贝伐单抗联合吉西他滨的抗肝癌的协同作用及机制研究
|
摘要
研究背景和目的:
原发性肝癌(hepatic cell carcinoma,HCC,以下简称肝癌)是世界上常见的恶性肿瘤之一,肝癌死亡率占我国恶性肿瘤死亡率的第二位。包括肝癌在内的多数恶性实体肿瘤的生长和转移依赖于血管生成,血管生成可为肿瘤细胞的增殖提供营养和代谢的通路,促进肿瘤的生长和转移,加快病情的恶化,故肿瘤血管生成现已成为热门的研究领域。
贝伐单抗(Bevacizumab,Avastin,rhuMAb-VEGF)是人工合成的一种针对血管内皮生长因子(vascular endothelial growth factor,VEGF)的重组人源化IgG1型单克隆抗体。Bevacizumab特异性结合于VEGF后能阻碍后者与内皮细胞表面受体Flt-1及KDR结合,阻断VEGF介导的下游信号通路,使VEGF不能发挥促进血管内皮细胞增殖以及肿瘤内血管新生的作用,从而阻断对肿瘤生长至关重要的血液、氧气和其他生长必需的营养供应,使肿瘤无法在体内生长和散播转移,令化疗能够发挥有效的抗肿瘤作用,进而延缓肿瘤的生长和转移。
Bevacizumab与放、化疗联合具有协同作用:①可使肿瘤区血管生成正常化,组织间高压减轻,利于化疗药物向肿瘤区输送。②放、化疗所致局部肿瘤区缺氧可促VEGF表达,帮助肿瘤细胞抵抗放、化疗的凋亡诱导机制;而联用Bevacizumab将预防此继发保护效应,使治疗反应增敏。
临床观察到联合VEGF单抗和化疗所建立的生物化疗模式取得的有效率不仅远高于单用化疗或VEGF单抗,而且超过二者代数和,特别是对一部份复发/转移病人,已经无效的化疗药物在使用Bevacizumab等分子靶向药物后重新恢复敏感性。似乎Bevacizumab的抗肿瘤作用除了抑制肿瘤血管形成以外,还包括增加肿瘤细胞对化疗药物的敏感性。国内外目前均无对这种现象的合理解释。
本课题研究的主要目的是探讨靶向药物与化疗药物单独及联合应用对肝癌的抑制作用,靶向药物与化疗的协同作用,为肝癌的综合治疗、提高疗效提供实验资料。
第一章贝伐单抗对肝癌细胞增殖的抑制作用
一、方法
1、采用RT-PCR、免疫细胞化学的方法分析肝癌细胞株中VEGF、Flt-1、KDR基因和蛋白水平的表达:选用肝癌细胞株HepG2、SMMC-7721为实验细胞并选择人脐静脉内皮细胞系ECV-304作为VEGF受体表达的阳性对照,采用独立样本t检验从中筛选VEGF mRNA表达量高,Flt-1、KDR阳性表达且mRNA表达量高的肝癌细胞株,用于本课题的研究;
2、MTT法检测Bevacizumab对肝癌细胞的增殖抑制作用:无血清条件下,0.1μg/ml、1μg/ml、10μg/ml、20μg/ml Bevacizumab作用于HepG2细胞48h,采用单因素方差分析(LSD法)比较不同浓度间及与对照组的增殖情况;
3、Annexin V-FITC/PI双染检测细胞凋亡:无血清条件下,10μg/mlBevacizumab作用于HepG2细胞48h后,经Annexin V-FITC/PI双染,流式细胞仪检测凋亡情况。采用独立样本t检验分析比较Bevacizumab组和对照组;
4、RT-PCR分析肝癌细胞株VEGF、Flt-1、KDR mRNA表达量的变化:无血清条件下,10μg/ml Bevacizumab作用于HepG2细胞48h后,行RT-PCR检测。采用独立样本t检验分析。
二、结果
1、RT-PCR结果示:肝癌细胞株HepG2中均有VEGF、Flt-1、KDR mRNA的表达,而SMMC-7721中只有VEGF、Flt-1 mRNA的表达;且HepG2中VEGF、Flt-1 mRNA的表达量均高于SMMC-7721(VEGF mRNA:P=0.001,t=9.056;Flt-1mRNA:P=0.000,F=55.975);免疫细胞化学结果示:HepG2见VEGF、Flt-1及KDR的表达,SMMC-7721见VEGF、Flt-1的表达,无KDR的表达。因此,我们选用VEGF、Flt-1和KDR均表达的HepG2细胞作为实验用细胞;
2、不同浓度Bevacizumab对肝癌细胞株HepG2的增殖有抑制作用。0.1μg/ml、1μg/ml、10μg/ml、20μg/ml组细胞增殖抑制率分别为8.8%、27%、32%、26%。其中1μg/ml、10μg/ml、20μg/ml组与对照组比较差异均有统计学意义(P值分别为0.001,0.000,0.001),但此三组间差异无统计学意义(P＞0.05);
3、Bevacizumab可诱导肝癌细胞株HepG2的凋亡。Bevacizumab作用于HepG2肝癌细胞48h后,10μg/ml Bevacizumab组的细胞凋亡率为(17.04±0.14)%,明显高于对照组(2.85±0.08)%(t=198.533,P=0.000);
4、Bevacizumab作用于肝癌细胞株HepG2后,其VEGF、KDR mRNA表达量均减少,与空白对照组比较差异具有统计学意义(t值分别为:12.476和5.071,P值分别为:0.000和0.007);而Flt-1 mRNA表达量与空白对照组比较无显著差异(t=2.449,P=0.07)。
三、结论
1、人肝癌细胞株中存在VEGF及其受体的共同表达;
2、Bevacizumab作用于肝癌细胞株HepG2后,其增殖受抑;细胞凋亡率增高;
3、Bevacizumab对HepG2的增殖抑制作用可能是通过KDR介导的,同时随着肿瘤细胞的增殖抑制,其VEGF表达减少;
4、Bevacizumab可能通过阻滞VEGF的自分泌作用而发挥对肝癌细胞的增殖抑制作用。
第二章贝伐单抗联合吉西他滨对肝癌细胞增殖的抑制作用
一、方法
1、MTT法检测Bevacizumab联合吉西他滨对肝癌细胞的增殖抑制作用:实验分组为:10μg/ml Bevacizumab组,0.01μg/ml、0.1μg/ml、1μg/ml、10μg/ml吉西他滨组和Bevacizumab+不同浓度吉西他滨联合组,对照组加入无血清RPMI-1640培养基。培养72h后行MTT检测。采用析因设计资料的方差分析;
2、Western Blot检测Bevacizumab联合吉西他滨对HepG2中ERK1/2、Bcl-2、Bcl-xl表达的影响:分别将Bevacizumab、吉西他滨、Bevacizumab联合吉西他滨及安慰剂作用于HepG2细胞,提取细胞蛋白后,行Western Blot检测。
二、结果
1、不同浓度吉西他滨对肝癌细胞株HepG2增殖的影响差异有显著性意义(F=1318.002,P=0.000),吉西他滨单用与Bevacizumab联用对HepG2增殖的影响差异有显著性意义(F=1663.503,P=0.000)。且两者之间交互效应显著(F=41.832,P=0.000)。0.01μg/ml、0.1μg/ml、1μg/ml、10μg/ml吉西他滨组的抑制率分别为2.72%、15.31%、36.42%、74.87%,联合10μg/ml Bevacizumab后其抑制率分别为31.2%、50.48%、63.43%、83.66%;
2、实验四组均在分子量为28 KD(Bcl-2、Bcl-xl)、44 KD和42 KD(ERK1/2)处出现特异性反应条带,其中空白组的Bcl-2及Bcl-xl的表达明显强于其余三组的表达。而Bcl-xl的表达在联合组最弱。空白组和单药Bevacizumab组的ERK表达与联合用药组及单药吉西他滨组比较明显减弱。
三、结论
1、Bevacizumab联合吉西他滨可增强吉西他滨单药对肝癌细胞的增殖抑制作用;
2、应用了化疗药物后,ERK基因的表达被明显抑制,进而促进细胞凋亡。无论化疗药物还是分子靶向药物,均可以下调Bcl-2及Bcl-xl的表达,而当两药联合时,Bcl-2及Bcl-xl的表达下降更为明显。提示两者协同显著增强对靶向药物及化疗敏感性的机制可能与促进细胞凋亡及调控Bcl-2及Bcl-xl的表达有关。
第三章贝伐单抗联合吉西他滨对肝癌裸鼠皮下移植瘤治疗作用的研究
一、方法
1、Bevacizumab联合吉西他滨对肝癌移植瘤体积和MVD的影响:建立人肝癌细胞HepG2裸鼠移植瘤模型,每隔3天观察一次,用游标卡尺测量肿瘤的大小。实验分A组:空白对照组,予以无菌生理盐水,0.2ml/只;B组:Bevacizumab组,5mg/kg:C组:吉西他滨组,100mg/kg:D组:Bevacizumab 5mg/kg+吉西他滨100mg/kg联合组。各组给药均为2次/周,共4周,腹腔注射。自接种后第19天起,每周检测肿瘤体积。连续给药4周后停药一周,处死裸鼠,取移植瘤组织进行CD34染色检测各组微血管密度(MVD)。对照组与各治疗组间移植瘤体积比较采用重复测量数据的方差分析;对照组与各治疗组间MVD的比较采用单因素方差分析;
2、Bevacizumab联合吉西他滨对荷瘤裸鼠皮下移植瘤VEGF、Flt-1、KDR、ERK、Bcl-2、Bcl-xl的影响:分别将Bevacizumab、吉西他滨、Bevacizumab联合吉西他滨及安慰剂作用于荷人肝癌HepG2裸鼠皮下移植瘤,各组留取新鲜活组织标本10%中性多聚甲醛固定,常规脱水,石蜡包埋,4μm厚连续切片,进行苏木素-伊红(HE)染色后制备成免疫组化切片,采用标准EnVision~(TM)法进行免疫组化流程染色;
3、Western Blot检测Bevacizumab联合吉西他滨对荷人肝癌HepG2裸鼠皮下移植瘤中ERK1/2、Bcl-2、Bcl-xl表达的影响:分别将Bevacizumab、吉西他滨、Bevacizumab联合吉西他滨及安慰剂作用于荷人肝癌HepG2裸鼠皮下移植瘤,提取组织蛋白后,行Western Blot检测。
二、结果
1、各治疗组给药后体积与对照组相比增长均较缓慢,其中Bevacizumab联合吉西他滨组最为明显。Bevacizumab联合吉西他滨组与Bevacizumab、吉西他滨组之间均有显著差异(P值分别为0.004,0.010),其抑瘤率分别为40.35%、45.61%、84.21%;
