CN112891354B - MDM2抑制剂Nutlin-3a在制备激活内质网应激诱导的癌细胞凋亡药物中的应用 - Google Patents
MDM2抑制剂Nutlin-3a在制备激活内质网应激诱导的癌细胞凋亡药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种MDM2抑制剂Nutlin‑3a在制备激活内质网应激诱导的癌细胞凋亡药物中的应用。通过实验证实,MDM2抑制剂Nutlin‑3a可通过增加胞质内钙离子浓度来诱导内质网应激的发生,从而激活死亡受体通路诱导结肠癌细胞凋亡。本发明还公开了一种MDM2抑制剂Nutlin‑3a与内质网应激激活剂的抗肿瘤的药物组合物,体内外实验均证实该药物组合物在结肠癌治疗方面具有显著的协同作用和良好的抗肿瘤效果。
Description
技术领域
本发明涉及结肠癌细胞内质网应激和联合用药领域,更具体地,涉及MDM2抑制剂Nutlin-3a在制备激活内质网应激诱导的癌细胞凋亡药物中的应用。
背景技术
结直肠癌(colorectal cancer,CRC)是消化系统最常见的恶性肿瘤之一,约占全世界每年确诊癌症和癌症相关死亡人数的10%,每年新增病例超过100万。结直肠癌的发生发展是一个多因素参与的复杂过程,如PI3K/AKT上调,P53基因突变等因素都可能诱导和促进结肠癌的发生。
鼠双微基因2(mouse double minute 2,MDM2)是p53最重要的负性调控因子。研究表明,许多癌症患者体内都存在着p53野生型功能的丧失,有两个重要的原因能导致p53抑癌功能的丧失,一个是p53蛋白内部结构发生突变,另一个是MDM2与p53形成负反馈,从而抑制p53功能。为此,近年来人们研发了多种MDM2抑制剂,其中Nutlin-3a为第一类MDM2小分子抑制剂,它可以与MDM2结合从而竞争性抑制MDM2与p53的结合,从而诱导p53积聚并恢复其转录活性,诱导肿瘤细胞发生凋亡。目前对于Nutlin-3a体内外研究已经被广泛报道,但是其对结肠癌生长和凋亡的调控研究的还很少。
内质网应激(Endoplasmic Reticulum Stress,ERS)是由于生化、生理和病理等刺激因素对内质网环境的刺激造成的内质网腔内错误折叠与未折叠蛋白聚集以及钙离子平衡紊乱等状况,从而激活未折叠蛋白反应、内质网超负荷反应和caspase-12介导的凋亡通路等信号途径的反应过程。内质网应激激活的未折叠蛋白反应最初是为了恢复内质网的稳态,对细胞起保护作用,但持续严重的内质网应激能够诱导肿瘤凋亡。最近的研究表明,很多抗肿瘤药物在发挥诱导肿瘤细胞凋亡的过程中也会引起内质网应激的发生。
细胞凋亡(Apoptosis)又称程序性细胞死亡,是一个生理性的细胞自主有序的自我毁灭过程,主要有外源性死亡受体途径、内源性线粒体凋亡途径和内质网应激三种凋亡途径。线粒体途径通过Bcl-2家族成员Bax和Bak激活。外源性死亡受体途径在促凋亡配体与TNF家族受体结合时被激活,并被诱饵受体(DcRs)的负调控。ERS介导的凋亡途径主要有GADD153/CHOP通路、ASK1-JNK通路和caspase-12通路三种。很多抗肿瘤药物通过诱导细胞凋亡的发生发挥其抗肿瘤作用,其中MDM2抑制剂Nutlin-3a通过外源性死亡受体途径诱导结肠癌细胞发生凋亡的报道鲜有报道。
另外,迄今为止,关于MDM2抑制剂Nutlin-3a激活内质网应激来调控外源性死亡受体凋亡途径诱导细胞凋亡的作用,国内外还未见报道。
发明内容
本发明的目的是提供MDM2抑制剂Nutlin-3a在制备激活内质网应激诱导的癌细胞凋亡的药物中的应用,且其与内质网应激激活剂联合使用可显著提高对癌细胞的杀伤作用;该MDM2抑制剂Nutlin-3a通过影响内质网应激相关蛋白CHOP的表达,从而激活外源性死亡受体凋亡途径,进而诱导结肠癌细胞发生凋亡。
为实现上述目的,本发明提供一种MDM2抑制剂Nutlin-3a在制备激活内质网应激诱导的癌细胞凋亡的药物中的应用。
优选地,所述MDM2抑制剂Nutlin-3a通过增加胞质内钙离子浓度激活内质网应激。
