腺病毒介导肝癌抑制基因-1联合5-氟尿嘧啶增强结直肠癌细胞杀伤作用及机制研究

目的 通过化学治疗药物5-氟尿嘧啶(5-FU)联合重组腺病毒(Ad)肝癌抑制基因-1(HCCS1)感染结直肠癌细胞,观察两者对结直肠癌细胞的联合杀伤作用.并初步探讨相关分子机制.方法 RT-PCR和Western印迹法检测Ad-HCCS1感染结直肠癌LoVo细胞后HCCS1的表达.活细胞计数试剂盒(CCK)-8法测定Ad-HCCS1联合5-FU处理时的细胞存活率.流式细胞仪检测各种处理后细胞凋亡的情况.Western印迹法检测各种处理后相关凋亡蛋白的变化.结果 ①Ad-HCCS1可成功介导HCCS1基因在LoVo细胞中的表达.②5-FU+Ad-HCCS1联合作用96 h时,LoVo细胞存活率为(11.23±4.61)%,与对照组比较差异有统计学意义[(92.23±3.77)%,P<0.01].③处理72 h后流式细胞仪分析结果 显示,5-FU+Ad-HCCS1联合组细胞凋亡率为(27.57±1.78)%,高于单用5-Fu组[(8.64±0.94)%]、单用Ad-HCCS1组[(13.19±1.32)%]和5-Fu+空载病毒组[(12.16±1.28)%],差异均有统计学意义(P值均<0.01).④在Ad-HCCS1感染LoVo细胞高表达HCCS1时,胞质蛋白中检测到组织蛋白酶(cathepsin)D;当5-FU加入后,胞质中的抗凋亡蛋白酶前体(procaspase)-8有所减少,而活性BH3结构域凋亡诱导蛋白(Bid)明显增加.在5-FU+Ad-HCCS1处理组,胞质中的Bax水平降低最为明显,而细胞色素C和活性凋亡蛋白酶(caspase)-3表达最多.结论 在5-FU基础上联合Ad-HCCS1可显著增强LoVo细胞的生长抑制和凋亡诱导,两种处理的联合作用点为Bax蛋白,为结直肠癌治疗提供了一种新策略.

Enhanced inhibitory effect and mechanisms of adenovirus-mediated hepatocellular carcinoma supressor gene 1 combined with 5-FU on growth of LoVo cells

Objective To observe the enhanced inhibitory effect of adenovirus (Ad)-mediated HCCS1 combined with 5-FU on the growth of LoVo cells, and to explore the molecular mechanisms.Methods RT-PCR and Western blot were used to detect the expression of HCCS1 in LoVo cells infected with Ad HCCS1. CCK-8 assay was applied to observe different inhibitory effects of different treatments on growth of LoVo cells. The apoptotic rates were detected by using flow cytometry. The apoptotic proteins were detected by using Western blot. Results ① The recombinant adenovirus, Ad HCCS1, could trigger the expression of HCCS1 in LoVo cell. ② In comparison with controls (92.23%±3.77%), the cell viability rate of LoVo was only (11.23±4.61 )% on 96 h after the combination treatment of 5-FU and Ad-HCCS1 (P<0. 01). ③ The apoptotic rate was (27.57±1.78)% on 72 h after the combination treatment, which was higher than that in 5-FU treated cells (8.64±0.94)%, Ad-HCCS1 treated cells (13.19±1.32)% and 5-FU Ad treated cells (12.16±1.28)%, (P<0. 01). ④ Cathepsin D was only detected in Ad HCCS1-infected cells. When treated with 5-FU, the procaspase-8 was decreased and the cleaved Bid was increased in cytosol. The lowest level of Bax and the highest level of cytoso C and cleaved caspase-3 were detected in cytosols of 5-FU+Ad HCCS1 treated cells. Conclusion The inhibitory and proapoptotic effects are significantly enhanced in LoVo cells when treated with Ad-HCCS1+5-FU. The key protein of the cross-talk is Bax and these data provided a new strategy to treat colorectal carcinomas.