gzhi Chen

Sodium arsenite and arsenic trioxide differently affect the oxidative stress, genotoxicity and apoptosis in A549 cells: An implication for the paradoxical mechanism

Although arsenic toxicity greatly depends on its chemical forms, few studies have taken into account the paradoxical phenomenon which is manifested by that sodium arsenite (NaAsO₂) acts as a potent carcinogen but arsenic trioxide (As₂O₃) serves as an effective therapeutic agent. In this study, we compared the in vitro effects of NaAsO₂ and As₂O₃ on cell viability, colony formation, cell cycle progression, apoptosis, genotoxicity and oxidative stress in human lung adenocarcinoma A549 cells. Our results demonstrated that both NaAsO₂ and As₂O₃ caused oxidative stress, genotoxicity, cytotoxicity, cell cycle arrest as well as apoptosis, while As₂O₃ induced higher production of reactive oxygen species (ROS) with a more remarkable decrease in superoxide dismutase (SOD) activities and intracellular levels of glutathione (GSH) than NaAsO₂. Moreover, the degree of DNA damage, chromosomal breakage, cell cycle arrest and apoptosis in As₂O₃-treated cells were more severe than those in NaAsO₂-treated cells. These findings suggest that differential effects and mechanisms of NaAsO₂ and As₂O₃ may responsible for the paradoxical effects of arsenic on the carcinogenesis and anticancer function.

Dual role of resveratrol in modulation of genotoxicity induced by sodium arsenite via oxidative stress and apoptosis.

The potential benefits of resveratrol as an anticancer (proapoptosis) and antioxidant (pro-survival) compound have been studied extensively. However, the role of resveratrol in modulation of the toxicity induced by sodium arsenite (NaAsO₂) is still unclear. In the present study, we examined the effects of resveratrol on NaAsO₂-induced cytotoxicity, DNA and chromosomal damage, cell cycle progression, apoptosis and oxidative stress in human lung adenocarcinoma epithelial (A549) cell line at concentrations from 1 to 20 μM after 24h exposure. Our results revealed that at 1 and 5 μM, resveratrol was found to exert benefit effects, promoting cell viability and proliferation over 24h NaAsO₂ exposure, whereas, resveratrol was showed to inhibit cell survival under the same condition at 20 μM. Corresponding to the opposing effect of resveratrol at low vs. high concentrations, DNA and chromosomal damage, cell apoptotic rate and level of oxidative stress were also alleviated by lower concentrations (1, 5 μM) of resveratrol, but exacerbated by higher concentration (20 μM) resveratrol. Our study implicates that resveratrol is the most beneficial to cells at 1 and 5 μM and caution should be taken in applying resveratrol as an anticancer therapeutic agent or nutraceutical supplement due to its concentration dependent effect.

Resveratrol Synergistically Triggers Apoptotic Cell Death with Arsenic Trioxide via Oxidative Stress in Human Lung Adenocarcinoma A549 Cells

Arsenic trioxide (As2O3) is a potent anticancer drug for the treatment of acute promyelocytic leukemia. However, the clinical applications of the agent to treat solid tumors are largely compromised by the drug resistance. Our previous study demonstrated that resveratrol, a plant-derived natural product, could potentiate the toxicity of arsenite in lung adenocarcinoma A549 cells at relatively high concentration, indicating that combination of resveratrol and As2O3 may be a helpful strategy to solve the drug resistance of As2O3 in tumor cells. To test this possibility, in the present study, we determined the combined effects of resveratrol and As2O3 in cultured A549 cells. Our results showed that co-treatment of resveratrol with As2O3 resulted in a synergistic augmentation of cytotoxicity and apoptosis in cells at the tested concentration. To further reveal the detailed mechanism of this synergistic effect on cytotoxicity and apoptosis, apoptosis-related proteins, DNA and chromosomal damage, and the level of oxidative stress were also evaluated. Our data revealed that co-treatment with resveratrol and As2O3 caused more genotoxicity and serious oxidative stress in A549 cells than that of single agent treatment. Moreover, resveratrol and As2O3 could also corporately enhance the release of cytochrome c and the expressions of death receptor Fas and FasL. Together, our results suggest that resveratrol and As2O3 synergistically increase the apoptotic cell death in A549 cells through induction of oxidative stress, indicating that the combination of resveratrol with As2O3 may be a promising strategy to increase the clinical efficacy of As2O3 in the treatment of lung tumor.

