桧木醇通过mTOR通路抑制自噬诱导卵巢癌SKOV3细胞凋亡

Hinokitiol induced apoptosis and inhibited autophagy of ovarian cancer cell SKOV3 via mTOR signaling pathway

目的:探究桧木醇(Hinokitiol)对卵巢癌SKOV3细胞自噬的作用及机制。方法:将SKOV3细胞分为SKOV3组、Hinokitiol(5μmol/L)组、Hinokitiol(10μmol/L)组和Hinokitiol(20μmol/L)组,分别用0、5、10、20μmol/L的桧木醇处理各组细胞,CCK8检测培养不同时间细胞的增殖倍数,克隆实验检测细胞增殖,流式检测细胞凋亡,免疫印迹检测自噬相关蛋白Beclin1、P62、LC3及通路蛋白雷帕霉素靶蛋白(mTOR)和UNC-51样激酶1(Ulk1)的表达,免疫荧光检测LC3的表达。结果:桧木醇处理细胞4d后,与SKOV3组比较,Hinokitiol(5、10、20μmol/L)组细胞增殖倍数均显著降低,克隆形成细胞数均明显减少,细胞凋亡率明显升高;同时,Hinokitiol(5、10、20μmol/L)组细胞Beclin1表达水平和LC3Ⅱ/LC3Ⅰ的比值明显低于SKOV3组,P62表达水平与SKOV3组比较明显升高,细胞质LC3阳性表达也明显减少;此外,Hinokitiol(5、10、20μmol/L)还能显著升高SKOV3细胞mTOR的表达水平,并抑制Ulk1表达。结论:桧木醇可通过抑制自噬诱导卵巢癌SKOV3细胞凋亡,作用机制与激活mTOR信号通路有关。

Objective: To investigate the effect and mechanism of hinokitiol on autophagy of ovarian cancer cell SKOV3. Methods: For this purpose in this study,SKOV3 cells were divided into SKOV3 group,Hinokitiol (5 μmol/L) group,Hinokitiol (10 μmol/L) group and Hinokitiol (20 μmol/L) group.Cells in each group were treated with 0,5,10,20 μmol/L Hinokitiol.CCK8 was used to detect cell multiplication at different time.Clone test was used to detect cell proliferation.Apoptosis was detected by flow cytometry.Western blot was used to detect the expression of autophagy-related proteins Beclin1,P62,LC3 and pathway proteins rapamycin (mTOR) and Ulk1.The expression of LC3 was detected by immunofluorescence. Results: After 4 days of treatment with birchol,compared with SKOV3 group,the cell proliferation multiple,the number of clonogenic cells and the apoptotic rate in Hinokitiol (5,10,20 μmol/L) group were significantly decreased,and the cell apoptosis rate was significantly increased.Meanwhile,the expression level of Beclin1 and the ratio of LC3Ⅱ/LC3Ⅰ in Hinokitiol (5,10,20 μmol/L) group were significantly lower than those in SKOV3 group,the expression level of P62 was significantly higher than that in SKOV3 group,and the positive expression of LC3 in cytoplasm was also significantly decreased.In addition,Hinokitiol (5,10,20 μmol/L) significantly increased the expression of mTOR and inhibited the expression of Ulk1 in SKOV3 cells. Conclusion: Hinokitiol can induce apoptosis of ovarian cancer SKOV3 cells by inhibiting autophagy,and the mechanism is related to activating mTOR signaling pathway.