Caki-1 cells Similarly treatment with sense or inverted sequence ODNs failed to reduce bFGF expression. This inhibitory effect was also found to be AS-ODNs dose dependent (Figure 3-8). While a low dose of 0 5 M B460 reduced the cellular bFGF l evel to about 80% of control a high dose of 5 M B460 led to a reduction by 65%. BFGF mRNA levels in different PS-ODNs treatment groups also were dete1 rnined (Figure 3 9) The results indicated a marked inhibition of bFGF mRNA after treatment with B460 which was absent in cells treated with scramble ODNs Again, this suggests a role for RNase Hin the efficacies of bFGF AS-ODNs B460 Because FGF can have mitogenic effects in renal cells (Gospodarowicz et al 1986 ; Issandou and Darbon 1991 ), the influence of antisense and control ODNs treatment on Caki-1 cell growth was investigated. Control ODNs or liposome vehicles showed no effect on Caki-1 cell growth (Figure 3 10). In contrast Caki-1 cell growth was significantly inhibited by AS-ODNs targeted against different regions of bFGF mRNA B460 was found to be the most effective while AS-ODNs targeting the 3 UTR (B93 l) or coding region (B471) showed le ss cell growth inhibition When comparing the data of

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60 Figures 37 and 3-10 it also i s apparent that the extent of Caki -1 cell g rowth inhibition by different AS-ODNs wa s closely related to their pot e nc y in down re g ulatin g bFGF e xpre ss ion In order to g ain a better under s tandin g of the underlyin g mechani s m s of th e observed growth inhibitory effect FGF rec e ptor expression was determined in Caki-1 cells (Figure 311 ) It was found that 3 out of 4 FGF rec e ptor s involved in the bFGF s ignal transduction pathway were expressed by Caki -1 cells, indicating that bFGF may play an autocrine role in Caki-1 cel l s Additional s tudie s indicated that B460 treatment had small but significant effects on Caki 1 apoptosis and c e ll cycle distribution (Figures 3-12 and 3-13) However c l onogenicity studies s how e d no s ignificant differ e nce b e tween B4 60 treated and control cells indicatin g that B4 60 treatment had no direct cell killing effect on Caki-1 cell s ( data not shown) These findin gs s u gges t that blockin g the bFGF s ignal tran s duction pathway may affect Caki -1 cell growth through cell cycle inhibition and induction of apoptotic cell d e ath Discussion Although dramatic advance s have been made in the treatment of cancer the de velo pment of efficacious anticancer a g ent s still lag s behind the rapid s tride s in our under s tanding of cancer biology especial l y with the advent of molecular biolo gy The continued progre ss in our knowledge of the biolo gy of neopl as m and in the identification cloning and sequencing of ge ne s critical to tumor cell function permit s th e exploitation of thi s information to develop s pecific a ge nt s that may dir ec tl y modulate th e function of the se ge ne s or their protein product s On e m e thodolo gy that takes direct advantage of

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61 molecular sequencing data involves the use of anti se nse oligonucleotides (Ho and Parkinson 1997) The antisense-mediated ge ne inhibition was first introduced in 1978 b y Stephenson and Zamecnik (Zamecnik and Stephen so n 1 978 ; Stephenson and Zamecnik, 1978) The underlying concept is relatively s traightforward : the u se of a se quence complementary by virtue of Watson-Crick basepair hybridization to a s pecific mRNA can inhibit its expression and then induce a blockade in the transfer of genetic information from DNA to protein The selection of an appropriate target sequence is the first step in the process of AS-ODNs drug development As a matter of fact the hybridization between AS-ODNs and the target mRNA which has a particular three dimensional shape re s ultin g from seco ndary and tertiary s tructure s depends on the accessibi li ty of the target se quence Only limited stretches of m.RNA se quences are actually available for heteroduplex formation with AS-ODNs Still there i s no s ure way to detern1ine a priori which AS ODN s sequence would work be st (Cohen, l 989;Woolf et al 1992 ; Brysch and Schlingensiepen 1994) The region surrounding the start codon (AUG) is probably the most popular target followed by 5 UTR, coding region s or s plicin g s ite s In the present s tudie s AS-ODNs sequence de s ign and se l ection focu se d mainly on the s tart codon region of VEGF and bFGF genes Thi s region i s al so common to al I isoforms of these two growth factors (Figure 3-1 and 3-6) The most commonly used AS-ODNs are 18-20 base s in len gt h According to s tati s tical calcuJations a particular se quenc e of 1 3 bases in RNA and of 17 ba ses in DNA s hould be found only once in the entire human genome thu s r e pr ese ntin g unique

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62 elements within the cell (Helene and Toulme 1990) It also has been found that the activities of AS-ODNs increased with AS-ODNs length but the increased thermodynamic stability of hybridization observed with AS-ODNs binding to non-target mRNA seq uence s that may be similar but not identical to the target sequence, results in reduced specificity (Monia et al ., 1992) Thus an AS-ODNs length of 20 -mer is usually considered optimal and therefore was used in the present studies for all the designed AS ODNs To ensure the specificity of the target sequence all designed AS-ODNs sequences were checked for global sequence comparison using the Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information (NCBI http :// www ncbi.nlm.nih .g ov/BLAST/) Only sequences specific for the VEGF/bFGF gene and having at least 4 miss-match bases with other genes were used In addition, polyguaosine (GGGG) which is known to exert non-antisense effects was avoided (Benimetskaya et al ., 1997) Lastly all the AS-ODNs desi g ned were examined for s econdary structures such as hairpins s elf-dimers and cro ss -dimers using Netprimer (PREMIER Biosoft International Palo Alto CA) Since cells contain a variety of exoand endonucleases that can degrade ODN s, nucleotide modifications have been made to make the AS-ODNs more resistant to nuclease digestion than the native ODNs that have phosphodie s ter linkages in their backbones The most widely explored analogues have been the phosphorothioates in which one of the nonbridging oxygen atoms in each internucleoside phosphate linkage i s replaced by a sulfur atom This modification is easily adapted to automated synthesis and confers metabolic stability becau se its resistance to degradation by DNase (Stein et al ., 1988) In addition these analogue s retain water s olubility and permit RNase H mediated

PAGE 70

63 hydrolysis of the target mRNA strand This modification has been successfully used in a variety of investigations (Galderisi et al ., 1999 ; Ho and Parkinson 1997 ; Crooke 1998) Based on these favorable properties and extensive information available for its application phosphorothioate modification was used in the current investigations of VEGF/bFGF AS-ODNs AS-ODNs may exert biological activity through a variety of mechanisms Although some of the mechanisms of inhibition have been characterized rigorous proof for others is still frequently lacking Two classes of AS-ODNs can be discerned : (a) the RNase-H dependent ODNs which induce the degradation of mRNA ; and (b) the steric blocker ODNs which physically prevent or inhibit the progress of splicing or translational machinery (Crooke, 1992 ~ Dias N and Stein 2002) The most commonly implicated antisense mechanisms relate to RNase H mediated hydrolysis of the target mRNA This is also the case for most of the anti sense drugs investigated in the clinic RNase His a ubiquitous enzyme that hydrolyzes the RNA strand of the RNA/DNA hybrid Thus the binding of AS-ODNs to its target mRNA may induce digestion of the message AS-ODNs assisted RNase H dependent reduction of target RNA expression can be quite efficient reaching 80-95% down-regulation of protein and mRNA expression (Dias N and Stein, 2002) Furthermore in contrast to the s teric-blocker ODNs RNase H dependent ODNs can inhibit protein expression when a much wider region of the mRNA is targeted Thus unlike most steric-blocker ODNs that are efficient only when targeted to certain 5 UTR or AUG start codon regions RN ase H dependent AS-ODNs can exert effects when targeted to widely separated areas in the coding region as well (Dean and McKay, 1994 ; Larrouy et al ., 1992) In the pre s ent

PAGE 71

64 s tudie s ) mRNA levels of VEGF and bFGF were significantly down-re g ulated after the AS-ODNs treatment (Figures 3-5 and 3 9) The se findings s u ggest that current AS-ODNs function mainly throu g h the RNase H me c hani s m Successful de s ign and evaluation of AS-ODNs relies on their efficient d e li very of into the cytoplasm where the y can exe rt th ei r anti se n se effect A cationic lipo so me (DOA TP : DOPE) based deli very syste m was used to d e li ver AS-0 DN s in the pre se nt studies The evaluation and optimization of thi s deli very system has been di scussed in detail in Chapter 2 Following the aforementioned guidelines in AS-ODNs de sign and selection effective AS-ODNs again s tVEGF and bFGF have be e n identified TreatmentofCaki -1 ce ll with the AS-ODNs led to significan t repression of VEGF and bFGF expression l evels (Figures 3-2 and 3 7) The se effec t s were OD Ns seque n ce specific d ose d e pend en t and could be achieved at low non-t ox ic do ses These re s ult s indicate that AS-ODN s can be u se d to efficiently modulate th e speci fic tar get ge ne exp r essio n s

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65 VEGF mRNA VEGF 121 1 2 3 4 VEGF 145 1 2 3 4 VEGF 165 1 2 3 4 VEGF 189 1 2 3 4 VEGF 206 1 I 21 3 I 4 ~~/4:--V515 Figure 3 -1 VEGF mRN A s tru c tu re showing all isofo r ms derived from a l ternative s pl i ci n g

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66 120 100 0 .. ., C 0 80 (,,) 0 ., 60 0 Q) > Q) 40 u. C) w 20 > 0 .:J--U nt reat ed DOTAP Sc r a,rble Sense Inverted V5 15 Treatment Figure 3 2 VEGF levels in culture medium of Caki-1 cells treated with different AS ODNs Caki-1 ce ll s were untreated treated with vehic l e (DOT AP) only, treated with 1 M control ODNs o r VEGF AS-DONs (V515) for 24 hr The 100 % VEGF expre s sion le v el of the untreate d group correspond s to ~ 800 p g/ ml / 10 6 cell s. Each bar represents the m e an + S E of 3 independent experiment s The s tar indicate s a statistically significant difference from the untreated group (p < 0 05 student's /-test)

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120 100 0 .. .., C 0 80 (,,) 0 60 0 G) > G) 40 (!) w > 20 0 0 1 67 D Corti r-..ied V515 treatmert 24 ty V515 treatment 2 3 4 Time (day) 5 6 7 8 Figure 3-3 VEGF levels in the culture medium of Caki-1 cells at different times after V515 treatment Media containing V515 were either or replaced with fresh medium after 24 hr Each datum point r e pre se nt s the mean S E. of 3 independent experiments

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68 1 2 0 100 e .. C 0 80 u 0 .. 0 60 Cl) > Cl) 40 u. (!) w > 20 * 0 0 1 2 3 4 5 Antisense PS ODNs dose (M) Figure 3-4 VEGF level s in culture medium of Caki -1 cells after tr eat ment with different doses of VS 15 The O do se repre se nts the Caki-1 cells tr ea ted with 5 M control sc ramble OD Ns Eac h datum point repre se nt s the mean S E of 3 independent expe riment s Stars indicate s tati s tical significance compared to the contro l ODN s treated ( p < 0.05, s tudent s t-te s t)

