CN117045802A - 蛋白磷酸酶pp2a抑制剂与吉西他滨联合在制备抗肿瘤药物中的应用 - Google Patents
蛋白磷酸酶pp2a抑制剂与吉西他滨联合在制备抗肿瘤药物中的应用 Download PDFInfo
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- CN117045802A CN117045802A CN202310899784.5A CN202310899784A CN117045802A CN 117045802 A CN117045802 A CN 117045802A CN 202310899784 A CN202310899784 A CN 202310899784A CN 117045802 A CN117045802 A CN 117045802A
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Abstract
本发明涉及生物医学领域，具体是蛋白磷酸酶PP2A抑制剂与吉西他滨联合在制备抗肿瘤药物中的应用。吉西他滨是一种脱氧胞苷类似的前体药物，需要被脱氧胞苷激酶DCK磷酸化激活；而PP2A可以在Ser74位点去磷酸化DCK，降低DCK的催化活性从而抑制吉西他滨的药效；因此，PP2A抑制剂可能与吉西他滨有协同抗肿瘤作用。本发明提供了一种靶向PP2A的联合用药方案，可有效地提升吉西他滨在多种肿瘤的治疗效果，易于临床使用推广。
Description
技术领域
本发明涉及生物医学领域，具体地说，是蛋白磷酸酶PP2A抑制剂与吉西他滨联合在制备抗肿瘤药物中的应用。
背景技术
吉西他滨(Gemcitabine)是一种脱氧胞苷类似的化疗药物，广泛用于各种实体肿瘤的一线或超适应症治疗，包括胰腺导管腺癌、胆道系统肿瘤、乳腺癌、卵巢癌和非小细胞肺癌等。尽管吉西他滨在临床上取得很大成功，但仍有超70％的晚期胆管癌患者和超90％的胰腺癌患者对吉西他滨的反应性不佳，极大地限制该药物的应用前景。因此，破译吉西他滨的耐药机制，开发新的联合治疗方案，以提高吉西他滨在肿瘤治疗中的客观反应率，对难以从靶向治疗和免疫治疗中获益的晚期肿瘤患者至关重要。
作为一种前体药物，吉西他滨发挥抗肿瘤作用需要经历磷酸化激活过程。经核苷转运体摄取进入细胞后，吉西他滨被脱氧胞苷激酶(deoxycytidine kinase,DCK)磷酸化为吉西他滨单磷酸(dFdCMP)，进一步转化为具有药理活性的吉西他滨二磷酸和三磷酸代谢物(dFdCDP和dFdCTP)，最终作为假核苷掺入DNA中，抑制肿瘤细胞的DNA复制并诱导细胞凋亡(Binenbaum,Y.,Na'ara,S.,and Gil,Z.(2015).Gemcitabine resistance in pancreaticductal adenocarcinoma.Drug Resist Updat 23,55-68.Mini,E.,Nobili,S.,Caciagli,B.,Landini,I.,and Mazzei,T.(2006).Cellular pharmacology of gemcitabine.AnnOncol 17Suppl 5,v7-12.)。DCK对吉西他滨的磷酸化是其活化的关键限速步骤；因此，提高DCK的活性将有助于增强吉西他滨的药效，对促进晚期肿瘤的化疗效果有重要意义。
DCK是哺乳动物细胞中脱氧核苷补救途径的限速酶，并在众多核苷类似物的抗肿瘤和抗病毒药物，如吉西他滨、氟达拉滨、克拉屈滨、扎西他滨和拉夫米定等的激活中起关键作用。已有研究报道，DCK的催化活性受自身磷酸化修饰调控，其中Ser74是主要的磷酸化位点；该位点的磷酸化，将显著提升DCK与吉西他滨等多种底物的亲和力，促进DCK的催化活性和吉西他滨的药效(Smal,C.,Vertommen,D.,Bertrand,L.,Ntamashimikiro,S.,Rider,M.H.,Van Den Neste,E.,and Bontemps,F.(2006).Identification of in vivophosphorylation sites on human deoxycytidine kinase.Role of Ser-74in thecontrol of enzyme activity.J Biol Chem 281,4887-4893.Zhong,R.,Xin,R.,Chen,Z.,Liang,N.,Liu,Y.,Ma,S.,and Liu,X.(2016).The Role of Deoxycytidine Kinase(dCK)in Radiation-Induced Cell Death.Int J Mol Sci 17.)。
