Study On Direct Inhibition Of Human Hepatoma Carcinoma Cell By Bevacizumab And Therapeutic Effects Of Bevacizumab Combined With Gemcitabine On Human Hepatoma Carcinoma Xenografts In Nude Mice

Bevacizumab is a humanized recombinant monoclonal antibody that targets vascular endothelial growth factor(VEGF).Recently,Bevacizumab has been approved by the FDA as first-line treatment of metastatic colorectal cancer, second-line treatment of metastatic colorectal cancer,first-line treatment of non-small cell lung cancer(NSCLC),metastatic HER2 negative breast cancer.Several PhaseⅠandⅡstudies to evaluate the effecacy of bevacizumab on hepatocellular carcinoma(HCC) showed that bevacizumab either as a single agent or in combination with cytotoxic or molecularly targeted agents has a good therapeutic effect.Tumor angiogenesis is a complex and dynamic process involving factors that are essential for the development of new tumor blood vessels,tumor growth,and metastasis.HCCs are vascular tumors,and increased levels of VEGF and microvessel density have been observed.High VEGF expression has been associated with inferior survival.VEGF is a major positive regulatory factor in the process of angiogenesis.VEGF exerts effect on endothelial cells by binding with specific receptors VEGFR-1(also referred to as fms-like tyrosine kinase 1[Flt-1]) and VEGFR-2(also referred to KDR,and the murine homologue,Flk-1).Now,the effect of Bevacizumab is attribute to inhibiting the proliferation of endothelial cells by blocking VEGF bind to VEGF receptors on endothelial cells.However,we know a little about whether anti-VEGF therapy has a direct effect on tumor cells.Several studies have reported the presence of VEGFRs on several tumor cells,exogenous VEGF may promote growth through stimulation of VEGFRs on tumors cells.It's confirmed that VEGFRs are expressed on leukemia,melanoma non-small-cell lung carcinoma,pancreatic cancer,prostate carcinoma and breast carcinoma and VEGF/VEGFR autocrine loop are presence in these tumor cells.Recent studies have shown that Flt-1/KDR is also presence in HCC cells that has a variance expression, and inhibiting the expression of VEGF could led to a decrease in tumor growth significantly.So we deduced that Bevacizumab may inhibit the growth of HCC cells by blocking VEGF bind to the VEGF receptors on HCC cells.Now,Bevacizumab alone has therapeutic effect for cancer,but several preclinic studies demonstrated that the combination thrapy of Bevacizumab and chemotherapy result in additive antitumor activity.Recently,the clinic studies about Gemcitabine treated with HCC are carrying out,but the effective power was less than 20%.However,Bevacizumab could increase the antitumor effects of traditional chemothepapeutics,including colon carcinoma and non-small cell lung cancer.Recently,there were also several clinic studies demonstrated the improved efficacy of Bevacizumab therapy in combination with Gemcitabine or other chemotherapeutics for HCC.Our research studied the effect and mechanism that Bevacizumab exerted to proliferation of huaman hepatoma carcinoma cell,and the therapeutic effects of bevacizumab combined with Gemcitabine on human hepatoma carcinoma xenografts in nude mice. The first part:Study on the VEGF autocrine loop in HCC cells Methods:1) Analyze the expression of VEGF,Fit-1,KDR in the gene and protein levels by the use of RT-PCR and immuocytochemistry:we chosed two HCC cell lines HepG2 and SMMC-7721,endothelial cell line ECV304 as positive control,and then tilted the HCC cell which expressed both Flt-1 and KDR using Indepent-Samples T Test.2) Exogenous recombinant human VEGF(rhVEGF) have effect on HCC cell: After HepG2 cell was treated in serum-free medium with rhVEGF,of which concentration was 12.5ng/ml,25 ng/ml,50 ng/ml,100 ng/ml for 72h,we detected the proliferation by the MTT assay,and analyzed the enhancive effect of different concentration on cell proliferation using One-way ANOVA;We also detected the expression ofVEGF,Flt-1,KDRmRNA afer HepG2 was treated with rhVEGF(100 ng/ml) by RT-PCR,and analyzed using Indepent-Samples T Test.Results1) Results of RT-PCR showed that all hepatoma cell lines expressed VEGF,Flt-1 and KDRmRNA,while SMMC-7721 only expressed VEGF and Flt-1mRNA.The expressing amount of VEGF and Flt-1mRNA in HepG2 are higher than that in SMMC-7721(VEGFmRNA:t=9.056,P=0.001;Flt-1mRNA:F=55.975,P=0.000); Results of immunocytochemistry:VEGF and Flt-1 expressed on both HCC cell lines, but KDR expressed only on HepG2.Therefore,we adopted HepG2 cells which expressed highly VEGF as well as Flt-1 and KDR as experimental cells which were used for analysing the Bevacizumab's direct effect on HCC cells.2) Different concentration of RhVEGF could promote the proliferation of hepatoma cell lines HepG2,OD value of groups with 12.Sng/ml,25 ng/ml,50 ng/ml, 100 ng/ml rhVEGF showed statistical significance compared with control group(P valued 0.023,0.002,0.000,0.000 respectively).Within a range from 25 to 100ng/ml, the concentration of rhVEGF increased following stronger promotion.Expression of VEGF and KDRmRNA in all groups increased(t valued 22.450 vs 9.086;P valued 0.000 vs 0.001),while that of Flt-1mRNA showed no significant change(t=1.225, P=0.288).Conclusions1) The VEGF and VEGFR are coexpressed by the HCC cell lines;2) rhVEGF may promote the proliferation of HepG2 