CN1458841A - 应用基质金属蛋白酶抑制剂治疗癌症 - Google Patents
应用基质金属蛋白酶抑制剂治疗癌症 Download PDFInfo
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- CN1458841A CN1458841A CN01815690A CN01815690A CN1458841A CN 1458841 A CN1458841 A CN 1458841A CN 01815690 A CN01815690 A CN 01815690A CN 01815690 A CN01815690 A CN 01815690A CN 1458841 A CN1458841 A CN 1458841A
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Abstract
本发明涉及应用活性剂、特别是抑制剂通过影响、最好是抑制基质金属蛋白酶表达来治疗癌症。该活性剂的靶尤其可以涉及基质金属蛋白酶-9(MMP-9)信号转导途径的下游调节剂。按照本发明,尤其可以应用p38β和p38γ以及促分裂原活化激酶激酶6(MKK6)或促分裂原活化激酶激酶3(MKK3)的抑制剂。
Description
本发明涉及基质金属蛋白酶表达的活性剂、特别是其抑制剂的应用。更具体地讲,本发明涉及应用这类药物治疗癌症、尤其是癌症侵入。
正如所知道的,胞外基质的降解是一个非常复杂的过程并且它是许多病理和生理过程的一部分。因此,胞外基质的蛋白酶降解在癌症侵入方面起关键作用,在非肿瘤组织重建过程中也起关键作用。癌症的侵袭表型主要取决于若干蛋白酶的活性和表达。已充分确定基质金属蛋白酶在这种肿瘤细胞介导的胞外基质蛋白水解中的作用。这些基质金属蛋白酶之一是MR92,000 IV型胶原蛋白酶(MMP-9)。MMP-9降解一种主要由IV型胶原蛋白组成的结构—基底膜,基底膜通常将上皮与基质区室分隔开(1,2)。许多生长因子通过与跨膜酪氨酸受体结合、进而激活信号转导途径来诱导MMP-9和其它蛋白酶的表达(3,4)。然而,某些细胞系甚至在缺乏这类生长因子时也产生大量的蛋白酶,提示信号级联的组成型激活是一种基础机制(5)。事实上,有关肾细胞癌、白血病以及多种其它癌症的信号转导途径的组成型激活已有描述。这些信号转导途径组成型激活的参考文献参见H.Oka等和S.C.Kim等的出版物(6,7)。
本领域技术人员众所周知的是,胁迫活化蛋白激酶和促分裂原活化蛋白激酶(SAPK和MAPK)在信号途径中起关键作用。有三个主要的亚家族,包括p38/RK、JNK/SAPK和p42/p44 MAPK’s/ERK’s。一般而言,促细胞分裂原和分化因子刺激ERK’s,而JNK和p38不仅被诸如紫外线的环境胁迫、渗透胁迫激活,而且被炎性细胞因子激活。所有三个亚家族都调控细胞凋亡,ERK’s是负调节剂,而JNK’s和p38’s是正调节剂(8)。迄今为止,p38的4种人类同种型已被克隆:p38α(9)、p38β(10)、p38γ(11)(也被称为SAPK3或ERK6)和p38δ(12)(也被称为SAPK4)。所述α和β同种型主要参与介导发至细胞核的促炎信号且调节细胞凋亡(8)。已经暗示p38γ在肌肉发育和对低氧胁迫应答方面起作用(13,14)。p38α和p38β广泛分布于人体组织中,发现其表达在脑和心脏中是最丰富的(10)。SAPK3主要存在于骨骼肌中(15,16)。关于SAPK4的功能知之甚少。发现在唾液、垂体和肾上腺组织中有高水平表达(12)。p38同种型重要的上游调节剂包括蛋白激酶MKK6和MKK3。
迄今为止,关于基质金属蛋白酶参与癌症侵入和转移的发现和研究集中于各种基质金属蛋白酶结构域的功能及其与抑制剂结构域的相互作用。例如,已知参与胞外基质降解的基质金属蛋白酶的蛋白水解活性必须准确地被其内源蛋白抑制剂—金属蛋白酶的组织抑制剂(TIMP)调节。这些金属蛋白酶组织抑制剂在肿瘤细胞降解基质时也起重要的作用。分析了几种癌中例如肾细胞癌以及胃癌中TIMP和MMP的活性。有关这些研究的参考文献参见A.Kugler和G.I.Murray等的出版物(17,18)。这些金属蛋白酶组织抑制剂是一个在调节分泌型金属蛋白酶活性方面起关键作用的分泌型蛋白质家族。迄今为止,其中三个已被表征(TIMP1、TIMP2和TIMP3)。它们影响金属蛋白酶原(prometalloprotease)的激活并且用来调节胞外基质的蛋白水解,重要的是在组织重建和炎症过程中起作用。有关这些金属蛋白酶组织抑制剂的特征参见D.T.Denhardt等的出版物(19)。也存在合成基质金属蛋白酶抑制剂如marimastat(BB-2516),这是一种IC50值在微摩尔浓度范围内的丁二酰胺(butanediamid)衍生物。C.Simon等(20)证明,在用乙酸肉豆蔻佛波醇(PMA)治疗后,在人鳞状细胞癌细胞系(UM-SCC-1)中增强的MMP-9分泌和体外侵袭力可以通过应用通用p38抑制剂SB203580而被抑制,PMA是一种广泛用于研究皮肤癌发生的已知肿瘤促进剂(综述参见(21))。
现在意外地发现,p38以及MKK6和/或MKK3途径的组成型活性在癌细胞内高水平表达MMP-9方面起关键作用。由于这些意外发现,使得有可能应用靶向/针对MMP-9表达的下游调节剂的活性剂来治疗癌症、尤其是癌症侵入。
因此,本发明制备用来治疗癌症的有效活性剂的问题通过按照权利要求1和2的应用而得以解决。优选的实施方案在所附权利要求3-17中给出。所有这些权利要求书的内容通过引用结合到本发明的说明书中。
按照本发明,使用至少一种活性剂来影响、特别是抑制基质金属蛋白酶在真核细胞中的表达,从而治疗癌症。这尤其还包括应用这种活性剂生产相应的药物或相应的药用组合物。按照本发明,所述活性剂可以任选以其药学上可接受的盐形式以及任选与药学上可接受的载体一起使用。
按照本发明使用的活性剂是最好影响、特别是抑制参与癌症、最好是癌症侵入的上述基质金属蛋白酶的那些活性剂。按照本发明,一种参与癌症侵入的优选基质金属蛋白酶是基质金属蛋白酶9。
