Bidirectional Adjustment Of Manganese Superoxide Dismutase Overexpression On Esophageal Carcinoma TE-1 Cell

Objective: Establish esophageal carcinoma cell line TE-1 of manganese superoxide dismutase (MnSOD) overexpression; and observe the effect of MnSOD with radiation and buthionine sulfoximine on esophageal carcinoma cell line.Methods: (1) Transfect MnSOD gene into human esophageal carcinoma cell TE-1 via lentiviral vector, and establish manganese superoxide dismutase overexpression stable cell lines: TE-1-MnSOD(L) (MnSOD moderate-overexpression), TE-1-MnSOD(H) (MnSOD high-overexpression), TE-1-neo (transfected with empty vector); (2) Colony-forming assay the plating efficiencies of TE-1, TE-1-neo, TE-1-MnSOD(L), TE-1-MnSOD(H); (3) Inhibition rate of proliferation was measured by MTT assay after the following several treatments:â‘ treated with different concentrations of BSO (0.1 mg/ ml, 0.25 mg/ml, 0.5 mg/ml, 0.75 mg/ml, 1 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml);â‘¡treated with different doses of radiation (1 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy);â‘¢treated with BSO (1 mg/ml) combined with radiation (2 Gy, 4 Gy);(4) Cell apoptosis was detected by flow cytometry; (5) MnSOD protein expression of the four kinds of cells was detected by Western blot; (6) MnSOD mRNA expression was detected by RT-PCR.Results: (1) The TE-1 cell lines of MnSOD protein overexpression was established successfully. Compared with TE-1and TE-1-neo, MnSOD protein expression of TE-1-MnSOD(L) and TE-1-MnSOD(H) was higher, and there were significant differences between TE-1-MnSOD(L) and TE-1-MnSOD(H) (P<0.05). (2) The plating efficiencies of TE-1, TE-1-neo, TE-1-MnSOD(L) and TE-1-MnSOD(H) were 34.67%, 31.67%, 24.33% and 43.00%, respectively. Compared with TE-1, plating efficiency of TE-1-MnSOD(L) and TE-1-MnSOD(H), the former reduced, the later increased, and there were significant differences between the two cell lines (P<0.05). (3) BSO in the concentration range of 0.1 and 2.5 mg/ml inhibited the proliferation of TE-1 cell and the transfected cells in some degree. The inhibition of BSO did not change depending on MnSOD protein expression. (4) Radiation in the range of 1 ~ 8 Gy inhibited the proliferation of the TE-1 cell and the transfected cells in a certain degree. With the dosage increasing, the inhibition of cell proliferation enhanced. TE-1-MnSOD(L) was sensitive than TE-1 on radiation, the inhibition was higer than TE-1 and TE-1-MnSOD(H) was resistent than TE-1 on radiation ,the inhibition was lower than TE-1 (P<0.05). (5) The inhibition of TE-1-MnSOD(L) treated with radiation and BSO was higher than TE-1, and the inhibition of TE-1-MnSOD(H) treated with radiation with BSO was lower than TE-1. (6) Apoptosis rate of TE-1 cell and the transfected cells the when ware treated with BSO, radiation and treated with radiation compared with BSO had increased compared control group, and the joint group which increased the most obviously. And apoptosis rate of TE-1-MnSOD(L) in each group was the largest, apoptosis rate of the TE-1-MnSOD(H) was the minimal. (7) MnSOD protein and mRNA expression in the treatment groups had reduced compared control group, and the joint group which reduced the most obviously.Conclusion:(1) The TE-1 cell lines of MnSOD protein overexpression at different levels was established successfully, and they were TE-1-MnSOD(L) TE-1-MnSOD(L) and TE-1-neo.(2) Compared with TE-1, plating efficiency of TE-1-MnSOD(L) decreased, and which of TE-1-MnSOD(H) increased when MnSOD gene was transfected into TE-1 cells via lentiviral vecto.(3) The inhibition of BSO did not change depending on MnSOD protein expression.(4) The radiation showed a bidirectional adjustment on TE-1 cell of MnSOD overexpression. Radiation sensitivity of TE-1-MnSOD(L) increased though increasing apoptosis rate, and radiation sensitivity of TE-1-MnSOD(H) decreased though decreasing apoptosis rate. The bidirectional adjustment mechanism could significantly increase combined with the BSO.