Effects Of CP-25 Combined With 5-FU Against Hepatocellular Carcinoma

Liver cancer is one of the most common malignant tumors.The GLOBOCAN2012(Estimated Cancer Incidence,Mortality and Prevalence Worldwide in 2012)reported by International Agency for Research on Cancer(IARC)shows that the liver cancer incidence and mortality among all cancers are respectively ranked 6th and 2nd,and the incidence of male is significantly higher than female incidence.Hepatocellular carcinoma(HCC)is the primary type of liver cancer,which has high malignance,strong metastatsis and poor prognosis.Surgery is the most important mean in many HCC therapies(including surgery,radiotherapy,chemotherapy and liver transplantation,etc.).But due to the stealthiness of HCC,most patients are diagnosed as late and missed the best operation time.Chemotherapy is the leading mean of HCC treatment except surgical removal.However,the poor selectivity,easy drug resistance and side effects of chemotherapy drugs lead to the unsatisfied treatment effect.Therefore,it is particularly important to seek a safe and effective HCC chemotherapy drug.CP-25,a new kind of active monomer drugs-benzene sulfonyl paeoniflorin(paeoniflorin-6-oxygen-benzene sulfonic acid ester),is got from active monomerpaeoniflorin after esterification modification by our resreach laboratory,which has good anti-inflammatory and immune regulation.However,whether CP-25 has the effect of anti-HCC,has not been reported.Our group found that CP-25 can inhibit hepatoma cell SMMC-7721 proliferation and tumor growth of H22 transplanted tumor mice.So we speculate that the CP-25 has certain anti-HCC effect.But the preliminary experimental results show that the anti-HCC effects are weak when using CP-25 alone,which may be differ from cytotoxic clinical drug.The present study found that the digestive system tumor treatment medicine 5-FU is now the most common drug against gastrointestinal tumor,using 5-fluorouracil alone has exactly curative effect of HCC,but which has severe side effects,including gastrointestinal reaction,hair loss and bone marrow suppression,etc.The present study found combining some anticancer drugs can get a synergistic anticancer,non-overlap the major side effects,so it has the characteristics of high efficiency and low toxicity.Speculation by reading literature and previous experiment,we found that the part mechanism of CP-25 and 5-FU has a similar coincidence.Therefore,this topic was based on previous studies,using in vivo and in vitro experiment to explore the effect of CP-25 combined with 5-FU against HCC and its possible mechanisms.Objective Exploring the effect of CP-25 combined with 5-FU against HCC and its possible mechanisms.Methods1.Male,2 weeks,50 C57BL/6J mice were randomly divided into five groups,including normal group,model-,CP-25-(70 mg·kg-1),5-FU-(20 mg·kg-1)and CP-25+5-FU group(CP-25 70 mg·kg-1,5-FU 20 mg·kg-1),each group of 10.Excepting the control group,mice were intraperitoneally injected with DEN(2 mg·kg-1),and the normal mice were intraperitoneally injected with equal volume solvent.After buliding16 weeks,medicine group were respectively given by gastric gavage(CP-25,once a day and six times a week)or intraperitoneally injection(5-FU,once three days and two times a week)corresponding subjects persisting 8 weeks,normal group and model group to give the same amount of solvent.At 44 weeks,the mice were killed by bloodletting,liver,spleen,lung specimens were removed and weighed,and macroscopically visible liver tumors and nodules on liver surface were recorded.HE staining was used to observe the pathology change in mice tissue.The mice serum AST,ALT,ALP levels were detected by Reitman Frankel assay.And the mice serum AFP level was detected by ELISA.Western blot and immunohistochemistry were used to assay the expression of P53,Bax,Bcl-2,Caspase-9,Cytochrome c(Cyt c),MMP-2,MMP-9 in mice liver.2.The CP-25,5-FU,CP-25+5-FU were respectively added into the selected HCC cell lines HepG2 and Bel-7402 medium and incubated for 24 and 48 h.Effects of CP-25 combined with 5-FU on the proliferation of were intraperitoneally injected with were measured by CCK-8 assay.The apoptosis rate was detected by flow cytometry and the invasion and metastasis ability of HepG2 and Bel-7402 were observed by scratch test and transwell.The expression of P53,Caspase-9,Cytochrome c,Bax,Bcl-2 in HepG2 and Bel-7402,and MMP-2 and MMP-9 in Bel-7402 were assayed by western blot.Results1 Treatment function of CP-25 combined with 5-FU on DEN-induced HCC mice1.1 Effects on nodules and tumor volume in DEN-induced HCC mice The liver images showed the surfaces of