![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
*
Nutrition/Metabolism Laboratory, Department of Surgery, Beth Israel-Deaconess Medical Center, Harvard Medical School, Boston, MA 02215;
Archer Daniels Midland Company, Decatur, IL 62521;
**
Department of Neurosurgery, Fuji City General Hospital, Fuji, Japan; and
The Arthur G. James Cancer Hospital and Research Institute, The Ohio State University, Comprehensive Cancer Center, Columbus, OH 43210
3To whom correspondence and reprint requests should be addressed.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
KEY WORDS: • soy • prostate cancer • apoptosis • angiogenesis • mice
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, Parkin and Muir 1992
). Among dietary factors, increased consumption of
soybean products has been hypothesized to contribute to reduced
prostate cancer risk (Hebert et al. 1998
, Messina et al. 1994
). Epidemiologic, in vitro and laboratory animal
studies provide evidence for the hypothesis that phytochemicals in soy
products have anticarcinogenic properties (Kennedy 1995
,
Messina et al. 1994
). Much of the attention has focused
upon genistein and daidzein, the predominant isoflavones found in soy
in amounts of ~1–3 mg/g (Wang and Murphy 1994b
). In addition, protease inhibitors, the
Bowman-Birk inhibitor, inositol hexaphosphate (phytic acid),
lignans, phytosterols and saponins found in soy products may also have
bioactivities relevant to the inhibition of carcinogenesis
(Kennedy 1995
, Messina et al. 1994
,
Rao and Sung 1995
, Shamsuddin 1995
).
Relatively few animal studies have been conducted to investigate the
role of soy components on prostate cancer tumorigenesis, and little is
known regarding possible in vivo mechanisms whereby bioactive
components in soy may influence the prostate. Earlier studies report
that soy-containing diets reduce the severity of prostatitis in
rats (Sharma et al. 1992
) and prevent the development of
dysplastic lesions of the prostate of neonatal
diethylstilbestrol-treated mice (Makela et al. 1995
). Rats consuming a soy flour–containing diet exhibit
reduced growth of well-differentiated transplantable Dunning R3327
prostatic adenocarcinoma compared with those fed a casein-based
control diet (Landstrom et al. 1998
, Zhang et al. 1997
). In contrast, a small study with genistein added to the
drinking water (intake not quantitated) or administered via
intraperitoneal injection of 0.143–0.428 mg genistein/kg body weight
had no effect on the growth of the subcutaneously implanted
MAT-LYLu prostate carcinoma in rats (Naik et al. 1994
). Overall, additional in vivo studies are required to
allow definitive conclusions regarding soy products or isoflavones on
prostate carcinogenesis, tumor progression and mechanism of action.
We report that concentrations of soy isoflavones exceeding those
typically observed in vivo are necessary to inhibit the growth of human
prostate cancer cell lines in vitro. In contrast, the proliferation of
vascular endothelial cells is significantly inhibited at concentrations
25 µmol/L. Feeding soy phytochemicals or soy protein
isolate inhibits the growth of the human LNCaP prostate cancer cell
line in vivo. The inhibition of tumor growth in vivo is correlated with
alterations in tumor biomarkers, including reduced proliferating cell
nuclear antigen
(PCNA)4
labeling as a marker of proliferation, increased apoptosis and reduced
microvessel density. Our observations suggest that dietary soy products
inhibit prostate cancer progression in vivo via multiple interacting
mechanisms.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Soy isoflavones (genistein, daidzein) were purchased from Sigma
Chemical (St. Louis, MO). The soy protein isolate (SPI, Supro 670HG,
Lot#C5C-XPC-001, Protein Technology International, St. Louis, MO)
contains 2.07 mg isoflavone aglycone equivalents (each isoflavone was
calculated to reflect only the weight of the aglycone because mixtures
of free isoflavones and glycosylated forms are found in the product),
including 1.22 mg genistein equivalents, 0.64 mg daidzein equivalents
and 0.21 mg glycitein equivalents per gram. A soy phytochemical
concentrate (SPC) was provided by Archer Daniels Midland Company
(Decatur, IL). Soy phytochemical concentrate was prepared as follows:
soybeans were cracked, dehulled and flaked by standard procedures
followed by a hexane extraction to remove the majority of lipid. The
resulting defatted soy flour was extracted with aqueous ethanol (60%,
v/v) to produce a mixture containing carbohydrates (0.6–0.7 g/g
material), isoflavones (0.02 g/g), fat (0.12 g/g), ash (0.04 g/g) and
protein (0.05 g/g). A proprietary extraction procedure was then
employed to remove the carbohydrates; the remaining material was
spray-dried to form a powder called SPC and analyzed for
isoflavones according to published methods (Wang and Murphy 1994a
). The final SPC employed in our studies contains
170 mg isoflavone aglycone equivalents per gram of material, which
includes 79.2 mg of genistein equivalents, 70.4 mg of daidzein
equivalents and 20.4 mg of glycitein equivalents. One gram of soy
phytochemical concentrate also contains 0.14 g of protein, 0.055 g
of fat, 0.027 g of ash and 0.065 g of moisture, with the remaining
matter undefined but apparently rich in saponins.
Prostate cancer cell culture studies.
