CN116942671A - ALKinhibitor2在制备治疗食管鳞癌的药物中的应用 - Google Patents
ALKinhibitor2在制备治疗食管鳞癌的药物中的应用 Download PDFInfo
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Abstract
本发明属于医药领域,具体涉及ALK inhibitor 2在制备治疗食管鳞癌的药物中的应用。本发明的究结果表明,ALK inhibitor 2能够高效抑制RSK4酶的表达,且能够高效的抑制食管鳞癌细胞的增殖、侵袭和迁移,与Afatinib联用,能够显著抑制食管鳞癌埃克替尼耐药株的增殖,该结果为食管鳞癌患者的治疗和预后提供了一种有效的技术手段。
Description
技术领域
本发明属于医药领域,具体涉及ALK inhibitor 2在制备治疗食管鳞癌的药物中的应用。
背景技术
食管鳞癌(esophageal squamous cell carcinoma,ESCC)是食管癌最常见的组织学类型。尽管临床上有针对ESCC的多种治疗方法,但晚期ESCC的5年总生存率仅为15%。放射治疗是ESCC一种重要的治疗方式,特别是对那些手术不可切除的晚期食管癌患者。遗憾的是,肿瘤细胞的放疗抵抗会导致ESCC的复发和治疗失败,大多数患者在病理缓解后仍有复发,放疗还会引起多种副作用。由于放疗的局限性,研制开发针对ESCC的靶向治疗药物十分必要。
ESCC分子靶向治疗主要的靶点有表皮生长因子受体(epidermal growth factorreceptor,EGFR)、人表皮生长因子受体2(human epidermal factor,VEGF)等,但目前关于靶向治疗的研究仍处于起始阶段,ESCC精准的治疗靶点还不十分明确,耐药性的出现使正常剂量的靶向药物不再发挥应有的抑癌作用。因此,开发新的化疗药物对ESCC患者的治疗和预后具有重要意义。
ALK inhibitor 2(compound 18)是一种有效的嘧啶类ALK抑制剂,具有睾丸特异性丝氨酸/苏氨酸激酶2(TSSK2)和粘着斑激酶(FAK)抑制活性,目前尚无ALK inhibitor 2在食管鳞癌中的报道。
发明内容
本发明的目的在于提供ALK inhibitor 2的新用途。
第一方面,本发明提供ALK inhibitor 2在制备治疗食管鳞癌的药物中的应用。
进一步的,所述ALK inhibitor 2抑制食管鳞癌细胞增殖。
进一步的,所述ALK inhibitor 2抑制食管鳞癌细胞侵袭和迁移。
进一步的,所述ALK inhibitor 2降低食管鳞癌细胞的耐药性。
进一步的,所述ALK inhibitor 2联合Afatinib用于制备治疗食管鳞癌的药物。
第二方面,本发明提供一种治疗食管鳞癌的药物,所述药物包括有效剂量的ALKinhibitor 2。
进一步的,ALK inhibitor 2为所述药物的唯一有效成分或有效成分之一。
更进一步的,所述药物抑制食管鳞癌细胞增殖、侵袭和迁移。
第三方面,本发明提供一种治疗食管鳞癌的药物组合,所述药物包括有效剂量的ALK inhibitor 2和Afatinib。
与现有技术相比,本发明具有如下有益效果:
本发明提供了ALK inhibitor 2的新用途,本发明的研究结果表明,ALKinhibitor 2能够高效抑制RSK4酶的表达,且能够高效的抑制食管鳞癌细胞的增殖、侵袭和迁移,与Afatinib联用,能够显著抑制食管鳞癌埃克替尼耐药株的增殖,该结果为ESCC患者的治疗和预后提供了一种有效的技术手段。
附图说明
图1为ALK inhibitor 2的化学结构式。
图2为ALK inhibitor 2对RSK4酶的半抑制曲线。
图3为Western Blot检测不同浓度ALK inhibitor 2对RSK4下游底物的磷酸化水平的影响。
图4为ALK inhibitor 2对食管鳞癌细胞(TE-10)的半抑制曲线。
图5为3μm ALK inhibitor 2处理下的食管鳞癌细胞(TE-10)的生长曲线。
图6为ALK inhibitor 2对食管鳞癌细胞(TE-10)侵袭和迁移的影响,其中,A为细胞吉姆萨染色图,B为A的定量统计结果。
