Chronic coexposure to arsenic and estrogen potentiates genotoxic estrogen metabolic pathway and hypermethylation of DNA glycosylase MBD4 in human prostate epithelial cells.

BACKGROUND: Previously we reported that arsenic and estrogen cause synergistic effects in the neoplastic transformation of human prostate epithelial cells. In addition to receptor-mediated pathways, DNA-reactive estrogen metabolites have also been shown to play a critical role in mutagenicity and carcinogenicity. Both estrogen and arsenic are known prostate carcinogens, however, the effect of coexposure to these two chemicals on genes involved in estrogen metabolism is not known. Therefore, the objective of this study was to evaluate the role of arsenic and estrogen coexposure on the expression of estrogen receptors and estrogen metabolism-associated genes. Earlier, we also reported the synergistic effect of arsenic and estrogen on decreased expression of MBD4 genes that play an important role in DNA repair through its DNA glycosylase activity. To further understand the mechanism, the promoter methylation of this gene was also analyzed. METHODS: Total RNA and protein were isolated from RWPE-1 human prostate epithelial cells that were coexposed to arsenic and estrogen for a chronic duration (6 months). The expression of estrogen receptors, estrogen metabolism associated phase I genes (CYP 1A1, 1A2, 3A4, and 1B1) and phase II gene catechol-O-methyltransferase (COMT), as well as antioxidant MnSOD, were analyzed either at the RNA level by quantitative reverse transcriptase-polymerase chain reaction or at the protein level by western blot. Promoter methylation of MBD4 was analyzed by pyrosequencing. RESULTS: Expression of MnSOD and phase I genes that convert E2 into genotoxic metabolites 2-OH-E2 and 4-OH-E2 were significantly increased, whereas the expression of phase II gene COMT that detoxifies estrogen metabolites was significantly decreased in arsenic and estrogen coexposed cells. MBD4 promoter was hypermethylated in arsenic and estrogen coexposed cells. Coexposure to arsenic and estrogen has synergistic effects on the expression of these genes as well as in MBD4 promoter hypermethylation. CONCLUSIONS: These novel findings suggest that coexposure to arsenic and estrogen acts synergistically in the activation of not only the estrogen receptors but also the genes associated with genotoxic estrogen metabolism and epigenetic inactivation of DNA glycosylase MBD4. Together, these genetic and epigenetic aberrations provide the molecular basis for the potentiation of carcinogenicity of arsenic and estrogen coexposure in prostate epithelial cells.

Higher Concentrations of Folic Acid Cause Oxidative Stress, Acute Cytotoxicity, and Long-Term Fibrogenic Changes in Kidney Epithelial Cells.

Kidney fibrosis is a common step during chronic kidney disease (CKD), and its incidence has been increasing worldwide. Aberrant recovery after repeated acute kidney injury leads to fibrosis. The mechanism of fibrogenic changes in the kidney is not fully understood. Folic acid-induced kidney fibrosis in mice is an established in vivo model to study kidney fibrosis, but the mechanism is poorly understood. Moreover, the effect of higher concentrations of folic acid on kidney epithelial cells in vitro has not yet been studied. Oxidative stress is a common property of nephrotoxicants. Therefore, this study evaluated the role of folic acid-induced oxidative stress in fibrogenic changes by using the in vitro renal proximal tubular epithelial cell culture model. To obtain comprehensive and robust data, three different cell lines derived from human and mouse kidney epithelium were treated with higher concentrations of folic acid for both acute and long-term durations, and the effects were determined at the cellular and molecular levels. The result of cell viability by the MTT assay and the measurement of reactive oxygen species (ROS) levels by the DCF assay revealed that folic acid caused cytotoxicity and increased levels of ROS in acute exposure. The cotreatment with antioxidant N-acetyl cysteine (NAC) protected the cytotoxic effect, suggesting the role of folic acid-induced oxidative stress in cytotoxicity. In contrast, the long-term exposure to folic acid caused increased growth, DNA damage, and changes in the expression of marker genes for EMT, fibrosis, oxidative stress, and oxidative DNA damage. Some of these changes, particularly the acute effects, were abrogated by cotreatment with antioxidant NAC. In summary, the novel findings of this study suggest that higher concentrations of folic acid-induced oxidative stress act as the driver of cytotoxicity as an acute effect and of fibrotic changes as a long-term effect in kidney epithelial cells.