Three human prostate cancer cell lines, LNCaP, PC-3 and DU 145
(American Type Culture Collection, Rockville, MD), were used for the
studies. Human prostate cancer cell lines were maintained as monolayer
cultures in Dulbecco's modified Eagle's medium (DMEM) supplemented
with 10% fetal bovine serum, 2 mmol L-glutamine/L, 1
x 105 U penicillin/L and 100 g
streptomycin/L in a 95% air, 5% CO2, and
water-saturated atmosphere. The in vitro growth studies were
completed with 5 x 103 cells/well, plated into 96-well
microplates, treated with soy isoflavones or soy phytochemical
concentrate dissolved in dimethyl sulfoxide (final dimethyl sulfoxide
concentration 0.1% by volume) and incubated for 72 h.
Dimethyl sulfoxide vehicle controls were used in all studies. Cell
numbers were quantitated by the XTT (sodium
3'-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis (4-methoxy-6-nitro)
benzene-sulfonic acid hydrate) assay (Roehm et al. 1991
). All assays were completed in triplicate, experiments
were replicated at least once and results were confirmed by direct cell
counting using a hemocytometer.
Analysis of prostate cancer cell cycle progression and DNA fragmentation.
Cells were grown under conditions as described above, harvested by trypsinization and centrifugation at 1500 x g for 5 min, washed with PBS and fixed with 80% ethanol. Cells were then washed with PBS, resuspended, stained by adding propidium iodide (at a final concentration of 50 mg/L) and RNase (at a final concentration of 50 mg/L), and incubated at 37°C for 30 min. Stained cells were analyzed by FACScans (Becton Dicknson, San Jose, CA) for DNA fragmentation and cell cycle using programs provided by Becton-Dickinson.
Endothelial cell proliferation studies.
Endothelial cell proliferation studies employed methods previously
described (Tanaka et al. 1997
). In brief, human
umbilical vein endothelial cells (American Type Culture Collection)
were maintained in Medium 199 containing 10% fetal bovine serum, 100
mg/L heparin, and 30 mg/L endothelial cell growth supplement
(Collaborative Biomedical Products, Bedford, MA). For assays, human
umbilical vein endothelial cells (5 x 104) were
plated in 12-well plates; genistein or SPC was added at defined
concentrations (0, 25 and 50 µmol/L). After 72 h, cells were
labeled with 3.7 x 104 Bq of
3H-thymidine, and incorporation into DNA was quantitated in
a scintillation counter 24 h later. Studies were completed in
quiescent conditions (no heparin) and with heparin stimulation.
Determination of serum insulin-like growth factor-1 (IGF-I).
Serum IGF-I was extracted and quantitated by RIA following the procedures provided by Nichols Institute Diagnostics (San Juan Capistrano, CA).
Diet formulations and treatment groups.
SPC and SPI were used to prepare the following six semipurified
experimental diets according to our formulation (Table 1
) by Research Diets (New Brunswick, NJ): 1) AIN-76 diet
as the control; 2) AIN-76 with casein replaced by SPI,
20% by weight, providing 415 mg isoflavone equivalents/kg diet;
3) AIN-76 with SPC at 0.2% of the diet providing 341 mg
isoflavone equivalents/kg; 4) AIN-76 with casein
replaced by SPI (20% of the diet) with addition of SPC (0.2% of the
diet), providing 756 mg isoflavone equivalents/kg; 5)
AIN-76 with SPC at 1.0% of the diet, providing 1705 mg isoflavone
equivalents/kg; and 6) AIN-76 with casein replaced by
SPI (20%) with addition of SPC (1.0% of the diet), providing 2120 mg
isoflavone equivalents/kg. Dietary isoflavone levels were confirmed by
HPLC analysis.
|
Forty-eight male SCID mice (8 wk old) were purchased from Harlan
Sprague Dawley (Indianapolis, IN). After 1 wk of adaptation to the
AIN-76 diet, mice were inoculated subcutaneously on the right flank
with a suspension of 2 x 106 LNCaP cells isolated
from subcutaneously grown LNCaP tumors from donor SCID mice. Recipient
mice were then randomly assigned into six groups (n
= 8) and fed one of the experimental diets. Food intake, body
weight and tumor diameters were measured three times weekly. Tumor
volumes were calculated by the following formula: tumor volume
(cm3) = 0.523 x [length (cm) x width2 (cm2)]. The experiment was terminated
at d 21 when mean tumor volumes in the control mice exceeded 2
cm3. An aliquot of tumor tissue was fixed in 10% buffer
neutralized formalin, embedded in paraffin, and cut into
4-µm sections for in situ histochemical detection of
apoptosis and immunohistochemical analyses of angiogenesis and
proliferation. All procedures with animals were reviewed and approved
by the Institutional Animal Care and Use Committee at Beth Israel
Deaconess Medical Center according to NIH guidelines (NRC
1985
).
Immunohistochemical determination of angiogenesis (microvessel density).
Immunohistochemical quantitation of microvessel density was used as a
marker for tumor angiogenesis following a previously described method
(Zhou et al. 1998
). In brief, after deparaffinization,
rehydration and washing in PBS, tissue sections were incubated with
trypsin at 37°C for 30 min, quenched with 88 mmol
H2O2/L of methanol for 30 min and blocked with
normal goat serum at 100 mL/L buffer (1.0 g bovine serum albumin and
0.1 mL Tween 20 in 100 mL PBS). The sections were then immunoreacted
with a rabbit polyclonal antibody directed against human Factor VIII
related antigen (DAKO, Carpinteria, CA, 1:100 dilution), and a
biotinylated "universal" horse anti-mouse/rabbit immunoglobulin
(Ig)G (Vector Laboratories, Burlingame, CA), followed by treatment with
avidin-biotin complex (Vector Laboratories) and 3–3'
diaminobenzidine. Sections were counterstained with methyl green and
mounted. Microvessel density was calculated by counting microvessels
under 200-fold magnification at three representative sites that did not
contain tumor necrosis.
