The Roles Of ATM/Chk-2Pathway Regulated By HIF-2α In The Promotion Effect Of Arsenic On Benzo(a)Pyrene-induced Cell Malignant Transformation

Currently, cancer is one of the most serious harm to human health, which is the result that cell malignant transformation is induced by a variety of environmental carcinogenic factors. Benzo (a) pyrene (BaP) is a common environmental carcinogen. Several studies have indicated that BaP exposure may cause the dysfunction of signaling pathways of DNA repair, which leads to cell malignant transformation. Although arsenic has been recognized as a human carcinogen, some investigations have reported that arsenic may promote carcinogenesis of other environmental carcinogens (such as cigarette) by inhibiting the repair of DNA damage. Hypoxia-inducible factors (HIFs), which are transcription factors with a variety of regulatory mechanisms, are induced the expressions by some environmental chemicals, which is linked to genetic instability and cell malignant transformation.In the past, the investigations on carcinogenesis and its molecular mechanisms of environmental pollutants were usually focused on single and (or) high levels of environmental carcinogens, however, the toxic effects of a mixture of low levels and its molecular mechanisms have rarely been reported. Due to environmental pollution accompanied with comprehensive features, studies effects of environmental pollutants and molecular mechanism of joint damage are more important for understanding the unhealthy of the body caused by environmental pollution hazard and exploring for preventive measures.Therefore, we are going to investigate the effects of the exposure to arsenite or/and BaP on cell malignant transformation of human bronchial epithelial (HBE) cells to arsenite and BaP promotes a transformed phenotype. The changes of DNA damage repair and cell malignancy will be observed by a variety of molecular biological methods. To investigate the roles of hypoxia-inducible factor-2α (HIF-2α) and its molecular mechanism in the promotion effect of a low level of arsenite on BaP-induced cell malignant transformation, we will study the effects of blockage or knockdown of HIF-2α using inhibitor or siRNA on some proteins and cell malignant transformation, which will contributed to understanding the mechanisms of the effects of exposure to arsenite or/and BaP on human health. Our results will help to understand the mechanisms of arsenic promoting cancer and to provide some new clues to find early biomarkers and new prevention measures of arsenic or/and BaP toxicities.Methods1. The effects of different levels of BaP and arsenite on DNA damage and cell apoptosis of HBE cellsHBE cells were exposed to0.0,0.5,1.0,5.0, or10.0μM BaP for24h, or co-exposed to1.0μM BaP for24h after being treated by0.0,0.5,1.0,5.0, or10.0μM arsenite for24h, and then they were repaired for0,3,24,48h. Alkaline Comet assay was used to quantify the DNA damage and Hoechst33258staining was used to detect cell apoptosis in HBE cells. We were going to investigate the effects of different levels of arsenite or/and BaP on DNA damage, cell apodosis, and DNA damage repair of HBE cells.2. The effects of a low level of arsenite on BaP-induced cell neoplastic transformation of HBE cellsCells were divided to four treatment groups:control group, arsenite treatment group, BaP treatment, as well as arsenite and BaP treatment group. Cells of different group were treated with1.0μM arsenite or/and1.0μM BaP for30passages. The anchorage-independent growth assay and the high-content screening device were used to quantify the neoplastic transformation and cell migration of treated HBE cells, respectively. We were going to investigate the effects of arsenite or/and BaP on cell neoplastic transformation and cell migration of HBE cells.3. The effects of a low level of arsenite on BaP-induced the genetic instability of HBE cellsFor chronic exposure, cells were treated with1.0μM arsenite or/and1.0μM BaP for30passages. Giemsa staining was used to examine chromosomal aberrations. Immunostaining and Western blotting were used to quantify the level of γ-H2AX of treated HBE cells. We were going to investigate the effects of arsenite or/and BaP on chromosomal aberrations and y-H2AX levels of HBE cells.4. The effects of arsenite and BaP treatment on signaling pathways of DNA repair in HBE cells After