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69 A Untreated Scramble V5 1 5 188 VEGF B 200 ti) 00 180 0 160 '$. Q) 140 > ca 1 20 Q) "100 Q) > 80 Q) 0 Untreated Scramble V 51 5 Treatment Figure 3 5 Message RNA l evels in Caki-1 cells e ither untreat e d treat e d with a 1 M do se of sc ramble ODN s or VEGF AS OD Ns (V515) A) Rep rese ntati ve relati ve RT-P C R results; B ) Re l ati ve VEGF mRNA leve l s of Caki-1 cel l s after th e treatment The sta r indicates a s tati s tical significance compared to untreated control (p < 0 05 student 's t-t es t )

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70 bFGF mRNA CUG CUG CUG CUG A U G 86 319 346 361 486 5 bFGF proteins I I I I I I I I Nuclear localization signal ST O P 951 I 18 kD 22kD I 22 5 kD I 24kD I 3 4k D Figure 3-6 Message RNA structure and protein product s of bFGF ge ne Modified from Okada-Ban ( Okada-Ban et al ., 2000)

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800 700 :::::600 E c, 500 a. G> 400 > Q) LL 300 (!) 200 100 71 * OJ.-Untreated OOTAP Scranille Sense Inverted 8460 8471 8931 Treatment Fi g ure 3-7 Cellular bFGF levels of Ca.kj-1 cells after AS-ODN s treatment Caki-1 cell s were either untreated treated with liposome alone 1 M control ODN s or bFGF AS ODNs for 24 hr Eac h bar represents the mean S E of 3 independent experiments Stars indicate s tatistically significant difference from the untreated contro l g roup (p < 0 0 5 s tudent 's t-te s t)

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120 100 Cl> 0) cu +,I C Cl> (.,) ... Cl> 0. Cl> u. (!) u. .0 80 60 40 20 0 72 * 0 1 2 3 4 5 8460 dose (M) Figure 3-8 Cellular bFGF levels in culture medium of Caki-1 cells after treatment with different doses of B460 The O dose Caki-1 cells treated with 5 M control scramb l e ODNs Each datum point represents the mean S E of 3 independent experiments Stars indicate statistically significant differences compared t o the control ODNs treated group (p < 0 05 student's I-test)

PAGE 80

A 18s bFGF B "' co .... 0 ..., Q) > ..., ns Q) ... Q) > Q) ct z 0:: E LL (!) LL .0 73 Untreated DOTAP Scramble B460 200 150 100 50 0 Untreated DOTAP Scramble B460 Treatment Figure 3 9 Message RNA levels in Caki-1 cells either untreated treated with a I M do se of scr amble ODNs or bFGF AS-ODNs (B460) A) Repre se ntative relativ e RT-PCR results~ B) Relative bFGF mRNA level s of Cakj-1 cells after the treatment The st ar i ndicate s a s tatistical l y significant difference compared to the untreated control group (p < 0 05 s tudent s /-test)

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120 G> 100 0, cu C G) G) Q. fl) G) u ,I 80 60 40 J 20 74 * o~Untreated OOTAP Scrarmle Sense Inverted 8460 8471 8931 Treatment Figure 3-10 Effect of AS-ODNs treatment on Caki-1 cell growth Caki-1 cells were either untreated, treated with lipo some alone 1 M control ODNs or bFGF AS -OD Ns for 4 days and the number of ce ll s determined Each bar represents the mean S E of 3 independent experiments Stars indicate statistically significant difference s compared to untr e ated co n trol group (p < 0 05 student s t-test)

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75 Figure 3-11 FGF receptor expression in Caki 1 cells RT-PCR results of FGF receptors 1-4 and control 18s expression in Caki-1 cells

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76 Figure 3-12 Apoptotic eel I d eat l1 in Cak i -1 ce ll s e ith e r untreated o r tr ea t e d with bFGF AS-ODNs A) R e pr ese nt a ti ve picture of u11tr ea t e d Caki-1 cells s tained with D ea dEnd Fl u oro m e tri c TUN E L Systern : B ) r e pr ese nt a ti ve picn1re of Caki1 ce ll s afte r treatment wit h B4 60 sta in e d with D eadEn ct Ttvt Fluorometric TUNEL System

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77 10 9 Q,) 0, s 8 .., C Q,) 7 u Q,) 6 Q. Cl) 5 Q,) u 4 u ...., 3 .s Q. 2 0 Q. < 1 Untreated DOTAP Sc r amble 8460 Treatment Figure 3-13 Apoptotic cell death in Caki-1 cells after AS-ODNs treatment Caki 1 cells were either untreated, treate d with l iposome alone treated with I M scramb l e control ODNs or bFGF AS-ODNs Each bar represent s the mean S E of 3 independent e xperiment s. The star indicated a statistical l y significant difference from the untreated control group (p < 0 05 st u dent 's / test)

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78 75 Untreated 18] Control 60 D B460 U) Cl) (.) .... 45 0 Cl) 0) !! C 30 Cl) (.) ... Cl) Q. 15 0-+---GO G 1 s G2M Cell cycle distribution Figure 3 14 Effect ofbFGF AS ODNs treatment on the ce l l cyc l e distribution of Caki-1 cel l s. Each bar represents t h e mean S E of 3 independent experiments. Stars indicate s tatistical significant differences compared to the untreated control group (p < 0 05, s tudent 's t test)

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CHAPTER4 ANTI-ANGIOGENIC EFFICACY STUDIES Int1oduction The greater understanding of the proces s of tumor angiogenesis coupled with the notion that tumors require a blood supply to grow and metastasize has fueled the re searc h for strategies that block or di sru pt the an g iogenic process Moreover because normal vascular endothelial cells turn over so s lowly conventional wisdom suggests that an anti-angiogenic approach to cancer therapy s hould offer improved efficacy and reduced toxicity with much less potential for drug resi st ance Angiogenesis is a complex process with multiple, sequential and interdependent steps (Fidler 1999) This comp l exity creates many potential targets for inhibition Key characteristic of the immature vascuJature of tumors have allowed the development of seve ral categories of anti-angiogenic agents (Kerbel 2000) Preclinical studies have identified agents that (i) inhibit endothelial cell activation (ii) inhibit endothelial proliferation/migration (iii) inhibit basement membrane degradation and (iv) inhibit inte g rin receptor activation Angiogenesis can be qualitatively and quantitatively measured in a large variety of in 1 1 itro and in vivo model systems As mentioned before the angiogenic cascade can be dissected into different sequential steps so that can be studied separately in vitro Research has mainly focused on the proliferation and migration of endothelial cells as key elements for angiogenic potential in vit,o For this research different endothelial cell s ources can be utilized For human tumor re searc h mo s t laboratories make u se of 79

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80 HUVECs Although readily available a major advantage the major drawback of these cells is their macrovascular origin which makes them less suitable for studies on angiogenesis a rnicrovascuJar process In recent years microvascuJar endothelial cells derived from different organs have been established and become commonly available Protocols for isolating purified tumor endothelia l cells also have been developed (St Croix et al ., 2000) Assays to study proliferation of endothelial cells are based on cell counting radio labeled thymidine incorporation or on colorimetric assays for measurement of mitochondrial activity Detection of cell death also is used to determine cell growth effects To measure endothelial cell migration Boyden chambers are primarily used Though an easier system based on wounding of a confluent monolayer of endothelial cells and measuring wound width or invading cells as a function of time is also available, it maybe physio l ogically less relevant to tumor angiogenesis In tl1e present studies we established a co-culture system based on modified boyden chambers to evaluate endothelial ce l l proliferation and migration potential (Figure 4-1 and 4-4) In order to mimic the in vi, o interaction of tumor and endothelial cells co-cultured tumor cells were the primary source of pro angiogenic growth factors for the endothelial cells The advantage of the in , itro assays is clearly the control that can be exerted over selected parameters However the angiogenic cascade consists of multiple steps in their entirety in vi, o investigations are needed The most frequently used in vivo assay systems are the chicken chorioallantoic membrane assay (Nguyen et al ., 1994a) the corneal pocket assay (Conrad et al ., 1994), transparent chamber preparations such as the dorsal skin-fold chamber (Algire G H ., 1943 Lichtenbeld et al 1998) the cheek pouch

PAGE 88

81 window (Shubik et al ., 1976) and polymer matrix implar1ts (Mahadevan et al ., 1 9 89 ; Plunkett a11d Hailey 1990) A s impler system using intradermal implantation of tumors cells is also available to study the tumor angiogenic process in vii o (Sidky and Auerbach 1976) This assay has been validated for evaluating tumor-induced angiogene sis in vivo using a variety of different tumor model s and treatment interventions (Lindner and Borden 1997 ; McMilla11 et al ., 1999 ; Daniel s en a11d Rof s tad 1998) and was applied in the present studies Through the utilization of both the co-culture system in vitro and the intrade11nal an g iogenesis assay in 1rivo objective and reasonable assessments of the anti-angiogenic efficacy of treatment interventions can be achieved. In the present studies the anti angiogenic efficacy of VEGF and bFGF AS-ODNs treatments were evaluated using these in vit,o and in vivo models Materials and Methods Cell Culture The clear cell RCC cell line Caki-1 was a gift from Dr Susan Knox (Stanford University, CA) Caki-1 cells were grown in Dulbecco's modified minimum essential medium (DMEM, Invitrogen Grand I s land NY) s upplemented with 10 % fetal bovine serum (PBS Invitrogen, Grand Island, NY) 1 % penicillin-streptomycin (Invitrogen, Grand Island, NY) a11d 1 % 200 mmol/L L-glutamine (Invitrogen, Grand Island NY) The mouse heart endothelial cell line (MHE) was a gift from Dr Robert Auerbach (University of Wisconsin, WI) MHE cells were grown in DMEM s upplemented with l 0% heat inactivated FBS 1 % penicillin-streptomycin and l % 200 mmol /L L-glutamine Human microvascular endothelial cell from the lung (HMVEC-L) cells were obtained

PAGE 89

82 from Clonerics (San Diego CA) HMVEC-L cells were grown in EBM-2-MV (Clonetics San Diego CA) supplemented with 5% FBS Antisense Phospborothioate Oligodeoxynucleotides (AS-ODNs) Anti se nse and control ODN s (20-mers) were custom synthesized by Geno Mechanix (Alachua, FL) The entire backbone of all ODN s was phosphorothioate modified AS-ODNs V515 was complementary to 5 UTRjust ups tream of the translation start site (AUG codon) of VEGF mRNA : 5' CTC ACC CGT CCA TGA GCC CG 3' Scramble sequence : 5' CAC CCT GCT CAC CGC ATG GC 3' ; se nse sequence : 5' CGG GCT CAT GGA CGG GTG AG 3' and an inverted seq uence : 5' GCC CGA GT ACCT GCC CAC TC 3', were u se d as ODN s controls AS-ODNs B46 0 was complementary to the translation start site (AUG codon) of bFGF m.RNA : 5' TCC CGG CTG CCA TGG TCC CT 3' Scramble sequence : 5' GCC TGG ACC CTG GCT CTC TC 3'; sense sequence : 5' AGG GAT GGC TGC CGG GA 3' and an inverted sequence : 5' TCC CTG GTA CCG TCG GCC CT 3' were u se d as controls All ODNs were suspended in sterile and e ndotoxin free water at a concentration of 1 mM, aliquoted and stored at -20 C DOTAP:DOPE Liposome Preparation Cationic liposomes were prepared using the method described by Tang (Tang and Hughes 1999) Briefly cationic lipid l ,2 -dioleoyloxy-3-(trimethylammonium) propane (DOT AP) was dissolved in chloroform and mixed with a helper lipid 1 2-dioleoyl-3-sn phosphatidylethanolamine (DOPE) (Avanti Polar-Lipids Alabaster Al) at a molar ratio of 1 : I The mixture was evaporated to drynes s in a round-bottomed flask u s ing a rotary evaporator at room temperature The re s ultin g lipid film was dried by nitrogen for an

