Effects Of Rh-Endostatin On Growth, Invasion And Metastasis Of Hepatocellular Carcinoma Cells In Vitro

Background and Objective:Hepatocellular carcinoma(HCC) is one of China's common malignancies with high mortality and the third rank of death induced by cancer,while occupied the second place in some rural areas.There are about 11 million people died from HCC each year in china,accounting for 45%of the death from HCC in worldwide.At present,HCC five-year survival rate has improved owing to early diagnosis and treatment means progress.But various means of the treatment of liver cancer is still not satisfied with the results.And the portal vein tumor thrombus formation, intrahepatic the invasion and distant metastasis are the main reason for poor treatment of HCC,the progress of the disease fast important reason.Therefore, further study of HCC,seeking new treatment methods and drugs are very necessary.Endostatin is the extracellular matrix of collagenâ…©â…§carboxyl-terminal fragment,is a kind of endogenous glycoproteins separated from the medium of the mouse vessel tumor cells initially.It has specific role in anti-angiogenesis,inhibits endothelial cell proliferation and transfer,and induces apoptosis directly.The anti-tumor effect mechanism by inhibiting vascular endothelial cells of endostatin has been widely confirmed.Recently,there is a preliminary study report that endostatin inhibits tumor cell growth,invasion and metastasis directly,but the research reports of endostatin effect on hepatoma cells directly has not been seen yet at home and abroad.Entremed produced endostatin by yeast expression system,and had carried out stageâ… andâ…¡clinical trials.But because of its instability in vitro and higher production cost,it was not continued to clinical application.A new endostatin agent - recombinant human vascular endostatin(rh-Endostatin,endostar^TM) produced by Jiangsu Simcere Pharmaceutical CO.,Ltd.,has resolved many problems which are faced abroad.A number of animal experiments have shown that it can effectively inhibit endothelial cell proliferation,angiogenesis and tumor growth,in the clinical application to lung cancer has also made an encouraging result.In our topic,we will apply rh-Endostatin for the main experimental reagent,to study its effects on proliferation,apoptosis,cell cycle and the invasive and metastasis ability of human hepatoma cell line HepG2 and high potential invasion human hepatoma cell line HCCLM6 in vitro,and try to explore its mechanism.So we will improve rh-Endostatin anti-tumor mechanisms,and understand the possibility of rh-Endostatin using in hepatocellular carcinoma treatment,and provide further theoretical basis for clinical application.Chapter 1:Effects of rh-Endostatin on proliferation,cell cycle and apoptosis of hepatocellular carcinoma cells in vitroMaterials and Methods:1.The HCCLM6 and HepG2 cells were digested,collected and counted,and then incubated withα5β1 integrin antibody(Anti-α5β1) and FITC-goat anti-mouse IgG.Then,Samples were analyzed with FACscan.2.Respectively,HCCLM6 and HepG2 cells were inoculated in confocal test plate,and removal of the medium after cells were fully adherent.Then cells were fixed by 4%paraformaldehyde and incubated withα5β1 integrin antibody (Anti-α5β1) and FITC-goat anti-mouse IgG.Hochest33258 were added into every plate for nuclear negative staining.The receptor of rh-Endostatinα5β1 integrin was determined in confocal laser fluorescence microscope in the expression of two cell characterization.3.Plastic 96-well fiat bottom assay plates were coated with rh-Endostatin or BSA.HCCLM6 and HepG2 cells were seeded in each well.The experiments were designed into BSA group,immobilized rh-Endostatin group and Anti-α5β1 interference Group.Adhesion was allowed to proceed for 4h.The unattached cells were aspirated.And the adherent cells were tested by MTT.Absorbance was determined at 570 nm on a microplate reader with microplate manager.4.HCCLM6 and HepG2 cells were treated with different final concentrations of rh-Endostatin(50,100,200 and 400μg/ml) respectively.Cells were continued to be incubated for 24h,48h,and 72h respectively.The cells were tested by MTT. Absorbance was determined at 570 nm on a microplate reader with microplate manager.We detected and analysis the effects of rh-Endostatin on proliferation of the two cell lines.5.The experiment groups were described as the former.HCCLM6 and HepG2 cells were treated with different final concentrations of rh-Endostatin(50,100,200 and 400μg/ml) respectively.After 24h,the effects of rh-Endostatin on cell cycle of the two cell lines were tested by flow cytometry.6.The experiment groups were described as the former.After 72h,the effects of rh-Endostatin on apoptosis of the two cell lines were tested by dual-fluorescence staining reagent(Annexin V-FITC and PI dye) and flow cytometry.Statistical methods: Experimental data handing statistical methods were performed in SPSS software version 11.5.Quantitative data were said to(?)