verlukast and 4-carboxyfluorescein

We have obtained a novel multidrug resistant cell line, derived from HT29 G(+) human colon carcinoma cells, by selection with gradually increasing concentrations of the anti-mitotic, microtubule-disrupting agent colchicine. This HT29(col) cell line displayed a 25-fold increase in colchicine resistance and exhibited cross-resistance to doxorubicin, VP16, vincristine and taxol. Immunoblotting, combined with RT-PCR showed that the multidrug resistance phenotype was conferred by specific overexpression of the multidrug resistance protein 1. Confocal scanning laser microscopy revealed that multidrug resistance protein 1 specifically localized in the plasma membrane of HT29(col) cells. In a functional assay, using the fluorescent multidrug resistance protein 1 substrate 5-carboxyfluorescein, an increased efflux activity of HT29(col) cells was measured, as compared to the wild-type HT29 G(+) cells. MK571, a specific inhibitor of multidrug resistance protein 1, blocked the 5-carboxyfluorescein efflux, but only partially reversed resistance to colchicine, indicating that additional multidrug resistance mechanisms operate in HT29(col) cells. In conclusion, these results show for the first time overexpression of a functional multidrug resistance protein 1 under colchicine pressure, indicating that colchicine is not a P-glycoprotein-specific substrate. Colchicine-induced overexpression of multidrug resistance protein 1 is accompanied by a changed sphingolipid composition, i.e., enhanced levels of glucosylceramide and galactosylceramide. In addition, ceramide, a lipid messenger molecule involved in apoptosis-related signal transduction processes, was much more abundant in HT29(col) cells, which is indicative of a stress response.

Topics: Antineoplastic Agents; ATP-Binding Cassette Transporters; Cell Survival; Ceramides; Colchicine; Colonic Neoplasms; Dose-Response Relationship, Drug; Doxorubicin; Drug Resistance, Multiple; Etoposide; Fluoresceins; Galactosylceramides; Glucosylceramides; HT29 Cells; Humans; Immunoblotting; Immunohistochemistry; Microscopy, Confocal; Microtubules; Mitosis; Multidrug Resistance-Associated Proteins; Paclitaxel; Propionates; Quinolines; Signal Transduction; Sphingolipids; Vincristine