liver were fruity,smooth and without nodule formation in normal group.The model mice liver had uneven surfaces and appeared obvious tumor nodules.Compared with the model group,CP-25,5-FU and CP-25+5-FU groups significantly reduced the number and volume of tumor nodules on liver surface,and compared with CP-25,5-FU alone groups,the number of tumor nodules in CP-25+5-FU group was obviously reduced.1.2 Effects on liver,spleen index in DEN-induced HCC mice The SPASS analysis showed that the model group mice liver,spleen indexes were increased significantly than normal group.Compared with model group,CP-25,5-FU and CP-25+5-FU significantly decreased the rised liver,spleen indexes induced by DEN.Compared with 5-FU alone group,the spleen index was significantly reduced while the liver index had no significant difference in CP-25+5-FU group.1.3 CP-25 combined with 5-FU significantly improved the liver pathological tissue damage induced by DEN HE pathological images showed that liver cells had clear nuclei,whole hepatic lobule structure,orderly hepatic cord in normal mice.However,in model mice,the size and morphology of liver cells were different,the structure of hepatic lobule was disorder and a large number of false flocculus and inflammatory cells infiltration could be found.CP-25,5-FU and CP-25+5-FU significantly improved liver damage,and reduce inflammatory cells infiltration and false flocculus form.Compared with CP-25 and 5-FU alone group,the CP-25+5-FU obviously improved liver pathological damage.1.4 CP-25 combined with 5-FU obviously decreased the rised levels of serum ALT,AST,ALP,AFP induced by DEN Compared with normal group,model group mice serum ALT,AST,ALP,AFP levels were significantly increased.CP-25,5-FU and CP-25+5-FU groups mice serum ALT,AST,ALP and AFP levels were significantly lower than model group.Compared with CP-25 and 5-FU alone group,the serum ALT,AST,ALP,AFP levels were obviously reduced in CP-25+5-FU.1.5 Effects on liver P53,Bax,Bcl-2,Cytochrome c,Caspase-9 in DEN-induced HCC mice Compared with the normal group,the model group mice liver tissue P53,Bax,Cytochrome c,Caspase-9 expression were significantly reduced and Bcl-2,MMP-2,MMP-9 expression were increased significantly.In CP-25,5-FU and CP-25+5-FU groups,P53,Bax,Cytochrome c,Caspase-9 expression were significantly increased and Bcl-2,MMP-2,MMP-9 expression were significantly reduced than model group.CP-25+5-FU mice liver tissue P53,Bax,Cytochrome c,Caspase-9 expression were obviously higher than model group,while Bcl-2,MMP-2,MMP-9 levels were obviously lower.2 Effects of CP-25 combined with 5-FU on HepG2 and Bel-74022.1 Effects on HepG2 and Bel-7402 growth Compared with control group,CP-25,5-FU and CP-25+5-FU groups all had growth inhibition effects on HepG2,Bel-7402,and the cell survival rate in CP-25+5-FU group was lower than CP-25,5-FU alone group.2.2 Effects on HepG2 and Bel-7402 apoptosis FCM detecting apoptosis rate showed that,compared with control group,CP-25,5-FU and CP-25+5-FU groups all induced the apoptosis of HepG2 and Bel-7402.After treated with CP-25+5-FU 48 h,the apoptotic rate of HepG2 and Bel-7402 cells in CP-25+5-FU group was significantly higher than CP-25 and 5-FU alone groups.2.3 Effects on the invasion and metastasis ability of Bel-7402 After treated with CP-25 combined with 5-FU 24 h,compared with control group,the invasion and metastasis ability of Bel-7402 in CP-25 group were significantly lower,while it was not obvious in 5-FU group.Compared with CP-25 and 5-FU alone groups,CP-25+5-FU group can obviously inhibit the invasion and metastasis ability of liver cancer cell.2.4 Effects on the expression of P53,Bax,Bcl-2,Cytochrome c,Caspase-9,MMP-2,MMP-9of HepG2 and Bel-7402 Western blot results showed that,compared with control group,the expression of Bax,P53,Caspase-9 and Cytochrome c in CP-25,5-FU and CP-25+5-FU groups were significantly increased,while the Bcl-2,MMP-2 and MMP-9 expression were decreased significantly.The expression of Bax,activated P53,Caspase-9 and Cytochrome c in CP-25+5-FU group cells were significantly higher than CP-25 and 5-FU alone groups,while the Bcl-2,MMP-2 and MMP-9 expression were significantly lower than CP-25 and 5-FU alone groups.Conclusion:1 CP-25 combined with 5-FU has an anti-HCC effect by inhibiting the occurrence and development of hepatocellular carcinoma(HCC)in mice induced by DEN.2 CP-25 combined with 5-FU can induce the hepatoma cells HepG2,Bel-7402 apoptosis and inhibit the invasion and metastasis of Bel-7402.3 CP-25 combined with 5-FU induce the HepG2,Bel-7402 apoptosis through activating the mitochondrial apoptosis induced by P53.