PP2A在细胞信号转导的稳态维持中发挥重要作用，通常被认为是一个肿瘤抑癌因子，参与调控细胞分裂、细胞周期、DNA损伤反应、应激反应(如缺氧)、生长因子刺激、细胞粘附、细胞存活与死亡等生物学过程。结构上，PP2A全酶由三个亚基组成：催化亚基“C”(PP2Ac)，结构亚基“A”(PP2Aa)和调节亚基“B”(PP2Ab)；其中，PP2A对下游底物的特异性识别，由特定的B亚基介导实现(Kaur,A.,and Westermarck,J.(2016).Regulation ofprotein phosphatase 2A(PP2A)tumor suppressor function by PME-1.Biochem SocTrans44,1683-1693.Sents,W.,Ivanova,E.,Lambrecht,C.,Haesen,D.,and Janssens,V.(2013).The biogenesis of active protein phosphatase 2A holoenzymes:a tightlyregulated process creating phosphatase specificity.FEBS J 280,644-661.)。目前，针对PP2A的小分子药物开发方兴未艾；研究表明，PP2A小分子激动剂和抑制剂可以通过不同的细胞信号转导途径，发挥抗肿瘤效应；其中，PP2A小分子抑制剂LB-100已通过I和II期临床研究，其安全性、耐受性和抗肿瘤活性得到了不错的验证(Chung,V.,Mansfield,A.S.,Braiteh,F.,Richards,D.,Durivage,H.,Ungerleider,R.S.,Johnson,F.,and Kovach,J.S.(2017).Safety,Tolerability,and Preliminary Activity of LB-100,anInhibitor of Protein Phosphatase 2A,in Patients with Relapsed Solid Tumors:AnOpen-Label,Dose Escalation,First-in-Human,Phase I Trial.Clin Cancer Res 23,3277-3284.)。
发明内容
本发明的目的在于提供一种新的抗肿瘤联合用药方案，即蛋白磷酸酶PP2A抑制剂与常规化疗药物吉西他滨的联合使用，以提高吉西他滨的抗肿瘤效果。
发明人前期通过免疫共沉淀联合质谱蛋白鉴定技术，发现丝苏氨酸蛋白磷酸酶PP2A与DCK在细胞中相互结合；通过纯化PP2A催化亚基(PP2Ac)与DCK蛋白进行体外去磷酸反应，发现PP2Ac可以在Ser74位点去磷酸化DCK。以上研究证实，PP2A参与DCK Ser74位点的磷酸化调控，靶向干预PP2A可能对吉西他滨的药效产生影响。
本发明的第一方面，提供蛋白磷酸酶PP2A抑制剂与吉西他滨联合在制备抗肿瘤药物中的应用。
进一步地，所述的蛋白磷酸酶PP2A抑制剂，是能抑制PP2A亚基表达、全酶组装与催化活性的物质，包括但不限于：小分子化合物Fostriecin、LB100等，以及其他能引起同样效应的物质例如PP2A各亚基的shRNA，或抑制PP2A各亚基表达的microRNA，或质粒、病毒表达载体等。
进一步地，所述的肿瘤为胆管癌、胰腺导管腺癌、宫颈癌、卵巢癌、乳腺癌或非小细胞肺癌。
更进一步地，所述的肿瘤是以吉西他滨为主要化疗方案的晚期肿瘤。
本发明的第二方面，提供蛋白磷酸酶PP2A抑制剂在制备促进脱氧胞苷激酶DCKSer74位点磷酸化、进而促进吉西他滨激活和药效的药物中的应用。
