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University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 1 The Effects of the Anti Cancer Agent Dasatinib on KHT Murine Sarcoma Cells Megan Lipcsey Sharon Lepler, Dr. Lori Rice, and Dr. Dietmar W Siemann College of Medicine, University of Florida Metastatic sarcomas originate in connective tissues and often spread to secondary organs such as the lungs. They prove significantly more difficult to treat once they have gained access to the circulatory system. This study assessed the efficacy of the molec ular targeting agent dasatinib to inhibit the phosphorylati on of Src, a non receptor tyrosine kinase and its down stream effector focal adhesion kinase (FAK). These proteins are often over expressed in malignant cells and they mediate key cellular transduction pathways that contribute to metastatic activity. In v itro studies were done using a KHT murine sarcoma cell line to assess the impact of dasatinib on cellular growth, proliferation, migration and invasion C hemotherapeutic impact at the protein level was also studied Treated cells demonstrated a dose dep endent decrease in phosphorylated Src (pSrc) and phosphorylated FAK (pFAK). Phenotypic changes included inhibition of proliferation, migration and invasion, and progression out of the G1 phase of cell cycle. Using immunofluorescence and standard biological assays, the Siemann lab has previously shown that the levels of pSrc and pFAK in a prostate cancer cell line decrease upon exposure to dasatinib. These findings come from literature reports of early clinical trials showing that dasatinib has promise as an anti cancer agent. This warrants further studies to fully understand how it affects the me tastatic cascade in particular. INTRODUCTION Sarcomas are malignant growths that arise from various connective tissues in the body and can range in severity from low to high grade. There are approximately 50 different types that can occur anywhere in the body, with the most commonly diagnosed in the smooth muscles of the abdomen and the soft tissues of the legs. The survival rate of a patient diagnosed with sarcoma depends on the stage and grade of the tumor as well as other underlying factors, with earlier stages and lower grades resulting in a higher survival rate. While surgical excision is generally the primary method of treatment, adjuvant chemotherapy and radi ation are frequently necessary. Despite the fact that sarcomas are rare compared to other cancers (10,660 new cases and 3,820 deaths in the U.S. last year) (American life deteriorate rapidly if th e tumor spreads to and invades secondary organ sites. The most prevalent secondary organs seen in metastatic sarcoma are the lungs, a diagnosis that consequentially drops the overall median survival down to only 15 months (Billingsley et al., 1999). While research is ongoing, scientists have identified genetic factors as well as environmental factors and viruses that cause sarcoma. Tumor growth and proliferation occur when normal cellular onco proteins are over expressed (Pollock et al., 1997), proto oncoge nes are upregulated, or the cell fails to properly regulate growth factors (Todaro and De Larco, 1978). The proto oncogene Src is a tyrosine kinase that is found in higher concentrations in tumor cells (Rosen et al., 1986) and is widely implicated in many aspects of tumorigenesis, invasion, and metastasis. One of the signal transduction molecules phosphorylated (activated) by Src is focal adhesion kinase (FAK), which is involved in cell adhesion and mobility and is also over expressed in various types of ca ncers (Owens et al., 1995). Because of their fundamental roles in metastasis, Src and its downstream effector FAK are promising molecular targets. The anti neoplastic agent dasatinib has multiple targets, including Src. It has already been approved by the FDA as a treatment for chronic myeloid leukemia (CML) and Philadelphia chromosome positive acute lymphoblastic leukemia (ALL) in adults (National Cancer Institute, 2009). It is currently in phase II clinical trials for advanced sarcomas (clinicaltrials.gov ). The purpose of this study was to assess the efficacy of dasatinib in interfering with the cellular transduction pathways of Src, and subsequently those of FAK, resulting in the decreased ability of sarcoma cells to proliferate, invade, and metastasize This would provide an alternative treatment strategy for sarcomas that are no longer responsive to existing therapeutic regimens as well as improve outcomes through local control.

