| Prostate cancer is one of the leading causes of death among males in the United States. In this study, we hypothesized that an activated PKC-iota-dependent anti-apoptotic pathway, drives the cell cycle proliferation and survival of prostate cancer cells.;We investigated the role of atypical PKC-iota (PKC-iota) in androgen-independent prostate DU-145 carcinoma, androgen-dependent prostate LNCaP carcinoma compared to transformed non-malignant prostate RWPE-1 cells. Western blotting and immunoprecipitations demonstrated that PKC-iota is associated with cyclin-dependent activating kinase (CAK/Cdk7) in androgen-dependent, RWPE-1 and LNCaP cells but not in androgen-independent DU-145 cells. Treatment of prostate RWPE-1 cells with PKC-iota silencing RNA (siRNA) decreased cell proliferation, cell cycle accumulation at G2/M phase and decreased phosphorylation of Cdk7 and cdk2. In addition, PKC-iota siRNA treatment provoked a decrease in phosphorylation of Bad and increased Bad/Bcl-xL heterodimerization, leading to cell apoptosis.;In DU-145 cells, PKC-iota is anti-apoptotic and still required for cell survival. Treatment with PKC-iota siRNA blocked an increase in cell number, and inhibited G1/S transition. In addition to cell cycle arrest, both RWPE-1 cells and DU-145 cells underwent apoptosis via mitochondria dysfunction and activating apoptosis cascades such as release of cytochrome c, activation of caspase-7, and poly-(ADP-ribose) polymerase (PARP) cleavage.;Mechanistic pathways involving aPKCs in the NF-kappaB survival pathway were established using pro-inflammatory cytokine, tumor necrosis factor alpha (TNFalpha). Results demonstrated that RWPE-1 cells and DU-145 cells are insensitive to TNFalpha whereas LNCaP cells are sensitive to TNFalpha treatment and undergo apoptosis. In DU-145 cells, TNFalpha induced PKC-iota activation of IkappaB kinase, IKKalpha/beta, while in RWPE-1 cells, PKC-zeta activates IKKalpha/beta. Both RWPE-1 and DU-145 show degradation of IkappaBalpha allowing NF-kappaB/p65 translocation to the nucleus. In LNCaP cells, the upstream kinase activation IKKalpha/beta was not observed, although there have been reports that LNCaP cells weakly activate IKKalpha and have NF-kappaB activation. In vivo kinase assay demonstrates that PKC-iota is the substrate of IKKalpha/beta. A putative PKC-iota inhibitor (ICA-1) inhibited activation of IKKalpha/beta in vivo .;Hence, PKC-iota is an antiapoptotic protein and this suggests that anti-PKC-iota therapy may be a viable option for prostate carcinoma cells. |
