CN113663075B - Pcsk9抑制剂在制备治疗或辅助治疗肿瘤的药物中的应用 - Google Patents
Pcsk9抑制剂在制备治疗或辅助治疗肿瘤的药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种PCSK9抑制剂在制备治疗或辅助治疗肿瘤的药物中的应用，本申请首次发现抑制肿瘤细胞中PCSK9的表达，显著降低了癌细胞的增殖、迁移能力，促进肿瘤细胞的凋亡，因此靶向PCSK9可用于治疗乳腺癌。另外，PCSK9抑制剂的使用可提高相关信号通路药物疗效、有效降低乳腺癌的死亡率，大大提高抗肿瘤药物抑制肿瘤细胞的效果。
Description
技术领域
本发明属于生物技术领域，具体涉及一种PCSK9抑制剂在制备治疗或辅助治疗肿瘤的药物中的应用。
背景技术
2020年世界卫生组织国际癌症研究机构分析了185个国家地区、36种癌症，主要分析2020年各癌症的发病率和致死率，以及各地区肿瘤的发病情况。发布的全球最新癌症数据显示，2020年全球1930万新增病例以及近1000万人死亡病例，在新增癌症分布类型上，乳腺癌新发病例数达226万人，首次超过肺癌(220万人)，成为全球最常见癌症。最终分析结果显示，乳腺癌约占所有新确诊癌症人数的11.7％，占女性新确诊癌症人数的24.5％，居女性癌症发病人数的首位，也是全球女性死亡人数最多的癌症。在新确诊的女性患者中，每4人就有1人是乳腺癌患者，每6人就有1人死于乳腺癌。
乳腺癌的早期发现和早期诊断是提高疗效的关键。现如今我国乳腺癌患者的五年生存率高达到83％。这和分期有关系，越早期越好。化疗是治疗恶性肿瘤最重要的手段之一，然而，肿瘤细胞对化疗药物产生耐药常常最终导致化疗失败。内分泌治疗依据不同乳腺癌分型的个体差异性较大。乳腺癌通常分为四种分子亚型Luminal A型(ER+/PR+,HER-2-、Luminal B型(ER+/PR+,HER-2+)、HER-2+型(ER-/PR-，HER-2+)和Basal-like/TNBC型(ER-/PR-/HER-2-)，不同分子亚型的乳腺癌临床治疗效果和患者生存期差异很大，目前个体化治疗和靶向治疗引起临床重视。
前蛋白转化酶枯草溶菌素9(Proprotein convertase subtilisin/kexin type9,PCSK9)于2003年在家族性高胆固醇血症中被发现，参与血脂代谢的调控，能够降解LDLR，使胆固醇不能被有效的清除，而抑制PCSK9的活性可以使血液中的LDL-C水平降低。PCSK9抑制剂类药物在临床已获批应用于降低血脂。但是PCSK9在乳腺癌预后和发生发展中的作用机制尚不明确。
发明内容
为了解决上述背景技术中所提出的问题，本发明的目的在于提供一种PCSK9抑制剂在制备治疗或辅助治疗肿瘤的药物中的应用。
为达到上述目的，本发明所采用的技术方案为：一方面，本发明提供了一种PCSK9抑制剂在制备治疗或辅助治疗肿瘤的药物中的应用。
另一方面，本发明提供了一种PCSK9抑制剂在制备降低肿瘤细胞增殖的药物中的应用。
另一方面，本发明提供了一种PCSK9抑制剂在制备降低肿瘤细胞迁移的药物中的应用。
再一方面，本发明提供了一种PCSK9抑制剂与PI3K抑制剂联用在制备治疗肿瘤的药物中的应用。
再一方面，本发明提供了一种PCSK9作为肿瘤的预后标志物在制备肿瘤预后评估的产品中的应用。
进一步地，所述肿瘤为乳腺癌。
再一方面，本发明提供了一种治疗或辅助治疗肿瘤的药物组合物，其含有作为活性物质的PCSK9抑制剂。
