The Roles Of Jnk/c-jun Signal Pathway In Cellular Proliferation Abnormity And Cell Cycle Disorder Induced By Low Concerntration Of Sodium Arsenite In Human Embryo Lung Fibroblast Cells

Arsenic is a metalloid element that is widely distributed in the environment. Sodium arsenite is the main inorganic form of arsenic in the environment. Available epidemiological data have shown that long-term exposure to high concentration of arsenide may induce various cancers including skin cancer, bladder cancer and liver cancer. Importantly, occupational inhalation of arsenic, which exists in nonferrous metal ore smelters and insecticide manufacturers, is associated with increased risks of human lung cancer. Arsenic is an identified human carcinogen. Sodium arsenite is demonstrated to induce cellular proliferation and cell cycle disorder both in cell culture model and animal model; however, its mechanisms are not well known.Cancer is one of diseases caused by environmental factors and genetic factors. Cell exposure to carcinogenic agents can lead to a series of events, such as oncogene reinforcement and anti-oncogene inhibition caused by gene mutation, dysfunction of cell signal transduction, deregulation of cell cycle and uncontrolled cellular proliferation, which cause eventually cancer. Of these, the abnormal cellular proliferation and cell cycle disorders are the bases of cancer.Some studies indicate that arsenite may induce biphase effects of cellular proliferation and cell apoptosis in human cells. Furthermore, ROS and MAPKs signal pathway may play crucial roles in cellular proliferation and cell apoptosis induced by difference concentration of sodium arsenite. However, molecular mechanisms underlying this phenomenon are not well understood.In the present study, we investigated the effects of different concentration of sodium arsenite on cellular proliferation, cell cycle, JNK activation, c-Jun activation and cycline D1 express and their relationship in human embryo lung fibroblast (HELF) cells. Furthermore, we studied the effects of blocking JNK/c-Jun signal pathway, which were caused by JNK inhibitor (SP600125), JNK siRNA, and c-Jun siRNA, on cellular proliferation abnormilty and cell cycle disorder induced by low concentration of sodium arsenite in HELF cells. By these, we demonstrated the roles of JNK/c-Jun signal pathway in cellular proliferation abnormity and cell cycle disorder induced by low concerntration of sodium arsenite in HELF cells.Methods1. The effects of different concentrantion of sodium arsenite on cellular proliferation and cell cycle in HELF cells To evaluate the effects of sodium arsenite on cellular proliferation and cell cycle in HELF cells, the HELF cells were exposed to 0.0, 0.1, 0.5 or 5.0μM of sodium arsenite for 12, 24 or 48 h, respectively. Cellular proliferation was evaluated by Cell Counting Kit-8 assay, and the proportion of cell cycle was analyzed by Flow Cytometry with propidium iodide staining.2. The effects of different concentrantion of sodium arsenite on cyclin D1 expression in HELF cellsTo examine cyclin D1 level induced by sodium arsenite in HELF cells, After HELF cells were treated with 0.0, 0.1, 0.5, or 5.0μM of sodium arsenite for 6, 12 or 24 h, cyclin D1 level was detected by western blot assay.3. The effects of different concentrantion of sodium arsenite on JNK/c-Jun signal pathway in HELF cellsWe investigated whether sodium arsenite induced JNK and c-Jun activation in HELF cells. After HELF cells were treated with 0.0, 0.1, 0.5, or 5.0μM of sodium arsenite for 6, 12 or 24 h, the levels of JNK, phospho-JNK, c-Jun and phospho-c-Jun were detected by western blot assay.4. The roles of JNK/c-Jun signal pathway in the cellular proliferation and cell cycle induced by low concentrantion of sodium arsenite in HELF cellsTo confirm the roles of JNK/c-Jun signal pathway in the cellular proliferation and the cell cycle induced by low concerntrantion of sodium arsenite, HELF cells were left untreated or pre-treated with 20μM of JNK inhibitor (SP600125) for 30 min or transfected with 20 nM of JNK siRNA or c-Jun siRNA for 24 h, then cells were exposed to 0.0, 0.1, 0.5 or 5.0μM of sodium arsenite for 24 h, respectively. Cellular proliferation and the proportion of cell cycle were measured with Cell Counting Kit-8 assay and Flow Cytometry, respectively.5. The roles of JNK/c-Jun signal pathway in the cyclin D1 expression induced by low concentrantion of sodium arseniteWe investigated whether JNK/c-Jun signal pathway was involved in sodium arsenite-induced cyclin D1 expression. HELF cells were left untreated or pre-treated with 20 ofμM JNK inhibitor (SP600125) for 30 min or transfected with 20 nM of JNK siRNA or c-Jun siRNA for 12 h, then cells were exposed to 0.0, 0.1, or 0.5μM of sodium arsenite for 24 h, respectively. The levels of JNK protein, phospho-JNK, c-Jun protein, phospho-c-Jun, and cyclin D1 were detected by western blot assay.Results1. The effects of different concentrantion of sodium arsenite on cellular proliferation and cell cycle in HELF cells(1) The effects of different concerntration of sodium arsenite on cellular proliferation in HELF cellsCellular proliferation was significantly increased by 0.1 or 0.5μM of sodium arseinte at 12, 24 or 48 h in HELF cells. The curve peak of cellular proliferation presented at 0.1μM of sodium arsenite and at exposure 24 h. In contrast, cellular proliferation was markedly decreased in 5.0μM of arsenite at each time. Data indicated that low concentarion of sodium arsenite increased cell proliferation but high concerntration of sodium arsenite inhibited cell growth at in HELF cells.