PARTIAL REVERSAL OF MULTIDRUG-RESISTANCE IN HUMAN BREAST-CANCER CELLSBY AN N-MYRISTOYLATED PROTEIN-KINASE C-ALPHA PSEUDOSUBSTRATE PEPTIDE

The predominant characteristics of multidrug resistant (MDR) cancer ce lls are broad spectrum resistance to chemotherapeutic agents and a pro nounced defect in intracellular accumulation of the drugs, in associat ion with overexpression of the drug efflux pump P-glycoprotein, Protei n kinase C (PKC) phosphorylates the linker region of P-glycoprotein. E vidence has been presented that the isozyme PKC-alpha may contribute t o the drug resistance phenotype of human breast cancer MCF7-MDR cells, PKC-alpha is markedly overexpressed in MCF7-MDR cells, and artificial overexpression of PRC-alpha in MCF7 constructs that overexpress P-gly coprotein significantly enhances the MDR phenotype of the cells in ass ociation with increased P-glycoprotein phosphorylation, Verapamil, cyc losporin A, and a number of other agents that compete with cytotoxic d rugs for binding sites on P-glycoprotein can potently reverse MDR, but this is accompanied by severe toxicity in vivo. In this report, we de monstrate that an N-myristoylated peptide that contains a sequence cor responding to the pseudosubstrate region of PKC-alpha (P1) partially r everses multidrug resistance in MCF7-MDR cells by a novel mechanism th at involves inhibition of PKC-alpha, P1 and two related PKC inhibitory N-myristoylated peptides restored intracellular accumulation of chemo therapeutic drugs in association with inhibition of the phosphorylatio n of three PKC-alpha substrates in MCF7-MDR cells: PKC-alpha, Raf-1 ki nase, and P-glycoprotein. A fourth N-myristoylated peptide substrate a nalog of PKC, P7, did not affect drug accumulation in the MCF7-MDR cel ls and failed to inhibit the phosphorylation of the PKC-alpha substrat es, The effects of P1 and verapamil on drug accumulation in MCF7-MDR c ells were additive. P1 did not affect P-glycoprotein expression, MCF7- MDR cells were not cross-resistant to P1, which suggests that the pept ide was not transported by P-glycoprotein. Furthermore, P1 was disting uished from MDR reversal agents such as verapamil and cyclosporin A by its inability to inhibit [H-3]azidopine photoaffinity labeling of P-g lycoprotein. P1 actually increased [H-3]azidopine photoaffinity labeli ng of P-glycoprotein in MCF7-MDR cells, providing evidence that the ef fects 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 b y a novel mechanism that involves inhibition of PKC-alpha-catalyzed P- glycoprotein phosphorylation.