Promoter demethylation and chromatin remodeling by green tea polyphenols leads to re-expression of GSTP1 in human prostate cancer cells

Abstract

Epigenetic silencing of gluthathione-S-transferase pi (GSTP1) is recognized as being a molecular hallmark of human prostate cancer. We investigated the effects of green tea polyphenols (GTPs) on GSTP1 re-expression and further elucidated its mechanism of action and long-term safety, compared with nucleoside-analog inhibitor of DNA methyltransferase (DNMT), 5-aza-2'-deoxycitidine. Exposure of human prostate cancer LNCaP cells to 1-10 microg/ml of GTP for 1-7 days caused a concentration- and time-dependent re-expression of GSTP1, which correlated with DNMT1 inhibition. Methyl-specific-PCR and sequencing revealed extensive demethylation in the proximal GSTP1 promoter and regions distal to the transcription factor binding sites. GTP exposure in a time-dependent fashion diminished the mRNA and protein levels of MBD1, MBD4 and MeCP2; HDAC 1-3 and increased the levels of acetylated histone H3 (LysH9/18) and H4. Chromatin immunoprecipitation assays demonstrated that cells treated with GTP have reduced MBD2 association with accessible Sp1 binding sites leading to increased binding and transcriptional activation of the GSTP1 gene. Exposure of cells to GTP did not result in global hypomethylation, as demonstrated by methyl-specific PCR for LINE-1 promoter; rather GTP promotes maintenance of genomic integrity. Furthermore, exposure of cells to GTP did not cause activation of the prometaststic gene S100P, a reverse response noted after exposure of cells to 5-aza-2'deoxycitidine. Our results, for the first time, demonstrate that GTP has dual potential to alter DNA methylation and chromatin modeling, the 2 global epigenetic mechanisms of gene regulation and their lack of toxicity makes them excellent candidates for the chemoprevention of prostate cancer.

Figure 1

Effect of green tea polyphenols (GTP), its major constituent, epigallocatechin-3-gallate (EGCG) and nucleoside DNA methyltransferase inhibitor, 5-aza-2′deoxycitidine (5-Aza-dC) on 5-cytosine DNA methyltransferase (DNMT) in human prostate cancer LNCaP cells. A, dose-dependent inhibition of DNMT activity by various concentrations of GTP 5–20μg/mL and 5–20μM of EGCG and 5-Aza-dC B, time-dependent inhibition of DNMT activity at 10μg/mL GTP and 10μM of EGCG and 5-Aza-dC for 1–7 days. The results are mean of 6–8 determinations and are analyzed by one way ANOVA, bars ± SD. C, dose- and time- dependent inhibition of DNMT1 protein expression after GTP treatment, anti-Oct-1 is used as loading control, and D, dose- and time- dependent inhibition of DNMT1 mRNA by GTP at indicated doses and times. The results of mRNA levels are mean ± SD of 6–8 determinations **P<0.001, compared to control. Dotted line in the graph represents DNMT1 mRNA levels expressed in normal human prostate epithelial cells. The details are described in ‘Materials and Methods’ section.

Figure 2

Effect of green tea polyphenols (GTP), its major constituent, epigallocatechin-3-gallate (EGCG) and nucleoside DNA methyltransferase inhibitor, 5-aza-2′deoxycitidine (5-Aza-dC) on glutathione-S-transferase pi (GSTP1) re-expression in human prostate cancer LNCaP cells. A, dose- and time- dependent re-expression of GSTP1 protein by 5–10μg/mL GTP, 5–10μM 5-Aza-dC and 10–20μM of EGCG by using ELISA assay from Biotrin International. The results are mean of 6–8 determinations and are analyzed by one way ANOVA, bars ± SD and **P<0.001, compared to control. B, dose- and time-dependent re-expression of GSTP1 protein at indicated doses and times of treatment with GTP, positive control used is total lysate from DU145 cells. C, GSTP1 re-expression at message level using 10μg/mL GTP for 1–7 days and 10μM 5-Aza-dC for 3 days, M, marker; TBP is used as internal control. D, immunofluorescence staining for GSTP1 after GTP treatment. Constitutive cytoplasmic expression of GSTP1 in DU145 cells, positive control. Lack of detectable GSTP1 expression in either the cytoplasm or nucleus of naïve human prostate cancer LNCaP cells (control) as evident from the complete absence of green fluorescence in the cells. Re-expression of GSTP1 in LNCaP cells after 1, 3 and 7 days of treatment with 10μg/ml concentration with GTP and with 10μM 5-Aza-dC for 3 days as evident by the green fluorescence in the cytoplasm surrounding the blue, DAPI-stained nucleus.

