Effect Of EGCG Derivative Y6 On Anti-angiogenesis And Synergistic Attenuated Daunorubicin Anti-hepatocarcinoma And Its Mechinsm In Vivo

Objectives:(1)To investigate the effects of EGCG derivative Y6 on angiogenesis in human hepatocellular carcinoma and its mechanism in vivo.(2)To study the effects and mechanism of EGCG derivative Y6 on the sensitization and attenuation of daunorubicin anti-hepatocarcinoma in vivo.Methods:BALB/C nude mice were injected subcutaneously with the suspension of HepG2 cells to establish the human hepatocellular carcinoma transplantation tumor model,and then were randomly divided into 8 groups(n=8,half male and half female),including the control group,EGCG group,Y6-M group,DNR group,DNR+Y6-H group,DNR+Y6-M group,DNR+Y6-L group and DNR+EGCG group.DNR was administered intraperitoneally to the animals every 4 days,while EGCG or Y6 was given orally to the animals daily.Mice were sacrificed and the tumor tissues were weighed 20 days after the treatment.The methods of part one: Endothelial cell specific marker CD34 was used to label vascular endothelial cells to calculate vascular density(MVD).Theexpression of VEGF mRNA was detected by RT-PCR.The expression of VEGF protein was measured by immunohistochemistry and Western blotting.The methods of part two:(1)Nude mice were weighed and the rate of tumor inhibition was calculated.The morphological changes of tumor tissue was observed by hematoxylin-eosin(HE)staining.The apoptosis of tumor cells were detected by a terminal deoxynucletidyl transferase-mediated dUTP neck end labeling(TUNEL assay).(2)The morphological changes of myocardium was observed by hematoxylin-eosin(HE)staining.Myocardial homogenates was prepared for the detection of SOD and MDA.(3)The expression of CBR1 mRNA was detected by RT-PCR.The expression of CBR1 protein was detected by immunohistochemistry and Western blotting.Chromatography(HPLC)was used to determine the concentration of daunorubicin metabolite daunorubicin in serum.The methods of part three: The expression of HIF-1? mRNA was detected by RT-PCR.The expression of HIF-1?protein was detected by immunohistochemistry.The expression of HIF-1?,ERK1/2/p-ERK1/2 and AKT/p-AKT proteins were measured by Western blotting.Results:Part I: Inhibitory effects of Y6 alone or in combination with DNR on angiogenesis was explored in HCC xenografts.(1)MVD results: Compared with control group,the MVD of EGCG group,Y6-M group or DNR group significantly decreased(P<0.05).Compared with DNR group,the MVD of tumor tissues in combination groups significantly reduced(P<0.01).The MVD of Y6-M alone or in combination with DNR group were significantly lower than that of EGCG alone or in combination with DNR(P<0.05 or P<0.01).Compared with DNR group,the MVD of tumor tissue in each combination group significantly reduced(P<0.01).The MVD of Y6-Malone or combined with DNR group was significantly lower than that of EGCG alone or combined with DNR(P<0.05 or P<0.01).With the increase of Y6 dose,the inhibitory effect of Y6 on tumor tissue MVD increased.(2)VEGF mRNA expression as follows: Compared with the control group,EGCG,Y6-M or DNR alone significantly significantly inhibited the expression of VEGF mRNA in tumor tissues(P<0.05 or P<0.01).Compared with DNR group,Y6 or EGCG combined with DNR decreased significantly the expression of VEGF mRNA.There was no significant difference in the expression of VEGF mRNA between Y6-M alone or in combination with DNR and EGCG alone or in combination with DNR.The inhibitory effect of Y6 on the expression of VEGF mRNA in tumor tissues was in an dose-dependent manner.(3)VEGF protein expression as follows: Compared with the control group,the expression of VEGF protein in tumor tissues of Y6-M group and DNR group decreased significantly(P<0.05 or P<0.01).Compared with DNR group,EGCG or Y6 combined with DNR was significantly down-regulated the expression of VEGF protein in tumor tissues(P<0.05 or P<0.01).Compared with EGCG alone or in combination with DNR,Y6-M alone or in combination with DNR decreased significantly the expression of VEGF protein(P<0.05 or P<0.01).The inhibitory effect of Y6 on the expression of VEGF was in an dose-dependent manner.Part II: The effect of EGCG derivative Y6 on the sensitization and attenuation of daunorubicin anti-hepatocarcinoma in vivo.1 Inhibitory effect of Y6 alone or combined with DNR on the growth of transplanted hepatocellular carcinoma in nude mice.(1)Tumor inhibition rate: The inhibitory effects of EGCG or Y6 alone was not very strong on the growth of transplanted tumors.However,it enhancedsignificantly the inhibitory effect of DNR on the growth of transplanted tumors.According to the Jin Zheng Jun method,the value of q was exceeded 1.15,Y6-M combined with DNR showed a synergistic effect on the growth of tumor.The inhibitory rate of Y6-M or Y6-H combined with DNR was significantly greater than that of EGCG combined with DNR.