CN114209696B - 去氢毛钩藤碱在制备抗慢性粒细胞白血病药物中的应用 - Google Patents
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Abstract
本发明提供了去氢毛钩藤碱在制备抗慢性粒细胞白血病药物中的应用,属于医药技术领域。本发明在人源慢性粒细胞白血病细胞模型上证明去氢毛钩藤碱能够通过靶向抑制鞘氨醇激酶1而抑制白血病细胞增殖,引起细胞周期阻滞,诱导细胞凋亡,发挥抗慢性粒细胞白血病活性,去氢毛钩藤碱在人源慢性粒细胞白血病治疗中具有较好的开发前景。
Description
技术领域
本发明属于医药技术领域,尤其是涉及一种去氢毛钩藤碱在制备抗慢性粒细胞白血病药物中的应用。
背景技术
慢性粒细胞白血病(Chronic Myeloid Leukemia,简称为CML)是血液系统的恶性克隆性疾病。研究发现CML的发病机制主要为费城染色体(Ph染色体)的形成。Ph染色体是由t(9;22)(q34;q11)染色体易位形成,其基因产物bcr-abl编码的致癌蛋白BCR-ABL具有持续的酪氨酸激酶活性,介导致癌信号的传递。目前,临床上治疗CML的分子靶向药物主要是酪氨酸激酶抑制剂伊马替尼(Imatinib)。伊马替尼显著延长了CML患者的生存期,但是原发和继发性耐药的发生以及药物本身的毒副作用提示开发新的抗CML药物仍是亟需解决的课题。
目前,尚无有关去氢毛钩藤碱抗CML活性的相关报道,去氢毛钩藤碱在抗CML药物领域的开发对部分CML患者具有重要意义。
发明内容
有鉴于此,为了克服现有药物存在的对CML亲本细胞和耐药细胞治疗选择性的差异,药物治疗疗效不甚满意的问题,本发明提出了去氢毛钩藤碱在制备抗慢性粒细胞白血病药物中的应用,去氢毛钩藤碱能够通过靶向抑制鞘氨醇激酶1达到抗CML的效果。
鞘氨醇激酶1(Sphingosine Kinase 1,简称为SPHK1)在包括白血病在内的多种肿瘤中表达水平较高,且与肿瘤预后密切相关,SPHK1也是调节鞘脂变阻器的关键酶,在肿瘤发生、发展过程中发挥重要作用。
为达到上述目的,本发明的技术方案是这样实现的:
本发明提供了去氢毛钩藤碱在制备抗慢性粒细胞白血病药物中的应用,所述去氢毛钩藤碱的结构式,如(I)所示:
化学式为C22H26N2O3;分子量为366.45。
进一步的,所述去氢毛钩藤碱具有抑制CML细胞增殖活性的作用,并呈现为剂量依赖性。
进一步的,所述去氢毛钩藤碱通过靶向抑制鞘氨醇激酶1抑制CML细胞增殖活性。
进一步的,所述去氢毛钩藤碱对人源CML亲本细胞K562的增殖抑制活性的IC50值为12.33μM,对耐药细胞K562/G01的增殖抑制活性的IC50值为12.77μM。
进一步的,所述去氢毛钩藤碱能够诱导CML细胞G2/M期周期阻滞。
进一步的,所述去氢毛钩藤碱能够诱导CML细胞凋亡。
本发明另一方面还提供了一种抗慢性粒细胞白血病药物,包括作为活性成分的去氢毛钩藤碱,优选的,还包括其药学上可接受的载体。
进一步的,所述药物的剂型为颗粒剂、片剂、胶囊剂、粉末剂、油滴剂、溶液剂或它们的组合。
相对于现有技术,本发明所述的去氢毛钩藤碱在制备抗慢性粒细胞白血病药物中的应用具有以下优势:
本发明所述的去氢毛钩藤碱在人源慢性粒细胞白血病(CML)细胞模型上能够通过靶向抑制鞘氨醇激酶1(SPHK1)而抑制白血病细胞增殖,引起细胞周期阻滞,诱导细胞凋亡,发挥抗慢性粒细胞白血病活性,去氢毛钩藤碱在CML治疗中具有较好的开发前景,可解决现有药物存在的对CML亲本细胞和耐药细胞治疗选择性的差异,药物治疗疗效不甚满意问题,为开发新的抗CML药物提供了新思路。
附图说明
图1为本发明实施例1中去氢毛钩藤碱抑制CML细胞增殖作用;
