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Carcinogenesis Advance Access originally published online on April 29, 2007
Carcinogenesis 2007 28(8):1710-1717; doi:10.1093/carcin/bgm103
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Akt–GSK-3 pathway as a target in genistein-induced inhibition of TRAMP prostate cancer progression toward a poorly differentiated phenotype

Lara H. El Touny and Partha P. Banerjee*

Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington, DC 20057, USA

* To whom correspondence should be addressed. Tel: +1 202 687 8611; Fax: +1 202 687 1823; Email: ppb{at}georgetown.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Anti-proliferative properties of genistein in prostate and other cancers have been studied extensively. However, the identification of genistein targets that may mediate its chemopreventive effects in vivo requires further elucidation. In this study, we have demonstrated that the incorporation of genistein in the diet of transgenic adenocarcinoma mouse prostate model (TRAMP/FVB) mice resulted in a reduction in prostate size and the incidence of poorly differentiated (PD) cancer ensuing in an accumulation of prostates at the prostatic intra-epithelial neoplasia (PIN) stage. TRAMP/FVB prostate cancer progression and the onset of PD cancer were characterized by the activation of acutely transforming retrovirus AKT8 in rodent T cell lymphoma (Akt), phosphorylation of glycogen synthase kinase 3-beta (GSK-3ß), post-transcriptional up-regulation of cyclin D1 and repression of cadherin-1 via snail-1 up-regulation. Incorporation of genistein in the diet significantly inhibited the activation of Akt, restored the activation of GSK-3ß, reduced cyclin D1 levels post-transcriptionally and maintained the expression of the cadherin-1 complex via down-regulation of snail-1. By identifying the Akt–GSK-3 pathway and subsequently its downstream effectors, as targets for genistein chemopreventive action, we have elucidated one possible mechanism by which genistein decreases the proliferative potential, retards cancer progression and maintains the integrity of the prostatic epithelial cells in vivo.

Abbreviations: Akt, acutely transforming retrovirus AKT8 in rodent T cell lymphoma; CaP, prostate cancer; DLP, dorsolateral prostate; EGF, epidermal growth factor; ER, estrogen receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSK-3ß, glycogen synthase kinase 3-beta; IGF, insulin-like growth factor; PD, poorly differentiated; PIN, prostatic intra-epithelial neoplasia; TRAMP, transgenic adenocarcinoma mouse prostate


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Genistein, the most abundant isoflavonoid in soybeans and a staple of the Asian diet, is under investigation because of the epidemiological observation linking soy consumption to a decreased incidence of hormone-dependent cancers in Asian populations as compared with their Western counterparts (1). Although the incidence of latent, non-infiltrative prostate carcinomas is similar in both populations, clinically relevant carcinomas and prostate cancer (CaP) mortality are profoundly higher in the West (2). This observation led to the initiation of several studies aimed at determining the chemopreventive effects of genistein specifically or soy in general in animal models of spontaneous and chemically induced CaP.

The transgenic adenocarcinoma mouse prostate model (TRAMP) was generated by using the prostate-specific rat probasin promoter (–426/+28) to drive the expression of the SV40 large and small (T, t) antigens (3). The age-dependent nature of the CaP progression in TRAMP makes it comparable with the age-associated human CaP. In TRAMP mice, development of prostatic intra-epithelial neoplasia (PIN) is followed by invasive, prostate adenocarcinoma that progresses into poorly differentiated (PD) carcinoma, followed by metastatic CaP to lymph nodes, lungs and to a lesser extent bone (3). These characteristics of the TRAMP model render it useful for the evaluation of possible chemopreventive effects of natural and therapeutic agents.

The molecular biology of CaP and its progression is characterized by aberrant activity of several regulatory pathways, within prostate epithelial cells and in the surrounding stromal tissue. One such pathway is the phosphoinositol-3-kinase–Akt pathway; Akt is a serine/threonine kinase that functionally modulates numerous substrates involved in the regulation of cell proliferation/survival, angiogenesis and tissue invasion (4). All these processes represent hallmarks of cancer, and a burgeoning literature is defining the importance of Akt in human cancer and experimental models of tumorigenesis. In fact, phosphatase and tensin homolog deleted in chromosome ten loss of function and Akt activation has been significantly correlated with the progression of CaP (5). Furthermore, transgenic mice expressing activated Akt-1 in the prostate develop PIN (6). Akt plays a major role in the proliferation of cancer cells (4). This is most prominent at the G1–S transition of the cell cycle via phosphorylation and inhibition of glycogen synthase kinase 3-beta (GSK-3ß). GSK-3ß activity is controlled by phosphorylation and subcellular localization (7). When phosphorylated by active Akt on serine 9, its kinase activity is inhibited and it loses the ability to prime cyclin D1 for degradation; thus, Akt-induced inactivation of GSK-3ß stabilizes cyclin D1, whereas inhibition of Akt by Wortmannin results in the accelerated degradation of cyclin D1 (8,9). Snail is an another target of GSK-3ß. GSK-3ß phosphorylates snail at two consensus motifs to regulate its function via cytoplasmic retention and ubiquitination (10). Furthermore, GSK-3ß has been shown to transcriptionally repress snail. In fact, silencing or inhibition of GSK-3 results in an increase in snail transcription (11). Snail is absent in normal epithelial cells, whereas its expression has been documented in epithelial tumors and correlates inversely with tumor grade (12). Snail negatively regulates E-cadherin (cadherin-1 in mice) transcription via binding to its promoter (13). Besides the importance of E-cadherin regulation during development, repression of E-cadherin transcription is particularly relevant in the transition from adenoma to carcinoma. Disruption of E-cadherin-mediated adhesion is considered a key step in progression toward the invasive phase of carcinoma (14) and down-regulation of E-cadherin is one of the most frequently reported phenomena in metastatic cancers (15). Although genistein has been shown previously to decrease the incidence of PD prostate carcinoma in TRAMP mice (16), a possible effect on Akt–GSK-3 pathway and its downstream effectors is unknown.