(2)Tumor tissue HE staining results: The tumor cells were markedly shaped,the proportion of nuclear plasma increased,and the nuclear dark stained.The degree of tumor necrosis in EGCG or Y6 combined with DNR group was more severe than DNR group.The area of tumor tissue necrosis increased with the increase of Y6 dose.(3)TUNEL staining results: The apoptotic cells in tumor tissues were stained by TUNEL and the nuclei were stained brownishly.Compared with the control group,the number of apoptotic cells in the EGCG group,Y6-M group and DNR group increased significantly(P<0.05 or P<0.01).Compared with DNR group,the number of apoptotic cells in the tumor tissue of each combination group significantly increased(P<0.01).Compared with DNR group,the number of apoptotic cells in combination groups increased significantly(P<0.01).The effect of Y6 alone or combined with DNR group on the apoptosis of tumor cells was significantly stronger than that of EGCG alone or in combination with DNR(P<0.05 or P<0.01),and was in an dose-dependent manner.2 Protective effect of EGCG derivative Y6 on daunorubicin induced myocardial toxicity in nude mice.(1)Myocardial tissue HE staining results: Compared with the control group,the myocardial cells in the EGCG group and Y6-M group were arranged neatly,the cell gap was normal without edema,and there was almost no toxicityto the myocardium of nude mice.In the DNR group,the myocardial fibers of the nude mice were disordered,the outline of the cells was unclear,and the cell gap was narrowed.EGCG and Y6 had protective effects on the myocardial tissue damage of nude mice caused by DNR.(2)Myocardial SOD activity and MDA content: Compared with the control group,SOD activity in the DNR group was significantly decreased(P<0.01),while MDA content was significantly increased(P<0.01).Compared with the DNR group,the SOD activity in the myocardial tissue of the combined treatment groups were significantly higher than that in the DNR group(P<0.01),and the MDA content was significantly reduced(P<0.01).The protective effect of Y6 on the DNR-induced cardiac toxicity was stronger than that of the EGCG with in a dose-dependent manner.3 EGCG derivative Y6 reduced the expression of CBR1 in tumor tissue and the production of daunorubicin alcohol.(1)CBR1 expression as follows: Compared with control,the expression of CBR1 mRNA decreased significantly in EGCG and Y6-M groups(P<0.05 or P<0.01).Compared with the DNR group,the expression of CBR1 mRNA in the tumor tissue of each combination group significantly decreased(P<0.01).The inhibitory effect of Y6 on the expression of CBR1 mRNA in the tumor tissue was in an dose-dependent manner.(2)VEGF protein expression as follows: Compared with the control group,the expression of CBR1 protein in Y6-M tumor tissue significantly reduced(P<0.05).Compared with DNR group,the expression of CBR1 protein in tumor tissue of EGCG or Y6 combined with DNR groups reduced significantly(P<0.01).(3)Daunorubicin alcohol concentration as follows: Both EGCG and Y6 could reduce the production of daunorubicin alcohol,and Y6 had a stronger inhibitory effect on DNR metabolism than EGCG.Serum daunorubicinol increased with Y6 dose reduced concentration.Part III:The effects of Y6 alone or combined with daunorubicin on the expression of HIF-1? in tumor tissues based on MAPK/ERK and PI3K/AKT signaling pathways.(1)HIF-1? expression as follows: The inhibitory effect of Y6 alone on HIF-1? gene and protein expression was significantly stronger than that of EGCG.There was not statistically significant between EGCG+DNR group and Y6-M+DNR group in the expression of HIF-1? gene in tumor tissue(P>0.05).Compared with EGCG+DNR group,the expression of HIF-1? protein in Y6-M+DNR group was significantly decreased(P<0.01).The expression of HIF-1? gene and protein were inhibited by Y6 in a dose-dependent manner.(2)ERK1/2/p-ERK1/2 and AKT/p-AKT proteins expression: EGCG,Y6 and DNR significantly inhibited the expression of p-ERK1/2 and p-AKT proteins in tumor tissues(P<0.05 or P<0.01),and Y6 was stronger than EGCG.Both EGCG and Y6 could enhance the inhibitory effect of DNR on the expression of p-ERK1/2 and p-AKT proteins in tumor tissues.The expression of p-ERK1/2 and p-AKT proteins were inhibited by Y6 in a dose-dependent manner.Conclusions:(1)EGCG derivative Y6 inhibits angiogenesis in human hepatocellular carcinoma xenografts.(2)EGCG derivative Y6 sensitizes and attenuates daunorubicin anti-hepatoma effect.(3)The effect of EGCG derivative Y6 on anti-angiogenesis and synergisticattenuated anthracycline antibiotics anti-hepatoma in vivo may be related to inhibition of MAPK/ERK and PI3K/AKT signal pathways down-regulating the expression of HIF-1?,CBR1 and VEGF.