| Arsenide is a well documented human carcinogen,and sodium arsenite(NaAsO2)is a main existing form of arsenide.Epidemiological evidences show that the chronic exposure to inorganic arsenic induces many kinds of cancer,such as tumors of skin,lung,and bladder.Although arsenide is confirmed as a group 1 of carcinogen by the International Agency for Research on Cancer(IARC),because there is no experimental model of arsenide-induced tumorigenesis,the mechanisms underlying arsenide in carcinogenesis are still not well understand.Recently,the arsenic-induced in vitro models,such as neoplastic transformation of human cells,DNA damages,and cell apoptosis,have been used to investigate the mechanisms of arsenide-caused carcinogenesis.Inflammation is a major risk factor for cancer development.The suppressor genes mutation and epigenetic changes induced by the production and release of cytokines and chemokines in inflammatory processes,which due to intracellular environment disorder involved in the progression and development of cancer.The internal genetics and epigenetics changes combined with the external cytokines and chemokines influence due to cancer initiation and progression by the kinds of tumorigenic potential factors.The molecular mechanism of inflammation regulated by arsenite is very important to investigate the arsenite carcinogenesis.Epithelial-to-mesenchymal transition(EMT)and acquirement of cancer stem cells(CSCs)-like properties are involved in a lot of diseases,especially in the generation and development of the cancer.Recently,the relationships between the EMT and acquirement of CSCs-like properties,and the malignancy degree and metastasis of cancer cells,have becoming the top research issues.However,it remains unclear that the effects and its molecular mechanisms of EMT and acquirement of CSCs-like properties in the cell neoplastic transformation and carcinogenesis induced by environmental carcinogens.So,it is important to pay more attention on the molecular mechanisms involved in the EMT and acquirement of CSCs-like properties induced by arsenide,which has important theoretical significance and practical value for finding the early biological markers of carcinogenesis induced by arsenide and for finding new prevention and treatment measures of arsenism.Part ? HIFs involved in malignant transformation and DNA damages of HBE cells induced by arseniteObjectiveTo investigate the molecular mechanisms underlying hypoxia-inducible factors(HIFs)expression induced by different levels of arsenite under normoxia,and the molecular mechanisms of HIFs involved in malignant transformation,DNA damages,and cell apoptosis induced by arsenite.MethodsTo establish the models including cell proliferation,malignant transformation,DNA damages,and cell apoptosis of human bronchial epithelial(HBE)cells induced arsenite.Anchorage-independent growth assays were performed,and tumorigenicity in intact animals was assessed to confirm arsenite-induced malignant transformation.Alkaline single-cell electrophoresis and Hoechest assay were used to quantify the DNA damage and apoptosis in HBE cells.Reverse-transcriptase polymerase chain reaction(RT-PCR)and Western blot were performed to determine the levels and functions of mitogen-activated protein kinase(MAPKs),HIFs,p53 and their relationships.The proteasome inhibitor MG 132 and co-treatment with arsenite to determine the ubiquitination status of HIFs protein by immunoprecipitation assay.By applying inhibitors,siRNA and plasmid transfection to investigate the molecular mechanisms of HIFs involved in malignant transformation,DNA damages,and cell apoptosis induced by arsenite.Results1.Effects of arsenite on the malignant transformation and DNA damages of HBE cellsHBE cells were treated by 0.0,1.0,5.0,or 10.0 ?M arsenite for 24 h,respectively;results showed that the percentages of cell proliferation were improved by 1.0 ?M arsenite.There were no marked increases of DNA migration for cells exposed to 1.0?M arsenite;however,DNA migration was significantly increased in cells treated with 10.0 ?M arsenite.And the ratio of apoptosis increased in