In this study, we investigated the effect of genistein on the onset and progression of CaP in the TRAMP/FVB model. We have shown that genistein does not prevent the onset of PIN but decreases the incidence of PD cancer in TRAMP/FVB mice. We propose that one mechanism by which genistein might slow the growth of CaP cells is via the inhibition of Akt, the subsequent activation of GSK-3ß, degradation of cyclin D1 but also the repression of snail, resulting in the maintenance of E-cadherin expression.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 References
 
Animal handling and treatment
TRAMP (The Jackson laboratory, Bar Harbor, ME) and FVB mice (Charles River Laboratories, Wilmington, MA) were maintained at the Georgetown University animal facilities. Male and female TRAMP mice were mated with FVB counterparts, and heterozygous male offspring were confirmed by genotyping as described previously (3). Four-week-old transgenic males were fed genistein-free purified AIN-76A pellets (Harlan Teklad, Indianapolis, IN) supplemented with 0, 250 and 1000 mg genistein per kilogram diet (n = 15/diet group) (Sigma, St Louis, MO) until 20 weeks of age. Another group of mice was kept on a regular diet and 10 mice were killed at 5, 9, 18 and 24 weeks of age (n = 10/age group). Animal care and treatments were conducted in accordance with established guidelines and protocols approved by the Georgetown University Animal Care and Use Committee.

Tissue preparation, histopathological examination and immunohistochemistry
After completion of genistein treatment (20 weeks) or reaching one of the endpoint ages, mice were killed, blood collected and various organs (prostate, seminal vesicles, hearts, livers, lungs, kidneys and testes) dissected out, weighed, fixed in 4% paraformaldehyde for 48 h, dehydrated and paraffin embedded. Portions of prostatic lobes (dorsolateral, ventral and anterior) were rapidly frozen on dry ice and stored at –80°C, until processed for mRNA and protein analysis.

For histopathological evaluation, 5 µm sections of paraffin-embedded prostatic lobes were cleared with xylene, stained with hematoxylin and eosin and examined under an Olympus BX41 light microscope (Olympus Imaging America, Center Valley, PA), the histopathological grade was recorded based on the most advanced pathology in each prostate specimen and scoring was done based on previously established methods in TRAMP (16).

For immunohistochemistry, tissue sections were heat treated (90°C for 10 min) in 10 mM sodium citrate buffer, pH 6.0 for antigen retrieval. Duplicate slides were incubated with rabbit anti-Ki-67 (NeoMarkers, Fremont, CA), mouse anti-cadherin-1 (BD Biosciences, San Jose, CA) and rabbit anti-snail-1 (Abcam, Cambridge, MA) overnight at 4°C followed by incubation with the appropriate Alexa Fluor-tagged secondary antibodies (for Ki-67) (one set of slides) and biotin-labeled secondary antibody (Invitrogen, Carlsbad, CA) for 1 h. Slides were then incubated with Vectastain ABC-Peroxidase (Vector Laboratories, Burlingame, CA) for 30 min and developed with 3,3'-diaminobenzidine. The same slide or the next section was stained with hematoxylin. Slides were photographed using a camera-equipped microscope (ZEISS AxioPlan2 Imaging System, Jena, Germany). Images were transferred to photoshop and all cells in each duct were counted from all prostate specimens from each treatment or age group in triplicates and evaluated for the percentage of Ki-67-positive cells.

Serum genistein, testosterone and estradiol levels
Collected trunk blood was allowed to clot for 24 h at 4°C, samples were centrifuged and serum was stored at –80°C until time of use. Serum genistein was unconjugated by incubation with acetate buffer (0.1 M, pH 5.0), containing 0.2 U/ml of ß-glucuronidase and 2 U/ml of sulfatase (Sigma). Total genistein was then doubly extracted in diethyl ether (Sigma) and measured by the time-resolved fluoroimmunoassay according to the manufacturer’s protocol (Labmaster TRF-genistein, Turku, Finland).

Serum testosterone and estradiol were measured with competitive enzyme immunoassay (EIA)-testosterone (sensitivity 6 pg/ml) and EIA-estradiol (sensitivity 8 pg/ml), respectively (Cayman Chemical, Ann Arbor, MI), according to the manufacturer's protocols.

Reverse transcription–polymerase chain reaction
RNA was extracted with TRIzol solution from homogenized prostatic tissue as suggested by the manufacturer (Invitrogen). After RNA quantification, genes were amplified using the Reverse-IT one-step kit (Abgene, Rochester, NY). Mouse-specific primers were designed through Primer Quest program (Integrated DNA Technologies, Coralville, IA). Cadherin-1-forward: 5'-AGTCAACGATCCTGACCAGCAGTT-3' and cadherin-1-reverse: 5'-GATTCCCGCCTTCATGCAGTTGTT-3', snail-1-forward: 5'-TCCAAACCCACTCGGATGTGAAGA-3' and snail-1-reverse: 5'-TGTACCTCAAAGAAGGTGGCCTGA-3' and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-forward 5'-GTGTTCCTACCCCCAATGTG-3' and GAPDH-reverse: 5'-CTTGCTCAGTGTCCTTGCTG-3'. Polymerase chain reactions were initiated at 94°C for 2 min, followed by 25 cycles of 94°C for 1 min, 1 min annealing temperature, 72°C for 1 min followed by final extension at 72°C for 5 min. Annealing temperatures for cadherin-1, snail-1 and GAPDH were 58°C and the yielded polymerase chain reaction products (856, 461 and 349 bp, respectively) were separated on 1.5% agarose gels and visualized by ethidium bromide fluorescence using the Fuji LAS-1000 imager (Tokyo, Japan). Images were then captured and imported to Photoshop.