图7为不同处理对食管鳞癌埃克替尼耐药株(KYSE450-IR)增殖的影响,其中,a为DMSO处理,b为2μm的Afatinib处理,c为2μm的ALK inhibitor 2处理,d为Afatinib+ALKinhibitor 2处理。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明,但不应理解为本发明的限制。如未特殊说明,下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段,下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
本发明披露了ALK inhibitor 2的新用途,所述ALK inhibitor 2的分子式为C23H28ClN7O3,CAS No.:761438-38-4,化学结构式如图1所示。
实施例1:ALK inhibitor 2对RSK4酶的抑制
1实验方法
本次实验利用ADP-Glo方法检测ALK inhibitor 2在RSK4酶上的作ALK inhibitor2起始浓度为10μM,5倍梯度稀释,2个重复,6个浓度。
(1)冰上解冻RSK4酶,RSK Substrate,kinase assay buffer III(5×缓冲液),DTT(0.1M)和ATP(10mM),并且以上试剂在整个实验过程中需要一直放置在冰上。
(2)将5×的缓冲液用去离子水配置成1×的缓冲液,并在其中加入DTT,DTT在1×缓冲液中的浓度为50μM;
(3)将1μl/孔5×待测化合物加入到白色微孔板中,微孔板在离心机上1000转离心1分钟;
阳性对照孔(Pos.Ctrl):1μl/孔化合物稀释溶剂;
空白对照孔(Blank):1μl/孔1×缓冲液。
(4)RSK4酶完全解冻后,使用1×的缓冲液将RSK4酶稀释到1ng/μl,取2μl/孔加入到白色微孔板中,此时每孔中RSK4的酶量为2ng;空白对照孔加入2μl/孔1×缓冲液;此步骤请在冰上进行,加完后,微孔板在离心机上1000转离心1分钟;
(5)配制RSK Substrate/ATP混合液:
RSK Substrate/ATP混合液:取130μl的RSK Substrate(1mg/ml)加入3.25μl 5mMATP和127μl的2×缓冲液(注意此处是按比例稀释),此时ATP浓度为62.5μM,RSK Substrate浓度为0.5mg/ml;此步骤请在冰上进行;
(6)取2μl/孔RSK Substrate/ATP的混合溶液到白色微孔板中,此时RSKSubstrate浓度为0.2mg/ml,ATP浓度为25μM,加完后微孔板1000转离心1分钟;
(7)离心完毕后,给微孔板贴上膜,压紧贴膜,在25℃中孵育1小时;
(8)将Promega的试剂盒中需要用的ADP-GloTM reagent和Kinase Detection相关试剂平衡到室温,并按照说明书将Kinase Detection buffer和Kinase DetectionSubstrate混合备用。
(9)结束孵育后,取5μl/孔ADP-GloTM reagent加入到白色微孔板中,微孔板1000转离心1分钟,25℃中孵育40分钟;
(10)结束孵育后,取Kinase Detection混合液10μl/孔加入到微孔板中,微孔板1000转离心1分钟,25℃中孵育30分钟;
(11)结束孵育后在读板器上进行化学发光(Luminescence)检测,读取发光值(RLU);
(12)酶抑制率计算:
%Inhibition=100-(RLU(Sample)-RLU(Blank))/(RLU(Pos.Ctrl)-RLU(Blank))×100%。
2实验结果
如图2所示,随着ALK inhibitor 2浓度的升高,ALK inhibitor 2对RSK4激酶活性的抑制作用也增强。采用GraphPad软件进行曲线拟合,得出IC50值为8.75nM。结果表明ALKinhibitor 2能够有效抑制RSK4的磷酸化,抑制RSK4的激活,是有效的RSK4抑制剂。
实施例2:ALK inhibitor 2对RSK4下游底物的磷酸化水平的影响
1实验方法