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MEGAN LIPCSEY SHARON L EPLER D R LORI R ICE & D R DIETMAR W S IEMANN University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 2 MATERIALS AND METHOD S Cell Culture and Reagents All experiments were do ne using a murine sarcoma KHT cell line (Rockwell and Kallman, 1972). The cells were maintained in Alpha modified minimum essential medium serum (FBS), 1% penicillin streptomycin, and 1% 200 gl utamine at 3 7 C. They were collected by trypsoniza trypso nize the cells, which were then centrifuged for 10 min. at 1000rpm. All reagents used were obtained from Life Technologies. Primary antibodies for immunofluorescence for pSr c were from Cell Signaling Technologies and for pFAK were from BioSource International. Secondary antibodies were from Invitrogen. Drug P reparation Dasatinib was provided by Bristol Myers Squibb p repared in dimethyl sulfoxide, was diluted to the appropriate concentration using phosphate buffered saline (PBS). The drug was kept frozen and prepared immediately before use at a dilution of 10ul/mL drug to media. In V itro Cell Growth KHT cells were p lated in triplicate at 10 4 in 60mm cell culture dishes. After 24 hours, the medium was then replaced and treated with dasatinib (0.1uM, 0.5uM, 1.0uM, 5.0uM, or 10uM). The cells were collected and counted using trypan blue staining 1, 2, 4 and 7 days after treatment. Adhered and floating cells were harvested and counted separately to account for the high numbers of detached cells. Migration Assay Appropriate concentrations of dasatinib (0.1uM, 0.5uM, 1.0uM, 5.0uM, or 10uM) were added to the top and botto m wells of modified Boyden migration chambers (BD Falcon) with 8m pore size. KHT cells were then plated in the top well at 10 3 cells/200mL and kept at 37 C for 48 hours. The inside of the upper wells were scraped and then stained with crystal violet dye. The number of cells that had migrated through the membrane was counted under a dissecting microscope. Invasion Assay BD BioCoat Matrigel Inv asion Chamber for 2 hours at 37 C. 2x10 4 cells treated with das atinib (0.1uM, 0.5uM, 1.0uM, 5.0uM, or 10uM) in 200 mL of the same media added to the bottom chamber. After 48 hour incubation at 37 C, the inside s of the upper wells were scraped and then stained with cr ystal violet. Cells were then counted using a dissecting microscope. Cell Cycle Analysis Subconfluent KHT cells were treated with dasatinib (0.1uM, 0.5uM, 1.0uM, 5.0uM and 10uM) and incubated at 37 C for 24 hours. They were collected using the previous ly stated protocol and prepared at 10 6 cells/3mL in a solution of 50% PBS and 50% cold 100% ethan ol. Cells were then stored at 4 C overnight. Cells were centrifuged and then treated with 1mg/ml RNase A for 30 min. with intermittent vortexing, after which they were spun for 10min. at 4 C at 1000rpm. Cells were stained with propidium iodide and analyzed using a FACScan flow cytometer, then analyzed using ModFitLT 3.0 software. Immunofluorescence and Confocal Microscopy Cells were plated at 2x10 4 cells/mL in 35mm tissue culture dishes (Fluorodish TM ) and treated with 10 M dasatinib after 24 hours. They were fixed with 3% formaldehyde in 1x Cytoskeleton Buffer (1mL 10X CB, 320mM Sucrose, 2mM EGTA, brought to volume with dH 2 O) (per 100mL of 10X CB: 1.95g MES, 0.285g MgCl 2 and 10.29g KCl, brought to volume with dH 2 O). Primary and secondary antibodies for pSrc and pFAK were administered according to company protocols. The cells were fixed with Vectashield mounting medium with DAPI to stain nuclear DNA. Imaging w as done using a confocal microscope at 64X magnification. RESULTS Dasatinib I nhibits C ell G rowth I n V itro Dasatinib inhibited KHT growth in a dose dependent fashion with results beginning 48 hours after treatment (Figure 1).

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THE E F FECTS OF THE ANTI CANCER AGENT D ASATI NIB ON KHT MURINE SA RCOMA C ELLS University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 3 Figure 1: Growth curve of sarcoma cells after treatment with dasatinib. Tx indicates treatment, which was administered 24 hours after cells were plated. Proliferation decreases as dosage increases, with clear effects at the 4 day point. Dasatinib I nhibits KHT M igration KHT cells were treated with dasatinib; the number of cells that had migrated across a Matrigel membrane was counted after staining with crystal violet. The number of cells per dosage was determined by averaging three wells. Figure 2 is the avera ge of 4 experiments. Migratory ability was inhibited in a dose dependent fashion. This i nhibition was significant ( p < .01) beginning with the 0.5 M treatment (Figure 2). Figure 2: Dasatinib inhibits migration. Increased concentrations of the drug resulted in a decrease in the average number of cells able to cross the membrane. indicates significance, with p <0.01

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MEGAN LIPCSEY SHARON L EPLER D R LORI R ICE & D R DIETMAR W S IEMANN University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 4 Dasatinib I nhibits KHT I nvasion Exposure to dasatinib resulted in a dose dependent decrease in the amount of KHT cells able to invade the Matrigel and cross the membrane. As seen in the migration, this inhibition was sig nificant beginning with the 0.5 M treatment (Fig ure 3). Figure 3: Dasatinib inhibits invasion. Increased concentrations of drug resulted in a sig nificant decrease in the average number of cells able to invade the Matrigel. indicates significance, with p < 0.01. Dasatinib T reatment R esults in an A ccumulation at the G1 P hase of the C ell C ycle The number of KHT cells that accumulated in the G1 p hase of the cell cycle was greater after dasatinib treatment, relative to controls, with a concomitant decrease in S and G2/M phases (Table 1). These cell cycle trends were dose dependent. The accumulation of cells in the G1 phase is evident in the DNA his to grams in Figure 4, with the 1.0 M treatment resulting in a greater than two fold increase in G1 phase than in the control Table 1: Amount of DNA in Different Stages of the Cell Cycle Determined Using Flow Cytometry Dasatinib (M) G1 Phase (%) G2/M Phase (%) S Phase (%) Control (0) 38.4 18.0 43.6 0.1 54.0 14.6 31.3 0.5 80.9 7.4 11.7 1.0 85.5 5.6 8.9