PAGE 90

83 additional 10 min to evaporate any residual chloroform The lipid film was re-suspended in sterile water to a final concentration of 1 mg/ml based on the weight of cationic lipid The resultant mixtures were shaken in a water bath at 35 C for 30 min The suspensions then were sonicated u sing a Sonic Dismembrator (Fisher Scientific Pittsburgh PA) for 1 min at room temperature to forn1 homogenized liposomes The particle-size distribution of liposomes was measured using a NICOMP 380 ZLS instrument (Santa Barbara, CA) The average particle diameter was 144 0 + 77 0 nm Liposomes were stored at 4 C and used within 3 months Co-culture Assay Transwell (Corning Corning NY) 6-wel l dishes with a membrane pore size of 0 4 M were used Caki1 cells were seeded at 5 x 10 4 in the transwell inserts and :Ml-IE or HMVEC-L cells were plated at 5 x 10 4 per well in the 6-well dishes and allowed to attach overnight The Caki-1 cell medium then was replaced with serum free medium containing 1 M VS 15 or B460 AS-ODNs or control ODNs delivered with liposome (DOTAP : DOPE) After a 5 hr of treatment medium containing 10% heat inactivated FBS was added to yield a final FBS concentration of 2 5% The transwells containing treated Caki -1 cells were assembled with 6-we ll dished containing MHE and HMVEC-L cells and incubated at 37 C for 72 hr at which time the numbers of MHE or HMVEC-L cells were determined by haemocytometer count (Figure 4-1 ) Migration Assay Caki-1 ce ll s were set at l x 10 5 per well in 24-well dishes and allowed to attach overnight The Caki-1 cells then were treated with I M V5 l 5 or B460 AS-ODNs or control ODNs for 24 hr HTS FluoroBlok inserts (Becton Dickinson Franklin Lakes, NJ)

PAGE 91

84 with a pore size of 8 0 m were assembled into the 24-well dish with the Caki-1 cells MHE or HMVEC-L cells were grown in T-150 flasks to about 80% confluence The e ndothelial cells were s tained in mediun1 containing l O / ml Di-I (Molecular Probes Eugene, OR) for 24 hr washed 4 times with PBS collected and added into th e FluoroBlok inserts (5 x 10 4 MHE or HMYEC-L) and incubated for another 24 hr The number of migrated endothelial cells then was determined b y direct measurement of the fluorescence in the bottom well u s ing a CytoFluor 4000 plate reader (Perceptive BioSy s tems St Paul MN) (Figure 4-4) Intiadermal Angiogenesis Assay Caki-1 cells were treated with AS-DONs for 5 hr in vitro as described before The cells were then collected and inoculated intradermally (5 x 10 4 ) in a volllil1e of 10 lat 4 sites on the ventral surface of nude mice One drop of 0 4% trypan blue was added to the cell suspension before injection, which making it lightly colored si mplified s ubsequent location of the sites of injection Three days later the mice were killed, the skin was carefully separated from the underlying muscle and the 11umber of vessels counted u si ng a dissecting micro scope (Sidky and Auerbach 1976) Scoring of all of the reaction areas was carried out at the same ma g nification (Sx) and only vesse l s readily detected at this magnification were counted The s ite s of injection recognized by local swe llin g and blue staining, were exposed by carefully removing fat or other tissue covering the area. All vesse l s that touched the edge of the tumor inoculates were counted All the animals in the experiments were pre-coded and vessel counts in each animal were sco red twice The resultant data point s for each treatment group were pooled for s tati sti cal analysis (Wilcoxon rank sum test)

PAGE 92

85 Results Anti-angiogenic efficacy of VEGF/bFGF AS-ODNs treatments was first evaluated in vitro The transwell co-culture system was used to examine the effect of AS ODNs treatment of Caki-1 cells on the proliferation of co-cultured endothelial cells (Figure 4-1 ) This setting allowed the constant exchange of growth factors without direct tumor-endothelial cell-cell interaction and mimicked the paracrine interaction between tumor and endothelial cells Since a human RCC tumor cell line and xenograft in nude mice were used in these studies both human (HMVEC-L) and mouse (MHE) endothelial cells were studied Caki-1 tumour cells were grown in transwells with 0 4 m membrane pores The effects of pretreating Caki-1 tumor cells with VEGF or bFGF AS-ODNs on endothelial cell proliferation then were determined (Figure 4-2 and 4-3) The results showed that compared to untreated Caki-1 cells, Caki-1 cells pre-treated with V515 or B460 significantly inhibited both HMVECV-L and .MHE cell proliferation. Once again, treating Caki-1 cells with a variety of control PS-ODNs had no effect on HMYEC-L or W-IE cell growth To test whether a reduction in VEGF or bFGF expression by tumor cells could affect endothelial cell migration, HMVEC-L or W-IE cells were stained with 10 / ml Di-I for 24 hours and added into Fluoroblok inserts placed into 24 well dishes containing Caki-1 tumour cells treated with V 515 The number of pre-labelled endothelial cells which migrated through the 8 m pore size membranes in a 24 hr period were quantified by determining the fluorescence intensity in the bottom well (Figure 4-4) The results showed (Figure 4-5) that 24 hr after co-culturing the two cell populations ~ 45% (p < 0 05 student s t-test) and 37% (p < 0 05, student's t-test) fewer MHE or HMYEC-L

PAGE 93

86 cells respectively migrated through the membrane in the presence ofVSlS treated Caki-1 cells compared to untreated or s cramble control AS-ODN s tr ea ted Caki-1 cells Similarly ~ 37% (p < 0 05 student s t-test) and ~ 33% (p < 0 05 student's /-test) fewer MHE or HMVEC-L cells re s pectivel y migrated throu g h th e membrane in the presence of B460 treated Caki -1 cells compared to untreated or sc ramble treated control AS-ODN s (F i g ure 4-6) Although the in vitro s tudie s indicated that treatin g Caki-1 tumor cells with VEGF or bFGF mRNA targeted AS-ODNs down-re g ulated VEGF/bFGF protein production s ufficiently to affect the proliferation and migration of endothelial cell s, it was important to demonstrate that s uch treatments also could affect Caki-1 cell induction of angio ge nesi s in vivo To examine thi s po ssi bility Caki-1 cells that had been treated with VSIS B460 or control ODN s were injected intradermally and the number of vessels induced were counted 3 day s later (Figure 4-7) While untreated Caki-1 cel l s and control ODN s treated Caki-1 cel l s had very s imilar angio ge nic potency in , ;, o (both groups induced ~ 44-4 6 new vessels in the assay period) the angio ge nic potential of Caki -1 cell s that had been pre-treated with VEGF AS-ODNs (VS IS) was found to be s ignificantly impaired ; only ~ 25 5 (p < 0 05 Wilcoxon rank sum te s t) new blood vessels were observed Similarly bFGF AS-ODNs (B460) treated tumor cells al so induced le ss vessels ~ 27 new blood vesse l s (p < 0 05 Wilcoxon rank sum te s t) More importantly the mo s t significant inhibition of formation of new blood vessels was observed when the Caki-1 cells were treated with both V515 and B460 only ~ 20 new vessels d eve loped (p < 0 05 Wilcoxon rank s um te s t)

PAGE 94

87 Discussion Anti-angiogenesis treatment strategies represent a new approach to cancer management. Given that solid tumors cannot progress effectively without the generation of new blood vessels, various tacks have been taken to interfere tumor angiogenesis One possible target which has received considerable attention i s the pro-angiogenic factor VEGF VEGF can induce endothelial cell proliferation and migration in , itro (Soker et al ., 1997 ; Hanahan and Folkman 1996a ; Hanahan and Folkman 19966) and angiogenesis in vi, o (Leung et al ., 1989 ; Plate et al ., 1992a ; Plate et al ., 19926 ) Its expression level has been associated with a variety of tumors and correlated to treatment outcome (Gasparini et al ., 1997 ; Maeda et al ., 1996a ; Maeda et al. 1996b ). Basic fibroblast growth factor (bFGF) is another important pro-angiogenic factor (Yoshida et al ., 1996 ; Hoying and Williams 1996) It also has been found to associated with different tumors and correlated to treatment outcome, especially RCC (Nguyen et al ., 1994b ; Nanus et al ., 1993 ; Miyake et al. 1996) Antisen se oligodeoxynucleotide technology provides an approach for inhibiting gene expression with tar ge t specificity as a particular advantage (Stein and Cheng, 1993 ; Engelhard, 1998a ; Engelhard, 1998b ) Effective AS ODNs against VEGF and bFGF have been identified and tested in vitro (Chapter 3). To evaluate the VEGF/bFGF suppression through the u se of AS-ODNs on Caki-1 tumor angiogenesis both in vitro and in vivo efficacies were s tudied The co-culture system which allows constant exchange of growth factors between tumor and endothelial cells was used to study the treatment on endothelial proliferation The tumor cells also serve d as primary source of pro-angiogenic growth factors for endothelial cells. This provid es a setting that closely mimics the in vivo situation Significantly impaired

PAGE 95

88 proliferation potential of both human and mouse microvascular endothelial cells were observed after VEGF/bFGF AS-ODNs treatment (Figure 4-2 and 4-3) In order to evaluate the AS-ODNs treatment on endothelial cell migration potential a very similar co culture system (FluoroBlok system) utilizing 24 well dishes and larger pore sized membranes (8 M) was used. The results showed that suppression ofVEGF/bFGF expression by AS-ODNs was sufficient to inhibit the migration of endothelial cells in response to pro-angiogenic growth factors produced by tumor cells (Figure 4-5 and 4-6) VEGF/bFGF AS-ODNs treatment of Caki -1 cells led to inhibition of endothelial cell proliferation and migration in both human and mouse microvascular endothelial cells (Figure 4-2 4-3 4-5 and 4-6) suggesting that such treatment s should also exert their effects in a mouse model In deed the anti-angiogenic efficacy of VEGF / bFGF AS ODNs was readily demonstrated in vivo using the intradermal angiogenesis assay (Figure 4-8). These results not only s upport the role of VEGF and bFGF as important pro angiogenic growth factors in Caki-1 cell induced angiogenesis but also clearly suggest that inhibition of cancer cell VEGF or bFGF expression may ultimately impact tumor growth The use of AS-ODNs against VEGF/bFGF was sufficient to illicit anti angiogenic effects both in vitro and in vi, o Taken together these findings suggest that AS-ODNs targeted to VEGF and bFGF are effective in inhibiting Caki-1 tumor cell induced angiogenesis They further implying that such a treatment strategy may have utility in the treatment of RCC

PAGE 96

89 I Tumor cells I I Endothelial cells I Figure 4-1 Transwell co-culture system for evaluating VEGF / bFGF AS-ODNs treatment of Cakil cells 011 endothelial cell proliferation

