CN108245524A - 隐丹参酮在抑制stat5蛋白磷酸化方面的应用 - Google Patents
隐丹参酮在抑制stat5蛋白磷酸化方面的应用 Download PDFInfo
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Abstract
本发明公开了隐丹参酮在抑制STAT5蛋白磷酸化方面的应用。本发明研究发现,隐丹参酮能够有效地抑制细胞内STAT5蛋白的磷酸化以及磷酸化STAT5蛋白所调控的下游蛋白的表达;该抑制作用具有特异性、显著性和迅速性。因此,隐丹参酮对STAT5蛋白磷酸化异常的细胞具有显著的生长抑制作用及凋亡诱导作用,基于本发明,隐丹参酮可以作为一种新的STAT5蛋白磷酸化的抑制剂,在STAT5蛋白磷酸化的高效抑制剂制备,以及STAT5蛋白异常磷酸化的疾病治疗等方面,都具有重要的科学意义与临床应用价值。
Description
技术领域
本发明属于生物医药技术领域。更具体地,涉及隐丹参酮在抑制STAT5蛋白磷酸化方面的应用。
背景技术
STAT5是信号转导与转录激活因子(Signal Transducer and Activator ofTranscription,STAT)家族中的一员,被激酶磷酸化后可作为转录因子在细胞信号通路的转导中起着承上启下的重要作用,磷酸化后的STAT5蛋白在生长发育、免疫调节、疾病的发生发展等生理病理过程中均具有重要作用。
在STAT5异常磷酸化所致的疾病中,异常磷酸化的STAT5蛋白可以显著上调其下游细胞信号通路中众多的增殖相关蛋白及凋亡抵抗相关蛋白的表达,使细胞呈现出快速增殖和不易凋亡的恶变特性,而有效抑制STAT5蛋白的异常磷酸化能够显著抑制恶变细胞的增殖并有效地诱导其凋亡。
因此,开发新的更有效的STAT5蛋白磷酸化的抑制剂,以及磷酸化STAT5蛋白所调控的通路中的下游蛋白的高效抑制剂已成为当今病变细胞靶向药物的热点。
发明内容
本发明要解决的技术问题是克服上述现有技术的缺陷和技术不足,提供一种STAT5蛋白磷酸化抑制剂。本发明研究发现,隐丹参酮能够有效抑制细胞内STAT5蛋白的磷酸化,有望开发成为STAT5蛋白的磷酸化抑制剂应用于试剂开发、药物制备和药效分析评价及临床检验中。
本发明的目的是提供隐丹参酮在作为或制备STAT5蛋白磷酸化的抑制剂中的应用。
本发明上述目的通过以下技术方案实现:
本发明研究发现,隐丹参酮在一定条件下能够有效抑制STAT5蛋白的磷酸化,并且进一步下调磷酸化STAT5蛋白所调控的信号通路下游蛋白的表达,从而显著地抑制细胞生长并有效地诱导细胞凋亡。在实验中,隐丹参酮有效的作用浓度为大于或等于1μmol/L,最佳的作用浓度为大于或等于20μmol/L;在20μmol/L隐丹参酮的浓度下,有效作用时间为大于或等于0.5小时,最佳作用时间为大于或等于4小时。通过研究,我们发现隐丹参酮是STAT5蛋白磷酸化的高效抑制剂,可用于抑制STAT5蛋白异常磷酸化细胞的增殖;采用包含隐丹参酮的制剂或与其他药物联合用于制备治疗由于STAT5蛋白异常磷酸化所导致疾病(如慢性髓性白血病等)的药物。
因此,以下应用均应在本发明的保护范围之内:
隐丹参酮在作为或制备STAT5蛋白磷酸化抑制剂中的应用,其特征在于,所述的隐丹参酮还包括其可药用的盐和酯、选择性取代的类似物或者包含隐丹参酮的一种或多种化合物的组合;还包括隐丹参酮的衍生物,或者其衍生物在药学上可接受的盐或盐的溶剂化物。
所述STAT5蛋白磷酸化抑制剂的作用对象不包括STAT5总蛋白、但包括磷酸化STAT5蛋白以及磷酸化STAT5蛋白所调控的信号通路下游的靶标分子。
隐丹参酮的有效剂量为大于或等于1μmol/L,最佳剂量为大于或等于20μmol/L。
隐丹参酮抑制人细胞中STAT5蛋白磷酸化的有效时间为大于或等于0.5小时,最佳时间为大于或等于4小时。
隐丹参酮在抑制STAT5蛋白异常磷酸化细胞的生长和/或在制备治疗STAT5蛋白异常磷酸化疾病的药物中的应用中的应用。