再一方面，本发明提供了一种降低肿瘤细胞增殖的药物组合物，其含有作为活性物质的PCSK9抑制剂。
再一方面，本发明提供了一种降低肿瘤细胞迁移的药物组合物，其含有作为活性物质的PCSK9抑制剂。
再一方面，本发明提供了一种治疗肿瘤的药物组合物，其含有作为活性物质的PCSK9抑制剂与PI3K抑制剂。
进一步地，所述肿瘤为乳腺癌。
本发明的有益效果是：
本发明首次发现乳腺癌中的PCSK9相比于正常人群存在高表达，且PCSK9高表达的乳腺癌患者比PCSK9低表达的乳腺癌患者的总生存期差，因此PCSK9可以作为标志物用来评估乳腺癌患者的预后情况。
本申请首次发现抑制肿瘤细胞中PCSK9的表达，显著降低了癌细胞的增殖、迁移能力，促进肿瘤细胞的凋亡，因此靶向PCSK9可用于治疗乳腺癌。PCSK9抑制剂的使用可提高相关信号通路药物疗效、有效降低乳腺癌的死亡率，大大提高抗肿瘤药物抑制肿瘤细胞的效果。
本申请发现PCSK9通过调控上皮间质转化和激活下游相关信号通路促进乳腺癌进展的作用机制，可为乳腺癌靶向药物的开发提供重要的理论基础，具有较高的临床应用前景。
附图说明
图1A为TCGA分析乳腺癌和正常人群PCSK9表达情况图；图1B为Kplan-Meierplotter分析乳腺癌中PCSK9高表达患者的总生存期状况图。
图2为本发明实施例1中PCSK9抑制剂R-IMPP作用于MCF-7和MDA-MB-231后PCSK9蛋白表达情况检测图。
图3A为本发明实施例2中抑制PCSK9后，MCF-7和MDA-MB-231细胞克隆形成能力检测图，与对照组相比，R-IMPP显著抑制MCF-7和MDA-MB-231细胞的克隆形成能力，R-IMPP浓度越高，抑制克隆形成能力越强；图3B为图3A中R-IMPP抑制MCF-7和MDA-MB-231细胞的克隆形成能力的量化直方统计图。
图4为本发明实施例3中抑制PCSK9后，MCF-7和MDA-MB-231细胞迁移能力检测图；其中图A为抑制PCSK9后，穿过Transwell小室的MCF-7和MDA-MB-231细胞展示图，图B为抑制PCSK9后，对图A穿过Transwell小室的MCF-7和MDA-MB-231细胞的量化直方统计图。
图5为本发明实施例4中小鼠移植瘤模型检测PCSK9抑制剂在乳腺癌发展中的作用；其中图A为不同处理组(对照组，单用PCSK9抑制剂，单用PI3K抑制剂和两者联用组)对小鼠移植瘤生长的影响，单用PCSK9抑制剂或者单用PI3K抑制剂均能抑制荷载MCF-7细胞的移植瘤的生长，两者联用对移植瘤生长具有协同抑制作用，图B为不同处理组(对照组，单用PCSK9抑制剂，单用PI3K抑制剂和两者联用组)处理后移植瘤的重量展示图。
图6为本发明实施例5中PCSK9抑制剂抗肿瘤的机制研究结果图；其中图A为MCF-7细胞中单用R-IMPP、TGX-221和R-IMPP与TGX-221两者联用处理后相关蛋白的表达情况，图B为MDA-MB-231细胞中单用R-IMPP、TGX-221和R-IMPP与TGX-221两者联用处理后相关蛋白的表达情况。
具体实施方式
下面结合具体实施例和附图对本发明的技术方案做进一步说明，但应该理解本发明的保护范围并不受具体实施例的限制。
本申请主要从以下几个方面进行研究：(1)通过TCGA数据库分析PCSK9在乳腺癌患者和正常人群中的表达情况，Kplan-Meier plotter分析PCSK9高表达患者的总生存期；(2)Western blot实验检测R-IMPP处理后降低PCSK9表达；(3)R-IMPP处理后，细胞的克隆形成能力和迁移能力降低；(4)R-IMPP可以抑制小鼠移植瘤的生长，且TGX-221与R-IMPP联用具有协同作用；(5)R-IMPP主要是促进MCF-7和MDA-MB-231细胞的凋亡蛋白表达的作用。综上所述，本申请阐述PCSK9可以作为乳腺癌分子标志物且靶向PCSK9可以有效抑制乳腺癌细胞的生长和迁移。
