Objective: To investigate the role of FOXO3a-Bim signaling in triptolide induced bladder cancer T24 cells apoptosis. Methods: 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. Results: 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 (P<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 (P<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(P<0.05). Conclusion: Triptolide induces T24 cells apoptosis through FOXO3a-Bim signaling pathway.
目的： 探讨FOXO3a-Bim信号通路在雷公藤内酯醇诱导膀胱癌T24细胞细胞凋亡的作用。 方法： 培养膀胱癌T24细胞，将实验分为空白对照组，雷公藤内酯醇组(50 nmol/L)，MK2206组(50 nmol/L雷公藤内酯醇＋5 μmol/L MK2206)，FOXO3a-siRNA组(50 nmol/L雷公藤内酯醇＋100 nmol/L FOXO3a-siRNA)和Bim-siRNA组(50 nmol/L雷公藤内酯醇＋100 nmol/L Bim-siRNA)，用噻唑蓝(MTT)法检测T24细胞生长抑制率，Annexin V/Propidium iodide (PI)双染法检测细胞凋亡，Western印迹方法检测p-Akt、Akt、p-FOXO3a、FOXO3a、Bim、Bax、cleaved-caspase 3表达。 结果： 25、50、100、250 nmol/L雷公藤内酯醇处理24 h(17%±9%、24%±5%、43%±8%、61%±8%)、48 h(20%±7%、34%±6%、56%±7%、74%±5%)和72 h(32%±8%、41%±7%、69%±7%、84%±3%)后细胞生长抑制率均明显高于对照组(P<0.05)。24、48、72 h的IC(50)分别为(113±10)、(91±8)、(68±5) nmol/L。24 h(10%±4%、15%±5%、29%±8%、46%±8%)、48 h(16%±5%、24%±6%、40%±7%、55%±9%)和72 h(27%±4%、38%±5%、50%±9%、65%±8%)后细胞凋亡率均明显高于对照组(均P<0.05)。25、50、100 nmol/L雷公藤内酯醇诱导细胞凋亡、降低p-Akt、p-FOXO3a表达同时增高Bim、Bax、cleaved-caspase 3表达。FOXO3a-siRNA及Bim-siRNA降低了雷公藤内酯醇诱导Bim、Bax、cleaved-caspase 3高表达。雷公藤内酯醇组细胞抑制率(30%±8%)明显高于空白对照组(P<0.05)。MK2206组(54%±6%)、FOXO3a-siRNA组(18%±7%)和Bim-siRNA组(11%±6%)细胞抑制率亦高于对照组，但与雷公藤内酯醇组相比，MK2206组细胞抑制率明显增高，FOXO3a-siRNA组和Bim-siRNA组细胞抑制率明显降低(P<0.05)。 结论： 雷公藤内酯醇通过激活FOXO3a-Bim信号通路诱导细胞凋亡，抑制T24细胞生长。.
Keywords: Apoptosis; Forkhead transcription factors; Genes, bcl-2; Tripterygium.