Research On The Synergism And Mechanism Of Bevacizumab Combined With Gemcitabine In The Treatment Of Primary Hepatic Carcinoma

Background and Objective:Primary hepatic carcinoma(PHC)is one of the common malignant tumors in the world,of which the mortality rate ranked second in our country.Growth and metastasis of most malignant solid tumors including PHC depend on angiogenesis which could provide the proliferation of tumor cell with nutrition and metabolism pathway,promote the growth and metastasis of tumor and aggravate the patients' condition.Consequently tumor angiogenesis now has became a popular area for studying.Bevacizumab(Avastin,rhuMAb-VEGF)is a synthetic recombinant humanized monoclonal IgG1 antibody aiming directly at VEGF.Bevacizumab specifically binds with VEGF,results in interfering with the combination of VEGF and Flt-1,KDR which are the endotheliocyte surface receptors,blocking the downstream signal pathway mediated by VEGF,preventing VEGF from promoting the proliferation of vascular endothelial cell and angiogenesis in tumor,thus block blood,oxygen and other nutrition supply necessary for the tumor growth,all of which make the tumor unable to grow,perfuse and metastasize,the chemotherapy function effectively to retard the growth and metastasis of the tumor.Bevacizumab combined with radiotherapy and chemotherapy has synergistic effects:①enable the normalization of angiogenesis in tumor regions,relive high pressure between tissues,help the transport of chemotherapeutics toward tumor regions.②regional tumor hypoxia led by radiotherapy and chemotherapy could promote the expression of VEGF,which help tumor cell to resist apoptosis-induced mechanism result from radiotherapy and chemotherapy;But the combination of radiotherapy and chemotherapy with bevacizumab can obviate this secondary protective effect and sensitize the therapeutic reaction.Clinical observation shows that the effective power of biologic chemotherapy mode constructed by the combination of VEGF monoclonal antibody and chemotherapy is far stronger than either single use of chemotherapy or VEGF monoclonal antibody,even surpass the sum of both,expecially for some patients with recurrent or metastatic tumors.These patients can regain saitivity for chemotherapeutics which are already invalid after applying molecule targeted drugs like bevacizumab.Besides inhibition of tumor angiogenesis,the anti-tumor effect of bevacizumab seems include increasing the sensitivities of tumor cells for chemotherapeutics.There's no rational explanation for this phenomenon at home and abroad which make it necessary for us to do researches to perfect the theoryetical basis of biologic chemotherapy mode and then give an impulse to the combined therapy of tumor.The main purpose of this research is to investigate the depressant effects on PHC which were caused by using targeted drugs or chemotherapeutics alone and the combination of two drugs,the synergistic action of targeted drugs plus chemotherapy, thus found a theoretic basis for the combined therapy of PHC and improving its effectiveness.Chapter 1 Inhibition of bevacizumab on the proliferation of PHC cellsMethods:1.Analyzed the expression of gene and protein of VEGF,Flt-1 and KDR in PHC cell lines using RT-PCR and immuocytochemistry:choose two PHC cell lines HepG2 and SMMC-7721 as experiment cells,while human allantoic vein endothelial cell line ECV-304 as positive controls of VEGF receptor expression,sieved out PHC cell lines of high VEGFmRNA expressing,positive Flt-1 and KDR expressing as well as high mRNA expressing with independent sample t test for this research.2.Evaluated by MTT assay the inhibition of PHC cell resulted from bevacizumab: with no blood serum,0.1μg/ml,1μg/ml,10μg/ml,20μg/ml bevacizumab separately acted on HepG2 cells for 48 hours,then compared the proliferation of control group with other groups of different concentration using one-way ANOVA(LSD test).3.Detected the apoptosis by Annexin V-FITC/PI staining:with no blood serum, 10μg/ml bevacizumab acted on HepG2 cells for 48 hours,then staining by Annexin V-FITC/PI,detected the apoptosis with flow cytometry.Then,differences between group of bevacizumab and control group were tested with Independent T Test.4.Analyzed expression changes of VEGF,Flt-1 and KDR mRNA in PHC cell lines by RT-PCR:with no blood serum,10μg/ml bevacizumab acted on HepG2 cells for 48 hours,then tested by RT-PCR.Analyzed the results by independent sample t test.Results:1.Result of RT-PCR:all PHC cell lines HepG2 expressed