优选地,所述MDM2抑制剂Nutlin-3a用于制备内质网应激相关蛋白XBP1α、BIP、p-EIF-2α、ATF4、CHOP的表达促进剂。
优选地,所述癌细胞是结肠癌细胞。
优选地,所述结肠癌细胞是人结肠癌细胞RKO、人结肠癌细胞HCT116或人结肠癌细胞LOVO。
本发明还提供一种用于治疗癌症的药物组合物,其主要活性成分包括上述任一项所述的MDM2抑制剂Nutlin-3a及内质网应激激活剂。
优选地,所述内质网应激激活剂是衣霉素(Tunicamycin)和/或毒胡萝卜素(Thapsigargin)。
优选地,所述MDM2抑制剂Nutlin-3a与所述衣霉素的用量比为1-3.6uM:1.2-2.4ug/ml;
所述MDM2抑制剂Nutlin-3a与所述毒胡萝卜素的摩尔比为1-3.6:1.2-2.4。
上述药物组合物能够用于制备治疗癌症的药物。
优选地,所述癌症为结肠癌。
对比现有技术,本发明的有益效果:
1、本发明提供了Nutlin-3a在制备激活内质网应激诱导的癌细胞凋亡的药物中的应用,Nutlin-3a通过诱导内质网应激促进结肠癌细胞凋亡。
2、Nutlin-3a可以增加细胞内钙离子浓度进而激活内质网应激相关蛋白CHOP的表达,从而激活外源性死亡受体凋亡途径,进而诱导结肠癌细胞发生凋亡。
3、MDM2抑制剂Nutlin-3a与内质网应激激活剂联合使用,可以实现协同增效抗肿瘤作用;将MDM2抑制剂Nutlin-3a分别与Tunicamycin和Thapsigargin联合使用,可以显著抑制结肠癌细胞的生长、增殖,诱导结肠癌细胞的凋亡;且MDM2抑制剂Nutlin-3a与内质网应激激活剂联合使用增强了裸鼠皮下瘤模型中的抗肿瘤效果,为内质网应激发挥抗肿瘤作用提供理论依据,还为内质网应激激活剂Tunicamycin和Thapsigargin的应用提供了新的领域。
附图说明
图1为不同浓度Nutlin-3a处理人结肠癌细胞LOVO后细胞存活的比例;
图2为不同浓度Nutlin-3a处理人结肠癌细胞LOVO对细胞克隆形成的影响;
图3为不同浓度Nutlin-3a处理人结肠癌细胞LOVO后,凋亡相关蛋白cleaved-caspase-3的变化的Westernblotting结果;
图4为不同浓度Nutlin-3a处理人结肠癌细胞LOVO后,流式细胞术检测细胞凋亡情况变化;
图5为不同浓度Nutlin-3a处理三种P53野生型细胞结肠癌细胞RKO、HCT16和LOVO后,利用实时荧光定量PCR检测细胞内死亡受体通路相关基因mRNA水平变化情况;
图6为不同浓度Nutlin-3a处理三种P53野生型细胞结肠癌细胞RKO、HCT16和LOVO后,利用Westernblotting检测死亡受体通路中DR5蛋白水平变化情况以及外源性凋亡的起始caspase蛋白cleaved-caspase-8变化情况;
图7为利用慢病毒构建稳定敲低Caspase-8的稳转结肠癌细胞株与正常结肠癌细胞,经Nutlin-3a处理后,检测凋亡相关蛋白cleaved-caspase-3变化情况;
图8为不同浓度Nutlin-3a处理正常结肠癌细胞和稳定敲低Caspase-8的稳转结肠癌细胞,对两种细胞存活率的影响;
图9为利用siRNA干扰技术干扰细胞内DR5表达后,不同浓度Nutlin-3a处理正常结肠癌细胞和干扰DR5后的结肠癌细胞,对两种细胞存活率的影响;
图10为利用siRNA干扰技术干扰细胞内DR5表达后,Nutlin-3a处理正常结肠癌细胞和干扰DR5后的结肠癌细胞,利用Westernblotting检测DR5蛋白水干扰情况以及外源性凋亡的起始caspase蛋白cleaved-caspase-8和凋亡相关蛋白cleaved-caspase-3变化情况;
图11为利用siRNA干扰技术干扰细胞内DR5表达后,Nutlin-3a处理正常结肠癌细胞和干扰DR5后的结肠癌细胞,流式细胞术检测细胞凋亡情况变化;
图12为不同浓度Nutlin-3a处理三种P53野生型细胞结肠癌细胞RKO、HCT16和LOVO后,利用Westernblotting检测内质网应激相关蛋白表达情况;
