The Roles Of JNK And ERK1/2 Signaling Pathways In Hormesis Induced By Sodium Arsenite In Human Embryo Lung Fibroblast Cells

Hormesis or biphasic effect induced by environmental agents is a dose-response phenomenon characterized by a low-dose stimulation and a high-dose inhibition when body exposuring environmental agents. The curve of hormetic dose-response relationship is aβ-shaped or a U-shaped curve. Recently, Calabrese and Baldwin et al have suggested that the majority dose-response modes caused by environmental agents might be neither the threshold mode nor the mode of non-threshold linear, but rather hormetic mode. Implicating the new theory of hormesis would cause that the chief principle of toxicology should be rethinked and many territories of environment, medical, public health should be changed.The effects of sodium arsenite on body are very multiplicity. Sodium arsenite is a known carcinogeon in human and is used as a chemotherapeutic agent in the treatment of some tumors, however, the mechanisms underlying carcinogesis and anti-cancer effect are not known. Some studies indicated that arsenite may induce cell proliferation and apoptosis via hormetic relationship in human cells; however, mechanisms underlying this phenomenon are not well understood.To establish the hormetic mode induced by sodium arsenite, it was investigated that the dose-effect and time-effect relationships of cell proliferation and apoptosis caused by sodium arsenite in human embryo lung fibroblasts (HELF) cells. To explain molecular mechanisms of sodium arsenite-induced hormesis, we investigate the roles of JNK and ERK1/2 signaling pathways in hormesis induced by sodium arsenite in HELF cells. We also construct JNK protein deficient cell strains to explore further the role of JNK signaling pathway in sodium arsenite-induced hormesis. The results of this investigation would provide the theory foundation to understand and accept the hormesis induced by environmental agents and to understand the effects of arsenic on human health.Methods1. The effects of sodium arsenite on cell proliferation and apoptosis in HELF cellsTo evaluate the dose-effect and time-effect relationships of cell proliferation and apoptosis induced by sodium arsenite in HELF cells, cell proliferation was evaluated by MTT assay and cell apoptosis was measured by Hoechst staining assay after HELF cells were treated with sodium arsenite of control, 0.1, 0.5, 1.0, 5.0, or 10.0μmol/L for exposure time of 3, 6, 12, 24, or 48 h, respectively.2. The effects of sodium arsenite on phospho-JNK level and phospho-ERK1/2 level in HELF cellsAfter HELF cells were treated with sodium arsenite of control, 0.1, 0.5, 1.0, 5.0, or 10.0μmol/L for exposure time of 3, 6, 12, 24, or 48 h, the total protein of HELF cells was extracted and the levels of JNK,phospho-JNK ERK1/2 and phospho-ERK1/2 were detected with Western blot assay. 3. The effects of JNK inhibitor and ERK1/2 inhibitor on sodium arsenite-induced cell proliferation and apoptosisHELF cells were left untreated or pre-treated with 20μmol/L JNK inhibitor, SP600125 or 100μmol/L ERK1/2 inhibitor, PD98059 for 30 min before exposure to control, 0.1, 0.5, 1.0, 5.0, or 10.0μmol/L arsenite for 24 h, respectively. Cell proliferation and apoptosis were measured.4. Construction of JNK deficient cell strains and observation of cell biologic charactersAfter extracting total RNA from HELF cells, the target DNA fragment was amplified by reverse transcript-PCR (RT-PCR) with specific primers. The JNK recombinant vectors of antisense RNA were constructed. HELF cells were transfected with eukaryotic expression vectors of JNK gene antisense RNA. Transfected cells were screened by 800μg/mL G418. The level of JNK protein in transfected cells was detected by Western blot assay to verify the efficacy of antisense inhibition. The cellular morphology and growth curve were observeated.Results1. The effects of sodium arsenite on cell proliferation and apoptosis in HELF cells(1)The effect of sodium arsenite on cell proliferation in HELF cells Cell proliferation was significantly increased in cells incubated with 0.1 or 0.5μmol/L sodium arsenite at 24h. The curve peak of cell proliferation presented at 0.5μmol/L sodium arsenite. In contrast, there were markedly decreased in cell proliferation in 5.0 or 10.0μmol/L sodium arsenite at 24 and 48 h. Data indicated that sodium arsenite increased cell proliferation at low concentrations, but inhibited cell growth at high amounts, which is reflected in concentration-responseβ-shaped curve.