Molecular Mechanisms Underlying The Neoplastic Transformation, DNA Damages, And Apoptosis Of Human Keratinocyte Induced By Arsenite

Arsenide is a well documented human carcinogen, and sodium arsenite(NaAsO2) is a main existing form of arsenide. The molecular mechanisms underlying the carcinogenesis effects induced by arsenide remain unclear, because the models of arsenide-induced tumorigenesis in experimental animals have not been successfully established. 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. It has been indicated that the carcinogenesis induced by different levels of arsenide may not the same. Low levels of arsenide improve the cell proliferation, which induce the neoplastic transformation of cells and the cancer. However, high levels of arsenide induce the increases of reactive oxygen species (ROS) levels, DNA damages, and cell apoptosis, which may be involved in the cell mutation, genomic instability, and the cancer.Epithelial-to-mesenchymal transition (EMT) and acquirement of tumor stem cells (TSCs)-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 TSCs-like properties, and the malignancy degree and metastasis of tumor cells, have becoming the top research issues. However, it remains unclear that the effects and its molecular mechanisms of EMT and acquirement of TSCs-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 cell neoplastic transformation, DNA damages, and cell apoptosis induced by different concentrations of arsenide, especially on the signal pathways underlying in the EMT and acquirement of TSCs-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.Methods1. To establish the models including cell proliferation, neoplastic transformation, DNA damages, and cell apoptosis of HaCaT cells induced arsenite.After HaCaT cells were exposed to1640medium containing0.0or1.0μM NaAsO2for30passages (about15weeks), we determined the cell proliferation and malignancy degree. After HaCaT cells were treated by0.0,0.5,1.0,2.0,5.0, or10.0μM NaAsO2for24or48h, respectively, the cell proliferation, DNA damages, and cell apoptosis were determined.2. Anchorage-independent growthSoft agar dishes were prepared with under-layers of0.70%agarose in fresh medium. To test for soft-agar colony growth capacity, cells were plated in triplicate at a density of1×104in2mL of0.35%agarose over the agar base. Cultures were fed every three days, after4weeks, colonies were examined microscopically.3. Tumorigenicity in nude miceTo confirm neoplastic transformation,1×107/0.2mL of treated cells were injected subcutaneously into the right armpit of nude/BalbC mice. Four weeks later, the tumor tissues were removed, fixed with4%formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and analyzed by light microscopy.4. Reverse-transcriptase polymerase chain reaction (RT-PCR)Total RNA was transcribed into cDNA by AMV reverse transcriptase. Primers for mot-2, mdm2, survivin, CD34, and K5were used for PCR amplification. The PCR was evaluated by checking the PCR products on2%w/v agarose gels.5. Western blotCell lysates were subjected to SDS-PAGE and transferred to NC membranes. Immune complexes were detected by enhanced chemiluminescence.6. Southwestern blotCell extracts were separated by SDS-PAGE and transferred to NC membranes. After transferring, the filters were hybridized with the biotin-labeled probe (κB DNA). The positions of the biotin-labeled oligonucleotides were detected by a chemiluminescent reaction.7. Co-immunoprecipitationCell extracts were incubated with IP antibody and subsequently with A+G sepharose beads at4℃overnight. To determine protein-protein interaction or to determine DNA-protein interaction, the immunoprecipitates were analyzed by Western blot or by Southwestern blot. 8. Immunostaining analyses of the nuclear translocation of p53Treated cells were stained with rabbit p53antibody for24h. They were then incubated with Cy3-conjugated goat-anti-rabbit secondary antibody for1h. To stain the nuclei, DAPI was added for15min, and the cells were observed under a fluorescence microscope. The images were analyzed by use of an Image-Pro Plus6.0.9. Spheroid formationIn non-adherent dishes, cells (1×104) were suspended in defined, serum-free medium composed of DMEM/F-12,10ng/ml of human recombinant basic fibroblast growth factor, and10ng/ml of epidermal growth factor. Cells were grown for10days and fed every48h. Total