Endothelial Cell Capillary-like Tube Formation Research Articles

Protein S-mediated signal transduction pathway regulates lung cancer cell proliferation, migration and angiogenesis

Objective/BackgroundProtein S (PS; encoded by the PROS1 gene), a key vitamin K-dependent anticoagulant protein, is emerging as a key structural and functional protein that is overexpressd in various malignancies, but how PS signals to promote lung cancer progression is unclear. MethodsWe used immortalized, nontumorigenic human lung epithelial cell line NL-20, A549 cells as experimental cellular models for lung cancer, and human microvascular endothelial cells (HMEC-1) as a model system for angiogenesis. A loss- and gain-of-function approach was then used to analyze the role of tumor-derived PS and their natural TAM receptors Tyro3 and MerTK in regulating cell proliferation, migration, anchorage-independent growth, and capillary-like tube formation, all prominent attributes of the metastatic phenotype of tumor cells. ResultsEvidence is now provided that regulation of PROS1 gene expression using either stable cell lines expressing lentiviral-short hairpin RNA (shRNAs) or a replication-incompetent adenovirus alters the phosphorylation of several major signaling pathways, including Erk, PKB/Akt, p38, and focal adhesion kinase (FAK), and modulates PS-dependent Tyro3- and MerTK-mediated cell migration, proliferation, and anchorage-independent growth of lung cancer cells, and endothelial cell capillary-like tube formation. ConclusionThese finding suggest that the PS-Tyro3 and -MerTK axis mediates important signaling pathways to promote lung cancer progression. Genetic inhibition of endogenous PS may serve as a promising target for anticancer drug development.

P11.31 EB1-dependent survival of glioblastoma tumor-bearing mice after treatment with the novel checkpoint activator BAL101553 is associated with inhibition of cancer stem-like cell-mediated tumor angiogenesis and astrocytic differentiation

Abstract BACKGROUND Glioblastoma (GBM) patients have limited treatment options. Cancer stem-like cells (CSLCs) contribute to GBM invasiveness, representing promising targets. BAL101553 is a novel small molecule tubulin-binding agent, promoting tumor cell death through spindle assembly checkpoint activation, which is currently in Phase 1/2a in advanced solid tumor patients including GBM. We have shown that overexpression of microtubule + end-binding 1-protein (EB1) correlates with GBM progression and poor survival. This study aimed to evaluate the effect of short-term IV and long-term daily oral BAL101553 treatment on mice orthotopically grafted with GBM CSLCs (GBM6) according to EB1 expression-level, and to decipher its mechanism of action on CSLCs. MATERIAL AND METHODS The A2B5+ GBM6 cell line was established from CSLCs isolated from a GBM patient. GBM6 cells display a mesenchymal phenotype with a high tumorigenicity and infiltrative pattern of migration. As EB1 is overexpressed in GBM6, we investigated drug activity on GBM6 according to EB1 expression, by using EB1-expressing cells (GBM6-GFP-sh0) and EB1-downregulated (GBM-GFP-shEB1) cells. BAL27862 is the active moiety of the prodrug BAL101553, which was developed to improve solubility. Two sets of animal experiments were performed with 4 groups randomized for GBM6-GFP-sh0- and GBM6-GFP-shEB1-bearing mice, receiving either BAL101553 (25 mg/kg) or vehicle intravenously on day 30, 33, and 36 after glioma implantation, or BAL101553 (30 mg/kg) or vehicle orally from day 35–135 post-grafting (5 days dosing/week). Survival and tumor invasion were analyzed. In vitro, BAL27862 activity was tested by using a cytotoxicity assay, and endothelial cell capillary-like tube formation assay. RESULTS Both treatment schedules provided an EB1-expression level-dependent survival benefit; with daily oral treatment this was almost one year longer than observed in vehicle-treated mice. Moreover, the anti-proliferative and anti-invasive effects of BAL101553 were more potent in mice bearing EB1-expressing tumors as compared to EB1-downregulated tumors. This was associated with inhibition of stem cell properties. Formation of vascular structures by the fluorescent GBM6-GFP-sh0 cells was observed in the brains of grafted mice. Drug treatment counteracted the formation of brain tumor vessels by inhibiting GBM6 trans-differentiation into tumor-derived endothelial cells, as well as VEGF secretion; thus, preventing normal endothelial cell migration activated by CSLCs. CONCLUSION BAL101553/BAL27862 triggers astrocytic differentiation and counteracts tumor angiogenesis by acting on CSLCs; thus, potentially adding to the direct anti-tumor cell effect. A high level of EB1 expression in CSLCs potentiates the drug’s effects, suggesting the potential of EB1 expression as a response-predictive biomarker for BAL101553 in GBM.