2、与对照组比较,单药吉西他滨组MVD无显著差异(P=0.267);单药Bevacizumab组及联合吉西他滨组的MVD均减少,与对照组比较差异均有统计学意义(P值均为0.000);Bevacizumab联合吉西他滨组的MVD减少较Bevacizumab组更为显著,差异有统计学意义(P=0.000);
3、Flt-1在四组中均有较高的表达,其中在空白对照组和联合用药组中的表达均为强阳性;单药吉西他滨组和单药Bevacizumab组中Flt-1的表达均为中度阳性;各组间的差异并不明显。KDR在空白对照组中的表达为强阳性;单药吉西他滨组中KDR的表达为弱阳性;联合用药组和单药Bevacizumab组中KDR为阴性;
4、实验四组均在分子量为28 KD(Bcl-2、Bcl-xl)、44 KD和42 KD(ERK1/2)处出现特异性反应条带,其中空白组的Bcl-2及Bcl-xl的表达明显强于其余三组的表达。而Bcl-2及Bcl-xl的表达均在联合组最弱。空白组和单药Bevacizumab组的ERK表达与联合用药组及单药吉西他滨组比较明显减弱。
三、结论
1、Bevacizumab与吉西他滨均可延缓肿瘤的生长,且Bevacizumab联合吉西他滨可显著增加二者单药的疗效;
2、Bevacizumab可抑制移植瘤新生血管的形成,联合吉西他滨治疗后的移植瘤组织中新生血管形成减少更为显著;
3、Bevacizumab极有可能直接封闭VEGF,阻断VEGF经由VEGFR-2进行的信号转导,阻止肿瘤细胞中新生血管的生成;
4、应用化疗药物后,ERK基因的表达被明显抑制,进而促进细胞凋亡。无论化疗药物还是分子靶向药物,均可以下调Bcl-2及Bcl-xl的表达,而当两药联合时,Bcl-2及Bcl-xl的表达下降更为明显。提示两者协同显著增强对靶向药物及化疗敏感性的机制可能与促进细胞凋亡及调控Bcl-2及Bcl-xl的表达有关。
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 cells
Methods:
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 gemcitabine
Methods:
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 HepG2
Methods:
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.
原发性肝癌(hepatic cell carcinoma,HCC,以下简称肝癌)是世界上常见的恶性肿瘤之一,肝癌死亡率占我国恶性肿瘤死亡率的第二位。包括肝癌在内的多数恶性实体肿瘤的生长和转移依赖于血管生成,血管生成可为肿瘤细胞的增殖提供营养和代谢的通路,促进肿瘤的生长和转移,加快病情的恶化,故肿瘤血管生成现已成为热门的研究领域。
贝伐单抗(Bevacizumab,Avastin,rhuMAb-VEGF)是人工合成的一种针对血管内皮生长因子(vascular endothelial growth factor,VEGF)的重组人源化IgG1型单克隆抗体。Bevacizumab特异性结合于VEGF后能阻碍后者与内皮细胞表面受体Flt-1及KDR结合,阻断VEGF介导的下游信号通路,使VEGF不能发挥促进血管内皮细胞增殖以及肿瘤内血管新生的作用,从而阻断对肿瘤生长至关重要的血液、氧气和其他生长必需的营养供应,使肿瘤无法在体内生长和散播转移,令化疗能够发挥有效的抗肿瘤作用,进而延缓肿瘤的生长和转移。
Bevacizumab与放、化疗联合具有协同作用:①可使肿瘤区血管生成正常化,组织间高压减轻,利于化疗药物向肿瘤区输送。②放、化疗所致局部肿瘤区缺氧可促VEGF表达,帮助肿瘤细胞抵抗放、化疗的凋亡诱导机制;而联用Bevacizumab将预防此继发保护效应,使治疗反应增敏。
临床观察到联合VEGF单抗和化疗所建立的生物化疗模式取得的有效率不仅远高于单用化疗或VEGF单抗,而且超过二者代数和,特别是对一部份复发/转移病人,已经无效的化疗药物在使用Bevacizumab等分子靶向药物后重新恢复敏感性。似乎Bevacizumab的抗肿瘤作用除了抑制肿瘤血管形成以外,还包括增加肿瘤细胞对化疗药物的敏感性。国内外目前均无对这种现象的合理解释。
本课题研究的主要目的是探讨靶向药物与化疗药物单独及联合应用对肝癌的抑制作用,靶向药物与化疗的协同作用,为肝癌的综合治疗、提高疗效提供实验资料。
第一章贝伐单抗对肝癌细胞增殖的抑制作用
一、方法
1、采用RT-PCR、免疫细胞化学的方法分析肝癌细胞株中VEGF、Flt-1、KDR基因和蛋白水平的表达:选用肝癌细胞株HepG2、SMMC-7721为实验细胞并选择人脐静脉内皮细胞系ECV-304作为VEGF受体表达的阳性对照,采用独立样本t检验从中筛选VEGF mRNA表达量高,Flt-1、KDR阳性表达且mRNA表达量高的肝癌细胞株,用于本课题的研究;
2、MTT法检测Bevacizumab对肝癌细胞的增殖抑制作用:无血清条件下,0.1μg/ml、1μg/ml、10μg/ml、20μg/ml Bevacizumab作用于HepG2细胞48h,采用单因素方差分析(LSD法)比较不同浓度间及与对照组的增殖情况;
3、Annexin V-FITC/PI双染检测细胞凋亡:无血清条件下,10μg/mlBevacizumab作用于HepG2细胞48h后,经Annexin V-FITC/PI双染,流式细胞仪检测凋亡情况。采用独立样本t检验分析比较Bevacizumab组和对照组;
4、RT-PCR分析肝癌细胞株VEGF、Flt-1、KDR mRNA表达量的变化:无血清条件下,10μg/ml Bevacizumab作用于HepG2细胞48h后,行RT-PCR检测。采用独立样本t检验分析。
二、结果
1、RT-PCR结果示:肝癌细胞株HepG2中均有VEGF、Flt-1、KDR mRNA的表达,而SMMC-7721中只有VEGF、Flt-1 mRNA的表达;且HepG2中VEGF、Flt-1 mRNA的表达量均高于SMMC-7721(VEGF mRNA:P=0.001,t=9.056;Flt-1mRNA:P=0.000,F=55.975);免疫细胞化学结果示:HepG2见VEGF、Flt-1及KDR的表达,SMMC-7721见VEGF、Flt-1的表达,无KDR的表达。因此,我们选用VEGF、Flt-1和KDR均表达的HepG2细胞作为实验用细胞;
2、不同浓度Bevacizumab对肝癌细胞株HepG2的增殖有抑制作用。0.1μg/ml、1μg/ml、10μg/ml、20μg/ml组细胞增殖抑制率分别为8.8%、27%、32%、26%。其中1μg/ml、10μg/ml、20μg/ml组与对照组比较差异均有统计学意义(P值分别为0.001,0.000,0.001),但此三组间差异无统计学意义(P＞0.05);
3、Bevacizumab可诱导肝癌细胞株HepG2的凋亡。Bevacizumab作用于HepG2肝癌细胞48h后,10μg/ml Bevacizumab组的细胞凋亡率为(17.04±0.14)%,明显高于对照组(2.85±0.08)%(t=198.533,P=0.000);
4、Bevacizumab作用于肝癌细胞株HepG2后,其VEGF、KDR mRNA表达量均减少,与空白对照组比较差异具有统计学意义(t值分别为:12.476和5.071,P值分别为:0.000和0.007);而Flt-1 mRNA表达量与空白对照组比较无显著差异(t=2.449,P=0.07)。
三、结论
1、人肝癌细胞株中存在VEGF及其受体的共同表达;
2、Bevacizumab作用于肝癌细胞株HepG2后,其增殖受抑;细胞凋亡率增高;
3、Bevacizumab对HepG2的增殖抑制作用可能是通过KDR介导的,同时随着肿瘤细胞的增殖抑制,其VEGF表达减少;
4、Bevacizumab可能通过阻滞VEGF的自分泌作用而发挥对肝癌细胞的增殖抑制作用。
第二章贝伐单抗联合吉西他滨对肝癌细胞增殖的抑制作用
一、方法
1、MTT法检测Bevacizumab联合吉西他滨对肝癌细胞的增殖抑制作用:实验分组为:10μg/ml Bevacizumab组,0.01μg/ml、0.1μg/ml、1μg/ml、10μg/ml吉西他滨组和Bevacizumab+不同浓度吉西他滨联合组,对照组加入无血清RPMI-1640培养基。培养72h后行MTT检测。采用析因设计资料的方差分析;
2、Western Blot检测Bevacizumab联合吉西他滨对HepG2中ERK1/2、Bcl-2、Bcl-xl表达的影响:分别将Bevacizumab、吉西他滨、Bevacizumab联合吉西他滨及安慰剂作用于HepG2细胞,提取细胞蛋白后,行Western Blot检测。
二、结果
1、不同浓度吉西他滨对肝癌细胞株HepG2增殖的影响差异有显著性意义(F=1318.002,P=0.000),吉西他滨单用与Bevacizumab联用对HepG2增殖的影响差异有显著性意义(F=1663.503,P=0.000)。且两者之间交互效应显著(F=41.832,P=0.000)。0.01μg/ml、0.1μg/ml、1μg/ml、10μg/ml吉西他滨组的抑制率分别为2.72%、15.31%、36.42%、74.87%,联合10μg/ml Bevacizumab后其抑制率分别为31.2%、50.48%、63.43%、83.66%;
2、实验四组均在分子量为28 KD(Bcl-2、Bcl-xl)、44 KD和42 KD(ERK1/2)处出现特异性反应条带,其中空白组的Bcl-2及Bcl-xl的表达明显强于其余三组的表达。而Bcl-xl的表达在联合组最弱。空白组和单药Bevacizumab组的ERK表达与联合用药组及单药吉西他滨组比较明显减弱。
三、结论
1、Bevacizumab联合吉西他滨可增强吉西他滨单药对肝癌细胞的增殖抑制作用;
2、应用了化疗药物后,ERK基因的表达被明显抑制,进而促进细胞凋亡。无论化疗药物还是分子靶向药物,均可以下调Bcl-2及Bcl-xl的表达,而当两药联合时,Bcl-2及Bcl-xl的表达下降更为明显。提示两者协同显著增强对靶向药物及化疗敏感性的机制可能与促进细胞凋亡及调控Bcl-2及Bcl-xl的表达有关。