Immunohistochemical determination of proliferation.
The proliferation index was evaluated by calculating the proportion of
cells with PCNA staining (Zhou et al. 1998
). In brief,
after deparaffinization, rehydration and washing in PBS, tumor sections
were soaked in 10 mmol citrate buffer/L and heat-treated for 5 min
in a microwave oven. Sections were then stained following the
procedures as described for factor VIII staining, using horse serum at
100 mL/L of the buffer (1.0 g bovine serum albumin and 0.1 mL Tween 20
in 100 mL PBS) for blocking and a PCNA mouse monoclonal antibody (DAKO)
as a primary antibody. Both PCNA-positive proliferating cells and
total tumor cells were counted in three nonnecrotic areas of each
section using light microscopy at 400-fold magnification.
In situ apoptotic cell detection.
Apoptotic cells were determined by a terminal deoxynucleotidyl
transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay
using the ApopTag in situ detection kit (Oncor, Gaithersburg, MD),
following the manufacture's procedures with modification (Zhou et al. 1998
). In brief, after deparaffinization, rehydration
and washing in PBS, sections were treated with 20 mg/L proteinase K for
20 min at room temperature and washed. Endogenous peroxidase activities
in sections were quenched with 0.88 mol H2O2/L
of PBS for 5 min. Sections were applied with terminal deoxynucleotidyl
transferase labeled with digoxygenin peroxidase and incubated for
1 h at 37°C; the reaction was stopped by stop and wash buffer.
Sections were then incubated with antidigoxygenin peroxidase for 30 min
at room temperature, washed, stained with 3–3' deaminobenzidine
substrate, counterstained with methyl green and mounted. Known positive
and negative control slides were used for comparison. Three
representative areas of each section without necrosis were selected,
and both apoptotic cells and total nuclei cells were counted under
light microscopy at 400-fold magnification. The apoptotic index was
expressed as the percentage of apoptotic nuclei to total nuclei.
Statistical analysis.
Results from cell culture studies, tumor volume, apoptotic index,
proliferation index and microvessel density were initially evaluated by
ANOVA followed by Fisher's protected least significant difference test
(Steel and Torrie 1980
) to evaluate pairwise comparisons
among treatment groups using the Statview 4.5 (Abacus Concepts,
Berkeley, CA) program. A probability level of P < 0.05 was considered significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Genistein at 25 and 50 µmol/L significantly inhibited
LNCaP cell growth by 33% (P < 0.001) and 50%
(P < 0.0001), respectively (Fig. 1
A). Genistein at 50 µmol/L also significantly inhibited the
growth of DU 145 cells by 23% (P < 0.01, Fig. 1
B) and PC-3 cells by 34% (P < 0.005, Fig. 1
C). In comparison to genistein, daidzein had weaker effects
on human prostate cancer cell lines in vitro. Daidzein at 50
µmol/L significantly inhibited the growth of LNCaP cells
by 40% (P < 0.05, Fig. 1
A). At lower
concentrations (5 or 10 µmol/L), daidzein tended (P = 0.15) to increase human prostate cancer cell numbers.
|
D) at concentrations between 5
µmol/L (18 µg/mL) and 100 µmol/L (360 µg/mL) of total aglycone
isoflavone equivalents (2.3–46 µmol/L of genistein
equivalents and 2.0–40 µmol/L of daidzein equivalents).
At 100 µmol/L (46 µmol/L genistein equivalents and 40
µmol/L daidzein equivalents), SPC inhibited LNCaP cells by
80% (P < 0.0001), DU 145 cells by 50% (P
< 0.001) and PC-3 cells by 25% (P < 0.05).
These data suggest that soy isoflavones probably account for much of
the inhibitory effects of SPC on prostate cancer cell growth in vitro. Cell cycle progression and DNA fragmentation of LNCaP cells treated with genistein and SPC in vitro.
Genistein at 50 µmol/L, but not at 10 µmol/L,
significantly affected cell cycle progression by arresting LNCaP cells
at G2-M phases (Table 2
, P < 0.005). Genistein at 50 µmol/L
induced DNA fragmentation, a marker for apoptosis, of LNCaP cells by
twofold (P = 0.45). Parallel studies (data not
shown) with SPC (0, 10, or 50 µmol/L) also showed DNA fragmentation
and cell cycle arrest in G2-M phases for LNCaP cells although the
magnitude of the response was attenuated compared with that of pure
genistein. Additional studies with PC-3 and DU 145 cells showed
statistically significant (P < 0.05) dose-dependent G2-M
arrest and enhanced DNA fragmentation at >50 µmol/L concentrations
(data not shown).
|
The effects of genistein or SPC on cell proliferation were examined
under quiescent (growth suppressed) conditions and after stimulation by
heparin (Fig. 2
). Quiescent cells showed a significant inhibition of incorporation of
3H-thymidine at 25 µmol/L genistein
(P < 0.001) or SPC (P < 0.05).