Artemisinic acid exhibits antitumor activity in MCF-7 breast cancer cells through the inhibition of angiogenesis, VEGF, m-TOR and AKT signalling pathways

<p>The aim of the present study was to evaluate the antitumor and anti-angiogenic effects of artemisinic acid in MCF-7 human breast cancer cells. Various cell signalling pathways (VEGF, m-TOR and AKT signalling pathways) and MTT assay were used. The in vivo antitumor activity of artemisinic acid was evaluated by means of tumor xenograft mouse model. Transwell cell migration assay was used to examine the chemotactic motility of the human umbilical vascular endothelial cells (HUVECs), while as endothelial cell capillary-like tube formation assay was used to evaluate the effect of artemisinic acid on the tube formation in HUVECs. We found that artemisinic acid considerably reduced both the volume and weight of concrete tumors and reduced angiogenesis in a xenograft mouse tumor model in vivo. Further, artemisinic acid suppressed the VEGF-induced cell migration and capillary-like tube formation of HUVECs in a dose-dependent manner. Artemisinic acid was found to suppress the VEGF-induced phosphorylation of VEGFR2 and also the activity of AKT and m-TOR.</p><p><strong>Video Clip:</strong></p><p><a href="https://youtube.com/v/w7jZqcDvMTc">Western blot assay:</a> 4 min 33 sec </p>

α1-Acid glycoprotein disrupts capillary-like tube formation of human lung microvascular endothelia

ABSTRACTPurpose: The acute phase protein, α1-acid glycoprotein, is expressed in the lung, and influences endothelial cell function. We asked whether it might regulate angiogenesis in human lung microvascular endothelia. Materials and Methods: α1-acid glycoprotein was isolated from human serum by HPLC ion exchange chromatography. Its effects on endothelial cell functions including capillary-like tube formation on Matrigel, migration in a wounding assay, chemotaxis in a modified Boyden chamber, adhesion, and transendothelial flux of the permeability tracer, 14C-albumin, were tested. Results: α1-acid glycoprotein dose-dependently inhibited capillary-like tube formation without loss of cell viability. At ≥0.50 mg/mL, it inhibited tube formation >70%, and at 0.75 mg/mL, >97%. α1-acid glycoprotein dose- and time-dependently restrained EC migration into a wound as early as 2 hours, and in washout studies, did so reversibly. It was inhibitory against vascular endothelial growth factor-A and fibroblast growth factor-2-driven migration but failed to inhibit chemotactic responsiveness. When α1-acid glycoprotein was added to preformed tubes, it provoked their almost immediate disassembly. As early as 15 minutes, it induced tube network collapse without endothelial cell–cell disruption. It exerted a biphasic effect on cell adhesion to the Matrigel substrate. At lower concentrations (0.05–0.25 mg/mL), it increased cell adhesion, whereas at higher concentrations (≥0.75 mg/mL) decreased adhesion. In contrast, it had no effect on transendothelial 14C-albumin flux. Conclusion: α1-acid glycoprotein, at concentrations found under physiological conditions, rapidly inhibits endothelial cell capillary-like tube formation that may be explained through diminished cell adhesion to the underlying matrix and/or reversibly decreased cell migration.