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THE E F FECTS OF THE ANTI CANCER AGENT D ASATI NIB ON KHT MURINE SA RCOMA C ELLS University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 5 Figure 4: Cell cycle analysis showing the DNA distribution in control and 1.0M dose cells. Shown here are representative histograms. Exposure to dasatinib resulted in cell accumulation in the G1 phase. There are fewer cel ls in the S and G2 phases seen in the 1.0M dasatinib histogram. Dasatinib D ecreased pSrc and pFAK at F ocal A dhesions S ites The control KHT cells showed pSrc localized at the periphery of the cell where the focal adhesion sites would be found (Figure 5A). In contrast, pSrc was delocalized throughout the cytoplasm in cells exposed to dasatinib (Figure 5B). The same results were demonstrated in cellular pFAK distribution (Figures 6A, 6 B). Figure 5 : Dasatinib decreased the amount of phosphoryl ated Src at focal adhesion sites. ( A) shows control phosphorylated Src (bright green) localized around the cell membranes. Nuclei are stained blue. (B) is a higher magnification showing phosphorylated Src redistributed throughout the cytosol of a cell afte r exposure to 1.0M dasatinib.

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MEGAN LIPCSEY SHARON L EPLER D R LORI R ICE & D R DIETMAR W S IEMANN University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 6 Figure 6: Dasatinib decreased the amount of phosphorylated FAK at focal adhesion sites. ( A) shows control phosphorylated FAK (bright green) localized around the cell membranes. (B) is a higher magnific ation of a single cell showing phosphorylated FAK redistributed throughout the cytosol of a cell after exposure to 1.0M dasatinib. DISCUSSION Metastatic potential in sarcoma cells is largely attributed to anomalies in the signaling pathways regulated by the Src family kinases ( Thomas & Brugge, 2007 ). Studies have confirmed elevated levels of Src as well as its downstream effector FAK in various malignant cell lines, including those of sarcoma (Rosen et al., 1986). Inhibiting the phosphorylation of Src and FAK using dasatinib would hinder pathways responsible for KHT cell growth, migration and invasion. Dasatinib is in phase II clinical trials to treat advanced stage sarcoma, but there is not extensive research focusing on its ability to prevent metastas is in particular. KHT sarcoma cells grow rapidly which enables the growth and development of tumors. Dasatinib effectively inhibited in vitro cell growth in a dose dependent manner. These results are consisten t with those found in pancreatic cell lines (C hang et al. 2008); however Buettner et al. (2008) found that dasatinib did not strongly affect proliferation in melanoma cells. The ability of dasatinib to inhibit KHT growth is corroborated by cell cycle analysis in which treated cells accumulated in th e G1 phase. The decrease in amount of DNA per cell undergoing replication in the S phase would help explain the inhibition observed in the growth curve. Various studies using inhibitory antibodies and agents that arrest cell cycle have shown the importance of Src in cell progression through the S and G2 phases into mitosis ( Mamidipudi et al. 2004 ; Roche et al. 1995). There are multiple regulatory mechanisms for Src tyrosine kinase. It is often activated by more than one of these regulation mechanisms (on e of which is phosphorylation) and then goes on to activate growth factor receptor proteins and mitotic kinase pathways such as the Ras MAP pathway (Thomas & Brugge, 2007). FAK is also involved in growth and proliferation through its interactions with gro wth factors and integrins. Dasatinib is known to effectively inhibit migration and invasion in several cancer models, and this study confirms this in the KHT sarcoma cell line. These results were further verified by immunofluorescence. Focal adhesion kinas e is recruited to focal adhesion sites by integrins, where it interacts with actin to cause a turnover of these sites that propels the cell along the substrate. The auto phosphorylation of FAK attracts activated pSrc, which then phosphorylates it at three tyrosine residues (Parsons, 2003). Sieg et al (1999) found that Fak deficient fibroblast mutants demonstrate retardation in migratory ability as well as morphological defects. Less pSrc and pFAK at the focal adhesion sites of cells after treatment (seen i n Fig ure 5 and 6) help to explain the inhibition of migration and invasion and emphasizes the importance of Src as an upstream activator of FAK. Additionally, the Siemann lab has used protein immunoblots to demonstrate decreased protein levels of activated Src and FAK after dasatinib