Differential sensitivity of renal carcinoma cells to doxorubicin and epigenetic therapeutics depends on the genetic background.

Differential sensitivity to chemotherapeutics is a limitation in chemotherapy of kidney cancer patients. Role of genetic background in chemotherapy is not fully understood. Therefore, this study evaluated the influence of genetic/epigenetic background of renal cancer cells on the sensitivity to chemotherapeutics. Two renal cell carcinoma (RCC) cell lines, Caki-1 and 786-0, with different genetic makeup of p53 and VHL were treated with doxorubicin either alone or in combination with epigenetic therapeutics 5-aza-2-dc and TSA. Sensitivity of RCC cells to these drugs was evaluated by cell viability and cell cycle analysis and was further confirmed by analysis of selected genes expression. Cell viability data revealed that 786-0 cells were more sensitive than Caki-1 to doxorubicin. Combination of doxorubicin with 5-aza-2-dc or TSA was more effective to inhibit growth of Caki-1 cells but not the 786-0. Data of cell cycle analysis and expression of representative genes for tumor suppressor, cell cycle and survival, drug transporter and DNA repair further provided the molecular basis for differential sensitivity of Caki-1 and 786-0 cell lines to doxorubicin. Important findings of this study suggest that doxorubicin is more cytotoxic to primary renal cancer 786-0 cells with mutant VHL and p53 than the metastatic Caki-1 cells with wild-type VHL and p53, and this differential response was independent of p53 expression level. This study suggests that combination of doxorubicin with epigenetic therapeutics could potentially be beneficial in clinical treatment of renal cancer patients with wild-type VHL and p53 but not in patients with mutant VHL and p53.

Arsenic induces fibrogenic changes in human kidney epithelial cells potentially through epigenetic alterations in DNA methylation.

Arsenic contamination is a significant public health issue, and kidney is one of the target organ for arsenic-induced adverse effects. Renal fibrosis is a well-known pathological stage frequently observed in progressive chronic kidney disease (CKD). Epidemiological studies implicate arsenic exposure to CKD, but the role of arsenic in kidney fibrosis and the underlying mechanism is still unclear. It is in this context that the current study evaluated the effects of long-term arsenic exposure on the cellular response in morphology, and marker genes expression with respect to fibrosis using human kidney 2 (HK-2) epithelial cells. Results of this study revealed that in addition to increased growth, HK-2 cells underwent phenotypic, biochemical and molecular changes indicative of epithelial-mesenchymal transition (EMT) in response to the exposure to arsenic. Most importantly, the arsenic-exposed cells acquired the pathogenic features of fibrosis as supported by increased expression of markers for fibrosis, such as Collagen I, Fibronectin, transforming growth factor ß, and α-smooth muscle actin. Upregulation of fibrosis associated signaling molecules such as tissue inhibitor of metalloproteinases-3 and matrix metalloproteinase-2 as well as activation of AKT was also observed. Additionally, the expression of epigenetic genes (DNA methyltransferases 3a and 3b; methyl-CpG binding domain 4) was increased in arsenic-exposed cells. Treatment with DNA methylation inhibitor 5-Aza-2'-dC reversed the EMT properties and restored the level of phospho-AKT. Together, these data for the first time suggest that long-term exposure to arsenic can increase the risk of kidney fibrosis. Additionally, our data suggest that the arsenic-induced fibrotic changes are, at least in part, mediated by DNA methylation and therefore potentially can be reversed by epigenetic therapeutics.

Nicotine-induced oxidative stress contributes to EMT and stemness during neoplastic transformation through epigenetic modifications in human kidney epithelial cells.

