Effects And Mechanisms Of Gambogic Acid On Tumor Vascular System

Background and Purpose:There is a close relationship between occurrence and development of tumor and vascular system within tumor.Angiogenesis has been recognized as a critical hallmark of tumor malignancy.On one hand,angiogenesis provides indispensible oxygen and nutrient for tumor growth,on the other hand,the new route for tumor metastasis can be opened by angiogenesis as well.Therefore,inhibiting tumor angiogenesis especially destroying the blood vessels with normal structure and function has become a new way in recent anti-tumor therapy.Gambogic acid(GA)is one of the major active ingredients extracted from Chinese medicine gamboges resin.Recent studies have shown that GA has potential antitumor effect,but its underlying mechanism remains to be elucidated.This study is to investigate the role of GA in tumor angiogenesis and to determine the effects and mechanism of GA on tumor vascular structure and function,in attempting to provide a novel insight for studying Chinese medicine on inhibiting tumor angiogenesisExperimental Approach:In vitro,MTS assay was used to determine the proliferative capability of Human umbilical vein endothelial cells(HUVECs)following GA treatment in terms of time and dose.The changes in number and morphology of HUVECs were assessed by bright field microscopy.Also,lactate dehydrogenase(LDH)assay was employed to evaluate the toxicity of GA to HUVECs.Wound-healing assay and Transwell assay were used to determine the horizontal and vertical migration of HUVECs in the presence of GA,respectively.Further,spheroid sprouting assay and aorta ring assay were both utilized to investigate the ability of HUVEC in sprouting angiogenesis.Irn addition,tube formation assay was to explore the capability of HUVECs in tube formation following the treatment of GA.In vivo,we investigated the effects of GA on physiological angiogenesis by a couple of models including Matrigel Plug assay,Chick embryo chorioallantoic membrane(CAM)assay and retina angiogenesis assay.Further,tumor angiogenesis model was employed to reveal the role of GA in tumor growth,vascular density and vascular structure and function.In terms of mechanism,western blot and immunofluorescence were utilized to determine the effects of GA on numerous signaling pathways including YAP/STAT3,VEGF/VEGFR2 and ANG2/TIE2.These methods in combination with siRNA transfection were used to confirm the target of GA,uncovering the detailed mechanisms of actionKey Results:GA was able to inhibit the proliferation of HUVECs in as dose-and time-dependent manner in vitro.Bright filed microscopy also confirmed that GA could reduce the number of HUVECs but had no impact on the morphology.LDH assay revealed that the inhibition effect of GA on HUVEC proliferation was not due to its cytotoxicity.Moreover,we found both the horizontal and vertical migration of HUVECs were suppressed by GA,as reflected by Wound-healing assay and Transwell migration assay,respectively.More interestingly,both spheroid sprouting assay and aorta ring assay showed that GA was capable of inhibiting the initial step of angiogenesis,vascular sprouting.Also,the tube formation ability of HUVECs was decreased in the presence of GA.In vivo,we found that massive CD31-positive blood vessels were produced after the Matrigel plugs were implanted into the adult mice,but GA was able to limit the angiogenesis process in a dose-dependent manner,this was substantiated by retina angiogenesis experiment and CAM assay.Of note,GA prevented the growth of B16F10 melanoma and MC38 colon cancer in mice.Vascular studies showed that both the number and area of CD31-positive blood vessels were decreased following GA treatment.More importantly,both the structure and function of blood vessels were destroyed after GA adminstration.In terms of mechanism,we found that the inhibition effect of GA on tumor angiogenesis was through targeting YAP,further blocking the YAP/STAT3 signaling axisConclusions and Implications:The findings of this study are as following:C? GA was able to inhibit a couple of biological behaviors of HUVECs including proliferation,migration,sprouting and tube formation;?GA could prevent the process of physiological angiogenesis in multiple models;?GA limited tumor growth through inhibiting the tumor angiogenesis and destroying the blood vessels with normal structure and function;?GA suppressed tumor angiogenesis via targeting transcriptional factor YAP,further blocking YAP/STAT3 signaling axis.However,it had no effects on traditional angiogenesis-associated signaling pathway including VEGF/VEGFR2.This project helped us illustrate the new mechanisms of GA on inhibiting tumor angiogenesis and provided a novel route and visual angle for us to study tumor metastasis.