Mitomycin C (MMC) is commonly used to treat bladder cancer, the third most prevalent malignant disease among males in USA. The clinical effectiveness of MMC is often limited by the emergence of drug resistant tumor cells through mechanism(s) not completely understood. The overall purpose of this research proposal is to gain insight into the mechanism(s) of resistance to MMC with particular emphasis on understanding the role of glutathione (GSH) and related enzymes in the process. GSH levels will be compared in sensitive and resistant bladder cancer cells and correlated with MMC cytotoxicity. In order to further substantiate that the GSH levels affect MMC response, effect of BSO induced GSH depletion on MMC cytotoxicity will be studied in vitro in colony forming assay and in vivo in nude mouse xenografts. In addition, sorting of subsets of cells on the basis of GSH content will also be attempted to seek correlation between GSH content and MMC cytotoxicity. The levels of GSH related enzymes, particularly GSH peroxidases including the novel monomeric form will be determined in these cells and correlated with MMC cytotoxicity. Since interrelationship of monomeric peroxidase with other peroxidases is not known, this enzyme will be purified and characterized. Expression and characteristics of GSH S-transferase (GST) isoenzymes of normal human bladder will be investigated to identify tissue specific GSTs. The levels of GST isoenzymes will be compared in sensitive and resistant cells in order to see alterations in specific class-toxicity. In order to gain insight into the mechanism by which GSTs may contribute to MMC resistance, GST mediated catalytic and non-catalytic detoxification of MMC will be studied in vitro using purified GST and in situ in sensitive and resistant cells. Studies will be performed to see if MMC resistance can be reversed by irreversible inhibitors of GST. MMC induced DNA cross-links will be quantitated in sensitive and resistant cells by alkaline elution assay. The extent and levels of enzymes involved in removal of DNA cross-link will also be compared in these cells. In addition, the contribution of MMC induced free radical scavengers on free radical generation by ESR spectroscopy and MMC cytotoxicity in colony forming assay. In long term, the proposed studies may be helpful in devising strategies to circumvent cellular resistance to MMC.