在本发明的一个优选实施方案中,所用的活性剂最好靶向基质金属蛋白酶信号转导途径的至少一个成员、特别是靶向MMP-9信号转导途径的一个成员。这种MMP-9信号转导途径的一个优选成员是所谓的p38蛋白质家族。在按照本发明应用时,这些p38蛋白之一是p38β蛋白,按照本发明应用的另一个优选成员是p38γ(SAPK3或PRK6)蛋白。按照本发明的活性剂的另一优选靶是促分裂原活化激酶激酶家族。该促分裂原活化激酶激酶家族的两个优选成员是促分裂原活化激酶激酶6(MKK6)和促分裂原活化激酶激酶3(MKK3)。在按照本发明应用时,MMP-9信号转导途径的一个成员可以单独地被所述活性剂所靶向,但也有可能是被所述活性剂所靶向的MMP-9信号转导途径的两个或三个或甚至更多个不同成员的随机组合。
在另一个优选的实施方案中,也有可能任选的是所述基质金属蛋白酶信号转导途径、最好是MM-9信号转导途径的激活剂、调节剂和/或生物学前体被所述活性剂所靶向和/或影响。这些激活剂、调节剂和/或生物学前体可以是:例如已知参与调节蛋白酶酶活性、转录因子如AP-1和负责蛋白酶表达水平的其它因子的激酶;负责激活金属蛋白酶原或组织抑制剂的蛋白酶;或者甚至可以被所述活性剂影响的迄今为止未知的化合物。
按照本发明,应用已知活性剂或者新型活性剂是可行的。在本发明的一个优选实施方案中,所述活性剂是一种针对基质金属蛋白酶信号转导途径、最好是MMP-9信号转导途径的至少一个成员具有特异性抑制能力的化合物。该活性剂最好是一种分子量(MW)低、尤其是MW<1000的相对小的分子。如果这种活性剂是咪唑衍生物,则它是更优选的。已知这类咪唑衍生物如SB203580(MW377,4)或SB202190(MW331,3)(两者均可得自Calbiochem,San Diego,Ca,USA)是有效的激酶表达抑制剂。在本发明的另一优选实施方案中,所述活性剂是p38蛋白抑制剂。这可以是p38蛋白的已知抑制剂或者也可以是又一种新型抑制剂。在本发明的另一优选实施方案中,所述活性剂是促分裂原活化激酶激酶家族的抑制剂。该抑制剂可以是所述促分裂原活化激酶激酶家族,如用本说明书中所用的标准分子生物学技术构建的激酶死亡突变体的肽类抑制剂,或者也可以是一种新型抑制剂化合物。数种抑制剂是已知的,人们可以在Y.Fukami等、J.C.Lee等和D.Fabbro等的出版物中找到其中的几种(22-24)。
在按照本发明的另一优选实施方案中,所述活性剂是所述基质金属蛋白酶信号转导途径的激活剂、调节剂和/或生物学前体的抑制剂,所述抑制剂可以是激酶抑制剂、转录因子抑制剂例如AP-1抑制剂、组织抑制剂、蛋白酶抑制剂以及基质金属蛋白酶信号转导途径的其它已知抑制剂或新型抑制剂。
在按照本发明的另一优选实施方案中,所述活性剂是编码抑制基质金属蛋白酶表达、最好是抑制p38和/或促分裂原活化激酶激酶活性的肽或多肽的多核苷酸。该肽可以是例如p38激酶缺陷型突变体、促分裂原活化激酶激酶死亡突变体和本领域技术人员已知的其它肽。
本发明可以用来治疗所有种类的癌症、尤其是具有基质金属蛋白酶过量表达并且因此具有高侵袭力和转移力的癌症。据报道MMP-9在头、颈、皮肤和胃的鳞状上皮癌中以及在胃纤维肉瘤中过量表达。也发现在结肠癌、乳癌和肝细胞癌患者血清中MMP-9水平增加。因此,在可治疗的疾病中,具体参考上述癌症。众所周知转移性疾病(而且通常也是侵袭性肿瘤生长本身)限制癌症患者的寿命。癌症中信号转导途径组成型激活的原因迄今为止尚未了解。它可能是由于生长因子受体基因突变诸如基因扩增或自分泌环即相同组织中配体和受体的表达所致。上述癌症是按照本发明活性剂的良好靶,因为癌症侵入是这些癌症中一个争论不休的问题。
按照本发明,有可能选择所述活性剂的给药形式。使这种形式可适用于患者的年龄、性别或其它特征、癌症的严重程度和其它参数。可以存在常规药用载体、稀释剂或常规添加剂。
剂量可以随临床情况和患者病症而随意选择。
最后,本发明包括用于治疗癌症的药用组合物或药物,所述药用组合物或药物含有至少一种影响、特别是抑制基质金属蛋白酶在真核细胞中表达的活性剂。有关这种组合物或药物各种特征的参考文献参见上述相应的说明书正文。
本发明的所述特征和其它特征可以根据以下优选实施方案的说明以及权利要求书加以总结。个别特征可以分别实现或以亚组合形式实现。材料和方法
载体:按照其它文献(31-33)所述,通过丝氨酸207和苏氨酸211被谷氨酸取代,产生MKK-6的组成型活性突变体;通过丝氨酸207和苏氨酸211被丙氨酸取代,产生显性失活MKK-6表型(34);以及通过在p38激酶的典型TGY序列中苏氨酸被丙氨酸取代以及酪氨酸被苯丙氨酸取代,产生激酶缺陷型p38突变体;按照其它文献(11,35,36)所述,将所有产生的c-DNA亚克隆到哺乳动物表达载体pcDNA3中。由MMP-9启动子的5’缺失型片段或突变型启动子(-79 AG-1 mt)驱动的CAT报道构建体在其它文献中已有描述(37)。TAM-67构建体编码缺失氨基酸3-122的突变型c-jun蛋白(38)。*5AP-1 pBLCAT构建体由前接5个AP-1重复序列的最小胸苷激酶启动子CAT报道构建体组成(39)。
组织培养和材料。UM-SCC-1细胞(技术人员已知的,可得自Thomas Carey博士,University of Michigan,Ann Arbor,MI)、Hlac82(技术人员已知的,可得自Hans Peter Zenner博士,University of Tübingen,德国)和NIH 3T3细胞(几乎由每个细胞生物学实验室保持,也可得自Hans Peter Zenner博士,参见上文),在补充10%胎牛血清(FBS,GibcoLife Technologies,Karlsruhe,德国)的McCoy’s 5A培养基中保持。为了收集条件培养基供酶谱分析(zymography)和蛋白质印迹法用,将80%汇合的UM-SCC-1、H1a82和NIH 3T3细胞分别在无血清培养基(McCoy’s 5A培养基,其组分对于技术人员是已知的,可得自Gibco LifeTechnologies,Karlsruhe,德国)中孵育48小时,而同时指示加入或不加入SB203580(Calbiochem,San Diego,CA)或载体(DMSO)时。下文“无血清培养基”也被缩写为“SFM”。收集所述培养基,在将细胞在0.2mg/ml MTT-活体染料中孵育并且通过分光光度法于570nm读出等份的DMSO溶解的甲晶体后,测定增殖。生长曲线的产生如(25)所述,在含血清和无血清条件下,采用在允许细胞附着12小时(第0天)后和此后至多4天(第1-4天)的同时加入各种量的SB203580。