spheres were then counted under a microscope.Results1. Effects of arsenite on the cell proliferation, neoplastic transformation, DNA damages, and cell apoptosis of HaCaT cellsHaCaT cells were treated by0.0、0.5,1.0,2.0,5.0, or10.0μM arsenite for24or48h, respectively; HaCaT cells were exposed to1640medium containing0.0or1.0μM arsenite for30passages (about15weeks). Our data showed that the percentages of cell proliferation were improved by0.5,1.0, and2.0μM arsenite, respectively, especially by1.0μM arsenite. The γ-H2AX levels were slightly up-expression in0.5,1.0, and2.0μM arsenite-treated groups. The levels of γ-H2AX and cleaved caspase3were increased by5.0and10.0μM arsenite. The mice injected with1.0μM arsenite-transformed HaCaT cells formed tumors, which were composed of undifferentiated, epithelial-like cells. These results suggest that low concentrations of arsenite induce the cell proliferation and neoplastic transformation; however, high concentrations of arsenite cause DNA damages and cell apoptosis. 2. Effects of arsenite on the p53/survivin, MAPKs, and NF-kB signal pathways in HaCaT cellsHaCaT cells were treated by0.0,0.5,1.0,2.0,5.0, or10.0μM arsenite for24, respectively, data showed that there were decreased p-p53level and increased levels of p-ERK, p-NF-κB/RelA, survivin, and mot-2in1.0μM arsenite-treated cells. However, there were decreased survivin level and increased levels of p-JNK, p-p38, and p-p53in5.0and10.0μM arsenite-treated cells. These results suggest that low concentrations of arsenite activate the ERK and NF-κB signal pathways, block p53function, and improve the levels of survivin and mot-2, however, high concentrations of arsenite activate the JNK and p38signal pathways, improve p53function, and decrease survivin level.3. The roles of MAPKs and NF-κB signal pathways in the effects of arsenite on p53After HaCaT cells were pre-treated by0.0and10.0μM ERK inhibitor, U0126, JNK inhibitor, SP600125, NF-κB inhibitor, Bay11-7082, or p38inhibitor, SB203580for6h, respectively, they were exposed to0.0,1.0, or10.0μM arsenite. Our data showed that, the inhibition of either ERK or NF-κB blocked the1.0μM arsenite-induced decreases of p-p53levels. The blockage of ERK also inhibited the1.0μM arsenite-induced decreases of nuclear p53fluorescence intensity. The inhibition of JNK blocked the10.0μM arsenite-induced increases of p-p53level and nuclear p53fluorescence intensity. These results suggest that ERK and NF-κB signal pathways are involved in the inactivation of p53induced by low concentrations of arsenite; however, JNK signal pathway is involved in p53activation induced by a high concentration of arsenite.4. The roles of ERK regulated mot-2and survivin via NF-kB in the neoplastic transformation of HaCaT induced by a low concentration of arsenite After HaCaT cells were pre-treated by20.0nM NF-KB/RelA-siRNA, or con-siRNA for12h, or by0.0or10.0μM ERK inhibitor, U0126for6h, respectively, they were exposed to0.0or1.0μM arsenite for24h. Our data showed that the inhibition of ERK signal pathway blocked the DNA-binding activity of NF-κB. The inhibition of either NF-κB or ERK blocked the increases of mot-2and survivin levels induced by1.0μM arsenite. These results suggest that in low concentrations of arsenite-treated HaCaT cells, ERK up-regulates the levels of mot-2and survivin via NF-κB.After HaCaT cells were pre-treated by0.0or1.0μM ERK inhibitor, U0126for6h, respectively, they were exposed to0.0or1.0μM arsenite for24h, which was continued for30passages (about15weeks). Our data showed that the inhibition of ERK blocked the1.0μM arsenite-induced cell proliferation, colony formation in soft agar, and tumorigenesis in nude mice. These results suggest that ERK is involved in the cell proliferation and neoplastic transformation induced by low concentrations of arsenite.5. The roles of JNK regulated survivin via p53in the DNA damages and cell apoptosis of HaCaT induced by high concentrations of arseniteAfter HaCaT cells were pre-treated by0.0or1.0μM JNK inhibitor, SP600125for6h, respectively, they were exposed to0.0or10.0μM arsenite for24h. Our data showed that the inhibition of JNK blocked the10.