图13为不同浓度Nutlin-3a处理结肠癌细胞,在不同时间段荧光显微镜下观察细胞内钙离子浓度变化情况;
图14为不同浓度Nutlin-3a处理结肠癌细胞8小时以后,利用流式细胞术检测细胞内钙离子浓度变化情况;
图15为单独应用钙离子螯合剂BAPTA-AM、单独应用Nutlin-3a、提前利用钙离子螯合剂BAPTA-AM处理后再加Nutlin-3a处理结肠癌细胞,利用流式细胞术检测细胞内钙离子浓度变化情况;
图16为单独应用钙离子螯合剂BAPTA-AM、单独应用Nutlin-3a、提前利用钙离子螯合剂BAPTA-AM处理后再加Nutlin-3a处理结肠癌细胞,利用Western blotting检测内质网应激相关蛋白表达情况;
图17为单独应用钙离子螯合剂BAPTA-AM、单独应用Nutlin-3a、提前利用钙离子螯合剂BAPTA-AM处理后再加Nutlin-3a处理结肠癌细胞,利用实时荧光定量PCR检测细胞内内质网应激相关蛋白Bip、CHOP和GADD34的mRNA水平变化;
图18为利用siRNA干扰技术干扰细胞内CHOP表达后,Nutlin-3a处理正常结肠癌细胞和干扰CHOP后的结肠癌细胞,流式细胞术检测细胞凋亡情况变化;
图19为利用siRNA干扰技术干扰细胞内CHOP表达后,Nutlin-3a处理正常结肠癌细胞和干扰CHOP后的结肠癌细胞,利用Western blotting检测CHOP蛋白水干扰情况以及死亡受体通路相关蛋白DR5和外源性凋亡的起始caspase蛋白cleaved-caspase-8和凋亡相关蛋白cleaved-caspase-3变化情况;
图20为内质网应激激活剂Tunicamycin、内质网应激激活剂Thapsigargin、Nutlin-3a、Nutlin-3a联合内质网应激激活剂Tunicamycin、Nutlin-3a联合内质网应激激活剂Thapsigargin分别处理三种P53野生型结肠癌细胞RKO、HCT116、LOVO后,对细胞存活率的影响;
图21为Nutlin-3a单独用药或分别联合内质网应激激活剂Tunicamycin和Thapsigargin处理结肠癌细胞后,利用流式细胞术检测细胞凋亡情况变化;
图22为Nutlin-3a单独用药或分别联合内质网应激激活剂Tunicamycin和Thapsigargin处理结肠癌细胞后,利用Western blotting检测外源性凋亡的起始caspase蛋白cleaved-caspase-8和凋亡相关蛋白cleaved-caspase-3变化情况;
图23-图24为Nutlin-3a单独用药或分别联合内质网应激激活剂Tunicamycin和Thapsigargin用药处理,对裸鼠皮下瘤生长的影响;
图25为MDM2抑制剂Nutlin-3a单独用药或或分别联合内质网应激激活剂Tunicamycin和Thapsigargin用药处理下裸鼠皮下结肠癌模型实体瘤TUNEL检测凋亡情况,图示标尺为100μm;
图26为MDM2抑制剂Nutlin-3a在结肠癌中通过激活内质网诱导结肠癌细胞通过死亡受体通路发生凋亡的作用机制图。
具体实施方式
下面通过结合具体实施案例进一步对本发明进行进一步详细说明。各实施例及试验例中所用的设备和试剂如无特殊说明,均可从商业途径得到。这些实施案例所描述的具体实施例仅用以解释本发明而用于限定本发明。为了更好地理解本发明而不是限制本发明的范围,在本发明中所用的表示用量、时间、百分比的所有数字、以及其他数值,在所有情况下都应理解为以词语“大约”所修饰。因此,除非特别说明,否则在说明书和所附权利要求书中所列出的数字参数都是近似值,其可能会根据试图获得的理想效果的不同而加以改变。
本发明实验所用材料与试剂
