CN113773368A - 一种foxm1拮抗多肽及其衍生物与应用 - Google Patents
一种foxm1拮抗多肽及其衍生物与应用 Download PDFInfo
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Abstract
本发明公开了一种FOXM1拮抗多肽及其衍生物与应用，所述FOXM1拮抗多肽的氨基酸序列如SEQ ID No:1所示。该拮抗多肽及其衍生物能结合FOXM1，通过与FOXM1的结合阻断其下游信号通路从而抑制肝癌细胞增殖，促进肝癌细胞发生凋亡，为肝癌等提供有效的治疗小分子药物，能够在医学与生物学领域得到广泛的应用。
Description
技术领域
本发明属于生物技术和生物医药技术领域，涉及一种FOXM1拮抗多肽及其衍生物与应用。
背景技术
肝细胞癌(HCC)是世界上常见的恶性肿瘤之一。世界卫生组织国际癌症研究中心(International Agency for Research on Cancer，IARC)报告指出，肝癌发病率位于肺癌、乳腺癌等之后居恶性肿瘤第5位，死亡率位于肺癌、结直肠之后，居恶性肿瘤第3位，是五大致死率最高的癌症中唯一一种年发病率上升的癌症。肝癌发病地域分布在世界范围内有显著差异性，发展中国家的肝病发病率较高。危险因素包括乙型肝炎病毒、丙型肝炎病毒、脂肪肝、酒精相关肝硬化、吸烟、肥胖、糖尿病、铁超载和各种饮食暴露。
肝癌预后差。只有5％到15％的患者有资格接受手术切除，这只适用于早期患者，并且由于肝脏再生能力降低，通常没有肝硬化；与左肝切除术相比，右肝切除术后并发症的风险更高。晚期肝癌的治疗方案包括：(a)经动脉化疗栓塞术(TACE)，与中晚期肝癌保守治疗相比，TACE可使2年生存率提高23％；(b)口服索拉非尼(一种激酶抑制剂)是晚期患者最常接受的选择。然而，只有不到三分之一的患者从该治疗中获益，并且在开始该方案后的六个月内，耐药性很明显。随着长期使用，化疗药物，如索拉非尼，会有如毒性或药物无效等问题的产生。因此，目前的消融治疗和化疗都不能有效改善这种毁灭性疾病的预后。进一步研究寻找更好的治疗肝癌的方法是必要的。
伴随着医学及分子生物学的不断发展及研究的日益深入，逐渐的认识到肝癌的发生、发展是多基因共同作用、多个信号通路参与而引起的“炎症-增生-癌变”的演变过程。国内外研究发现，肝癌是一种分子发病机制较为复杂的实体瘤，其发生发展涉及多条信号通路的失调，与多种基因变异有关。据COSMIC Database资料，西方人群肝癌基因变异谱中8个基因突变频率超过5％，主要集中在基因组不稳定性、永生化和Wnt等信号通路。据GenomiCare资料，中国人群中有20多个基因变异频率超过5％，主要集中在基因组不稳定性、血管生成、细胞周期失控、生长因子、PI3K-AKT-mTOR等信号通路。
除放疗和化疗外，随着靶向治疗、基因治疗、新辅助化疗、免疫治疗等治疗手段的发现与应用，为肝癌的治疗提供了更多更好的选择，也成为肝癌治疗重要的组成部分。分子靶向药物治疗在特定患者中疗效显著，患者生存获益。但靶向药物的应用存在局限性，主要因为目前靶向药物的有效的靶点还较少，同时在选择适宜的靶向治疗药物前，需要对患者进行相应的分子靶点检测。目前，与肝癌治疗相关的靶点主要有VEGFR1-3、FGFR1-4、PDGFRɑ、RTK、KIT、RET，均已用于临床。因此，肝癌靶向治疗选用药物的关键是对与发病机制相关的信号通路驱动及肝癌特异性转录因子进行鉴别。
