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Division of Hematology and Oncology, Department of Internal Medicine, The Ohio State University College of Medicine and Public Health, Columbus, OH 43210
3To whom correspondence should be addressed. E-mail: clinton-1{at}medctr.osu.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • IGF-I • tomato • soy • polyphenols • prostate
Prostate carcinogenesis is a complex process extending over decades and involving interactions among many environmental and lifestyle factors coupled with inheritance. A number of investigations, using diverse approaches, suggest that the insulin-like growth factor-I (IGF-I) network may be a key stimulant of prostate carcinogenesis and a target for dietary, chemopreventive and pharmacologic interventions. Several epidemiological studies have reported that higher plasma concentrations of IGF-I are associated with increased risk of prostate cancer (1–3). In support of observations of humans, overexpression of IGF-I in the prostate stimulates carcinogenesis in genetically manipulated mice (4). Several in vitro cell culture systems suggest that IGF-I has mitogenic and survival effects on normal and malignant prostate epithelial cells (PrEC) (5,6). Furthermore, we have shown that dietary energy restriction or feeding soy isoflavones will reduce prostate cancer progression in vivo in association with a lower plasma IGF-I (7,8).
Cellular and molecular mechanisms regarding how IGF-I acts on prostate and other cells are beginning to be discovered. IGF-I binds to its membrane receptor, activating the receptor tyrosine kinase (RTK), which subsequently phosphorylates intercellular substrates including the insulin receptor substrate-1 (IRS-1) (9), which in turn activates a cascade of downstream signals. Phosphatidylinositide 3-kinase (PI3K)/AKT and RAS/Raf/mitogen-activated protein kinase (MAPK) are the two best characterized intracellular signaling pathways downstream to IGF-I receptor (IGF-IR), and both pathways are postulated to stimulate cell proliferation and enhance resistance to apoptosis (10). Other signaling pathways, such as protein kinase C and Crk, may also be involved in IGF-I–mediated biological responses (10). In addition to showing mitogenic and survival responses in cancer cells, IGF-I may also contribute to activation of differentiation pathways in some cell types. Indeed, a high cellular density of the IGF-IR and low levels or an absence of IRS-I favor transmission of differentiation signaling leading to growth arrest and apoptosis as opposed to mitogenic effects (11,12). It is unclear whether IGF-IR and IRS-1 levels, their ratio or functional status (phosphorylation) can be regulated by dietary variables or chemopreventive regents.
The profound geographic variation in prostate cancer risk and the shift in risk with migration in low to high incidence countries have stimulated epidemiologic and experimental investigations into how diet and nutrition mediate prostate carcinogenesis (13,14). Evidence points to specific vegetables as contributing to a lower risk of prostate cancer. Soy products are associated with lower risk in Asian populations, and this theory is supported by our observations in a murine model (8). Tomatoes are another food hypothesized to lower the risk of prostate cancer based on epidemiologic (15,16) and rat studies (17). Each of these foods contains a number of components that could influence prostate cancer risk, such as protease inhibitors in soy and lycopene in tomato products. However, the studies reported herein focused on several representatives of a diverse array of small molecules in soy and tomatoes collectively referred to as polyphenols. Studies in various in vitro and in vivo models of cancer have suggested that these compounds potentially inhibit carcinogenesis via a variety of mechanisms including antioxidant activity, changes in carcinogen metabolism, modulation of cell cycle progression, alterations in intracellular signaling and inhibition of angiogenesis (13,18). However, much of the data relevant to mechanisms of action have been derived from systems other than prostate cancer and thus may not be relevant to the unique processes involved in prostate carcinogenesis.
