Shiyan Gu

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.

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.

N6-methyladenosine mediates the cellular proliferation and apoptosis via microRNAs in arsenite-transformed cells

N6-methyladenosine (m6A) modification is implicated to play an important role in cellular biological processes, but its regulatory mechanisms in arsenite-induced carcinogenesis are largely unknown. Here, human bronchial epithelial (HBE) cells were chronically treated with 2.5 μM arsenite sodium (NaAsO2) for about 13 weeks and these cells were identified with malignant phenotype which was demonstrated by increased levels of cellular proliferation, percentages of plate colony formation and soft agar clone formation, and high potential of resistance to apoptotic induction. Our results firstly demonstrated that m6A modification on RNA was significantly increased in arsenite-transformed cells and this modification may be synergistically regulated by methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms tumor 1-associated protein (WTAP) and Fat mass and obesity-associated protein (FTO). In addition, knocking down of METTL3 in arsenite-transformed cells can dramatically reverse the malignant phenotype, which was manifested by lower percentages of clone and colony formation as well as higher rates of apoptotic induction. Given the critical roles of miRNAs in cellular proliferation and apoptosis, miRNAs regulated by m6A in arsenite-transformed cells were analyzed by Venn diagram and KEGG pathway in this study. The results showed that these m6A-mediated miRNAs can regulate pathways which are closely associated with cellular proliferation and apoptosis, implicating that these miRNAs may be the critical bridge by which m6A mediates dysregulation of cell survival and apoptosis in arsenite-transformed cells. Taken together, our results firstly demonstrated the significant role of m6A in the prevention of tumor occurrence and progression induced by arsenite.

The selectivity of artemisinin-based drugs on human lung normal and cancer cells

Artemisinin-based drugs are documented to possess anticancer potential that is selectively effective to cancer cells. However, this selectivity is disputable in different studies and the mechanism is still unclear. To clarify this discrepancy, this study employed five assays to evaluate the cytotoxic effects of artemisinin and artesunate on normal human bronchial epithelial (HBE) cells and lung adenocarcinoma A549 cells. The results of five cytotoxic assays coherently showed that artemisinin and artesunate caused dose-dependent cytotoxicity in both HBE and A549 cells with a slight selectivity to A549 cells. Further, both HBE cells and A549 cells demonstrated elevated levels of intracellular reactive oxygen species (ROS) and increased DNA damage. Since artemisinin and artesunate exerted significant cytotoxic effect on both normal cells and cancer cells via the same pathway of ROS-mediated DNA damage, the side effects of artemisinin and artesunate on normal cell cannot be ignored when developing their antitumor effects.

Alterations of miRNAs and Their Potential Roles in Arsenite-Induced Transformation of Human Bronchial Epithelial Cells

The alterations of micro RNAs (miRNAs) and their potential roles in arsenite-induced tumorigenesis are still poorly understood. In this study, miRNA Array was used to detect the expression level of miRNAs in human bronchial epithelial (HBE) cells that were transformed by 2.5 μM arsenite for 13 weeks. These cells exhibited a neoplastic phenotype manifested by increased levels of cellular proliferation and migration and clone formation. Subsequently, 191 dysregulated miRNAs were identified to be associated with arsenite-induced transformation by miRNA Array. Among them, six miRNAs were validated by their expression levels with quantitative real-time polymerase chain reaction (qPCR), and 17 miRNAs were further explored via their target genes as well as regulatory network. Three databases, TargetMiner, miRDB, and TarBase, were used to predict the target genes of the 17 miRNAs, and a total of 954 common genes were sorted. Results of Gene Ontology (GO) analyses showed that the 954 genes were involved in diverse terms of GO categories, such as positive regulation of macroautophagy, epithelial cell maturation, and synaptic vesicle clustering. Moreover, results of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses demonstrated that most of these target genes were enriched in various cancer-related pathways, including non-small cell lung cancer, Wnt signaling pathway, cell cycle, and p53 signaling pathway. The miRNA-gene regulatory network, which was constructed by cytoscape software with miRNAs and their target genes, showed that miR-15b-5p, miR-106b-5p, and miR-320d were the core hubs. Collectively, our results provide new insights into miRNA-mediated mechanisms underlying arsenite-induced transformation, although more experimental verification is still needed to prove these predictions.

Dihydroartemisinin Sensitizes Human Lung Adenocarcinoma A549 Cells to Arsenic Trioxide via Apoptosis

Recent studies have shown that arsenic trioxide (ATO) is an effective anti-cancer drug for treatment of acute promyelocytic leukemia and other types of human cancer. However, we have found that lung cancer cells constantly develop a high level of resistance to ATO. In this study, we have explored a possibility of combination of dihydroartemisinin (DHA) and ATO treatments to reduce ATO resistance of lung cancer cells. We determined the combinatory effects of DHA and ATO on cytotoxicity of human lung adenocarcinoma (A549) cells. We showed that co-exposure to DHA and ATO of A549 cells synergistically increased the cytotoxicity and apoptotic cell death in the cells. We found that the synergistic effect of DHA and ATO in promoting apoptosis mainly resulted from increased cellular level of reactive oxygen species (ROS) and DNA damage. ATO alone only exerted moderate growth inhibitory effects on A549 cells. The results indicate that DHA can significantly sensitize ATO-induced cytotoxicity of A549 lung cancer cells through apoptosis mediated by ROS-induced DNA damage. Interestingly, we found that the combinatory treatment of DHA and ATO did not result in significant adverse effects in normal human bronchial epithelial (HBE) cells. Our results further provide evidence for the potential application of combinatory effects of DHA and ATO as a safe therapy for human lung cancer.

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.