Heparin stimulation increased 3H-thymidine
incorporation ~10-fold. Incorporation of label was reduced by >50%
by 25 µmol/L genistein (P < 0.001) or SPC
(P < 0.001) and by >80% (P < 0.001)
by either of these soy products at a concentration of 50
µmol/L.
|
Subcutaneous growth of the human prostate cancer cell line LNCaP in
male SCID mice was used as an in vivo model to evaluate the effects of
dietary soy products SPC and SPI on prostate tumor growth. The
experiment was terminated when mean tumor volume of the control group
reached 2.3 ± 0.3 cm3. Dietary soy products
did not significantly alter food intake or body weight (Table 3
). Tumor volumes at d 21 (Fig. 3
and Table 4
) from mice treated with diets containing SPI (20% of the diet) alone,
SPC (0.2%) alone, SPI (20%) with SPC (0.2%), SPC (1.0%) alone and
SPI (20%) with SPC (1.0%) were reduced by 11% (P = 0.45), 19% (P = 0.17), 28% (P < 0.05), 30% (P < 0.05) or 40% (P < 0.005), respectively, compared with those of casein-fed control
mice. Factorial analysis indicated that there was a significant main
effect of SPC on prostate tumor growth (P < 0.05),
whereas there was no significant main effect of soy protein as protein
source (P = 0.09).
|
|
|
Formalin-fixed tumor tissues were processed to prepare tissue slides
and used for in situ histochemical detection of apoptosis by TUNEL
assay, proliferation by PCNA staining, and angiogenesis by microvessel
density quantitation (factor VIII staining). The results are presented
in Table 4
. Compared with controls, tumors from mice treated with the
diets containing SPI (20%) alone, SPC (0.2%) alone, SPI with SPC
(0.2%), SPC (1.0%) alone and SPI with SPC (1.0%) showed a lower
proliferation index by 6% (P = 0.24), 14%
(P < 0.01), 15% (P < 0.005), 18%
(P < 0.001) and 21% (P < 0.0001)
respectively (Table 4)
. In contrast, tumor cell apoptosis rates were
greater in mice fed the above diets by 33% (P = 0.28),
84% (P < 0.05), 80% (P < 0.01),
60% (P < 0.05) and 136% (P < 0.0001) respectively, compared with controls. The microvessel densities
of tumors derived from mice fed the above diets were reduced by 43%
(P < 0.01), 29% (P = 0.07), 49%
(P < 0.005), 51% (P < 0.005), and
61% (P < 0.0001) respectively, compared with that of
control group.
Multiple linear regression analysis was applied to determine the correlations between tumor biomarkers and tumor volumes. The analysis resulted in the following correlation equation: tumor volume (cm3) = 2.52 - 0.09(apoptotic index) + 0.138(microvessel density) - 0.008(proliferation index) (R = 0.673, P < 0.001). Using this model, the lower tumor volume in soy-fed mice was associated with increased tumor cell apoptosis (P < 0.05) and reduced tumor microvessel density (P < 0.0001), but not significantly associated with tumor proliferation (P = 0.64). These results suggest that both apoptotic index and microvessel density may serve as biomarkers in evaluating the effects of soy products on prostate tumor growth, whereas proliferation index may be a dependent biomarker.
Effects of soy products on serum IGF-I levels.
To explore the possible effects of dietary soy products on IGF-I, we quantitated IGF-I concentrations in the serums derived from mice fed control diet or the diet containing 20% SPI with 1.0% SPC. Serum IGF-I levels in mice fed the 20% SPI diet with 1.0% SPC (223 ± 61 ng/mL, means ± SEM, n = 5) were significantly lower (P < 0.05) than those in control group (349 ± 40 ng/mL, n = 4). IGF-I levels in other groups were not measured.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The
angiogenic process depends upon the growth of endothelial cells.
Inhibition of endothelial cell proliferation is one of the
antiangiogenic strategies to inhibit tumor growth and metastasis. Our
in vitro studies with endothelial cells indicate that they are more
sensitive to the growth inhibitory effects of soy phytochemicals than
the relatively resistant human prostate tumor cells, supporting the
hypothesis that the antiangiogenic effects of soy isoflavones may be
one of the key mechanisms altering tumor growth.
Soy isoflavones inhibit the proliferation of several malignant cell
lines, including prostate cancer cell lines in vitro (Geller et al. 1998
, Messina et al. 1994
, Naik et al. 1994
, Onozawa et al. 1998
, Peterson and Barnes 1993
, Santibanez et al. 1997
). Under
ideal in vitro growth conditions, we observed that prostate cancer
cells are relatively resistant to growth inhibition by soy-derived
products. The 50% inhibitory concentrations
(IC50) exceeded 50 µmol/L, a
concentration not likely to occur in vivo due to consumption of soy
products. We found that the effect of genistein on proliferation of
LNCaP cells in vitro was associated with cell cycle arrest at the G2-M
phases, a phenomenon observed in other cell lines (Choi et al. 1998
, Pagliacci et al. 1994
, Shao et al. 1998
, Yanagihara et al. 1996
, Zhou et al. 1998
). The underlying molecular mechanisms by which prostate
cancer cell lines have different responses to soybean isoflavones have
not been determined. It is possible that the expression of
oncogenes/tumor suppressor genes may contribute in part to this cell
type–specific response to soy isoflavone treatment. For example, the
LNCaP cell line has wild-type p53, DU 145 has mutant p53, whereas
p53 in the PC-3 cell line is deleted. This cell type–specific response
may be useful for studying the molecular mechanisms of action.