酶谱分析。完全如(20,25)所述,采用含有0.1%(wt/vol)明胶的SDS-PAGE凝胶测定MMP-9,进行酶谱分析。依赖MMP的蛋白水解检测为暗视野中的白色区。
蛋白质印迹法。为了检测条件培养基中的MMP-9,使来自相等细胞数的培养基在无还原剂的情况下变性,蛋白质通过SDS-PAGE分离,然后转移至硝化纤维素滤膜。所述滤膜用3%BSA封闭,与抗基质金属蛋白酶的小鼠单克隆抗体(#IM37L Oncogene Research Products,Calbiochem,Cambridge,MA)一起保温。接下来,将印迹与辣根过氧化物酶缀合的抗兔IgG一起保温,通过市售免疫印迹检测系统ECL(增强化学发光)如生产商(Amersham Life Science,Arlington Heights,IL)所述来显现免疫反应性条带。采用同时识别磷酸-p38和去磷酸-p38的单克隆抗体(sc-535-G和sc-6023,Santa Cruz,Santa Cruz,CA),检测p38α和SAPK3蛋白。简而言之,细胞在含有PMSF(100mg/ml)和原钒酸钠(1mM)的RIPA缓冲液中提取。用SDS-PAGE分离在变性条件提取的蛋白质。滤膜用3%BSA封闭,随后与第一抗体一起保温过夜。为了显现免疫反应性条带,再次使用ECL系统。
供p38α活性和SAPK3活性测定用的凝胶内(in-gel)激酶测定。如(20)所述进行p38α活性的激酶测定。简而言之,细胞用缓冲液A[1%NP40(辛基苯氧基聚乙氧基乙醇)、25mM Tris-HCl(pH7.4)、25mMNaCl、1mM钒酸钠、10mM NaF、10nM焦磷酸钠、10nM冈田酸、0.5mM EGTA和1mM苯甲基磺酰氯]提取。将提取的蛋白质与2μg与人和小鼠p38α有免疫反应性的抗p38α抗体(sc-535-G,Santa Cruz,Santa Cruz,CA)和A蛋白琼脂糖微珠(2mg)一起保温以进行免疫沉淀。微珠用缓冲液A洗涤,重悬于2X样品缓冲液中,免疫复合物在含有髓鞘碱性蛋白的聚丙烯酰胺凝胶中电泳。凝胶顺序用含20%2-丙醇、5mM 2-疏基乙醇、6M盐酸胍和0.04%Tween 40-5mM 2-疏基乙醇的缓冲液处理。然后将凝胶与10μM ATP和25μCi[32P]ATP在含有2mM二硫苏糖醇-0.1mM EGTA-5mM MgCl2的缓冲液中于25℃保温1小时,在含有5%三氯乙酸和1%焦磷酸钠的溶液中洗涤,干燥,然后放射自显影。对于SAPK3活性,细胞在缓冲液A中提取,将提取的蛋白质与2μg与人和小鼠SAPK3有免疫反应性的抗SAPK3抗体(06-603,Upstate Biotechnology,Lake Placid,NY,USA)和G蛋白琼脂糖微珠一起孵育。微珠在缓冲液A和激酶缓冲液(50mM HEPES、0.1mMEDTA、0.0001%Brij35、0.0001%β-疏基乙醇、150mM NaCl、O1mg/ml牛血清白蛋白)中洗涤,然后与作为底物的1μg ATF2(sc-4007,SantaCruz,Santa Cruz,CA,USA)在40μl反应缓冲液(激酶缓冲液,0.3mMATP,0.4M MgCl2)中于30℃进行激酶反应30分钟。通过加入2X还原样品缓冲液且加热至100℃达5分钟而终止反应。通过离心除去微珠。如上所述,用抗磷酸-ATF2-抗体对上清液进行免疫印迹分析。采用ECL系统显现免疫反应性条带。
体外侵入测定。如(20,25)所述,采用用1/3稀释的Matrigel/SFM(Becton Dickinson,Bedford,MA)包被的8μm孔径滤膜进行侵入测定。将细胞以相似浓度平板接种到含有SB203580或DMSO的SFM、SB203580载体中。对于使用小鼠单克隆MMP-9抗体(#IM09L,OncogeneResearch Products,Cambridge,MA)(0.5μg/ml、1μg/ml和10μg/ml)的实验,将细胞平板接种到SFM加上SFM的抗体加上相似量的免疫前血清中。侵入量基于滤膜下表面上的MTT活性来测定,以室中总活性的百分比表示。
瞬时转染与后续CAT-ELISA。采用供瞬时转染用的Lipofectamin(GIBCO,Life Technologies,Karlsruhe,德国)如生产商所述进行瞬时转染。将UM-SCC-1和NIH3T3细胞在70%汇合时用下述构建体或突变体一起进行共转染:含有670bp包括转录起始位点的MMP9野生型启动子的CAT报道构建体或无启动子的CAT构建体(SV0)(3μg)以及如(26)所述的pCDNA3-MKK-6或pCDNA3-MKK-3组成型活性突变体(0.03-3μg)或显性失活p38α、p38β、SAPK3、SAPK4或MKK-6突变体与1倍或2倍摩尔过量的所述启动子构建体(由J.Han博士,ScrippsResearch Institute,La Jolla,CA友好提供)。采用模拟对照载体(pCDNA3),使每个样品中经转染的DNA量相等。按照生产商的说明(Roche Diagnostics,Mannheim,德国)进行CAT ELISA,从而测量CAT蛋白表达。
附图说明
·图1:SB203580对UM-SCC-1细胞的MMP-9表达(A)、体外侵入(B)和生长(C)的影响。
·图2:在不同的细胞系中体外侵入对MMP-9分泌的需求(A)、MMP-9在不同细胞系中的表达(B)和在与抗MMP-9抗体一起孵育后的体外侵入百分率(C)。