人结肠癌细胞RKO、HCT116、LOVO,Nutlin-3a(Sigma)、Tunicamycin(MCE)、Thapsigargin(MCE)、BAPTA-AM(MCE)、BALB/c裸鼠(北京维通利华)、Casp8-RNAi慢病毒(吉凯基因)、DMEM(Gibco)、胎牛血清(Gibco)、胰蛋白酶(索莱宝)、Trizol(Thermo)、CCK-8试剂盒(江苏凯基生物)、RNA反转录试剂盒(Thermo)、实时荧光定量PCR试剂盒(Thermo)、AnnexinV-FITC细胞凋亡检测试剂盒(同仁)、ECL化学发光试剂盒(赛默飞世尔科技公司)、lipofectamine 2000(Invitrogen)、OptiMEM培养基(Invitrogen)、Steri-Cycle i160CO2恒温培养箱(Thermo)、CytoFLEX流式细胞仪(贝克曼公司)、MutisKan FC酶标仪(Thermo)、DYY-2C垂直电泳仪电源(北京市六一仪器厂)、JY-SCZ2+型双垂直电泳槽(北京市君意东方仪器厂)、TE70XP半干转转膜仪(Amersham Biosciences)、Fluo-4AM钙离子检测试剂盒(碧云天)、TUNEL检测试剂盒(碧云天)。
实施例1
Nutlin-3a能够有效降低细胞存活率、抑制细胞增殖、诱导结肠癌细胞凋亡。
1、Nutlin-3a是一种高效的、非肽类MDM2-p53小分子抑制剂,分子式为:C30H30Cl2N4O4,分子量为581.49,结构式如下:
上述化合物作为MDM2-p53小分子抑制剂应用。该化合物的商用名为Nutlin-3a,可以从美国Sigma-Aldrich生化试剂公司购买获得。
2、结肠癌细胞体外培养
本实验所用细胞为P53野生型人结肠癌细胞LOVO,使用含质量分数10%胎牛血清(fetal bovine serum,FBS,Gibco)的DMEM和含质量分数1%青霉素-链霉素双抗的培养基,于37℃、5%CO2培养箱中进行细胞培养,待细胞生长至占培养皿底部80%左右时,使用质量分数0.1%胰酶消化并传代。
3、不同浓度Nutlin-3a对结肠癌细胞存活率的影响
将处于对数生长期的人结肠癌细胞LOVO消化,混匀垂悬于含质量分数10%FBS的DMEM,显微镜下计数,以1.2×104个细胞/孔均匀接种于96孔板中,每组设置6个副孔,共设置5组,细胞完全贴壁后,将Nutlin-3a用DMEM稀释成12.5uM、25μM、50μM、100μM,100uL/孔加入相应96孔板中在培养箱中培养20小时,每孔加入10uLCCK8,避光孵育2小时,在450nm处用酶标仪检测吸光值。结果如图1所示,随着Nutlin-3a作用浓度的增加,结肠癌细胞LOVO的存活率逐渐降低。
4、不同浓度Nutlin-3a对结肠癌细胞克隆形成能力的影响
在6孔板中以每孔300个细胞将人结肠癌细胞LOVO均匀铺好,待细胞贴壁后,更换含有0uM、2uM、4uM的Nutlin-3a的培养基,在37℃培养箱中继续培养,待孔板中出现肉眼可见克隆时终止培养,弃去培养基,4%的组织固定液固定20分钟以后,用结晶紫染色20分钟,PBS清洗掉多余结晶紫后,六孔板风干拍照。结果如图2所示,Nutlin-3a可以显著抑制结肠癌细胞克隆形成能力。
5、Nutlin-3a诱导结肠癌细胞发生凋亡的作用
将结肠癌细胞以5×106/皿均匀接种到4个6cm的小皿中,细胞完全贴壁后更换含有不同浓度Nutlin-3a的新鲜培养基,按照Nutlin-3a加药浓度共分为四组:0μM、35μM、50μM、75μM;培养箱中继续培养20小时,3mLPBS清洗两遍,用胰酶消化并收集细胞悬液,2000rpm离心1.5分钟,弃去培养基,根据细胞量加入适量细胞裂解液,冰上裂解20分钟,4°离心后取上清,利用BCA法测蛋白浓度,将各组蛋白上样量调成一致,在蛋白样品中加入适量5×的蛋白上样缓冲液,使5×的蛋白上样缓冲液在蛋白中稀释成1×,混匀,置于金属浴中,100℃加热10min变性。取20-30ug样品蛋白在120V稳压下用10%SDS-PAGE电泳分离,然后将蛋白转移到聚偏二氟乙烯(PVDF)膜上,用质量分数5%脱脂牛奶封闭1小时。然后用5%脱脂牛奶稀释的一抗体在4℃下孵育,再用二抗体孵育1小时,最后用辣根过氧化物酶(HRP)检测目的蛋白的表达情况。相关抗体信息见表1。