Nicotine is a component of cigarette smoke and mounting evidence suggests toxicity and carcinogenicity of tobacco smoke in kidney. Carcinogenicity of nicotine itself in kidney and the underlying molecular mechanisms are not well-understood. Hence, the objective of this study was to determine the carcinogenic effects of chronic nicotine exposure in Hk-2 human kidney epithelial cells. The effects of nicotine exposure on the expression of genes for cellular reprogramming, redox status, and growth signaling pathways were also evaluated to understand the molecular mechanisms. Results revealed that chronic exposure to nicotine induced growth and neoplastic transformation in HK-2 cells. Increased levels of intracellular reactive oxygen species (ROS), acquired stem cell-like sphere formation, and epithelial-mesenchymal-transition (EMT) changes were observed in nicotine exposed cells. Treatment with antioxidant N-acetyl cysteine (NAC) resulted in abrogation of EMT and stemness in HK-2 cells, indicating the role of nicotine-induced ROS in these morphological changes. The result also suggests that ROS controls the stemness through regulation of AKT pathway during early stages of carcinogenesis. Additionally, the expression of epigenetic regulatory genes was altered in nicotine-exposed cells and the changes were reversed by NAC. The epigenetic therapeutics 5-aza-2'-deoxycytidine and Trichostatin A also abrogated the stemness. This suggests the nicotine-induced oxidative stress caused epigenetic alterations contributing to stemness during neoplastic transformation. To our knowledge, this is the first report showing the ROS-mediated epigenetic modifications as the underlying mechanism for carcinogenicity of nicotine in human kidney epithelial cells. This study further suggests the potential of epigenetic therapeutics for pharmacological intervention in nicotine-induced kidney cancer.

Arsenic-Induced Neoplastic Transformation Involves Epithelial-Mesenchymal Transition and Activation of the ß-Catenin/c-Myc Pathway in Human Kidney Epithelial Cells.

Arsenic contamination is a serious environmental and public health issue worldwide including the United States. Accumulating evidence suggests that kidney is one of the target organs for arsenic-induced carcinogenesis. However, the mechanism of arsenic-induced renal carcinogenesis is not well understood. Therefore, the objective of this study was to evaluate the carcinogenicity of chronic exposure to an environmentally relevant concentration of arsenic on kidney epithelial cells and identify the molecular mechanism underlying this process. HK-2 kidney epithelial cells were treated with arsenic for acute, long-term, and chronic durations, and cellular responses to arsenic exposure at these time points were evaluated by the changes in growth, morphology, and expression of genes. The results revealed a significant growth increase after long-term and chronic exposure to arsenic in HK-2 cells. The morphological changes of EMT and stem cell sphere formation were also observed in long-term arsenic exposed cells. The anchorage-independent growth assay for colony formation and cell maintenance in cancer stem cell medium further confirmed neoplastic transformation and the induced cancer stem cell properties of arsenic-exposed cells. Additionally, the expression of marker genes confirmed the increased growth, EMT, and stemness during arsenic-induced carcinogenesis. Moreover, the increase expression of ß-catenin and c-Myc further suggested the role of these signaling molecules during carcinogenesis in HK-2 cells. In summary, results of this study suggest that chronic exposure to arsenic even at a relatively lower concentration can induce neoplastic transformation through acquisitions of EMT, stemness, and MET phenotypes, which might be related to the ß-catenin/c-Myc signaling pathway.

Duration-dependent effects of nicotine exposure on growth and AKT activation in human kidney epithelial cells.

Exposure to nicotine is known to cause adverse effects in many target organs including kidney. Epidemiological studies suggest that nicotine-induced kidney diseases are prevalent worldwide. However, the impact of duration of exposure on the nicotine-induced adverse effects in normal kidney cells and the underlying molecular mechanism is still unclear. Hence, the objective of this study was to evaluate both acute and long-term effects of nicotine in normal human kidney epithelial cells (HK-2). Cells were treated with 1 and 10 µM nicotine for acute and long-term duration. The result of cell viability showed that the acute exposure to 1 µM nicotine has no significant effect on growth. However, the 10 µM nicotine caused significant decrease in the growth of HK-2 cells. The long-term exposure resulted in significantly increased cell growth in both 1 and 10 µM nicotine-treated groups. Analysis of cell cycle and expression of marker genes related to proliferation and apoptosis further confirmed the effects of nicotine. Additionally, the analysis of growth signaling pathway revealed the decreased level of pAKT in cells with acute exposure whereas the increased level of pAKT in long-term nicotine-exposed cells. This suggests that nicotine, through modulating the AKT pathway, controls the duration-dependent effects on the growth of HK-2 cells. In summary, this is the first report showing long-duration exposure to nicotine causes increased proliferation of human kidney epithelial cells through activation of AKT pathway.

