Reduced pHi Inhibits Heat-Induced HSP,Enhances Apoptosis

描述（由申请人提供）：该补充项目（原项目3）补充了资助的计划项目补助金中的其他项目，并通过测试高温期间急性酸化将下调热休克蛋白的诱导，将增强细胞凋亡并使生长在低pH值下的黑色素瘤细胞对高温敏感的假设来服务于该计划。该项目的目标保持不变。人黑色素瘤细胞在pH 6.7的体外生长，肿瘤的酸性微环境的特征，导致热休克蛋白（HSP）在37 ℃和耐42 ℃的过度表达。HSP 70、27和90通过阻止凋亡信号传导来调节凋亡。相反，急性酸化下调HSP的热诱导，因此是增加细胞凋亡和增加细胞毒性剂如高热的有效性的策略。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下生长的黑色素瘤细胞中急性酸化下调42 xC对HSP的诱导的分子机制; 2）定量急性酸化与热和某些其他方式组合以增加与热休克蛋白下调相关的细胞凋亡的能力; 3）确定CNCn的有效浓度（a-氰基-4-羟基-肉桂酸），MCT抑制剂（H+连接的单羧酸转运）和MIBG（间碘苄基胍），一种呼吸抑制剂，与轻度急性细胞外酸化结合，使pHi降低至6.5以下，抑制HSP诱导，增强在低pH下生长的加热的黑素瘤细胞中的细胞凋亡和增强细胞毒性;和4）在体内用酸化加高温处理的黑素瘤中建立细胞凋亡和肿瘤生长延迟的预测因子。将选择用hsp 70 B启动子控制下的绿色荧光蛋白报道基因转染的DB-1人黑素瘤细胞的克隆并在体外表征。从这些克隆生长的异种移植物将被处理和分析，以评估我们的假设在体内。HSP表达和凋亡指数将在经处理的DB-1异种移植物的细胞或组织切片中确定，作为体内生长延迟的决定因素。在慢性低pH环境中通过选择性下调HSP来增强肿瘤细胞凋亡的策略具有增加人类肿瘤对涉及热疗法（例如热放疗）的多模式方案的响应的潜力。

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.