Epigenetic alterations impede epithelial-mesenchymal transition by modulating centrosome amplification and Myc/RAS axis in triple negative breast cancer cells.

Alterations in centrosome proteins may result in centrosome abnormalities such as disorganized spindles and centrosome amplification, leading to aneuploidy and genomic instability. Centrosomes exhibit unique epigenetic properties in which structural or positional information is propagated through somatic lineage by non-genetic pathways. Excessive centrosome amplification in breast cancer is accompanied by efficient clustering and loss of E-cadherin, indicating an important adaptive mechanism of cancer. This study sought to elucidate the effect of epigenetic alterations on centrosome amplification, epithelial-mesenchymal transition (EMT) and apoptosis in triple negative human breast adenocarcinoma derived MDA-MB-231 cell line. The results obtained here show that siRNA mediated silencing of DNMT1 and specific inhibition of HDAC1 & HDAC2 by Tricostatin A (TSA) synergistically inhibit cell proliferation through modulation of centrosome proteins γ-tubulin, TUBGCP2 and pericentrin. In addition, induction of apoptosis was observed by downregulation of Bcl2, upregulation of Bax and activation of PARP cleavage. Inhibition of EMT was confirmed through upregulation of E-cadherin and downregulation of N-cadherin and vimentin. Similarly, downregulation of Myc, RAS and CDK2, which plays important roles in proliferation and survival, was observed. Nuclear protein analysis revealed downregulation in the nuclear translocation of E2F1, which regulates centrosome amplification and metastasis in breast cancer. In conclusion, this study confirmed the role of epigenetic regulators in centrosome amplification and suggests that inhibition of DNA methylation and histone deacetylation-mediated chromatin remodelling synergistically disrupt EMT through modulation of centrosome amplification and Myc/RAS axis to potentiate apoptosis and attenuate cell proliferation in triple negative breast cancer cells.

SWI/SNF chromatin remodelling complex contributes to clearance of cytoplasmic protein aggregates and regulates unfolded protein response in Saccharomyces cerevisiae.

Chromatin remodelling complexes are multi-subunit assemblies, each containing a catalytic ATPase and translocase that is capable of mobilizing nucleosomes to alter the chromatin structure. SWI/SNF remodelling complexes with higher DNA translocation efficiency evict histones or slide the nucleosomes away from each other making DNA accessible for transcription and repair machinery. Chromatin remodelling at the promoter of stress-responsive genes by SWI/SNF becomes necessary during the heat and proteotoxic stress. While the involvement of SWI/SNF in transcription of stress-responsive genes has been studied extensively, the regulation of proteostasis by SWI/SNF is not well understood. This study demonstrates critical functions of SWI/SNF in response to cadmium-induced proteotoxic stress. Deletion of either ATPase-translocase subunit of SWI/SNF complex (Swi2/Snf2) or a regulatory subunit Swi3 abrogates the clearance of cadmium-induced protein aggregates. Our results suggest that Snf2 and Swi3 regulate the protein folding in endoplasmic reticulum (ER) that reduces the chances of forming unfolded protein aggregates under the proteotoxic stress of cadmium. The Ire1-mediated unfolded protein response (UPR) maintains ER homeostasis by upregulating the expression of chaperones and ER-associated degradation (ERAD) components. We found that Snf2 maintains normal oxidative environment essential for Ire1 activity. Deletion of SNF2 reduced the Ire1 activity and UPR, indicating involvement of Snf2 in Ire1-mediated ER proteostasis. Together, these findings suggest that SWI/SNF complex regulates ER homeostasis and protein folding crucial for tolerating proteotoxic stress.

Paederia foetida induces anticancer activity by modulating chromatin modification enzymes and altering pro-inflammatory cytokine gene expression in human prostate cancer cells.

Aberrant epigenetic modifications are responsible for tumor development and cancer progression; however, readily reversible. Bioactive molecules from diets are promising to cure cancer by modulating epigenetic marks and changing immune response. These compounds specifically target the activity of DNMTs and HDACs to cure various human cancers. In view of this, we investigated the anticancer and epigenetic regulatory activities of an edible-plant Paederia foetida. The efficacy of methanolic extract of P. foetida leaves (MEPL) was tested for the modulation of epigenetic factors in gene silencing, i.e. DNMT and HDAC and expression pattern of certain tumor-suppressor genes. After treatment of prostate cancer cells (PC-3 and DU-145) with MEPL, lupeol and ß-sitosterol; induction of apoptosis, decrease in cellular-viability and inhibition of cellular-migration were noticed. Simultaneously there was inhibition of DNMT1, HDACs and pro-inflammatory, IL-6, IL1-ß, TNF-α and anti-inflammatory, IL-10 genes in cancer and THP1 cell lines. The DNMT1 protein content, enzyme activity and Bcl2 expression decreased significantly; however, expression of E-cadherin (CDH1) and pro-apoptotic gene Bax increased significantly after the treatment of cells with drugs. We conclude plant-derived compounds can be considered to target epigenetic machineries involved with malignant transformation and can open new avenues for cancer therapeutics provoking immune response.

