Effects of sphingosine stereoisomers on P-glycoprotein phosphorylation and vinblastine accumulation in multidrug-resistant MCF-7 cells

Clifford W. Sachs, Larry M. Balks, S. Wayne Mascarella, Ahmad Safa, Anita H. Lewin, Carson Loomis, F. Ivy Carroll, Robert M. Bell, Robert L. Fine

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

ABSTRACT. To investigate the role of protein kinase C (PKC) in the regulation of multidrug resistance and P-glycoprotein (P-gp) phosphorylation, the natural isomer of sphingosine (SPH), D-erythro sphingosine (De SPH), and its three unnatural stereoisomers were synthesized. The SPH isomers showed similar potencies as inhibitors of in vitro PKC activity and phorbol binding, with IC50 values of approximately 50 μM in both assays. Treatment of multidrug-resistant MCF-7ADR cells with SPH stereoisomers increased vinblastine (VLB) accumulation up to 6-fold at 50 μM but did not alter VLB accumulation in drug-sensitive MCF-7 wild-type (WT) cells or accumulation of 5-fluorouracil in either cell line. Phorbol dibutyrate treatment of MCF-7ADR cells increased phosphorylation of P-gp, and this increase was inhibited by prior treatment with SPH stereoisomers. Treatment of MCF-7 cells with SPH stereoisomers decreased basal phosphorylation of the P-gp, suggesting inhibition of PKC-mediated phosphorylation of P-gp. Most drugs that are known to reverse multidrug resistance, including several PKC inhibitors, have been shown to directly interact with P-gp and inhibit drug binding. SPH stereoisomers did not inhibit specific binding of [3H] VLB to MCF-7ADR cell membranes or [3H]azidopine photoaffinity labeling of P-gp or alter 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 phosphorylation of P-gp.

Original languageEnglish (US)
Pages (from-to)603-612
Number of pages10
JournalBiochemical Pharmacology
Volume52
Issue number4
StatePublished - 1996
Externally publishedYes

Fingerprint

Phosphorylation
Stereoisomerism
Sphingosine
Vinblastine
MCF-7 Cells
P-Glycoprotein
Protein Kinase C
Isomers
Multiple Drug Resistance
Pharmaceutical Preparations
Protein C Inhibitor
Cell membranes
Protein Kinase Inhibitors
Fluorouracil
Labeling
Inhibitory Concentration 50
Adenosine Triphosphatases
Assays
Cells
Cell Membrane

Keywords

  • Multidrug resistance
  • P-glycoprotein
  • Protein kinase C
  • Sphingosine

ASJC Scopus subject areas

  • Pharmacology

Cite this

Sachs, C. W., Balks, L. M., Mascarella, S. W., Safa, A., Lewin, A. H., Loomis, C., ... Fine, R. L. (1996). Effects of sphingosine stereoisomers on P-glycoprotein phosphorylation and vinblastine accumulation in multidrug-resistant MCF-7 cells. Biochemical Pharmacology, 52(4), 603-612.

Effects of sphingosine stereoisomers on P-glycoprotein phosphorylation and vinblastine accumulation in multidrug-resistant MCF-7 cells. / Sachs, Clifford W.; Balks, Larry M.; Mascarella, S. Wayne; Safa, Ahmad; Lewin, Anita H.; Loomis, Carson; Carroll, F. Ivy; Bell, Robert M.; Fine, Robert L.

In: Biochemical Pharmacology, Vol. 52, No. 4, 1996, p. 603-612.

Research output: Contribution to journalArticle

Sachs, CW, Balks, LM, Mascarella, SW, Safa, A, Lewin, AH, Loomis, C, Carroll, FI, Bell, RM & Fine, RL 1996, 'Effects of sphingosine stereoisomers on P-glycoprotein phosphorylation and vinblastine accumulation in multidrug-resistant MCF-7 cells', Biochemical Pharmacology, vol. 52, no. 4, pp. 603-612.
Sachs, Clifford W. ; Balks, Larry M. ; Mascarella, S. Wayne ; Safa, Ahmad ; Lewin, Anita H. ; Loomis, Carson ; Carroll, F. Ivy ; Bell, Robert M. ; Fine, Robert L. / Effects of sphingosine stereoisomers on P-glycoprotein phosphorylation and vinblastine accumulation in multidrug-resistant MCF-7 cells. In: Biochemical Pharmacology. 1996 ; Vol. 52, No. 4. pp. 603-612.
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AU - Sachs, Clifford W.

AU - Balks, Larry M.

AU - Mascarella, S. Wayne

AU - Safa, Ahmad

AU - Lewin, Anita H.

AU - Loomis, Carson

AU - Carroll, F. Ivy

AU - Bell, Robert M.

AU - Fine, Robert L.

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N2 - ABSTRACT. To investigate the role of protein kinase C (PKC) in the regulation of multidrug resistance and P-glycoprotein (P-gp) phosphorylation, the natural isomer of sphingosine (SPH), D-erythro sphingosine (De SPH), and its three unnatural stereoisomers were synthesized. The SPH isomers showed similar potencies as inhibitors of in vitro PKC activity and phorbol binding, with IC50 values of approximately 50 μM in both assays. Treatment of multidrug-resistant MCF-7ADR cells with SPH stereoisomers increased vinblastine (VLB) accumulation up to 6-fold at 50 μM but did not alter VLB accumulation in drug-sensitive MCF-7 wild-type (WT) cells or accumulation of 5-fluorouracil in either cell line. Phorbol dibutyrate treatment of MCF-7ADR cells increased phosphorylation of P-gp, and this increase was inhibited by prior treatment with SPH stereoisomers. Treatment of MCF-7 cells with SPH stereoisomers decreased basal phosphorylation of the P-gp, suggesting inhibition of PKC-mediated phosphorylation of P-gp. Most drugs that are known to reverse multidrug resistance, including several PKC inhibitors, have been shown to directly interact with P-gp and inhibit drug binding. SPH stereoisomers did not inhibit specific binding of [3H] VLB to MCF-7ADR cell membranes or [3H]azidopine photoaffinity labeling of P-gp or alter 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 phosphorylation of P-gp.

AB - ABSTRACT. To investigate the role of protein kinase C (PKC) in the regulation of multidrug resistance and P-glycoprotein (P-gp) phosphorylation, the natural isomer of sphingosine (SPH), D-erythro sphingosine (De SPH), and its three unnatural stereoisomers were synthesized. The SPH isomers showed similar potencies as inhibitors of in vitro PKC activity and phorbol binding, with IC50 values of approximately 50 μM in both assays. Treatment of multidrug-resistant MCF-7ADR cells with SPH stereoisomers increased vinblastine (VLB) accumulation up to 6-fold at 50 μM but did not alter VLB accumulation in drug-sensitive MCF-7 wild-type (WT) cells or accumulation of 5-fluorouracil in either cell line. Phorbol dibutyrate treatment of MCF-7ADR cells increased phosphorylation of P-gp, and this increase was inhibited by prior treatment with SPH stereoisomers. Treatment of MCF-7 cells with SPH stereoisomers decreased basal phosphorylation of the P-gp, suggesting inhibition of PKC-mediated phosphorylation of P-gp. Most drugs that are known to reverse multidrug resistance, including several PKC inhibitors, have been shown to directly interact with P-gp and inhibit drug binding. SPH stereoisomers did not inhibit specific binding of [3H] VLB to MCF-7ADR cell membranes or [3H]azidopine photoaffinity labeling of P-gp or alter 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 phosphorylation of P-gp.

KW - Multidrug resistance

KW - P-glycoprotein

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