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目的:探讨FADD和Caspase-8表达缺失在肝细胞癌TRAIL耐受机制中的作用.方法:免疫组织化学方法检测肝细胞癌(hepatocellular carcinoma,HCC)组织中FADD蛋白的表达,原位杂交方法检测HCC中Caspase-8的表达,并结合临床资料进行分析.采用TRAIL联合应用亚毒性剂量的化疗药及细胞因子,观察其对于肝癌细胞株(HepG2、SMMC-7721)的胞毒作用,荧光分光光度计检测Caspase-8活性变化,Westernblot检测FADD表达的变化.结果:60例HCC中FADD阳性率,显著低于癌旁肝组织FADD阳性率(25%vs70%,P<0.01);HCC中33例Caspase-8表达阳性率,低于癌旁组织阳性率(19/20,95%).亚毒性剂量的化疗药(丝裂霉素、5-氟尿嘧啶、放线菌素D)可显著增强TRAIL的细胞毒活性,治疗后Caspase-8活性及FADD表达量显著增高.结论:HCC中FADD、Caspase-8的表达下调可能参与TRAIL耐受的机制,化疗药物可通过上调FADD的表达、增强Caspase-8活性来加强TRAIL的抗癌作用.
Objective: To investigate the role of FADD and Caspase-8 in the mechanism of TRAIL tolerance in hepatocellular carcinoma.Methods: Immunohistochemical method was used to detect the expression of FADD protein in hepatocellular carcinoma (HCC) tissues and the in situ hybridization Caspase-8 expression in HCC and clinical data were analyzed.Using TRAIL in combination with sub-toxic doses of chemotherapeutic drugs and cytokines, cytotoxicity of HepG2 and SMMC-7721 cells was observed, and fluorescence spectrophotometry The changes of Caspase-8 activity and the expression of FADD were detected by Western blotting.Results: The positive rate of FADD in 60 cases of HCC was significantly lower than that of para-cancerous liver tissue (25% vs 70%, P <0.01) The positive rate of Caspase-8 expression was lower than that of paracancerous tissue (19/20, 95%). The sub-toxic chemotherapy drugs (mitomycin, 5-fluorouracil and actinomycin D) Cytotoxic activity and the expression of Caspase-8 and FADD were significantly increased after treatment.Conclusion: The down-regulation of FADD and Caspase-8 in HCC may participate in the mechanism of TRAIL tolerance. Chemotherapy can enhance the expression of FADD and enhance the expression of Caspase-8 Activity to enhance the anti-cancer effect of TRAIL.