Epigenetic silencing of genes enhanced by collective role of reactive oxygen species and MAPK signaling downstream ERK/Snail axis: Ectopic application of hydrogen peroxide repress CDH1 gene by enhanced DNA methyltransferase activity in human breast cancer.

Loss of E-cadherin and epithelial to mesenchymal transition (EMT) are key steps in cancer progression. Reactive oxygen species (ROS) play significant roles in cellular physiology and homeostasis. Roles of E-cadherin (CDH1), EMT and ROS are intriguingly illustrated in many cancers without 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 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 beta-sitosterol. DNMT inhibitor, AZA prevented the H2O2 induced 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 beta-sitosterol may be tested for therapeutic intervention of breast cancer. This work broadens the amplitude of epigenome and open avenues for investigations on conjoint effects of canonical and intrinsic metabolite signaling and epigenetic modulations in cancer.

DNA methylation regulates Microtubule-associated tumor suppressor 1 in human non-small cell lung carcinoma.

Microtubule associated tumor suppressor 1 (MTUS1) has been recognized as a tumor suppressor gene in multiple cancers. However, the molecular mechanisms underlying the regulation of MTUS1 are yet to be investigated. This study aimed to clarify the significance of DNA methylation in silencing MTUS1 expression. We report that MTUS1 acts as tumor suppressor in non-small cell lung carcinoma (NSCLC). Analysis of in silico database and subsequent knockdown of DNMT1 suggested an inverse correlation between DNMT1 and MTUS1 function. Interestingly, increased methylation at MTUS1 promoter is associated with low expression of MTUS1. Treatment with DNA methyltransferases (DNMTs) inhibitor, 5-aza-2'-deoxycytidine (AZA) leads to both reduced promoter methylation accompanied with enrichment of H3K9Ac and enhanced MTUS1 expression. Remarkably, knockdown of MTUS1 showed increased proliferation and migration of NSCLC cells in contrast to diminished proliferation and migration, upon treatment with AZA. We concluded that low expression of MTUS1 correlates to DNA methylation and histone deacetylation in human NSCLC.

Curcumin Modulates Glycolytic Metabolism and Inflammatory Cytokines via Nrf 2 in Dalton's Lymphoma Ascites Cells In Vivo.

BACKGROUND: Warburg effect is characterized by the upregulation of HIF-1 and c-Myc regulated LDH-A, even aerobically owing to hypoxic environment and alterations in oncogenes or tumor suppressor genes in cancer. Reduced antioxidant defence system in transformed cells favors higher ROS production, which plays a significant role in carcinogenesis and acts as an important regulator of NF-κB. In addition, various proinflammatory cytokines play active roles in maintenance and progression of cancer. OBJECTIVE: In continuation with our previous studies illustrating the long-term effect of curcumin using a liver tissue, present study was aimed to elucidate the anti-cancer effect of curcumin due to its long-term effect in the regulation of glycolytic metabolism, NF-κB activation, expression of proinflammatory cytokines in Dalton's lymphoma ascites cells in vivo. METHOD: Spectrophotometric assays, RT-PCR and EMSA were performed to address the problems. RESULTS: Results revealed that curcumin-induced activation of antioxidant enzymes, Nrf2 and downstream signaling gene NQO1. Reduction of oxidative stress, down-regulation of NADPH: Oxidase, decline in ROS and H2O2 levels were also observed. Activation of NF-κB, expression of COX2, HIF-1α and cMyc, as well as expression and activity of LDH-A were significantly reduced by curcumin. Besides, expression of proinflammatory cytokines was significantly down-regulated via reducing binding of nuclear protein with AP-1, NF-IL6, ETS and NF-κB binding elements of IL-1α, IL-1ß, TNF-α and IL-6 promoters, respectively. CONCLUSION: Curcumin downregulates glycolytic metabolism via modulation of stress-activated genes and reduces oxidative stress by enhancing antioxidant defence system, which inhibits activation of NF-κB signaling and expression of proinflammatory cytokines in Dalton's lymphoma ascites cells in vivo.

