Reduced pHi Inhibits Heat-Induced HSP,Enhances Apoptosis

降低 pHi 抑制热诱导的 HSP，增强细胞凋亡

• 批准号: 6812676
• 负责人: DENNIS B. LEEPER
• 金额: $ 30.56万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6812676
• 关键词: Reduced; pHi; Inhibits; Heat; Induced

DESCRIPTION (provided by applicant): This Supplemental Project (former Project 3) complements the other projects in the funded Program Project grant and serves the Program by testing the hypothesis that acute acidification during hyperthermia will downregulate induction of heat shock proteins, will enhance apoptosis and will sensitize melanoma cells grown at low pH to hyperthermia. The goals of this project remain unchanged. In vitro growth of human melanoma cells at pH 6.7, characteristic of the acidic microenvironment of tumors, results in overexpression of heat shock proteins (HSP) at 37xC and resistance to 42xC. HSP70, 27, and 90 regulate apoptosis by preventing apoptotic signaling. In contrast, acute acidification down regulates thermal induction of HSP and, therefore, is a strategy to increase apoptosis and increase effectiveness of cytotoxic agents such as hyperthermia. Specific aims designed to test this hypothesis will validate concepts and define parameters predicting response in cultured cells and tumors. The specific aims are: 1) Characterize molecular mechanisms whereby acute acidification downregulates induction of HSP by 42xC in melanoma cells grown at low pH; 2) Quantify the ability of acute acidification combined with heat and certain other modalities to increase apoptosis linked to downregulation of heat shock proteins; 3) Determine the effective concentrations of CNCn (a-cyano-4-hydroxy-cinnamic acid), an inhibitor of MCT (H+-linked monocarboxylate transport) and MIBG (meta-iodobenzylguanidine), an inhibitor of respiration, which, in combination with a mild acute extracellular acidification, reduce pHi below 6.5, inhibit induction of HSP, enhance apoptosis and enhance cytotoxicity in heated melanoma cells grown at low pH; and 4) Establish predictors of apoptosis and tumor growth delay in melanomas treated with acidification plus hyperthermia in vivo. Clones of DB-1 human melanoma cells transfected with the green fluorescence protein reporter gene under control of the hsp70B promoter will be selected and characterized in vitro. Xenografts grown from these clones will be treated and analyzed to evaluate our hypothesis in vivo. HSP expression and apoptotic index will be determined in cells or histologic sections of treated DB-1 xenografts as determinants of growth delay in vivo. The strategy to enhance apoptosis in tumor cells in a chronic low pH environment by selective downregulation of HSP has the potential to increase the response of human tumors to multimodal regimens involving thermal therapy, such as thermoradiotherapy.

描述（由申请人提供）：该补充项目（前项目 3）补充了受资助的计划项目拨款中的其他项目，并通过测试以下假设来为该计划服务：高温期间的急性酸化将下调热休克蛋白的诱导，将增强细胞凋亡和会使在低 pH 值下生长的黑色素瘤细胞对高温敏感。该项目的目标保持不变。人类黑色素瘤细胞在 pH 6.7（肿瘤酸性微环境的特征）下进行体外生长，导致热休克蛋白 (HSP) 在 37xC 下过度表达并耐受 42xC。 HSP70、27 和 90 通过阻止细胞凋亡信号传导来调节细胞凋亡。相反，急性酸化下调HSP的热诱导，因此是增加细胞凋亡和提高细胞毒剂（例如热疗）有效性的策略。旨在检验这一假设的具体目标将验证概念并定义预测培养细胞和肿瘤反应的参数。 具体目标是： 1) 表征在低 pH 条件下生长的黑色素瘤细胞中，急性酸化通过 42xC 下调 HSP 诱导的分子机制； 2) 量化急性酸化与热和某些其他方式相结合增加与热休克蛋白下调相关的细胞凋亡的能力； 3) 确定 CNCn（a-氰基-4-羟基肉桂酸）、MCT（H+-连接单羧酸转运）抑制剂和 MIBG（间碘苄基胍）、呼吸抑制剂的有效浓度，其与轻度急性细胞外酸化，将 pHi 降低至 6.5 以下，抑制 HSP 的诱导，增强细胞凋亡并增强在低 pH 条件下生长的加热黑色素瘤细胞的细胞毒性； 4) 在体内用酸化加高温治疗的黑色素瘤中建立细胞凋亡和肿瘤生长延迟的预测因子。将在体外选择并表征用 hsp70B 启动子控制下的绿色荧光蛋白报告基因转染的 DB-1 人黑色素瘤细胞的克隆。对这些克隆生长的异种移植物进行处理和分析，以在体内评估我们的假设。将在处理的 DB-1 异种移植物的细胞或组织学切片中测定 HSP 表达和细胞凋亡指数，作为体内生长延迟的决定因素。通过选择性下调 HSP 来增强慢性低 pH 环境中肿瘤细胞凋亡的策略有可能增加人类肿瘤对涉及热疗法（例如热放射疗法）的多模式治疗方案的反应。