第三章贝伐单抗联合吉西他滨对肝癌裸鼠皮下移植瘤治疗作用的研究
一、方法
1、Bevacizumab联合吉西他滨对肝癌移植瘤体积和MVD的影响:建立人肝癌细胞HepG2裸鼠移植瘤模型,每隔3天观察一次,用游标卡尺测量肿瘤的大小。实验分A组:空白对照组,予以无菌生理盐水,0.2ml/只;B组:Bevacizumab组,5mg/kg:C组:吉西他滨组,100mg/kg:D组:Bevacizumab 5mg/kg+吉西他滨100mg/kg联合组。各组给药均为2次/周,共4周,腹腔注射。自接种后第19天起,每周检测肿瘤体积。连续给药4周后停药一周,处死裸鼠,取移植瘤组织进行CD34染色检测各组微血管密度(MVD)。对照组与各治疗组间移植瘤体积比较采用重复测量数据的方差分析;对照组与各治疗组间MVD的比较采用单因素方差分析;
2、Bevacizumab联合吉西他滨对荷瘤裸鼠皮下移植瘤VEGF、Flt-1、KDR、ERK、Bcl-2、Bcl-xl的影响:分别将Bevacizumab、吉西他滨、Bevacizumab联合吉西他滨及安慰剂作用于荷人肝癌HepG2裸鼠皮下移植瘤,各组留取新鲜活组织标本10%中性多聚甲醛固定,常规脱水,石蜡包埋,4μm厚连续切片,进行苏木素-伊红(HE)染色后制备成免疫组化切片,采用标准EnVision~(TM)法进行免疫组化流程染色;
3、Western Blot检测Bevacizumab联合吉西他滨对荷人肝癌HepG2裸鼠皮下移植瘤中ERK1/2、Bcl-2、Bcl-xl表达的影响:分别将Bevacizumab、吉西他滨、Bevacizumab联合吉西他滨及安慰剂作用于荷人肝癌HepG2裸鼠皮下移植瘤,提取组织蛋白后,行Western Blot检测。
二、结果
1、各治疗组给药后体积与对照组相比增长均较缓慢,其中Bevacizumab联合吉西他滨组最为明显。Bevacizumab联合吉西他滨组与Bevacizumab、吉西他滨组之间均有显著差异(P值分别为0.004,0.010),其抑瘤率分别为40.35%、45.61%、84.21%;
2、与对照组比较,单药吉西他滨组MVD无显著差异(P=0.267);单药Bevacizumab组及联合吉西他滨组的MVD均减少,与对照组比较差异均有统计学意义(P值均为0.000);Bevacizumab联合吉西他滨组的MVD减少较Bevacizumab组更为显著,差异有统计学意义(P=0.000);
3、Flt-1在四组中均有较高的表达,其中在空白对照组和联合用药组中的表达均为强阳性;单药吉西他滨组和单药Bevacizumab组中Flt-1的表达均为中度阳性;各组间的差异并不明显。KDR在空白对照组中的表达为强阳性;单药吉西他滨组中KDR的表达为弱阳性;联合用药组和单药Bevacizumab组中KDR为阴性;
4、实验四组均在分子量为28 KD(Bcl-2、Bcl-xl)、44 KD和42 KD(ERK1/2)处出现特异性反应条带,其中空白组的Bcl-2及Bcl-xl的表达明显强于其余三组的表达。而Bcl-2及Bcl-xl的表达均在联合组最弱。空白组和单药Bevacizumab组的ERK表达与联合用药组及单药吉西他滨组比较明显减弱。
三、结论
1、Bevacizumab与吉西他滨均可延缓肿瘤的生长,且Bevacizumab联合吉西他滨可显著增加二者单药的疗效;
2、Bevacizumab可抑制移植瘤新生血管的形成,联合吉西他滨治疗后的移植瘤组织中新生血管形成减少更为显著;
3、Bevacizumab极有可能直接封闭VEGF,阻断VEGF经由VEGFR-2进行的信号转导,阻止肿瘤细胞中新生血管的生成;
4、应用化疗药物后,ERK基因的表达被明显抑制,进而促进细胞凋亡。无论化疗药物还是分子靶向药物,均可以下调Bcl-2及Bcl-xl的表达,而当两药联合时,Bcl-2及Bcl-xl的表达下降更为明显。提示两者协同显著增强对靶向药物及化疗敏感性的机制可能与促进细胞凋亡及调控Bcl-2及Bcl-xl的表达有关。
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 cells
Methods:
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 gemcitabine
Methods:
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 HepG2
Methods:
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.
引文
[1]Neufeld G,Cohen T,Gengrinovitch S,et al.Vascular endothelial growth factor (VEGF) and its receptors[J].FASEB J,1999;13:9-22.
[2]Rosen LS.Clinical experience with angiogenesis signaling inhibitors:focus on vascular endothelial growth factor(VEGF) blockers[J].Cancer Control 2002Mar-Apr;9(2 Suppl):36-44.
[3]Evelyne D,Patricia H,Aude V,et al.Tumor angiogenesis and tissue factor expression during hepatocellular carcinoma progression in a transgenic mouse model[J].J Hepatol,2003;38(6):793-802.
[4]高峨嵋,徐建明.VEGF靶向药Avastin治疗实体瘤的研究进展[J].中国肿瘤临床与康复,2006.
[5]Jain RK.Delivery of novel therapeutic agents in tumors:physiological barriers and strategies[J].J Natl Cancer Inst,1989,81:570-576.
[6]Warren RS,Yaun H,Matli MR.Regulation by vascular endothelial growth factor of human colon cancer tumorigenesis in a mouse model of experimental liver metastasis[J].J Clin Invest,1995,95:1789-1797.
[7]Kim KJ,Li B,Winer J,et al.Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo[J].Nature,1993,362:841-844.
[8]Napoleone F.Vascular endothelial growth factor as a target for anticancer therapy[J].Oncologist,2004,9(supp 11):2210.
[9]Jain RK.Normalization of tumor vasculature:an emerging concep tin antiangiogenic therapy[J].Science,2005,307:58-62.and Lee CG,Heijn M, di Tomaso E,et al.Antivascular endothelial growth factor treatment augments tumor radiation response under normoxic or hypoxic conditions[J].Cancer Res,2000,60:5565-5570.
[10]Guan Z.Prospective randomised phase Ⅱ study of gemcitabine at standard or fixed dose rates cheduleinun resectable hepatocellular carcinoma[J].Br J Cancer.2003;89(10):1865.
[11]Fuchs CS.A phase Ⅱ trial of gemcitabine in patients with advanced hepatocellular carcinoma.Cancer[J],2002;94(12):3186.
[12]Zhu AX,Blaszkowsky LS,Ryan DP,et al.Phase Ⅱ study of gemcitabine and oxaliplatin in combination with bevacizumab in patients with advanced hepatocellular carcinoma[J].J Clin Oncol.2006;24:1898-1903.
[13]Kindler HL,Friberg G,Singh DA,et al.Phase ⅡTrial of Bevacizumab Plus Gemcitabine in Patients With Advanced Pancreatic Cancer[J].J Clin Oncol.2005Nov 1;23(31):8033-40.
[1]陈锡山,苏建堂,崔毓桂,等,膀胱移行细胞癌survivin与血管内皮生长因子表达相关性及其临床意义[J].南京医科大学学报,2005,25(8):561-563.
[2]刘晓,范萍,王水,等.血管内皮生长因子及其受体蛋白在乳腺癌组织中的表达及意义[J].南京医科大学学报,2005,25(11):777-779.
