紫草素通过PI3K/Akt/mTOR信号通路诱导人宫颈癌HeLa细胞凋亡和自噬

目的:观察紫草素(shikonin)对人宫颈癌HeLa细胞凋亡(apoptosis)和自噬(autophagy)的影响,并初步探讨PI3K/Akt/mTOR信号通路在其中的可能作用。方法:以紫草素作用于HeLa细胞后,采用CCK-8检测细胞活力;Annexin V/PI双染法检测细胞凋亡;GFP-LC3质粒转染HeLa细胞后观察自噬小体;紫草素分别与自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)和凋亡抑制剂Z-DEVD-FMK共同作用后,Western blot分析检测细胞内自噬和凋亡相关蛋白微管相关蛋白1轻链3 (LC3)和cleaved caspase-3表达的变化,并检测PI3K/Akt/mTOR信号通路中磷酸化PI3K(p-PI3K)、磷酸化Akt(p-Akt)和磷酸化mTOR(p-mTOR)蛋白表达的变化。结果:紫草素显著抑制HeLa细胞活力(P<0.05)。与对照组比较,紫草素可诱导HeLa细胞凋亡(P<0.05)。GFP-LC3质粒转染分析结果显示,HeLa细胞经紫草素作用后,细胞质中出现绿色点状聚集的自噬小体,而对照组细胞中极少观察到点状聚集的自噬小体形成。与紫草素组比较,紫草素+3-MA组中LC3-II/LC3-I显著降低,而cleaved caspase-3表达显著升高(P<0.05);与紫草素组比较,紫草素+Z-DEVD-FMK组LC3-II/LC3-I显著升高,而cleaved caspase-3表达显著降低(P<0.05)。与对照组比较,紫草素可使p-PI3K、p-Akt和p-mTOR表达明显降低(P<0.05)。结论:论紫草素能诱导HeLa细胞凋亡和自噬,且其凋亡及自噬具有协同作用,其机制可能与抑制PI3K/Akt/mTOR信号通路有关。

Shikonin induces apoptosis and autophagy of human cervical cancer HeLa cells by PI3K/Akt/mTOR signaling pathway

AIM: To observe the effects of shikonin on the apoptosis and autophagy of human cervical cancer He La cells,and to explore the possible role of PI3 K/Akt/m TOR signaling pathway in these processes. METHODS: The He La cells were treated with shikonin,and the cell viability was detected by CCK-8 assay. The apoptosis was detected by Annexin V/PI double staining. The autophagosome was observed by transfection with GFP-LC3 into the He La cells. After the treatment with shikonin combined with autophagy inhibitor 3-methyladenine( 3-MA) or apoptosis inhibitor Z-DEVDFMK,the protein levels of autophagy-and apoptosis-related molecules microtuble-associated protein 1 light chain 3( LC3)and cleaved caspase-3 in the He La cells were determined by Western blot. The protein levels of phosphorylated PI3 K( pPI3 K),phosphorylated Akt( p-Akt) and phosphorylated m TOR( p-mTOR) were also determined by Western blot.RESULTS: Shikonin significantly inhibited the viability of He La cells( P < 0. 05). Compared with control group,shikonin significantly induced apoptosis of He La cells( P < 0. 05). The results of GFP-LC3 plasmid transfection analysis showed that green dot-like congregate autophagosomes appeared in the cytoplasm of the He La cells after shikonin treatment,while the autophagosomes were rarely observed in control group. Compared with shikonin group,LC3-II/LC3-I was significantly decreased and cleaved caspase-3 was significantly increased in shikonin + 3-MA group( P < 0. 05). Compared with shikonin group,LC3-II/LC3-I was significantly increased and cleaved caspase-3 was significantly decreased in shikonin + Z-DEVDFMK group( P < 0. 05). Compared with control group,shikonin significantly decreased the protein levels of p-PI3 K,p-Akt and p-mTOR( P < 0. 05). CONCLUSION: The apoptosis and autophagy of the He La cells are induced by shikonin,these two processes are complementary. The mechanism may be related to inhibition of PI3 K/Akt/m TOR signaling pathway.