Chronic Oxidative Stress Increases Resistance to Doxorubicin-Induced Cytotoxicity in Renal Carcinoma Cells Potentially Through Epigenetic Mechanism.

Renal cell carcinoma is the most common form of kidney cancer and is highly resistant to chemotherapy. Although the role of oxidative stress in kidney cancer is known, the chemotherapeutic response of cancer cells adapted to chronic oxidative stress is not clear. Hence, the effect of oxidative stress on sensitivity to doxorubicin-induced cytotoxicity was evaluated using an in vitro model of human kidney cancer cells adapted to chronic oxidative stress. Results of MTT- and anchorage-independent growth assays and cell cycle analysis revealed significant decrease in sensitivity to doxorubicin in Caki-1 cells adapted to oxidative stress. Changes in the expression of genes involved in drug transport, cell survival, and DNA repair-dependent apoptosis further confirmed increased resistance to doxorubicin-induced cytotoxicity in these cells. Decreased expression of mismatch repair (MMR) gene MSH2 in cells exposed to oxidative stress suggests that loss of MMR-dependent apoptosis could be a potential mechanism for increased resistance to doxorubicin-induced cytotoxicity. Additionally, downregulation of HDAC1, an increase in the level of histone H3 acetylation, and hypermethylation of MSH2 promoter were also observed in Caki-1 cells adapted to chronic oxidative stress. DNA-demethylating agent 5-Aza-2dC significantly restored the expression of MSH2 and doxorubicin-induced cytotoxicity in Caki-1 cells adapted to chronic oxidative stress, suggesting the role of DNA hypermethylation in inactivation of MSH2 expression and consequently MMR-dependent apoptosis in these cells. In summary, this study for the first time provides direct evidence for the role of oxidative stress in chemotherapeutic resistance in renal carcinoma cells potentially through epigenetic mechanism.

Chronic oxidative stress leads to malignant transformation along with acquisition of stem cell characteristics, and epithelial to mesenchymal transition in human renal epithelial cells.

Oxidative injury to cellular macromolecules has been suggested as a common pathway shared by multiple etiological factors for kidney cancer. Whether the chronic oxidative stress alone is sufficient to induce malignant transformation in human kidney cells is not clear. Therefore, the objective of this study was to evaluate the effect of H2O2-induced chronic oxidative stress on growth, and malignant transformation of HK-2 normal kidney epithelial cells. This study revealed that chronic oxidative stress causes increased growth and neoplastic transformation in normal kidney epithelial cells at non-cytotoxic dose and increased adaptation to cytotoxic level. This was confirmed by gene expression changes, cell cycle analysis, anchorage independent growth assay and in vivo tumorigenicity in nude mice. Stem cells characteristics as revealed by up-regulation of stem cell marker genes, and morphological changes indicative of EMT with up regulation of mesenchymal markers were also observed in cells exposed to chronic oxidative stress. Antioxidant NAC did not reverse the chronic oxidative stress-induced growth, and adaptation suggesting that perturbed biological function in these cells are permanent. Partial reversal of oxidative stress-induced growth, and adaptation by silencing of Oct 4 and Snail1, respectively, suggest that these changes are mediated by acquisition of stem cell and EMT characteristics. In summary, this study for the first time suggests that chronic exposure to elevated levels of oxidative stress is sufficient to induce malignant transformation in kidney epithelial cells through acquisition of stem cell characteristics. Additionally, the EMT plays an important role in increased adaptive response of renal cells to oxidative stress.

