Limits...
Resveratrol regulates the PTEN/AKT pathway through androgen receptor-dependent and -independent mechanisms in prostate cancer cell lines.

Wang Y, Romigh T, He X, Orloff MS, Silverman RH, Heston WD, Eng C - Hum. Mol. Genet. (2010)

Bottom Line: In contrast, resveratrol directly binds epidermal growth factor receptor (EGFR) rapidly inhibiting EGFR phosphorylation, resulting in decreased AKT phosphorylation, in an AR-independent manner.Thus, resveratrol may act as potential adjunctive treatment for late-stage hormone refractory prostate cancer.More importantly, for the first time, our study demonstrates the mechanism by which AR regulates PTEN expression at the transcription level, indicating the direct link between a nuclear receptor and the PI3K/AKT pathway.

View Article: PubMed Central - PubMed

Affiliation: Genomic Medicine Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA.

ABSTRACT
The tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10) and the androgen receptor (AR) play important roles in tumor development and progression in prostate carcinogenesis. Among many functions, PTEN negatively regulates the cytoplasmic phosphatidylinositol-3-kinase/AKT anti-apoptotic pathway; and nuclear PTEN affects the cell cycle by also negatively regulating the MAPK pathway via cyclin D. Decreased PTEN expression is correlated with prostate cancer progression. Over-expression of AR and upregulation of AR transcriptional activity are often observed in the later stages of prostate cancer. Recent studies indicate that PTEN regulates AR activity and stability. However, the mechanism of how AR regulates PTEN has never been studied. Furthermore, resveratrol, a phytoalexin enriched in red grapes, strawberries and peanuts, has been shown to inhibit AR transcriptional activity in prostate cancer cells. In this study, we use prostate cancer cell lines to test the hypothesis that resveratrol inhibits cellular proliferation in both AR-dependent and -independent mechanisms. We show that resveratrol inhibits AR transcriptional activity in both androgen-dependent and -independent prostate cancer cells. Additionally, resveratrol stimulates PTEN expression through AR inhibition. In contrast, resveratrol directly binds epidermal growth factor receptor (EGFR) rapidly inhibiting EGFR phosphorylation, resulting in decreased AKT phosphorylation, in an AR-independent manner. Thus, resveratrol may act as potential adjunctive treatment for late-stage hormone refractory prostate cancer. More importantly, for the first time, our study demonstrates the mechanism by which AR regulates PTEN expression at the transcription level, indicating the direct link between a nuclear receptor and the PI3K/AKT pathway.

Show MeSH

Related in: MedlinePlus

Resveratrol-induced PTEN promoter activity is mediated by AR. (A) C4-2 cells were co-transfected with plasmids expressing PTEN-Luc/Renilla-Luc and treated as indicated for 48 h. PTEN promoter activity was measured by dual luciferase assay. AR-ligand DHT inhibited PTEN promoter activity in a dose-dependent manner and resveratrol stimulated PTEN promoter activity. In contrast, AR-antagonist Casodex was shown to stimulate PTEN promoter activity, with resveratrol only adding slightly to this effect (*P < 0.005). (B) Knockdown of AR results in increased PTEN promoter activity. C4-2 cells were co-transfected with control siRNA or anti-AR siRNA and PTEN-luc/Renilla-Luc plasmids. After 48 h of treatment, PTEN promoter activity was measured by luciferase assay. The insert shows the knockdown of AR protein expression (*P < 0.005). (C) DU145 and C4-2 cells were transfected with PTEN-Luc/Renilla-Luc plasmids. Cells were treated with DMSO, or 10 µm resveratrol for 48 h. PTEN promoter activity was measured by luciferase assay. The structures of the −1134 to −1 and truncated −1134 to −1001 PTEN promoters are shown on the left panel. (D) AR-negative DU145 and AR-positive CWR22rv1 cells were treated with DMSO, 10, 20 or 50 µm resveratrol for 48 h. Western blots show PTEN, phospho-AKT, total-AKT and Actin levels. The quantification of phospho-AKT is shown in Supplementary Material, Fig. S3. Note that resveratrol did not change PTEN levels in DU145 cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2957324&req=5

DDQ354F3: Resveratrol-induced PTEN promoter activity is mediated by AR. (A) C4-2 cells were co-transfected with plasmids expressing PTEN-Luc/Renilla-Luc and treated as indicated for 48 h. PTEN promoter activity was measured by dual luciferase assay. AR-ligand DHT inhibited PTEN promoter activity in a dose-dependent manner and resveratrol stimulated PTEN promoter activity. In contrast, AR-antagonist Casodex was shown to stimulate PTEN promoter activity, with resveratrol only adding slightly to this effect (*P < 0.005). (B) Knockdown of AR results in increased PTEN promoter activity. C4-2 cells were co-transfected with control siRNA or anti-AR siRNA and PTEN-luc/Renilla-Luc plasmids. After 48 h of treatment, PTEN promoter activity was measured by luciferase assay. The insert shows the knockdown of AR protein expression (*P < 0.005). (C) DU145 and C4-2 cells were transfected with PTEN-Luc/Renilla-Luc plasmids. Cells were treated with DMSO, or 10 µm resveratrol for 48 h. PTEN promoter activity was measured by luciferase assay. The structures of the −1134 to −1 and truncated −1134 to −1001 PTEN promoters are shown on the left panel. (D) AR-negative DU145 and AR-positive CWR22rv1 cells were treated with DMSO, 10, 20 or 50 µm resveratrol for 48 h. Western blots show PTEN, phospho-AKT, total-AKT and Actin levels. The quantification of phospho-AKT is shown in Supplementary Material, Fig. S3. Note that resveratrol did not change PTEN levels in DU145 cells.

