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-ι) 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-ι 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-ι silencing RNA (siRNA) decreased cell proliferation, cell cycle accumulation at G2/M phase and decreased phosphorylation of Cdk7 and cdk2. In addition, PKC-ι siRNA treatment provoked a decrease in phosphorylation of Bad and increased Bad/Bcl-xL heterodimerization, leading to cell apoptosis.
In DU-145 cells, PKC-ι is anti-apoptotic and still required for cell survival. Treatment with PKC-ι 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-κB survival pathway were established using pro-inflammatory cytokine, tumor necrosis factor alpha (TNFα). Results demonstrated that RWPE-1 cells and DU-145 cells are insensitive to TNFα whereas LNCaP cells are sensitive to TNFα treatment and undergo apoptosis. In DU-145 cells, TNFα induced PKC-ι activation of IκB kinase, IKKα/ß, while in RWPE-1 cells, PKC-ζ activates IKKα/ß. Both RWPE-1 and DU-145 show degradation of IκBα allowing NF-κB/p65 translocation to the nucleus. In LNCaP cells, the upstream kinase activation IKKα/ß was not observed, although there have been reports that LNCaP cells weakly activate IKKα and have NF-κB activation. In vivo kinase assay demonstrates that PKC-ι is the substrate of IKKα/ß. A putative PKC-ι inhibitor (ICA-1) inhibited activation of IKKα/ß in vivo.
Hence, PKC-ι is an antiapoptotic protein and this suggests that anti-PKC-ι therapy may be a viable option for prostate carcinoma cells.
| Identifer | oai:union.ndltd.org:USF/oai:scholarcommons.usf.edu:etd-1568 |
| Date | 05 February 2008 |
| Creators | Win, Hla Yee |
| Publisher | Scholar Commons |
| Source Sets | University of South Flordia |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Theses and Dissertations |
| Rights | default |
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