Chronic oxidative stress causes estrogen-independent aggressive phenotype, and epigenetic inactivation of estrogen receptor alpha in MCF-7 breast cancer cells.

The role of chronic oxidative stress in the development and aggressive growth of estrogen receptor (ER)-positive breast cancer is well known; however, the mechanistic understanding is not clear. Estrogen-independent growth is one of the features of aggressive subtype of breast cancer. Therefore, the objective of this study was to evaluate the effect of oxidative stress on estrogen sensitivity and expression of nuclear estrogen receptors in ER-positive breast cancer cells. MCF-7 cells chronically exposed to hydrogen peroxide were used as a cell model in this study, and their growth in response to 17-ß estradiol was evaluated by cell viability, cell cycle, and cell migration analysis. Results were further confirmed at molecular level by analysis of gene expressions at transcript and protein levels. Histone H3 modifications, expression of epigenetic regulatory genes, and the effect of DNA demethylation were also analyzed. Loss of growth in response to estrogen with a decrease in ERα expression was observed in MCF-7 cells adapted to chronic oxidative stress. Increases in mtTFA and NRF1 in these cells further suggested the role of mitochondria-dependent redox-sensitive growth signaling as an alternative pathway to estrogen-dependent growth. Changes in expression of epigenetic regulatory genes, levels of histone H3 modifications as well as significant restorations of both ERα expression and estrogen response by 5-Aza-2'-deoxycytidine further confirmed the epigenetic basis for estrogen-independent growth in these cells. In conclusion, results of this study suggest that chronic oxidative stress can convert estrogen-dependent nonaggressive breast cancer cells into estrogen-independent aggressive form potentially by epigenetic mechanism.

Potentiation of growth inhibition and epigenetic modulation by combination of green tea polyphenol and 5-aza-2'-deoxycytidine in human breast cancer cells.

Epigenetic therapy by DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza 2'dC) is clinically effective in acute myeloid leukemia; however, it has shown limited results in treatment of breast cancer and has significant toxicity to normal cells. Green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) has anti-cancer and DNA demethylating properties with no significant toxicity toward normal cells. Therefore, the objective of this study was to evaluate the therapeutic efficacy of a combination of non-toxic, low dose of 5-aza 2' dC with EGCG, on growth inhibition of breast cancer cells. Human breast cancer cell lines (MCF-7, MDA-MB 231) and non-tumorigenic MCF-10A breast epithelial cells were treated with either 5-aza 2' dC, EGCG, or their combination for 7 days. Cell growth inhibition was determined by cell count, cell viability, cell cycle, and soft agar assay, whereas genes expression changes were determined by quantitative real-time PCR and/or Western blot analysis. Histone modifications and global DNA methylation changes were determined by Western blot and RAPD-PCR, respectively. The results revealed significantly greater inhibition of growth of breast cancer cells by co-treatment with 5-aza 2' dC and EGCG compared to individual treatments, whereas it has no significant toxicity to MCF-10A cells. This was further confirmed by gene expression analysis. Changes in DNA methylation and histone modifications were also greater in cells with combination treatment. Findings of this study suggest that potentiation of growth inhibition of breast cancer cells by 5-aza 2' dC and EGCG combination treatment, at least in part, is mediated by epigenetic mechanism.

Estrogen-induced reactive oxygen species, through epigenetic reprogramming, causes increased growth in breast cancer cells.

