Group Title: BMC Cancer
Title: Phase II trial of isoflavone in prostate-specific antigen recurrent prostate cancer after previous local therapy
CITATION PDF VIEWER THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00099972/00001
 Material Information
Title: Phase II trial of isoflavone in prostate-specific antigen recurrent prostate cancer after previous local therapy
Physical Description: Book
Language: English
Creator: Pendleton, John
Tan, Winston
Anai, Satoshi
Chang, Myron
Hou, Wei
Shiverick, Kathleen
Rosser, Charles
Publisher: BMC Cancer
Publication Date: 2008
 Notes
Abstract: BACKGROUND-:Data exist that demonstrate isoflavones' potent antiproliferative effects on prostate cancer cells. We evaluated the efficacy of isoflavone in patients with PSA recurrent prostate cancer after prior therapy. We postulated that isoflavone therapy would slow the rate of rise of serum PSA.METHODS-:Twenty patients with rising PSA after prior local therapy were enrolled in this open-labeled, Phase II, nonrandomized trial (Trial registration # NCT00596895). Patients were treated with soy milk containing 47 mg of isoflavonoid per 8 oz serving three times per day for 12 months. Serum PSA, testosterone, lipids, isoflavone levels (genistein, daidzein, and equol), and quality of life (QOL) were measured at various time points from 0 to 12 months. PSA outcome was evaluated.RESULTS-:Within the mixed regression model, it was estimated that PSA had increased 56% per year before study entry and only increased 20% per year for the 12-month study period (p = 0.05). Specifically, the slope of PSA after study entry was significantly lower than that before study entry in 6 patients and the slope of PSA after study entry was significantly higher than before study entry in 2 patients. For the remaining 12 patients, the change in slope was statistically insignificant. Nearly two thirds of the patients were noted to have significant levels of free equol in their serum while on therapy.CONCLUSION-:Dietary intervention with isoflavone supplementation may have biologic activity in men with biochemical recurrent prostate cancer as shown by a decline in the slope of PSA. This study may lend support to the literature that nutritional supplements have biologic activity in prostate cancer and therefore, further studies with these agents in randomized clinical trials should be encouraged.
General Note: Periodical Abbreviation:BMC Cancer
General Note: Start page 132
General Note: M3: 10.1186/1471-2407-8-132
 Record Information
Bibliographic ID: UF00099972
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: Open Access: http://www.biomedcentral.com/info/about/openaccess/
Resource Identifier: issn - 1471-2407
http://www.biomedcentral.com/1471-2407/8/132

Downloads

This item has the following downloads:

PDF ( PDF )


Full Text



BMC CancerBioMed Central



Research article

Phase II trial of isoflavone in prostate-specific antigen recurrent
prostate cancer after previous local therapy
John M Pendleton', Winston W Tan2, Satoshi Anail, Myron Chang3,
Wei Hou3, Kathleen T Shiverick4 and Charles J Rosser* I


Address: 'Division of Urology, The University of Florida, Jacksonville, Florida 32209, USA, 2Department of Hematology-Oncology, Mayo Clinic,
Jacksonville, FL 32224, USA, 3Department of Epidemiology and Health Policy Research, The University of Florida, Gainesville, Florida 32610, USA
and 4Department of Pharmacology, The University of Florida, Gainesville, Florida 32610, USA
Email: John M Pendleton john.pendleton@jax.ufl.edu; Winston W Tan winston.tan@mayo.edu; Satoshi Anai satoshi@urology.ufl.edu;
Myron Chang MChang@childrensoncologygroup.org; Wei Hou whou@biostat.ufl.edu; Kathleen T Shiverick kshiverick@ufl.edu;
Charles J Rosser* charles.rosser@urology.ufl.edu
* Corresponding author



Published: I I May 2008 Received: 17 December 2007
BMC Cancer 2008, 8:132 doi:10.1186/1471-2407-8-132 Accepted: II May 2008
This article is available from: http://www.biomedcentral.com/1471-2407/8/132
2008 Pendleton et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background-: Data exist that demonstrate isoflavones' potent antiproliferative effects on
prostate cancer cells. We evaluated the efficacy of isoflavone in patients with PSA recurrent
prostate cancer after prior therapy. We postulated that isoflavone therapy would slow the rate of
rise of serum PSA.
Methods-: Twenty patients with rising PSA after prior local therapy were enrolled in this open-
labeled, Phase II, nonrandomized trial (Trial registration # NCT00596895). Patients were treated
with soy milk containing 47 mg of isoflavonoid per 8 oz serving three times per day for 12 months.
Serum PSA, testosterone, lipids, isoflavone levels (genistein, daidzein, and equol), and quality of life
(QOL) were measured at various time points from 0 to 12 months. PSA outcome was evaluated.
Results-: Within the mixed regression model, it was estimated that PSA had increased 56% per
year before study entry and only increased 20% per year for the 12-month study period (p = 0.05).
Specifically, the slope of PSA after study entry was significantly lower than that before study entry
in 6 patients and the slope of PSA after study entry was significantly higher than before study entry
in 2 patients. For the remaining 12 patients, the change in slope was statistically insignificant. Nearly
two thirds of the patients were noted to have significant levels of free equol in their serum while
on therapy.
Conclusion-: Dietary intervention with isoflavone supplementation may have biologic activity in
men with biochemical recurrent prostate cancer as shown by a decline in the slope of PSA. This
study may lend support to the literature that nutritional supplements have biologic activity in
prostate cancer and therefore, further studies with these agents in randomized clinical trials should
be encouraged.