±s.Multiple groups were compared in the way of One-Way ANOVA analysis(using LSD when variance was equal,Otherwise using Tamhane);the proliferation data which treated with different drugs concentration and different times were performed by factorial analysis;P≤0.05 for statistical significance.Results:1.Detected by FCM,α5β1 integrin was expressed in the cells HCCLM6 were at the rate of 85.3%,while 18.8%in HepG2 cells.2.In the observation of confocal laser fluorescence microscope,α5β1 integrin was expressed mainly on the membrane surface of the two cell lines.3.HCCLM6 and HepG2 cells could significantly bind with the coating solidified rh-Endostatin,compared with BSA group,the differences were significant (P=0.000).The HCCLM6 cells which were pre-incubated by Anti-α5β1 combined with rh-Endostatin much less than untreated cells(P=0.000).While HepG2 which were pre-incubated by Anti-α5β1 combinated with rh-Endostatin has no significant difference with the untreated group(P=0.077).It was concluded that Anti-α5β1 could blocke the combination between rh-Endostatin and HCCLM6 cells with high expression ofα5β1 integrin obviously,while the interception effect on HepG2 cells with low expression ofα5β1 integrin was unobvious.This also concluded thatαSβ1 integrin may become the function receptor of rh-Endostatin on the liver cancer cells.4.The growth inhibition effects of rh-Endostatin on the two cell lines were in the manner of the dose and time dependent.After 24 and 48h,rh-Endostatin has not obviously effect for the proliferation of HCCLM6,but after 72h inhibition effects were markedly improved and the highest inhibition rate was 42.3%.While every dose groups of rh-Endostatin at every time on the proliferation rate of HepG2 cells were fairly low.there was a certain effect only in high concentrations after 72h.5.With the development of drug concentration(50～400μg/ml),Rh-Endostatin could increase the proportion of HCCLM6 in G₁ phase,S phase decreasing and G₂ phase increasing slightly.Compared with the control group,the proportions in G₁ phase of 200,400μg/ml were significant difference(P=0.000),and the 100μg/ml group also had statistical significance(P= 0.031);the difference of S phase cells proportion on 400μg/ml group were obviously(P= 0.004).But rh-Endostatin had no obviously effect for every cycle of HepG₂ cells.The proportion of cells in G₁ phase were slightly increased only in the high-dose 400μg/ml,and had a statistical significance compared with the control group(P= 0.043).6.HCCLM6 cells which treated with rh-Endostatin were measured by flow cytometry showed that rh-Endostatin could induce apoptosis,and with the increase of dose,the rate of apoptosis increased.When the dose of rh-Endostatin were from 50 to 400μg/ml,the rate of apoptosis from 12.5±0.8%to 31.0±1.4%,and the difference was obviously compared with the control group(P＜0.01).Rh-Endostatin had a lower role on the apoptosis of HepG2 cells,only in doses of 200 and 400μg/ml, the apoptosis rates were 11.2±0.9%and 11.6±1.1%,and compared with the control group,there were a statistical significance(P = 0.025,P = 0.13).Chapter 2:Effects of rh-Endostatin on adhesion,invasion and metastasis of hepatocellular carcinoma cells in vitroMaterials and Methods:1.The HCCLM6 and HepG2 cells were treated with different final concentrations of rh-Endostatin(0,50,100,200 and 400μg/ml) respectively and added into the 96-well plates coated with matrigel.Cells were continued to be incubated for 2h.The unattached cells were aspirated and the adherent cells were tested by MTT.Absorbance was determined at 570 nm on a microplate reader with microplate manager.We counted out the inhibition of adhesion rate and detected the effects of rh-Endostatin on adhesion ability of the two cell lines.2.The experiment groups were described as the former.HCCLM6 and HepG2 cells were seeded on the upper chamber of transwell coated with matrigel.15%FBS in DMEM medium(500μl) was added to the lower chamber.After incubation for 24h at 37℃,the cells on the upper surface of the filters were removed by swabbing with a cotton swab.The cells that had migrated to the reverse side were fixed with methanol and stained with Giemsa,and were counted in 5 random fields under a microscope at×100 magnification.We detected and analysis the effects of rh-Endostatin on invasion ability of the two cell lines.3.In vitro cell migration analysis was the same as invasion analysis described before,but without matrigel on the upper chamber.We detected and analysis the effects of rh-Endostatin on migration ability of the two cell lines in this way.4.Matrix metalloproteinases(MMPs) activity were detected by gelatin zymography:Gelatin zymography was performed in 10%SD-SPAGE that had been cast in the presence of 0.1%gelatin.Samples were prepared in non reducing loading buffer.After electrophoresis,SDS was removed by 2.5%Triton X-100 to renature gelatinases.Gels were then incubated at 37℃for 42h in an incubation buffer (50mmol/L Tris-HCl,5mmol/CaCl₂,1μmol/LZnCl₂,0.02%Brij-35,PH7.6),and then were stained with 0.5%Coomassie Blue R-250 for 3h.The zones were scaned by gel imaging device;gray value of the total protein and the percentage of gray were detected with BandScan reading of the image analysis system.MMP activity was calculated to reflect the effects of rh-Endostatin on MMP of the two cell lines.Statistical methods:Experimental data handing statistical methods were performed in SPSS software version 11.5.Quantitative data were said to(?)