本次实验采用ALK inhibitor 2不同浓度刺激食管鳞癌细胞系(TE10,ECA109)Western blot检测RSK4下游底物的磷酸化水平的影响。
(1)蛋白提取,使用康为世纪RIPA裂解液。细胞培养瓶置于冰上裂解20min,来回摇动,可用枪头吹打。用细胞刮刀刮下细胞,转移至EP管中,4℃离心。可吸取10-20μl进行蛋白定量。加入上样缓冲液,并煮沸5min,12000rpm离心5min,于超低温冰箱保存。组织蛋白提取:液氮研磨,融化后转入EP管中,冰上裂解20min,4℃离心。
(2)蛋白定量,使用康为世纪BCA定量试剂盒。每个样品做两个复孔,取平均值做标准曲线及计算样品浓度。
(3)蛋白电泳,使用康为世纪SDS-PAGE凝胶制备试剂盒。分离胶浓度为8%,浓缩胶浓度为5%。分离胶距前玻璃板上沿1.5cm,注水封胶。用滤纸吸干水封液,灌入浓缩胶,插梳子。电压100V恒压1h30min至溴酚蓝跑出停止电泳。
(4)转膜,将胶、滤纸、海绵垫浸泡于4℃预冷的转膜缓冲液中平衡10min,将PVDF膜用甲醇浸润5min,充分湿润后转移至转膜缓冲液浸润10min。安装转膜夹顺序:黑色版、海绵、3层滤纸、胶、膜、3层滤纸、海绵、白色版。每步都需赶出气泡,一次性放好膜。在冰浴中300mA恒流转膜100min。
(5)封闭,使用5%脱脂奶粉室温摇床封闭1h。用TBST漂洗10minx3次。
(6)一抗孵育,孵育抗体时膜与胶接触一面始终朝上,4℃摇床孵育过夜。次日用TBST漂洗。
(7)二抗孵育,室温孵育1h。用TBST漂洗。
(8)发光,使用康为世纪化学发光检测试剂盒,用化学发光成像仪扫描检测结果。
(9)使用Photoshop软件对图像进行半定量灰度分析,计算蛋白相对表达量。
2实验结果
如图3所示,使用不同浓度ALK inhibitor 2刺激食管鳞癌细胞系,通过WesternBlot检测发现ALK inhibitor 2能够抑制RSK4下游底物磷酸化,下调P-RPS6(S235/S236)以及P-GSK3β(ser9)的表达。
实施例3:ALK inhibitor 2对食管鳞癌细胞生长的调控
1实验方法
本次实验采用不同浓度ALK inhibitor 2刺激食管鳞癌细胞系(TE10),CCK8检测ALK inhibitor 2对食管鳞癌细胞增殖的影响,Transwell检测ALK inhibitor 2对食管鳞癌细胞迁移的影响,同时以不加药物作为空白对照(Black或Vector)。
1.1CCK8检测步骤:
使用上海陶术生物科技有限公司的CCK-8细胞增殖试剂盒完成。
(1)96孔板接种细胞悬液,每孔100μL,每孔2,000个细胞。
(2)按照实验需要,进行培养或给与药物,处理适当时间。
(3)每孔加入CCK-8溶液10μL,37℃孵育。
(4)酶标仪选择450nm波长测定吸光度值。以时间和吸光度值绘制生长曲线。
1.2细胞迁移:
(1)将培养好的TE10细胞消化制备成单细胞悬液,用PBS洗一遍。
(2)对照组使用加有DMSO的DMEM培养基重悬至5×105个/ml,实验组使用3μM ALKinhibitor 2的DMEM培养基重悬至5×105个/ml。
(3)24孔板小室的下室加入500μl含10%胎牛血清的DMEM培养基,上室分别加入200μl实验组和对照组的细胞重悬液。
(4)常规培养24h。
(5)吸出小室内培养基,用无水甲醇固定小室细胞15min,PBS漂洗。
(6)24孔板中加入吉姆萨染色液,染色20min,使用PBS清洗。
(7)用棉签小心擦去膜内层细胞,将膜风干。
(8)显微镜下拍照,取5个高倍视野计数,取平均值。
1.3细胞侵袭:
(1)将冻存于-80℃冰箱中的BD Matrigel胶置于4℃过夜,解冻为液态。
(2)使用无血清培养基按照1:8稀释浓度稀释为50μg/ml Matrigel,充分混匀后,将60μl Matrigel胶加入到Transwell小室的上室。
(3)将培养好的TE10细胞消化制备成单细胞悬液,用PBS洗一遍。
(4)对照组使用加有DMSO的DMEM培养基重悬至5×105个/ml,实验组使用3μM ALKinhibitor 2的DMEM培养基重悬至5×105个/ml。
