New leads on vitamin E succinate-induced apoptosis signaling in prostate cancer cells

Proc Amer Assoc Cancer Res, Volume 45, 2004 1390 Previously α-tocopheryl succinate (vitamin E succinate, VES) has been reported to induce caspase-mediated apoptosis in PC-3 human prostate cancer cells (Zu K and Ip C. Cancer Res. 63: 6988-95, 2003). Caspase-9 was among several initiator caspases activated by VES, suggesting that the Bcl-2 family proteins, working via the mitochondrial pathway, were likely to be involved. To assess the effect of VES on mitochondrial regulation of apoptosis, several Bcl-2 member proteins were examined by Western blotting. The results showed that VES treatment increased the expression of some pro-apoptotic proteins, such as Bax and Bak. Although the expression level of Bcl-2 and Bad was unchanged by VES, the phosphorylation status of these two proteins was modulated in a manner consistent with an enhanced apoptotic response. The finding that VES caused changes in phosphorylated Bcl-2 and Bad implies that other kinase-type signaling molecules might also be recruited in the process. Microarray analysis was therefore carried out to identify additional VES targets that may contribute to apoptosis signaling. Akt1, an important cell survival molecule, emerged as a potential candidate, since it is known to phosphorylate Bad. Western blotting analysis comfirmed that phospho-Akt1, the active form of Akt1, was reduced by VES treatment in a time- and dose-dependent manner. Phospho-Forkhead transcription factor (FKHR), another downstream target of Akt1 associated with survival, was also decreased. Interestingly, the activity of PTEN and phosphoinositide-dependent kinase 1 (PDK 1), two upstream regulators of Akt1, was not affected. Nonetheless, the regulation of phospho-Akt1 by VES offers tantalizing clues with respect to the integration of the survival pathway and the mitochondrial control of apoptosis in VES signaling. Our microarray data also provided evidence that VES was able to activate the stress-induced MAPK cascade. Western blotting analysis showed increased protein level of Apoptosis signal-regulating kinase 1 (Ask1), JNK/Erk kinase (Sek1) and its cofactor GADD45, as well as increased phosphorylation of Sek1 and JNK. Further experimentation is currently underway to assess the functional significance of JNK pathway activation in VES-induced apoptosis. In summary, our study has generated a number of new leads regarding the molecular mechanism of action of VES in apoptosis induction.