[3]朱宏,施瑞华,张国新,等.环氧合酶-2、血管内皮生长因子和微血管密度与食管鳞癌病理特征的关系[J].南京医科大学学报,2005,25(2):84-86.
[4]Jordi Graells,Antonia Vinyals,Agnes Figueras,et al.Over production of VEGF_(165)Concomitantly Expressed with its Receptors Promotes Growth and Survival of Melanoma Cells through MAPK and PI3K Signaling[J].The journal of investigative dermatology,2004,123(6):1151-1161.
[5]Forster Y,Meye A,Krause S,et al.Antisense-mediated VEGF suppression in bladder and breast cancer cells[J].Cancer Lett,2004,212(1):95-103.
[6]Takei Y,Kadomatsu K,Yuzawa Y,et al.A small interfering RNA targeting vascular endothelial growth factor as cancer therapeutics[J].Cancer Res,2004, 64(10):3365-3370.
[7]ShenBQ,LeeDY, Gerber HP,et al.Homologous up-regulation of KDR/Flk receptors expression by vaseular endotheliai growth factor in vitro[J].J Biol Chem,1998:273(45):29979-29985.
[8] Yan Wu, Andrea T. Hooper, Zhaojing Zhong.The vascular endothelial growth factor receptor (VEGFR-1) supports growth and survival of human breast carcinoma[J]. Int. J. Cancer, 2006,119(7): 1519-1529.
[9] Barr MP, Bouchier-Hayes DJ, Harmey JJ. Vascular endothelial growth factor is an autocrine survival factor for breast tumour cells under hypoxia[J]. Int J Oncol,2008,32(1):41-8.
[1]GuanZ.Prospective randomized phase II study of gemcitabine at standard or fixed dose rate schedule in unresectable hepatocellular carcinoma [J]. Br J Cancer. 2003;89 (10): 1865.
[2]Andrew X. Zhu,Development of sorafenib and other molecularly targeted agents in hepatocellular carcinoma[J]. Cancer,2008,l 12(2):250-259.
[3]Zhu AX, Blaszkowsky LS, Ryan DP, et al. Phase II study of gemcitabine and oxaliplatin in combination with bevacizumab in patients with advanced hepatocellular carcinoma[J].J Clin Oncol. 2006;24:1898-1903.
[4]L. Placidi, E.C. Scott, D. Eckoff, et al. Metabolic drug interactions between angiogenic inhibitor, TNP-470 and anticancer agents in primary cultured hepatocytes and microsomes[J]. Drug Metab Dispos,1999:623-626.
[5]Bos JL. All in the family New insight s and questions regarding interconnectivity of Ras, Rapl and Ral [J]. EMBO J, 1998; 17(23):6776.
[6]Gysin S, Lee SH, Dean NM, et al. Pharmacologic inhibition of RAF→MEK→ERK signaling elicit s pancreatic cancer cell cycle arrest t hrough induced expression of p27 Kipl [J]. Cancer Res,2005; 65 (11):4870.
[7] Ito Y, Sasaki Y, Horimoto M, et al . Activation of mitogen-activated protein kinases/ext- racellular signal-regulated kinases in human hepatocellular carcinoma [J]. Hepatology, 1998;27(4):951.
[8]Adeyinka A, Nui Y, Cherlet T, et al. Activated mitogen-activated protein kinase expression during human breast tumorigenesis and breast cancer progression [J].Clin Cancer Res, 2002;8 (6): 1747.
[9]Albanell J, Codony-Servat J, Rojo F, et al. Activated extracellular signal-regulated kinases: association with epidermal growth factor receptor/transforming growth factor alpha expression in head and neck squamous carcinoma and inhibition by anti-epidermal growth factor receptor treatments [J]. Cancer Res, 2001; 61(17):6500.
[10]Uzgare AR, Kaplan PJ, Greenberg NM. Differential expression and/or activation of P38MAPK, erk1/2 , and jnk during the initiation and progression of prostate cancer [J]. Prostate, 2003;55 (2) :128.
[11]Rincon-Arano H, Rosales R, Mora N, et al. R-Ras promotes tumor growth of cervical epithelial cells [J]. Cancer, 2003; 97(3):575.
[12]Gioeli D, Mandell JW, Pet roni GR, et al. Activation of mitogen-activated protein kinase associated wit h prostate cancer progression [J]. Cancer Res, 1999;59 (2):279.
[13]Carson DA, Ribeiro JM. Apoptosis and disease. Lancet, 1993 ,341:1251-1254.
[14]Kim R, Tanabe K, Uchida Y,et al. Current status of t he molecular mechanisms of anticancer drug2induced apoptosis. The cont ribution of molecular21evel analysis to cancer chemot herapy[J] . Cancer Chemot her Pharmacol ,2002 ,50(5) :343-352.
[15]RJ Bold , J Chandra , DJ McConkey ,et al. Gemcitabine-in-duced programmed cell deat h (apoptosis) of human pancreatic carcinoma is determined by Bcl-2 content [J]. Annals of Surgical Oncology, 1999,6 (3) .279-285.
[16]Zhou JR,Mukher-jee P,Gugger ET,et al.Inhibition of murine bladder tumorigenesis by soy isoflavone via alterations in the cell cycle,apop-tosis,and angiogenesis[J].Cancer Res,1998;58(22):5231-5238.
[17]Carroll H P,Ali S,Kirby JA,et al.Accelerating the induction of Fas-mediated T cell apoptosis:a strategy for transplant tolerance[J].Clin Exp Immunol,2001;72(8):1423-1432.
[18]Wong BR,BesserD,Kim N,et al.TRANCE,a TNF fam ily m em ber,activates Akt/PKB through signaling complex involving TRAF6 and c-src[J].Molecular cell,1999;17(4): 1041-1049.
[19]Leech SH,Olie RA,Gaut schi O,et al. Induction of apoptosis in lung cancer cells following bcl-xl antisense treatment[J]. Int J Cancer ,2000 ,86 :570-576.
[1]Weidner N,Semple JP, Welch WR,et al.Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma[J]. N Engl J Med. 1991,324(1):1-8.
[2] Takahashi Y,Kitadai Y,Bucana CD, et al. Expression of vascular endothelial growt h factor and it s receptor,KDR,correlates with vascularity,metastasis and proliferatin of human colon cancer [J]. Cancer Res ,1995 ,55 :3964-3968.
[3]Breasalier RS,HO SB,Schoeppner HL,et al.Enhanced sialylation of mucin-associated carbohydrate structures in human colon cancer metastasis[J].Gastroenterology,1996,110(5):1354-67.
[4]Folkman J.Tumor angiogenesis:therapeutic implications[J].N Engl J Med 1971,28(5):1182-1186.
[5]Hanahan D,Weinberg RA.The hallmarks of cancer[J].Cell 2000,100:57-70.
[6]Rafii S,Lyden D,Benezra R,et al.Vascular and haematopoietic stem cells:Novel targets for anti-angiogenesis therapy[J].Nat Rev Cancer 2002,2:826-835.
[7]Zhu AX,Blaszkowsky LS,Ryan DP,et al.Phase Ⅱ study of gemcitabine and oxaliplatin in combination with bevacizumab in patients with advanced hepatocellular carcinoma[J].J Clin Oncol.2006;24:1898-1903.
[8]Parikh PM,Fuloria J,Babu G,et al.A phase Ⅱ study of gemcitabine and cisplatin in patients with advanced hepatocellular carcinoma[J].Trop Gastroenterol,2005;26(3):115.
[9]Sun W,Hailer DG,Mykulowycz K,et al.Combination of capecitabine,oxaliplatin with bevacizumab in treatment of advanced hepatocellular carcinoma(HCC):a phase Ⅱ study.J Clin Oncol(Meeting Abstracts).2007;25(18S).Abstract 4574.
[10]Kindler HL,Friberg G,Singh DA,et al.PhaseⅡ Trial of Bevacizumab Plus Gemcitabine in Patients With Advanced Pancreatic Cancer[J].J Clin Oncol.2005Nov 1;23(31):8033-40.
[11]Ranieri G,Gasparini G.Angiogenesis and angiogenesis inhibitors:a new potential anticancer therapeutic strategy[J].Curr Drug Targets Immune Endocr Metabol Disord,2001,1(3):241-253.
[12]Dudek AZ,Pawlak WZ,Kirstein MN.Molecular targets in the inhibition of angiogenesis[J].ExpertOp in Ther Targets,2003,7(4):527-541. 理学杂志,2003,19(4):401-404.
[14]Suzuki K,Hayashi N,Miyamoto Y,et al.Expression of vascular permeability factor/vascular endothelial growth factor in human hepatocellular carcinoma[J].Cancer Res,1996,56(13):3004.
[15]李晓明,汤钊猷,周铬,等.血管内皮生长因子的表达与肝细胞癌侵袭和转移的关系[J].肿瘤,1998,20:12-14.
[16]Poon RT,Lau C,Yu WC,et al.High serum levels of vascular endothelial growth factor predict poor response to transarterial chemoembolization in hepatocellular carcinoma:a prospective study[J].Oncol Rep.2004 May;11(5):1077-84.
[17]罗荣城,韩焕兴,主编.肿瘤生物治疗学[M],北京.人民卫生出版社(第二版).90-1.
[18]Borgstrom P,Gold DP,Hillan KJ,et al.Imprortance of VEGF for breast cancer angiogenesis in vivo:implications from intravital microscopy of combination treatments with an anti-VEGF neutralizing monoclonal antibody and doxorubicin[J].Anticancer Res,1999,19(5B):4203-14.
