CN116531382A - Apy29在制备治疗食管鳞癌的药物中的应用 - Google Patents
Apy29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APY29在制备治疗食管鳞癌的药物中的应用。本发明的研究结果表明,APY29能够高效抑制RSK4酶的表达,且能够高效的促进食管鳞癌细胞凋亡,抑制食管鳞癌细胞的增殖、侵袭和迁移,还可减轻食管鳞癌细胞的放疗抵抗,具有作为食管鳞癌放疗增敏剂的潜力,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APY29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。
APY29是一种I类IRE1α抑制剂,与IRE1α的ATP结合位点相结合,抑制其自我磷酸化(IC50=280nM)、增强其RNase作用(EC50=460nM),目前尚无APY29在食管鳞癌中的报道。
发明内容
本发明的目的在于提供APY29的新用途。
第一方面,本发明提供APY29在制备治疗食管鳞癌的药物中的应用。
进一步的,所述APY29促进食管鳞癌细胞凋亡。
进一步的,所述APY29抑制食管鳞癌细胞增殖。
进一步的,所述APY29抑制食管鳞癌细胞侵袭和迁移。
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APY29。
进一步的,APY29为所述药物的唯一有效成分或有效成分之一。
更进一步的,所述药物促进食管鳞癌细胞凋亡,抑制食管鳞癌细胞增殖、侵袭和迁移。
第三方面,本发明提供APY29在制备食管鳞癌放疗增敏剂中的应用。
第四方面,本发明提供一种食管鳞癌放疗增敏剂,所述食管鳞癌放疗增敏剂包括有效剂量的APY29。
进一步的,所述APY29抑制食管鳞癌细胞的放疗抵抗。
与现有技术相比,本发明具有如下有益效果:
本发明提供了APY29的新用途,本发明的研究结果表明,APY29能够高效抑制RSK4酶的表达,且能够高效的促进食管鳞癌细胞凋亡,抑制食管鳞癌细胞的增殖、侵袭和迁移,还可减轻食管鳞癌细胞的放疗抵抗,具有作为食管鳞癌放疗增敏剂的潜力,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APY29的化学结构式。
图2为APY29对RSK4酶的半抑制曲线。
图3为经APY29刺激的食管鳞癌细胞(TE-10)的周期分布。
图4为3μm APY29处理下的食管鳞癌细胞(TE-10)的生长曲线。
图5为APY29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6为APY29对食管鳞癌细胞(TE-10)的放疗抵抗的作用。
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。
本发明披露了化合物APY29的新用途,所述APY29的分子式为C17H16N8,CAS No.:1216665-49-4,化学结构式如图1所示。
实施例1:APY29对RSK4酶的抑制
1实验方法
本次实验利用ADP-Glo方法检测APY29在RSK4酶上的作APY29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APY29浓度的升高,APY29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55.10nM。结果表明APY29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:APY29对食管鳞癌细胞生长的调控
1实验方法
本次实验采用APY29刺激食管鳞癌细胞系(TE10),CCK8检测APY29对食管鳞癌细胞增殖的影响,流式细胞术检测食管鳞癌细胞周期分布,Transwell检测APY29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)。
1.1流式细胞术检测步骤:
(1)在TE10细胞状态良好的对数生长期进行试验,保证瓶细胞数量少为1x106个/ml。配制固定液比例为1×PBS:无水乙醇=1:2,每瓶细胞至少1ml固定液。
(2)用4℃预冷的1×PBS洗两次,尽量吸净1×PBS后加入固定液重悬,上流式细胞仪进行检测。
(3)将结果数据整理并进行统计。
1.2CCK8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。
1.3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3μMAPY29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。
1.4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3μMAPY29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3-5所示,与对照组相比,经APY29刺激的S期TE10细胞显著增多,提示APY29使TE10细胞发生S期阻滞,促进细胞凋亡,且TE10细胞的增殖、侵袭和迁移能力均受到显著的抑制作用。
实施例3:APY29对食管鳞癌细胞的放疗抵抗的作用
1实验方法
辐射克隆形成实验
(1)将TE10细胞置于含10%胎牛血清的1640培养基,实验组加3μMAPY29,对照组不加,将不同组细胞进行0Gy或8Gy剂量的X线照射后,正常培养24h。
(2)调整细胞浓度至1×103个/ml,取200μl接种至6cm培养皿,加10ml培养基。
(3)常规培养2周,出现肉眼可见集落形成时停止培养。
(4)弃去培养基,PBS洗涤2次。
(5)无水甲醇5ml固定15min,弃固定液。
(6)加入吉姆萨染液染色20min,自来水冲洗,空气干燥。
(7)拍照,并计数肉眼可见的克隆数,计算克隆形成率=克隆数/接种细胞数。
2实验结果
如图6所示,APY29处理可减轻TE10细胞的放疗抵抗,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.APY29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APY29促进食管鳞癌细胞凋亡。
3.根据权利要求1所述的应用,其特征在于,所述APY29抑制食管鳞癌细胞增殖。
4.根据权利要求1所述的应用,其特征在于,所述APY29抑制食管鳞癌细胞侵袭和迁移。
5.一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APY29。
6.根据权利要求5所述的药物,其特征在于,APY29为所述药物的唯一有效成分或有效成分之一。
7.根据权利要求6所述的药物,其特征在于,所述药物促进食管鳞癌细胞凋亡,抑制食管鳞癌细胞增殖、侵袭和迁移。
8.APY29在制备食管鳞癌放疗增敏剂中的应用。
9.一种食管鳞癌放疗增敏剂,其特征在于,所述食管鳞癌放疗增敏剂包括有效剂量的APY29。
10.根据权利要求9所述的食管鳞癌放疗增敏剂,其特征在于,所述APY29抑制食管鳞癌细胞的放疗抵抗。
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