AN N-MYRISTOYLATED PROTEIN-KINASE C-ALPHA PSEUDOSUBSTRATE PEPTIDE THAT FUNCTIONS AS A MULTIDRUG-RESISTANCE REVERSAL AGENT IN HUMAN BREAST-CANCER CELLS IS NOT A P-GLYCOPROTEIN SUBSTRATE

Protein kinase C-alpha (PKC-alpha) activation is an important contribu ting factor in human breast cancer MCF-7 MDR cell drug resistance. We recently reported the use of N-myristoylated PKC-alpha pseudosubstrate peptides with potent PKC-alpha inhibitory activity as reversal agents of drug resistance in MCF-7 MDR cells. The peptides potently inhibit phosphorylation of the PKC-alpha substrates P-glycoprotein (P-gp), raf kinase and PKC-alpha itself in MCF-7 MDR cells in association with a severalfold induction of intracellular uptake of P-gp substrate chemot herapeutics and a statistically significant twofold increase in cellul ar chemosensitivity. We now report that the N-myristoylated PKC-alpha pseudosubstrate peptide N-myristoyl-RFARKGALRQKNV (P3) is not a P-gp s ubstrate in MCF-7 MDR cells based on a comparison of the cellular upta ke of [I-125]-radiorabeled P3 in MCF-7 MDR vs MCF-7 WT eels. The exten t of cellular uptake of the radiolabeled peptide in the drug-resistant cell line MCF-7 MDR was either greater than or equivalent to the upta ke in the parental drug-sensitive MCF-7 WT cell line over a time cours e of 30 min to 6 h, and across a peptide concentration range of 25-100 mu M. Additionally, treatment of the MCF-7 MDR cells with verapamil ( VPL), a known P-gp efflux inhibitor, had no effect on the cellular acc umulation of radiolabeled P3. Our results provide direct evidence that the N-myristoylated pseudosubstrate peptide is taken up equivalently by drug-sensitive and MDR cancer cells and therefore has potential val ue as an MDR reversal agent that operates by a novel mechanism.