[19]Yamaguchi R,Yano H,Iemura A,et al.Expression of vascular endothelial growth factor in human hepatocellular carcinoma[J].Hepatology,1998,28:68-77.
[20]Yao DF,Wu XH,Zhu Y,et al.Quantitative analysis of vascular endothelial growth factor,microvascular density and their clinicopathologic features in human hepatocellular carcinoma[J].Hepatobiliary Pancreat Dis Int.2005May;4(2):220-6.
[21]徐洪雨,毕莉,杨幼林,等.血管内皮生长因子及微血管密度在肝癌中的表达[J].中国肿瘤杂志,2000,4:249-251.
[22]Chao Y,Li CP,Chau GY,et al.Prognostic significance of vascular endothelial growth factor,basic fibroblast growth factor,and angiogenin in patients with resectable hepatocellular carcinoma after surgery[J].Ann Surg Oncol,2003,10:355-362.
[23]Poon RT,Ho JW,Tong CS,et al.Prognostic significance of serum vascular endothelial growth factor and endostatin in patients with hepatocellular carcinoma[J].Br J Surg,2004,91:1354-1360.
[24]Jeng KS,Sheen IS,Wang YC,et al.Prognostic significance of preoperative circulating vascular endothelial growth factor messenger RNA expression in resectable hepatocellular carcinoma:a prospective study[J].World J Gastroenterol,2004,10:643-648.
[25]Kwak BK,Shim HJ,Park ES,et al.Hepatocellular carcinoma:correlation between vascular endothelial growth factor level and degree of enhancement by multiphase contrast enhanced computer tomography[J].Invest Radiol,2001,36:487-489.
[26]Luttun A,Tjwa M,Moons L,et al.Revascularization of ischemic tissues by PIGF treatment,and inhibition of tumor angiogenesis,arthritis and atherosclerosis by anti-Fltl[J].Nat Med,2002,8(8):831-840.
[27]Hiratsuka S,Maru Y,okada A,et al.Involvement of Flt-1 tyrosine kinase(vascular endothelial gowth faetor receptor-1)in patholgieal anogenesis[J].Caneer Res,2001,61(3):1207-1213.
[28]Wu Y,Zhong Z,Huber J,et al.Anti-vascular endothelial growth faetor recptor-1antagonist antibody as a therapeutie agent for cancer[J].Clin Canee:Res,2006,12(21):6573-6584.
[29]赵爱志,窦科峰,李开宗等.VEGF、KDR的表达对肝癌的血管生成、生长、转移的影响[J].中华外科杂志.2000,6(38):454-455.
[30]刘金洋,李康华,廖前德.骨肉瘤中Bcl-2、Fas蛋白表达的相关性研究[J].医学临床研究,2006,23(4):581-582.
[31]Voutsadakis IA.Apoptosis and the pathogenesis of lymphoma[J].Acta Oncol,2000,39(2):151-156.
[32]Zhao M,Zhang NX,Economou M,et al.Immunohistochemical detection of Bcl-2 protein in liver lesions:Bcl-2 protein is expressed in hepatocellular carcinomas but not in liver cell dysplasia[J].Histopathology,1994,25(3):237.
[33]秦军,李敏,孙丽娟,等.Bcl-2蛋白在肝胆癌及慢性肝病中的表达[J].实用医药杂志,2002,19(4):265.
[34]Gazzaniga P,Gandini O,Gradilone A,et al.Detection of basic fibroblast growth facter mRNA in urinary bladder cancer:correclation with local relapses[J].IntJ Oncol,1999,14:1123.
[35]李清龙,李永国,杨竹林.慢性肝病及肝癌组织中Bcl-2蛋白表达及意义[J].临床研究.2007,9(24):1501-1503.
[36]赵建平,王媛媛,阎玉虎.吉西他滨对人非小细胞肺癌A549细胞增殖与凋亡的影响.肿瘤防治研究[J].2006,(33)2:100-102.
[37]Kar S,Wang M,et al.Transient and sustained ERK phosphorylation and nuclear translocation in growth control[J].Cell Physiol,2002;192(2):151-9.
[38]唐承薇.奥曲肽对胃癌细胞转录活化蛋白-1的抑制作用[J].癌症,2002;21(8):850-854.
[39]Steinmetz R,Wagoner HA,Zeng P,et al.Mechanisms regulating the constitutive activation of the ERK signaling pat hway in ovarian cancer and the effect of RNAi for ERK1/2 on cancer cell proliferation[J].Mol Endocrino,2004;15(3):1004.
[40]Wu TT,Wang J S,Jiaan BP,et al.Role of p21~(WAF1) and p27~(K1P1) in predicting biochemical recurrence for organ-confined prostate adenocarcinoma[J].J Chin Med Assoc,2007;70(1):11.
[41]Handra-Luca A,Bilal H,Bert rand JC,et al.Extra-cellular signal-regulated ERK-1/ERK-2 pathway activation in human salivary gland mucoepidermoid carcinoma : association to aggressive tumor behavior and tumor cell proliferation [J]. Am J Pat hoi, 2003;163 (3):957.
[42]Hoshino R, Chatani Y, Yamori T, et al. Constitutive activation of the 41-/ 43-kDa mitogen-activated protein kinase signaling pathway in human tumors [J].Oncogene, 1999; 18(3):813.
[43]Mishima K, Inoue K, Hayashi Y. Overexpression of extracellular-signal regulated kinases on oral squamous cell carcinoma[J]. Oral Oncol, 2002 ; 38 (5):468.
[44]Gerber HP,Dixit V,Ferrara N.Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells[J].J Biol Chem,1998,273(21):13313-6.
[45]Harmey JH.VEGF and Cancer[M].New York,USA:Kluwer Academic/Plenum Publishers,2004:29.
[1]Leung DW,Cachines G,Kuang WJ,et al.Vascular endothelial growth factor issecreted angiogenic mitogen[J].Science,1989,246:1306.
[2]Senger DR,Van de Water L,Brown LF,et al.Vascular permeability factor(VPF,VEGF) in tumor biology[J].Cancer Metastasis Rev,1993,12(3-4):303-324.
[3]Ferara N,Smyth TD.The biology of vascular endotheilial growth factor[J].Endocrine Rev 1997,18:4-25.
[4]Bussolino F,Mantovani A,Persico G.Molecular mechanisms of blood vessel formation[J]. TIBS, 1997,22:251.
[5] Shibuya M. Role of VEGF-FLT receptor system in normal and tumor angiogenesis[J]. Adv Cancer Res, 1995,67:281.
[6] Brown LF,Detmar M,Claffey K,et al.Vasclular permeability factor/vascular endothelial growth factor: A multifunctional angiogenic cytokine[J]. Exs,1997,79:233.
[7] Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease [J]. Nature Med,1995,l:27-31.
[8] Lohn E, Liotta L. Molecular insights into cancer invasion: strategies for prevention and intervention [J]. Cancer Res, 1995,55:1865.
[9] Ferrara N,Davis-Smyth T.The biology of vascular endotelial growth factor.Endocr Rev 1997;18:4-25.
[10] Gordon M S,Margolin K, TalpazM,et al. Phase I safety and pharmacokinetic study of recombinat human anti-vascular endothelial growth factor in patients with advanced cancer[J].J Clin Oncol,2001, 19(4):843-850.
[11] Napoleone F. Vascular endothelial growth factor as a target for anticancer therapy[J].Oncologist,2004,9(suppl 1):2-10.
[12] HurwitzH, Fehrenbacher L, NovotnyW, et al.Bevacizumab plus irinotecan,fluorouraci,l and leucovorin for metastatic colorectal cancer[J].N Engl JMed, 2004, 350: 2335-2342.
[13] Langer CJ,Natale RB.The emerging role of vascular endothelial growth factor receptor tyrosine kinase inhibitors[J].Semin Oncol,2005,32(6 Suppl 10):S23-29.
[14] SandlerAB,Gray R, Brahmer J, et al. Randomized phase II/III trial ofpaclitaxel(P) plus carboplatin(C) with or without bevacizumab (NSE#704865) in patients with advanced non-squa-mous non-small cell lung cancer(NSCLC):An Eastern Cooperative Oncology Group (ECOG)Trial-E4599[J]. ProcAm Soc Clin Oncol , 2005, 23: 2(AbstrLBA4).
[15]Sandler A, Gray R, Brahmer J. et al. Randomized phase II/III trial of paclitaxel plus carboplatin with or without bevacizumab in patients with advanced non-squamous non-small cell lung cancer: An Eastern Cooperative Oncology Group Trial-E4599[J].J Clin Onco. 2005;23:abstract LBA4,2.
[16]Brahmer JR, Gray R, Schiller JH, et al. ECOG 4599 phase III trial of carboplatin and paclitaxel ± bevacizumab: subset analysis of survival by gender[JI. Proc Am Soc Clin Oncol. 2006;24:373s. Abstract 7036.
[17]Dowlati A, Gray R, Johnson DH, et al. Prospective correlative assessment of biomarkers in E4599 randomized phase II /III trial of carboplatin and paclitaxel ± bevacizumab in advanced non-small cell lung cancer (NSCLC)[J]. Proc Am Soc Clin Oncol. 2006;24:370s. Abstract 7027.
[18]Manegold C, von Pawel J, Zatloukal P, et al. Randomized, double-blind multicentre phaseIII study of bevacizumab combination with cisplatin and gemcitabine in chemotherapy-naive patients with advanced or recurrent non-squamous non-small cell lung cancer (NSCLC). Program and abstracts of the 43rd American Society of Clinical Oncology Annual Meeting; June 1-5,2007; Chicago, Illinois. Abstract LBA7514.