The above observations form the basis of our hypothesis that polyphenols from tomato and soy products may reduce prostate carcinogenesis by altering the response of prostate cancer cells to IGF-I. We first defined an in vitro system using a prostate cancer cell line (rat AT6.3) in which proliferation and survival are clearly stimulated by IGF-I, thus eliminating the interactive effects of a multitude of growth factors and inhibitors typically found in serum. We next used specific inhibitors of intracellular signaling pathways to demonstrate the importance of certain pathways in IGF-I responses in AT6.3 prostate cancer cells. We then addressed the hypothesis that polyphenols from tomato and soy may inhibit IGF-I signaling cascades.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Polyphenols (Fig. 1) were purchased from Sigma (St. Louis, MO), dissolved in dimethyl sulfoxide as stock solutions and diluted in medium to designated concentrations. The MAPK/extracellular signal–regulated kinase (ERK) kinase (MEK) inhibitor PD98059 and PI3K inhibitor wortmannin were from Calbiochem (San Diego, CA). Recombinant human IGF-I was purchased from R & D Systems (Minneapolis, MN). Monoclonal murine anti-phosphorylated tyrosine was from Transduction Laboratories (Lexington, KY). Polyclonal rabbit anti-phosphorylated AKT and anti-phosphorylated ERK1/2 antibodies were from New England Biolabs (Beverly, MA). The polyclonal rabbit anti-IRS-1 antibody was from Upstate Biotechnology (Lake Placid, NY). Polyclonal rabbit anti-IGF-IRß subunit, anti-rabbit IgG, anti-mouse IgG, horseradish peroxidase–conjugated secondary antibodies and chemiluminescence reagents were from Santa Cruz Biotechnology (Santa Cruz, CA).
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The androgen-independent Dunning AT6.3 rat prostate adenocarcinoma cell line (19) was provided by Dr. John T. Isaacs (Johns Hopkins University, Baltimore, MD). Optimal growth is achieved in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 250 nmol/L dexamethasone, 2 mmol/L L-glutamine, 20,000 IU/L penicillin and 20 mg/L streptomycin, in a 5% CO2 humidified atmosphere at 37°C (20). Cells used in these studies were propagated for less than eight passages. PrEC were obtained from Clonetics (San Diego, CA) and grown in media supplemented with a mixture of growth factors provided by the manufacturer (21).
Cell viability and proliferation assays.
AT6.3 cells were plated at a density of 750 
1000 cells/well in 96-well plates and incubated in complete medium for 24 h. Similarly, PrEC cells were seeded in 96-well plates with a density of 3000 cells/well. Monolayers were then rinsed twice with HBSS and treated with vehicle or designated concentrations of polyphenols for 72 h. Cell viability was quantified by the [3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (MTS) assay according to the manufacturer’s instructions (Promega, Madison, WI), as described previously (22). Significant effects were confirmed by direct counting of cell number employing trypan blue exclusion analysis (23).
Cell cycle analysis.
Propidium iodide (PI) staining and flow cytometry were used to examine cell cycle progression (8,22). AT6.3 cells (4 x 105) were grown in T-75 flasks for 24 h, rinsed with HBSS twice and placed in serum-free medium (SFM) with IGF-I (50 µg/L) with or without polyphenols for 24, 48 or 72 h. Flow cytometry was performed on an Elite flow cytometer (Beckman-Coulter, Miami, FL), and cell cycle analysis was performed using Modfit Software (Verity Software House, Topsham, ME). A minimum of 20,000 cells was examined for each sample; all conditions were tested in duplicate; and the means presented represent at least two or more independent experiments.
Annexin V analysis.
AT6.3 cells were treated as described above. Adherent and floating cells were harvested and pooled, and then simultaneously stained with fluorescein isothiocyanate (FITC)-Annexin V and PI (Clontech, Palo Alto, CA). The apoptotic (FITC-stained or both FITC- and PI-stained) and necrotic (PI-stained) cells were detected by flow cytometric analyses (24). A minimum of 10,000 cells was examined for each sample; all assays were done in duplicate; and means presented represent at least two independent experiments.
Terminal deoxynucleotidyltransferase–mediated deoxyuridine 5'-triphosphate nick end labeling (TUNEL) analysis.
Cells were treated as above, fixed with 1% paraformaldehyde and then stored in 70% ethanol at -20°C for at least 2 h. DNA fragments from the apoptotic cells were labeled with fluorescein-nucleotides by the terminal deoxynucleotidyl transferase enzyme according to the protocols of the manufacturer (Intergen, Purchase, NY). Flow cytometry was performed to determine the percentage of cells exhibiting positive labeling (25). A minimum of 20,000 cells was examined for each sample; each assay condition was tested in duplicate; and means presented represent at least two independent experiments.
4',6-diamidino-2-phenylindole (DAPI) staining.
