抑制自噬对5-氟尿嘧啶治疗肝癌疗效的影响及机制研究

目的研究自噬特异性抑制剂3-甲基腺嘌呤（3-MA）对5-氟尿嘧啶（5-FU）诱导肝癌细胞系SMMC7721凋亡的影响,并初步探讨其机制。方法利用单丹（磺）酰戊二胺（MDC）染色技术,在荧光显微镜下对细胞自噬进行定性观察;以CCK8法检测3-MA抑制细胞自噬前后经5-FU诱导SMMC7721细胞的存活,凋亡以AnnexinⅤ/PI流式细胞分析法检测;以Western blot法分别检测自噬特异性蛋白LC3及凋亡蛋白caspase-3活化片段和PARP裂解片段的表达。结果 5-FU处理肝癌SMMC7721细胞48 h后,可诱导其发生自噬,细胞存活率为（60.73±2.65）%,凋亡率为（40.42±2.34）%;联合应用3-MA处理48 h后,可使肝癌SMMC7721细胞存活率明显降低（P〈0.01）,为（42.31±1.32）%,而细胞凋亡率显著增加（P〈0.01）,为（60.92±2.99）%,同时引起自噬特异性蛋白LC3-Ⅱ及凋亡蛋白caspase-3活化片段和PARP裂解片段表达增加,其灰度值比较差异均有统计学意义（均P〈0.01）。结论自噬在5-FU诱导肝癌细胞系SMMC7721凋亡过程中起保护性作用,抑制自噬可提高肝癌SMMC7721细胞对5-FU的敏感性,其可能主要通过激活caspase-3及剪切PARP来实现的。因此,自噬特异性抑制剂3-MA可能为提高肝癌对5-FU的敏感性提供新思路。

Influence of Autophagy inhibition on 5-Fluorouracil-Induced Apoptosis of Hepatocellular Carcinoma and Its Mechanism

Objective To investigate the effects and mechanism of 3-methyladenine（3-MA,an autophagy inhibitor）on the apoptosis of hepatocellular carcinoma cell line SMMC7721 induced by 5-fluorouracil（5-FU）.Methods The autophagy was observed using fluorescent microscope by monodansyicadaverin（MDC）staining.The viability of SMMC7721 cell induced by 5-FU was measured using CCK8 assay before and after autophagy inhibited by 3-MA,meanwhile the apoptosis of SMMC7721 cell was determined via Annexin Ⅴ/PI assay.The light chain 3 protein（LC3,the autophagy specific protein）and caspase-3,PARP protein were detected by Western blot.Results The autophagy of SMMC7721 cell could be induced by 5-FU after treatment for 48 h,the cell survival rate was（60.73±2.65）%,and the apoptosis rate was（40.42±2.34）%.Compared with the group of 5-FU treatment,the survival rate of SMMC7721 cell in the combination of 5-FU and 3-MA after treatment for 48 h decreased to（42.31±1.32）%（P0.01）,and the apoptosis rate increased to（60.92±2.99）%（P0.01）,meanwhile the expressions of LC3-Ⅱ,activation fragment of caspase-3,and cleavage fragment of PARP significantly increased（P0.01）.Conclusions Autophagy is a protective phenomenon during the SMMC7721 cell line apoptosis induced by 5-FU,and autophagy inhibition may enhance the sensitivity of SMMC7721 cell line to 5-FU treatment,which is probably associated with the activation of caspase-3 and cleavage of PARP.Therefore,autophagy inhibition could be a promising strategy for adjuvant chemotherapy in hepatocellular carcinoma.