恶性胶质瘤中EGFR和VEGFR2分子靶向联合治疗的实验研究
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摘要
目的采用传代的人脑多形性胶质母细胞瘤(glioblastoma multiforme,GBM)新鲜手术标本来建立呈侵袭性生长及表皮生长因子受体(epidermal growth factor receptor,EGFR)高表达的人脑GBM裸小鼠脑原位移植模型,然后在此已建立的能保持亲本肿瘤生物学特性的人脑恶性胶质瘤原位移植模型基础上,把EGFR抑制剂与常规化疗药物或与血管内皮生长因子受体-2(vascular endothelial growth factor receptor-2,VEGFR2)抑制剂联合应用来治疗人脑GBM移植瘤,以探讨不同作用机理的化学治疗药物联合应用后对GBM的疗效。
方法将传代的新鲜人脑GBM手术标本行裸小鼠右尾状核接种后第3d用于分组实验。(1)40只鼠随机分为4组,10只/组,各组鼠分别应用靶向EGFR的蛋白酪氨酸激酶抑制剂AG1478(25mg/kg.次)、CDDP(3mg/kg.次)、AG1478+CDDP(25mg/kg.次+3mg/kg.次)及磷酸盐缓冲液(PBS,0.1ml/只.次)。PBS和药物均腹腔注射,每2d一次,共4次,用药后分别观察各组荷瘤鼠生存期。再取40只鼠,重复上述实验,肿瘤移植后14d处死所有组鼠取脑,HE染色后观察各组移植瘤形态及体积变化;EnVision和Western Blot法检测移植瘤中EGFR、磷酸化EGFR(P-EGFR)、增殖活性(Ki-67 labelling index,即Ki-67 LI)、血管内皮生长因子(vascular endothelial growth factor ,VEGF)、白细胞介素-8(IL-8)和微血管密度(microvessel density,MVD)变化;TUNEL法检测移植瘤细胞凋亡(apoptotic index即AI)变化。(2)40只荷瘤鼠随机分为4组,10只/组,分别应用靶向EGFR的单克隆抗体C225(50mg/kg.次)、靶向VEGFR-2的单克隆抗体DC101(40mg/kg.次)、C225+DC101 (50mg/kg.次+40 mg/kg.次)及PBS(0.1ml/只.次)。PBS组和药物均腹腔注射,每2d一次,共4次。肿瘤移植后14d,处死所有组鼠取脑,HE染色观察各组移植瘤形态及体积变化;EnVision法检测移植瘤中基质金属蛋白酶-1(matrix metalloproteinase,MMP1)、MVD及增值活性变化;TUNEL法检测肿瘤细胞凋亡变化。
结果(1)与对照组生存期为19.4±0.6d比较,单用CDDP或AG1478后荷瘤鼠生存期分别为20.7±0.4d和20.8±0.6d(P>0.05),而CDDP+AG1478组荷瘤鼠生存期为33.6±0.9d(P<0.05);CDDP+AG1478能明显减小移植瘤体积、降低增殖活性及促进肿瘤细胞凋亡,而单独用药对此不明显。AG1478及CDDP+AG1478抑制了EGFR磷酸化,使P-EGFR表达下降,并降低VEGF、IL-8表达量及减少MVD。(2)与对照组比较,DC101组移植瘤体积下降了59.7%(P<0.05),DC101+C225组移植瘤体积下降了62.9%(P<0.05),而C225组对移植瘤体积几乎无影响(P>0.05);DC101组移植瘤侵袭性加强,移植瘤中MMP1表达为(++);C225组移植瘤侵袭性下降,移植瘤中MMP1表达阳性(+);C225+DC101组移植瘤侵袭性也下降,移植瘤中MMP1表达阳性(+);DC101组移植瘤中MVD减少了64(%P<0.05),C225+DC101组MVD减少了68(%P<0.05),C225组的MVD无明显变化(P>0.05);DC101组肿瘤细胞Ki-67 LI下降了53.2%(P<0.05),DC101+C225联合用药后Ki-67 LI下降了56.4%(P<0.05),而C225组下降不明显(P>0.05);DC101诱导细胞AI增加了66.7%(P<0.05),DC101+C225联合用药后AI增加了75%(P<0.05),而C225用药后增加不明显(P>0.05)。
结论(1)AG1478与CDDP联合应用比单独用药有显著的抗肿瘤生长疗效,并在一定程度上抑制了肿瘤的血管生成,这为EGFR抑制剂和常规化疗药物联合治疗恶性胶质瘤提供了实验依据。(2)DC101与C225联合用药显著抑制GBM移植瘤生长,并降低了移植瘤侵袭和转移特性,两药合用疗效优于单用的疗效,这为抗肿瘤血管生成药物与抗肿瘤侵袭药物合用治疗恶性胶质瘤提供了实验依据。
PartⅠExploring the effect on tumor growth and angiogenesis after AG1478 combined with chemotherapy for treating the orthotopic xenografts of human glioblastoma in nude mice
Objective To evaluate the efficacy of antitumor activity and influence on angiogenesis after AG1478 combined with Cisplatin for treating the orthotopic xenografts of human gliobastoma multiforme.Methords Establishing orthotopic xenograft model of human gliobastoma multiforme with stable EGFR expression as described previously.On day 3 postinnoculation,40 mice were divided into 4 groups randomly with 10 every group,then 4 groups of mice received i.p. injections of AG1478(25mg/kg every time)、Cisplatin (3mg/kg every time)、AG1478+ Cisplatin(25mg/kg every time +3mg/kg every time)and PBS(0.1ml/20g every time)respectively with every 2 days for 4 times,then survival time were observed until mice bearing xenografts were natural death.The experiment was repeated,in which the same types of treatment were initiated on day 3.On day 14 after innnoculation ,every group of mice were sacrificed. Mouse brains were removed from the cranial cavity, fixed in formalin, bisected coronally, and embedded in paraffin.Serial sections were stained with H&E,then tumor appearance and volume were observed and measured.The expression of EGFR,phosphorylated EGFR (P-EGFR) were detected by immunochemical or Western blot methods;VEGF,IL-8 or