Mentions: Resveratrol has recently been shown to induce PTEN protein expression in MCF-7, a breast cancer cell line (17). Clinical observations note that AR levels correlated with PTEN protein expression and that the ratio between the two proteins by immunohistochemistry is correlated with patient survival and outcome (21). At the same time, AR over-expression and hyper-activity are often observed in androgen-independent prostate cancer in response to anti-androgen therapy (3,22). Our data above demonstrate that resveratrol (often considered a phyto-estrogen), acting as a weak estrogen, inhibits AR transcriptional activity in both androgen-dependent LNCaP cells and androgen-independent C4-2 cells (Fig. 2B and C). We then set out to determine the relationship of resveratrol-regulated AR activity on PTEN transcription. Androgen-independent C4-2 cells were transfected with PTEN promoter reporter plasmid and treated with 10 µm resveratrol combined with different concentrations of DHT or with Casodex. Resveratrol significantly stimulated PTEN promoter activity 3-fold (Fig. 3A). Interestingly, we found that DHT decreased PTEN promoter activity and mitigated resveratrol-induced PTEN promoter activity in a dose-dependent manner (Fig. 3A). In contrast, Casodex alone dramatically increased PTEN promoter activity to similar levels as that of resveratrol alone or both Casodex and resveratrol (Fig. 3A). To further confirm our observations here suggesting that AR regulates the PTEN promoter, we knocked down AR by transfecting C4-2 cells with anti-AR siRNA. AR siRNA significantly decreased AR expression (Fig. 3B, insert) and increased PTEN promoter activity 2.5-fold (Fig. 3B). Furthermore, additional treatment with DHT or Casodex after AR knockdown could not further regulate PTEN promoter activity (Fig. 3B). Taken together, therefore, our data here suggest that resveratrol-induced PTEN transcription is mediated by AR inhibition, at least in prostate cancer cells.Figure 3.


Resveratrol regulates the PTEN/AKT pathway through androgen receptor-dependent and -independent mechanisms in prostate cancer cell lines.

Wang Y, Romigh T, He X, Orloff MS, Silverman RH, Heston WD, Eng C - Hum. Mol. Genet. (2010)

Resveratrol-induced PTEN promoter activity is mediated by AR. (A) C4-2 cells were co-transfected with plasmids expressing PTEN-Luc/Renilla-Luc and treated as indicated for 48 h. PTEN promoter activity was measured by dual luciferase assay. AR-ligand DHT inhibited PTEN promoter activity in a dose-dependent manner and resveratrol stimulated PTEN promoter activity. In contrast, AR-antagonist Casodex was shown to stimulate PTEN promoter activity, with resveratrol only adding slightly to this effect (*P < 0.005). (B) Knockdown of AR results in increased PTEN promoter activity. C4-2 cells were co-transfected with control siRNA or anti-AR siRNA and PTEN-luc/Renilla-Luc plasmids. After 48 h of treatment, PTEN promoter activity was measured by luciferase assay. The insert shows the knockdown of AR protein expression (*P < 0.005). (C) DU145 and C4-2 cells were transfected with PTEN-Luc/Renilla-Luc plasmids. Cells were treated with DMSO, or 10 µm resveratrol for 48 h. PTEN promoter activity was measured by luciferase assay. The structures of the −1134 to −1 and truncated −1134 to −1001 PTEN promoters are shown on the left panel. (D) AR-negative DU145 and AR-positive CWR22rv1 cells were treated with DMSO, 10, 20 or 50 µm resveratrol for 48 h. Western blots show PTEN, phospho-AKT, total-AKT and Actin levels. The quantification of phospho-AKT is shown in Supplementary Material, Fig. S3. Note that resveratrol did not change PTEN levels in DU145 cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2957324&req=5

