Stewart John H, Tran Thai-Lan, Levi Nicole, Tsai Wilson S, Schrump David S, Nguyen Dao M
Department of Surgery, Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA.
J Surg Res. 2007 Jul;141(1):120-31. doi: 10.1016/j.jss.2007.03.048.
Cytotoxic chemotherapeutic drugs such as cisplatin (CDDP) synergistically interact with soluble Fas ligand (sFasL) to mediate profound induction of apoptosis in cancer cells, particularly those refractory to this death-inducing ligand. The goal of this study was to evaluate the roles of the mitochondria-dependent apoptotic cascade and the CDDP-generated reactive oxygen species (ROS) in mediating the supra-additive enhancement of cytotoxicity and apoptosis in combination-treated malignant pleural mesothelioma (MPM) cells. MPM cells were treated with sequential CDDP/sFasL in vitro. Cell viability and apoptosis were determined by MTT and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays. Stable transfectants expressing high levels of Bcl2 were created by retroviral gene transfer. Specific proteolytic activity of caspases 3, 8, and 9 were measured using fluorescent substrates. Pretreating MPM cells with CDDP increased their susceptibility to sFasL by 2- to more than 20-fold. Overexpression of either Bcl-2, the selective caspase 9 inhibitor z-LEHD-fmk, or the antioxidant N-acetylcysteine significantly abrogated combination-induced cytotoxicity and apoptosis. Moreover, the robust activation of caspase 8 in combination-treated cells was completely suppressed by Bcl-2 overexpression, thus implicating a mitochondria-mediated amplification feedback loop. As an in vivo correlate, sequential intraperitoneal administration of CDDP and sFasL significantly inhibited the growth of intraperitoneal MPM human xenografts in nude mice. Our data indicate that the mitochondria-dependent feedback loop of the caspase activation cascade and the generation of ROS are both essential in mediating profound cytotoxicity and apoptosis of MPM cells treated with CDDP and sFasL. This mechanistic study establishes a the translational framework for the clinical application of sequential CDDP/sFasL in the treatment of MPM.
细胞毒性化疗药物，如顺铂（CDDP），可与可溶性Fas配体（sFasL）协同作用，介导癌细胞中深刻的凋亡诱导，尤其是那些对这种死亡诱导配体难治的癌细胞。本研究的目的是评估线粒体依赖性凋亡级联反应和CDDP产生的活性氧（ROS）在介导联合治疗的恶性胸膜间皮瘤（MPM）细胞中细胞毒性和凋亡的超加成增强中的作用。MPM细胞在体外接受序贯CDDP/sFasL处理。通过MTT和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记试验测定细胞活力和凋亡。通过逆转录病毒基因转移创建表达高水平Bcl2的稳定转染子。使用荧光底物测量半胱天冬酶3、8和9的特异性蛋白水解活性。用CDDP预处理MPM细胞可使其对sFasL的敏感性提高2至20倍以上。Bcl-2、选择性半胱天冬酶9抑制剂z-LEHD-fmk或抗氧化剂N-乙酰半胱氨酸的过表达均显著消除联合诱导的细胞毒性和凋亡。此外，联合处理细胞中半胱天冬酶8的强烈激活被Bcl-2过表达完全抑制，从而暗示线粒体介导的放大反馈环。作为体内相关性，序贯腹腔注射CDDP和sFasL可显著抑制裸鼠腹腔内MPM人异种移植瘤的生长。我们的数据表明，半胱天冬酶激活级联反应的线粒体依赖性反馈环和ROS的产生在介导用CDDP和sFasL处理的MPM细胞的深刻细胞毒性和凋亡中均至关重要。这项机制研究为序贯CDDP/sFasL在MPM治疗中的临床应用建立了转化框架。
