TRAIL-induced apoptosis in prostate cancer

描述（由申请人提供）：前列腺癌是美国男性的一个重大健康问题。晚期疾病的治愈性治疗目前不可用，需要新的治疗方法。重组形式的肿瘤坏死因子相关凋亡诱导因子（tumor necrosis factor related apoptosis inducing factor，TRAIL）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相对耐受。通过亚毒性剂量的多柔比星（其下调抗凋亡蛋白c-FLIP）或通过表达膜TRAIL的腺病毒克服抗性。这两种方法对恶性细胞都没有选择性，并且需要将多柔比星或TRAIL限制于特定的靶群体。我们的假设是，阿霉素与前列腺特异性启动子probasin的组织特异性表达TRAIL的组合将是一种有效的治疗前列腺癌的方法。为了支持这一假设并确定前列腺细胞中TRAIL抗性的潜在机制，将研究以下具体目标：（1）为了检验多柔比星介导的c-FLIP下调的机制涉及细胞周期依赖性泛素化的假设。将分析分选为G 0/G1、S和G2/M群的细胞的c-FLIP、泛素化和TRAIL易感性水平。(2)为了检验表达TRAIL的腺病毒（AdTRAIL）通过允许配体和激动性TRAIL受体的细胞内相互作用导致细胞内死亡诱导信号复合物的形成而克服抗性的假设。将在稳定过表达蛋白质的长和短同种型的PC 3细胞中研究c-FLIP在提供对AdTRAIL的抗性中的作用。将通过共聚焦显微镜分析TRAIL受体的细胞内定位以及与配体、半胱天冬酶和c-FLIP的相互作用。蛋白质相互作用也通过免疫沉淀/免疫印迹测定。(3)验证化疗联合前列腺特异性基因治疗是治疗前列腺癌的有效方法的假设。将在具有功能性雄激素受体的细胞和非前列腺细胞中测试来自前列腺特异性启动子ARR 2 PB的腺病毒表达TRAIL的特异性的有效性。将在体外测定单独的病毒或与阿霉素组合的病毒的凋亡潜力。AdARR 2 PBTRAIL单独或与多柔比星组合的毒性和有效性也将在无胸腺小鼠的皮下异种移植物中测试。

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.