所述STAT5蛋白异常磷酸化细胞为肿瘤细胞,所述STAT5蛋白异常磷酸化疾病为肿瘤;所述肿瘤为血液肿瘤或固体瘤。
优选地,所述肿瘤为慢性髓性白血病、慢性粒细胞性白血病、急性淋巴细胞性白血病、急性髓性白血病、神经胶质细胞瘤、乳腺癌、结肠癌、肝癌、宫颈癌、Burkitt's淋巴瘤或黑色素瘤等。
优选地,所述药物可口服给药或注射给药,包括可接受的药用载体制成各种药学上可接受的制剂,如针或冻干粉针制剂、丸剂、汤剂、片剂、颗粒剂、硬胶囊、软胶囊、控释、缓释制剂、注射剂或口服制剂,还包括一种以上的药学上可接受的赋形剂。
另外,所述药物作为STAT5蛋白的磷酸化抑制剂可以单独使用或与其他药物联合配伍使用。
针对STAT5蛋白异常磷酸化的疾病,在制药和临床治疗中将隐丹参酮用于制备STAT5蛋白磷酸化的抑制剂以及抑制磷酸化STAT5蛋白所调控的下游蛋白的预防、治疗、辅助治疗和/或预后防复发的药物及保健品。隐丹参酮在针对磷酸化STAT5蛋白及其下游蛋白异常表达的疾病的临床检验和药效检测评估中的应用。隐丹参酮在开发、制备磷酸化STAT5蛋白的相关抑制剂或开发、制备用于抑制STAT5蛋白磷酸化的分析试剂或生化试剂或检测试剂中的应用。隐丹参酮在细胞与分子生物学基础研究中作为抑制磷酸化STAT5蛋白和干预其靶标蛋白表达的细胞增殖抑制剂或细胞凋亡诱导剂的应用。
隐丹参酮作为STAT5蛋白磷酸化抑制剂在制备提高机体免疫力的药物、保健药品或保健食品方面的应用;以及隐丹参酮在设计、开发、制备针对磷酸化STAT5蛋白的相关抑制剂或将之作为生化试剂、分析试剂或检测试剂中的应用;以及隐丹参酮作为STAT5蛋白磷酸化抑制剂在药效检测评估、临床检验以及相关药理、药效和临床研究中的应用,均应在本发明的保护范围之内。
具体可在基础研究中将隐丹参酮作为抑制STAT5蛋白磷酸化的细胞增殖抑制剂和细胞凋亡诱导剂的应用,以及将隐丹参酮作为研究STAT5蛋白的磷酸化、磷酸化调控过程、磷酸化后的STAT5蛋白所调控的细胞信号通路以及与上述通路相关的细胞信号通路的生物学功能提供了的新技术、新方法和新手段。
本发明具有以下有益效果:
本发明公开了隐丹参酮在作为或制备STAT5蛋白磷酸化的抑制剂中的应用。基于本发明,用隐丹参酮制备STAT5蛋白磷酸化的抑制试剂,隐丹参酮是一种天然的二萜醌类化合物,主要来源于中国传统草药丹参,易于制备,且对人体毒性较低,可应用性强。
本发明用隐丹参酮制备的抑制剂不仅高效低毒,还能够抑制磷酸化STAT5蛋白所调控的众多下游靶蛋白。该制剂专一性强、见效快且易于操作,为靶向治疗疾病提供了思路,并为实验研究STAT5蛋白及其磷酸化蛋白以及它们所调控的下游靶蛋白的生物学功能提供了新技术、新方法和新手段。
在多种疾病中,隐丹参酮制备的抑制剂可抑制STAT5蛋白的磷酸化以及磷酸化STAT5蛋白所介导的与增殖、凋亡密切相关的细胞信号通路网络,从转录水平抑制众多下游蛋白的表达,最终抑制细胞生长、促使细胞发生凋亡。这种抑增殖、促凋亡的特性在肿瘤治疗中尤为突出,其抗癌活性对多种肿瘤细胞和个体均有显著效果。
附图说明
图1. 实施例1中不同浓度的隐丹参酮对STAT5总蛋白与STAT5磷酸化蛋白的作用。
图2. 实施例2中20µM隐丹参酮对STAT5总蛋白与STAT5磷酸化蛋白在不同时间点的作用。
图3. 实施例3中隐丹参酮对K562细胞的增殖抑制作用。
具体实施方式
以下结合具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。除非特别说明,本发明所用试剂和材料均为市购。
以下实施例中所用试剂如下:
隐丹参酮:购自SelleckChemicals公司,纯度经高效液相色谱(HPLC)测定大于99%。使用二甲基亚枫(DMSO)配置成5µmol/L的储液,冻于-80度,每次使用时用培养基稀释至工作浓度。
K562细胞系:购自中国科学院上海生命科学研究院细胞资源中心。
抗体:p-STAT5(Tyr694)和GAPDH抗体均购自美国Cell Signaling Technology公司。