Epigenetics of reproductive infertility.

Infertility is a complex pathophysiological condition. It may caused by specific or multiple physical and physiological factors, including abnormalities in homeostasis, hormonal imbalances and genetic alterations. In recent times various studies implicated that, aberrant epigenetic mechanisms are associated with reproductive infertility. There might be transgenerational effects associated with epigenetic modifications of gametes and studies suggest the importance of alterations in epigenetic modification at early and late stages of gametogenesis. To determine the causes of infertility it is necessary to understand the altered epigenetic modifications of associated gene and mechanisms involved therein. This review is devoted to elucidate the recent mechanistic advances in regulation of genes by epigenetic modification and emphasizes their possible role related to reproductive infertility. It includes environmental, nutritional, hormonal and physiological factors and influence of internal structural architecture of chromatin nucleosomes affecting DNA and histone modifications in both male and female gametes, early embryogenesis and offspring. Finally, we would like to emphasize that research on human infertility by gene knock out of epigenetic modifiers genes must be relied upon animal models.

Long term effect of curcumin in restoration of tumour suppressor p53 and phase-II antioxidant enzymes via activation of Nrf2 signalling and modulation of inflammation in prevention of cancer.

Inhibition of carcinogenesis may be a consequence of attenuation of oxidative stress via activation of antioxidant defence system, restoration and stabilization of tumour suppressor proteins along with modulation of inflammatory mediators. Previously we have delineated significant role of curcumin during its long term effect in regulation of glycolytic pathway and angiogenesis, which in turn results in prevention of cancer via modulation of stress activated genes. Present study was designed to investigate long term effect of curcumin in regulation of Nrf2 mediated phase-II antioxidant enzymes, tumour suppressor p53 and inflammation under oxidative tumour microenvironment in liver of T-cell lymphoma bearing mice. Inhibition of Nrf2 signalling observed during lymphoma progression, resulted in down regulation of phase II antioxidant enzymes, p53 as well as activation of inflammatory signals. Curcumin potentiated significant increase in Nrf2 activation. It restored activity of phase-II antioxidant enzymes like GST, GR, NQO1, and tumour suppressor p53 level. In addition, curcumin modulated inflammation via upregulation of TGF-ß and reciprocal regulation of iNOS and COX2. The study suggests that during long term effect, curcumin leads to prevention of cancer by inducing phase-II antioxidant enzymes via activation of Nrf2 signalling, restoration of tumour suppressor p53 and modulation of inflammatory mediators like iNOS and COX2 in liver of lymphoma bearing mice.

Long term effect of curcumin in regulation of glycolytic pathway and angiogenesis via modulation of stress activated genes in prevention of cancer.

Oxidative stress, an important factor in modulation of glycolytic pathway and induction of stress activated genes, is further augmented due to reduced antioxidant defense system, which promotes cancer progression via inducing angiogenesis. Curcumin, a naturally occurring chemopreventive phytochemical, is reported to inhibit carcinogenesis in various experimental animal models. However, the underlying mechanism involved in anticarcinogenic action of curcumin due to its long term effect is still to be reported because of its rapid metabolism, although metabolites are accumulated in tissues and remain for a longer time. Therefore, the long term effect of curcumin needs thorough investigation. The present study aimed to analyze the anticarcinogenic action of curcumin in liver, even after withdrawal of treatment in Dalton's lymphoma bearing mice. Oxidative stress observed during lymphoma progression reduced antioxidant enzyme activities, and induced angiogenesis as well as activation of early stress activated genes and glycolytic pathway. Curcumin treatment resulted in activation of antioxidant enzyme super oxide dismutase and down regulation of ROS level as well as activity of ROS producing enzyme NADPH:oxidase, expression of stress activated genes HIF-1α, cMyc and LDH activity towards normal level. Further, it lead to significant inhibition of angiogenesis, observed via MMPs activity, PKCα and VEGF level, as well as by matrigel plug assay. Thus findings of this study conclude that the long term effect of curcumin shows anticarcinogenic potential via induction of antioxidant defense system and inhibition of angiogenesis via down regulation of stress activated genes and glycolytic pathway in liver of lymphoma bearing mice.

Curcumin attenuates carcinogenesis by down regulating proinflammatory cytokine interleukin-1 (IL-1α and IL-1ß) via modulation of AP-1 and NF-IL6 in lymphoma bearing mice.

