Yang L L, Wang X Y, Zheng L Y, Fang S J, Xu M, Zhao Z W, Ji J S
Department of Anesthesiology, the Fifth Affiliated Hospital of Wenzhou Medical University, Lishui Hospital of Zhejiang University, Lishui Municipal Central Hospital, Lishui 323000, China.
Zhonghua Yi Xue Za Zhi. 2017 Apr 18;97(15):1187-1190. doi: 10.3760/cma.j.issn.0376-2491.2017.15.016.
To investigate the role of FOXO3a-Bim signaling in triptolide induced bladder cancer T24 cells apoptosis. T24 cells were used and divided into control group, triptolide group(50 nmol/L), MK2206 group(50 nmol/L triptolide+ 5 μmol/L MK2206), FOXO3a-siRNA group(50 nmol/L triptolide+ 100 nmol/L FOXO3a-siRNA), Bim-siRNA group (50 nmol/L triptolide+ 100 nmol/L Bim-siRNA). MTT assay was used to analyze the cells growth inhibition.Annexin V/PI staining was implemented to detect cell apoptosis rate, the expression of p-Akt, Akt, p-FOXO3a, FOXO3a, Bim, Bax.Cleaved-caspase 3 was analyzed by Western blot. After treatment with triptolide 25, 50, 100, 250 nmol/L, the cell growth inhibition rates at 24 hours(17%±9%, 24%±5%, 43%±8%, 61%±8%), 48 hours (20%±7%, 34%±6%, 56%±7%, 74%±5%) and 72 hours(32%±8%, 41%±7%, 69%±7%, 84%±3%) were significantly higher than control group respectively.The IC(50) at 24, 48, 72 hours were (113±10), (91±8), (68±5) nmol/L; the cell apoptosis rates at 24 hours (10%±4%, 15%±5%, 29%±8%, 46%±8%), 48 hours (16%±5%, 24%±6%, 40%±7%, 55%±9%) and 72 hours (27%±4%, 38%±5%, 50%±9%, 65%±8%) were significantly increased (<0.05). Western blot showed that triptolide reduced the expression of p-Akt, p-FOXO3a and increased the expression of Bim, Bax, cleaved-caspase 3.The cell inhibition rate in Triptolide group (30%±8%) was significantly higher than that in the control group (<0.05) and the rates in MK2206 group (54% ±6%), FOXO3a-siRNA group (18%±7%) and Bim-siRNA group (11%±6%) were also higher than the control group.Compared with the triptolide group, the inhibition rate in MK2206 group was significantly increased, but decreased in FOXO3a-siRNA group and Bim-siRNA group(<0.05). Triptolide induces T24 cells apoptosis through FOXO3a-Bim signaling pathway.
探讨FOXO3a - Bim信号通路在雷公藤甲素诱导膀胱癌T24细胞凋亡中的作用。采用T24细胞，分为对照组、雷公藤甲素组（50 nmol/L）、MK2206组（50 nmol/L雷公藤甲素 + 5 μmol/L MK2206）、FOXO3a - siRNA组（50 nmol/L雷公藤甲素 + 100 nmol/L FOXO3a - siRNA）、Bim - siRNA组（50 nmol/L雷公藤甲素 + 100 nmol/L Bim - siRNA）。采用MTT法分析细胞生长抑制情况。采用Annexin V/PI染色检测细胞凋亡率，检测p - Akt、Akt、p - FOXO3a、FOXO3a、Bim、Bax、Cleaved - caspase 3的表达。采用蛋白质免疫印迹法分析Cleaved - caspase 3。用25、50、100、250 nmol/L雷公藤甲素处理后，24小时（17%±9%，24%±5%，43%±8%，61%±8%）、48小时（20%±7%，34%±6%，56%±7%，74%±5%）和72小时（32%±8%，41%±7%，69%±7%，84%±3%）的细胞生长抑制率分别显著高于对照组。24、48、72小时的半数抑制浓度（IC50）分别为（113±10）、（91±8）、（68±5）nmol/L；24小时（10%±4%，15%±5%，29%±8%，46%±8%）、48小时（16%±5%，24%±6%，40%±7%，55%±9%）和72小时（27%±4%，38%±5%，50%±9%，65%±8%）的细胞凋亡率显著升高（P<0.05）。蛋白质免疫印迹法显示，雷公藤甲素降低p - Akt、p - FOXO3a的表达，增加Bim、Bax、Cleaved - caspase 3的表达。雷公藤甲素组的细胞抑制率（30%±8%）显著高于对照组（P<0.05），MK2206组（54%±6%）、FOXO3a - siRNA组（18%±7%）和Bim - siRNA组（11%±6%）的细胞抑制率也高于对照组。与雷公藤甲素组相比，MK2206组的抑制率显著升高，而FOXO3a - siRNA组和Bim - siRNA组的抑制率降低（P<0.05）。雷公藤甲素通过FOXO3a - Bim信号通路诱导T24细胞凋亡。