Cells were treated as described above and harvested. After washing with PBS, the cells were fixed in 70% ethanol for 20 min at room temperature, and then washed again with PBS. The cells were stained with DAPI (1 g/L) nuclear stain (Sigma) at 1:1000 dilution in the dark for 12 min (26). Cells were washed again with PBS, placed on glass slides and examined by fluorescence microscopy (Nikon Eclipse E800; Nikon Instruments, Melville, NY).
Western blotting analysis.
The cellular content of IGF-IRß and IRS-1 was examined by Western blotting in studies of 1.5 million cells initially seeded in 150-mm dishes for 24 h, then washed with HBSS and treated with specified agents and polyphenols for an additional 72 h. For the evaluation of tyrosine phosphorylation by Western blotting, cells (4 x 106) were seeded in 150-mm dishes with complete medium for 24 h. After rinsing with HBSS twice, cells were incubated in SFM with vehicle or 50 µmol/L polyphenols for 24 h, then stimulated by IGF-I at designated concentrations and harvested at specified time intervals. The MEK inhibitor PD98059 (50 µmol/L) or the PI3K inhibitor wortmannin (1µmol/L) were added 30 min prior to IGF-I stimulation in selected studies.
Treated cells were washed with ice-cold PBS twice, harvested, pelleted and immediately placed in ice-cold lysis buffer (50 mmol/L Tris-HCl, pH 7.4, 2 mmol/L EGTA, 10 mmol/L EDTA, 100 mmol/L NaF, 1 mmol/L Na4P2O7, 2 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride, 20 mg/L leupeptine, 20 mg/L aprotinin and 0.4% Triton X-100). Homogenates were centrifuged at 12,000 x g for 10 min at 4°C, and the Triton X-100 soluble fraction was collected. The protein concentration of each sample was determined with the Bio-Rad (Hercules, CA) protein assay kit. An equal amount of protein was loaded on lanes and separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride (PDVF membranes) (Bio-Rad). Membranes were blocked with 10 g/L bovine serum albumin in Tris-buffered saline, 0.1% Tween-20 for 1 h at room temperature for anti-phosphotyrosine antibody, whereas 50 g/L nonfat dry milk was the blocking agent for other antibodies. Membranes were incubated with specific antibodies overnight at 4°C. Immunoreactive proteins were identified by horseradish peroxidase-conjugated secondary antibodies, followed by enhanced chemiluminescence reagents and exposure to film. The membranes were scanned and quantified by densitometry (AlphaImager 2000; Alpha Innotech,San Leandro,CA). The relative expression levels were expressed as the percentage of ß-actin levels.
Statistical analysis.
Data were initially analyzed by ANOVA using Statview 4.5 software (Abacus Concepts, Berkeley, CA). In some studies, square-root transformation was completed if data were not normally distributed prior to ANOVA. If a significant effect of treatment was detected by ANOVA, Fisher’s protected least significant difference test was subsequently used to evaluate pairwise comparisons. A value of P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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IGF-I enhanced proliferation with a maximum at 50 µg/L when added to SFM. The cell number in incubations with 50 µg/L IGF-1 was greater than vehicle-treated cells in SFM (P < 0.001) and was 82% of the number when cells were treated with complete medium (P < 0.05) (Fig. 2). Cells grown in SFM achieved only 30% (P < 0.001) of the cell number found in complete medium at 72 h. The results of MTS analysis were confirmed by direct counting (data not shown). On the basis of these results, we selected IGF-I at 50 µg/L in serum-free conditions for the subsequent experiments.
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Genistein (10 µmol/L) inhibited IGF-I–induced proliferation by 24% (P < 0.05) compared with vehicle-treated cells, whereas other polyphenols required higher concentrations to inhibit proliferation (Fig. 3A). At 50 µmol/L, genistein potently inhibited IGF-I–stimulated proliferation (P < 0.001 vs. vehicle) with viability even lower than that of cells grown in SFM alone. Biochanin A, quercetin and kaempferol also inhibited IGF-I–induced AT6.3 cell proliferation (P < 0.001 vs. vehicle) with the 50% inhibitory concentration (IC50) for these polyphenols between 25 and 40 µmol/L. Daidzein and rutin were less potent with IC50 of >60 µmol/L.