FOXM1作为调控细胞周期和细胞进程重要的转录因子，已证实在39种人类恶性肿瘤中，FOXM1调控网络是18000例癌症患者不良预后的主要预测因子，这也揭示了FOXM1在癌症中的重要作用。然而，FOXM1究竟是如何在人体细胞中发挥其致癌活性的，目前尚不完全清楚。一种机制与FOXM1靶点的转录激活有关，这种激活导致多种促肿瘤效应，包括增强细胞增殖。另外，FOXM1可能通过与其他蛋白质相互作用作为癌基因，从而支持不同的致癌途径。FOXM1调控着肿瘤发生和发展的关键过程。作为一种典型的增殖相关转录因子，FOXM1在转录水平上直接或间接激活靶基因的表达，并表现出时空模式，其失调几乎涉及到肿瘤细胞的所有特征。并且大量的数据和实验证实，在多种不同肝细胞癌中FOXM1的表达量均上调，并且通过对病人数据分析显示，FOXM1的表达量与肝癌病人的预后密切相关。FOXM1被认为是肿瘤的致命伤，并成为判断肿瘤发生发展的新的肿瘤标志物及抗肿瘤药物开发的重要靶标。目前国内以FOXM1为靶标的相关药物治疗仍在临床前研究阶段，并无任何一种直接靶向FOXM1的抗肿瘤药物上市或应用于临床。
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FOXM1拮抗多肽及其衍生物与应用。该拮抗多肽及其衍生物与受体FOXM1具有特异性高亲和力，能够通过与FOXM1的结合来阻断FOXM1的信号通路，该拮抗多肽及其衍生物在靶向抑制肝癌细胞增殖、促进肝癌细胞凋亡等方面起着重要的作用，其在肝癌靶向治疗方面具有巨大的应用价值。
为了达到上述目的，本发明所采用的技术方案为：一方面，本发明提供了一种FOXM1拮抗多肽，所述FOXM1拮抗多肽的氨基酸序列如SEQ ID No:1所示。
另一方面，本发明提供了一种FOXM1拮抗多肽的衍生物，所述FOXM1拮抗多肽的衍生物为上述所述的FOXM1拮抗多肽氨基酸侧链基团上，和/或，上述所述的FOXM1拮抗多肽片段的氨基端或羧基端进行常规修饰得到的产物；
或者为上述所述的FOXM1拮抗多肽上连接用于多肽或蛋白检测或纯化的标签所得到的产物。
进一步地，所述常规修饰包括氨基化、酰胺化、羟基化、羧基化、羰基化、烷基化、乙酰化、磷酸化、酯化、糖基化、环化、生物素化、荧光基团修饰、聚乙二醇PEG修饰或固定化修饰；
优选地，所述标签包括His6、GST、EGFP、MBP、Nus、HA、IgG、FLAG、c-Myc或ProfinityeXact。
进一步地，所述FOXM1拮抗多肽的衍生物为上述所述的FOXM1拮抗多肽末端进行酰胺化修饰得到的产物。
进一步地，所述FOXM1拮抗多肽及其衍生物的获得，采用现有技术中的公知方法进行，既可以用多肽自动合成仪进行化学合成；通过将短肽序列推导出核苷酸序列，然后克隆到载体中进行生物合成；也可以从现有存在的生物体内进行大量提取和纯化。
进一步地，所述FOXM1拮抗多肽及其衍生物可以来源于哺乳类动物或者鸟类，例如灵长类动物(人类)；啮齿类动物，包括小鼠，大鼠，仓鼠，兔，马，牛，犬类，猫等。
另一方面，本发明提供了一种多聚核苷酸，其编码上述所述的FOXM1拮抗多肽或上述所述的FOXM1拮抗多肽的衍生物。
再一方面，本发明提供了一种载体，其包含上述所述的多聚核苷酸。
再一方面，本发明提供了一种宿主细胞，其转染了上述所述的载体。
再一方面，本发明提供了一种上述所述的FOXM1拮抗多肽或上述所述的FOXM1拮抗多肽的衍生物在制备预防和/或治疗高表达FOXM1的肿瘤的药物中的应用。
再一方面，本发明提供了一种上述所述的FOXM1拮抗多肽或上述所述的FOXM1拮抗多肽的衍生物在制备抑制高表达FOXM1的肿瘤细胞增殖和/或促进高表达FOXM1的肿瘤细胞凋亡的药物中的应用。
进一步地，所述高表达FOXM1的肿瘤包括肝癌、肺癌、乳腺癌、结直肠癌，优选为肝癌。
再一方面，本发明提供了一种药物组合物，包含上述所述的FOXM1拮抗多肽、上述所述的FOXM1拮抗多肽的衍生物中的至少一种。
进一步地，所述药物组合物含有一种或者是多种药学上可以接受的载体。
进一步地，所述药学上可以接受的载体包括稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、吸附载体、表面活性剂或润滑剂等。
