Ellagic acid mitigates arsenic-trioxide-induced mitochondrial dysfunction and cytotoxicity in SH-SY5Y cells.

In the current study, neuroprotective significance of ellagic acid (EA, a polyohenol) was explored by primarily studying its antioxidant and antiapoptotic potential against arsenic trioxide (As2 O3 )-induced toxicity in SH-SY5Y human neuroblastoma cell lines. The mitigatory effects of EA with particular reference to cell viability and cytotoxicity, the generation of reactive oxygen species, DNA damage, and mitochondrial dynamics were studied. Pretreatment of SH-SY5Y cells with EA (10 and 20 µM) for 60 min followed by exposure to 2 µM As2 O3 protected the SH-SY5Y cells against the harmful effects of the second. Also, EA pre-treated groups expressed improved viability, repaired DNA, reduced free radical generation, and maintained altered mitochondrial membrane potential than those exposed to As2 O3 alone. EA supplementation also inhibited As2 O3 -induced cytochrome c expression that is an important hallmark for determining mitochondrial dynamics. Thus, the current investigations are more convinced for EA as a promising candidate in modulating As2 O3 -induced mitochondria-mediated neuronal toxicity under in vitro system.

Antagonistic effect of N-ethylmaleimide on arsenic-mediated oxidative stress-induced poly(ADP-ribosyl)ation and cytotoxicity.

Long-term exposure to arsenic has been known to induce neoplastic initiation and progression in several organs; however, the role of arsenic (As2 O3 ) in oxidative stress-mediated DNA damage remains elusive. One of the immediate cellular responses to DNA damage is poly(ADP-ribosyl)ation (PARylation), which mediates DNA repair and enhances cell survival. In this study, we found that oxidative stress (H2 O2 )-induced PARylation was suppressed by As2 O3 exposure in different human cancer cells. Moreover, As2 O3 treatment promoted H2 O2 -induced DNA damage and apoptosis, leading to increased cell death. We found that N-ethylmaleimide (NEM), an organic compound derived from maleic acid, could reverse As2 O3 -mediated effects, thus enhancing PARylation with attenuated cell death and increased cell survival. Pharmacologic inhibition of glutathione with l-buthionine-sulfoximine blocked the antagonistic effect of NEM on As2 O3 , thereby continuing As2 O3 -mediated suppression of PARylation and causing DNA damage. Our findings identify NEM as a potential antidote against As2 O3 -mediated DNA damage in a glutathione-dependent manner. Copyright © 2016 John Wiley & Sons, Ltd.

Endoplasmic reticulum stress mediates the arsenic trioxide-induced apoptosis in human hepatocellular carcinoma cells.

Arsenic trioxide has been proven to trigger apoptosis in human hepatocellular carcinoma cells. Endoplasmic reticulum stress has been known to be involved in apoptosis through the induction of CCAAT/enhancer-binding protein homologous protein. However, it is unknown whether endoplasmic reticulum stress mediates arsenic trioxide-induced apoptosis in human hepatocellular carcinoma cells. Our data showed that arsenic trioxide significantly induced apoptosis in human hepatocellular carcinoma cells. Furthermore, arsenic trioxide triggered endoplasmic reticulum stress, as indicated by endoplasmic reticulum dilation, upregulation of glucose-regulated protein 78 and CCAAT/enhancer-binding protein homologous protein. We further found that 4-phenylbutyric acid, an inhibitor of endoplasmic reticulum stress, alleviated arsenic trioxide-induced expression of CCAAT/enhancer-binding protein homologous protein. More important, knockdown of CCAAT/enhancer-binding protein homologous protein by siRNA or inhibition of endoplasmic reticulum stress by 4-phenylbutyric acid alleviated apoptosis induced by arsenic trioxide. Consequently, our results suggested that arsenic trioxide could induce endoplasmic reticulum stress-mediated apoptosis in hepatocellular carcinoma cells, and that CCAAT/enhancer-binding protein homologous protein might play an important role in this process.

Differential in vivo genotoxicity of arsenic trioxide in glutathione depleted mouse bone marrow cells: expressions of Nrf2/Keap1/P62.

Generation of reactive oxygen species is one of the major contributors in arsenic-induced genotoxicity where reduced glutathione (GSH) could be an important determining factor. To understand the role of endogenous GSH, arsenic trioxide (As2O3) was administered in buthionine sulfoximine (BSO)- and N-acetyl-L-cysteine (NAC)-treated mice. As2O3-induced significant chromosome aberrations (CAs) in all treatment groups compared with the control. BSO-treated mouse bone marrow cells showed significant CAs at a dose of 2 mg As2O3 kg(-1) b.w. Similar induction was not evident at 4 mg As2O3 kg(-1) b.w. and exhibited antagonistic effect at 8 mg As2O3 kg(-1) b.w. To understand this differential effect, expression pattern of Nrf2 was observed. Nrf2 expression increased following As2O3 treatment in a dose-dependent manner up to 4 mg As2O3 kg(-1) b.w after which no further increase was noticed. NAC pre-treatment significantly reduced the extent of As2O3-induced CAs suggesting the protective role of endogenous GSH against arsenic-induced genotoxicity.

