Dietary isothiocyanates inhibit cancer progression by modulation of epigenome.

Cell cycle, growth, survival and metabolism are tightly regulated together and failure in cellular regulation leads to carcinogenesis. Several signaling pathways like the PI3K, WNT, MAPK and NFKb pathway exhibit aberrations in cancer and help achieve hallmark capabilities. Clinical research and in vitro studies have highlighted the role of epigenetic alterations in cancer onset and development. Altered gene expression patterns enabled by changes in DNA methylation, histone modifications and RNA processing have proven roles in cancer hallmark acquisition. The reversible nature of epigenetic processes offers robust therapeutic targets. Dietary bioactive compounds offer a vast compendium of effective therapeutic moieties. Isothiocyanates (ITCs) sourced from cruciferous vegetables demonstrate anti-proliferative, pro-apoptotic, anti-inflammatory, anti-migratory and anti-angiogenic effect against several cancers. ITCs also modulate the redox environment, modulate signaling pathways including PI3K, MAPK, WNT, and NFkB. They also modulate the epigenetic machinery by regulating the expression and activity of DNA methyltransferases, histone modifiers and miRNA. This further enhances their transcriptional modulation of key cellular regulators. In this review, we comprehensively assess the impact of ITCs such as sulforaphane, phenethyl isothiocyanate, benzyl isothiocyanate and allyl isothiocyanate on cancer and document their effect on various molecular targets. Overall, this will facilitate consolidation of the current understanding of the anti-cancer and epigenetic modulatory potential of these compounds and recognize the gaps in literature. Further, we discuss avenues of future research to develop these compounds as potential therapeutic entities.

Fisetin Deters Cell Proliferation, Induces Apoptosis, Alleviates Oxidative Stress and Inflammation in Human Cancer Cells, HeLa.

BACKGROUND: Fisetin, a flavonol profusely found in vegetables and fruits, exhibited a myriad of properties in preclinical studies to impede cancer growth. PURPOSE: This study was proposed to delineate molecular mechanisms through analysing the modulated expression of various molecular targets in HeLa cells involved in proliferation, apoptosis and inflammation. METHODS: MTT assay, flow cytometry, nuclear morphology, DNA fragmentation and Annexin-Pi were performed to evaluate the anti-cancer potential of fisetin. Furthermore, qPCR and proteome profiler were performed to analyse the expression of variety of gene related to cell death, cell proliferation, oxidative stress and inflammation and cancer pathways. RESULTS: Fisetin demonstrated apoptotic inducing ability in HeLa cells, which was quite evident through nuclear morphology, DNA ladder pattern, decreased TMRE fluorescent intensity, cell cycle arrest at G2/M and increased early and late apoptosis. Furthermore, fisetin treatment modulated pro-apoptotic genes such as APAF1, Bad, Bax, Bid and BIK at both transcript and protein levels and anti-apoptotic gene Bcl-2, BIRC8, MCL-1, XIAP/BIRC4, Livin/BIRC7, clap-2/BIRC3, etc. at protein levels to mitigate cell proliferation and induce apoptosis. Interestingly, the aforementioned alterations consequently led to an elevated level of Caspase-3, Caspase-8 and Caspase-9, which was found to be consistent with the transcript and protein level expression. Moreover, fisetin downregulated the expression of AKT and MAPK pathways to avert proliferation and enhance apoptosis of cancer cells. Fisetin treatment also improves oxidative stress and alleviates inflammation by regulating JAK-STAT/NF-kB pathways. CONCLUSION: Together, these studies established that fisetin deters human cervical cancer cell proliferation, enhances apoptosis and ameliorates inflammation through regulating various signalling pathways that may be used as a therapeutic regime for better cancer management.

Genistein Modulates Signaling Pathways and Targets Several Epigenetic Markers in HeLa Cells.