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5 4.0x10 5 3.5x10 3.0x10 5 c 2.sx10 5 ::, 0 5 c., 2.0x10 G> 5 0 1.5x10 1.0x10 5 5.0x10 4 90 MHE Cell lines [[jJ Untreated DOTAP (ZJ Control 0 V515 HMVEC-L Figure 4-2 Treatment of Caki-1 cells with VEGF AS-ODNs on the growth of co cultured endothelial cell Caki-1 cells were either untreated, treated with liposome alone, treated with I M scramble control ODNs or VEGF AS-ODNs Each bar represents the mean S E of 3 independent experiments Stars indicate statistical significance compared to untreated control group (p < 0 05, student's t-test)

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4.0x10 5 3 5x10 5 3 0x10 5 1: 2 5x10 5 :::, 0 5 CJ 2 0x10 G> 5 0 1 5x10 1.0x10 5 5.0 x 10 4 MHE 91 Cell lines ... ... ... ... ... ... W Urtreated DOTAP Control 0 8460 Figure 4-3 Treatment of Caki-1 cell s with bFGF AS-ODN s on the growth of co cultured endothelial ce l l Caki 1 cells were either untreated treated with liposome alone treated with l M sc r amble contro l ODNs or bFGF AS-ODNs Each bar represents the mean S E of 3 independen t experiments Sta r s indicate stati s tical s ignificance compared to untreated control g roup (p < 0 05, student 's t-test)

PAGE 99

92 0 I Endothelial cells I Labeled with D iI Fluorescence opaque membrane Q o,,.., /at,,,/cd ""'1. m41-""" / /,,-OUflh the mombrllfli,. thoy .,. no /on(IM .Jiktld,,d from the light soorr.,, I/ltd l
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900 800 700 .. ,n C 600 G) .. C G) 500 (.) C 400 G) (.) ,n 300 0 ::, 200 LL 100 93 EE8 Untreated DOTAP ~ Scr a mble D vs1s MHE Cell l i ne ., ., ., ., ., ., ., ., ., HMVEC L Figure 4-5 Treatment of Caki 1 cel l s with VEGF AS-ODNs on the migration potential of co cultured endothelial cells Caki-1 cells were either untreated treated with liposome alone treated with 1 M scramble control ODNs or VEGF AS ODNs. Each bar represents the mean S E of 3 independent experiments. Stars indicate statistical significance compared to untreated contro l group (p < 0 05 student s t test).

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U) C B C Cl) u U) e 0 ::, LL 900 800 700 600 500 400 300 200 100 EE3 Untreated DOTAP Scr a mble 0 B460 M HE 94 -----, .... , .... , ... , , ... ... , .... , .... .... , .... .... , ... ... , ... , .... , .... , ... .... , .... , .... .... , .... , ..... , , ... ~. HMVEC-L Cell line Figure 4 6 Treatment of Caki1 ce l ls with bFGF AS-ODNs on the migration potential of co cultured endothelial cel l s Caki-1 cells were either untreated, treated with liposome alone treated with l M scramble control ODNs or bFGF AS-ODNs Each bar represents the mean S E of 3 independent experiment s. Stars indicate statistical significance compared to untreated control group (p < 0 05 student s test)

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Caki-1 cell s treated iii vitro Inject intradermally 0 0 3 days lat e r Di sse cting Microscope 95 Figure 47. Intrad enna) ang i ogenesis assay Cak i-1 cells were tr eated in vitfo w it h AS ODNs and were th en injected intrade nn a ll y tnto the ventral surface of nud e mice Three da ys lat e r th e skin was removed and the vesse l s ind u ce d we r e coun t e d u si n g a dissecting mi croscope Ri g ht : rep r ese nt a ti ve pictures of lLtmor induced angiogenesis in an intrad e rmal angiogenesis assay

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96 60 50 -l!-45 J. 44 I ..., 40 C :, .. 0 r* (J 30 G> 27 en 25 5 en G> > 20 20 10 0-t------------------. Untreated Scramble V515 8460 V 5 1 5 + 8460 Treatment Figure 4-8 Number of blood vessels induced 3 days after injecting Caki 1 cells Caki-1 cells were either untreated, or pre treated with 1 M scramble control ODNs VEGF AS ODNs bFGF AS ODNs or both Each datum point presents one injection site Bar represents the median of 16 injection sites in each group Stars indicate statistical significance compared to untreated control group (p < 0 05 Wilcoxon rank sum test)

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CHAPTERS EFFICACY OF AS-ODNS TREATMENT IN CAKI-1 XENOGRAFTS Innoduction Renal cell carcinoma (RCC) is the most common malignancy of the kidney in adults and accounts for about 3% of all adult malignancies (Jemal et al ., 2002) Unless discovered at an early stage at a time when it i s a still a resectable neoplasm RCC has a very unfavorable treatment outcome to conventional measures Unfortunately RCC i s characterized by a lack of early warning signs, resulting in a high proportion of patients with metastases at diagnosis As a consequence RCC remain s fatal in nearly 80% of its patients (Tsui et al ., 2000a ; Tsui et al ., 2000b ; Lau et al ., 2002 ; Delahunt et al ., 2002) Histopathologic evaluations of RCC reveal it to be a highly vascularized neoplasm demonstratin g clear evidence of abundant angiogene s is and abnormal blood vesse l de v elopment (Yoshino et al 2000 ; Slaton et al ., 2001) Not surprisingly, several studies have pointed to an important role for pro-angiogenic growth factors in RCC Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) have often been implicated Both factors have been shown to be expressed in renal cell carcinoma tissues and renal cell carcinoma cell lines (Mydlo et al ., 1993 ; Gospodarowicz et al 1986 ; Sato et al ., 1999 ; Hemmerlein et al ., 2001 ; McLaughlin and Lipworth 2000 ; Ferrara and Keyt 1997) Serum levels of VEGF and bFGF often are elevated in RCC patients (Nguyen et al ., 1994b ; Fujimoto et al 1991 ; Wechsel et al. 1999 ; Tomisawa et al ., 1999 ; Paradis et al ., 2000) and renal cell carcinoma VEGF and bFGF mRNA levels have been reported to be much higher than tho se found in s urrounding nor1r1al tissues 97

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98 (Tricarico et al ., 1999 ; Thelen et al ., 1999 ; Tsuchiya et al ., 2001 ; Eguchi et al ., 1 992) In addition elevated serum/urine bFGF levels have been shown to associated with mali gnan t progression and poor treatment outcome (Song et al ., 2001 ; Jacobsen et al. 2000 ; Rasmuson et al 2001; Edgren et al ., 1999 ; Do s quet et al ., 1 997 ; Fujimoto et al 1995 ; Plunkett and Hailey 19 90 ; Miyake et al ., 1 996 ; Nguyen et al ., 1994a; Huang et al ., 1996) Taken together these fmding s s ug ges t that VEGF and bFGF are the important factors involved in the angiogenic proce ss of RCC Currently, there is great interest in anti-angiogenic therapies for the treatment of RCC For example, interferon-a, a peptide known to have anti-angiogenic effects likely due to s uppression of bFGF expression (Singh et al ., 1995) has been shown to prolong s urvi val in patients with RCC Interleukin-12 a cytokine with immuno-regulatory and anti-angiogenic activity (Yoest et al ., 1995) also has demon s trated antitumor activity in RCC (Motzer et al ., 1998) Other dru gs developed principally as angiogenesis inhibitors and stt1died in RCC include the fumigillin analog TNP-470 thalidomide and a monoclonal antibody to VEGF (Stadler et al ., 1999 ; Chow e t al ., 1996) AS-ODNs treatment is another effective measure to s uppres s the expression of pro-angiogenic factors Previous studies have identified effective AS-ODNs sequences again VEGF and bFGF (Chapter 3), and their anti-angiogenic efficacy has been demonstrated ;n nt r o and ;n vivo (Chapter 4) In the present investigations AS-ODNs complementary to VEGF or bFGF mRNA were tested for their anti-tumor efficacy through systemic administration in a solid tumor model ofRCC (Cak:i-1 xenografts)

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99 Materials and Methods Caki-1 Xenografts FemaJe nude mice (NCR, nu/nu) age 6 8 weeks were maintained under specific pathogen-free conditions (University of Florida HeaJth Science Center) with food and water supplied ad libitum Initial Caki-1 xenografts were established by inoculating 5 x 10 6 Caki-1 tumor cells subcutaneously into the flanks of nude mice Because of the rather long time for the xenografts to appear and grow and the relatively low take rate s ub se quent passages of Caki-1 tumors were achieved by implanting tumor pieces subcutaneously in the flanks of recipient animals When the tumor s reached a s ize ~ 200 mrn 3 animals were randomly assigned to the different treatment groups All animal experiments have been carried out with IACUC committee approval. Antisense Phosphorothioate Oligodeoxynucleotides (AS-ODNs) Antisense and control ODN s (20-mers) were customer sy nthesized by Geno Mechanix (Alachua, FL) AS-ODN s V515 was complementary to 5 UTRjust up-stream of the translation start site (AUG codon) ofVEGF mRNA : 5' CTC ACC CGT CCA TGA GCC CG 3' AS ODNs B460 was complementary to the translation s tart si te (AUG codon) ofbFGF mRNA : 5' TCC CGGCTGCCA TGGTCC CT 3' A scramble ODN s ofV515 : 5' -C AC CCT GCT CAC CGC ATG GC 3' was used as OD Ns controls In the tumor up-take s tudie s Fluorescein isothioicyanate (FITC) was labelled at 5 end ofVEGF scramble ODN s. All AS-ODNs were sus pended in sterile and endotoxin free water at a concentration of 1 mM, aliquoted and sto red at -20 C

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100 DOT AP:DOPE Liposome Preparation Cationic liposomes were prepared using the method described by Tang (Tang and Hughes 1999) Briefly, cationic lipid 1 ,2 -dioleoyloxy-3-(trimethy]ammonium) propane (DOT AP) was dissolved in chloroform and mixed with a helper lipid 1 2-dioleoyl-3-sn phosphatidylethanolamine (DOPE) (Avanti Polar-Lipids Alabaster Al) at a molar ratio of 1 : I The mixture was evaporated to dryness in a round-bottomed flask using a rotary evaporator at room temperature The resulting lipid film was dried by nitrogen for an additional 10 min to evaporate any re s idual chloroform The lipid film was re-suspended in sterile water to a final concentration of I mg / ml based on the weight of cationic lipid The resultant mixtures were shaken in a water bath at 35 C for 30 min The suspensions then were sonicated using a Sonic Dismembrator (Fisher Scientific Pittsburgh PA) for I min at room temperature to form homogenized liposomes The particle-size distribution of liposomes was measured using a NI COMP 3 80 ZLS instrument (Santa Barbara CA) The average particle diameter was 144 0 77 0 nm Liposome s were stored at 4 C and used within 3 months FACS Analysis of ODNs Tissue Distribution I,, Vivo FITC labeled control ODNs were mixed with DOTAP : DOPE in 200 l 5% dextrose and injected into Caki-1 xenograft bearing mice via the tail vein at a dose of 20 mg/kg. 24 hr later, the mice were killed by CO 2 asphyxiation various tissues were collected, including sp l een liver kidney lung and tumor The ti ssues were enzymatically dissociated into single cell suspension using an enzyme cocktail of 0.02o/o DNase 0 025% collagenase and 0 025% pronase (Allalunis-Tumer and Siemann 1986) All sa mple s were then fixed in I% p-formaldehyde for 24 hours and kept in dark The

