Abstract 4079: Proanthocyanidins reactivate silenced tumor suppressor genes p16INK4 a and Cip1/p21 by reducing DNA methylation and increasing histone acetylation in human non-small cell lung cancer cells

Abstract

Abstract Non-small cell lung cancer (NSCLC) represents α80% of all types of lung cancer and includes squamous cell carcinoma, adenocarcinoma and large-cell carcinomas. The risk of NSCLC is greatly affected by epigenetic modulations in lung cells. Cancer epigenetics deal with important processes including DNA methylation and histone modification patterns that can either activate oncogenes or inactivate tumor suppressor genes, the crucial steps in tumor development. The study of epigenetic regulations has become a new target for early detection, risk assessment, prognosis and cancer therapy. Therefore, to develop more effective therapeutic agents for lung cancer, we sought to determine the effect of grape seed proanthocyanidins (GSPs), which have been shown to have anti-carcinogenic effects in some tumor models, on epigenetic modulation in NSCLC cells. For this purpose two genetically different NSCLC cell lines, A549 (p53 wild type) and H1299 (p53 mutant), were selected and treated with lower concentrations of GSPs (5, 10, 20µg/ml) for six days. The status of DNA global methylation patterns was determined by immunostaining using a monoclonal antibody specific to 5-methylcytosine (5-mC) and global DNA methylation assay. Treatment of cells with GSPs resulted in a dose-dependent decrease in 5-mC-positive cells as compared to non-GSPs-treated controls. These data were verified by dot-blot analysis. Global methylation levels of DNA as obtained from quantitative DNA methylation assay reduced to 36±5% in A549 cells and 31±5% in H1299 cells with the treatment of GSPs. The expression levels of the DNA methyltransferases, e.g., DNMT1, DNMT3a and DNMT3b, were higher in non-GSPs treated cells and reduced with GSPs treatment as analyzed by western blotting and real-time PCR. The hypermethylation of DNA in NSCLC cells may lead to silencing of tumor suppressor genes, thereby contributing to the risk of lung cancer. Therefore, we analyzed the methylation status of the various tumor suppressor genes, like p16INK4a, Cip1/p21 and RASSF1A through methylation specific PCR and the relative repression levels of their mRNA were analyzed using real-time PCR, which indicated that the DNA methylation levels of these tumor suppressor genes were reduced in GSPs treated cells, and that resulted in re-expression of the mRNA and proteins of silenced tumor suppressor genes compared to non-GSPs treated cells. GSPs decreased histone deacetylase activity and increased the levels of histone acetylation which included the acetylated lysine 9 and 14 on histone H3 (H3-Lys 9 and 14) and acetylated lysine 5 and 12 on histone H4 but decreased the levels of methylated H3-Lys 9. Together, our study provides new insight into the epigenetic mechanism of action of GSPs that may have important implications for epigenetic therapy of lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4079. doi:1538-7445.AM2012-4079