Page 1 of 10
(page number not for citation purposes)







http://www.biomedcentral.com/1471-2407/8/132


Background
Environmental factors have been investigated as possible
etiologic influences for the development of prostate can-
cer, including occupational exposures, smoking, alcohol
consumption, sexual practices, venereal transmitted dis-
eases, and vasectomy. Only dietary influences have shown
any promise[ 1]. The most provocative data supporting the
influence of dietary factors on the clinical incidence of
prostate cancer come from international studies and from
studies of disease expression in immigrant populations.
Historically, the incidence of prostate cancer in Japan has
been extremely low. However, as Japanese men migrate to
Hawaii or the U.S. mainland and subsequently adopt a
western culture, their incidence of prostate cancer rapidly
approaches that of Caucasian Americans [3,4]. The diets
of native Chinese and Japanese are rich in fiber and low in
saturated fat. These cultural differences may contribute to
the lower rates of clinical prostate cancer in the Far East
compared to Northern Europe and North America. In
addition, recent evidence suggests that soy consumption
contributes to the lower clinical incidence of prostate can-
cer in Asian countries [5].

One of the several compounds in soy is isoflavone, a soy-
bean protease inhibitor. In vitro experimental data suggest
that isoflavones are cancer preventive agents [6]. Genis-
tein glycosides account for more than two-thirds of the
total soybean isoflavone content [7]. Genistein is known
to inhibit both androgen-dependent and androgen-inde-
pendent prostate cancer cell growth in vitro. It is specu-
lated that the mechanisms by which genistein exerts its
effect are related to the inhibition of protein tyrosine
kinase, DNA topoisomerases, angiogenesis, as well as its
effect on cellular differentiation and apoptosis [8].

Daidzein is another major constituent of total soybean
isoflavone content. It is speculated that some humans
possess the necessary intestinal flora to convert daidzein
to equol, a molecule similar to estrogen [9]. Thus, isofla-
vones may act through hormonal manipulation. If isofla-
vones change the hormonal milieu, it is quite possible
that their mechanism of action is centered on the andro-
gen receptor (AR). AR activity has been implicated as piv-
otal in several phases of prostate cancer: origin, transition
to androgen independent status, and progression. Linkage
studies suggest that the activity of the AR might be corre-
lated with tumor incidence and aggressiveness. Research-
ers have found that a decreased repeat length of an AR
polymorphism correlates with an increased risk of and
aggressiveness of prostate cancer [10].

We set out to determine if dietary supplementation is
indeed a potential therapeutic option, by evaluating the
efficacy of isoflavone in subjects with prostate-specific
antigen (PSA) recurrent prostate cancer after radiation


therapy or radical prostatectomy. Herein, we report the
serum PSA, CAG androgen receptor polymorphism, and
quality of life results of our phase II, prospective study.

Methods
This study was performed after review and approval by the
Institutional Review Board of the University of Florida at
Jacksonville, an inner-city hospital serving Duval County.
The study was an investigator initiated, industry spon-
sored (Hain-Celestial, Denver CO), phase II, non-rand-
omized clinical trial. The study was registered with
National Institute of Health (registration #
NCT00596895).

Study Design
Sample size determination to detect a reduction of 10% in
PSA from initial levels [actual calculation as logo0 (PSA)
with a standard deviation of 37% of PSA level] required
27 patients to have a significance ofp = 0.05 and a power
of 60%. However, due to poor accrual, the study was
closed after the enrollment of 20 patients. With the sam-
ple size of 20, a reduction of 12% in PSA can be detected
with the same significance level and power.

Twenty patients were enrolled in this study from May
2004 to January 2007. Eligibility criteria included: able to
give informed consent, adenocarcinoma of the prostate
(no small cell component), and prior treatment with radi-
ation (at least 6200 cGy, n = 9) or radical prostatectomy
(n = 11) for clinically localized disease (clinical TI or T2).
Table 1 shows the demographic, clinical, and pathologic
characteristics of the study cohort. All patients had dem-
onstrated evidence of biochemical failure within a
median of 6 3 years after treatment. Biochemical failure
was defined as a) detectable and rising PSA after surgery or
b) three consecutive PSA rises after radiation therapy [ 11].

Patients could not have demonstrable and/or histologi-
cally confirmed metastatic or locally recurrent disease
demonstrated on bone scan, computed tomography or
transrectal ultrasound, or be clinically symptomatic at the
time of enrollment. Patients could have received andro-
gen deprivation therapy (ADT), but not within 12 months
of entry into the study. In addition, patients had to have a
life expectancy of at least one year and performance status
of<2 of Zubrod scale. Patients with a known allergic reac-
tion to milk or soy products were excluded.

Twenty men who have had evidence of biochemical
relapse after radiation therapy and/or prostatectomy com-
prised the study cohort. These patients ingested Soy
Dream Enriched, Original or Vanilla, soy milk which pro-
vided 47 mg ofisoflavonoid per 8 oz serving. The patients
received three 8 oz servings per day in an open label, non-
blinded fashion. No dose escalation or reduction was


Page 2 of 10
(page number not for citation purposes)


BMC Cancer 2008, 8:132








http://www.biomedcentral.com/1471-2407/8/132


Table I: Demographic, Clinical, and Pathologic Characteristics of 20 men with biochemical recurrent prostate cancer after previous
therapy


Patient No. Age (yr.)


