Activation of caspase-3-like proteases in apoptosis induced by sphingosineand other long-chain bases in Hep3B hepatoma cells

Sphingosine and other long-chain bases (including sphinganine, dimethylsphi ngosine and stearylamine), but not ocytlamine (a short-chain analogue of sp hinganine), induced apoptosis in Hep3B hepatoma cells. Because both D- and L-erythrosphingosine and stearylamine exert potent apoptotic effects on Hep 3B cells, it is possible that these long-chain bases map activate apoptosis by inhibiting protein kinase C (PKC) activity. However, pretreatment with the PKC activator PMA could not rescue cells from apoptosis triggered by lo ng-chain bases. Therefore the involvement of PKC in this apoptotic process requires further characterization. We also investigated whether these long- chain bases might be metabolized into ceramide in order to elicit their apo ptotic action. We found that long-chain bases acted independently of cerami de in the induction of apoptosis, since addition of fumonisin B1, a fungal agent which effectively inhibits ceramide synthesis from sphingosine, did n ot protect against apoptosis. Additionally, we found that sphingosine-induc ed apoptosis was accompanied by activation of caspases. The functional role of caspases in this apoptotic process was examined by using specific caspa se inhibitors. The general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone, which exhibits a broad specificity for caspase-family proteases, effectively blocked sphingosine-induced apoptosis. Furthermore, our results indicate that caspase-3-like proteases, but not caspase-1, are activated during apoptosis triggered by sphingosine. Enhancement of caspase -3-like activity and cleavage of poly(ADP-ribose) polymerase, an in vivo su bstrate for caspase-3, was clearly demonstrated in sphingosine-treated Hep3 B cells. Considered together, these results suggest that caspase-3-like pro teases participate in apoptotic cell death induced by sphingosine.