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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
1R29CA050638-01A1
Application #
3459576
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Project Start
1990-04-01
Project End
1995-03-31
Budget Start
1990-04-01
Budget End
1991-03-31
Support Year
1
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Miami School of Medicine
Department
Type
Schools of Medicine
DUNS #
City
Miami
State
FL
Country
United States
Zip Code
33146
Singh, S V; Xu, B H; Tkalcevic, G T et al. (1994) Glutathione-linked detoxification pathway in normal and malignant human bladder tissue. Cancer Lett 77:15-24
Xu, B H; Gupta, V; Singh, S V (1994) Mitomycin C sensitivity in human bladder cancer cells: possible role of glutathione and glutathione transferase in resistance. Arch Biochem Biophys 308:164-70
Xu, B H; Gupta, V; Singh, S V (1994) Characterization of a human bladder cancer cell line selected for resistance to mitomycin C. Int J Cancer 58:686-92
Xu, B H; Gupta, V; Singh, S V (1994) Mechanism of differential sensitivity of human bladder cancer cells to mitomycin C and its analogue. Br J Cancer 69:242-6
Jani, J P; Specht, S; Stemmler, N et al. (1993) Metastasis of B16F10 mouse melanoma inhibited by lovastatin, an inhibitor of cholesterol biosynthesis. Invasion Metastasis 13:314-24
Singh, S V; Xu, B H; Maurya, A K et al. (1992) Modulation of mitomycin C resistance by glutathione transferase inhibitor ethacrynic acid. Biochim Biophys Acta 1137:257-63
Xu, B H; Singh, S V (1992) Effect of buthionine sulfoximine and ethacrynic acid on cytotoxic activity of mitomycin C analogues BMY 25282 and BMY 25067. Cancer Res 52:6666-70
Xu, B H; Singh, S V (1992) Potentiation of mitomycin C cytotoxicity by glutathione depletion in a multi-drug resistant mouse leukemia cell line. Cancer Lett 66:49-53
Singh, S V; Roberts, B; Gudi, V A et al. (1991) Immunohistochemical localization, purification, and characterization of human urinary bladder glutathione S-transferases. Biochim Biophys Acta 1074:363-70
Nair, S; Singh, S V; Krishan, A (1991) Flow cytometric monitoring of glutathione content and anthracycline retention in tumor cells. Cytometry 12:336-42