本发明的第三方面，提供蛋白磷酸酶PP2A抑制剂在制备促进吉西他滨的药物敏感性的试剂中的应用。
本发明的第四方面，提供一种新的联合用药物，具体的是一种治疗肿瘤的联合用药物，所述的联合用药物的活性成分为蛋白磷酸酶PP2A抑制剂和吉西他滨。
进一步地，所述的蛋白磷酸酶PP2A抑制剂，是能促进脱氧胞苷激酶DCK Ser74位点磷酸化、进而促进吉西他滨激活和药效的物质。
本发明所述的联合、联用、联合用药物，包括同时或顺序的施用有效量的蛋白磷酸酶PP2A抑制剂和有效量的吉西他滨。所述的顺序可以是先用蛋白磷酸酶PP2A抑制剂，再用吉西他滨；也可以是先用吉西他滨，再用蛋白磷酸酶PP2A抑制剂。
进一步地，所述的联合用药物，还可以包含一种或两种以上药用载体或辅料。
本发明所述的药用载体，即药学上可接受的载体，是指药学领域中常用的除活性成分以外添加物，例如稀释剂(淀粉类、糖类、纤维素类和无机盐类)、赋形剂等，填充剂如淀粉蔗糖、粘合剂如水、乙醇、纤维素衍生物、明胶和聚乙烯吡咯烷酮，崩解剂如干淀粉、羧甲基淀粉钠，增溶剂如聚山梨酯类和聚氧乙烯脂肪酸酯类等，吸收促进剂、表面活性剂如吐温、司盘，吸附载体、润滑剂如硬脂酸镁、微粉硅胶等。另外，还可以在组合物中加入其它辅料如香味剂、甜味剂等。
本发明所述的联合用药物，可以药物组合物的形式通过口服、鼻吸入、直肠、肠胃外或经皮给药的方式施用于需要这种治疗的患者。用于口服时，可将其制成常规的固体制剂如片剂、粉剂、颗粒剂、胶囊剂、丸剂、缓释微丸、固体分散体、包合物等，制成的液体制剂如混悬剂、乳剂、熔胶剂、糖浆剂、合剂、溶液剂等，用于肠胃外给药时，可将其制成注射用的溶液、水或油性混悬剂、乳剂、脂质体、微囊、微球、毫微粒等，也可将其制成各种缓释、控释制剂。优选的形式是片剂、包衣片剂、胶囊、微丸、栓剂和注射剂，特别优选特定部位靶向释放的制剂。
在本发明的优选实施例中，蛋白磷酸酶PP2A抑制剂和吉西他滨的给药方式包括静脉注射、口服和局部给药等。
本发明所述的联合用药物的施用量可根据用药途径、患者年龄、体重、所治疗的肿瘤种类和严重程度等变化，本发明所述的有效量，可以是0.001-1000mg/kg体重，优选日剂量可以是0.01-100mg/kg体重，更优选为0.1-50mg/kg体重。可以一次或多次施用。
本发明所述的联合用药物，用于治疗肿瘤，优选用于治疗胆管癌、胰腺导管腺癌、宫颈癌、卵巢癌、乳腺癌和非小细胞肺癌。
本发明的有益效果在于：
1、吉西他滨是一种脱氧胞苷类似的前体药物，需要被脱氧胞苷激酶DCK磷酸化激活；而PP2A可以在Ser74位点去磷酸化DCK，降低DCK的催化活性从而抑制吉西他滨的药效。本发明根据前期研究基础，解析了PP2A调控DCK磷酸化进而影响吉西他滨药效的机制，创造性地使用PP2A抑制剂来增强吉西他滨的药物敏感性，提出联合用药的新策略，可使更多接受以吉西他滨为主要化疗方案的晚期肿瘤患者获益。
2、本发明的联合蛋白磷酸酶PP2A抑制剂和吉西他滨的方法，能促进脱氧胞苷激酶DCK Ser74位点磷酸化，实验结果显示，PP2A抑制剂与吉西他滨在抗肿瘤治疗中起协同作用，PP2A抑制剂可以促进吉西他滨的药效。本发明提供的靶向PP2A的联合用药方案，可有效提升吉西他滨对多种肿瘤的治疗效果，克服吉西他滨的化疗耐药，有利于临床使用推广。
附图说明
图1显示差异表达PP2A催化亚基(PP2Ac)影响脱氧胞苷激酶DCK Ser74位点磷酸化和吉西他滨药物敏感性。其中，A和B分别显示在胆管癌CCLP1细胞中，过表达PP2Ac抑制吉西他滨诱导的DCK Ser74位点磷酸化，敲低PP2Ac表达则促进吉西他滨诱导的DCK Ser74位点磷酸化；C为细胞活力曲线，显示在CCLP1细胞中过表达PP2Ac抑制吉西他滨的药效，而敲低PP2Ac表达则促进吉西他滨的药效。
图2显示PP2A小分子激动剂和抑制剂影响脱氧胞苷激酶DCK Ser74位点磷酸化和吉西他滨药物敏感性。其中，A和B显示在胆管癌CCLP1细胞中，PP2A小分子激动剂SMAP和iHAP1抑制DCK Ser74位点磷酸化，而PP2A小分子抑制剂LB100促进DCK Ser74位点的磷酸化水平；C为细胞活力曲线，显示在CCLP1细胞中PP2A小分子激动剂SMAP和iHAP1抑制吉西他滨的药物敏感性，而小分子抑制剂LB100促进吉西他滨的药物敏感性；D为细胞克隆生长情况，显示SMAP和iHAP1抑制，而LB100则促进吉西他滨的药效。