进一步地，所述药物组合物可以制成片剂、粒剂、胶囊、口服液或注射剂等多种形式，各种剂型的药物可以按照药学领域的常规方法制备。
再一方面，本发明提供了一种检测试剂，包含上述所述的FOXM1拮抗多肽、上述所述的FOXM1拮抗多肽的衍生物中的至少一种。
本发明的有益效果是：
(1)本发明提供了一种FOXM1拮抗多肽及其衍生物，所述的拮抗多肽及其衍生物能够专一性与FOXM1结合，并特异性与FOXM1结合抑制FOXM1信号通路。
(2)本发明提供的FOXM1拮抗多肽及其衍生物可以通过阻断FOXM1的信号通路抑制癌细胞增殖，促进癌细胞发生凋亡，可以作为FOXM1结合位点的生物类多肽药物，可用于预防和/或治疗肿瘤。能够在医学与生物学领域得到广泛的应用，并产生巨大的社会与经济效益。
附图说明
图1为本发明FOXM1拮抗多肽FIP-1的亲水性检测结果图；
图2为本发明实施例1中FIP-1与HepG2细胞中FOXM1结合能力的结果图；其中图A中蓝色为细胞核，绿色为TAT、红色为FOXM1，以及合并图，图B中蓝色为细胞核，绿色为FIP-1、红色为FOXM1，以及合并图；
图3为本发明实施例2中FIP-1抑制肝癌细胞HepG2的增殖作用结果图；其中图A为本发明实施例2中HepG2细胞克隆形成能力检测图，图B为图A中HepG2细胞的克隆形成能力的量化直方统计图；
图4为FIP-1抑制细胞活性，促进HepG2发生凋亡的结果图。
具体实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。
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。
以下实施例中使用氨基酸序列如SEQ ID No:1所示的FOXM1拮抗多肽，并以穿膜肽TAT作为载体(样品序列为Gly-Leu-Ser-Ser-Met-His-Ser-Ala-Pro-Pro-Leu-Arg——Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Gln，以FIP-1表示)由上海楚肽生物科技有限公司合成。通过强耀生物公司官网对FIP-1序列进行亲水性分析，得到如图1所示的结果，从图1可以看出，该多肽具有良好的亲水性，可能与FOXM1有效结合。
实施例1FIP-1可以和HepG2细胞中的FOXM1结合
将状态良好的HepG2细胞接种在含有爬片的12孔板中，加入新鲜培养基培养过夜，第二天早晨加入标记的两种多肽，FIP-1和对照组多肽TAT，培养6h，在培养板中将已爬好细胞的玻片用PBS浸洗3次，每次3min；用4％的多聚甲醛固定爬片15min，PBS浸洗玻片3次，每次3min；0.5％Triton X-100(PBS配制)室温通透20min(细胞膜上表达的抗原省略此步骤)；PBS浸洗玻片3次，每次3min，吸水纸吸干PBS，在玻片上滴加正常山羊血清，室温封闭30min；吸水纸吸掉封闭液，不洗，每张玻片滴加足够量的稀释好的一抗并放入湿盒，4℃孵育过夜；第二天加荧光二抗：PBST浸洗爬片3次，每次3min，吸水纸吸干爬片上多余液体后滴加稀释好的荧光二抗，湿盒中20-37℃孵育1h，PBST浸洗切片3次，每次3min；复染核：滴加DAPI避光孵育5min，对标本进行染核，PBST 5min×4次洗去多余的DAPI；用吸水纸吸干爬片上的液体，用含抗荧光淬灭剂的封片液封片，然后在荧光显微镜下观察采集图像。结果如图2，从图中可以看出，与对照组相比，实验组中的绿色的FIP-1与红色的FOXM1能有效重叠，证实FIP-1可以和FOXM1有效结合。
实施例2FIP-1能抑制HepG2细胞的增殖
