Soumen Bera

Does a role for selenium in DNA damage repair explain apparent controversies in its use in chemoprevention

The trace element selenium is an essential micronutrient that has received considerable attention for its potential use in the prevention of cancer. In spite of this interest, the mechanism(s) by which selenium might function as a chemopreventive remain to be determined. Considerable experimental evidence indicates that one possible mechanism by which selenium supplementation may exert its benefits is by enhancing the DNA damage repair response, and this includes data obtained using cultured cells, animal models as well as in human clinical studies. In these studies, selenium supplementation has been shown to be beneficial in reducing the frequency of DNA adducts and chromosome breaks, consequentially reducing the likelihood of detrimental mutations that ultimately contribute to carcinogenesis. The benefits of selenium can be envisioned as being due, at least in part, to it being a critical constituent of selenoproteins such as glutathione peroxidases and thioredoxin reductases, proteins that play important roles in antioxidant defence and maintaining the cellular reducing environment. Selenium, therefore, may be protective by preventing DNA damage from occurring as well as by increasing the activity of repair enzymes such as DNA glycosylases and DNA damage repair pathways that involve p53, BRCA1 and Gadd45. An improved understanding of the mechanism of seleniums impact on DNA repair processes may help to resolve the apparently contradicting data obtained from decades of animal work, human epidemiology and more recently, clinical supplementation studies.

Natural Allelic Variations in Glutathione Peroxidase-1 Affect Its Subcellular Localization and Function

Glutathione peroxidase 1 (GPx-1) has been implicated in the etiology of several common diseases due to the association between specific allelic variations and cancer risk. The most common among these variations are the codon 198 polymorphism that results in either a leucine or proline and the number of alanine repeat codons in the coding sequence. The molecular and biologic consequences of these variations remain to be characterized. Toward achieving this goal, we have examined the cellular location of GPx-1 encoded by allelic variants by ectopically expressing these genes in MCF-7 human breast carcinoma cells that produce undetectable levels of GPx-1, thus achieving exclusive expression in the same cellular environment. A differential distribution between the cytoplasm and mitochondria was observed, with the allele expressing the leucine-198 polymorphism and 7 alanine repeats being more cytoplasmically located than the other alleles examined. To assess whether the distribution of GPx-1 between the cytoplasm and mitochondria had a biologic consequence, we engineered derivative GPx-1 proteins that were targeted to the mitochondria by the addition of a mitochondria targeting sequence and expressed these proteins in MCF-7 cells. These cells were examined for their response to oxidative stress, energy metabolism, and impact on cancer-associated signaling molecules. The results obtained indicated that both primary GPx-1 sequence and cellular location have a profound impact on cellular biology and offer feasible hypotheses about how expression of distinct GPx-1 alleles can affect cancer risk. Cancer Res; 74(18); 5118-26. ©2014 AACR.

The effects of selenium and the GPx-1 selenoprotein on the phosphorylation of H2AX

BACKGROUND Significant data supports the health benefits of selenium although supplementation trials have yielded mixed results. GPx-1, whose levels are responsive to selenium availability, is implicated in cancer etiology by human genetic data. Seleniums ability to alter the phosphorylation of the H2AX, a histone protein that functions in the reduction of DNA damage by recruiting repair proteins to the damage site, following exposure to ionizing radiation and bleomycin was investigated. METHODS Human cell lines that were either exposed to selenium or were transfected with a GPx-1 expression construct were exposed to ionizing radiation or bleomycin. Phosphorylation of histone H2AX was quantified by flow cytometry and survival by the MTT assay. Phosphorylation of the Chk1 and Chk2 checkpoint proteins was quantified by western blotting. RESULTS In colon-derived cells, selenium increases GPx-1 and attenuated H2AX phosphorylation following genotoxic exposures while the viability of these cells was unaffected. MCF-7 cells and transfectants that express high GPx-1 levels were exposed to ionizing radiation and bleomycin, and H2AX phosphorylation and cell viability were assessed. GPx-1 increased H2AX phosphorylation and viability following the induction of DNA damage while enhancing the levels of activated Chk1 and Chk2. CONCLUSIONS Exposure of mammalian cells to selenium can alter the DNA damage response and do so by mechanisms that are dependent and independent of its effect on GPx-1. GENERAL SIGNIFICANCE Selenium and GPx-1 may stimulate the repair of genotoxic DNA damage and this may account for some of the benefits attributed to selenium intake and elevated GPx-1 activity.