in a dose-dependent manner,which is mediated through KDR.With the proliferation of tumor cell,the VEGF is luther expressed,and the interaction between VEGF and receptor KDR is enhanced;3) The VEGF autocrine loop may exist in the HCC cells,which was mediated through receptor KDR.The second part:Study on the growth inhibiton of human hepatoma cell line by bevacizumab in vitroMethods:1) Detected the inhibited effect of bevacizumab on hepatoma cells by MTT assay:HepG2 cells were treated in serum-free medium with bevacizumab for 48h, of which concentration was 0.1μg/ml,1μg/ml,10μg/ml,20μg/ml,then differences between groups of different concentrations and control group were tested with One-way ANOVA(LSD Test).2) Detected the apoptosis of cells by Annexin V-FITC/PI:HepG2 cells were treated in serum-free medium with 10μg/ml bevacizumab for 48h,then differences between group of bevacizumab and control group were tested with Independent T Test.3) Analyzed the cell cycle by flow cytometry:HepG2 cells were treated in serum-free medium with 10μg/ml bevacizumab for 48h,then stained by PI,detected by flow cytometry,calculate the proportions of cells in each stage.Differences between group of bevacizumab and control group were tested with Independent T Test.4) Analyzed changes of expressions of VEGF,Flt-1,KDRmRNA in hepatoma cell line by RT-PCR:HepG2 cells were treated in serum-free medium with 10μg/ml bevacizumab for 48h,then detected by RT-PCR.Differences between group of bevacizumab and control group were tested with Independent T Test. Results:1) Bevacizumab of different concentrations may inhibit the proliferation of hepatoma cell line HepG2.The inhibition ratio of cell proliferation in groups with 0.1μg/ml,1μg/ml,10μg/ml,20μg/ml bevacizumab were 8.8%,27%,32%,26%, respectively.There was statistical significances between groups with 1μg/ml, 10μg/ml,20μg/ml bevacizumab and countrol group(P valued 0.001,0.000,0.001 respectively),but there were no significant differences among the three Bevacizumab groups(P＞0.05).2) Bevacizumab may induce the apoptosis of HCC cell HepG2.The apoptosis rates of HepG2 cells in bevacizumab groups were(17.04±0.14)%,higher than that of control group(2.85±0.08)%(t=198.533,P=0.000).3) Bevacizumab may cause the change of cell cycle in HCC cell HepG2.S stage of HepG2 cells had no significant change(t=0.477,P=0.646),cells in G1 stage increased(t=13.457,P=0.000),while that in G2 stage decreased(t=16.787,P=0.000) compared with the control group.4) Expression of VEGF and KDRmRNA in hepatoma cell line HepG2 decreased after treatment of bevacizumab(t valued 12.476 and 5.071,P valued 0.000 and 0.007).Expression of Flt-1mRNA has no significant different with that of control group(t=2.449,P=0.07).Conclusions:1) Proliferation of hepatoma cell line HepG2 was inhibited after treatment of bevacizumab,while the apoptosis rate increased. 2) Inhibited effect of bevacizumab on HepG2 cell proliferation may be mediated by KDR.With the cell proliferation inhibited,the expression of VEGF decreased.3) Bevacizumab may inhibited hepatoma cell proliferation by blocking the autocrine of VEGF.The third part:Study on therapeutical effects of Bevacizumab combined with gemcitabine on human hepatoma carcinoma xenografts in nude miceMethods:1) Constructed nude mice model of transplantation tumor with human hepatoma cell HepG2,obsered every three days and measured the sizes of tumors with vemier caliper.Put the mice into experiment as long as the diameter of tumor increased to 0.4～0.6cm.Groups were assigned as follows:group A as blank control group,given stroke-physiological saline solution,0.2ml each.Group B as bevacizumab group, given 5mg/kg bevacizumab.Group C as gemcitabine group,given 100 mg/kg gemcitabine.Group D given 5mg/kg bevacizumab combined with 100mg/kg gemcitabine.All groups were intraperitoneal injected twice a week for four weeks.Measured the volumes of tumors every week since the 10th day after xenografting.Withdrawalled drugs for one week after four weeks successive administration.Then killed the nude mice,got the tumor tissues and measured the microvessel density(MVD) of each groups by CD34 staining.Compared the volumes of transplantation tumors in control group with other groups using repeated measure ANOVA,the MVD using one-way ANOVA.Results:1) Volumes in all experiment groups growed slower after administration compared with that in control group,the inhibition ratio were 40.35%,45.61%, 84.21%respectively in bevacizumab,gemcitabine and combination of both group, the effect was most significant in combination goup.There were significant differences between combination goup and bevacizumab,gemcitabine group(P valued 0.004 vs 0.010). 2) MVD in group of gemcitabine alone showed no significant difference compared with control group(P=0.267),while that of bevacizumab group and combination group decreased and showed statistical significance compared with control group(P valued 0.000 all).Decrease of MVD in gemcitabine plus bevacizumab group was more significant than that in bevacizumab group.The difference showed statistical significance(P=0.000).Conclusions:1) Bevacizumab could inhibit the growth of HCC xenograft,it could augment the effects when combined with gemcitabine;2) Bevacizumab have therapeutic effects on HCC xenograft in nude mice, mainly through reducing angiogenesis.