·图3:在用显性失活p38同种型蛋白处理后对MMP-9启动子活性的影响(A)以及p38α和p38γ在两种不同细胞系中的表达(B-E)。
·图4:MMP-9在两种不同细胞系中的表达(A),在用激酶缺陷型MKK6(B)和组成型活性MKK6和MKK3(C)突变体处理后对MMP-9启动子活性的影响。
·图5:MMP-9启动子活性被MKK-6诱导(A)以及AP-1基序中的点突变对MKK-6依赖性启动子激活的影响(B)。
·图6:组成型活化MKK-6对CAT-报道分子的影响(A)以及在用不同载体处理后的MKK-6依赖性MMP-9启动子激活(B)。
实验1
UM-SCC-1细胞平板接种于补充10%胎牛血清(FBS,Gibco LifeTechnologies,Karlsruhe,德国)的McCoy’s 5A培养基中,次日再补充含SB 203580(10μM,Calbiochem,San Diego,CA)或载体(二甲亚砜DMSO,0.01%)的无血清培养基。48小时后,收获条件培养基,用溴化3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑鎓(MTT)测定增殖率。根据增殖差异标准化的等份条件培养基采用单克隆抗MMP-9抗体进行免疫印迹分析。接下来,将印迹与辣根过氧化物酶缀合的抗兔IgG一起孵育,如生产商(Amersham,Arlington Heights,IL)所述,通过ECL显现免疫反应性条带。按照光密度测定法观察到蛋白质表达降低70%。数据代表三个重复实验。
实验1结果示于图1中。
图1A显示组成型产生大量MMP-9且表现出体外和体内侵袭表型的鳞状细胞癌细胞系UM-SCC-1受咪唑衍生物SB203580处理影响。如免疫印迹分析证明,SB203580降低MMP-9蛋白表达,于浓度10μM时降低约70%。
在图1B中,将UM-SCC-1细胞平板接种于预先用Matrigel包被的在无血清培养基中的滤膜上,然后与不同量的SB203580一起孵育60小时,以测定体外侵入。将载体(DMSO)浓度保持在0.1%。侵入以侵入通过Matrigel的细胞百分率表示。用不同浓度的SB 203580处理后的侵入以平均百分数+/-S.E.表示,代表每组3个平皿。数据代表三个重复实验。图1B显示,采用浓度5μM和10μM,体外侵入有分别为43+/-9%和69+/-8%的剂量依赖性降低。
图1C显示将UM-SCC-1细胞暴露于5-和10-所述p38抑制剂至多5天,不影响细胞生长,从而排除了所述化合物有引起对体外侵入的抑制效应的抗有丝分裂效应。各种p38同种型在其对SB203580的敏感性方面有所不同。对于p38α报道的IC50为0.1μM,而对于p38β报道的IC50为5-10μM。p38γ和p38δ不被所述咪唑衍生物抑制(27)。因此,降低MMP-9表达和UM-SCC-1细胞的体外侵入所需的SB 203580浓度与p38β的IC50接近一致。在该实验中,让所述细胞在含有10%FBS、加入各种量的SB 203580的培养基中生长5天。在指定天数用0.2mg/ml MTT-活体染料并且通过分光光度法于570nm连续读出DMSO溶解的甲晶体来测定活细胞数。数据点代表三个重复实验。
因此,实验1和相关的图1表明,p38 SAPK抑制剂SB 203580降低MMP-9的高水平表达和UM-SCC-1细胞的体外侵入,但对细胞生长无任何影响。实验2
在该实验中,将不同的细胞系平板接种于预先用Matrigel包被的在无血清培养基中的滤膜上并进行孵育。侵入以平均百分数+/-S.E.表示,代表每组3个平皿。数据代表三个重复实验。为了解决UM-SCC-1细胞的体外侵入是否需要MMP-9的问题,首先使三个不同细胞系(UM-SCC-1、NIH3T3、Hlac82)的表达水平与其体外侵入行为相关联。图2A显示条件培养基中的MMP-9量几乎与所述细胞系的侵袭力平行。UM-SCC-1细胞产生最大量蛋白酶且表现出最大侵袭表型,而不分泌任何可检测MMP-9的NIH3T3细胞在Matrigel包被的滤膜上的侵袭力要小得多。有趣的是,在MMP-2分泌和被测细胞系侵袭力之间并无相关性。这可能是由于MMP-2的活化需要不同膜型基质金属蛋白酶(MT-MM)所致(21),该蛋白在NIH3T3细胞上可能并被表达。
按照图2B,给不同细胞系更换为无血清培养基,然后培养48小时。收获条件培养基,采用MTT测定细胞数。如(20,25)所述和本领域技术人员已知的酶谱分析,测定根据细胞数差异校正的等份条件培养基的MMP-9活性。数据代表三个重复实验。只有表达最高量MMP-9的细胞系在酶谱分析中才显示出清晰的条带。
为了最终证实UM-SCC-1细胞的体外侵袭表型需要MMP-9活性,在图2C中,将UM-SCC-1细胞平板接种于预先用Matrigel包被的在无血清培养基中的滤膜上,然后与各种量的抗MMP-9抗体(0.5μg/ml、1μg/ml、10μg/ml)或等量的所述生产商提供的免疫前血清一起孵育60小时。侵入以细胞侵入通过Matrigel的百分率表示。在用不同浓度的识别活性形式和潜伏形式的MMP-9的抗体或由所述生产商提供的免疫前血清处理后,侵入以平均百分数+/-S.E.表示,代表每组3个平皿。数据代表三个重复实验。图2C显示体外侵入随抗体浓度增加的剂量依赖性降低。
因此,实验2和相关的图2表明,所述细胞系的体外侵入需要MMP-9分泌到UM-SCC-1细胞的条件培养基中。因此,可以得出的结论是,SB203580通过降低通过p38信号途径的MMP-9表达,抑制UM-SCC-1细胞的体外侵入。实验3
