Partial reversal of multidrug resistance in human breast cancer cells by an N-myristoylated protein kinase C-alpha pseudosubstrate peptide.
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.
Gupta
K P
KP
Department of Cell Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
Ward
N E
NE
Gravitt
K R
KR
Bergman
P J
PJ
O'Brian
C A
CA
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000345:Affinity Labels / Q000737:chemistry; D000595:Amino Acid Sequence; D001386:Azides / Q000737:chemistry; D001943:Breast Neoplasms / Q000473:pathology*; D004095:Dihydropyridines / Q000737:chemistry; D018432:Drug Resistance, Multiple; D005260:Female; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids; D009842:Oligopeptides / Q000737:chemistry; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D051571:Protein Kinase C-alpha; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D001386:Azides; D004095:Dihydropyridines; D007527:Isoenzymes; D009227:Myristic Acids; D009842:Oligopeptides; D011518:Proto-Oncogene Proteins; D019814:Myristic Acid; C041130:azidopine; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha