Effect of treatment duration and glutathione depletion on mitomycin C cytotoxicity in vitro.

Glutathione (GSH) has been shown to modulate the cytotoxicity of a variety of chemotherapeutic agents. The effect of mitomycin C (MMC) treatment duration and the effect of GSH depletion on in vitro cytotoxicity against the human colon cancer cell line HT-29 was studied under aerobic conditions. Continuous-exposure experiments revealed that the cytotoxicity of 0.1 microM MMC, as measured by clonogenic cell survival, exhibited a shoulder until exposure time was at least 12 h, after which time exponential cytotoxicity was observed. Lowering GSH levels to less than 3% of control using buthionine sulfoximine (BSO) did not enhance cytotoxicity of MMC given for 1 h or continuously for less than 12 h. However, GSH depletion did enhance cytotoxicity of MMC given continuously for at least 12 h, with a dose-modifying factor at 1% survival of 1.4 for a 24-h treatment. GSH depletion under these conditions enhanced cytotoxicity of even minimally cytotoxic MMC concentrations (0.02 microM). Absolute levels of GSH-related enzymes, including glutathione-S-transferase, and the MMC-metabolizing enzyme DT-diaphorase did not change appreciably. A tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay was used to verify the results further and to determine the optimal sequence of BSO administration with a 24-h MMC treatment. BSO added simultaneously with MMC did not increase cytotoxicity, compared to MMC alone. BSO added and then removed prior to MMC was effective (dose-modifying factor at 50% survival = 1.3), but the greatest cytotoxicity was noted when BSO was present before and during MMC treatment (dose-modifying factor = 1.5). GSH depletion in another cell line (SW480) showed similar enhancement of 24-h MMC cytotoxicity. These studies show that aerobic cytotoxicity of MMC is improved by administration of the drug in continuous fashion for at least 12 h, as opposed to continuous administration for shorter periods or 1-h bolus administration. Cytotoxicity of continuous (at least 12-h) MMC treatment can be modestly enhanced by GSH depletion, which must precede MMC exposure in order to be effective.