由于MMP-9的表达几乎只在启动子水平上受调控(28,29),并且为了进一步表征不同p38同种型在调节MMP-9表达中的作用,所以采用Lipofectamin(Gibco Life Technologies,Karlsruhe,德国)如生产商所述以及用由野生型MMP-9启动子驱动的氯霉素乙酰转移酶(CAT)报道分子或无启动子的CAT构建体(SV0)和指定量的(其中2是效应子质粒相对于所述报道构建体2倍摩尔过量)包括显性失活p38α、p38β、p38γ和p38δ的表达载体或空载体(pCDNA3),瞬时转染UM-SCC-1细胞。转染DNA量的差异用空载体归一化。采用CAT-ELISA按照生产商所述方法(Roche Diagnostics,Mannheim,德国),测定根据蛋白质量差异标准化的细胞提取物的CAT表达。数据以对照平均百分数+/-S.E.表示,代表每组2个平皿,以3个独立的实验进行。用本领域技术人员已知的标准分子生物学技术,在p38激酶的典型TGY序列中通过苏氨酸被丙氨酸取代和酪氨酸被苯丙氨酸取代,产生激酶缺陷型突变体,然后将所有产生的cDNA克隆到哺乳动物表达载体pcDNA3中。
按照图3A,发现SB203580敏感性同种型突变体p38β阻抑MMP-9启动子驱动的CAT报道分子的活性,在2倍摩尔过量时阻抑62+/-20%,相比之下,p38α突变体仅降低MMP-9启动子活性21+/-20%。p38δ突变体抑制MMP-9启动子55+/-9%。用p38γ突变体转染,实际上使MMP-9启动子沉默,即启动子活性被阻抑99.9+/-0.5%。在无启动子的CAT构建体转染后,没有观察到显著的CAT表达。
图3A显示,除p38β和p38δ之外,是p38γ而非p38α显性失活表达构建体降低MMP-9启动子活性。该实验进一步支持是p38β而不是p38α参与MMP-9启动子组成型激活,另外,它们有力地表明p38δ和最重要的是p38γ在MMP-9启动子组成型激活中的作用。
在图3B-E中,将UM-SCC-1细胞和NIH3T3细胞保持在含有10%FBS的培养基中。蛋白提取物(相等蛋白)和凝胶内激酶测定如上所述制备,然后或者采用多克隆抗p38α抗体或多克隆抗SAPK3抗体进行免疫印迹(B)和(D),或者采用MBP为底物进行凝胶内激酶测定(C),或者采用重组ATF2为底物进行免疫激酶反应(E)。数据代表三个重复实验。根据这些实验,可以排除的是,在p38α被激酶缺陷型突变体抑制后观察到的MMP-9启动子活性适度降低也可能是由于缺乏所述激酶的表达和/或活性所致。为此,测定UM-SCC-1细胞和NIH3T3细胞(阴性对照)中p38α的活性和表达。也通过用多克隆SAPK3抗体免疫沉淀,和随后在相似的实验中的激酶反应,分析p38γ的活性和表达。
可以根据图3B-E来推断,注意到在两种细胞系中均存在p38α和p38γ两种蛋白质(图3B和3C)。然而,发现两种p38同种型的酶活性在UM-SCC-1细胞中高,与NIH3T3细胞中不可检测相反。因此,在UM-SCC-1细胞中存在p38α和p38γ且是组成型活性的。实验4
在此将UM-SCC-1细胞和NIH3T3细胞更换为无血清培养基,然后培养48小时。收获条件培养基,采用MTT测定细胞数。通过酶谱分析测定根据细胞数差异校正的等份条件培养中的MMP-9活性。数据代表三个重复实验。
图4A显示,是UM-SCC-1细胞而非NIH3T3细胞,表达基质金属蛋白酶9(MMP-9)。
由于MKK6蛋白激酶广泛激活p38同种型(与MKK3相反,它作为p38α和SAPK4/p38δ同种型的相当特异性的激活剂起作用)(26,30),因此为了测定MKK6在调节MMP-9中的作用,在图4B中,采用由野生型MMP-9启动子驱动的CAT报道分子或无启动子的CAT构建体(SV0)和指定量的(其中2是效应子质粒相对于所述报道构建体2倍摩尔过量)编码激酶死亡MKK6突变体的表达载体,瞬时转染UM-SCC-1细胞。按照上述出版物(26),通过用丙氨酸取代丝氨酸151和苏氨酸155产生所述激酶死亡表型。观察到强降低MMP-9启动子活性99+/-0.5%。这些数据说明,MKK6激酶缺陷型突变体阻抑UM-SCC-1细胞中的MMP-9启动子活性,并且MKK6是MMP-9的上游调节物,MMP-9可能通过p38同种型发送信号。
为了进一步表征MKK6在调节MMP-9启动子中的作用,检查MKK6激酶组成型激活的效应。在图4C中,采用由野生型MMP-9启动子驱动的CAT报道分子或无启动子的CAT构建体(SV0)和指定量的(其中2是效应子质粒相对于所述报道构建体2倍摩尔过量)编码组成型活化MKK6和MKK3蛋白的表达载体或所述空载体,瞬时转染NIH3T3细胞。转染DNA量的差异用空载体归一化。采用CAT-ELISA,测定根据蛋白质量差异标准化的细胞提取物的CAT表达。数据以对照平均百分数+/-S.E.表示,代表每组2个平皿,以3个独立的实验进行。按照J.Hahn等的出版物(26),丝氨酸151和苏氨酸155被谷氨酸取代,实现MKK6的组成型活化。为了避免内源刺激物干扰所述启动子的激活,利用不表达内源MMP-9的NIH3T3细胞(图4A)。在用CAT报道分子和1倍摩尔过量的MKK6突变体共转染后,观察到5倍的MMP-9启动子激活。用相似构建的MKK3突变体重复相同实验。以相似的摩尔过量,仅观察到2.8倍启动子诱导。
图4C说明,MKK6对所有p38同种型(包括p38γ)多少有些非特异性活化,而MKK3更窄地靶向p38α和p38δ。这些结果支持p38γ在调节MMP-9中的作用。实验5
在该实验中,NIH3T3细胞用由野生型MMP-9启动子5’侧翼区驱动的CAT报道分子(3μg)和编码组成型活化MKK-6蛋白(MKK6(Glu))的表达载体(0.4μg)以0.1∶1的效应子质粒相对于所述670 bp-CAT报道分子的摩尔比来瞬时转染。采用CAT-ELISA,测定根据蛋白质量差异标准化的细胞提取物的CAT表达。数据以相对于对照(MMP-9 670bp-CAT构建体)的平均诱导倍数±SE表示,代表3个独立的实验(图5A)。在图5B中,NIH3T3细胞用由野生型MMP-9启动子驱动的或含有在AP-1基序列中位于-79点突变的MMP-9启动子驱动的CAT报道分子(3μg)和组成型活化MKK-6构建体(MKK-6(Glu))(0.4μg)以0.1∶1的效应子质粒相对于CAT构建体的摩尔比瞬时转染。转染DNA量的差异用空载体归一化。采用CAT-ELISA,测定根据蛋白质量差异标准化的细胞提取物的CAT表达。显示相对于对照(MMP-9 670bp-CAT构建体)的CAT表达平均诱导倍数±SE,数据代表3个独立的实验。
