Phosphorylation of Fas‐associated Death Domain Contributes to Enhancement of Etoposide‐induced Apoptosis in Prostate Cancer Cells

Fas‐associated death domain (FADD) plays an important role as an adapter molecule in Fas (CD95/APO‐l)‐mediated apoptosis and contributes to anticancer drug‐induced cytotoxicity. We treated three human prostate cancer cell lines with etoposide, a toposiomerase II inhibitor with activity against various tumors including prostate cancer. We found that the overexpression of FADD sensitizes etoposide‐induced apoptosis through a rapid activation of c‐Jun NH(2)‐terminal kinase (JNK) and, subsequently, of caspase 3. In addition, phosphorylation of FADD at serine 194 coincided with this sensitization. Treatment with the caspase 3 inhibitor, N‐acetyl‐Asp‐Glu‐Val‐Asp‐aldehyde (DEVD‐CHO), or overexpression of either mitogen‐activated protein kinase kinase (MKK) 7 or Bcl‐xL canceled FADD‐mediated sensitization to etoposide‐induced apoptosis. Moreover, treatment with the caspase 8 inhibitor, benzyloxy‐carbonyl‐Val‐Ala‐Asp‐fluoromethylketone (z‐IETD‐fmk), or overexpression of viral FLICE/caspase‐8‐inhibitory protein (FLIP) from equine herpesvirus type 2 E8 also had an inhibitory effect, supporting a major involvement of a caspase 8‐dependent mitochondrial pathway. Interestingly, FADD was phosphorylated, and etoposide‐induced JNK/caspase activation and apoptosis were enhanced in the cells arrested at G2/M transition, but not in those overexpressing mutant FADD, in which 194 serine was replaced by alanine. Our results demonstrate that phosphorylated FADD‐dependent activation of the JNK/caspase pathway plays a pivotal role in sensitization to etoposide‐induced apoptosis in prostate cancer cells.