Critical roles for JNK, c-Jun, and Fas/FasL-Signaling in vitamin E analog-induced apoptosis in human prostate cancer cells.

Background: Alpha-tocopherol ether-linked acetic acid (alpha-TEA), an analog of vitamin E (RRR-alpha-tocopherol), is a potent pro-apoptotic agent for human cancer cells in vivo and in vitro.

Methods: alpha-TEA-induced apoptosis was investigated in LNCaP and PC-3 human prostate cancer cells. Apoptosis was measured by DAPI-staining and FACS analyses of the sub-G1 fraction. Signaling molecules involved in apoptosis were measured by Western immunoblot analyses with or without prior immunoprecipitation, FACS analyses of cell surface membrane expression, RT-PCR analyses of mRNA levels, and chromatin immunoprecipitation. Functional significance was determined using siRNAs, dominant negative mutant, chemical inhibitor, or neutralizing antibody.

Results: Alpha-TEA treatment increased Fas and Fas ligand mRNA and protein levels; as well as, levels of cell surface membrane Fas in both cell lines. Blockage of Fas signaling attenuated alpha-TEA-induced apoptosis. alpha-TEA treatment also produced prolonged, elevated levels of activated (phosphorylated) c-Jun N-terminal kinase (JNK) and its substrate c-Jun, both of which were demonstrated to be necessary for alpha-TEA-induced apoptosis. Chromatin immunoprecipitation results showed binding of c-Jun to the promoters of both Fas and FasL in alpha-TEA treated cells. Investigations of alpha-TEA-triggered apoptosis showed dual signaling from Fas with essential roles for both FADD and Daxx with FADD initiating the classical pathway mediated by caspase-8 activation and Daxx initiating an alternate pathway involving activation of JNK, c-Jun, and increased levels of Fas and FasL.

Conclusions: Collectively, data support critical roles for JNK, c-Jun, and dual signaling from Fas/FasL via FADD and Daxx in alpha-TEA-induced apoptosis of human prostate cancer cells.