CN114432319A - 一种1,8萘啶衍生物的新用途 - Google Patents
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Abstract
本发明公开了一种1,8萘啶衍生物的新用途,即其在制备促进死亡受体表达和/或抑制抗凋亡蛋白表达药物中的应用,通过细胞生物学实验结合药理研究表明,1,8萘啶衍生物能够激活死亡受体5的表达,抑制抗凋亡蛋白c‑FLIP和XIAP的表达;本发明有助于1,8萘啶衍生物从机制转向临床研究,有望成为解决肿瘤治疗耐药性的新型药物,也为癌患者的治疗提供了更多的选择。
Description
技术领域
本发明属于医药技术领域,具体涉及一种1,8萘啶衍生物在制备调控死亡受体和抗凋亡蛋白药物中的用途。
背景技术
凋亡是理想的肿瘤细胞死亡方式,因为凋亡不会引发炎症反应所以被广泛关注。细胞凋亡包含两种途径分别是:外源性凋亡和线粒体介导的内源性凋亡。外源性凋亡途径是通过死亡受体与配体结合,募集大量接头蛋白FADD(Fas-associated death domian),然后通过死亡效应结合域(Death Effect Domain,DED)与无活性前体蛋白酶原Procaspase-8结合,形成死亡诱导信号复合物(death-inducing signaling complex,DISC),DISC会诱导Procaspase-8活化并自切割形成具有酶催化活性的Caspase-8,直接激活下游凋亡执行蛋白Caspase-3/6/7诱导细胞凋亡。
死亡受体是TNFR超家族(TNFR superfamily, TNFRSF)成员,其胞外区主要是半胱氨酸富集区(Cysteine rich domain, CRD),胞内含有传递凋亡信号的死亡结构域(deathdomain, DD)。死亡受体常见的主要有TNFR1、Fas、DR4和DR5。除此之外还存在诱骗受体DcR1/2,它们会与DR竞争结合配体,从而阻碍凋亡的开始。死亡受体DR在许多癌细胞中均有表达,并且DR的结合和聚集是有效的凋亡启动的先决条件,因此死亡受体已经逐渐成为一种潜在有效的抗肿瘤治疗的靶标蛋白。
在凋亡信号通路中存在抗凋亡蛋白c-FLIP以及XIAP,其中c-FLIP是大多数肿瘤产生耐药性的主要原因。c-FLIP最早是从病毒中发现,该蛋白位于凋亡信号通路的上游。由于c-FLIP 与Procaspase-8具有相似的结构,会竞争性的与FADD、Procaspase-8结合,阻碍死亡诱导信号复合物DISC形成及Caspases联级反应,从而中断凋亡信号传导,使癌细胞具有耐药,促进癌细胞的生长存活。在很多恶性肿瘤中均发现c-FLIP的高表达,例如黑色素瘤、肝癌、结肠癌以及前列腺癌等。c-FLIP不仅对肿瘤细胞凋亡产生抗性,同时也可参与了细胞的繁殖和存活通路。研究表明该蛋白促进肿瘤细胞的存活,这也是许多恶性肿瘤具有耐药性的主要原因。以上事实证明面对肿瘤的耐药性,急需能够有效杀伤肿瘤细胞的新型药物。
发明内容
本发明提供了一种1,8萘啶衍生物3u的新用途,即其在制备促进死亡受体表达和抑制抗凋亡蛋白表达药物中的应用,该化合物通过靶向死亡受体以及抑制抗凋亡蛋白,激活凋亡相关蛋白从而达到降低肿瘤耐药性的作用。
所述1,8萘啶衍生物3u的化学结构式如下:
本发明所述的促进死亡受体表达和抑制抗凋亡蛋白表达药物的成分(或有效成分)为1,8萘啶衍生物3u,还可以加入一种或多种药物或食品上可接受的辅料,以改善药物或食品吸收效果或便于服用,如制成胶囊或丸剂、粉剂、片剂、粒剂、口服液和注射液等,即制成药剂学上适宜的使用剂型,或食品领域适宜的食用方式。
本发明化合物参照申请号为201310091607.0中的方法制得。
本发明方法的优点和技术效果:
本发明通过蛋白免疫印迹实验验证了1,8萘啶衍生物的功能,实验结果显示1,8萘啶衍生物通过促进死亡受体表达,激活凋亡相关蛋白Caspase-8/3、PARP1,从而抑制癌细胞,导致癌细胞凋亡;1,8萘啶衍生物通过抑制抗凋亡蛋白c-FLIP和XIAP的表达,阻碍了c-FLIP与Procaspase-8的竞争性结合,增加了癌症细胞对化合物诱导凋亡的敏感性;本发明通过流式细胞术实验证明了本发明的化合物诱导癌细胞死亡呈现浓度依赖的方式,加药浓度越高治疗效果越好;本发明化合物与凋亡蛋白相关抑制剂ZVAD、IETD联合作用,能降低癌细胞对化合物的敏感性,增加了IC50值,说明本发明化合物通过凋亡途径杀死肿瘤细胞;
