雷帕霉素对肝癌化疗的增敏作用及相关机制研究

Chemosensitivity enhancement of rapamycin on hepatocellular carcinoma and related mechanism

目的探讨雷帕霉素(rapamycin)对阿霉素(DOX)治疗肝癌效果的影响以及相关机制。方法 DOX单独或与rapamycin联合处理HepG2、Hep3B 2种肝癌细胞48 h,用MTT法测定细胞的增殖情况;DOX、rapamycin以及两者联合处理HepG2、Hep3B细胞24 h,以流式细胞术检测细胞凋亡率,用Western blot法检测PI3K/Akt/mTOR通路相关蛋白(p-Akt、p-mTOR、p21和p53)的表达。结果 DOX能浓度依赖性抑制HepG2和Hep3B细胞的增殖,且对HepG2细胞的抑制作用明显强于Hep3B细胞;与单用DOX比较,DOX与rapamycin联用对2种细胞增殖的抑制作用明显增加,且增加Hep3B细胞对DOX敏感性的作用尤为显著。DOX作用HepG2和Hep3B细胞24 h,可诱导2种细胞发生不同程度的凋亡,rapamycin单独作用的促凋亡作用不明显,但与DOX联用后能明显增加DOX的促凋亡作用,2种细胞的凋亡率均明显增加。Western blot结果显示,rapamycin能明显上调p-Akt、p21和下调p-mTOR在2种细胞的表达水平,轻度上调p53在HepG2中的表达;DOX能轻度上调p-Akt、p21和下调p-mTOR在2种细胞的表达水平,明显上调p53在HepG2中的表达,Hep3B中无p53表达;两药合用对上述蛋白表达的影响有协同作用。结论 rapamycin能增加DOX对不同p53状态肝癌细胞的增殖抑制和促凋亡作用及肝癌对DOX化疗的敏感性,其机制可能与rapamycin激活凋亡的p53和不依赖p53的p21途径有关。

Objective To investigate chemosensitivity enhancing effect of rapamycin on doxorubicin（DOX） for hepatocellular carcinoma（HCC） and related mechanism.Methods The proliferation of HepG2 and Hep3B cells was determined by MTT assay after both cells were treated with DOX alone or the combination of DOX and rapamycin for 48 h.The cell apoptosis and the related proteins（p-Akt,p-mTOR,p21 and p53） of PI3K/Akt/mTOR signaling pathway in HepG2 and Hep3B cells were determined by flow cytometry and Western blot respectively after both cells were exposed to DOX,rapamycin or the combination of DOX and rapamycin for 24 h.Results DOX inhibited the proliferation of both HepG2 and Hep3B cells in a concentration dependent manner.The proliferation inhibition effect of DOX on HepG2 cell was significantly stronger than that on Hep3B cells.The combined treatment of DOX and rapamycin significantly increased the proliferation inhibition in both cells,especially in Hep3B cells,compared with the DOX treatment alone.Apoptosis was induced in both cells to different extents after exposure to DOX for 24 h.Although rapamycin treatment alone had no notable apoptosis inducing effect,it significantly increased the apoptosis inducing effect of DOX in both cells after the combination treatment.Western blot demonstrated rapamycin significantly upregulated p-Akt and p21,downregulated p-mTOR expressions,and slightly upregulated p53 expression.DOX slightly upregulated p-Akt and p21,downregulated p-mTOR expressions,and significantly upregulated p53 expression.The combina-tion of DOX and rapamycin had a synergetic effect on the expression of the above proteins.Conclusion Rapamycin can increase the sensitivity of HCC to doxorubicin chemotherapy,and the probable mechanism may be the activation of p53-dependent and-independent p21 pathway to apoptosis.