±s.Multiple groups were compared in the way of One-Way ANOVA analysis(using LSD when variance was equal,Otherwise using Tamhane);P≤0.05 for statistical significance.Results:1.Rh-Endostatin can inhibit adhesion ability of HCCLM6 cells to matrigel obviously.There was significant difference that experimental groups compared with control group(P=0.000),and the inhibitory rates were from 26.4%to 49.4%.But rh-Endostatin inhibition effects on adhesion ability of HepG2 cells were not obvious. There were some inhibition effects only in the dose of 200μg/ml and 400μg/ml, which rates were 13.3%and 17.5%.The effect of rh-Endostatin on adhesion of the two cell lines was a certain dose-dependent manner.Inhibition rate of adhesion increased with concentration increased.2.Compared with the control group,The HCCLM6 cells that had migrated to the reverse side of artificial basement membrane were significantly reduced after treated with rh-Endostatin(P＜0.05).The invasion inhibition rates of rh-Endostatin on the concentration from 50μg/ml to 400μg/ml were between 17.1%and 56%.But rh-Endostatin inhibition effects on adhesion ability of HepG2 cells were not obvious. The invasion inhibition rates were only between 4.1%and 15.1%;and compared with the control group,treating group with 200μg/ml and 400μg/ml rh-Endostatin were significant differences(P=0.004,0.035respectively).Similarly,the effect of rh-Endostatin on adhesion of the two cell lines was a certain dose-dependent manner.3.Rh-Endostatin could inhibit HCCLM6 and HepG2 cells migrating through the polycarbonate membrane in dose-dependent manner.Migration inhibitory rate of HCCLM6 cells were from 7.8%to 41.1%.The effects of inhibition on HepG2 cells were much weaker than the former and the inhibitory rate was only 9%in the largest concentration of 400μg/ml.For HCCLM6 cells,the differences were significant between higher concentration group of rh-Endostatin(200 and 400μg/ml) and control group(P were 0.000),and the difference was a statistical significance between concentration group of 100μg/ml and the control group(P=0.031);for HepG2 cells,there was a significant difference only in concentration group of 400μg/ml(P=0.013).4.Activity of MMP-2 in medium:There were two obvious bands in PAGE gel of HCCLM6 and HepG2 cells culture medium after treated with rh-Endostatin.The two bands were pro-MMP-2 zone and aMMP-2 zone,and the pro-MMP-9 zones were looming in every group lanes of HCCLM6 media.In both of the two cell lines medium,there were no significant differences in percentage of MMP-2(pro-MMP-2 + aMMP-2);The gray and width of aMMP-2 were decreased with the increasing of rh-Endostatin concentrations.In the concentrations 200 and 400μg/ml,there were no aMMP-2 bands in culture medium of the two cell lines.The differences of aMMP-2 activity ratio(aMMP-2/MMP-2) were significant between higher concentration group of rh-Endostatin(50 and 100μg/ml) and control group.This showed that rh-Endostatin could significantly inhibit the activation of HCCLM6 and HepG2 cells MMP-2,but not the production of MMP-2.Conclusion:α5β1 integrins were expressed highly in high-potential invasion human hepatoma cell line-HCCLM6,but expressed slightly lower in HepG2 cells; Rh-Endostatin could inhibit the proliferation,form resistance Lag of G1 phase and induce apoptosis on HCCLM6 cells with high expression ofα5β1 integrins in the manner of dose-dependent.Rh-Endostatin could significantly inhibit the adhesion, invasion and migration of HCCLM6 cells with high expression ofα5β1 integrins in vitro.While rh-Endostatin had insignificant effects for HepG2 cells which with low expression ofα5β1 integrins in these respects.Rh-Endostatin could inhibit the activation of MMP-2 to achieve anti-tumor metastasis role.In addition to the ability of anti-angiogenesis we proved that rh-Endostatin had a direct role in inhibiting the growth and metastasis of liver cancer cells to achieve the effect of anti-tumor in vitro for the first time.This found also improved the anti-tumor mechanisms of rh-Endostatin to further and provided basis theoretical for the clinical treatment application of rh-Endostatin on liver cancer in the future.