Figure 3

Effect of green tea polyphenols (GTP) on reversal of methylation in human prostate cancer LNCaP cells. A, Map of the GSTP1 gene and putative CpG islands. The relative position and sizes of the 7 exons (solid bars) of the GSTP1 are depicted. B, MS-PCR for GSTP1 promoter on genomic DNA isolated from cells after treatment with 10μg/mL GTP for 7 days and 10μM 5-Aza-dC for 3 days. Displayed are the products generated with primers specific for unmethylated GSTP1 CpG island alleles (U) and for hypermethylated GSTP1 CpG island alleles (M). C, methylation analysis of individual clones from proximal and D, distal GSTP1 promoter after GTP and 5-Aza-dC treatment. Reduction in GSTP1 CpG island hypermethylation was noted in LNCaP DNA by these treatments. At least 15 PCR clones from each treatment were sequenced; the CpG island DNA methylation pattern for selected clones are displayed. Closed circles indicate a methylated CpG and open circles indicate an unmethylated CpG. The details are described in ‘Materials and Methods’ section.

Figure 4

Effect of green tea polyphenols (GTP) on methyl-CpG binding proteins and chromatin accessibility by transcription factor in human prostate cancer LNCaP cells. A, protein expression and densitometry of MBD1, MeCP2 and MBD4 in LNCaP cells after 1, 3 and 7 days of treatment with 10 μg/ml concentration with GTP and with 10μM 5-Aza-dC for 3 days. B, alterations in message expression of MBD1, MeCP2 and MBD4 after similar treatments. The results of mRNA levels are mean ± SD of 6–8 determinations **P<0.001, compared to control. Dotted line in the graph represents corresponding MBDs mRNA levels expressed in normal human prostate epithelial cells. C, ChIP assay for MBD2 association with GSTP1 promoter, D, ChIP assay for Sp1 promoter accessibility to the DNA by PCR analysis. GTP treatment to LNCaP cells caused decrease association with MBD2 and an increase in Sp1 binding to the GSTP1 proximal promoter, an effect not observed after treatment with 5-Aza-dC. The details are described in ‘Materials and Methods’ section.

Figure 5

Effect of green tea polyphenols (GTP) and histone deacetylase inhibitor, Trichostatin A (TSA) on histone deacetylases (HDACs) and histone modification in human prostate cancer LNCaP cells. A, time-dependent inhibition of total HDAC activity after 10μg/mL GTP treatment and 10nM TSA for 24 h. B, protein expression and densitometry of HDAC1, HDAC2 and HDAC3 in LNCaP cells after 1, 3 and 7 days of treatment with 10μg/ml concentration with GTP and with 10nM TSA for 24 h. C, alterations in messenger expression of HDAC1, HDAC2 and HDAC3 after similar treatments. The results of mRNA levels are mean ± SD of 6–8 determinations **P<0.001, compared to control. Dotted line in the graph represents corresponding HDACs mRNA levels expressed in normal human prostate epithelial cells. D, histone acetylation after GTP treatment. Increase in acetylation of H3 at lysine 9 and 18 and total H4 acetylation was observed with10μg/ml concentration with GTP and with 10nM trichostatin A, an inhibitor or histone deacetylase. The details are described in ‘Materials and Methods’ section.

Figure 6

Effect of green tea polyphenols (GTP) on cellular toxicity. A, global hypomethylation in human prostate cancer LNCaP cells was assessed by the methylation status of LINE-1 promoter. MS-PCR analysis of CpG island within LINE-1 promoter after treatment of cells with10μg/ml concentration with GTP and with 10μM 5-Aza-dC for 3 days was assessed. A decrease in unmethylated sequences was observed after GTP treatment whereas 5-Aza-dC treatment caused LINE-1 hypomethylation. M, methylated sequences and U, unmethylated sequences. B, 5-Aza-dC treatment to LNCaP cells caused severe cytotoxicity as evident by morphological changes whereas with10μg/ml concentration of GTP did not cause cytotoxicity. C, S100P mRNA expression by RT-PCR analysis after treatment with 5-Aza-dC and GTP. A significant decrease in S100P mRNA expression was observed after GTP treatment but not with 5-Aza-dC. Graph represents mRNA levels normalized to GAPDH. Results are mean ± SD of 3 different experiments. **P<0.001, compared to control. The details are described in ‘Materials and Methods’ section.