BACKGROUND: Several epigenetic changes are responsible for transcriptional alterations of signaling pathways and tumour suppressor genes (TSGs) contributing to carcinogenesis. This study was aimed to examine the effect of the phytochemical, genistein on various molecular targets in HeLa cells. METHODS: Quantitative PCR was used to analyze the expression of various molecular targets. Biochemical assays were employed to study the epigenetic enzymes. To correlate the transcriptional status of the selected TSGs and epigenetic modulation, their promoter 5'CpG methylation levels were evaluated by quantitative methylation array followed by methylation specific restriction digestion. RESULTS: The expression of several genes involved in the cell cycle regulation, migration, inflammation, phosphatidylinositol 3-kinase (PI3K) and mitogen activated kinase-like protein (MAPK) pathway were found to be modulated including CCNB1, TWIST1, MMP14, TERT, AKT1, PTPRR, FOS and IL1A. Genistein modulated the expression of DNA methyltransferases (DNMTs), histone deacetylases (HDACs), histone methyltransferases (HMTs), demethylases, and histone phosphorylases. Furthermore, genistein decreased the activity of DNMTs, HDACs, and HMTs and reduced global DNA methylation levels. Promoter methylation of several TSGs, including FHIT, RUNX3, CDH1, PTEN, and SOC51, was lowered with corresponding transcriptional increase. Network analysis indicated similar effect of genistein. CONCLUSION: This study presents a comprehensive mechanism of action of genistein showcasing effective epigenetic modulation and widespread transcriptional changes resulting in restoration of tumour suppressor gene expression. This study corroborates the development of genistein as a candidate for anti-cancer therapy.

Genistein Induces Alterations of Epigenetic Modulatory Signatures in Human Cervical Cancer Cells.

INTRODUCTION: Epidemiological studies indicate that diet rich in fruits and vegetables is associated with decreased cancer risk thereby indicating that dietary polyphenols can be potential chemo-preventive agents. The reversible nature of epigenetic modifications makes them a favorable target for cancer prevention. Polyphenols have been shown to reverse aberrant epigenetic patterns by targeting the regulatory enzymes, DNA methyltransferases (DNMTs) and histone deacetylases (HDACs). In vitro and in silico studies of DNMTs and HDACs were planned to examine genistein's role as a natural epigenetic modifier in human cervical cancer cells, HeLa. METHODS: Expression of the tumour suppressor genes (TSGs) [MGMT, RARß, p21, E-cadherin, DAPK1] as well the methylation status of their promoters were examined alongwith the activity levels of DNMT and HDAC enzymes after treatment with genistein. Expression of DNMTs and HDACs was also studied. In-silico studies were performed to determine the interaction of genistein with DNMTs and HDACs. RESULTS: Genistein treatment significantly reduced the expression and enzymatic activity of both DNMTs and HDACs in a time-dependent way. Molecular modeling data suggest that genistein can interact with various members of DNMT and HDAC families and support genistein mediated inhibition of their activity. Timedependent exposure of genistein reversed the promoter region methylation of the TSGs and re-established their expression. CONCLUSIONS: In this study, we find that genistein is able to reinstate the expression of the TSGs studied by inhibiting the action of DNMTs and HDACs. This shows that genistein could be an important arsenal in the development of epigenetic based cancer therapy.

(-)-Epigallocatechin-3-gallate reverses the expression of various tumor-suppressor genes by inhibiting DNA methyltransferases and histone deacetylases in human cervical cancer cells.

There has been increasing evidence that numerous bioactive dietary agents can hamper the process of carcinogenesis by targeting epigenetic alterations including DNA methylation. This therapeutic approach is considered as a significant goal for cancer therapy due to the reversible nature of epigenetic-mediated gene silencing and warrants further attention. One such dietary agent, green tea catechin, (-)-epigallocatechin-3-gallate (EGCG) has been shown to modulate many cancer-related pathways. Thus, the present study was designed to investigate the role of EGCG as an epigenetic modifier in HeLa cells. DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibition assays were conducted, and the transcription levels of DNMT3B and HDAC1 were assessed by enzymatic activity assay and RT-PCR, respectively. Furthermore, we studied the binding interaction of EGCG with DNMT3B and HDAC1 by molecular modeling as well as promoter DNA methylation and expression of retinoic acid receptor-ß (RARß), cadherin 1 (CDH1) and death-associated protein kinase-1 (DAPK1) in EGCG-treated HeLa cells by RT-PCR and MS-PCR. In the present study, time-dependent EGCG-treated HeLa cells were found to have a significant reduction in the enzymatic activity of DNMT and HDAC. However, the expression of DNMT3B was significantly decreased in a time-dependent manner whereas there was no significant change in HDAC1 expression. Molecular modeling data also supported the EGCG-mediated DNMT3B and HDAC1 activity inhibition. Furthermore, time-dependent exposure to EGCG resulted in reactivation of known tumor-suppressor genes (TSGs) in HeLa cells due to marked changes in the methylation of the promoter regions of these genes. Overall, the present study suggests that EGCG may have a significant impact on the development of novel epigenetic-based therapy.