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THE E F FECTS OF THE ANTI CANCER AGENT D ASATI NIB ON KHT MURINE SA RCOMA C ELLS University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 7 treatment in other cancer cell lines. Shor e t al (2007) found that dasatinib also inhibits migration and invasion in various human sarcoma cell lines. Preventing the spread of cancer is crucial to patient survival, mainly beca use treatment options are limited after secondary organ involvement. These experiments show that the anti cancer drug dasatinib is a potent inhibitor of murine sarcoma cell migration and invasion, which are key components of metastasis. Consistent with rel ated research, it also inhibits proliferation and causes these cells to accumulate in the G1 phase of the cell cycle. Further research on the efficacy of this agent in preventing metastasis in animal models is needed. However, dasatinib has the potential t o treat fibroblast sarcoma as well as prevent tumor formation in secondary organs. LITERATURE CITED American Cancer Society Detailed guide: Sarcoma Adult Soft Tissue C ancer [Internet] Altanta (GA). American Cancer Society; 2011 [updated 2011 Ju n 6; cited 2010 Jan 5th]. Available fro m: http://www.cancer.org/ Billingsley KG Burt ME Jara E Ginsberg RJ, Woodruff DH Leung Y, et al. Pulmonary metastases from soft tissue sarcoma: Analysis of patterns of disease and postmetastasis survival Ann Surg [Internet]. 1999 May [ cited 2010 Jan 10 ] ; 229(5): 602. Available from: http://www.ncbi. nlm.nih.gov/pmc/articles/PMC1420804/ Buettner R, Mesa T, Vultur A, Lee F, & Jove R Inhibition of S rc family kinases with dasatinib blocks migration and invasion of human melanoma cells. Mol Cancer Res [Internet]. 2008 Nov [cited 2010 Jan 15 ]; 6(11): 1766 74. Available from: http://mcr.aacrjournals.org/content/6/11/1766.full Chang Q, Jorgensen C, Pawson T, & Hedley DW. Effects of dasatinib on EphA2 receptor tyrosine kinase activity and downstream signaling in pancreatic ca ncer. Br J of Cancer [Internet]. 2008 Sept 16 [cited 2010 Jan 10 ]; 99(7): 1074 82. Available from: http://www.ncbi. nlm.nih.gov/pmc/articles/ PMC2567084 Laird AD, Li G, Moss KG, Blake RA, Broome MA, Cherrington JM, et al. Src family kinase activity is required for signal transducer and activator of transcription 3 and focal adhesion kinase phosphorylation and vascular endothelial growth factor signaling in vivo and for anchorage dependent and independent growth of human tumor cells. Mol Cancer Ther [Internet]. 2003 May [cited 2010 Jan 15 ]; 2(5): 461 Available from: http://mct.aacrjournals.org/content/2/5/461.short Ma midipudi V, Zhang J Lee KC, & Cartwright CA. RACK1 regulates G 1/S progression by suppressing S rc kinase activity. Mol Cell Biol [Internet]. 2004 Aug [cited 2010 Jan 15 ]; 24(15): 6788 98. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC444846 Owens LV, Xu L, Craven RJ, Dent GA, Weiner TM, Kornberg L, et al. Overexpression of the focal adhesion kinase (p125FAK) in invasi ve human tumors. Cancer Res [Internet]. 1995 July 1 [cited 2010 Jan 15 ]; 55(13): 2752 5 Available from: http://can cerres.aacrjournals.org/content/55/13/2752.long Parsons JT Focal adhesion kinase: the first ten years. J Cell Sci [Internet]. 2003 April 15 [cited 2010 Jan 15 ] ; 116: 1409 16. Available from: http://jcs.biologists.org/content/116/8/1409.long Pollock RE, L ang A, El Naggar AK, Radinsky R, & Hung M C. Enhanced MDM2 oncoprotein expression in soft tissue sarcoma: Several possible regulatory mechanisms. Sarcoma [Internet]. 1997 March [cited 2010 Jan 7]; 1(1): 23 9. Available from: http://www.ncbi.nlm.nih.gov/pmc / articles/PMC2373579 Roche S, Fumagalli S, & Courtneidge S. Requirement for S rc family protein tyrosine kinases in G2 for fibroblast cell division. Science [Internet]. 1995 Sept 15 [cited 2010 Jan 10]; 269(5230): 1567 9. Available from: http ://www.sciencemag.org/content/269/5230/1567.long Rockwell S, Kallman RF. Growth and cell population kinetics of single and multiple KHT sarcomas. Cell Proliferation [Internet]. 2008 May 1 [cited 20 10 Jan 10] ; 5(6): 449 57. Available from: http://onlinelibrary wiley.com/doi/ 10.1111/j.1365 2184.1972.tb00383.x/abstract Rosen N, Bolen JB Schwartz AM, Cohen P, DeSeau V, & Israel M A. Analysis of pp60c src protein kinase activity in human tumor cell lin es and tissues. J Biol Chem [Internet]. 1986 Oct 15 [cited 2010 Jan 15]; 261(29): 13754 9. Available from: http://www.jbc.org/content/261/29/13754.full.pdf+html Shor AC, Keschman EA, Lee FY, Muro Cacho C Letson GD, Trent JC et al. Dasatinib inhibits migr ation and invasion in diverse human sarcoma cell lines and induces apoptosis in b one sarcoma cells dependent on S rc kinase for survival. Cancer Res [Internet]. 2007 Mar 15 [cited 2010 Jan 7]; 67(6): 2800 Available from: http://cancerres.aacrjournals.org/c ontent/67/6/2800 Sieg D Hauck C, & Schlaepfer D. Required role of focal adhesion kinase (FAK) for integrin stimulated cell migration. J Cell Sci [Internet]. 1999 Aug 15 [cited 2010 Jan 15]; 112(16): 2677 91. Available from: http://jcs.biologists.org/ cont ent /112/16/2677.long Thomas SM, & Brugge JS. C ellular functions regulated by Src family kinases. A n nu Rev Cell Dev Biol [Internet]. 1997 Nov [cited 2010 Jan 15]; 13: 513 609. Available from: http://www.sut.ac.th/iat/biotech/Montarop/transfer/ signalling/S rc.annurev.pdf Todaro GJ, & De Larco JE. Growth factors produced by sarcoma virus transformed cells Cancer Res [Internet]. 1978 Nov [cited 2010 Jan 15]; 38: 4147 Available from: http://cancerres.aacrjournals.org/content/38/11 _Part_2 /4147.short