图3显示PP2A小分子抑制剂LB100与吉西他滨联合应用在小鼠体内的抗肿瘤效果。其中，A和B为胆管癌CCLP1细胞的皮下肿瘤和用药后的肿瘤生长曲线；C为宫颈癌HeLa细胞的皮下肿瘤和用药5周后的肿瘤体积统计；以上结果均显示，小分子抑制剂LB100单独使用几乎不产生抗肿瘤效果，但与吉西他滨联合应用，可以极大地促进吉西他滨在上述肿瘤中的治疗效果。
具体实施方式
下面结合实施例对本发明提供的具体实施方式作详细说明。为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整的描述。实施例中未注明具体条件者，按照常规条件或者制造商建议的条件进行。
PP2Ac质粒(CF-PP2Ac)购自北京义翘神州，PP2Ac siRNA购自上海吉玛生物；PP2A小分子抑制剂LB100、激活剂SMAP和iHAP1购自Selleck公司；细胞活力检测试剂盒CellTiter-Glo购自Promega；结晶紫染料购自碧云天生物；β-actin抗体购自ABclonal(AC026)，DCK抗体购自Proteintech Group(17758-1-Ig)，Flag-tag抗体购自ABclonal(AE005)，p-DCK Ser74购自武汉普健生物(AtaGenix)；Matrigel基质胶购自BD公司；人胆管癌细胞CCLP1和宫颈癌细胞HeLa购自中国科学院上海细胞库；6周龄BALB/c(nu/nu)雌性裸鼠(SPF级)购自上海必凯生物。其他所用试剂或者仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1：差异表达PP2A催化亚基(PP2Ac)影响脱氧胞苷激酶DCK Ser74位点磷酸化和吉西他滨药物敏感性
本发明通过免疫印迹实验检测细胞中蛋白表达情况。使用6孔板培养CCLP1细胞，在细胞融合度50％时，使用Lipo800分别转染3μg GFP和CF-PP2Ac质粒或10μL siNC和siPP2Ac；转染48h后加入20μM吉西他滨，继续培养3小时后加入100μL IP裂解液提取细胞蛋白。蛋白样本经超声、离心、BCA定量分析后，用1×SDS上样缓冲液中制备成等浓度的蛋白样品；随后，蛋白样品经SDS-PAGE电泳，转膜和膜封闭后使用特异性一抗进行孵育(4℃，过夜)，然后使用荧光素偶联的二抗孵育(37℃，60分钟)，洗涤后使用奥德赛荧光扫描仪(Li-Cor)检测荧光信号。免疫印迹的一抗稀释比例通常为1:1000，二抗稀释比例为1:5000。免疫印迹中p-DCK Ser74的相对荧光强度由Image J软件定量分析。
细胞活力检测：本发明使用Promega公司的CellTiter-Glo试剂盒检测差异表达PP2Ac的CCLP1细胞接受药物处理后的细胞存活情况。实验步骤如下：1)将肿瘤细胞按照5000个/100μL培养基/孔的量接种至不透明的96孔细胞培养板中，并给予相应的药物处理，然后将细胞置于37℃、5％ CO2的孵箱中培养相应的时间；2)解冻CellTiter-Glo试剂，按照和培养基1:1的量加入到细胞培养孔中；3)室温避光孵育10-15分钟；使用Synergy 2酶标仪(BioTek)读取发光信号值，随后进行归一化处理，绘制细胞存活曲线并计算细胞增殖半数抑制(GI50)值。
图1.A和B显示，在CCLP1细胞中过表达PP2Ac(CF-PP2Ac)，可以抑制吉西他滨诱导的DCK磷酸化激活，而干扰PP2Ac则促进上述效应。图1.C显示，在CCLP1细胞中过表达PP2Ac抑制吉西他滨的药物敏感性，而干扰PP2Ac显著促进吉西他滨的药物敏感性。
实施例2：PP2A小分子激动剂和抑制剂调控脱氧胞苷激酶DCK Ser74位点磷酸化和吉西他滨的药物敏感性