HBE cells were treated with1.0μM arsenite or/and1.0μM BaP for6,12, and24h, the levels and phosphorylations of ATM、ATR、Chk1, and Chk2were analyzed by western blotting. After HBE cells were pretreated with0.0or1.0μM arsenite for24h, they were treated with0.0or1.0μM BaP for24h., Immunoloreaction assays were used to quantify the level of8-oxoguanine glycosylase-1(8-hOGGl). We were going to investigate the effects of arsenite or/and BaP on the activations of some proteins in signaling pathways of DNA repair in HBE cells.5. The effects of arsenite and BaP treatment on the levels and functions of HIF-2a in HBE cellsHBE cells were treated with1.0μM arsenite or/and1.0μM BaP for30passages, or HBE cells were exposed to1.0μM arsenite or/and1.0μM BaP for6,12, or24h. The levels of HIF-1α and HIF-2α were detected by Western blotting assay. After HBE cells were pretreated with0.0or1.0μM arsenite for24h, they were treated with0.0or1.0μM BaP for24h. The quantitative real-time polymerase chain reaction (qRT-PCR) was used to quantify the levels of the HIF target genes, such as VEGF, PGK, and Oct4. We are going to investigate the effects of arsenite or/and BaP on the levels of HIF-1α, HIF-2a, and their target molecules in HBE cells.6. The roles of HIF-2a in the blocking effects of arsenite on BaP-induced activations of DNA repair pathways in HBE cellsHBE cells were incubated with1.0μM arsenite or/and1.0μM BaP for24h after they were treated by control siRNA (20nM) or HIF-2α siRNA (10nM) for24h. Western blotting were used to quantify the levels and phosphorylations of ATM、 Chk-2, and y-H2AX. Immunostaining was used to quantify the level of y-H2AX to observed the roles of HIF-2a in the blocking effects of arsenite on BaP-induced activation of the DNA repair pathway in HBE cells.7. The roles of HIF-2a in the promotion effect of arsenite on benzo(a)pyrene-induced cell transformationHBE cells were exposed to topotecan (an inhibitor of HIF-2a) for3h per passage, then treated with1.0μM arsenite or/and1.0μM BaP for30passages. The anchorage-independent growth assay was used to quantify the neoplastic transformation and Giemsa staining was used to examine chromosomal aberrations.. We were going to investigate the roles of HIF-2a in the promotion effect of arsenite on BaP-induced cell transformation and chromosomal aberrations in HBE cells.Results1. The effects of different levels of BaP and arsenite on DNA damage and cell apoptosis of HBE cellsIn the BaP-treated HBE cells, there was a dose-dependent increase of Olive tail spur. HBE cells were exposed to different levels of arsenite and1.0μM BaP showed a dose-dependent increase of Olive tail spur. However, there were not significant among the cell apoptosis ratio of cells treated by0.5or1.0μM arsenite with co-exposure to1.0μM Bap and control cells. For HBE cells exposed to1.0μM arsenite with co-exposure of1.0μM BaP, their DNA repairs were very little. Data indicate that BaP induces DNA damage; a low level of arsenite promotes BaP-induced DNA damage via inhibiting DNA repair in HBE cells. 2. The effects of a low level of arsenite on BaP-induced cell neoplastic transformation of HBE cellsThe colonies in agar of HBE cells exposed to BaP and HBE cells co-exposed to arsenite and BaP were formed more than passage control HBE cell. The colonies in agar of HBE cells exposed to co-expose to arsenite and BaP were formed more than cells exposed to BaP or arsenite alone. The cell motility and migration of cells treated by arsenite or/and BaP were greater than that of passage control cells. The motility and migration treated by arsenite and BaP was greater than that of arsenite or BaP alone. Data indicate that chronic treatment of BaP induces cell neoplastic transformation and a low level of arsenite may promote the effects and cell migration in HBE cells.3. The effects of arsenite on BaP-induced genetic instability in HBE cells.HBE cells treated by BaP or/and arsenite have appeared some types of chromosomal aberrations, such as chromatic breaks, ring chromosomes, chromosome breaks, and telemetric fusions. The ratios of chromosomal aberrations in HBE cells treated by BaP or/and arsenite were markedly higher than that in control HBE cells. The chromosomal aberrations in HBE cells treated by BaP and arsenite were more serious than that in HBE cells treated by arsenite or BaP alone. Immunofluorescence intensity