bFGF单抗抗血管新生及抗肿瘤作用研究
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摘要
研究背景和目的
碱性成纤维细胞生长因子(basic fibroblast growth fator, bFGF)是成纤维细胞生长因子家族中的一员,它作为一种广泛的有丝分裂原,可通过自分泌或旁分泌的方式促进多种细胞,包括肿瘤细胞,血管内皮细胞的增殖、分化。FGF家族成员主要通过与其高亲和力受体的结合向细胞内传递信号,发挥其生物学活性。高亲和力受体又称受体酪氨酸激酶(Receptor tyrosine kinase, RTKs),存在FGFR1, FGFR2, FGFR3, FGFR4四种蛋白。bFGF除不与FGFR2b结合外可与其他几类FGFR亚型包括FGFRlb, FGFR1c, FGFR2c, FGFR3b, FGFR3c,FGR4结合。其中FGFR-1是bFGF的主要受体,高表达于血管内皮细胞及多种肿瘤细胞表面,bFGF与其结合后,FGFR通过二聚化而发生自身磷酸化,活化的FGFR可招募一些接头蛋白,从而激活下游的信号通路,包括MAPKs/ERKs, PI3K/AKT, PLCγ/PKC等,后者又活化转录因子及与细胞周期,凋亡／抗凋亡,细胞骨架等相关的蛋白,从而调节细胞的生物学活性。bFGF及FGFR高表达于包括黑色素瘤,肺癌在内的多种肿瘤组织,且与肿瘤的转移、分期、预后相关,bFGF/FGFR系统成为抗肿瘤治疗的新靶点。
血管新生过程是从已存在血管中长出新血管的过程。肿瘤血管新生不仅为肿瘤生长提供营养,更是肿瘤发生转移的物质基础。肿瘤血管新生的过程包括内皮细胞增殖,内皮干细胞的迁移,内皮细胞的迁移和侵入,内皮细胞的增殖,内皮细胞在管状结构中的组织,循环系统的形成,血管的成熟,血管的退化等。bFGF作为重要的促血管新生因子在血管新生过程的多个环节发挥作用。
黑色素瘤是皮肤癌中最致命的一种,近年来在西方国家其发病率和死亡率持续增加。肺癌在我国及世界许多国家高发,且成为癌症的第一杀手。黑色素瘤对放化疗药物中度敏感,且容易产生耐药,黑素素瘤的耐药性及其高转移率是导致其很难治愈的重要原因。因此新的治疗手段尤其是靶向生物疗法备受关注。bFGF及FGFR在黑色素瘤组织中高表达,与其转移及预后密切相关,有学者甚至认为病人血清及组织中bFGF的水平可作为肿瘤预后的重要指标。bFGF可通过自分泌的方式促进黑色素瘤细胞及肺癌细胞的增殖。正常黑素细胞不表达bFGF,然而其体外培养则必须有外源性bFGF的加入,而且将bFGF基因转入正常黑素细胞使其高表达bFGF,可出现类似于黑色素瘤早期的表型；除此之外bFGF还是具有抗凋亡作用,可抵抗饥恶及化疗药物诱导的靶细胞凋亡。
肺癌的发病率和死亡率在我国乃至世界许多国家居于首位,其高转移率及对放化疗药物的耐药是肺癌患者死亡的重要原因。且目前传统的治疗方法主要针对早期肺癌患者,对于晚期患者则难以治愈。因此近年来新的治疗手段,尤其是靶向抗体治疗备受关注。有研究表明bFGF在非小细胞系肺癌组织中表达率高于50%,且与淋巴结转移及分期相关,其在伴有淋巴结转移及中晚期肺癌组织中的表达明显高于早期。有研究证明bFGF是多种肿瘤的广谱耐药机制。bFGF导致肺癌细胞对化疗药物耐药的原因在于bFGF不仅能促进肺癌细胞的增殖,还可使其逃避化疗药物诱导的凋亡。因此bFGF可能成为肺癌治疗的良好的靶点。
因此本研究以血管内皮细胞,黑色素瘤细胞,肺癌细胞为研究对象研究bFGF单抗的体内外抗血管新生,及抗肿瘤作用。
方法
1.bFGF单抗抗血管新生作用
1)将本室保存的多株产生抗bFGF单抗的杂交瘤细胞克隆化后,扩大培养,注射福氏不完全佐剂处理过的小鼠腹腔,制备腹水型单克隆抗体。蛋白G亲和层析柱纯化bFGF单抗,间接ELISA法检测其与bFGF的结合活性及与aFGF、VEGF是否存在交叉反应。另外将天然及巯基乙醇处理后热失活的bFGF包板,用间接ELISA法检测单抗识别抗原表位。只与天然bFGF反应,而不与热失活的bFGF反应的单抗识别的是bFGF的空间表位；相反可与二者都结合的bFGF单抗识别的bFGF的线性表位。
2)将bFGF单抗与FGF高亲和力受体FGFR-1βⅢC混合后加入已包被有bFGF的ELISA板中,通过竞争ELISA法检测bFGF单抗是否能够抑制bFGF与其高亲和力受体FGFR-1βⅢC的结合。如果能抑制说明bFGF单抗识别bFGF受体结合相关区域。另外将bFGF单抗与肝素混合混合后加入已包被有bFGF的ELISA板中,通过竞争ELISA法检测bFGF单抗是否能够抑制bFGF与肝素的结合,如果抑制说明bFGF单抗识别bFGF肝素结合相关区域。
3)取原代脐静脉血管内皮细胞以bFGF刺激其增殖,同时加入bFGF单抗,CCK-8法检测此单抗对bFGF促进的血管内皮细胞增殖的影响；
4)将血管内皮细胞接种到已铺上基质胶的细胞培板中,加入bFGF及bFGF和单抗的混合物,培养2-5h后镜下计数封闭的管状结构,以检测bFGF单抗对血管内皮细胞成管的影响；
5) Transwell法检测单抗对血管内皮细胞迁移的影响,将血管内皮细胞加入transwell小室的上室中,下室加入含10ng/mlbFGF的M199完全培养基,诱导上室中血管内皮细胞向下室的迁移,另外加入bFGF单抗检测其对迁移的影响,20h后吉姆萨染色,擦去上层细胞计数迁移的细胞数；
6)96孔板中接种血管内皮细胞,贴壁后加入bFGF单抗作用48h,收集黑色素瘤细胞,加入单抗处理过的血管内皮细胞中,15min后洗板,光镜下已粘附的黑色素瘤细胞数,以检测bFGF单抗对血管内皮细胞与黑色素瘤细胞粘附的影响；
7)取7日龄鸡胚,以牙科钻开窗,暴露尿囊膜,以中速定性滤纸为载体加入bFGF和bFGF单抗,作用4天后,滴加等量甲醇和丙酮混合液以杀死鸡胚并固定尿囊膜,15-20 min后,待CAM血管中的血液凝固后,剪取尿囊膜,放入水中展平,拍照,计数滤纸周围呈放射状排列的血管数,检测bFGF单抗的抗血管新生作用。
2.bFGF单抗抗黑色瘤生长作用
1)收集B16和B16F10细胞,提取RNA,以RT-PCR法检测B16和B16F10细胞表达bFGF, FGFR及Glypicanl, Glypican5, Syndecanl, Syndecan4情况。
2)收集B16细胞,铺板,加入bFGF单抗作用96h,CCK-8法检测bFGF单抗对体外培养的黑色素瘤细胞增殖的影响；
3)收集bFGF单抗处理和未处理的B16细胞,以AnnexinV/PI双染,流式细胞仪检测bFGF单抗诱导黑色素瘤细胞凋亡的情况,AnnexinV+/PI-:表示细胞发生早期凋亡,AnnexinV+/PI+表示细胞发生晚期凋亡,
4) Transwell法检测bFGF单抗对B16F10细胞迁移的影响,将B16F10细胞加入transwell小室的上室中,下室加入含RPMI1640完全培养基,诱导上室中黑色素细胞向下室的迁移,另外加入bFGF单抗检测其对迁移的影响,20h后吉姆萨染色,擦去上层细胞计数迁移的细胞数；
5)使用C57 BL／6小鼠建立黑色素移植瘤小鼠,并检测尾静脉和瘤周皮下注射抗体及不同给药剂量对荷瘤小鼠肿瘤生长、瘤重及小鼠体重的影响。免疫组化法检测肿瘤组织CD31表达(微血管密度),TUNEL法检测肿瘤组织凋亡的情况,以初步探讨bFGF单抗的抗肿瘤机制。
6)以5中同样的方法建立黑色素瘤小鼠模型,持续给药,直至小鼠死亡,记录小鼠生存时间,绘制小鼠生存曲线,计算各组小鼠中位生存时间。
3. bFGF单抗抗Lewis肺癌生长及转移作用使用C57 BL／6小鼠建立肺癌原位转移模型,瘤周皮下注射bFGF单抗,三天一次连续六次,并检测其对肿瘤生长、瘤重、及小鼠体重的影响；给药六次后处死小鼠取完整双肺,计数肺表面转移灶结节数,并用HE染色肺组织确认癌转移灶。用免疫组化法检测肿瘤组织微血管密度,初步探讨bFGF单抗抗肿瘤转移机制。
结果
1.bFGF单抗抗血管新生作用
1) bFGF单抗不与aFGF和VEGF发生交叉反应,单抗MabF7识别bFGF的线性表位,单抗MabF10, MabF12识别bFGF空间表位。
2) bFGF单抗MabF7, MabF10, MabF12可抑制bFGF与其高亲和力受体FGFR-1βⅢC的结合。IC50分别为7.5μg／ml、65μg／ml、50μg／ml。然而三株单抗对bFGF与肝素的结合则无抑制作用,说明其识别bFGF受体结合区域的表位。
3)通过促增殖实验发现,MabF7, MabF10, MabF12均可抑制bFGF诱导的血管内皮细胞的增殖。IC50分别为50μg／ml、100μg／ml和150μg／ml。
4)体外基质胶模拟血管内皮细胞成管作用发现三株单抗均可抑制内皮细胞成管过程,通过计数完整的管状结构的个数计算管状结构形成率,无抗体组,对照抗体组,MabF7, MabF10, MabF12组管状结构形成率分别为：100%,105.93±3.85%,56.53±4.35%,29.23±6.45%,以及12.77±2.67%,结果显示MabF7,MabF10, MabF12与无抗体组(Medium)相比均有显著差异(P=0.000)；而对照IgG组与无抗体组(Medium)之间无显著差异(P=0.104)。
5) Transwell模拟血管内皮细胞迁移作用,发现三株单抗均可抑制内皮细胞迁移过程,通过计数迁移的细胞数计算抑制率,无抗体组,对照抗体组,MabF7, MabF10, MabF12组细胞迁移率分别为100%,106.25±7.89%,69.50±6.86%,74.00±4.16%,67.75±3.30%,单抗组与无抗体对照组相比差异显著,具有统计学意义(P<0.05)。
6)光镜下计数bFGF单抗对血管内皮细胞与黑色素瘤细胞粘附作用发现,bFGF单抗对二者间的粘附作用无显著影响(P>0.05)。
7)计数尿囊膜上加药滤纸周围呈放射状的血管条数。计数后发现bFGF组,bFGF+MabF7组,bFGF+MabF10组,bFGF+MabF12呈放射状的血管条数分别为15±0.82,7.5±1.29,13.5±3.10,8.5±0.58,bFGF+MabF7组,显示bFGF+MabF7, bFGF+MabF12与bFGF组相比均有显著差异(P<0.01)；而bFGF+MabF10与bFGF组之间无显著差异(P=0.896)。
2. bFGF单抗抗黑色瘤生长作用
1) bFGF单抗可抑制黑色素瘤细胞的生长,200μg／ml,作用96h后MabF7组,MabF10组,MabF12组抑制率分别为42.37±2.38%,40.71±3.90%,41.48±2.76%。相同剂量的对照IgG对B16细胞生长则无显著抑制作用。
2) bFGF单抗可诱导黑色素瘤细胞发生凋亡,200μg／ml MabF7作用96h,凋亡率(以Annexin V+细胞计算)为73.20±10%,与对照IgG组及无抗体处理组相比差异显著,具有统计学意义(P<0.05)。
3) bFGF单抗可抑制黑色素瘤细胞发生迁移,无抗体处理组,对照IgG, MabF7, MabF10以及MabF12组的迁移率分别为100%,109.00±9.56%,72.14±13.57%,34.68±14.37%,和36.56±13.60%。MabF7,MabF10,MabF12与无抗体组(Medium)相比均有显著差异(P<0.05)；而对照IgG组与无抗体组(Medium)之间无显著差异(P=0.295)。
4)三株单抗MabF7, MabF10, MabF12给药5次后对黑色素瘤生长的抑制率分别为46.40%,23.24%,19.89%。将不同浓度的MabF7注射小鼠发现MabF7可以浓度依赖的方式抑制黑色素瘤的生长,PBS组,Control IgG组,MabF7高,中,低剂量组的瘤重分别为6.303±1.196,5.529±0.658,3.842±1.060,4.778±0.888,5.061±0.922。结果显示MabF7高中低组与PBS组相比均有显著差异(P<0.05)；而对照IgG组与PBS之间无显著差异(P=0.143)
5) bFGF单抗MabF7可抑制瘤组织微血管密度。MabF7组微血管密度为24.67±5.20,对照IgG组为45.38±12.59,具有显著差异(P<0.05)。使用TUNEL对瘤组织的细胞凋亡情况进行检测,发现MabF7处理组瘤组织存在细胞凋亡染色,而对照IgG组则少见。
4)bFGF单抗MabF7可提高荷瘤小鼠生存期,PBS组,对照IgG组,MabF7组小鼠的中位生存期分别为24,26,32天。
3. bFGF单抗抑制肺癌的转移
1)bFGF单抗MabF7可抑制肺癌皮下移植瘤的生长,MabF7组瘤重为2.64±1.05 g显著低于PBS组的5.15±1.31 g(P=0.013),抑瘤率为48.23%；对照IgG组瘤重为4.67±0.40与PBS组相比无显著差异(P=0.781),抑瘤率为8.43%；MabF7与对照IgG组比较差异显著(P=0.010)。
2) bFGF单抗MabF7可抑制肺癌发生原位转移,给药六次后bFGF单抗MabF7组肺转移结节数为3.00±2.10与PBS组的12.29±2.63相比差异显著(P=0.000),抑制率为75.59%；对照IgG组的转移结节数为11.80±3.56,与PBS组相比无显著差异(P=0.768)
3)bFGF单抗MabF7可降低肺癌组织的微血管密度,结果显示bFGF单抗MabF7组MVD 22.00±2.94显著低于PBS组的43.5±11.59(P=0.003)；对照IgG组MVD为45.75±5.44与PBS组相比无显著差异(P=0.718)。MabF7组与对照IgG组相比差异显著(P=0.002)。
结论
1.三株单抗MabF7, MabF10, MabF 12均能抑制bFGF与FGFR1的结合。三株单抗均可一定程度的抑制血管内皮细胞的增殖,迁移及成管过程,但鸡胚尿囊膜实验则显示,只有MabF7和MabF 12具有显著的抑制尿囊膜血管新生作用,提示bFGF单抗可用于抗血管新生作用。
2.以黑色素瘤为模型探讨bFGF单抗对肿瘤生长的作用,发现三株单抗均可抑制黑色素瘤的生长,并诱导其发生凋亡。但通过建立C57 BL／6小鼠黑色素抑制瘤模型研究其体内抗肿瘤效果则发现,只有MabF7具有显著的抑制肿瘤生长的作用。MabF7对体内黑色素瘤生长的抑制作用可能与其诱导肿瘤凋亡及降低微血管密度有关。提示抗体的体内治疗可能与抗体的识别表为,亲和力,及药代动力学均有关系。
3.以Lewis细胞建立肺癌原位转移模型,发现MabF7可抑制肺癌的生长及转移,此作用与MabF7抑制肿瘤微血管密度有关。
BACKGROUND & OBJECTIVE
Basic fibroblast growth fator (bFGF, FGF-2) belongs to the family of heparin-binding growth factors. Acting as a broad-spectrum mitogen and potent angiogenic agent, bFGF mediates a variety of cellular responses including cell proliferation and differentiation. bFGF functions mainly by interacting with high affinity tyrosine kinase FGF receptors (FGFRs) on the surface of target cells. There are four Fgf receptor genes in mammals, FgfR1-FgfR4, encoding several splice variants that increase the signaling repertoire. bFGF could bind to all FGFR including FGFRlb, FGFR1c, FGFR2c, FGFR3b, FGFR3c, FGR4 except for FGFR2b. Upon binding to bFGF, FGFRs dimerize and are tyrosine phosphorylated, leading to the activation of downstream signaling pathways including MAPKs (mitogen-activated protein kinases)/ERKs (extracellular signal-regulated kinases), PI3K (phosphoinositide 3-kinase)/AKT (protein kinase B), and PLCy(Phospholipase C gamma)/PKC(protein kinase).bFGF and FGFR are highly expressed in many tumor tissue including melanoma and lung cancer, which play important role in tumor development and progression. All of these reports suggest the potential of bFGF as a target for tumor therapy.
