Enzastaurin inhibits ABCB1-mediated drug efflux independently of effects on protein kinase C signalling and the cellular p53 status

• Published in: European journal of cancer (1990) Vol. 69; p. S72
• Main Authors: Michaelis, M, Rothweiler, F, Loeschmann, N, Sharifi, M, Ghafourian, T, Cinatl, J
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Science Ltd 01-12-2016
• Subjects: ABC transporter; Adenosine triphosphatase; Biotechnology; Cancer therapies; Cell lines; Cells; Clinical trials; Cytotoxicity; Docking; Efflux; Fluorescence; Hematology, Oncology and Palliative Medicine; Intracellular levels; Intracellular signalling; Kinases; Medical research; Neuroblastoma; Neuroblasts; p53 Protein; Phosphorylation; Protein kinase C; Protein transport; Proteins; Rhabdomyosarcoma; Rhodamine; Signal transduction; Substrate inhibition; Substrates; Verapamil; Vincristine
• ISSN: 0959-8049; 1879-0852
• DOI: 10.1016/S0959-8049(16)32805-2

Background: Enzastaurin is a PKCβ inhibitor that has been tested in clinical trials. Here, enzastaurin was tested in neuroblastoma and rhabdomyosarcoma cell lines. Material and Methods: Cell viability in response to enzastaurin was tested in the project cell lines, their vincristine-resistant sub-lines, primary neuroblastoma cells, ABCB1-transduced, ABCG2-transduced, and p53-depleted cells. Pathway activation was determined through detection of protein phosphorylation. ABC transporter function was studied using cytotoxic and fluorescent substrates, specific inhibitors, determination of ABC transporter ATPase activity. The interaction of enzastaurin and ABCB1 was studied by in silico docking studies. Results: Enzastaurin IC50s ranged from 3.3 to 9.5µM in cell lines and primary cells independently of the ABCB1, ABCG2, or p53 status. Enzastaurin 0.3125µM interfered with ABCB1-mediated drug transport. PKCα and PKCβ may phosphorylate and activate ABCB1 under the control of p53. However, enzastaurin exerted similar effects on ABCB1 in the presence or absence of functional p53. Also, enzastaurin inhibited PKC signalling only in concentrations ≥1.25µM. The investigated cell lines did not express PKCβ. PKCα depletion reduced PKC signalling but did not affect ABCB1 activity. Intracellular levels of the fluorescent ABCB1 substrate rhodamine 123 rapidly decreased after wash-out of extracellular enzastaurin, and enzastaurin induced ABCB1 ATPase activity resembling the ABCB1 substrate verapamil. Computational docking experiments detected a direct interaction of enzastaurin and ABCB1. These data suggest that enzastaurin directly interferes with ABCB1 function. Enzastaurin further inhibited ABCG2-mediated drug transport but by a different mechanism since it reduced ABCG2 ATPase activity. Conclusions: The interaction of enzastaurin with ABC transporters needs to be considered for the further development of therapies combining enzastaurin with ABC transporter substrates.