CN113813257A - 双咪唑盐及载药体系在作为抗癌剂中的应用和抗癌制剂 - Google Patents
双咪唑盐及载药体系在作为抗癌剂中的应用和抗癌制剂 Download PDFInfo
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Abstract
本发明提供了双咪唑盐及载药体系在作为抗癌剂中的应用和抗癌制剂,涉及抗癌药技术领域。本发明提供作为糖基转移酶抑制剂的双咪唑盐C20/C22和包含所述双咪唑盐C20/C22的载药体系,研究了其针对人脐静脉内皮细胞系(HUVEC)和一系列癌细胞系的选择性细胞毒性;结果表明C20/C22及其载药体系具有作为新型抗癌剂的临床潜力,并且作为辅助性的抗癌药物值得进一步发展。
Description
技术领域
本发明属于抗癌药技术领域,具体涉及双咪唑盐及载药体系在作为抗癌剂中的应用和抗癌制剂。
背景技术
癌症是一种危及生命的疾病,目前治疗癌症的药物多为广谱性化疗药,因其对正常细胞的杀伤作用非常明显,会严重降低癌症患者的生存质量。因此,发现具有选择性细胞毒性,新作用机制和较少毒副作用的新型抗癌药物是迫切需求。
蛋白糖基化是最重要的翻译后修饰过程,据不完全统计,超过一半的人类蛋白都经过糖基化修饰(PMID:24339177)。机体生物功能紊乱,蛋白质折叠异常都会导致糖基化异常;异常糖基化与很多疾病息息相关,越来越多的证据表明糖基化在肿瘤的形成、迁移过程扮演着重要角色(PMID:31550741)。细胞表面糖蛋白与很多生物学功能有关,包括细胞增殖、分化、迁移;细胞与细胞之间的相互作用、相互识别;细胞外基质与宿主-病原体相互作用;免疫调节;信号转导等(PMID:19530676)。
糖基转移酶是涉及聚糖生物合成的一大类酶(家族),其催化高度特异性的糖基转移反应,部分糖基转移酶在癌细胞中表达异常(PMID:19902428)。目前已上市的糖基转移酶抑制剂如下:Deoxymannojirimycin(脱氧甘露糖野尻霉素)Swainsonine(八氢吲嗪三醇)是α1,2-甘露糖苷转移酶抑制剂,α1,2-甘露糖苷转移酶是N-糖基化关键酶,抑制其活性会引起ER Stress最终导致癌症细胞凋亡。GlcNAcβ1-2(4,6-di-O-methyl-)Manα1-6Glcβ-pnp(GlcNAc N-乙酰葡糖胺基转移酶V抑制剂)N-乙酰葡糖胺基转移酶催化GIcNAcβ1-6分支加到N-glycans,该酶催化的最终产物会促进肿瘤细胞的迁移和侵袭。2-deoxy-Manα1-6(Gnβ1-2Manα1-3)Manβ-octyl:甘露糖(α1,3)糖蛋白β1,2-N乙酰葡糖氨基转移酶2抑制剂,该酶控制低聚糖转化为复杂的和杂交的天门冬酰胺结合聚糖。Manα1-6(6-O-methyl-Manα1-3)Manβ-octyl:甘露糖(α1,3)糖蛋白β1,2-N乙酰葡糖氨基转移酶1抑制剂,是底物类似物,竞争性抑制酶活,促进细胞凋亡。但是目前抑制糖基转移酶的基因药物仍存在风险,且竞争性糖基转移酶的分子量大,将它们作为抗癌制剂仍存在技术困难,所以需要小分子量的抗癌制剂。
发明内容
有鉴于此,本发明的目的在于提供双咪唑盐及载药体系在作为抗癌剂中的应用和抗癌制剂,利用具有糖基转移酶抑制剂作用的小分子双咪唑盐(C20/C22)或载药体系用于抗癌,可抑制癌细胞生长并引起细胞周期阻滞,激活多条凋亡通路导致癌细胞凋亡。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种抗癌作用的含双咪唑盐的载药体系,所述双咪唑盐包括带正电的双咪唑二价盐,结构如式I所示:
其中n为20或22。
优选的,所述载药体系包括利用白蛋白纳米粒包封所述双咪唑盐。
优选的,所述载药体系的载药量不低于7%。