Interleukin-1 (IL-1α and IL-1ß) is a prototypic, potent, multifunctional proinflammatory cytokine affecting almost all cell types. Expression of IL-1 is up regulated in different tumor phenotypes and is implicated as an important factor in tumor progression via expression of metastatic, angiogenic genes and growth factors. Therefore, down regulation of expression of IL-1 may be able to inhibit cancer progression. Mechanism of transcriptional regulation of mouse IL-1α is not yet reported. AP-1 binding site at -12 to -6 on human IL-1α promotor is highly conserved in rat IL-1α gene and regulates its expression. Based on in silico analysis, regions -12 to -6bp is found to be conserved in human and mouse IL-1α gene promotor and therefore selected to study activation of IL-1α. Further, the regions -12 to -6bp in mouse IL-1α gene promotor corresponding to AP-1 binding element show 3'→5' orientation, necessary for AP-1 binding. The present work is focused on long term effect of curcumin on expression of IL-1α and IL-1ß in liver of lymphoma bearing mice. Transcriptional regulation of IL-1α and IL-1ß was analyzed by AP-1 and NF-IL-6 respectively. Elevated expression and protein level of IL-1α and IL-1ß were found in lymphoma bearing mice compared to normal, which were significantly down regulated by curcumin treatment. Similarly, curcumin treatment down regulated activation of IL-1α and IL-1ß via AP-1 and NF-IL-6 respectively. The findings conclude that curcumin attenuates carcinogenesis by down regulating proinflammatory cytokine interleukin-1 (IL-1α and IL-1ß) via modulation of AP-1 and NF-IL6 respectively in lymphoma bearing mice.

Long-term effect of curcumin down-regulates expression of tumor necrosis factor-α and interleukin-6 via modulation of E26 transformation-specific protein and nuclear factor-κB transcription factors in livers of lymphoma bearing mice.

Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are typical multifunctional proinflammatory cytokines involved actively in the regulation of immunity, hematopoiesis, inflammation and carcinogenesis. Coordinated regulation of these cytokines could be required for effective regulation of early phase inflammation and cancer progression. The present work was aimed to analyze the anti-carcinogenic action of curcumin on the expression of TNF-α and IL-6 even after withdrawal of treatment. Up-regulated expressions of TNF-α and IL-6 in terms of mRNA and protein levels in lymphoma bearing mice were significantly down-regulated by curcumin as compared to normal. Electrophoretic mobility shift assay (EMSA) results revealed that curcumin reduced binding of nuclear protein with ETS and NF-κB binding elements of TNF-α and IL-6 promoters, respectively. The anti-carcinogenic effect of curcumin against lymphoma progression has been reported previously. In continuation, the present study suggests that the long-term effect of curcumin may contribute to attenuate cancer progression via the down-regulation of TNF-α and IL-6 modulated by E26 transformation-specific protein (ETS) and nuclear factor-κB (NF-κB), respectively.

Anti-carcinogenic action of curcumin by activation of antioxidant defence system and inhibition of NF-κB signalling in lymphoma-bearing mice.

NF-κB (nuclear factor κB) plays a significant role in inflammation, immunity, cell proliferation, apoptosis and malignancy. ROS (reactive oxygen species) are among the most important regulating factors of NF-κB. Intracellular ROS are mainly regulated by an endogenous antioxidant defence system. Any disruption of redox balance leads to oxidative stress, which causes a number of pathological conditions including inflammation and malignancy. Increased metabolic activity in cancerous cells leads to oxidative stress, which is further enhanced due to depletion of the endogenous antioxidant defence system. However, the activation and signalling of NF-κB are reported to be inhibited by overexpression and induced activity of antioxidant enzymes. Therefore the present study focuses on the correlation between the endogenous antioxidant defence system, ROS and NF-κB activation during lymphoma growth in mice. The study highlights the anti-carcinogenic role of curcumin by modulation of NF-κB activation and oxidative stress via the endogenous antioxidant defence system. Oxidative stress was monitored by lipid peroxidation, protein carbonylation and antioxidant enzyme activity. NF-κB-mediated signalling was tested by DNA-binding activity. The results reflect that intracellular production of H2O2 in oxidative tumour micro-environment regulates NF-κB activation. Curcumin inhibits oxidative state in the liver of lymphoma-bearing mice by enhancing the transcription and activities of antioxidant enzymes, which in turn modulate activation of NF-κB, leading to a decrease in lymphoma growth. Morphological changes as well as cell proliferation and cell survival assays confirmed reduced lymphoma growth. Thus curcumin contributes to cancer prevention by disrupting the vicious cycle of constant ROS production, responsible for a high oxidative micro-environment for tumour growth.