An Approach To Synthesis,Anti-tumor Activity And Mechanism For Asiatic Acid Derivatives

Asiatic acid(AA),one of the active pentacyclic triterpenoids found in Centalla asiatica,can be easily prepared from hydrolysis of asiaticoside.Besides its traditional usage to treat skin defect,AA also has other biological effects including anti-tumor,anti-inflammation,hepatoprotective,anti-depression,and anti-Alzheimer’s disease activities.It’s well known that various modification have been attempted to improve its potency.Here AA was separated and purified from Centella Asiatical(L).A series of AA derivatives were synthesized and biologically evaluated in anti-tumor and anti-angiogenic activities.And further researches were made on the mechanisms underlying the anti-tumor/anti-angiogenic property of N-(2α,3β,23-Acetoxyurs-12-en-28-oyl)-L-proline Methyl Ester(AA-PMe).a high potent derivative of them.In addtion,we explored absorption,metabolism and toxicity of AA-PMe on both cell and zebrafish models in order to provide evidence to reveal mechanism of drug action.Firstly,we separated and purified asiatic acid from Centella Asiatical(L)and successfully developed a reversed-phase high-performance liquid chromatography(HPLC)method with UV detection for quantitative determination of asiatic acid.Then,a series of AA derivatives were synthesized by substituting seven different amio acids at position of C-28.Their cytotoxic activities were then evaluated in vitro using seven cancer cell lines(HeLa,HepG2.B16F10,SGC7901.A549,MCF7 and PC3).We then sought to evaluate the anti-angiogenic activity of the derivatives using Tg(flil:EGFP)zebrafish.Results showed that acetylation of the C-2.C-3.and C-23 hydroxy groups in conjunction with a substituted amino group at C-28.resulted in derivatives having both stronger cell growth inhibitory and anti-angiogenic activities relative to their parent compound AA.We found a high potent deivative AA-PMe.Secondly,we sought to elucidate the effects of AA-PMe on the malignant phenotype of gastric cancer cells and the underlying mechanisms.As a result,we indicated that AA-PMe efficiently inhibited proliferation,induced apoptosis and cell cycle arrest in G0/G1 phase,blocked Gl-S transition,and suppressed migration and invasion of gastric cancer cells.Crucially,these effects were apparent with almost no toxicity to normal cells.Besides these,AA-PMe has the ability to suppress constitutive STAT3 activation in gastric cancer cells.This inhibition was mediated by blockade of Janus-activated kinase 2(JAK2).Additionally,AA-PMe modulated the expression of STAT3-regulated factors,containing Bcl-2,cyclin D1,c-Myc,Bax,MMP-2 and MMP-9.Finally,transfection with STAT3 shRNA reversed the AA-PMe-driven modulation of STAT3-downstream gene products,which suggested that AA-PMe is a novel inhibtior of STAT3 and has the potential in gastric cancer prevention and therapy.Thirdly,we investigated the anti-angiogenic activity of AA-PMe in zebrafish embryos and human umbilical vein endothelial cells(HUVECs).Our results showed that AA-PMe disrupted the formation of inter-segmental vessels,the dorsal aorta and the posterior cardinal vein in transgenic zebrafish embryos.In HUVECs,AA-PMe inhibited cell viability,migration and tube formation in a dose-dependent manner.Compared to AA,AA-PMe showed a remarkable ability to inhibit angiogensis in vitro and in vivo via suppressing VEGF-induced phosphorylation of VEGFR2.Our results further indicated that AA-PMe inhibited angiogenesis via modulating VEGFR2-mediated signaling pathway with the involvement of some key kinases such as ERK and AKT.Together,this study reveals,for the first time,that AA-PMe acts as a potent VEGFR2 kinase inhibitor,exerts the anti-angiogenic activity at least partly through VEGFR2-mediated signaling pathway and may be a candidate anti-angiogenic agent for further research.Addtionally,we made research on drug absorption,metabolism and toxicity of zebrafish.established a reliable HPLC method to measure the content of AA-PMe in intra-and extra-of cell and zebrafish culture.The results showed the absorption of AA-PMe in cells and zebrafish was in a dose-dependent manner at low concentration,when the concentration of AA-PMe increased to a certain degree,the content of AA-PMe in intra-cell and zebrafish culture tended to be stable.In addtion,we found the toxicity of AA was stronger than AA-PMe in zebrafish.The acute toxicity dose of AA-PMe was above 25μM,while AA was above 10μM,and the chronic toxicity of AA-PMe was below 25μM.while AA was below 10μM.Morphological abnormalities including edema of the periphery and/or abdomen,hemorrhage,abnormal body shape,large yolk sac,and motility reduction were been observed in AA-PMe developmental toxicity assay.The pharmacokinetic study of AA-PMe provided a theoretical basis for revealing the mechanism of drug action.In conclusion,we deeply researched on AA and its derivatives in pharmacochemistry,pharmacology and pharmacokinetics aspects,which had a guiding significance in structure transformation of asiatic acid and activity evaluation of its derivatives in future.