Despite the progress made in cancer diagnosis and treatment, breast cancer remains the second leading cause of cancer-related death among the women. Exposure to elevated levels of endogenous estrogen or environmental estrogenic chemicals is an important risk factor for breast cancer. Estrogen metabolites and ROS generated during estrogen metabolism are known to play a critical role in estrogen carcinogenesis. However, the molecular mechanisms through which estrogen-induced ROS regulate gene expression is not clear. Epigenetic changes of DNA methylation and histone modifications are known to regulate genes expression. Therefore, the objective of this study was to evaluate whether estrogen-induced ROS, through aberrant expression of epigenetic regulatory genes and epigenetic reprogramming, causes growth of breast cancer cells. Estrogen responsive MCF-7 and T47D human breast cancer cells were exposed to natural estrogen 17 beta-estradiol (E2) and synthetic estrogen Diethylstilbestrol (DES) both alone and in combination with antioxidant N-acetyl cysteine. Effects of NAC-mediated scavenging of estrogen-induced ROS on cell growth, gene expression, and histone modifications were measured. The result of MTT and cell cycle analysis revealed significant abrogation of E2 and DES-induced growth by scavenging ROS through NAC. E2 and DES caused significant changes in expression of epigenetic regulatory genes for DNA methylation and histone modifications as well as changes in both gene activating and repressive marks in the Histone H3. NAC restored the expression of epigenetic regulatory genes and changes in histone marks. Novel findings of this study suggest that estrogen can induce growth of breast cancer cells through ROS-dependent regulation of epigenetic regulatory genes and epigenetic reprogramming of histone marks.

Epigallocatechin-3-gallate attenuates arsenic-induced fibrogenic changes in human kidney epithelial cells through reversal of epigenetic aberrations and antioxidant activities.

Renal fibrosis is a pathogenic intermediate stage of chronic kidney disease (CKD). Nephrotoxicants including arsenic can cause kidney fibrosis through induction of oxidative stress and epigenetic aberrations. Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, is known to have antioxidant and epigenetic modulation properties. Whether EGCG, through its antioxidant and epigenetic modulating activities, can attenuate fibrogenesis is not known. Therefore, the objective of this study was to determine whether EGCG can attenuate arsenic-induced acute injury and long-term exposure associated fibrogenicity in kidney epithelial cells. To address this question, two human kidney epithelial cell lines Caki-1 and HK-2 exposed to arsenic for both acute and long-term durations were treated with EGCG. The protective effect of EGCG on arsenic-induced cytotoxicity and fibrogenicity were evaluated by measuring the cell growth, reactive oxygen species (ROS) production, genes expression, and epigenetic changes in histone marks. Results revealed that EGCG has a protective effect in arsenic-induced acute cytotoxicity in these cells. EGCG scavenges the increased levels of ROS in arsenic exposed cells. Aberrant expression of fibrogenic genes in arsenic exposed cells were restored by EGCG. Abrogation of arsenic-induced fibrogenic changes was also associated with EGCG-mediated restoration of arsenic-induced aberrant expression of epigenetic regulatory proteins and histone marks. Novel findings of this study suggest that EGCG, through its antioxidant and epigenetic modulation capacities, has protective effects against arsenic-induced cytotoxicity and fibrogenic changes in kidney epithelial cells.

Manufacturing and preclinical toxicity of GLP grade gene deleted attenuated Leishmania donovani parasite vaccine.

Centrin1 gene deleted Leishmania donovani parasite (LdCen1-/-) was developed and extensively tested experimentally as an intracellular stage-specific attenuated and immunoprotective live parasite vaccine candidate ex vivo using human PBMCs and in vivo in animals. Here we report manufacturing and pre-clinical evaluation of current Good-Laboratory Practice (cGLP) grade LdCen1-/- parasites, as a prerequisite before proceeding with clinical trials. We screened three batches of LdCen1-/- parasites manufactured in bioreactors under cGLP conditions, for their consistency in genetic stability, attenuation, and safety. One such batch was preclinically tested using human PBMCs and animals (hamsters and dogs) for its safety and protective immunogenicity. The immunogenicity of the CGLP grade LdCen1-/- parasites was similar to one grown under laboratory conditions. The cGLP grade LdCen1-/- parasites were found to be safe and non-toxic in hamsters and dogs even at 3 times the anticipated vaccine dose. When PBMCs from healed visceral leishmaniasis (VL) cases were infected with cGLP LdCen1-/-, there was a significant increase in the stimulation of cytokines that contribute to protective responses against VL. This effect, measured by multiplex ELISA, was greater than that observed in PBMCs from healthy individuals. These results suggest that cGLP grade LdCen1-/- manufactured under cGMP complaint conditions can be suitable for future clinical trials.

Chronic exposure to arsenic, estrogen, and their combination causes increased growth and transformation in human prostate epithelial cells potentially by hypermethylation-mediated silencing of MLH1.