Ki-67 in the xenografts were detected by immunochemical methods,and apoptosis were detected by TUNEL method.Results The expression of P-EGFR were decreased because phosphorylation of EGFR were inhibited by AG1478,accordingly the expression of VEGF or IL-8 were decreased,and microvessel destiny was also declined.Compared with PBS group,the suvival were strikingly prolonged,and volume of xenografts were reduced by the combination of Cisplatin with AG1478;meanwhile, the combined application could significantly decrease proliferative activity and increase apoptotic rate,but no effect in drugs alone.Conclusion The combination of AG1478 with Cisplatin significantly increased the activities against tumor growth and inhibited the angiogenesis,which provided a rationale for its clinical evaluation in combination with both chemotherapy and other molecular target drugs for treating malignant gliomas.
PartⅡAntitumor treatment efficiency by targeting epidermal growth factor receptor and vascular endothelial growth factor receptor-2 in an orthotopic human glioblastoma model
Objective: To explore the inhibitive effect on transplanted tumor of human glioblastoma multiforme in the brain of nude mice after t reatment of C225 and DC101 alone and combined application.Methods: Establishing the human GBM orthotopic models with high expression of EGFR and VEFR in the brain of nude according to the previous method.On day 3 postinnoculation,40 mice were divided into 4 groups randomly with 10 every group,then 4 groups of mice received i.p. injections of C225(50mg/kg)、DC101(40mg/kg)、CD225+DC101 (50 +40)mg/kg or PBS(0.1ml/20g)respectively with every 2 days for 4 times.On day 14 after innnoculation ,every group of mice were killed.Mouse brains were removed from the cranial cavity, fixed in formalin, bisected coronally, and embedded in paraffin. Serial sections were stained with H&E to observe tumor appearance and to measure the maximum or minimun diameter under light microscope so as to work out tumor volume.The expression of MMP1,MVD or Ki-67 were detected by immunochemical methods,and apoptosis were detected by TUNEL method.Results:When compared with controls(PBS) group,the xenograft volumes were decreased by 59.7% and 62.9% in the DC101 and the DC101+C225 group respectively(P<0.05),but no effect in the C225group(P>0.05);The invasion ability of xenograft was improved,and expression of MMP1 was increased to (++) in the DC101group. The invasion ability of xenograft was declined,and expression of MMP1 was decreased to (+) in the C225 group,which was also founded in the DC101+C225 group.MVD were reduced by 64% and 68% in the DC101 and the DC101+C225 group respectively(P<0.05), but no effect in the C225group(P>0.05);Ki-67 labelling index(LI) was decreased by 53.2% and 56.4% in the DC101 and the DC101+C225 group respectively(P<0.05), but no effect in the C225 group(P>0.05);Apoptotic index(AI) was increased by 66.7% and 75% in the DC101 and the DC101+C225 group respectively(P<0.05), but no effect in the C225 group(P>0.05).Results:The combined C225 and DC101 can inhibit the growth and invasion ability of transplanted tumor of human glioblastoma multiforme in nude mice. The effect of C225 + DC101 is better than that of C225 and DC101 alone,which provided a experimental foundation for treatmenting malignant glioma by the method of unifying drugs with different pharmacological features.