Western blot analysis
Protein extracts were prepared from prostatic tissue of genistein-fed mice or mice in different age groups by homogenization in tissue lysis buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate and 5 mM ethylenediaminetetraacetic acid) using a polytronic tissue tearer (BioSpec Products, Bartlesville, OK). Equal amounts of protein extracts were resolved on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis according to our previously published methods (17). Membranes were probed with anti-cyclin D1, anti-GSK-3{alpha}/ß (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Akt, anti-phospho-Akt (S473), anti-pGSK-3{alpha}/ß (S21/9), anti-pCyclinD1 (T286) (in vitro experiment) (Cell Signaling Technology, Danvers, MA), anti-cadherin-1 (BD Biosciences) and anti-snail-1 (Abcam). Membranes were stripped and re-probed with GAPDH antibody (1/10 000 dilution) (Abcam) to ensure for equal loading. Molecular weight markers (Invitrogen) were run on each gel confirming the molecular size of immunoreactive proteins.

Cell culture and GSK-3 inhibitors treatment
TRAMP-C2 cell line [gift from Dr Norman M.Greenberg, Fred Hutchinson Cancer Research Center, University of Washington (18)] was maintained at 37°C with 5% CO2 in phenol red-free Iscove's minimal essential medium (Biofluids, Rockville, MD) supplemented with 10% fetal bovine serum (Quality Biologicals, Gaithersburg, MD), 2 mM glutamine, 100 U/ml penicillin G sodium and 100 µg/ml streptomycin sulfate (Sigma). Twenty-four hours after seeding (4 x 105 cells/100 mm plate), cells were treated with 100 nM of GSK-3 inhibitors IX [(2'Z, 3'E)-6-bromoindirubin-3'-acetoxime] or X [(2'Z, 3'E)-6-bromoindirubin-3'-oxime) (EMD Biosciences, San Diego, CA) for 72 h or 50 nM Wortmannin (Tocris, Ellisville, MO) for 24 h. Protein lysates and western blot analysis were prepared as described previously.

Statistical analyses
Data analyses were carried out between the various age groups by using one-way analysis of variance in prism 3 (GraphPad Software, San Diego, CA). One-way analysis of variance was also used to compare the various parameters measured between the three following groups: transgenic mice on AIN-76A diet only, transgenic mice fed genistein (250 mg/kg diet) and transgenic mice fed genistein (1000 mg/kg diet).

For comparison of testosterone and estradiol concentrations, five serum samples from each treatment group were assayed without pooling in duplicates; data analysis for polymerase chain reaction and western blots were from all dorsolateral prostates (DLPs) per treatment or age group. For Ki-67 analysis, all different specimens were used from each age (total n = 40) or treatment group (total n = 45), and in vitro experiments were repeated thrice. Data were analyzed using Prism 3 GraphPad software and presented as means ± SEMs. The one-way analysis of variance was used followed by a Dunnett test to determine significant differences between groups, with an assigned confidence interval of 95%. Western blots and agarose gel band intensities were captured by the Fuji LAS-1000 imager from three independent experiments, quantified by using the ImageJ software (National Institutes of Health, Bethesda, MD) and presented as means ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 References
 
Genistein in the diet does not prevent the initiation of carcinogenesis but retards the onset of PD cancer of TRAMP/FVB mice in a dose-dependent manner
To determine the effect of genistein on the prostatic growth in TRAMP/FVB mice, we characterized the age-dependent progression of CaP in TRAMP/FVB-crossed mice in terms of prostate weight and histopathology. Prostates from 40 TRAMP/FVB mice (fed a regular diet) killed at various ages (5, 9, 18 and 24 weeks) (10 per group) were weighed individually and examined histologically. We observed an age-dependent increase in prostate weights (0.05 ± 0.001, 0.101 ± 0.001, 1.008 ± 0.26 and 1.800 ± 0.730 g) (Figure 1A) as well as an age-dependent shift toward more advanced stages in histopathological scoring (Figure 1C, left). In fact, all DLP specimens from 5-week-old TRAMP/FVB mice were normal as opposed to 25% of prostates from 9-week-old TRAMP/FVB, with the remaining 75% exhibiting PIN. Meanwhile, the majority of prostates (71 and 80%) from 18- and 24-week old TRAMP/FVB mice exhibited PD cancer, respectively. On the other hand, TRAMP/FVB mice fed the 250 and 1000 mg genistein per kilogram diet exhibited a decrease in prostate weight (Figure 1B) that achieved statistical significance (P < 0.05) with the 1000 mg/kg dose (Figure 1B). Histological examination revealed a dose-dependent decrease in the incidence of PD cancer (70% in control-fed TRAMP/FVB versus 38 and 19% in TRAMP/FVB mice fed 250 and 1000 mg genistein/kg diet, respectively) (Figure 1C, right). Interestingly, we observed a significant dose-dependent increase in the incidence of PIN in DLPs from genistein-fed TRAMP/FVB (56 and 81% for 250 and 1000 mg genistein per kilogram groups as opposed to only 16% of control diet-fed mice). This result suggests that genistein incorporation in the diet does not prevent the onset of PIN but rather prevents/retards the progression toward a poorly differentiated phenotype. It is noteworthy to mention that we have opted to use in our study and in all the following experiments, the TRAMP/FVB crossbreed, as we and others [L.H.El Touny and P.P.Banerjee, unpublished data, (19)] have observed incidences of seminal vesicle tumors in the pure TRAMP C57BL/6 background, that we have not observed in the TRAMP/FVB cross.


Figure 1
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  		Fig. 1. Genistein in the diet reduces TRAMP/FVB prostate weights and retards the onset of PD cancer with accumulation at the PIN stage, in a dose-dependent fashion. (A) Prostate weight average of (i) 10 TRAMP/FVB mice per age group kept on a regular diet and killed at different ages (5, 9, 18 and 24 weeks) (left panel) and (ii) three groups of TRAMP/FVB mice (n = 15/diet group) fed a diet containing 0, 250 and 1000 mg genistein per kg AIN-76A diet from 4–20 weeks of age. Values are presented as means ± SEM, *indicates P < 0.05. (B) Histological distribution of prostate samples from (i) the different age groups of TRAMP/FVB mice fed a regular diet and killed at different ages (5, 9, 18 and 24 weeks) (left) and (ii) TRAMP/FVB mice groups (n = 15, each) fed a diet containing 0, 250 and 1000 mg genistein per kg AIN-76A diet from 4–20 weeks of age (right).