图2为本发明实施例2中去氢毛钩藤碱诱导CML细胞周期阻滞作用;
图3为本发明实施例3中去氢毛钩藤碱诱导CML细胞凋亡作用;
图4为本发明实施例4中去氢毛钩藤碱抑制CML细胞中鞘氨醇激酶1及BCR-ABL/PI3K/Akt通路;
图5为本发明实施例5中去氢毛钩藤碱通过靶向抑制SPHK1发挥抗CML作用。
具体实施方式
除有定义外,以下实施例中所用的技术术语具有与本发明所属领域技术人员普遍理解的相同含义。以下实施例中所用的试验试剂,如无特殊说明,均为常规生化试剂;所述实验方法,如无特殊说明,均为常规方法。
下面结合实施例及附图来详细说明本发明。
实施例1
去氢毛钩藤碱抑制CML细胞增殖测试
采用MTT法检测去氢毛钩藤碱抑制细胞增殖效果,选取人源CML亲本细胞K562及其多药耐药细胞K562/G01。
细胞培养:人源CML亲本细胞K562及其多药耐药细胞K562/G01,采用10%FBS的RPMI-1640培养基培养,置于37℃、5%CO2的培养箱中。对于K562/G01细胞耐药培养,显微镜下观察细胞状态良好后,无菌条件下收集细胞进行传代,每次传代完毕后向培养基中加入Imatinib,其浓度从6μM逐渐增加至10μM,然后脱药培养两周,状态稳定后可用于后续实验。
细胞铺板:取对数生长期K562和K562/G01细胞进行计数,以密度为4×104细胞/ml,每孔200μl,接种于96孔板中,设定空白对照,即不含细胞悬液的空培基。铺好板后将其放入37℃、5%CO2培养箱中。
给药处理:设置药物处理组和对照组,对照组加入0.5μl DMSO,药物处理组给予0.5μl终浓度为4、8、16、32、64、100μM的去氢毛钩藤碱DMSO溶液,每个浓度设置5个复孔,药物与细胞共孵育48h。
检测:96孔板中每孔加入20μl MTT溶液,轻轻拍打混匀,孵育4h后离心,1000rpm,5min,吸去150μl上清,再给各孔加150μl DMSO,吹打混匀,酶标仪450nm波长下检测吸光度值。
数据分析:细胞存活率=(药物处理组OD值-空白对照组OD值)/(DMSO对照组OD值-空白对照组OD值)×100%
结果:见图1所示:去氢毛钩藤碱对K562以及K562/G01均有显著的增殖抑制作用,具有剂量依赖性,其半数抑制率IC50分别为12.33μM,12.77μM,去氢毛钩藤碱对亲本细胞和耐药细胞抑制增殖的效果相当,无显著差异。
实施例2
去氢毛钩藤碱诱导CML细胞G2/M期周期阻滞测试
细胞培养同实施例1。
细胞铺板:收集对数生长期细胞K562和K562/G01,计数,以密度为4×105细胞/ml,每孔2ml,接种于六孔板,摇匀。
给药处理:设置药物处理组和对照组,对照组加入2μl DMSO,药物处理组给予2μl终浓度为8、16、32μM的去氢毛钩藤碱DMSO溶液,摇匀后在培养箱中共孵育48h。
固定:收集细胞并离心,1000rpm,5min,细胞沉淀用PBS重悬,离心1000rpm,5min,加入250μl PBS重悬,将细胞悬液逐滴加入750μl冰乙醇中,置4℃过夜。
染色:取出已固定的细胞,2500rpm离心5min,弃上清,1mL冰PBS清洗一遍,2500rpm离心5min,弃上清,细胞沉淀用100μl PBS重悬,再加入50μg/mL的PI染液200μl,混匀,4℃避光孵育1h。
测定:尼龙网过滤待测细胞悬液后,采用流式细胞仪检测细胞周期分布情况。
Western blot检测相关蛋白:细胞铺板及给药方式同上,共孵育48h后收集细胞,加入RIPA裂解液和蛋白酶抑制剂,提取总蛋白,BCA法进行蛋白定量,蛋白样品与上样缓冲液混匀,95℃处理5min,进行SDS-PAGE,60V,30min,100V,1h。电泳完成后将其转移到PVDF膜上,5%脱脂奶粉室温封闭1h;将PVDF膜和一定浓度的一抗密封于杂交袋后在4℃摇床过夜孵育,1×TBST清洗四次,每次10min;室温孵育二抗1h,1×TBST清洗四次,每次10min;加ECL显影。