Soy isoflavones exhibit different in vitro potencies. Daidzein is a
major soy isoflavone, contributing ~40% of total soy isoflavones. We
observed that daidzein was less potent than genistein in inhibiting the
growth of LNCaP and DU 145 cells in vitro. Similar results have been
found in a number of cancer cell lines from different tissues,
including bladder cancer (Zhou et al. 1998
), breast
cancer (Constantinou et al. 1996
, Peterson and Barnes 1991
, Scholar and Toews 1994
), melanoma
(Menon et al. 1998
) and prostate cancer (Peterson and Barnes 1993
).
Our studies suggest that human prostate cancer cell lines are sensitive to the growth-inhibitory effects of soy components, but that relatively high concentrations are required for in vitro effects. At genistein concentrations <25 µmol/L, little change in proliferative rates is observed. In vivo concentrations of soy isoflavones after consumption of soy products are typically ~10 µmol/L, and the compounds are rapidly cleared from the serum. In contrast to this relative resistance observed in vitro, we detected significant effects on tumor growth and biomarkers in vivo. We hypothesize that the optimal growth conditions employed in the in vitro studies with carefully controlled media conditions, temperature and oxygenation are not predictive of the complex and harsh in vivo conditions in the tumor microenvironment, in which hypoxia, necrosis, and suboptimal perfusion and diffusion limit nutrient availability and removal of metabolic waste. Our studies indicate that the ability of soy products to modulate prostate tumor cell function is more profound in vivo than in vitro.
In addition to direct effects on tumor cells, soy products may modulate
a number of other host processes, indirectly influencing prostate tumor
growth. For example, others have reported that genistein inhibits
endothelial cell proliferation in response to growth factors in vitro.
Our studies show that pure genistein and SPC inhibit
heparin-stimulated endothelial cell proliferation in vitro by
>50% with concentrations 25 µmol/L. The in vivo assessment of
prostate tumor microvessel density as a biomarker of tumor angiogenesis
shows a reduced vascularity in mice fed soy products. The inhibition of
tumor angiogenesis is typically associated with enhanced apoptosis and
has little effect on proliferation index (Folkman 1995
).
The significant increase in tumor apoptosis may represent a combined
direct effect on tumor cell function and a secondary indirect effect
related to inhibition of tumor angiogenesis.
The antiangiogenesis mechanisms influenced by soy isoflavones may be
multiple. First, soy isoflavones may inhibit the production and/or
bioactivity of angiogenic factors. Angiogenesis factors control
vascular endothelial cell proliferation and migration within the
growing tumor matrix. Among them, vascular endothelial growth factor
(VEGF) is believed to play an important role in angiogenesis. Genistein
inhibits VEGF level by post-transcriptional regulation of its
expression (Levy et al. 1996
) in vitro. Soy isoflavones
may also directly inhibit endothelial cell proliferation. Genistein has
been shown to be more potent than other isoflavones in inhibiting
endothelial cell proliferation in vitro (Fotsis et al. 1993
, Xia et al. 1996
).
IGF-I is also a growth factor associated with enhanced angiogenesis
(Nakao-Hayashi et al. 1992
). Furthermore, circulating
IGF-I concentrations are positively associated with prostate cancer
risk in human studies (Chan et al. 1998
,
Mantzoros et al. 1997
, Wolk et al. 1998
).
Our studies provide the foundation for the hypothesis that soy may
inhibit prostate angiogenesis both by direct effects on endothelial
cells and by reducing circulating concentrations of critical growth
factors.
We chose SPC as the major source of soybean bioactive components for
our studies because it contains a diverse array of biologically active
compounds that could potentially interact to provide more potent
anti-prostate cancer activity. These hypothetical benefits would
not be appreciated in studies of pure compounds. The in vivo inhibition
of cancer incidence or progression by soy products or pure isoflavones
has been reported for gastric cancer (Yanagihara et al. 1993
), leukemia cells (Jing et al. 1993
), breast
cancer (Hawrylewicz et al. 1991 and 1995
) and others
(Messina et al. 1994
). In contrast, some studies have
not found in vivo inhibitory effects of soy on tumorigenesis
(Clinton et al. 1979
, Messina et al. 1994
, Naik et al. 1994
). Of concern, some
studies report that soy-based dietary treatments had
tumor-promoting effects (McIntosh et al. 1995
,
Rao et al. 1997
). In addition, it has been hypothesized
that the estrogenic properties of soy isoflavones may stimulate breast
tumor growth under some conditions (Hsieh et al. 1998
).
Investigators, clinicians and commercial enterprises should use caution
in universally recommending soy supplements enriched in isoflavones for
cancer prevention or therapy except in the context of clinical studies.
In summary, we observed that dietary soy phytochemicals inhibited the growth of LNCaP tumor in mice associated with reduced proliferation, enhanced tumor cell apoptosis and reduced tumor angiogenesis. These observations were supported by in vitro studies showing that soy isoflavones or soy phytochemicals inhibited LNCaP cell growth, blocked cell cycle progression at G2-M phases and enhanced DNA fragmentation, a marker for apoptosis. Our studies provide evidence that dietary soy phytochemical–containing soybean products should be developed further as agents for the prevention and treatment of prostate cancer.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Supported in part by National Institutes of
Health grants F32 CA71161 and Harvard Clinical Nutrition Research
Center, NIH Grant #P30DK40561 to J.-R.Z., and KO7 CA01680 and RO1
CA72482 to S.K.C.
