CN112933239A - 激活肿瘤细胞内源性pd-1的试剂在制备抗肿瘤药物中的用途 - Google Patents
激活肿瘤细胞内源性pd-1的试剂在制备抗肿瘤药物中的用途 Download PDFInfo
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Abstract
本发明涉及激活肿瘤细胞内源性PD‑1的试剂在制备抗肿瘤药物中的用途。具体地,本发明涉及组蛋白去乙酰化酶抑制剂与激活p53通路的抗肿瘤剂的组合在制备用于治疗受试者的肿瘤的药物中的用途。另一方面,本发明涉及一种用于体外增强肿瘤细胞内源性PD‑1表达的方法,包括:1)在37℃恒温细胞培养箱中培养肿瘤细胞1天;2)向步骤1)的肿瘤细胞中加入激活p53通路的抗肿瘤剂,将肿瘤细胞继续培养18小时;和3)向步骤2)的肿瘤细胞中加入曲古柳菌素A和烟酰胺处理细胞6小时。
Description
技术领域
本发明涉及药物化学技术领域,具体提供一种靶向肿瘤细胞关键分子的联合给药方式。根据本发明的方法,可显著提高肿瘤细胞内源性PD-1表达。
背景技术
最新全球癌症数据显示,全球癌症死亡病例近千万例,癌症一直是人类生命的第一杀手。我国始终将国民生命安全和身体健康放在首位,重视科技和医疗发展。随着科技和医疗卫生的不断发展,癌症治疗已不局限于普通的手术、放疗、化疗或靶向治疗。
近年来,免疫检查点抑制剂PD-1/PD-L1的单克隆抗体免疫疗法在临床上取得显著疗效。在晚期黑色素瘤、肺癌、乳腺癌、皮肤癌和淋巴瘤等在内的近20种实体肿瘤中,PD-1/PD-L1单抗通过阻断免疫细胞表面的PD-1受体和肿瘤细胞表面的PD-L1配体之间的相互作用,帮助免疫细胞中的T细胞恢复活力,从而杀伤癌细胞,实质性的改善了晚期肿瘤患者的生存期。但随着临床治疗病例增加,患者间差异性很大,耐药性、副作用或无应答逐渐明显,这成为免疫疗法的一大阻碍,因此,基于PD-1为靶点的治疗方案亟需进一步完善。
近期研究发现,肿瘤细胞中也表达PD-1,并且以一种免疫非依赖的方式调控肿瘤细胞的生物学行为。在黑色素瘤细胞、肝癌细胞、胰腺癌细胞和肺癌细胞中,均发现肿瘤细胞内源性PD-1参与调控肿瘤细胞的增殖。由于肿瘤细胞内源性PD-1表达量极低,因此需要研发一种激活肿瘤细胞内源性PD-1的方法,为基于肿瘤细胞PD-1的治疗方法提供新的用药策略。
此外,研究发现在一些癌症中组蛋白去乙酰化酶(HDAC)水平明显异常;HDAC可以去除组蛋白乙酰基使染色体更加紧密,从而抑制基因的转录。因此,HDAC抑制剂(HDACi)可以通过提高乙酰化水平来促进基因表达以达到治疗癌症的效果。2006年FDA审批通过了默克尔公司研发的HDACi——伏立诺他——用于治疗皮肤T淋巴细胞瘤。但HDACi的选择性不强,因此多采用联合其他肿瘤药物的方式应用于肿瘤治疗。
目前,仍然需要开发能有效***的组合药物,其既可以靶向肿瘤,并且能够有效增强目前抗肿瘤药物的有效性。
发明内容
本发明是基于以下意想不到的发现:Nutlin-3A或喜树碱(Camptothecin,CPT)与曲古柳菌素A(Trichostatin A,TSA)和烟酰胺(Nicotinamide,NAM)组合在体外处理肿瘤细胞后,可显著增加肿瘤细胞内源性PD-1的表达,并有效地抑制肿瘤细胞的生长。
Nutlin-3A是Nutlin-3的活性异构体,为一种鼠双微体2(MDM2)拮抗剂,抑制MDM2-p53相互作用,且稳定p53蛋白,因此诱导细胞周期停滞和细胞凋亡。
