| BackgroundHepatocellular carcinoma(HCC)is the most common primary liver tumor,mainly occurring in chronic liver disease patients with cirrhosis,and is the sixth common malignancy in the world.Many studies have found that HCC is the most common cause of death in this population,with cancer deaths ranking third among all malignancies.HCC is one of the most important medical problems,which has unique epidemiological features with a variable prevalence and progression of different risk factors,and the distribution is very uneven in the world.The incidence of HCC is the highest in Asia and Africa,while the prevalence rate of HCC in China ranks first in the world,and most cases of HCC are closely related to HBV.In developed countries,the incidence of HCC is closely related to HCV infection.In the past few decades,great progress has been achieved in the study and treatment of liver cancer.At diagnosis,however,most patients have been at advanced stage of the disease,therefore,the prognosis of patients with HCC is still not satisfactory.Surgical resection is the preferred treatment for a single tumour without cirrhosis,allowing extensive resection and indicating a lower risk of complications.Other treatments include liver transplantation,ablation techniques,chemoembolization,systemic chemotherapy,intrahepatic radiation,immunotherapy,and so on.At present,treatment options for advanced hepatocellular carcinoma are limited,and the design of reasonable chemotherapy protocols and targeted therapies has become the focus of clinical treatment of HCC.Oral administration of multiple kinase inhibitor of regorafenib can potentially inhibit tumor growth by antiangiogenesis.Several vascular kinases,such as VEGFR1/2,PDGFR-b and FGFR1,are considered to be the main target of regorafenib for cancer therapy.The FDA approved regorafenib in 2012 for the treatment of metastatic colorectal cancer and advanced gastrointestinal stromal tumors.Since the chemical structure of regorafenib is very similar to the chemical structure of sorafenib,it is also the first and the only small molecule targeted therapy approved for HCC treatment,and several in vitro studies have been initiated abroad and Clinical trials to assess the efficacy of regorafenib on HCC.HCC patients had a safe dose of 160 mg/day orally.In 2016,Bruitz reported in the Lancet that the efficacy of the regorafenib was better than that of the first-line drug sorafenib.That's provided that regorafenib has a more potent potential pharmacological activity.Oncogenic signal transducer and activator of transcription protein 3(STAT3)is a component of the JAK/STAT3 signaling pathway,involving in a variety of physiological and pathophysiological processes of the organism.Activation of the JAK/STAT signaling pathway stimulates cell proliferation,differentiation,migration,and apoptosis.This signaling pathway plays an important role in hematopoiesis,immune development,mammary gland development and lactation,adipogenesis,and gender duality growth.STAT3 could bind with recptor though cytokine factor and non-receptor tyrosine kinase binding,and with the receptor-coupled JAK kinase interaction in the cytoplasm of JAK phosphorylation,and further phosphorylation of STAT3,the formation of activated STAT3 dimer,nuclear translocation,followed by activation of various STAT3 target genes,which regulat the cell survival,migration,invasion,immune escape and angiogenesis.STAT3 is also a cytoplasmic signaling molecule and nuclear transcription factor,which is activated by phosphorylation of the tyrosine residue at its carboxyl terminal via the Jak kinase response to a variety of cytokines(including IL-6,IFN,epidermal growth factor FGF,and so on.).In the nucleus,STAT3 regulates the expression of proteins that mediate mitochondrial mediated apoptosis,such as Bcl-2,Mcl-1,and cIAP2.So far,the effection and mechanism of regorafenib on the HCC are still unclear.We have not found the relative referance in the database such as PubMed,China Knowledge Network and Wanfang database.ObjectiveTo explore the effection and mechanism of regorafenib on the occurrence,development and invasion of HCC cells via JAK/STAT3 signal pathway,expressions of p-STAT3 protein and STAT3 mRNA were detected in human hepatocellular carcinoma tissues and non tumor tissue.The relationship between the p-STAT3 and STAT3 mRNA with the occurrence and progression of HCC was