(2) The effects of different concerntration of sodium arsenite on cell cycle in HELF cellsThe cell proportion in S phase was significantly elevated by 0.1 or 0.5μM of sodium arsenite at 24 h, as well as the cell proportion in G1 phase was markedly decreased. The curve peak was present at 0.1μM sodium arsenite group, 5.0μM of sodium arsenite induced significantly the increases of the cell proportion in G2/M phase at 24 h. Data indicated that low concentration of sodium arsenite promoted cell cycle transition from G1 to S phases. However, high concerntration of sodium arsenite induced cell cycle arrest at G2/M phase.2. The effects of different concerntration of sodium arsenite on cyclin D1 expression in HELF cellsCyclin D1 expression levels were markedly increased by 0.1 or 0.5μM of sodium arsenite at 6, 12, or 24 h in HELF cells. In contrast, 5.0μM of sodium arsenite showed no effect on cyclin D1 expression in HELF cells. Data indicated that cyclin D1 expression was inducted by low concentration of sodium arsenite in HELF cells.3. The effects of different concentrantion of sodium arsenite on JNK/c-Jun signal pathway in HELF cells(1) The effects of different concerntration of sodium arsenite on phosho-JNK levels in HELF cellsPhopho-JNK levels were dramatically increased by 0.1 or 0.5μM of arsenite at 6, 12, or 24 h in HELF cells, whereas 5.0μM of arsenite did not show any effect on phospo-JNK in HELF cells. Data suggested that JNK signal pathway could be activated by low concerntration of arsenite in HELF cells, furthermore, the change similar to the cyclin D1 expression induction by sodium arsenite.(2) The effects of different concerntration of sodium arsenite on phosho-c-Jun levels in HELF cellsPhopho-c-Jun levels were dramatically increased after HELF cells incubated by 0.1 or 0.5μM of sodium arsenite at 6, 12, or 24 h, however, although 5.0μM of sodium arsenite may also increased phopho-c-Jun level, the effect was weakly. Data suggested c-Jun activation was induced by low concentration of sodium arsenite in HELF cells.4. The roles of JNK/c-Jun signal pathway in cellular proliferation and cell cycle induced by low concentrantion of sodium arsenite in HELF cells(1) The effects of blocking JNK/c-Jun signal pathway on cellular proliferation induced by low concerntration of sodium arseniteJNK inhibitor, SP600125, significantly blocked increases of cellular proliferation induced by 0.1 or 0.5μM of sodium arsenite in HELF cells. JNK siRNA and c-Jun siRNA also markedly suppressed the increases of cellular proliferation induced by 0.1 or 0.5μM of sodium arsenite in HELF cells. Data suggested that JNK/c-Jun signal pathway play a critical role on cellular proliferation induced by low concerntration of sodium arsenite in HELF cells.(2) The effects of JNK/c-Jun knockdown on cell cycle induced by low concerntration of sodium arseniteThe elevation of cell proportion in S phase induced by low concerntration of sodium arsenite was markedly inhibited by the JNK siRNA. Furthermore, the c-Jun siRNA inhibited dramatically the sodium arsenite-dependent transition of G1 to S phases in HELF. Data suggested that JNK/c-Jun signal pathway involvedin the cell cycle transition induced by low concerntration of sodium arsenite in HELF cells.5. The roles of JNK/c-Jun signal pathway in cyclin D1 expression induced by low concentrantion of sodium arsenite in HELF cells(1) The effects of blocking JNK signal pathway on c-Jun activation and cyclin D1 expression induced by low concerntration of sodium arsenitePretreatment of HELF cells with SP600125, resulted in a dramatic inhibition of the phosphorylation of JNK and c-Jun. Furthermore, inhibition of JNK signal pathway by SP600125 resulted in a significant decrease in sodium arsenite-induced cyclin D1 expression. Similarly, with JNK expression was dramatically knockdown by JNK siRNA, the phospho-c-Jun levels and cyclin D1 expression were significantly decreased as well. Data demonstrated that activation of JNK signal pathway may be implicated in c-Jun activation and cyclin D1 expression induction by low concerntrantion of sodium arsenite in HELF cells. (2) The effects of c-Jun kockdown on cyclin D1 expression induced by low concerntration of sodium arseniteKnockdown of c-Jun expression by transfection of HELF cells with c-Jun siRNA resulted in a significant decrease in cyclin D1 expression induced by low concerntration of sodium arsenite. Whereas, c-Jun siRNA did not show any inhibitory effect on phospho-JNK levels which induction by the same concerntration of sodium arsenite in HELF cells. Data suggested that induction of cyclin D1 expression by low concerntration of sodium arsenite in HELF cells requires JNK/c-Jun signal pathway.Conclusions:1. The low concerntration of sodium arsenite induces the increases of cellular proliferation; however, the high concerntration of sodium arsenite inhibites cellular proliferation in HELF cells.2. The low concerntration of sodium arsenite prometes cell cycle transition from G1 to S phases, which induces cell cycle disorder in HELF cells3. Activation of JNK/c-Jun pathway and the cyclin D1 protein expression are induced by low concerntration of sodium arsenite in HELF cells.4. JNK/c-Jun pathway and cyclin D1 play critical roles in cellular proliferation abnormity and cell cycle disorder induced by low concerntration of sodium arsenite in HELF cells.