cells with 10.0 ?M arsenite exposure,but there were no changes in cells exposed to 1.0 or 5.0 ?M arsenite.HBE cells were exposed to 0.0 or 1.0 ?M arsenite for 30 passages(about 15 weeks).Our data showed that the colonies in agar of HBE cells exposed to arsenite were formed more than passage control cell,and the mice injected with arsenite-transformed HBE cells formed tumors.These results suggest that low levels of arsenite induce the cell proliferation and neoplastic transformation,while high levels of arsenite cause DNA damages and cell apoptosis,2.Effects of arsenite on MAPKs and HIFs of HBE cellsHBE cells were treated by 0.0,1.0,5.0,or 10.0 ?M arsenite for 24 h,respectively;results showed there were increased levels of p-ERK and upregulation of HIF-2?,in 1.0?M arsenite-treated cells.However,there were increased levels of p-JNK and upregulation of HIF-1?,in 10.0 ?M arsenite-treated cells.These results suggest that low levels of arsenite activate the ERK and up-regulate HIF-2?;however,high levels of arsenite activate the JNK and increase HIF-1? expression.3.The roles of ERK regulated HIF-2? in the dysfunction of p53 in HBE cells induced by a low level of arseniteAfter HBE cells were pre-treated by 10.0 ?M ERK inhibitor U0126 for 3h,or 10.0?M JNK inhibitor SP600125 for 3 h,respectively,they were exposed to 0.0 or 1.0 ?M arsenite for 24 h.Our data showed that the inhibition of ERK signal pathway releases the block in degradation of ubiquitinated HIF-2? induced by low levels of 1.0 ?M arsenite;the levels of p-p53 were lower in arsenite-transformed cells and in arsenite-exposed HBE cells for 3,6,12 and 24 h.After HBE cells were pre-treated by 10.0 nM HIF-1?-siRNA,HIF-2?-siRNA or con-siRNA for 24 h,they were exposed to 0.0 or 1.0 ?M arsenite for 24 h.Data showed that the inhibition of HIF-2? expression blocked dysfunction of p53 induced by 1.0 ?M arsenite.4.The roles of ERK regulated HIF-2? in the malignant transformation of HBE induced by a low level of arseniteAfter HBE cells were pre-treated by 10.0 nM HIF-1?-siRNA,HIF-2?-siRNA or con-siRNA for 24 h,they were exposed to 0.0 or 1.0 ?M arsenite for 24 h.Our data showed that inhibition of HIF-2? expression could reduce the proliferation induced by arsenite and inhibition of HIF-2? expression in arsenite transformed cells reduced proliferation and anchorage-independent growth of arsenite-transformed HBE cells.After over-expressed HIF-2? by plasmids transfection in ERK-inhibited cells,the decrease of cell proliferation by blocking of ERK was reversed.After HBE cells were pre-treated by 0.0 or 1.0 ?M HIFs inhibitor,Topotecan for 3h,respectively,they were exposed to 0.0 or 1.0 ?M arsenite for 24 h,which was continued for 30 passages(about 15 weeks).Our data showed that the inhibition of HIF-2? expression blocked the 1.0?M arsenite-induced cell proliferation,colony formation in soft agar,and tumorgenesis in nude mice.These results suggest that HIF-2? is involved in the cell proliferation and malignant transformation induced by low level of arsenite.5.The roles of JNK regulated HIF-1? in the DNA damages and cell apoptosis of HBE induced by high levels of arseniteAfter HBE cells were pre-treated by 10.0 ?M JNK inhibitor SP600125 for 3h,they were exposed to 0.0 or 10.0 ?M arsenite for 24 h.Our data showed the inhibition of JNK signal pathway reverse the block in degradation of ubiquitinated HIF-1? induced by high levels of 10.0 ?M arsenite,and the inhibition of JNK blocked the 10.0 ?M arsenite-induced increase of apoptosis and decrease DNA damage.After the JNK-inhibited cells were over-expressed HIF-1? by transfecting plasmids,the increase of the number of yH2AX foci and the reduction of apoptosis by inhibition of JNK were reversed.These results indicate that HIF-1? dependent on JNK is necessary for DNA damage and apoptosis in HBE cells exposed to a high level of arsenite.ConclusionLow levels of arsenite induce the cell proliferation and malignant transformation,however,high levels of arsenite cause DNA damage and cell apoptosis;the stimulation by the low level of arsenite provokes a response to suppress ubiquitinated-HIF-2?degradation depending on the activation of ERK,which inhibits p53 function,is involved in the cell proliferation and malignant transformation of HBE cells induced by low levels of arsenite;JNK is responsible for ubiquitin-mediated stabilization of HIF-1? protein,which plays an important roles in the DNA damage and cell apoptosis induced by high level of arsenite in HBE cells.Part ? HIFs mediated inflammation to EMT and CSCs involved in malignant transformation of HBE cells induced by arseniteObjectiveTo investigate the molecular mechanism of HIF-2?