4 Abbreviations used: IGF-I: insulin-like
growth factor-I; PCNA: proliferating cell nuclear antigen; PSA:
prostate specific antigen; SCID: severe combined immune deficient; SPC:
soy phytochemical concentrate; SPI: soy protein isolate; TUNEL:
terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end
labeling; VEGF: vascular endothelial growth factor.
Manuscript received February 3, 1999. Initial review completed March 5, 1999. Revision accepted June 8, 1999.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1. American Institute of Nutrition 1977 Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 107: 1340–1348.
2.
Chan J. M., Stampfer M. J., Giovannucci E., Gann P. H., Ma J., Wilkinson P., Hennekens C. H., Pollak M. Plasma insulin-like growth factor-I and prostate cancer risk: a prospective study. Science (Washington, DC) 1998;279:563-566
3. Choi Y. H., Zhang L., Lee W. H., Park K. Y. Genistein-induced G2/M arrest is associated with the inhibition of cyclin B1 and the induction of p21 in human breast carcinoma cells. Int. J. Oncol. 1998;13:391-396[Medline]
4. Clinton S. K., Destree R., Anderson D. B., Truex C. R., Imrey P. B., Visek W. J. 1,2-Dimethylhydrazine-induced colon cancer in rats fed beef or vegetable protein. Nutr. Rep. Int. 1979;20:335-342
5. Constantinou A. I., Mehta R. G., Vaughan A. Inhibition of N-methyl-N-nitrosourea-induced mammary tumors in rats by the soybean isoflavones. Anticancer Res 1996;16:3293-3298[Medline]
6. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. Med. 1995;1:27-31[Medline]
7.
Fotsis T., Pepper M., Adlercreutz H., Fleischmann G., Hase T., Montesano R., Schweigerer L. Genistein, a dietary-derived inhibitor of in vitro angiogenesis. Proc. Natl. Acad. Sci. U.S.A. 1993;90:2690-2694
8. Geller J., Sionit L., Partido C., Li L., Tan X., Youngkin T., Nachtsheim D., Hoffman R. M. Genistein inhibits the growth of human-patient BPH and prostate cancer in histoculture. Prostate 1998;34:75-79[Medline]
9. Hawrylewicz E. J., Huang H. H., Blair W. H. Dietary soybean isolate and methionine supplementation affect mammary tumor progression in rats. J. Nutr. 1991;121:1693-1698
10. Hawrylewicz E. J., Zapata J. J., Blair W. H. Soy and experimental cancer: animal studies. J. Nutr. 1995;125:698S-708S
11.
Hebert J. R., Hurley T. G., Olendzki B. C., Teas J., Ma Y., Hampl J. S. Nutritional and socioeconomic factors in relation to prostate cancer mortality: a cross-national study. J. Natl. Cancer Inst. 1998;90:1637-1647
12.
Hsieh C.-Y., Santell R. C., Haslam S. Z., Helferich W. G. Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer (MCF-7) cells in vitro and in vivo. Cancer Res 1998;58:3833-3838
13. Jing Y., Nakaya K., Han R. Differentiation of promyelocytic leukemia cells HL-60 induced by daidzein in vitro and in vivo. Anticancer Res 1993;13:1049-1054[Medline]
14. Kennedy A. R. The evidence for soybean products as cancer preventive agents. J. Nutr. 1995;125:733S-743S
15. Landstrom M., Zhang J.-X., Hallmans G., Aman P., Bergh A., Damber J.-E., Mazur W., Wahala K., Adlercreutz H. Inhibitory effects of soy and rye diets on the development of Dunning R3327 prostate adenocarcinoma in rats. Prostate 1998;36:151-161[Medline]
16.
Levy A. P., Levy N. S., Goldberg M. A. Post-transcriptional regulation of vascular endothelial growth factor by hypoxia. J. Biol. Chem. 1996;271:2746-2753
17. Makela S., Santti R., Salo L., McLachlan J. A. Phytoestrogens are partial estrogen agonists in the adult male mouse. Environ. Health Perspect. 1995;7:123-127
18. Mantzoros C. S., Tzonou A., Signorello L. B., Stampfer M., Trichopoulos D., Adami H. O. Insulin-like growth factor 1 in relation to prostate cancer and benign prostatic hyperplasia. Br. J. Cancer 1997;76:1115-1118[Medline]
19. McIntosh G. H., Regester G. O., Le Leu R. K., Royle P. J., Smithers G. W. Dairy proteins protect against dimethylhydrazine-induced intestinal cancers in rats. J. Nutr. 1995;125:809-816
20. Menon L. G., Kuttan R., Nair M. G., Chang Y. C., Kuttan G. Effect of isoflavones genistein and daidzein in the inhibition of lung metastasis in mice induced by B16F-10 melanoma cells. Nutr. Cancer 1998;30:74-77[Medline]
21. Messina M. J., Persky V., Setchell K. D., Barnes S. Soy intake and cancer risk: a review of the in vitro and in vivo data. Nutr. Cancer 1994;21:113-131[Medline]
22. Morton M. S., Griffiths K., Blacklock N. The preventive role of diet in prostatic disease. Br. J. Urol. 1996;77:481-493[Medline]
23. Naik H. R., Lehr J. E., Pienta K. J. An in vitro and in vivo study of antitumor effects of genistein on hormone refractory prostate cancer. Anticancer Res 1994;14:2617-2619[Medline]
24. Nakao-Hayashi J., Ito H., Kanayasu T., Morita I., Murota S. Stimulatory effects of insulin and insulin-like growth factor I on migration and tube formation by vascular cells. Atherosclerosis 1992;92:141-149[Medline]
25. National Research Council (1985) Guide for the Care and Use of Laboratory Animals. Publication no. 85–23 (rev.), National Institutes of Health, Bethesda, MD.