(2)The effect of sodium arsenite on cell apoptosis in HELF cellsCell apoptosis was significantly decreased in cells incubated with 0.5 μmol/L sodium arsenite at 3, 6, 12, 24, or 48 h. In contrast, there were markedly increased in cell apoptosis in 5.0 or 10.0μmol/L sodium arsenite at 24 or 48 h. Data indicated that sodium arsenite decreased cell apoptosis at low concentrations, but increased cell apoptosis at high amounts, which is reflected in concentration-response U-shaped curve.2. The effects of sodium arsenite on phospho-JNK level and phosphor-ERK1/2 level in HELF cells(1)The effects of sodium arsenite on phospho-JNK level in HELF cellsPhospho-JNK levels were significantly increased with 0.1, 0.5, or 10.0μmol/L sodium arsenite at 3, 6, or 12 h, respectively. Phospho-JNK levels were significantly increased with 0.5 and 10.0μmol/L sodium arsenite at 24 h, respectively. Data indicated that low and high concentrations of sodium arsenite activated JNK signaling pathway, however, middle concentration of sodium arsenite did not activate the signaling pathway.(2)The effects of sodium arsenite on phospho-ERK1/2 level in HELF cellsPhospho-ERK1/2 levels were significantly increased with 0.1, 0.5 1.0, 5.0, or 10.0μmol/L sodium arsenite at 6 or 12 h, respectively. Phospho-ERK1/2 levels were significantly increased with 5.0 or 10.0μmol/L sodium arsenite at 24 h, respectively. Data suggested that different concentrations of sodium arsenite activated JNK signaling pathway, however, there were significant differences among different concentrations of sodium arsenite.3. The effects of JNK inhibitor and ERK1/2 inhibitor on sodium arsenite-induced cell proliferation and apoptosis(1)The effects of JNK inhibitor and ERK1/2 inhibitor on sodium arsenite-induced cell proliferationJNK inhibitor, SP600125, significantly blocked increases of cell proliferation induced by 0.1 or 0.5μmol/L sodium arsenite and the decrease in cell proliferation induced by 5.0, or 10.0μmol/L sodium arsenite. ERK1/2 inhibitor, PD98059, significantly inhibited the increases in cell proliferation induced by 0.1 and 0.5μmol/L sodium arsenite and enhanced markedly the decreases in cell proliferation induced by 5.0, or 10.0μmol/L sodium arsenite. Data indicated that SP600125 blocked significantly sodium arsenite- induced hormesis, however, PD98059 antagonized markedly the increases of cell proliferation induced by low concentration of sodium arsenite and improved the decreases of cell proliferation induced by high concentration of sodium arsenite.(2)The effects of JNK inhibitor and ERK1/2 inhibitor on sodium arsenite-induced cell apoptosisJNK inhibitor, SP600125, significantly blocked decrease of cell apoptosis induced by 0.5μmol/L sodium arsenite and the increases in cell apoptosis induced by 5.0, or 10.0μmol/L sodium arsenite. Treated with ERK1/2 inhibitor, PD98059, the decreases in cell apoptosis induced by 0.1 and 0.5μmol/L sodium arsenite and the incerases in cell apoptosis induced by 5.0, or 10.0μmol/L sodium arsenite were also existent. Data indicated that SP600125 blocked significantly sodium arsenite-induced hormesis. However, the effects of different concentrations of sodium arsenite on cell apoptosis were not influenced by PD98059.4. Construction of JNK deficient cell strains and observation of cell biologic characters(1)Construction of eukaryotic expression vectors of JNK gene antisense RNA.387 bp and 326 bp target DNA fragments were observated after pEGFP-C1-asJNK(top) vector and pEGFP-C1-asJNK(mid) vector were analysised by restriction endonucleases, respctively. The sequencing results showed that the target DNA sequence accorded with that of JNK gene cDNA in Genbank by 100.0%. Data indicated that two eukaryotic expression vectors of JNK gene antisense RNA were successfully constructed.(2)Identification of JNK protein deficient cell strains The results of Western blot showed that JNK protein levels in asJNK-HELF(top) cells and asJNK-HELF(mid) cells were 41% and 13% of that in C1-HELF cells, respectively, or JNK protein levels in asJNK-HELF(top) cells and asJNK-HELF(mid) cells were decreased 59% and 87%, which indicated that two cell strains of JNK protein deficience were successfully constructed and JNK inhibition in asJNK-HELF(top) cells is more effective than sJNK-HELF(mid) cells.(3)Observation of biologic characters of JNK protein deficient cell strainsAt the beginning of transfection, the morphology of asJNK-HELF(top) cells, asJNK-HELF(mid) cells and C1-HELF cells changed obviously when compared with that of HELF cells. 14 days later, no obvious differences were observed among the four cells. The growth curves of asJNK-HELF(top) cells, asJNK-HELF(mid) cells, C1-HELF cells and HELF cells were all "S" shape. There were not any significant differences. The results indicated that there was no significant effect of JNK protein deficiency on growth speed of cell.Conclusions1. Hormesis or biphasic effect of cell proliferation and apoptosis were induced by sodium arsenite in HELF cells.2. JNK signaling pathway and ERK1/2 signaling pathway play important roles in sodium arsenite-induced hormesis of cell proliferation and apoptosis in HELF cells. JNK signaling pathway seems to play a more critical role than ERK1/2 signaling pathway in the hormesis.3. Two JNK protein deficient cell strains were successfully constructed and JNK inhibition in asJNK-HELF(top) cells is more effective than sJNK-HELF(mid) cells.