Anti-Cancer Effect And Its Associated Mechanism Of TMEA,an Active Compound Of Sanguisorba Officinalis L.,through Angiogenesis Inhibition Cytotoxicity

Objective:The purpose of this study is to screen the anti-angiogenesis and cytotoxic active component from components of ellagic acid from Sanguisorba officinalis L.,and to explore anti-tumor characteristics of active component and its associated mechanism in vivo and in vitro.Methods:?1?We screen the active component from the tannins of Sanguisorba officinalis L.through the cytotoxic activity and anti-angiogenesis assays:The previously separated and prepared 10 kinds of ellagic acid were investigated by MTS using HUVEC,HepG2,A549,SW620 cell proliferation model on anti-angiogenesis and cytotoxic activity.?2?The isolation and structure identification of TMEA in vivo and in vitro:TMEA was prepared by using silica gel column with ethyl acetate and petroleum ether to flush.We collected the fractions and determined the content of the TMEA by HPLC.The structure of TMEA was identified by UHPLC-TOF-MS,¹³C-NMR and ¹H-NMR.?3?The anti-angiogenesis and cytotoxic activity of TMEA:Effects of TMEA on proliferation,migration and tube formation of HUVEC were studied by the MTS assay,wound healing assays and the tube formation respectively.Time-concentration effect of TMEA on the proliferation of HepG2,A549 and SW620cell were determined by MTS.The cell viability of TMEA on LO-2,HEK-293 and BMC were measured by MTS.Then,to investigate the effect of TMEA on SW620tumor transplanted in nude mice,the mice were randomly divided into five groups according to tumor volume,including Model,5-FU,and three groups of TMEA?200mg/kg,100 mg/kg and 50 mg/kg?.The mice were successively oral administered for21days,and growth of tumor volumes were measured by vernier caliper on the 0,3rd,7th,10th,14th,17th,21st day.The tumor tissue weighing and organ index were measured on the last day?21st day?.The protein expressions of CD31,Bax,Bcl-2,Caspase-3 were detected by immunohistochemistry on tumor tissue in vivo.?4?The mechanism of TMEA on inhibiton of angiogenesis and cytotoxic activity:The molecule docking was used to investigate combination TMEA to VEGFR2.The mRNA expressions of VEGF,PI3K,mTOR and Caspase-3,Bax,Bcl-2 were detectd by RT-PCR in HUVEC cell and SW620 cell,respectively.Moreover,the expressions of VEGF,PI3K/AKT/mTOR,Bax,Bcl-2,Caspase-3 were determined by western blotting.Results:?1?The proliferation of HUVEC,HepG2,A549 and SW620 cell was significantly inhibited by TMEA which is components of ellagic acid from Sanguisorba officinalis L.?P<0.01?,compared to control group.?2?The sample was prepared with ethyl acetate and petroleum ether?8:2?to flush silica gel column and its structure was identified as TMEA by UHPLC-TOF-MS,¹³C-NMR,¹H-NMR.?3?TMEA?5,10,20,40?g/mL?inhibited the proliferation,migration of HUVEC cells?P<0.01 or P<0.05?;TMEA?1,5,10,20,40?g/mL?may inhibit tube formation of HUVEC?P<0.01 or P<0.05?,compared to control group.Meanwhile,our study investigated that the proliferation of HepG2,A549 cells were significantly inhibited by TMEA on the concentration-depended manner?P<0.01 or P<0.05?,the proliferation of SW620 cell was significantly inhibited by TMEA?P<0.01 or P<0.05?.TMEA showed diffirent toxic effect on LO-2,HEK-293 and BMC?P<0.01or P<0.05?.The two groups of TMEA?200,100 mg/kg?could significantly inhibit the growth of tumor volume in SW620 transplanted tumor mice?P<0.01 or P<0.05?,and TMEA has no influence on organ index while decreased the spleen index.In the SW620 transplant tumor mice,the two groups of TMEA?200 mg/kg,100 mg/kg?can promote the expression of Caspase-3,Bax,and inhibit the expression of Bcl-2 and CD31?P<0.01 or P<0.05?,compared to model.?4?The molecule docking indicated that TMEA may combine with VEGFR2 in the active pockets of Asn223A,Gly922A and Leu840A.RT-PCR suggested that TMEA treatment decrease the expression of VEGF,PI3K,mTOR mRNA in HUVEC?P<0.01 or P<0.05?,compared to the control group.And the mRNA expression of Caspase-3 and Bax were significantly increased while mRNA expression of Bcl-2 mRNA was significantly inhibited in SW620 cell?P<0.01 or P<0.05?,Compared to control group.Western blotting results showed that TMEAsignificantlyinhibitedVEGFexpressioninHUVECcells,p-PI3Kp85?Tyr458?/PI3K,p-AKT?Ser473?/AKT,p-mTOR?Ser2448?/mTOR were decreased after TMEA treatment?P<0.01 or P<0.05?,compared to control group.The expression of Bax and Caspase-3 were significantly increased while Bcl-2 was significantly declined after TMEA treatment in SW620?P<0.01 or P<0.05?,compared to control group.Conclusions:TMEA could inhibit tumor angiogenesis and tumor cell proliferation simultaneously,whose mechanism is associated with the VEGF/PI3K/AKT/mTOR pathway,also is related to promote Bax,Caspase-3 protein expression and inhibit Bcl-2 expression to induce apoptosis of tumor cells.