| Hepatocellular carcinoma(HCC)is a high-occurrence regional tumor in Guangxi and one of the most common malignant tumor in the world,and so it seriously endangers people's health and living quality.The high priority is to seek for much more safer and effective anti-hepatoma effects drugs.As the highest content of catechins in tea,(-)-epigallocatechin-3-gallate(EGCG)has the anti-tumor,reversing multidrug resistance of HCC,anti-oxidation,inhibiting angiogenesis and extensive pharmacological activities.EGCG can inhibit the growth of hepatoma carcinoma cells and angiogenesis of HCC by inhibiting HIF-1 alpha,reverse multidrug resistance(MDR)of HCC and inhibit the metabolism of anthracycline.But because of the special characters of such as instable,bad fat-soluble,poor bioavailability,fast metabolism and short action time,the potency of EGCG should be improved.This study was to synthesize EGCG derivatives of structural modification,screen their anti-hepatocarcinoma effects and investigate its mechanism in vitro,research the synergy and attenuation action on anthracycline drugs by using different techniques involving molecular biology,cell biology and experimental zoology.All these results will provide an experiment and theoretical basis for finding EGCG derivatives that are much more effective,safe and stable than EGCG,enlarging the anti-hepatocarcinoma application of EGCG and investigating new multi-targeted anti-hepatocarcinoma drugs.Part I:Preparation of EGCG derivatives of structural modificationObjective:To prepare EGCG ethylated derivatives with EGCG as a lead compound by modifying the chemical structural.Methods:EGCG reacted with(CH3CH2O)2SO2 to produce crude products of EGCG ethylated derivatives with nitrogen as protection,K2C03 as catalyst and acetone as reaction medium.The reaction products were separated and purified by medium pressure preparative liquid chromatography.Chemical structures of the purified compounds were identified by 1H-NMR,13C-NMR,2DNMR and mass spectrometry.Results:Six compounds were separated and purified.They were EGCG ethylated derivatives of different degree of ethyl group substitutions.There chemical structures were identified respectively as:(1)5,7,3',4',5',3",4",5"-eight-O-ethyl-EGCG(Y1);(2)5,7,3',4',3",4",5"-seven-O-ethyl-EGCG(Y2);(3)5,7,4',5',3",4",5"-seven-O-ethyl-EGCG(Y3);(4)5,3',4',5',3",4",5"-seven-O-ethyl-EGCG(Y4);(5)5,7,3',4',3",4"-six-O-ethyl-EGCG(Y5);(6)3',4',3",4",5"-six-O-ethyl-EGCG(Y6).Conclusion:Six new compounds that had been prepared were EGCG ethylated derivatives.Part ?:Study on anti-hepatocarcinoma effect and its mechanism of EGCG derivatives of structural modification in vitroObjective:To screen anti-hepatocarcinoma effect of EGCG ethylated derivatives in vitro,analyze the mechanism of action and structure-function relationship preliminarily in order to obtain EGCG derivatives that are much more effective or stable than EGCGMethods:Hepatocellular carcinoma cell line SMMC-7721 and HepG2 were used,MTT assay was performed to examine the inhibitory effects of EGCG ethylated derivatives on the proliferation of cells and IC50 was calculated.And then real-time PCR and western blot were used to detect HIF-1?,VEGF and CBR1 genes and protein expression level under normal condition and hypoxic condition in vitro respectively.Molecular docking and ADME prediction were carried out by computer software.Results:(1)EGCG ethylated derivative Y5 and Y6 can dose-dependently inhibit the proliferation of SMMC-7721 and HepG2 cells in vitro by MTT assay.In SMMC-7721 cells,the IC50 of EGCG ethylated derivative Y5 and Y6 were 232.9?mol/L and 61.4?mol/L,respectively,and that were lower than EGCG's(247.8?mol/L).In HepG2 cells,The IC50 of EGCG ethylated derivative Y5 and Y6 were 229.9?mol/L and 43.5mol/L respectively,and that also were lower than EGCG's(357.1?mol/L),especially the IC50 of Y6 was the lowest among them.