喜树碱(Camptothecin,CPT)是从珙桐科落叶植物喜树的种子或根皮中提出的一种生物碱,能直接破坏DNA结构,与DNA结合而使DNA易受内切酶的攻击,同时抑制DNA聚合酶而影响DNA的复制,主要对增殖细胞敏感,为细胞周期特异性药物,作用于S期,对G1、G2与M期细胞有轻微的杀伤力。
曲古柳菌素A(Trichostatin A,TSA)是一种组蛋白去乙酰化酶抑制剂(histonedeacetylase inhibitor,HDACi)。
烟酰胺(Nicotinamide;NAM),又称尼克酰胺,是烟酸的酰胺化合物。为白色的结晶性粉末;无臭或几乎无臭,味苦;略有引湿性。在水或乙醇中易溶,在甘油中溶解。临床上主要用于防治糙皮病、口炎、舌炎,病态窦房结综合征,房室传导阻滞等问题。另外,烟酰胺作为组蛋白去乙酰化酶Sir2α的抑制剂,可以抑制Sir2α介导的p53去乙酰化,使得p53处于乙酰化状态,对于发挥p53功能起到重要作用(参见Luo,J.,等人,2001.'Negative controlof p53 by Sir2alpha promotes cell survival under stress',Cell,107:137-48)。
本发明人意想不到地发现,肿瘤细胞内源性PD-1参与p53依赖性肿瘤抑制通路,基于此,本发明人提出了以下方案。
因而,在第一方面,本发明提供了组蛋白去乙酰化酶抑制剂(histonedeacetylase inhibitor,HDACi)与激活p53通路的抗肿瘤剂的组合在制备用于***的药物中的用途,优选地,所述药物抑制受试者中的肿瘤生长。
在优选的实施方案中,所述组蛋白去乙酰化酶抑制剂包括但不限于:曲古柳菌素A(Trichostatin A,TSA)、烟酰胺、trapoxin B、苯基丁酸盐(phenylbutyrate)、丙戊酸、伏立诺他(vorinostat,suberanilohydroxamic acid或SAHA,作为)销售、贝利司他(belinostat,PXD101,作为销售)、帕比司他(panobinostat,作为销售)、达西司他(dacinostat,LAQ824)、恩替司他(entinostat,SNDX-275或MS-275)、他地那兰(tacedinaline,CI994)和莫西司他(mocetinostat,MGCD0103)。
进一步优选地,所述激活p53通路的抗肿瘤剂MDM2-p53抑制剂或细胞毒性剂,例如,Nutlin-3A,或喜树碱。
所述肿瘤为非小细胞肺癌、骨肉瘤、乳腺癌、大肠癌、胃癌、肝癌、卵巢癌、***、淋巴瘤、白血病、***癌、黑色素瘤、子宫内膜癌、神经母细胞瘤、神经胶质瘤、肉瘤或/和胆管细胞癌;优选为非小细胞肺癌或骨肉瘤。
优选地,根据本发明的受试者为哺乳动物受试者,优选地,所述受试者为人。
一种靶向肿瘤细胞关键分子的联合给药以提高肿瘤细胞内源性PD-1表达的方法。
另一方面,本发明提供了一种用于体外增强肿瘤细胞内源性PD-1表达的方法,所述方法包括以下:
1)在37℃恒温细胞培养箱中培养肿瘤细胞1天;
2)向步骤1)的肿瘤细胞中加入激活p53通路的抗肿瘤剂,将肿瘤细胞继续培养18小时;和
3)向步骤2)的肿瘤细胞中加入曲古柳菌素A和烟酰胺,处理细胞6小时。
在优选的实施方案中,其中当所述肿瘤细胞为p53-Tet-on可诱导p53稳定表达的H1299细胞系时,步骤2)中的激活p53通路的抗肿瘤剂为强力霉素;当所述肿瘤细胞为骨肉瘤细胞U2OS时,步骤2)中的激活p53通路的抗肿瘤剂选自喜树碱或Nutlin-3A。