将肝癌细胞HepG2以100个/孔接种于6孔细胞培养板中，每孔培养基体积为2mL，培养24h；之后加入100μM FIP-1和适量新鲜培养基，并以未加药组作为对照，每两天更换培养基和药物，连续培养至，细胞团含有50个以上细胞，用甲醇固定实验组和对照组细胞后，0.1％的结晶紫染色，统计每组中的克隆形成数，结果如图3，从图中可以看出，与对照组相比，FIP-1能有效抑制细胞的克隆数，抑制克隆的形成，证实FIP-1可以有效抑制细胞增殖。
实施例3CCK-8检测FIP-1对HepG2细胞的抑制作用
接种HepG2细胞于10cm培养皿中，用10％FBS的DMEM培养基培养至密度95％，待细胞达到所需密度后，吸掉培养基，用PBS清洗2次，加入1mL胰酶，37度孵育消化1min，之后加入1mL 10％FBS的DMEM培养基，捶打分散细胞，吸取10μL细胞悬液加入到1mL DMEM培养基中制成细胞悬液，用细胞计数板计数所制备的细胞悬液中的细胞数量，然后按每孔5×103个接种于96孔板中培养24小时。次日，弃掉细胞培养基，向培养板加入新鲜培养基和不同浓度的FIP-1，将培养板在培养箱孵育48小时，向每孔加入10μL CCK-8溶液(注意不要在孔中生成气泡，它们会影响OD值的读数)将培养板在培养箱内孵育1-4小时，用酶标仪测定在450nm处的吸光度，然后按照下述公式计算细胞活力，并绘制抑制曲线。细胞活力(％)＝[A(加药)-A(空白)]/[A(0加药)-A(空白)]×100，A(加药)：具有细胞、CCK-8溶液和药物溶液的孔的OD值，A(0加药)：具有细胞、CCK-8溶液而没有药物溶液的孔的OD值，A(空白)：没有细胞的孔的OD值。结果见图4。由图4可以看出，随着药物浓度的增加，细胞活力不断下降，并且细胞的IC50值约为90μM。
SEQUENCE LISTING
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Claims (10)
1.一种FOXM1拮抗多肽，其特征在于，所述FOXM1拮抗多肽的氨基酸序列如SEQ ID No:1所示。
2.一种FOXM1拮抗多肽的衍生物，其特征在于，所述FOXM1拮抗多肽的衍生物为权利要求1所述的FOXM1拮抗多肽氨基酸侧链基团上，和/或，权利要求1所述的FOXM1拮抗多肽片段的氨基端或羧基端进行常规修饰得到的产物；
或者为权利要求1所述的FOXM1拮抗多肽上连接用于多肽或蛋白检测或纯化的标签所得到的产物；
优选地，所述常规修饰包括氨基化、酰胺化、羟基化、羧基化、羰基化、烷基化、乙酰化、磷酸化、酯化、糖基化、环化、生物素化、荧光基团修饰、聚乙二醇PEG修饰或固定化修饰；
优选地，所述标签包括His6、GST、EGFP、MBP、Nus、HA、IgG、FLAG、c-Myc或ProfinityeXact。
3.一种多聚核苷酸，其特征在于，其编码权利要求1所述的FOXM1拮抗多肽或权利要求2所述的FOXM1拮抗多肽的衍生物。
4.一种载体，其特征在于，其包含权利要求3所述的多聚核苷酸。
5.一种宿主细胞，其特征在于，其转染了权利要求4所述的载体。
6.权利要求1所述的FOXM1拮抗多肽或权利要求2所述的FOXM1拮抗多肽的衍生物在制备预防和/或治疗高表达FOXM1的肿瘤的药物中的应用。
7.权利要求1所述的FOXM1拮抗多肽或权利要求2所述的FOXM1拮抗多肽的衍生物在制备抑制高表达FOXM1的肿瘤细胞增殖和/或促进高表达FOXM1的肿瘤细胞凋亡的药物中的应用。
8.根据权利要求6或7所述的应用，其特征在于，所述高表达FOXM1的肿瘤包括肝癌、肺癌、乳腺癌、结直肠癌，优选为肝癌。
9.一种药物组合物，其特征在于，包含权利要求1所述的FOXM1拮抗多肽、权利要求2所述的FOXM1拮抗多肽的衍生物中的至少一种。
10.一种检测试剂，其特征在于，包含权利要求1所述的FOXM1拮抗多肽、权利要求2所述的FOXM1拮抗多肽的衍生物中的至少一种。
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