Serum PSA at
Initial Diagnosis
(ng/ml)


1 62 White
2 67 White

3 67 White
4 78 White


African American
White
White
White
White
White
White


12 68 White

13 76 African American
14 79 White

15 73 White
16 60 White


17
18
19*
20
Median


White
White
African American
White


UNK
4.8

UNK
4.7

14.08
8.28
10.36
UNK
UNK
8.1
UNK

UNK

11.13
4.0

10
9.2

17.3
9.8
4.1
8.7
9.2


Gleason Score at
Initial Diagnosis


3+4 = 7
3+3 = 6

3+3 = 6
3+4 = 7

UNK
4+5 = 9
3+3 = 6
UNK
4+4 = 8
4+5 = 9
2+3 = 5

4+4 = 8

3+3 = 6
3+4 = 7

3+2 = 5
3+4 = 7

4+3 = 7
3+3 = 6
3+4 = 7
UNK
7


Clinical Stage at
Initial Diagnosis


T3aNxMx
T2xNxMx

T2xNxMx
T2aNxMx

T3aNxMx
T2cNxMx
TIcNxMx
UNK
T2bNxMx
T3bNxMx
TIcNxMx

T2aNxMx

T2aNxMx
TIcNxMx

TIcNxMx
TIcNxMx

TIcNxMx
T2xNxMx
T2bNxMx
T2aNxMx


Initial Treatment



RP
Brachy/EBRT/
ADT
RP
Brachy/EBRT/
ADT
RP/EBRT
ADT/RP
EBRT
RP/Salvage EBRT
EBRT/ADT
RP
EBRT

RP/Adjuvant
EBRT
EBRT
RP/Adjuvant
EBRT
EBRT
RP/Adjuvant
EBRT
EBRT
RP/salvage EBRT
RP
EBRT


Length of F/U
months


12
12

12
6, started ADT

12
12
12
12
12
3, started ADT
9, withdrew due
to side effects
6, started ADT

12
12

12
12

12
12
6, lost to F/U
6, lost to F/U
12


*, noncompliant
Brachy, brachytherapy
EBRT, external beam radiation therapy
RP, radical prostatectomy
ADT, androgen deprivation therapy
F/U, follow-up


planned. Compliance was assessed by counting empty soy
milk containers and verified by presence of soy compo-
nents in serum samples.

Pretreatment evaluation included a complete medical his-
tory, physical examination (including digital rectal exam-
ination), serum PSA, free/total testosterone, lipids, serum
isoflavone levels (genistein, daidzein, and equol), and
assessment of quality of life (Functional Assessment of
Cancer Treatment-Prostate, FACT-P questionnaire). Fur-
thermore, whole blood was obtained prior to initiation of
the study to assess for DNA polymorphism. Follow-up
serum PSA levels to assess efficacy were obtained at 3, 6,
9, and 12 months after initiation of treatment. Medical
history, physical examination, serum testosterone, lipids,
isoflavone, and quality of life were assessed at 6 and 12
months after initiation of treatment.


Quality of life prior to and during therapy was measured
by the FACT-P questionnaire, which consists of 38 items
calculated to evaluate functional impairment and the per-
ceived effect of that impairment on quality of life. The
instrument assesses overall and specific aspects of quality
of life in five domains (i.e. physical, functional, social,
emotional, and prostate related). These include self-
reported ability to perform normal physical and social
activities, attitude towards self and future, level of physical
well-being, and quality of support from friends, family
and health care providers. The measure was used to assess
the impact of the dietary intervention on quality of life.

At least three pretherapy serum PSAs were available for
each subject to compute pretherapy PSA slope. Next, PSA
had been measured within days of the time the patient
was enrolled in the study. This level was used as study



Page 3 of 10
(page number not for citation purposes)


BMC Cancer 2008, 8:132







http://www.biomedcentral.com/1471-2407/8/132


entry level. PSA serum level was measured again 3 months
after enrollment in the study, after 6 months, and after 12
months. PSA outcome was evaluated in two ways: a) how
response changed over time [i.e. slope of line log (PSA)
study entry versus 3 months, versus 6 months, versus 12
months of treatment], and b) as a change on the calcu-
lated PSA doubling time before treatment versus PSA dou-
bling time after 3 months, 6 months, and 12 months of
treatment. PSA doubling time was calculated using the
following formula: PSA doubling time = log 2 x t/
[log(final PSA) log (initial PSA)]. Because pretherapy
PSA levels were compared to posttherapy PSA levels, each
subject could serve as his own control.

Patients were allowed to continue on therapy until a) they
exhibited evidence of PSA progression (as defined by two
successive increases in their PSA with an absolute increase
of at least 30% above baseline), b) they had clinical evi-
dence or radiographic evidence of distant metastases, or c)
patient/physician opted to stop therapy.

Isoflavone Analysis
A complete LC-MS analysis of patient serum samples for
genistein, daidzein and equol (total and aglycone) was
performed. The serum samples were both sulfate and glu-
curonide conjugates which requires hydrolysis with a
mixed 3-glucuronidase and sulfatase enzyme preparation
(Helix pomatia; Sigma Chemicals, St Louis, MO) to deter-
mine total versus free isoflavone. For each sample, an aliq-
uot was analyzed directly for free isoflavones, while
another was enzymatically hydrolysed to determine total
isoflavones. Quantitative isoflavone analysis was accom-
plished using reversed-phase HPLC with UV and mass
spectral detection in series [12]. Briefly, samples were pre-
pared for analysis by mixing with ammonium acetate
buffer and formic acid prior to multiple extractions in
ethyl acetate. Pooled extracts were dried under nitrogen
and resuspended in 0.5 ml of 50:50 acetonitrile: 0.2%
aqueous formic acid. Samples were then injected onto an
Apollo C18 column (Alltech, Deerfield, IL) with Alltech
Adsorbosphere HS C18 guard column in a Hewlett-Pack-
ard 1100 series liquid chromatograph system (Wilming-
ton, DE). Separation occurred under a linear gradient with
mobile phase B (0.1% formic acid in acetonitrile) increas-
ing from 40% to 60% over 60 min. Mobile phase A was
0.1% formic acid in water. The flow rate was 0.40 ml/min
with column temperature of 40 o C. Free isoflavones and
deconjugated glucosides were detected for quantitation
using an HP variable wavelength UV detector at 260 nm
and quantified against an external standard series. Identi-
fication was confirmed using the Finnigan LCQ Ion Trap
Mass Spectrometer (Finnigan MAT, San Jose, CA) in posi-
tive ion mode with electrospray ionization. Extraction
efficiency was determined using the isoflavone biochanin


A, with phenolphthalein-glucuronide as deconjugation
surrogate.