实施例1 隐丹参酮对磷酸化STAT5蛋白的抑制呈剂量依赖性
1、实验方法
(1)获取细胞
取培养至对数生长期的慢性髓性白血病K562细胞,750rpm离心3分钟进行收集,用仅含10%胎牛血清(Fetal Bovine Serum,简称FBS)的RPMI-1640培养基重悬细胞,并将细胞悬液的浓度调至3×105个/ml,以500µL/孔的量将上述细胞悬液接入48孔板内,置于37℃,5% CO2培养箱中培养24小时;
(2)加药处理
取配好的5mM隐丹参酮母液用DMSO溶液稀释至工作浓度(0µM,1µM,2.5µM,5µM,7.5µM,10µM,15µM,20µM和30µM)的100倍,故配制浓度分别为0mM,0.1mM,0.25mM,0.5mM,0.75mM,1mM,1.5mM,2mM和3mM,吹打混匀,并加入相应浓度的孔中。而后,将细胞培养板置于37℃,5%CO2培养箱中培养24小时;
(3)收集细胞,进行Western blotting实验
3000rpm离心5分钟收集细胞,依据细胞沉淀量的多少加入相应量的1×SDS LoadingBuffer 于99℃煮10分钟,裂解蛋白并通过Western blotting实验检测磷酸化后的STAT5蛋白的表达情况。
2、实验结果:
随着浓度的增加,隐丹参酮能够逐步抑制STAT5蛋白的磷酸化。在药物浓度达到20μmol/L时,抑制效率显著提高,且抑制效果会随药物浓度的增加而进一步增强。
实施例2 隐丹参酮对磷酸化STAT5蛋白的抑制呈时间依赖性
1、实验方法
(1)获取细胞
取培养至对数生长期的慢性髓性白血病K562细胞,750rpm离心3分钟进行收集,用仅含10%胎牛血清(Fetal Bovine Serum,简称FBS)的RPMI-1640培养基重悬细胞,并将细胞悬液的浓度调至3×105个/ml,以500µL/孔的量将上述细胞悬液接入48孔板内,置于37℃,5% CO2培养箱中培养24小时;
(2)加药处理
取配好的5mM隐丹参酮母液用DMSO溶液稀释至工作浓度20µM的100倍,即2mM,吹打混匀,并加入各孔中;对照组(0µM隐丹参酮)加入相应量的DMSO。而后,将细胞培养板置于37℃,5% CO2培养箱中培养;
(3)收集细胞,进行Western blotting实验
分别于加药后0.5、1、2、4、6、12和24小时以3000rpm离心5分钟收集相应组别的细胞,对照组(0µM隐丹参酮)与24小时组的样品一起收集。依据细胞沉淀量的多少加入相应量的1×SDS Loading Buffer 于99°C煮10分钟,裂解蛋白并通过Western blotting实验检测磷酸化后的STAT5蛋白的表达情况。
2、实验结果:
20µM隐丹参酮在0.5小时可发挥抑制STAT5蛋白的磷酸化作用;随着作用时间的延长,隐丹参酮能够显著抑制STAT5蛋白的磷酸化,并于12小时后清空该蛋白。
实施例3 隐丹参酮显著抑制K562细胞的增殖
1、实验方法
(1)获取细胞
取培养至对数生长期的慢性髓性白血病K562细胞,750rpm离心3分钟进行收集,用仅含10%胎牛血清(Fetal Bovine Serum,简称FBS)的RPMI-1640培养基重悬细胞,并将细胞悬液的浓度调至3×105个/ml,以100µL/孔的量将上述细胞悬液接入96孔板内,置于37℃,5% CO2培养箱中培养24小时。
(2)加药处理
取配好的5mM隐丹参酮母液用DMSO溶液稀释至工作浓度(0µM,1µM,2.5µM,5µM,7.5µM,10µM,15µM,20µM和30µM)的100倍,故配制浓度分别为0mM,0.1mM,0.25mM,0.5mM,0.75mM,1mM,1.5mM,2mM和3mM,吹打混匀,并加入相应浓度的孔中。而后,将细胞培养板置于37℃,5%CO2培养箱中培养24小时;
(3)在各细胞培养孔内加入15µL MTT溶液(MTT试剂盒购自Promega公司,USA),置于37℃,5% CO2培养箱中培养4小时;
(4)在(3)中各孔内加入100µL STOP溶液,置于37℃,5% CO2培养箱中培养24小时;