本发明还提供了双咪唑盐或上述载药体系在制备抗癌剂中的应用,所述双咪唑盐包括带正电的双咪唑二价盐,结构如式I所示:
其中n为20或22。
优选的,所述抗癌剂抗转移性癌症。
优选的,所述抗癌剂抗肝癌、乳腺癌、肺癌、前列腺癌、结肠癌和宫颈癌中的一种或多种。
本发明还提供了一种抑制癌细胞增殖的制剂,所述制剂包括双咪唑盐或上述载药体系,所述双咪唑盐包括带正电的双咪唑二价盐,结构如式I所示:
其中n为20或22。
本发明还提供了一种抑制癌细胞迁移和侵袭的制剂,所述制剂包括双咪唑盐或上述载药体系,所述双咪唑盐包括带正电的双咪唑二价盐,结构如式I所示:
其中n为20或22。
本发明还提供了一种诱导癌细胞凋亡的制剂,所述制剂包括双咪唑盐或上述载药体系,所述双咪唑盐包括带正电的双咪唑二价盐,结构如式I所示:
其中n为20或22。
本发明还提供了一种诱导癌细胞周期停滞的制剂,所述制剂包括双咪唑盐或上述载药体系,所述双咪唑盐包括带正电的双咪唑二价盐,结构如式I所示:
其中n为20或22。
有益效果:本发明提供了作为糖基转移酶抑制剂的双咪唑盐C20/C22(PMID:23375091)和包含所述双咪唑盐C20/C22的载药体系,研究了其针对人脐静脉内皮细胞系(HUVEC)和一系列癌细胞系的选择性细胞毒性;并在乳腺癌(MCF-7/MDA-MB-231)和肝癌细胞(HepG2)中研究C20/C22的抗增殖和细胞凋亡特性。结果表明C20/C22具有作为新型抗癌剂的临床潜力,并且作为辅助性的抗癌药物值得进一步发展。
本发明实施例中还以白蛋白纳米粒包封所述双咪唑盐作为载药体系(Am-C20/C22)进行相应研究,Am-C20/C22表现出缓释作用和更高的药物水溶性,减少过敏及免疫排斥反应,并且Am-C20/C22相比于游离的C20/C22,对癌细胞表现出更高的细胞毒性(p<0.001)。实施例实验结果显示用C20/C22和Am-C20/C22处理癌细胞,引起了癌细胞G2/M期阻滞,促使癌细胞发生内质网应激反应,并上调了TRAIL受体DR4/DR5表达,下调了促存活蛋白Bcl-2的表达,并增加了促凋亡蛋白CHOP、Bax、BaK、Caspase-3、Caspase-7、Caspase-8和Caspase-9表达。因此,C20/C22和Am-C20/C22优先抑制癌细胞生长,引起细胞周期阻滞,并能激活多条凋亡通路导致癌细胞凋亡,可作为抗癌药物辅助药物。
附图说明
图1为经C20/C22处理的癌细胞,在穿越基质胶覆盖的小室孔实验结果;
图2为对照组与经C20/C22处理的癌细胞的免疫荧光分析结果;
图3为经C20/C22处理的癌细胞对P-Selectin和E-Selectin黏附能力变化;
图4为C20/C22对癌细胞活性氧产生的影响;
图5为C20/C22在细胞周期中的作用;
图6为C20/C22对癌细胞凋亡的影响;
图7为C20/C22处理后,受体非依赖性细胞凋亡的相关凋亡因子的表达水平;
图8为C20/C22处理后,TRAIL信号通路DR4和DR5受体在细胞内外表达水平。
具体实施方式
本发明提供了一种抗癌作用的含双咪唑盐的载药体系,所述双咪唑盐包括带正电的双咪唑二价盐,结构如式I所示:
其中n为20或22。
本发明所述载药体系(Am-C20/C22)优选包括利用白蛋白纳米粒包封所述双咪唑盐(C20/C22)。在本发明中,所述白蛋白纳米粒包封优选包括:将白蛋白纳米粒(Albuminnanoparticles,AmNps,简写Am)与所述双咪唑盐进行混合,所述白蛋白纳米粒与双咪唑盐混合的重量比优选为(5~20):1,更优选为10:1。本发明所述Am-C20/C22的包封效率(EE)与混合的重量比有关,当Am:C20/C22(w/w)的比例在5:1,10:1和20:1时,EE值分别为75.92%,89.86%和92.95%;当药脂比为1:10时,载药量约为8.98%。本发明所述包封可增强C20/C22在水溶液中的溶解度;并且在盐水和含有10%FBS的PBS中,EE(%)随时间降低,表明C20/C22可以在生理条件下从Am-C20/C22缓慢释放以表达其抗癌作用,如在pH为5.5、6.5和7.4的条件下C20/C22的累积释放,在酸性环境(例如肿瘤区域)中释放更多C20/C22。
