[PubMed] [Google Scholar] 18. malignancy cells, such as human gastric malignancy and bladder malignancy cells [32, 33]. Gamabufotalin (R)-BAY1238097 (CS-6), a major bufadienolide isolated from using human umbilical vein endothelial cells (HUVECs) proliferation, migration and tube formation assays, by aortic ring assay and by matrigel plug assay mouse models. Our results exhibited that CS-6 inhibited VEGF-mediated angiogenesis in endothelial cells, which suggesting that CS-6 could be used as a potential anti-angiogenesis agent that targets VEGF/VEGFR-2 signaling pathways. RESULTS CS-6 inhibits VEGF-induced cell proliferation of HUVECs As an importance of proliferation for endothelial angiogenesis and tumor growth [35], we firstly investigated the influence of CS-6 (Fig. ?(Fig.1A)1A) in human endothelial cells proliferation. After treatment for HUVECs with a range of Rabbit Polyclonal to Cyclin C CS-6 (0, 1, 10, 25, 50 and 75 nM) for 12 h and 24 h and cell viability was determined by MTT assay. The results revealed CS-6 has moderate inhibition for HUVECs proliferation and showed no obvious cytotoxicity at low concentration (Fig. ?(Fig.1B1B). Open in a separate window Physique 1 CS-6 inhibits VEGF-induced proliferation of HUVECsA. Chemical structure of gamabufotalin (CS-6). B. Viability inhibition of CS-6 on HUVECs under normal culture condition. HUVECs were exposed to CS-6 at the indicated doses and occasions, and viability was measured by MTT assay. Data were represented as percentage of vehicle-treated control. C. CS-6 inhibits the proliferation of VEGF-induced HUVECs. HUVECs were treated with CS-6 with or without 50 ng/mL (R)-BAY1238097 VEGF for 24 h or 48 h, and viability was measured by MTT assay. Three impartial experiments were performed (*p 0.05, CS-6-treated group vs. DMSO group; ##p 0.01, VEGF-treated group vs. Solvent; ***p 0.001, VEGF and CS-6-treated group vs. VEGF-treated group). We further decided whether CS-6 inhibited VEGF-induced HUVECs cell growth using MTT assay. As shown in Fig. ?Fig.1C,1C, the number of HUVECs stimulated with VEGF for 24 h and 48 h increased about 1.25 folds and 1.9 folds, respectively. These results showed that CS-6 could inhibit VEGF-induced cell growth in a dose-dependent and time-dependent manner; however we observed a greater inhibition by the CS-6 in VEGF stimulated HUVECs proliferation in comparison with absence of VEGF (Fig. ?(Fig.1C).1C). Taken together, our data showed that CS-6 was a potent inhibitor of VEGF-activated endothelial cell proliferation. CS-6 inhibits VEGF-induced endothelial cell migration and invasion Cell migration and invasion are essential for endothelial cells during angiogenesis. We next investigated the effects of CS-6 on cell migration and invasion by wound healing assays and Transwell assays, respectively. The results showed that CS-6 significantly inhibited the migrating and invasive properties of VEGF-induced endothelial cells in a dose-dependent manner (Fig. ?(Fig.2A2A and ?and2B2B). Open in a separate windows Physique 2 CS-6 inhibits VEGF-induced endothelial cell migration and invasionA. CS-6 inhibits HUVECs migration induced by VEGF in wound healing assay. HUVECs were plated, scratched and then incubated with CS-6 with or without 50 ng/mL VEGF. Cell migration was measured by manual counting. Initial magnification, 100 B. CS-6 inhibits (R)-BAY1238097 HUVECs invasion in Transwell assay. HUVECs were plated in Transwell pre-coated with matrigel. Cell migrated to the bottom of the membrane were counted by using an inverted microscope. Initial magnification, 40 C. CS-6 suppressed VEGF-induced stress fibre formation in endothelial cells. HUVECs were exposed to 50 nM CS-6 for 0.5 h, and then stimulated with or without VEGF for 15 min. F-actin of cells was visualized by dyLightTM 554 palloidin staining and imaged by Leica confocal microscopy. D. CS-6 inhibited.