白细胞介素12通过抑制survivin表达加强肿瘤坏死因子相关凋亡诱导配体诱导的肝癌细胞凋亡

目的 探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)对肝细胞癌(HCC)的治疗作用,以及TRAIL耐药的可能机制和逆转耐药方案。方法 采用原位杂交方法观察HCC与正常肝组织中TRAIL的表达差异。采用不同浓度的sTRAIL(可溶性)处理HCC细胞株及真核表达质粒pIRES-EGFP-sTRAIL转染HCC细胞株,观察sTRAIL的抑癌疗效。建立裸鼠肝癌模型,观察sTRAIL的体内抑癌作用。进一步,检测HCC中survivin的表达并采用反义寡核苷酸封闭治疗。最后,观测sTRAIL和IL-12联合抗癌效果。结果 肝癌组织DR表达量显著强于正常肝组织DR表达量。60例肝癌组织中54例不表达诱捕受体DcRl,25例不表达DcR2,而20例正常肝组织均表达DcR。两种．HCC细胞株中DcR1表达缺失。经sTRAIL(100ng/ml)处理24h,HCC细胞凋亡发生率约10％,而Jurkat细胞凋亡率达70％以上。体外pIRES-EGFP-sTRAIL转染对肝癌细胞杀伤作用不敏感。体内直接瘤体注射pIRES-EGFP-sTRAIL可对裸鼠肝癌无明显抑制作用。HCC高表达survivin,反义寡核苷酸封闭可部分逆转TRAIL耐药。IL-12使survivin表达明显下调,显著加强TRAIL对HCC细胞的杀伤作用。结论 HCC对TRAIL诱导的凋亡有耐药现象。survivin参与HCC对TRAIL的耐药机制,反义寡核苷酸封闭可部分逆转TRAIL耐药。IL-12可通过抑制siurvivin表达增强TRAIL对HCC杀癌作用。联合基因治疗(如TRAIL和IL-12基因)可能成为一种有前途HCC治疗方案。

Interleukin-12 enhanced tumor necrosis factor related apoptosis-inducing ligand TRAIL-induced apoptosis in human hepatocellular carcinoma by inhibiting expression of survivin

OBJECTIVE: To investigate therapeutic potential of TRAIL in hepatocellular carcinoma (HCC) and the mechanism of sTRAIL resistance and to reverse the resistance to sTRAIL-inducing apoptosis. METHODS: The expression profiles of TRAILR were determined 60 HCC samples, in 20 normal liver tissues and 2 HCC cell lines HepG2 and SMMC-7721 by in situ hybridization. Cellular effects of sTRAIL in promoting apoptosis on HCC cell lines HepG2 and SMMC-7721 were analyzed after exposure to recombinant protein and after transfection with a cDNA expression construct. In vivo effects of sTRAIL on tumor growth were investigated using a nude mice HCC model of hepG2. Furthermore, the expression of survivin in HCC was detected, and treatment with antisence oligonucleotide was accepted. Finally, therapeutic effect on HCC by combining sTRAIL and interleukin-12 (IL-12) was detected. RESULTS: Both DR4 and DR5 were present in all HCC tissues as well as normal hepatic tissues. In contrast, 54 HCC tissues did not express DcR1 and 25 did not express DcR2. But both DcR were detectable in all of the normal liver tissues. The expression patterns of DR and DcR in HCC samples were quite different from those in normal tissue. DR5, DR4, and DcR2 expressed in both cell lines, while no DcR1 expression was detected. Recombinant sTRAIL alone was found to have a slight activity as it killed a maximum of 15% of HCC cells within 24 h while killing over 70% of Jurkat cells. In vivo administration of the TRAIL gene couldn't inhibit tumor growth in a nude mice HCC model. Mostly, HCC tissue and both HCC cell lines expressed survivin, whereas normal liver tissue did not express survivin. Treatment with antisence oligonucleotide enhanced sTRAIL-inducing apoptosis. IL-12 significantly augmented sTRAIL-inducing apoptosis and inhibited survivin expression. CONCLUSIONS: HCC cells are insensitive towards TRAIL-mediated apoptosis. Survivin may play a role in resistance to TRAIL-induced apoptosis in HCC, and antisence oligonucleotide could partly reverse the resistance to TRAIL-inducing apoptosis. IL-12 may sensitize HCC cells to TRAIL-induced apoptosis by preventing survivin. Combining gene therapy strategy such as combining gene therapy of TRAIL with IL-12 may be a promising maneuver to HCC.