further analyzed.Subsequently,regorafenib was added on two primary HCC cell lines(HUH7 and LM3)to observe its effect on proliferation,apoptosis,invasion,and the mechanism through the JAK/STAT3 signaling pathway.Meanwhile,the model of nude mice transplanted tumor model and lung metastatic model of hepatocellular carcinoma cell line LM3 and LM3-STAT3 were studied to further explore the effect of regorafenib on proliferation,apoptosis and invasion of HCC cells via JAK/STAT3 signaling pathway and its action mechanism.This study was divided into the following three parts.Main Content:The first part:Expression and Significance of STAT3 in Hepatocellular CarcinomaMethods1.Immunohistochemistry,Western blot and reverse transcription polymerase chain reaction(RT-PCR)were used to detect the expression of p-STAT3 protein and STAT3 mRNA in 78 cases of human HCC tissues and 50 cases of non tumor tissues.2.Statistical analysis:SPSS 19.0 statistical software was applied for statistical analysis,associated with ?2 test,t test and analysis of variance.Inspection level:a=0.05.ResultsResults of immunohistochemistry,Western blot,and RT-PCR showed that:1.The expression of p-STAT3 protein in HCC tissues was higher than that non tumor tissues,and the difference was statistically significant(p<0.05).Western blot showed that the expression of STAT3 has not different(p>0.05)but the p-STAT3(p<0.05).And STAT3 mRNA in the HCC tissues had no difference with the non tumor tissues(p>0.05).2.The expression of p-STAT3 protein and STAT3 mRNA was independent of age and sex difference in patients with HCC(p>0.05).3.The expression of p-STAT3 protein and STAT3 mRNA increased with the decrease of histological differentiation of HCC cells,with statistical significant difference(p<0.05).4.There was significant difference that the expression of p-STAT3 protein and STAT3 mRNA in patients with lymph node metastasis was higher than that in patients without metastasis(p<0.05).5.The expression of p-STAT3 protein and STAT3mRNA increased with the increase of TNM stage,and the difference was statistically significant(p<0.05).The second part:Effection and Mechanism of Regorafenib on the Characters of Hepatocellular Carcinoma CellsMethods1.The expression of JAK1,JAK2,STAT3,p-STAT3,MMP2 and MMP9 was detected by Western blot in HCC cell lines of HepG2,Hep3B,LM3 and HUH7.Two HCC cell lines(LM3 and HUH7)with higher protein expression were selected for the next study.2.The two HCC cell lines(LM3 and HUH7)were cultured in 96-well plate.Different concentration of regorafenib(0?mol/L,5?mol/L,10?mol/L,20?mol/L,30?mol/L,40?mol/L and 50?mol/L)were used to explore inhibitory effect rate,and the IC50 of the drug were determined for further experiment.Five time gradients of Oh,24h,48h,72h,96h were preset to observe the inhibition of HCC cell lines by regorafenib before and after treatment.3.The inhibitory effect of regorafenib on HCC cell lines was examined by colony formation assay and EdU proliferation test.4.Flow cytometry evaluated the influence of regorafenib on the cell cycle and apoptosis of HCC cell lines,and apoptosis of HCC cells was detected by caspase3/caspase9 apoptosis kit.5.Transwell assay was applied to detect the inhibitory effect of regorafenib on tumor invasion and migration in HCC cell lines.6.Expressions of JAK1,JAK2 and STAT3 mRNA were tested by RT-PCR in HCC cell lines treated by regorafenib.7.Western blot was involved to detect protein expressions of JAK1,p-JAK1,JAK2,p-JAK2,STAT3,p-STAT3,casepase3,cleved-casepase3,caspase9,cleaved-caspase9,MMP2 and MMP9 in HCC cell lines.Results1.In HCC cell lines,the expression of JAK1,JAK2,STAT3,p-STAT3,MMP2 and MMP9 were relatively abundant in LM3 and HUH7 cell lines.Then,we use LM3 and HUH7 cell lines for the next study.2.After the treatment of different concentration of regorafenib(0?mol/L,5?mol/L,10?mol/L,20?mol/L,30?mol/L,40?mol/L and 50?mol/L)on HCC cell lines(LM3 and HUH7),the inhibitory effect of regorafenib on M3 and HUH7 increased gradually with the elevation of drug concentration,indicating a dose-dependent manner.The IC50 of the HUH7 is 9.887?mol/L(R2=0.9328),and the LM3 is 9.914?mol/L(R2=0.9096).This inhibitory effect was most pronounced at 20umol,and lOumol was used for further study.3.The results of colony forming assay and EdU proliferation test showed that the proliferation of HCC cells(10?mol/L regorafenib)was obviously inhibited,and