Role of Protein Kinase C-Iota in Prostate Cancer Win, Hla 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 IKKkappa/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. English
Role of Protein Kinase C-Iota in Prostate Cancer
Win, Hla
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 IKKkappa/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.
English