Abstract
Prostate cancer is one of the most frequently diagnosed cancers among men. Dietary intake of nutrients is considered crucial for preventing the initiation of events leading to the development of carcinoma. Many dietary compounds have been considered to contribute to cancer prevention including zinc, which has a pivotal role in modulating apoptosis. However, the mechanism for zinc-mediated prostate cancer chemoprevention remains enigmatic. In this study, we investigated the therapeutic effect of zinc in prostate cancer chemoprevention for the first time. Exposure to zinc induced apoptosis and resulted in transactivation of p21WAF1/Cip1 in a Smad-dependent and p53-independent manner in prostate cancer cells. Smad2 and PIAS1 proteins were significantly upregulated resulting in dramatically increased interactions between Smad2/4 and PIAS1 in the presence of zinc in LNCaP cells. Furthermore, it was found that the zinc-induced Smad4/2/PIAS1 transcriptional complex is responsible for Smad4 binding to SBE1 and SBE3 regions within the p21WAF1/Cip1 promoter. Exogenous expression of Smad2/4 and PIAS1 promotes zinc-induced apoptosis concomitant with Smad4 nuclear translocation, whereas endogenous Smad2/4 silencing inhibited zinc-induced apoptosis accompanying apparent p21WAF1/Cip1 reduction. Moreover, the knockdown of PIAS1 expression attenuated the zinc-induced recruitment of Smad4 on the p21WAF1/Cip1 promoter. The colony formation experiments demonstrate that PIAS1 and Smad2/4 silencing could attenuate zinc apoptotic effects, with a proliferation of promoting effects. We further demonstrate the correlation of apoptotic sensitivity to zinc and Smad4 and PIAS1 in multiple cancer cell lines, demonstrating that the important roles of PIAS1, Smad2, and Smad4 in zinc-induced cell death and p21WAF1/Cip1 transactivation were common biological events in different cancer cell lines. Our results suggest a new avenue for regulation of zinc-induced apoptosis, and provide a model that demonstrates zinc endorses the Smad2/4/PIAS1 complex to activate the p21WAF1/Cip1 gene that mediates apoptosis.
Highlights
We examined the effect of zinc (150 mM) on Smad[3], Smad[4], PIAS2 and p53, and observed that zinc did not alter the expression of these proteins within this time frame (Figures 1b and c)
These results indicated the possible role of Smad[2] and PIAS1 in zinc-induced apoptosis
Smad[2] gene for 24 h for immunoblot analysis with the specific antibody against Smad[4] or Smad[2], using pBSU6 vector group as control. (b) LNCaP transfected with increased amounts of Smad[4] or Smad[2] plasmids for 24 h for immunoblot analysis with p21WAF1/Cip[1] antibody. (c) The percentage of cells of sub-G1 region induced by zinc with Smad[2] or Smad[4] silence in flow cytometry analysis
Summary
Normal human prostate glands accumulate almost 10 times more zinc than other soft tissues, such as liver and kidney, and significantly decreased zinc levels are shown in malignant prostate tissues.[3,4,5,6,7] Most human prostate cancer develops from the peripheral zone of the prostate, where high levels of zinc are identified in the normal epithelium, but dramatically lower levels in the tumor.[3,4,5,6,7] Exogenous zinc showed an anti-proliferation effect in prostate cancer cells via induction of mitochondrial apoptogenesis.[3,8] Increasing evidence has shown a link between the reduction of intracellular zinc concentrations and human tumor development.[7]. Zinc affects cell cycle and apoptosis by increasing the ratio of Bax to Bcl-2, which further upregulates the p21WAF1/Cip[1] mRNA level in prostate cancer.[8,9,10] The regulation of the cell cycle through modulation of p21WAF1/Cip[1] is considered to be an intrinsic characteristic of many tumor suppressor proteins, including p53, BRAC1, and Smads.[9,11,12,13,14,15] TGF-b-activating. R-Smad/Co-Smad complex directly activates the promoter region of the p21WAF1/Cip[1] gene and upregulates cyclindependent kinase (CDK) inhibitors to promote G1–S cell cycle arrest.[13,14] Impairment of the Smad pathway causes escape from growth inhibition and leads to the promotion of cell proliferation, thereby contributing to carcinogenesis.[16,17,18,19]. It has been shown that protein inhibitors of activated signal transducers and activators of transcription (PIAS) proteins interact with the TGF-b pathway and regulate Smad-mediated transcriptional activity.[21,22,23] The PIAS proteins are implicated
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