-mediated inflammation to epithelial-to-mesenchymal transition(EMT)and acquirement of cancer stem cells(CSCs)-like properties during malignant transformation of cells induced by chronic exposure to arsenite.MethodsBasing on the models of malignant transformation of human bronchial epithelial(HBE)cells induced arsenite chronic exposure.Enzyme linked immunosorbent assay(ELISA)were used to determine the levels of IL-6,IL-8,IL-1? and TNF-?.Anchorage-independent growth assays were performed to confirm arsenite-induced malignant transformation.RT-PCR and Western blot were performed to determine the levels and functions of STAT3,moleculars about EMT,CSCs and their relationships.Immunofluorescence microscopy was used to confirm the EMT-associated shift in the localization of markers.Spheroid formation assays and analyses of side populations(SPs)were performed to confirm that arsenite induces the EMT and acquired CSCs-like phenotype.Southwestern,reporter assays,and chromatin immunoprecipitation were performed to determine if HIF-2? directly bind to Twistl and Bmil promoter.By applying siRNA and IL-6-neutralizing antibody to investigate the molecular mechanisms of HIF-2?-mediated inflammation to EMT and CSCs-like properties during malignant transformation of cells induced by arsenite.Results1.Effects of arsenite on inflammation of HBE cellsHBE cells were treated by 0.0 or 1.0 ?M arsenite for 3,6,12,or 24h,respectively;results showed that arsenite increases the mRNA and secretion levels of pro-inflammatory cytokines IL-6 and IL-8.HBE cells were exposed to 0.0 or 1.0 ?M arsenite for 30 passages(about 15 weeks).Our data showed that the mRNA and secreted levels of IL-6 and IL-8 are up-regulated in arsenite-transformed HBE cells,and the levels of IL-6 and IL-8 increased with increased exposure to arsenite.These results suggest that arsenite induced inflammation in HBE cells.2.Arsenite induce EMT and acquirement of CSCs-like properties in HBE cellsHBE cells were exposed to 0.0 or 1.0 ?M arsenite for 30 passages.Our data showed that,in cells exposed to 1.0 ?M arsenite for 20 passages,the cells acquired a fibroblast-like,mesenchymal appearance consistent with EMT;with increased time of exposure to arsenite,after 20 passages,the E-cadherin level was decreased;in contrast,N-cadherin,vimentin,CD133,and CD44 levels were increased;the percentage of SP cells increased in the arsenite-induced EMT of HBE cells and these cells acquired the ability for formed the spheroids.These results suggest that the chronic exposure of HBE cells to arsenite undergo an EMT and acquire the CSCs-like properties.3.The roles of HIF-2? in the increase of Twistl and Bmil involved in EMT andacquirement of CSCs-like properties induced by arsenite in HBE cellsHBE cells were exposed to 0.0 or 1.0 ?M arsenite for 6,12,and 24h,respectively;data showed that arsenite increases the expression of Twist 1 and Bmi;.HBE cells were exposed to 0.0 or 1.0 ?M arsenite for 30 passages(about 15 weeks),in cells exposed to 1.0 ?M arsenite for 10 passages,there were increased levels of Twistl and Bmil.With longer times of arsenite exposure,such changes were more significant As determined by reporter assays,the HIF-2?