为了测定被特异性p38信号转导途径激活剂MKK-6刺激所需的启动子区,将NIH3T3细胞用由92-kDa胶原蛋白酶野生型启动子5’缺失型片段驱动的CAT报道分子共转染。包括MMP-9启动子的144-bp片段在内的所有被测构建体被MKK-6类似激活(在MKK-6构建体相对于MMP-9启动子构建体的摩尔比为0.1时,为3.5倍)。相反,如果使用由90bp或73bp的5’侧翼序列驱动的CAT报道构建体,即使有刺激,也是很少的,提示在-144和转录起始位点之间区域中的某些转录因子结合位点是MKK-6依赖性MMP-9启动子激活所需的(图5A)。搜索该部分的序列表明,在-79存在一个AP-1(活化转录激活因子1)结合位点(37)。AP-1是由Fos(c-Fos、FosB、Fra1、Fra2)和Jun(c-Jun、JunD、JunB)家族成员组成的蛋白二聚体。所产生的复合物与称为AP-1位点或十四烷酰佛波醇(TPA)效应元件(TRE)的特定DNA序列结合。该术语是指TPA由于增加蛋白表达和磷酸化而有效刺激AP-1的DNA结合(40)。MMP-9启动子含有这类TRE元件。发现一个AP-1位点位于-79,第二个位于离开主要转录起始位点的-540(37,41)。然后测定该TRE元件在MMP-9启动子被MKK-6激活中的作用。在AP-1位点(-79)中引入点突变(TGAGTCA→TTTGTCA)(37)完全消除了MMK-6对全长野生型MMP-9启动子的诱导性。这表明,MKK-6依赖性反式激活需要该MMP-9启动子区(图5B),所述区包含在MMP-9启动子的近端144bp 5’侧翼区内。实验6:
在该实验中,用编码组成型活化MKK-6突变蛋白(MKK6(Glu))的构建体(1μg)、由含最小胸苷激酶启动子和5个AP-1基序重复序列(5*AP-1)构成的启动子驱动的CAT报道分子(1μg)和编码激酶缺陷型p38蛋白同种型突变体(p38α、p38β、p38γ、p38δ)的载体(2μg),瞬时转染NIH3T3细胞。为了检验5*AP-1构建体中最小胸苷激酶启动子的效应,使用相似量的缺乏AP-1重复序列的质粒(1μg,pBLCAT)。转染DNA量的差异用空载体(pcDNA3)归一化。采用CAT-ELISA,测定根据蛋白质量差异标准化的细胞提取物的CAT表达。数据以相对于对照(5*AP-1CAT报道分子)的CAT表达诱导倍数表示,数据代表2个独立的实验(图6A)。在图6B中,用编码组成型活化MKK-6突变蛋白(MKK6(Glu))的构建体(4μg)、由670bp野生型MMP-9启动子驱动的CAT报道分子(3μg)和编码缺乏反式激活结构域的c-jun蛋白的载体(TAM67)(2μg),瞬时转染NIH3T3细胞。转染DNA量的差异用空载体(分别为pcDNA3、CMV5)归一化。采用CAT-ELISA,测定根据蛋白质量差异标准化的细胞提取物的CAT表达。数据以相对于对照(MMP-9野生型启动子CAT构建体)的CAT表达诱导倍数表示,实验代表2个独立的实验。
MKK-6依赖性诱导需要在MMP-9野生型启动子的近端区中AP-1的存在和完整性,这表明MKK-6是AP-1依赖性转录的激活剂。因此,将组成型活性MKK-6构建体与由后接最小胸苷激酶启动子的5次重复的AP-1共有位点驱动的CAT报道分子一起共转染到NIH 3T3细胞中。发现MKK-6强激活5*AP-1CAT报道构建体。该激活或者通过从所述启动子中去除AP-1重复序列,或者通过任一种p38同种型显性失活突变体的共转染而被消除。因此,MKK-6确实可以通过需要p38激酶活性的途径来激活AP-1依赖性转录(图6A)。
缺乏其反式激活结构域的c-jun(TAM67)的表达,消除MKK-6依赖性MMP-9启动子的激活。这表明MKK-6依赖性MMP-9启动子的反式激活需要近端TRE元件(-79)和MKK-6激活通过若干个p38激酶同种型的AP-1依赖性转录的能力。因此,显然MKK-6通过活化转录因子AP-1诱导MMP-9野生型启动子活性。为了证明这一点,将由此作为显性失活AP-1突变体的缺乏其反式激活结构域(42)的c-jun蛋白与活化MKK-6突变体一起共转染到NH 3T3细胞中。瞬时表达的蛋白与fos结合且产生反式激活缺陷型AP-1复合体,与完整AP-1竞争与MMP-9启动子中的所述TRE元件结合。该突变蛋白的表达引起几乎完全抑制MKK-6依赖性MMP-9启动子的激活,而与相对于全长MMP-9启动子CAT报道分子量的摩尔比已经为0.5∶1时的对照(空载体)相反,证明所推定的MKK-6依赖性MMP-9启动子的反式激活需要AP-1复合体(图6B)。
实验5和实验6的结果表明,MKK-6可以诱导AP-1依赖性转录活性,而这是MMP-9启动子的诱导所需的,并且MKK-6介导的激活需要MMP-9启动子中的近端AP-1位点。
所有实验结果清楚地表明,按照本发明,通过给予影响、特别是抑制MMP-9信号转导途径下游调节剂的活性剂,将正面影响癌症、最好是癌症转移的侵袭力。
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Claims (17)
1.一种用于影响、特别是抑制基质金属蛋白酶在真核细胞中表达的活性剂在制备治疗癌症的药物或药用组合物方面的应用。
2.治疗癌症的方法,其特征在于真核细胞用影响、特别是抑制基质金属蛋白酶表达的活性剂来治疗。
3.权利要求1或2的应用或方法,其特征在于所述基质金属蛋白酶是基质金属蛋白酶-9(MMP-9)。
4.任一前述权利要求的应用或方法,其特征在于所述活性剂靶向所述基质金属蛋白酶信号转导途径的至少一个成员。
5.权利要求4的应用或方法,其特征在于所述成员是p38蛋白质家族的一个成员。
6.权利要求5的应用或方法,其特征在于所述p38蛋白是p38β蛋白。
7.权利要求5的应用或方法,其特征在于所述p38蛋白是p38γ(SAPK3或ERK6)蛋白。
8.权利要求4的应用或方法,其特征在于所述成员是促分裂原活化激酶激酶家族的一个成员。
