Effects of Chronic Ethacrynic Acid Exposure on Glutathione Conjugation and MRP Expression in Human Colon Tumor Cells

Abstract

Chronic exposure to ethacrynic acid of a subcloned HT29 human colon cancer cell line produces a 3- to 4-fold increase in the level of resistance to this agent. The resistant cells (HT6-8) have an enhanced capacity to metabolize the parent drug and efflux it from the cell. This is reflected in a 5-fold enhanced decompositioning rate constant for ethacrynic acid in HT6-8 (3.47 × 10⁻³min⁻¹) versus wild type cells (1.58 × 10⁻²min⁻¹). We observed that the glutathione conjugate of ethacrynic acid is an effective competitive inhibitor for binding to the multidrug resistance-associated protein by [³⁵S]azidophenacyl-glutathione, a photoaffinity analog of glutathione. In addition, the HT6-8 cells overexpressed multidrug resistance-associated transcript 2- to 3-fold. These results suggest that resistance to ethacrynic acid results from a concerted, coordinate increase in defense mechanisms which detoxify the drug and remove its conjugate via plasma membrane efflux.