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.

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.

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.

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.

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.

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.

Preclinical validation of a live attenuated dermotropic Leishmania vaccine against vector transmitted fatal visceral leishmaniasis.

Visceral Leishmaniasis (VL), a potentially fatal disease is caused by Leishmania donovani parasites with no vaccine available. Here we produced a dermotropic live attenuated centrin gene deleted Leishmania major (LmCen-/-) vaccine under Good Laboratory Practices and demonstrated that a single intradermal injection confers robust and durable protection against lethal VL transmitted naturally via bites of L. donovani-infected sand flies and prevents mortality. Surprisingly, immunogenicity characteristics of LmCen-/- parasites revealed activation of common immune pathways like L. major wild type parasites. Spleen cells from LmCen-/- immunized and L. donovani challenged hamsters produced significantly higher Th1-associated cytokines including IFN-γ, TNF-α, and reduced expression of the anti-inflammatory cytokines like IL-10, IL-21, compared to non-immunized challenged animals. PBMCs, isolated from healthy people from non-endemic region, upon LmCen-/- infection also induced more IFN-γ compared to IL-10, consistent with our immunogenicity data in LmCen-/- immunized hamsters. This study demonstrates that the LmCen-/- parasites are safe and efficacious against VL and is a strong candidate vaccine to be tested in a human clinical trial.

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.

Pressurized liquid extraction followed by liquid chromatography coupled to a fluorescence detector and atmospheric pressure chemical ionization mass spectrometry for the determination of benzo(a)pyrene metabolites in liver tissue of an animal model of colon cancer.

Since metabolism is implicated in the carcinogenesis of toxicants, an efficient extraction method together with an analytical method is warranted to quantify tissue burdens of a carcinogen and/or its metabolites. Therefore, the aim of this study was to validate a pressurized liquid extraction (PLE) method for measuring metabolites of benzo(a)pyrene [B(a)P; a food-borne carcinogen] from tissue samples. The sample extraction was performed separately by PLE and liquid-liquid extraction (LLE). PLE followed by high-performance liquid chromatography coupled to online fluorescence detector (HPLC-FLD) was used to quantify separated analytes; and by ultra-high-performance liquid chromatography (UHPLC) coupled to atmospheric pressure chemical ionization tandem mass spectrometry (UHPLC-APCI-MS/MS) were used for confirmation purposes. The UHPLC-MS/MS was set-up in the atmospheric pressure chemical ionization (APCI) positive interface with selective reaction monitoring (SRM). The analytical performance characteristics of the PLE technique was assessed at different temperatures, pressure, number of cycles and solvent types. A methanol + chloroform + water mixture (30:15:10, v/v/v) yielded greater recoveries at an extraction temperature range of 60-80°C, pressure of 10 MPa and an extraction time of 10 min. The PLE method was validated by the analysis of spiked tissue samples and measuring recoveries and limits of quantitation for the analytes of interest using HPLC-FLD equipment. The optimized PLE-HPLC-FLD method was used to quantify the concentrations of B(a)P metabolites in liver samples obtained from a colon cancer animal model. Overall, PLE performed better in terms of extraction efficiency, recovery of B(a)P metabolites and shortened sample preparation time when compared with the classic LLE method.

Carbon nanotubes affect early growth, flowering time and phytohormones in tomato.

Carbon nanotube (CNT) applications are increasing in consumer products, including agriculture devices, making them an important contaminant to study in the field of plant nanotoxicology. Several studies have observed the uptake and effects of CNTs in plants. However, in other studies differing results were observed on growth and physiology depending on the plant species and type of CNT. This study focused on the effects of CNTs on plant phenotype with growth, time to flowering, fruiting time as endpoints, and physiology, through amino acid and phytohormone content, in tomato after exposure to multiple types of CNTs. Plants grown in CNT-contaminated soil exhibited a delay in early growth and flowering (especially in treatments of 1 mg/kg multi-walled nanotubes (MWNTs), 10 mg/kg MWNTs, and 1 mg/kg MWNTs-COOH). However, CNTs did not affect plant growth or height later in the life cycle. No significant differences in abscisic acid (ABA) and citrulline content were observed between the treated and control plants. However, single-walled nanotube (SWNT) exposure significantly increased salicylic acid (SA) content in tomato. These results suggest that SWNTs may elicit a stress response in tomatoes. Results from this study offer more insight into how plants respond and acclimate to CNTs. These results will lead to a better understanding of CNT impact on plant phenotype and physiology.