Resveratrol protects against arsenic trioxide-induced oxidative damage through maintenance of glutathione homeostasis and inhibition of apoptotic progression

Arsenic trioxide (As2O3) is commonly used to treat acute promyelocytic leukemia and solid tumors. However, the clinical application of the agent is limited by its cyto‐ and genotoxic effects on normal cells. Thus, relief of As2O3 toxicity in normal cells is essentially necessary for improvement of As2O3‐mediated chemotherapy. In this study, we have identified a series of protective effects of resveratrol against As2O3‐induced oxidative damage in normal human bronchial epithelial (HBE) cells. We showed that treatment of HBE cells with resveratrol significantly reduced cellular levels of DNA damage, chromosomal breakage, and apoptosis induced by As2O3. The effect of resveratrol against DNA damage was associated with a decreased level of reactive oxygen species and lipid peroxidation in cells treated by As2O3, suggesting that resveratrol protects against As2O3 toxicity via a cellular anti‐oxidative stress pathway. Further analysis of the roles of resveratrol demonstrated that it modulated biosynthesis, recycling, and consumption of glutathione (GSH), thereby promoting GSH homeostasis in HBE cells treated by As2O3. This was further supported by results showing that resveratrol prevented an increase in the activities and levels of caspases, Fas, Fas‐L, and cytochrome c proteins induced by As2O3. Our study indicates that resveratrol relieves As2O3‐induced oxidative damage in normal human lung cells via maintenance of GSH homeostasis and suppression of apoptosis. Environ. Mol. Mutagen. 56:333–346, 2015.

Critical role of cellular glutathione homeostasis for trivalent inorganic arsenite-induced oxidative damage in human bronchial epithelial cells.

Trivalent inorganic arsenic (iAs(3+)) is a powerful carcinogen that enhances the risk of lung cancer. Paradoxically, iAs(3+) also shows substantial efficacy in the treatment of lung tumors. However, the exact molecular mechanisms underlying iAs(3+)-induced toxicity and therapeutic effect in lung remain unclear. In this study, the effects of iAs(3+), sodium arsenite (NaAsO2) and arsenic trioxide (As2O3), on cell viability, apoptosis, genotoxicity and oxidative stress in cultured human bronchial epithelial cells were observed. Our results showed that NaAsO2 and As2O3 exposure could result in defects in cell proliferation and greatly enhance the level of oxidative damage. To clarify the critical role of glutathione (GSH) homeostasis in oxidative damage induced by iAs(3+), we further measured the content of GSH, ratio of GSH to GSSG, and the activities of GSH-related enzymes involved in the process of GSH synthesis, recycling and utilization. Our data demonstrated that NaAsO2 and As2O3 disrupted the balance of GSH homeostasis, and NaAsO2- and As2O3-induced oxidative damage was closely associated with the imbalance in GSH synthesis, recycling and utilization. To better understand the physiologic significance of Nrf2 in maintaining GSH-homeostasis, the expression level of Nrf2 was measured after iAs(3+) exposure. We found that the protein expression levels of Nrf2 were increased in both NaAsO2- and As2O3-treated cells. Collectively, our findings suggest that disturbed Nrf2-regulated GSH-homeostasis is associated with the oxidative damage triggered by iAs(3+), and loss of GSH homeostasis might implicate in both the pathogenesis of iAs(3+)-induced lung diseases and anticancer activity of iAs(3+).

Nuclear translocation of nuclear factor kappa B is regulated by G protein signaling pathway in arsenite-induced apoptosis in HBE cell line

Arsenite is a certainly apoptosis inducer in various cell types. However, the detailed mechanism underlying how arsenite trigger apoptosis remains elusive. In this study, using human bronchial epithelial cell as a culture system, we demonstrated that arsenite‐induced nuclear translocation of nuclear factor kappa B (NF‐κB) resulted in the release of cytochrome c, the modulation of Fas and FasL, caspase activation, and ultimately leading to cell apoptosis. Importantly, we showed for the first time that the NF‐κB‐mediated apoptosis induced by arsenite was regulated by G protein‐adenylate cyclase (AC)‐cyclic adenosine monophosphate (cAMP)‐protein kinase A (PKA) pathway. Inhibition of this classical G protein signaling pathway by a typical PKA inhibitor, H‐89, caused the inactivation of NF‐κB, the depletion of caspase‐3, 8 and 9 activities, and thus reducing the level of cell apoptosis. Taken together, our results indicate that arsenite is able to trigger cell apoptosis in human bronchial epithelial cells through the nuclear translocation of NF‐κB, which can be modulated by G protein signaling pathway. These findings further suggest that inhibition of G protein‐mediated pathway by specific inhibitors may be a potential strategy for the prevention of arsenite toxicity.

MicroRNA-155 regulates arsenite-induced malignant transformation by targeting Nrf2-mediated oxidative damage in human bronchial epithelial cells

Arsenite is a well-documented human lung carcinogen but the detailed mechanisms of carcinogenesis remain unclear. In this study, human bronchial epithelial (16-HBE) cells were continuously exposed to 2.5μM arsenite for about 13 weeks to induce the phenotypes of malignant transformation. Our results showed that Nrf2 expression was gradually decreased whereas no significant change was observed on NF-κB activation with increased time of arsenite exposure. To test the roles of Nrf2-meidtaed oxidative damage in the arsenite-induced malignant transformation, we compared the levels of cGMP, PKG and oxidative damage-related indicators between arsenic-transformed cells and control cells. Our data demonstrated there were no significantly differences on the contents of cGMP, PKG, MDA and the production of ROS, but the levels of GSH and NO, the activities of SOD, tNOS and iNOS were significantly enhanced in the arsenic-transformed cells. Importantly, Nrf2 inactivation could be modulated by miR-155, and inhibition of miR-155 remarkably attenuated the malignant phenotypes and promoted apoptotic cell death in the arsenic-transformed cells. Together, our findings provide the novel mechanism that miR-155 may regulate arsenite-induced cell malignant transformation by targeting Nrf2-mediated oxidative damage, indicating that inhibition of miR-155 may be a potential strategy against lung carcinogenesis of arsenite.

