Ng Chuen-Pei, Zisman Amnon, Bonavida Benjamin
Department of Microbiology, Immunology, and Molecular Genetics, UCLA School of Medicine and Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California 90095-1747, USA.
Prostate. 2002 Dec 1;53(4):286-99. doi: 10.1002/pros.10155.
Tumors have an inherent immunogenicity that can be exploited by immunotherapy. However, often tumors develop mechanisms that render them resistant to most immunologic cytotoxic effector mechanisms. This study examines the underlying mechanism of resistance to Apo2L/TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-mediated apoptosis.
We studied prostate tumor cell lines for their sensitivity to Apo2L/TRAIL-mediated apoptosis in the presence and absence of the sensitizing agent actinomycin D (Act D). Apoptosis was determined by flow cytometry and signaling for apoptosis by Western blot.
Treatment with subtoxic concentrations of Act D significantly sensitizes the tumor cells (CL-1, DU-145, and PC-3 prostate tumor cells) to Apo2L/TRAIL-mediated apoptosis. The cytotoxicity of Act D-sensitized prostate tumor cells was a result of synergistic activation of caspases (caspase-3, -9, and -8), detectable after 6 hr of treatment. Treatment with Apo2L/TRAIL alone, although it was insufficient to induce apoptosis, resulted in the loss of mitochondrial membrane potential and release of cytochrome c from the mitochondria into the cytoplasm in the absence of significant caspases activation. These findings suggested that a major apoptosis resistance factor blocking the Apo2L/TRAIL apoptotic signaling events is present downstream of the mitochondrial activation. The expression of receptors and anti-apoptotic proteins were examined in Act D-sensitized CL-1 cells. The earliest and the most pronounced change induced by Act D was down-regulation of X-linked inhibitor of apoptosis (XIAP) and up-regulation of Bcl-xL/-xS proteins. The role of XIAP in resistance was demonstrated by overexpression of Smac/DIABLO, which inhibited inhibitors of apoptosis (IAPs) and sensitized the cells to Apo2L/TRAIL. Apo2L/TRAIL receptors (DR4, DR5, DcR1, and DcR2), c-FLIP, Bcl-2, and other IAP members (c-IAP1 and c-IAP2) were marginally affected at later times in the cells sensitized by Act D.
This study suggests that the combination of Act D-induced down-regulation of XIAP (Signal I) and Apo2L/TRAIL-induced release of cytochrome c (Signal II) leads to the reversal of resistance to Apo2L/TRAIL-mediated apoptosis in the tumor cells. The sensitization of tumor cells to Apo2L/TRAIL by Act D is of potential clinical application in the immunotherapy of drug/Apo2L/TRAIL refractory tumors.
肿瘤具有内在免疫原性，可被免疫疗法利用。然而，肿瘤常常会形成使其对大多数免疫细胞毒性效应机制产生抗性的机制。本研究探讨了对Apo2L/TRAIL（肿瘤坏死因子相关凋亡诱导配体）介导的凋亡产生抗性的潜在机制。
我们研究了前列腺肿瘤细胞系在存在和不存在致敏剂放线菌素D（Act D）的情况下对Apo2L/TRAIL介导的凋亡的敏感性。通过流式细胞术测定凋亡，并通过蛋白质印迹法检测凋亡信号。
用亚毒性浓度的Act D处理可显著使肿瘤细胞（CL-1、DU-145和PC-3前列腺肿瘤细胞）对Apo2L/TRAIL介导的凋亡敏感。Act D致敏的前列腺肿瘤细胞的细胞毒性是半胱天冬酶（半胱天冬酶-3、-9和-8）协同激活的结果，在处理6小时后可检测到。单独用Apo2L/TRAIL处理，尽管不足以诱导凋亡，但在没有明显的半胱天冬酶激活的情况下，导致线粒体膜电位丧失和细胞色素c从线粒体释放到细胞质中。这些发现表明，阻断Apo2L/TRAIL凋亡信号事件的主要凋亡抗性因子存在于线粒体激活的下游。在Act D致敏的CL-1细胞中检测了受体和抗凋亡蛋白的表达。Act D诱导的最早且最明显的变化是X连锁凋亡抑制蛋白（XIAP）的下调和Bcl-xL/-xS蛋白的上调。Smac/DIABLO的过表达证明了XIAP在抗性中的作用，其抑制凋亡抑制因子（IAPs）并使细胞对Apo2L/TRAIL敏感。在Act D致敏的细胞中，Apo2L/TRAIL受体（DR4、DR5、DcR1和DcR2）、c-FLIP、Bcl-2和其他IAP成员（c-IAP1和c-IAP2）在后期受到的影响较小。
本研究表明，Act D诱导的XIAP下调（信号I）和Apo2L/TRAIL诱导的细胞色素c释放（信号II）相结合，导致肿瘤细胞对Apo2L/TRAIL介导的凋亡的抗性逆转。Act D使肿瘤细胞对Apo2L/TRAIL敏感，在药物/Apo2L/TRAIL难治性肿瘤的免疫治疗中具有潜在的临床应用价值。
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