μM arsenite-induced increase of survivin level; moreover, SP600125improved the binding of p53with mdm2in HaCaT cells exposed to10.0μM arsenite; further, the inhibition of JNK blocked the arsenite-induced increase of cleaved caspase3level, but improved the arsenite-induced increase of y-H2AX level. These results suggest that NK down-regulates survivin level via p53, which is involved in the DNA damages and cell apoptosis in high concentrations of arsenite-treated HaCaT cells. 6. Low concentrations of arsenite induce EMT and acquirement of TSCs-like properties in HaCaT cellsHaCaT cells were exposed to1640medium containing0.0or1.0μM arsenite for30passages. Our data showed that, in cells exposed to1.0μM arsenite for20passages, the cells acquired a fibroblast-like, mesenchymal appearance consistent with EMT; with increased time of exposure to arsenite, there was greater attenuation of cellular adhesive capacity; further, after20passages, the E-cadherin level was decreased; in contrast, N-cadherin, vimentin, CD34, and K5levels were increased; cells acquired the ability for formed the spheroids. With longer times of arsenite exposure, such changes were more significant. These results suggest that the chronic exposure of HaCaT cells to low concentrations of arsenite undergo an EMT and acquire the TSCs-like properties.7. Effects of low concentrations of arsenite on NF-κB and snail in HaCaT cellsHaCaT cells were exposed to1640medium containing0.0or1.0μM arsenite for30passages. Our data showed that, in cells exposed to1.0μM arsenite for10passages, there were increased levels of p-IKKβ, p-IκBα, p-NF-κB/RelA in classical NF-κB signal pathway, and snail. With longer times of arsenite exposure, such changes were more significant. These results suggest that the chronic exposure to low concentrations of arsenite induces the activation of classical NF-κB signal pathway, which up-regulats snail level.8. The roles of NF-κB in the increase of snail induced by low concentrations of arseniten in HaCaT cellsHaCaT cells were exposed to0.0or1.0μM arsenite for24or48h, respectively. After HaCaT cells were pre-treated by0.0or1.0μM NF-κB inhibitor, Bay11-7082for6h, or by20.0nM IKKβ-siRNA, IKKa-siRNA, or NF-κB/RelA-siRNA for12h, respectively, they were exposed to0.0or1.0μM arsenite for24h. Our data showed that the increases of snail levels with a time-dependent relationship were induced by acute treatment of1.0μM arsenite. The blockage of classical NF-κB signal pathway inhibited the increase of snail level induced by acute treatment of1.0μM arsenite These results suggest that low concentrations of arsenite up-regulates snail level by the classical NF-κB signal pathway in HaCaT cells.9. The roles of NF-κB in the EMT, acquirement of TSCs-like properties, and tumorigenesis induced by low concentrations of arsenite in HaCaT cellsAfter HaCaT cells were pre-treated by0.0or1.0μM NF-κB inhibitor, Bayl1-7082for6h, respectively, they were exposed to0.0or1.0μM arsenite for24h, which was continued for30passages. Our data showed that the inhibition of NF-κB blocked1.0μM arsenite-induced decreases of E-cadherin level and cellular adhesive capacity; moreover, Bayl1-7082blocked arsenite-caused increased levels of N-cadherin, vimentin, CD34, and K5; further, the inhibition of NF-κB blocked the arsenite-induced acquirement of a fibroblast-like appearance, the ability for formed the spheroids, and the ability for the tumorigenesis in nude mice. These results suggest that NF-κB is involved in the EMT, acquirement of TSCs-like properties, and oncogenicity induced by a low concentration of arsenite.Conclusions1. Low concentrations of arsenite induce the cell proliferation and neoplastic transformation; however, high concentrations of arsenite cause DNA damage and cell apoptosis.2. The activation of NF-κB by ERK, which inhibits p53function and decrease survivin level, is involved in the cell proliferation and neoplastic transformation of HaCaT cells induced by low concentrations of arsenite3. The up-regulation of survivin level by JNK via p53activation plays an important roles in the DNA damage and cell apoptosis induced by high concentrations of arsenite in HaCaT celle4. HaCaT cells undergo an EMT and acquire the TSCs-like properties during the cell neoplastic transformation induced by the chronic treatment of low concentrations of arsenite.5. The up-regulation of snail level by the activation of classical NF-κB signal pathway is involved in the EMT, acquirement of TSCs-like properties, and cell neoplastic transformation induced by low concentrations of arsenite in HaCaT cells.