以下实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1 TCGA分析乳腺癌和正常人群PCSK9表达情况；Kplan-Meier plotter分析乳腺癌中PCSK9高表达患者的总生存期状况1)分别对来源于TCGA数据库中的“ZhaoBreast”、“Curtis Breast”、“TCGA Breast”和“Richardson Breast”研究，分析乳腺癌和正常人群PCSK9表达情况，结果如图1A所示，从图1A可以看出乳腺癌患者中的PCSK9相比于正常人群存在高表达。
2)Kplan-Meier plotter分析乳腺癌中PCSK9高表达患者的总生存期状况，结果如图1B所示，从图1B可以看出PCSK9高表达患者的总生存期比PCSK9低表达患者的总生存期差。
实施例2 PCSK9抑制剂R-IMPP作用于MCF-7和MDA-MB-231后PCSK9蛋白表达情况检测
1)Westernblot实验
1.1蛋白提取
将PCSK9抑制剂R-IMPP处理48h后的MCF-7和MDA-MB-231细胞，胰酶消化，收集，离心，PBS洗三遍，加入裂解液和蛋白酶抑制剂，冰上孵育30min，100℃煮沸30min，短时间内-20℃保存。
1.2 SDS-PAGE凝胶电泳
配制12％的分离胶和5％的浓缩胶。每孔加入20微克蛋白样品及蛋白Marker 10μL，调节电压90V，30min。蛋白样品进入分离胶后，调节电压至120V，1h，至蛋白样品中的指示剂-溴酚蓝接近凝胶底部时停止电泳。
1.3蛋白转印
将PVDF膜浸泡在甲醇中活化1min，滤纸浸泡在转膜缓冲液中。取出凝胶，对照Marker切下包含目的蛋白的凝胶。按照湿法转膜的要求：滤纸-PVDF-凝胶膜-滤纸的顺序铺好，200mA，转印1.5h。
1.4抗体孵育
取出PVDF膜，浸泡在5％的脱脂牛奶中，封闭1h；TBST洗3遍，根据蛋白Marker将含目的蛋白和β-actin的PVDF膜剪开，分别孵育anti-PCSK9抗体，anti-LDLR抗体，anti-PI3K抗体，anti-AKT抗体，anti-p-AKT抗体，anti-BCL2抗体，anti-p-BCL2抗体，anti-CASPASE3抗体及anti-actin抗体，摇床上4℃缓慢摆动，孵育过夜；TBST洗3边，加入anti-rabbit或anti-mouse二抗，室温下孵育1h。
1.5发光与成像分析
TBST洗3遍，加入ECL发光液，将膜放入凝胶成像仪，软件成像并记录分析。
2)R-IMPP作用肿瘤细胞后相关蛋白的表达情况
R-IMPP作用于乳腺癌细胞MCF-7和MDA-MB-231后，检测发现PCSK9表达显著降低，LDLR表达升高。因此R-IMPP作为PCSK9的抑制剂效果被证实，可以用于后续实验。
结果如图2所示，经R-IMPP处理后，PCSK9在MCF7和MDA-MB-231细胞中低表达，同时LDLR在MCF7和MDA-MB-231细胞中高表达，因为PCSK9通过与LDLR结合引起LDLR在溶酶体的降解。PCSK9表达降低，则LDLR表达升高。
实施例3抑制PCSK9后，MCF-7和MDA-MB-231细胞克隆形成能力检测
1)细胞克隆形成能力的检测
