| Objective: Human hepatocellular carcinoma(HCC)is known as the king of all in many cancer in many cancer,The key problem for the treatment of hepatocellular carcinoma is easy to transfer and make multi drug resistance(MDR)to chemotherapeutic drugs.It has been reported that(-)-Epigallocatechin gallate(EGCG)can inhibit the carbonyl reductase activity in the metabolism of anthracyclines,thereby increasing the sensitivity of its anti liver cancer and reducing its cardiac toxicity.However,due to the unstable nature of EGCG,short duration,low bioavailability,and the effect of the strength to be improved,it is necessary to optimize the structural modification.To this end,the subject group in the previous experiment independently synthesized acetylation(such as AcEGCG),methyl of,ethyl of(such as y6)and propyl of EGCG and so on many kinds of EGCG derivatives by activity screening found that acetylation and ethylated EGCG derivatives than the equivalent dose of EGCG collaborative daunorubicin inhibiting effect on liver cancer cells stronger,but with EGCG whether the same mechanism is still unclear.Therefore,in order to further verify the EGCG derivatives of EGCG with the same target to the mechanism of action in this study,in order to find out the than EGCG is more stable,efficient,nontoxic derivatives,to discover and create EGCG derivatives become new target to the anti hepatoma drugs,improve the effect of chemotherapy and provide a theoretical basis and experimental evidence.PartⅠ: The effect of EGCG derivatives of carbonyl reductase activityObjective: To Study whether EGCG derivate inhibit CBR1 enzymes reduce human hepatocelluar carcinoma(HCC)generated the metabolites of DNR in vitroMethods: To determined the influence of EGCG 、AcEGCG and ethylation Y6 on DNR production of DNROL metabolites in Hep G2 and SMMC-7721 cells lysis liquid by HPLC.Results: EGCG and its derivatives Y6 in cell lysis liquid DNR metabolism has a certain degree of inhibition,the SMMC7721 cell lysis solution to produce DNROL was 13.86 ng/mgprotein/min,compared with normal control group,the generation of DNROL in the EGCG low,medium and high(15μM 、30μM、 60μM)-dose group were reduced by 13.39%,25.75%,42.78%,the generation of DNROLin the AcEGCG high,medium and low(15μM 、30μM、 60μM)-dose group were reduced by10.73%,21.9%,37%,the generation of DNROL in the Y6 low,medium and high(15μM 、30μM、 60μM)-dose group were reduced by 18.7%,36.37%,53.25%.The results of AcEGCG medium and high-dose group and all dose groups of EGCG and Y6 have significant differences(P<0.05),but the results of low AcEGCG-dose group was no statistical significance(P>0.05).In the same reaction system,the Hep G2 cell lysis solution to produce DNROL was 13.13ng/mgprotein/min,compared with the control group,the production of DNROL in the EGCG low,medium and high(at the same concentrations of SMMC7721 cell)-dose group were decreased by13.43%,27.8%,44.32%,the production of DNROL in the AcEGCG low,medium and high-dose group were decreased by 11.59%,23.26%,41.01%,the production of DNROL in the Y6 low,medium and high-dose group were decreased by 18.75%,37.87%,55.9%.The results of AcEGCG medium and high-dose group and all dose groups of EGCG and Y6 have significant differences(P<0.05),but the results of low AcEGCG-dose group was no statistical significance(P>0.05).Conclusion: AcEGCG and Y6 can inhibit the enzymatic activity of CBR1 directly,to reduce the he production of DNROL,and the Y6 is the most strong.PartⅡ: The effect of EGCG derivatives of CBR1 protein expression by MAPK/AKT and PI3K/AKT signaling pathwayObjective: To study the effect of EGCG derivatives of CBR1 protein expression by MAPK/AKT and PI3K/AKT signaling pathway in vitroMethods: Immunohistochemical method was used to detect under normal condition and hypoxic condition Y6 influence the HIF-1 a and CBR1 protein expression on hepatocellular carcinoma cell SMMC-7721;Western Blot detection under cultured in the normal condition and hypoxic condition,EGCG and its derivatives for hepatocellular carcinoma cells in the MAPK/ERK signaling pathway(and join ERK1/2 inhibitor PD98059)changes the expression