Angiogenesis is the process of new blood vessel formation from pre-existing ones, which play a key role in various physiological and pathological conditions, including embryonic development, wound repair, inflammation, and tumor growth. Tumor growth is absolutely dependent on the induction of a neovascular network.Tumor cells recruit new blood vessels that serve as conduits for delivery of nutrients and for tumor metastsis. Typically, angiogenesis occurs through endothelial cell sprouting. The first stage involves the degradation of the basement membrane around a blood vessel and of the interstitial matrix. Endothelial cells migrate in response to bFGF and proliferate along a leading migratory edge.Canalization or lumen formation, as well as branch and loop formation, lead to anastamosis of these spouts allowing blood flow.The final stage involve structural inclusion of pericytes and formation of a new basement membrane surrounding the new vessel.
Malignant melanoma is one of the most aggressive Human tumors. In recent decades, the incidence of malignant melanoma has steadily increased. A particularly worrying feature of the tumor is its increasing incidence and its capacity for rapid metastatic spread. Because of their resistance to current therapies, such as surgical excision, systemic chemotherapy with dacarbazine, and immunotherapy with vaccines, melanomas remain a significant cause of mortality. To achieve improvement in overall survival, it is important to develop effective new therapies. The expression of bFGF is absent in normal melanocytes, which require exogenous bFGF to maintain the growth. bFGF transduced normal melanocytes exhibit transformed phenotype resembling early-stage melanoma, indicating a critical role of bFGF in the transformation of melanocytes to melanoma Most melanomas express high levels of bFGF and FGFRs, which form an autocrine loop and are critical for the survival and growth of melanoma cells; melanoma growth can be arrested by interfering with the production or biological activity of bFGF alone. bFGF has long been regarded as an important tumor angiogenic factor, and the specific roles of bFGF in angiogenesis and spontaneous metastasis of melanoma have been revealed too. All of these reports suggest the potential of bFGF as a target for melanoma therapy. Antisense targeting of bFGF/FGFR-1 in primary and metastatic melanoma cells blocks the tumor growth by inducing apoptosis without activation or increased production of VEGF. In addition, bFGF peptide vaccines also show significant inhibition on angiogenesis and tumor growth.
Lung cancer is the major cause of death from all Human malignancies in CHINA and the other countries, and is frequently associated with poor prognosis. Overall 5-year relative survival rate of lung cancer for all stage is very low. This is mainly due to ineffective detection of lung cancer at its early stage. A particularly worrying feature of the tumor is its drug resistance and its capacity for rapid metastatic spread. bFGF is one of the epigenetic mechanisms explaining the multidrug resistance (MDR) of tumors. Some previous studies have shown that bFGF prevents chemotherapy induced apoptosis, which result in chemoresistance in many cancers such as small cell lung cancer. This anti-apoptotic effect is mediated by MAPK/ERKs, PI3K/AKT, and/or PKC that increase the expression and activation of several anti-apoptotic proteins including Bcl-2, Bcl-XL, and XIAP. All of these reports suggest the potential of bFGF as a target for lung cancer therapy.
In this study, HUVECs, melanoma cells, and lung caner cells were used as targeting cells for anti-angiogenesis and anti-tumor therapy by anti-bFGF monoclonal antibodies.
METHODS
1. Anti-angiogenesis activity by anti-bFGF mAbs
1) The hybridomas cells secreting monoclonal antibodies against bFGF were cloning. Ascites were produced by injecting hybridomas cells into Balb/c mouse. The antibodies were purified by a protein G column, and the isotyping of antibodies was carried out using ELISA. Anti-bFGF mAbs were incubated with rhFGF-2, rhFGF-1or rhVEGF165 immobilized onto 96-well plates and bound proteins were detected with a HRP-conjugated second antibody. To determine whether the anti-bFGF mAbs recognize conformational or linear epitopes of bFGF molecule, native or heat-denured bFGF was used in ELISA.
2) In the competitive FGFR1/FGF-2 blocking assay,96-well plate was coated with 0.5μg/ml bFGF. Then FGFR-1βⅢC-Fc at a fixed amount (50 ng) was mixed with anti-bFGF mAbs at increasing concentrations and onto the coated plate. After incubation at 37℃for 1h, bound FGFR-1βⅢC-Fc were detected. In the competitive heparin/FGF-2 blocking assay,96-well plate was coated with 0.5μg/ml bFGF. Then anti-bFGF mAbs at a fixed amount was mixed with heparin at increasing concentrations and onto the coated plate. After incubation at 37℃for 1h, bound anti-bFGF mAbs were detected.
3) HUVECs were seeded in 96-well plates. After overnight culture at 37℃under 5% CO2 in a humidified incubator, serial dilutions of purified anti-bFGF mAbs with lOng/ml rhFGF-2 were added to the wells. After the cells were incubated at 37℃for 4 more days,10μl CCK-8 reagent was added into each well.
4) ECM gel-induced capillary tube formation assay was used as an in vitro measurement of angiogenesis. HUVECs were added onto each well in the presence of anti-bFGF mAbs with or without bFGF.
5) HUVECs were harvested from culture flasks by trypsinization; Aliquot of 100μl cell suspension was added to each transwell insert of 8-μm-pore membrane. The inserts were then placed into the wells of the 24-well plates containing 0.6 ml of M199 with or without rhFGF-2. After incubation for 20 h at 37℃, cells remaining at the upper surface of the membrane were removed using a cotton swab, while the cells that migrated across the membrane to the lower surface were fixed with ethanol and stained with Giemsa solution. The number of stained cells was counted under several high-power microscopic fields.
6) HUVECs were treated with anti-bFGF mAbs for 48h. B16F10 cells were added to HUVECs. After 15min incubation, the B16F10 cells adhering to HUVECs were counted.
7) The 7-day-old embryos with intact yolks were placed in a bowl and incubated at 37℃with 3% CO2. Anti-bFGF mAbs or control mIgG were separately applied to the CAM of individual embryos. After 24 hours of incubation, neovascularization in chicken CAMs was observed with a stereomicroscope.
2. Anti-melanoma growth activity by anti-bFGF mAbs
1) Expression of bFGF, FGFR, Glypicanl, Glypican5, Syndecanl, Syndecan4 were detected by RT-PCR.
2) Inhibition of B16 cells growth were assayed by CCK-8 kit.
3) B16 cells were incubated for 96 h with anti-bFGF mAbs, or mouse normal IgG. The fraction of apoptotic cells were marked with FITC-conjugated Annexin-V and propodium iodide (PI). After 15 min of incubation at room temperature, the samples were analyzed by flow cytometry.
4) B16F10 cells migration was assayed using a Transwell chamber as preciously described.
5) Six-to seven-week-old male and female C57 BL/6 mice were subcutaneously inoculated in the right flank with 1×105 B16 cells; One week later, when palpable tumors (≥5 mm in diameter) developed, the mice were subcutaneously (around tumors) injected with anti-bFGF mAbs every 3 days for a total of five injections. Tumor size was measured every 3 days in two dimensions using a vernier caliper. Mice were sacrificed 3 weeks after tumor cell inoculation, and tumor weights were measured. The expression of CD31 in tumor tissues was analyzed to determine the intratumoral microvessel density. Apoptotic cells in tumor sections were detected using TUNEL assay kit.
6) C57 BL/6 mice were injected with B16 cells as before mentioned. Mouse survival was monitored from the day of tumor inoculation until the day of animal death.
3. Anti-lung cancer growth activity by anti-bFGF mAbs
The mouse lung cancer graft model was established by injection of Lewis lung carcinoma cells subcutaneously into the the right flank of C57BL/6 mice. When palpable tumors (≥5mm in diameter) developed, the mice were subcutaneously (around tumors) injected with anti-bFGF mAbs in 200μl PBS. Control mice received equal amounts of control IgG or PBS. Tumor volume and metastases of Lewis lung carcinoma were counted. The microvessel density (MVD) in tumor mass were measured by imunohistochemistry staining (IHCS).
RESULTS
1. Anti-angiogenesis activity by anti-bFGF mAbs
1) No binding of purified anti-bFGF antibodies to aFGF and VEGF were detected. The result of MabF7 binding to both native and denatured bFGF implies that MabF7 recognizes a continuous epitope. MabF10 and MabF12 do not recognize the heat-denatured bFGF suggesting that they both recognize conformation-dependent epitopes on bFGF.
2) Binding of bFGF to FGFR-1 was inhibited by anti-bFGF mAbs. IC50 of MabF7, MabF10 and MabF12 were 7.5μg/ml、65μg/ml、50μg/ml respectively. However anti-bFGF mAbs could not inhibit bFGF binding to heparin.
3) Anti-bFGF mAbs could inhibit HUVECs proliferation induced by bFGF. IC50 of MabF7, MabF10 and MabF12 were 50μg/ml、100μg/ml和150μg/ml respectively.
4) The tube formation in groups with no IgG, control IgG, MabF7, MabF10, and MabF12 are 100%,105.93±3.85%,56.53±4.35%,29.23±6.45%,12.77±2.67%, respectively. It indicates that anti-bFGF mAbs significantly inhibit the tube formation of HUVECs; however, control IgG shows no effect.
5) The number of migrating cells in the presence of mAbs reduces significantly compared to the control group (treatment without supplement), and the migration ratio in groups treated with no IgG, control IgG, MabF7, MabF10, and MabF12 are100%,106.25±7.89%,69.50±6.86%,74.00±4.16%,67.75±3.30%, respectively.
6) Anti-bFGF mAbs could not inhibit attaching of B16F10 to HUVECs.
7)The blood vessel of CAM in groups treated with bFGF, bFGF+MabF7, bFGF+MabF 10, and bFGF+MabF 12 are 15±0.82,7.5±1.29,13.5±3.10,8.5±0.58 respectively.
2. Anti-melanoma growth activity by anti-bFGF mAbs
1) Anti-bFGF mAbs could inhibit B16 cells proliferation. Inhibition of MabF7, MabF10 and MabF12 were 42.37±2.38%,40.71±3.90%,41.48±2.76% respectively.