表1相关抗体信息表
抗体名称 | 抗体厂家 | 抗体货号 |
Tublin | SIGMA | T8203 |
Cleaved caspase 3 | Cell Signaling | 9661s |
DR5 | Cell Signaling | 8074s |
Cleaved caspase 8 | Cell Signaling | 9496s |
XBP1s | Cell Signaling | 12782s |
Bip | Cell Signaling | 3183s |
Phospho-eIF2α | Cell Signaling | 3597s |
ATF-4 | Cell Signaling | 11815s |
CHOP | Cell Signaling | 2895s |
Anti-Rabbit | abm | SH026 |
Anti-Mouse | abm | SH024 |
结果如图3所示,Nutlin-3a可有效诱导结肠癌细胞凋亡相关蛋白cleavedcaspase-3的表达。
将结肠癌细胞以4×105/细胞均匀铺到六孔板中,按照Nutlin-3a加药浓度共分为四组:0μM、35μM、50μM、75μM;培养箱中继续培养,待最大加药浓度组的细胞大概有50%皱缩、漂浮时,收集细胞,根据凋亡检测试剂盒的方法进行Annexin V和PI染色,1小时内利用流式细胞仪检测各组细胞的凋亡情况。结果如图4所示,随着Nutlin-3a浓度的增加,结肠癌细胞发生凋亡的比例也在增加,图中Q1-LR代表早期凋亡,Q1-UR代表晚期凋亡和坏死细胞,Q1-UL代表损伤细胞数,Q1-LL代表正常活细胞数。
实施例2
Nutlin-3a通过外源性死亡受体凋亡通路诱导结肠癌细胞凋亡。
1、Nutlin-3a可以诱导死亡受体通路相关基因表达水平上调
将对数生长期结肠癌细胞RKO、HCT116和LOVO细胞用胰酶消化后,以以4×105/细胞均匀铺到六孔板中,细胞完全贴壁后,更换为新鲜培养基稀释的浓度为0μM、35μM、50μM、75μM的Nutlin-3a,培养箱中继续培养20小时,收集细胞,用Trizol法提取细胞RNA,利用Nano Drop 2000分光光度计测RNA浓度,取1ugRNA根据PrimeScriptTMRT Master Mix(Perfect Real Time)逆转录试剂盒说明书逆转为cDNA,根据Premix Ex TaqTMII(Tli RNaseH Plus)qRT-PCR试剂盒说明书成分及条件,每个样品的体系为10ul,以cDNA为模板进行PCR反应,每组样品设置3个副孔,计算出每组的2-ΔΔCt表示实验组与对照组中目的基因的表达情况。结果如图5,经过不同浓度Nutlin-3a处理后,死亡受体通路相关基因表达大多都有所增高,其中DR5在三种不同结肠癌细胞中表达均增高。
同样方法处理结肠癌细胞RKO、HCT116和LOVO后,收集细胞并提蛋白,利用Westernblotting检测死亡受体通路中DR5蛋白水平变化情况以及外源性凋亡的起始caspase蛋白cleaved-caspase-8变化情况。相关引物序列见表2。
表2相关引物序列表
结果(图6)表明,DR5蛋白和cleaved-caspase-8蛋白水平随着加药浓度的增加而上调。
2、Caspase-8蛋白在Nutlin-3a诱导的经外源性死亡受体通路发生的凋亡中至关重要
将生长状态良好的结肠癌细胞HCT116以4×105个/孔的密度接种于6孔板中,待细胞完全贴壁后根据慢病毒使用说明书提供的MOI值,利用公式:MOI=(病毒滴度x病毒体积)/细胞数目,计算感染需用的病毒体积和感染增强液类型,用阴性对照组和Casp8-RNAi慢病毒感染细胞,37℃培养12-16小时后更换为完全培养基继续培养,感染72小时后,加入含2ug/ml嘌呤霉素的培养基对细胞进行筛选,直至再无细胞出现死亡。将阴性对照组和Casp8-RNAi组细胞分别以5×106/皿均匀接种到4个6cm的小皿中,实验分组为:阴性对照组不加药,阴性对照组加Nutlin-3a(50uM),Casp8-RNAi组不加药,Casp8-RNAi组加Nutlin-3a(50uM);细胞完全贴壁后更换含有50uM浓度Nutlin-3a的新鲜培养基,加药后20小时收集细胞,利用Western blotting检测Caspase-8干扰效果,并检测凋亡相关蛋白cleaved-caspase-3变化情况。结果如图7,干扰Caspase-8可减弱Nutlin-3a诱导的细胞凋亡。
