The role of hypoxia in the resistance of cancer cells to DNA damaging agents has been clearly demonstrated in in vitro and in vivo. Several clinical studies have also demonstrated poor therapeutic outcome for hypoxic tumors. DNA repair, repair protein Kf, and intracellular GSH detoxification play a major role in cellular response to DNA damaging topoisomerase II inhibitors and gamma radiation. Although the functions of these factors can be inhibited by oxidation of functional thiols, cells resist thiol redox changes by producing NADPH through oxidative pentose phosphate cycle (OPPC). For example hydroxyethyldisulfide (HEDS), a unique non-toxic thiol oxidant, is not effective in modifying the cellular thiol redox in normal CHO cells with OPPC. Our preliminary studies indicated, for the first time, that CHO mutants impaired with OPPC activity are the ideal candidates for HEDS-mediated sensitization to DNA damaging agents. This unique property of OPPC, which eliminates HEDS-mediated thiol redox modification and DNA damage sensitization, will be exploited to sensitize hypoxic cancer cells deprived of glucose, a substrate for OPPC, without affecting glucose proficient normal cells. Glucose deprivation is common in hypoxic cells of several solid tumors because of poor vascularization and higher metabolic activity. We hypothesize that glucose/OPPC depleted hypoxic cancer cells are susceptible to HEDS-mediated redox [Protein thiol (PSH) & glutathione (GSH)] modification and sensitization to """"""""and tumor and DNA damaging"""""""" Topo II inhibitors and gamma radiation through multiple mechanisms that include inhibition of DNA repair, DNA repair protein function, GSH detoxification, and anti-apoptotic factors. We will test our hypothesis in hypoxic human cancer cells using biochemical and molecular approaches. First, we will determine the application of thiol redox modification in the sensitization of glucose deprived hypoxic cancer cells to topo II inhibitors and gamma radiation. Additional studies will also be conducted to test the efficacy of HEDS in low glucose hypoxic tumor in in vivo. Second, using siRNA and antibody technologies, we will determine whether specific inhibitors of OPPC can also induce HEDS-mediated redox modification, and subsequent sensitization of hypoxic cancer cells to DNA damaging agents. Finally, we will determine the mechanisms of redox modification- mediated hypoxic sensitization by determining its effect on DNA double strand breaks, DNA repair protein Kf, GSH detoxification and pro-apoptotic factors.