BACKGROUND: Chronic exposure to arsenic and estrogen is associated with risk of prostate cancer, but their mechanism is not fully understood. Additionally, the carcinogenic effects of their co-exposure are not known. Therefore, the objective of this study was to evaluate the effects of chronic exposure to arsenic, estrogen, and their combination, on cell growth and transformation, and identify the mechanism behind these effects. METHODS: RWPE-1 human prostate epithelial cells were chronically exposed to arsenic and estrogen alone and in combination. Cell growth was measured by cell count and cell cycle, whereas cell transformation was evaluated by colony formation assay. Gene expression was measured by quantitative real-time PCR and confirmed at protein level by Western blot analysis. MLH1 promoter methylation was determined by pyrosequencing method. RESULTS: Exposure to arsenic, estrogen, and their combinations increases cell growth and transformation in RWPE-1 cells. Increased expression of Cyclin D1 and Bcl2, whereas decreased expression of mismatch repair genes MSH4, MSH6, and MLH1 was also observed. Hypermethylation of MLH1 promoter further suggested the epigenetic inactivation of MLH1 expression in arsenic and estrogen treated cells. Arsenic and estrogen combination caused greater changes than their individual treatments. CONCLUSIONS: Findings of this study for the first time suggest that arsenic and estrogen exposures cause increased cell growth and survival potentially through epigenetic inactivation of MLH1 resulting in decreased MLH1-mediated apoptotic response, and consequently increased cellular transformation.

Distribution of live and dead cells in pellets of an actinomycete Amycolatopsis balhimycina and its correlation with balhimycin productivity.

Secondary metabolites such as antibiotics are typically produced by actinomycetes as a response to growth limiting stress conditions. Several studies have shown that secondary metabolite production is correlated with changes observed in actinomycete pellet morphology. Therefore, we investigated the correlation between the production of balhimycin and the spatio-temporal distribution of live and dead cells in pellets of Amycolatopsis balhimycina in submerged cultures. To this end, we used laser scanning confocal microscopy to analyze pellets from balhimycin producing and nonproducing media containing 0.2 and 1.0 g l(-1) of potassium di-hydrogen phosphate, respectively. We observed a substantially higher fraction of live cells in pellets from cultures yielding larger amounts of balhimycin. Moreover, in media that resulted in no balhimycin production, the pellets exhibit an initial death phase which commences from the centre of the pellet and extends in the radial direction. A second growth phase was observed in these pellets, where live mycelia are seen to appear in the dead core of the pellets. This secondary growth was absent in pellets from media producing higher amounts of balhimycin. These results suggest that distribution of live and dead cells and its correlation with antibiotic production in the non-sporulating A. balhimycina differs markedly than that observed in Streptomycetes.

Epigenetic mechanism of therapeutic resistance and potential of epigenetic therapeutics in chemorefractory prostate cancer.

Prostate cancer is the second leading cause of cancer death among men in the United States. Depending upon the histopathological subtypes of prostate cancers, various therapeutic options, such as androgen deprivation therapy (ADT), androgen receptor signaling inhibitors (ARSI), immunotherapy, and chemotherapy, are available to treat prostate cancer. While these therapeutics are effective in the initial stages during treatments, the tumors subsequently develop resistance to these therapies. Despite all the progress made so far, therapeutic resistance remains a major challenge in the treatment of prostate cancer. Although various mechanisms have been reported for the resistance development in prostate cancer, altered expression of genes either directly or indirectly involved in drug response pathways is a common event. In addition to the genetic basis of gene regulation such as mutations and gene amplifications, epigenetic alterations involved in the aberrant expression of genes have frequently been shown to be associated not only with cancer initiation and progression but also with therapeutic resistance development. There are several review articles compiling reports on genetic mechanisms involved in therapeutic resistance in prostate cancer. However, epigenetic mechanisms for the therapeutic resistance development in prostate cancer have not yet been summarized in a review article. Therefore, the objective of this article is to compile various reports and provide a comprehensive review of the epigenetic aberrations, and aberrant expression of genes by epigenetic mechanisms involved in CRPCs and therapeutic resistance development in prostate cancer. Additionally, the potential of epigenetic-based therapeutics in the treatment of chemorefractory prostate cancer as evidenced by clinical trials has also been discussed.

