P-glycoprotein (P-gp) is a transmembrane protein that transports a variety of structurally and functionally diverse drugs. We recently found that the interaction of drugs with P-gp promoted invasion and metastasis. In this study, we sought to determine the mechanism by which the interaction of P-gp with its substrates leads to the earliest membrane changes associated with cellular invasion, i.e., membrane ruffling. We focused on the activation of phosphatidylinositol-3-kinase (PI-3-kinase), a lipid kinase that regulates actin cytoskeletal organization and cell movement. Sensitive or multidrug-resistant (MDR) MCF-7 (human breast cancer) or KB (human oral carcinoma) cells were treated with drugs or vehicle, and then were stained with phalloidin-tetramethyl-rhodamine isothiocyanate. Membrane ruffles were visualized using a fluorescence microscope. PI-3-kinase activity was determined by an in vitro immune-complex kinase assay and thin-layer chromatography. Drugs transported by P-gp, vinblastine and trans-flupenthixol, increased membrane ruffling and PI-3-kinase activity in the MDR cell lines, MCF-7/AdrR and KBV-1, which overexpress P-gp. This effect was not seen with mechlorethamine, a drug that is not transported by P-gp, and was not detected in sensitive parental cell lines that do not express P-gp. A similar effect was also observed in the MDR1 transfectant, MCF-7/BC-19. Wortmannin, an inhibitor of PI-3-kinase, blocked the effect of VBL and tFPT on membrane ruffling and the activity of PI-3-kinase in MDR cells. These results indicate that drugs transported by P-gp induce membrane ruffling, an early indicator of cellular motility and metastatic potential, in cancer cells overexpressing P-gp and that this effect may be mediated through activation of PI-3-kinase.
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