阿司匹林对溶瘤腺病毒抗肝癌细胞HepG2生长协同作用研究

Enhanced antitumor effect of oncolytic adenovirus combining with aspirin on hepatocellular carcinoma HepG2 cells in vitro

目的溶瘤腺病毒具有溶瘤性和靶向性成为肿瘤治疗的热点之一。阿司匹林具有防治消化系统肿瘤的作用,能降低肿瘤发病风险和病死率抑制肿瘤细胞生长。通过阿司匹林联合溶瘤腺病毒ZD55-TRAIL,研究阿司匹林增强溶瘤腺病毒介导的基因表达、体外抑制肝癌细胞生长和诱导细胞凋亡的效应。方法将实验分为空白对照、阿司匹林、ZD55-TRAIL和阿司匹林联合ZD55-TRAIL共4组。通过蛋白质印迹法检测病毒E1A蛋白和外源抗癌基因TRAIL表达,四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)法检测细胞的生存率变化和协同抗癌效果结晶紫染色法检测细胞病理变化,Hoeschst33342染色和流式细胞仪分别检测HepG2细胞的凋亡形态学变化和细胞凋亡率。结果阿司匹林能提高ZD55-TRAIL感染HepG2细胞后介导的病毒E1A和外源抗癌基因TRAIL的表达。与ZD55-TRAIL处理HepG2细胞组相比阿司匹林联合ZD55-TRAIL处理组E1A蛋白表达量增加(133.6±19.8)%,差异有统计学意义,F=4 346.965,P<0.001。与阿司匹林和ZD55-TRAIL组相比,联合组TRAIL蛋白表达量分别升高(287.9±23.8)%和(54.8±3.8)%,3组间差异有统计学意义,F=3 725.864,P<0.001。单独阿司匹林和溶瘤腺病毒ZD55-TRAIL均具有良好的抑制肝癌细胞HepG2增殖能力且呈剂量依赖性两者联合能更有效杀伤肝癌细胞;其中阿司匹林5 mmol/L和ZD55-TRAIL 5 MOI联合组细胞存活率为(25.6±4.1)%,单独ZD55-TRAIL 5 MOI组为(42.2±3.7)%,单独阿司匹林5 mmol/L组为(56.0±5.9)%。联合组相比2个单独组存活率均下降,3组间差异有统计学意义,F=31.676,P<0.001。运用CalcuSyn软件计算联合处理组的模拟指数值表明联合使用两种药物处理具有协同作用。结晶紫染色结果发现,单独阿司匹林和ZD55-TRAIL处理对HepG2细胞具有明显的细胞毒性效应,两者联合表现出更强的细胞杀伤效果,与MTT结果一致。进一步研究发现,相对于阿司匹林和ZD55-TRAIL单一处理组,阿司匹林联合ZD55-TRAIL能够诱导肝癌细胞形成更多的染色质浓缩、核碎裂和凋亡小体;阿司匹林与ZD55-TRAIL联合处理组细胞凋亡率为(63.7±4.8)%,单独ZD55-TRAIL组为(46.8±1.9)%,单独阿司匹林组为(15.0±1.5)%,联合组相比2个单独组细胞凋亡率均增加,4组间差异均有统计学意义(F=291.021,P<0.001),表明联合处理更能诱导肝癌细胞凋亡。结论阿司匹林能够提高溶瘤腺病毒介导的E1A和TRAIL蛋白表达,两者具有协同的肝癌细胞增殖抑制作用且联合处理能有效增强诱导肝癌细胞的死亡率为肝癌的靶向治疗提供了新的思路。

OBJECTIVE Oncolytic adenovirus has become one of the hottest fields in tumor therapy due to its oncolytic and targeted properties. Aspirin can prevent and treat tumors in the digestive system,reduce the risk and mortality,and inhibit the growth of tumor cells. The effects of growth inhibition and induction of apoptosis were investigated by oncolytic adenovirus ZD55-TRAIL combined with aspirin in hepatocellular carcinoma( HCC). METHODS The experiment was divided as four groups including mock, aspirin, ZD55-TRAIL, and the combination of aspirin and ZD55-TRAIL. The expression of virus E1A protein and exogenous anti-cancer gene TRAIL was detected by Western Blot. The cell survival rate and synergistic anti-cancer effect were detected by MTT assay. The cytopathologic change was detected by crystal violet staining. RESULTS Aspirin can improve the expression of the virus E1A and the exogenous anticancer gene TRAIL in HCC HepG2 cells infected with ZD55-TRAIL. As compared with ZD55-TRAIL, the expression level of E1A in HepG2 cells treated with aspirin plus ZD55-TRAIL was increased by (133. 6±19. 8)%,and ANOVA analysis showed that the difference between the two groups was statistically significant (F=4 346. 965,P<0.001);in addition, as compared with aspirin and ZD55-TRAIL alone, the expression level of TRAIL in combination group was up-regulated by ( 287. 9± 23. 8)%,and (54. 8±3. 8)%,respectively. ANOVA analysis showed the difference between the two groups was statistically significant (F= 3 725. 864,PV0. 001). ZD55-TRAIL alone or aspirin alone has good inhibition ability in HepG2 cell proliferation with dose-dependent manner, while their combination can more effectively kill HCC cells. Among them, as compared with the cell viability rate of HepG2 cells treated with 5 MOI ZD55-TRAIL (42. 2±3. 7)% alone and 5 mmol/L aspirin alone (56. 0±5. 9)%, the cell viability rate (25. 6土 4. 1)% in their combination group was decreased. Further ANOVA analysis showed that the difference among three groups was significant when treated by aspirin 5 mmol/L and ZD55-TRAIL 5 MOI(F=31. 676,P = 0. 001). CalcuSyn software was used to calculate the analog index value of the combined treatment group, indicating that the combined treatment has synergistic effect. The crystal violet staining showed that single aspirin and ZD55-TRAIL treatment had obvious cytotoxic effect on HepG2 cells, and the combination showed stronger cell killing effect,which was consistent with MTT results. Further studies found that compared with the single treatment,the combination treatment can induce HCC cells to form more chromatin concentration,nuclear fragmentation and apoptotic bodies. As compared with ZD55-TRAIL alone (46.8±1. 9)% and aspirin alone (15.0±1.5)%,the cell apoptotic rate of their combination treatment ( 63. 7±4. 8)% was more effectively increased;Further ANOVA analysis showed the difference among four groups was significant (F=291. 021,P<0. 001). The results showed that the combination treatment could induce more HCC cell apoptosis. CONCLUSIONS Aspirin can improve the El A and TRAIL expression in HepG2 cells treated by oncolytic adenovirus, which has synergistic inhibitory effect on the proliferation of HCC cells. The combination treatment can effectively enhance the death rate of HCC cells, which provides a new strategy for the targeted therapy of liver cancer.