Amplification of TRAIL-Induced Apoptosis in Mesothelioma

描述（由申请人提供）：死亡受体的激活提供了一种可能的机制，可以绕过耐药位点，直接参与凋亡的caspase机制。死亡受体通路还以未知的方式与其他信号合作，放大细胞凋亡。对于一些高度耐药的间皮瘤系，包括M28， tnf相关的凋亡诱导配体（TRAIL）诱导细胞凋亡，这种细胞凋亡通过与化疗药物（如依托泊苷）或蛋白酶体抑制剂同时治疗而大大放大。在我们的建议中，TRAIL诱导细胞凋亡的机制可以被放大，这些发现与体内间皮瘤模型的相关性将被探索。首先，依托opo苷放大trail诱导的细胞凋亡的特定途径将被研究，特别关注线粒体的作用，通过Bcl-XL或显性阴性caspase 9过表达阻断线粒体途径，通过检测线粒体对死亡受体信号tBid的敏感性，以及通过分析线粒体bh3蛋白。应激激活途径JNK/SAPK的可能作用，将在M28细胞中被主要的JNK1和/或JNK2阴性或MEKK4刺激的稳定阻断。其次，蛋白酶体抑制剂放大TRAIL诱导的细胞凋亡的机制将被研究。蛋白酶体抑制剂与化疗药物不同，会增加TRAIL受体DR5的表达。这表明蛋白酶体在死亡受体的降解和表达调控中的新作用。蛋白酶体在DR5降解中的作用、DR5的特异性以及增加的DR5对扩增的细胞凋亡的贡献将被确定。最后，将测试体外研究结果与体内间皮瘤模型的相关性，在M28细胞皮下肿瘤的裸鼠中，将评估全身TRAIL和化疗、蛋白酶体抑制剂或两者同时给予的系统性TRAIL对肿瘤大小、凋亡指数和DR5受体表达的影响。在个体患者的间皮瘤肿瘤中，作为肿瘤碎片球状体，对TRAIL和/或依托泊苷的反应，了解trail诱导的细胞凋亡扩增的机制可以帮助我们了解恶性细胞逃避细胞凋亡的途径，以及绕过这些耐药位点的方法，从而改善治疗策略

DESCRIPTION (provided by applicant): Activation of death receptors offers a possible mechanism of bypassing sites of resistance and engaging the caspase machinery of apoptosis directly Death receptor pathways also cooperate in unknown ways with other signals to amplify apoptosis. For several highly resistant mesothelioma lines, including M28, the TNF-related apoptosis inducing ligand (TRAIL) induces apoptosis, an apoptosis greatly amplified by concurrent treatment either with chemotherapeutic agents, such as etoposide, or with proteasome inhibitors In our proposal, the mechanism(s) by which TRAIL-induced apoptosis can be amplified and the relevance of these findings to in vivo models of mesothelioma will be explored. First, specific pathways by which etoposide amplifies TRAIL-induced apoptosis will be investigated, with particular attention to the role of mitochondria, by blocking mitochondrial pathways with overexpression of Bcl-XL or dominant negative caspase 9, by testing mitochondrial sensitivity to the death receptor signal tBid, and by analysis of mitochondrial BH3-containing proteins. The possible role of the stress activated pathway, JNK/SAPK, will be examined in M28 cells with stable blockade with dominant negative JNK1 and/or JNK2 or with stimulation of JNK by MEKK4 Secondly, the mechanism(s) by which proteasome inhibitors amplify TRAIL-induced apoptosis will be investigated Proteasome inhibitors, unlike chemotherapeutic agents, increase expression of the TRAIL receptor, DR5. This suggests a novel role for the proteasome in the degradation and regulation of expression of a death receptor. The role of the proteasome in DR5 degradation, the specificity for DR5 and the contribution of increased DR5 to the amplified apoptosis will be determined. Finally, the in vitro findings will be tested lot their relevance to in vivo mesothelioma models In nude mice with subcutaneous tumors of M28 cells, systemic TRAIL and either chemotherapy, proteasome inhibitors or both given systemically will be assessed for effects on tumor size, apoptotic index and expression of the DR5 receptor In human mesothelioma tumors from individual patients studied as tumor fragment spheroids, response to TRAIL and/or etoposide, proteasome inhibitors or both will also be determined Understanding the mechanisms of amplification of TRAIL-induced apoptosis can provide insight into the means by which malignant cells evade apoptosis and ways to bypass those sites of resistance and thereby improve treatment strategies