Studies accomplished during the initial funding period of this grant have led to the discovery of a new in vivo pathway of cisplatin metabolism, Cisplatin, one of the most commmonly used drugs in the treatment of human tumors, is nephrotoxic. The new evidence reveals that rats produce a circulating metabolite of cisplatin that is activated to a potent nephrotoxin by the enzyme gamma-glutamyl transpeptidase (GGT), located on the luminal surface of the proximal tubule cells. The therapeutic effect of cisplatin is attribute to its ability to kill dividing cell by damaging DNA. However, the types of tumors that respond to cisplatin contain GGT- positive cells. The GGT-dependent mechanism responsible for acute nephrotoxicity may be the dominant mechanism by which cisplatin kills tumor cells. The goal of the studies proposed in this renewal application is to determine whether the GGT-dependent pathway of cisplatin metabolism is a major component of the therapeutic effect of cisplatin.
Three specific aims are proposed: First, the nephrotoxic metabolite of cisplatin will be isolated, purified and identified. A novel in vitro assay system that identify compounds which are converted to biologically active toxins by GGT will be used to monitor the isolation of the nephrotoxic metabolite. Second, experiments are proposed to test whether GGT-positive tumors are more sensitive than GGT-negative tumors to the in vivo toxicity of cisplatin. In brief, this will be done transfecting GGT into two human tumor cell lines, injecting the cells into nude mice, treating the resulting tumors with cisplatin and assessing the response of the GGT- positive and GGT-negative tumors. Third, methods for inducing GGT expression in human epithelial ovarian tumors will be studied. Ovarian tumor which initially respond to cisplatin then develop resistance, are a mixture of GGT-positive and GGT-negative cells. The resistant tumor may be the result of an outgrowth of GGT-negative cells. Preliminary studies establish that several chemotherapeutic agents induce GGT expression in human ovarian tumor cell in vitro. These new results could lead to chemotherapeutic protocols that sensitize tumors to cisplatin and profoundly increase the number of tumors that can be cured. Introduction of cisplatin into clinical use increased the cure rate for testicular tumors, which are strongly GGT-positive, from less than 20% to greater than 90%. Other tumors show initial response to cisplatin, but become resistant. The research out lined in this proposal will offer important new insights on cisplatin metabolism and may define combination chemotherapy protocols that will restore the initial promise of cisplatin as a cure for many human tumors.
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