| Objective1.In vitro experiments were conducted to observe the effect of evodiamine on the proliferation capacity of human liver cancer cell lines HepG2,human liver cancer cell line MHCC97H and human liver cancer cell line MHCC97L.Two cell lines were selected to further study the effect of evodiamine on proliferation and apoptosis-related phenotypes and proteins,and to explore the interaction mechanism of evodiamine against tumor proliferation and apoptosis.2.In vivo experiments were conducted to establish subcutaneous tumor-bearing models of HepG2 hepatocellular carcinoma cells in nude mice,to observe the effect of evodiamine on subcutaneous tumor-bearing models of hepatocellular carcinoma cells in nude mice,and to further verify the effect and interaction mechanism of evodiamine on tumor proliferation and apoptosis through in vivo experiments.Methods1.The effect of evodiamine on the proliferation of human hepatocellular carcinoma cell lines HepG2,MHCC97H and MHCC97L was determined by MTT assay with concentrations of 5 ? mol/mL,2.5 ? mol/mL,1.25 ? mol/mL,0.625 ? mol/mL,0.3125 ? mol/mL,0.15625 ? mol/mL and 0 ? mol/mL.Subsequently,human liver cancer cell lines HepG2 and MHCC97H were selected for follow-up experiments.In this paper,the effect of evodiamine on long-term proliferation inhibition of HepG2 and MHCC97H human hepatocellular carcinoma cell lines was detected by plate clonal formation assay.To observe the effect of evodiamine on HepG2 and MHCC97H morphology of human hepatocellular carcinoma cell lines.Hoechst 33342 staining method was used for detecting the apoptosis effect of evodiamine on HepG2 and MHCC97H human liver cancer cell lines.For purpose of further validation of evodiamine and the correlation of human liver cancer cell lines HepG2,MHCC97H and apoptosis,in this article,using flow cytometry to detect evodiamine influence on human liver cancer cell apoptosis,and at the same time in order to verify the evodiamine associated with human liver cancer cell lines HepG2 and MHCC97H proliferation,in this essay,using flow cytometry to detect evodiamine effects on human liver cancer cell cycle.Western blot was performed to detect the effect of evodiamine on protein expression levels of Cleaved caspase3,Cleaved parp,Parp,P-erk,Erk,P-p38,P38,Bel-2,CyclinB1 and P53.In order to further study the interaction mechanism of evodiamine on human liver cancer cells,this paper selected human liver cancer cell line MHCC97H for discussion.According to the previous experiments,this paper used Erk inhibitor U0126 and P38 small molecule interfering RNA(Si p38 RNA)to explore the relevant mechanism,and used MTT method and WB to select the appropriate concentration of U0126 and Si p38 RNA.Western-blot was used to detect the effect of evodiamine+U0126 or Si P38 RNA on Cleaved parp,Parp,P-erk,Erk,P-P38,P38 protein expression levels in cells.2.The HepG2 cells were inoculated under the skin of the right costal waist of nude mice to establish the subcutaneous tumor-bearing model of liver cancer in nude mice.After successful modeling,the nude mice were randomly divided into three groups according to tumor volume:The blank group,sorafenib group,evodiamine group,respectively give 0.5%carboxyl cellulose solution,sorafenib(30mg/kg/d),evodiamine(30 mg/kg/d)oral lavage,weighing at least every three days weight and size of tumors,and after the experiment,put to death at least remove subcutaneous tumor,weighing the subcutaneous tumor weight,observe the changes of tumor weight.Western blot was used to analyze the effect of evodiamine on expression levels of Cleaved parp,Parp,P-erk,Erk,P-p38,P38 and CyclinB1 protein in tumor tissues.Immunohistochemical was used for examination of evodiamine on expression levels of Cleaved parp,P-erk and P-p38 related proteins in tumor tissues.Results1.(1)MTT results showed that at the concentration of 1.25 ? mol/ml for 48h,evodiamine could evidently inhibit the proliferation of HCC cells,which was obviously different from the control group(F=58.278,P=0.000;F=37.523,P=0.000;F=45.824,P=0.000),especially HepG2 and MHCC97H,which changed significantly with the increase of time and dose.The IC50 of HepG2,MHCC97H and MHCC97L 48H were 1.49 ?mol/ml,0.96 ? mol/ml and 3.27 ? mol/ml,respectively.(2)tablet clone forming experiments show that evodiamine can apparently inhibit HepG2 and MHCC97H cells of long-term proliferation,and increased with the increase of the dose inhibit obviously depend on a concentration,the drug concentration of 1.25 ? mol/ml,evodiamine can clearly inhibit the appreciation of liver cancer cells,compared with the blank group with significant difference(F=21.686,P=0.028;F=113.869,P=0.000).(3)in 48 h after drug intervention,compared with the blank group,obvious changes have taken place in drug group of cell morphology,from the original spindle type,polygons,irregular shape,dough stick wall growth of cells and gradually shrink,deformation,fall off,cell volume smaller,refraction,cell membrane bubbles,the distance between cells significantly larger,and the morphology changes with the increase of dosage and more obvious.(4)the Hoechst 33342 staining method showed that,compared with the blank group,the drug group showed significant changes in apoptosis morphology with the increase of dose,and obvious apoptosis characteristics were observed:nuclear retraction,apoptotic corpuscles,membrane vacuoles,etc.,with fragmented nuclei and dense,bright blue hyperchromatic nuclei.