Reduced pHi Inhibits Heat-Induced HSP,Enhances Apoptosis

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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.