TRAIL-induced apoptosis in prostate cancer

描述(申请人提供)：前列腺癌是美国男性的一个重要健康问题。目前还没有治疗晚期疾病的根治方法，需要新的治疗方法。重组形式的TRAIL(肿瘤坏死因子相关凋亡诱导因子)被认为是一种新型的抗肿瘤药物，但前列腺癌细胞对可溶性TRAIL相对耐药。抗药性可以通过亚毒性剂量的阿霉素(它下调抗凋亡蛋白c-flip)或通过表达膜TRAIL的腺病毒来克服。这两种方法对恶性细胞都不是选择性的，需要限制阿霉素或TRAIL到特定的靶群。我们的假设是，联合使用阿霉素和前列腺特异性启动子前池中组织特异性表达的TRAIL将是治疗前列腺癌的有效方法。为了支持这一假说并确定前列腺细胞TRAIL耐药的潜在机制，我们将研究以下具体目标：(1)验证阿霉素介导的c-FLIP下调机制涉及细胞周期依赖泛素化的假说。分类为G0/G1、S和G2/M群体的细胞将被分析c-Flip、泛素化和TRAIL敏感性水平。(2)验证TRAIL表达型腺病毒(AdTRAIL)通过细胞内配体与激动型TRAIL受体相互作用，形成细胞内死亡诱导信号复合体，从而克服耐药性的假说。C-FLIP在提供对AdTRAIL的抗性中的作用将在稳定高表达该蛋白的长、短异构体的PC3细胞中进行研究。TRAIL受体在细胞内的定位以及与配体、caspase和c-flip的相互作用将通过共聚焦显微镜进行分析。蛋白质的相互作用也通过免疫沉淀/免疫印迹来确定。(3)验证化疗与前列腺特异性基因治疗相结合是治疗前列腺癌的有效方法的假设。来自前列腺特异性启动子ARR2PB的腺病毒表达TRAIL的特异性的有效性将在具有功能性雄激素受体的细胞和非前列腺癌细胞中进行测试。病毒单独或与阿霉素联合使用的凋亡潜力将在体外确定。AdARR2PBTRAIL单独或与阿霉素联合使用的毒性和有效性也将在裸鼠皮下移植中进行测试。

DESCRIPTION (provided by applicant): Prostate cancer is a significant health problem among American men. Curative treatments for advanced disease are currently not available and novel treatment approaches are needed. Recombinant forms of TRAIL (tumor necrosis factor related apoptosis inducing factor) have been considered as novel anti-tumor agents but prostate cancer cells are relative resistant to soluble TRAIL. Resistance is overcome by sub-toxic doses of doxorubicin which downregulate the anti-apoptotic protein c-FLIP or by an adenovirus expressing membrane TRAIL. Neither approach is selective for malignant cells and will require restriction of either doxorubicin or TRAIL to a specific target population. It is our hypothesis that combination of doxorubicin with tissue-specific expression of TRAIL from the prostate-specific promotor probasin will be an effective therapeutic approach against prostate cancer. To support this hypothesis and identify underlying mechanisms of TRAIL resistance in prostate cells, the following specific aims will be investigated: (1) To test the hypothesis that the mechanism of doxorubicin-mediated downregulation of c-FLIP involves cell cycle dependent ubiquitination. Cells that are sorted into G0/G1, S and G2/M populations will be analyzed for levels of c-FLIP, ubiquitination, and TRAIL susceptibility. (2) To test the hypothesis that the TRAIL expressing adenovirus (AdTRAIL) overcomes resistance by permitting intracellular interaction of ligand and agonistic TRAIL receptors resulting in formation of an intracellular death inducing signaling complex. The role of c-FLIP in providing resistance to AdTRAIL will be investigated in PC3 cells stably overexpressing long and short isoforms of the protein. Intracellular location of TRAIL receptors and interaction with ligand, caspases and c-FLIP will be analyzed by confocal microscopy. Protein interactions are also determined by immunoprecipitation/immunoblotting. (3) To test the hypothesis that combination of chemotherapy and prostate-specific gene therapy is an effective approach against prostate cancer. The effectiveness of specificity of an adenovirus-expressing TRAIL from the prostate-specific promotor ARR2PB will be tested in cells with a functional androgen receptor and non-prostate cells. The apoptotic potential of virus alone or in combination with doxorubicin will be determined in vitro. The toxicity and effectiveness of AdARR2PBTRAIL alone or in combination with doxorubicin will also be tested in subcutaneous xenografts in athymic mice.