[19]Sandler A, Dahlberg SE, Dowlati A, et al. A phase II study of cisplatin plus etopside plus bevacizumab for previously untreated extensive stage small cell lung cancer by the Eastern Coopertive Oncology Group(ECOG 3501).Program and abstracts of the 43rd American Society of Clinical Oncology Annual Meeting; June 1-5,2007; Chicago, Illnois. Abstract 7564.
[20]Tortora G, Caputo R, Damiano V,et al. Combined targeted inhibition of Bcl-2, Bcl-xl, epidermal growth factor receptor, and protein kinase A type I causes potent antitumor, apoptotic and antiangiogenic activity[J].Clin Cancer Res 2003; 9: 866-871.
[21] Herbst RS, Johnson DH, Mininberg E, et al. Phase I / II trial evaluating the anti-vascular endothelial growth factor monoclonal antibody bevacizumab in combination with the HER-1/epidermal growth factor receptor tyrosine kinase inhibitor erlotinib for patients with recurrent non-small-cell lung cancer[J]. J Clin Oncol, 2005, 23:2544-2555.
[22] Nathan P, Chao D, Brock C, Savage P, Harries M, Gore M, Eisen T.Br J Cancer. 2006 May 8;94(9):1217-20.
[23] J. D. Hainsworth, J. A. Sosman, D. R. Spigel, R. C. Schwert, D. L. Carrell,F. Hubbard and F. A. Greco.Journal of Clinical Oncology, 2004 ASCO Annual Meeting Proceedings (Post-Meeting Edition).Vol 22, No 14S (July 15 Supplement), 2004: 4502.
[24] Yamaguchi R,Yano H, Iemura A,et al. Expression of vascular endothelial growth factor in human hepatocellular carcinoma[J].Hepatology,1998,28:68-77.
[25]Chao Y,Li CP,Chau GY,et al. Prognostic significance of vascular endothelial growth factor, basic fibroblast growth factor,and angiogenin in patients with resectable hepatocellular carcinoma after surgery[J]. Ann Surg Oncol,2003,10:355-362.
[26] Poon RT,Ho JW,Tong CS,et al. Prognostic significance of serum vascular endothelial growth factor and endostatin in patients with hepatocellular carcinoma[J]. Br J Surg,2004,91:1354-1360.
[27] Jeng KS,Sheen IS,Wang YC,et al. Prognostic significance of preoperative circulating vascular endothelial growth factor messenger RNA expression in resectable hepatocellular carcinoma:a prospective study[J].World J Gastroenterol,2004,10:643-648.
[28]Schwartz JD,Schwartz M,Sung M,et al. Bevacizumab in unresectable hepatocellular carcinoma (HCC) for patients without metastasis and without invasion of the portal vein. American Society of Clinical Oncology Gastrointestinal Cancers Symposium,San Francisco,CA,January 26-28,2006.
[29] Zhu AX,Blaszkowsky LS,Ryan D?,et al. Phase II study of gemcitabine and oxaliplatin in combination with bevacizumab in patients with advanced hepatocellular carcinoma[J]. J Clin Oncol,2006, 24:1898-1903.
[30] Hen HX. Expanding the clinical development of bevacizumab[J].Oncologist,2004,9(Suppl 1):27-35.
[31] Dlaz-rubio E. New chemotherapeutic advances in pancreatic, colorectal,and gastric cancer[J].Oncologist,2004,9(3):282-294.
[2]Rosen LS.Clinical experience with angiogenesis signaling inhibitors:focus on vascular endothelial growth factor(VEGF) blockers[J].Cancer Control 2002Mar-Apr;9(2 Suppl):36-44.
[3]Evelyne D,Patricia H,Aude V,et al.Tumor angiogenesis and tissue factor expression during hepatocellular carcinoma progression in a transgenic mouse model[J].J Hepatol,2003;38(6):793-802.
[4]高峨嵋,徐建明.VEGF靶向药Avastin治疗实体瘤的研究进展[J].中国肿瘤临床与康复,2006.
[5]Jain RK.Delivery of novel therapeutic agents in tumors:physiological barriers and strategies[J].J Natl Cancer Inst,1989,81:570-576.
[6]Warren RS,Yaun H,Matli MR.Regulation by vascular endothelial growth factor of human colon cancer tumorigenesis in a mouse model of experimental liver metastasis[J].J Clin Invest,1995,95:1789-1797.
[7]Kim KJ,Li B,Winer J,et al.Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo[J].Nature,1993,362:841-844.
[8]Napoleone F.Vascular endothelial growth factor as a target for anticancer therapy[J].Oncologist,2004,9(supp 11):2210.
[9]Jain RK.Normalization of tumor vasculature:an emerging concep tin antiangiogenic therapy[J].Science,2005,307:58-62.and Lee CG,Heijn M, di Tomaso E,et al.Antivascular endothelial growth factor treatment augments tumor radiation response under normoxic or hypoxic conditions[J].Cancer Res,2000,60:5565-5570.
[10]Guan Z.Prospective randomised phase Ⅱ study of gemcitabine at standard or fixed dose rates cheduleinun resectable hepatocellular carcinoma[J].Br J Cancer.2003;89(10):1865.
[11]Fuchs CS.A phase Ⅱ trial of gemcitabine in patients with advanced hepatocellular carcinoma.Cancer[J],2002;94(12):3186.
[12]Zhu AX,Blaszkowsky LS,Ryan DP,et al.Phase Ⅱ study of gemcitabine and oxaliplatin in combination with bevacizumab in patients with advanced hepatocellular carcinoma[J].J Clin Oncol.2006;24:1898-1903.
[13]Kindler HL,Friberg G,Singh DA,et al.Phase ⅡTrial of Bevacizumab Plus Gemcitabine in Patients With Advanced Pancreatic Cancer[J].J Clin Oncol.2005Nov 1;23(31):8033-40.
[1]陈锡山,苏建堂,崔毓桂,等,膀胱移行细胞癌survivin与血管内皮生长因子表达相关性及其临床意义[J].南京医科大学学报,2005,25(8):561-563.
[2]刘晓,范萍,王水,等.血管内皮生长因子及其受体蛋白在乳腺癌组织中的表达及意义[J].南京医科大学学报,2005,25(11):777-779.
[3]朱宏,施瑞华,张国新,等.环氧合酶-2、血管内皮生长因子和微血管密度与食管鳞癌病理特征的关系[J].南京医科大学学报,2005,25(2):84-86.
[4]Jordi Graells,Antonia Vinyals,Agnes Figueras,et al.Over production of VEGF_(165)Concomitantly Expressed with its Receptors Promotes Growth and Survival of Melanoma Cells through MAPK and PI3K Signaling[J].The journal of investigative dermatology,2004,123(6):1151-1161.
[5]Forster Y,Meye A,Krause S,et al.Antisense-mediated VEGF suppression in bladder and breast cancer cells[J].Cancer Lett,2004,212(1):95-103.
[6]Takei Y,Kadomatsu K,Yuzawa Y,et al.A small interfering RNA targeting vascular endothelial growth factor as cancer therapeutics[J].Cancer Res,2004, 64(10):3365-3370.
[7]ShenBQ,LeeDY, Gerber HP,et al.Homologous up-regulation of KDR/Flk receptors expression by vaseular endotheliai growth factor in vitro[J].J Biol Chem,1998:273(45):29979-29985.
[8] Yan Wu, Andrea T. Hooper, Zhaojing Zhong.The vascular endothelial growth factor receptor (VEGFR-1) supports growth and survival of human breast carcinoma[J]. Int. J. Cancer, 2006,119(7): 1519-1529.
[9] Barr MP, Bouchier-Hayes DJ, Harmey JJ. Vascular endothelial growth factor is an autocrine survival factor for breast tumour cells under hypoxia[J]. Int J Oncol,2008,32(1):41-8.
[1]GuanZ.Prospective randomized phase II study of gemcitabine at standard or fixed dose rate schedule in unresectable hepatocellular carcinoma [J]. Br J Cancer. 2003;89 (10): 1865.
[2]Andrew X. Zhu,Development of sorafenib and other molecularly targeted agents in hepatocellular carcinoma[J]. Cancer,2008,l 12(2):250-259.
[3]Zhu AX, Blaszkowsky LS, Ryan DP, et al. Phase II study of gemcitabine and oxaliplatin in combination with bevacizumab in patients with advanced hepatocellular carcinoma[J].J Clin Oncol. 2006;24:1898-1903.
[4]L. Placidi, E.C. Scott, D. Eckoff, et al. Metabolic drug interactions between angiogenic inhibitor, TNP-470 and anticancer agents in primary cultured hepatocytes and microsomes[J]. Drug Metab Dispos,1999:623-626.
[5]Bos JL. All in the family New insight s and questions regarding interconnectivity of Ras, Rapl and Ral [J]. EMBO J, 1998; 17(23):6776.
[6]Gysin S, Lee SH, Dean NM, et al. Pharmacologic inhibition of RAF→MEK→ERK signaling elicit s pancreatic cancer cell cycle arrest t hrough induced expression of p27 Kipl [J]. Cancer Res,2005; 65 (11):4870.
[7] Ito Y, Sasaki Y, Horimoto M, et al . Activation of mitogen-activated protein kinases/ext- racellular signal-regulated kinases in human hepatocellular carcinoma [J]. Hepatology, 1998;27(4):951.
[8]Adeyinka A, Nui Y, Cherlet T, et al. Activated mitogen-activated protein kinase expression during human breast tumorigenesis and breast cancer progression [J].Clin Cancer Res, 2002;8 (6): 1747.