优选地,步骤2)中所述的强力霉素使用水溶解,工作液浓度为1μg/mL;喜树碱或Nutlin-3A使用DMSO溶解/水稀释,喜树碱的工作液浓度为0.1-1μM,Nutlin-3A的工作液浓度为10μM。
优选地,步骤3)中曲古柳菌素A为DMSO溶解/水稀释,工作液浓度为1μM;烟酰胺用水溶解,工作液浓度为5mM。
附图说明
图1A和图1B:强力霉素联合HDACi显著增强非小细胞肺癌细胞中内源性PD-1的表达。图1A:强力霉素(Doxy)处理24小时和/或TSA+NAM处理最后6小时,RT-qPCR技术检测H1299 p53-Tet-on细胞中PD-1的mRNA表达水平;图1B:强力霉素(Doxy)处理24小时和/或TSA+NAM处理最后6小时蛋白质免疫印迹技术检测H1299p53-Tet-on细胞中PD-1的蛋白质表达水平。
图2A至图2D:CPT或Nutlin-3A联合HDACi显著增强骨肉瘤U2OS细胞中内源性PD-1的表达。图2A:CPT单独及联合TSA+NAM处理的U2OS细胞中PD-1的mRNA水平的改变;图2B:CPT单独及联合TSA+NAM处理的U2OS细胞中PD-1的蛋白质水平的改变;图2C:Nutlin-3A单独及联合TSA+NAM处理的U2OS细胞中PD-1的mRNA水平的改变;图2D:Nutlin-3A单独及联合TSA+NAM处理的U2OS细胞中PD-1的蛋白质水平(D)的改变。
图3A至图3C:肿瘤细胞中PD-1以一种免疫非依赖的方式调控肿瘤细胞生长。图3A:H1299-EV和H1299-PD-1稳转细胞系进行的小鼠荷瘤实验30天后的小鼠及瘤子的生长情况;图3B荷瘤实验(图3A)的瘤子的体积变化趋势;图3C:荷瘤实验(图3A)的瘤子的重量。
图4A至图4C:肿瘤细胞内源性PD-1参与p53依赖的肿瘤抑制。图4A:蛋白质免疫印迹检测H1299 p53-Tet-on细胞系中PD-1的敲减效率;图4B:细胞增殖实验分析H1299 p53-Tet-on sh-Ctr和sh-PD-1细胞在Doxy和/或TSA+NAM药物作用下的生长情况;图4C:根据图4B的结果进行的定量分析。
具体实施方式
为了更详细的说明本发明,本说明书提供了以下具体实施方案,并结合附图说明这些具体实施方案,但本发明的方案并非仅限于此。本领域技术人员可以结合本领域的公知常识,对本发明的方法、用途进行适当的改变,只要其能够实现本发明所述的功能,即应落入本发明的范围内。
实施例中未注明具体条件的实验方法,通常按照常规条件、或按照试剂、细胞或试剂盒的供应商所建议的条件进行。未注明具体来源的试剂,为市场购买的常规试剂。
实施例1:强力霉素联合HDACi显著增强非小细胞肺癌细胞中内源性PD-1的表达
利用p53缺陷的非小细胞肺癌H1299细胞系(购自国家实验细胞资源共享服务平台,资源编号:3111C0001CCC000469),构建p53-Tet-on可诱导表达的稳转细胞系(方法如Jiang,L.等人,2015.Ferroptosis as a p53-mediated activity during tumoursuppression',Nature,520:57-62所述)。接种该稳转细胞系于6孔板中,放于37℃恒温细胞培养箱中培养,一天后加入药物处理,以不加药的为阴性对照。
具体实施步骤如下:接种p53-Tet-on稳转细胞系于6孔板,培养于DMEM嘌呤霉素抗性培养基(含有2μg/mL嘌呤霉素、100U/mL青霉素、100μg/mL链霉素和10%FBS)中,放于细胞培养箱中培养。次日,加入强力霉素1μg/mL处理24小时,联合用药处理组在强力霉素处理18hrs时加入HDACi药物(1μM TSA和5μM NAM)再处理6小时,另设置强力霉素、HDACi药物单独处理组。最后,收取细胞进行mRNA和蛋白质水平的检测。
