Partial inhibition of multidrug resistance by safingol is independent of modulation of P-glycoprotein substrate activities and correlated with inhibition of protein kinase C

Abstract

Safingol is a lysosphingolipid protein kinase C (PKC) inhibitor that competitively interacts at the regulatory phorbol binding domain of PKC. We investigated the effects of safingol on antineoplastic drug sensitivity and PKC activity of MCF-7 tumor cell lines. Safingol treatment of 32P-labeled MCF-7 WT and MCF-7 DOX(R) cells inhibited phosphorylation of the myristoylated alanine-rich protein kinase C substrate in both cell lines, suggesting inhibition of cellular PKC. However, only in MCF-7 DOX(R) cells did safingol treatment increase accumulation of [3H]vinblastine and enhance toxicity of Vinca alkaloids and anthracyclines. Drug accumulation changes in MCF-7 DOX(R) cells treated with safingol were accompanied by inhibition of basal and phorbol 12,13-dibutyrate-stimulated phosphorylation of P- glycoprotein (P-gp). Expression of P-gp and levels of mdr1 message in MCF-7 DOX(R) cells were not altered by safingol treatment alone or in combination with vinblastine. Treatment of MCF-7 DOX(R) cell membranes with safingol did not inhibit [3H]vinblastine binding or [3H]azidopine photoaffinity labeling of P-gp. Furthermore, safingol did not stimulate P-gp ATPase activity in membranes prepared from MCF-7 DOX(R) cells. We conclude that enhanced drug accumulation and sensitivity in MCF-7 DOX(R) cells treated with safingol are correlated with inhibition of PKC rather than competitive interference with P-gp drug binding through direct interaction with P-glycoprotein.