TRAIL-induced apoptosis in prostate cancer

TRAIL 诱导前列腺癌细胞凋亡

• 批准号: 6863702
• 负责人: CHRISTINA VOELKEL-JOHNSON
• 金额: $ 23万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6863702
• 关键词: adeno associated virus group; apoptosis; athymic mouse; cell cycle; cell population study; combination cancer therapy; confocal scanning microscopy; cysteine endopeptidases; doxorubicin; enzyme inhibitors; flow cytometry; gene delivery system; gene expression; gene therapy; genetic regulation; genetically modified animals; immunoprecipitation; prostate neoplasms; protein localization; protein protein interaction; transfection /expression vector; tumor necrosis factor alpha; ubiquitin; western blottings

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.

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。目前尚无针对晚期疾病的治愈方法，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）被认为是新型抗肿瘤药物，但前列腺癌细胞对可溶性 TRAIL 相对耐药。通过下调抗凋亡蛋白 c-FLIP 的亚毒剂量阿霉素或表达膜 TRAIL 的腺病毒可以克服耐药性。这两种方法都对恶性细胞没有选择性，并且需要将阿霉素或 TRAIL 限制于特定的目标群体。我们假设阿霉素与来自前列腺特异性启动子 Probasin 的 TRAIL 的组织特异性表达相结合将是针对前列腺癌的有效治疗方法。为了支持这一假设并确定前列腺细胞中 TRAIL 耐药的潜在机制，将研究以下具体目标：（1）检验阿霉素介导的 c-FLIP 下调机制涉及细胞周期依赖性泛素化的假设。将分析分选为 G0/G1、S 和 G2/M 群体的细胞的 c-FLIP、泛素化和 TRAIL 敏感性水平。 (2) 检验以下假设：表达 TRAIL 的腺病毒 (AdTRAIL) 通过允许配体和激动性 TRAIL 受体在细胞内相互作用，导致细胞内死亡诱导信号复合物的形成来克服耐药性。 c-FLIP 在提供 AdTRAIL 抗性方面的作用将在稳定过表达该蛋白长亚型和短亚型的 PC3 细胞中进行研究。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。蛋白质相互作用也可通过免疫沉淀/免疫印迹测定。 (3) 检验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 的特异性有效性将在具有功能性雄激素受体的细胞和非前列腺细胞中进行测试。将在体外测定病毒单独或与阿霉素组合的凋亡潜力。 AdARR2PBTRAIL 单独使用或与阿霉素联合使用的毒性和有效性也将在无胸腺小鼠的皮下异种移植物中进行测试。