The overall objectives of this project are (a) to evaluate the role of cellular thiols and glutathione (GSH)-dependent detoxifying enzymes in intrinsic and acquired resistance of human colon cancer cells to BCNU and melphalan, and (b) to determine the limits to which resistance can be reversed by modulation of thiol metabolism. These objectives will be accomplished in 6 specific aims.
Specific aim 1 is to characterize levels of GSH, non-protein thiols, total protein thiols, and activities of GSH transferases, peroxidase and reductase in sensitive and resistant cell lines. The importance of thiol-related resistance mechanisms relative to other resistance mechanisms will be evaluated through collaborative interactions of the project directors.
Specific aim 2 is to analyze the effectiveness of buthionine sulfoximine, N-methylformamide, dimethylformamide, and GSH transferase inhibitors, such as ethacrynic acid and piriprost, in reversing alkylator resistance.
In Specific aim 3 the effects of modulators of other resistance mechanisms on thiol metabolism will be evaluated, and the potential for additive or synergistic interactions between modulators of thiol metabolism and modulators of other resistance mechanisms will be explored. The effectiveness of GSH and/or GST modulation in vivo will be assessed in Specific aim 4, through collaborative interactions with Projects 1 and 4.
Specific aim 5 deals with in vitro studies of the response of human colon cancer cells to thiol chemoprotectors, and Specific aim 6 is to evaluate the elevated GSH as well as levels and activities of GSH transferases for resistance of patient tumors to chemotherapy. Since recent data suggests that alpha and mu transferase isozymes may be more important for detoxifying alkylating agents that are the pi isozymes which are elevated in many colon tumors, emphasis will be on developing assays for these three major classes of transferases in biopsy tissue as well as in tumor-derived cell lines. The health relatedness of this study is to improvement of control of colon cancer in patients through a thorough understanding of alkylator resistance mechanisms.
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