(5)flow cytometry showed that apoptosis was significantly increased in the drug group with the increase of drug dose.At the drug concentrations of 1.25 ?mol/ml,evodiamine could apparently inhibit the proliferation of human liver cancer cells,which was obviously different from the blank group(F=364.535,P=0.000;P=0.031).The average apoptosis rate of HepG2 cells was 3.13%,9.50%,22.20%,32.60%,and 7.33%,42.57%,56.20%,58.97%,respectively,for MHCC97H cells.The apoptosis of MHCC97H cells was significantly increased atl.25? mol/ml,but the change was not obvious with the increase of dose.And HepG2 cells changed evidently with the increase of dose.(6)flow cytometry showed that G0/G1 phase and S phase decreased with the increase of drug dose,G0/G1 phase of HepG2 decreased from 62.84%to 36.33%,S phase 27.93%to 0.00%,MHCC97H G0/G1 phase 50.29%to 39.62,S phase 33.26%to 12.90%,and G2/M clearly increased,G2/M phase of HepG2 increased from 9.22%to 63.67%,and G2/M phase of MHCC97H increased from 16.44%to 47.48%.(7)The results of western-blot showed that:evodiamine could obviously regulate the expression of Cleaved caspase3,Cleaved parp,p-erk,p-p38,CyclinB1 and P53 protein in human liver cancer cell line HepG2,which was statistically significant compared with the blank group(P=3.071,P=0.041;F=14.768,P=0.001;P=0.038;F=13.452,P=0.002;F=2.078,P=0.044;F=3.336,P=0.037);The expression level of the apoptotic inhibitory protein Bcl-2 was significantly reduced,which was statistically significant compared with the blank group(F=6.978,P=0.013).However,there was no significant effect on Parp,Erk and P38 protein,which was not statistically significant compared with the blank group(F=0.722,P=0.567;P=0.091;F=0.051,P=0.981);Evodiamine can distinguishly regulate the expression of Cleaved caspase3,Cleaved parp,P-erk,P-p38,CyclinB1 and P53 protein in human liver cancer cell line MHCC97H,which was statistically significant compared with the blank group(P=0.027;F=15.791,P=0.001;F=2.905,P=0.043;F=2.902,P=0.043;F=6.641,P=0.015;F=4.803,P=0.034);The expression level of apoptotic inhibitory protein Bcl-2 was significantly reduced,which was statistically significant compared with the blank group(F=3.079,P=0.042).However,there was no significant effect on Parp,Erk and P38 protein,which was not statistically significant compared with the blank group(F=0.031,P=0.991;F=0.255,P=0.856,F=0.020,P=0.996).Using Erk inhibitor U0126 and P38 small molecule interfering RNA,the results showed that the expression of Cleaved parp protein was reduced by combining evodiamine with U0126 or Si p38 RNA,which was clearly different from the control group(F=25.568,P=0.000;F=327.293,P=0.000).2.The results of the tumor bearing nude mice model of HepG2 cells showed that the body weight of the tumor-bearing nude mice in the dose group of evodiamine increased significantly,which was obviously distinguish from the control group(F=8.672,P=0.039).The subcutaneous tumor weight of the mice in the dose-group of evodiamine and the dose-group of sorafenib was significantly reduced,which was clearly different from normol group(F=28.114,P=0.043;F=28.114,P=0.000).The expression levels of Cleaved parp,P-erk,P-p38,and CyclinB1 in tumor tissues were up-regulated by evdiamine,which was significantly differed from the control group(F=51.030,P=0.000;F=4.259,P=0.042;F=16.792,P=0.001;F=23.076,P=0.001),but had no significant effect on Parp,Erk,and P38 proteins,which were not statistically significant(F=0.097,P=0.752;F=0.439,P=0.941;F=0.537,P=0.605).Sorafenib upregulated Cleaved parp,P-p38,and CyclinB1 protein expression in tumor tissues,which were obviously distinguish from the blank group(F=51.030,P=0.000;F=16.792,P=0.016;F=23.076,P=0.001),but had no significant effect on Parp,Erk,and P38 protein,which was not statistically significant compared with the control group(F=0.097,P=0.691;F=0.439,P=0.470;F=0.537,P=0.641).Conclusion1.Evodiamine can obviously inhibit the proliferation of hepatocellular carcinoma cells.The results of MTT assay and plate cloning assay indicated that evodiamine could inhibit the proliferation of HCC cells in short or long term.Flow cytometry showed that evodiamine could block hepatocellular carcinoma cells in G2/M phase.WB results showed that evodiamine can significantly up-regulate the protein expression of CyclinB1,which is closely related to G2/M phase.2.Evodiamine can induce apoptosis of liver cancer cells.The results of MTT experiment,morphological observation and Hoechst 33342 staining indicated that evodiamine may promote the death of liver cancer cells through apoptosis.Further flow cytometry showed that evodiamine could induce apoptosis in hepatocellular carcinoma cells.WB results showed that evdiamine could obviously up-regulate the expression levels of Cleaved caspase3 and Cleaved parp protein in liver cancer cells.It also down-regulated the expression of Bcl-2 protein,which inhibited apoptosis.3.Both evodiamine and sorafenib can induce apoptosis of liver cancer cells through the MAPK pathway and play a role in tumor treatment,but the specific mechanism is different.The results of in vivo and in vitro experiments showed that evdiamine mainly up-regulated the expression of Cleaved parp protein through the activation of P-erk and P-p38 in MAPK,and promoted the apoptosis of liver cancer cells.In vivo experiments showed that sorafenib mainly up-regulated the expression of Cleaved parp protein by activating P-p38 in MAPK,so as to promote the apoptosis of liver cancer cells.4.The anti-tumor effect of evodiamine is the result of multi-target and multi-pathway.It can affect the tumor from many aspects,so as to play the role of anti-tumor. |
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