Genomic DNA Isolation & Evaluation of AR CAG
Polymorphisms
Whole blood was obtained from patients and DNA
extracted utilizing QiAamp DNA blood kit (Qiagen,
Valencia, CA). Nested PCR reaction was utilized to
amplify the CAG polymorphism on the AR gene. Primers
for AR gene were designed using Primer Express software
(PE-Applied Biosystems). Outside primers for CAG was
constructed: 5'-GTGCGCGAAGTGATCCAGAA-3' and 5'-
TCTGGGACGCAACCTCTCTC-3' and inside primers 5'-
AGAGGCCGCGAGCGCAGCACCTC-3'-fam and 5'-GCT-
GTGAAGGTTGCTGTTCCTCAT-3'. The inside forward
primer was labeled with fluorescent 6-carboxy-fluorescein
(FAM). The first PCR reaction consisted of 17 cycles (94 o C
for 1 min, 55C for 1 min, and 72C for 30 sec). The
nested PCR used 1 gil of the first PCR product and ampli-
fied further for 28 cycles (94 o C for 1 min, 66 o C for 1 min,
and 720C for 1.5 min). Screening for AR mutations was
performed using single-strand conformation polymor-
phism (SSCP) of PCR amplified AR genomic DNA. The
radio-labeled PCR fragment identified above was ana-
lyzed by nondenaturing polyacrylamide gel electrophore-
sis [13]. Fragments showing an aberrant PCR-SSCP
pattern on gel were subjected to direct DNA sequencing to
document the exact nucleotide base deletion, addition, or
change.

Statistical analysis
A mixed regression model was used to compare the slope
of PSA after study entry to that before study entry. The PSA
levels for a given patient were treated as repeated measure-
ments and the uniform correlation between PSA measure-
ments was assumed. The slopes before and after study
entry were globally compared using all data points in the
mixed model. In addition, the slope of PSA after study
entry was compared to that before study entry for each
individual patient by regular regression analysis. The PSA
doubling time before study entry was computed by tlog2/
[log(PSA at entry) log(initial PSA)] and the PSA dou-
bling time after study entry was computed by tlog2/
[log(last PSA) log(PSA at entry)]. Note that the PSA dou-
bling time can be negative if the denominator is negative.
The PSA doubling times before and after study entry were
compared by the sign test based on paired data. The Wil-
coxon signed rank test was performed for other chemis-
tries (Table 2) and QOL. The correlation between AR gene
CAG polymorphisms and PSA levels at 12 months after
study entry was evaluated by the Spearman's correlation
coefficient. All reported p-values were 2-sided. All data
were analyzed using SAS version 9.1.3 software.




Page 4 of 10
(page number not for citation purposes)


BMC Cancer 2008, 8:132








http://www.biomedcentral.com/1471-2407/8/132


Table 2: Serum PSA, Testosterone, and Cholesterol Before and During Study


Pt No. PSA at
entry
(ng/ml)


1
2
3
4
5
6
7
8
9
10
II
12
13
14
15
16
17
18
19
20
Median


0.57
0.50
1.76
1.17
6.87
5.94
1.18
0.67
0.15
0.4
18.86
11.2 1
0.74
3.49
1.36
0.50
2.67
0.59
1.04
0.79
1.11I


PSA at
12
months
(ng/ml)


1.93
0.35
1.62
N/A
I 1.23
13.41
1.73
N/A
0.12
N/A
N/A
N/A
0.67
3.90
1.59
0.50
5.15
1.10
N/A
1.05
1.6 1


P value 0.081


Free PSA
at entry
(ng/ml)



0.10
0.08
N/A
0.11
N/A
0.83
N/A
0.08
0.01
N/A
N/A
1.49
0.07
0.22
N/A
N/A
0.15
N/A
N/A
N/A
0.11


Free PSA
at 12
months
(ng/ml)


0.381
0.007
N/A
N/A
1.690
2.250
0.280
N/A
N/A
N/A
N/A
N/A
N/A
3.902
N/A
0.020
0.290
0.100
N/A
N/A
0.19


0.285


Testoster
one at
entry (ng/
ml)


493
331
259
508
324
N/A
264
264
68
360
137
204
565
280
521
N/A
287
333
N/A
299
299


Testoster
one at 12
months
(ng/ml)


468
382
263
N/A
299
418
118
N/A
68
N/A
N/A
N/A
N/A
265
487
281
382
317
N/A
N/A
308


Free
Testoster
one at
entry (ng/
ml)

N/A
46.7
11.9
9.3
N/A
N/A
N/A
10.2
3.4
N/A
4.6
N/A
15.6
10.4
19.3
N/A
9.8
4.0
N/A
10.5
10.3


Free Choleste
Testostero rol at


ne at 12
months
(ng/ml)


0.064


Results
Subjects' characteristics
Twenty men with biochemical recurrent prostate cancer
were enrolled. Table 1 shows the demographic, clinical,
and pathologic characteristics of the cohort. Compliance
was estimated to be high with only two subjects noted not
to ingest the required amount of soy milk. Six men did not
complete the study: 1 due to side effects, 2 were lost to fol-
low-up, and 3 began ADT. One of the men who had initi-
ation of ADT did so despite no significant increase in
serum PSA. The other two patients had a significant
increase in serum PSA (PSA doubling time < 3 months).
The two non-compliant patients were among the six men
who dropped out of the study.

PSA level Reduction
Six patients achieved 8%, 9%, 20%, 23%, 30% and 70%0
reduction in PSA after the treatment, respectively. In addi-
tion, one patient had no change in PSA. The remaining 13
patients had increased PSA.

