Prostate cancer (PCA) remains the most commonly diagnosed visceral cancer in men with more than 230,000 newly diagnosed cases in 2005, and is the second leading cause of cancer deaths among US men. Epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) are potent mitogens that regulate proliferation and survival of PCA cells via autocrine and paracrine loops involving MAPK, Akt and NF-κB-mediated signaling pathways. Recently we showed that inositol hexaphosphate (IP6) inhibits human PCA DU145 xenograft growth in male nude mice, which was associated with its anti-proliferative and apoptotic effects in both in vivo and in vitro PCA systems; however, its mechanism of efficacy is not known completely. Here, we performed detailed mechanistic studies with IP6 to assess and define its effect on growth inhibition and apoptosis in human PCA PC3 cells cultured under serum or starved-condition without or with growth factor stimulation. Cell growth and proliferation were assayed by cell counting, BrdU and MTT assays. Apoptosis was analyzed by YO-PRO staining and FACS analysis. Western blotting was performed for phospho- or total protein levels, and electrophoretic mobility shift assay was done for NF-κB and AP1 DNA binding activity. Pre-treatment of cells, grown under serum or starved-condition without or with growth factor-stimulation, with IP6 (2 mM dose) strongly inhibited cell proliferation and induced apoptosis. IP6 strongly inhibited constitutive and ligand (EGF or IGF-I)-activated ERK1/2 and Akt phosphorylation suggesting their roles in IP6 efficacy targeting mitogen and survival signaling pathways. Consistent with a marked increase in apoptosis, IP6 also enhanced the levels of cleaved caspase-3 and PARP, but surprisingly it increased NF-κB DNA binding activity too. In accord with this observation, nuclear levels of p65 and p50 sub-units of NF-κB were also elevated by IP6 with a strong decrease in phospho- and total IκBα, p65 and p50 protein levels in cytosol; however this NF-κB activation cascade by IP6 was also associated with strong apoptosis induction suggesting a pro-apoptotic role of NF-κB under our study conditions. Additional studies identifying mechanism of a strong decrease in total IκBα levels by IP6 showed that it causes caspase- and proteosome-mediated degradation of IκBα leading to NF-κB activation. In other studies, IP6 strongly inhibited the levels of AP1 DNA binding activity together with a decrease in nuclear levels of phospho- and total c-fos and c-Jun, which were consistent with its inhibitory effect on MAPK/ERK1/2 signaling. These findings suggest that IP6 abrogates both mitogenic and cell survival signaling (ERK1/2 and Akt involving AP1), and activates NF-κB signaling as a stress response in its growth inhibitory and apoptotic efficacy against prostate carcinoma cells.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]