P-glycoprotein-independent decrease in drug accumulation by phorbol ester treatment of tumor cells.
The effect of a change in the phosphorylation state of the drug transporter P-glycoprotein (P-gp) on its drug transport activity was studied for the substrates daunorubicin (DNR), etoposide (VP-16), and calcein acetoxymethyl ester (Cal-AM). Phorbol ester (PMA), added to stimulate phosphorylation of P-gp by protein kinase C (PKC), caused a decrease in the cellular accumulation of DNR and VP-16, both in multidrug-resistant (MDR) P-gp-overexpressing cells and in wild-type cells. Since treatment of cells with kinase inhibitor staurosporine (ST) reversed this effect of PMA and the non-PKC-stimulating phorbol ester 4alpha-phorbol, 12,13-didecanoate (4alphaPDD) did not result in a decreased DNR accumulation, we conclude that this effect is the result of kinase activity. The concentration dependence of the inhibition of P-gp by verapamil (Vp) was not influenced by PMA. Accumulation of the P-gp substrate Cal-AM was not influenced by PMA in wild-type cells. Therefore, Cal-AM was used to study the effect of PMA-induced phosphorylation of P-gp on its transport activity. Activation of PKC with PMA or inhibition of protein phosphatase 1/2A (PP1/PP2A) with okadaic acid (OA) did not affect the accumulation of Cal-AM in the MDR cells or wild-type cells. The kinase inhibitor ST increased the Cal-AM accumulation only in the MDR cells. Neither stimulating PKC with PMA nor inhibiting PP1/PP2A with OA led to a decreased inhibition of P-gp by ST, indicating that ST inhibits P-gp directly. From these experiments, we conclude that PKC and PP1/PP2A activity do not regulate the drug transport activity of P-gp. However, these studies provide evidence that PMA-induced PKC activity decreases cellular drug accumulation in a P-gp-independent manner.
Wielinga
P R
PR
University Hospital Vrije Universiteit, Department of Medical Oncology, Amsterdam, The Netherlands.
Heijn
M
M
Broxterman
H J
HJ
Lankelma
J
J
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000502:physiology*; D000970:Antineoplastic Agents / Q000493:pharmacokinetics*; D003630:Daunorubicin / Q000493:pharmacokinetics; D004305:Dose-Response Relationship, Drug; D018432:Drug Resistance, Multiple; D005047:Etoposide / Q000493:pharmacokinetics; D005452:Fluoresceins / Q000493:pharmacokinetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007624:KB Cells; D011493:Protein Kinase C / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D005452:Fluoresceins; C085925:calcein AM; D005047:Etoposide; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D003630:Daunorubicin