通过免疫印迹和细胞活力检测，分别检测PP2A小分子激动剂和抑制剂对DCKSer74位点磷酸化和吉西他滨药物敏感性的影响；在免疫印迹实验中，分别使用SMAP(5μM)、iHAP1(5μM)和LB100(10μM)与CCLP1细胞孵育3h，随后收集细胞蛋白检测p-DCK Ser74的表达情况；在细胞活力检测中，分别使用SMAP(5μM)、iHAP1(5μM)和LB100(10μM)与不同浓度梯度的吉西他滨联合，在CCLP1细胞中孵育72h后检测细胞存活情况。此外还进行细胞克隆形成实验，将SMAP(3μM)、iHAP1(1μM)和LB100(5μM)分别与不同浓度梯度的吉西他滨联合处理CCLP1细胞，通过结晶紫染色评估120h后的克隆形成情况。
图2.A和B显示在胆管癌CCLP1细胞中，PP2A小分子激动剂SMAP和iHAP1抑制吉西他滨诱导的DCK Ser74位点磷酸化，而抑制剂LB100则促进该磷酸化；图2.C显示在CCLP1细胞中SMAP和iHAP1抑制吉西他滨的药物敏感性，而LB100促进该药物敏感性；图2.D通过细胞克隆形成实验证实与图2.C相同的结论。
实施例3：PP2A小分子抑制剂LB100促进吉西他滨在小鼠体内的抗肿瘤效果
小鼠皮下荷瘤模型建立与药物处理：将生理盐水和Matrigel按照体积比3：1混合，将肿瘤细胞重悬于混合物中，然后将肿瘤细胞接种到6周龄雌性裸鼠皮下，每只小鼠接种细胞量为1×106。2周左右观察肿瘤生长情况；当肿瘤体积达到100mm3时，将小鼠随机分组(n≥5)，分别给予不同药物(吉西他滨、LB100或吉西他滨与LB100联合)治疗或等量的生理盐水处理。吉西他滨通过腹腔注射，给药剂量为10mg/kg；LB100通过腹腔注射，给药剂量为2mg/kg；联合用药组的给药剂量和方式与单独用药组一致。每周给药3次，治疗持续4-5周。每3天测量一次肿瘤大小，并计算肿瘤体积(0.5×L×W2，其中L为肿瘤最长直径，W为肿瘤最短直径)。当肿瘤达到1500mm3时，用二氧化碳窒息法将小鼠安乐死，然后石蜡包埋肿瘤标本，并绘制肿瘤生长曲线。
图3.A和B显示，单用LB100治疗，来自CCLP1的皮下肿瘤生长无明显变化；单用吉西他滨治疗，肿瘤体积有一定的缩小；同时联用LB100和吉西他滨，肿瘤体积较单用吉西他滨组显著缩小，提示PP2A抑制剂LB100能够提高吉西他滨的治疗效果。图3.C显示，在HeLa细胞形成的皮下肿瘤中，单用LB100或吉西他滨均没有明显的治疗效果，而联用LB100和吉西他滨，肿瘤体积显著缩小，提示LB100可促进吉西他滨的疗效。以上结果表明LB100与吉西他滨在抗肿瘤治疗中起协同作用，PP2A抑制剂可以促进吉西他滨的药效。
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
Claims (9)
1.蛋白磷酸酶PP2A抑制剂与吉西他滨联合在制备抗肿瘤药物中的应用。
2.根据权利要求1所述的蛋白磷酸酶PP2A抑制剂与吉西他滨联合在制备抗肿瘤药物中的应用，其特征在于，所述的蛋白磷酸酶PP2A抑制剂，是能抑制PP2A亚基表达、全酶组装与催化活性的物质。
3.根据权利要求2所述的蛋白磷酸酶PP2A抑制剂与吉西他滨联合在制备抗肿瘤药物中的应用，其特征在于，所述的蛋白磷酸酶PP2A抑制剂包括但不限于：小分子化合物Fostriecin、LB100，PP2A各亚基的shRNA，或抑制PP2A各亚基表达的microRNA，或质粒、病毒表达载体。
4.根据权利要求1所述的蛋白磷酸酶PP2A抑制剂与吉西他滨联合在制备抗肿瘤药物中的应用，其特征在于，所述的肿瘤为胆管癌、胰腺导管腺癌、宫颈癌、卵巢癌、乳腺癌或非小细胞肺癌。
5.蛋白磷酸酶PP2A抑制剂在制备促进脱氧胞苷激酶DCK Ser74位点磷酸化、进而促进吉西他滨激活和药效的药物中的应用。
6.蛋白磷酸酶PP2A抑制剂在制备促进吉西他滨的药物敏感性的试剂中的应用。
7.一种治疗肿瘤的联合用药物，其特征在于，所述的联合用药物的活性成分为蛋白磷酸酶PP2A抑制剂和吉西他滨。
8.根据权利要求7所述的治疗肿瘤的联合用药物，其特征在于，所述的联合用药物，还包含一种或两种以上药用载体或辅料。
9.根据权利要求7所述的治疗肿瘤的联合用药物，其特征在于，所述的联合用药物，用于治疗胆管癌、胰腺导管腺癌、宫颈癌、卵巢癌、乳腺癌或非小细胞肺癌。
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