2) Apoptosis was analyzed by flow cytometry with Annexin V/PI staining. With mAbs at a concentration of 200μg/ml,73.20±10% of B16 cells show apoptosis after 96 h of incubation, whereas IgG control group shows no effect compared with medium control
3) The number of migrating cells in the presence of mAbs reduces significantly compared to the control group (treatment without supplement), and the migration ratio in groups treated with no IgG, control IgG, MabF7, MabF10, and MabF12 are100%,109.00±9.56%,72.14±13.57%,34.68±14.37%, and 36.56±13.60%, respectively.
4) MabF7 remarkably reduces tumor burdens and suppresses tumor growth by 46.40% at the concentration of 5 mg/ml. However, the inhibitions by MabF10 and MabF12 are 23.24% and 19.89%.
5) The microvessel density in tumor is determined by staining the tumor section with anti-CD31 antibody, and the numbers of microvessel density in tumor reduces significantly in MabF7 group (24.67±5.20) in comparison with that in IgG control group (45.38±12.59).
6) More TUNEL-positive cells with deep brown stained nuclei are detected in MabF7 treated tumors than that in control IgG treated group.
7) To investigate whether the inhibition of tumor growth by MabF7 impacts the survival of animals, a survival assay has been performed as shown in the Kaolan-Meier survival curve in Fig.8. The median survival time of mice treated with MabF7 (32 days) is longer than that of mice treated with PBS (24 days) or control IgG (26 days)
3. Anti-Lewis lung cancer growth activity by anti-bFGF mAbs
Anti-bFGF mAb could inhibit tumor growth in C57BL/6 mice Lewis lung cancer model. Tumor weight in MabF7 group is 2.64±1.05 g which is lower than that of PBS group (5.15±1.31 g) (P<0.05). The mean number of metastatic lung nodules in MabF7 group is 3.00±2.10 which is lower than that of PBS group (12.29±2.63) (P<0.05). Moreover MabF7 reduce the expression of MVD in the tumor tissue.
CONCLUSIONS
1. Binding of bFGF to FGFR-1 was inhibited by anti-bFGF mAbs MabF7, MabF10, MabF12. All of them could also inhibit HUVECs proliferation, tube formation, and migration. However in CAM assay, only MabF7 and MabF12 could inhibit angiogenesis.
2. MabF7, MabF10, MabF12 could inhibit melanoma cells proliferation, migration, and induce apoptosis in melanoma cells in vitro. However only MabF7 could inhibit melanoma growth in vivo through inhibition of the angiogenesis and induction of the apoptosis in tumor.
3. Anti-bFGF mAb could inhibit tumor growth and pulmonary metastasis in C57BL/6 mice Lewis lung cancer model, and suppress angiogenesis of tumor tissue.
碱性成纤维细胞生长因子(basic fibroblast growth fator, bFGF)是成纤维细胞生长因子家族中的一员,它作为一种广泛的有丝分裂原,可通过自分泌或旁分泌的方式促进多种细胞,包括肿瘤细胞,血管内皮细胞的增殖、分化。FGF家族成员主要通过与其高亲和力受体的结合向细胞内传递信号,发挥其生物学活性。高亲和力受体又称受体酪氨酸激酶(Receptor tyrosine kinase, RTKs),存在FGFR1, FGFR2, FGFR3, FGFR4四种蛋白。bFGF除不与FGFR2b结合外可与其他几类FGFR亚型包括FGFRlb, FGFR1c, FGFR2c, FGFR3b, FGFR3c,FGR4结合。其中FGFR-1是bFGF的主要受体,高表达于血管内皮细胞及多种肿瘤细胞表面,bFGF与其结合后,FGFR通过二聚化而发生自身磷酸化,活化的FGFR可招募一些接头蛋白,从而激活下游的信号通路,包括MAPKs/ERKs, PI3K/AKT, PLCγ/PKC等,后者又活化转录因子及与细胞周期,凋亡／抗凋亡,细胞骨架等相关的蛋白,从而调节细胞的生物学活性。bFGF及FGFR高表达于包括黑色素瘤,肺癌在内的多种肿瘤组织,且与肿瘤的转移、分期、预后相关,bFGF/FGFR系统成为抗肿瘤治疗的新靶点。
血管新生过程是从已存在血管中长出新血管的过程。肿瘤血管新生不仅为肿瘤生长提供营养,更是肿瘤发生转移的物质基础。肿瘤血管新生的过程包括内皮细胞增殖,内皮干细胞的迁移,内皮细胞的迁移和侵入,内皮细胞的增殖,内皮细胞在管状结构中的组织,循环系统的形成,血管的成熟,血管的退化等。bFGF作为重要的促血管新生因子在血管新生过程的多个环节发挥作用。
黑色素瘤是皮肤癌中最致命的一种,近年来在西方国家其发病率和死亡率持续增加。肺癌在我国及世界许多国家高发,且成为癌症的第一杀手。黑色素瘤对放化疗药物中度敏感,且容易产生耐药,黑素素瘤的耐药性及其高转移率是导致其很难治愈的重要原因。因此新的治疗手段尤其是靶向生物疗法备受关注。bFGF及FGFR在黑色素瘤组织中高表达,与其转移及预后密切相关,有学者甚至认为病人血清及组织中bFGF的水平可作为肿瘤预后的重要指标。bFGF可通过自分泌的方式促进黑色素瘤细胞及肺癌细胞的增殖。正常黑素细胞不表达bFGF,然而其体外培养则必须有外源性bFGF的加入,而且将bFGF基因转入正常黑素细胞使其高表达bFGF,可出现类似于黑色素瘤早期的表型；除此之外bFGF还是具有抗凋亡作用,可抵抗饥恶及化疗药物诱导的靶细胞凋亡。
肺癌的发病率和死亡率在我国乃至世界许多国家居于首位,其高转移率及对放化疗药物的耐药是肺癌患者死亡的重要原因。且目前传统的治疗方法主要针对早期肺癌患者,对于晚期患者则难以治愈。因此近年来新的治疗手段,尤其是靶向抗体治疗备受关注。有研究表明bFGF在非小细胞系肺癌组织中表达率高于50%,且与淋巴结转移及分期相关,其在伴有淋巴结转移及中晚期肺癌组织中的表达明显高于早期。有研究证明bFGF是多种肿瘤的广谱耐药机制。bFGF导致肺癌细胞对化疗药物耐药的原因在于bFGF不仅能促进肺癌细胞的增殖,还可使其逃避化疗药物诱导的凋亡。因此bFGF可能成为肺癌治疗的良好的靶点。
因此本研究以血管内皮细胞,黑色素瘤细胞,肺癌细胞为研究对象研究bFGF单抗的体内外抗血管新生,及抗肿瘤作用。
方法
1.bFGF单抗抗血管新生作用
1)将本室保存的多株产生抗bFGF单抗的杂交瘤细胞克隆化后,扩大培养,注射福氏不完全佐剂处理过的小鼠腹腔,制备腹水型单克隆抗体。蛋白G亲和层析柱纯化bFGF单抗,间接ELISA法检测其与bFGF的结合活性及与aFGF、VEGF是否存在交叉反应。另外将天然及巯基乙醇处理后热失活的bFGF包板,用间接ELISA法检测单抗识别抗原表位。只与天然bFGF反应,而不与热失活的bFGF反应的单抗识别的是bFGF的空间表位；相反可与二者都结合的bFGF单抗识别的bFGF的线性表位。
2)将bFGF单抗与FGF高亲和力受体FGFR-1βⅢC混合后加入已包被有bFGF的ELISA板中,通过竞争ELISA法检测bFGF单抗是否能够抑制bFGF与其高亲和力受体FGFR-1βⅢC的结合。如果能抑制说明bFGF单抗识别bFGF受体结合相关区域。另外将bFGF单抗与肝素混合混合后加入已包被有bFGF的ELISA板中,通过竞争ELISA法检测bFGF单抗是否能够抑制bFGF与肝素的结合,如果抑制说明bFGF单抗识别bFGF肝素结合相关区域。
3)取原代脐静脉血管内皮细胞以bFGF刺激其增殖,同时加入bFGF单抗,CCK-8法检测此单抗对bFGF促进的血管内皮细胞增殖的影响；
4)将血管内皮细胞接种到已铺上基质胶的细胞培板中,加入bFGF及bFGF和单抗的混合物,培养2-5h后镜下计数封闭的管状结构,以检测bFGF单抗对血管内皮细胞成管的影响；
5) Transwell法检测单抗对血管内皮细胞迁移的影响,将血管内皮细胞加入transwell小室的上室中,下室加入含10ng/mlbFGF的M199完全培养基,诱导上室中血管内皮细胞向下室的迁移,另外加入bFGF单抗检测其对迁移的影响,20h后吉姆萨染色,擦去上层细胞计数迁移的细胞数；
6)96孔板中接种血管内皮细胞,贴壁后加入bFGF单抗作用48h,收集黑色素瘤细胞,加入单抗处理过的血管内皮细胞中,15min后洗板,光镜下已粘附的黑色素瘤细胞数,以检测bFGF单抗对血管内皮细胞与黑色素瘤细胞粘附的影响；
7)取7日龄鸡胚,以牙科钻开窗,暴露尿囊膜,以中速定性滤纸为载体加入bFGF和bFGF单抗,作用4天后,滴加等量甲醇和丙酮混合液以杀死鸡胚并固定尿囊膜,15-20 min后,待CAM血管中的血液凝固后,剪取尿囊膜,放入水中展平,拍照,计数滤纸周围呈放射状排列的血管数,检测bFGF单抗的抗血管新生作用。
2.bFGF单抗抗黑色瘤生长作用
1)收集B16和B16F10细胞,提取RNA,以RT-PCR法检测B16和B16F10细胞表达bFGF, FGFR及Glypicanl, Glypican5, Syndecanl, Syndecan4情况。
2)收集B16细胞,铺板,加入bFGF单抗作用96h,CCK-8法检测bFGF单抗对体外培养的黑色素瘤细胞增殖的影响；
3)收集bFGF单抗处理和未处理的B16细胞,以AnnexinV/PI双染,流式细胞仪检测bFGF单抗诱导黑色素瘤细胞凋亡的情况,AnnexinV+/PI-:表示细胞发生早期凋亡,AnnexinV+/PI+表示细胞发生晚期凋亡,
4) Transwell法检测bFGF单抗对B16F10细胞迁移的影响,将B16F10细胞加入transwell小室的上室中,下室加入含RPMI1640完全培养基,诱导上室中黑色素细胞向下室的迁移,另外加入bFGF单抗检测其对迁移的影响,20h后吉姆萨染色,擦去上层细胞计数迁移的细胞数；
5)使用C57 BL／6小鼠建立黑色素移植瘤小鼠,并检测尾静脉和瘤周皮下注射抗体及不同给药剂量对荷瘤小鼠肿瘤生长、瘤重及小鼠体重的影响。免疫组化法检测肿瘤组织CD31表达(微血管密度),TUNEL法检测肿瘤组织凋亡的情况,以初步探讨bFGF单抗的抗肿瘤机制。
6)以5中同样的方法建立黑色素瘤小鼠模型,持续给药,直至小鼠死亡,记录小鼠生存时间,绘制小鼠生存曲线,计算各组小鼠中位生存时间。
3. bFGF单抗抗Lewis肺癌生长及转移作用使用C57 BL／6小鼠建立肺癌原位转移模型,瘤周皮下注射bFGF单抗,三天一次连续六次,并检测其对肿瘤生长、瘤重、及小鼠体重的影响；给药六次后处死小鼠取完整双肺,计数肺表面转移灶结节数,并用HE染色肺组织确认癌转移灶。用免疫组化法检测肿瘤组织微血管密度,初步探讨bFGF单抗抗肿瘤转移机制。
结果
1.bFGF单抗抗血管新生作用
1) bFGF单抗不与aFGF和VEGF发生交叉反应,单抗MabF7识别bFGF的线性表位,单抗MabF10, MabF12识别bFGF空间表位。
2) bFGF单抗MabF7, MabF10, MabF12可抑制bFGF与其高亲和力受体FGFR-1βⅢC的结合。IC50分别为7.5μg／ml、65μg／ml、50μg／ml。然而三株单抗对bFGF与肝素的结合则无抑制作用,说明其识别bFGF受体结合区域的表位。
3)通过促增殖实验发现,MabF7, MabF10, MabF12均可抑制bFGF诱导的血管内皮细胞的增殖。IC50分别为50μg／ml、100μg／ml和150μg／ml。
4)体外基质胶模拟血管内皮细胞成管作用发现三株单抗均可抑制内皮细胞成管过程,通过计数完整的管状结构的个数计算管状结构形成率,无抗体组,对照抗体组,MabF7, MabF10, MabF12组管状结构形成率分别为：100%,105.93±3.85%,56.53±4.35%,29.23±6.45%,以及12.77±2.67%,结果显示MabF7,MabF10, MabF12与无抗体组(Medium)相比均有显著差异(P=0.000)；而对照IgG组与无抗体组(Medium)之间无显著差异(P=0.104)。
5) Transwell模拟血管内皮细胞迁移作用,发现三株单抗均可抑制内皮细胞迁移过程,通过计数迁移的细胞数计算抑制率,无抗体组,对照抗体组,MabF7, MabF10, MabF12组细胞迁移率分别为100%,106.25±7.89%,69.50±6.86%,74.00±4.16%,67.75±3.30%,单抗组与无抗体对照组相比差异显著,具有统计学意义(P<0.05)。
6)光镜下计数bFGF单抗对血管内皮细胞与黑色素瘤细胞粘附作用发现,bFGF单抗对二者间的粘附作用无显著影响(P>0.05)。
7)计数尿囊膜上加药滤纸周围呈放射状的血管条数。计数后发现bFGF组,bFGF+MabF7组,bFGF+MabF10组,bFGF+MabF12呈放射状的血管条数分别为15±0.82,7.5±1.29,13.5±3.10,8.5±0.58,bFGF+MabF7组,显示bFGF+MabF7, bFGF+MabF12与bFGF组相比均有显著差异(P<0.01)；而bFGF+MabF10与bFGF组之间无显著差异(P=0.896)。
2. bFGF单抗抗黑色瘤生长作用
1) bFGF单抗可抑制黑色素瘤细胞的生长,200μg／ml,作用96h后MabF7组,MabF10组,MabF12组抑制率分别为42.37±2.38%,40.71±3.90%,41.48±2.76%。相同剂量的对照IgG对B16细胞生长则无显著抑制作用。
2) bFGF单抗可诱导黑色素瘤细胞发生凋亡,200μg／ml MabF7作用96h,凋亡率(以Annexin V+细胞计算)为73.20±10%,与对照IgG组及无抗体处理组相比差异显著,具有统计学意义(P<0.05)。
3) bFGF单抗可抑制黑色素瘤细胞发生迁移,无抗体处理组,对照IgG, MabF7, MabF10以及MabF12组的迁移率分别为100%,109.00±9.56%,72.14±13.57%,34.68±14.37%,和36.56±13.60%。MabF7,MabF10,MabF12与无抗体组(Medium)相比均有显著差异(P<0.05)；而对照IgG组与无抗体组(Medium)之间无显著差异(P=0.295)。
4)三株单抗MabF7, MabF10, MabF12给药5次后对黑色素瘤生长的抑制率分别为46.40%,23.24%,19.89%。将不同浓度的MabF7注射小鼠发现MabF7可以浓度依赖的方式抑制黑色素瘤的生长,PBS组,Control IgG组,MabF7高,中,低剂量组的瘤重分别为6.303±1.196,5.529±0.658,3.842±1.060,4.778±0.888,5.061±0.922。结果显示MabF7高中低组与PBS组相比均有显著差异(P<0.05)；而对照IgG组与PBS之间无显著差异(P=0.143)
5) bFGF单抗MabF7可抑制瘤组织微血管密度。MabF7组微血管密度为24.67±5.20,对照IgG组为45.38±12.59,具有显著差异(P<0.05)。使用TUNEL对瘤组织的细胞凋亡情况进行检测,发现MabF7处理组瘤组织存在细胞凋亡染色,而对照IgG组则少见。
4)bFGF单抗MabF7可提高荷瘤小鼠生存期,PBS组,对照IgG组,MabF7组小鼠的中位生存期分别为24,26,32天。
3. bFGF单抗抑制肺癌的转移
1)bFGF单抗MabF7可抑制肺癌皮下移植瘤的生长,MabF7组瘤重为2.64±1.05 g显著低于PBS组的5.15±1.31 g(P=0.013),抑瘤率为48.23%；对照IgG组瘤重为4.67±0.40与PBS组相比无显著差异(P=0.781),抑瘤率为8.43%；MabF7与对照IgG组比较差异显著(P=0.010)。
2) bFGF单抗MabF7可抑制肺癌发生原位转移,给药六次后bFGF单抗MabF7组肺转移结节数为3.00±2.10与PBS组的12.29±2.63相比差异显著(P=0.000),抑制率为75.59%；对照IgG组的转移结节数为11.80±3.56,与PBS组相比无显著差异(P=0.768)
3)bFGF单抗MabF7可降低肺癌组织的微血管密度,结果显示bFGF单抗MabF7组MVD 22.00±2.94显著低于PBS组的43.5±11.59(P=0.003)；对照IgG组MVD为45.75±5.44与PBS组相比无显著差异(P=0.718)。MabF7组与对照IgG组相比差异显著(P=0.002)。
结论
1.三株单抗MabF7, MabF10, MabF 12均能抑制bFGF与FGFR1的结合。三株单抗均可一定程度的抑制血管内皮细胞的增殖,迁移及成管过程,但鸡胚尿囊膜实验则显示,只有MabF7和MabF 12具有显著的抑制尿囊膜血管新生作用,提示bFGF单抗可用于抗血管新生作用。
2.以黑色素瘤为模型探讨bFGF单抗对肿瘤生长的作用,发现三株单抗均可抑制黑色素瘤的生长,并诱导其发生凋亡。但通过建立C57 BL／6小鼠黑色素抑制瘤模型研究其体内抗肿瘤效果则发现,只有MabF7具有显著的抑制肿瘤生长的作用。MabF7对体内黑色素瘤生长的抑制作用可能与其诱导肿瘤凋亡及降低微血管密度有关。提示抗体的体内治疗可能与抗体的识别表为,亲和力,及药代动力学均有关系。
3.以Lewis细胞建立肺癌原位转移模型,发现MabF7可抑制肺癌的生长及转移,此作用与MabF7抑制肿瘤微血管密度有关。
BACKGROUND & OBJECTIVE
