FADD,Caspase-8的表达缺失在肝细胞癌TRAIL耐受机制中的作用

Influence of loss of FADD and Caspase8 on TRAIL resistance in hepatocellular carcinoma

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.

AIM： To investigate the influence of loss of FADD and Caspase-8 on TRAIL resistance in hepatocellular carcinoma （HCC）.METHODS： Immunohistochemistry method was used to detect FADD expression in HCC, and in situ hybridization was employed to determine Caspase-8 expression. The effects of TRAIL in combination with chemotherapeutic agents or anticancer cytokines on promoting apoptosis in the HCC cell lines were analyzed, the Caspase-8 activity was detected by Caspase-8 Fluorescent Assay Kit and FADD expression was detected by Western blot before and after treatment. RESULTS： Fifteen out of 60 HCC cases were found to express FADD protein. The positive rate was significantly lower in HCC than in non-cancerous adjacent liver tissues （25% vs 70%, P 〈 0.01）. Caspase-8 positive rate （19/20） was higher in normal liver tissues than in HCC （33/60） （P 〈 0.01）. Chemotherapeutic agents （mitomycin, 5-FU or actinomycin D） dramatically augmented TRAIL-induced apoptosis in HCC cell lines by increasing FADD and Caspase-8 expression. CONCLUSION： The down-regulated expression of FADD and loss of Caspase-8 expression might play important roles in resistance to TRAIL-induced apoptosis.