将阴性对照组细胞、Casp8-RNAi组细胞分别计数,以1.2×104个细胞/孔均匀接种于96孔板中,每组设置6个副孔,共设置5组,将Nutlin-3a用DMEM稀释成12.5uM、25μM、50μM、100μM,100uL/孔加入相应96孔板中,在培养箱中培养20小时后每孔加入10uLCCK8,避光孵育2小时,用酶标仪在450nm处检测吸光值。结果(图8)表明,经不同浓度Nutlin-3a处理两种结肠癌细胞,敲低Caspase-8后的结肠癌细胞存活率比对照组的高。
3、DR5在Nutlin-3a诱导的经外源性死亡受体通路发生的凋亡中至关重要
对将对数生长期结肠癌细胞RKO以50%密度接种于4个6cm小皿中,实验分组为:NC组不加药、NC组加Nutlin-3a(50uM)、si-DR5组不加药、si-DR5组加Nutlin-3a(50uM);待细胞完全贴壁后,进行细胞转染。转染前将培养基提前两个小时更换为3ml的Opti-MEM无血清培养基,根据实验分组分别配制siRNA和Lipofectamin2000稀释液:100pmol siRNA稀释于500ul Opti-MEM培养基,5ul Lipofectamin2000稀释于500ul Opti-MEM培养基,上下颠倒混匀后室温静置5分钟,将两种稀释液混匀后静置15分钟,轻柔滴加到相应小皿中,6-8小时更换为正常无双抗培养基继续培养18个小时,按照实验分组对转染后细胞进行加药处理,加药后20小时收集细胞,利用Westernblotting检测DR5干扰效果,并检测cleaved-caspase-3和cleaved-caspase-8蛋白变化情况。结果(图10)表明,干扰DR5可以减轻Nutlin-3a诱导的凋亡相关蛋白Cleaved caspase-3、Cleaved caspase8的表达。
同样方法处理细胞后,收集细胞,根据凋亡检测试剂盒的方法进行Annexin V和PI染色,并利用流式细胞仪检测各组细胞的凋亡情况。结果如图11所示,干扰DR5组细胞Nutlin-3a诱导的凋亡率与对照组相比明显降低。
按照上述方法转染RKO细胞,转染24小时后收集细胞,并将对照组细胞和si-DR5组细胞分别计数,以1.2×104个细胞/孔均匀接种于96孔板中,每组设置6个副孔,共设置5组,将Nutlin-3a用DMEM稀释成12.5uM、25μM、50μM、100μM,100uL/孔加入相应96孔板中,在培养箱中培养20小时后每孔加入10uLCCK8,避光孵育2小时,用酶标仪在450nm处检测吸光值。结果(图9)表明,经同样浓度Nutlin-3a处理两种结肠癌细胞,干扰DR5后的结肠癌细胞存活率比对照组的高。
实施例3
Nutlin-3a通过增加细胞内钙离子浓度诱导内质网应激的发生
1、Nutlin-3a可以诱导内质网应激相关蛋白上调
取对数生长期的RKO、HCT116和LOVO细胞用胰酶消化后,以5×106/皿分别均匀接种到4个6cm的小皿中,按照Nutlin-3a加药浓度共分为四组:0μM、35μM、50μM、75μM;培养箱中继续培养20小时,收集细胞并提蛋白,利用Westernblotting方法检测内质网应激相关蛋白XBP1α、BIP、p-EIF-2α、ATF4、CHOP表达情况。结果图12所示,Nutlin-3a可有效上调内质网应激相关蛋白的表达水平并且呈浓度依赖性。
2、Nutlin-3a可以增加细胞内钙离子浓度