Basic fibroblast growth fator (bFGF, FGF-2) belongs to the family of heparin-binding growth factors. Acting as a broad-spectrum mitogen and potent angiogenic agent, bFGF mediates a variety of cellular responses including cell proliferation and differentiation. bFGF functions mainly by interacting with high affinity tyrosine kinase FGF receptors (FGFRs) on the surface of target cells. There are four Fgf receptor genes in mammals, FgfR1-FgfR4, encoding several splice variants that increase the signaling repertoire. bFGF could bind to all FGFR including FGFRlb, FGFR1c, FGFR2c, FGFR3b, FGFR3c, FGR4 except for FGFR2b. Upon binding to bFGF, FGFRs dimerize and are tyrosine phosphorylated, leading to the activation of downstream signaling pathways including MAPKs (mitogen-activated protein kinases)/ERKs (extracellular signal-regulated kinases), PI3K (phosphoinositide 3-kinase)/AKT (protein kinase B), and PLCy(Phospholipase C gamma)/PKC(protein kinase).bFGF and FGFR are highly expressed in many tumor tissue including melanoma and lung cancer, which play important role in tumor development and progression. All of these reports suggest the potential of bFGF as a target for tumor therapy.
Angiogenesis is the process of new blood vessel formation from pre-existing ones, which play a key role in various physiological and pathological conditions, including embryonic development, wound repair, inflammation, and tumor growth. Tumor growth is absolutely dependent on the induction of a neovascular network.Tumor cells recruit new blood vessels that serve as conduits for delivery of nutrients and for tumor metastsis. Typically, angiogenesis occurs through endothelial cell sprouting. The first stage involves the degradation of the basement membrane around a blood vessel and of the interstitial matrix. Endothelial cells migrate in response to bFGF and proliferate along a leading migratory edge.Canalization or lumen formation, as well as branch and loop formation, lead to anastamosis of these spouts allowing blood flow.The final stage involve structural inclusion of pericytes and formation of a new basement membrane surrounding the new vessel.
Malignant melanoma is one of the most aggressive Human tumors. In recent decades, the incidence of malignant melanoma has steadily increased. A particularly worrying feature of the tumor is its increasing incidence and its capacity for rapid metastatic spread. Because of their resistance to current therapies, such as surgical excision, systemic chemotherapy with dacarbazine, and immunotherapy with vaccines, melanomas remain a significant cause of mortality. To achieve improvement in overall survival, it is important to develop effective new therapies. The expression of bFGF is absent in normal melanocytes, which require exogenous bFGF to maintain the growth. bFGF transduced normal melanocytes exhibit transformed phenotype resembling early-stage melanoma, indicating a critical role of bFGF in the transformation of melanocytes to melanoma Most melanomas express high levels of bFGF and FGFRs, which form an autocrine loop and are critical for the survival and growth of melanoma cells; melanoma growth can be arrested by interfering with the production or biological activity of bFGF alone. bFGF has long been regarded as an important tumor angiogenic factor, and the specific roles of bFGF in angiogenesis and spontaneous metastasis of melanoma have been revealed too. All of these reports suggest the potential of bFGF as a target for melanoma therapy. Antisense targeting of bFGF/FGFR-1 in primary and metastatic melanoma cells blocks the tumor growth by inducing apoptosis without activation or increased production of VEGF. In addition, bFGF peptide vaccines also show significant inhibition on angiogenesis and tumor growth.
Lung cancer is the major cause of death from all Human malignancies in CHINA and the other countries, and is frequently associated with poor prognosis. Overall 5-year relative survival rate of lung cancer for all stage is very low. This is mainly due to ineffective detection of lung cancer at its early stage. A particularly worrying feature of the tumor is its drug resistance and its capacity for rapid metastatic spread. bFGF is one of the epigenetic mechanisms explaining the multidrug resistance (MDR) of tumors. Some previous studies have shown that bFGF prevents chemotherapy induced apoptosis, which result in chemoresistance in many cancers such as small cell lung cancer. This anti-apoptotic effect is mediated by MAPK/ERKs, PI3K/AKT, and/or PKC that increase the expression and activation of several anti-apoptotic proteins including Bcl-2, Bcl-XL, and XIAP. All of these reports suggest the potential of bFGF as a target for lung cancer therapy.
In this study, HUVECs, melanoma cells, and lung caner cells were used as targeting cells for anti-angiogenesis and anti-tumor therapy by anti-bFGF monoclonal antibodies.
METHODS
1. Anti-angiogenesis activity by anti-bFGF mAbs
1) The hybridomas cells secreting monoclonal antibodies against bFGF were cloning. Ascites were produced by injecting hybridomas cells into Balb/c mouse. The antibodies were purified by a protein G column, and the isotyping of antibodies was carried out using ELISA. Anti-bFGF mAbs were incubated with rhFGF-2, rhFGF-1or rhVEGF165 immobilized onto 96-well plates and bound proteins were detected with a HRP-conjugated second antibody. To determine whether the anti-bFGF mAbs recognize conformational or linear epitopes of bFGF molecule, native or heat-denured bFGF was used in ELISA.
2) In the competitive FGFR1/FGF-2 blocking assay,96-well plate was coated with 0.5μg/ml bFGF. Then FGFR-1βⅢC-Fc at a fixed amount (50 ng) was mixed with anti-bFGF mAbs at increasing concentrations and onto the coated plate. After incubation at 37℃for 1h, bound FGFR-1βⅢC-Fc were detected. In the competitive heparin/FGF-2 blocking assay,96-well plate was coated with 0.5μg/ml bFGF. Then anti-bFGF mAbs at a fixed amount was mixed with heparin at increasing concentrations and onto the coated plate. After incubation at 37℃for 1h, bound anti-bFGF mAbs were detected.
3) HUVECs were seeded in 96-well plates. After overnight culture at 37℃under 5% CO2 in a humidified incubator, serial dilutions of purified anti-bFGF mAbs with lOng/ml rhFGF-2 were added to the wells. After the cells were incubated at 37℃for 4 more days,10μl CCK-8 reagent was added into each well.
4) ECM gel-induced capillary tube formation assay was used as an in vitro measurement of angiogenesis. HUVECs were added onto each well in the presence of anti-bFGF mAbs with or without bFGF.
5) HUVECs were harvested from culture flasks by trypsinization; Aliquot of 100μl cell suspension was added to each transwell insert of 8-μm-pore membrane. The inserts were then placed into the wells of the 24-well plates containing 0.6 ml of M199 with or without rhFGF-2. After incubation for 20 h at 37℃, cells remaining at the upper surface of the membrane were removed using a cotton swab, while the cells that migrated across the membrane to the lower surface were fixed with ethanol and stained with Giemsa solution. The number of stained cells was counted under several high-power microscopic fields.
6) HUVECs were treated with anti-bFGF mAbs for 48h. B16F10 cells were added to HUVECs. After 15min incubation, the B16F10 cells adhering to HUVECs were counted.
7) The 7-day-old embryos with intact yolks were placed in a bowl and incubated at 37℃with 3% CO2. Anti-bFGF mAbs or control mIgG were separately applied to the CAM of individual embryos. After 24 hours of incubation, neovascularization in chicken CAMs was observed with a stereomicroscope.
2. Anti-melanoma growth activity by anti-bFGF mAbs
1) Expression of bFGF, FGFR, Glypicanl, Glypican5, Syndecanl, Syndecan4 were detected by RT-PCR.
2) Inhibition of B16 cells growth were assayed by CCK-8 kit.
3) B16 cells were incubated for 96 h with anti-bFGF mAbs, or mouse normal IgG. The fraction of apoptotic cells were marked with FITC-conjugated Annexin-V and propodium iodide (PI). After 15 min of incubation at room temperature, the samples were analyzed by flow cytometry.
4) B16F10 cells migration was assayed using a Transwell chamber as preciously described.
5) Six-to seven-week-old male and female C57 BL/6 mice were subcutaneously inoculated in the right flank with 1×105 B16 cells; One week later, when palpable tumors (≥5 mm in diameter) developed, the mice were subcutaneously (around tumors) injected with anti-bFGF mAbs every 3 days for a total of five injections. Tumor size was measured every 3 days in two dimensions using a vernier caliper. Mice were sacrificed 3 weeks after tumor cell inoculation, and tumor weights were measured. The expression of CD31 in tumor tissues was analyzed to determine the intratumoral microvessel density. Apoptotic cells in tumor sections were detected using TUNEL assay kit.
6) C57 BL/6 mice were injected with B16 cells as before mentioned. Mouse survival was monitored from the day of tumor inoculation until the day of animal death.