Summer Focus on Medical Research : The Effects of the Anti-Cancer Agent Dasatinib on KHT Murine Sarcoma Cells
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Title: Summer Focus on Medical Research : The Effects of the Anti-Cancer Agent Dasatinib on KHT Murine Sarcoma Cells
Series Title: Journal of Undergraduate Research
Physical Description: Serial
Language: English
Creator: Lipcsey, Megan
Lepler, Sharon
Rice, Lori
Sieman, Dietmar W.
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011
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Genre: serial   ( sobekcm )
 Notes
Abstract: Metastatic sarcomas originate in connective tissues and often spread to secondary organs, such as the lungs. They prove significantly more difficult to treat once they have gained access to the circulatory system. This study assessed the efficacy of the molecular targeting agent dasatinib to inhibit the phosphorylation of Src, a non-receptor tyrosine kinase, and its downstream effector focal adhesion kinase (FAK). These proteins are often over-expressed in malignant cells and they mediate key cellular transduction pathways that contribute to metastatic activity. In vitro studies were done using a KHT murine sarcoma cell line to assess the impact of dasatinib on cellular growth, proliferation, migration, and invasion. Chemotherapeutic impact at the protein level was also studied. Treated cells demonstrated a dose-dependent decrease in phosphorylated-Src (pSrc) and phosphorylated-FAK (pFAK). Phenotypic changes included inhibition of proliferation, migration and invasion, and progression out of the G1 phase of cell cycle. Using immunofluorescence and standard biological assays, the Siemann lab has previously shown that the levels of pSrc and pFAK in a prostate cancer cell line decrease upon exposure to dasatinib. These findings come from literature reports of early clinical trials showing that dasatinib has promise as an anti-cancer agent. This warrants further studies to fully understand how it affects the metastatic cascade in particular.
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The Effects of the Anti-Cancer Agent Dasatinib on KHT Murine
Sarcoma Cells


Megan Lipcsey, Sharon Lepler, Dr. Lori Rice, and Dr. Dietmar W. Siemann

College of Medicine, University of Florida

Metastatic sarcomas originate in connective tissues and often spread to secondary organs, such as the lungs. They prove significantly
more difficult to treat once they have gained access to the circulatory system. This study assessed the efficacy of the molecular
targeting agent dasatinib to inhibit the phosphorylation of Src, a non-receptor tyrosine kinase, and its downstream effector focal
adhesion kinase (FAK). These proteins are often over-expressed in malignant cells and they mediate key cellular transduction
pathways that contribute to metastatic activity. In vitro studies were done using a KHT murine sarcoma cell line to assess the impact
of dasatinib on cellular growth, proliferation, migration, and invasion. Chemotherapeutic impact at the protein level was also studied.
Treated cells demonstrated a dose-dependent decrease in phosphorylated-Src (pSrc) and phosphorylated-FAK (pFAK). Phenotypic
changes included inhibition of proliferation, migration and invasion, and progression out of the G1 phase of cell cycle. Using
immunofluorescence and standard biological assays, the Siemann lab has previously shown that the levels of pSrc and pFAK in a
prostate cancer cell line decrease upon exposure to dasatinib. These findings come from literature reports of early clinical trials
showing that dasatinib has promise as an anti-cancer agent. This warrants further studies to fully understand how it affects the
metastatic cascade in particular.


