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.

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.

Inhibition of As(III) and Hg(II) caused aortic hypercontraction by eugenol, linalool and carvone.

Acute and chronic exposure to arsenic and mercury is known to produce vasoconstriction. There is, however, no clarity concerning the pathways leading to this increased contraction. In this study we elicit and compare maximum contractility of rat aortas under resting conditions in the presence of arsenic and mercury, and delineate pathways mediating this effect. Phenylephrine (PE) induced hypercontraction of 37% and 32% were obtained when isolated aortic segments were exposed to 25 ?M As(III) and 6 nM Hg(II), respectively. Isometric contraction measurements in presence of apocynin, verapamil and sodium nitroprusside indicates that the major causes of increased contraction are reactive oxygen species (ROS) and depletion of nitric oxide (NO). Calcium influx plays a minor role in arsenic and mercury caused hypercontraction. In unexposed aorta, eugenol causes relaxation by inhibiting ROS and elevating NO, linalool by blocking voltage dependent calcium channel (VDCC) and elevating NO, and carvone by blocking calcium influx through VDDC. Since the arsenic and mercury hypercontraction is mediated by increased ROS and depleted NO, we hypothesize that molecules which neutralize ROS or elevate NO will be better ameliorators. In line with this argument, we found eugenol to be the best ameliorator of arsenic and mercury hypercontraction followed by linalool and carvone.

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.

Potentiated homeopathic drug Arsenicum Album 30C inhibits intracellular reactive oxygen species generation and up-regulates expression of arsenic resistance gene in arsenite-exposed bacteria Escherichia coli.

OBJECTIVE: To examine if potentiated homeopathic drug Arsenicum Album 30C (Ars Alb 30C) can reduce sodium arsenite-induced toxicity in Escherichia coli. METHODS: E. coli were exposed to low arsenite insult after they grew up to log phase in standard Luria-Bertani medium. E. coli were treated with 1 or 2 mmol/L sodium arsenite alone (control), or Ars Alb 30C was added to the medium of a subset of sodium arsenite-treated bacteria (drug-treated), or homeopathically agitated alcohol was added to the medium containing a subset of sodium arsenite-treated bacteria (placebo-treated). A sub-set of untreated E. coli served as the negative control. Glucose uptake, specific activities of hexokinase, lipid peroxidase (LPO), superoxide dismutase (SOD) and catalase, intra- and extra-cellular sodium arsenite content, cell growth, cell membrane potential, DNA damage, intracellular reactive oxygen species (ROS), adenosine triphosphate (ATP) and free glutathione content and expressions of arsB and ptsG gene in normal control, sodium arsenite-treated, drug-treated and placebo-treated E. coli were analyzed. Treatments were blinded and randomized. RESULTS: In sodium arsenite-treated E. coli, glucose uptake, intracellular ROS, LPO and DNA damage increased along with decrease in the specific activities of hexokinase, SOD and catalase, intracellular ATP and free glutathione contents and cell membrane potential and growth, and there were increases in expression levels of arsB gene and ptsG gene. Ars Alb 30C administration reduced arsenic toxicity in E. coli by inhibiting generation of ROS and increasing tolerance to arsenite toxicity and cell growth. CONCLUSION: Ars Alb 30C ameliorated arsenic toxicity and DNA damage, validating efficacy of ultra-highly diluted remedies used in homeopathy.

Potent reducing effects of vitamin D3 on the frequency of apoptosis induced by arsenic trioxide in NB4 cell line.

BACKGROUND: Arsenic trioxide and 1,25-(OH)2D3 (vitamin D3) are used for the treatment of lymphocytic leukemia. However, the effects of combined treatment of these drugs are controversial. In this study, the combined effects of these drugs on the induction of apoptosis in NB4 cells were investigated using the neutral comet assay. METHODS: NB4 cells were treated with various doses of arsenic trioxide (0.1 - 3 microM) and vitamin D3 (100 - 600 nM (alone or in combination. Twenty-four hours after treatment, neutral comet assay was performed and apoptotic cells were scored under a fluorescent microscope following staining with ethidium bromide. RESULTS: Results show that all doses of arsenic trioxide used in this study induced apoptosis in NB4 cells. The frequency of induced apoptosis was dose dependent and significantly higher than the controls (P<0.05 - 0.01). In contrast, vitamin D3 at concentrations of 100 - 600 nM produced no significant effect on apoptosis induction compared to the controls. Treatment of NB4 cells with a combination of arsenic trioxide and vitamin D3 resulted in reduction of apoptosis induced by arsenic trioxide which was not dependent on the dose of vitamin D3 (P<0.05). CONCLUSION: Results indicate that arsenic trioxide is a potent inducer of apoptosis in NB4 cells and vitamin D3 significantly decreased the sensitivity of cells to the induction of apoptosis by arsenic trioxide. These findings suggest that 1,25-(OH)2D3 might be involved in anti-apoptotic processes via reactive oxygen species scavenging or other mechanisms not yet known.

