Expression and shedding of endothelial protein C receptor in prostate cancer cells.

Cancer cell international (2011-02-16)
Mario Menschikowski, Albert Hagelgans, Oliver Tiebel, Ludwig Klinsmann, Graeme Eisenhofer, Gabriele Siegert

Increasing evidences show that beyond its role in coagulation, endothelial protein C receptor (EPCR) interferes with carcinogenesis. Pro-carcinogenic effects of EPCR were linked with a raised generation of activated protein C (aPC) and anti-apoptotic signalling. This study was carried out to analyze the expression, cell surface exposition, and shedding of EPCR in normal and malignant prostate cell lines. EPCR expression is up-regulated both at the mRNA and protein levels in invasive prostate DU-145 and PC-3 cells in comparison to normal prostate epithelial cells (PrEC) and less-invasive LNCaP cells. Release of soluble EPCR (sEPCR) is induced by 12-myristate 13-acetate, ionomycin, H2O2, and disruptor of lipid rafts in PrEC, DU-145, and PC-3 cells. Furthermore, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), but not interleukin-6 or interferon-γ increase sEPCR release. In LNCaP cells, neither pharmacological agents nor IL-1β or TNF-α result in a significant increase of sEPCR release. The effects of IL-1β and TNF-α on EPCR shedding in DU-145 cells are mediated by MEK/ERK 1/2, JNK, and p38 MAPK signalling cascades. In PC-3 cells, however, the MEK/ERK 1/2 pathway is down-regulated and incubation with cytokines did not elevate the phosphorylated ERK-1/2 fraction as in the case of DU-145 cells. Treatment with 4-aminophenylmercuric acetate (APMA), an activator of metalloproteases, causes a disproportionately large increase of sEPCR release in DU-145 and PC-3 cells, compared to PrEC and LNCaP cells. Finally, an increased release of sEPCR mediated by APMA treatment is shown to be connected with reduced generation of activated protein C indicating the functionality of EPCR in these cells. The study demonstrates a number of substantial differences in expression and shedding of EPCR in prostate cancer cell lines in comparison with normal cells that may be relevant for understanding the role of this receptor in carcinogenesis.

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4-Aminophenylmercuric acetate, ≥90% (titration)
Methyl-β-cyclodextrin, produced by Wacker Chemie AG, Burghausen, Germany, ≥95.0% cyclodextrin basis (calculated)
Methyl-β-cyclodextrin, Produced by Wacker Chemie AG, Burghausen, Germany, Life Science, ≥98.0% cyclodextrin basis
Methyl-β-cyclodextrin, powder, BioReagent, suitable for cell culture
Methyl-β-cyclodextrin, average Mn 1310