Mechanisms for high methoxymorpholino doxorubicin cytotoxicity in doxorubicin-resistant tumor cell lines

Methoxymorpholino doxorubicin (MMRDX) is an anthracycline analogue that is able to overcome tumor cell resistance to classical anthracyclines. Mechanisms for increased MMRDX cytotoxicity were analyzed in a small cell lung carcinoma cell line (GLC(4)), its 300-fold doxorubicin-resistant and multidrug resistance-associated protein (MRP)-over-expressing subline (GLC(4)/ADR), an ovarian carcinoma cell line (A2780) and its 100-fold doxorubicin resistant and P-glycoprotein (P-gp)-over-expressing subline A2780AD. Cross-resistance, measured with the MTT assay at MMRDX concentration resulting in 50% growth inhibition, was 1.8-fold in GLC(4)/ADR and 4.5-fold in A2780AD compared to their respective parental cell lines. Cellular MM RDX accumulation was equal in GLC(4) and GLC(4)/ADR and 2-fold lower in A2780AD compared to A2780. Doxorubicin fluorescence was analyzed with confocal laser scan microscopy. Fluorescence was nuclear in sensitive, and cytoplasmic in resistant, cell lines, while MMRDX fluorescence was found in the nucleus in all cell lines. Pre-incubation with the MRP blocker MK571 restored in GLC(4)/ADR cells the nuclear doxorubicin fluorescence pattern, as observed in GLC(4) cells. MMRDX, thus, can largely overcome cross-resistance in these P-gp- and MRP-overexpressing doxorubicin-resistant cell lines. Our results suggest that MMRDX is not a substrate for MRP-mediated resistance. (C) 1997 Wiley-Liss, Inc.