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We subsequently demonstrated similar antiproliferative effects on normal PrEC in vitro (Fig. 3B). MTS assays demonstrated that genistein, biochanin A, quercetin and kaempferol inhibited PrEC growth with IC50 of <25 µmol/L (P < 0.001 for all, compared with vehicle). At 25 µmol/L, quercetin was more potent than other polyphenols (P < 0.05 vs. others). Daidzein and rutin had minimal activity. PrEC require a proprietary medium with a mixture of growth factors, including pituitary extract and insulin. Additional studies (data not shown) showed that replacement of insulin with IGF-I (50 µg/L) resulted in equivalent PrEC growth, and polyphenols showed similar inhibition patterns under those conditions.
Cell cycle progression in prostate cancer cells stimulated with IGF-I and treated with polyphenols.
As predicted, IGF-I initially increased the proportion of cells in S phase; the effect was optimal at 48 h when cells remained in log phase growth (Table 1). Serum deprivation arrested cells in G0/G1, whereas serum or IGF-I stimulation increased the proportion of cells in S (P < 0.01). Cell cycle analysis at 48 h showed a profound G2/M arrest by genistein and kaempferol at 50 µmol/L (P < 0.001) (Table 1, Fig. 4A), an effect even more pronounced at 72 h (data not shown). Quercetin and biochanin A produced a more subtle but significant G2/M arrest (both P < 0.05). Daidzein and rutin did not alter the proportion of cells in specific phases of the cell cycle (Table 1).
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Examination of the sub-G0 peak by flow cytometry provided the first clue that polyphenols antagonize the prosurvival signals of IGF-I (Table 1, Fig. 4A). The sub-G0 peak represents a late stage of cell death, in which the nucleus has fragmented into smaller packets containing less than the 2N complement of DNA. AT6.3 cells grown in SFM exhibited a sub-G0 peak of 5.66% compared with only 0.45% in those grown in serum (P < 0.001). Addition of IGF-I (50 µg/L) to SFM reduced the sub-G0 peak to 1.36% (P < 0.01 vs. cells in SFM). However, genistein or kaempferol at 50 µmol/L produced a sub-G0 peak (P < 0.05 vs. IGF-I or serum).
Flow cytometric and TUNEL analysis were also employed as a biomarker of DNA degradation associated with the activation of effector components of the apoptotic cascade. Cells grown in SFM showed increased TUNEL staining intensity by >60-fold after 72 h, which was almost completely prevented by 50 µg/L IGF-I (P < 0.001) (Table 1, Fig. 4B). Genistein antagonized IGF-I at 10 µmol/L (P < 0.001) as assessed by TUNEL staining. Genistein, biochanin A, quercetin and kaempferol at 50 µmol/L all increased TUNEL staining in the presence of IGF-I (P < 0.001). Daidzein and rutin had no effect.
Annexin V and PI analysis was another tool used to assess cellular changes associated with activation of apoptotic cascades. IGF-I (50 µg/L) reduced Annexin V staining compared with that of cells in SFM alone (P < 0.01) (Table 1). Genistein, biochanin A, quercetin and kaempferol (50 µmol/L) increased Annexin V positivity (P < 0.001 vs. vehicle controls) (Table 1, Fig. 4C). Genistein also had a significant effect at 10 µmol/L. DAPI staining and fluorescence microscopy were also employed to confirm the presence of early nuclear morphologic changes characteristic of apoptosis (Fig. 4D). For example, genistein (50 µmol/L)-treated AT6.3 cells showed typical changes associated with chromatin condensation and/or nuclear fragmentation after 72 h. In summary, data from cell cycle analysis, TUNEL, Annexin V, and DAPI staining supported the conclusion that genistein, biochanin A, quercetin and kaempferol counteract the survival and antiapoptotic effects of IGF-I in prostate cancer cells.
Polyphenols alter the ratio of IGF-IR to IRS-1.