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101 fluorescent intensities of the samples were analyzed by FACS on a Beckman Dickinson flow cytometer made available through the University of Florida Core Facility for Flow Cytometry. Fluorescent Microscopy of ODNs Up-take I,, Vivo FITC labeled control PS-ODNs were mixed with DOT AP : DOPE in 200 l 5% dextrose and injected into Caki-1 xenograft bearing mice via the tail vein at a dose of 20 mg/kg Three hours later the mice were killed by CO 2 asphyxiation lung, liver spleen and kidney as well as the tumors removed, quick frozen in liquid nitrogen and 20 m sections were cut Fluorescent field pictures of these section s were taken using a Zeiss Axioplan 2 Florescence Microscope (Zeiss Thorn.wood NY) within 3 days VEGF and bFGF Weste1n Blots VEGF AS-ODNs V515 bFGF AS-ODN s B460 or scramble control ODNs were injected v;a tail vein at a dose of IO mg/kg At various times after injection (24, 48 and 72 hr) the mice were killed the tumors excised and quick frozen in liquid nitrogen The tumors were then homogenized (Dounce tissue grinder, Wheaton Millville NJ) and the homogenates were lysed on ice for 30 min with I ml of hypotonic buffer (20 mM Tris HCI pH 6 8 1 mM MgC1 2 2 mM EGTA 0.5 % Nonidet P-40 2 mM Phenylmethanesulphonyl fluoride (PMSF) 200 U / ml Approtinin 2 g/ml leupetin) (Giaonakakou et al ., 1998) per 0 1 g tissue Following a brief but vigorous vortex the samples were centrifuged at 14 000 rpm for 10 min at 4 C A 30 I aloquot of each sample was mixed with 10 l 4x SDS-PAGE s ample buffer (0 3 M Tris-HCI pH 6 8 45% glycerol 20% f3-mercaptoethanol 9 2% SDS and 0.04 g / lOOml bromophenol blue) and heated at I 00 C for IO min. 30 l of each sample wa s then analyzed by SDS-PAGE

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102 on a 12% separating gel and 3% stacking gel Following transfer the membrane was immunoblotted using a VEGF primary antibody (Sigma, Saint Louis Missouri) 1 : 1000 or bFGF primary antibody (Upstate Biotechnology Lake Placid, NY) I : I 000 diluted in antibody solution (3o/o diy milk, 25 rnM Tris pH 7 5 0 5 M NaCl 0 05% Tween20) overnight at 4 C After washing a secondary antibody labeled with horseradish peroxidase was applied and incubated at room temperature for 1 hr Protein bands were visualized and densitometry was performed Tumo1 Growth Delay Assay Once the Caki-1 xenografts reached a size of ~ 200 mm 3 animals were assigned randomly to various treatment groups VS 15 B460 or control ODNs were administrated via the tail vein with DOT AP : DOPE liposomes at do s es of 5 mg/kg or IO mg/kg on day 1 and day 4. Tumors were measured using callipers and volumes were approximated by the formula volume=J / 6nub 2 with a and b represent two perpendicular tumor diameters The times for the tumors in the various treatment groups to grow from 200 mm 3 to 1000 mm 3 were recorded and compared (Wilcoxon rank sum test) Results The cationic liposome based delivery system was previously shown (Chapter 2) to efficiently deliver AS-ODNs into cultured Caki-1 cells To evaluate the tissue distribution and tumor up-take of AS-ODNs FITC labeled ODNs were mixed with cationic liposome DOTAP : DOPE in 5% dextrose and were injected via the tail vein at a dose of 20 mg/kg into Caki -1 xenograft bearing nude mice. 24 hr later significant up-take of FITC-ODNs indicated by increased fluorescent intensity were observed in lung liver kidney spleen and tumor, with liver having the highest up-take (Figure 5-1 ) Frozen sections prepared 3

PAGE 110

103 hr later also showed that the FITC-ODNs were efficiently taken up in th e Caki-1 tumor (Figure 5-2) The heterogeneous ODNs distribution likely reflects the inhomogeneous blood vessel network inside the tumor Significant up-take of FITC-ODNs was also observed in norn1al tissues such as lung liver kidney and s pleen (Figure 5-3) Unlike tumor no1111al tissue up-take appeared to be rather unifor1n These findings suggested that the cationic liposome (DOT AP : DOPE) based deli very system can also serve as an efficient vehicle for systemic delivery of AS-ODN s in vivo To investigate whether the delivery of AS-ODN s was s ufficient to s uppre ss the target g rowth factor expression level s, tumor s ample s were collected at various times after systemic delivery of B4 60 and V5 l 5 We stern blot analysis of these sam ple s s howed significant reduction s in VEGF level s at 24 48 and 72 hr post injection, with the maximum depression occurring at 48 hr after treatment (Figure 5-4) When B460 was administrated, significant and s imilar reduction s in bFGF levels were observed at 48 and 72 hr (Figure 5-5) These finding s clearly indicate that the delivery of VEGF and bFGF antisense to tumors can lead to significant reduction s in VEGF and bFGF expression level s in Caki-1 xenografts To deterrnine whether the se reductions of pro-angiogenic g rowth factor production by AS-ODNs was s ufficient to have anti-tumor efficacy in , ;, o the effect of VEGF/bFGF AS-ODNs treatments on Caki-1 tumor growth was examined Caki-1 xenografts-bearing mice were treated with two dose s ofVEGF (V515) or bFGF (B460) AS-ODNs (5 or 10 mg/kg) l and 4 days after the tumors reached a s ize of ~ 200 mm 3 The time for the tumors to grow from 200 mm 3 to l 000 mm 3 then was recorded (Figure 5-6) The results showed that the median time for the tumor s to grow to 5 times the

PAGE 111

104 starting size was significantly prolonged in the VS 15 or B460 treated groups Administrating two doses of 5 mg/kg VSI 5 caused a growth delay of ~ 4 days (p < 0 05 Wilcoxon rank sum test) while treatment with two 10 mg/kg doses led to a tumor growth delay of ~ 6 5 days Two doses of 5 mg/kg B460 caused a growth delay of ~ 5 5 days (p < 0 05, Wilcoxon rank sum test) whereas two 10 mg/kg doses led to a tumor growth delay of ~ 10 5 days (p < 0 05, Wilcoxon rank sum test) The higher dose treatments of both V515 and B460 therefore resulted in an approximately doubled the response of the tumors compared to the tumors of untreated or scramble ODNs treated mice to grow to the 5x starting size endpoint (Figure 5-6) Discussion The increased understanding of the biology of neoplasia and the identification, cloning and sequencing of genes critical to tumor cell function per1nits the exploitation of this infortnation to develop specific agents that may directly modulate the function of the s e genes or their protein products Antisense oligonucleotides (AS-ODNs) are being investigated in this manner. However ODNs are negatively charged large molecules that would result in poor cellular uptake and intracellular distribution A reasonable solution to this problem is the use of cationic liposome based delivery methods which have become the standard for in vitro AS-ODNs experiments (Capaccioli et al 1993 ; Lappalainen et al ., 1994) (see also Chapter 2) However for in vivo delivery of AS-ODNs methods are stiU being standardized To date in , ;, o ODNs solutions delivery approaches have relied on saline (Agrawal et al ., 1995; Dean and McKay 1994; Nesterova and Cho-Chung, 1995 ; Akhtar and Agrawal 1997 ; Yazaki et al 1996 ; Whitesell et al 1991 ) Despite the apparent success of some ongoing trials using such

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105 approaches it is generally believed that improved delivery to specific tissues will prove advantageous and lead to higher success rates Cationic liposomes which have been used successfully in vitro are possible candidates for in l il o delivery of AS-ODNs Evidence for their utility already exists (Zhang et al ., 2000 ; Chen et al. 2000; Yang et al 1998) This approach may be even more preferable for the treatment of solid tumors Small molecule agents have a large volume of distribution after i v administration (Chabner B A and Longo D L ., 1996) Unfortunately the result of this is often a narrow therapeutic index due to a high level of toxicity in healthy tissues By encapsulating the AS-ODNs with liposomes the complexes become macromolecular The volume of distribution therefore is significantly reduced and may result in a decrease of nonspecific toxicities There also is believed to be an increase in the amount of drug that can be effectively delivered to the tumor (Lasic and Papahadjopoulos 1995 ; Gabizon and Martin 1997) Under optimal conditions the drug is carried within liposomal aqueous space while in the circulation but leaks out at a sufficient rate to become hie-available on arrival at the tumor The liposome protects the drug from metabolism and inactivation in the plasma and due to the size limitations in the transport of large molecules across healthy endothelium the drug accumulates to a reduced extent in healthy tissues (Mayer et al ., 1989 ; Vaage et al ., 1994). The junctions in the vascular endothelium of healthy tissues vary depending on the types of tissue (Seymour 1992) In most tissues including connective tissue and tissues of muscle heart brain, and lung intercellular tight junctions result in openings < 2 nm. Though these openings can approach 6 nm in post-capillary venules they are still considerably smaller than the size of liposomes (usually 65-150 nm) (Seymour 1992 ; Lum and Malik 1994 ). For organs or tissues with discontinuous

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106 en dothelium such as fenestrated endothelium of the kidney glomerulus or the sinusoidal endothelium of the liver and sp leen they can have junctions ranging from 40-60 nm for the for1ner and up to 150 nm for the latter (Seymour 1992) Still most liposome formulations are larger than the threshold required for g Jom eruJar filtration However liposomes are able to enter tumors effectively due to a discontinuous tumor microvasculature where pore sizes vaty between 100 to 780 nm in size (Yuan et al. 1995 ; Hobbs et al ., 1998) The selective accumulation in tumors is thus made possible by the impervious nature of the endothelium of most healthy tissues. Bearing this belief in mind experiments were carried out to evaluate delivery efficiency ofFITC-ODNs by cationic liposome (DOTAP : DOPE) in Caki-1 xenograft tumor bearing mice Fluorescent intensity studies of different tissues showed that AS ODNs were delivered into multiple organs and most importantly into the xenograft tumor as well (Figure 5-1 ) The histological section of the tissues further confirmed that efficient deliveries were achieved in liver kidney lun g and spleen (Figure 5-3) Significant up-take of AS-ODNs though heterogeneous was al s o observed in Caki-1 tumors This is believed to be due to the heterogeneous distribution of the tumor vascular network (Figure 5-2) Nonethe le ss cationic liposomes cleared proved to be an efficient delivery vehicle for AS-ODNs in vivo Western blot studies of the tumor homogenates confirmed that using such delivery system, sufficient AS-ODNs were taken-up by the tumors to suppress VEGF and bFGF protein expression {Figure 5-4 and 5-5) The fact that maximum suppression of VEGF protein levels was observed earlier than that of bFGF lev e l s is believed due to the sho rt half life of VEGF in vivo (Ferrara, 1999) More importantly this suppression of

PAGE 114

107 pro-angiogenic growth factors was focused to result i n significant tumor growth delays in the Caki-1 tumor model (Figure 5-6) These re s ults provide the first experimental evidence that the systemic treatment of mice bearing macroscopic tumors with bFGF AS ODNs can have substantial antitumor efficacy, and that VEGF AS-ODNs are effective in treating a VEGF independent tumor model In both cases significant tumor growth delays could be achieved without overt toxicity and with doses well below the LD 10 dose (Figure 5-6) The results of this study indicate a key role for the VEGF and bFGF signalling pathway in RCC angiogenesis It is clear that interfering with VEGF and bFGF signaling can disrupt the tumor induced angiogenesis which in turn leads to tumor growth inhibition Specially, AS-ODNs treatment s provided a viable measure to modulate t11mor angiogenesis and resulted in significant growth delays in this VEGF independent RCC tumor model Taken together these findings suggest that AS-ODNs targeted to VEGF and bFGF may have utility in the management ofRCC It may al s o possibly provide therapeutic benefit when used in conjunction with other anti-cancer therapies

