Abstract A229: Mechanism of action of perifosine on the mantle cell lymphoma line, Granta-519.

Abstract

Abstract Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and sphingolipids and provides a scaffold for signal transduction. Altered raft assembly has been implicated in cancer progression. Alkylphospholipids have been used with promising specific cytotoxic effects in different types of cancer cells. These alkylphospholipids act by interaction with cell membranes and protein lipid rafts. Cell viability was determined by trypan blue assay. Cell cycle was evaluated by flow cytometry. Lipid rafts were isolated using sucrose density-gradient centrifugation after 12 hours of treatment with perifosine. Antibody microarray and western blotting was used to determine levels of key cellular proteins in special on cell signaling proteins. We showed that perifosine, an alkylphospholipid, targets raft-like domains in model membranes and induces apoptosis in mantle cell lymphomas (ED-50 20 μM - Granta-519) by activation of both extrinsic and intrinsic pathways. We observed an effect on the organization of the cell cycle, with increase of the population at G2/M phase (8,6% ± 0,6% to 27,4% ± 2,4%). Cyclin D1 decrease was detected at 24 hours of treatment. We also showed that perifosine downregulates NTAL/LAB (Non-T-cell activation linker/ Linker for activation of B-cells), an adaptor protein that is targeted to rafts by palmitoylation and specifically expressed in hematopoietic tissues. Moreover, perifosine induced a loss of NTAL/LAB in the lipid rafts of Granta-519 cells after 12 hours of treatment. Since NTAL/LAB may function as an adaptor protein in AKT signalization, we evaluate the effect of perifosine on the ATK pathway. We showed that perifosine lead to dephosphorylation of AKT and downstream components of AKT signaling. Moreover, in functional experiments, perifosine inhibited AKT activation by CD40L or IL-4 in Granta-519 cells, after few minutes of incubation, in a way similar to the specific PI3K inhibitor wortmannin. Treatment with methyl-β-cyclodextrin, a compound for cholesterol depletion, potentiated this effect. This suggests that NTAL/LAB translation is highly regulated and dependent on a functional AKT pathway. Our results indicate that perifosine acts on NTAL/LAB presumable by interference with its protein-lipid interactions and consequently AKT signaling. Our results demonstrate that a lipid raft targeting drug may present several effects on signal transduction, causing a severe toxicity to mantle cell lymphoma. Moreover, adaptor proteins like NTAL/LAB emerge as possible new therapeutic targets in lymphoma. This research was supported by FAPESP, CNPq and CAPES. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A229. Citation Format: Germano A. Ferreira, Carolina H. Thome, Guilherme A. dos Santos, Priscila S. Scheucher, Andreia M. Leopoldino, Ana M. Simão, Clarice Izume, Rodrigo A. Panepucci, Pietro Ciancaglini, Eduardo M. Rego, Vitor M. Faça, Lewis J. Greene. Mechanism of action of perifosine on the mantle cell lymphoma line, Granta-519. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A229.