方法将传代的新鲜人脑GBM手术标本行裸小鼠右尾状核接种后第3d用于分组实验。(1)40只鼠随机分为4组,10只/组,各组鼠分别应用靶向EGFR的蛋白酪氨酸激酶抑制剂AG1478(25mg/kg.次)、CDDP(3mg/kg.次)、AG1478+CDDP(25mg/kg.次+3mg/kg.次)及磷酸盐缓冲液(PBS,0.1ml/只.次)。PBS和药物均腹腔注射,每2d一次,共4次,用药后分别观察各组荷瘤鼠生存期。再取40只鼠,重复上述实验,肿瘤移植后14d处死所有组鼠取脑,HE染色后观察各组移植瘤形态及体积变化;EnVision和Western Blot法检测移植瘤中EGFR、磷酸化EGFR(P-EGFR)、增殖活性(Ki-67 labelling index,即Ki-67 LI)、血管内皮生长因子(vascular endothelial growth factor ,VEGF)、白细胞介素-8(IL-8)和微血管密度(microvessel density,MVD)变化;TUNEL法检测移植瘤细胞凋亡(apoptotic index即AI)变化。(2)40只荷瘤鼠随机分为4组,10只/组,分别应用靶向EGFR的单克隆抗体C225(50mg/kg.次)、靶向VEGFR-2的单克隆抗体DC101(40mg/kg.次)、C225+DC101 (50mg/kg.次+40 mg/kg.次)及PBS(0.1ml/只.次)。PBS组和药物均腹腔注射,每2d一次,共4次。肿瘤移植后14d,处死所有组鼠取脑,HE染色观察各组移植瘤形态及体积变化;EnVision法检测移植瘤中基质金属蛋白酶-1(matrix metalloproteinase,MMP1)、MVD及增值活性变化;TUNEL法检测肿瘤细胞凋亡变化。
结果(1)与对照组生存期为19.4±0.6d比较,单用CDDP或AG1478后荷瘤鼠生存期分别为20.7±0.4d和20.8±0.6d(P>0.05),而CDDP+AG1478组荷瘤鼠生存期为33.6±0.9d(P<0.05);CDDP+AG1478能明显减小移植瘤体积、降低增殖活性及促进肿瘤细胞凋亡,而单独用药对此不明显。AG1478及CDDP+AG1478抑制了EGFR磷酸化,使P-EGFR表达下降,并降低VEGF、IL-8表达量及减少MVD。(2)与对照组比较,DC101组移植瘤体积下降了59.7%(P<0.05),DC101+C225组移植瘤体积下降了62.9%(P<0.05),而C225组对移植瘤体积几乎无影响(P>0.05);DC101组移植瘤侵袭性加强,移植瘤中MMP1表达为(++);C225组移植瘤侵袭性下降,移植瘤中MMP1表达阳性(+);C225+DC101组移植瘤侵袭性也下降,移植瘤中MMP1表达阳性(+);DC101组移植瘤中MVD减少了64(%P<0.05),C225+DC101组MVD减少了68(%P<0.05),C225组的MVD无明显变化(P>0.05);DC101组肿瘤细胞Ki-67 LI下降了53.2%(P<0.05),DC101+C225联合用药后Ki-67 LI下降了56.4%(P<0.05),而C225组下降不明显(P>0.05);DC101诱导细胞AI增加了66.7%(P<0.05),DC101+C225联合用药后AI增加了75%(P<0.05),而C225用药后增加不明显(P>0.05)。
结论(1)AG1478与CDDP联合应用比单独用药有显著的抗肿瘤生长疗效,并在一定程度上抑制了肿瘤的血管生成,这为EGFR抑制剂和常规化疗药物联合治疗恶性胶质瘤提供了实验依据。(2)DC101与C225联合用药显著抑制GBM移植瘤生长,并降低了移植瘤侵袭和转移特性,两药合用疗效优于单用的疗效,这为抗肿瘤血管生成药物与抗肿瘤侵袭药物合用治疗恶性胶质瘤提供了实验依据。
PartⅠExploring the effect on tumor growth and angiogenesis after AG1478 combined with chemotherapy for treating the orthotopic xenografts of human glioblastoma in nude mice
Objective To evaluate the efficacy of antitumor activity and influence on angiogenesis after AG1478 combined with Cisplatin for treating the orthotopic xenografts of human gliobastoma multiforme.Methords Establishing orthotopic xenograft model of human gliobastoma multiforme with stable EGFR expression as described previously.On day 3 postinnoculation,40 mice were divided into 4 groups randomly with 10 every group,then 4 groups of mice received i.p. injections of AG1478(25mg/kg every time)、Cisplatin (3mg/kg every time)、AG1478+ Cisplatin(25mg/kg every time +3mg/kg every time)and PBS(0.1ml/20g every time)respectively with every 2 days for 4 times,then survival time were observed until mice bearing xenografts were natural death.The experiment was repeated,in which the same types of treatment were initiated on day 3.On day 14 after innnoculation ,every group of mice were sacrificed. Mouse brains were removed from the cranial cavity, fixed in formalin, bisected coronally, and embedded in paraffin.Serial sections were stained with H&E,then tumor appearance and volume were observed and measured.The expression of EGFR,phosphorylated EGFR (P-EGFR) were detected by immunochemical or Western blot methods;VEGF,IL-8 or Ki-67 in the xenografts were detected by immunochemical methods,and apoptosis were detected by TUNEL method.Results The expression of P-EGFR were decreased because phosphorylation of EGFR were inhibited by AG1478,accordingly the expression of VEGF or IL-8 were decreased,and microvessel destiny was also declined.Compared with PBS group,the suvival were strikingly prolonged,and volume of xenografts were reduced by the combination of Cisplatin with AG1478;meanwhile, the combined application could significantly decrease proliferative activity and increase apoptotic rate,but no effect in drugs alone.Conclusion The combination of AG1478 with Cisplatin significantly increased the activities against tumor growth and inhibited the angiogenesis,which provided a rationale for its clinical evaluation in combination with both chemotherapy and other molecular target drugs for treating malignant gliomas.
