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Objective:To explore the role of nuclear transcription factor erythrocyte line-2p45 (NF-E2) related factor-2 (NRF2) on autophagy during malignant transformation of immortalized human keratinocytes (HaCaT) induced by sodium arsenite (NaAsO 2). Methods:Using cell culture methods, long-term cultured HaCaT cells in DMEM high-glucose medium containing 0.0 (control group) and 1.0 μmol/L NaAsO 2 (arsenic-exposed group) to the 35th generation were used to construct a cell malignant transformation model, and 0, 1, 7, 14, 21, 28 and 35th generation cells of control group and arsenic-exposed group were collected during establishment of cell malignant transformation model. The NRF2 siRNA, phosphatidylinositol-3-hydroxykinase (PI3K) inhibitor LY294002 and mammalian target of rapamycin (mTOR) inhibitor Rapamycin were used to treat the 35th generation of malignant transformed HaCaT cells in arsenic-exposed group (T-HaCaT). The protein expressions of NRF2, PI3K-protein kinase B (Akt)-mTOR signaling pathway related indicators PI3K, Akt, mTOR, phosphorylated (p)-PI3K, p-Akt, p-mTOR, autophagy-related proteins p62, Beclin1, microtubule-associated protein-1 light chain (LC)3Ⅰ, and LC3Ⅱof different generations HaCaT cells in control group and arsenic-exposed group, and T-HaCaT cells of each treatment group were determined by Western blotting. Results:There were significant differences in the NRF2 protein and the ratios of p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR between different generations HaCaT cells in arsenic-exposed group ( F = 9.371, 16.035, 15.932, 27.739, P < 0.05), and they were higher than NRF2 protein and ratio of p-mTOR/ mTOR of the same generation in control group ( P < 0.05). Compared with HaCaT cells of the same generation, the expressions of NRF2, p-PI3K, p-Akt, p-mTOR and p62 proteins in T-HaCaT cells were significantly higher, Beclin1 protein expression and the ratio of LC3Ⅱ/LC3Ⅰ were significantly lower ( P < 0.05). The NRF2 silenced T-HaCaT cells had higher expression of Beclin1 and the ratio of LC3Ⅱ/LC3Ⅰ, and lower expressions of NRF2, p-mTOR and p62 than the corresponding control siRNA (Con siRNA) group ( P < 0.05). The T-HaCaT cells in LY294002 treatment group had higher expression of Beclin1 and the ratio of LC3Ⅱ/LC3Ⅰ, and lower expressions of NRF2, p-PI3K, p-Akt and p-mTOR proteins than the corresponding non-treatment group ( P < 0.05). The T-HaCaT cells in Rapamycin treatment group had higher expression of Beclin1 and the ratio of LC3Ⅱ/LC3Ⅰ, and lower expression of p-mTOR protein than the corresponding non-treatment group ( P < 0.05). Conclusions:During the arsenic-induced malignant transformation of HaCaT cells, NRF2 can act as a downstream factor of PI3K-Akt and an upstream factor of mTOR in PI3K-Akt-mTOR signaling pathway, an important regulatory mechanism of autophagy. This abnormal expression of autophagy may eventually lead to malignant transformation of cells.
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Objective To explore the mechanism of cell apoptosis of immortalized human keratinocytes (HaCaT cells) and protein expression related to this process after long term exposure to sodium arsenite (NaAsO2,1.0 μmol/L).Methods Malignant transformation model was set up through long-term exposure of HaCaT cells to 1.0 μmol/L NaAsO2.Cell passage for 0,1,7,14,21,28 and 35 generations in the process of malignant transformation were collected for measurement of cell apoptosis rate by flow cytometry,and apoptosis related proteins by Western blotting,including activation of cysteine protease 3,8 (cleaved-caspase-3,8),C/EBP homologous protein (CHOP),B-cell leukemia/lymphoma 2 (Bcl-2),and Bcl-2 associated X protein (Bax).Results Along with the arsenite treatment,the apoptosis levels were significantly decreased (F =26.770,all P ＜ 0.05),the apoptosis levels (0.307 ± 0.049,0.213 ± 0.055,0.163 ± 0.057,0.147 ± 0.035,0.053 ± 0.012) of the 7th,14th,21st,28th and 35thgenerations of cells after arsenite treatment were lower than that of control group of the 0th generation (0.393 ±0.021,all P ＜ 0.05).Compared between generations,there were statistical differences of the protein expression levels of cleaved-caspase-3,Chop,Bax and Bcl-2 in arsenite group (cleaved-caspase-3:1.000 ± 0.000,1.030 ± 0.027,1.104 ± 0.069,1.016 ± 0.087,0.838 ± 0.075,0.753 ± 0.082,0.677 ± 0.073;Chop:1.000 ± 0.000,1.059 ± 0.018,0.934 ± 0.095,0.976 ± 0.216,0.793 ± 0.136,0.651 ± 0.042,0.564 ± 0.056;Bax:1.000 ± 0.000,1.069 ± 0.037,1.028 ± 0.042,0.954 ± 0.118,0.641 ± 0.135,0.531 ± 0.132,0.429 ± 0.085;Bcl-2:1.000 ± 0.000,1.072 ± 0.023,1.249 ± 0.134,1.334 ± 0.143,1.633 ± 0.221,1.507 ± 0.152,1.461 ± 0.145,F =7.730,7.355,27.802,12.438,all P ＜ 0.05),compared with control group of the 0th generation (1.000 ± 0.000) and the same generation control group (1.000 ± 0.000),after the 21st generation,the differences were statistically significant (all P ＜ 0.05),while there was no difference of the protein expression levels of cleaved-caspase-8 (F =0.832,P ＞ 0.05).Conclusion In the process of malignant transformation,the apoptosis levels of HaCaT cells are inhibited after long term sodium arsenite exposure through mitochondria and endoplasmic reticulum (ER) stress signaling pathways.