Our previous work has shown that a number of sphingolipid metabolites inclu
ding sphingosine, sphinganine, and other long-chain bases potently induced
apoptosis in human hepatoma cells. in this study, we examined the possibili
ty that sphingosine may trigger apoptosis in human hepatoma cells via inhib
ition of anti-apoptotic pathways. We investigated the effect of sphingosine
on AKT kinase, a serine/threonine kinase which was found to protect cells
from apoptosis induced by a variety of extracellular stresses. Our results
indicated that sphingosine inhibited basal and serum-stimulated AKT kinase
activity in a dose-dependent manner in hepatoma cells. Additionally, sphing
osine-induced inhibition of AKT kinase was correlated with induction of apo
ptosis in these cells. Pretreatment of insulin, a potent stimulator of AKT
kinase, partially reversed the inhibition of AKT kinase by sphingosine and
counteracted the apoptotic action of this sphingolipid. Expression of activ
ated AKT kinase partially protected cells from sphingosine-induced apoptosi
s, whereas expression of kinase-dead AKT kinase had no effect. The molecula
r mechanism by which AKT kinase suppressed the apoptotic action of sphingos
ine was investigated. Our results showed that increased release of cytochro
me C from mitochondria and subsequent activation of caspase-3 were detected
in sphingosine-treated hepatoma cells. On the contrary, expression of acti
vated AKT kinase in Hep3B cells attenuated cytochrome C release and caspase
-3 activation induced by sphingosine. Taken together, these findings sugges
t that suppression of AKT kinase is one of the mechanisms by which sphingos
ine induces apoptosis in hepatoma cells and activation of AKT kinase may in
hibit sphingosine-induced apoptosis by blocking a step upstream of cytochro
me C release and caspase-3 activation. (C) 2001 Wiley-Liss, Inc.