本发明所述C20/C22已在文献(PMID:23375091)中作为小分子糖基转移酶抑制剂进行公开。在本发明实施例中,所述双咪唑盐优选为亚硫酸盐,使用的C20的结构检测数据优选如下:1H NMR(300MHz,DMSO-d6):d1.15–1.40(m,32H),1.65–1.85(m,4H),2.31(s,6H),3.85(s,6H),4.15(t,J=9.2Hz,4H),7.71(s,2H),7.78(s,2H),9.14(s,2H);13C NMR(100MHz,DMSO-d6):d 25.5,28.4,28.8,28.9,29.00,29.04(3C),29.4,35.7,溶剂峰,48.7,122.2,123.6,136.6;HRMS(ESI)[M-O3SCH3]+计算:C29H55N4O3S:539.3994;实测:539.3991;使用的C22的结构检测数据如下:1H NMR(400MHz,DMSO-d6):d 1.12–1.42(m,36H),1.68–1.85(m,4H),2.29(s,6H),3.84(s,6H),4.14(t,J=7.2Hz,4H),7.70(s,2H),7.76(s,2H),9.11(s,2H);13CNMR(100MHz,DMSO-d6):d 25.5,28.3,28.8,28.9,29.0,29.1(4C),29.3,35.7,溶剂峰,48.7,122.2,123.6,136.5;HRMS(ESI)[M-O3SCH3]+计算C31H59N4O3S:567.4307;实测:567.4305。
本发明还提供了双咪唑盐或上述载药体系在制备抗癌剂中的应用,所述双咪唑盐包括带正电的双咪唑二价盐,结构如式I所示:
其中n为20或22。
本发明所述抗癌剂优选抗转移性癌症,更优选包括抗肝癌、乳腺癌、肺癌、前列腺癌、结肠癌和/或宫颈癌。在本发明实施例中,利用肝癌细胞系(SMMC-7721,HepG2和Huh7),乳腺癌细胞系(MCF-7和MDA-MB-231),肺癌细胞系(A549),前列腺癌细胞系(PC3),结肠癌细胞系(SW480),宫颈癌细胞系(Hela)和人脐静脉内皮细胞系(HUVEC)进行研究,证实了所述抗癌剂对癌症细胞的选择性毒性。
本发明还提供了一种抑制癌细胞增殖的制剂,所述制剂包括双咪唑盐或上述载药体系,所述双咪唑盐包括带正电的双咪唑二价盐,结构如式I所示:
其中n为20或22。
在本发明实施例中,用正常HUVEC细胞样品观察到C20/C22的最高IC50值为5μM,而用癌细胞观察到最低IC50值为2μM。对C20/C22敏感的癌细胞系半数抑制浓度条件下,对人源HUVEC细胞的抑制率不到10%,对小鼠海马神经元细胞HT22细胞抑制率不到15%。表明在一定治疗窗范围内,C20/C22可作为特异性细胞毒性剂,对癌细胞具有较强的抑制增殖及杀伤作用,对正常细胞的毒性较小。
本发明还提供了一种抑制癌细胞迁移和侵袭的制剂,所述制剂包括双咪唑盐或上述载药体系,所述双咪唑盐包括带正电的双咪唑二价盐,结构如式I所示:
其中n为20或22。
在本发明实施例中,经C20/C22处理的癌细胞,在划痕实验中能显著抑制癌细胞迁移,且随着给药浓度的增加,抑制效果更加明显;经C20/C22处理的癌细胞,在穿越基质胶覆盖的小室孔实验中能显著抑制癌细胞侵袭,C20和C22对癌细胞的侵袭均起到抑制作用,随着给药浓度增加,抑制效果更加明显,该结果表明所述制剂可以很好的抑制细胞侵袭,对预防癌细胞跨内皮迁移进而发生癌转移具有潜在的临床应用价值。