3. Anti-lung cancer growth activity by anti-bFGF mAbs
The mouse lung cancer graft model was established by injection of Lewis lung carcinoma cells subcutaneously into the the right flank of C57BL/6 mice. When palpable tumors (≥5mm in diameter) developed, the mice were subcutaneously (around tumors) injected with anti-bFGF mAbs in 200μl PBS. Control mice received equal amounts of control IgG or PBS. Tumor volume and metastases of Lewis lung carcinoma were counted. The microvessel density (MVD) in tumor mass were measured by imunohistochemistry staining (IHCS).
RESULTS
1. Anti-angiogenesis activity by anti-bFGF mAbs
1) No binding of purified anti-bFGF antibodies to aFGF and VEGF were detected. The result of MabF7 binding to both native and denatured bFGF implies that MabF7 recognizes a continuous epitope. MabF10 and MabF12 do not recognize the heat-denatured bFGF suggesting that they both recognize conformation-dependent epitopes on bFGF.
2) Binding of bFGF to FGFR-1 was inhibited by anti-bFGF mAbs. IC50 of MabF7, MabF10 and MabF12 were 7.5μg/ml、65μg/ml、50μg/ml respectively. However anti-bFGF mAbs could not inhibit bFGF binding to heparin.
3) Anti-bFGF mAbs could inhibit HUVECs proliferation induced by bFGF. IC50 of MabF7, MabF10 and MabF12 were 50μg/ml、100μg/ml和150μg/ml respectively.
4) The tube formation in groups with no IgG, control IgG, MabF7, MabF10, and MabF12 are 100%,105.93±3.85%,56.53±4.35%,29.23±6.45%,12.77±2.67%, respectively. It indicates that anti-bFGF mAbs significantly inhibit the tube formation of HUVECs; however, control IgG shows no effect.
5) The number of migrating cells in the presence of mAbs reduces significantly compared to the control group (treatment without supplement), and the migration ratio in groups treated with no IgG, control IgG, MabF7, MabF10, and MabF12 are100%,106.25±7.89%,69.50±6.86%,74.00±4.16%,67.75±3.30%, respectively.
6) Anti-bFGF mAbs could not inhibit attaching of B16F10 to HUVECs.
7)The blood vessel of CAM in groups treated with bFGF, bFGF+MabF7, bFGF+MabF 10, and bFGF+MabF 12 are 15±0.82,7.5±1.29,13.5±3.10,8.5±0.58 respectively.
2. Anti-melanoma growth activity by anti-bFGF mAbs
1) Anti-bFGF mAbs could inhibit B16 cells proliferation. Inhibition of MabF7, MabF10 and MabF12 were 42.37±2.38%,40.71±3.90%,41.48±2.76% respectively.
2) Apoptosis was analyzed by flow cytometry with Annexin V/PI staining. With mAbs at a concentration of 200μg/ml,73.20±10% of B16 cells show apoptosis after 96 h of incubation, whereas IgG control group shows no effect compared with medium control
3) The number of migrating cells in the presence of mAbs reduces significantly compared to the control group (treatment without supplement), and the migration ratio in groups treated with no IgG, control IgG, MabF7, MabF10, and MabF12 are100%,109.00±9.56%,72.14±13.57%,34.68±14.37%, and 36.56±13.60%, respectively.
4) MabF7 remarkably reduces tumor burdens and suppresses tumor growth by 46.40% at the concentration of 5 mg/ml. However, the inhibitions by MabF10 and MabF12 are 23.24% and 19.89%.
5) The microvessel density in tumor is determined by staining the tumor section with anti-CD31 antibody, and the numbers of microvessel density in tumor reduces significantly in MabF7 group (24.67±5.20) in comparison with that in IgG control group (45.38±12.59).
6) More TUNEL-positive cells with deep brown stained nuclei are detected in MabF7 treated tumors than that in control IgG treated group.
7) To investigate whether the inhibition of tumor growth by MabF7 impacts the survival of animals, a survival assay has been performed as shown in the Kaolan-Meier survival curve in Fig.8. The median survival time of mice treated with MabF7 (32 days) is longer than that of mice treated with PBS (24 days) or control IgG (26 days)
3. Anti-Lewis lung cancer growth activity by anti-bFGF mAbs
Anti-bFGF mAb could inhibit tumor growth in C57BL/6 mice Lewis lung cancer model. Tumor weight in MabF7 group is 2.64±1.05 g which is lower than that of PBS group (5.15±1.31 g) (P<0.05). The mean number of metastatic lung nodules in MabF7 group is 3.00±2.10 which is lower than that of PBS group (12.29±2.63) (P<0.05). Moreover MabF7 reduce the expression of MVD in the tumor tissue.
CONCLUSIONS
1. Binding of bFGF to FGFR-1 was inhibited by anti-bFGF mAbs MabF7, MabF10, MabF12. All of them could also inhibit HUVECs proliferation, tube formation, and migration. However in CAM assay, only MabF7 and MabF12 could inhibit angiogenesis.
2. MabF7, MabF10, MabF12 could inhibit melanoma cells proliferation, migration, and induce apoptosis in melanoma cells in vitro. However only MabF7 could inhibit melanoma growth in vivo through inhibition of the angiogenesis and induction of the apoptosis in tumor.
3. Anti-bFGF mAb could inhibit tumor growth and pulmonary metastasis in C57BL/6 mice Lewis lung cancer model, and suppress angiogenesis of tumor tissue.
引文
1. Ornitz DM, Itoh N. Fibroblast growth factors [J]. Genome Biol,2001,2:Reviews 3005.1-3005.12
2. McNeil PL, Muthukrishnan L, Warder E, et al.Growth factors are released by mechanically wounded endothelial cells [J]. The Journal of Cell Biology,1989, 109:811-822.
3. Florkiewicz R, Majak RA, Buechler RD. Quantitative export of FGF-2 occurs through an alternative, energy dependent, non-ER/Golgi pathway [J]. J Cell Physiol,1995,162:388-399.
4. Vlodawsky I. ExtracelluLar sequestration and release of fibroblast growth factor:a reguLatory mechanism[J]. Trends Biochem Sci,1991,16:268-71.
5. Omitz DM, Xu J, Colvin JS, et al. Receptor specificity of the fibroblast growth factor family [J]. J Biol Chem,1996,271:15292-7.
6. Werner S, Duan DSR, Peters K, et al. Differential splicing in the extra cellular region of fibroblast growth factor receptor-1 generates receptor variants with different ligand-binding specificities [J]. Mol Cell Biol,1992,12:82-88.
7. Groth C, Lardelli M. The structure and function of vertebrate fibroblast growth factor receptor 1 [J].Int J Dev Biol,2002,46(4):393-400.
8. Fantl WJ, Johnson DE, Williams LT. Signalling by receptor tyrosine kinases [J].Annu Rev Biochem,1993,62:453-481.
9. Eswarakumar VP, Lax I and Schlessinger J. Cellular signaling by fibroblast growth factor receptors[J]. Cytokine Growth Factor Rev,16:139-149,2005.
10. Ong SH, Guy GR, Hadari YR, et al.FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors[J]. Mol Cell Biol,2000,20:979-89.
11. Dhalluin C, Yan K, Plotnikova O, et al. Structural basis of SNT PTB domain interactions with distinct neurotrophic receptors[J]. Mol Cell,2000,6:921-9.
12. Wang JK, Xu H, HC Li, et al. Broadly expressed SNT-like proteins link FGF receptor stimuLation to activators of Ras[J]. Oncogene,1996,13:721-729.
13. Kouhara H, Hadari YR, Spivak-Kroizman T, et al. A lipid-anchored Grb2-binding protein that links FGF-receptor activation of the Ras/MAPK signaling pathway [J]. Cell,1997,89:693-702.
14. Pellegrini L. Role of heparin sulfate in fibroblast growth factor signaling:a structural view [J]. Curr Opin Struct Biol,2001,11:629-634.
15. Schlessinger J, Plotnikov AN, Ibrahimi OA, et al. Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization[J]. Mol Cell,2000,6:743-50.
16. Rusnati M, Urbinati C, Presta M. Inernalization of basic fibrobalst growth factor (bFGF) in cultured endothelial cells:role of the low affinity heparin-like bFGF receptors [J].J Cell Physiol,1993,154:152-161.
17. Rusnati M, Presta M. Interaction of angiogenesis basic fibroblast growth factor with endothelial cell heparin sulfate proteoglycans biological implications in neovascularization [J]. Int J Clin Lab Res,1996,26:15-23.
18. Horowitz A, Tkachenko E, Simons M. Fibroblast growth factor specific modulation of cellular response by syndecan-4[J]. J Cell Biol,2002,157:715-725.
19. Chua CC, Rahimi N, Forsten-Williams K, et al. Heparan sulfate proteoglycans function as receptors for fibroblast growth factor-2 activation of extracellular signal-regulated kinase 1 and 2[J]. Circ Res,2004,94:316-323.
20. Adams RH, Alitalo K. Molecular regulation of angiogenesis and lym phangiogen--esis [J]. Nat. Rev. Mol. Cell Biol,2007,8:464-478
21. Hiratsuka, S. Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice [J]. Proc. Natl. Acad. Sci,1998,95: 9349-9354
22. Cross MJ, Claesson-Welsh L. FGF and VEGF function in angiogenesis:signalling pathways, biological responses and therapeutic inhibition [J]. Trends Pharmacol Sci,2000,22:201-7.
23. Presta M, Tiberio L, Rusnati M, et al. Basic fibroblast growth factor requires a long-lasting activation of protein kinase C to induce cell proliferation in transformed fetal bovine aortic endothelial cells [J]. Cell Regul,1991,2:719-26.
24. Hiraoka N, Allen E, Apel IJ,et al. Matrix metalloproteinases regulate neovascularization by acting as pericellular fibrinolysins[J]. Cell,1998; 95:365-77.
25.高春芳,陆伦根,孔宪涛,等.纤维化疾病的基础和临床[M].上海,上海科学技术出版社,2004.
26. Dell'Era P, Mohammadi M, Presta M. Different tyrosine autophosphorylation requirements in fibroblast growth factor receptor-1 mediate urokinase-type plasminogen activator induction and mitogenesis[J]. Mol Biol Cell,1999; 10:23-33.
27. Nakoman C, Resmi H, Ay 0, et al. Effects of basic fibroblast factor (bFGF) on MMP-2, TIMP-2, and type-Ⅰ collagen levels in Human lung carcinoma fibroblasts [J].Biochimie,2005,87:343-351.
28. Hirano T, Gong Y, Kato Y, et al. Influence of angiogenetic factors and matrix metalloproteinases upon tumour progression in non-small-cell lung cancer [J]. British Journal of Cancer,2001,85:1706-1712.
29. Presta M, Dell'Era P, Mitola S,et al. Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis[J]. Cytokine Growth Factor Rev,2005,16: 159-178.
30. Eliceiri BP. Integrin and growth factor receptor crosstalk[J]. Circ Res,2001,89: 1104-1110.
31.Tanghetti E, Ria R, DellEra P, et al. Biological activity of substrate-bound basic fibroblast growth factor (FGF2):recruitment of FGF receptor-1 in endothelial cell adhesion contacts [J]. Oncogene,2002,21:3889-3897.
32. Sahni A, Francis CW. Stimulation of endothelial cell proliferation by FGF-2 in the presence of fibrinogen requires alpha v beta 3[J]. Blood,2004,104:3635-3641.
33. Scatena M, Almeida M, Chaisson ML, et al. NF-kappa B mediates alphavbeta3 integrin-induced endothelial cell survival. J Cell Biol [J],1998,141:1083-1093.
34. Landgren E, Klint P, Yokote K, et al. Fibroblast growth factor receptor-1 mediates chemotaxis independently of direct SH2-domain protein binding [J]. Oncogene, 1998,17:283-91.
35. Kanda S, Lerner EC, Tsuda S, et al. The non receptor protein-tyrosine kinase c-Fes is involved in fibroblast growth factor-2-induced chemotaxis of murine brain capillary endothelial cells [J]. J Biol Chem 2000,275:10105-10111.
36. Hori A, Sasada R, Matsutani E, et al. Suppression of solid tumor growth by immunoneutralizing monoclonal antibody against Human basic fibroblast growth factor[J].Cancer Res,1991,51:6180-6184.
37. MuraiN, Ueba T, Takahashi JA,et al. Apoptosis of Human glioma cells in vitro and in vivo induced by a neutralizing antibody against Human basic fibroblast growth factor[J].J Neurosurg,1996,85:1072-1077.
38.谢庆祥,周最明,韩聪祥,等.bFGF及其抗体对裸鼠皮下移植膀胱瘤生长影响的研究[J].肿瘤防治研究,2004,31：521-522.
39.林卫,杨延林,彭芝兰,等.bFGF单克隆抗体对卵巢癌移植瘤血管生成的抑制作用[J].现代妇产科进展,2005,14：281-284.
40. Hersey P and Zhang XD. How melanoma cells evade trail-induce apoptosis [J]. Nat Rev Cancer,2001,1:42-150.
41. Ugurel S, Rappl G, Tilgen W, et al. Increased Serum concentration of angiogenic factors in malignant melanoma patients correlates with tumor progression and survival [J]. J Clin Oncol,2001,19:577-583
42. Xerri L, Battyani Z, Gmb JJ, et al. Expression of FGF1 and FGFR1 in Human melanoma tissues [J]. Melanoma Res,1996,6:223-230.
43. Halaban R, Ghosh S, Baird A. bFGF is the putative natural growth factor for Human melanocytes [J]. In Vitro Cellar & Developmental Biology,1987,23: 47-52.