实验结果显示,强力霉素单独处理组诱导p53表达,同时检测到肺癌细胞中内源性PD-1mRNA(图1A)和蛋白质表达水平(图1B)增加;强力霉素和HDACi药物联合处理组,PD-1的表达显著增加;而HDACi单独处理组,只有微弱的增加。由此表明,靶向肿瘤细胞p53分子联合HDACi的给药能激活肺癌细胞内源性PD-1的表达。
实施例2:CPT或Nutlin-3A联合HDACi显著增强骨肉瘤U2OS细胞中内源性PD-1的表达
具体实施步骤如下:接种骨肉瘤细胞U2OS(购自国家实验细胞资源共享服务平台,资源编号:3111C0001CCC000028)于6孔板中,培养于DMEM培养基(含10%FBS)中,放于细胞培养箱中培养。次日,加入CPT 1μM或Nutlin-3A 10μM处理24小时,联合用药处理组在CPT或Nutlin-3A处理18小时加入HDACi药物(1μM TSA和5μM NAM)再处理6小时,另设置CPT/Nutlin-3A、HDACi药物单独处理组。最后,收取细胞进行mRNA和蛋白质水平的检测。
实验结果参见图2A至图2D,其显示CPT或Nutlin-3A单独处理组均能靶向增加p53表达,同时检测到骨肉瘤U2OS细胞中内源性PD-1mRNA和蛋白水平表达增加;在CPT或Nutlin-3A与HDACi药物的联合处理组中,PD-1的表达增加更为明显;而在HDACi药物的单独处理组中,PD-1的表达仅有微弱增加。由此表明,靶向肿瘤细胞p53的分子联合HDACi的给药也能增强骨肉瘤U2OS细胞中内源性PD-1的表达。
实施例3:肿瘤细胞中PD-1以免疫非依赖的方式调控肿瘤细胞生长
具体步骤如下:
(1)利用p53缺陷的H1299细胞系构建PD-1过表达的稳转细胞系,具体步骤如Chu,B.等人,2019.ALOX12 is required for p53-mediated tumour suppression through adistinct ferroptosis pathway,Nat Cell Biol,21:579-91所述;
(2)将3*106个PD-1过表达或对照细胞分别和基底胶以1.5:1的比例混匀,注射200μL细胞基底胶混合液到免疫全缺陷的6周龄B-NDG小鼠皮下,每只小鼠的左侧接种对照细胞,右侧接种PD-1过表达细胞;
(3)一周后统计瘤子的长和宽,按照公式:长*(宽)2除以2,计算出瘤子体积,之后每2-3天统计一次;
(4)在荷瘤实验进行到30天时终止实验,收取小鼠皮下的瘤子进行拍照、称重及数据分析。
实验结果显示(如图3A至图3C所示),在免疫全缺陷的小鼠中开展的荷瘤实验,不论是从瘤子的重量还是从瘤子的生长趋势来看,PD-1的表达对肿瘤细胞生长起到抑制作用,而且这种抑制作用是一种免疫非依赖的形式。
实施例4:肿瘤内源性PD-1参与p53依赖的肿瘤抑制
具体步骤如下:
(1)在H1299 p53-Tet-on稳转细胞系中,利用慢病毒干扰PD-1的表达,筛选出H1299 p53-Tet-on sh-PD-1和sh-Ctr的细胞系,方法如Jiang,L.,等人,2015.Ferroptosisas a p53-mediated activity during tumour suppression,Nature,520:57-62所示。