本发明有助于增强临床一线癌症的治疗效果,为临床治疗的耐药性提供一种新的策略,对于癌患者的治疗有着重大意义。
附图说明
图1为化合物3u处理后结直肠癌细胞HCT116后的抗凋亡蛋白和死亡受体的表达情况图;
图2为化合物3u处理后结直肠癌细胞HCT116后的凋亡相关蛋白的表达情况图;
图3为MTT检测化合物3u处理后细胞的活力结果;
图4为流式细胞术检测被Annexin v和/或PI染色的细胞群分布实验中的正常细胞的细胞群分布;
图5为被4μmol/L浓度的3u处理后的细胞群分布图;
图6为被8μmol/L浓度的3u处理后的细胞群分布图;
图7为被12μmol/L浓度的3u处理后的细胞群分布图;
图8为被20μmol/L浓度的3u处理后的细胞群分布图;
图9为被40μmol/L浓度的3u处理后的细胞群分布图。
具体实施方式
下面结合附图和实施例对本发明作进一步详细说明,但本发明保护范围不局限于所述内容,实施例中使用的试剂,如无特殊说明,均为常规市售试剂或按常规方法配制的试剂,实施例中使用方法,如无特殊说明均为常规实验方法。
实施例1:采用蛋白免疫印迹法检测化合物3u处理后细胞内死亡受体及凋亡相关蛋白表达情况
1、化合物3u处理后结直肠癌细胞HCT116的总蛋白的提取
(1)加药前一天将HCT116细胞铺进6孔板中,细胞量为每孔60万;
(2)设置空白组和加药组,第二天待细胞贴壁以后,用培养基配制不同浓度的药物(0μmol/L、4μmol/L、12μmol/L、20μmol/L、40μmol/L),然后加入六孔板中,放在37℃、5% CO2培养箱中处理3h;
(3)3h后取出,收集细胞,加入碧云天RIPA-S裂解液和PMSF(1mmol/L);
(4)每5min震荡一次,30min之后,12000r、4℃离心10min,留下上清;
2、用碧云天BCA蛋白浓度检测试剂盒测定蛋白浓度
(1)配制BSA标准品:0.025、0.05、0.1、0.2、0.3、0.4、0.5mg/mL;
(2)分别取20μL的样品稀释液和20μL BSA标准品加入96孔板中,然后加入200μLBCA工作液,放置在37℃恒温振荡器中孵育30min;
(3)使用酶标仪在560nm处测定吸光度;
(4)根据标准品的吸光度绘制出一次函数,然后计算样品浓度值,-80℃保存;
3、SDS-PAGE电泳
(1)使用12%或15%的分离胶、5%的浓缩胶;
(2)按照30μg的上样量取样品,加入5×Loading buffer,充分混匀后,95℃煮5min,4℃下离心10min,将处理好的样品加入凝胶孔中;
(3)先80V处理30min或待marker开始分离,调节电压至110V处理2h或待marker完全分离后,电泳结束,除去玻璃板,取出凝胶;
4、转膜
(1)根据凝胶大小,裁剪PVDF膜,将PVDF膜先用甲醇进行活化;将凝胶、PVDF膜、海绵垫、滤纸浸泡在转膜缓冲液中,按照海绵、滤纸、PVDF膜、凝胶、滤纸、海绵的顺序依次叠放好,需要注意在操作过程中去除气泡;
(2)组装好后,放入电泳槽中,使用DYY-7C电泳仪,110V处理2h,转膜的过程中需要放置在冰水中;
5、免疫印迹反应
(1)转膜结束后,用5%脱脂牛奶将PVDF膜进行封闭,室温2h;
(2)封闭结束以后,用1×PBST进行清洗,然后加入已经按照说明书稀释好的一抗,用薄膜封好后,4℃孵育过夜;
(3)用1×TBST洗PVDF膜,洗三次,每次10min;
(4)加入已经按照说明书稀释好的二抗,二抗的稀释比例为1:2500,将PVDF膜用薄膜封好并加入二抗,室温孵育2h;
(5)用1×TBST洗PVDF膜,洗三次,每次10min;
(6)二抗结束后,配制ECL显影液,根据tanon设定的程序进行曝光显影;
