Drug resistance induced by ouabain via the stimulation of MDR1 gene expression in human carcinomatous pulmonary cells

The inhibition of the Na+M+-ATPase by cardiotonic drugs like ouabain deeply perturbs both the properties of the cell membrane and the ionic compositio n of the cytoplasm and hence alters fundamental cell reactions. These three types of reactions may be involved in the stimulation of multidrug resista nce 1 (MDR-1) gene expression and the synthesis of permeability glycoprotei n [P-glycoprotein (P-gp)]. We have determined whether ouabain, which binds to an extracellular motif of the Na+/KC+ ATPase, stimulates MDR-1 gene expr ession by measuring both mRNA and protein and whether the resulting P-gp ex trudes hydrophobic compounds and causes resistance to antimitotic agents. T he experiments were performed on Calu-3 cells, a human cell line from a pul monary carcinoma. Northern blotting showed that treating the cells with sub micromolar concentrations of ouabain stimulated MDR-1 gene expression withi n 24 h, The ouabain-induced stimulation of MDR-1 expression was not restric ted to Calu-3 cells but also occurred in human carcinomatous colon (T-84 an d HT-29) and hepatic (H7V3) cells, However, it is not ubiquitous because it was not found in HeLa cells. The stimulation was reproduced by other Na+/K +-ATPase inhibitors and occurred via enhanced gene transcription, apparentl y due to the increased cytosolic calcium concentration. Ouabain also increa sed the membrane content of P-gp, as detected by immunoblotting and immunoh istology, We have developed a microvideo assay based on the properties of a cetoxymethyl ester calcein and calcein to show that this P-gp extruded the hydrophobic acetoxymethyl ester calcein, Ouabain also caused the Calu-3 cel ls to become resistant to doxorubicin and vinblastine. Thus, although ouaha in acts extracellularly, it may stimulate MDR-1 gene expression and P-gp sy nthesis and make cells resistant to hydrophobic cytotoxic compounds.