Effect of dentin powder on the antimicrobial properties of hyperpure chlorine-dioxide and its comparison to conventional endodontic disinfecting agents.

INTRODUCTION: Previously we found that the high purity chlorine-dioxide(ClO2) has a very potent disinfectant efficacy on oral pathogenic microorganisms and as a root canal irrigant it is able to eliminate the experimental Enterococcus faecalis(E. faecalis) infection from the root canal system. This study examines whether the presence of dentin powder influences the antibacterial efficacy of ClO 2. METHODS: In an in vitro dentin powder model the following irrigants were tested against planktonic E. faecalis: 2% chlorhexidine (CHX), 2.5% sodium hypochlorite (NaOCl), 0.12%ClO2 (Solumium) and one local root canal medicament: saturated Ca(OH)2. Survival of bacteria exposed to agents without and with human dentin powder or preincubated with dentin powder was investigated. The effect of the dentin powder on ClO2 concentration was investigated by titrations. RESULTS: Without dentin powder ClO 2 killed all E. faecalis and delivered the best result already after 1 minute; however, after longer contact time with dentin the difference between the disinfectants disappeared. The presence of dentin powder decreased the concentration of ClO 2 and attenuated the antibacterial efficiency of ClO2 and Ca(OH)2, but did not decrease of CHX and NaOCl.Preincubation with dentin powder caused significant loss of antibacterial activity of all investigated agents, ClO2 and Ca(OH)2 having the highest reduction. CONCLUSION: As the presence of dentin powder had a negative effect on the efficacy of disinfectants, the importance of elimination of dentin scrapings and smear layer from the root canal system during endodontic treatments is highly recommended. ClO 2 can be effective for a final rinse.

Upregulation of ID2 antagonizes arsenic trioxide-induced antitumor effects in cancer cells.

AIMS AND BACKGROUND: Arsenic trioxide (ATO) strongly induces apoptosis and differentiation in acute promyelocytic leukemia, and induces cell cycle arrest in most solid tumors. Although many signaling pathways are involved in its antitumor mechanism, a detailed investigation of the transforming growth factor beta-bone morphogenetic protein signaling pathway has not been performed. METHODS AND STUDY DESIGN: A microarray containing 113 genes associated with the pathway was used to screen important molecules that participate in the antitumor effects of ATO. The expression levels of the inhibitors of DNA binding-2 (ID2) in 4 different types of cancer cells were determined by quantitative reverse transcription PCR and Western blotting. Human esophageal squamous cell carcinoma cell line Eca109 and pancreatic carcinoma cell line BxPC3 cells were transfected with siRNAs targeting ID2 and scrambled control siRNA. Cell proliferation was evaluated by methyl thiazolyl tetrazolium assay. RESULTS: Eighteen upregulated and 12 downregulated genes were identified. After verification at the transcriptional and translational levels in 4 different cancer cells, ID2 was identified as an ATO antitumor-associated protein. In addition, specific silencing of ID2 could enhance ATO-induced cell proliferation inhibition in cancer cells. CONCLUSIONS: A combination of ATO and ID2-targeted agents may have considerable therapeutic benefits in cancers.

Azidothymidine hinders arsenic trioxide-induced apoptosis in acute promyelocytic leukemia cells by induction of p21 and attenuation of G2/M arrest.

To enhance anticancer efficacy of the arsenic trioxide (ATO), the combination of ATO and azidothymidine (AZT), with convergence anti-telomerase activity, were examined on acute promyelocytic leukemia (APL) cell line, NB4. In spite of an induction of apoptosis by both drugs separately and a synergistic effect of them on hTERT down-regulation and telomerase inhibition, the ATO-induced cytotoxicity was reduced when it was used in combination with AZT. AZT attenuated the ATO effects on viability, metabolic activity, DNA synthesis, and apoptosis. These observations, despite the deflection from the main goal of this study, dedicate an especial opportunity to elucidate the importance of some of the mechanisms that have been suggested by which ATO induces apoptosis. Cell cycle distribution, ROS level, and caspase-3 activation analyses suggest that AZT reduced the ATO-induced cytotoxic effect possibly via relative induction and diminution of cells accumulated in (G1, S) and (G2/M) phase, respectively, as well as through attenuation of ROS generation and subsequent caspase-3 inhibition. QRT-PCR assay revealed that induction of p21expression by the combined AZT/ATO compared to ATO alone could be a reason for the relative decline of cells accumulation in G2/M and the increase of cells in G1 and S phases. Therefore, the G2/M arrest and ROS generation are likely principle mediators for the ATO-induced apoptosis and can be used as a guide to design rational combinatorial strategies involving ATO and agents with G2/M arrest or ROS generation capacity to intensify ATO-induced apoptosis.