The effect of arsenic exposure and the efficacy of DMSA on the proteins and lipids of the gill tissues of Labeo rohita.

Arsenic is a naturally occurring, highly toxic environmental pollutant. Fourier transform infrared (FT-IR) spectroscopy is a non-disturbing technique which provides quantitative information about the molecular composition of biological samples. The aim of this work is to study the compositional and structural changes at the molecular level occurring in gill tissues of Labeo rohita fingerlings due to arsenic exposure for various exposure periods by using FT-IR spectroscopic technique. The results of the present study suggest that arsenic exposure causes significant changes on the major biochemical constituents such as proteins, lipids and nucleic acids in the gill tissues of L. rohita. The changes are more pronounced as the period of exposure is increased. The significant decrease in the intensity and area of the amide I peak and CH(3) asymmetric stretching band suggests an alteration in the protein profile and lipid levels respectively, due to arsenic exposure. The amide A peak shifts suggests a change in the level of protein amide hydrogen bonding due to arsenic exposure. Further, the treatment with meso-2,3-dimercaptosuccinic acid (DMSA) improves the levels of biochemical constituents significantly, which suggest that DMSA treatment reduces the toxic effects and helps the recovery of gill tissues and its return to the level of the control.

Resveratrol protects against arsenic trioxide-induced cardiotoxicity in vitro and in vivo.

BACKGROUND AND PURPOSE: The clinical use of arsenic trioxide (As(2)O(3)), a potent antineoplastic agent, is limited by its severe cardiotoxic effects. QT interval prolongation and apoptosis have been implicated in the cardiotoxicity of As(2)O(3). The present study was designed to evaluate the effects of resveratrol on As(2)O(3)-induced apoptosis and cardiac injury. EXPERIMENTAL APPROACH: In a mouse model of As(2)O(3)-induced cardiomyopathy in vivo, QT intervals and plasma enzyme activities were measured; cardiac tissues were examined histologically and apoptosis assessed. In H9c2 cardiomyocyte cells, viability, apoptosis, generation of reactive oxygen species (ROS) and cellular calcium levels were measured. KEY RESULTS: In the mouse model, resveratrol reduced As(2)O(3)-induced QT interval prolongation and cardiomyocyte injury (apoptosis, myofibrillar loss and vacuolization). In addition, increased lactate dehydrogenase activity and decreased activities of glutathione peroxidase, catalase and superoxide dismutase were observed in the plasma of As(2)O(3)-treated mice; these changes were prevented by pretreatment with resveratrol. In As(2)O(3)-treated H9c2 cardiomyocytes, resveratrol significantly increased cardiomyocyte viability and attenuated cell apoptosis as measured by acridine orange/ethidium bromide staining, TdT-mediated dUTP nick end labelling assay and caspase-3 activity. As(2)O(3)-induced generation of ROS and intracellular calcium mobilization in H9c2 cells was also suppressed by pretreatment with resveratrol. CONCLUSIONS AND IMPLICATIONS: Our results showed that resveratrol significantly attenuated As(2)O(3)-induced QT prolongation, structural abnormalities and oxidative damage in the heart. In H9c2 cardiomyocytes, resveratrol also decreased apoptosis, production of ROS and intracellular calcium mobilization induced by treatment with As(2)O(3). These observations suggested that resveratrol has the potential to protect against cardiotoxicity in As(2)O(3)-exposed patients.

An approach to elucidate potential mechanism of renal toxicity of arsenic trioxide.