1.1取处于对数生长期的MCF-7和MDA-MB-231细胞，用0.25％胰酶消化下来后并将其吹打成单个细胞，制成细胞悬液备用。细胞计数后，以400个细胞/皿接种于6孔板中并转动，使细胞分散均匀。R-IMPP按0、10μM和20μM浓度分别加入培养皿中，每3天换新鲜含有各浓度R-IMPP的培养基，每个浓度设3个复孔，置于37℃5％CO2环境下，静置培养2周。
1.2经常观察，待培养皿底部出现肉眼可见的克隆时，终止培养。弃去旧的培养液，用PBS浸洗2次。加入甲醇4mL/皿，固定15分钟，去固定液，然后PBS洗3次。加2mL 0.1％结晶紫染色液染色10分钟，PBS洗3次，然后用自来水缓慢洗去染色液，置于空气中干燥。
1.3将培养皿倒置于一张白纸上，用肉眼直接计数克隆，最后计算克隆形成率。公式：克隆形成率＝克隆平均数/接种细胞数×100％。
2)R-IMPP对MCF-7和MDA-MB-231克隆形成能力的影响
0.1％结晶紫染色结果如图3所示，MCF-7和MDA-MB-231细胞经R-IMPP处理后的克隆数明显较少，且20μM R-IMPP的克隆形成数目显著少于10μM，说明R-IMPP降低MCF-7和MDA-MB-231的克隆形成能力，MCF-7和MDA-MB-231细胞对R-IMPP存在浓度依赖性。
实施例4抑制PCSK9后，MCF-7和MDA-MB-231细胞迁移能力检测
1)细胞迁移能力检测
1.1待MCF-7和MDA-MB-231细胞培养至对数生长期，再用R-IMPP处理48h后消化细胞，用PBS和无血清培养基先后洗涤1次，收集细胞并用无血清培养基悬浮细胞，计数，调整浓度为2×105/ml。
1.2在下室(即24孔板底部)加入600－800μL含10％血清的培养基，Transwell小室加入100－150μL细胞悬液，继续在孵箱培养24h。
1.3将transwell小室取出并浸泡在4％PFA中，固定30min，用结晶紫染色，用棉棒轻轻擦去小室内未穿膜的细胞然后镜检，并计算小室膜下表面的细胞数，计算中间和四周5个视野，取平均值。镜下对染色后的细胞进行计数，计数值高，则该组细胞迁移能力强。
2)R-IMPP对MCF-7和MDA-MB-231迁移能力的影响
图4结果显示R-IMPP显著抑制MCF-7和MDA-MB-231细胞的迁移能力。
实施例5小鼠移植瘤模型检测PCSK9抑制剂在乳腺癌发展中的作用
1)小鼠移植瘤模型
4～6周龄的BalB/C裸鼠饲养于SPF级实验动物中心，适应环境一周；取对数生长期的MCF-7细胞，胰酶消化方法收集细胞，尽量用PBS清洗两到三次，计数后依据5×106个细胞/只裸鼠最终用1×PBS重悬至适当体积，每管1ml分装，立即放置冰盒运输至动物房；
用酒精棉球消毒裸鼠后肢背侧，每只裸鼠皮下接种0.2ml细胞混悬液。接种第1～4天每天观察肿瘤结节出现情况，第4天开始出现肉眼可见的移植瘤，用游标卡尺测定移植瘤的体积，待其体积生长至50～100mm3，我们随机将其分为4组，5只/每组：空白对照组、R-IMPP治疗组、TGX-221治疗组、R-IMPP与TGX-221联合治疗组。开始治疗，记为0天，分别对四组腹腔注射给药，3天1次，持续3周。每次给药前对各小鼠称重并测量各组瘤体的瘤长径(L)和短径(S)，根据公式V(cm3)＝L×S2×0.5(V:瘤体积，L:长径，S:短径)，绘制瘤体体积-时间变化曲线。治疗三周后，将裸鼠脱颈处死并剖下肿瘤组织以备后用。
2)R-IMPP对小鼠移植瘤的影响