(2)Real-time PCR showed that the mRNA expression of HIF-1?,VEGF and CBR1 remarkably increased in SMMC-7721 and HepG2 cells under hypoxic condition(P<0.01),compared with that under normal condition.EGCG ethylated derivative Y6 and AcEGCG remarkably inhibited the mRNA expression of HIF-1?,VEGF and CBR1(P<0.01 or P<0.05)in SMMC-7721 cell.In HepG2 cell,EGCG ethylated derivative Y6 and AcEGCG remarkably inhibited the mRNA expression of HIF-1? and-VEGF(P<0.01 or P<0.05),the high dose of drugs depressed the mRNA expression of CBR1.(3)Western blot showed that the protein expression of HIF-1?,VEGF and CBR1 remarkably increased in SMMC-7721 and HepG2 cells under hypoxic condition(P<0.01).EGCG ethylated derivative Y6 and AcEGCG remarkably inhibited the protein expression of HIF-1?,VEGF and CBR1(P<0.01 or P<0.05)in SMMC-7721 cell,but the inhibition effect was not significant in low dose of drugs.In HepG2 cell,EGCG ethylated derivative Y6 and AcEGCG inhibited the protein expression of HIF-1? and VEGF(P<0.01 or P<0.05),but had no effect on the protein expression of CBR1.(4)Molecular docking results expressed that the affinity in sequence from high to low was:Y6,Y5,AcEGCG and EGCG on HIF-1? and VEGFR,and was:Y6,Y5,EGCG and AcEGCG on CBR1.(5)ADME prediction results showed that lipophicity in sequence from high to low was:Y6,Y5,AcEGCG and EGCGConclusion:EGCG ethylated derivative Y5 and Y6 have more higher anti-hepatocarcinoma effect in vitro than EGCG The mechanism of anti-hepatocarcinoma effect may be related to HIF-1? mediated signaling pathways.The affinity between EGCG derivatives of structural modification and HIF-1??VEGFR and CBR1 are stronger than EGCG.And the lipophicity of EGCG derivatives of structural modification are higher than EGCGPart ?:The effect of EGCG derivatives of structural modification on angiogenesisObjective:To study the effect of EGCG derivatives of structural modification on angiogenesis in vivo and in vitro,and then discuss the mechanism of anti-hepatocarcinoma effect.Methods:Human umbilicus vein endothelial cell(HUVEC)was used in vitro,MTT assay was performed to examine the inhibitory effects of EGCG ethylated derivatives on the proliferation of cells and IC50 was calculated.And then the chicken chorioallantoic membrane(CAM)vascular models were used to observe the inhibition of angiogenesis in vivo.Results:(1)EGCG,AcEGCG and EGCG ethylated derivative Y6 can dose-dependently inhibit the proliferation of HUVEC in vitro by MTT assay.The IC50 of AcEGCG and EGCG ethylated derivative Y6 in HUVEC were 97.9?mol/L and 62.7?mol/L respectively,and that were lower than EGCG's(209.3 ?mol/L),while the IC50 of Y6 was the lowest among them.(2)In CAM test,the result showed that AcEGCG and EGCG ethylated derivative Y6 could dose-dependently notably inhibit angiogenesis,especially for the second and third grade capillaries,which had great significance compared with that of negative group(P<0.05 or P<0.01).There were no significant differences between Y6,AcEGCG and the same number of moles of EGCG in anti-angiogenesis effect.Conclusion:AcEGCG and EGCG ethylated derivative Y6 can inhibit angiogenesis in vivo and in vitro,and they are more powerful than EGCG in vitro,but there is no significant difference in vivo.These results suggest that anti-angiogenesis may relate to its anti-hepatocarcinoma effect.Part IV:Experimental study on the synergistic effect of EGCG derivatives of structural modification combined with anthracycline drugs for HCC in vitroObjective:To study the synergistic