Serum-starved AT6.3 cells had increased expression of IGF-IR (Fig. 5). Subsequent stimulation with IGF-I dramatically down-regulated the receptor content, a characteristic response to IGF-I in other cell lines (27,28). Genistein, biochanin A, quercetin and kaempferol each counteracted (P < 0.05) the down-regulation of IGF-IR after binding of the ligand by 47, 16, 24 and 37%, respectively, compared with vehicle alone (standardized to 100%). Daidzein and rutin did not prevent the down-regulation of the IGF-IR after IGF-I activation. Similar to IGF-IR, the expression of IRS-1 was high in AT6.3 cells grown in SFM. IGF-I incubation modestly reduced IRS-1 content by 40% (P < 0.01 vs. SFM). Interestingly, genistein, biochanin A, quercetin and kaempferol further reduced IRS-1 concentrations to 27, 34, 29 and 26% of that of cells grown under serum-free conditions (all P < 0.05, compared with IGF-I or serum free). Daidzein and rutin treatment had no effect compared with vehicle alone. Because we hypothesized that the relationship of IGF-IR to IRS-I influences the biological responses, we calculated the ratio based on densitometric evaluation for descriptive purposes only (data not shown). The agents most effective in reducing growth and enhancing apoptosis had a high ratio of the IGF-1R to IRS-1.
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PI3K and MEK inhibitors blocked proliferation in prostate cancer cells treated with IGF-I (P < 0.001 for PD098059 or PD098059 with wortmannin-treated cells; P < 0.01 for wortmannin-treated cells, compared with cells in IGF-I without inhibitor) (Fig. 6A). We next documented the ability of these inhibitors to alter the phosphorylation of the target proteins in AT6.3 cells (Fig. 6B). The MEK inhibitor PD098059 (50 µmol/L) abolished the phosphorylation of the downstream protein ERK1/2 but had no effect on AKT phosphorylation. The PI3K inhibitor wortmannin (1µmol/L) completely blocked the phosphorylation of AKT after IGF-I stimulation, with no inhibition of ERK1/2 phosphorylation. The two inhibitors together completely inhibited phosphorylation of both AKT and ERK1/2. These studies suggested that constitutively activated ERK1/2 in association with IGF-I–activated AKT pathways are critical for the proliferation of AT6.3 cells.
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IRS-1 phosphorylation was reduced by kaempferol, biochanin A, genistein and quercetin (Fig. 8A). Phosphorylated AKT was not detected in cells starved for 24 h, but was readily detected after IGF-I stimulation (Fig. 7). Compared with IGF-I–stimulated cells, AKT phosphorylation was inhibited in those treated with genistein (50%, P < 0.001), biochanin A (34%, P < 0.01), kaempferol (40%, P < 0.01) and quercetin (14%, P < 0.05) (Fig. 8A and B). MEK was constitutively active in AT6.3 cells, as demonstrated by the high level of phosphorylated ERK1/2 in the cells starved in SFM for 24 h. Interestingly, compared with the IGF-I vehicle—treated cells, ERK1/2 phosphorylation was suppressed in those exposed to genistein (30%, P < 0.01), kaempferol (19%, P < 0.05) and biochanin A (52%, P < 0.001). Quercetin, rutin and daidzein did not inhibit the phosphorylation of ERK1/2 (Fig. 8A and C). Overall, these studies suggest that the polyphenols have slightly different abilities to target specific components of the IGF-I signaling cascade.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In vitro and in vivo studies have shown that IGF-I stimulates proliferation and inhibits apoptosis in a variety of normal and cancer cells (24,29,30), although few studies thus far have focused on prostate cells (31–33). We observed that rat AT6.3 cells entered quiescence with a tendency for a small percentage of cells to exhibit markers of apoptosis when grown under serum-free conditions. IGF-I rapidly stimulated prostate cell cycle progression with a greater proportion of cells in the S phase over 24 to 48 h after exposure to the growth factor. Our cell proliferation data and cell cycle analysis support previous studies showing IGF-I stimulation of thymidine incorporation in human DU145 prostate cancer cells (32) and a mitogenic effect in rat PA-III cells (34). We observed that IGF-I completely reversed the tendency of AT6.3 cells to express markers of apoptosis, such as sub-G0 DNA cell fragments, TUNEL positivity and Annexin V staining under serum-free conditions. Overall, these observations support the hypothesis that IGF-I may be one of several critical growth factors stimulating proliferation and survival in prostate cancer cells.