Production of leishmanin skin test antigen from Leishmania donovani for future reintroduction in the field.

The leishmanin skin test was used for almost a century to detect exposure and immunity to Leishmania, the causative agent of leishmaniasis, a major neglected tropical disease. Due to a lack of antigen used for the intradermal injection, the leishmanin skin test is no longer available. As leishmaniasis control programs are advancing and new vaccines are entering clinical trials, it is essential to re-introduce the leishmanin skin test. Here we establish a Leishmania donovani strain and describe the production, under Good Laboratory Practice conditions, of leishmanin soluble antigen used to induce the leishmanin skin test in animal models of infection and vaccination. Using a mouse model of cutaneous leishmaniasis and a hamster model of visceral leishmaniasis, soluble antigen induces a leishmanin skin test response following infection and vaccination with live attenuated Leishmania major (LmCen-/-). Both the CD4+ and CD8+ T-cells are necessary for the leishmanin skin test response. This study demonstrates the feasibility of large-scale production of leishmanin antigen addressing a major bottleneck for performing the leishmanin skin test in future surveillance and vaccine clinical trials.

Correlation between pellet morphology and glycopeptide antibiotic balhimycin production by Amycolatopsis balhimycina DSM 5908.

Actinomycetes, a class of filamentous bacteria, are an important source of several industrially relevant secondary metabolites. Several environmental factors including the media composition affect both biomass growth and product formation. Likewise, several studies have shown that environmental factors cause changes in cellular morphology. However, the relationship between morphology and product formation is not well understood. In this study, we first characterized the effect of varying concentrations of phosphate and ammonia in defined media on pellet morphology for an actinomycete Amycolatopsis balhimycina DSM 5908, which produces balhimycin, a glycopeptide antibiotic. Our results show that higher balhimycin productivity is correlated with the following morphological features: (1) higher pellet fraction in the biomass, (2) small elongated pellets, and (3) shorter filaments in hyphal growth in the periphery of the pellets. The correlation between morphology and product formation was also observed in industrially relevant complex media. Although balhimycin production starts after 72 h with maximum production around 168 h, the morphological changes in pellets are observed as early as 24 h after commencing of the batch. Therefore, morphology may be used as an early predictor of the end-of-batch productivity. We argue that a similar strategy can be developed for other strains where morphological indicators may be used as a batch monitoring tool.

Recent advances in the molecular basis of chemotherapy resistance and potential application of epigenetic therapeutics in chemorefractory renal cell carcinoma.

Among various types of cancers, kidney cancer is unique with respect to a low frequency of mutations and a relatively higher level of chemotherapy resistance. Resistance to chemotherapy is a major challenge in kidney cancer treatment in the clinic. Tremendous progress has been made in identifying the molecular changes associated with chemotherapy resistance in RCC. However, the exact contribution of these molecular changes to the acquisition of chemotherapy resistance is not fully understood. In addition to genetic changes, epigenetic alterations have been shown to contribute to various pathways associated with chemotherapy resistance, such as increased cell proliferation and survival, regulation of drug influx and efflux transporters, increased DNA repair, loss of DNA-damage-dependent apoptotic potential, cellular dedifferentiation to cancer stem cell, and epithelial-mesenchymal transitions (EMT). Moreover, recent studies suggest that epigenetic aberrations that can be reversed by epigenetic therapeutics can potentially be targeted to restore chemosensitivity in chemorefractory kidney cancer. This review article highlights current knowledge of the role of genetic and epigenetic aberrations as well as the physiological and metabolic changes associated with chemotherapeutic resistance. Additionally, current approaches and future directions for overcoming chemotherapeutic resistance including the potential of epigenetic therapeutic in chemorefractory kidney cancer have also been discussed. This article is categorized under: Cancer > Genetics/Genomics/Epigenetics.