结果:见图2所示:流式细胞术结果显示,去氢毛钩藤碱能够诱导CML细胞G2/M期周期阻滞。32μM去氢毛钩藤碱引起K562细胞G2/M期细胞比例升高到45.9%,K562/G01细胞G2/M期细胞比例升高到38.8%;Western blot结果显示,去氢毛钩藤碱干预K562和K562/G01细胞后,细胞周期G2/M期关键因子CDC2和cyclin B1表达呈现剂量依赖性降低。
实施例3
去氢毛钩藤碱诱导CML细胞凋亡测试
细胞培养、细胞铺板和给药处理方法同实施例2。
检测方法:收集细胞,离心,1000rpm,5min,弃上清,PBS清洗一次,100μl 1×Binding Buffer重悬细胞沉淀,将对照组细胞分成4组,即Blank、Annexin-V单阳组、PI单阳组、Annexin-V/PI双阳组,按要求分别加入Annexin-V和PI;药物处理组加入5μl Annexin-V和5μl PI染液,室温下避光孵育15min,反应完成后,再加入100μl 1×Binding Buffer,尼龙网膜过滤后进行流式细胞检测。采用Western blot检测凋亡相关蛋白caspase3和PARP的表达情况:方法同实施例2种Western blot法。
结果:见图3所示:去氢毛钩藤碱可以剂量依赖性诱导K562和K562/G01细胞发生凋亡,32μM去氢毛钩藤碱处理后,K562中凋亡细胞比例升高到29.2%,K562/G01中凋亡细胞比例升高到35.88%;Western blot结果显示,去氢毛钩藤碱干预K562和K562/G01细胞后,caspase 3和PARP均发生裂解活化,caspase 3和PARP蛋白表达水平降低,cleaved caspase3和cleaved PARP表达水平升高。
实施例4
去氢毛钩藤碱抑制CML细胞中鞘氨醇激酶1及BCR-ABL/PI3K/Akt通路测试
细胞培养、细胞铺板和给药处理方法同实施例2。
反转录PCR:收集细胞,PBS清洗一次,细胞沉淀中加入500μl Trizol,混匀后室温静置5min,加入150μl氯仿,涡旋震荡15s至无明显分层,室温静置5min,4℃下12000g离心15min,吸取上层水相150μl,加入150μl异丙醇,混合均匀后静置10分钟,4℃下12000g离心15min,弃上清,采用75%乙醇(DEPC水配制)清洗2次,7500g离心5min,弃上清,沉淀静置5min,采用DEPC水溶解后测定RNA浓度。取RNA 500ng,采用Takara逆转录试剂盒进行反转录;
荧光定量PCR:
实验所用S1PR1引物序列为:正向5’-GAGAACAGCATTAAACTGACC-3’;反向5’-CCAGGATGATAAAGCAGCAG-3’;GAPDH引物序列为正向5’-CATGAGAAGTATGACAACAGCCT-3’;反向5’-AGTCCTTCCACGATACCAAAGT-3’。配制PCR反应溶液,向PCR小管中加入引物、cDNA、DEPC水以及TB Green Premix Ex Taq II,反应体系总体积为25μl,采用CFX96系统按照试剂盒说明书设定反应条件:第一步:95℃预变性30s;第二步:95℃变性5s,60℃退火30s,40个循环;第三步:溶解曲线。利用2-△△Ct相对定量法进行结果分析。相对表达量=2-(给药组待测基因Ct平均值-给药组GAPDH Ct平均值)/对照组待测基因Ct平均值-对照组GAPDH Ct平均值)。
