| ObjectiveTo investigate the dynamic changes of the cellular redox state and Keap1/Nrf2-ARE antioxidant pathway related genes in the process of malignant transformation of HBE(human bronchial epithelial) cells induced by low doses of sodium arsenite, providing a new avenue for the study of the molecular mechanism of arsenic carcinogenesis.Methods1. Identification of the malignant transformation of HBE cellsHBE cells continuous exposed to 1 μmol/L sodium arsenite, at the same time,the passage control group was cultured. The malignant transformation of HBE cells was identified by morphological observation, cell growth kinetics decided by cell counting assay, matrix metalloproteinase-9(MMP-9) detected by Elisa assay and soft agar assay.2. Detection of the expression of Keap1/Nrf2-ARE signaling pathway related indexes in the process of malignant transformation of HBE cellsThe group treated with 1 μmol/L sodium arsenite was named the exposure group, the untreated group was called the passage control group. After the malignant transformation of HBE cells induced by low doses of sodium arsenite, at passage 1,8,15,22,29,36,43, the flow cytometry was used to detect the levels of reactive oxygen species(ROS),thiobarbituric acid colorimetric method was used to detect the levels of malondialdehyde(MDA), WST-1 kit was used to detect the activity of superoxide dismutase(SOD) and Western blot was used to determine the expression of Keap/Nrf2-ARE pathway related Nrf2 protein, Keap1 protein and downstream related HO-1 protein.3. Detection of the expression of Keap1/Nrf2-ARE signaling pathway related indexes of T-HBE cellsThe arsenic-transformed cells(T-HBE cells) from anchorage-independent colonies(≥30 cells) were picked up and continued to grow in DMEM. Once again, ROS, MDA,SOD, Nrf2, Keap1 and HO-1 were detected for T-HBE cells and passage control group via the above assays.4. The function of transcription factor Nrf2 on the malignant transformation of HBE cellsAccording to the experimental results above, T-HBE cells were transfected with Nrf2 si RNA,meanwhile,the passage control group, transfected with Con si RNA group and T-HBE group as control groups. Western blot was used to decide the silencing of Nrf2,simultaneously detected the expression of HO-1 protein. After that, cell proliferation was tested via MTT assay, cell migration was detected via Wound-healing cell migration assay,cell anchorage independent growth was investigated via soft agar assay for the four groups.Results1. Identification of the malignant transformation of HBE cells(1) Compared with the passage control group, the boundaries between the nucleus and cytoplasm in the treated cells were indistinct, the colors of the cytoplasm were more turbid,the numbers of nucleolus were obviously increased, the boundaries between cells were not smooth, but fold, and showed invasive growth.(2) 104 cells of passage 29, 36, 43 were seeded in 24-well plates, the cells were counted separately after 24, 48, 72, 96, 120 h. The results showed that the treated cells grew faster outstandingly than the control cells at 96,120 h at passage 36(p<0.05), furthermore, accelerated obviously than the control cells at 72,96, 120 h at passage 43(p<0.05). In addition, the doubling time of treated cells of passage43 was 22.56±0.96 h, significantly reduced compared with the passage control cells(31.41±3.78 h)(p<0.05).(3) 2×104cells of passage 29, 36, 43 were cultured to detect the secretion of MMP-9. Results displayed that the secretion of MMP-9 for the treated cells of passage 36 [(2.651±0.246) ng/ml], passage 43 [(2.610±0.242) ng/ml] were obviously more than the passage control group(p<0.05).(4) Soft agar assay was carried out for the cells of passage 43, results showed that the colonies of treated cells[(273±90) numbers ] were significantly more than the control cells [(20±4) numbers ](p<0.05).2. Changes of the expression of Keap1/Nrf2-ARE signaling pathway related indexes in the process of malignant transformation of HBE cellsDuring the period of malignant transformation, the cells of passage 1, 8, 15, 22, 29, 36,43 were detected. Results showed that compared to the normal group( passage 0), the levels of ROS, MDA and SOD of the treated cells displayed a wavy tendency of up-regulation first and down-regulation second before passage 22. However, after passage29, ROS and MDA were gradually declined to a lower level, especially the levels of ROS,yet SOD was continuously lifted. In the whole cell, nucleus and cytoplasm, the expression of HO-1 proteins were all maintained at a high level, and showed a distinct increasing tendency in the nucleus after passage 29. In the whole cell and the nucleus, the expression of Nrf2 proteins started to display a trend of up-regulation at passage 8-22, subsequently continuously increased after passage 29, however, decreased gradually in the cytoplasm. In the whole cell, nucleus and cytoplasm, the expression of Keap1 proteins all showed a tendency of reduction continuously, furthermore, displayed a much lower expression in the cytoplasm after passage 22.3. Changes of the expression of Keap1/Nrf2-ARE signaling pathway related indexes of T-HBE cellsT-HBE cells and the passage control cells were detected. Results showed that compared to the passage control cells, the levels of ROS and MDA were decreased significantly, while the activity of SOD was increased obviously in T-HBE cells(p<0.05).The expression of Nrf2 proteins in the whole cell and the nucleus was increased evidently, but decreased sharply in the cytoplasm(p<0.05). In the whole cell, nucleus and cytoplasm, the expression of Keap1 proteins all was reduced, while the expression of HO-1proteins all was increased(p<0.05).4. The function of transcription factor Nrf2 on the malignant transformation of HBE cells(1) T-HBE cells were transfected with Nrf2 si RNA. Results showed that, compared to T-HBE cells, the expression of Nrf2 and HO-1 proteins were both decreased evidently in the whole cell and the nucleus of the transfected and passage control cells(p<0.05).(2) For the passage control cells, T-HBE cells, T-HBE cells transfected with Nrf2 si RNA, T-HBE cells transfected with Con si RNA, MTT assay was carried out to detect cell proliferation.Results showed that compared to T-HBE cells, the relative cell proliferation of T-HBE cells transfected with Nrf2 si RNA and the passage control cells were decreased sharply(p<0.05).(3) Moreover, Wound-healing cell migration assay was carried out for the fourgroup cells. The results showed that cell migration rate of T-HBE cells transfected with Nrf2 si RNA [(50.7±4.1)%] and the passage control cells [(52.1±11.2)%] were significantly reduced compared with T-HBE cells [(79.5±7.3)%](p<0.05).(4) In addition, soft agar assay was performed for the four group cells. The results showed that the colony numbers of T-HBE cells transfected with Nrf2 si RNA [(85±9) numbers] and the passage control cells [(12±7) numbers] were distinctly decreased compared with T-HBE cells [(301±29)numbers](p<0.05).ConclusionThe continuous over-expression of Nrf2 in Keap1/Nrf2-ARE signaling pathway may promote cell proliferation, which is involved in the malignant transformation of HBE cells induced by low doses of sodium arsenite. |
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