雷公藤红素体内外诱导人宫颈癌HeLa细胞自噬作用及分子机制

Celastrol Triggers Autophagy in Human Cervical Cancer Hela Cells in Vitro and in Vivo

目的探讨雷公藤红素诱导人宫颈癌HeLa细胞自噬的分子机制。方法采用四甲基偶氮唑蓝(MTT)法检测细胞增殖、流式细胞仪检测细胞周期及凋亡率、荧光显微镜检测细胞自噬、Western blotting检测相关蛋白的表达、小鼠皮下移植肿瘤模型检测肿瘤生长。结果实验结果表明,雷公藤红素体外抑制人宫颈癌HeLa细胞的增殖并诱导细胞自噬及细胞周期阻滞于G_0/G_1期,但对细胞凋亡无影响;雷公藤红素促进自噬标志性蛋白LC3Ⅰ转化为LC3Ⅱ以及Beclin 1上调;此外,雷公藤红素促进PTEN、p-ERK1/2、p-MEK1/2的表达,但抑制Akt、p70S6K及mTOR的磷酸化;用自噬抑制剂3-methyladenine(5 mmol·L^(-1))预处理可逆转雷公藤红素对HeLa细胞的抗增殖及自噬诱导作用。动物实验结果表明,雷公藤红素剂量依赖性地抑制皮下移植肿瘤的生长,且抑制肿瘤组织中p-AKT、p-m TOR等蛋白的表达并上调自噬标志性蛋白Beclin-1及LC3Ⅱ的表达。结论雷公藤红素在体内外的抗肿瘤作用是通过抑制Akt信号通路并诱导HeLa细胞自噬产生的。

OBJECTIVE To investigage the effects of celastrol-triggered HeLa cells autophagy and the molecular mechanisms in vitro and in vivo. METHODS The antiproliferative effect of celastrol was detected using MTT assay. Apoptotic rate and cell cycle were evaluated using flow cytometric analysis. Autophagy was detected using fluorescence microscope. Protein expression was evaluated using Western blotting. Tumor growth was evaluated by subcutaneous xenograft model in vivo. RESULTS Celastrol inhibited HeLa cells proliferation and induced HeLa cells autophagy and cell cycle arrest at G0/G1 phase, but not induced HeLa cell apoptosis in vitro. The protein expression of Beclin 1 was up-regulated and the conversion from LC3 Ⅰ to LC3 Ⅱ was increase in HeLa cells in vitro after treatment with celastrol. Moreover, celastrol promoted the protein expression of PTEN、p-ERK1/2、p-MEK1/2 and inhibited the phosphorylated of Akt, p70S6K and mTOR in HeLa cells. After pretreatment with 3-methyladenine （5 mmol·L-1）, the antiproliferative and induced-autophagy effects of celastrol were reversed. Furthermore, celastrol inhibited tumor growth and the protein expression of p-Akt and p-mTOR, but up-regulated the protein expression of LC3 Ⅱ and Beclin 1 in vivo. CONCLUSION Antitumor effect of celastrol dependent on cells autophagy in HeLa cells via inhibition of Akt/mTOR signaling pathway in vitro and in vivo.