Amplification of TRIAL-Indcued Apoptosis in Mesothelioma

描述（由申请人提供）：死亡受体的激活提供了绕过抗性位点并直接参与细胞凋亡的半胱天冬酶机制的可能机制。死亡受体途径还以未知的方式与其他信号合作以放大细胞凋亡。对于几种高度耐药的间皮瘤细胞系，包括M28，TNF-相关的凋亡诱导配体（TRAIL）诱导凋亡，通过与化疗剂如依托泊苷或蛋白酶体抑制剂同时治疗，凋亡大大放大。在我们的建议中，TRAIL诱导的凋亡可以被放大的机制和这些发现与间皮瘤体内模型的相关性将被探索。 首先，将研究依托泊苷放大TRAIL诱导的细胞凋亡的特定途径，特别注意线粒体的作用，通过用Bcl-XL或显性负性胱天蛋白酶9的过表达阻断线粒体途径，通过测试线粒体对死亡受体信号tBid的敏感性，以及通过分析线粒体含BH 3的蛋白质。 将在M28细胞中检查应激激活途径JNK/SAPK的可能作用，所述M28细胞用显性负性JNK 1和/或JNK 2稳定阻断或用MEKK 4刺激JNK。其次，将研究蛋白酶体抑制剂放大TRAIL诱导的细胞凋亡的机制。 这表明蛋白酶体在死亡受体的降解和表达调控中具有新的作用。 将确定蛋白酶体在DR 5降解中的作用、对DR 5的特异性以及增加的DR 5对扩增的细胞凋亡的贡献。 最后，将测试体外发现与体内间皮瘤模型的相关性。在具有M28细胞皮下肿瘤的裸鼠中，将评估全身给予的全身性TRAIL和化疗、蛋白酶体抑制剂或两者对肿瘤大小、凋亡指数和DR 5受体表达的影响。对TRAIL和/或依托泊苷、蛋白酶体抑制剂或两者的反应也将被确定。理解TRAIL诱导的细胞凋亡的放大机制可以提供对恶性细胞逃避细胞凋亡的手段和绕过这些抗性位点的方法的深入了解，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