DDQ354F3: Resveratrol-induced PTEN promoter activity is mediated by AR. (A) C4-2 cells were co-transfected with plasmids expressing PTEN-Luc/Renilla-Luc and treated as indicated for 48 h. PTEN promoter activity was measured by dual luciferase assay. AR-ligand DHT inhibited PTEN promoter activity in a dose-dependent manner and resveratrol stimulated PTEN promoter activity. In contrast, AR-antagonist Casodex was shown to stimulate PTEN promoter activity, with resveratrol only adding slightly to this effect (*P < 0.005). (B) Knockdown of AR results in increased PTEN promoter activity. C4-2 cells were co-transfected with control siRNA or anti-AR siRNA and PTEN-luc/Renilla-Luc plasmids. After 48 h of treatment, PTEN promoter activity was measured by luciferase assay. The insert shows the knockdown of AR protein expression (*P < 0.005). (C) DU145 and C4-2 cells were transfected with PTEN-Luc/Renilla-Luc plasmids. Cells were treated with DMSO, or 10 µm resveratrol for 48 h. PTEN promoter activity was measured by luciferase assay. The structures of the −1134 to −1 and truncated −1134 to −1001 PTEN promoters are shown on the left panel. (D) AR-negative DU145 and AR-positive CWR22rv1 cells were treated with DMSO, 10, 20 or 50 µm resveratrol for 48 h. Western blots show PTEN, phospho-AKT, total-AKT and Actin levels. The quantification of phospho-AKT is shown in Supplementary Material, Fig. S3. Note that resveratrol did not change PTEN levels in DU145 cells.
Mentions: Resveratrol has recently been shown to induce PTEN protein expression in MCF-7, a breast cancer cell line (17). Clinical observations note that AR levels correlated with PTEN protein expression and that the ratio between the two proteins by immunohistochemistry is correlated with patient survival and outcome (21). At the same time, AR over-expression and hyper-activity are often observed in androgen-independent prostate cancer in response to anti-androgen therapy (3,22). Our data above demonstrate that resveratrol (often considered a phyto-estrogen), acting as a weak estrogen, inhibits AR transcriptional activity in both androgen-dependent LNCaP cells and androgen-independent C4-2 cells (Fig. 2B and C). We then set out to determine the relationship of resveratrol-regulated AR activity on PTEN transcription. Androgen-independent C4-2 cells were transfected with PTEN promoter reporter plasmid and treated with 10 µm resveratrol combined with different concentrations of DHT or with Casodex. Resveratrol significantly stimulated PTEN promoter activity 3-fold (Fig. 3A). Interestingly, we found that DHT decreased PTEN promoter activity and mitigated resveratrol-induced PTEN promoter activity in a dose-dependent manner (Fig. 3A). In contrast, Casodex alone dramatically increased PTEN promoter activity to similar levels as that of resveratrol alone or both Casodex and resveratrol (Fig. 3A). To further confirm our observations here suggesting that AR regulates the PTEN promoter, we knocked down AR by transfecting C4-2 cells with anti-AR siRNA. AR siRNA significantly decreased AR expression (Fig. 3B, insert) and increased PTEN promoter activity 2.5-fold (Fig. 3B). Furthermore, additional treatment with DHT or Casodex after AR knockdown could not further regulate PTEN promoter activity (Fig. 3B). Taken together, therefore, our data here suggest that resveratrol-induced PTEN transcription is mediated by AR inhibition, at least in prostate cancer cells.Figure 3.

Bottom Line: In contrast, resveratrol directly binds epidermal growth factor receptor (EGFR) rapidly inhibiting EGFR phosphorylation, resulting in decreased AKT phosphorylation, in an AR-independent manner.Thus, resveratrol may act as potential adjunctive treatment for late-stage hormone refractory prostate cancer.More importantly, for the first time, our study demonstrates the mechanism by which AR regulates PTEN expression at the transcription level, indicating the direct link between a nuclear receptor and the PI3K/AKT pathway.

View Article: PubMed Central - PubMed

Affiliation: Genomic Medicine Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA.

ABSTRACT
The tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10) and the androgen receptor (AR) play important roles in tumor development and progression in prostate carcinogenesis. Among many functions, PTEN negatively regulates the cytoplasmic phosphatidylinositol-3-kinase/AKT anti-apoptotic pathway; and nuclear PTEN affects the cell cycle by also negatively regulating the MAPK pathway via cyclin D. Decreased PTEN expression is correlated with prostate cancer progression. Over-expression of AR and upregulation of AR transcriptional activity are often observed in the later stages of prostate cancer. Recent studies indicate that PTEN regulates AR activity and stability. However, the mechanism of how AR regulates PTEN has never been studied. Furthermore, resveratrol, a phytoalexin enriched in red grapes, strawberries and peanuts, has been shown to inhibit AR transcriptional activity in prostate cancer cells. In this study, we use prostate cancer cell lines to test the hypothesis that resveratrol inhibits cellular proliferation in both AR-dependent and -independent mechanisms. We show that resveratrol inhibits AR transcriptional activity in both androgen-dependent and -independent prostate cancer cells. Additionally, resveratrol stimulates PTEN expression through AR inhibition. In contrast, resveratrol directly binds epidermal growth factor receptor (EGFR) rapidly inhibiting EGFR phosphorylation, resulting in decreased AKT phosphorylation, in an AR-independent manner. Thus, resveratrol may act as potential adjunctive treatment for late-stage hormone refractory prostate cancer. More importantly, for the first time, our study demonstrates the mechanism by which AR regulates PTEN expression at the transcription level, indicating the direct link between a nuclear receptor and the PI3K/AKT pathway.

Show MeSH
Related in: MedlinePlus