Trans-3,4,5,4′-tetramethoxystilbene, a resveratrol analog, potently inhibits angiogenesis in vitro and in vivo
Abstract
Aim: Trans-3,4,5,4′-tetramethoxystilbene (DMU-212) has shown strong antiproliferative activities against a variety of cancer cells. The aim of this study was to investigate the anti-angiogenic effects of DMU-212 in vitro and in vivo.
Methods: Human umbilical vein endothelial cells (HUVECs) were used in this study. Cell viability was studied with MTT assay, and cell apoptosis was evaluated using TUNEL assay and morphological observation. The expression of the related genes and proteins was analyzed with qRT-PCR and Western blot, respectively. Angiogenesis of HUVECs were studied using cell migration and capillary-like tube formation assays in vitro, and mouse Matrigel plug assay and chick chorioallantoic membrane (CAM) assay in vivo. The tyrosine kinase activities of VEGFR1 and VEGFR2 were measured using commercial kits.
Results: DMU-212 (5–80 μmol/L) significantly inhibited VEGF-stimulated proliferation of HUVECs (IC50 value was approximately 20 μmol/L), and induced apoptosis. Furthermore, DMU-212 concentration-dependently inhibited VEGF-induced migration of HUVECs and capillary-like structure formation in vitro. DMU-212 also inhibited VEGF-induced generation of new vasculature in Matrigel plugs in vivo with significantly decreased area of infiltrating CD31-positive endothelial cells, and inhibited newly formed microvessels in chick CAMs. Moreover, DMU-212 concentration-dependently suppressed VEGF-induced phosphorylation of VEGFR2, and inhibited phosphorylation of multiple downstream signaling components in the VEGFR2 pathway, including c-Src, FAK, Erk1/2, Akt, mTOR, and p70S6K in HUVECs. DMU-212 had no effect on VEGF-induced phosphorylation of VEGFR1.
Conclusion: DMU-212 is a potent inhibitor of angiogenesis that exerts anti-angiogenic activity at least in part through the VEGFR2 signaling pathway.
Keywords:
Methods: Human umbilical vein endothelial cells (HUVECs) were used in this study. Cell viability was studied with MTT assay, and cell apoptosis was evaluated using TUNEL assay and morphological observation. The expression of the related genes and proteins was analyzed with qRT-PCR and Western blot, respectively. Angiogenesis of HUVECs were studied using cell migration and capillary-like tube formation assays in vitro, and mouse Matrigel plug assay and chick chorioallantoic membrane (CAM) assay in vivo. The tyrosine kinase activities of VEGFR1 and VEGFR2 were measured using commercial kits.
Results: DMU-212 (5–80 μmol/L) significantly inhibited VEGF-stimulated proliferation of HUVECs (IC50 value was approximately 20 μmol/L), and induced apoptosis. Furthermore, DMU-212 concentration-dependently inhibited VEGF-induced migration of HUVECs and capillary-like structure formation in vitro. DMU-212 also inhibited VEGF-induced generation of new vasculature in Matrigel plugs in vivo with significantly decreased area of infiltrating CD31-positive endothelial cells, and inhibited newly formed microvessels in chick CAMs. Moreover, DMU-212 concentration-dependently suppressed VEGF-induced phosphorylation of VEGFR2, and inhibited phosphorylation of multiple downstream signaling components in the VEGFR2 pathway, including c-Src, FAK, Erk1/2, Akt, mTOR, and p70S6K in HUVECs. DMU-212 had no effect on VEGF-induced phosphorylation of VEGFR1.
Conclusion: DMU-212 is a potent inhibitor of angiogenesis that exerts anti-angiogenic activity at least in part through the VEGFR2 signaling pathway.