PAGE 115

600 500 ,,, C S 400 C CJ) CJ 300 C CJ) CJ e 0 ::, LL 200 100 o-'--_c= 108 D Control FITC-ODNs * Lung Liver Spleen Kidney Tumor Organ Figure 5-1 F I uo r esce n ce intens i ty of diffe r ent organs before an d after i v injectio n of 20 mg/kg FI'l'C l abe l e d OD Ns Each bar r ep r ese n ts the mean S E of samp l es from 6 anima l s Stars in d icate s t atistica l significance compared to untrea t ed contro l anima l s (p < 0 05 st ud e nt s t -t est)

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)09 Figure 5 2 Representative picture of uptake and distribution of FITC-labeled ODNs in Caki-1 xenografts determined 3 hr after i.v injection of a 20 mg/kg dose

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11 0 Figure 5 -3 R ep r ese ntati ve pictures of upta ke and distribution of F ITC-l abe l e d ODNs in Li ve r lun g, kidne y and sp l ee n determined 3 hr after i v injection of a 20 m g/kg do se

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I I I A Caki-1 24Hr 48 Hr 72 Hr B 120 Cl) a, "' .., 100 C: Cl) e Cl) 80 Q. Cl) > Cl) 60 IL. (!) w 40 > Cl) > .. ... .., 20 "' Cl) it: 0 Control 24hr 48hr 72hr Different time point after V515 treatment Figure 5 4 VEGF p r otein l evels in Caki 1 xenografts at variou s times after treatment with 10 mg/kg VEGF AS-ODN s A) repre s entative VEGF we s tern blot results ; B) relative VEGF leve l s in Caki-1 tumo r s Each bar rep r esents the mean S E of s ix tumors Stars indicate statistical significance compared to untreated control tumors (p < 0 05 student s !-test)

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A B Cl) C, J! C Cl) (.) .. Cl) 0. u. 0 u. .c Control 120 100 80 60 40 20 112 * 0 hr 24 hr 48 hr 72 hr Time after 8460 treatment Figure 5-5 Protein levels of bFGF in Caki-1 xenografts at differet times after treatment with 10 mg/kg bFGF AS-ODNs A) representative bFGF western blot results ; B) relative bFGF levels of Caki-1 tumors Each bar represents the mean + S E of six tumors. Stars indicate statistical significance compared to untreated control tumors (p < 0 05 student s te s t)

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113 25 Med ian IX) 25-75/4 :r: 10-90/4 c,s 20 20 "C Q) N 16 ,,, 15 15 c,s 13 5 .., C )( 10 9.5 II) 0 8 .. Q) E 5 Io~------.----,-----..----r-----,-----, Untreated Scramble 5mi;>'kg 10 rrgkg 5ng/kg 10,rg/kg V515 8460 Treatment Figure 5-6 Tumor response of Caki -1 tumor s treat e d systemical ly with VEGF or bFGF AS -OD Ns or contro l s Each group contains IO animal s. Stars indicate s tat is tical sign ificance compared to untreated control (p < 0 05 Wilcoxon rank s um test)

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CHAPTER6 CO:MBINATION STUDIES Introduction It has been established that solid tumors cannot grow beyond microscopic size without inducting a new vascular network Development of novel anti-tumor agents that exploit this observation and suppress angiogenesis is an active area of cancer research Angiogenesis is a complex process with multiple sequential and interdependent steps this complexity also creates many potential targets for therapeutic intervention Many agents that are antiangiogenic have been identified and characterized (Folkman, 1985 ; Moses and Langer 1991; Schweigerer 1995 ; Scott and Harris 1994 ; Fan et al ., 1995 ; Kerbel 2000 ; Sauer et al ., 2002) Each of these affect at least one of the several stages involved in new vessel formation i e basement membrane degradation, endothelial cell migration endothelial cell proliferation and tube formation Although strategies aimed at compromising tumor angiogenesis have demonstrated significant ant-tumor effect in preclinical investigations achieving tumor cures in patients with anti-angiogenic agents is likely to be extremely difficult (Folkman 1985 ; Moses and Langer 1991 ; Schweigerer 1995 ; Scott and Harris, 1994; Fan et al ., 1995) The complexity of pathways available for angiogenesis probably means that disrupting only a single aspect probably will not be enough It would seem likely that targeting one factor for example VEGF, would result in the eventual selection of tumor cell variants which could induce angiogenesis through production of other growth factors (Yoshiji et al ., 1997) Indeed there is evidence that as tumors progress the number of I I 4

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115 different pro-angiogenic growth factors produced by the tumor increases (Relf et al ., l 997 ~ Eggert et al ., 2000) Moreover in addition to angiogenesis dependent tumor growt h angiogenesis independent tumor growt h can occur in human tumors (Maniatis et al 1 999) One study of non-small-cell lun g tumor s found up to 1 6% of the tumor s appeared to g row along pre-exi s tin g blood vessels. Th e tumor s did not exhibit signs of angiogenesi s but the microvessel den s ity appeared s imilar to that seen in angiogenic tumors (Pez ze lla et al 1997) Other s tudie s also revealed '' co-opt ' of vessels durin g early growth by tumors where evidence of an gi ogenesis was absent (Holash et al. 1999 ; Burke and DeNardo 2001). Therefore s trategie s aimed at multipl e s tep s and differ en t growth factors involved in angiogenesis may offer enhanced efficacy and long lasting effects Moreover, the over-arching g oal of anti-angiogenic strategies i s to interfere with pro-angiogenic balance between tumor stromal and endothelial cells in order to sta bilize the g rowth of the tumor by preventing further development of a functional vessel network Therefore, theoretically the optimaJ treatment outcome of anti-angiog e nic strategies would be tumor growtl1 arrest rather than tumor killing In order to eliminate the existing pockets of tumor cells, cytotoxic agents need to be used in conjunction with s uch therapies These considerations alon g with the extensive clinical use of conventional anti-cancer therapy make the thorough investigation of s trategie s combining different anti-angiogenic approaches as well as combination s with conventional treatment modality imperative In the current studies the anti-tumor efficacy of VEGF/bFGF AS-ODNs treatment in combinations with other anti-angiogenic strategies as well as a conventional cytotoxic therapy radiotherapy were evaluated

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116 Materials and Methods Caki-1 Xenografts Female nude mice (NCR, nu/nu) age 6 8 weeks were maintained under specific pathogen-free conditions (University of Florida Health Science Center) with food and water supplied ad libiti,m. Initial Caki-1 xenografts were established by inoculating 5 x 10 6 Caki-1 tumor cells subcutaneously into the flanks of nude mice Because of the rather long time for the xenografts to appear and grow and the relatively low take rate subsequent passages of Caki-1 tumors were achieved by implanting tumor pieces subcutaneously in the flanks of recipient animals. To initiate tumors in the leg muscle, animals were inoculated intramuscularly in the left hind leg with 2 x 10 6 tumor cells suspended in 20 l 0.9% saline chloride When the tumors reached a size ~ 200 rnm 3 animals were randomly assigned to the different treatment groups. All animal experiments have been carried out with IACUC committee approval Antisense Phosphorothioate Oligodeoxynucleotides (AS-ODNs) Antisense and control ODNs (20-mers) were customer synthesized by Geno Mech anix (Alachua FL) AS-ODNs V515 was complementary to 5 UTRjust up-stream of the translation start site (AUG codon) ofVEGF mRNA : 5' CTC ACC CGT CCA TGA GCC CG 3' AS-ODNs B460 was complementary to the translation start site (AUG codon) ofbFGF tnRNA : 5' TCC CGGCTGCCA TGGTCC CT 3'. A scramble sequence ofV515 : 5' CAC CCT GCT CAC CGC ATGGC 3' was used as control ODNs All AS-ODNs were suspended in steri l e and endotoxin free water at a concentration of 1 mM, aliquoted and stored at -20 C

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117 DOTAP:DOPE Liposome Preparation Cationic liposomes were prepared using the method described by Tang (Tang and Hughes 1999) Briefly cationic lipid l 2-dioleoyloxy-3-(trimethylammonium) propane (DOT AP) was dissolved in chloroforrn and mixed with a helper lipid 1 2-dioleoyl-3-sn phosphatidylethanolamine (DOPE) (Avanti Polar-Lipids Alabaster, Al) at a molar ratio of 1 : 1 The mixture was evaporated to dryness in a round-bottomed flask using a rotary evaporator at room temperature The re sulting lipid film was dried by nitrogen for an additional IO min to evaporate any re s idual chloroform The lipid ftlm was re-suspended in steri le water to a final concentration of I m g/ml based on the weight of cationic lipid The resultant mixtures were shaken in a water bath at 35 C for 30 min The suspensions then were sonicated using a Sonic Dismembrator (Fisher Scientific Pittsburgh PA) for I min at room temperature to forrn homogenized liposome s. The particle-size distribution of liposomes was measured using a NICO:rvn> 380 ZLS instrument (Santa Barbara, CA) The average particle diameter was 144 0 + 77 0 nm Liposomes were stored at 4 C and used within 3 months PD0203359-0002 D1ug Prepaiation PD0203359-0002 (Pfizer Warner Lambert Ann Harbor MI) 1 tert-butyl-3-[6{2-chloro-3 5-dimethoxy-phenyl)-2-( 4-diethylarnino-butylamino ) -pyrido [2 3-d] pyrimidin-7-yl]-urea hydrochloride was prepared in 0.05 M sodium lactate buffer (pH 4 0) at a concentration of 10 mg/ml The drug so lution was stored at 4 C and used within a week

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118 Local Tumor Irradiation Irradiations were performed using a 6MV Clinac 600c linear accelerator (Varian Oncology Systems, Palo AJto CA) operating at a dose rate of 400 cGy / min on unanesthetized mice confined in plastic jigs The animals tumor-bearing legs extended through openings in the sides of the jigs allowing the tumors to be irradiated locally Tumo1 Growth Delay Assay Tumor response to treatment was determined using a growth delay assay Tumor s initiated in the hind legs were measured every second day by passing the tumor-bearing leg through a series o f increasing diameter holes in a plastic plate. The smallest diameter hole the tumor-bearing leg could pass through was recorded and converted to a tumor volume using the formula : tumor volume = l / 6nd 3 -100 where d = hole diameter and 100 represent a volume correction factor determined for a mouse leg without a tumor Tumors established subcutaneously in the flank were measured using callipers and volumes were approximated by the formula, tumor volum e= l l 6.7lllb 2 with a and b represent two perpendicular tumor diameters The times for the tumors in the various treatment groups to grow to 200 mm 3 and from 200 mm 3 to 1000 mm 3 were recorded and compared (Wilcoxon rank sum test) Results Given the encouraging positive results of systemic administration ofVEGF/bFGF AS -ODN s in the human tumor xenograft model of RCC (chapter 5) experiments were first carried out to examine the efficacy of combining VEGF and bFGF AS-ODNs treatments However, combining 2 doses of 10 mg/kg V5 l 5 and B460 did not yield a significantly greater tumor response than did B460 treatment alone (Figure 6 -1 ) Since