26.
Onozawa M., Fukuda K., Ohtani M., Akaza H., Sugimura T., Wakabayashi K. Effects of soybean isoflavones on cell growth and apoptosis of human prostatic cancer cell line LNCaP. Jpn. J. Clin. Oncol. 1998;28:360-363
27. Pagliacci M. C., Smacchia M., Migliorati G., Grignani F., Riccardi C., Nicoletti I. Growth-inhibitory effects of the natural phyto-oestrogen genistein in MCF-7 human breast cancer cells. Eur. J. Cancer 1994;30A:1675-1682
28. Parkin D. M., Muir C. S. Cancer incidence in five continents. Comparability and quality of data. IARC Sci. Publ. 1992;120:45-173
29. Peterson G., Barnes S. Genistein inhibition of the growth of human breast cancer cells: independence from estrogen receptors and the multi-drug resistance gene. Biochem. Biophys. Res. Commun. 1991;179:661-667[Medline]
30. Peterson G., Barnes S. Genistein and biochanin A inhibit the growth of human prostate cancer cells but not epidermal growth factor receptor tyrosine autophosphorylation. Prostate 1993;22:335-345[Medline]
31. Rao A. V., Sung M.-K. Saponins as anticarcinogens. J. Nutr. 1995;125:717S-724S
32.
Rao C. V., Wang C. X., Simi B., Lubet R., Kelloff G., Steele V., Reddy B. S. Enhancement of experimental colon cancer by genistein. Cancer Res 1997;57:3717-3722
33. Roehm N. W., Rodgers G. H., Hatfield S. M., Glasebrook A. L. An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT. J. Immunol. Methods 1991;142:257-265[Medline]
34. Santibanez J. F., Navarro A., Martinez J. Genistein inhibits proliferation and in vitro invasive potential of human prostatic cancer cell lines. Anticancer Res 1997;17:1199-1204[Medline]
35. Scholar E. M., Toews M. L. Inhibition of invasion of murine mammary carcinoma cells by the tyrosine kinase inhibitor genistein. Cancer Lett 1994;87:159-162[Medline]
36. Shamsuddin A. M. Inositol phosphates have novel anticancer function. J. Nutr. 1995;125:725S-732S
37. Shao Z. M., Alpaugh M. L., Fontana J. A., Barsky S. H. Genistein inhibits proliferation similarly in estrogen receptor-positive and negative human breast carcinoma cell lines characterized by P21WAF1/CIP1 induction, G2/M arrest, and apoptosis. J. Cell. Biochem. 1998;69:44-54[Medline]
38. Sharma O. P., Adlercreutz H., Strandberg J. D., Zirkin B. R., Coffey D. S., Ewing L. L. Soy of dietary source plays a preventive role against the pathogenesis of prostatitis in rats. J. Steroid Biochem. Mol. Biol. 1992;43:557-564[Medline]
39. Steel R.G., D & Torrie J. H. Principles and Procedures of Statistics: A Biometrical Approach 2nd ed. 1980 McGraw-Hill New York, NY.
40. Tanaka T., Manome Y., Wen P., Kufe D. W., Fine H. A. Viral vector-mediated transduction of a modified platelet factor 4 cDNA inhibits angiogenesis and tumor growth. Nat. Med. 1997;3:437-442[Medline]
41. Wang H., Murphy P. A. Isoflavone content in commercial soybean foods. J. Agric. Food Chem. 1994;42:1666-1673
42. Wang H., Murphy P. A. Isoflavone composition of American and Japanese soybean in Iowa: effects of variety, crop year, and location. J. Agric. Food Chem. 1994;42:1674-1677
43.
Wolk A., Mantzoros C. S., Andersson S. O., Bergstrom R., Signorello L. B., Lagiou P., Adami H. O., Trichopoulos D. Insulin-like growth factor 1 and prostate cancer risk: a population-based, case-control study. J. Natl. Cancer Inst. 1998;90:911-915
44. Xia P., Aiello L. P., Ishii H., Jiang Z. Y., Park D. J., Robinson G. S., Takagi H., Newsome W. P., Jirousek M. R., King G. L. Characterization of vascular endothelial growth factor's effect on the activation of protein kinase C, its isoforms, and endothelial cell growth. J. Clin. Investig. 1996;98:2018-2026[Medline]
45.
Yanagihara K., Ito A., Toge T., Numoto M. Antiproliferative effects of isoflavones on human cancer cell lines established from the gastrointestinal tract. Cancer Res 1993;53:5815-5821
46. Yanagihara K., Numoto M., Tauchi H., Akama Y., Yokozaki H., Tahara E., Kamiya K., Seito T. Genetic status of p53 and induction of apoptosis by radiation or isoflavones in human gastric carcinoma cell lines. Int. J. Oncol. 1996;9:95-102
47. Zhang J. X., Hallmans G., Landstrom M., Bergh A., Damber J. E., Aman P., Adlercreutz H. Soy and rye diets inhibit the development of Dunning R3327 prostatic adenocarcinoma in rats. Cancer Lett 1997;114:313-314[Medline]
48.
