粉防己碱通过抑制PI3K/AKT/mTOR信号通路诱导卵巢癌细胞自噬

Tetrandrine induces autophagy of ovarian cancer cells by inhibiting PI3K/Akt/m TOR signaling pathway

目的:研究粉防己碱(tetrandrine)对人卵巢浆液性囊腺癌SKOV3细胞活力及自噬的影响,并初步探讨其作用机制。方法:采用MTT法检测不同作用浓度的粉防己碱对SKOV3细胞活力的影响。采用吖啶橙染色法观察粉防己碱对SKOV3细胞中自噬溶酶体形成的影响。Western blot法检测粉防己碱处理前后SKOV3细胞自噬相关蛋白LC3的表达情况及相关信号通路的变化。最后,采用MTT法检测粉防己碱单用及联合自噬抑制剂3-甲基腺嘌呤(3-MA)对SKOV3细胞活力的影响。结果:粉防己碱可显著抑制SKOV3细胞的活力(P<0.01),作用24 h其半数抑制浓度为15.21μmol/L。吖啶橙染色结果显示,粉防己碱明显促进SKOV3细胞中自噬溶酶体的生成。Western blot实验结果显示,粉防己碱干预后,SKOV3细胞中LC3-Ⅱ和P62蛋白的表达水平明显上调;同时,粉防己碱能明显降低PI3K/AKT/mTOR信号通路中p-mTOR和p-AKT蛋白的水平(P<0.01)。自噬抑制剂3-MA增强了粉防己碱对SKOV3细胞活力的抑制作用。结论:粉防己碱可通过抑制PI3K/AKT/mTOR信号通路诱导卵巢癌SKOV3细胞自噬,同时,自噬活化参与了粉防己碱对SKOV3细胞生长的抑制作用。

AIM:To investigate the effect of tetrandrine on the autophagy of human ovarian serous cystadenocarcinoma SKOV3 cells,and to explore its molecular mechanism.METHODS:The SKOV3 cells were treated with various concentrations of tetrandrine,and the cell viability was measured by MTT assay. The formation of autophagolysosomes was observed by acridine orange staining under fluorescence microscope. The protein levels of LC3,mTOR,pmTOR,Akt and p-Akt in the SKOV3 cells were determined by Western blot. The viability of the SKOV3 cells treated with tetrandrine alone or combined with autophagy inhibitor 3-methyladenine(3-MA)were measured by MTT assay.RESULTS:Tetrandrine significantly inhibited the viability of SKOV3 cells in a dose-dependent manner(P<0. 01). The results of acridine orange fluorescence staining showed that the number of intracellular autophagolysosomes with bright red fluorescence in the SKOV3 cells was significantly increased after tetrandrine treatment,while the autophagolysosomes were rarely observed in control group. The protein levels of LC3-II and P62 in the SKOV3 cells were significantly increased after tetrandrine treatment(P<0. 01). Furthermore,treatment with tetrandrine resulted in significant down-regulation of phosphorylated form of mTOR and AKT in the SKOV3 cells(P<0. 01),while total mTOR and AKT protein levels were not changed. Finally,combination of tetrandrine and 3-MA significantly decreased the cell viability compared with using tetrandrine alone(P<0. 01).CONCLUSION:The autophagy of human ovarian cancer SKOV3 cells were induced by tetrandrine and the molecular mechanism may be related to inhibition of PI3 K/Akt/mTOR signaling pathway.