Investigation of molecular mechanisms of cell death induced by combination treatment with TRAIL and chemotherapy in malignant gliomas

我们研究了联合应用TRAIL和DNA损伤性化疗药物诱导脑胶质瘤细胞死亡信号的分子途径。FADD在胶质瘤细胞中表达，显性阴性的FADD的表达显著抑制了联合治疗的协同细胞毒作用。与死亡受体形成盘状结构的启动子caspase-8被切割并被联合处理激活。Caspase-3的重要效应因子caspase-3也被切割并激活，导致caspase-3的内源底物PARP被切割，这表明联合处理诱导的细胞死亡是通过FADD直接激活caspase途径介导的。细胞色素c和凋亡诱导因子(AIF)在联合作用下的BID裂解和释放进入细胞质，提示线粒体的凋亡途径在其协同细胞毒作用中起重要作用。T…的组合更多的RAIL和X射线放射治疗(XRT)在人脑胶质瘤细胞中表现出相似的增强细胞毒性。10GyXRT可上调DR5的表达，TRAIL中和DR5-Fc的细胞毒作用可被消除，提示DR5的诱导可能在这一作用中起重要作用。与化疗相似，XRT/TRAIL联合治疗可激活FADD/caspase-8介导的caspase级联直接激活和线粒体凋亡通路。野生型p53的U87 MG细胞对TRAIL/XRT耐药。引入显性阴性的p53，p53DD，并没有使U87 MG细胞对联合治疗敏感，这表明野生型p53的功能不是增强细胞毒性所必需的。TRAIL/XRT联合处理不影响正常人脑星形胶质细胞的活性。TRAIL/化疗和TRAIL/XRT联合用药对胶质瘤细胞的协同细胞毒作用与转录因子NF-kB无关。我们的结果表明，TRAIL可以有效地与DNA损伤化疗和XRT这两种人类恶性胶质瘤的常规治疗方案相结合，从而为这种联合治疗可能成为治疗难治性恶性胶质瘤的潜在新疗法提供证据。较少

We investigated the molecular pathways involved in death signals induced by combination treatment with TRAIL and DNA damaging chemotherapeutic agents in glioma cells. FADD was expressed in glioma cells and expression of a dominat-negtive form of FADD significantly suppressed the synergistic cytotoxicity of the combination treatment. An initiator caspase, caspase-8, which forms DISC with death receptors, was cleaved and activated by the combination treatment. An essential effector caspase, caspase-3, was also cleaved and activated, resulting in cleavage of PARP, an intrinsic substrate of caspase-3, suggesting that cell death induced by the combination treatment is mediated by direct caspase activation pathways through FADD. Bid cleavage and release of cytochrome c and apoptosis-inducing factor (AIF) into cytoplasm upon the combination treatment were also observed, suggesting that the mitochondrial apoptosis pathways play an important role in its synergistic cytotoxiciy. Combination of T … More RAIL and X-ray radiotherapy (XRT) exhibited similar enhanced cytotoxicity in human glioma cells. XRT at 10 Gy upregulated DR5 expression and the cytotoxicity was abrogated in the presence of TRAIL neutralizing DR5-Fc, indicating that DR5 induction may be important in this effect. Similar to chemotherapy, XRT/TRAIL combination treatment activated FADD/caspase-8 mediated direct activation of caspase cascade and mitochondrial apoptosis pathways as well. U87MG cells which is wild type for p53 showed resistance to TRAIL/XRT therapy. Introduction of a dominant-negative p53, p53DD, did not sensitize U87MG cells to the combination treatment, suggesting that wild-type p53 function is not required for the enhanced cytotoxicity. TRAIL/XRT combination treatment did not affect viability of normal human astrocytes. Function of a transcription factor NF-kB was not involved in the synergistic cytotoxic effects of both TRAIL/chemotherapy and TRAIL/XRT combination in glioma cells. Our results suggest that TRAIL could be effectively combined with both DNA damaging chemotherapy and XRT, conventional therapeutic modalities of human malignant gliomas, and thus provide evidence that such combination treatment could be potential novel therapeutics against intractable malignant gliomas. Less