Protection of Nrf2 against arsenite-induced oxidative damage is regulated by the cyclic guanosine monophosphate-protein kinase G signaling pathway.

Arsenite has been shown to induce a variety of oxidative damage in mammalian cells. However, the mechanisms underlying cellular responses to its adverse effects remain unknown. We previously showed that the level of Nrf2, a nuclear transcription factor significantly increased in arsenite‐treated human bronchial epithelial (HBE) cells suggesting that Nrf2 is involved in responding to arsenite‐induced oxidative damage. To explore how Nrf2 can impact arsenite‐induced oxidative damage, in this study, we examined Nrf2 activation and its regulation upon cellular arsenite exposure as well as its effects on arsenite‐induced oxidative damage in HBE cells. We found that Nrf2 mRNA and protein levels were significantly increased by arsenite in a dose‐ and time‐dependent manner. Furthermore, we showed that over‐expression of Nrf2 significantly reduced the level of arsenite‐induced oxidative damage in HBE cells including DNA damage, chromosomal breakage, lipid peroxidation and depletion of antioxidants. This indicates a protective role of Nrf2 against arsenite toxicity. This was further supported by the fact that activation of Nrf2 by its agonists, tertiary butylhydroquinone (t‐BHQ) and sulforaphane (SFN) resulted in the same protective effects against arsenite toxicity. Moreover, we demonstrated that arsenite‐induced activation of Nrf2 was mediated by the cyclic guanosine monophosphate (cGMP)‐protein kinase G (PKG) signaling pathway. This is the first evidence showing that Nrf2 protects against arsenite‐induced oxidative damage through the cGMP‐PKG pathway. Our study suggests that activation of Nrf2 through the cGMP‐PKG signaling pathway in HBE cells may be developed as a new strategy for prevention of arsenite toxicity.

Arsenite-induced endoplasmic reticulum-dependent apoptosis through disturbance of calcium homeostasis in HBE cell line

Calcium (Ca2+) is a ubiquitous cell signal responsible for multiple fundamental cellular functions, including apoptosis. Whether the homeostasis of Ca2+ is involved in arsenite‐induced apoptosis remains unclear. In this study, we observed that arsenite significantly elevated the intracellular Ca2+ concentration in a dose‐ and time‐dependent manner. By using the Ca2+‐ATPase inhibitor, thapsigargin, and the inositol 1,4,5‐ trisphosphate receptors (IP3Rs) inhibitor, heparin, we further confirmed that the disturbance of endoplasmic reticulum (ER) Ca2+ homeostasis caused Ca2+ overload in the cells. Moreover, loss of ER Ca2+ homeostasis also led to ER stress, mitochondrial dysfunction, and NF‐κB activation. Importantly, pretreatment of cells with heparin remarkably attenuated the elevated cell apoptosis induced by arsenite, but inhibition of ER Ca2+ uptake with thapsigargin exacerbated arsenite‐induced cell damage significantly. Together, we demonstrated for the first time that arsenite disturbed the Ca2+ homeostasis in ER, which subsequently led to ER stress, mitochondrial dysfunction, and NF‐κB nuclear translocation, and thus consequently triggering cell apoptosis. Our findings indicate regulation of disrupted Ca2+ homeostasis in ER may be a potential strategy for prevention of arsenite toxicity.

Regulation of ABCG2 by nuclear factor kappa B affects the sensitivity of human lung adenocarcinoma A549 cells to arsenic trioxide

Arsenic trioxide (As2O3) is successfully used as an anticancer agent against acute promyelocytic leukemia and some solid tumors. However, the application of As2O3 is largely limited by its drug resistance in the treatment of non-small cell lung carcinoma (NSCLC). Therefore, it is an urgent task to enhance the sensitivity of lung cancer cells to As2O3. In this study, using human lung adenocarcinoma A549 cells as a cell culture model, we demonstrated that an adenosine triphosphate binding cassette (ABC) transporter, ABCG2, was significantly increased by As2O3 treatment, while other ABC transporters, ABCB1 and ABCC1 showed no remarkable change in the response to As2O3. After inhibition of ABCG2 by its specific inhibitor, the drug sensitivity of As2O3 to A549 cells was significantly enhanced, manifested by decreased cell viability and colony formation as well as the increased ROS production and cell apoptosis. To further understand the molecular mechanism underlying the elevation of ABCG2 expression in As2O3-treated cells, we detected the activation state of nuclear factor kappa B (NF-κB) pathway and its relationship with ABCG2 expression. Our results revealed that the increased expression of ABCG2 was regulated by NF-κB, and thus affecting the cell death of As2O3-treated A549 cells. These findings indicate that inhibition of NF-κB/ABCG2 pathway by specific inhibitors may be a new strategy for the improvement of As2O3 sensitivity in NSCLC treatment.