9.权利要求8的应用或方法,其特征在于所述促分裂原活化激酶激酶是促分裂原活化激酶激酶6(MKK6)或促分裂原活化激酶激酶3(MKK3)。
10.任一前述权利要求的应用或方法,其特征在于所述活性剂靶向所述基质金属蛋白酶信号转导途径的激活剂、调节剂和/或生物学前体。
11.任一前述权利要求的应用或方法,其特征在于所述活性剂是一种小分子化合物,最好是一种分子量(MW)<1000的小分子化合物。
12.权利要求11的应用或方法,其特征在于所述小分子化合物是一种咪唑衍生物,其中所述咪唑衍生物最好是SB203580或SB202190。
13.任一前述权利要求的应用或方法,其特征在于所述活性剂是一种编码影响、最好是抑制基质金属蛋白酶表达的肽、最好是多肽的多核苷酸。
14.任一前述权利要求的应用或方法,其特征在于所述癌症具有侵袭表型。
15.任一前述权利要求的应用或方法,其特征在于所述癌症是:
a)鳞状上皮癌,最好是头、颈、皮肤或胃的鳞状上皮癌,或者
b)结肠癌、乳癌或肝细胞癌,或者
c)胃纤维肉瘤。
16.药用组合物,所述药用组合物包含适合量的至少一种活性剂并且任选还包含一种药学上可接受的载体,其中所述活性剂影响、最好是抑制基质金属蛋白酶在真核细胞中的表达。
17.权利要求16的药用组合物,其进一步的特征为权利要求4-13中任一项限定的活性剂。
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EP00114909A EP1174129A1 (en) | 2000-07-17 | 2000-07-17 | Use of a matrix-metalloprotease inhibitor for the treatment of cancer |
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EP (2) | EP1174129A1 (zh) |
JP (1) | JP2004503583A (zh) |
CN (1) | CN1458841A (zh) |
AU (1) | AU2001287632A1 (zh) |
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Cited By (2)
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CN109528720A (zh) * | 2019-01-08 | 2019-03-29 | 浙江大学 | Sb203580在制备抗肿瘤药物中的应用及抗肿瘤药物 |
CN111742369A (zh) * | 2017-12-20 | 2020-10-02 | 皇家飞利浦有限公司 | 使用靶基因表达的数学建模评估mapk-ap-1细胞信号传导途径活性 |
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GB0224013D0 (en) * | 2002-10-15 | 2002-11-27 | Oxford Glycosciences Uk Ltd | A protein involved in therapy |
CA2502685A1 (en) * | 2002-10-23 | 2004-05-06 | Exelixis, Inc. | Cdkl1 as modifier of branching morphogenesis and methods of use |
WO2006085828A1 (en) * | 2005-02-11 | 2006-08-17 | Agency For Science, Technology And Research | Methods for the detection of hepatocellular carcinoma |
CA2754635C (en) * | 2009-03-06 | 2018-06-19 | Universite Paris Descartes | Use of gingival fibroblast conditioned media to treat metastatic cancer |
WO2014100779A1 (en) | 2012-12-21 | 2014-06-26 | Advanced Cell Technology, Inc. | Methods ofr production of platelets from pluripotent stem cells and compositions thereof |
US20170038382A1 (en) * | 2014-01-24 | 2017-02-09 | Ntercept, Llc | Methods and compositions for immune dis-inhibition |
EA035898B1 (ru) | 2014-10-03 | 2020-08-28 | НАНОТИКС, ЭлЭлСи | Композиции и способы для ингибирования биологической активности растворимых биомолекул |
KR20180034619A (ko) | 2015-07-29 | 2018-04-04 | 나노틱스 엘엘씨 | 가용성 생체분자를 소거하기 위한 모듈 조성물 및 이와 관련된 방법 |
WO2018129207A1 (en) | 2017-01-04 | 2018-07-12 | Nanotics, Llc | Methods for assembling scavenging particles |
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JP2877509B2 (ja) * | 1989-05-19 | 1999-03-31 | アムジエン・インコーポレーテツド | メタロプロテイナーゼ阻害剤 |