CAF cellular glycolysis: linking cancer cells with the microenvironment

Cancers have long being hallmarked as cells relying heavily on their glycolysis for energy generation in spite of having functional mitochondria. The metabolic status of the cancer cells have been revisited time and again to get better insight into the overall carcinogenesis process which revealed the apparent crosstalks between the cancer cells with the fibroblasts present in the tumour microenvironment. This review focuses on the mechanisms of transformations of normal fibroblasts to cancer-associated fibroblasts (CAF), the participation of the CAF in tumour progression with special interest to the role of CAF cellular glycolysis in the overall tumorigenesis. The fibroblasts, when undergoes the transformation process, distinctly switches to a more glycolytic phenotype in order to provide the metabolic intermediates necessary for carrying out the mitochondrial pathways of ATP generation in cancer cells. This review will also discuss the molecular mechanisms responsible for this metabolic make over promoting glycolysis in CAF cells. A thorough investigation of the pathways and molecules involved will not only help in understanding the process of activation and metabolic reprogramming in CAF cells but also might open up new targets for cancer therapy.

Translational Regulation of GPx-1 and GPx-4 by the mTOR Pathway

Glutathione peroxidase activity was previously determined to be elevated in lymphocytes obtained from patients treated with the Bcr-Abl kinase inhibitor imatinib mesylate. In order to expand upon this observation, the established chronic myelogenous leukemia cell lines KU812 and MEG-01 were treated with imatinib and the effect on several anti-oxidant proteins was determined. The levels of GPx-1 were significantly increased following treatment with imatinib. This increase was not due to altered steady-state mRNA levels, and appeared to be dependent on the expression of Bcr-Abl, as no increases were observed following imatinib treatment of cells that did not express the fusion protein. The nutrient-sensing signaling protein, mammalian target of rapamycin (mTOR), can be activated by Bcr-Abl and its activity regulates the translation of many different proteins. Treatment of those same cells used in the imatinib studies with rapamycin, an inhibitor of mTOR, resulted in elevated GPx-1 and GPx-4 protein levels independent of Bcr-Abl expression. These proteins all belong to the selenoprotein family of peptides that contain the UGA-encoded amino acid selenocysteine. Collectively, these data provide evidence of a novel means of regulating anti-oxidants of the selenoprotein family via the mTOR pathway.

Gallic Acid and Gallates in Human Health and Disease: Do Mitochondria Hold the Key to Success?

Gallic acid and gallate esters are widely used as dietary supplements or additives with clinical significances. Over the last few decades, a large number of publications have been reported stating the antioxidative, antiapoptotic, cardioprotective, neuroprotective, and anticancer properties of gallic acid and gallates, and mostly demonstrated their antioxidative or prooxidative properties influencing the reactive oxygen species (ROS) signaling networks. However, very little focus has been paid to clinical trials, and this restricted their use as a prescribed preventative supplement. Since mitochondria are the principal organelles responsible for ROS generation, we reviewed the existing literature of mitochondria-specific effects of gallates including ROS production, respiration, mitochondrial biogenesis, apoptosis, and the physico-chemical parameters affecting the outcome of gallate supplementation to various health scenarios such as cardiovascular diseases, neurodegeneration, hepatic ailments, or cancers. The major signaling pathways and the molecules targeted by gallic acid and its derivatives have also been discussed with emphasis on mitochondria as the target site. This review provides a better understanding of the effect of gallic acid and gallate esters on mitochondrial functions and in designing effective preventative measures against the onset of various diseases.

Allele-specific interaction between glutathione peroxidase 1 and manganese superoxide dismutase affects the levels of Bcl-2, Sirt3 and E-cadherin

Abstract Manganese superoxide dismutase (MnSOD) is a mitochondrial-resident enzyme that reduces superoxide to hydrogen peroxide (H2O2), which can be further reduced to water by glutathione peroxidase (GPX1). Data from human studies have indicated that common polymorphisms in both of these proteins are associated with the risk of several cancers, including breast cancer. Moreover, polymorphisms in MnSOD and GPX1 were shown to interact to increase the risk of breast cancer. To gain an understanding of the molecular mechanisms behind these observations, we engineered human MCF-7 breast cancer cells to exclusively express GPX1 and/or MnSOD alleles and investigated the consequences on the expression of several proteins associated with cancer aetiology. Little or no effect was observed on the ectopic expression of these genes on the phosphorylation of Akt, although allele-specific effects and interactions were observed for the impact on the levels of Bcl-2, E-cadherin and Sirt3. The patterns observed were not consistent with the steady-state levels of H2O2 determined in the transfected cells. These results indicate plausible contributing factors to the effects of allelic variations on cancer risk observed in human epidemiological studies.