PSA level Comparison of Slopes
The slopes of PSA after study entry were compared to that
before study entry in a mixed regression model as
described in the statistical analysis section. The PSA levels


Cholester
ol at 12


entry months
(mg/dl) (mg/dl)


0.806


for a given patient were treated as repeated measurements
and the uniform correlation between PSA measurements
were assumed. Within the mixed regression model, it was
estimated that PSA had increased 56% per year before
study entry and PSA increased 20% per year for the 12-
month study period (p = 0.05). There were 6 drop-outs:
four had PSA measurements up to 9 months and two had
PSA measurements up to 6 months. Since the mixed
regression model has the ability to incorporate missing
data, the six drop-outs were included in the analysis.

The slope of PSA after study entry was also compared to
that before study entry for each individual patient. It was
found that the slope of PSA after study entry was signifi-
cantly lower than that before study entry in 6 patients (p
< 0.05) and the slope of PSA after study entry was signifi-
cantly higher than that before study entry in 2 patients (p
< 0.05). For the remaining 12 patients, the change in slope
was insignificant (Figure 1). In two drop-outs, the PSA
slopes after study entry were significantly higher than that
before study entry; in other two drop-outs, the PSA slopes
after study entry were significantly lower than that before
study entry; in the remaining two drop-outs, the differ-
ences in PSA slope were not statistically significant. Drop-
outs are noted by "D" on Figure 1.



Page 5 of 10
(page number not for citation purposes)


BMC Cancer 2008, 8:132







http://www.biomedcentral.com/1471-2407/8/132


5


0




E
0


DI)


0
"~0

UI)
0-


0 12 -60


0 12 -60


0 12 -60


0 12 -60


Time (months)



Figure I
PSA levels in logarithm scale for each patient. Blue line (N = 6) represents the slope of PSA during isoflavone therapy
was significantly lower than that before the therapy, red line (N = 2) represents the slope of PSA during isoflavone therapy was
significantly higher than that before the therapy and black line (N = 12) represents the slope of PSA during isoflavone therapy
was unchanged than that before the therapy. CAG, CAG androgen receptor polymorphisms. D, subject who dropped out
before completion of the therapy.


PSA doubling time
Changes in PSA after study entry were compared to that
before study entry in terms of doubling time. The PSA
doubling times before and after study entry were com-
puted for each patient. PSA doubling times were positive
in all patients (median = 15 months) before study entry,
i.e. PSA levels at study entry were higher than the initial
PSA levels. In contrast, the PSA doubling time was nega-
tive during the therapy in 6 patients. Note that the dou-
bling time was negative if the last PSA level was lower than
the PSA level at study entry. In addition, one patient had
no change in PSA and the doubling time was undefined.
The PSA doubling time was positive during the therapy for
the remaining 13 patients (median = 15 months). Among
the 13 patients, 7 had PSA doubling times longer than


that before study entry. Improvements have been seen in
14 patients in terms of PSA doubling time (p = 0.044, sign
test).

Other chemistries
Free testosterone decreased while on therapy (median
10.3 vs. 9.7 ng/ml, p = 0.031). However, neither total tes-
tosterone nor cholesterol level was significantly manipu-
lated during isoflavone therapy (Table 2).

Isoflavone Level and AR Molecular Weight
Median total genistein, daidzein, and equol prior to the
start of therapy were <0.002, 0.012, and 0.023 lig/ml,
respectively. Serum isoflavone levels measured at 6
months are depicted in Table 3. Among 16 men with


Page 6 of 10
(page number not for citation purposes)


OAG 13 OAG 16 OAG =16 D OAG =16D OAG =17






OAG 19 OAG 19 OAG =20 OAG 21 OAG =21






OAG 21 OAG =21 D OAG =21 OAG 21 OAG =24 D






OAG 24 OAG =26 OAG =30 D OAG= D OAG =


BMC Cancer 2008, 8:132








http://www.biomedcentral.com/1471-2407/8/132


Table 3: Total and Free Serum Isoflavones and Androgen Receptor CAG repeat


Patient No. Total Genistein ((tg/ml)


I
2
3
4
5
6
7
8
9
10
II
12
13
14
15
16
17
18
19
20
Median


0.709
1.284
0.189
0.555
0.102
0.739
2.071
N/A
0.022
N/A
0.023
0.186
<0.002
0.058
1.049
0.492
1.989
<0.002
N/A
N/A
0.524


Total Daidzein ((tg/ml)


0.735
0.660
0.468
0.409
0.572
0.468
0.602
N/A
0.014
N/A
0.568
0.063
0.005
0.192
0.580
0.170
0.817
0.02 I
N/A
N/A
0.468


Total Equol (tg/ml)


Androgen Receptor CAG repeat


0.115
0.063
0.331
0.306
0.523
0.039
0.329
N/A
0.020
N/A
<0.001
0.061
0.048
0.050
0.188
0.062
0.492
0.119
N/A
N/A
0.115


N/A, not assessed (adequate sample unable to be obtained)
tg, microgram
mL, milliliter
MW, molecular weight

available data, 12 men (75%) demonstrated significant (>
2 x CTL, CTL = 0.023 pig/ml) levels of total equol and 10
of 16 (63%) had significant (> 2 x CTL, CTL = 0.009 |ig/
ml) levels of free equol present in serum. In the study
cohort, median genistein level was 0.524 lig/ml (range <
0.002 to 2.071), median daidzein levels was 0.468 lig/ml
(range 0.005 to 0.817), and median equol level was 0.115
lig/ml (range <0.001 to 0.523). Median free genistein
level was 0.012 lig/ml (range <0.002 to 0.019), median
free daidzein level 0.016 pig/ml (range 0.003 to0.434),
and median free equol level was 0.040 lig/ml (range
<0.001 to 1.328). The correlations of free equol and total
equol with PSA level at 12 months were 0.11 and 0.39,
respectively (p-values = 0.71 and 0.19). Correlations of
genistein (free and total) and daidzen (free and total) with
PSA level at 12 months were not significant either.