本发明还提供了一种诱导癌细胞凋亡的制剂,所述制剂包括双咪唑盐或上述载药体系,所述双咪唑盐包括带正电的双咪唑二价盐,结构如式I所示:
其中n为20或22。
在本发明实施例中,所述制剂可激活多种途径诱导癌症细胞凋亡,如C20/C22可能会引起癌细胞的线粒体应激及内质网应激反应进而引起细胞凋亡,用C20/C22处理的细胞中内源性促凋亡蛋白Cytochrome c、CHOP、Bax、BAK和ER-stress标志性蛋白(GRP-78、IRE1α、ATF6、PERK和P-JNK等)TRAIL凋亡通路DR4,R5受体在细胞内表达量和细胞表面分布均增加。启动子(Caspase-8和Caspase-9)和效应器Caspase(Caspase-3,Caspase-6和Caspase-7)表达水平升高。同时,C20/C22会使抗凋亡蛋白Bcl-2的表达量降低,C20/C22通过多条凋亡通路诱导癌细胞凋亡。因此,C20/C22均表现出潜在的抗癌作用。
本发明还提供了一种诱导癌细胞周期停滞的制剂,所述制剂包括双咪唑盐或上述载药体系,所述双咪唑盐包括带正电的双咪唑二价盐,结构如式I所示:
其中n为20或22。
在本发明实施例中,C20/C22显著增加了癌细胞在G2/M期的百分比(p<0.001),这表明用C20/C22处理会导致G2/M期细胞周期停滞,进而引起内质网应激导致细胞凋亡。
下面结合实施例对本发明提供的双咪唑盐及载药体系在作为抗癌剂中的应用和抗癌制剂进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
1、实验材料
磷酸盐缓冲盐水(PBS,Thermo Fisher Scientific,上海,中国)和胰蛋白酶-EDTA溶液(新赛美,北京,中国)。
Bcl-2、Bax、Caspase-3、Caspace-8和Caspace-9等抗体获自Affinity(北京,中国)。
抗LewisA、Lewis B、SleX和Lewis Y抗体获自Abcam(美国)。
TRAIL凋亡诱导因子获自Santa Cruz Biotechnology,Inc.(加拿大)。
Super ECL检测试剂(Yeasen Biotech Co,Ltd),细胞凋亡检测试剂盒(TransDetect Annexin V-FITC/PI,Yeasen Biotech,上海,中国)和细胞周期检测试剂盒(PI染色,上海,中国)。
2、C20/C22的合成
根据文献(PMID:23375091)的记载,进行C20/C22的合成,随后通过1H NMR和LC-ESI-MS分析化合物结构,完成对各种糖基转移酶抑制活性研究。
3、细胞培养
肝癌(SMMC-7721、HepG2和Huh7),乳腺癌(MCF-7和MDA-MB-231),肺癌(A549)、前列腺癌(PC3)、结肠癌(SW480)、宫颈癌(Hela)和HUVEC细胞系,均获自中国科学院类型培养物保藏委员会的细胞库(上海,中国)。
含有10%胎牛血清(FBS,Thermo Fisher Scientific,上海,中国)和1%青霉素/链霉素(Thermo Fisher Scientific,上海,中国)的细胞生长完全培养基RPMI 1640和DMEM(Thermo Fisher Scientific,上海,中国)用于生长细胞。
将PC3,HUVEC和SW480细胞系在RPMI 1640培养基中培养。同时,在DMEM培养基中培养MCF-7,MDA-MB-231,A549,SMMC-7721,HepG2和Huh7细胞系。在37℃含5%CO2无菌培养箱进行培养。
4、细胞毒性检测
将处于对数生长期(1×104)的细胞接种到含有完全生长培养基的96孔板上,在CO2培养箱中孵育24h。随后在无血清体条件下用不同浓度的C20/C22处理细胞24h,以0.01%DMSO为空白对照。之后在每孔加入10μLCCK8溶液(翊圣,上海,中国),37℃孵育2h,酶标仪检测450nm处吸光度值(OD450),并且每次测量重复6次。根据下式计算细胞活力。
