Significance of NE-κB activation and therapeutic strategies for targeting its pathway in androgen-independent prostate cancer

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Introduction: Although androgen ablation is the most effective therapy for androgen-dependent (AD) prostate cancer, it eventually fails with time and then a portion of the cancer relapses to androgen-independent (AI) prostate cancer. Recently it has become clear that constitutive activation of NF-κB is detrimental to a number of human malignancies including prostate cancer. Several reports have showed that NE-κB is constitutively activated in AI PC-3 and DU145 but not in AD LNCaP cell lines. LNCaP cell model is an excellent choice for the study of AI progression for the reason that derivative LNCaP AI sublines have been established. To elucidate the mechanisms responsible for an early step of AI progression, we determined the status of NE-κB activity in LNCaP cells after a brief androgen deprivation. Materials and Method: Transient transfections and luciferase assay were performed to compare NE-κB promoter activity in LNCaP cells with and without R1881, a synthetic analogue of androgen … More . Western blot analysis of NE-κB subunits and their inhibitor IκBα proteins, ELISA assay of NE-κB-dependent cytokines, and immunocytochemical localization of NF-κB subunit p65 in LNCaP cells were also performed. To verify that NE-κB activation can promote the growth of LNCaP cells, an adenoviral vector bearing superrepressor of IκBα (Ad-SR- IκBα), a dominant negative inhibitor of NF-κB, was assessed. Results: After R1881 deprivation, NE-κB promoter activity was markedly elevated (5 to 6-fold), and phosphorylated IκBα expression increased approximately 10-fold with time There was no significant change in the expression of p65, p50, or IκBα protein, and no evidence of altered IL-6, TNF-a, or IL-1β secretion up to 4 days after R1881 withdrawal. Immunostaining revealed that R1881 removal promoted the translocation of p65 from cytoplasm to cell nucleus, even though almost all p65 localized only to cytoplasm in the presence of R1881. Without R1881, blockade of NE-κB activity by Ad-SR- IκBα accelerated apoptosis and decreased cell growth as demonstrated by TUNEL and MTT assay, respectively. Conclusions: Our data suggest that NE-κB activation occurs rapidly after androgen deprivation in cultured human prostate cancer cells. NE-κB activation could arise as an early event for AI progression. We propose that increased IκBα phosphorylation and concomitant NE-KB activation account for the critical step of AI progression. Blocking NF-κB activation with Ad-SR- IκBα offers a novel therapeutic approach to induce the death of prostate cancer cells undergoing AI progression. Less