Bioreduction Rate Research Articles

Modulation of mitomycin C resistance by glutathione transferase inhibitor ethacrynic acid

This study was undertaken to elucidate the mechanism(s) of cross-resistance (4.9-fold) to mitomycin C(MMC) in a multi-drug-resistant cell line, P388/R-84. Intracellular accumulation of MMC by sensitive (P388/S) and P388/R-84 cells was comparable. Despite a 32% reduction in NADPH cytochrome P-450 reductase activity (responsible for MMC activation) in P388/R-84 cells, the rate of MMC bio-reduction by sensitive and resistant cells was similar. These results suggested that MMC resistance in P388/R-84 cell line must depend on factors other than impaired drug accumulation or bio-activation. Recent studies suggest that glutathione transferase (GST) dependent drug detoxification also contributes to cellular resistance of a variety of alkylating agents. Even though overexpression of GST have been noted in some MMC resistant tumor cells, it is not known if its level affects sensitivity to MMC. We have, therefore, determined the effect of ethacrynic acid (an inhibitor of GST activity) treatment on MMC cytotoxicity in P388/R-84 cells, which have about 2-fold higher GST activity than P388/S cells. The IC 50 value for the inhibition of GST activity in vitro by ethacrynic acid (EA) was 16.5 μM (5 μg/ml). A depletion in intracellular GSH was also observed by treating P388/R-84 cells with EA alone or in combination with MMC. A non-toxic concentration of EA (1 μg/ml); 3.3 μM) increased MMC cytotoxicity by 36% in P388/R-84 cells. MMC cytotoxicity was increased 2-fold by EA treatment in glutathione (GSH)-depleted P388/R-84 cells. These results suggest that GST mediated drug inactivation may represent another important mechanism of MMC resistance.

Measurement of bioreduction rates of cells with distinct responses to ionizing radiation and cisplatin

A low frequency electron paramagnetic resonance (EPR) spectrometer has been used to measure the bioreduction rate of an exogenously added nitroxide free radical species. Measurements have been made in a well controlled, in vitro system using an X-ray and cisplatin sensitive Chinese hamster ovary (CHO) cell line, xrs-5, and partial revertants which display wild-type levels of sensitivity to X-rays but retain xrs-5 levels of cisplatin sensitivity. The xrs-5 cells reduce this radical species at a rate which is approx. 50% that of the wild-type CHO cell line, K1. The partial revertants maintain this defect in bioreduction despite their decrease in radiosensitivity. However, the bioreduction rate observed in these cells correlates with their sensitivity to the chemotherapeutic drug cisplatin. Low frequency EPR allows measurements and imaging of living tissue and may be of value as a predictive assay of human tumor response to chemotherapy.