Metallothionein prevention of arsenic trioxide-induced cardiac cell death is associated with its inhibition of mitogen-activated protein kinases activation in vitro and in vivo.

Cardiotoxicity induced by arsenic trioxide has become a serious blockade of clinical applications of this effective anticancer agent. The general mechanism responsible for arsenic cardiotoxicity has been attributed to its induction of oxidative stress. Metallothionein (MT) has been extensively proven to be a potent endogenous antioxidant that protects heart against oxidative stress-induced cardiac damage. To investigate whether and how MT protects against arsenic cardiotoxicity, MT-overexpressing H9c2 (MT-H9c2) cardiac cells and transgenic (MT-TG) mice with their corresponding controls were exposed to the clinical relevant dose of arsenic trioxide. Cardiac cell apoptosis was detected by molecular indices, including the cleavage of caspase 3 and caspase 12, Bax/Bcl2 expression ratio, CHOP expression and/or confirmed by a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Arsenic trioxide dose- and time-dependently induced cardiac cell death in H9c2 cells with a significant activation of major MAPK subfamily members such as ERK1/2, JNK and p38, but not in MT-H9c2 cells. Importantly, the protective effect of MT on arsenic trioxide-induced apoptotic cell death was completely recaptured in the heart of MT-TG with a significant prevention of MAPKs activation. These results indicate that arsenic trioxide-upregulated MAPKs might play important role in arsenic trioxide-induced apoptotic cell death in cardiac cells both in vivo and in vitro, and MT's suppression of arsenic trioxide apoptotic effect was associated with the inhibition of MAPK activation. Therefore, selective elevation of cardiac MT levels with pharmacological approaches may be a potential strategy for the prevention of arsenic cardiotoxicity.

Rho-kinase inhibitor Y-27632 attenuates arsenic trioxide toxicity in H9c2 cardiomyoblastoma cells.

The purpose of this study is to examine the molecular mechanism underlying the toxicity of arsenic trioxide (ATO) in cardiac cells. H9c2 rat cardiomyoblastoma cells undergo apoptosis during exposure to the concentrations of ATO > 10 µM for 24 h. The process is accompanied by the activation of caspases and is suppressed by the pan-caspase inhibitor z-VAD. Since ATO-induced H9c2 cell death is suppressed by Rho-kinase (ROCK) inhibitor Y-27632, but not by any antioxidants tested, apoptosis by ATO seems to be initiated through a ROCK-dependent and reactive oxygen species-independent mechanism. During the execution of apoptosis by ATO, the induction of autophagy is also observed. Importantly, autophagy is accelerated in cells treated with ATO plus Y-27632, although Y-27632 alone does not induce autophagy. The cytoprotective effect of Y-27632 against ATO toxicity is abrogated by the co-administration of an autophagy inhibitor, 3-methyladenine, suggesting that autophagy contributes to the cytoprotection by Y-27632. Taken together, the data indicate that the activation of ROCK is required for apoptotic H9c2 cardiomyoblastoma cell death by ATO, and that the ROCK inhibition not only inhibits caspase-dependent apoptotic machinery, but also causes a rise in the cytoprotective autophagy processes during ATO exposure.

Arsenic trioxide toxicity in H9c2 myoblasts--damage to cell organelles and possible amelioration with Boerhavia diffusa.

Arsenic trioxide (ATO) has been long used as a chemotherapeutic agent because of its significant anticancer property. Unfortunately, the use of ATO is limited due to its cardiotoxic effects. The present study evaluates the protective property of ethanolic extract of Boerhavia diffusa (BDE) against ATO-induced toxicity on various cell organelles in H9c2 cardiomyocytes. The effects of different concentrations of ATO (5, 7.5 and 10 µM) on cell organelles like mitochondria, endoplasmic reticulum (ER), lysosome and actin, generation of reactive oxygen species, antioxidant enzyme status and intracellular calcium overload were evaluated. ATO significantly (P ≤ 0.05) altered mitochondrial transmembrane potential, intracellular calcium level, ER, lysosomal activity and F-actin network in addition to induction of oxidative stress. Co-treatment with BDE protected the cardiomyocytes from the adverse effects of ATO, especially at 5 µM concentration, which was evident from decreased activity of lactate dehydrogenase (5 µM ATO + 20 µg/mL BDE: 6.61 ± 1.97 µU/mL, respective control group: 16.15 ± 1.92 µU/mL), reduced oxidative stress, calcium influx and organelle damage. Results obtained from the present study allow for a better characterization of the effects of ATO on H9c2 myoblasts. In conclusion, our data suggest that cell organelles are also the targets of ATO-induced cardiotoxicity in addition to other reported targets like ion channels, and BDE has the potential to protect the cardiotoxicity induced by ATO.