 
Genistein in the diet reduces the proliferative index of TRAMP/FVB prostates while resulting in serum levels comparable with Asian populations with no significant changes in serum hormone levels
The incorporation of genistein in the diet of TRAMP/FVB mice resulted in a dose-dependent increase in total serum genistein levels (327.88 ± 76.96 and 3237.95 ± 67.10 in 250 and 1000 mg/kg diet compared with 3.962 ± 1.00 nmol/l in phytoestrogen-free diet) with no significant alterations in serum testosterone or estradiol (supplementary Figure 1A and B, available at Carcinogenesis Online). These genistein levels did not incur any significant changes on total body and organ (testes, liver, kidney, lung and heart) weights (supplementary Figure 1B, available at Carcinogenesis Online) nor did they incur any abnormal histopathological effects on the above-mentioned organs (data not shown).

To determine the effect of genistein on the proliferation of prostate cells, we examined Ki-67 immunoreactivity in the various stages of TRAMP/FVB CaP. We observed a dose-dependent increase in the number of Ki-67 immunoreactive cells (15.75 ± 1.3, 32.59 ± 1.95, 63.2 ± 5.4 and 81.25 ± 2.8% in prostate sections from 5-, 9-, 18- and 24-week old TRAMP/FVB mice, respectively) (Figure 2A). This result demonstrates that Ki-67 immunoreactivity is useful for evaluating the proliferation of DLPs, especially with the literature linking increased Ki-67 indices with the presence of advanced stage disease or increased tumor grade (20). The immunostaining specificity was further proven by the absence of elevated Ki-67 immunoreactivity in the anterior prostates of these TRAMP/FVB mice, which do not develop CaP in this model (data not shown). Immunofluorescence staining results were confirmed with similar Vectastain ABC-Peroxidase immunohistochemical staining results (Vector Laboratories) (data not shown).


Figure 2
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  		Fig. 2. Genistein in the diet reduces the prostatic proliferative index of TRAMP/FVB DLPs. Ki-67 staining in representative prostate sample from (A) each TRAMP/FVB age group or (B) each genistein treatment group. Five micrometer sections of DLPs placed on permafrost slides were deparaffinized, hydrated, treated for epitope retrieval by the heated citrate buffer method, blocked in 0.1% bovine serum albumin and incubated with an antibody against Ki-67, washed, incubated with the appropriate secondary antibody-conjugated to Alexa-488, counterstained with propidium iodide and mounted and visualized under a fluorescent microscope with a x20 magnification. Quantification of Ki-67 labeling from DLPs in each age or treatment group is presented in the respective right panels. The number of Ki-67-labeled cells was counted in each duct of prostatic tissue section and was presented as a percentage to total number of propidium iodide-stained cells in each duct analyzed. Three different sections from all DLPs in each group were quantified and values are presented as mean percentage ± SEM. *,** and ***indicate P < 0.05, P < 0.01 and P < 0.001, respectively.

 
On the other hand, dietary genistein resulted in a dose-dependent reduction in Ki-67 immunoreactivity in DLPs of 20-week-old TRAMP/FVB mice (Figure 2B). Approximately 74% of prostate cells from DLPs of control TRAMP/FVB mice were Ki-67 positive as opposed to 48 and 37% of cells from DLPs of 250 and 1000 mg/kg genistein-fed TRAMP/FVB mice, respectively. This result suggests that the decreased incidence of PD cancer and significant accumulation at the PIN stage may be due to decreased proliferation.

Dietary genistein prevents Akt activation and decreases GSK-3ß phosphorylation resulting in the post-transcriptional decrease in cyclin D1
The loss of dependence upon exogenous growth factors in the tumor environment is an essential feature of carcinogenesis. This is partly achieved by mutations in intracellular signaling pathways that constitutively activate survival/anti-apoptotic and proliferative pathways. Akt is a critical protein in one signaling pathway that appears to be frequently activated in prostate carcinogenesis (21). We observed an age-dependent activation of Akt in TRAMP/FVB DLPs that achieved significance by 18 weeks of age (Figure 3A). This activation, evidenced by increased phosphorylation on serine 473, was accompanied by a consistent increase in the inhibitory phosphorylation of GSK-3ß on serine 9 (Figure 3A), suggesting that the Akt pathway is functionally activated in the progression of TRAMP/FVB CaP. We next evaluated the effect of dietary genistein on the age-dependent activation of the Akt pathway by examining total and phosphorylated Akt and GSK-3ß levels in genistein-fed 20-week-old TRAMP/FVB mice (Figure 3B). We observed a drastic and significant decrease in phospho-Akt (S473) reaching >75% in the 1000 mg/kg diet with no effect on total Akt levels. Concomitant with the decrease in active Akt, we observed a similar pattern of reduction in the GSK-3ß phosphorylation on serine 9 with no significant effect on total levels. Having established the reduced Akt activation by dietary genistein and the subsequent decrease in p-GSK-3ß (Ser9), we aimed at determining the effect of this inactivation on the levels of cyclin D1, one major post-transcriptional target of GSK-3ß. In fact, active GSK-3ß is known to phosphorylate cyclin D1 triggering its proteolysis. We quantified cyclin D1 protein levels in DLP lysates from TRAMP/FVB mice of the various age groups and observed an age-dependent increase in cyclin D1 protein levels by >3.0-fold by 18 and 24 weeks of age with no change in mRNA levels (Figure 3C and data not shown). On the other hand, genistein consumption resulted in a dose-dependent decrease in cyclin D1 protein levels up to 75% in the 1000 mg/kg group with no alteration in mRNA levels (Figure 3D and data not shown). Since the decrease in cyclin D1 seems to be post-transcriptional and is very similar to the pattern of decreased Akt and GSK-3ß phosphorylation, it is plausible that the inhibition of Akt and subsequent activation of GSK-3ß results in the degradation of cyclin D1 and thereby contributing to the decreased proliferation in TRAMP/FVB DLPs by genistein.