44. Coleman AB and Lugo TG. Normal Human melanocytes that express a bFGF transgene still require exogenous bFGF for growth in vitro [J]. J Invest Dermatol 110:793-9,1998.
45. Nesbit M, Nesbit HK, Bennett J, et al. Basic fibroblast growth factor induces a transformed phenotype in normal Human melanocytes [J]. Oncogene,1999 18:6469-6476.
46. Valesky M, Spang AJ, Fisher GW, et al. Noninvasive dynamic fluorescence imaging of Human melanomas reveals that targeted inhibition of bFGF or FGFR-1 in melanoma cells blocks tumor growth by apoptosis[J]. Mol Med,2002, 8:103-112.
47. Ackermann J, Beermann F. The fibroblast growth factor-2 is not essential for melanoma formation in a transgenic mouse model [J]. Pigment Cell Res,2005,18; 315-319.
48. Alanko T, Rosenberg M, Saksela O. FGF expression allows nevus cells to survive in three-dimensional collagen gel under conditions that induce apoptosis in normal Human melanocytes [J]. J Invest Dermatol,1999,113:111-116.
49. Dotto G P, Gisela M, Sikha G, et al. Transfornation of murine melanocytes by basic fibroblast growth factor cDNA and oncogenes and selective suppression of the transformed phenotype in a reconstituted cutaneous environment [J]. The Journal of cell biology,1989,109:3115-3128.
50. Wang Y, Becker D. Antisense targeting of basic fibroblast growth factor and fibroblast growth factor receptor-1 in Human melanomas blocks intratumoral angiogenesis an d tumor growth[J]. Nat Med,1997,3:887-893.
51. Plum SM, Holaday JW, Ruiz A, et al. Administration of a liposomal FGF-2 peptide vaccine leads to abrogation of FGF-2-mediated angiogenesis and tumor development [J]. Vaccine,8:1294-1303,2000.
52. He QM, Wei YQ, Tian L, et al. Inhibition of tumor growth with a vaccine based on xenogeneic homologous fibroblast growth factor receptor-1 in mice[J]. The journal of Biological chemistry,2003,278:21831-21836.
53. Rofstad EK, Halsor EF. Halsor.Vascular edothrlial growth factor, interleukin 8, platelet-derived endothelial cell growth factor, and basic fibroblast growth factor promote angiogenesis and metastasis in Human melanoma xenografts[J]. Cancer Res,2000,60:4932-4938.
54. Futami J, Seno M, Ueda M, et al. Inhibition of cell growth by a fused protein of Human ribonuclease 1 and Human basic fibroblast growth factor [J]. Protein Eng,1999,12:1013-1019.
55. Davol PA, Bizuneh A, Frackelton AR Jr. Wortmannin, a phosphoinositide 3-kinase inhibitor, selectively enhances cytotoxicity of receptor-directed-toxin chimeras in vitro and in vivo[J]. Anticancer Res,1999,19:1705-1713.
56.毛友生,高燕宁,赫捷,等.肺癌分子生物学特性与转移和预后的关系[J].中华肿瘤杂志,2006,28：632-634.
57.夏钰弘,杨国仪,洪建刚,等.IGFBP-3、bFGF、Ki-67蛋白在非小细胞肺癌组 织中的表达及意义[J].江苏医药,2009,35：41-43.
58.高双荣,钟雪云,向军俭.LN, FN, bFGF及EGFR与肺癌转移[J].广东医学,2002，23：909-911.
59.余荣环,张杰,向明,等.非小细胞肺癌碱性成纤维细胞生长因子表达与微血管密度测定的临床意义[J].临床肺科杂志,2006,11：288-289.
60. Iwasaki A, Kuwahara M, Yoshinaga Y, et al.Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor(VEGF) level, as prognostic indicators in NSCLC[J]. European Journal of cardio-thoracis surgery,2004,25:443-448.
61. Pardo OE, Wellbrock C, Khanzada UK, et al. FGF-2 protects small cell lung cancer cells from apoptosis through a complex involving PKCε, B-Raf and S6K2 [J]. EMBO J,2006,25:3078-3088.
62. Pardo OE, Latigo J, Jeffery RE, et al. The Fibroblast Growth Factor Receptor Inhibitor PD 173074 Blocks Small Cell Lung Cancer Growth In vitro and In vivo [J]. Cancer Research,2009,69:8645-8651
[1]毛友生,高燕宁,赫捷,等.肺癌分子生物学特性与转移和预后的关系[J].中华肿瘤杂志,2006,28(8)：632-634.
[2]Bikfalvi A, Klein S, Pintucci G, et al. Biological roles of fibroblast growth factor-2 [J]. Endocr. Rev,1997,18(1):26-45.
[3]Presta M, Dell'Era P, Mitola S,et al. Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis [J]. Cytokine Growth Factor Rev,2005,16(2):159-178.
[4]Cox G, Jones JL, Walker RA, et al. Angiogenesis and non-small cell lung cancer [J]. Lung Cancer,2000,27(2):81-100.
[5]夏钰弘,杨国仪,洪建刚,等.IGFBP-3、bFGF、Ki-67蛋白在非小细胞肺癌组织中的表达及意义[J].江苏医药,2009,35(1)：41-43.
[6]高双荣,钟雪云,向军俭.LN, FN, bFGF及EGFR与肺癌转移[J].广东医学,2002,23(9)：909-911.
[7]Joensuu H, Anttonen A, Eriksson M, et al. Soluble Syndecan-1 and Serum Basic Fibroblast Growth Factor Are New Prognostic Factors in Lung Cancer [J]. Cancer Research,2002,62(18):5210-5217.
[8]Kuhn H, Kopff C, Konrad J, et al. Influence of basic fibroblast growth factor on the proliferation of non-small cell lung cancer cell lines [J]. Lung Cancer,2004,44(22): 167-174.
[9]Pardo OE, Wellbrock C, Khanzada UK, et al. FGF-2 protects small cell lung cancer cells from apoptosis through a complex involving PKCε, B-Raf and S6K2 [J]. EMBO J,2006, 25(13):3078-3088.
[10]Pardo OE, Latigo J, Jeffery RE, et al. The Fibroblast Growth Factor Receptor Inhibitor PD173074 Blocks Small Cell Lung Cancer Growth In vitro and In vivo [J]. Cancer Research,2009,69(22):8645-8651
[11]Hori A, Sasada R, Matsutani E, et al. Suppression of solid tumor growth by immunoneutralizing monoclonal antibody against Human basic fibroblast growth factor [J]. Cancer Res,1991,51(22):6180-6184.
[12]Rofstad EK and Halsor EF. Vascular endothelial growth factor, interleukin 8, platelet-derived endothelial cell growth factor, and basic fibroblast growth factor promote angiogenesis and metastasis in Human melanoma xenografts [J]. Cancer Res,2000, 60(17):4932-4938.
[13]向军俭,靳英杰,黄红亮等.人bFGF单克隆抗体的制备及MabF7对黑色素瘤B16的体外抗瘤效应[J].中华肿瘤防治杂志,2008,15(1)：19-22
[14]刘李登,王宏,向军俭等.bFGF单抗与顺铂体外联合移植黑色素瘤B16细胞增殖效应的研究[J].中华肿瘤防治杂志,2010,17(1)：1-4
[15]Kuhn H, Konrad J, Holtz S, et al. Enhanced expression of VEGF following bFGF inhibition in non small cell lung cancer cell lines [J].Lung Cancer,2006,54(2):149-153.
[16]Giavazzi R, Senninno B, Coltrini D, et al. Distinct role of fibroblast growth factor 2 and vascular endothelial growth factor on tumor growth and angiogenesis [J].Am J Pathol, 2003,162(6):1913-1926.a
1. Dale L Ludwig, Daniel S Pereiral, Zhen ping Zhul,et al. Monoclonal antibody therapeutics and apoptosis[J]. Oncogene,2003,22:9097-9106
2. Younes, A. Kadin, M.E. Emerging applications of the tumor necrosis factor family of ligands and receptors in cancer therapy[J]. Journal of Clinical Oncology, 2003,21,3526-3534
3. Toshiaki Ohtsuka, Donald Buchsbaum, Patsy Oliver,et al. Synergistic induction of tumor cell apoptosis by death receptor antibody and chemotherapy agent through JNK/p38 and mitochondrial death pathway [J]. Oncogene,2003,22:2034-2044
4. Toshiaki Ohtsuka(?),Tong Zhou. Bisindolylmaleimide Ⅷ Enhances DR5-mediated Apoptosis through the MKK4/JNK/p38 Kinase and the Mitochondrial Pathways[J]. The Journal of Biological Chemistry,2002,277(32): 29294-29303
5. S Kaliberov, MA Stackhouse, L Kaliberova,et al. Enhanced apoptosis following treatment with TRA-8 anti-Human DR5 monoclonal antibody and overexpression of exogenous Bax in Human glioma cells[J]. Gene Therapy,2004,11:658-667
6.Georgios V. Georgakis, Yang Li, Robin Humphreys, et al. Activity of selective fully Human agonistic antibodies to the TRAIL death receptors TRAIL-R1 and TRAIL-R2 in primary and cultured lymphoma cells:induction of apoptosis and enhancement of doxorubicin-and bortezomib-induced cell death[J]. British Journal of Haematology,2005,130:501-510
7. Masa Yuki Fujino,Xiao-kang Li, Yusuke Kitazawa,et al. Distinct Pathways of Apoptosis Triggered by FTY720, Etoposide, and Anti-Fas Antibody in Human T-Lymphoma Cell Line (Jurkat Cells) [J].The Journal of Pharmacology and Experimental Therapeutics,2002,300(3),939-945.
8. Lacronique V, Mignon A, Fabre M, et al. Bcl-2 protects from lethal hepatic apoptosis induced by an anti-Fas antibody in mice[J]. Nat Med,1996,2(1):80-6.
9. Igor Tamm, Toyoko Kikuchi, Arturo Zychlinskyt. Acidic and basic fibroblast growth factors are survival factors with distinctive activity in quiescent BALB/c 3T3 murine fibroblasts[J].Cell Biology April,1991,88:3372-3376,
10. Patrik Georgii-Hemming, Helena Jernberg Wiklund, Osten Ljunggren,et al. Insulin-Like Growth Factor I Is a Growth and Survival Factor in Human Multiple Myeloma Cell Lines[J]. Blood,1996,88(6):2250-2258
11. Anne-Tove Brenne, Torstein Baade Ro, Anders Waage, et al. Interleukin-21 is a growth and survival factor for Human myeloma cells[J]. Blood,2002,99(10): 3756-3762
12. Ping Yang, Jessica L. Wiser, James J. Peairs, et al. Human RPE Expression of Cell Survival Factors [J]. Investigative Ophthalmology & Visual Science,2005, 46(5):1755-1764
13. Jacques E. No" r, Joan Christensen,David J. Mooney, et al. Vascular Endothelial Growth Factor (VEGF)-Mediated Angiogenesis Is Associated with Enhanced Endothelial Cell Survival and Induction of Bcl-2 Expression[J]. Am J Pathol,1999, 154(2):375-384
14. Lewis C. Cantley The Phosphoinositide 3-Kinase Pathway [J]. Science,2002, 296(5573):1655-1657
15. Tsuyoshi Miyazaki, Hideki Katagiri, Yumi Kanegae,et al. Reciprocal Role of ERK and NFkB Pathways in Survival and Activation of Osteoclasts[J]. The Journal of Cell Biology,2002,148(2):333-342
16.Thomas F Franke, Christoph P Hornik, Lisa Segev,et al. PI3K/Akt and apoptosis: size matters Oncogene (2003) 22,8983-8998
17 Douglas Burtrum, Zhenping Zhu, Dan Lu A. Fully Human Monoclonal Antibody to the Insulin-Like Growth Factor I Receptor Blocks Ligand-Dependent Signaling and Inhibits Human Tumor Growth in Vivo.Cancer Reseach.(2003) 63,8912-8921
18. Jing Yang, Jianfei Qian, Michele Wezeman, et al. Targeting b2-microglobulin for induction of tumor apoptosis in Human hematological malignancies. Cancer Cell (2006)10,295-307,
19 Masayasu Iwase, Sayaka Takaoka, Makiko Uchida, et al. Epidermal growth factor receptor inhibitors enhance susceptibility to Fas-mediated apoptosis in oral squamous cell carcinoma cells. Oral Oncol (2007)
20.Gregory B. Carey, Laura Tonnetti, David W.Scott, et al. The Role of the PI3K Pathway in Anti-IgM(Anti-μ)- Sensitive and-Resistant B-cell Lymphomas. 《Molecular Mechanisms of Programmed Cell Death》 37-47
21 Sclabas G.M, Fujioka S, Schmidt C,et al. Restoring Apoptosis in Pancreatic Cancer Cells by Targeting the Nuclear Factor-κB Signaling Pathway With the Anti-Epidermal Growth Factor Antibody IMC-C225 Journal of Gastrointestinal Surgery (2002)7,37-43.
22. Liang You, Biao He, Zhidong Xu, et al. An Anti-Wnt-2 Monoclonal Antibody Induces Apoptosis in Malignant Melanoma Cells and Inhibits Tumor Growth. Cancer Research(2004) 64,5385-5389.
23. Jing Yang, Xiang Zhang, Ji Wang, Anti-β2-microglobulin monoclonal antibodies induce apoptosis in myeloma cells by recruiting MHC class I to and excluding growth and survival cytokine receptors from lipid rafts. Blood. 2007(110),3028-3035.
24.Rakesh Raoa, Barbara Loganb, Kathy Forrest,et al. Lipid rafts in cytokine signaling Cytokine & Growth Factor Reviews. (2004)15 103-110.
