Partial Inhibition of Multidrug Resistance by Safingol Is Independent of Modulation of P-glycoprotein Substrate Activities and Correlated with Inhibition of Protein Kinase C

Clifford W Sachs,Ahmad R Safa,Steadman D Harrison,Robert L Fine

doi:10.1074/jbc.270.44.26639

Clifford W Sachs, Ahmad R Safa + Show 2 more

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https://doi.org/10.1074/jbc.270.44.26639

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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 DOXR 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 DOXR cells did safingol treatment increase accumulation of [3H]vinblastine and enhance toxicity of Vinca alkaloids and anthracyclines. Drug accumulation changes in MCF-7 DOXR 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 DOXR cells were not altered by safingol treatment alone or in combination with vinblastine. Treatment of MCF-7 DOXR 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 DOXR cells. We conclude that enhanced drug accumulation and sensitivity in MCF-7 DOXR 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.

Highlights

Safingol is a lysosphingolipid protein kinase C (PKC) inhibitor that competitively interacts at the regulatory phorbol binding domain of PKC
We conclude that enhanced drug accumulation and sensitivity in MCF-7 DOXR 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
The results indicate that safingol inhibits protein kinase C and partially inhibits the MDR phenotype of MCF-7 DOXR cells but does not exhibit properties of a substrate for P-gp or alter P-gp expression

Summary

Introduction

Safingol is a lysosphingolipid protein kinase C (PKC) inhibitor that competitively interacts at the regulatory phorbol binding domain of PKC. Treatment of MCF-7 DOXR cell membranes with safingol did not inhibit [3H]vinblastine binding or [3H]azidopine photoaffinity labeling of P-gp. We conclude that enhanced drug accumulation and sensitivity in MCF-7 DOXR 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. Most modulators of multidrug resistance inhibit specific binding of drugs to P-gp in membranes derived from MDR cells (4 –7) and photoaffinity labeling of P-gp by photoactive drug analogs [4, 8, 9]. These results suggest that competitive inhibition of P-gp drug binding is a mechanism by which P-gp function and multidrug resistance may be inhibited. In studies that use PKC inhibitors that inhibit drug binding to P-gp, the following question is raised: Does inhibition of P-glycoprotein function by PKC inhibitors bound by P-gp result from inhibition of drug binding or from inhibition of P-glycoprotein phosphorylation by PKC?

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