Sphingolipids are putative intracellular signal mediators in cell differentiation, growth inhibition, and apoptosis. Sphingosine, sphinganine, and phytosphingosine are structural analogs of sphingolipids and are classified as long-chain sphingoid bases. Sphingosine and sphinganine are known to play important roles in apoptosis. In the present study, we examined the phytosphingosineinduced apoptosis mechanism, focusing on mitochondria in human T-cell lymphoma Jurkat cells. Phytosphingosine significantly induced chromatin DNA fragmentation, which is a hallmark of apoptosis. Enzymatic activity measurements of caspases revealed that caspase-3 and caspase-9 are activated in phytosphingosineinduced apoptosis, but there is little activation of caspase-8 suggesting that phytosphingosine influences mitochondrial functions. In agreement with this hypothesis, a decrease in ∆ ∆ ∆ ∆Ψ Ψ Ψ Ψ m and the release of cytochrome c to the cytosol were observed upon phytosphingosine treatment. Furthermore, overexpression of mitochondria-localized anti-apoptotic protein Bcl-2 prevented phytosphingosine apoptotic stimuli. Western blot assays revealed that phytosphingosine decreases phosphorylated Akt and p70 O rganelle mitochondria play an important role in the induction of apoptosis machinery. Upon apoptosis, mitochondria undergo PT with the opening of an apoptosis-related pore called a mitochondrial PT pore complex, which is the basis for mitochondria-mediated apoptosis.(1) PT pore opening results in a reduction of ∆Ψ m and the release of AIF, Smac/ DIABLO, and cytochrome c to the cytosol.(2−6) Released AIF directly affects nuclei and triggers chromatin condensation.(4) Cytochrome c binds to Apaf-1 and then activates initiator caspase, caspase-9. Activated caspase-9 in turn activates the effector caspase, caspase-3, and activated caspase-3 cleaves a variety of substrates to execute apoptosis by means of chromatin DNA fragmentation, morphological changes, and a loss of cell volume. (7,8) Smac/ DIABLO promotes this cytochrome c-dependent caspase activation by binding to the inhibitor of apoptotic proteins, IAP.(5,6) The anti-apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-x L , inhibit apoptosis by preventing opening of the PT pore.(1) Apoptotic signals are transmitted by many pathways to mitochondria. One such pathway induced by most anticancer drugs involves direct or indirect induction of PT by affecting mitochondria.(9,10) Another pathway involves inhibition of Bcl-2 and Bcl-x L anti-apoptotic effects by specific binding with the pro-apoptotic Bcl-2 family protein (e.g. Bax, Bid, and Bad) migrating from the cytosol. In p53-mediated apoptosis, cytosolic Bax is redistributed to the mitochondria to induce PT.(2) In Fas/ CD95-mediated apoptosis, caspase-8 is activated, followed by cleavage of Bid. Cleaved (activated) Bid translocates from the cytosol to the mitochondria and executes the postmitochondrial apoptotic pathway.(11) In the depletion of survival factormediated apoptosis, the PI 3-kinase pathway is attenuated. Ser/ Thr kinase Ak...
