Ceramide mediates nanovesicle shedding and cell death in response to phosphatidylinositol ether lipid analogs and perifosine

J J Gills,J Lopiccolo,M Tsokos,T Goldkorn,C Zhang,C Marceau,M S Abu-Asab,P A Dennis,S S Castillo,A P Kozikowski

doi:10.1038/cddis.2012.72

Abstract

Anticancer phospholipids that inhibit Akt such as the alkylphospholipid perifosine (Per) and phosphatidylinositol ether lipid analogs (PIAs) promote cellular detachment and apoptosis and have a similar cytotoxicity profile against cancer cell lines in the NCI60 panel. While investigating the mechanism of Akt inhibition, we found that short-term incubation with these compounds induced rapid shedding of cellular nanovesicles containing EGFR, IGFR and p-Akt that occurred in vitro and in vivo, while prolonged incubation led to cell detachment and death that depended on sphingomyelinase-mediated generation of ceramide. Pretreatment with sphingomyelinase inhibitors blocked ceramide generation, decreases in phospho-Akt, nanovesicle release and cell detachment in response to alkylphospholipids and PIAs in non-small cell lung cancer cell lines. Similarly, exogenous ceramide also decreased active Akt and induced nanovesicle release. Knockdown of neutral sphingomyelinase decreased, whereas overexpression of neutral or acid sphingomyelinase increased cell detachment and death in response to the compounds. When transferred in vitro, PIA or Per-induced nanovesicles increased ceramide levels and death in recipient cells. These results indicate ceramide generation underlies the Akt inhibition and cytotoxicity of this group of agents, and suggests nanovesicle shedding and uptake might potentially propagate their cytotoxicity in vivo.

Highlights

Most Akt inhibitors in development target the ATP-binding region, whereas lipid-based inhibitors such as phosphatidylinositol ether lipid analogs (PIAs) and the alkylphospholipids perifosine (Per) and miltefosine were designed to interfere with the pleckstrin homology (PH) domain of Akt
The poor response rates of a majority of common solid tumors to Per as a single agent has lowered expectations and prompted further investigation into its mechanism of action.[6]. We show both types of compounds stimulate release of nanovesicles containing active Akt, EGFR and IGF-IR and the tetraspanins CD151 and CD81 from tumor cells in vitro and in vivo through sphingomyelinase-mediated generation of ceramide
Our initial aim was to investigate whether PIAs could inhibit growth factor induced, as well as endogenous Akt activation in cancer cells

Summary

Introduction

Most Akt inhibitors in development target the ATP-binding region, whereas lipid-based inhibitors such as phosphatidylinositol ether lipid analogs (PIAs) and the alkylphospholipids perifosine (Per) and miltefosine were designed to interfere with the PH domain of Akt. Alkylphospholipids can activate JNK, target cell membranes, induce p21 in a p53-independent manner and disturb lipid rafts.[6] Single-agent activity with Per has been observed in sarcoma and Waldenstrom Macroglobinulemia (WM) patients. The poor response rates of a majority of common solid tumors to Per as a single agent has lowered expectations and prompted further investigation into its mechanism of action.[6]. We show both types of compounds stimulate release of nanovesicles containing active Akt, EGFR and IGF-IR and the tetraspanins CD151 and CD81 from tumor cells in vitro and in vivo through sphingomyelinase-mediated generation of ceramide. PIA and Per induced decreases in P-Akt, cell detachment and death depended on ceramide generation.

Methods

Results

Conclusion