Zhou J.-R., Mukherjee P., Gugger E. T., Tanaka T., Blackburn G. L., Clinton S. K. The inhibition of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis. Cancer Res 1998;58:5231-5238
This article has been cited by other articles:
|
|
 |
|
  T. T.Y. Wang, T. S. Hudson, T.-C. Wang, C. M. Remsberg, N. M. Davies, Y. Takahashi, Y. S. Kim, H. Seifried, B. T. Vinyard, S. N. Perkins, et al. Differential effects of resveratrol on androgen-responsive LNCaP human prostate cancer cells in vitro and in vivo Carcinogenesis, October 1, 2008; 29(10): 2001 - 2010. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Gallo, G. F. Zannoni, I. De Stefano, M. Mosca, C. Ferlini, E. Mantuano, and G. Scambia Soy Phytochemicals Decrease Nonsmall Cell Lung Cancer Growth In Female Athymic Mice J. Nutr., July 1, 2008; 138(7): 1360 - 1364. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Alschuler and D. Rubin Integrative Tumor Board: Pancreatic Cancer Naturopathic Medicine Integr Cancer Ther, June 1, 2008; 7(2): 105 - 108. [PDF]
|
|
|
|
 |
|
 R J. Barnard Prostate cancer prevention by nutritional means to alleviate metabolic syndrome Am. J. Clinical Nutrition, September 1, 2007; 86(3): 889S - 893S. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Gallo, E. Mantuano, M. Fabrizi, C. Ferlini, S. Mozzetti, I. De Stefano, and G. Scambia Effects of a phytoestrogen-containing soy extract on the growth-inhibitory activity of ICI 182 780 in an experimental model of estrogen-dependent breast cancer Endocr. Relat. Cancer, June 1, 2007; 14(2): 317 - 324. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Mai, G. L. Blackburn, and J.-R. Zhou Soy phytochemicals synergistically enhance the preventive effect of tamoxifen on the growth of estrogen-dependent human breast carcinoma in mice Carcinogenesis, June 1, 2007; 28(6): 1217 - 1223. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Rice, R. Handayani, Y. Cui, T. Medrano, V. Samedi, H. Baker, N. J. Szabo, C. J. Rosser, S. Goodison, and K. T. Shiverick Soy Isoflavones Exert Differential Effects on Androgen Responsive Genes in LNCaP Human Prostate Cancer Cells J. Nutr., April 1, 2007; 137(4): 964 - 972. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. B. Michaud, J. P. Karpinski, K. L. Jones, and J. Espirito Dietary supplements in patients with cancer: Risks and key concepts, part 2 Am. J. Health Syst. Pharm., March 1, 2007; 64(5): 467 - 480. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Vrieling, M. A. Rookus, E. Kampman, J. M. G. Bonfrer, C. M. Korse, J. van Doorn, J. W. Lampe, A. Cats, B. J. M. Witteman, F. E. van Leeuwen, et al. Isolated Isoflavones Do Not Affect the Circulating Insulin-Like Growth Factor System in Men at Increased Colorectal Cancer Risk J. Nutr., February 1, 2007; 137(2): 379 - 383. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Li, O. Kucuk, M. Hussain, J. Abrams, M. L. Cher, and F. H. Sarkar Antitumor and Antimetastatic Activities of Docetaxel Are Enhanced by Genistein through Regulation of Osteoprotegerin/Receptor Activator of Nuclear Factor-{kappa}B (RANK)/RANK Ligand/MMP-9 Signaling in Prostate Cancer. Cancer Res., May 1, 2006; 66(9): 4816 - 4825. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Jagadeesh, S. Kyo, and P. P. Banerjee Genistein Represses Telomerase Activity via Both Transcriptional and Posttranslational Mechanisms in Human Prostate Cancer Cells Cancer Res., February 15, 2006; 66(4): 2107 - 2115. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Singh, A. A. Franke, G. L. Blackburn, and J.-R. Zhou Soy Phytochemicals Prevent Orthotopic Growth and Metastasis of Bladder Cancer in Mice by Alterations of Cancer Cell Proliferation and Apoptosis and Tumor Angiogenesis Cancer Res., February 1, 2006; 66(3): 1851 - 1858. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Handayani, L. Rice, Y. Cui, T. A. Medrano, V. G. Samedi, H. V. Baker, N. J. Szabo, and K. T. Shiverick Soy Isoflavones Alter Expression of Genes Associated with Cancer Progression, Including Interleukin-8, in Androgen-Independent PC-3 Human Prostate Cancer Cells J. Nutr., January 1, 2006; 136(1): 75 - 82. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Z. Fang, D. Chen, Y. Sun, Z. Jin, J. K. Christman, and C. S. Yang Reversal of Hypermethylation and Reactivation of p16INK4a, RAR{beta}, and MGMT Genes by Genistein and Other Isoflavones from Soy Clin. Cancer Res., October 1, 2005; 11(19): 7033 - 7041. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Mentor-Marcel, C. A. Lamartiniere, I. A. Eltoum, N. M. Greenberg, and A. Elgavish Dietary Genistein Improves Survival and Reduces Expression of Osteopontin in the Prostate of Transgenic Mice with Prostatic Adenocarcinoma (TRAMP) J. Nutr., May 1, 2005; 135(5): 989 - 995. [Abstract] [Full Text] [PDF]
|
|