Figure 3
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  		Fig. 3. Activation of the Akt pathway in the age-dependent CaP progression in TRAMP/FVB and its inactivation by dietary genistein. Western blot analysis of total and phosphorylated levels of Akt, GSK-3 (S473 and S9, respectively) and cyclin D1 in three representative samples (labeled 1, 2 and 3) from (A) age group of TRAMP/FVB mice on a regular diet or (B) genistein treatment group [two representative samples from each treatment group (labeled 1 and 2)]. Fifty microgram proteins from tissue lysates were run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and immunoblots were probed with the appropriate antibodies and their horseradish peroxidase-conjugated secondary antibodies. Immunoblots were stripped and re-probed for GAPDH to ensure equal loading. The quantitative analyses of immunoblots are shown on the right. Values are the mean relative protein levels ± SEMs from three different blots. *and **indicate P < 0.05 and P < 0.01.

 
Genistein in the diet results in the retention of cadherin-1 expression via a dose-dependent decrease in snail-1 transcription
Many studies have shown that the alterations in cadherin-1 expression levels are linked to loss of cellular differentiation and acquisition of invasive and metastatic potential in a multitude of human tumors, including CaP (22,23). Since dietary genistein resulted in a dose-dependent decrease in the incidence of PD cancer, we aimed at determining the effect of genistein on cadherin-1 expression in TRAMP/FVB mice. Interestingly, cadherin-1 is an indirect target of GSK-3ß. In fact, active GSK-3ß represses snail-1, a transcriptional repressor of cadherin-1, resulting in cadherin-1 accumulation (12,13). We characterized the age-dependent progression of snail-1 and cadherin-1 at the message and protein levels (Figure 4A and B). We observed a significant decrease in cadherin-1 levels that coincided with an increase in snail-1 levels by 18 weeks of age; this result was further corroborated by a significant decrease in cadherin-1 membranous staining by 18 weeks, which was accompanied by an increase in snail-1 nuclear staining (Figure 4C). On the other hand, cadherin-1 levels (mRNA and proteins) were increased dose-dependently by dietary genistein (Figure 5A and B). The increase in cadherin-1 (up to 2.5-fold protein increase in the 1000 mg/kg genistein group) was accompanied by a significant dose-dependent decrease in snail-1 (>2-fold reduction in the same diet group).


Figure 4
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  		Fig. 4. Age-dependent modulation of snail-1 and cadherin-1 expression levels in TRAMP/FVB mice. The relative mRNA (A) and protein (B) levels of snail-1 and cadherin-1 in the different age groups. Snail-1 and cadherin levels were determined by (A) reverse transcription–polymerase chain reaction: RNA was extracted from all prostate specimens from mice necropsied at different ages, via the TRIzol method, quantified and 500 ng RNA were subjected to one-step reverse transcription–polymerase chain reaction with appropriate cadherin-1, snail-1 and GAPDH primers to ensure for equal RNA loading. Photograph in (A) is a representative of three samples (designated 1, 2 and 3) from each age group. Snail-1 and cadherin-1 protein levels were determined by running 50 µg proteins from tissue lysates on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferring onto nitrocellulose membranes and probing immunoblots with snail-1 and cadherin-1 antibody and their horseradish peroxidase-conjugated secondary antibodies. Immunoblots were stripped and re-probed for GAPDH to ensure equal loading. Photograph in (B) is representative of three samples (designated 1, 2 and 3) from each age group. Values are the mean relative protein levels ± SEMs from three different blots. *, **and ***indicate P < 0.05, P < 0.01 and P < 0.001, respectively. (C) Snail-1 and cadherin-1 staining was determined in representative sections from prostates of TRAMP/FVB mice from the indicated ages. Slides were deparaffinized, subjected to the heated citrate buffer epitope retrieval method, blocked in 0.1% bovine serum albumin, incubated with snail-1 or cadherin-1 antibodies and then the appropriate secondary antibody, developed with the ABC-P method, counterstained with hematoxylin and visualized under a light microscope at an original magnification of x20 (inset represents magnified pictures to show cellular localization).

 


Figure 5
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  		Fig. 5. Effect of genistein on the mRNA and protein levels of snail-1 and cadherin-1. (A) Five hundred nanograms of RNA from all samples of each genistein treatment group as well as from the control group were subjected to one-step reverse transcription–polymerase chain reaction with primers for cadherin-1 and snail-1 as well as GAPDH to ensure for equal RNA amounts. The quantitative analyses of polymerase chain reaction products are on the right. Values are the mean relative message levels ± SEMs from three different agarose gels. **Indicate P < 0.01. (B) For protein levels, 50 µg of tissue lysates from two samples of each genistein treatment group as well as control group were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and iummunoblots were probed with snail-1 and cadherin-1 antibodies, stripped and re-probed with antibodies against GAPDH to ensure for equal loading. The quantitative analyses of immunoblots are on the right. Values are the mean relative protein levels ± SEMs from three different immunoblots. **Indicates P < 0.01.

 
Pharmacological inhibition of GSK-3 increases cyclin D1 and snail-1 levels in TRAMP cells
In order to demonstrate the link between Akt activation/GSK-3ß inactivation and the modulation of cyclin D1 and snail-1 levels in our cell system, we used two pharmacological GSK-3ß inhibitors and examined their effect on cyclin D1 and snail-1 protein levels in the TRAMP-derived cell line. Using a dose that significantly decreased the activity of GSK-3, as evidenced by decreased phosphorylation of cyclin D1 on Thr 286 (supplementary Figure 2A, available at Carcinogenesis Online), we observed a significant increase in cyclin D1 and snail-1 protein levels (up to 2.0-fold) (Figure 6). On the other hand, inhibition of Akt (>70%) (supplementary Figure 2B, available at Carcinogenesis Online) by Wortmannin decreased these levels by >2.0-fold.


