The Expression Of Urokinase Type Plasminogen Activator And Vacuolar-type H(+)-ATPases In Hepatocellular Carcinoma And Their Relationship With The Invasion And Metastasis Of Hepatocellular Carcinoma

Background ang Objective:Hepatocellular carcinoma(HCC)is a kind of high malignant tumor with a highly morbility in China,especially in Guangxi province.The relaps and metastasis of HCC is the key problem for poor treatment and bad prognosis.The process of metastasis mainly includes that cancer cell's detachment from the primary tumor,infiltration to the extracellular matrix(ECM),invadion into the lymphotic and blood vessel,adhere to distant endothelial cell where is far away from the primary and then get out of the blood vessel to grow a new metastasis in other organ.The degradation of the ECM is the necessary steps for the tumor metastasis. The proteolysis of ECM is realized mainly through catepsin/uPA/uPAR/MMPs/ profibrinolysin network system,uPA(urokinase type plasminogen activator,uPA) plays a very important role in these system,uPA is activated first by cathepsin,then profibrinolysin and Matrix Metalloproteinases(MMPs)will be activated thereafter,so that the function to degrade ECM will be carried out.The over expression of the uPA and MMPs is association with the metastasis in many tumors closely.Most of the solid tumors are hypoxia.Tumor cells obtain energy from glycolysis which will bring much acid metabolite.Cancer cell,especially those cells with high metastasis potency,usually pump H⁺ out of the cells by the proton pump(H^- -V-ATPase)so as to maintain a stable pH value inside the cells and to avoid apoptosis caused by acidosis.It has been shown that cancer cells which survived in the acid environment have a strong ability to invading.in addition,catepsin,uPA,and MMPs will get increasing their secretion and redistribution in the acid environment.More secretion,which is beneficial to the infiltration and metastasis,will be in the cell surface and the invading border of cancer cells.The inhibition of V-ATPase will minimize the output of H⁺,which will resulte in intracellular acidosis then apoptosis,weaken the acidity surrounding the cancer cells,and also will be unfavourable to the secretion of proteinase and the degradation of the ECM..The previous study about HCC was not focus on the effect of the microenvironment on cancer but the oncogene and the pass way.Now,we sduty the relationship between uPA and V-ATPase with development and metastasis of HCC.Materials and Methods:1.The source of samples:45 cases(39 males and 6 females,range from 21 to 72 years old)of surgical specimens of HCC and the adjacent liver tissues (from Dec.2004 to Apr.2006)were obtained from the department of liver and gall surgery of the first affiliated hospital of GuangXi Medical University. Tissues which are 2cm away from the tumor were definited as paracancer tissues.7 cases of liver parahemangioma tissues were taken as control group. The lowly metastatic cell line(HCC97L)was purchased from liver cancer Research Institute of Zhongshan Hospital of Fudan University.2.Reverse Transcription Polymerase Chain Reaction(RT-PCR)and Immunohistochemistry were used to detect the expression of the uPA,MMP-9 and V-ATPase at the level of the mRNA and proteins respectively,the correlation among them was evaluated;and then the correlation between the experimental results with clinical data was evaluated,including metastasis and cancer embolus in vein,infection of HBV,pathologic grade,the level of AFP in plasma,the size of the tumors.3.uPA RNA was extracted from Hepatocarcinoma tissues,prokaryotic expression plasmid was constructed,and was identified by enzyme digest and sequenced.4.pCMV-uPA-HA eukaryotic expression plasmid was constracted and DNA sequence was detected.and then transfecting the lowly metastatic cell line (HCC97L),selecting the stable expression transfectes,the eukaryotic expression plasmid was identified by Enzyme-cut,RT-PCR and Western blot.5.RT-PCR,Western blot and Immunocytochemistry were used to detect the expression of uPA and V-ATPase at the level of mRNA and proteins in the cells trasfected with pCMV-uPA-HA and with pCMV-HA respectively.6.Migratory ability of HCC97L-pCMV-uPA-HA and HCC97L-pCMV-HA cells were assessed in a wounded monolayer model.7.Evaluating the difference of migratory ability of the HCC97L pCMV-uPA-HA cells in the culture with 50 and 100nM Bafilomycin A 18.Culturing cells in medium with 300,400 and 500nM Bafilomycin A₁ respectively and harvesting cells after 12 hours,detecting apoptosis by Flow cytometry9.Culturing cells in medium with 500,600 and 700nM Bafilomycin A₁ respectively,detecting depression of cells by CKK-8 kit.Result:1.There are 20 cases with vein tumor thrombus and metastasis in the 45 HCC tissues,and 25 without,Aslo the HCC cases were divided into different groups according to pathologic grade,size of tumors,level of AFP in plasma, and size of tumors. 