VEGF,Flt-1 and KDR mRNA,while SMMC-7721 only expressed VEGF and Flt-1 mRNA.The expression of VEGF and Flt-1 mRNA in HepG2 are higher than that in SMMC-7721(VEGF mRNA:P=0.001,t=9.056;Flt-1mRNA:P=0.000,F=55.975);Results of immunocytochemistry:VEGF and Flt-1 expressed in both cell lines,but KDR expressed only in HepG2.Therefore,we adopted HepG2 cells for experiments because of their high VEGF expression as well as Flt-1 and KDR expression.2.Bevacizumab of different concentration could inhibit the proliferation of PHC cell lines HepG2,cytostasis rate of groups with 0.1μg/ml,1μg/ml,10μg/ml,20μg/ml bevacizumab were 8.8%,27%,32%,26%,respectively.Compared with control group, cytostasis rate in groups with 1μg/ml,10μg/ml,20μg/ml bevacizumab showed statistical significance.(P valued 0.001,0.000,0.001). 3.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).4.Expression of VEGF and KDR mRNA in PHC cell lines HepG2 declined after effecting by bevacizumab.Compared with that of blank control group,the differences showed no statistical significance(t valued 12.476 vs 5.071,P valued 0.000 vs 0.007). Expression of Flt-1 mRNA was not significant different with that of blank control group(t=2.449,P=0.07).Conclusions:1.Expression of VEGF and its receptors both existed in all human PHC cell lines.2.Effecting by bevacizumab,the proliferation of PHC cell lines HepG2 was inhibited with the apoptosis rate increased.3.Depressant effects on the proliferation of HepG2 by bevacizumab could be mediated by KDR.Expression of VEGF decreased with the inhibition of proliferation in tumor cells.4.Bevacizumab may inhibit the proliferation of PHC cells by blocking the autocrine of VEGF.Chapter 2 Effects on PHC cell lines HepG2 by bevacizumab combined with gemcitabineMethods:1.Evaluated the inhibition of proliferation in PHC cells with bevacizumab plus gemcitabine by MTT assay:Experiment groups were dealt with 10μg/ml bevacizumab or 0.01μg/ml,0.1μg/ml,1μg/ml,10μg/ml gemcitabine or bevacizumab combined with gemcitabine of different concentration,while control group with RPMI-1640 medium of no blood serum.Detected by MTT assay after 72 hours cultivation and analyzed with factorial analysis. 2.Evaluated by Western Blot the effects on expression of ERK1/2,Bcl-2 and Bcl-xl in HepG2 cells after using bevacizumab plus gemcitabine:using bevacizumab, gemcitabine,combination of both and placebo separately in HepG2 cells,then extracted cell proteins and evaluated by Western Blot.Results:1.Effects on the proliferation of PHC cell lines HepG2 by gemcitabine of different concentration showed significant differences(F=1318.002,P=0.000).Effects on proliferation of HepG2 with gemcitabine alone and that plus bevacizumab showed significant differences(F=1663.503,P=0.000).The reciprocation between gemcitabine and bevacizumab is significant(F=41.832,P=0.000).Inhibition ratios of groups with 0.01μg/ml,0.1μg/ml,1μg/ml,10μg/ml gemcitabine were respectively 2.72%,15.31%,36.42%,74.87%,while combining with 10μg/ml bevacizumab,the inhibition ratios were respectively 31.2%,50.48%,63.43%,83.66%.2.Specific reaction strap appeared at 28KD Mol wt(Bcl-2,Bcl-xl),44KD and 42KD(ERK1/2) in all four groups.Expression of Bcl-2 and Bcl-xl in blank control group was significantly stronger than that of the other three groups.Bcl-xl expressed the weakest in the group with combination of bevacizumab and gemcitabine.ERK expressed significantly weaker in blank group and bevacizumab group than combination group and gemcitabine group.Conclusions:1.The combination with bevacizumab could amplify depressant effect of gemcitabine on PHC cells.2.Expression of ERK gene was evidently depressed with chemotherapeutics, which led to the promotion of apoptosis.Either chemotherapeutics or molecule targeted drugs can down regulate the expression of Bcl-2 and Bcl-xl.Furthermore, this action was far clearer with the combination of two drugs.These results suggested that the synergic mechanism of increasing patients' sensitivities for targeted drugs and chemotherapeutics by combining two drugs may be related with the promotion of apoptosis as well as regulation of Bcl-2 and Bcl-xl expression. Chapter 3 Treatment effect of bevacizumab combined with gemcitabine on subcutaneous transplantation tumor of nude mice bearing PHC cells HepG2Methods:1.Effects on the volumes and MVD of PHC transplantation tumors Treated with bevacizumab plus gemcitabine:constructed nude mice model of transplantation tumor with human PHC cells HepG2,obsered every three days and measured the sizes of