INHIBITION OF MAP KINASE BY SPHINGOSINE AND ITS METHYLATED DERIVATIVE, N,N-DIMETHYLSPHINGOSINE - A CORRELATION WITH INDUCTION OF APOPTOSIS IN SOLID TUMOR-CELLS

Endogenous sphingolipid metabolites such as ceramides and sphingosines have been increasingly recognized as lipid mediators of cell growth, differentiation and apoptosis. We have previously studied the ability of sphingosine (Sph) and N,N-dimethylsphingosine (DMS) to induce apopt osis in a variety of solid tumor cell lines. Here we report that in tu mor cell lines displaying high mitogen-activated protein kinase activi ty (MAPK), treatment with 5 mu M of these sphingolipids significantly inhibited MAPK activity within 2-5 min (p < 0.005-0.01 as compared to controls) and induced apoptosis within hours. In contrast, untransform ed cells and those tumor cell lines with low MAPK activity showed no s ignificant change in activity and no apoptosis. High concentrations of C2-ceramide (50-100 mM), which induced apoptosis in the solid tumor c ells, did not show significant effect on MAPK activity. MAPK activity was not directly inhibited in vitro, but tyrosine phosphatase activity was increased 2-4 fold in solid tumor cells by Sph or DMS (p < 0.01-0 .05), suggesting that a phosphatase may play an important role in sphi ngolipid-directed MAPK regulation. Sph/DMS-induced apoptosis, but not MAPK inhibition, was blocked by protease inhibitors, indicating that M APK inhibition is an earlier step of Sph/DMS-induced apoptosis than pr oteolysis. Furthermore, in human breast carcinoma MDA468 cells and hum an epidermal carcinoma A431 cells, both of which overexpress the epide rmal growth factor (EGF) receptor, 20-200 nM EGF inhibited MAPK (p < 0 .005-0.01) and induced apoptosis. These observations suggest that inhi bition of the MAPK cascade may be involved in apoptotic signaling by S ph/DMS in some solid tumor cells, or by EGF in some cancer cells which overexpress the EGF receptor. Finally, the PKC-specific inhibitor, ca lphostin C, under conditions in which PKC is completely suppressed, in hibited MAPK activity and induced apoptosis only weakly in these solid tumor cells, whereas the non-specific PKC inhibitor staurosporine ind uced both apoptosis and MAPK inhibition significantly, suggesting that MAPK inhibition and apoptosis by Sph/DMS occurs independently of PKC in these cell lines, although these pathways may act cooperatively in other cell types. This study provides insight into possible mechanisms involved in sphingolipid-induced apoptosis in solid cancer tumor cell lines.