结果见图1、2,c-FLIP被认为是凋亡最主要的抑制蛋白,包括c-FLIPL、c-FLIPS,从图1中可以看出在化合物3u作用后的HCT116细胞中,抗凋亡蛋白XIAP、c-FLIP表达量减少,死亡受体5表达增加,并且伴随着化合物3u的浓度增加抗凋亡蛋白的抑制效果越好,呈现浓度依赖的方式抑制抗凋亡蛋白表达,说明化合物3u抑制抗凋亡蛋白XIAP、c-FLIP的表达效果与浓度相关;图2结果显示化合物3u作用后的HCT116细胞中,凋亡相关蛋白Caspase-8/3、PARP1表达量增加,结合死亡受体5表达增加,说明化合物3u通过死亡受体激活凋亡通路信号,化合物3u能通过调控凋亡方式抑制结直肠癌细胞。
实施例2:化合物3u处理细胞后的活力测定
采用不同浓度的化合物3u处理HCT116细胞,如果使用相关抑制剂则提前一个小时先用抑制剂进行处理后,再加入化合物3u,采用MTT实验技术对细胞活性进行检测。
在用药处理的前一天将对数生长期的HCT116均匀铺进96孔板中,每孔5000~10000个细胞。
待第二天细胞贴壁以后,用培养基配制不同浓度的化合物3u及抑制剂,设置空白组,按照0、1、5、10、25、50μmol/L,每孔180μL且每个浓度至少三个平行复孔,培养箱培养44h后,加入5mg/mL的MTT溶液,每孔20μL,继续培养4h,4h后从培养箱取出,去除上清液时小心不要吸到细胞,随后加入150μL DMSO,室温孵育15min,待结晶物充分溶解,用酶标仪测量490nm下的OD值,用graphpad6.0进行IC50的计算。
MTT法检测化合物3u对HCT116细胞的影响,结果见图3,从图中可以看出凋亡蛋白抑制剂ZVAD/IETD的加入显著增加了IC50,同时化合物3u对HCT116细胞非常敏感,只需要2.6μmol/L就可以达到半数细胞的死亡;但是当提前用ZVAD抑制Caspase-8和Caspase-3的蛋白活性,它的半数致死量提高了近3倍,降低了化合物对癌细胞的敏感性,同时也证明是通过凋亡路径诱导结直肠癌细胞的死亡;
但是与坏死相关抑制剂共同作用于结直肠癌HCT116时,抑制剂的加入并没有明显影响化合物3u对HCT116的敏感性,IC50几乎不变,这也表明化合物3u不会引起能够产生炎症反应的细胞坏死机制的发生,只会诱导凋亡的发生。
实施例3:流式细胞术检测被Annexin v和/或PI染色的细胞群分布
将对数生长期的HCT116细胞用PBS洗三遍之后,用0.25%胰酶消化3min,然后加入含10%血清的RPMI1640培养基终止消化,将细胞从培养瓶中收集至离心管中,离心后再重悬细胞。用血球计数板计数,将细胞铺进6孔板中,每孔细胞数量为60万个,次日加入用培养基稀释的不同浓度的化合物3u,将加了药的6孔板放入37℃、5%二氧化碳的培养箱中培养,3h后取出,收集细胞,用PBS清洗三次后,用300μLPBS重悬细胞;
设置双阴组、单染Annexin V组(只加5μL Annexin V)、单染PI组(只加5μL PI)、双染组(5μL Annexin V和5μL PI);加完后室温避光孵育15min后,使用流式细胞仪(BDAccuriTM C6 Flow Cytometer, USA)检测Annexin V/PI的细胞比例;Annexin V横坐标指示,PI纵坐标指示,左下指示正常细胞群(Annexin V和PI均没染上),右下指示凋亡早期细胞(只染上Annexin V),左上指示坏死细胞群(只染上PI),右上指示凋亡后期细胞群(Annexin V和PI均染上);
结果见图4-9,结果显示化合物3u以浓度依赖的方式调控Annexin V/PI的细胞群比例,化合物3u的浓度越高,凋亡早期的细胞数量也越来越多,当达到40μmol/L的时候凋亡后期的细胞数量已经达到51%,这也说明浓度越高凋亡的进展越快,进一步证明化合物3u通过凋亡的方式来抑制结直肠癌细胞的发展并且用药浓度越高反应越快。
以上说明本发明化合物3u具有三重靶向的作用,不仅能够通过靶向死亡受体激活外源性凋亡途径诱导癌细胞死亡,除此之外,还发现该化合物对抗凋亡蛋白c-FLIP和XIAP具有显著抑制效果,这也为解决黑色素瘤、肝癌、结肠癌以及前列腺癌等肿瘤耐药性问题提供了新的解决思路。
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