PI3K／AKT／mTOR信号通路在肝癌细胞自体吞噬中的作用研究

Role of PI3K/AKT/mTOR pathway in autophagy of hepatcellular carcinoma cells

目的探讨在肝癌细胞中PI3K／AKT／mTOR信号通路在氨基酸缺乏诱导的自体吞噬中的作用。方法观察HCCLM3、MHCC97L及SMMC-7721肝癌细胞在氨基酸缺乏情况下及与P13K抑制剂3-MA、Wortmannin及LY294002，mTOR抑制剂雷帕霉素协同作用下细胞活力变化。GFP—LC3荧光法观察细胞内自噬体的形成。Western印迹检测LC3-Ⅱ蛋白的表达。结果经无氨基酸培养液EBSS处理后48h，HCCLM3、MHCC97L及SMMC-7721细胞存活率分别为（78．9±3．8）％、（57．2±13．1）％及（3．4±3．0）％（P〈0．01）。与HCCLM3、MHCC97L相比，细胞中的自噬体在SMMC-7721细胞中明显增多，分别为（13．1±3．5）％、（20．0±1．1）％及（48．9±4．5）％（P〈0．01）。Western印迹显示在EBSS处理后早期即有LC3—Ⅱ蛋白的明显表达。在EBSS基础上雷帕霉素20μmol／L处理24h，3种细胞株活力明显下降。3-MA、Wortmannin及LY294002处理也加速EBSS诱导的细胞死亡。结论高转移潜能肝癌细胞比较耐受自噬样细胞死亡。P13K／AKT／mTOR信号通路对自噬样细胞死亡可能起重要调节作用。

Objective To evaluate the role of PI3K/AKT/mTOR pathway in autophagy of hepatocellular carcinoma cells induced by amino acid starvation. Methods The cell viability was measured after amino acid starvation, PI3K inhibitors （3-MA, Wortmannin, LY294002） and roTOR inhibitor Rapamycin treatments in hepatocellular carcinoma cell lines with different metastatic potentials. The autophagosome in hepatocellular carcinoma cells was observed by using GFP-LC3 fluorometric method and LC3-U protein expression by Western blotting. Results The cell viability after amino acid starvation for 48 hours was （78.9 ± 3.8 ） % for HCCLM3, （57.2 ± 13.1 ） % for MHCC97L and （3.4 ± 3. 0） % for SMMC-7721, with statistical difference （ P 〈 0. 01 ）. The autophagosome in HCCLM3 was significantly lower than in MHCC97L and SMMC-772 （ P 〈 0. 01 ） , which was （ 13.1 ± 3.5 ） %, （ 20.0 ± 1.1 ） % and （48.9 ± 4.5 ） % respectively. Western blotting showed that LC3-11 protein was highly expressed in SMMC-7721 cell early after amino acid starvation. After 24 hours of treatment with rapamycin at 20 p, moL/L on EBSS, 3 cell lines showed marked decrease of viability. The mTOR inhibitor Rapamycin and PI3K inhibitor 3-MA, Wortmannin and LY294002 could obviously accelerate amino acid starvation induced cell death in all 3 cell lines. Conclusions The hepatocellular carcinoma cells with high metastatic potential is more resistant to amino acid starvation induced autophagic cell death. The PI3K/AKT/mTOR pathway may regulate the autophagic cell death in Hepatoeellular carcinoma cells.