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

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.

Ceramide conversion to sphingosine-1-phosphate is essential for survival in C3H10T1/2 cells.

Ceramide and sphingosine-1-phosphate (S1P) are important dietary lipids involved in cell growth, differentiation, apoptosis and cell survival. Treatment of C3H10T1/2 murine fibroblast cells (10T1/2) with ceramide did not induce apoptosis, a commonly observed effect of ceramide treatment. To determine whether the metabolism of ceramide played a role in this resistance to apoptosis, inhibitors of ceramidase and sphingosine kinase, two important enzymes in sphingolipid metabolism, were used. Treatment of 10T1/2 cells both without or with ceramide plus N-oleoyl-ethanolamine (NOE) and (1S,2R)-D-erythro-s-(N-myristoylamino)-1-phenol-1-propanol (MAPP), two ceramidase inhibitors, resulted in fourfold and eightfold increases, respectively, in apoptosis. Cells treated without or with ceramide plus N,N-dimethylsphingosine (DMS), a potent sphingosine kinase inhibitor, resulted in fourfold and sixfold increases, respectively, in apoptosis. In all treatments the induction of apoptosis was prevented by the addition of S1P. With the addition of S1P with NOE and MAPP as well as with ceramide, treatments reduced the apoptotic response by 30 and 35%, respectively; whereas the addition of S1P with the DMS only and ceramide with DMS treatments reduced the apoptotic response by 60 and 70%, respectively. Studies using labeled ceramide demonstrated ceramide was metabolized to S1P. In addition, a 14-fold increase in apoptosis occurred in cells treated with a nonhydrolyzable analog of ceramide, ceramine, compared with vehicle control. Because inhibiting the conversion of ceramide to S1P resulted in apoptosis, the lack of an apoptotic response to ceramide alone for C3H10T1/2 cells is attributable to the conversion of this pro-apoptotic sphingolipid to the anti-apoptotic metabolite S1P, which is essential for cell survival.