Membrane vesicle formation due to acquired mitoxantrone resistance in human gastric carcinoma cell line EPG85-257

Abstract

A newly established gastric carcinoma cell line (EPG85-257P) exhibited a high sensitivity to mitoxantrone (DHAD) as determined by a monolayer proliferation assay. The concentration to inhibit cell growth to 50% of controls (IC50) was 0.0022 micrograms/ml culture medium. The cells were continuously incubated for more than 4 months in the presence of stepwise increased concentrations of DHAD, and the IC50 was increased to 0.41 micrograms/ml, i.e., 186.4-fold. This resistant variant was named EPG85-257RNOV. The EPG85-257RNOV cells became cross-resistant to Adriamycin with enhanced IC50 by 10.5-fold and to daunomycin with enhanced IC50 by 3.9-fold. No distinct resistance was observed to vinblastine, vincristine, and colchicine. Verapamil (10(-6), 4 X 10(-6) and 10(-5) M) and cyclosporin A (10(-6), 3 X 10(-6) and 10(-5) M) did not reverse DHAD resistance. As shown by immunocytochemistry (monoclonal antibodies: C219 and JSB-1) and Northern blot analysis, DHAD resistance was not associated with the appearance of the multidrug resistance (MDR)-associated (Mr 170,000) P-glycoprotein or the overexpression of P-glycoprotein mRNA. The data indicate a chemoresistance pattern unlike typical MDR (often called "atypical" MDR). The phenotypes of parent and resistant EPG85-257 cells were compared using interference contrast microscopy, electron microscopy, and immunocytochemistry. After DHAD application the following structural characteristics were found to be associated with emergence of resistance: (a) intensive formation of surface vesicles in the resistant variant. Such vesicles were almost absent in sensitive cells; (b) the vesicles contained the selecting DHAD which was visualized by its blue color; and (c) in electron microscopy the vesicles were formed by an inner and an outer double membrane, presumably derived from the plasmalemma. These observations suggest a complex cellular mechanism responsible for DHAD resistance which includes formation of membrane vesicles, vesicular drug binding, and drug compartmentalization.