Figure 6
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  		Fig. 6. Pharmacological inhibitors of GSK-3 increase the expression of cyclin D1 and snail-1. TRAMP-C2 cells were treated with 0 and 100 nM GSK-3 inhibitors (2'Z, 3'E)-6-bromoindirubin-3'-oxime (inhibitor IX) or (2'Z, 3'E)-6-bromoindirubin-3'-acetoxime (inhibitor X) for 72 h or 50 nM Wortmannin for 24 h. Fifty micrograms of cell lysates from three independent experiments were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and iummunoblots were probed with cyclin D1 and snail-1, stripped and re-probed with antibodies against GAPDH to ensure for equal loading. The quantitative analyses of immunoblots are on the right. Values are the mean relative protein levels ± SEMs from three different immunoblots. **Indicates P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 References
 
We have examined the effects of dietary genistein on initiation and progression of CaP in the TRAMP/FVB model. We have shown that dietary genistein consumption reduces the incidence of PD CaP but cannot prevent the development of PIN. TRAMP/FVB CaP progression was accompanied by an age-dependent activation of Akt, GSK-3ß phosphorylation and increase in cyclin D1 and proliferation. This trend in Akt activation, phosphorylation of GSK-3ß and up-regulation of cyclin D1 was most striking by 18 weeks of age, which coincides with a 70% incidence of PD cancer in TRAMP/FVB. We have also demonstrated the up-regulation of snail-1, concomitant to cadherin-1 down-regulation, during the age-dependent progression of PD prostate carcinoma in TRAMP/FVB. On the other hand, incorporation of genistein in the diet resulted in a drastic reduction in Akt activation, significant dephosphorylation of GSK-3ß (Serine 9) culminating in a dose-dependent post-transcriptional reduction in cyclin D1 levels and proliferation. Dietary genistein also induced a dose-dependent snail-1 down-regulation accompanied by cadherin-1 up-regulation. In vitro experiments have also demonstrated that GSK-3 inhibition increases cyclin D1 and snail-1 expression, potentially mimicking the in vivo cancer progression scenario.

This work has demonstrated that genistein consumption does not reduce the incidence of PIN suggesting that the reduced incidence of PD cancer is not attributable to an inhibitory effect of genistein on tumor initiation. This is consistent with the epidemiological observation of a similar incidence of non-infiltrative prostate tumors in Western and Eastern populations (2), suggesting that long-term genistein consumption is not effective in inhibiting prostate tumor initiation. In this study, dietary genistein resulted in serum genistein levels comparable with those documented in Eastern populations consuming a soy-rich diet (276 nmol/l) and in healthy men taking soy supplements (up to 3000 nmol/l) (24). The serum genistein levels recorded in TRAMP/FVB mice were in fact 2.0-fold lower than those achieved in humans (>7 µmol/l) by daily consumption of 300 mg genistein for 84 days with no evidence of genotoxicity (25).

The decreased incidence of PD cancer and accumulation of DLPs at the PIN stage by genistein may be due to (i) reduced proliferation and delayed progression into a less differentiated phenotype and/or (ii) interference with the activation/inhibition of specific pathways involved in the progression of PIN stage to PD cancer. Accumulating evidence suggests that constitutive activation of the phosphoinositol-3-kinase–Akt-signaling pathway is one of the mechanisms involved in the progression of various types of cancer including CaP (26). Activation of the Akt pathway confers a survival/growth advantage to cancer cells via the positive regulation of cyclin D1 via GSK-3 (9), interference with apoptosis (27–29), increased transcription of estrogen receptor (ER)-responsive genes (30) as well as activation of telomerase (31). We have observed an increase in phosphorylated Akt by 9 weeks of age, when >70% of prostates exhibited PIN pathology. This result is in agreement with the reported surge in phospho-Akt at the transition from histologically normal epithelium to PIN in human prostatic tissue (32). This increase was also accompanied by an increase in total Akt-1 by 9 weeks of age when compared with younger TRAMP/FVB mice (5 weeks old) with normal DLPs. To the best of our knowledge, this is the first report of an age-dependent increase in total Akt-1 levels in DLPs of TRAMP/FVB mice. It also further validates the TRAMP/FVB model, by corroborating with reports documenting increases in total Akt-1 levels in human CaP tissues (33). On the other hand, dietary genistein resulted in a reduction in Akt activation. The inhibition of Akt by genistein has been reported in a multitude of cancer cells including but not limited to prostatic cell lines (34–37). However, the reported Akt inhibition has been achieved with supraphysiological levels (25–50 µM) and therefore does not reflect the activities of physiological levels of genistein in vivo. Several mechanisms can underlie the inactivation of Akt by genistein in DLPs of TRAMP/FVB mice such as the inhibition of upstream receptor activation and/or activation of phosphatases that modulate Akt phosphorylation. The phosphatase and tensin homolog deleted in chromosome ten may be a likely target of genistein and has been shown to be induced by it in cultured breast cancer cells (38); however, we did not detect any modulation of phosphatase and tensin homolog deleted in chromosome ten protein levels by genistein in TRAMP/FVB DLPs (data not shown). The epidermal growth factor (EGF) receptor and ErbB2 over-expression, as well as the activation of the insulin-like growth factor (IGF)-1 -signaling pathways have been documented in TRAMP CaP progression; furthermore, genistein has been shown to decrease the expression of EGF receptor and IGF receptor-1 but not ErbB2 or IGF-1 in DLPs of genistein-fed (C57/BL6) TRAMP mice by 12 weeks of age (39). However, the inactivation of the EGF receptor/IGF receptor-signaling pathways in the DLPs of genistein-fed TRAMP/FVB remains to be verified.