To our knowledge, these studies are the first to demonstrate that polyphenols specifically block IGF-I–regulated events in prostate cancer cells. We selected a series of polyphenols from tomatoes and soy that exhibit slightly different structural characteristics (Fig. 1). As expected, the glycoside rutin did not modulate AT6.3 cell growth, apoptosis, or IGF-I–regulated intracellular signaling events. These observations emphasize that the polyphenols typically found in food as glycosides must be hydrolyzed in vivo to demonstrate anti-prostate cancer activity. Furthermore, one of the compounds evaluated, daidzein, showed essentially no activity under the conditions of our studies, demonstrating that small structural changes have a profound effect on bioactivity relative to IGF-I signaling. Genistein and biochanin A, both found in soy, and quercetin and kaempferol, representative of polyphenols in tomatoes and other fruits and vegetables, all could antagonize IGF-I, but with slightly different capacities to modulate specific components of the signaling cascade. Furthermore, the findings that each polyphenol exhibited a different potency in modulating specific intracellular events suggest that combinations of polyphenols may be more effective in slowing prostate cancer progression than single agents. These findings support our belief that combinations of dietary interventions, particularly whole-food products that provide an array of bioactive components with a broad spectrum of molecular targets, may have advantages over single chemopreventive agents.
We included representative data from studies employing normal human PrEC. These cells are very sensitive to changes in medium and cannot be propagated under conditions in which IGF-I is the only survival and proliferative signal, as can be done with the malignant AT6.3 cells. However, we feel that it is important to address the question concerning the selectivity and potency of the polyphenols to modulate PrEC growth in comparison with that of prostate cancer cells. We concluded that those polyphenols active in human (8) and rat prostate cell lines are also capable of inhibiting the proliferation of PrEC, thus demonstrating no clear selectivity. How these data should be interpreted relative to cancer prevention is open to debate. It is our opinion that factors enhancing the proliferation of normal epithelial cells, either external variables such as nutrients or internal variables such as hormones and growth factors, will increase the pool of cells at risk for genetic damage and thereby contribute to promoting prostate carcinogenesis (35,36). Therefore, exposure of epithelial cells to polyphenols may inhibit early steps in initiation as well as slow the progression of established lesions that are present decades prior to detection of clinically important disease (37).
Under conditions in which IGF-I was the driving force for proliferation and survival of AT6.3 prostate cancer cells, we documented the ability of several polyphenols to arrest the cell cycle. Genistein and kaempferol demonstrated a G2/M arrest at the time points we examined, whereas biochanin A and quercetin showed a weaker but similar effect. These observations support the earlier reports on genistein and prostate cancer cells, as well as other cell types, grown in various types of bovine serum (8,38–40). How genistein or kaempferol specifically block cell cycle progression has not been clearly elucidated. However, cyclin-dependent kinases (CDK) and cyclins (41,42) are crucial regulators of cell cycle progression, and the G2/M transition is regulated by CDK1 in combination with cyclins A and B (42). Alterations in the accumulation or degradation of cyclin B1 in response to polyphenols may be one mechanism whereby these compounds influence the G2/M transition, because B1 plays a critical role in stimulating progression from G2 through the exit of M phase (43,44). Genistein has also been shown to inhibit the growth of LNCaP cells in vitro in association with a suppression of cyclin B expression and enhanced expression of the CDK inhibitor p21/WAF1 (45,46). Quercetin causes growth inhibition and apoptosis of MCF-7 breast carcinoma cells by mechanisms that also appear to be dependent on the up-regulation of p21/WAF1 (47). In DU145 prostate cancer cells, quercetin has also been shown to increase p21 expression in association with reduced proliferation (48). Interestingly, biochanin A, which typically causes a G0/G1 arrest, was reported to decrease the expression of both cyclin B and p21 in LNCaP cells (49). These and other studies suggest that polyphenols act through processes that ultimately alter the pattern of key cell cycle regulators, although a comprehensive and detailed understanding of these events remains to be elucidated.