PartⅡAntitumor treatment efficiency by targeting epidermal growth factor receptor and vascular endothelial growth factor receptor-2 in an orthotopic human glioblastoma model
Objective: To explore the inhibitive effect on transplanted tumor of human glioblastoma multiforme in the brain of nude mice after t reatment of C225 and DC101 alone and combined application.Methods: Establishing the human GBM orthotopic models with high expression of EGFR and VEFR in the brain of nude according to the previous method.On day 3 postinnoculation,40 mice were divided into 4 groups randomly with 10 every group,then 4 groups of mice received i.p. injections of C225(50mg/kg)、DC101(40mg/kg)、CD225+DC101 (50 +40)mg/kg or PBS(0.1ml/20g)respectively with every 2 days for 4 times.On day 14 after innnoculation ,every group of mice were killed.Mouse brains were removed from the cranial cavity, fixed in formalin, bisected coronally, and embedded in paraffin. Serial sections were stained with H&E to observe tumor appearance and to measure the maximum or minimun diameter under light microscope so as to work out tumor volume.The expression of MMP1,MVD or Ki-67 were detected by immunochemical methods,and apoptosis were detected by TUNEL method.Results:When compared with controls(PBS) group,the xenograft volumes were decreased by 59.7% and 62.9% in the DC101 and the DC101+C225 group respectively(P<0.05),but no effect in the C225group(P>0.05);The invasion ability of xenograft was improved,and expression of MMP1 was increased to (++) in the DC101group. The invasion ability of xenograft was declined,and expression of MMP1 was decreased to (+) in the C225 group,which was also founded in the DC101+C225 group.MVD were reduced by 64% and 68% in the DC101 and the DC101+C225 group respectively(P<0.05), but no effect in the C225group(P>0.05);Ki-67 labelling index(LI) was decreased by 53.2% and 56.4% in the DC101 and the DC101+C225 group respectively(P<0.05), but no effect in the C225 group(P>0.05);Apoptotic index(AI) was increased by 66.7% and 75% in the DC101 and the DC101+C225 group respectively(P<0.05), but no effect in the C225 group(P>0.05).Results:The combined C225 and DC101 can inhibit the growth and invasion ability of transplanted tumor of human glioblastoma multiforme in nude mice. The effect of C225 + DC101 is better than that of C225 and DC101 alone,which provided a experimental foundation for treatmenting malignant glioma by the method of unifying drugs with different pharmacological features.
引文
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43. Eller JL, Longo SL, Hicklin DJ, Canute GW. Activity of anti-epidermal growth factor receptor monoclonal antibody C225 against glioblastoma multiforme.Neurosurgery, 2002;51:1005-1013.
44. Eller JL, Longo SL, Kyle MM,et al. Anti-epidermal growth factor receptor monoclonal antibody cetuximab augments radiation effects in glioblastoma multiforme in vitro and in vivo.Neurosurgery 2005,56:155-162.
45. Penar PL, Khoshyomn S, Bhushan A, et al.Inhibition of epidermal growth factor receptor-associated tyrosine kinase blocks glioblastoma invasion of the brain. Neurosurgery,1997,40:141-151.
46. Brockmann MA, Ulbricht U, Gruner K, et al.Glioblastoma and cerebral microvascular endothelial cell migration in response to tumor-associated growth factors. Neurosurgery,2003,52:1391-1399.
47. Lal A, Glazer CA, Martinson HM,et al. Mutant epidermal growth factor receptor up-regulates molecular effectors of tumor invasion.Cancer Res,2002,62:3335-3339.
1 Kleihues P, Soylemezoglu F, Schauble B, et al:Histopathology, classification,and grading of gliomas.Glia,1995,15:211-221.
2 Wong ET, Hess KR, Gleason MJ et al.Outcomes and prognostic factors in recurrent glioma patients enrolled onto phase II clinical trials. J Clin Oncol,1999,17:2572-2578.
3 J Shiotsu Y.Current screening for molecular target therapy of cancer.Japanese Journal of Cancer&Chemotherapy,2003,30(12):1863-1872.
4 Thrupson EW,Price JT.Mechanisms of tumor invasion and metastasis:emerging targets for therapyExp opin ther tar,2002,6(2):217-233.
5 KurokiM, Arakawa F, Khare PD, et al. Specific targeting strategies of cancer gene therapy using a single - chain variable fragment( scFv)with a high affinity for CEA . Anticancer Res,2002,20 (6A):4067～4071.
6 Arteaga CL.The epidermal growth factor receptor: from mutant oncogene in non human cancers to therapeutic target in human neoplasia. J Clin Oncol,2001,19(Suppl 1):32S-40S.
7 Arteaga C: Targeting HER1/EGFR: a molecular approach to cancer therapy. Sem Oncol,2003,30(Suppl 7):3-14.
8 Moghal N ,Sternberg PW.Multiple positive and negative regulators of signaling by the EGR-receptor.Current Opinion in Cell Biology ,1999 ,11 :190.
