Expression of P-glycoprotein in HeLa cells confers resistance to ceramide cytotoxicity.
The role of glucosylceramide synthase (GCS) in regulating ceramide-induced apoptosis has been widely studied. The purpose of this investigation was to evaluate the role of P-glycoprotein (P-gp) in regulating ceramide cytotoxicity by using C6-ceramide. To accomplish this, we employed HeLa cells with conditional expression of the multidrug resistance gene 1/P-gp. HeLa cells expressing P-gp (P-gp/on cells) challenged with [14C]C6-ceramide (6 µM), synthesized 4.5-fold the amount of C6-glucosylceramide (GC) compared to HeLa cells with suppressed expression of P-gp (P-gp/off cells), whereas the generated levels of C6-sphingomyelin were almost equal (33 and 29% of intracellular 14C, respectively). Tamoxifen, a P-gp antagonist, decreased the C6-GC levels from 3.5-1.0% in the P-gp/off and from 17-2.8% of the total lipid 14C levels in the P-gp/on cells. Tamoxifen did not inhibit cell-free C6-GC synthesis in the P-gp/off or P-gp/on homogenates. However, a specific GCS inhibitor, ethylenedioxy-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (ethylenedioxy-P4), blocked synthesis by 90%. In the cytotoxicity assays, the P-gp/off cells were sensitive to C6-ceramide and the P-gp/on cells were resistant. Resistance to C6-ceramide in the P-gp/on cells was reversed by tamoxifen but not by ethylenedioxy-P4. Experiments in another cervical cancer model showed that multidrug-resistant P-gp-rich KB-V1 cells synthesized 3-fold more C6-GC from C6-ceramide than the parental, P-gp-poor KB-3-1 cells, and whereas tamoxifen had no effect on the C6-GC synthesis in the KB-3-1 cells, it inhibited synthesis by 70% in the KB-V1 cells. This study demonstrates that P-gp potentiates C6-ceramide glycosylation and if antagonized augments C6-ceramide sensitivity, both features previously ascribed to GCS. We propose that P-gp can be an effective target for enhancing short-chain ceramide cytotoxicity in the treatment of drug-resistant cancer.
John Wayne Cancer Institute at Saint John's Health Center, Santa Monica, CA 90404, USA.
Jacqueline V
JV
Chapman
Valérie
V
Gouazé-Andersson
Myles C
MC
Cabot
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D000970:Antineoplastic Agents / Q000494:pharmacology; D002277:Carcinoma / Q000235:genetics* / Q000473:pathology; D002470:Cell Survival; D002518:Ceramides / Q000494:pharmacology*; D016572:Cyclosporine / Q000494:pharmacology; D003603:Cytotoxins / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D013629:Tamoxifen / Q000494:pharmacology; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000473:pathology; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D002518:Ceramides; D003603:Cytotoxins; D013629:Tamoxifen; D016572:Cyclosporine; D014700:Verapamil; C504327:N-(alpha-hydroxyoctadecanoyl)phytosphingosine