Resistance to apoptosis is an essential characteristic of cancer cells compared with normal epithelial cells (35). However, the resistance to apoptosis is not absolute but is best conceptualized as a change in sensitivity that can be modulated by growth factors and hormones, and perhaps other variables in the tumor microenvironment. We demonstrated that IGF-I promoted survival and enhanced the resistance of AT6.3 prostate cancer cells to apoptosis. We employed several different biomarkers of apoptosis, each of which demonstrated a slightly different feature of the apoptotic cascade. Annexin V staining detects early cell membrane changes, while the TUNEL assay documents cleavage of chromosomal DNA into smaller fragments, and flow cytometric evaluation of the sub-G0 peak as well as DAPI staining document the dissolution of the nucleus into smaller fragments characteristic of the final phases of apoptotic cell death. We documented that genistein, kaempferol, quercetin and biochanin A reversed the IGF-I survival benefits based on consistent changes in the expression of these apoptotic biomarkers. To our knowledge, few studies have examined the ability of polyphenols to modulate IGF-I–induced survival in cancer cells. One published example used the human MCF-7 beast cancer cell line and showed that IGF-I protects cells from apoptosis in the presence of the protein synthesis inhibitor cycloheximide, whereas genistein reverses the survival benefits of IGF-I (50). In summary, these studies document that several polyphenols commonly in foods can alter the apoptotic threshold of prostate cancer cells in vitro, a mechanism that we hypothesize may slow the progression of human prostate cancer.
Investigators are gradually determining the receptor-activated intracellular pathways that mediate the biological activity of IGF-I, although very few studies specifically focus on prostate cells. The IGF-IR is a glycoprotein complex consisting of two transmembrane ß subunits and two extracellular 
subunits (10). The ligand binding specificity is conferred by subunits, whereas ß subunits contain the tyrosine kinase (10). The expression of receptor subunits appears to be transcriptionally down-regulated by IGF-I through a negative feedback inhibition mechanism (27,28). Binding of IGF-I to its receptor causes activation of the RTK and autophosphorylation (51). IRS-1 is a critical substrate for the IGF-I RTK and contains multiple phosphorylation sites (52). IRS-1 functions as an adaptor protein for other SH2 proteins like PI3K (53). IGF-IR not only mediates the mitogenic and antiapoptotic actions of IGF-I but also may stimulate differentiation programs, depending on the cell type and the pattern of other regulators in the cellular environment (54). The relative effects of IGF-I on a cell, such as a predominant proliferation versus differentiation pathway, may also depend on the status of postreceptor signaling cascades (12,54–56). For example, it has been postulated that the ratio of IGF-IR to IRS-1 defines the type of cellular response to IGF-I. When the cellular content of IGF-IR is high, sufficient IRS-1 is required for cells to inhibit the differentiation program while favoring proliferation (54–56). When IRS-1 is low, a dominant differentiation program is activated (54–56). Consistent with these findings, transformed PrEC show a loss of IGF-IR expression, and reexpression of IGF-IR using a retroviral vector causes reduced tumorigenicity and inhibition of the malignant phenotype (57). In human and murine prostate cancers, IGF-IR expression is typically decreased or lost with advanced metastatic or androgen-independent lesions (58–60). However, few studies have examined factors that may influence the ratio of IGF-IR to IRS-1. Interestingly, our results showed that genistein, kaempferol, biochanin A and quercetin decreased the overall cell content of IRS-I based on Western blotting, and inhibited the down-regulation of IGF-IR expression induced by IGF-I stimulation. The high IGF-IR and low IRS-I in AT6.3 prostate cancer cells after exposure to polyphenols may enhance the terminal differentiation pathways that contribute to eventual cell death. Consistent with this hypothesis, genistein has been reported to enhance differentiation or maturation markers in several cancer cell lines, including the MCF-7 and MDA-MB-468 breast cancer and N2A neuroblastoma lines (61,62). Thus, our data support the hypothesis that the ability of bioactive polyphenols to alter the ratio of IGF-IR to IRS-I may be one mechanism enhancing apoptosis and contributing to an inhibition of the prostate cancer cascade.
Few studies have attempted to characterize IGF-I–mediated postreceptor downstream intracellular signaling cascades in prostate cancer cells. However, studies in other cell types have provided some insight into IGF-I signaling processes that may be relevant to prostate cancer. Evidence suggests that PI3K/AKT and MAPK are important pathways in transmitting IGF-I mitogenic and antiapoptotic signals (10,24,63). Our studies using specific inhibitors of MEK and PI3K demonstrated an inhibition of AT6.3 cell proliferation in parallel with an inhibition of the phosphorylation of ERK1/2 and AKT. Interestingly, the high phosphorylation of ERK1/2 in cells grown in serum-free conditions indicates that AT6.3 prostate cancer cells have acquired a defect in intracellular signaling that leads to constitutive activation of MAPK growth-promoting or survival pathways. This may occur in part via cell-cell interaction or aberrant expression of an autocrine-secreted growth factor(s), or through mutations of genes encoding ERK1/2 or upstream signaling proteins (64). For example, a PTEN mutation in LNCaP cells leads to constitutive phosphorylation of AKT in some prostate cancers (65). In contrast to ERK1/2, the phospho-AKT in AT6.3 cells was undetectable after 24 h in SFM, while stimulation with IGF-I rapidly increased AKT phosphorylation, which remained high for >24 h. Overall, our results suggest that activation of AKT may be one of the key pathways transmitting IGF-I proliferative and survival signals in AT6.3 cells and that constitutive activation of ERK1/2 also contributes to the survival of these cells in vitro.