9 Moulder SL ,Yakes FM,M uthuswaniy SK,et al .Epidermal growth factor receptor(HER1)tyrosine kinase inhibitor Gefitinib inhibits HER2/neu (erbB2) overexpressing breast cancer cells in vitro and in vivo. Cancer Res,2001,61(24):8887-8895.
10 Wells A.EGF receptor.Review.Int J Biochem Cell.1999,31:637-643.
11 Mendelsohn J , Baselga J . The EGF receptor family as targets for cancer therapy.Oncogene,2000,19:6550-6565
12 Shinojima N, Tada K, Shiraishi S,et al. Prognostic value of epidermal growth factor receptor in patients with glioblastoma multiforme. Cancer Res,2003,63:6962-6970.
13 Van Beusechem VW,Grill J,Mastenbroek DC,et a1.Effieient and selective gene transfer into primary human brain tumors by using single-chain antibody-targeted adenoviral vectors with native tropism abolished.J Viml,2002,76(6):2753-2762.
14 Goldstein NI, Prewett M, Zuklys K, et al. Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model.Clin Cancer Res,1995;1:1311-1318.
15 Mendelsohn J.Antibody-mediated EGF receptor blockade as an anticancer therapy:from the laboratory to the clinic.Cancer Immunol Immunother,2003,52:342-346.
16 Herbst RS, Kim ES, Harari PM. IMC-C225, an anti-epidermal growth factor receptor monoclonal antibody, for treatment of head and neck cancer.Expert Opin Biol Ther,2001,1:719-732.
17 Warshall P,Oh lsson I,Biberfeld P,et a1.Intratumoral infusion of the monoclonal antibody-mAb 425-against the epidermal growth factor receptor in patients with advanced malignant glioma.Cancer human Immune Ther,1997,44( 3):157.
18 Mendelsohn J.Targeting the epidermal growth factor receptor for cancer therapy.J Clin Oncol,2002,20(18 Suppl):1S-13S.
19 RabenD , Bi ancoC , D amia noV , et al .Antitumor activity of ZD6126, a novel vascular-targeting agent, is enhanced when combined with Gefitinib,an epidermal growth f actor receptor tyrosine kinase inhibitor, and potentiates the effects of radiation in a human non-small cell lung cancer xenograft model .Mol Cancer Ther,2004,3 (8):9 77-983.
20 BaselgaJ, Averbuch SD. Gefitinib as an anticancer agent. Drugs,2000 60 ( Supp1 1 ):33-40.
21 Klohs WD,Fry DW,Kraker AJ.Inhibitor of tyrosine kinase.Curr Opin Oncol,1997,9:562-568.
22 Anita Lal, Chad A. Glazer,et al.Mutant epidermal growth factor receptor up-regulates molecular effectors of tumor invasion.Cancer Res,2002,62:3335-3339.
23 Johns TG, Luwor RB, Murone C,et al.Carmel Murone,Antitumor efficacy of cytotoxic drugs and the monoclonal antibody 806 is enhanced by the EGF receptor inhibitor AG1478.Proc Natl Acad Sci U S A,2003,100(26):15871-15876.
24 Guillamo J-S, Leuraud P, de Bouard S,et al.Anti-proliferative and anti-invasive EGFR amplification dependent and antiangiogenic EGFR independent activity of ZD1839 (‘Iressa’)tyrosine kinase inhibitor on human glioblastomas. Proc Am Assoc Cancer Res ,2003,44: (abstract 1009).
25 Learn CA, Archer GE, Riggins GJ,et al.Inhibition of cell cycle progression and invasion of cells expressing epidermal growth factor receptor (EGFR) but not mutant EGFR variant III(EGFRvIII) by the specific EGFR tyrosine kinase inhibitor (TKI) Gefitinib (Iressa, ZD1839). Proc Am Assoc Cancer Res, 2003,44:(abstract 6199).
26 Li B, Chang C-M, Yuan M,et al. Resistance to small molecule inhibitors of epidermal growth factor receptor in malignant gliomas. Society for neuro-oncology eighth annual meeting,2003,291(abstract CB-13).
27 Folkman J. Tumor angiogenesis: therapeutic implications. N. Engl. J. Med.,1971,285:1182-1186.
28 Patan S.Vasculogenesis and angiogenesis as mechanisms of vascular network formation,growth and remodeling.J Neuro Oncol,2000,50:1-15.
29 Dachs GU ,Tozer GM. Hypoxia modulated gene expression:angiogenesis , metastasis and therapeutic exploitation. Eur J Cancer ,2000 ,36 :1649.
30 Machein MR,Plate KH.VEGF in brain tumors.J Neurooncol,2000,50:109-120.
31 Dunn IF,Heese O,Black PMcl.Growth factors in glioma angiogenesis :FGFs,PDGF,EGFand TGFs.J Neurooncol,2000,50:121-137.
32 Daniel JB,Erwin G.Glomeruloid microvascular proliferation orchestrated by VPF/VEGF.Am J Pathol,2001,158(3):789-796.
33 Mueller MM,Werbowetski,Del Maestro RF.Soluble factors involved in glioma invasion.Acta Neurochir(Wien),2003,145(11):999-l008.