[9]Albanell J, Codony-Servat J, Rojo F, et al. Activated extracellular signal-regulated kinases: association with epidermal growth factor receptor/transforming growth factor alpha expression in head and neck squamous carcinoma and inhibition by anti-epidermal growth factor receptor treatments [J]. Cancer Res, 2001; 61(17):6500.
[10]Uzgare AR, Kaplan PJ, Greenberg NM. Differential expression and/or activation of P38MAPK, erk1/2 , and jnk during the initiation and progression of prostate cancer [J]. Prostate, 2003;55 (2) :128.
[11]Rincon-Arano H, Rosales R, Mora N, et al. R-Ras promotes tumor growth of cervical epithelial cells [J]. Cancer, 2003; 97(3):575.
[12]Gioeli D, Mandell JW, Pet roni GR, et al. Activation of mitogen-activated protein kinase associated wit h prostate cancer progression [J]. Cancer Res, 1999;59 (2):279.
[13]Carson DA, Ribeiro JM. Apoptosis and disease. Lancet, 1993 ,341:1251-1254.
[14]Kim R, Tanabe K, Uchida Y,et al. Current status of t he molecular mechanisms of anticancer drug2induced apoptosis. The cont ribution of molecular21evel analysis to cancer chemot herapy[J] . Cancer Chemot her Pharmacol ,2002 ,50(5) :343-352.
[15]RJ Bold , J Chandra , DJ McConkey ,et al. Gemcitabine-in-duced programmed cell deat h (apoptosis) of human pancreatic carcinoma is determined by Bcl-2 content [J]. Annals of Surgical Oncology, 1999,6 (3) .279-285.
[16]Zhou JR,Mukher-jee P,Gugger ET,et al.Inhibition of murine bladder tumorigenesis by soy isoflavone via alterations in the cell cycle,apop-tosis,and angiogenesis[J].Cancer Res,1998;58(22):5231-5238.
[17]Carroll H P,Ali S,Kirby JA,et al.Accelerating the induction of Fas-mediated T cell apoptosis:a strategy for transplant tolerance[J].Clin Exp Immunol,2001;72(8):1423-1432.
[18]Wong BR,BesserD,Kim N,et al.TRANCE,a TNF fam ily m em ber,activates Akt/PKB through signaling complex involving TRAF6 and c-src[J].Molecular cell,1999;17(4): 1041-1049.
[19]Leech SH,Olie RA,Gaut schi O,et al. Induction of apoptosis in lung cancer cells following bcl-xl antisense treatment[J]. Int J Cancer ,2000 ,86 :570-576.
[1]Weidner N,Semple JP, Welch WR,et al.Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma[J]. N Engl J Med. 1991,324(1):1-8.
[2] Takahashi Y,Kitadai Y,Bucana CD, et al. Expression of vascular endothelial growt h factor and it s receptor,KDR,correlates with vascularity,metastasis and proliferatin of human colon cancer [J]. Cancer Res ,1995 ,55 :3964-3968.
[3]Breasalier RS,HO SB,Schoeppner HL,et al.Enhanced sialylation of mucin-associated carbohydrate structures in human colon cancer metastasis[J].Gastroenterology,1996,110(5):1354-67.
[4]Folkman J.Tumor angiogenesis:therapeutic implications[J].N Engl J Med 1971,28(5):1182-1186.
[5]Hanahan D,Weinberg RA.The hallmarks of cancer[J].Cell 2000,100:57-70.
[6]Rafii S,Lyden D,Benezra R,et al.Vascular and haematopoietic stem cells:Novel targets for anti-angiogenesis therapy[J].Nat Rev Cancer 2002,2:826-835.
[7]Zhu AX,Blaszkowsky LS,Ryan DP,et al.Phase Ⅱ study of gemcitabine and oxaliplatin in combination with bevacizumab in patients with advanced hepatocellular carcinoma[J].J Clin Oncol.2006;24:1898-1903.
[8]Parikh PM,Fuloria J,Babu G,et al.A phase Ⅱ study of gemcitabine and cisplatin in patients with advanced hepatocellular carcinoma[J].Trop Gastroenterol,2005;26(3):115.
[9]Sun W,Hailer DG,Mykulowycz K,et al.Combination of capecitabine,oxaliplatin with bevacizumab in treatment of advanced hepatocellular carcinoma(HCC):a phase Ⅱ study.J Clin Oncol(Meeting Abstracts).2007;25(18S).Abstract 4574.
[10]Kindler HL,Friberg G,Singh DA,et al.PhaseⅡ Trial of Bevacizumab Plus Gemcitabine in Patients With Advanced Pancreatic Cancer[J].J Clin Oncol.2005Nov 1;23(31):8033-40.
[11]Ranieri G,Gasparini G.Angiogenesis and angiogenesis inhibitors:a new potential anticancer therapeutic strategy[J].Curr Drug Targets Immune Endocr Metabol Disord,2001,1(3):241-253.
[12]Dudek AZ,Pawlak WZ,Kirstein MN.Molecular targets in the inhibition of angiogenesis[J].ExpertOp in Ther Targets,2003,7(4):527-541. 理学杂志,2003,19(4):401-404.
[14]Suzuki K,Hayashi N,Miyamoto Y,et al.Expression of vascular permeability factor/vascular endothelial growth factor in human hepatocellular carcinoma[J].Cancer Res,1996,56(13):3004.
[15]李晓明,汤钊猷,周铬,等.血管内皮生长因子的表达与肝细胞癌侵袭和转移的关系[J].肿瘤,1998,20:12-14.
[16]Poon RT,Lau C,Yu WC,et al.High serum levels of vascular endothelial growth factor predict poor response to transarterial chemoembolization in hepatocellular carcinoma:a prospective study[J].Oncol Rep.2004 May;11(5):1077-84.
[17]罗荣城,韩焕兴,主编.肿瘤生物治疗学[M],北京.人民卫生出版社(第二版).90-1.
[18]Borgstrom P,Gold DP,Hillan KJ,et al.Imprortance of VEGF for breast cancer angiogenesis in vivo:implications from intravital microscopy of combination treatments with an anti-VEGF neutralizing monoclonal antibody and doxorubicin[J].Anticancer Res,1999,19(5B):4203-14.
[19]Yamaguchi R,Yano H,Iemura A,et al.Expression of vascular endothelial growth factor in human hepatocellular carcinoma[J].Hepatology,1998,28:68-77.
[20]Yao DF,Wu XH,Zhu Y,et al.Quantitative analysis of vascular endothelial growth factor,microvascular density and their clinicopathologic features in human hepatocellular carcinoma[J].Hepatobiliary Pancreat Dis Int.2005May;4(2):220-6.
[21]徐洪雨,毕莉,杨幼林,等.血管内皮生长因子及微血管密度在肝癌中的表达[J].中国肿瘤杂志,2000,4:249-251.
[22]Chao Y,Li CP,Chau GY,et al.Prognostic significance of vascular endothelial growth factor,basic fibroblast growth factor,and angiogenin in patients with resectable hepatocellular carcinoma after surgery[J].Ann Surg Oncol,2003,10:355-362.
[23]Poon RT,Ho JW,Tong CS,et al.Prognostic significance of serum vascular endothelial growth factor and endostatin in patients with hepatocellular carcinoma[J].Br J Surg,2004,91:1354-1360.
[24]Jeng KS,Sheen IS,Wang YC,et al.Prognostic significance of preoperative circulating vascular endothelial growth factor messenger RNA expression in resectable hepatocellular carcinoma:a prospective study[J].World J Gastroenterol,2004,10:643-648.
[25]Kwak BK,Shim HJ,Park ES,et al.Hepatocellular carcinoma:correlation between vascular endothelial growth factor level and degree of enhancement by multiphase contrast enhanced computer tomography[J].Invest Radiol,2001,36:487-489.
[26]Luttun A,Tjwa M,Moons L,et al.Revascularization of ischemic tissues by PIGF treatment,and inhibition of tumor angiogenesis,arthritis and atherosclerosis by anti-Fltl[J].Nat Med,2002,8(8):831-840.
[27]Hiratsuka S,Maru Y,okada A,et al.Involvement of Flt-1 tyrosine kinase(vascular endothelial gowth faetor receptor-1)in patholgieal anogenesis[J].Caneer Res,2001,61(3):1207-1213.
[28]Wu Y,Zhong Z,Huber J,et al.Anti-vascular endothelial growth faetor recptor-1antagonist antibody as a therapeutie agent for cancer[J].Clin Canee:Res,2006,12(21):6573-6584.
[29]赵爱志,窦科峰,李开宗等.VEGF、KDR的表达对肝癌的血管生成、生长、转移的影响[J].中华外科杂志.2000,6(38):454-455.
[30]刘金洋,李康华,廖前德.骨肉瘤中Bcl-2、Fas蛋白表达的相关性研究[J].医学临床研究,2006,23(4):581-582.
[31]Voutsadakis IA.Apoptosis and the pathogenesis of lymphoma[J].Acta Oncol,2000,39(2):151-156.
[32]Zhao M,Zhang NX,Economou M,et al.Immunohistochemical detection of Bcl-2 protein in liver lesions:Bcl-2 protein is expressed in hepatocellular carcinomas but not in liver cell dysplasia[J].Histopathology,1994,25(3):237.
[33]秦军,李敏,孙丽娟,等.Bcl-2蛋白在肝胆癌及慢性肝病中的表达[J].实用医药杂志,2002,19(4):265.
[34]Gazzaniga P,Gandini O,Gradilone A,et al.Detection of basic fibroblast growth facter mRNA in urinary bladder cancer:correclation with local relapses[J].IntJ Oncol,1999,14:1123.