effect of EGCG ethylated derivative Y6 combined with anthracycline drugs for HCC in vitro.Methods:Hepatocellular carcinoma cell line SMMC-7721 and HepG2 were used in vitro.MTT assay was performed to examine the inhibitory effects of EGCG ethylated derivative Y6 combined with daunorubicin(DNR)on the proliferation of cells.Analysis of cell apoptosis induced by EGCG ethylated derivative Y6 and DNR combination by flow-cytometry.Results:(1)MTT assay demonstrated that the growth inhibition rates in SMMC-7721 and HepG2 cells by different concentration of Y6/DNR combination were significantly higher than that treated with DNR alone.And the IC50 of combination Y6/DNR was lower than that of DNR alone.There was no obvious difference in the growth inhibition rates between Y6/DNR and EGCG/DNR,but the dose of Y6(23.9?mol/L)was lower than EGCG(65.4?mol/L).(2)Flow-cytometry assay showed that apoptosis rate of SMMC-7721 is 10.59%when cell treated with DNR alone.The apoptosis rates were increased significantly when combined two drugs treatment(EGCG/DNR:16.31%,Y6/DNR:16.19%)(P<0.05).There was no obvious difference in the apoptosis rates between Y6/DNR and EGCG/DNR,but the combination dose of Y6(23.9?mol/L)was lower than EGCG(65.4?mol/L).The apoptosis rate of HepG2 is 27.19%when cell treated with DNR alone.The apoptosis rates were increased significantly when combined two drugs treatment(EGCG/DNR:35.23%,Y6/DNR:33.16%)(P<0.05).There was no obvious difference in the apoptosis rates between Y6/DNR and EGCG/DNR,but the combination dose of Y6(12.8?mol/L)was lower than EGCG(65.4?mol/L).Conclusion:EGCG ethylated derivative Y6 can enhance the effect of DNR on inhibition of HCC cells proliferation,and increase the apoptosis rates of HCC cells.And the synergistic anticancer effect of Y6 is superior to EGCG.Part V:Experimental study on the attenuation action of EGCG derivatives of structural modification on anthracycline drugs-induced cardiotoxicityObjective:To study the effects of EGCG derivatives of structural modification on anthracycline drugs-induced cardiotoxicity and preliminary discusse the mode of action.Methods:Zebrafish embryos were induced by doxorubicin(DOX)as models for analysis of heart toxicity and were treated with EGCG derivatives of structural modification,and then morphological and functional changes of embryos hearts were observed.Mice were induced by DNR as myocardium injury models and were treated with EGCG and AcEGCG The electrocardiogram,activities of myocardial enzymes and the levels of cardiac troponin T hs(TNT-Hs)in serum,the activities of SOD and the contents of MDA in serum and myocardium were measured,and the cardiac ultrastructural changes were observed with scanning electron microscope.Results:(1)Cardiac toxicity of zebrafish embryos assay demonstrated that EGCG derivatives of structural modification can reduce cardiac toxicity induced by DOX in different extent,improve the morphological changes of embryos hearts and increase stroke volume,especially the attenuation action of Y6 was the strongest among them.(2)Cardiac toxicity of mice assay showed that EGCG and AcEGCG can improve the general growth status of mice,decrease the incidence of arrhythmia,inhibit the activities of myocardial enzymes such as ALT?AST?CK?CK-MB?LDH and ?-HBDH,decrease the levels of TNT-Hs in serum,improve the activities of SOD,decrease the contents of MDA in serum and myocardium.The ultrastructure of myocardium showed attenuated injury in mice of AcEGCG and EGCG group,compared to model group.Conclusion:EGCG derivatives of structural modification have protective effects against anthracycline drugs-induced cardiotoxicity... |
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