细胞活力(%)=(实验组OD450-空白OD450)/(对照组的OD450-空白OD450)×100%
5、流式细胞术
将从对数生长期(3×105)收集的细胞接种到6孔板上过夜孵育,无血清培养基条件下加入C20/22。在含有C20/C22的培养基中孵育24h后,用磷酸盐缓冲盐水(PBS)洗涤细胞,无EDTA胰酶消化。
细胞凋亡和细胞周期检测试剂盒(BD Biosciences,San Jose,CA)的说明对细胞进行染色,流式细胞仪检测细胞活性氧产生,细胞凋亡及周期阻滞情况。
6、蛋白质印迹分析
将细胞于含有C20/C22的培养基中孵育6、12和24h,然后提取总蛋白质并使用BCA试剂盒检测蛋白质浓度(翊圣,上海,中国)。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分离蛋白质,并转印至聚偏二氟乙烯(PVDF)膜(Invitrogen)。将膜与一抗4℃条件过夜孵育,TBST洗膜三次,加二抗(1000×稀释),室温孵育2h。TBST洗膜三次,将增强的化学发光(ECL)显影溶液(Affinity,Biosciences)加入PVDF膜中,记录结果并用Gapdh作为对照进行分析。
7、免疫荧光分析
24孔板中放入已TC处理的玻片,紫外照射灭菌,每孔接种1×105个细胞,过夜孵育,加入无血清培养基配制的半数抑制浓度的C20/C22溶液,继续37℃,5%CO2条件下孵育24h,弃上清,预冷PBST洗涤3次,4%多聚甲醛固定,预冷PBST洗涤3次,检测胞内蛋白需进行通透处理,细胞表面蛋白直接用1%BSA室温封闭1h,弃封闭液,加1:100稀释的一抗,4℃过夜孵育,弃上清,预冷PBST洗涤3次,加FITC标记二抗室温避光孵育1h,预冷PBST避光洗涤3次,加DAPI染核,PBST洗涤一次,制片,避光-20℃保存,或直接用共聚焦显微镜进行检测。
8、凝集素检测糖原表达分析
凝集素染色评估细胞表面糖基化,按照文献(PMID:22843320)记载的方法进行。96孔板中每孔接种1×105个细胞,加入C20/C22处理24h,以0.01%DMSO作为对照,给药后固定,加入生物素标记的凝集素,碱性磷酸酶标记的亲和素和硝基苯-磷酸盐反应底物一起孵育。用酶标仪测量OD405处颜色变化。强度归一化为1.0×105cells的强度。使用Student's t检验比较平均值之间的差异。在所有检测中,p<0.05被认为有统计学意义。每个样本至少要分析6次。
9、癌细胞迁移侵袭能力分析
9.1划痕实验检测细胞迁移能力:对数生长期细胞胰酶消化,完全培养基终止消化反应,弃上清,加完全培养基重悬细胞,六孔板每孔接种细胞,过夜孵育后弃上清,加入1mLPBS洗涤一次,洗涤后用10μL枪头快速划痕,划痕后加入对应浓度的C20/C22溶液,以0.1%的DMSO作为空白对照,分别于划痕后0/24/48小时进行划痕处拍照,24/48h划痕宽度减0h划痕宽度即为各时间点迁移距离。
9.2穿孔实验检测细胞侵袭能力:细胞实验前进行无血清培养基培养,饥饿24h后进行实验。基质胶用预冷的无血清培养基稀释,稀释之后加到transwell小室,37℃封胶。配药:按照给药浓度的2倍进行配药。封胶后进行细胞处理,把饥饿后的细胞胰酶消化,基础培养基重悬,计数,取等体积细胞悬液和C20/C22溶液预混,轻轻吹打到预先封胶的小室中,小室外侧24孔板中完全培养基,37℃,5%CO2条件下孵育24h。培养后倒掉小室内培养基,抠掉基质胶,脱脂棉签轻轻擦掉小室内细胞,预冷PBS洗涤两次,把小室放在乙醇中固定,固定后用PBS洗涤两次,每次洗涤后用脱脂棉签轻轻擦掉小室内部,乙醇完全挥发后放在结晶紫中染色,染色后用脱脂棉签轻轻擦掉小室内部,吸干水分之后拍照记录Transwell结果。
