DNA demethylation and histone deacetylation inhibition co‐operate to re‐express estrogen receptor beta and induce apoptosis in prostate cancer cell‐lines

Abstract

tom@pelhamcourt.clara.co.uk Introduction Epigenetic silencing mechanisms are increasingly thought to play a major role in the development of human cancers, including prostate cancer. The two major epigenetic regulatory mechanisms involve alterations in DNA methylation and histone acetylation status. Promoter CpG island hypermethylation and histone hypoacetylation, catalysed by DNA methyltransferase (DNMT) and histone deacetylase (HDAC) respectively, are associated with transcriptional repression. Evidence in other cancers suggests the two mechanisms are dynamically linked, yet few studies have examined the potential interaction of the two pathways in prostate cancer. Here we report a synergistic, apoptotic effect of treatment with a demethylating agent and a histone deacetylase inhibitor on cultured prostate cancer cells, with associated re-expression of the putative antiproliferative agent oestrogen receptor beta.Methods LNCaP, DU-145 and PC-3 cells were pre-treated with the DNMT inhibitor 5'-aza-2'-deoxycytidine (5-AZAC) for 72 hours followed by 24 hours combined treatment with 5-AZAC and the HDAC inhibitor trichostatin A (TSA). Following treatment, cells were either processed for cell proliferation, cell toxicity, cell viability and apoptosis assays, or harvested for quantitative real-time RT-PCR gene expression analyses. Assessed target genes were oestrogen receptor beta (ERβ), androgen receptor (AR), progesterone receptor (PGR) and prostate specific antigen (PSA). ResultsIn all cell-lines co-treatment with 5-AZAC and TSA was associated with significantly reduced cell proliferation when compared with control groups (p<0.05); associated reduced cell viability and increased cell death was seen in all cases. Caspase activation was significantly increased in the co-treatment group, indicating that apoptosis was a major mechanism of cell death. Most marked effects were seen in the androgen-dependent, AR-positive LNCaP cell-line. In all cell-lines a marked synergistic re-expression of ERβ was identified in the co-treatment group, a finding which was not seen for either AR or PSA. A similar re-expression of PGR was also identified. ConclusionsAt concentrations associated with gene re-expression, the DNA demethylating agent 5-AZAC and the HDAC inhibitor TSA co-operate in an additive and synergistic way to induce apoptosis in prostate cancer cell-lines. Increased apoptosis in the co-treatment group was associated with marked re-expression of ERβ, which is an intriguing finding, raising the possibility of further targeting of prostate cancer cells with ERβ-selective agents such as phytooestrogens and selective oestrogen receptor modulators (SERMs).

Effect of selective estrogen receptor modulators on cell proliferation and estrogen receptor activities in normal human prostate stromal and epithelial cells

“…Hurtado et al 26 for example showed that overexpression of ERb1 (ERb) induced a cell cycle arrest in the early G1 phase in LNCaP cells. Walton et al 27 showed that re-expression of ERb using a DNA demethylating agent and a histone deacetylase inhibitor caused increased apoptosis in prostate cancer cells. Thus, carcinogenesis in prostate seems to be characterized by a loss of ERb expression.…”

Histone deacetylase inhibitors in cancer therapy. A review

“…1) HDAC inhibitors were combined with other epigenetic modifiers. Inhibitors of DNA methyl transferases 5-azacytidine (azacitidine) and 5-aza-2'-deoxycytidine (decitabine) had increased antitumor effects when used with HDAC inhibitors [60][61][62][63][64] . Decitabine and VPA both induced apoptosis and the combination increased their effects both in vitro and in vivo 65,66 .…”

Nucleoside analogs: molecular mechanisms signaling cell death

“…In contrast, deacetylation of these residues is associated with a repressive state (Rice and Allis, 2001). Consequently, combinations of histone deacetylase inhibitors with hypomethylating agents results in reactivation of gene expression (Richon and O'Brien, 2002), cell cycle arrest and apoptosis (Zhu et al, 2001b;Tang et al, 2004;Schmelz et al, 2005;Walton et al, 2008). The azanucleosides analogs have also shown synergistic activity with conventional nucleoside analog chemotherapeutic agents such as 5-fluorouracil, which is based on their ability to reactivate previously silenced proapoptotic genes (Kanda et al, 2005;Morita et al, 2006).…”