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γ分泌酶抑制剂阻断Notch信号通路对人卵巢癌SKOV3细胞增殖和凋亡的影响
Notch Signaling Pathway Blocked by γ-secretase Inhibitor and Its Effect on the Growth and Apoptosis of SKOV3 Cells
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摘要: 目的 观察γ分泌酶抑制剂 （DAPT）对人宫颈癌SKOV3细胞增殖和凋亡的影响，并探讨其作用机制。方法 不同浓度DAPT对SKOV3细胞作用后，采用CCK-8法检测对SKOV3细胞的增殖抑制作用，吖啶橙/溴化乙锭（AO/EB）荧光双染色法及流式细胞术检测细胞凋亡，RT-PCR、Western blot检测SKOV3细胞Notch1 mRNA和蛋白表达水平的变化。结果 与空白对照组相比，5 μmol/L、10 μmol/L、20 μmol/L浓度的DAPT对SKOV3细胞均有一定的增殖抑制作用（P<0.05)，且其抑制作用呈剂量依耐性增加（P<0.05)，24 h抑制率分别为19.87%、28.38%、46.67%。与空白对照组相比，不同浓度的DAPT作用SKOV3细胞24 h后凋亡率增加（P<0.05)，且随DAPT浓度增加，凋亡细胞逐渐从早期凋亡过渡至晚期凋亡；Notch1 mRNA和蛋白表达抑制，其抑制率分别为 （10.23%、20.50％、38.83％）和 （12.89%、27.47％、49.84％）。结论 γ分泌酶抑制剂DAPT可阻断Notch信号通路，能抑制卵巢癌SKOV3细胞的增殖，促进细胞凋亡，其作用机制可能与下调Notch1表达有关。Abstract: Objective To determine the effect and mechanism of γ-secretase inhibitor DAPT on the growth and apoptosis of human ovarian carcinoma SKOV3 cells. Methods The effect of γ-secretase inhibitor DAPT was tested in vitro using SKOV3 cells. Its inhibition effect on cell proliferation was determined by CCK-8 assay. The cell apoptosis was detected by AO/EB double staining and flow cytometry. The expression of Notch1 mRNA and protein was detected by RT-PCR and Western blot. Results Compared with controls, 5 μmol/L, 10 μmol/L and 20 μmol/L of DAPT showed an effect of cell growth inhibition in a dose-dependent manner, with 19.87%, 28.38%, and 46.67% of 24 h inhibitory rates, respectively. Dose-dependent effect of DAPT on cell apoptosis was also evident, with (5.80 ± 0.98)%, (12.96 ± 4.99)%, (30.88 ± 7.63)%, and (42.98 ± 1.46)% apoptosis rates for the control, 5 μmol/L, 10 μmol/L and 20 μmol/L DAPT groups, respectively. RT-PCR analysis demonstrated that the expression of Notch1 mRNA decreased significantly in the DAPT groups, with an inhibition rate of 10.23%, 20.50％, and 38.83％ for the three DAPT groups, respectively. Western blot results demonstrated that the expression of Notch1 protein decreased significantly, with an inhibition rate of 12.89%, 27.47%, and 49.84% for the three DAPT groups, respectively. Conclusion γ-secretase inhibitor DAPT can block Notch signaling pathway, inhibit proliferation, and induce apoptosis of SKOV3 cells through down-regulation of the expression of Notch1.
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