The predominant characteristics of multidrug resistant (MDR) cancer cells are broad spectrum resistance to chemotherapeutic agents and a pronounced defect in intracellular accumulation of the drugs, in association with overexpression of the drug efflux pump P-glycoprotein. Protein kinase C (PKC) phosphorylates the linker region of P-glycoprotein. Evidence has been presented that the isozyme PKC- may contribute to the drug resistance phenotype of human breast cancer MCF7-MDR cells. PKC- is markedly overexpressed in MCF7-MDR cells, and artificial overexpression of PKC- in MCF7 constructs that overexpress P-glycoprotein significantly enhances the MDR phenotype of the cells in association with increased P-glycoprotein phosphorylation. Verapamil, cyclosporin A, and a number of other agents that compete with cytotoxic drugs for binding sites on P-glycoprotein can potently reverse MDR, but this is accompanied by severe toxicity in vivo. In this report, we demonstrate that an N-myristoylated peptide that contains a sequence corresponding to the pseudosubstrate region of PKC- (P1) partially reverses multidrug resistance in MCF7-MDR cells by a novel mechanism that involves inhibition of PKC-. P1 and two related PKC inhibitory N-myristoylated peptides restored intracellular accumulation of chemotherapeutic drugs in association with inhibition of the phosphorylation of three PKC- substrates in MCF7-MDR cells: PKC-, Raf-1 kinase, and P-glycoprotein. A fourth N-myristoylated peptide substrate analog of PKC, P7, did not affect drug accumulation in the MCF7-MDR cells and failed to inhibit the phosphorylation of the PKC- substrates. The effects of P1 and verapamil on drug accumulation in MCF7-MDR cells were additive. P1 did not affect P-glycoprotein expression. MCF7-MDR cells were not cross-resistant to P1, which suggests that the peptide was not transported by P-glycoprotein. Furthermore, P1 was distinguished from MDR reversal agents such as verapamil and cyclosporin A by its inability to inhibit [H]azidopine photoaffinity labeling of P-glycoprotein. P1 actually increased [H]azidopine photoaffinity labeling of P-glycoprotein in MCF7-MDR cells, providing evidence that the effects of P1 on P-glycoprotein in MCF7-MDR cells are not restricted to inhibition of the phosphorylation of the pump. P1 may provide a basis for developing a new generation of MDR reversal agents that function by a novel mechanism that involves inhibition of PKC--catalyzed P-glycoprotein phosphorylation.

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