Although dietary genistein did not significantly alter serum testosterone and estradiol levels in this study, its known ability to interact with the ER{alpha} and ß suggests that these may be possible targets of action in the prostate, resulting in decreased proliferation; either directly or via growth factor receptor signaling crosstalk. In fact, while androgen receptor and ER levels have not been reported to be altered in human CaP, the up-regulation of ER{alpha} and the androgen receptor, but not ERß transcripts has been reported in TRAMP cancer progression, suggesting that increased steroid signaling may contribute to tumorigenesis in this model (39). We have in fact observed a significant reduction in ER{alpha} and progesterone receptor with no effect on androgen receptor message levels by genistein (data not shown) suggesting that ER signaling might be repressed by dietary genistein, which could contribute to Akt inhibition. However, the available data related to modulation of sex steroid signaling by genistein still requires further elucidation.

The first identified substrate of Akt is GSK-3 (40). Akt has been shown to phosphorylate GSK-3 ({alpha}/ß) on serine 21 and 9, respectively, upon activation by EGF, insulin growth factor-1, nerve growth factor and insulin (41). Additionally to its role in glucose metabolism, GSK-3 plays a role in cell proliferation via regulation of genes involved in cell cycle progression and survival. In this work, the kinetics of GSK-3ß age-dependent phosphorylation and genistein-induced dephosphorylation in the TRAMP/FVB DLPs closely follow those of Akt, suggesting that GSK-3ß is under the control of Akt in the TRAMP/FVB model. We are led to this hypothesis due to evidence that Akt-induced phosphorylation of GSK-3 could be mediated by IGF-1 signaling, with both IGF-1 and IGF receptor-1 up-regulated in TRAMP CaP progression (39). However, since Akt is not the only kinase that phosphorylates GSK-3, we cannot eliminate the possible involvement of kinases such as p90rsk, p70 S6 kinase and protein kinase C (7), which remains to be determined. The significance of GSK-3 modulation by genistein is highlighted via its effects on cyclin D1 and snail-1. The regulation of cyclin D1 levels in TRAMP/FVB progression as well as by genistein does not seem to be transcriptional and may therefore be a result of translational (via activation or inhibition of the elongation/ initiation factor eIF4E (42) or post-translational effects (phosphorylation or absence of phosphorylation) of cyclin D1 by GSK-3 (9). The reduced adhesion in tumorigenesis involves loss of cadherin-1 expression via transcriptional repression among other mechanisms (43). The Snail family of proteins is implicated in cadherin-1 repression. Interestingly, active GSK-3ß is known to inhibit snail-1 transcription (11). We, in fact, have observed an increase in snail-1 levels that accompanied the age-dependent increase in Akt activation and GSK-3ß inactivation. More interestingly, genistein consumption resulted in a dose-dependent decrease in snail-1 levels concomitant with cadherin-1 accumulation. To our knowledge, this is the first report characterizing snail-1 kinetics during CaP progression and its modulation by genistein. This work has provided evidence suggesting that the Akt–GSK-3 pathway may be one of the pathways targeted by genistein in its prevention of PD cancer. These findings could pave the way to a better understanding of its chemopreventive capacities via modulation of Akt targets.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
References
 
Supplementary Figures 1 and 2 can be found at http://carcin.oxfordjournals.org/


   Acknowledgments
 
Grant support: This work was supported by National Institutes of Health grant R01 DK060875 to P.P.B.