OBJECTIVE: To investigate arsenic trioxide's renal toxicity, we analyzed the gene-expression patterns of primary renal and human kidney cells (HEK293 cell line) following exposure to arsenic trioxide. Moreover, we examined a potential renal toxic mechanism(s) of arsenic trioxide by using a toxicity-related gene and investigated potential treatments to reduce the renal toxicity of arsenic trioxide. MATERIALS AND METHODS: Arsenic trioxide was exposed to primary renal and HEK293 cells, and the gene-expression analysis was conducted using DNA microarray. Then, reactive oxygen species inhibitors or alpha-lipoic acid were added to HEK293 cells exposed arsenic trioxide and cell viability was determined. RESULTS: Expression of HMOX1 mRNA increased in a time- and dose-dependent manner, and translation of heme oxygenase 1 protein was also induced. Arsenic trioxide-induced cytotoxicity was inhibited by reactive oxygen species inhibitors. Moreover, superoxide anion was detected in arsenic trioxide-treated HEK293 cells. alpha-Lipoic acid ameliorated arsenic trioxide-induced cytotoxicity and reduced superoxide anion production in HEK293 cells, whereas it had no effect in promyelocytic leukemia cells (HL-60 cells and NB4 cells) and myeloma cells (KMS12BM cells and U266 cells). CONCLUSIONS: Arsenic trioxide-induced renal toxicity is strongly associated with the increased expression of HMOX1, and the cytotoxic mechanisms of arsenic trioxide involves reactive oxygen species production as well as another pathway. These preliminary results suggest that alpha-lipoic acid may be a suitable agent for prevention or treatment of arsenic trioxide-induced renal toxicity.

Effect of PAO (phenylarsine oxide) on the inhibitory effect of insulin and IGF-1 on insulin release from INS-1 cells.

Phenylarsine oxide (PAO) which complexes vicinal thiol groups is a valuable pharmacological tool to investigate the interaction of peptides such as insulin with their receptors and the signal transduction from the receptor to the cell interior. This tool was now used to elucidate the inhibitory effects of insulin and IGF-1 on insulin secretion via their receptors. Insulin and IGF-1 inhibited insulin release from INS-1 cells, an insulin secreting cell line. PAO was able to reverse this inhibitory effect of both hormones. Dimercaptopropanol (DMP), which is well known to antagonize PAO effects, inhibited the abolishment of PAO effect on the inhibitory effect of insulin and IGF-1 regarding insulin release. Membrane bound GLUT2 in INS-1 cells was increased by either insulin and IGF-1 which is counteracted by PAO. Thus the inhibitory effect of insulin and IGF-1 on insulin release is operative and can be disturbed by a thiol interacting compound such as PAO. This may happen at the receptor level or at the sub-receptor level.

Targeting Hsp90 by 17-AAG in leukemia cells: mechanisms for synergistic and antagonistic drug combinations with arsenic trioxide and Ara-C.

17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a new anticancer agent currently in clinical trials. The ability of 17-AAG to abrogate the function of heat-shock protein Hsp90 and modulate cellular sensitivity to anticancer agents has prompted recent research to use this compound in drug combination therapy. Here we report that 17-AAG has striking opposite effects on the activity of arsenic trioxide (ATO) and ara-C. Combination of 17-AAG with ATO exhibited a synergistic effect in leukemia cells, whereas coincubation of 17-AAG and ara-C showed antagonistic activity. Mechanistic studies revealed that ATO exerted cytotoxic action by reactive oxygen species generation, and activated Akt survival pathway. 17-AAG abrogated Akt activation and enhanced the activity of ATO. In contrast, treatment of leukemia cells with 17-AAG caused a G1 arrest, a decrease in DNA synthesis and reduced ara-C incorporation into DNA, leading to antagonism. The ability of 17-AAG to enhance the antileukemia activity of ATO was further demonstrated in primary leukemia cells isolated from patients with acute myeloid leukemia and chronic lymphocytic leukemia, including cells from refractory patients. Our data suggest that combination of 17-AAG and ATO may be an effective therapeutic regimen. Caution should be exercised in using 17-AAG together with ara-C, as their combination effects are schedule dependent.

As2O3-induced c-Src/EGFR/ERK signaling is via Sp1 binding sites to stimulate p21WAF1/CIP1 expression in human epidermoid carcinoma A431 cells.

