Deciphering the epigenetic mechanism of gene regulation and evaluation of medicinal plant products in modulation of chromatin modifications in cancer.

The development and progression of cancer is predominantly regulated by both tumor suppressor genes as well as oncogenes. Either gene mutation causes hyper-activation of ‘oncogenes’ and suppression of ‘cell cycle control and tumor suppressor genes’; or alleles of these sets of genes expression profiles are modulated by epigenetic mechanisms. Hence, cancer is the manifestation of both genetic and epigenetic alternations modulating the metabolic profile and cellular homeostasis. Since cellular metabolism is mostly controlled by enzymatic activities and enhanced during the disease progression, chemo-preventive bioactive molecules from edible and medicinal plants are being increasingly evaluated which could be one of the desired ‘toxicity free’ therapeutic strategies against cancer. This thesis attempts to investigate the anticancer and epigenetic regulatory activity of Paederia foetida (P. foetida) and its active constituents; lupeol and β-sitosterol. The regulation mechanism(s) of tumor suppressor gene E-cadherin (CDH1) is tested considering multiple modalities of epigenetic modulations; like, DNA methylation, histone methylation, acetylation and phosphorylation. The cellular environment(s)/metabolism(s) were experimentally modulated by ectopic application of drugs and/or H2O2 with the cell culture media/buffer executing in vitro cell cultures. Crude methanolic leaf extract of P. foetida (MEPL) and two of the identified products, lupeol and β-sitosterol exhibit anticancer activity through inhibition of ‘cellular viability and migration’ by up regulation of CDH1 gene expression. Along with this, it also induces apoptosis through up regulation of pro-apoptotic gene Bax and down regulation of the anti-apoptotic gene Bcl2 in human prostate (PC-3 and DU-145) and breast cancer (MCF-7 and MDA-MB-231) cell lines. Ectopic applications of MEPL, lupeol and β-sitosterol also significantly down regulated the expression of DNMTs and HDACs as well as reduced the total DNMT and HDAC activity. Loss of CDH1 protein and epithelial to mesenchymal transition (EMT) are key steps in cancer progression. Reactive oxygen species (ROS) within cells are the products of metabolic reactions and play some crucial roles in cellular metabolism, physiological functions and homeostasis. Roles of E-cadherin, EMT and ROS are discretely and intriguingly illustrated in many cancers focusing their collective concert during cancer progression. We report that hydrogen peroxide (H2O2) treatment modulate CDH1 gene expression by epigenetic modification(s). Sublethal dosage of H2O2 treatment decrease of E-cadherin, increase DNMT1, HDAC1, Snail, Slug and enrich H3K9me3 and H3K27me3 in the CDH1 promoter. The effect of H2O2 was attenuated by ROS scavengers; NAC, lupeol and β-sitosterol. DNMT inhibitor, AZA prevented the H2O2induced promoter-CpG-island methylation of CDH1. Treatment of cells with U0126 (inhibitor of ERK) reduced the expression of DNMT1, Snail and Slug, increased CDH1. This implicates that CDH1 is synergistically repressed by histone methylation, DNA methylation and histone deacetylation mediated chromatin remodelling and activation of Snail and Slug through ERK pathway. Increased ROS leads to activation of epigenetic machineries and EMT activators Snail/Slug which in their course of action inactivates CDH1 gene and lack of E-cadherin protein promotes EMT in breast cancer cells. ROS and ERK signaling facilitate epigenetic silencing and support the fact that subtle increase of ROS above basal level act as key cell signaling molecules. Free radical scavengers, lupeol and β-sitosterol may be tested for therapeutic intervention of breast cancer. In aggressive tumors, there is increase expression of enhancer of zeste homologue (EZH2) and silencing of CDH1 genes. EZH2 is histone-lysine N-methyltransferase enzyme encoded by EZH2 gene. It participates in histone methylation and, ultimately, transcriptional repression. EZH2 specifically catalyses the addition of methyl groups to histone H3 at lysine 27(H3K27). The role of oxidative stress upon EZH2 and EZH2 mediated CDH1 regulation is still not explored. To decipher this, this thesis aimed to investigate the effect of H2O2 induced ROS on EZH2 expression. Data obtained reveals the induced expression of EZH2 in H2O2 treated cells suggesting the role of oxidative stress in EZH2 expression. Again treatment with EZH2 siRNA results in enhanced expression of CDH1 and reduced expression of EZH2 suggesting the role of EZH2 in negative regulation of CDH1 (CDH1) expression. Simultaneous treatment with EZH2 siRNA and H2O2 results in enhanced CDH1 (CDH1) expression and decrease expression of EZH2 which reveals the involvement of oxidative stress in enhanced expression of EZH2 and thus reduced the expression of CDH1 in breast cancer. Moreover, other studies also demonstrate the regulation of CDH1 and EZH2 by DNA methylation and histone modifications. DNMT1 is overexpressed and inversely correlated with CDH1 expression in breast cancer. Our thesis here demonstrates the negative regulation of CDH1 by EZH2. Most importantly it was found that, by inhibiting DNMT1 with 5-AZA-cytidine, it induced the expression of CDH1 indicating DNA methylation as well as EZH2 methylation is responsible for CDH1 down regulation in breast cancer. H3K27me3 also helps in the methylation of EZH2 and EZH2 mediated trimethylation of H3K27 is a prerequisite for promoter DNA methylation. This thesis demonstrates the expression of EZH2 and occupancy H3K27me3 is notably higher in untreated control cells as compared to EZH2 siRNA treated cells suggesting that CDH1 expression is regulated by higher expression of EZH2 thus results in H3K27 hyper-methylation. Inhibition of histone deacetylation by TSA (HDAC inhibitor) also restores the expression of CDH1 by inhibiting the EZH2 expression. This suggests that, histone deacetylation also plays an important role in regulation of EZH2 expression. Collectively, this work broadens the amplitude of the epigenome, enhance the understanding of the modulation of the epigenome and open avenues for investigations on conjoint effects of canonical and intrinsic metabolite signaling and epigenetic modulations of genes during tumor development and cancer progression.