of phosphorylation protein p-ERK1/2 and PI3K/AKT signaling pathway(and join AKT inhibitors LY294002)p-AKT phosphorylated protein expression changes and effects on CBR1 protein expression.Results: Immunohistochemical staining showed that the HIF-1α and CBR1 protein is mainly distributed in cytoplasm and part of the nuclear membrane,a few located in the nucleus in SMMC-7721 cells,the protein expression of HIF-1α and CBR1 remarkably increased in SMMC-7721 cells under hypoxic condition,EGCG ethylated derivative Y6 remarkably inhibited the protein expression of HIF-1α and CBR1.Western blot showed that the protein expression of HIF-1αand CBR1 remarkably increased in SMMC-7721 cells under hypoxic condition(P﹤0.01).EGCG ethylated derivative Y6 remarkably inhibited the protein expression of CBR1(P﹤0.05)in SMMC-7721 cell;compared with the normal condition and hypoxic condition the protein expression of ERK1/2 and AKT were not significant differences,EGCG and Y6 have not effect on the expression of ERK1/2 and AKT,however,compared with the normal condition,the protein expression of p-ERK1/2 and p-AKT remarkably increased in SMMC-7721 cells under hypoxic condition,which has statistical significance(P﹤0.05),EGCG and its derivatives Y6 can reduce hepatocellular carcinoma MAPK/ERK signaling pathway in the expression of p-ERK1/2 protein and the p-AKT of PI3K/AKT signal channel protein expression,after joining the MAPK/ERK signaling pathway inhibitors(PD98059),p-ERK1/2 content decreased Rapidly,the levels of CBR1 expression are significantly reduced,similarly,after joining the PI3K/AKT signaling pathway inhibitors(LY294002),p-AKT content decreased significantly,CBR1 levels are significantly reduced,to explain the MAPK/ERK and PI3K/AKT signaling pathways are regulate the expression of CBR1 protein.Conclusion: The hepatocellular carcinoma cells increasing the high expression HIF-1α to stimulate CBR1 content with hypoxia induced,lead to the DNR drug resistance;EGCG and its derivatives Y6 can reduce the CBR1 expression to strengthen daunorubicin antitumor activity.EGCG and its derivatives Y6 remarkably inhibited the protein expression of CBR1 by HIF-1α-MAPK/ERK and PI3K/AKT signaling pathway in SMMC-7721 cells.PartⅢ: The experimental research of EGCG derivatives sensitization attenuated anthracycline-based antibiotic resistance to HCCObjective: To study the effect of EGCG derivatives on DNR induced cardiac toxicity and tumor weight in HCC Hep G2 xenograft nude mouseMethods: To select 80 nude mouse with normal electrocardiogram and set up HCC Hep G2 xenograft Model.Then randomly divided into 8 groups with 10 mousees(half male and half female)per group:(1)normal control;(2)EGCG(40mg/kg);(3)Y6-M(55 mg/kg);(4)DNR(2 mg/kg)model;(5)DNR(2 mg/kg)+EGCG(40mg/kg);(6)DNR(2 mg/kg)+Y6-L(27.5 mg/kg);(7)DNR(2 mg/kg)+ Y6-M(55 mg/kg);(8)DNR(2 mg/kg)+ Y6-H(110 mg/kg).The Electrocardiogr-am,longes and shortest of tumor diameter,body weight and tumor weight were measured.Results: The results of drug efficacy,EGCG,Y6 and DNR had effect on inhibiting tumor growth.But the effection of EGCG and Y6 were not strong,While the EGCG and Y6 respectively with DNR combined effect is greater than when they used alone.The results of drug toxicity,the body weight of EGCG and Y6 used alone compared with the normal group had no obvious change(P>0.05);compared with the normal group,the body weight of DNR group was decreased(P﹤0.05);The body weigh change between control group and DNR group of EGCG and Y6 respectively by the cooperative use of DNR.In addition,we also adopt electrocardiogram to detect the nude mouse heart toxicity,the results show that EGCG and Y6 had not influenced on electrocardiogram,but after giving DNR,nude mouse in a certain degree of cardiac toxicity,the electrocardiogram of EGCG,Y6 and DNR togethere respectively had obvious improvement.The results suggest that EGCG and Y6 can protect the myocardial cells,antagonism DNR cardiac toxicity.Conclusion: EGCG derivatives Y6 can enhance DNR resistance to cancer of the function of HCC and lower DNR cardiac toxicity. |
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