(2)接种H1299 p53-Tet-on sh-PD-1和sh-Ctr细胞于6孔板中,设计不加药组作为阴性对照,处理组分别为Doxy、TSA+NAM以及Doxy联合TSA+NAM处理,各组有3个重复。
(3)加药培养2天后,用4%多聚甲醛室温固定20分钟,再用0.1%结晶紫染色、拍照。
(4)将结晶紫染色的细胞用10%的乙酸溶解30分钟,通过酶标仪检测吸光值给各组细胞进行相对计数及统计学分析。
实验结果如图4A至图4C所示,其显示Doxy单独给药时,p53表达能抑制肿瘤细胞生长;TSA+NAM单独给药时,对肿瘤细胞生长的抑制作用较弱;而Doxy和TSA+NAM联合给药时,即肿瘤细胞内同时激活p53和PD-1时,显著抑制了肿瘤细胞的生长。与对照组细胞相比,敲减PD-1会降低药物对肿瘤细胞生长的抑制作用。由此表明,肿瘤细胞内源性PD-1参与p53依赖的肿瘤抑制。
Claims (10)
1.组蛋白去乙酰化酶抑制剂与激活p53通路的抗肿瘤剂的组合在制备用于治疗受试者的肿瘤的药物中的用途。
2.根据权利要求1所述的用途,其中所述组蛋白去乙酰化酶抑制剂选自曲古柳菌素A、烟酰胺、trapoxin B、苯基丁酸盐、丙戊酸、伏立诺他、贝利司他、帕比司他、达西司他、恩替司他、他地那兰和莫西司他中的一种或多种,优选为曲古柳菌素A和烟酰胺。
3.根据权利要求1或2所述的用途,其中所述激活p53通路的抗肿瘤剂选自Nutlin-3A或喜树碱。
4.根据权利要求1-3中任一项所述的用途,其中所述肿瘤选自非小细胞肺癌、骨肉瘤、乳腺癌、大肠癌、胃癌、肝癌、卵巢癌、***、淋巴瘤、白血病、***癌、黑色素瘤、子宫内膜癌、神经母细胞瘤、神经胶质瘤、肉瘤或/和胆管细胞癌;优选为非小细胞肺癌或骨肉瘤。
5.激活p53通路的抗肿瘤剂、曲古柳菌素A和烟酰胺的组合在制备用于治疗受试者的肿瘤的药物中的用途。
6.根据权利要求1-5中任一项所述的用途,其中所述药物抑制受试者中的肿瘤生长。
7.根据权利要求1-6中任一项所述的用途,其中所述受试者为人。
8.一种用于体外增强肿瘤细胞内源性PD-1表达的方法,所述方法包括以下步骤:
1)在37℃恒温细胞培养箱中培养肿瘤细胞1天;
2)向步骤1)的肿瘤细胞中加入激活p53通路的抗肿瘤剂,将肿瘤细胞继续培养18小时;和
3)向步骤2)的肿瘤细胞中加入曲古柳菌素A和烟酰胺处理细胞6小时。
9.根据权利要求8所述的方法,其中当所述肿瘤细胞为p53-Tet-on***可诱导p53稳定表达的H1299细胞系时,步骤2)中的激活p53通路的抗肿瘤剂为强力霉素;当所述肿瘤细胞为骨肉瘤细胞U2OS时,步骤2)中的激活p53通路的抗肿瘤剂选自喜树碱或Nutlin-3A。
10.根据权利要求9所述的方法,其中步骤2)中所述的强力霉素使用水溶解,工作液浓度为1μg/mL;喜树碱或Nutlin-3A使用DMSO溶解/水稀释,喜树碱的工作液浓度为0.1-1μM,Nutlin-3A的工作液浓度为10μM;
步骤3)中曲古柳菌素A为DMSO溶解/水稀释,工作液浓度为1μM;烟酰胺用水溶解,工作液浓度为5mM。
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CN115975942B (zh) * | 2023-02-27 | 2023-09-22 | 山东第一医科大学附属肿瘤医院(山东省肿瘤防治研究院、山东省肿瘤医院) | 一种胰腺癌免疫治疗耐药细胞系及其制备方法和应用 |
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