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119 B460 AS-ODNs treatment affects Caki-1 cell proliferation and apoptosis (chapter 4) it is apparent that thjs treatment has direct anti-tumor effects in addition to the anti-angiogenjc effects. It was postulated that multiple exposures might be required to demonstrate the effect This hypothesis was confinned in the experiment with more treatment doses. When 4 doses of V515 and B460 were administrated, a moderate but significant enhancement in tumor growth delay was observed (Figure 6-2) The results showed that combination of two different AS-ODNs (V5 l 5 and B460) could provide a moderate increase in the tumor response However this approach may lead to increased toxicity and side effects due to the common delivery vehicle utilized and common metabolic pathway involved. It would appear more reasonab l e and appealing to combine AS-ODNs treatments with other anti-angiogenic therapies or conventional anti-cancer treatment VEGF and bFGF signaling play central roles in tumor angiogenesis Besides directly targeting VEGF /bFGF proteins through the use of AS -OD Ns blocking their specific receptor tyrosine kinase activities offers another way to disrupt the signaling pathway The experimental compound PD0203359-0002 (Figure 6-3) was found to specifically inhibit the VEGF and bFGF receptor tyrosine kinase activities in vitro and in , ;, o Oral administration of this compound at doses of 20 mg/kg and 40 mg / kg to Caki-1 xenograft bearing mice for 14 days resulted in dose dependent tumor growth delay (Figure 6-4) However, considerable side effects and toxicity were observed in the higher dose treatment group as indicated by significant weight loss (up to 30%), which limited its potential use However, when the lower dose treatment of PD0203359-0002 (20 mg/kg) was administrated in conjunction with VEGF or bFGF AS -O NDs, significantly

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120 enhanced tumor responses were observed (Figure 6-5) These effects were achieved at non-toxic doses of either treatment Lastly, the VEGF and bFGF AS-ODNs treatment in combination of with radiation was evaluated VEGF and bFGF AS-ODNs were given as 2 doses after locally irradiating the tumors with a single dose of IO Gy The re s ult sl1owed that the combination treatment led to significant ly prolonged tumor responses (Figure 6-6) Discussion It has been suggested that combining anti-angiogenic agents that work via different mechanisms may augment anti-tumor efficacy For example a VEGF receptor antibodies might be administrated with a conventional chemotherapeutic agents that are dosed in an '' anti-angiogenic '' schedule to enhance the tumor response (Browder et al ., 2000 ; Hanahan et al 2000 ; Klement et al ., 2000) It is not surprising given the complexity of tumor angiogenesis and redundancy of pro-angiogenic growth factors made by tumor cells, that multiple steps or targets of the angiogenic process need to be controlled in order to achieve long-term tumor control. In the RCC tumor, multiple pro angiogenic growth factors are found to be over-expressed These include VEGF bFGF PDGF etc (Wechsel et al ., 2000 ; Takahashi et al 1994; Mydlo et al. 1988 ; Relf et al. 1997) Therefore exploring therapies in this model seems very logical Combining VEGF and bFGF AS-ODNs was found effective only when multiple doses were used over a prolonged treatment course (Figure 6 -1 and Figure 6 -2 ) Because VEGF and bFGF are considered to be the most important pro-angioge11ic g rowth factor in tumors the combination studies were somewhat disappointing. Other studies have shown that VEGF and bFGF possess sy ner gistic effects in the induction of angiogenesis (Goto et

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121 al 1993; Seghezzi et al ., 1998 ; Mandriota and Pepper 1997 ; Yoshiji et al 2002) Suppression of VEGF by neutralizing antibody or AS -OD Ns also resulted in inhibition of bFGF induced angiogenesis (Seghezzi et al ., 1998 ; Majima et al ., 2000). These findings suggest possible interactions between these two growth factors or involvement of the same signal transduction pathway Moreover, since both factors were targeted using the AS-ODNs, it is possible that the delivery and up-take of AS-ODNs into the tumors was saturated Besides bFGF also plays an important role in the RCC cell growth Blocking bFGF expression would lead to cell cycle inhibition of Cakj-1 cells and also induce apoptotic cell death ( chapter 3 ) Therefore combining the AS-ODNs targeted to both VEGF and bFGF may only yield a moderate gain especially compared to bFGF AS ODNs treatment alone Agents that target different steps in angiogenic process or work under different mechanisms should provide increased efficacy without possible increases . 10 tOXIClty One such approach is to combine AS-ODNs targeted to VEGF/bFGF with a strategy to specially block VEGF/bFGF receptors This was achieved by combining V515/B460 AS-ODNs treatment with PD0203359-0002 administration This approach targeted two different cell populations and two different aspects of the angiogenic process by inhibiting the growth factors produced by tumor cells and the receptor function of the endothelial cells Results showed additive enhancement of tumor responses with such combination (Figure 6-5) Importantly the enhanced responses were achieved at the low, non-toxic dose of PD0203359-0002 The se findings support the principle that targeting multiple angiogenic steps using different therapeutic approaches may offer greater anti hlIDor effects without increasing the risks of toxicity

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122 Since anti-angiogenic therapie s should not result in tumor kill on their own the resumption of tumor growth when therapy is discontinued represent s a significant potential problem A logical solution to this problem is the combination of such treatments with conventional cytotoxic agents such as chemotherapy or radiotherapy Evidence already exists that combining anti-angiogenic agents with chemotherapeutic agents can improve the treatment efficacy (Lee et al ., 1987 ; Bertolini et al ., 2000 ; Hanahan 1998; Klement et al ., 2002; Inoue et al ., 2000) However RCC is usually resistant to chemotherapy A review of 83 clinical trial s with 4,542 patients showed a low overall response to treatment (6 8 %; 4.7 % partial re spo n se rate and 1 3% complete response rate) (Vogelzang and Stadler 1998) It therefore seems more reasonable to combine the current anti-angiogenic therapy with cytotoxic therapy like radiation in the tumor model of RCC Although RCC is typically considered a radio-resistant tumor as well it has been proposed that radiation therapy may provide some benefits to patients Pre-operative radiation therapy may provide benefits s uch as : 1) reduction of the ri s k of tumor dissemination at the time of surgery ; 2) reduction the size of primary tumor ; 3) increased resectability ; 4) reduction of tumor vasculature (Cox et al ., 1970; Kortinann et al., I 999). Moreover postoperative radiation therapy has the theoretical benefit of providing local tumor control in patients with positive surgical margins incompletely resected tumor or lymph node involvement (Makarewicz et al ., 1998 ; Stein et al ., 1992 ; Riches 1966) Besides, tumor pro g ression during the course of treatment is a major reason for radiotherapy failures And the ability of tumor s to progres s i s dependent on the formation of new blood vessels Consequently the application of anti-angiogenic strategies that disrupt the pro-angiogenic balance between neoplastic stromal and

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123 endothelial cell may result in growth stabilization of the tumor by preventing further development of a functional vessel network. This concept was confirmed in the present studies When VEGF and bFGF AS ODNs treatment were combined with a single local radiation dose of 10 Gy, augmented tumor responses were observed (Figure 6 6). The effect appears to be additive The mechanisms underlying the enhancement of the radiation response by the anti-angiogenic therapies likely include an increase in tumor oxygenation (Lee et al ., 2000 ; Hess et al ., 2001 ; Teicher et al ., 1995) a decrease in vascular density (Lee et al ., 2000) and possibly the radio-sensitization of endothelial cells (Gorski et al 1999 ; Mauceri et al ., 1998) While there has been some concern that the inhibition of tumor angiogenesis might increase the fraction of hypoxic tumor cells and thereby induce radiation resistance this concern is not relevant in the present studies since the anti-angiogenic agents (V515 and B460) were administrated after the radiation exposure Also the concern of induction of hypoxia is not supported by evidence of improved tumor oxygenation after treatment (Lee et al 2000 ; Hess et al 2001 ; Teicher et al ., 1995) as well as studies showing that blocking the VEGF signaling pathway improves radiotherapy response under both fully oxygenated and hypoxic conditions (Lee et al ., 2000) Still it is an important issue that is strongly dependent on the agent under investigation and one that will warrant rigorous scrutiny These preliminary results clearly indicate that combining different anti-angiogenic agents or combining anti-angiogenic with conventional anti-cancer therapies may provide significant improvements in treatment outcomes and hence should be thoroughly explored.

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124 30 Median IX) 25-75/4 I 10-90% 25 ca "CS * Cl> 20 5 N 20 1 9 .5 fl) ca 16 ..., 15 C )( II) 0 10 9 .., 8 Cl> E 5 t0 -+-----.---------r---~----------, Un t reated Sc ra mble V515 B460 V515 + b460 Treatment Figure 6-1 Tumor responses of Caki 1 xenografts after treatment with VEGF and bFGF AS ODNs Two doses ofV515 and B460 (10 mg/kg each) were given on day 1 and day 4 Each group contained 10 animals. Stars indicate statistical significance compared to untreated contro l animals (p < 0 05, Wilcoxon rank sum test)

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125 35 Median IXJ 25 75% I 10 90% 30 "O 27 25 23 ,n 20 ns 18 C 15 >< It) 0 10 Cl) 9 9 E 5 0 -+-----.----,-----,------.---------, Untreated Scramble V515 B460 V515+B460 Treatment Figure 6-2. Tumor responses of Caki-1 xenografts after treatment with VEGF and bFGF AS-ODNs V515 an d B460 were given as 4 doses of 10 mg/kg on day 1 4, 7 an d 10 Each group con t ained IO animals Stars indicate statistical significance compared to untreated contro l anima l s (p < 0 05 Wi l coxon rank sum test). V5 l 5 + B 460 treated group is statistical l y significant compared to V515 or B460 trea t ed groups (p < 0 05 Wilcoxon rank sum test)

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126 """'o N N Cl 0 N PD 0203359-0002 Figure 6-3 Chemical structure of VEGF/bFGF receptor tyro s ine kinase inhibitor PD0203 3 59 0002

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127 25 Median IX] 25-75% I 10-90% 20 cu '0 17 0 15 cu .., C 11 >< 10 It) 0 9 .., Q) E 5 i= 0-+-----------------------, Lactate buffer 20mg/kg 40mg/kg PD0203359-0002 Treatment Figure 6 4 Tumor responses of Caki-1 xenografts after treatment with PD0203 3 5 9 0002 PD0203359-002 was given daily at the doses indicated for 14 days Each group contained 10 animals Stars i ndi cate statistical sign i ficance compared to vehicle treated control animals (lactate buffer) (p < 0 05 Wilcoxon rank sum test)

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128 35 Med i an IX) 25-75% 30 I 10-90% ca "'O CD 25 N 22.5 "' 20 ca 19.5 .., 17 C 15 15.5 >< II) 0 11 5 .., 10 CD 8 5 E I5 0 +------,----r-----r---r----~----,---r----, Figure 6 -5 Tumor re s pon ses of Caki-1 xenografts after treatment wi th VEGF/bFGF AS ODNs and PD020335 9-0002 V515 and B4 60 were given at 2 do ses of 10 m g/kg on day 1 and d ay 4 PD020335 9 -0002 was give n daily at a do se of 20 mg/kg for 14 days Each group contained 10 animal s. Stars indicate statistica l significance compared to untreated control animals (p < 0 05 Wilcoxon rank sum test) V515 + PD treated gro up is sign ificantly different compared to V515 or PD treated gro up s ~ B4 60+ PD treated group is significan tly different compared to B5 60 or PD treat e d g roup s (p < 0 05 Wilcoxon rank sum test)

