Human endostatin inhibits growth of human non-small-cell lung cancer in a murine xenotransplant model.

Overall prognosis in human NSCLC remains poor. Antiangiogenic treatment has become a promising concept for the treatment of solid malignancies. Our purpose was to evaluate the efficacy of recombinant HSENDO for the treatment of human NSCLC in an orthotopic murine xenotransplantation model. The efficacy of HSENDO was tested in vitro in cell-proliferation, cell-migration and tube-formation assays. In vivo, the effect of HSENDO on tumor growth was tested in s.c. xenotransplanted human NSCLC and on intrapulmonary induced human NSCLC. In vitro, HSENDO inhibited both human and rodent endothelial cell proliferation in a time- and dose-dependent fashion. Endothelial cell migration was inhibited by 97%. Tube formation of murine endothelial cells was inhibited and preexisting tubes degenerated after HSENDO exposure. In vivo, HSENDO delayed growth of s.c. xenotransplanted tumors. Immunohistochemic staining demonstrated no change in microvessel density but a significant reduction of proliferating tumor cells and an increase in bFGF and VEGF expression, reflecting the antiangiogenic effect of HSENDO. Intrapulmonary tumor induction caused death subsequent to metastatic disease. Systemic HSENDO application extended survival significantly. HSENDO was demonstrated to inhibit endothelial cell proliferation, migration and tube formation effectively. In vivo growth of s.c. transplanted tumors was delayed and survival extended by 32% and 69%, respectively, after intrapulmonary NSCLC induction.

The microbial receptor CEACAM3 is linked to the calprotectin complex in granulocytes.

Engulfment of foreign pathogens is an evolutionary ancient host cell endocytic response. Signaling pathways effecting phagocytosis are divergent and largely depend on the structural features of the cell surface receptor utilized. CEACAM3, a member of the CD66 complex on human neutrophils, has been implicated as a cellular receptor promoting phagocytosis of microorganisms. The cytoplasmic domain of CEACAM3 (CEACAM3(cyt)) contains an immunoreceptor tyrosine-based activation motif. In this study we demonstrate that CEACAM3(cyt) is phosphorylated by protein kinase C, casein kinase I, and Src-kinase in vitro. To identify molecules binding to CEACAM3(cyt) in vivo, we used differentially phosphorylated recombinant expressed CEACAM cytoplasmic domains to isolate CEACAM3(cyt)-associated proteins from granulocyte extracts. Calprotectin, which modulates neutrophil integrin-mediated adhesion and leukocyte trafficking and displays antimicrobial activity, interacts specifically with CEACAM3(cyt). This interaction is calcium-modulated but independent of phosphorylation of CEACAM3(cyt). Although tyrosine-phosphorylated CEACAM3(cyt) binds and stimulates Src-kinases in vitro, no CEACAM3(cyt)-associated phosphokinase activity was copurified.

cis Interaction of the cell adhesion molecule CEACAM1 with integrin beta(3).

CEACAM1 is a cell adhesion molecule that has been implicated in a number of physiological processes (eg, tumor suppressor in epithelial tissues, potent angiogenic factor in microvessel formation, microbial receptor in human granulocytes and epithelial cells). The mechanism of CEACAM1 action is still largely unresolved but recent findings demonstrated that the cytoplasmic CEACAM1 domain is linked indirectly to the actin-based cytoskeleton. We have isolated integrin beta(3) as an associated protein using CEACAM1 tail affinity purification. This association depends on phosphorylation of Tyr-488 in the CEACAM1 cytoplasmic domain. Confocal laser scanning microscopy confirmed in vivo colocalization of both molecules in human granulocytes and epithelial cells. Furthermore, the concentrated colocalization at the tumor-stroma interface of invading melanoma masses suggests a functional role of CEACAM1-integrin beta(3) interaction in melanoma invasion. Moreover, colocalization of the two adhesion molecules is also found at the apical surface of glandular cells of pregnancy endometrium. Colocalization of CEACAM1 and integrin beta(3) at the transitional zone from proliferative to invasive extravillous trophoblast of the maternal-fetal interface supports a role for CEACAM1/integrin beta(3) complexes in cell invasion.

Endostatin inhibits angiogenesis by stabilization of newly formed endothelial tubes.

Endostatin decreased vascular endothelial growth factor (VEGF)-induced formation of endothelial tubes and microvessels sprouting from aortic rings and blocked their network. After cessation of treatment, the survival time of endostatin plus VEGF-treated tubes was approximately doubled in comparison to VEGF alone. Endostatin antibody blocked VEGF-induced endothelial tube formation and disrupted existing tubes. Endostatin immunostaining was localized between endothelium and basement membrane and in inter-endothelial junctions of new, but not of quiescent, blood vessels. In tumors grown in SCID mice, endostatin immunostaining was stronger accompanying blood vessel maturation and was significantly prominent in vessels of tumor marginal zone where angiogenesis is highly active. These data indicate a new antiangiogenic action of endostatin stabilizing and maturating endothelial tubes of newly formed blood vessels. Thus, strategies accelerating vascular stabilization and maturation could be promising in tumor therapy.

Cell adhesion molecule CEACAM1 associates with paxillin in granulocytes and epithelial and endothelial cells.

CEACAM1 functions as an epithelial tumor suppressor and as an angiogenic growth factor. In the present study, utilizing differentially (serine/threonine or tyrosine) phosphorylated cytoplasmic domains of CEACAM1 and CEACAM3 as bait to isolate associated proteins from granulocyte extracts, we have identified human paxillin as a binding partner of the tyrosine-phosphorylated cytoplasmic CEACAM1 domain. CEACAM1-paxillin complexes were coimmunoprecipitated from extracts of granulocytes, the colonic cell line HT29, and HUVECs. We identified phosphorylated Tyr-488-a residue in the cytoplasmic CEACAM1 domain known to be essential for the tumor suppressive effect-to be necessary for this association. The CEACAM1-paxillin interaction was confirmed using laser scanning confocal microscopy analyses in granulocytes and HT29 cells, where CEACAM1 colocalizes with paxillin at the plasma membrane. In HUVECs a highly polarized expression pattern and colocalization of paxillin and CEACAM1 was observed. These findings support the findings that CEACAM1 is linked to the actin-based cytoskeleton.