9.3选择素识别实验检测细胞侵袭能力:
对ELISA板进行选择素涂层,4℃过夜,弃上清,洗涤两次,加封闭液,4℃封闭过夜,弃掉封闭液,洗涤两次,加入C20/C22处理预先处理的细胞悬液,37℃孵育2h,弃上清,加10%甲醛固定,结晶紫染色,去离子水洗涤细胞4次,显微镜下拍照,加含甲酸的甲醇溶液溶解,酶标仪检测590nm处吸光度。
9.4凝集素染色
按照文献(PMID:22843320)记载的方法进行凝集素染色评估细胞表面糖基化:96孔板中每孔接种105个细胞,加入C20/C22处理24h,以0.01%DMSO作为对照,给药后固定,加入生物素标记的凝集素,碱性磷酸酶标记的亲和素和硝基苯-磷酸盐反应底物一起孵育。用酶标仪测量OD405处颜色变化。强度归一化为1.0×105cells的强度。使用Student's t检验比较平均值之间的差异。在所有检测中,p<0.05被认为有统计学意义。每个样本至少要分析6次。
10、统计分析
在本发明中使用Graphpad Prism 8进行统计分析,数据表示为平均值±标准偏差(SD)。在我们的数据中*p<0.05,**p<0.01,***p<0.001(使用Student's t检验和单向ANOVA)表示统计学意义。
二、结果分析
1、C20/C22的结构分析
C20的结构检测数据如下:1H NMR(300MHz,DMSO-d6):d 1.15–1.40(m,32H),1.65–1.85(m,4H),2.31(s,6H),3.85(s,6H),4.15(t,J=9.2Hz,4H),7.71(s,2H),7.78(s,2H),9.14(s,2H);13C NMR(100MHz,DMSO-d6):d 25.5,28.4,28.8,28.9,29.00,29.04(3C),29.4,35.7,溶剂峰,48.7,122.2,123.6,136.6;HRMS(ESI)[M-O3SCH3]+计算:C29H55N4O3S:539.3994,实测:539.3991。
C22的结构检测数据如下:1H NMR(400MHz,DMSO-d6):d 1.12–1.42(m,36H),1.68–1.85(m,4H),2.29(s,6H),3.84(s,6H),4.14(t,J=7.2Hz,4H),7.70(s,2H),7.76(s,2H),9.11(s,2H);13C NMR(100MHz,DMSO-d6):d 25.5,28.3,28.8,28.9,29.0,29.1(4C),29.3,35.7,溶剂峰,48.7,122.2,123.6,136.5;HRMS(ESI)[M-O3SCH3]+计算C31H59N4O3S:567.4307,实测:567.4305。
2、C20/C22抑制癌细胞增殖
通过CCK8测定评估C20/C22对癌细胞的细胞毒性作用。通过GraphPad Prism 8计算的所有细胞系的半数抑制浓度(IC50)。在上述细胞系中,用正常HUVEC细胞样品观察到C20/C22的最高IC50值=5μM,而用癌细胞观察到最低IC50值=2μM。对C20/C22敏感的癌细胞系半数抑制浓度条件下,对人源HUVEC细胞的抑制率不到10%,对小鼠海马神经元细胞HT22细胞抑制率不到15%。表明在一定治疗窗范围内,C20/C22可作为特异性细胞毒性剂,对癌细胞具有较强的抑制增殖及杀伤作用,对正常细胞的毒性较小。
3、C20/C22抑制癌细胞迁移和侵袭效果及机制分析
经C20/C22处理的癌细胞,在划痕实验中能显著抑制癌细胞迁移,例如C20处理MCF-7细胞24h及48h后,给药24h后,对照组迁移平均距离约为0.4mm,C201μM组迁移平均距离约为0.2mm,C202μM组迁移平均距离约为0.18mm,给药48h后对照组迁移距离约为1.3mm,C201μM组迁移平均距离约为0.35mm,C202μM组迁移平均距离约为0.3mm,该结果显示C20对MCF-7细胞有明显的抑制细胞迁移的作用,且随着给药浓度的增加,抑制效果更加明显;