Analysis of Toxicants-Induced Alterations in DNA Methylation by Methylation-Sensitive-Random Amplified Polymorphic DNA-Polymerase Chain Reaction (MS-RAPD-PCR).

Overwhelming evidence suggests that in addition to the genetic changes of DNA mutations, epigenetic changes of DNA methylation and histone modifications play important role in regulation of gene expression. DNA methylation is the most frequent epigenetic alteration observed in mammalian genomes, and generally it is negatively correlated with gene expression. Various methods are available for the detection of DNA methylation changes. Although the recent high-throughput methods for DNA methylation analysis have various advantages, they require high levels of technical expertise, costly equipment, and reagents. Because of these reasons, many of the global DNA methylation analysis methods are mainly performed at core facility, and laboratories with limited resources and expertise are not able to use these methods. Methylation-Sensitive-Random Amplified Polymorphic DNA-Polymerase Chain Reaction (MS-RAPD-PCR) is a restriction enzyme digestion and PCR-based method for the analysis of DNA methylation changes. This method is cost-effective, requires simple and basic instrumentation, and therefore can easily be performed in any laboratory with basic setup having a regular DNA thermal cycler and DNA gel electrophoresis system. Additional advantages of this method over other methods for DNA methylation analysis are that it requires very less amount of DNA and can screen DNA methylation changes globally at genome-wide level with high sensitivity. This method has been successfully used to detect changes in DNA methylation either occurring naturally or induced by various toxicants and environmental factors. A detail experimental protocol for MS-RAPD-PCR is described in this chapter.

Epigenetic reprogramming and potential application of epigenetic-modifying drugs in acquired chemotherapeutic resistance.

Chemotherapy is the most common clinical choice of treatment for cancer, however, acquired chemoresistance is a major challenge that limits the successful outcome of this option. Systematic review of in vitro, in vivo, preclinical and clinical studies suggests that acquired chemoresistance is polygenic, progressive, and involve both genetic and epigenetic heterogeneities and perturbations. Various mechanisms that confer resistance to chemotherapy are tightly controlled by epigenetic regulations. Poised epigenetic plasticity and temporal increase in epigenetic alterations upon chemotherapy make chemoresistance likely an epigenetic-driven process. The transient and reversible nature of epigenetic modulations enable ways to intervene the epigenetic re-programing associated with acquired chemoresistance via application of epigenetic modifying drugs. This review discusses recent understandings behind the various mechanisms of acquired chemoresistance that are under the control of epigenetic drivers, potential application of epigenetic-based drugs in resensitizing refractory cancers to chemotherapy, the limitations and future scope for clinical application of epigenetic therapeutics in successfully addressing chemoresistance.

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.

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 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.

Role of cellular reprogramming and epigenetic dysregulation in acquired chemoresistance in breast cancer.

Acquired resistance to chemotherapy is a major limitation in clinical treatment for breast cancer. Accumulating evidence from in vitro, in vivo and clinical studies suggest that acquired chemoresistance is progressive, multifactorial and involve genetic and epigenetic aberrations. Among various mechanisms that contribute to chemoresistance, cellular reprogramming has extensively been implicated in breast cancer resistance lately. Cellular reprogramming events such as acquisition of epithelial to mesenchymal transition (EMT) and cancer stemness (CSCs) not only provide cancer cells with reversible phenotypic plasticity and survival advantage against cytotoxicity but also leads to aggressiveness, metastasis, clinical resistance, tumor recurrence and poor survival. The transient and reversible nature of cellular reprogramming processes and their controlled interaction with epigenetic regulatory complexes strongly support the involvement of dynamic epigenetic regulatory network in governing the cellular reprogramming and associated acquired chemoresistance. Further, epigenetic modulations are also gaining interest as promising interventions addressing the cancer cell reprogramming machinery to overcome acquired chemoresistance. This review discusses the previous reports and our recent findings that lead to current understanding of epigenetic dysregulation dictating the cellular reprogramming processes such as acquisition of EMT and CSCs phenotype and how they co-ordinate to establish acquired drug resistance in breast cancer.