-dependent transcriptional activity in HBE cells is activated by arsenite,and Twistl-Luc and Bmil-Luc respond to arsenite stimulation,suggested that HIF-2? regulates Twistl and Bmil expression by directly binding to its promoter.After HBE cells were pre-treated by 0.0 or 1.0 ?M HIFs inhibitor,Topotecan for 3h,respectively,they were exposed to 0.0 or 1.0 ?M arsenite for 24 h,which was continued for 30 passages(about 15 weeks).Our data showed that the inhibition of HIF-2? blocked 1.0 ?M arsenite-induced decreases of E-cadherin level,moreover,blocked arsenite-caused increased levels of N-cadherin,vimentin,CD 133 and CD44.These results suggest that HIF-2? mediated Twistl and Bmil is involved in the EMT,acquirement of CSCs-like properties induced by arsenite.4.Effects of arsenite on STAT3 signal pathways in HBE cellsHBE cells were treated by 0.0 or 1.0 ?M arsenite for 15,30 min and 1,3,6,12,24 h,respectively,data showed that 1.0 ?M arsenite activated STAT3 after incubation for 3 h and that phospho-STAT3 levels reached a maximum level after 12h;HBE cells were exposed to 0.0 or 1.0 ?M arsenite for 30 passages(about 15 weeks),in cells exposed to 1.0 ?M arsenite for 10 passages,phospho-STAT3 levels have been activated,with longer times of arsenite exposure,such changes were more significant.HBE cells were treated by the medium from arsenite-transformed HBE cells for 15,30,60 min,data showed that the medium from arsenite-transformed HBE cells induced activation of STAT3.And this process was blocked by pretreatment of the cells with an IL-6 neutralizing antibody.These data indicate that,in HBE cells,IL-6 is involved in the arsenite-induced activation of STAT3.5.The roles of IL-6/STAT3 involved in EMT induced by arsenite in HBE cellsHBE cells were exposed to 0.0 or 1.0 ?M arsenite for 30 passages(about 15 weeks),then cells were treated by 0.5?g/mL IL-6 neutralizing antibody for 3 days,Our data showed that alteration from epithelial to spindle-like mesenchymal morphology is a manifestation of arsenite-induced transformation.With inhibition of IL-6 in malignant transformed cells,the epithelial morphology was not changed relative to untransformed cells;moreover,the blockaged of IL-6 levels inhibited 1.0 ?M arsenite-induced decreases of E-cadherin level,moreover,blocked arsenite-caused increased levels of N-cadherin and vimentin.These results suggest that IL-6 is involved in the EMT induced by arsenite.6.The roles of HIF-2? in the increase of IL-6 and IL-8 involved in inflammation to malignant transformation of HBE cells induced by arseniteAfter HBE cells were pre-treated by 10.0 nM HIF-2?-siRNA or con-siRNA for 24 h,they were exposed to 0.0 or 1.0 ?M arsenite for 24 h.Our data showed that the inhibition of HIF-2? blocked 1.0 ?M arsenite-induced increases of IL-6 and IL-8 levels.HBE cells were exposed to 0.0 or 1.0 ?M arsenite for 30 passages(about 15 weeks),then cells were treated by 10.0 nM HIF-2?-siRNA or con-siRNA for 24 h,data showed that the expression and secretion of IL-6 and IL-8 have been diminished with HIF-2?knockdown in the arsenite-transformed HBE cells.HBE cells were exposed to 0.0 or 1.0 ?M arsenite for 30 passages(about 15 weeks),then cells were treated by 10.0 nM HIF-2?-siRNA or con-siRNA for 24 h and applyed 10 ng/mL human recombinant IL-6 and IL-8 proteins,data showed that silencing HIF-2? reduced anchorage independent growth of arsenite-transformed HBE cells,but the decrease of anchorage independent growth was reversed by adding recombinant IL-6 and IL-8.After HBE cells treated by 1.0 ?M arsenite for 10 passages were cultured in the presence of 10 ng/mL human recombinant IL-6 and IL-8,data showed that in HBE cells treated by 1.0 ?M arsenite for 10 passages,Up-regulation of IL-6 and IL-8 induced the increases of formed colonies in agar,in contrast,HBE cells were exposed to 0.0 or 1.0?M arsenite for 30 passages(about 15 weeks),then cells were treated by 0.5?g/mL IL-6 and IL-8 neutralizing antibody for 3 days,arsenite-transformed HBE cells depleted of IL-6 and IL-8 by these antibodies developed fewer colonies relative to untreated cells.All these results demonstrate that HIF-2? regulated IL-6 and IL-8 are involved in the arsenite-induced transformation of HBE cells.ConclusionArsenite could induce inflammation in HBE cells;HBE cells undergo an EMT and acquire the CSCs-like properties during the cell malignant transformation induced by the chronic exposure to arsenite.HIF-2? up-regulated Twistl and Bmil is involved in EMT and acquirement of