C22处理MCF-7细胞24h及48h,给药24h后,对照组迁移平均距离约为0.5mm,C221μM组迁移平均距离约为0.25mm,C222μM组迁移平均距离约为0.15mm,给药48h后对照组迁移距离约为1.4mm,C221μM组迁移平均距离约为0.65mm,C222μM组迁移平均距离约为0.5mm,该结果显示C22对MCF-7细胞有明显的抑制细胞迁移的作用,且随着给药浓度的增加,抑制迁移效果更加明显。在其他癌细胞系表现出了相同抑制趋势。
如图1所示,经C20/C22处理的癌细胞,在穿越基质胶覆盖的小室孔实验中能显著抑制癌细胞侵袭,C20和C22对MCF-7等癌细胞的侵袭均起到抑制作用,随着给药浓度增加,抑制效果更加明显,该结果表明该药物可以很好的抑制细胞侵袭,对预防癌细胞跨内皮迁移进而发生癌转移具有潜在的临床应用价值。
癌细胞表面路易斯寡糖SleX与选择素结合促进了癌细胞从循环系统转移到其他组织,经C20/C22处理的癌细胞对选择素黏附能力影响实验结果显示,C20/C22处理细胞显著抑制了细胞对选择素的黏附能力,抑制癌细胞向其他组织转移的能力。
免疫荧光分析结果如图2所示,与对照组相比经C20/C22处理的癌细胞,细胞表面SleX及Lewis Y表达量明显降低,而LewisA和Lewis B表达量略增加,可能是由于β4GalNAT与识别相同的底物,SleX及Lewis Y合成是由β4GalNAT催化,由于该酶活性被抑制,其竞争性酶β3GalNAT会识别更多的底物,进而促进其产物LewisA和Lewis B表达量增加。SleX及Lewis Y表达对癌细胞转移的至关重要,上述两种路易斯寡糖表达量降低会抑制癌细胞转移,减少癌细胞恶性程度,因此C20/C22有望为转移型癌症患者带来福音。
如图3所示,经C20/C22处理的癌细胞对P-Selectin和E-Selectin黏附能力均有所降低,表明上述两种抑制剂可通过抑制SleX和Core2合成降低其跨内皮迁移能力。
4、C20/C22对癌细胞活性氧(ROS)产生的影响
C20/C22处理后的细胞,DCFH-DA染色,流式细胞仪检测ROS,结果如图4所示引起了癌细胞内ROS表达量增加。这一结果表明C20/C22可能会引起癌细胞的线粒体应激及内质网应激反应进而引起细胞凋亡。
5、C20/C22抑制癌细胞表面糖基化过程
C20/C22处理后的癌细胞,RCAⅠ识别的Gal,PHA-L识别的Galβ4GlcNAcβ6以及MALⅠ识别的Galβ4GlcNAc表达量降低(p<0.01),而WGA识别的GlcNAc表达量升高。
6、C20/C22在细胞周期中的作用
将C20/C22处理的癌细胞系用PI染色,并通过流式细胞仪进行细胞周期分析。如图5所示,C20/C22显著增加了乳腺癌细胞MCF-7等在G2/M期的百分比(p<0.001),这表明用C20/C22处理会导致G2/M期细胞周期停滞。进而引起内质网应激导致细胞凋亡。
7、C20/C22对癌细胞凋亡的影响
如图6所示,Annexin V-FITC和碘化丙啶(PI)染色用于测定乳腺癌MCF-7等癌细胞凋亡水平,较低给药浓度条件引起了癌细胞不同程度凋亡,且随着给药浓度增加,癌细胞凋亡比例亦增加,说明C20/C22是剂量依赖性细胞毒性制剂。对于其他的癌细胞系例如肝癌(HepG2)等细胞表现出相同的细胞毒性作用。
8、癌细胞中抗凋亡因子,促凋亡因子和caspase酶家族分子的表达
