| Objective: The wide range of molecular kinase inhibitory effects of sorafenib can suggest that combined of antiviral drug new type cytokine IFN-Lambda 3 to enhance the clinical benefit of hepatocellular carcinoma.Here,we first evaluated the combined use of IFN-Lambda 3 as an adjuvant therapy for sorafenib in HCC.In the first part of the study,we aim to investigate experimental studies on human HCC cell lines in vitro,observing the combined drug use patterns of two drugs,whether there is a synergistic inhibitory effect on the proliferation of human HCC cell lines and stimulate the pathways of apoptosis,and to explore its potential underlying mechanisms.At the same time,in order to better explore whether the combination of the two drugs has potential anti-HCC effect and clinical application value.In the second part of the study,we selected that express IFN-Lambda receptor complexes and application of human hepatocellular carcinoma cell line from above experiments,to establish an immunodeficient nude mice xenograft tumor model and observe the growth of nude mice transplanted for the final evaluation.Methods:(1)Human hepatocellular carcinoma cell line include Hep G2,SMMC-7721,SK-HEP-1,BEL-7402 and normal human liver cell line L-02 were cultured and human liver cancer tissues were collected from HCC patients,real-time quantitative-PCR screening of specific IFN-Lambda receptor complexes(IL-28R1 and IL-10R2)gene expression level was detected,the expression of IL-28R1,IL-10R2 m RNA in cell lines was determined,with two human hepatocellular carcinoma cell lines as target cells,followed by the subsequent parallel independent experiments;(2)The experimentally determined cell were cultured,using sorafenib and IFNLambda 3 as the in vitro drug intervention,experimental groups were as follows: 1)Control group: cells were cultured under normal conditions;2)IFN-Lambda 3 treatment group: hepatocellular carcinoma cell lines were cultured,drug treatment cells with 10 ng/m L;3)Sorafenib treatment group: hepatocellular carcinoma cell line was cultured,drug treatment cells with 3 ?mol/L;4)IFN-Lambda 3+Sorafenib combined treatment group: hepatocellular carcinoma cell line was cultured,3 ?mol/L Sorafenib and 10 ng/m L IFN-Lambda 3 treatment cells.The two cell lines selected above experiment,cultured 24 and 48 hours later,cells were collected,the survival rate was detected by CCK-8,cell cloning assay detected colony formation ability of cells in the two groups;(3)24,48 hours later,flow cytometry was used to detect the cell cycle and cell apoptosis,the changes of mitochondrial membrane potential and intracellular reactive oxygen species in the two groups of hepatocellular carcinoma cell lines;(4)The above-mentioned two groups of hepatocellular carcinoma cell lines were cultured,and the cells were collected after 48 hours.The expression levels of p21,cyclin D1 and cleaved-caspase-3 were detected by Western blot;(5)Human hepatocellular carcinoma cell line SMMC-7721,select the logarithmic growth state of the cells to establish nude mice subcutaneous transplantation,according to the different methods of drug intervention treatment,16 nude mice can be divided into the following four groups: 1)Control group: 2 m L/kg saline once per day for 28 days;2)IFN-Lambda 3 treatment group: 0.1 mg/kg once per day for 28 days;3)Sorafenib treatment group: 5 mg/kg once per day for 28 days;4)IFN-Lambda 3+Sorafenib combine treatment group: 0.1 mg/kg IFN-Lambda 3 and 5 mg/kg sorafenib once per day for 28 days;(6)Observing the general situation of transplanted nude mice under Sorafenib and IFN-Lambda 3 and the volume of transplanted tumor;After 28 days,histopathological observation and proliferating protein Ki-67 were detected by pathology H-E and immunohistochemical staining in nude mice,the number of apoptotic cells was observed by Tunel staining.Results: The Hep G2,SMMC7721,BEL-7402 cell lines and human hepatocellular carcinoma specimens were drawn at the level of genes,which have expression m RNA of IFN-Lambda receptor complexes,but the expression of SK-Hep-1 was negative;CCK-8 and colony formation assay show that sorafenib and IFN-Lambda 3 have synergistic inhibition of growth of human hepatocellular carcinoma cells,flow cytometry showed that sorafenib and IFN-Lambda 3 have synergistic induction of apoptosis and G0/G1 phase arrest;and sorafenib and IFN-lambda 3 promotes reactive oxygen loss of mitochondrial membrane potential and intracellular production of p21 induced cleaved-caspase-3 and expression of cyclin D1;After treatment four weeks later,tumor volume increased in control group,and in other drug treatment groups it was suppressed obviously.The mice treated with one or both of the drugs formed substantially smaller tumors than the control mice(p < 0.01),and those treated with both sorafenib and IFN-Lambda 3 formed substantially smaller tumors than the individual sorafenib or IFN-Lambda 3 treated mice(p < 0.05).In addition,immunostaining was used to analyze Ki-67 protein expression in resected tumor tissues.Tumors formed in mice treated with sorafenib,IFN-Lambda 3,or a combination of sorafenib and IFN-Lambda 3 exhibited decreased positivity for Ki-67 compared to tumors in the control group;mice treated with both sorafenib and IFN-Lambda 3 exhibited the most decreased Ki-67 positivity(p < 0.05).Furthermore,TUNEL staining was performed to detect apoptosis in resected tumor tissues.Mice treated with sorafenib,IFN-Lambda 3,or a combination of sorafenib and IFN-Lambda 3 exhibited high apoptotic rates in their tumors compared with the control group,and mice treated with both sorafenib and IFN-Lambda 3 exhibited the highest rate of apoptosis(p < 0.05).Conclusion: Sorafenib and IFN-Lambda 3 have synergistic effects on inhibiting proliferation and inducing apoptosis of human hepatocellular carcinoma cell lines(Hep G2 and SMMC-7721)in vitro,the mechanism may be to inhibit cell proliferation and stimulate cell growth apoptosis pathway,arrest G0/G1 phase and promote the loss of mitochondrial membrane potential and induce ROS production,and influence of cell cycle regulated proteins(p21 and cyclin D1)and the expression of apoptosis protein(cleaved-caspase-3);Meanwhile both drugs can significant synergistic inhibit the growth of human liver cancer SMMC-7721 cells xenografts in a nude mouse model,via suppressing tumor cell proliferation and induce apoptosis,confirmed that sorafenib and IFN-Lambda 3 act synergistically to suppress tumor growth in vivo and treatment of HCC in the body. |
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