There were 9 documented AR gene CAG polymorphisms
in our cohort (Table 3). Eight men had < 20 CAG repeat.
A longer CAG polymorphism was associated with a lower
PSA level at 12 months after study entry (Spearman's cor-
relation coefficient = -0.63, p = 0.05) and a slower rise in
PSA over time (Figure 2).

Quality of Life
There was no significant difference in the five domains of
the FACT-P between pretherapy and on therapy (Figure
3). Side effects were minimal. One patient of 20 (5%)


PSA
17.53






5.91



30


. T,,ne


23
CAG 16
9


Figure 2
Effect of PSA vs. CAG Polymorphism MW over
Time. MW, molecular weight.



reported diarrhea and withdrew from the study. No other
side effects were reported.

Discussion
Numerous phase I and II clinical trials have studied men
with biochemical recurrence (PSA only) prostate cancer
after previous therapy. To date no randomized clinical
trial has demonstrated a survival advantage of salvage



Page 7 of 10
(page number not for citation purposes)


BMC Cancer 2008, 8:132







http://www.biomedcentral.com/1471-2407/8/132


PWB SWB EWB FWB PCS


Figure 3
Baseline (white) and 12 month (black) FA(
ity of Life questionnaire in 20 men with bi<
recurrent prostate cancer after previous ti
(PWB, physical well being; SWB, social well bein
emotional well being; FWB, functional well being
tate cancer specific).


therapies in this cohort. Thus, these men pos
therapeutic dilemma. The majority of them a
matic. Therapeutic maneuvers with hormon
chemotherapy, and experimental agents may
cant side effects. It is a difficult to persuade a
matic man to begin a potential toxic reg
unknown benefit. Because of these reasons, m
only recurrence are excellent candidates for eff
tic regimens. The goal of such treatment is to d
vent disease progression and thereby proloi
until ADT. Furthermore, slowing the rate of P
have an effect on overall survival.

We treated 20 men with 141 mg of isoflavone
12 months. Our dose regimen was based on
omized studies performed to assess cardiov
ease[14] and 2) ease of administration (1-8
soy milk for breakfast, lunch, and dinner).
only sign of recurrent disease after failed their
ing serum PSA profile, PSA response was the
point measure of our study. In general, serun
at study entry were not significantly differ
months of therapy (mean 3.02 vs. mean 3
tively). Of the 20 patients, the slope of PSA
entry was significantly lower than that before
in 6 patients, significantly higher than that b
entry in 2 patients, and unchanged in the re
patients. Thus, we were able to demonstrate
the rise of serum PSA after the initiation of so'
demonstrating isoflavones biologic activity
cancer patients. Furthermore responders sta
lower serum PSA level.


Several novel phase II non-randomized clinical trials have
been reported in patients with biochemical recurrent
prostate cancer. 1,25-dihydroxyvitamin D3 can result in
differentiated cancer cells and induce apoptosis [15].
Gross and others demonstrated that 0.5 |ig-2.5 |ig of 1,25-
0 Baseline dihydroxyvitamin D3 (Calitriol) daily slowed the rate of
S12 month serum PSA rise in 6 of 7 patients. Unfortunately, hypercal-
ciuria developed in all patients, with one developing a
renal calculus and another developing renal insufficiency
[ 16]. It is possible that the precursor to 1,25-dihydroxyvi-
tamin D3, 25 hydroxyvitamin D3 may be administered
with significantly less side effects. Research suggests that
the prostate, similar to the kidney, possesses the enzyme
T-P Qual- to convert 25-hydroxyvitamin D3 to 1,25-dihydroxyvita-
ochemical min D3, thus enabling it to exert their effect [17]. Next,
herapy. Woo reported 9 out of 15 patients with PSA relapse pros-
g; EWB, tate cancer after definitive therapy had stable or decreas-
PCS, pros- ing serum PSA levels when treated with 2,000 IU (50 rig)
of 25-dihydroxyvitamin D3 cholecalciferoll) daily. No
adverse side effects were reported [18]. Pruthi and others
reported the effects of celecoxib (COX-2 inhibitor) 400
e a difficult mg given orally twice daily in 40 men with PSA recurrent
re asympto- prostate cancer after definitive treatment. Eleven men had
al ablation, a decline in serum PSA and 8 had a stabilization of PSA.
have signifi- One patient was removed from the study for a presumed
n asympto- transient ischemic attack [19]. Researchers from Wake
;imen with Forest reported that 15-120 mg/day of lycopene supple-
en with PSA mentation was safe and well tolerated in a cohort of 36
active holis- men with PSA recurrent prostate cancer. However, no
lelay or pre- serum PSA responses were observed and 37% of patients
ng the time had PSA progression [20].
SA rise may
Hussain and others reported on a heterogeneous group of
men with prostate cancer who were treated with 100 mg
per day for of soy isoflavone (Novasoy) twice daily for a maximum of
1) 22 rand- 6 months. There was a decrease in the rate of the rise of
ascular dis- serum PSA in men with PSA recurrent disease. The rates of
oz glass of rise decreased from 14% to 6% while on study [21]. These
Because the results are consistent with our study, where the rates of
apy was ris- rise decreased from 56% to 20% while on study. Our
primaryy end more favorable results may be due to longer treatment
i PSA levels duration (6 months vs. 12 months). Taken together, the
nt after 12 data from the two clinical trials in men with PSA recurrent
.17, respec- prostate cancer treated with isoflavone are encouraging
after study and should be further studied. Ideally these studies
study entry should be combined with genomics or proteomics assays
before study to 1) determine the profile of treatment responders, and
maining 12 2) attempt to elucidate mechanistic pathways that can be
a decline in exploited by combining these agents with other holistic
y milk, thus drugs or with a more conventional therapy.
in prostate
rted with a Isoflavones in the soy milk included genistein and daid-
zein. Numerous studies have demonstrated the cytostatic
and cytotoxic effect of genistein in various malignancies
including breast, lung, melanoma, prostate, head and