25. Kelly Davison, Koren K. Mann, Samuel Waxman, et al. JNK activation is a mediator of arsenic trioxide-induced apoptosis in acute promyelocytic leukemia cells. Blood., (2004)103,3496-3502.
26. Maundrell, K, AntonssonB., Magnenat E, et al. Bcl-2 undergoes phosphorylation by c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the constitutively active GTP-binding protein Racl. J. Biol. Chem. (1997) 272, 25238-25242
27. Bong-Jo Kim, Seung-Wook Ryu, and Byoung-Joon Song,et al. JNK-and p38Kinase-mediated Phosphorylation of Bax Leads to Its Activation and Mitochondrial Translocation and to Apoptosis of Human Hepatoma HepG2 Cells. The Journal of Biological Chemistry (2006) 281,21256-21265
28. Dharminder Chauhan, Guilan Li, Teru Hideshima, et al. JNK-dependent Release of Mitochondrial Protein, Smac,during Apoptosis in MultipleMyeloma (MM) Cells. The Journal of Biological Chemistry. (2003) 278,17593-17596.
29. Mitchell R Smith. Rituximab (monoclonal anti-CD20 antibody):mechanisms of action and Resistance[J]. Oncogene,2003,22(47):7359-68
30.姜勇,罗深秋《信号转导》161-164
31. Wei Aixinjueluo, Keiko Furukawa, Qing Zhang. Mechanisms for the Apoptosis of Small Cell Lung Cancer Cells Induced by Anti-GD2 Monoclonal Antibodies. The Journal of Biological Chemistry[J].2005,280(33),29828-29836
32.Dylan Daniel, Becky Yang, David A. Lawrence, et al. Cooperation of the pro-apoptotic receptor agonist rhApo2L/TRAIL with the CD20 antibody rituximab against non-Hodgkin's lymphoma xenografts[J]. Blood,2007,110(12):4037-46
33. Gigi N.C. Chiul, Lincoln A. Edwardsl, Anita I. Kapanen,et al. Modulation of cancer cell survival pathways using multivalent liposomal therapeutic antibody constructs[J]. Molecular Cancer Therapeutics.2007,6(3):844-855
34. Kare'ne Mahtouk, Michel Jourdan, John De Vos,et al. An inhibitor of the EGF receptor family blocks myeloma cell growth factor activity of HB-EGF and potentiates dexamethasone or anti-IL-6 antibody-induced apoptosis[J]. Blood, 2004,103(5):1829-1837
35. Mitchell R. Smith, Fang Jin, and Indira Joshi. Enhanced efficacy of therapy with antisense BCL-2 oligonucleotides plus anti-CD20 monoclonal antibody in scid mouse/Human lymphoma xenografts[J]. Mol Cancer Ther,2004,3(12):1693-9.
2. McNeil PL, Muthukrishnan L, Warder E, et al.Growth factors are released by mechanically wounded endothelial cells [J]. The Journal of Cell Biology,1989, 109:811-822.
3. Florkiewicz R, Majak RA, Buechler RD. Quantitative export of FGF-2 occurs through an alternative, energy dependent, non-ER/Golgi pathway [J]. J Cell Physiol,1995,162:388-399.
4. Vlodawsky I. ExtracelluLar sequestration and release of fibroblast growth factor:a reguLatory mechanism[J]. Trends Biochem Sci,1991,16:268-71.
5. Omitz DM, Xu J, Colvin JS, et al. Receptor specificity of the fibroblast growth factor family [J]. J Biol Chem,1996,271:15292-7.
6. Werner S, Duan DSR, Peters K, et al. Differential splicing in the extra cellular region of fibroblast growth factor receptor-1 generates receptor variants with different ligand-binding specificities [J]. Mol Cell Biol,1992,12:82-88.
7. Groth C, Lardelli M. The structure and function of vertebrate fibroblast growth factor receptor 1 [J].Int J Dev Biol,2002,46(4):393-400.
8. Fantl WJ, Johnson DE, Williams LT. Signalling by receptor tyrosine kinases [J].Annu Rev Biochem,1993,62:453-481.
9. Eswarakumar VP, Lax I and Schlessinger J. Cellular signaling by fibroblast growth factor receptors[J]. Cytokine Growth Factor Rev,16:139-149,2005.
10. Ong SH, Guy GR, Hadari YR, et al.FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors[J]. Mol Cell Biol,2000,20:979-89.
11. Dhalluin C, Yan K, Plotnikova O, et al. Structural basis of SNT PTB domain interactions with distinct neurotrophic receptors[J]. Mol Cell,2000,6:921-9.
12. Wang JK, Xu H, HC Li, et al. Broadly expressed SNT-like proteins link FGF receptor stimuLation to activators of Ras[J]. Oncogene,1996,13:721-729.
13. Kouhara H, Hadari YR, Spivak-Kroizman T, et al. A lipid-anchored Grb2-binding protein that links FGF-receptor activation of the Ras/MAPK signaling pathway [J]. Cell,1997,89:693-702.
14. Pellegrini L. Role of heparin sulfate in fibroblast growth factor signaling:a structural view [J]. Curr Opin Struct Biol,2001,11:629-634.
15. Schlessinger J, Plotnikov AN, Ibrahimi OA, et al. Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization[J]. Mol Cell,2000,6:743-50.
16. Rusnati M, Urbinati C, Presta M. Inernalization of basic fibrobalst growth factor (bFGF) in cultured endothelial cells:role of the low affinity heparin-like bFGF receptors [J].J Cell Physiol,1993,154:152-161.
17. Rusnati M, Presta M. Interaction of angiogenesis basic fibroblast growth factor with endothelial cell heparin sulfate proteoglycans biological implications in neovascularization [J]. Int J Clin Lab Res,1996,26:15-23.
18. Horowitz A, Tkachenko E, Simons M. Fibroblast growth factor specific modulation of cellular response by syndecan-4[J]. J Cell Biol,2002,157:715-725.
19. Chua CC, Rahimi N, Forsten-Williams K, et al. Heparan sulfate proteoglycans function as receptors for fibroblast growth factor-2 activation of extracellular signal-regulated kinase 1 and 2[J]. Circ Res,2004,94:316-323.
20. Adams RH, Alitalo K. Molecular regulation of angiogenesis and lym phangiogen--esis [J]. Nat. Rev. Mol. Cell Biol,2007,8:464-478
21. Hiratsuka, S. Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice [J]. Proc. Natl. Acad. Sci,1998,95: 9349-9354
22. Cross MJ, Claesson-Welsh L. FGF and VEGF function in angiogenesis:signalling pathways, biological responses and therapeutic inhibition [J]. Trends Pharmacol Sci,2000,22:201-7.
23. Presta M, Tiberio L, Rusnati M, et al. Basic fibroblast growth factor requires a long-lasting activation of protein kinase C to induce cell proliferation in transformed fetal bovine aortic endothelial cells [J]. Cell Regul,1991,2:719-26.
24. Hiraoka N, Allen E, Apel IJ,et al. Matrix metalloproteinases regulate neovascularization by acting as pericellular fibrinolysins[J]. Cell,1998; 95:365-77.
25.高春芳,陆伦根,孔宪涛,等.纤维化疾病的基础和临床[M].上海,上海科学技术出版社,2004.
26. Dell'Era P, Mohammadi M, Presta M. Different tyrosine autophosphorylation requirements in fibroblast growth factor receptor-1 mediate urokinase-type plasminogen activator induction and mitogenesis[J]. Mol Biol Cell,1999; 10:23-33.
27. Nakoman C, Resmi H, Ay 0, et al. Effects of basic fibroblast factor (bFGF) on MMP-2, TIMP-2, and type-Ⅰ collagen levels in Human lung carcinoma fibroblasts [J].Biochimie,2005,87:343-351.
28. Hirano T, Gong Y, Kato Y, et al. Influence of angiogenetic factors and matrix metalloproteinases upon tumour progression in non-small-cell lung cancer [J]. British Journal of Cancer,2001,85:1706-1712.
29. Presta M, Dell'Era P, Mitola S,et al. Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis[J]. Cytokine Growth Factor Rev,2005,16: 159-178.
30. Eliceiri BP. Integrin and growth factor receptor crosstalk[J]. Circ Res,2001,89: 1104-1110.
31.Tanghetti E, Ria R, DellEra P, et al. Biological activity of substrate-bound basic fibroblast growth factor (FGF2):recruitment of FGF receptor-1 in endothelial cell adhesion contacts [J]. Oncogene,2002,21:3889-3897.
32. Sahni A, Francis CW. Stimulation of endothelial cell proliferation by FGF-2 in the presence of fibrinogen requires alpha v beta 3[J]. Blood,2004,104:3635-3641.
33. Scatena M, Almeida M, Chaisson ML, et al. NF-kappa B mediates alphavbeta3 integrin-induced endothelial cell survival. J Cell Biol [J],1998,141:1083-1093.
34. Landgren E, Klint P, Yokote K, et al. Fibroblast growth factor receptor-1 mediates chemotaxis independently of direct SH2-domain protein binding [J]. Oncogene, 1998,17:283-91.
35. Kanda S, Lerner EC, Tsuda S, et al. The non receptor protein-tyrosine kinase c-Fes is involved in fibroblast growth factor-2-induced chemotaxis of murine brain capillary endothelial cells [J]. J Biol Chem 2000,275:10105-10111.
36. Hori A, Sasada R, Matsutani E, et al. Suppression of solid tumor growth by immunoneutralizing monoclonal antibody against Human basic fibroblast growth factor[J].Cancer Res,1991,51:6180-6184.
37. MuraiN, Ueba T, Takahashi JA,et al. Apoptosis of Human glioma cells in vitro and in vivo induced by a neutralizing antibody against Human basic fibroblast growth factor[J].J Neurosurg,1996,85:1072-1077.
38.谢庆祥,周最明,韩聪祥,等.bFGF及其抗体对裸鼠皮下移植膀胱瘤生长影响的研究[J].肿瘤防治研究,2004,31：521-522.
39.林卫,杨延林,彭芝兰,等.bFGF单克隆抗体对卵巢癌移植瘤血管生成的抑制作用[J].现代妇产科进展,2005,14：281-284.
40. Hersey P and Zhang XD. How melanoma cells evade trail-induce apoptosis [J]. Nat Rev Cancer,2001,1:42-150.
41. Ugurel S, Rappl G, Tilgen W, et al. Increased Serum concentration of angiogenic factors in malignant melanoma patients correlates with tumor progression and survival [J]. J Clin Oncol,2001,19:577-583
42. Xerri L, Battyani Z, Gmb JJ, et al. Expression of FGF1 and FGFR1 in Human melanoma tissues [J]. Melanoma Res,1996,6:223-230.
43. Halaban R, Ghosh S, Baird A. bFGF is the putative natural growth factor for Human melanocytes [J]. In Vitro Cellar & Developmental Biology,1987,23: 47-52.
44. Coleman AB and Lugo TG. Normal Human melanocytes that express a bFGF transgene still require exogenous bFGF for growth in vitro [J]. J Invest Dermatol 110:793-9,1998.
45. Nesbit M, Nesbit HK, Bennett J, et al. Basic fibroblast growth factor induces a transformed phenotype in normal Human melanocytes [J]. Oncogene,1999 18:6469-6476.
46. Valesky M, Spang AJ, Fisher GW, et al. Noninvasive dynamic fluorescence imaging of Human melanomas reveals that targeted inhibition of bFGF or FGFR-1 in melanoma cells blocks tumor growth by apoptosis[J]. Mol Med,2002, 8:103-112.
47. Ackermann J, Beermann F. The fibroblast growth factor-2 is not essential for melanoma formation in a transgenic mouse model [J]. Pigment Cell Res,2005,18; 315-319.
48. Alanko T, Rosenberg M, Saksela O. FGF expression allows nevus cells to survive in three-dimensional collagen gel under conditions that induce apoptosis in normal Human melanocytes [J]. J Invest Dermatol,1999,113:111-116.
49. Dotto G P, Gisela M, Sikha G, et al. Transfornation of murine melanocytes by basic fibroblast growth factor cDNA and oncogenes and selective suppression of the transformed phenotype in a reconstituted cutaneous environment [J]. The Journal of cell biology,1989,109:3115-3128.
50. Wang Y, Becker D. Antisense targeting of basic fibroblast growth factor and fibroblast growth factor receptor-1 in Human melanomas blocks intratumoral angiogenesis an d tumor growth[J]. Nat Med,1997,3:887-893.
51. Plum SM, Holaday JW, Ruiz A, et al. Administration of a liposomal FGF-2 peptide vaccine leads to abrogation of FGF-2-mediated angiogenesis and tumor development [J]. Vaccine,8:1294-1303,2000.
52. He QM, Wei YQ, Tian L, et al. Inhibition of tumor growth with a vaccine based on xenogeneic homologous fibroblast growth factor receptor-1 in mice[J]. The journal of Biological chemistry,2003,278:21831-21836.
53. Rofstad EK, Halsor EF. Halsor.Vascular edothrlial growth factor, interleukin 8, platelet-derived endothelial cell growth factor, and basic fibroblast growth factor promote angiogenesis and metastasis in Human melanoma xenografts[J]. Cancer Res,2000,60:4932-4938.
54. Futami J, Seno M, Ueda M, et al. Inhibition of cell growth by a fused protein of Human ribonuclease 1 and Human basic fibroblast growth factor [J]. Protein Eng,1999,12:1013-1019.
55. Davol PA, Bizuneh A, Frackelton AR Jr. Wortmannin, a phosphoinositide 3-kinase inhibitor, selectively enhances cytotoxicity of receptor-directed-toxin chimeras in vitro and in vivo[J]. Anticancer Res,1999,19:1705-1713.
