Antiangiogenic Property Of Four Natural Compounds And Antihypertensive Effect Of Total Flavones Extracted From Hippophae Rhamnoides L.

1.Four Natural Compounds Suppress Tumor Angiogenesis and Tumor GrowthTumor angiogenesis is the hallmark of tumor growth and tumor metastasis,and it has been a novel and potential target for tumor therapy and drug development.How to select angiogenesis inhibitors which are effective,safe,affordable and with clear molecular basis is a great challenge to modern drug discovery,and also it is a key problem in caner therapy.Recently,most of anti-angiogenic reagents used in clinical are antibodies or soluble peptides,which can not successfully inhibit tumor growth. Therefore,identifying novel tumor angiogenesis inhibitors,especially from traditional herb medicine,has become a promising topic and new direction.Making clear of underlying molecular targets of certain drugs is the primary criterion to exploit angiogenesis inhibitors.Because the signaling pathways involved in tumor angiogenesis are complex and interactional,it is difficult to identify their detailed mechanism.Based on the well-known endothelial cell signaling pathways, we selected four angiogenesis inhibitors（Morelloflavone,acetyl-11-keto-β-boswellic acid,Celastrol and l’-acetoxychavicol acetates） from natural compounds and reported their underlying mechanisms for the first time.Through working on different platforms that are in vitro,ex vivo and in vivo models,we systematically investigated the biological activities of compounds on tumor angiogenesis.In in vitro models,we evaluated the anti-proliferative effect of compounds on primarily cultured human umbilical vascular endothelial cells（HUVECs） and human prostate cancer cells（PC-3） through MTT assay,flow cytometry and apoptosis protein detection.And also,we evaluated the inhibitory effects of compounds on biological functions of endothelial cells using wound-healing migration assay,invasion assay and capillary-structure formation assay.In ex vivo model,we carried out rat aortic ring assay to investigate the inhibitory function of indicated compounds on microvessel sprouting.And,further in in vivo mouse models,we used Matrigel plug assay and xenograft human prostate tumor mice to evaluate the inhibitory actions of compounds on infiltrating blood vessel formation and tumor growth.Finally,in molecular and biochemistry study,we used western blotting,pull-down assay and in vitro kinase assay to reveal the underlying mechanism.Morelloflavone,a biflavonoid extracted from Garcinia dulcis.However,the function and the mechanism of this compound in cancer treatment and tumor angiogenesis have not been elucidated to date.We demonstrated that morelloflavone could inhibit the activation of both RhoA-GTPases and Racl-GTPases,but have little effect on the activation of Cdc42-GTPase.Additionally,morelloflavone inhibited the phosphorylation and activation of Raf/MEK/ERK pathway kinases without affecting VEGFR2 activity,by which morelloflavone inhibits cell proliferation,migration, invasion,and capillary-like tube formation of HUVECs in a dose-dependent manner. Morelloflavone effectively inhibited microvessel sprouting of endothelial cells in the aortic ring assay and the formation of new blood microvessels induced by VEGF in the mouse Matrigel plug assay.Furthermore,Morelloflavone（8mg/kg·d,15d,n=5） could inhibit tumor growth and tumor angiogenesis by PC-3 in xenograft mouse tumor model in vivo,without toxic effect on mice body weight.These findings are the first to reveal the novel functions of Morelloflavone in tumor angiogenesis and its molecular basis for the anticancer action.VEGF/VEGF receptor signaling plays critical role in angiogenesis.In this study, we also demonstrated that Acetyl-11-keto-β-boswellic acid（AKBA）,an active component from an Ayurvedic medicinal plant（Boswellia serrata）,could strongly inhibit tumor angiogenesis.Western blot analysis and in vitro kinase assay revealed that AKBA suppressed VEGF-induced phosphorylation of VEGF receptor 2 kinase （KDR/Flk-1） with IC50 of 1.68μmol/L.Specifically,AKBA suppressed the downstream protein kinases of VEGFR2,including Src family kinase,focal adhesion kinase,extracellular signal-related kinase,AKT,mammalian target of rapamycin （mTOR） and ribosomal protein S6 kinase（S6K）.AKBA significantly inhibited blood vessel formation in the Matrigel plug assay in mice and effectively suppressed microvessel sprouting in rat aortic ring assay ex vivo.Importantly,AKBA inhibited tumor growth in the human prostate tumor xenograft mice treated daily（10 mg/kg, n=5） and the inhibitory effect of AKBA on tumor growth was well-correlated with suppression of angiogenesis.Understanding molecular basis and target of traditional medicine is critical for modern drug development.Although Celastrol,derived from Trypterygium wilfordii Hook F.