Page 8 of 10
(page number not for citation purposes)


BMC Cancer 2008, 8:132







http://www.biomedcentral.com/1471-2407/8/132


neck squamous cell carcinoma, leukemia, and lymphoma
[22]. Genistein has a heterocyclic diphenolic structure
similar to estrogen [22]. Because of the similar structure,
it is proposed that soy (genistein) exerts its effect on cells
by interacting with hormone receptors. The exact mecha-
nism of genistein's anti-tumoral effects is unknown. Pro-
posed mechanisms are perturbations in cell cycle,
decreased proliferation, decreased angiogenesis, and
increased apoptosis. In addition to genistein and daid-
zein, another metabolite of isoflavone is equol. Equol is
produced via the bacterial conversion of the soy isofla-
vone daidzein in the intestines [23]. It is estimated that
approximately 30% of Americans can convert diadzein to
equol, however the real percentage is unknown seeing
that no large study has assessed this in the general Ameri-
can population. The percentages are higher (> 80%) in
Chinese and Japanese. Similar to the other isoflavone
metabolite, equol has a chemical structure related to
estrogen and is known to interact with estrogen receptors
[23 ]. Over two-thirds of the men in our study were able to
convert daidzein to equol. Overall, median total equol
level was 0.115 gg/ml (range <0.001 to 0.523) and
median free equol level was 0.040 gg/ml (range <0.001 to
1.328). There could be genetic differences in isoflavone
metabolism in each racial group, however due to the
small numbers this could not be assessed.

The CAG repeat polymorphisms of the androgen receptor
gene have been associated with an increased prostate can-
cer risk and the repeat length has been correlated with
cancer stage and grade at presentation [24]. Investigators
have also correlated CAG repeat polymorphism of the AR
gene to response to ADT [25]. Similarly, in this small
study, we demonstrated that subjects with CAG polymor-
phisms molecular weight > 22 were more likely to
respond to the experimental therapy (Figure 2). In future
studies, CAG polymorphisms may be used to determine
eligibility into these trials (i.e., subjects with CAG poly-
morphisms > 22 will be enrolled). Due to the small num-
bers in the current study, we could not correlate CAG
polymorphisms with equol production which could be a
fascinating correlation.

There are several limitations to this study. First, this is a
small study that was terminated early due to poor accrual.
The planned power of 60% was not attained. It is possible
that with a larger number of subjects, a more significant
change in serum PSA while on therapy could have been
demonstrated. Furthermore there was a substantial drop
out rate in this study. Previous studies have commented
on the difficulty of performing clinical trials in an inner-
city population such as ours [26,27]. In addition, this
phase II study did not have a control group and the ther-
apy was neither randomized nor blinded. Though not
ideal, this study design which was employed by other


groups [16,18-20] would provide useful data on the feasi-
bility and utility of isoflavone in this cohort prior to
embarking on a larger, costly phase II or even phase III
study. Last, serum PSA was the primary endpoint of this
study. We acknowledge the limitations of serum PSA in
this study (i.e., fluctuations). But to date there are no
other prognostic markers for prostate cancer in this
cohort. To decrease the chance of detecting 'noise' (or
common fluctuations of serum PSA levels), we assessed
not absolute PSA but PSA trends. Nevertheless, the results
of this study suggest biologic activity with the isoflavone
diet in men with biochemical recurrent prostate cancer

Conclusion
Our findings show that isoflavone was a well-tolerated
alternative to expectant management or early ADT in men
with biochemical recurrent prostate cancer. Isoflavone
may slow the rate of increase of serum PSA in a subset of
patients with biochemical progression after radiation
therapy or radical prostatectomy. The findings reported
herein warrant further investigation in a larger cohort of
patients. In these larger studies, correlative biomarkers
should be explored to assist with elucidating a possible
mechanism of action with isoflavones in prostate cancer.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
JMP was study coordinator, WWT physician of 9 patients
who assisted with recruitment and follow-up, SA per-
formed CAG portion of the project, MC and WH were stat-
isticians, KTS assisted with interpretation of isoflavone
serum levels, CJR was study PI.

Acknowledgements
20 men who participated in this study & Hain-Celestial, Denver CO for
providing the soy milk.

References
I. Pienta KJ, Esper PS: Risk factors for prostate cancer. Ann Intern
Med 1993, I 18:793.
2. Dunn JE: Cancer epidemiology in population of the United
States. Cancer Res 1975, 35:240.
3. Haenszel W, Kurihoro M: Studies of Japanese migrants. J NatI
Cancer Inst 1968, 40:43.
4. Shemizer H, Ross RK, Berstein L, Yatane R, Henderson BE, Mack TM:
Cancer of prostate and breast among Japanese and white
immigrants in Los Angeles county. BrJ Cancer 1991, 63:963.
5. Messina M, Persky V, Setchell KDR, Barnes S: Soy intake and can-
cer risk: a review of in vitro and in vivo data. Nutr Cancer 1994,
2 1:113.
6. Akiyama T, Ogawara H: Use and specificity of genistein as an
inhibitor of protein-tyrosine kinases. Methods Enzymol 1991,
201:362.
7. Coward L, Barnes NC, Setchell KDR, Barnes S: Genistein and daid-
zein and their B-glycoside conjugates; anti-tumor isoflavones
in soybean foods of the American and Asian diets. J Agric Food
Sci 1993,41:1961.
8. Watanabe T, Shiraishi T, Saasaki H, Oishi M: Inhibitors for protein
tyrosine kinases, ST638 and genistein, induce differentiation


