Reactive oxygen species ROS like
Reactive oxygen species (ROS), like hydrogen peroxide (H2O2) play a major role in carcinogenesis by DNA damage and activation of pro-inflammatory signaling pathways. Overproduction and accumulation of ROS is associated with increase in nucleotide modifications, DNA strand break, deletions and chromosomal rearrangement. Depending on its intracellular concentration and localization, H2O2 exhibits either pro- or anti-apoptotic activities. In comparison to normal cells, cancer GSK126 are characterized by an increased H2O2 production rate and an impaired redox balance thereby affecting the microenvironment as well as the anti-tumoral immune response. Emerging evidences suggests ROS acts as critical signaling stimuli involving various cellular functions such as apoptosis, migration, invasion and EMT in the tumor microenvironment [27,28]. Small molecule second messengers (for examples, calcium and hydrogen peroxide) propagate information quickly owing to their much larger diffusion coefficient [29,30]. The roles of E-cadherin, EMT and ROS in many cancers are illustrated discreetly. The molecular mechanisms, signaling and/or network interdependence of their action in tumor development and cancer progression are not studied adequately. To decipher the mechanisms and pathways of actions further, we thoroughly investigate whether ROS, like hydrogen peroxide may modulate E-cadherin expression! And if it does so, which mechanism(s)/epigenetic modification(s) are involved in the inactivation of CDH1 gene promoter? We have executed bioinformatics analyzes and cell culture experiments. Cells were cultured and treaded with sub lethal dosage of H2O2 mixed in culture media. The expression profile of E-cadherin, DNA methyltransferase 1 (DNMT1), histone deacetylase 1 (HDAC1), and EMT factors Snail and Slug were recorded. Of notice, CDH1 was silenced, and DNMT1, HDAC1, Snail and Slug were overexpressed by H2O2 treatment. Application of ROS scavengers; NAC, lupeol and beta sitosterol attenuated this effect. Additionally, application of DNMT inhibitor 5-Aza-2′-deoxycytidine (AZA) prevented the H2O2 induced downregulation of E-cadherin, grossly suggesting promoter -CpG- methylation of CDH1 gene. Methylation of CDH1 was confirmed by MS-PCR experiments. Then we used ERK inhibitor, U0126 in cell culture which reduced the expression of DNMT1, Snail and Slug, but expression of CDH1 was enhanced. This simple experiment depicts that the expression of CDH1 is synergistically regulated by ERK pathway downstream effectors DNMT1 and HDAC1 overexpression (causing CDH1 promoter DNA methylation and inactivation), Snail and Slug over expression and occupancy of CDH1 promoter for keeping it silent.
Materials and methods
Discussion Various aberrant epigenetic modulations like chromatin modifications, hypermethylation of CpG-island and transcriptional repressions are involved in silencing or inhibition of the expression of genes, including CDH1, CAV1, CLU in different cancers [6,7,33,34]. The impact of E-cadherin expression on overall survival of patients was analyzed using online KM plotter, suggesting poor prognosis owing to lower expression of E-cadherin in both estrogen positive and estrogen negative breast cancer patients (Fig. 1). To decipher the correlation of gene function and intrinsic metabolites we standardized first Cell survival and migration capability of breast cancer cell lines by ectopic application of ROS producer, like H2O2 and ROS scavengers like, NAC, Lupeol and beta-sitosterol (Fig. 2, Fig. 3). Hydrogen peroxide (H2O2) is involved in various signal transduction pathways and cell fate decisions. The most prominent member of this class is the superoxide anion, largely produced by either the mitochondrial electron transport chain, in particular its complexes I, II and III, or by NAD(P)H oxidases. Here, we found H2O2 induced cellular migration via downregulation of CDH1 gene and absence of E-cadherin protein and enhanced expression of panel cooperatively active genes, including DNMT1, HDAC, Snail and Slug. Treatment of cells with H2O2 augmented vigorous ROS production which favors cellular proliferation and migration. The effects of H2O2 were reversed by treatment of cells with NAC, lupeol and beta-sitosterol, through scavenging ROS generated by H2O2 (Fig. 2, Fig. 3). Question may arise that this regulation mechanism would not be the effect of ROS. Hence, we detected H2O2 induced ROS production by fluorescence measurement using DCFDA dye and clarified. Important to watch the reduction of free radical generation by the application of same amount of H2O2when antioxidants NAC, lupeol and beta sitosterol were simultaneously used in both MCF-7 and MDA-MB-231 breast cancer cells. MCF-7 and MDA-MB-231 cells were treated with H2O2 in the presence or absence of different drugs and NAC for 24 h and ROS level was detected using DCFDA dye by fluorescence microscopy as seen in the representative photographic images.