the difference was statistically significant between the control group and experimental group(p<0.05).4.Flow cytometry results showed that the G2/M cycle of the cell(LM3 and HUH7)was arrested in the experimental group(lO?mol/L regorafenib),and the difference between the two groups was statistically significant(p<0.05).5.Flow cytometry results showed that there was statistical difference in the apoptosism,which was markedly enhanced in the experimental group(1O?mol/L regorafenib)(p<0.05).6.Caspase3/caspase9 apoptosis kit detection indicated that caspase3/caspase9 in the HCC cell lines were obviously activated in the experimental group(10?mol/L regorafenib),compared with that in the control group(without administration)(p<0.05).7.Results of Transwell assay showed that regorafenib could reduce the migration and invasive ability of HCC cells,and the difference was statistically difference between the experimental group(10?mol/L regorafenib)and the control group(without administration)(p<0.05).8.There was no apparent difference in the expression of JAK1,JAK2 and STAT3 mRNA between the control group and the treatment group in HCC cell lines by RT-PCR(p>0.05).9.Western blot results suggested that there were not different in the protein expressions of STAT3,JAK1 and JAK2 between the control group and the treatment group in the LM3 cell line.Howeve,the expression of p-STAT3,p-JAK1,p-JAK2,caspase3,caspase9,MMP2 and MMP9 were all decreased in the experimental group than that in the control group(p<0.05).The expression of cleaved-casepase3 and cleaved-casepase9 were increased in the treatment group(p<0.01).In LM3-STAT3 cell line,the expression of JAK1 and JAK2 was decreased in the experimental group(p<0.05),and the STAT3,p-STAT3,JAK1,JAK2,casepase3 and casepase9,cleaved-casepase3 and cleaved-casepase9,MMP2 and MMP9 have no significant different between the control group and experimental group(p>0.05).The third part:Study on the Effect of Regorafenib in Nude Mice Tumor Model and its MechanismMethods1.Construct the tumor model of nude mice with the hepatoma cell line LM3 and LM3-STAT3.2.Comparison of tumor size between the experimental group(regorafenib)and the control group(saline)in the tumor model.3.Construct the lung metastases model of nude mice with the hepatoma cell line LM3 and LM3-STAT3.4.Comparison of the amount of tumors between the experimental group(regorafenib)and the control group(saline)in the lung metastases model.5.Immunohistochemistry was used to analyze the expression of p-STAT3 between the experimental group(regorafenib)and the control group(saline)in the tumor model.6.Western blot was applied to compare the expression of JAK1,p-JAK1 JAK2,p-JAK2,STAT3,p-STAT3,MMP9 and MMP2 between the experimental group(regorafenib)and the control group(saline)in the tumor model.Results1.Compared with the control group,Regorafenib could significantly inhibit the tumor size after treatment of regorafenib in nude mice,and there was a significant difference(p<0.05).2.Immunohistochemistry results showed that compared with the control group,the expression of p-STAT3 was reduced in the tumors of nude mice treated by regorafenib treated,with significant difference(p<0.05).3.Western blot results indicated that in the LM3 tumor model,compared with the control group,JAK1,JAK2,and STAT3 expressions have no significant different after treatment of regorafenib(p>0.05),but the p-JAK1,p-JAK2,p-STAT3 MMP2 and MMP9 was significant reduced(p<0.05).In the LM3-STAT3 tumor model,compared with the control group,the expressions of JAK1,JAK2,STAT3,p-STAT3,MMP2 and MMP9 have no significant different after treatment of regorafenib(p>0.05),but the p-JAK1 and p-JAK2 was significant reduced(p<0.05).Conclusion1.The overexpresion of p-STAT3 protein and STAT3 mRNA in HCC tissues,were associated with the occurrence,development,invasion and metastasis of HCC.2.Regorafenib can inhibit cell proliferation,migration and invasion of HCC cells.Regorafenib can also promote the apoptosis of HCC.3.In vivo and vitro,regorafenib can inhibit the phosphation of p-JAK1,p-JAK2 and p-STAT3,up-regulate cleaved-caspase3 and cleaved-caspase9,and suppress the the MMP2 and MMP9.This supports that the regorafenib could inhibit the proliferation,promote apoptosis and suppress the migration or invasion of HCC cells by JAK/STAT3 signal pathway. |
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