Molecular targets of nuclear factor kappa B (NF-κB) inhibitors in metastatic prostate cancer.

B79

The nuclear factor kappa B (NF-kB) family of transcription factors regulates the expression of a wide spectrum of genes that are involved in the regulation of the immune response and inflammation. Members of the NF-kB family are comprised of dimeric combinations and are activated by a number of receptor-mediated signaling pathways. In almost all cells types, NF-kB complexes are localized in the cytoplasm where they are bound to a family of inhibitors (I-kB). Stimulated I-kB proteins are rapidly phosphorylated by two I-kB kinases, IKKα and IKKβ. The I-kB proteins are then degraded via the ubiquitin-proteosome pathway. Degradation of I-kB proteins allow the NF-kB p50/p60 heterodimer to localize into the nucleus, where it then binds to transcription binding sites of various genes, enhancing the transcription of several genes involved in inflammation. However, regulation by its agonists and antagonists in different stages of cancer, particularly of metastatic prostate cancer, is not well understood. In the present study, we investigate the following: (a) the expression of NF-kB and its correlation with the progression of prostate cancer, (b) the mechanism of action against prostate cancer cells by synthetic and natural NF-kB inhibitors and (c) the main targets of NF-kB regulation. Using NF-kB inhibitors we demonstrate that metastatic prostate cancer cells express a higher amount of NF-kB. Our studies utilized two types of metastatic prostate cancer cells: 59R, derived from transgenic adenocarcinoma of the mouse prostate (TRAMP), and PC-3, representing human metastatic prostate adenocarcinoma. Similar treatments of NF-kB inhibitors were compared with cells representing human benign prostate hyperplasia (BPH). The inhibitory effects mediated by NF-kB inhibitors were significant in the metastatic prostate cancer cells contrary to the BPH cells. Exposure to different concentrations of a synthetic NF-kB inhibitor, andographolide, revealed reduced DNA binding activity determined by gel shift assays. NF-kB inhibitors were also found to induce cell growth inhibition and apoptosis in a time dose dependent manner in metastatic prostate cancer cells. Western blot analysis of proteins involved inflammation revealed down regulation of NF-kBp65 and cyclin D1 proteins, however, TNF-α and Caveolin-1 protein levels were not affected. Studies are in progress to determine whether natural inhibitors of NF-kB exhibit similar or different mechanisms of action compared to synthetic inhibitors, while other studies in progress will establish whether there is a direct association between NF-kB expression and other cell proinflammatory cytokines promoting metastatic prostate cancer. Findings from this study will help to determine whether potential NF-kB inhibitors can be further tested for their efficacy against prostate cancer.