Abstract 3796: Inhibition of epigenetic chromatin-modification enzymes: histone deacetylases and DNA methyltransferases by silibinin in human NSCLC H1299 cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Epigenetic modifications of histones (e.g. hypoacetylation) and DNA (e.g. hypermethylation) cause reversible alterations in chromatin structure leading to aberrant changes in gene expression, which eventually impact cellular processes like cell cycle, apoptosis, and invasion during malignant transformation of cells. Several tumor suppressor genes like CDH1, RARB, RASSF1A, MGMT etc are silenced by these interlinked mechanisms in human lung cancer; the leading diagnosed and lethal malignancy world wide. Hence, the impetus has been on developing novel agents that target ‘epigenome’ for both cancer therapy and chemoprevention. Silibinin, a flavanolignan isolated from the seeds of milk thistle exerts strong anti-cancer efficacy against many epithelial cancers including lung. Recently, we showed that silibinin inhibits p53 null-H1299 cell growth through G1 arrest by modulating the expression and function of associated key cell-cycle regulators. Herein, we assessed the effect of silibinin on major chromatin-modification enzymes to delineate their possible involvement in the observed silibinin effects. A time response study for histone deacetylase (HDAC) activity in total cell extract showed that silibinin treatment causes a dose-dependent decrease in enzyme activity from 6h [(100 µM; p<0.05)]-72h [(75-100 µM; p<0.001)]. Concomitantly, under similar treatment conditions, a sustained accumulation of acetylated histones (H3 and H4) in total cellular chromatin was observed between 6-24h time points. We also addressed the question whether silibinin directly inhibits HDAC activity in a cell free system. In both total and nuclear cell extracts, a decreasing trend in enzyme activity was observed by silibinin addition in incubation reactions, but the concentration for 50% inhibition (IC50) was ∼ 400 µM, suggesting an indirect effect of silibinin in regulating HDAC activity in cell free system. Consistent with these results, silibinin (75-100 µM) also caused a decrease in the protein levels of HDAC1, HDAC2, HDAC3, DNMT1 and DNMT3A in nuclear and total extracts after 24, 48 and 72h of treatment. Next, we evaluated the expression profile of 84 key genes encoding chromatin-modification enzymes in H1299 cells after silibinin treatment (100 µM; 72h). Real time RT-PCR array analysis identified a subset of fifteen genes, primarily including DNMT3A, HDAC1, HDAC6, SET domain proteins (SETD1A, D4, D6) and lysine specific demethylases (KDM 5B, 5C, 4A) which were significantly down-regulated (∼2-4 fold) in silibinin treated samples compared to vehicle controls. Collectively, these results show that silibinin inhibits specific histone deacetylases, histone demethylases and DNA methyltransferases, which might cooperatively contribute to the anti-cancer efficacy of this non-toxic phytochemical. (Supported by CA113876) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3796.