56.毛友生,高燕宁,赫捷,等.肺癌分子生物学特性与转移和预后的关系[J].中华肿瘤杂志,2006,28：632-634.
57.夏钰弘,杨国仪,洪建刚,等.IGFBP-3、bFGF、Ki-67蛋白在非小细胞肺癌组 织中的表达及意义[J].江苏医药,2009,35：41-43.
58.高双荣,钟雪云,向军俭.LN, FN, bFGF及EGFR与肺癌转移[J].广东医学,2002，23：909-911.
59.余荣环,张杰,向明,等.非小细胞肺癌碱性成纤维细胞生长因子表达与微血管密度测定的临床意义[J].临床肺科杂志,2006,11：288-289.
60. Iwasaki A, Kuwahara M, Yoshinaga Y, et al.Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor(VEGF) level, as prognostic indicators in NSCLC[J]. European Journal of cardio-thoracis surgery,2004,25:443-448.
61. Pardo OE, Wellbrock C, Khanzada UK, et al. FGF-2 protects small cell lung cancer cells from apoptosis through a complex involving PKCε, B-Raf and S6K2 [J]. EMBO J,2006,25:3078-3088.
62. Pardo OE, Latigo J, Jeffery RE, et al. The Fibroblast Growth Factor Receptor Inhibitor PD 173074 Blocks Small Cell Lung Cancer Growth In vitro and In vivo [J]. Cancer Research,2009,69:8645-8651
[1]毛友生,高燕宁,赫捷,等.肺癌分子生物学特性与转移和预后的关系[J].中华肿瘤杂志,2006,28(8)：632-634.
[2]Bikfalvi A, Klein S, Pintucci G, et al. Biological roles of fibroblast growth factor-2 [J]. Endocr. Rev,1997,18(1):26-45.
[3]Presta M, Dell'Era P, Mitola S,et al. Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis [J]. Cytokine Growth Factor Rev,2005,16(2):159-178.
[4]Cox G, Jones JL, Walker RA, et al. Angiogenesis and non-small cell lung cancer [J]. Lung Cancer,2000,27(2):81-100.
[5]夏钰弘,杨国仪,洪建刚,等.IGFBP-3、bFGF、Ki-67蛋白在非小细胞肺癌组织中的表达及意义[J].江苏医药,2009,35(1)：41-43.
[6]高双荣,钟雪云,向军俭.LN, FN, bFGF及EGFR与肺癌转移[J].广东医学,2002,23(9)：909-911.
[7]Joensuu H, Anttonen A, Eriksson M, et al. Soluble Syndecan-1 and Serum Basic Fibroblast Growth Factor Are New Prognostic Factors in Lung Cancer [J]. Cancer Research,2002,62(18):5210-5217.
[8]Kuhn H, Kopff C, Konrad J, et al. Influence of basic fibroblast growth factor on the proliferation of non-small cell lung cancer cell lines [J]. Lung Cancer,2004,44(22): 167-174.
[9]Pardo OE, Wellbrock C, Khanzada UK, et al. FGF-2 protects small cell lung cancer cells from apoptosis through a complex involving PKCε, B-Raf and S6K2 [J]. EMBO J,2006, 25(13):3078-3088.
[10]Pardo OE, Latigo J, Jeffery RE, et al. The Fibroblast Growth Factor Receptor Inhibitor PD173074 Blocks Small Cell Lung Cancer Growth In vitro and In vivo [J]. Cancer Research,2009,69(22):8645-8651
[11]Hori A, Sasada R, Matsutani E, et al. Suppression of solid tumor growth by immunoneutralizing monoclonal antibody against Human basic fibroblast growth factor [J]. Cancer Res,1991,51(22):6180-6184.
[12]Rofstad EK and Halsor EF. Vascular endothelial growth factor, interleukin 8, platelet-derived endothelial cell growth factor, and basic fibroblast growth factor promote angiogenesis and metastasis in Human melanoma xenografts [J]. Cancer Res,2000, 60(17):4932-4938.
[13]向军俭,靳英杰,黄红亮等.人bFGF单克隆抗体的制备及MabF7对黑色素瘤B16的体外抗瘤效应[J].中华肿瘤防治杂志,2008,15(1)：19-22
[14]刘李登,王宏,向军俭等.bFGF单抗与顺铂体外联合移植黑色素瘤B16细胞增殖效应的研究[J].中华肿瘤防治杂志,2010,17(1)：1-4
[15]Kuhn H, Konrad J, Holtz S, et al. Enhanced expression of VEGF following bFGF inhibition in non small cell lung cancer cell lines [J].Lung Cancer,2006,54(2):149-153.
[16]Giavazzi R, Senninno B, Coltrini D, et al. Distinct role of fibroblast growth factor 2 and vascular endothelial growth factor on tumor growth and angiogenesis [J].Am J Pathol, 2003,162(6):1913-1926.a
1. Dale L Ludwig, Daniel S Pereiral, Zhen ping Zhul,et al. Monoclonal antibody therapeutics and apoptosis[J]. Oncogene,2003,22:9097-9106
2. Younes, A. Kadin, M.E. Emerging applications of the tumor necrosis factor family of ligands and receptors in cancer therapy[J]. Journal of Clinical Oncology, 2003,21,3526-3534
3. Toshiaki Ohtsuka, Donald Buchsbaum, Patsy Oliver,et al. Synergistic induction of tumor cell apoptosis by death receptor antibody and chemotherapy agent through JNK/p38 and mitochondrial death pathway [J]. Oncogene,2003,22:2034-2044
4. Toshiaki Ohtsuka(?),Tong Zhou. Bisindolylmaleimide Ⅷ Enhances DR5-mediated Apoptosis through the MKK4/JNK/p38 Kinase and the Mitochondrial Pathways[J]. The Journal of Biological Chemistry,2002,277(32): 29294-29303
5. S Kaliberov, MA Stackhouse, L Kaliberova,et al. Enhanced apoptosis following treatment with TRA-8 anti-Human DR5 monoclonal antibody and overexpression of exogenous Bax in Human glioma cells[J]. Gene Therapy,2004,11:658-667
6.Georgios V. Georgakis, Yang Li, Robin Humphreys, et al. Activity of selective fully Human agonistic antibodies to the TRAIL death receptors TRAIL-R1 and TRAIL-R2 in primary and cultured lymphoma cells:induction of apoptosis and enhancement of doxorubicin-and bortezomib-induced cell death[J]. British Journal of Haematology,2005,130:501-510
7. Masa Yuki Fujino,Xiao-kang Li, Yusuke Kitazawa,et al. Distinct Pathways of Apoptosis Triggered by FTY720, Etoposide, and Anti-Fas Antibody in Human T-Lymphoma Cell Line (Jurkat Cells) [J].The Journal of Pharmacology and Experimental Therapeutics,2002,300(3),939-945.
8. Lacronique V, Mignon A, Fabre M, et al. Bcl-2 protects from lethal hepatic apoptosis induced by an anti-Fas antibody in mice[J]. Nat Med,1996,2(1):80-6.
9. Igor Tamm, Toyoko Kikuchi, Arturo Zychlinskyt. Acidic and basic fibroblast growth factors are survival factors with distinctive activity in quiescent BALB/c 3T3 murine fibroblasts[J].Cell Biology April,1991,88:3372-3376,
10. Patrik Georgii-Hemming, Helena Jernberg Wiklund, Osten Ljunggren,et al. Insulin-Like Growth Factor I Is a Growth and Survival Factor in Human Multiple Myeloma Cell Lines[J]. Blood,1996,88(6):2250-2258
11. Anne-Tove Brenne, Torstein Baade Ro, Anders Waage, et al. Interleukin-21 is a growth and survival factor for Human myeloma cells[J]. Blood,2002,99(10): 3756-3762
12. Ping Yang, Jessica L. Wiser, James J. Peairs, et al. Human RPE Expression of Cell Survival Factors [J]. Investigative Ophthalmology & Visual Science,2005, 46(5):1755-1764
13. Jacques E. No" r, Joan Christensen,David J. Mooney, et al. Vascular Endothelial Growth Factor (VEGF)-Mediated Angiogenesis Is Associated with Enhanced Endothelial Cell Survival and Induction of Bcl-2 Expression[J]. Am J Pathol,1999, 154(2):375-384
14. Lewis C. Cantley The Phosphoinositide 3-Kinase Pathway [J]. Science,2002, 296(5573):1655-1657
15. Tsuyoshi Miyazaki, Hideki Katagiri, Yumi Kanegae,et al. Reciprocal Role of ERK and NFkB Pathways in Survival and Activation of Osteoclasts[J]. The Journal of Cell Biology,2002,148(2):333-342
16.Thomas F Franke, Christoph P Hornik, Lisa Segev,et al. PI3K/Akt and apoptosis: size matters Oncogene (2003) 22,8983-8998
17 Douglas Burtrum, Zhenping Zhu, Dan Lu A. Fully Human Monoclonal Antibody to the Insulin-Like Growth Factor I Receptor Blocks Ligand-Dependent Signaling and Inhibits Human Tumor Growth in Vivo.Cancer Reseach.(2003) 63,8912-8921
18. Jing Yang, Jianfei Qian, Michele Wezeman, et al. Targeting b2-microglobulin for induction of tumor apoptosis in Human hematological malignancies. Cancer Cell (2006)10,295-307,
19 Masayasu Iwase, Sayaka Takaoka, Makiko Uchida, et al. Epidermal growth factor receptor inhibitors enhance susceptibility to Fas-mediated apoptosis in oral squamous cell carcinoma cells. Oral Oncol (2007)
20.Gregory B. Carey, Laura Tonnetti, David W.Scott, et al. The Role of the PI3K Pathway in Anti-IgM(Anti-μ)- Sensitive and-Resistant B-cell Lymphomas. 《Molecular Mechanisms of Programmed Cell Death》 37-47
21 Sclabas G.M, Fujioka S, Schmidt C,et al. Restoring Apoptosis in Pancreatic Cancer Cells by Targeting the Nuclear Factor-κB Signaling Pathway With the Anti-Epidermal Growth Factor Antibody IMC-C225 Journal of Gastrointestinal Surgery (2002)7,37-43.
22. Liang You, Biao He, Zhidong Xu, et al. An Anti-Wnt-2 Monoclonal Antibody Induces Apoptosis in Malignant Melanoma Cells and Inhibits Tumor Growth. Cancer Research(2004) 64,5385-5389.
23. Jing Yang, Xiang Zhang, Ji Wang, Anti-β2-microglobulin monoclonal antibodies induce apoptosis in myeloma cells by recruiting MHC class I to and excluding growth and survival cytokine receptors from lipid rafts. Blood. 2007(110),3028-3035.
24.Rakesh Raoa, Barbara Loganb, Kathy Forrest,et al. Lipid rafts in cytokine signaling Cytokine & Growth Factor Reviews. (2004)15 103-110.
25. Kelly Davison, Koren K. Mann, Samuel Waxman, et al. JNK activation is a mediator of arsenic trioxide-induced apoptosis in acute promyelocytic leukemia cells. Blood., (2004)103,3496-3502.
26. Maundrell, K, AntonssonB., Magnenat E, et al. Bcl-2 undergoes phosphorylation by c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the constitutively active GTP-binding protein Racl. J. Biol. Chem. (1997) 272, 25238-25242
27. Bong-Jo Kim, Seung-Wook Ryu, and Byoung-Joon Song,et al. JNK-and p38Kinase-mediated Phosphorylation of Bax Leads to Its Activation and Mitochondrial Translocation and to Apoptosis of Human Hepatoma HepG2 Cells. The Journal of Biological Chemistry (2006) 281,21256-21265
28. Dharminder Chauhan, Guilan Li, Teru Hideshima, et al. JNK-dependent Release of Mitochondrial Protein, Smac,during Apoptosis in MultipleMyeloma (MM) Cells. The Journal of Biological Chemistry. (2003) 278,17593-17596.
29. Mitchell R Smith. Rituximab (monoclonal anti-CD20 antibody):mechanisms of action and Resistance[J]. Oncogene,2003,22(47):7359-68
30.姜勇,罗深秋《信号转导》161-164
31. Wei Aixinjueluo, Keiko Furukawa, Qing Zhang. Mechanisms for the Apoptosis of Small Cell Lung Cancer Cells Induced by Anti-GD2 Monoclonal Antibodies. The Journal of Biological Chemistry[J].2005,280(33),29828-29836
32.Dylan Daniel, Becky Yang, David A. Lawrence, et al. Cooperation of the pro-apoptotic receptor agonist rhApo2L/TRAIL with the CD20 antibody rituximab against non-Hodgkin's lymphoma xenografts[J]. Blood,2007,110(12):4037-46
33. Gigi N.C. Chiul, Lincoln A. Edwardsl, Anita I. Kapanen,et al. Modulation of cancer cell survival pathways using multivalent liposomal therapeutic antibody constructs[J]. Molecular Cancer Therapeutics.2007,6(3):844-855
34. Kare'ne Mahtouk, Michel Jourdan, John De Vos,et al. An inhibitor of the EGF receptor family blocks myeloma cell growth factor activity of HB-EGF and potentiates dexamethasone or anti-IL-6 antibody-induced apoptosis[J]. Blood, 2004,103(5):1829-1837
35. Mitchell R. Smith, Fang Jin, and Indira Joshi. Enhanced efficacy of therapy with antisense BCL-2 oligonucleotides plus anti-CD20 monoclonal antibody in scid mouse/Human lymphoma xenografts[J]. Mol Cancer Ther,2004,3(12):1693-9.