CSCs-like properties;IL-6 activated STAT3 is involved in the EMT induced by arsenite;HIF-2? mediated arsenite-induced IL-6 and IL-8 expression were essential for the malignant progression of arsenite-transformed HBE cells that such inflammation is involved in arsenite-induced malignant transformation of HBE cells.Part ? HIFs involved in inflammation of ICR mice induced by arseniteObjectiveTo investigate the effect of arsenite on the inflammation of ICR mice and the mechanism of HIFs involved in the inflammation induced by subchronic exposure to arserute.MethodsSubchronic exposure to different levels of arsenite established the models of including inflammation of ICR mice.Histological examination by Hematoxylin Eosin(H&E)stains were used to determine the pulmonary inflammatory changes in ICR mice;bronchoalveolar lavage detected the inflammatory cell infiltrate in pulmonary;ELISA and RT-PCR were used to determine the levels of pro-inflammatory cytokines,IL-6,IL-8,IL-1? and TNF-?.Western blot and immunochemistry were performed to determine the levels of moleculars about HIFs.The expression of HIFs and IL-6 was analyzed by Pearson correlation analysis.Results1.Effects of different levels of arsenite on inflammation of ICR miceForty healthy male mice were randomly divided into four groups.They were administrated with arsnite at the concentration of 0.0,50.0,100.0,and 200.0 ?M in the drinking water for 3 months,results showed that arsenite treated mice showed marked inflammatory and the pathological sections of lung were infiltrated by eosinophile granulocyte mostly and other inflammatory cell;the expression of pro-inflammatory cytokines IL-6,IL-8 and TNF-? were increased and the levels of IL-6 and TNF-? were increased in serum.These results suggest that arsenite induced inflammation in ICR mice.2.Effects of different levels of arsenite on HIFs expression in the pulmonary of ICR miceForty healthy male mice were randomly divided into four groups.They were administrated with arsnite at the concentration of 0.0,50.0,100.0,and 200.0?M in the drinking water for 3 months,results show that different levels of arsenite induced upregulation of HIF-1? and HIF-2?.3.Pearson correlation analysis on expression of HIF-1? and HIF-2? and pro-inflammatory cytokines IL-6 expression in the pulmonary of ICR miceForty healthy male mice were randomly divided into four groups.They were administrated with arsnite at the concentration of 0.0,50.0,100.0,and 200.0?M in the drinking water for 3 months,results showed that the level of HIFs and IL-6 has a positive correlation relationship.These results suggest that the increase of IL-6 level attributed partly to the overexpression of HIFs induced by arsenite.ConclusionDifferent levels of arsenite treated mice showed marked inflammatory,the expression and levels of pro-inflammatory cytokines IL-6 and TNF-? were increased;the level of HIFs and IL-6 has a positive correlation relationship.In summary,the novel findings of this study are as follows:1.Low levels of arsenite induce the cell proliferation and malignant transformation,however,high levels of arsenite cause DNA damage and cell apoptosis.2.ERK is responsible for ubiquitin-mediated stabilization of HIF-2a protein,which inhibits p53 function,is involved in the cell proliferation and malignant transformation of HBE cells induced by low levels of arsenite3.JNK is responsible for ubiquitin-mediated stabilization of HIF-1? protein,which plays important roles in the DNA damage and cell apoptosis induced by high level of arsenite in HBE cells.4.HBE cells undergo an EMT and acquire the CSCs-like properties during the cell malignant transformation induced by the chronic exposure to arsenite,HIF-2?up-regulated Twistl and Bmil is involved in EMT and acquirement of CSCs-like properties.5.Arsenite induced inflammation to EMT and acquire the CSCs-like properties involved in cell malignant transformation.6.Different levels of arsenite treated mice showed marked inflammatory,the level of HIFs and pro-inflammatory cytokine has a positive correlation relationship. |
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