INTRODUCTION

Sarcomas are malignant growths that arise from various
connective tissues in the body and can range in severity
from low to high grade. There are approximately 50
different types that can occur anywhere in the body, with
the most commonly diagnosed in the smooth muscles of
the abdomen and the soft tissues of the legs. The survival
rate of a patient diagnosed with sarcoma depends on the
stage and grade of the tumor as well as other underlying
factors, with earlier stages and lower grades resulting in a
higher survival rate. While surgical excision is generally
the primary method of treatment, adjuvant chemotherapy
and radiation are frequently necessary. Despite the fact that
sarcomas are rare compared to other cancers (10,660 new
cases and 3,820 deaths in the U.S. last year) (American
Cancer Society, 2009), a patient's prognosis and quality of
life deteriorate rapidly if the tumor spreads to and invades
secondary organ sites. The most prevalent secondary
organs seen in metastatic sarcoma are the lungs, a
diagnosis that consequentially drops the overall median
survival down to only 15 months (Billingsley et al., 1999).
While research is ongoing, scientists have identified
genetic factors as well as environmental factors and viruses
that cause sarcoma. Tumor growth and proliferation occur
when normal cellular onco-proteins are over-expressed
(Pollock et al., 1997), proto-oncogenes are upregulated, or


the cell fails to properly regulate growth factors (Todaro
and De Larco, 1978). The proto-oncogene Src is a tyrosine
kinase that is found in higher concentrations in tumor cells
(Rosen et al., 1986) and is widely implicated in many
aspects of tumorigenesis, invasion, and metastasis. One of
the signal transduction molecules phosphorylated
(activated) by Src is focal adhesion kinase (FAK), which is
involved in cell adhesion and mobility and is also over-
expressed in various types of cancers (Owens et al., 1995).
Because of their fundamental roles in metastasis, Src and
its downstream effector FAK are promising molecular
targets. The anti-neoplastic agent dasatinib has multiple
targets, including Src. It has already been approved by the
FDA as a treatment for chronic myeloid leukemia (CML)
and Philadelphia chromosome-positive acute lymphoblastic
leukemia (ALL) in adults (National Cancer Institute,
2009). It is currently in phase II clinical trials for advanced
sarcomas (clinicaltrials.gov).
The purpose of this study was to assess the efficacy of
dasatinib in interfering with the cellular transduction
path\\ a s of Src, and subsequently those of FAK, resulting
in the decreased ability of sarcoma cells to proliferate,
invade, and metastasize. This would provide an alternative
treatment strategy for sarcomas that are no longer
responsive to existing therapeutic regimens as well as
improve outcomes through local control.


University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 I Summer 2011
1




MEGAN LIPCSEY, SHARON LEPLER, DR. LORI RICE, & DR. DIETMAR W. SIEMANN


MATERIALS AND METHODS

Cell Culture and Reagents

All experiments were done using a murine sarcoma KHT
cell line (Rockwell and Kallman, 1972). The cells were
maintained in Alpha modified minimum essential medium
(aMEM, Invitrogen) supplemented with 10% fetal bovine
serum (FBS), 1% penicillin-streptomycin, and 1% 200-
mmol/L L-glutamine at 37 OC. They were collected by
trypsonization. aMEM was used to de-trypsonize the cells,
which were then centrifuged for 10 min. at 1000rpm. All
reagents used were obtained from Life Technologies.
Primary antibodies for immunofluorescence for pSrc were
from Cell Signaling Technologies and for pFAK were from
BioSource International. Secondary antibodies were from
Invitrogen.

Drug Preparation

Dasatinib was provided by Bristol-Myers Squibb
Pharmaceutical Research Institute. A 10mM stock solution,
prepared in dimethyl sulfoxide, was diluted to the
appropriate concentration using phosphate buffered saline
(PBS). The drug was kept frozen and prepared immediately
before use at a dilution of lOul/mL drug to media.

In Vitro Cell Growth

KHT cells were plated in triplicate at 104 in 60mm cell
culture dishes. After 24 hours, the medium was then
replaced and treated with dasatinib (0.luM, 0.5uM, 1.OuM,
5.0uM, or 10uM). The cells were collected and counted
using trypan blue staining 1, 2, 4, and 7 days after
treatment. Adhered and floating cells were harvested and
counted separately to account for the high numbers of
detached cells.

Migration Assay

Appropriate concentrations of dasatinib (0.luM, 0.5uM,
l.OuM, 5.0uM, or 10uM) were added to the top and bottom
wells of modified Boyden migration chambers (BD
Falcon) with 8im pore size. KHT cells were then plated in
the top well at 103 cells/200mL and kept at 37 OC for 48
hours. The inside of the upper wells were scraped and then
stained with crystal violet dye. The number of cells that
had migrated through the membrane was counted under a
dissecting microscope.