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>. C'G ,:, a, N "' C'G .., C >< II) 0 .., a, E I129 70 Median IX) 25-75/o 60 I 10-90 % 50 45 40 38 5 30 28 20 17 5 14 10 9 9 5 o,_ __________________ ___ ~s~s 6 460 ~s~s+~ 64 6o+~ Treatment Figure 6 -6 Tumor responses of Caki-1 xenografts after treatment with VEGF/bFGF AS ODNs and radiation V515 and B460 were given as 2 doses of 10 mg/kg on day 1 and day 4 Local radiation of 10 Gy was given 1 hr before the first do se of AS -OD Ns Each group contained IO animals Stars indicate statistical significance compared to untreated control animals (p < 0 05 Wilcoxon rank sum test) V515 + Rx and B460 + Rx treated groups are significantly different compared to Rx, V515 or B460 treated groups (p < 0 05, Wilcoxon rank s um test)

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CHAPTER 7 SUMMARY AND PERSPECTIVE Angiogenesis is a critical pathogenic step in the growth and metastatic dissemination of solid tumors (Folkman, 2001 ) It is a complex process involving multiple steps and pathways highly dependent on the local balance between positive and negative regulatory factors It is now well established that the majority of tumor blood vessels are newly for111ed as a result of angiogenesis triggered by the release of stimulators such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) Strategies for therapeutic suppression of angiogenesis have focused extensively on the various aspects of the process of angiogenesis Many agents that are anti-angiogenic have been identified and characterized; each of these affect at least one of the several stages involved in new vessel forrnation i.e basement membrane degradation, endothelial cell migration, endothelial cell proliferation and tube formation Pro-angiogenic growth factors VEGF and bFGF are primaiy targets because of they play central roles in tumor angiogenesis Strategies targeting VEGF/bFGF signaling vary from using antibodies against VEGF /bFGF proteins or their receptors over expressing soluble receptors and interference with VEGF/bFGF binding to their receptors to inhibit their receptor tyrosine kinase activities Antisense technology offers another way to specifically target these growth factors producted by the tumor cells The efficacy of anti sense oligodeoxynucleotides targeted against VEGF /bFGF treatments on the angiogenesis and growth of tumors was evaluated in this dessertation 130

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131 Renal cell carcinoma (RCC) is the most common malignancy of the kidney Because of lacking specific symptoms for early detection and unfavorable treatment outcomes to conventional anti-cancer therapies in the advanced disease it remains a challenge in modem oncology A characteristic feature ofRCC is evidence of abundant angiogenesi s and abnormal blood vessel development. VEGF and bFGF are known contributors in the regulation of RCC initiated angiogenesis Their expression levels also associated with the progression and prognosis of RCC In the present studies we evaluated the effects of blocking VEGF and bFGF production by AS-ODNs on the growth and angiogenic activity in a pre-clinical model of RCC (Caki-1 ) In order to achieve therapeutic effect sufficient cellular uptake of AS-ODNs is required A cationic liposome (DOT AP:DOPE) based delivery system was developed to enhance the delivery efficiency By optimizing the lipo some composition and liposome to ODNs charge ratio, efficient delivery of ODNs both in vitro and in 1 ivo was achieved with a safe non-toxic dose AS-ODNs sequences against all isoforms of VEGF and bFGF were designed and effective seq u ences were identified (VEGF AS-ODNs VSl 5 and bFGF AS-ODNs, B460) In vitro studies showed that treating Caki -1 cells with antisense AS-ODNs directed against VEGF or bFGF mRNA led to a reduction in the levels of VEGF /bFGF expression. This suppression of these pro-angiogenic growth factors was sufficient to impair the proJif eration and migration of co-cultured endothelial cells In addition, bFGF AS-ODNs treatment also exerted a direct effect on Caki-1 cell growth. The observed effects were antisense sequence spec ific dose dependent and could be achieved at a low non-toxic concentration of AS-ODNs When VEGF/bFGF AS-ODNs treated Caki-1 cells

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132 were injected into nude mice and evaluated for their an g iogenic potential in an intradermal angiogenesis assay the number of vessels initiated were approximately half that initiated by untreated Caki-1 cell s. These results d e mon s trated that AS-ODNs treatment could be used to modulate the expre s sion of pro-angio g enic growth factor s, like VEGF and bFGF in vil o It therefore wa s concluded that the s uppre s sion of VEGF or bFGF due to AS-ODNs treatment was s ufficient to impair th e tumor angio g enic proces s both in vitro and in vi1 o To evaluate the anti-tumor effect s of AS ODN s treatment VEGF/bFGF AS ODNs V5 l 5 and B460 were administrated systemically by tail vein to nude mice bearing macroscopic Caki-1 xenografts The results showed that two do s es ofVEGF or bFGF AS-ODNs given 1 and 4 days after the tumors reached a size of ~ 200 mm 3 approximately doubled the time required for tumors to grow to 5 times the s ize at the beginning of treatment These findings indicate that a significant anti-nimor effect could be achieved with AS ODNs targeted to pro angiogenic growth factors Given the complexity of tumor angiogenesis target one molecule or step in the process may ultimately not be sufficient to impair tumor g rowth Increased efficacy without increased toxicity can be achieved when combinin g anti-angiogenic approache s against different aspects of angiogenesis or combining anti-angiogenic strategies with conventional anti cancer therapies. When multiple doses of VEGF and bFGF AS-ODNs were combined, enhanced tumor growth delay was observed however the overall gain was moderate. This is believed to be due to the fact that both agents were delivered by and acted through the same mechanism, and this could saturate the effect that could be achie ved In contrast the combination ofVEGF and bFGF AS-ODNs with VEGF/bFGF

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133 receptor tyrosine kinase inhibitor (PD0203359-0002) showed significantly longer tumor growth delay without toxicity Moreover when VEGF/bFGF AS-ODNs treatment was combined with the conventional anti-cancer therapy radiotherapy an a additive response was observed The findings indicate that using AS-ODNs as an anti-angiogenic strategy not only has utility as an effective anti-tumor treatment on it s own but also provides significant benefit in combination with other new or conventional anti-cancer therapie s. Given the encouraging re s ults, there are still areas of interest that could be pursued in future preclinical studies For example combination ofVEGF/bFGF AS ODNs with other anti-angiogenic agents such as endogenous angiogenic inhibitors like endostatin angiogstatin The combination ofVEGF/bFGF AS-ODNs was evaluated with single dose radiation therapy Future studies might focus on combination with fractionated radiation therapy which is more relevant to the clinical setting Further studies also need to carefully evaluate the impact of anti-angiogenic therapies on tumor oxygenation and whether or how they could affect the effects of radiotherapy. Another important question is what will be the best schedule for administrating of radiotherapy with anti-angiogenic therapie s. Also even though RCC i s a chemo-resistant tumor the possible benefits of combining VEGF/bFGF AS-ODNs treatment with chemotherapy in sensitive or responsive tumor models needs to be examined Finally the assessment of the effects of VEGF/bFGF AS-ODNs treatment on tumor metastasis will be worthwhile and ought to be tested in future preclinical investigations The research described in this dissertation has been presented at 6 national and international meetings. Portions of the studies have been published in the Briti s h Journal of C ancer (Shi and Siemann 2002) and Sert1inars in Radiation Oncology (Siemann and

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134 Shi 2002) Another manu s cript based on the s tudie s of thi s work has been submitted for publication to Anticanc e r R ese ar c h

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REFERENCES Abe T Suzuki S Hatta T, Takai K Yokota T Takaku H (1998) Specific inhibition of influenza virus RNA polymerase and nucleoprotein gene expression by liposomally encapsulated antisense phosphorothioate oligonucleotides in MDCK cells Antivir C hem C he,nother 9 : 253-262 Abraham JA, Whang JL, Tumolo A Mergia A Friedman J, Gospodarowicz D Fiddes JC (1986) Human basic fibroblast growth factor : nucleotide sequence and genomic organization EMBO J 5: 2523-2528 Abraham JA, Mergia A Whang JL, Tumolo A Friedman J Hjerrild KA, Gospodarowicz D Fiddes JC (1986) Nucleotide sequence of a bovine clone encoding the angiogenic protein basic fibroblast growth factor Science 233 ; 545-548 Agrawal S Zhang X, Lu Z Zhao H, Tamburin JM, Yan J, Cai H Diasio RB Habus I, Jiang Z (1995) Absorption tissue distribution and in vivo stability in rats of a hybrid antisense oligonucleotide following oral administration Bioch e m Phamzacol 50 : 571-576 Agrawal S Iyer RP ( 1997) Perspectives in antisen s e therapeutics Pharmaco/ Ther 76 : 151-160 Agrawal S Zhao Q Jiang Z Oliver C, Giles H Heath J Serota D (1997) Toxicologic effects of an oligodeoxynucleotide phosphorothioate and its analogs following intravenous administration in rats Antisen s e N1,clei c Acid Dn1g D e 1 1 7 : 575-584 Ajmani PS Tang F Krishnaswami S Meyer EM, Sumners C Hughes JA (1999) Enhanced transgene expression in rat brain cell cultures with a disulfide-containing cationic lipid Neuro s ci Lett 277 : 141-144 Akhtar S Agrawal S (1997) In vivo studies with antisense oligonucleotides Trends Pharmaco/S c i18 : 12-18 Algire G H ( 1943) An adaptation of the transparant chamber technique to the mouse. J Natl Cancer Inst 1-11 Allalunis-Tumer MJ Siemann DW (1986) Recovery of cell subpopulations from human tumour xenografts following dissociation with different enzymes Br J Cancer 54 : 615622 American cancer society Cancer facts and figures 2001. 2002 135

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BIOGRAPHICAL SKETCH Wenyin Shi was born on April 2 1 97 4 in Shanghai P.R China, to Dr Xuehui Shi and Dr Ying Wang He received hi s elementary and sec ond ary education in Shanghai P R China and graduated high school in 1992 He received a Bachelor of Medicine degree in Clinical Medicine from Shanghai Medical University Shanghai P R .C hina, in 1997 He finished one year of residency in the Department of Surgery at Huashan Hosptial Shanghai Medical University before he began his doctoral studies in the Interdisciplinary Program in College of Medicine University of Florida, Gainesville Florida, in August 1998 In 1999 he joined the Depar trnent of Pharmacology and Therapeutics and continued his doctoral studies under the mentorship of Dr Dietmar W Siemann After graduation, he will continue his research in anti-cancer therapy 171

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I certify that I have read this study and that in my opinion it confo1ms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Dietmar W Siemann Chair Professor of Phannacology and Therapeutics I certify that I have read this study and that in my opinion it confor1ns to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy M. Ian Phillips Professor of Physiology I certify that I have read this study and that in my opinion it confor1ns to acceptable standards of scholarly presentation and is fully ad 0 ,,_,, e and quality, as a dissertation for the degree of Doctor of PhilosophY---t,c;.--:, Stephen P. Su e Professor of Anatomy and Cell Biology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Edwin M. Meyer Associate Professor of Pharmacology and Therapeutics This dissertation was submitted to the Graduate Faculty of the College of Medicine and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy August 2002 Dean ollege of Medicine Dean Graduate School

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