34 Bemsen HJ,Rijken PE,Peter JP,et al .Delayed vascular changes after antiangiogeic therapy with anti-VEGF antibodies in human glioma xenografts in nudemice.Neurosurgery,1998,43(3):570-575.
35 Dhanabal M ,Ramchandran R ,Waterman MJ , et al . Endostatin induces endothelial cell apoptosis. J Biol Chem ,1999 ,274 :11721.
36 Moser TL ,Stack MS ,Asplin I , et al . Angiostatin binds ATP synthase on the surface of human endothelial cells. Proc Natl Acad Sci USA ,1999 ,96 :2811.
37 Ma HI,Lin SZ,Chiang YH,et al.Intratumoral gene therapy of malignant brain tumor in a rat model with angiostatin delivered by adeno-associated viral vector.Gene Ther,2002,9( 1):2 -11.
38 Kerr JS ,Wexler RS,Mousa SA,et al.Noval small molecule alpha V integrin antagonsts:comparative anti-cancer efficacy with known angiogenesis inhibitors.Anticancer Res,1999,19(2A):959-968.
39 Wen PY,Kesari S,Drappatz,et al.Malignant gliomas:strategies to increase the effectiveness of targeted molecular treatment.Expert Rev Anticancer Ther,2006,6(5):733-754.
40 Jeffrey J. Raizer.HER1/EGFR tyrosine kinase inhibitors for the treatment of glioblastoma multiforme.J Neuro-Oncology ,2005,74:77-86.
41 Amir Abdollahi, Kenneth E. Lipson, Axel Sckell,et al.Combined therapy with direct and indirect angiogenesis inhibition results in enhanced antiangiogenic and antitumor effects.Cancer Res,2003,63:8890-8898.
42 Katrin Lamszus,Marc A. Brockmann,Carmen Eckerich, et al.Inhibition of glioblastoma angiogenesis and invasion by combined treatments directed against vascular endothelial growth factor receptor-2, epidermal growth factor receptor,and vascular endothelial-cadherin.Clin Cancer Res 2005,11(13):4934-4940.
43 Reardon DA,Quinn JA,Vredenburgh JJ,et al.PhaseⅠtrial of Gefitinib plus sirolimus in adults with recurrent malignant glioma.Clin Cancer Res,2006,12:860-868.
44 De Groot JF,Gilbert MR,Hess KR,et al.Phase II study of combination carboplatin and erlotinib in patients with recurrent glioblastoma multiforme.J Clin Oncol.2007,25:2024
45 Vredenburgh JJ,Desjardins A,Hemdon JE ,et a1.Phase II trial of bevacizumab and irinotecan in re-current malignant glioma.Clin Cancer Res,2007,13(4):1253-1259.
46 Baumann F,Bjeljac M,Kollias SS,et a1.Combined thalidomide and temozolomide treatment in patients with glioblastoma multiforme.J Neurooncol,2004,67(1-2):191-200.
47 Groves MD,Puduvalli VK,Chang SM,et al.A North Aerican brain tumor consortium (NABTC 99-04) phase II trial of temozo1omide plus thalidomide for recurrent glioblastoma multiforme.J Neuro oncol,2007,81(3):271-277.
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5. Raizer JJ.HER1/EGFR tyrosine kinase inhibitors for the treatment of glioblastoma multiforme.J Neurooncol,2005,74:77-86.
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8. Casanova ML ,Larcher F,Casanova B,et al .A critical role for ras-mediated,epidermal growth factor receptor-dependent angiogenesis in mouse skin carcinogenesis.Cancer Res ,2002 ,62(12) :3402
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14.刁艺,黄强,董军,李如军,兰青,李晓楠.高侵袭和EGFR过表达的人脑胶质瘤组织异种原位移植模型.中华神经外科杂志,2008,24(11):822-825.
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41. Maeda K, Chung YS, Takatsuka S, et al. Tumor angiogenesis and tumour cell proliferation as prognostic indicators in gastric carcinoma. Br J Cancer, 1995, 72: 319-323.
42. Grundwald V, Hidalgo M. Developing inhibitors of the epidermal growth factor receptor for cancer Treatment.J Natl Cancer Inst,2003,95:851-867.
43. Eller JL, Longo SL, Hicklin DJ, Canute GW. Activity of anti-epidermal growth factor receptor monoclonal antibody C225 against glioblastoma multiforme.Neurosurgery, 2002;51:1005-1013.
44. Eller JL, Longo SL, Kyle MM,et al. Anti-epidermal growth factor receptor monoclonal antibody cetuximab augments radiation effects in glioblastoma multiforme in vitro and in vivo.Neurosurgery 2005,56:155-162.
45. Penar PL, Khoshyomn S, Bhushan A, et al.Inhibition of epidermal growth factor receptor-associated tyrosine kinase blocks glioblastoma invasion of the brain. Neurosurgery,1997,40:141-151.