取对数生长期结肠癌细胞,以2x105个/孔接种于24孔板中,按照Nutlin-3a加药浓度共分为六组:0μM、15uM、35μM、55μM、65μM、85μM;每组设置三个副孔,按照分组用含不同浓度Nutlin-3a的新鲜培养基处理细胞,分别在3小时、6小时、12小时去除培养基,用PBS清洗3次,加入含2uM浓度Fluo-4AM工作液,37℃孵育30分钟进行荧光探针装载,随后用荧光显微镜(图13)和流式细胞仪检测Fluo-4的荧光情况(图14)。结果表明,Nutlin-3a可以呈浓度依赖性和时间依赖性刺激细胞内钙离子浓度的增加。
3、钙离子螯合剂BAPTA可以降低Nutlin-3a诱导的内质网应激
取对数生长期结肠癌细胞,以5×106/皿分别均匀接种到4个6cm的小皿中,实验分组为:对照组、BAPTA组(2uM)、Nutlin-3a组(50uM)、BAPTA+Nutlin-3a组(2uM BAPTA+50uMNutlin-3a);按照分组对细胞进行不同加药处理12小时后,去除培养基,用PBS清洗3次,加入含2uM浓度Fluo-4AM工作液,37℃孵育30分钟进行荧光探针装载,随后用流式细胞仪检测Fluo-4的荧光情况(图15)。结果表明,BAPTA可以有效降低Nutlin-3a刺激引起的细胞内钙离子浓度的增高。
同样分组以及加药方式处理结肠癌细胞,收集细胞提取蛋白和RNA,利用Westernblotting方法检测内质网应激相关蛋白Bip、ATF4、CHOP表达情况(图16),利用实时定量PCR检测内质网应激相关基因Bip、CHOP、GADD34变化情况(图17)。结果表明,抑制细胞内钙离子可以减弱Nutlin-3a诱导的内质网应激。
实施例4
Nutlin-3a通过内质网应激相关CHOP调控DR5诱导结肠癌细胞发生凋亡
将对数生长期结肠癌细胞RKO以50%密度接种于4个6cm小皿中,实验分组为:NC组不加药、NC组加Nutlin-3a(50uM)、si-CHOP组不加药、si-CHOP组加Nutlin-3a(50uM);待细胞完全贴壁后,按相同方法进行细胞转染,6-8小时更换为正常无双抗培养基继续培养18个小时,按照实验分组对转染后细胞进行加药处理,加药后20小时收集细胞,提取蛋白,利用Western blotting检测CHOP干扰效果,并检测DR5、cleaved-caspase-3和cleaved-caspase-8蛋白变化情况。结果如图19所示,干扰CHOP以后,Nutlin-3a诱导的DR5、cleaved-caspase-3和cleaved-caspase-8蛋白水平也随之降低。
同样方法处理细胞,收集细胞,根据凋亡检测试剂盒的方法进行Annexin V和PI染色,并利用流式细胞仪检测各组细胞的凋亡情况。结果如图18所示,干扰CHOP组细胞Nutlin-3a诱导的凋亡率与对照组相比明显降低。
MDM2抑制剂Nutlin-3a在结肠癌中通过激活内质网诱导结肠癌细胞通过死亡受体通路发生凋亡的作用机制图如图26所示。
实施例5
Nutlin-3a与内质网应激激活剂联合使用可显著降低细胞存活率、诱导结肠癌细胞凋亡。
1、Nutlin-3a与内质网应激激活剂联合使用可显著降低细胞存活率
取对数生长期的RKO、HCT116和LOVO细胞,胰酶消化后用完全培养基垂悬细胞并计数,以1.2×104个细胞/孔均匀接种于96孔板中,每组设置6个副孔,共设置5组,实验分组为:对照组、Tunicamycin组(15-30ug/ml)、Thapsigargin组(15-30uM)、Nutlin-3a组(12.5-45uM)、Tunicamycin+Nutlin-3a组(15-30ug/ml Tunicamycin+12.5-45uM Nutlin-3a)、Thapsigargi+Nutlin-3a组(15-30uM Thapsigargin+12.5-45uMNutlin-3a);按照上述实验分组处理细胞,其中内质网应激激活剂Tunicamycin和Thapsigargin提前30分钟处理细胞,诱导结肠癌细胞内质网应激的发生,加药处理后细胞继续培养20小时,每孔加入10uLCCK8,避光孵育2小时,在450nm处用酶标仪检测吸光值。结果如图20所示,Nutlin-3a与内质网应激激活剂联合使用可以显著降低细胞存活率。