Revealing the dual role of gallic acid in modulating ampicillin sensitivity of Pseudomonas aeruginosa biofilms

AIM To understand the effects of gallic acid (GA) on ampicillin (Amp) sensitive or resistant strain of Pseudomonas sp. and also in modulating the corresponding biofilms. METHODOLOGY The cell viability was determined by broth dilution, dry weight and CFU assays. Biofilm formation was measured by crystal violet assay while oxygen consumption rate was measured to verify the metabolic status of the cells. The membrane damage and drug efflux/accumulation were studied by fluorimetric assays. RESULTS GA transformed the Amp resistant cells, both planktonic and biofilms, into highly sensitive one by inducing membrane damage and enhancing accumulation of drug, whereas the Amp sensitive cells gained resistance against Amp. CONCLUSION Use of GA as an antimicrobial compound should be analyzed more critically depending on the drug dosages, drug sensitivity as well as types of bacterial strains being studied.

Abstract 225: Allelic variations in MnSOD and GPx-1 affect metabolism, mitochondrial membrane potential and expression of signaling proteins

MnSOD detoxifies superoxide and impacts tumor biology by generating H 2 O 2 which can diffuse through the mitochondrial membrane and affect metabolism and apoptosis. The selenium-dependent enzyme GPx-1 localizes to both the cytoplasm and the mitochondria and reduces H 2 O 2 to water and may therefore modulate the impact of MnSOD on carcinogenesis. A val to ala polymorphism in codon 16 of the MnSOD gene has been shown to be associated with increased prostate cancer risk in men with the lowest level of dietary antioxidant intake and individuals who consumed less dietary antioxidants, including selenium, had the greatest risk of prostate cancer (Li et al, 2005). A polymorphism in the GPx-1 gene resulting in a leucine (L) instead of a proline (P) at position 198 has frequently been reported to be associated with elevated cancer risk. To examine the molecular mechanism behind the epidemiological observation that genotypes in GPx-1 gene modify elevated risk of cancer associated with MnSOD genotypes, MCF-7 human breast cancer cells null for GPx-1 and having negligible levels of endogenous MnSOD were transfected with GPx-1 and MnSOD allele-specific expression constructs to determine outcomes related to cellular signaling and metabolism. MnSOD and GPx-1 alleles differentially interacted to modulate the expression of the anti-oxidant stress response regulator Nrf2, the cell adhesion protein E-Cadherin, the cell signaling protein pAkt, the anti-apoptotic protein Bcl-2 and the mitochondria located deacetylase Sirt3. Also, co-expression of the GPx-1 A7L and either MnSOD val or MnSOD ala increased oxidative phosphorylation as measured by O 2 uptake using the Seahorse XF24 XF analyzer, which simultaneously determines relative mitochondrial respiration and glycolysis. In order to assess whether GPx-1 and MnSOD allelic variants modulate mitochondrial membrane potential, CMX-ROS fluorescence was measured. Independent and irrespective of which allele was evaluated, MnSOD and GPx-1 individually decreased mitochondrial potential as compared to that seen using MCF-7 control cells. In addition, co-expression of GPx-1 A7L with either MnSOD val or MnSOD ala further decreased membrane potential above that observed for either MnSOD and GPx-1 alone. In order to determine if MnSOD and GPx-1 levels are associated with a higher risk for biochemical recurrence of prostate cancer after radical prostatectomy, immunohistochemistry of human prostate tissue cores will be performed. Preliminary results indicate that a high MnSOD/ GPx-1 ratio was observed in prostate cancer tissue compared to adjacent normal tissue. Citation Format: Dede N. Ekoue, Soumen Bera, Emmanuel Ansong, Peter Hart, Virgilia Macias, Andre Kajdacsy-Balla, Marcelo Bonini, Alan M. Diamond. Allelic variations in MnSOD and GPx-1 affect metabolism, mitochondrial membrane potential and expression of signaling proteins. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 225.

Exposure of chronic myelogenous leukemia cells to imatinib results in the post-transcriptional induction of manganese superoxide dismutase

Abstract The treatment of chronic myelogenous leukemia (CML) with specific tyrosine kinase inhibitors typically results in clinical success, although therapeutic failure frequently occurs. In order to investigate the biological consequences of treating CML cells with such drugs, we previously reported that the antioxidant selenoprotein glutathione peroxidase-1 (GPx-1) was induced by imatinib in both patient samples and cultured cells. Here, we extend these findings to demonstrate that the treatment of CML cell lines, but not non-CML cells, results in an approximately four-fold increase in the levels of another important antioxidant protein, manganese superoxide dismutase (MnSOD), without altering the steady state levels of the corresponding transcript.