2.The positive rates of uPA mRNA in HCC,paracancer and control groups are 100%(45/45),84.44%(38/45)and 28.57%(2/7)respectively.The significant difference can be seen between the HCC with paracancer tissues groups,and with control group(P＜0.01,P＜0.05),the expression of uPA proteins are 75.56%(34/450,26.67%(12/45),28.57%(2/7)respectivly,The significant difference can be seen between the HCC with paracancer tissues groups,and with control group(P＜0.01,P＜0.05),the expression of uPA mRNA and proteins were significantly correlated with thrombus in portal vein and metastasis(P＜0.01),but not with the infection of hepatitis B virus,pathological stage,the level of serum alpha-fetoprotein(AFP)and the size of tumor(P＞0.05).3.The positive rates of MMP-9 mRNA in HCC,paracancer and control groups are 100%(45/45),86.67%(39/45),57.14%(4/7)respectively.The significant difference can be seen between the HCC with paracancer tissues groups,and with control group(P＜0.01,P＜0.05),the expression of MMP-9 proteins was 66.67%(30/45),22.22%(10/45,14.29%(1/7)respectivly,The significant difference can be seen between the HCC with paracancer tissues groups,and with control group(P＜0.01,P＜0.05),the expression of MMP-9 mRNA and proteins were significantly correlated with thrombus in portal vein and metastasis(P＜0.01),but not with the infection of hepatitis B virus,pathological stage,the level of serum alpha-fetoprotein(AFP)and the size of tumor(P＞0.05).4.The positive rates of V-ATPase mRNA in HCC,paracancer and control. groups are 100%(45/45),86.67%(39/45,71.43%(5/7)respectively.The significant difference can be seen between the HCC with paracancer tissues groups,and with control group(P＜0.01,P＜0.05);the expression of V-ATPase proteins are 82.22%(30/45),51.11%(10/45),42.86%(3/7)respectivly,The significant difference can be seen between the HCC with paracancer tissues group,and with control group(P＜0.01,P＜0.05),the expression of V-AYPase mRNA and protein were significantly correlated with thrombus in portal vein and metastasis(P＜0.01),but not with the infection of hepatitis B virus, pathological stage,the level of serum alpha-fetoprotein(AFP)and the size of tumor(P＞0.05)the expression of uPA mRNA shows positive correlation with MMP-9 mRNA,the same can be seen between uPA with MMP-9 proteins,and uPA with V-ATP-ase mRNA,and uPA with V-AYP-ase protein.5.The clone of uPA gene was abtained from human liver tissues by PCR,and its sequence was detected,uPA eukaryotic expression plasmid was abtained and was sequenced,and then transfecting the low metastatic cell line(HCC97L),the stable expression transfectes was obtained,the expression of the uPA mRNA and proteins in pCMV- uPA-HA-cells were obvious stronger than that of in pCMV-HA-cells and in untransfected cells by RT-PCR and Western blot.6.the expression of uPA,V-ATPase mRNA and proteins in pCMV-uPA-HA-cells was obvious stronger than that of in pCMV-HA-cells by RT-PCR and Western blot respectively.Immunocytochemistry shows the positive stain of uPA is stronger in pCMV-uPA-HA-cells than that of in pCMV-HA-cells,the same can be seen with V-ATPase,in addition,the distribution of V-ATPase on plasma membrane is prominent in pCMV-uPA-HA-cells and inconspicuous in pCMV-HA-cells.7.Migration test in wounded monolayers revealed Migration was faster in uPA-HA-cells than in pCMV-HA-cells,it suggest that increasing secretion and strong expression of uPA would promote migration,invasion and metastasis.8.The speed of covering the Scraping become slow after Bafilomycin A 1 was put into culture medium,Suppressing effects of 100 nM was stronger than that of 50 nM.This suggested that Bafilomycin A₁ could decrease the migratory ability.9.Detecting apoptosis by flow cytometry:in the control groups and in the groups with 300,400,500nM Bafilomycin A₁ respectivly,the rate of apoptosis were 1.22%,4.31%,7.48å’Œ20.6%respectivly; 10.Detecting suppresson by CKK-8 Kit:in the groups with 300, 400,500nM Bafilomycin A₁,the rate of cells' suppressing were 32.63%, 51.14%,58.67%respectivly。conclusion:1.The positive rates of uPA,MMP-9 and V-ATPase mRNA and proteins in HCC group were much higher than in paracancer and control groups.The high expression was correlated with hepatocarcinogenesis and metastasis.2.With the increasing of metastatic potentiality of hepatocellular carcinoma cells,the V-ATPase's expression and distribution on membrane increased.3.Hepatocarcinoma cells's migratory ability would decrease when V-AYPase was inhibited by Bafilomycin A₁,and cancer cells were surppressed and then apoptosis followed when the concentration of Bafilomycin A₁ was raised.4.Inhibition of V-ATPase will result in the decreasing of effluence H^+'s effluence,and cancer cells will die of acidosis,The inhibition of V-ATPase could be considered as a new target for HCC.