Genistein modulates signaling pathways and targets several epigenetic markers in hela cells

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. 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

(-)-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 DNMT3β and HDAC1 were assessed by enzymatic activity assay and RT-PCR, respectively. Furthermore, we studied the binding interaction of EGCG with DNMT3β and HDAC1 by molecular modeling as well as promoter DNA methylation and expression of retinoic acid receptor-â (RARâ), 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 DNMT3β 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 DNMT3β 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

Sulforaphane Reverses the Expression of Various Tumor Suppressor Genes by Targeting DNMT3B and HDAC1 in Human Cervical Cancer Cells

© 2015 Munawwar Ali Khan et al. Sulforaphane (SFN) may hinder carcinogenesis by altering epigenetic events in the cells; however, its molecular mechanisms are unclear. The present study investigates the role of SFN in modifying epigenetic events in human cervical cancer cells, HeLa. HeLa cells were treated with SFN (2.5 μM) for a period of 0, 24, 48, and 72 hours for all experiments. After treatment, expressions of DNMT3B, HDAC1, RARβ, CDH1, DAPK1, and GSTP1 were studied using RT-PCR while promoter DNA methylation of tumor suppressor genes (TSGs) was studied using MS-PCR. Inhibition assays of DNA methyl transferases (DNMTs) and histone deacetylases (HDACs) were performed at varying time points. Molecular modeling and docking studies were performed to explore the possible interaction of SFN with HDAC1 and DNMT3B. Time-dependent exposure to SFN decreases the expression of DNMT3B and HDAC1 and significantly reduces the enzymatic activity of DNMTs and HDACs. Molecular modeling data suggests that SFN may interact directly with DNMT3B and HDAC1 which may explain the inhibitory action of SFN. Interestingly, time-dependent reactivation of the studied TSGs via reversal of methylation in SFN treated cells correlates well with its impact on the epigenetic alterations accumulated during cancer development. Thus, SFN may have significant implications for epigenetic based therapy

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

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

Sulforaphane Reverses the Expression of Various Tumor Suppressor Genes by Targeting DNMT3B and HDAC1 in Human Cervical Cancer Cells

Sulforaphane (SFN) may hinder carcinogenesis by altering epigenetic events in the cells; however, its molecular mechanisms are unclear. The present study investigates the role of SFN in modifying epigenetic events in human cervical cancer cells, HeLa. HeLa cells were treated with SFN (2.5 µM) for a period of 0, 24, 48, and 72 hours for all experiments. After treatment, expressions of DNMT3B, HDAC1, RARβ, CDH1, DAPK1, and GSTP1 were studied using RT-PCR while promoter DNA methylation of tumor suppressor genes (TSGs) was studied using MS-PCR. Inhibition assays of DNA methyl transferases (DNMTs) and histone deacetylases (HDACs) were performed at varying time points. Molecular modeling and docking studies were performed to explore the possible interaction of SFN with HDAC1 and DNMT3B. Time-dependent exposure to SFN decreases the expression of DNMT3B and HDAC1 and significantly reduces the enzymatic activity of DNMTs and HDACs. Molecular modeling data suggests that SFN may interact directly with DNMT3B and HDAC1 which may explain the inhibitory action of SFN. Interestingly, time-dependent reactivation of the studied TSGs via reversal of methylation in SFN treated cells correlates well with its impact on the epigenetic alterations accumulated during cancer development. Thus, SFN may have significant implications for epigenetic based therapy