46. Brockmann MA, Ulbricht U, Gruner K, et al.Glioblastoma and cerebral microvascular endothelial cell migration in response to tumor-associated growth factors. Neurosurgery,2003,52:1391-1399.
47. Lal A, Glazer CA, Martinson HM,et al. Mutant epidermal growth factor receptor up-regulates molecular effectors of tumor invasion.Cancer Res,2002,62:3335-3339.
1 Kleihues P, Soylemezoglu F, Schauble B, et al:Histopathology, classification,and grading of gliomas.Glia,1995,15:211-221.
2 Wong ET, Hess KR, Gleason MJ et al.Outcomes and prognostic factors in recurrent glioma patients enrolled onto phase II clinical trials. J Clin Oncol,1999,17:2572-2578.
3 J Shiotsu Y.Current screening for molecular target therapy of cancer.Japanese Journal of Cancer&Chemotherapy,2003,30(12):1863-1872.
4 Thrupson EW,Price JT.Mechanisms of tumor invasion and metastasis:emerging targets for therapyExp opin ther tar,2002,6(2):217-233.
5 KurokiM, Arakawa F, Khare PD, et al. Specific targeting strategies of cancer gene therapy using a single - chain variable fragment( scFv)with a high affinity for CEA . Anticancer Res,2002,20 (6A):4067～4071.
6 Arteaga CL.The epidermal growth factor receptor: from mutant oncogene in non human cancers to therapeutic target in human neoplasia. J Clin Oncol,2001,19(Suppl 1):32S-40S.
7 Arteaga C: Targeting HER1/EGFR: a molecular approach to cancer therapy. Sem Oncol,2003,30(Suppl 7):3-14.
8 Moghal N ,Sternberg PW.Multiple positive and negative regulators of signaling by the EGR-receptor.Current Opinion in Cell Biology ,1999 ,11 :190.
9 Moulder SL ,Yakes FM,M uthuswaniy SK,et al .Epidermal growth factor receptor(HER1)tyrosine kinase inhibitor Gefitinib inhibits HER2/neu (erbB2) overexpressing breast cancer cells in vitro and in vivo. Cancer Res,2001,61(24):8887-8895.
10 Wells A.EGF receptor.Review.Int J Biochem Cell.1999,31:637-643.
11 Mendelsohn J , Baselga J . The EGF receptor family as targets for cancer therapy.Oncogene,2000,19:6550-6565
12 Shinojima N, Tada K, Shiraishi S,et al. Prognostic value of epidermal growth factor receptor in patients with glioblastoma multiforme. Cancer Res,2003,63:6962-6970.
13 Van Beusechem VW,Grill J,Mastenbroek DC,et a1.Effieient and selective gene transfer into primary human brain tumors by using single-chain antibody-targeted adenoviral vectors with native tropism abolished.J Viml,2002,76(6):2753-2762.
14 Goldstein NI, Prewett M, Zuklys K, et al. Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model.Clin Cancer Res,1995;1:1311-1318.
15 Mendelsohn J.Antibody-mediated EGF receptor blockade as an anticancer therapy:from the laboratory to the clinic.Cancer Immunol Immunother,2003,52:342-346.
16 Herbst RS, Kim ES, Harari PM. IMC-C225, an anti-epidermal growth factor receptor monoclonal antibody, for treatment of head and neck cancer.Expert Opin Biol Ther,2001,1:719-732.
17 Warshall P,Oh lsson I,Biberfeld P,et a1.Intratumoral infusion of the monoclonal antibody-mAb 425-against the epidermal growth factor receptor in patients with advanced malignant glioma.Cancer human Immune Ther,1997,44( 3):157.
18 Mendelsohn J.Targeting the epidermal growth factor receptor for cancer therapy.J Clin Oncol,2002,20(18 Suppl):1S-13S.
19 RabenD , Bi ancoC , D amia noV , et al .Antitumor activity of ZD6126, a novel vascular-targeting agent, is enhanced when combined with Gefitinib,an epidermal growth f actor receptor tyrosine kinase inhibitor, and potentiates the effects of radiation in a human non-small cell lung cancer xenograft model .Mol Cancer Ther,2004,3 (8):9 77-983.
20 BaselgaJ, Averbuch SD. Gefitinib as an anticancer agent. Drugs,2000 60 ( Supp1 1 ):33-40.
21 Klohs WD,Fry DW,Kraker AJ.Inhibitor of tyrosine kinase.Curr Opin Oncol,1997,9:562-568.
22 Anita Lal, Chad A. Glazer,et al.Mutant epidermal growth factor receptor up-regulates molecular effectors of tumor invasion.Cancer Res,2002,62:3335-3339.
23 Johns TG, Luwor RB, Murone C,et al.Carmel Murone,Antitumor efficacy of cytotoxic drugs and the monoclonal antibody 806 is enhanced by the EGF receptor inhibitor AG1478.Proc Natl Acad Sci U S A,2003,100(26):15871-15876.