and protein levels of y-H2AX in HBE cells treated by BaP and arsenite were higher than that in HBE cells treated by BaP alone. Data indicate that a low level of arsenite improves BaP-induced chromosomal aberrations and blocks the increases of α-H2AX level induced by BaP. 4. The effects of arsenite and BaP treatment on DNA repair pathway in HBE cellsIn HBE cells treated by BaP alone, the levels of p-ATM and p-Chk-2pathway were increased, which showed a time-dependent increase. In HBE cells treated by BaP and arsenite, the levels of p-ATM and p-Chk-2were increased at6h, then which were decreased after6h. The levels of8-hOGGl in HBE cells incubated with arsenite and BaP were markedly lower than that in HBE cells treated by BaP alone. Data indicated that arsenite inhibits the repair of BaP-induced DNA damage by blocking the ATM/Chk-2pathway in HBE cells. Data indicate that a low level of arsenite may inhibit BaP-induced activation of ATM-Chk2pathway, one of DNA repair pathways, and increase of8-hOGG1level in HBE cells.5. The effects of arsenite and BaP treatment on HIF-2a levels and functions in HBE cellsThe levels of HIF-2a protein, VEGF mRNA, PGK mRNA, and Oct4mRNA in HBE cells treated by arsenite and BaP were significantly higher than that in passage control HBE cells and BaP-treated HBE cells. The HIF-la levels in all HBE cells were not appreciably expressed. HIF-2a level in arsenite-treated HBE cells showed a time-dependent increase. The HIF-2a levels in HBE treated by BaP and arsenite were increased at6h, then they were decreased after6h. Data indicate that a low level of arsenite increases the levels of HIF-2a, Oct4mRNA, and VEGF mRNA in HBE cells, which may inhibited by BaP.6. The roles of HIF-2a in the blocking effects of arsenite on BaP-induced activations of DNA repair pathways in HBE cells The levels of p-ATM, p-Chk2, and y-H2AX and the immunofluorescence intensity of y-H2AX in HBE cells treated by BaP were higher than that in control HBE cells. The activations of ATM, Chk2, and y-H2AX in HBE cells treated by arsenite and BaP were lower than that in BaP alone. After HEB cells were treated by HIF-2a siRNA, the activations of ATM, Chk2, and y-H2AX in HBE cells treated by arsenite and BaP were re-increased. Data indicate that arsenite may inhibit BaP-induced activation of ATM-Chk2pathway and increase of γ-H2AX level in HBE cells, in which, HIF-2a plays an important roles.7. The roles of HIF-2a in the promotion effect of arsenite on benzo(a)pyrene-induced cell transformationWith the topotecan (HIF-2a inhibitor), the colonies in agar and chromosomal aberrations of HBE cells exposed to BaP were markedly higher than passage control HBE cells. With the topotecan, the colonies in agar of HBE cells co-exposed to arsenite and BaP were similar to passage control HBE cells. With the topotecan, the chromosomal aberrations of HBE cells co-exposed to arsenite and BaP were less than that of HBE cells treated by BaP alone. Data indicate that HIF-2a plays an important role in the promoting effects of arsenite on BaP-induced cell neoplastic transformation and chromosomal aberration.Conclusions1. BaP may induce DNA damages of HEB cells and a low level of arsenite promotes BaP-induced DNA damage and chromosomal aberrations and inhibits BaP-induced DNA repair in HBE cells.2. BaP may induce cell neoplastic transformation of HEB cells and a low level of arsenite promotes BaP-induced cell neoplastic transformation and cell migration in HBE cells.3. For chronic treatment, a low level of arsenite induces the increases of HIF-2α level and the activations of its function.4. HIF-2α is involved in the blocking effects of a low level of arsenite on the activation of ATM-Chk2pathway and the increases ofγ-H2AX (DNA repair protein) level in HBE cells.5. HIF-2α plays an important role in the promoting effects of arsenite on BaP-induced cell neoplastic transformation in HBE cells.In summary, data showed that a low level of arsenite increased HIF-2α level. The treatment of BaP induced DNA damage and activated ATM-Chk-2signaling pathway for DNA repair. When the pathway was inhibited by HIF-2α, DNA damages were effectively repaired, which induced cell neoplastic transformation. Our results have indicated that the roles of ATM/Chk-2pathway regulated by HIF-2α in the promotion effect of arsenic on benzo(a)pyrene-induced cell malignant transformation.