(5)取出96孔培养板,分别于570nm波长和630nm波长的激发光下检测各孔的吸光值,A570-A630,即为各孔真实的吸光值,反应的是各孔中细胞的存活率,据此可算出各孔细胞在药物作用下的增殖抑制情况。
2、实验结果:
结果如附图3所示,隐丹参酮可以显著抑制K562细胞的增殖,半致死剂量约为15~20µM。
综上结果显示,本发明研究发现,隐丹参酮对于细胞中STAT5蛋白的磷酸化具有显著的抑制作用,且该抑制作用呈现明显的剂量依赖性和时间依赖性。当隐丹参酮的浓度大于或等于1μmol/L时,抑制效果即可显现出来,且随着药物浓度的增加,抑制作用不断增强,在药物浓度大于等于20μmol/L时,抑制效率显著提高,且抑制效果会随药物浓度的增加而进一步增强。隐丹参酮对STAT5蛋白的磷酸化的抑制作用,在用药物处理后0.5小时即可显现出来,且随着作用时间的延长,抑制作用会持续进行,在作用时间大于4小时后效果愈加明显。以隐丹参酮制备的STAT5蛋白的磷酸化抑制剂,对于抑制磷酸化STAT5蛋白的下游靶蛋白也具有良好的效果。
因此可有如下应用:隐丹参酮在制备治疗STAT5蛋白异常磷酸化疾病的药物中的应用,如:制备抑制肿瘤细胞增殖和抗肿瘤(包括血液肿瘤和固体瘤)的药物;隐丹参酮在设计、开发、制备STAT5蛋白磷酸化的相关抑制剂或将之作为生化试剂、分析试剂或检测试剂中的应用;隐丹参酮作为STAT5蛋白磷酸化的抑制剂在药效检测评估、临床检验以及相关药理、药效和临床研究中的应用;将隐丹参酮作为抑制STAT5蛋白磷酸化的细胞增殖抑制剂和细胞凋亡诱导剂在生物与医学基础研究中的应用,以及将隐丹参酮作为研究STAT5蛋白及其磷酸化蛋白以及它们调控的细胞信号通路中相关靶蛋白的生物学功能的新技术、新方法和新手段。
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 (10)
1.隐丹参酮在作为或制备STAT5蛋白磷酸化抑制剂中的应用,其特征在于,所述的隐丹参酮还包括其可药用的盐和酯、选择性取代的类似物或者包含隐丹参酮的一种或多种化合物的组合;还包括隐丹参酮的衍生物,或者其衍生物在药学上可接受的盐或盐的溶剂化物。
2.根据根据权利要求1所述的应用,其特征在于,所述STAT5蛋白磷酸化抑制剂的作用对象不包括STAT5总蛋白、但包括磷酸化STAT5蛋白以及磷酸化STAT5蛋白所调控的信号通路下游的靶标分子。
3.根据权利要求1所述的应用,其特征在于,隐丹参酮的有效剂量为不低于1μmol/L,最佳剂量为不低于20μmol/L;隐丹参酮抑制人细胞中STAT5蛋白磷酸化的有效时间为不低于0.5小时,最佳时间为不低于4小时。
4.隐丹参酮在抑制STAT5蛋白异常磷酸化细胞的生长和/或在制备治疗STAT5蛋白异常磷酸化疾病的药物中的应用。
5.根据权利要求4所述的应用,其特征在于,所述STAT5蛋白异常磷酸化细胞为肿瘤细胞,所述STAT5蛋白异常磷酸化疾病为肿瘤;所述肿瘤为血液肿瘤或固体瘤。
6.根据权利要求5所述的应用,其特征在于,所述肿瘤为慢性髓性白血病、慢性粒细胞性白血病、急性淋巴细胞性白血病、急性髓性白血病、神经胶质细胞瘤、乳腺癌、结肠癌、肝癌、宫颈癌、Burkitt's淋巴瘤或黑色素瘤。
7.根据权利要求4所述的应用,其特征在于,所述药物作为STAT5蛋白的磷酸化抑制剂可以单独使用或与其他药物联合配伍使用。
8.隐丹参酮作为STAT5蛋白磷酸化抑制剂在制备提高机体免疫力的药物、保健药品或保健食品方面的应用。
9.隐丹参酮在设计、开发、制备针对磷酸化STAT5蛋白的相关抑制剂或将之作为生化试剂、分析试剂或检测试剂中的应用,或隐丹参酮作为研究磷酸化STAT5蛋白调控的细胞信号通路及其相关靶蛋白生物学功能的新方法和技术方面的应用。
10.隐丹参酮作为STAT5蛋白磷酸化抑制剂在药效检测评估、临床检验以及相关药理、药效和临床研究中的应用。
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