通过流式细胞仪分析AnnexinV-FITC和PI染色的癌细胞的细胞膜和核形态变化,结果表明C20/C22处理可以诱导癌细胞凋亡。为了进一步揭示由C20/C22触发的凋亡途径,如图7所示通过蛋白质印迹分析研究了受体非依赖性细胞凋亡的相关凋亡因子的表达水平,TRAIL凋亡通路以及内质网应激(ER-Stress)情况。当用含有C20/C22无血清培养基处理癌细胞时,Bcl-2家族分子的表达水平以时间依赖性方式改变,而在所有癌细胞中促凋亡因子Cytochrome C,Bax,BAK的表达增加,TRAIL信号通路DR4,DR5受体表达增加,ER-Stress标志性蛋白GRP78、ATF6、IRE1α、P-JNK和CHOP表达量增加;而发现抗凋亡因子Bcl-2被抑制。此外Caspase-8和Caspase-9的表达随着C20/C22孵育时间的增加而增加,与Caspase-3(37kDa)的情况相同。免疫荧光方法分析了TRAIL信号通路DR4,DR5受体在细胞内外表达均有增加,如图8所示,经C20/C22处理后的细胞加入TRAIL配体诱导明显增加了癌细胞凋亡比例。该分析表明C20/C22通过上调促凋亡蛋白因子Bax,BAK刺激癌细胞中的凋亡途径(p<0.05),下调抗凋亡因子Bcl-2,激活凋亡启动子Caspase-9,最终导致效应器Caspace-3的活化并以时间依赖的方式增加切割的Caspase-3的水平。
9、C20/C22白蛋白纳米粒的性质
制备具有不同白蛋白:C20/C22(w/w)比例的白蛋白纳米粒(Albuminnanoparticles,AmNps),以增强C20/C22在水溶液中的溶解度,并对其物理化学性质进行了分析,包括粒度,多分散指数(PDI),zeta电位,包封效率(EE)和药物负载能力(LC)。结果表明具有较高的Am:C20/C22(w/w)比例的具有小的粒度分布,例如10:1和20:1。此外当Am:C20/C22比例为10:1也表现出较小的多分散指数(PDI)。
当Am:C20/C22(w/w)的比例在1:1,5:1,10:1和20:1时,EE值分别为58%,75.92%,89.86%和92.95%。最后,选择以10:1的比例制备Am-C20/C22 NPs用于进一步分析其稳定性和细胞毒性。
Am-C20/C22的稳定性测量方法为测量其在盐水和含有10%FBS的PBS中在4℃和37℃下,随着时间的延长其粒径的变化。散点图显示粒径没有显著变化,并且可以维持(没有发生聚集)至少一周。PDI数据绘制在中,其显示了Am-C20/C22的窄尺寸分布。
在盐水和含有10%FBS的PBS中,EE(%)随时间降低,表明C20/C22可以在生理条件下从Am-C20/C22缓慢释放以表达其抗癌作用。根据在pH为5.5、6.5和7.4的条件下C20/C22的累积释放,可以看出在酸性环境(例如肿瘤区域)中释放更多C20/C22。
10、Am-C20/C22的细胞毒性
采用MTT方法检测各种浓度的Am-C20/C22复合物(10:1)处理了24h,其对癌细胞系的细胞毒性。在培养24h后,Am-C20/C22对癌细胞IC50约为0.9μM,明显低于游离C20/C22组IC50(2~3μM)。因此表明Am-C20/C22对所有这些测试的癌细胞表现出比游离的C20/C22更高的细胞毒性(p<0.001)。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
2.根据权利要求1所述应用,其特征在于,所述载药体系包括利用白蛋白纳米粒包封所述双咪唑盐。
3.根据权利要求1或2所述应用,其特征在于,所述载药体系的载药量不低于7%。
5.根据权利要求4所述应用,其特征在于,所述抗癌剂抗转移性癌症。
6.根据权利要求4或5所述应用,其特征在于,所述抗癌剂抗肝癌、乳腺癌、肺癌、前列腺癌、结肠癌和宫颈癌中的一种或多种。
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