[35]李清龙,李永国,杨竹林.慢性肝病及肝癌组织中Bcl-2蛋白表达及意义[J].临床研究.2007,9(24):1501-1503.
[36]赵建平,王媛媛,阎玉虎.吉西他滨对人非小细胞肺癌A549细胞增殖与凋亡的影响.肿瘤防治研究[J].2006,(33)2:100-102.
[37]Kar S,Wang M,et al.Transient and sustained ERK phosphorylation and nuclear translocation in growth control[J].Cell Physiol,2002;192(2):151-9.
[38]唐承薇.奥曲肽对胃癌细胞转录活化蛋白-1的抑制作用[J].癌症,2002;21(8):850-854.
[39]Steinmetz R,Wagoner HA,Zeng P,et al.Mechanisms regulating the constitutive activation of the ERK signaling pat hway in ovarian cancer and the effect of RNAi for ERK1/2 on cancer cell proliferation[J].Mol Endocrino,2004;15(3):1004.
[40]Wu TT,Wang J S,Jiaan BP,et al.Role of p21~(WAF1) and p27~(K1P1) in predicting biochemical recurrence for organ-confined prostate adenocarcinoma[J].J Chin Med Assoc,2007;70(1):11.
[41]Handra-Luca A,Bilal H,Bert rand JC,et al.Extra-cellular signal-regulated ERK-1/ERK-2 pathway activation in human salivary gland mucoepidermoid carcinoma : association to aggressive tumor behavior and tumor cell proliferation [J]. Am J Pat hoi, 2003;163 (3):957.
[42]Hoshino R, Chatani Y, Yamori T, et al. Constitutive activation of the 41-/ 43-kDa mitogen-activated protein kinase signaling pathway in human tumors [J].Oncogene, 1999; 18(3):813.
[43]Mishima K, Inoue K, Hayashi Y. Overexpression of extracellular-signal regulated kinases on oral squamous cell carcinoma[J]. Oral Oncol, 2002 ; 38 (5):468.
[44]Gerber HP,Dixit V,Ferrara N.Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells[J].J Biol Chem,1998,273(21):13313-6.
[45]Harmey JH.VEGF and Cancer[M].New York,USA:Kluwer Academic/Plenum Publishers,2004:29.
[1]Leung DW,Cachines G,Kuang WJ,et al.Vascular endothelial growth factor issecreted angiogenic mitogen[J].Science,1989,246:1306.
[2]Senger DR,Van de Water L,Brown LF,et al.Vascular permeability factor(VPF,VEGF) in tumor biology[J].Cancer Metastasis Rev,1993,12(3-4):303-324.
[3]Ferara N,Smyth TD.The biology of vascular endotheilial growth factor[J].Endocrine Rev 1997,18:4-25.
[4]Bussolino F,Mantovani A,Persico G.Molecular mechanisms of blood vessel formation[J]. TIBS, 1997,22:251.
[5] Shibuya M. Role of VEGF-FLT receptor system in normal and tumor angiogenesis[J]. Adv Cancer Res, 1995,67:281.
[6] Brown LF,Detmar M,Claffey K,et al.Vasclular permeability factor/vascular endothelial growth factor: A multifunctional angiogenic cytokine[J]. Exs,1997,79:233.
[7] Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease [J]. Nature Med,1995,l:27-31.
[8] Lohn E, Liotta L. Molecular insights into cancer invasion: strategies for prevention and intervention [J]. Cancer Res, 1995,55:1865.
[9] Ferrara N,Davis-Smyth T.The biology of vascular endotelial growth factor.Endocr Rev 1997;18:4-25.
[10] Gordon M S,Margolin K, TalpazM,et al. Phase I safety and pharmacokinetic study of recombinat human anti-vascular endothelial growth factor in patients with advanced cancer[J].J Clin Oncol,2001, 19(4):843-850.
[11] Napoleone F. Vascular endothelial growth factor as a target for anticancer therapy[J].Oncologist,2004,9(suppl 1):2-10.
[12] HurwitzH, Fehrenbacher L, NovotnyW, et al.Bevacizumab plus irinotecan,fluorouraci,l and leucovorin for metastatic colorectal cancer[J].N Engl JMed, 2004, 350: 2335-2342.
[13] Langer CJ,Natale RB.The emerging role of vascular endothelial growth factor receptor tyrosine kinase inhibitors[J].Semin Oncol,2005,32(6 Suppl 10):S23-29.
[14] SandlerAB,Gray R, Brahmer J, et al. Randomized phase II/III trial ofpaclitaxel(P) plus carboplatin(C) with or without bevacizumab (NSE#704865) in patients with advanced non-squa-mous non-small cell lung cancer(NSCLC):An Eastern Cooperative Oncology Group (ECOG)Trial-E4599[J]. ProcAm Soc Clin Oncol , 2005, 23: 2(AbstrLBA4).
[15]Sandler A, Gray R, Brahmer J. et al. Randomized phase II/III trial of paclitaxel plus carboplatin with or without bevacizumab in patients with advanced non-squamous non-small cell lung cancer: An Eastern Cooperative Oncology Group Trial-E4599[J].J Clin Onco. 2005;23:abstract LBA4,2.
[16]Brahmer JR, Gray R, Schiller JH, et al. ECOG 4599 phase III trial of carboplatin and paclitaxel ± bevacizumab: subset analysis of survival by gender[JI. Proc Am Soc Clin Oncol. 2006;24:373s. Abstract 7036.
[17]Dowlati A, Gray R, Johnson DH, et al. Prospective correlative assessment of biomarkers in E4599 randomized phase II /III trial of carboplatin and paclitaxel ± bevacizumab in advanced non-small cell lung cancer (NSCLC)[J]. Proc Am Soc Clin Oncol. 2006;24:370s. Abstract 7027.
[18]Manegold C, von Pawel J, Zatloukal P, et al. Randomized, double-blind multicentre phaseIII study of bevacizumab combination with cisplatin and gemcitabine in chemotherapy-naive patients with advanced or recurrent non-squamous non-small cell lung cancer (NSCLC). Program and abstracts of the 43rd American Society of Clinical Oncology Annual Meeting; June 1-5,2007; Chicago, Illinois. Abstract LBA7514.
[19]Sandler A, Dahlberg SE, Dowlati A, et al. A phase II study of cisplatin plus etopside plus bevacizumab for previously untreated extensive stage small cell lung cancer by the Eastern Coopertive Oncology Group(ECOG 3501).Program and abstracts of the 43rd American Society of Clinical Oncology Annual Meeting; June 1-5,2007; Chicago, Illnois. Abstract 7564.
[20]Tortora G, Caputo R, Damiano V,et al. Combined targeted inhibition of Bcl-2, Bcl-xl, epidermal growth factor receptor, and protein kinase A type I causes potent antitumor, apoptotic and antiangiogenic activity[J].Clin Cancer Res 2003; 9: 866-871.
[21] Herbst RS, Johnson DH, Mininberg E, et al. Phase I / II trial evaluating the anti-vascular endothelial growth factor monoclonal antibody bevacizumab in combination with the HER-1/epidermal growth factor receptor tyrosine kinase inhibitor erlotinib for patients with recurrent non-small-cell lung cancer[J]. J Clin Oncol, 2005, 23:2544-2555.
[22] Nathan P, Chao D, Brock C, Savage P, Harries M, Gore M, Eisen T.Br J Cancer. 2006 May 8;94(9):1217-20.
[23] J. D. Hainsworth, J. A. Sosman, D. R. Spigel, R. C. Schwert, D. L. Carrell,F. Hubbard and F. A. Greco.Journal of Clinical Oncology, 2004 ASCO Annual Meeting Proceedings (Post-Meeting Edition).Vol 22, No 14S (July 15 Supplement), 2004: 4502.
[24] Yamaguchi R,Yano H, Iemura A,et al. Expression of vascular endothelial growth factor in human hepatocellular carcinoma[J].Hepatology,1998,28:68-77.
[25]Chao Y,Li CP,Chau GY,et al. Prognostic significance of vascular endothelial growth factor, basic fibroblast growth factor,and angiogenin in patients with resectable hepatocellular carcinoma after surgery[J]. Ann Surg Oncol,2003,10:355-362.
[26] Poon RT,Ho JW,Tong CS,et al. Prognostic significance of serum vascular endothelial growth factor and endostatin in patients with hepatocellular carcinoma[J]. Br J Surg,2004,91:1354-1360.
[27] Jeng KS,Sheen IS,Wang YC,et al. Prognostic significance of preoperative circulating vascular endothelial growth factor messenger RNA expression in resectable hepatocellular carcinoma:a prospective study[J].World J Gastroenterol,2004,10:643-648.
[28]Schwartz JD,Schwartz M,Sung M,et al. Bevacizumab in unresectable hepatocellular carcinoma (HCC) for patients without metastasis and without invasion of the portal vein. American Society of Clinical Oncology Gastrointestinal Cancers Symposium,San Francisco,CA,January 26-28,2006.
[29] Zhu AX,Blaszkowsky LS,Ryan D?,et al. Phase II study of gemcitabine and oxaliplatin in combination with bevacizumab in patients with advanced hepatocellular carcinoma[J]. J Clin Oncol,2006, 24:1898-1903.
[30] Hen HX. Expanding the clinical development of bevacizumab[J].Oncologist,2004,9(Suppl 1):27-35.
[31] Dlaz-rubio E. New chemotherapeutic advances in pancreatic, colorectal,and gastric cancer[J].Oncologist,2004,9(3):282-294.