Interestingly, the six polyphenols showed different abilities to target the phosphorylation of the signaling proteins examined. Genistein, biochanin A, kaempferol and quercetin potently suppressed the phosphorylation of IRS-1 and AKT. Genistein, biochanin A, kaempferol, but not quercetin, potently inhibited ERK1/2 phosphorylation. Daidzein and rutin had no effect on the phosphorylation of IRS-1, AKT or ERK1/2. These results suggest that the suppression of several tyrosine kinase–mediated signaling pathways is strongly associated with the ability of specific polyphenols to inhibit IGF-I growth and survival signals in prostate cancer cells.
A key question that remains is the relevance of these findings to in vivo conditions. The in vitro system provides an elegant approach for identifying IGF-I–regulated processes without confounding by interacting growth factors and hormones as well as cell-to-cell interactions, and cell-to-matrix interactions found in a tumor microenvironment in vivo. The data generated from our in vitro studies must now be validated in the more complex in vivo rodent or human systems (66). It is our hope that our findings in cell cultures will provide biomarkers that are relevant to human intervention trials with tomato or soy products, or perhaps studies of energy balance in which the IGF-I axis may be perturbed. An additional critical issue regarding these data concerns the relevance of dose-response findings under in vitro conditions to the in vivo environment. The blood concentrations of many polyphenols are often in the range of 1–10 µmol/L under conditions of typical consumption, and little information is yet available regarding tissue concentrations (66). We have previously hypothesized that in vitro conditions for cancer cells are optimized for growth and survival, whereas the in vivo microenvironment is suboptimal, perhaps best characterized as harsh, due to hypoxia, accumulated metabolic waste, increased interstitial pressure and poor perfusion of nutrients associated with chaotic vascularity (8). We propose that the harsh conditions in vivo allow for enhanced sensitivity of tumor cells to antiproliferative and proapoptotic polyphenols and perhaps other dietary components that have anticancer properties. Certainly, our studies with mice bearing a transplantable prostate tumor and fed a dietary soy concentrate support this hypothesis (8). We suggest that caution should be used when attempting to directly extrapolate in vitro findings, relative to specific concentrations, to in vivo expectations.
Finally, our studies suggest that various polyphenols have slightly different abilities to modulate growth factor signaling cascades. The implication of these observations is that tomato and soy products provide a diverse array of compounds that together, over a long period of dietary exposure, may provide an important opportunity to slow prostate carcinogenesis. Obviously, these concepts must be rigorously tested in translational investigations.
In conclusion, these studies support the hypothesis that dietary polyphenols derived from tomato and soy products may suppress the progression of prostate carcinogenesis via inhibition of IGF-I–stimulated intracellular signaling cascades critical for growth and survival of prostate cancer cells.
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FOOTNOTES |
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2 Supported by National Institutes of Health/National Cancer Institute R01 CA72482-03 and by P30 CA16058.
4 Abbreviations used: CDK, cyclin-dependent kinase; DAPI, 4',6-diamidino-2-phenylindole; ERK, extracellular signal–regulated kinase; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; IC50, 50% inhibitory concentration; IGF-I, insulin-like growth factor-I; IGF-IR, IGF-I receptor; IRS-1, insulin receptor substrate-1; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MTS, [3-(4,5-dimethylthiazol-zyl-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]; PI, propidium iodide; PI3K, phosphatidylinositide 3-kinase; RTK, receptor tyrosine kinase; SFM, serum-free medium; TUNEL, terminal deoxynucleotidyltransferase–mediated deoxyuridine 5'-triphosphate nick end labeling.
Manuscript received 13 January 2003. Initial review completed 19 February 2003. Revision accepted 18 April 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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