tumors with vernier caliper.Groups were assigned as follows:group A,as blank control group,gave stroke-physiological saline solution,0.2ml each.Group B, as bevacizumab group,gave bevacizumab 5mg/kg.Group C,as gemcitabine group, gave gemcitabine 100mg/kg.Group D,combination group,gave bevacizumab 5mg/kg combined with gemcitabine 100mg/kg.All groups were intraperitoneal injected twice a week for four weeks.Measured the volumes of tumors every week since the 19th day after inoculation.Withdrawalled drugs for one week after four weeks' successive administration.Then put the nude mice to death,got transplantation tumor tissues and measured the microvessel density(MVD) of each groups by CD34 dyeing.Compared the volumes of transplantation tumors in control group and other groups with repeated measure ANOVA,the MVD with one-way ANOVA.2.Effects on VEGE Flt-1,KDR,Bcl-2 and Bcl-xl of subcutaneous transplatation tumor in nude mice beating cancer with bevacizumab plus gemcitabine:use bevacizumab,gemcitabine,combination of both and normal saline in the subcutaneous transplantation tumors in nude mice bearing human PHC cells HepG2, separately took fresh living tissues in all groups,then fixed with 10%neutral paraformaldehyde,dehydrated conventionally,paraffin imbedded,serial sectioned in 4μm and dyeing by HE,finally made into immunohistochemical slices and dyeing immunohistochemically by standard En Vision^TM method.3.Evaluated effects on expression of ERK1/2,Bcl-2 and Bcl-xl in subcutaneous transplatation tumor in nude mice bearing human PHC cells HepG2 with bevacizumab plus gemcitabine by Western Blot:separately gave bevacizumab, gemcitabine,combination of both and normal saline to the subcutaneous transplantation tumors in nude mice bearing human PHC cells HepG2,then evaluated by Western Blot after extracting proteins in tissues.Results:1.Volumes in all experiment groups growed slower after administration compared with that in the control group.The effect was most significant in group with bevacizumab plus gemcitabine.There were significant differences among groups with bevacizumab,gemcitabine and combination of both(P valued 0.004 vs 0.010).The inhibition ratio of experiment groups were 40.35%,45.61%,84.21% respectively.2.MVD of group with gemcitabine alone showed no significant difference compared with control group(P=0.267),while that of groups with bevacizumab and combination of both decreased and showed statistical significance compared with control group(P all valued 0.000).Decrease of MVD in group with gemcitabine plus bevacizumab was more significant than that in bevacizumab group and the difference between two groups showed statistical significance(P=0.000).3.Flt-1 expressed relatively high in all four groups.Expression of Flt-1 in blank control and combination group showed strongly positive,while that in gemcitabine and bevacizumab group showed midrange positive.The differences among groups were not significant.Expression of KDR in blank control group showed strongly positive,while that in gemcitabine group showed weakly positive,bevacizumab and combination group negative.4.Specific reaction strap appeared at 28KD Mol wt(Bcl-2,Bcl-xl),44KD and 42KD(ERK1/2) in all four groups.Expression of Bcl-2 and Bcl-xl in blank control group was significantly stronger than that of the other three groups.Bcl-xl expressed the weakest in the group with combination of bevacizumab and gemcitabine.ERK expressed significantly weaker in blank group and bevacizumab group than combination group and gemcitabine group.Conclusions: 1.Both bevacizumab and gemcitabine could depress the growth of tumor,the combination of two drugs can amply the effects.2.Bevacizumab inhibited the formation of new vessels in transplantation tumors. The effects became more evident with the combination of gemcitabine.3.There was a strong likelihood that bevacizumab block the signal transduction of VEGF which was conducted by VEGFR-2 and prevent the formation of new vessels in tumors.4.Expression of ERK gene had been obviously depressed after using chemotherapeutics,thus promoted the apoptosis.Both chemotherapeutics and molecule targeted drugs can down regulated the expression of Bcl-2 and Bcl-xl.This down regulation became more evident with the combination of two drugs,which indicated that the synergic mechanism of increasing patients' sensitivities for targeted drugs and chemotherapeutics with the combination of two drugs may be related with the promotion of apoptosis as well as regulation of Bcl-2 and Bcl-xl expression.