2、Nutlin-3a与内质网应激激活剂联合使用可显著增加Nutlin-3a诱导凋亡的效果。
将对数生长期结肠癌细胞以5×106/皿接种于6个6cm小皿中,实验分组为:对照组、Tunicamycin组(15ug/ml)、Thapsigargin组(15uM)、Nutlin-3a组(45uM)、Tunicamycin+Nutlin-3a组(15ug/ml Tunicamycin+45uM Nutlin-3a)、Thapsigargi+Nutlin-3a组(15uMThapsigargin+45uM Nutlin-3a);按照上述实验方法处理细胞,20小时后收集细胞,根据凋亡检测试剂盒的方法进行AnnexinV和PI染色,并利用流式细胞仪检测各组细胞的凋亡情况如图21。
同样方法处理细胞后收集细胞提蛋白,利用Western blotting检测凋亡相关蛋白Cleaved caspase-3以及外源性凋亡的起始caspase蛋白cleaved-caspase-8变化情况(图22)。
结果表明,Nutlin-3a与内质网应激激活剂联合使用能够显著增加Nutlin-3a诱导凋亡的效果。
实施例6
Nutlin-3a与内质网应激激活剂联合应用在动物水平上的抗肿瘤效果
实验用动物为6-7周龄的雌性BALB/c-nu裸鼠,将处于对数生长阶段的HCT116细胞收集在200uL无血清培养基中,每200uL无血清培养基约1x107个细胞,分别在每只小鼠左腹股沟区皮下注射细胞悬液,7天后将小鼠随机分4组,分别为PBS组(注射,100ul/天)、Nutlin-3a组(口服,150mg/kg/天)、Tunicamycin+Nutlin-3a组(Tunicamycin(注射,0.3mg/kg);Nutlin-3a(口服,150mg/kg/天);Thapsigargin+Nutlin-3a组(Thapsigargin(注射,1ug/g;Nutlin-3a(口服,150mg/kg/天);)每天对肿瘤进行监测,每三天用卡尺测量一次最长和垂直宽度(a、b),根据公式(v):v=0.5ab2,计算每只小鼠的肿瘤体积并记录。
图23的结果显示随着药物处理天数的增加,PBS组的裸鼠肿瘤体积增长的最快,其次是Nutlin-3a组,而Nutlin-3a与Tunicamycin和Thapsigargin药物联合组经过15天的测量,肿瘤体积增长最为缓慢,说明药物联合处理对裸鼠体内的肿瘤具有很好的抑制作用。
图24为经药物处理15天后各组肿瘤的最终形态。
肿瘤组织的石蜡组织切片TUNEL染色显示药物联用的肿瘤组织中TUNEL荧光强度明显增强,凋亡率增加(图25)。
以上实验表明,Nutlin-3a与内质网应激激活剂联合应用可以显著增强Nutlin-3a的抗肿瘤效果。
需要说明的是,Nutlin-3a能够与一种内质网应激激活剂联合使用,也可以与多种内质网应激激活剂联合使用,其具体用量可以根据实验调整得到,如Nutlin-3a:衣霉素:毒胡萝卜素=6-12uM:2-6ug:1-4uM等,但该用量并不是对Nutlin-3a与内质网应激激活剂的用量进行限制。
以上公开的仅为本发明的具体实施例,但是,本发明实施例并非局限于此,任何本领域的技术人员能思之的变化都应落入本发明的保护范围。
Claims (3)
1.一种用于治疗癌症的药物组合物,其特征在于,其主要活性成分为MDM2抑制剂Nutlin-3a及内质网应激激活剂;
所述内质网应激激活剂是衣霉素和/或毒胡萝卜素;
所述MDM2抑制剂Nutlin-3a与所述衣霉素的用量比为1-3.6uM:1.2-2.4ug/ml;
所述MDM2抑制剂Nutlin-3a与所述毒胡萝卜素的摩尔比为1-3.6:1.2-2.4。
2.权利要求1所述的药物组合物在制备治疗癌症药物中的应用。
3.根据权利要求2所述的应用,其特征在于,所述癌症为结肠癌。
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