US5892112A (en) * | 1990-11-21 | 1999-04-06 | Glycomed Incorporated | Process for preparing synthetic matrix metalloprotease inhibitors |
WO1998015618A1 (en) * | 1996-10-09 | 1998-04-16 | Medical Research Council | Map kinases: polypeptides, polynucleotides and uses thereof |
EP1023313A1 (en) * | 1997-05-28 | 2000-08-02 | Daniel A. Mercola | Inhibition of stress activated protein kinase (sapk) pathway and sensitization of cells to cancer therapies |
TR200002224T2 (tr) * | 1998-02-04 | 2000-12-21 | Novartis Ag | Matris-bozucu metalloproteinasları inhibe eden sülfonilamino türevleri. |
FI980604A0 (fi) * | 1998-03-18 | 1998-03-18 | Univ Helsinki Licensing | Nya matrismetalloproteinasinhibitorer och -regulatorer |
US6277061B1 (en) * | 1998-03-31 | 2001-08-21 | The Research Foundation Of State University Of New York | Method of inhibiting membrane-type matrix metalloproteinase |
GB9809869D0 (en) * | 1998-05-09 | 1998-07-08 | Medical Res Council | Inhibition of protein kinases |
RU2001106631A (ru) * | 1998-09-11 | 2004-03-20 | Адзиномото Ко., Инк. (Jp) | Производные бензола и их фармацевтическое применение |
GB9902696D0 (en) * | 1999-02-09 | 1999-03-31 | Medical Res Council | Screening methods |
AU3003701A (en) * | 1999-11-19 | 2001-05-30 | Axxima Pharmaceuticals Ag | Inhibitors of helicobacter pylori induced gastrointestinal diseases |
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- 2001-07-17 JP JP2002511725A patent/JP2004503583A/ja active Pending
- 2001-07-17 EP EP01967193A patent/EP1301182A2/en not_active Withdrawn
- 2001-07-17 WO PCT/EP2001/008234 patent/WO2002005792A2/en not_active Application Discontinuation
- 2001-07-17 CA CA002418146A patent/CA2418146A1/en not_active Abandoned
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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CN111742369A (zh) * | 2017-12-20 | 2020-10-02 | 皇家飞利浦有限公司 | 使用靶基因表达的数学建模评估mapk-ap-1细胞信号传导途径活性 |
CN109528720A (zh) * | 2019-01-08 | 2019-03-29 | 浙江大学 | Sb203580在制备抗肿瘤药物中的应用及抗肿瘤药物 |
CN110624108A (zh) * | 2019-01-08 | 2019-12-31 | 浙江大学 | Fas或其配体FasL作为靶点在制备抗肿瘤药物中的应用 |
CN109528720B (zh) * | 2019-01-08 | 2021-03-30 | 浙江大学 | Sb203580在制备抗肿瘤药物中的应用及抗肿瘤药物 |
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WO2002005792A2 (en) | 2002-01-24 |
AU2001287632A1 (en) | 2002-01-30 |
WO2002005792A9 (en) | 2002-09-19 |
WO2002005792A3 (en) | 2002-05-30 |
EP1301182A2 (en) | 2003-04-16 |
JP2004503583A (ja) | 2004-02-05 |
EP1174129A1 (en) | 2002-01-23 |
RU2003104511A (ru) | 2004-08-20 |
CA2418146A1 (en) | 2002-01-24 |
US20040067883A1 (en) | 2004-04-08 |
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