24 Guillamo J-S, Leuraud P, de Bouard S,et al.Anti-proliferative and anti-invasive EGFR amplification dependent and antiangiogenic EGFR independent activity of ZD1839 (‘Iressa’)tyrosine kinase inhibitor on human glioblastomas. Proc Am Assoc Cancer Res ,2003,44: (abstract 1009).
25 Learn CA, Archer GE, Riggins GJ,et al.Inhibition of cell cycle progression and invasion of cells expressing epidermal growth factor receptor (EGFR) but not mutant EGFR variant III(EGFRvIII) by the specific EGFR tyrosine kinase inhibitor (TKI) Gefitinib (Iressa, ZD1839). Proc Am Assoc Cancer Res, 2003,44:(abstract 6199).
26 Li B, Chang C-M, Yuan M,et al. Resistance to small molecule inhibitors of epidermal growth factor receptor in malignant gliomas. Society for neuro-oncology eighth annual meeting,2003,291(abstract CB-13).
27 Folkman J. Tumor angiogenesis: therapeutic implications. N. Engl. J. Med.,1971,285:1182-1186.
28 Patan S.Vasculogenesis and angiogenesis as mechanisms of vascular network formation,growth and remodeling.J Neuro Oncol,2000,50:1-15.
29 Dachs GU ,Tozer GM. Hypoxia modulated gene expression:angiogenesis , metastasis and therapeutic exploitation. Eur J Cancer ,2000 ,36 :1649.
30 Machein MR,Plate KH.VEGF in brain tumors.J Neurooncol,2000,50:109-120.
31 Dunn IF,Heese O,Black PMcl.Growth factors in glioma angiogenesis :FGFs,PDGF,EGFand TGFs.J Neurooncol,2000,50:121-137.
32 Daniel JB,Erwin G.Glomeruloid microvascular proliferation orchestrated by VPF/VEGF.Am J Pathol,2001,158(3):789-796.
33 Mueller MM,Werbowetski,Del Maestro RF.Soluble factors involved in glioma invasion.Acta Neurochir(Wien),2003,145(11):999-l008.
34 Bemsen HJ,Rijken PE,Peter JP,et al .Delayed vascular changes after antiangiogeic therapy with anti-VEGF antibodies in human glioma xenografts in nudemice.Neurosurgery,1998,43(3):570-575.
35 Dhanabal M ,Ramchandran R ,Waterman MJ , et al . Endostatin induces endothelial cell apoptosis. J Biol Chem ,1999 ,274 :11721.
36 Moser TL ,Stack MS ,Asplin I , et al . Angiostatin binds ATP synthase on the surface of human endothelial cells. Proc Natl Acad Sci USA ,1999 ,96 :2811.
37 Ma HI,Lin SZ,Chiang YH,et al.Intratumoral gene therapy of malignant brain tumor in a rat model with angiostatin delivered by adeno-associated viral vector.Gene Ther,2002,9( 1):2 -11.
38 Kerr JS ,Wexler RS,Mousa SA,et al.Noval small molecule alpha V integrin antagonsts:comparative anti-cancer efficacy with known angiogenesis inhibitors.Anticancer Res,1999,19(2A):959-968.
39 Wen PY,Kesari S,Drappatz,et al.Malignant gliomas:strategies to increase the effectiveness of targeted molecular treatment.Expert Rev Anticancer Ther,2006,6(5):733-754.
40 Jeffrey J. Raizer.HER1/EGFR tyrosine kinase inhibitors for the treatment of glioblastoma multiforme.J Neuro-Oncology ,2005,74:77-86.
41 Amir Abdollahi, Kenneth E. Lipson, Axel Sckell,et al.Combined therapy with direct and indirect angiogenesis inhibition results in enhanced antiangiogenic and antitumor effects.Cancer Res,2003,63:8890-8898.
42 Katrin Lamszus,Marc A. Brockmann,Carmen Eckerich, et al.Inhibition of glioblastoma angiogenesis and invasion by combined treatments directed against vascular endothelial growth factor receptor-2, epidermal growth factor receptor,and vascular endothelial-cadherin.Clin Cancer Res 2005,11(13):4934-4940.
43 Reardon DA,Quinn JA,Vredenburgh JJ,et al.PhaseⅠtrial of Gefitinib plus sirolimus in adults with recurrent malignant glioma.Clin Cancer Res,2006,12:860-868.
44 De Groot JF,Gilbert MR,Hess KR,et al.Phase II study of combination carboplatin and erlotinib in patients with recurrent glioblastoma multiforme.J Clin Oncol.2007,25:2024
45 Vredenburgh JJ,Desjardins A,Hemdon JE ,et a1.Phase II trial of bevacizumab and irinotecan in re-current malignant glioma.Clin Cancer Res,2007,13(4):1253-1259.
46 Baumann F,Bjeljac M,Kollias SS,et a1.Combined thalidomide and temozolomide treatment in patients with glioblastoma multiforme.J Neurooncol,2004,67(1-2):191-200.
47 Groves MD,Puduvalli VK,Chang SM,et al.A North Aerican brain tumor consortium (NABTC 99-04) phase II trial of temozo1omide plus thalidomide for recurrent glioblastoma multiforme.J Neuro oncol,2007,81(3):271-277.