Partial Reversal of Multidrug Resistance in Human Breast Cancer Cells by an N-Myristoylated Protein Kinase C-α Pseudosubstrate Peptide

Krishna P Gupta,Nancy E Ward,Karen R Gravitt,Philip J Bergman,Catherine A O'Brian

doi:10.1074/jbc.271.4.2102

Abstract

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-alpha may contribute to the drug resistance phenotype of human breast cancer MCF7-MDR cells, PKC-alpha is markedly overexpressed in MCF7-MDR cells, and artificial overexpression of PKC-alpha 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-alpha (P1) partially reverses multidrug resistance in MCF7-MDR cells by a novel mechanism that involves inhibition of PKC-alpha. 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-alpha substrates in MCF7-MDR cells: PKC-alpha, 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-alpha 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 suggest 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 [3H]azidopine photoaffinity labeling of P-glycoprotein. P1 actually increased [3H] 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-alpha-catalyzed P-glycoprotein phosphorylation.

Highlights

For developing a new generation of Multidrug resistant (MDR) reversal agents that function by a novel mechanism that involves inhibition of Protein kinase C (PKC)-␣-catalyzed P-glycoprotein phosphorylation
We show that the peptide P1 reverses MDR by a novel mechanism that is associated with a sharp increase in intracellular drug accumulation and inhibition of the phosphorylation of P-glycoprotein and two other PKC-␣ substrates, Raf-1 kinase and PKC-␣ itself
P7 is an N-myristoylated peptide with a sequence that corresponds to a PKC phosphorylation site in P-glycoprotein [13]

Summary

Introduction

For developing a new generation of MDR reversal agents that function by a novel mechanism that involves inhibition of PKC-␣-catalyzed P-glycoprotein phosphorylation. Selective phorbol-ester PKC activators induce resistance in cancer cells to multiple cytotoxic drugs that are P-glycoprotein substrates in association with a sharp reduction in the intracellular accumulation of the drugs, providing evidence that PKC activation contributes to MDR. We show that the peptide P1 reverses MDR by a novel mechanism that is associated with a sharp increase in intracellular drug accumulation and inhibition of the phosphorylation of P-glycoprotein and two other PKC-␣ substrates, Raf-1 kinase and PKC-␣ itself. The N-myristoylated PKC-␣ pseudosubstrate peptide P1 may be a valuable starting point for developing a new generation of MDR reversal agents that function by a novel mechanism that involves inhibition of PKC-␣-catalyzed P-glycoprotein phosphorylation

Methods

Results

Conclusion