Thiophilic-interaction chromatography of enzymatically active tissue prostate-specific antigen (T-PSA) and its modulation by zinc ions

Abstract

Prostate-specific antigen (PSA) is a serine protease secreted both by normal prostate glandular epithelia cells and prostate cancer cells. We explored "thiophilic-interaction chromatography" (TIC) to isolate tissue prostate-specific antigen (T-PSA) from fresh human prostate cancer tissue harvested by radical prostatectomy for the purpose to characterize T-PSA for its enzymatic activity and sensitivity to zinc ions. We have shown, for the first time, that T-PSA has strong affinity for the thiophilic gel (T-gel). The average recovery of T-PSA from T-gel is over 87%. The presence of PSA in the column eluate was confirmed by ELISA and SDS/PAGE. Western blot developed with monoclonal antibody to PSA revealed that T-PSA was predominantly in the "free" form having a molecular weight of 33kDA. Furthermore, T-PSA was found to be enzymatically active. T-PSA was found to be less enzymatically active as compared to seminal plasma PSA. The inhibition of enzymatic activity of both f-PSA and T PSA over a wide range of concentrations of Zn 2+ ions (10 nM to 50 μM) was comparable. In contrast, the enzymatic activity of chymotrypsin, another serineprotease, was affected differently. At higher concentrations of Zn 2+ (10 μM and higher) the enzymatic activity of chymotrypsin was inhibited, whereas, at lower concentrations of Zn 2+ (5 μM and lower), the enzymatic activity was enhanced.

Enzymatic activity of free‐prostate‐specific antigen (f‐PSA) is not required for some of its physiological activities

“…f-PSA was incubated with a series of concentrations of zinc chloride, and demonstrated a dose-dependent inhibition of enzymatic activity by zinc. Inhibition of >95% was achieved at a concentration of 50 μM zinc chloride, which confirmed an earlier report [40]. …”

Anti-angiogenic activity of PSA-derived peptides

“…Consequently, the anti‐tumorigenic activity of PSA, as for the degradation of semenogelins, was proposed to be related to the serine protease enzymatic activity of PSA in that denatured PSA did not demonstrate biological activity in these assays . However, our group demonstrated in CaP cells (PC‐3M) that both purified, enzymatically active PSA (free‐PSA: f‐PSA) and enzymatically inactive PSA, where enzymatic activity was inactivated by incubation with Zinc 2 + , equivalently down‐regulated expression of pro‐angiogenic factors/cancer‐related genes/proteins, including VEGF, EphA2, Bcl2, Pim‐1 oncogene, CYR61 and uPA, and up‐regulated expression of anti‐angiogenic genes/proteins, including interferon (IFN) and IFN‐related genes . In addition, both enzymatically active and enzymatically inactive f‐PSA comparably modulated gene expression in human umbilical vein endothelial cells (HUVEC) of multiple growth factors, including suppression of expression of bFGF and VEGF, and up‐regulation of IFN, and of proteins that have a direct role in blood vessel development, including FAK, FLT, KDR, TWIST‐1, P‐38, and Cathepsin‐D.…”