Invasion Assay

aMEM media with 0.1% FBS was used to rehydrate a
BD BioCoat Matrigel Invasion Chamber for 2 hours at 37
OC. 2x104 cells treated with dasatinib (0.luM, 0.5uM,
1.OuM, 5.0uM, or 10uM) in 200 mL of the same media
were added to the top wells. aMEM with 10% FBS was
added to the bottom chamber. After 48-hour incubation at
37 OC, the insides of the upper wells were scraped and then
stained with crystal violet. Cells were then counted using a
dissecting microscope.

Cell Cycle Analysis

Subconfluent KHT cells were treated with dasatinib
(O.luM, 0.5uM, 1.OuM, 5.0uM, and 10uM) and incubated
at 37 OC for 24 hours. They were collected using the
previously stated protocol and prepared at 106 cells/3mL in
a solution of 50% PBS and 50% cold 100% ethanol. Cells
were then stored at 4 OC overnight. Cells were centrifuged
and then treated with lmg/ml RNase-A for 30 min. with
intermittent vortexing, after which they were spun for
10min. at 4 OC at 1000rpm. Cells were stained with
propidium iodide and analyzed using a FACScan flow
cytometer, then analyzed using ModFitLT 3.0 software.

Immunofluorescence and Confocal Microscopy

Cells were plated at 2x104 cells/mL in 35mm tissue
culture dishes (FluorodishTM) and treated with 10iM
dasatinib after 24 hours. They were fixed with 3%
formaldehyde in lx Cytoskeleton Buffer (ImL 10X CB,
320mM Sucrose, 2mM EGTA, brought to volume with
dH20) (per 100mL of 10X CB: 1.95g MES, 0.285g MgCl2,
and 10.29g KC1, brought to volume with dH20). Primary
and secondary antibodies for pSrc and pFAK were
administered according to company protocols. The cells
were fixed with Vectashield mounting medium with DAPI
to stain nuclear DNA. Imaging was done using a confocal
microscope at 64X magnification.

RESULTS

Dasatinib Inhibits Cell Growth In Vitro

Dasatinib inhibited KHT growth in a dose-dependent
fashion with results beginning 48 hours after treatment
(Figure 1).


University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 1 Summer 2011
2





THE EFFECTS OF THE ANTI-CANCER AGENT DASATINIB ON KHT MARINE SARCOMA CELLS


0 1 2 3 4 5 6 7 8


Day


Figure 1: Growth curve of sarcoma cells after treatment with dasatinib. Tx indicates treatment, which
was administered 24 hours after cells were plated. Proliferation decreases as dosage increases, with
clear effects at the 4 day point.


Dasatinib Inhibits KHT Migration

KHT cells were treated with dasatinib; the number of
cells that had migrated across a Matrigel membrane was
counted after staining with crystal violet. The number of
cells per dosage was determined by averaging three wells.


120-


100.


S80.
0

| 60
E

S40-


20,


04-


Figure 2 is the average of 4 experiments. Migratory ability
was inhibited in a dose-dependent fashion. This inhibition
was significant (p < .01) beginning with the 0.5iM
treatment (Figure 2).


I I


Dasatinib concentration (uM)



Figure 2: Dasatinib inhibits migration. Increased concentrations of the drug resulted in a decrease
in the average number of cells able to cross the membrane. indicates significance, with p <0.01.


University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 I Summer 2011
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MEGAN LIPCSEY, SHARON LEPLER, DR. LORI RICE, & DR. DIETMAR W. SIEMANN


Dasatinib Inhibits KHT Invasion

Exposure to dasatinib resulted in a dose-dependent
decrease in the amount of KHT cells able to invade


160.

140.

120.
u)
100.

80
E

o60.

< 40

20.


0 0.1


the Matrigel and cross the membrane. As seen in the
migration, this inhibition was significant beginning
with the 0.5 IM treatment (Figure 3).


0.5 1 5 10
Dasatinib concentration (uM)


Figure 3: Dasatinib inhibits invasion. Increased concentrations of drug resulted in a significant
decrease in the average number of cells able to invade the Matrigel. indicates significance, with p
< 0.01.


Dasatinib Treatment Results in an Accumulation
at the G1 Phase of the Cell Cycle

The number of KHT cells that accumulated in the G1
phase of the cell cycle was greater after dasatinib
treatment, relative to controls, with a concomitant decrease


in S and G2/M phases (Table 1). These cell cycle trends
were dose dependent. The accumulation of cells in the G1
phase is evident in the DNA histograms in Figure 4, with
the 1.0iM treatment resulting in a greater than twofold
increase in G1 phase than in the control.


Table 1: Amount of DNA in Different Stages of the Cell Cycle Determined Using
Flow Cytometry

Dasatinib ([LM) Gl Phase (%) G2/M Phase (%) S Phase (%)
Control (0) 38.4 18.0 43.6
0.1 54.0 14.6 31.3
0.5 80.9 7.4 11.7
1.0 85.5 5.6 8.9


University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 1 Summer 2011
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THE EFFECTS OF THE ANTI-CANCER AGENT DASATINIB ON KHT MURINE SARCOMA CELLS


DNA Content control


DNA Content- 1.0uM dasatinib


Figure 4: Cell cycle analysis showing the DNA distribution in control and 1.0pM dose cells. Shown here are representative
histograms. Exposure to dasatinib resulted in cell accumulation in the G1 phase. There are fewer cells in the S and G2 phases seen
in the 1.0pM dasatinib histogram.