ELISA:细胞培养、铺板和给药处理方法同实施例2。检测SPHK1活性和神经酰胺(Ceramide,Cer)水平时,收集细胞,1000rpm离心5min,PBS清洗一次,加入600μl PBS重悬,反复冻融三次,12000rpm离心10min,将上清移入新的EP管中,然后按照试剂盒说明书进行实验;检测S1P水平时,收集细胞,1000rpm离心5min,吸取上清液,检测培基中S1P水平,检测方法严格按照试剂盒说明书操作。
Western blot检测蛋白表达:方法同实施例2中Western blot法
结果:见图4所示,在给予去氢毛钩藤碱处理后,SPHK1蛋白表达水平及酶活性显著降低,BCR-ABL/PI3K/Akt通路中BCR-ABL、p-BCR-ABL(Tyr412)、PI3K p110α、p-Akt(Ser473)均被下调,同时ELISA结果显示去氢毛钩藤碱下调S1P含量,上调Cer含量,提示其对鞘脂变阻器的调控作用,以上结果表明,去氢毛钩藤碱能够抑制CML细胞中鞘氨醇激酶1及BCR-ABL/PI3K/Akt通路。
实施例5
去氢毛钩藤碱通过靶向抑制SPHK1发挥抗CML作用测试
构建SPHK1过表达质粒(上海吉凯),将其转染进入K562和K562/G01细胞,对照组转染空质粒,同时,选取SPHK1特异性抑制剂PF543作为阳性对照,进行了细胞增殖能力以及SPHK1下游相关蛋白水平的检测。
转染:收集对数生长期细胞K562和K562/G01,计数,采用无血清1640培养基以密度为2×105细胞/ml,每孔1ml,接种于12孔板,摇匀,采用Opti-MEM培基制备转染复合物,其中DNA与lipo2000比例为1:1,向每个孔中加入50μl转染试剂,同时设置阴性对照,转染6h后,弃去无血清培基,加入含10%FBS新鲜1640培基培养过夜,第二天进行加药处理。设置阴性对照组(NC),SPHK1过表达组,去氢毛钩藤碱处理组(HST,32μM),SPHK1过表达和去氢毛钩藤碱联合处理组。
MTT法检测细胞增殖能力,方法同实施例2中MTT法。
Western blot检测SPHK1及其下游相关蛋白水平,方法同实施例2中Western blot法。
结果:见图5所示,去氢毛钩藤碱可显著抑制CML细胞增殖,在SPHK1过表达时该增殖抑制活性被明显减弱。相较于去氢毛钩藤碱处理组,SPHK1过表达与去氢毛钩藤碱联合处理组细胞中SPHK1、BCR-ABL、p-BCR-ABL(Tyr412)、PI3K 110α、p-Akt(Ser473)水平均有所回升,表明SPHK1过表达明显削弱了去氢毛钩藤碱对SPHK1及BCR-ABL/PI3K/Akt通路的抑制作用。以上结果表明,去氢毛钩藤碱通过靶向抑制SPHK1发挥抗CML作用。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (6)
1.去氢毛钩藤碱在制备抗慢性粒细胞白血病药物中的应用。
2.根据权利要求1所述的应用,其特征在于:所述去氢毛钩藤碱抑制CML细胞增殖活性。
3.根据权利要求2所述的应用,其特征在于:所述去氢毛钩藤碱通过靶向抑制鞘氨醇激酶1抑制CML细胞增殖活性。
4.根据权利要求2所述的应用,其特征在于:所述去氢毛钩藤碱对人源CML亲本细胞K562的增殖抑制活性的IC50值为12.33μM,对耐药细胞K562/G01的增殖抑制活性的IC50值为12.77μM。
5.根据权利要求1所述的应用,其特征在于:所述去氢毛钩藤碱诱导CML细胞G2/M期周期阻滞。
6.根据权利要求1所述的应用,其特征在于:所述去氢毛钩藤碱诱导CML细胞凋亡。
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