Reversal of epigenetic aberrations associated with the acquisition of doxorubicin resistance restores drug sensitivity in breast cancer cells.

Acquired resistance against doxorubicin is a major limitation in clinical treatment of breast cancer. The molecular mechanism behind the aberrant expression of genes leading to doxorubicin resistance is not clear. Epigenetic changes play an important role in the regulation of gene expression. Therefore, the objective of this study was to identify the epigenetic mechanism underlying acquired doxorubicin resistance in breast cancer cells. Doxorubicin-resistant cells were selected by repeated exposure of MCF-7 and MDA-MB-231 breast cancer cell lines to clinically relevant doses of doxorubicin for 18 months. MTT assay, cell cycle analysis, colony formation, qRT-PCR, and Western blot analyses were used to characterize the epigenetic and molecular mechanism. Pyrosequencing was used to detect MSH2 promoter hypermethylation. Aberrant expression of epigenetic regulatory genes, a significant increase in H3 acetylation and methylation, as well as promoter hypermethylation-mediated inactivation of MSH2 gene were associated with the acquired resistant phenotype. Demethylating agent 5-Aza-deoxycytidine and HDAC inhibitor Trichostatin A significantly re-sensitized resistant cells to doxorubicin. Findings of this study revealed that epigenetic aberrations including promoter hypermethylation-mediated inactivation MSH2 contribute to the acquisition of doxorubicin resistance in breast cancer cells. Additionally, our data suggest that some of these epigenetic aberrations are progressive during resistance development and therefore can potentially be used as biomarkers for early detection of resistance. These epigenetic aberrations, being reversible, can also serve as targets for epigenetic therapy to re-sensitize doxorubicin-resistant breast cancer cells. Epigenetic inactivation of mismatch repair gene MSH2 further suggests that loss of MMR-dependent apoptotic potential could be a novel mechanistic basis for the acquisition of doxorubicin resistance in breast cancer cells.

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.

Treatment schedule and estrogen receptor-status influence acquisition of doxorubicin resistance in breast cancer cells.

Breast cancer is the most common cancer in women for which doxorubicin is still the mainstay treatment. However, chemotherapy resistance is a major limitation in breast cancer treatment. Role of treatment schedule and estrogen receptor (ER) status in subtypes of breast cancers in acquired resistance development is not clear. Therefore, objective of this study was to evaluate whether the treatment schedule and ER status in breast cancer cells influence the doxorubicin resistance. To address these questions, ER-positive MCF-7 and triple-negative MDA-MB-231 breast cancer cell lines were given either continuous or intermittent exposure with clinically relevant concentration of doxorubicin and the influence of these two treatment strategies on resistance to drug sensitivity was evaluated. Results revealed that intermittent treatment but not the continuous treatment induced resistance in breast cancer cells against doxorubicin. MCF-7 cells developed relatively earlier and high level of resistance when compared to MDA-MB-231 cells. Acquisition of epithelial to mesenchymal transition (EMT) and cancer stem cell-like phenotype was also observed during resistance development in MCF-7 cells. Changes in the expression of selected marker genes including drug transporters confirmed doxorubicin resistance in these cells. In summary, this study suggests that acquisition of resistance against doxorubicin depends on the treatment schedule of this drug as well as the estrogen receptor-based subtypes of breast cancer. Additionally, acquisition of EMT and stem cell-like phenotype further provided a molecular basis for breast cancer subtype-dependent chemotherapeutic resistance development. Findings of this study will have significant clinical implications in optimizing the chemotherapy schedule to minimize chemoresistance in breast cancer patients.