Dasatinib Decreased pSrc and pFAK at Focal
Adhesions Sites

The control KHT cells showed pSrc localized at the
periphery of the cell where the focal adhesion sites would


be found (Figure 5A). In contrast, pSrc was delocalized
throughout the cytoplasm in cells exposed to dasatinib
(Figure 5B). The same results were demonstrated in
cellular pFAK distribution (Figures 6A, 6B).


Figure 5: Dasatinib decreased the amount of phosphorylated-Src at focal adhesion sites. (A) shows control
phosphorylated-Src (bright green) localized around the cell membranes. Nuclei are stained blue. (B) is a higher
magnification showing phosphorylated-Src redistributed throughout the cytosol of a cell after exposure to 1.0pM
dasatinib.




University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 I Summer 2011
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MEGAN LIPCSEY, SHARON LEPLER, DR. LORI RICE, & DR. DIETMAR W. SIEMANN


Figure 6: Dasatinib decreased the amount of phosphorylated-FAK at focal adhesion sites. (A) shows control
phosphorylated-FAK (bright green) localized around the cell membranes. (B) is a higher magnification of a single
cell showing phosphorylated-FAK redistributed throughout the cytosol of a cell after exposure to 1.0pM dasatinib.


DISCUSSION

Metastatic potential in sarcoma cells is largely attributed
to anomalies in the signaling pathl\ a s regulated by the Src
family kinases (Thomas & Brugge, 2007). Studies have
confirmed elevated levels of Src as well as its downstream
effector FAK in various malignant cell lines, including
those of sarcoma (Rosen et al., 1986). Inhibiting the
phosphorylation of Src and FAK using dasatinib would
hinder pathl as\\ responsible for KHT cell growth,
migration and invasion. Dasatinib is in phase II clinical
trials to treat advanced stage sarcoma, but there is not
extensive research focusing on its ability to prevent
metastasis in particular.
KHT sarcoma cells grow rapidly, which enables the
growth and development of tumors. Dasatinib effectively
inhibited in vitro cell growth in a dose-dependent manner.
These results are consistent with those found in pancreatic
cell lines (Chang et al., 2008); however, Buettner et al.
(2008) found that dasatinib did not strongly affect
proliferation in melanoma cells. The ability of dasatinib to
inhibit KHT growth is corroborated by cell cycle analysis
in which treated cells accumulated in the G1 phase. The
decrease in amount of DNA per cell undergoing replication
in the S phase would help explain the inhibition observed
in the growth curve. Various studies using inhibitory
antibodies and agents that arrest cell cycle have shown the
importance of Src in cell progression through the S and G2


phases into mitosis (Mamidipudi et al., 2004; Roche et al.,
1995).
There are multiple regulatory mechanisms for Src
tyrosine kinase. It is often activated by more than one of
these regulation mechanisms (one of which is
phosphorylation), and then goes on to activate growth
factor receptor proteins and mitotic kinase path\\ a s such
as the Ras-MAP pathway (Thomas & Brugge, 2007). FAK
is also involved in growth and proliferation through its
interactions with growth factors and integrins.
Dasatinib is known to effectively inhibit migration and
invasion in several cancer models, and this study confirms
this in the KHT sarcoma cell line. These results were
further verified by immunofluorescence. Focal adhesion
kinase is recruited to focal adhesion sites by integrins,
where it interacts with actin to cause a turnover of these
sites that propels the cell along the substrate. The auto-
phosphorylation of FAK attracts activated pSrc, which then
phosphorylates it at three tyrosine residues (Parsons, 2003).
Sieg et al. (1999) found that Fak-deficient fibroblast
mutants demonstrate retardation in migratory ability as
well as morphological defects. Less pSrc and pFAK at the
focal adhesion sites of cells after treatment (seen in Figure
5 and 6) help to explain the inhibition of migration and
invasion and emphasizes the importance of Src as an
upstream activator of FAK. Additionally, the Siemann lab
has used protein immunoblots to demonstrate decreased
protein levels of activated Src and FAK after dasatinib


University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 1 Summer 2011
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THE EFFECTS OF THE ANTI-CANCER AGENT DASATINIB ON KHT MURINE SARCOMA CELLS


treatment in other cancer cell lines. Shor et al. (2007) found
that dasatinib also inhibits migration and invasion in
various human sarcoma cell lines.
Preventing the spread of cancer is crucial to patient
survival, mainly because treatment options are limited after
secondary organ involvement. These experiments show
that the anti-cancer drug dasatinib is a potent inhibitor of
marine sarcoma cell migration and invasion, which are key



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