Decreased drug accumulation in a mitoxantrone-resistant gastric carcinoma cell line in the absence of P-glycoprotein

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Abstract
An established gastric‐carcinoma cell line, EPG85‐257P, is extremely sensitive to mitoxantrone (IC50, 0.12 ng/ml). Stepwise selection with mitoxantrone for 3 years resulted in a cell line (EPG85‐257RN) that is 7,056‐fold resistant to mitoxantrone (IC50, 846 ng/ml) and displays cross‐resistance to the topoisomerase(topo)‐II poisons ametantrone (411×), etoposide (112×) and teniposide (60×) as well as the topo‐I poisons 7‐ethyl‐10‐hydroxycamptothecin (331×) and topotecan (58×). We now show that this resistance is multifactorial. Western blotting revealed a 5‐fold decrease in topo‐IIα polypeptide in the mitoxantrone‐resistant cells. Immunohistochemistry and Western blotting failed to demonstrate P‐glycoprotein over‐expression. Formation of trapped topo‐II–DNA complexes in the resistant cells required higher mitoxantrone concentrations than in parental cells, even though nuclei isolated from the EPG85‐257RN cells formed cleavage complexes normally. In agreement with these observations, which suggest the possibility of a defect in mitoxantrone accumulation, examination of mitoxantrone accumulation in both cell lines by confocal laser microscopy revealed that the EPG85‐257RN cells accumulate less mitoxantrone at steady state. From these results, we propose that mitoxantrone accumulation, along with alterations in topo‐IIα expression, contribute to the resistance to mitoxantrone observed in these cells.Int. J. Cancer 71:817‐824, 1997. © 1997 Wiley‐Liss Inc.

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