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 contributing 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 chemotherapeutics and a statistically significant twofold increase in cellular chemosensitivity. We now report that the N-myristoylated PKC-alpha pseudosubstrate peptide N-myristoyl-RFARKGALRQKNV (P3) is not a P-gp substrate in MCF-7 MDR cells based on a comparison of the cellular uptake of [125I]-radiolabeled P3 in MCF-7 MDR vs MCF-7 WT cells. The extent of cellular uptake of the radiolabeled peptide in the drug-resistant cell line MCF-7 MDR was either greater than or equivalent to the uptake in the parental drug-sensitive MCF-7 WT cell line over a time course of 30 min to 6 h, and across a peptide concentration range of 25-100 microM. Additionally, treatment of the MCF-7 MDR cells with verapamil (VPL), a known P-gp efflux inhibitor, had no effect on the cellular accumulation 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 value as an MDR reversal agent that operates by a novel mechanism.