检测结果如图5所示，相比于空白对照组，R-IMPP治疗组可以抑制肿瘤的生长，肿瘤的体积和重量均小于空白对照组；当TGX-221与R-IMPP联合用药治疗小鼠后，肿瘤的体积和重量显著低于空白对照组和单用R-IMPP或TGX-221治疗组，说明TGX-221与R-IMPP对于抗肿瘤生长具有协同性。
实施例6 PCSK9抑制剂抗肿瘤的机制研究
R-IMPP作用于乳腺癌细胞MCF-7和MDA-MB-231后，细胞的增殖能力和迁移能力均被抑制，说明PCSK9影响乳腺癌细胞的生长和迁移。小鼠移植瘤模型显示，抑制PCSK9后可以抑制肿瘤的生长，但是具体的原因未曾知道。因为肿瘤的发生发展是生长、迁移、侵袭和凋亡共同作用的结果，所以申请人探讨了具体是什么机制导致肿瘤生长被抑制。
MCF-7和MDA-MB-231细胞生长至对数期分别设置对照组，R-IMPP组，TGX-221组和R-IMPP与TGX-221联用组处理后，收集蛋白进行免疫印迹，对相关蛋白的表达情况进行检测。
结果如图6所示，经R-IMPP处理后，与对照组相比，抗凋亡蛋白BCL2在MCF7和MDA-MB-231细胞中表达降低，磷酸化p-BCL2表达升高，CASPASE-3蛋白表达升高，且TGX-221与R-IMPP联用处理细胞后，BCL2的表达显著低于单用R-IMPP或TGX-221，磷酸化BCL2和CASPASE3的表达显著高于单用R-IMPP或TGX-221，显示R-IMPP与TGX-221联用对MCF-7和MDA-MB-231促进凋亡表达具有协同性。说明R-IMPP能促进MCF7和MDA-MB-231细胞发生凋亡，且是通过抑制PI3K/Akt信号通路活化导致R-IMPP作用更明显。
虽然早已报道，靶向PCSK9在降血脂中的应用，但是PCSK9在乳腺癌中的作用及相关机制并未报道。本发明通过数据库分析发现PCSK9在乳腺癌细胞中表达升高，且其高表达的乳腺癌患者的总生存期差。之后申请人使用PCSK9的抑制剂R-IMPP处理MCF-7和MDA-MB-231细胞后，发现MCF-7和MDA-MB-231细胞的增殖能力、迁移能力降低，且凋亡能力升高。明确了R-IMPP对MCF-7和MDA-MB-231细胞体外的影响后，进一步通过小鼠移植瘤模型，发现R-IMPP可以抑制体内肿瘤的生长(见图5)。因此，申请人确定PCSK9对乳腺癌的发生发展有一定的影响。申请人想知道PCSK9在乳腺癌中具体是如何发挥其作用，进一步使用免疫印迹对相关蛋白进行检测，发现(见图6)R-IMPP可以降低抗凋亡蛋白BCL2的表达，增加磷酸化BCL2和CASPASE3蛋白的表达进而发挥促凋亡作用。PCSK9是通过与PI3K/Akt信号通路相互作用进而调控乳腺癌的进展。此结果给予我们提示，PCSK9通过对细胞中低密度脂蛋白的调控与PI3K/Akt信号通路相互作用参与肿瘤细胞增殖及迁移侵袭等方面。本发明通过分析乳腺癌患者和正常人群中PCSK9的表达以及PCSK9高低表达的乳腺癌患者的总生存期发现其可以作为乳腺癌预后的分子标志物，并且深入阐述PCSK9对PI3K/Akt下游信号的调控机制，有助于将PCSK9及其调控的下游信号通路作为肿瘤治疗的靶标，为临床肿瘤治疗提供新途径。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (2)
1.PCSK9抑制剂在制备降低乳腺癌细胞MDA-MB-231增殖和/或迁移的药物中的应用。
2.PCSK9抑制剂与PI3K抑制剂联用在制备降低乳腺癌细胞MDA-MB-231增殖和/或迁移的药物中的应用。
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