Thus, it is very tempting to speculate that EPCRs protective effect against tumor growth comes through its anti-inflammatory effect in tumor microenvironment

Thus, it is very tempting to speculate that EPCRs protective effect against tumor growth comes through its anti-inflammatory effect in tumor microenvironment. MPM cells attenuated their growth potential, whereas EPCR silencing in non-aggressive MPM cells designed to overexpress TF increased their tumorigenicity. Immunohistochemical analyses revealed that EPCR expression in tumor cells reduced tumor cell proliferation and enhanced apoptosis. Overall, our results enlighten the mechanism by which TF promotes tumor growth through PAR1, and they show how EPCR can attenuate the growth of TF-expressing tumor cells. studies gave conflicting data as EPCR-APC signaling decreased lung metastasis in melanoma model by preventing tumor cell migration through enhancement of endothelial CCT251455 barrier function (27, 28), whereas EPCR over expression increased metastasis in lung adenocarcinoma by promoting tumor cell survival (29). To date, there is no information on whether EPCR directly influences tumor growth. In the present study, we show that MPM cells that express TF and PAR1 but not PAR2 generate large tumors in the thoracic cavity. Suppression of either TF or PAR1 reduces tumor growth CCT251455 in this model. However, overexpression of TF in less aggressive MPM cells that lack TF but express PAR1 failed to induce an aggressive phenotype. Interestingly, we found no EPCR expression in aggressive MPM cells whereas abundant EPCR expression was found in non-aggressive MPM cells. Introduction of EPCR expression to aggressive MPM cells by EPCR knock-in completely attenuated their tumorigenicity whereas the knock-down of EPCR expression in non-aggressive MPM cells designed to overexpress TF markedly increased their tumorigenicity. The present study is the first to report that EPCR suppresses TF-driven tumor growth of mesothelioma. Materials and Methods, (for detailed methods see Supplemental Material) Cell lines REN cells were from S. Albelda, University of Pennsylvania, MS-1 cells were from S-M. Hsu, The University of Texas Health Science Center at Houston, and M9K cells were from B. Gerwin, NIH. All three MPM cell types were obtained from the above investigators before 2008. Characterization of these cells when they were first used in our tumorigenesis model showed an epitheloid phenotype in culture and retained classical MPM markers, confirming their MPM origin (29, 30). Generation of stable transfectants of MPM cells expressing/lacking TF, EPCR or PAR1 TF or PAR1 expression in REN MPM cells was selectively knocked-down by specific shRNA constructs cloned into pSilencer 2.1 U6-Puro expression vector. For generation of EPCR expressing REN cells, REN MPM cells were transfected with pZeoSV plasmid made up of human EPCR cDNA (20). MS-1 and M9K MPM cells were stably transfected with pcDNA 3.1 containing TF cDNA. To suppress EPCR expression in MS-1 and M9K cells, native MS-1 and M9K cells or MS-1 and M9K cells designed to overexpress TF were stably transfected with EPCR-specific shRNA constructs. Tissue factor activity The procoagulant activity of TF on intact cell CCT251455 surface of wild-type and stable transfectants was measured in a factor activation assay (31). Measurement of cytosolic Ca2+ release Fluorescence microscopy was used for measurement of cytosolic Ca2+ release as described earlier (32). Orthotopic murine model of thoracic human MPM One hundred l of MPM cell suspension made up of 1 106 cells were injected into the pleural cavity of nude mice as described earlier CCT251455 (30) with a few minor modifications. Mouse monoclonal to ALCAM Mice were sacrificed between 28 and 30 days following tumor cell implantation, and tumor growth was evaluated as described earlier (30). Histology and immunohistochemistry Tissues were processed for thin sectioning using standard procedures. Rehydrated tissue sections were processed for hematoxylin-eosin (H&E), elastin, collagen staining, or immunostaining for TF, EPCR, Ki67 or TUNEL staining. Statistical analysis Nonparametric statistical assessments, Kruskal-Wallis or MannCWhitney test, were used for determination of statistical significance. Results Status of TF, EPCR, PAR1, PAR2, TM and TFPI expression levels in.