Arsenic has been effectively used to treat acute promyelocytic leukemia, and can induce cell cycle arrest or apoptosis in human solid tumors. Previously, we have demonstrated that As2O3 can induce p21WAF1/CIP1 (p21) expression in A431 cells and then due to cellular cytotoxicity. Presently, we have clarified these signaling events and compared them with EGF. Using reporter assay, RT-PCR and Western blotting, we show that c-Src activation might be a prerequisite for As2O3-induced EGFR/Ras/Raf/ERK signaling. Furthermore, with the aids of 5'-deletion and site-directed mutagenesis, we demonstrate that Sp1 binding sites, ranging from -64 to -84 bp, are essential for As2O3- or EGF-regulated p21 expression. Finally, our experiments utilizing cycloheximide prompt the suggestion that the stability of mRNA or protein also contributes to As2O3- or EGF-induced p21 expression. Taken together, we conclude that the Sp1 binding sites are required for As2O3-induced p21 gene transcription through c-Src/EGFR/Ras/Raf/ERK pathway. Furthermore, post-transcriptional or post-translational stabilization mechanism is also essential for As2O3-induced p21 expression. EGF-induced p21 expression may involve similar mechanisms as those that operate in the As2O3-mediated reactions in A431 cells.

Role of the alkali labile sites, reactive oxygen species and antioxidants in DNA damage induced by methylated trivalent metabolites of inorganic arsenic.

In the last decade arsenic metabolism has become an important matter of discussion. Methylation of inorganic arsenic (iAs) to monomethylarsonic acid (MMA(V)) and dimethylarsinic acid (DMA(V)) is considered to decrease arsenic toxicity. However, in addition to these pentavalent metabolites, the trivalent metabolites monomethylarsonous (MMA(III)) and dimethylarsinous acid (DMA(III)) have been identified recently as intermediates in the metabolic pathway of arsenic in cultured human cells. To examine the role of oxidative damage in the generation of DNA strand breaks by methylated trivalent arsenic metabolites, we treated human lymphocytes with both metabolites at non-cytotoxic concentrations. We further tested whether these effects are sensitive to modulation by the antioxidants ascorbate (Vitamin C) and selenomethionine (Se-Met). Both trivalent metabolites produced oxidative stress related DNA damage, consisting of single strand breaks and alkali-labile sites, with MMA(III) being more potent at low concentrations than DMA(III). Neither MMA(III) nor DMA(III) induced DNA-double strand breaks. The oxidative stress response profiles of the metabolites were parallel as determined by lipid peroxidation induction. MMA(III) induced peroxidation from the lowest concentration tested, while effects of DMA(III) were apparent only at concentrations above 10 muM. The antioxidant Se-Met exhibited a more pronounced inhibition of trivalent arsenic metabolite-induced oxidative-DNA damage than did vitamin C. The present findings suggest that DNA damage by methylated trivalent metabolites at non-cytotoxic concentrations may be mediated by a mix of reactive oxygen and nitrogen oxidized species.

Arsenic trioxide-induced hela cell death is partially prevented by K+ channel blockers.

AIM: To investigate the effects of K+ channel blockers on arsenic trioxide-induced HeLa cell death. METHODS: Viability of HeLa cells was assessed by mitochondrial dehydrogenase activity using colorimetric MTT assay and the voltage-dependent K+ currents were recorded by using patch-clamp technique. RESULTS: Exposure of As2O3 (5 micromol x L(-1)) for 24 h caused marked HeLa cell death. The rest living cells after As2O3 24 h-incubation showed significant increase of K+ currents densities. At +80 mV, the densities of K+ currents (61 +/- 18) pA/10 pF (n = 8) in As2O3 24 h-incubation group were significantly more than that in the control group (38 +/- 10) pA/10 pF (n = 8, P < 0.05). The HeLa cells were prevented partially from As2O3-induced cell death by co-application for 24 h with typical voltage-dependent K+ channel blockers, 4-aminopyridine (3 mmol x L(-1)) or tetraethylammonium (5 mmol x L(-1)). 4-Aminopyridine (3 mmol x L(-1)) or tetraethylammonium (5 mmol x L(-1)) did not show any toxic effects on HeLa cells. CONCLUSION: Chronic treatment with As2O3 increased voltage-dependent K+ currents in HeLa cells and the cell death induced by As2O3 was reduced partially by voltage-dependent K+ channel blockers, 4-aminopyridine or tetraethylammonium.