Conflict of Interest statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

  1. Severson RK, et al. A prospective study of demographics, diet, and prostate cancer among men of Japanese ancestry in Hawaii. Cancer Res. (1989) 49:1857–1860.[Abstract/Free Full Text]
  2. Yatani R, et al. Geographic pathology of latent prostatic carcinoma. Int. J. Cancer (1982) 29:611–616.[Web of Science][Medline]
  3. Greenberg NM, et al. Prostate cancer in a transgenic mouse. Proc. Natl Acad. Sci. USA (1995) 92:3439–3443.[Abstract/Free Full Text]
  4. Liang J, et al. Multiple roles of the PI3K/PKB (Akt) pathway in cell cycle progression. Cell Cycle (2003) 2:339–345.[Medline]
  5. Bertram J, et al. Loss of PTEN is associated with progression to androgen independence. Prostate (2006) 66:895–902.[CrossRef][Web of Science][Medline]
  6. Majumder PK, et al. Prostate intraepithelial neoplasia induced by prostate restricted Akt activation: the MPAKT model. Proc. Natl Acad. Sci. USA (2003) 100:7841–7846.[Abstract/Free Full Text]
  7. Jope RS, et al. The glamour and gloom of glycogen synthase kinase-3. Trends Biochem. Sci. (2004) 29:95–102.[CrossRef][Web of Science][Medline]
  8. Diehl JA, et al. Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev. (1998) 12:3499–3511.[Abstract/Free Full Text]
  9. Alt JR, et al. Phosphorylation-dependent regulation of cyclin D1 nuclear export and cyclin D1-dependent cellular transformation. Genes Dev. (2000) 14:3102–3114.[Abstract/Free Full Text]
  10. Zhou BP, et al. Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat. Cell Biol. (2004) 6:931–940.[CrossRef][Web of Science][Medline]
  11. Bachelder RE, et al. Glycogen synthase kinase-3 is an endogenous inhibitor of Snail transcription: implications for the epithelial-mesenchymal transition. J. Cell Biol. (2005) 168:29–33.[Abstract/Free Full Text]
  12. Blanco MJ, et al. Correlation of Snail expression with histological grade and lymph node status in breast carcinomas. Oncogene (2002) 21:3241–3246.[CrossRef][Web of Science][Medline]
  13. Batlle E, et al. The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat. Cell Biol. (2000) 2:84–89.[CrossRef][Web of Science][Medline]
  14. Peinado H, et al. Transcriptional regulation of cadherins during development and carcinogenesis. Int. J. Dev. Biol. (2004) 48:365–375.[CrossRef][Web of Science][Medline]
  15. Hirohashi S. Inactivation of the E-cadherin-mediated cell adhesion system in human cancers. Am. J. Pathol. (1998) 153:333–339.[Abstract/Free Full Text]
  16. Mentor-Marcel R, et al. Genistein in the diet reduces the incidence of poorly differentiated prostatic adenocarcinoma in transgenic mice (TRAMP). Cancer Res. (2001) 61:6777–6782.[Abstract/Free Full Text]
  17. Touny LH, et al. Identification of both Myt-1 and Wee-1 as necessary mediators of the p21-independent inactivation of the cdc-2/cyclin B1 complex and growth inhibition of TRAMP cancer cells by genistein. Prostate (2006) 66:1542–1555.[CrossRef][Web of Science][Medline]
  18. Foster B, et al. Characterization of prostatic epithelial cell lines derived from transgenic adenocarcinoma mouse prostate (TRAMP) model. Cancer Res. (1997) 57:3325–3330.[Abstract/Free Full Text]
  19. Tani Y, et al. Epithelial-stromal tumor of the seminal vesicles in the transgenic adenocarcinoma mouse prostate model. Vet. Pathol. (2005) 42:306–314.[Abstract/Free Full Text]
  20. Quinn DI, et al. Molecular markers of prostate cancer outcome. Eur. J. Cancer (2005) 41:858–887.[CrossRef][Web of Science][Medline]
  21. Liao Y, et al. Increase of AKT/PKB expression correlates with gleason pattern in human prostate cancer. Int. J. Cancer (2003) 107:676–680.[CrossRef][Web of Science][Medline]
  22. Paul R, et al. The cadherin cell-cell adhesion pathway in prostate cancer progression. Br. J. Urol. (1997) 79:37–43.[Web of Science][Medline]
  23. Christofori G, et al. The role of the cell-adhesion molecule E-cadherin as a tumour-suppressor gene. Trends Biochem. Sci. (1999) 24:73–76.[CrossRef][Web of Science][Medline]
  24. Busby MG, et al. Clinical characteristics and pharmacokinetics of purified soy isoflavones: single-dose administration to healthy men. Am. J. Clin. Nutr. (2002) 75:126–136.[Abstract/Free Full Text]
  25. Miltyk W, et al. Lack of significant genotoxicity of purified soy isoflavones (genistein, daidzein, and glycitein) in 20 patients with prostate cancer. Am. J. Clin. Nutr. (2003) 77:875–882.[Abstract/Free Full Text]
  26. Li L, et al. The emerging role of the PI3-K-Akt pathway in prostate cancer progression. Prostate Cancer Prostatic Dis. (2005) 8:108–118.[CrossRef][Web of Science][Medline]
  27. Del Peso L, et al. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science (1997) 278:687–689.[Abstract/Free Full Text]
  28. Dijkers PF, et al. Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1. Curr. Biol. (2000) 10:1201–1204.[CrossRef][Web of Science][Medline]
  29. Lin HK, et al. Akt suppresses androgen-induced apoptosis by phosphorylating and inhibiting androgen receptor. Proc. Natl Acad. Sci. USA (2001) 98:7200–7205.[Abstract/Free Full Text]
  30. Banerjee S, et al. 17alpha-estradiol-induced VEGF-A expression in rat pituitary tumor cells is mediated through ER independent but PI3K-Akt dependent signaling pathway. Biochem. Biophys. Res. Commun. (2003) 300:209–215.[CrossRef][Web of Science][Medline]
  31. Kang SS, et al. Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase subunit. J. Biol. Chem. (1999) 274:13085–13090.[Abstract/Free Full Text]
  32. Paweletz CP, et al. Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front. Oncogene (2001) 20:1981–1989.[CrossRef][Web of Science][Medline]
  33. Le Page C, et al. Expression and localisation of Akt-1, Akt-2 and Akt-3 correlate with clinical outcome of prostate cancer patients. Br. J. Cancer (2006) 94:1906–1912.[CrossRef][Web of Science][Medline]
  34. Li Y, et al. Inhibition of nuclear factor kappaB activation in PC3 cells by genistein is mediated via Akt signaling pathway. Clin. Cancer Res. (2002) 7:2369–2377.
  35. Kim EJ, et al. Genistein inhibits insulin-like growth factor-I receptor signaling in HT-29 human colon cancer cells: a possible mechanism of the growth inhibitory effect of Genistein. J. Med. Food (2005) 8:431–438.[Web of Science][Medline]
  36. Gong L, et al. Inactivation of NF-kappaB by genistein is mediated via Akt signaling pathway in breast cancer cells. Oncogene (2003) 22:4702–4709.[CrossRef][Web of Science][Medline]
  37. Alhasan SA, et al. Genistein elicits pleiotropic molecular effects on head and neck cancer cells. Clin. Cancer Res. (2001) 12:4174–4181.
  38. Dave B, et al. The soy isoflavone genistein promotes apoptosis in mammary epithelial cells by inducing the tumor suppressor PTEN. Carcinogenesis (2005) 10:1793–1803.
  39. Wang J, et al. Genistein alters growth factor signaling in transgenic prostate model (TRAMP). Mol. Cell. Endocrinol. (2004) 219:171–180.[CrossRef][Web of Science][Medline]
  40. Cross DA, et al. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature (1995) 378:785–789.[CrossRef][Medline]
  41. Grimes CA, et al. The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Prog. Neurobiol. (2001) 65:391–426.[CrossRef][Web of Science][Medline]
  42. Muise-Helmericks RC, et al. Cyclin D expression is controlled post-transcriptionally via a phosphatidylinositol 3-kinase/Akt-dependent pathway. J. Biol. Chem. (1998) 273:29864–29872.[Abstract/Free Full Text]
  43. Cano A, et al. The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat. Cell Biol. (2000) 2:76–83.[CrossRef][Web of Science][Medline]
Received February 16, 2007; revised April 5, 2007; accepted April 19, 2007.


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