Page 9 of 10
(page number not for citation purposes)


BMC Cancer 2008, 8:132








http://www.biomedcentral.com/1471-2407/8/132


of mouse erythroleukemia cells in a synergistic manner. Exp
Cell Res 1989, 183:335.
9. Magee PJ, Raschke M, Steiner C, Duffin JG, Pool-Zobel BL, Jokela T,
et al.: Equol: a comparison of the effects of the racemic com-
pound with that of the purified S-enantiomer on the growth,
invasion, and DNA integrity of breast and prostate cells in
vitro. Nutr Cancer 2006, 54:232.
10. Andersson P, Varenhorst E, Soderkvist P: Androgen receptor and
vitamin D receptor gene polymorphisms and prostate can-
cer risk. Eurj Cancer 2006, 42(16):2833-7.
I I. American Society for Therapeutic Radiology and Oncology Consen-
sus Panel: Consensus statement: guidelines for PSA following
radiation therapy. IntJ Radiat Oncol Biol Phys 1997, 37:1035.
12. Coward L, Kirk M, Albin N, Barnes S: Analysis of plasma isofla-
vones by reversed-phase HPLC multiple reaction ion moni-
toring-mass spectrometry. Clin Chim Acta 1996, 247:121.
13. Ruizeveld de Winter JA, Janssen PLJ, Sleddens HM, Verleun-Mooijman
MC, Trapman J: Androgen receptor status in localized and
locally progressive hormone refractory human prostate can-
cer. AmJ Path 1994, 144:735.
14. Sacks FM, Lichtenstein A, Van Horn L, Harris W, Kris-Etherton P,
Winston M: American Heart Association Nutrition Commit-
tee. Soy protein, isoflavones, and cardiovascular health: an
American Heart Association Science Advisory for profes-
sionals from the Nutrition Committee. Circulation 2006,
113:1034.
15. Whitlatch LW, Young MV, Schwartz GG, Flanagan JN, Burnstein KL,
Lokeshwar BL, et al.: 5-Hydroxyvitamin D-lalpha-hydroxylase
activity is diminished in human prostate cancer cells and is
enhanced by gene transfer. J Steroid Biochem Mol Biol 2002,
81(12):135.
16. Gross C, Stamey T, Hancock S, Feldman D: Treatment of early
recurrent prostate cancer with 1,25-dihydroxyvitamin D3
(Calcitriol). J Urol 1998, 159:2035.
17. Schwartz GG, Whitlatch LW, Chen TC, Lokeshwar BL, Holick MF:
Human prostate cells synthesize 1,25-dihydroxyvitamin D3
from 25-hydroxyvitamin D3. Cancer Epidemiol Biomarkers Prev
1998, 7(5):391.
18. Woo TC, Choo R, Jamieson M, Chander S, Vieth R: Pilot study:
potential role of Vitamin D (Cholecalciferol) in patients with
PSA relapse after definitive therapy. Nutr Cancer 2005, 5 1:32.
19. Pruthi RS, DerksenJE, Moore D, Carson CC, Grigson G, Watkins C,
et al.: Phase II trial of celecoxib in prostate-specific antigen
recurrent prostate cancer after definitive radiation therapy
or radical prostatectomy. Clin Cancer Res 2006, 12:2172.
20. Clark PE, Hall MC, Borden LS Jr, Miller AA, Hu JJ, Lee WR, et al.:
Phase 1-11 prospective dose-escalating trial of lycopene in
patients with biochemical relapse of prostate cancer after
definitive local therapy. j Urol 2006, 67:1257.
21. Hussain M, Banerjee M, Sarkar FH, Djuric Z, Pollack MN, Doerge D,
et al.: Soy isoflavones in the treatment of prostate cancer.
Nutr Cancer 2003, 47:111 I I.
22. Adlercreutz CH, Goldin BR, Gorbach SL, Hockerstedt KA, Watanabe
S, Hamalainen EK, et al.: Soybean phytoestrogen intake and can-
cer risk. J Nutr 1995, 125:757S. Erratum in: J Nutr 1995; 125(7):
1960
23. Niculescu MD, Pop EA, Fisher LM, Zeisel SH: Dietary isoflavones
differentially induce gene expression changes in lymphocytes
from postmenopausal women who form equol as compared
with those who do not. J Nutr Biochem in press. 2006; Sept 7
24. Nam RK, Elhaji Y, Krahn MD, Hakimi J, Ho M, Chu W, et al.: Signif-
icance of the CAG repeat polymorphism of the androgen
receptor gene in prostate cancer progression. J Urol 2000,
164(2):567.
25. Klotz L, Correia A, Zhang W: The relationship between the
androgen receptor CAG repeat polymorphism length and
the response to intermittent androgen suppression therapy
for advanced prostate cancer. Prostate Cancer Prostatic Dis 2005,
8:179.
26. Baquet CR, Commiskey P, Daniel Mullins C, Mishra Sl: Recruitment
and participation in clinical trials: socio-demographic, rural/
urban, and health care access predictors. Cancer Detect Prev
2006, 30:24-33.
27. Blumenthal DS, Sung Coates R, Williams J, Liff J: Recruitment and
retention of subjects for a longitudinal cancer prevention


study in an inner-city black community. Health Serv Res 1995,
30:197-205.

Pre-publication history
The pre-publication history for this paper can be accessed
here:


http://www.biomedcentral.com/1471-2407/8/132/pre


Page 10 of 10
(page number not for citation purposes)


BMC Cancer 2008, 8:132


Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours you keep the copyright
Submit your manuscript here: BioMedcentral
http://www.biomedcentral.com/info/publishing adv.asp




University of Florida Home Page
© 2004 - 2010 University of Florida George A. Smathers Libraries.
All rights reserved.

Acceptable Use, Copyright, and Disclaimer Statement
Last updated October 10, 2010 - - mvs