(5)在Transwell小室的下室加入500μl含10%胎牛血清的DMEM培养基,上室分别加入200μl实验组和对照组的细胞重悬液。
(6)将Transwell小室置于恒温细胞培养箱中培养24h。
(7)培养结束后,取出Transwell小室,使用蘸有PBS的棉签擦洗上室,去除Matrigel胶;
(8)将小室放入到甲醇中,固定20min;
(9)24孔板中加入吉姆萨染色液,染色20min,使用PBS清洗。
(10)显微镜下拍照,取5个高倍视野计数,取平均值。
2实验结果
如图4-6所示,ALK inhibitor 2对TE10的半抑制浓度为0.3018μM,与空白对照相比,ALK inhibitor 2能够显著抑制TE10细胞的增殖、侵袭和迁移。
实施例4:ALK inhibitor 2联合Afatinib对对食管鳞癌埃克替尼(Icotinib)耐药株(KYSE450-IR)增殖的调控
1实验方法
对多种ESCC细胞株(T1、T10、T11、KYSE150、KYSE450、ECA109、EC9706)中EGFR的表达水平和对Icotinib的敏感性进行了检测,选取高表达EGFR且对Icotinib敏感(低IC50)的细胞系KYSE450,采用Icotinib药物浓度递增的作用方式进行诱导KYSE450细胞使之产生耐药性,并取得稳定的对Icotinib耐药的细胞系。参照文献报道以及在正式诱导培养前所做的预实验中得出的最佳诱导Icotinib的药物作用浓度梯度为:100nM,300nM,500nM,800nM,1μM和5μM。本实验初始诱导剂量为100nM,最高诱导剂量为5μM。诱导期间每2天更换含Icotinib培养液一次。当初始诱导剂量诱导两周,细胞生长稳定后,开始倍增药物诱导剂量,在到达最高诱导剂量之前每个剂量保持培养14天。经10个月诱导培养所得耐药细胞命名KYSE450-IR。
采用ALK inhibitor 2(2μm)、Afatinib(2μm)或ALK inhibitor 2+Afatinib(2μm,二者质量比为1:1)刺激KYSE450-IR细胞,以DMSO作为空白对照,CCK8检测(方法同实施例3)不同药物对KYSE450-IR增殖的影响。
2实验结果
如图7所示,单独用ALK inhibitor 2或Afatinib均能够抑制KYSE450-IR的增殖,但是Afatinib的抑制作用极低,说明KYSE450-IR对Afatinib产生耐药性,而ALK inhibitor2和Afatinib联合使用时,对KYSE450-IR的抑制作用显著强于单独使用ALK inhibitor 2刺激,说明ALK inhibitor 2和Afatinib能够起到协同增效的作用,该结果为食管鳞癌的治疗提供新的思路。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (10)
1.ALK inhibitor 2在制备治疗食管鳞癌的药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述ALK inhibitor 2抑制食管鳞癌细胞增殖。
3.根据权利要求1所述的应用,其特征在于,所述ALK inhibitor 2抑制食管鳞癌细胞侵袭和迁移。
4.根据权利要求1所述的应用,其特征在于,所述ALK inhibitor 2降低食管鳞癌细胞的耐药性。
5.根据权利要求1所述的应用,其特征在于,所述ALK inhibitor 2联合Afatinib用于制备治疗食管鳞癌的药物。
6.一种治疗食管鳞癌的药物,其特征在于,所述药物包括有效剂量的ALK inhibitor2。
7.根据权利要求6所述的药物,其特征在于,ALK inhibitor 2为所述药物的唯一有效成分或有效成分之一。
8.根据权利要求7所述的药物,其特征在于,所述药物抑制食管鳞癌细胞增殖、侵袭和迁移。
9.一种治疗食管鳞癌的药物组合,其特征在于,所述药物包括有效剂量的ALKinhibitor 2和Afatinib。
10.根据权利要求9所述的药物组合,其特征在于,所述药物组合降低食管鳞癌细胞对Icotinib的耐药性。
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