Regulation of PSA secretion and survival signaling by calcium-independent phopholipase A(2)beta in prostate cancer cells.

BACKGROUND: Serum prostate specific antigen (PSA) levels in prostate cancer patients serve as a useful biomarker for diagnosing and monitoring prostate cancer. Recently, secreted PSA has been characterized as an autocrine survival factor through activation of Akt and induction of AR. In the normal prostate, PSA is secreted in the lumen of prostatic ducts to lyse proteins in the seminal coagulum. METHODS: However, the mechanism for constitutive PSA secretion from benign prostate and its transport across the prostate-blood barrier into serum are unknown. Regulation of peptide secretion by iPLA(2)-beta has been reported in non-prostatic tissue and in prostate tissue iPLA(2)-beta is reported to be under androgen regulation. We investigated whether iPLA(2) plays a role for in PSA secretion by comparing iPLA(2) activity and expression in normal prostate epithelial RWPE-1 cells and in LNCaP prostate cancer cells. Expression of the two active iPLA(2)-beta mRNA splice variants, LH-iPLA(2) and SH-iPLA(2), were increased and the inhibitory ankyrin-iPLA(2) isoform was markedly reduced in LNCaP cells as compared to normal prostate epithelial RWPE-1 cells. RESULTS: These changes are consistent with a higher enzymatic activity in LNCaP cells. The iPLA(2)-beta-specific inhibitor BEL inhibited PSA secretion and induced apoptosis in LNCaP cells. iPLA(2) knockdown using SiRNA inhibited PSA secretion, downregulated AR and induced apoptosis. Exogenous PSA suppressed BEL-induced apoptosis and neutralizing anti-PSA antibody blocked the survival effect of PSA. CONCLUSIONS: These data indicate that iPLA(2)-beta participates in regulating PSA secretion and supports the concept that secreted PSA provides an autocrine survival function in LNCaP cells.

Ascorbate inhibits NADPH oxidase subunit p47phox expression in microvascular endothelial cells.

The production of reactive oxygen species (ROS) is central to the etiology of endothelial dysfunction in sepsis. Endothelial cells respond to infection by activating NADPH oxidases that are sources of intracellular ROS and potential targets for therapeutic administration of antioxidants. Ascorbate is an antioxidant that accumulates in these cells and improves capillary blood flow, vascular reactivity, arterial blood pressure, and survival in experimental sepsis. Therefore, the present study tested the hypothesis that ascorbate regulates NADPH oxidases in microvascular endothelial cells exposed to septic insult. We observed that incubation with Escherichia coli lipopolysaccharide (LPS) and interferon-gamma (IFNgamma) increased NADPH oxidase activity and expression of the enzyme subunit p47phox in mouse microvascular endothelial cells of skeletal muscle origin. Pretreatment of the cells with ascorbate prevented these increases. Polyethylene glycol-conjugated catalase and selective inhibitors of Jak2 also abrogated induction of p47phox. Exogenous hydrogen peroxide induced p47phox expression that was prevented by pretreatment of the cells with ascorbate. LPS+IFNgamma or hydrogen peroxide activated the Jak2/Stat1/IRF1 pathway and this effect was also inhibited by ascorbate. In conclusion, ascorbate blocks the stimulation by septic insult of redox-sensitive Jak2/Stat1/IRF1 signaling, p47phox expression, and NADPH oxidase activity in microvascular endothelial cells. Because endothelial NADPH oxidases produce ROS that can cause endothelial dysfunction, their inhibition by ascorbate may represent a new strategy for sepsis therapy.