（"Thunder of God Vine"） of traditional Chinese medicine,could potentiate apoptosis in many kinds of tumor,its mechanism is still unclear.In this study,when administration with Celastrol（2 mg/kg） in xenograft human prostate cancer mice （n=5）,we found Celastrol effectively suppressed the volume and the weight of solid tumors.In angiogenesis study,we found Celastrol significantly inhibited VEGF-triggered inflating neovessels in the Matrigel plug assay in vivo and microvessel sprouts in a rat aortic ring assay ex vivo.Our western blotting results showed that Celastrol could consistently suppress the phosphorylation of AKT,mTOR and S6K in treated HUVECs and PC-3 cells with a dose-dependent manner.Taken together,these findings suggest that Celastrol targets AKT/mTOR/S6K pathway, leading to effective suppression of tumor angiogenesis and tumor growth.The complex of Src family kinase and focal adhesion kinaes and small Rho GTPase are key components to regulate cell cytoskeleton,cell migration and vascular permeability.In this study,we found small molecular compound l’-acetoxychavicol acetates（ACA）,extracted from Alpinia galangal,could effectively inhibit the activation of Src and FAK in a concentration-dependent and a time-dependent in HUVECs.Additionally,the activities of small Rho GTPase,especially Cdc42-GTPase and Racl-GTPase,were suppressed by ACA.Through the above molecular mechanisms,ACA could regulate endothelial cell migration,proliferation and survival. Furthermore,microvessel sprouting of endothelial cells in the aortic ring assay and the formation of new blood microvessels induced by VEGF in the mouse Matrigel plug assay could all be suppressed by ACA’s treatment,l’-acetoxychavicol acetates could also inhibit tumor growth in human prostate tumor xenograft mice treated daily （6 mg/kg,n=5） and the immunochemistry analysis reveled that the inhibitory effect on tumor growth was accompanied with suppression of angiogenesis.These findings suggest that l’-acetoxychavicol acetates inhibits tumor angiogenesis through targeting Src/focal adhesion kinaes and small Rho GTPases.2.Antihypertensive Effect of Total Flavones Extracted from Hippophae rhamnoides L.The total flavones are active ingredient of Hippophae rhamnoides L.（TFH） and have diverse pharmacological functions.However,its protection on the cardiovascular system is still limited.Seed residues of Hippophae rhamnoides L.are rubbishes of primary manufacture in the industry.In order to deeply explore the resource of Hippophae rhamnoides L.and understand the biological function of TFH on hypertension,we used two kinds of hypertensive rat model to evaluate the cardio-protection of the total flavones extracted from Seed residues of Hippophae rhamnoides L.（TFH-SR）.One model is insulin resistant and mild hypertensive rat model（IRH）,and the other is spontaneously hypertensive rat model（SHR）.We successfully set up IRH rat model through high-sucrose diet feeding by observation and comparison of systolic blood presser（SBP）,serum insulin,lipid metabolism,and kidney and endothelial function.Further,we treated those IRH rats with different doses of TFH-SR（50mg/kg·d,100 mg/kg·d and 150mg/kg·d） for 8 weeks.The results showed that hypertension,hyperinsulinemia,dyslipidemia,and activated angiotensinⅡprovoked by the high sucrose diet all could be ameliorated or modulated by TFH-SR,and the effective dose is 150mg/kg·d.These data suggests that the total flavones might prove useful in the treatment and/or prevention of insulin resistance in non-diabetic state with cardiovascular disease by decreasing insulin resistance and blocking angiotensinⅡpathway.To further examine and confirm the antihypertensive action of TFH-SR,we treated SHR with TFH-SR at a dose of 100mg/kg·d.Though detection of SBP, circulatory and local angiotensinⅡlevel,and semi-analysis of the expression level of angiotensinⅡtype 1 receptor（AT₁R） using immunochemistry,we found TFH-SR could effectively decrease SBP,the formation of angiotensinⅡand the expression of AT₁R.These results indicate that TFH-SR could decrease blood pressure and protects target organs through inhibiting angiotensin converting enzyme activity and down-regulating the expression of AT₁R in SHR.