EFFECTS OF SPHINGOSINE STEREOISOMERS ON P-GLYCOPROTEIN PHOSPHORYLATION AND VINBLASTINE ACCUMULATION IN MULTIDRUG-RESISTANT MCF-7 CELLS

To investigate the role of protein kinase C (PKC) in the regulation of multidrug resistance and P-glycoprotein (P-gp) phosphorylation, the n atural isomer of sphingosine (SPH), D-erythro sphingosine (De SPH), an d its three unnatural stereoisomers were synthesized. The SPH isomers showed similar potencies as inhibitors of in vitro PKC activity and ph orbol binding, with IC50 values of approximately 50 mu M in both assay s. Treatment of multidrug-resistant MCF-7(ADR) cells with SPH stereois omers increased vinblastine (VLB) accumulation up to 6-fold at 50 mu M but did not alter VLB accumulation in drug-sensitive MCF-7 wild-type (WT) cells or accumulation of 5-fluorouracil in either cell line. Phor bol dibutyrate treatment of MCF-7(ADR) cells increased phosphorylation of P-gp, and this increase was inhibited by prior treatment with SPH stereoisomers. Treatment of MCF-7(ADR) cells with SPH stereoisomers de creased basal phosphorylation of the P-gp, suggesting inhibition of PK C-mediated phosphorylation of P-gp. Most drugs that are known to rever se multidrug resistance, including several PKC inhibitors, have been s hown to directly interact with P-gp and inhibit drug binding. SPH ster eoisomers did not inhibit specific binding of [H-3] VLB to MCF-7(ADR) cell membranes or [3H]azidopine photoaffinity labeling of P-gp or alte r P-gp ATPase activity. These results suggest that SPH isomers are not substrates of P-gp and suggest that modulation of VLB accumulation by SPH stereoisomers is associated with inhibition of PKC-mediated phosp horylation of P-gp.