The essential role of the mitochondria-dependent death-signaling cascade in chemotherapy-induced potentiation of Apo2L/TRAIL cytotoxicity in cultured thoracic cancer cells: Amplified caspase 8 is indispensable for combination-mediated massive cell death
The essential role of the mitochondria-dependent death-signaling cascade in chemotherapy-induced potentiation of Apo2L/TRAIL cytotoxicity in cultured thoracic cancer cells: Amplified caspase 8 is indispensable for combination-mediated massive cell death
复制标题
DOI：
10.1097/00130404-200607000-00004
复制
发表时间：
2006-07-01
期刊：
影响因子：
2.2
通讯作者：
Schrump, David S.
中科院分区：
文献类型：
--
作者：
Nguyen, Dao M.;Yeow, Wen-Shuz;Schrump, David S.
目的尽管充分表达了肿瘤坏死因子受体凋亡诱导配体（TRAIL）的功能性受体，但许多培养的肿瘤细胞对该配体的细胞毒性作用具有抵抗力。细胞毒性化疗药物已被证明可以与 Apo2L/TRAIL 协同作用，介导癌细胞凋亡。本研究的主要目的是评估顺铂或紫杉醇这两种常用的实体瘤化疗药物在小组培养的胸部癌细胞中增强 Apo2L/TRAIL 细胞毒性的作用，并检查线粒体依赖性 caspase 激活级联在介导联合治疗细胞凋亡中的作用。 方法用顺铂/Apo2L/TRAIL 或体外紫杉醇/Apo2L/TRAIL顺序组合。通过4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法测定细胞活力和细胞凋亡。通过逆转录病毒基因转移产生表达高水平 Bcl-2 的稳定转染子。使用荧光底物通过市售试剂盒测量 caspase 3、6、8 和 9 的特异性蛋白水解活性。 结果 所有细胞系优先表达高水平的 DR4 和/或 DR5 以及低水平的 DcR1/DcR2；所有这些都没有因化疗药物治疗而改变。用亚致死浓度的顺铂或紫杉醇预处理这些癌细胞，使它们对 Apo2L/TRAIL 的敏感性增加两倍至 20 倍以上。在联合处理的细胞中观察到对细胞凋亡的显着协同诱导。原代正常细胞的活力不受 Apo2L/TRAIL 或化疗与 Apo2L/TRAIL 组合的影响。 Bcl-2 的过度表达或 caspase 9 活性的抑制完全消除了组合诱导的细胞毒性和细胞凋亡，表明线粒体依赖性死亡信号级联在此过程中的重要作用。通过 Bcl-2 过表达或通过阻断线粒体调节的 caspase 9 的活性，联合处理的细胞中 caspase 8 的强力激活被完全抑制，从而确定放大反馈环路是 caspase 8 活性升高的来源。最后，线粒体介导的 caspase 8 活性放大对于 caspase 的完全激活和细胞凋亡的完全执行是必不可少的，因为使用选择性 caspase 6 抑制剂（位于反馈环路中 caspase 3 的下游，但位于 caspase 8 的上游）抑制其活性，不仅会严重抑制 caspase 8 的活性，而且还会抑制 caspase 9 和 3 的活性，并完全保护癌细胞免受组合诱导的细胞毒性。 结论 顺铂或紫杉醇与 Apo2L/TRAIL 协同相互作用，介导细胞凋亡的深度诱导。线粒体依赖性半胱天冬酶激活级联和放大反馈回路对于细胞死亡程序的完整执行至关重要。此外，我们的数据将线粒体确定为开发更精细策略的直接目标，以增强 Apo2L/TRAIL 作为抗癌剂的治疗效果。
PURPOSE Despite adequately expressing functional receptors for tumor necrosis factor receptor apoptosis-inducing ligand (TRAIL), many cultured tumor cells are refractory to the cytotoxic effect of this ligand. Cytotoxic chemotherapeutic drugs have been shown to synergize with Apo2L/TRAIL to mediate apoptosis in cancer cells. The main goal of this study was to evaluate the effect of either cisplatin or paclitaxel, two common used chemotherapeutic agents for solid tumors, on enhancing Apo2L/TRAIL cytotoxicity in a panel-cultured thoracic cancer cells and to examine the role of the mitochondria-dependent caspase activation cascade in mediating apoptosis of combination-treated cells.METHODS Cultured thoracic cancer cells were treated with cisplatin/Apo2L/TRAIL or paclitaxel/Apo2L/TRAIL sequential combinations in vitro. Cell viability and apoptosis were determined by 4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assays. Stable transfectants expressing high levels of Bcl-2 were created by retroviral gene transfer. Specific proteolytic activity of caspases 3, 6, 8, and 9 were measured by commercially available kits using fluorescent substrates.RESULTS All cell lines preferentially expressed high levels of DR4 and/or DR5 and low levels of DcR1/DcR2; all of which were not altered by chemotherapeutic drug treatments. Pretreatment of these cancer cells with sublethal concentrations of either cisplatin or paclitaxel increased their susceptibility to Apo2L/TRAIL by twofold to > 20-fold. Profound synergistic induction of apoptosis was observed in combination-treated cells. Viability of primary normal cells was affected by neither Apo2L/TRAIL nor the combinations of chemotherapy and Apo2L/TRAIL. Overexpression of Bcl-2 or inhibition of caspase 9 activity completely abrogated combination-induced cytotoxicity and apoptosis, indicating the essential role of the mitochondria-dependent death signaling cascade in this process. Robust activation of caspase 8 in combination-treated cells was completely suppressed either by Bcl-2 overexpression or by blocking of the activity of the mitochondria-regulated caspase 9, thus identifying the amplification feedback loop as the source of elevated caspase 8 activity. Finally, mitochondria-mediated amplification of caspase 8 activity was indispensable for complete caspase activation and full execution of apoptosis, because suppression of its activity using the selective caspase 6 inhibitor (located downstream of the caspase 3 but upstream of the caspase 8 in the feedback loop) resulted in profound suppression of not only caspase 8 activity but also those of caspases 9 and 3, as well as complete protection of cancer cells from combination-induced cytotoxicity.CONCLUSION Cisplatin or paclitaxel synergistically interacts with Apo2L/TRAIL to mediate profound induction of apoptosis. The mitochondria-dependent caspase activation cascade and the amplification feedback loop are essential for the complete execution of the cell death program. Furthermore, our data identify mitochondria as the direct target for the development of more refined strategies to enhance the therapeutic effect of Apo2L/TRAIL as an anticancer agent.