CN115089589A - 激酶抑制剂gsk1838705a在制备抑制食管癌的药物中的应用 - Google Patents
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Abstract
本发明公开了激酶抑制剂GSK1838705A在制备抑制食管癌的药物中的应用,涉及生物医药技术领域。本发明的激酶抑制剂GSK1838705A在制备抑制食管癌的药物中的应用,具体的是抑制食管癌相关成纤维细胞系的增殖的药物,通过激酶抑制剂GSK1838705A能够抑制食管癌相关成纤维细胞系的增殖,阻滞食管癌相关成纤维细胞系的细胞周期在G2/M期,并诱导食管癌相关成纤维细胞系中活性氧的产生,并诱导其凋亡,同时激酶抑制剂GSK1838705A能显著抑制食管癌相关成纤维细胞系中α‑SMA的表达,因此,激酶抑制剂GSK1838705A能够用于制备抑制食管癌的药物。
Description
技术领域
本发明涉及生物医药技术领域,尤其涉及激酶抑制剂GSK1838705A在制备抑制食管癌的药物中的应用。
背景技术
食管癌是人类常见的恶性肿瘤之一,发病率位居世界第八,死亡率居世界第六,且具有地域性,在亚洲,鳞状细胞癌是食管癌患者的主要类型,而腺癌多见于美国及西欧。食管癌早期症状不明显,临床确诊时约70%-80%的患者已丧失手术机会,化疗及放疗成为局部晚期食管癌的主要治疗手段。目前,食管癌患者的五年生存率尚不足20%,究其主要原因为食管癌对其常用的化疗药物如顺铂、紫杉醇等均表现出极其有限的敏感性,且食管癌易对放疗有明显的抵抗性,导致患者治疗结束后易出现肿瘤复发或转移。目前,国内外针对食管癌耐治疗抵抗的分子机制研究多着眼于癌细胞本身,而对于肿瘤微环境的研究则较少。
肿瘤微环境由癌肿瘤相关成纤维细胞(cancer-associated fibroblasts,CAFs)、免疫细胞、内皮细胞及细胞外基质等多种组分组成。越来越多的研究显示肿瘤微环境与肿瘤的发生发展密切相关。尤其是CAFs,其作为肿瘤微环境的主要成员,是肿瘤微环境对肿瘤组织发挥影响作用的主要执行者。已知CAFs存在于90%的上皮来源的肿瘤中,且细胞比例约占肿瘤微环境70%,可促进肿瘤组织的放化疗抵抗性、转移侵袭、免疫逃逸等多种恶性表型。诸多研究显示CAFs在食管癌中发挥了及其重要的促肿瘤作用,如CAFs可作为食管癌患者术后及新辅助治疗后的预后因子,并与食管癌患者淋巴转移显著相关,同时,CAFs可介导食管癌细胞产生显著的放化疗抵抗性。此外,CAFs与食管癌患者肿瘤组织中免疫细胞浸润显著相关,如CAFs可抑制细胞毒T细胞在肿瘤组织中的浸润,而促进免疫抑制的调节性T细胞浸润肿瘤组织,从而构建了免疫抑制的肿瘤微环境。
通过发明人长期对CAFs在食管癌中的促肿瘤作用及其机制研究,发现CAFs可介导食管癌细胞产生广谱耐药,并可通过调控食管癌细胞放疗后DNA损伤修复响应,介导食管癌细胞在体内外水平产生显著的放疗抵抗性。因此,CAFs作为食管癌治疗的有效靶点,消除或抑制CAFs的活性对于食管癌的根治性治疗具有极其重要的意义。
CAFs具有强大的抗氧化能力和DNA修复能力,对放化疗治疗极其抵抗,因而放化疗后残留的CAFs可介导食管癌组织的复发及转移,极大地限制了放化疗对于食管癌患者的治疗作用。提供一种能抑制CAFs活性的特异性抑制剂有望能显著地提高食管癌患者的治疗效果,解决食管癌患者治疗目前遇到的瓶颈问题。然而,目前能有效抑制CAFs活性的小分子抑制剂尚未见报道。
发明内容
针对上述问题,本发明的目的在于设计提供激酶抑制剂GSK1838705A在制备抑制食管癌的药物中的应用。
本发明通过以下技术手段解决上述技术问题:
激酶抑制剂GSK1838705A在制备抑制食管癌的药物中的应用,所述激酶抑制剂GSK1838705A的化学结构如下:
进一步,所述抑制食管癌的药物具体是抑制食管癌相关成纤维细胞系的增殖的药物。
进一步,所述激酶抑制剂GSK1838705A诱导食管癌相关成纤维细胞系中活性氧的产生。
进一步,所述激酶抑制剂GSK1838705A诱导食管癌相关成纤维细胞系的凋亡。
进一步,所述激酶抑制剂GSK1838705A抑制食管癌相关成纤维细胞系中α-SMA的表达。其中,α-SMA即激活的癌肿瘤相关成纤维细胞(CAFs)的标志物。
进一步,所述激酶抑制剂GSK1838705A阻滞食管癌相关成纤维细胞系的细胞周期在G2/M期。
进一步,所述抑制食管癌的药物是以激酶抑制剂GSK1838705A为唯一活性成分,或包括激酶抑制剂GSK1838705A的药物组合物。
进一步,所述包括激酶抑制剂GSK1838705A的药物组合物是指激酶抑制剂GSK1838705A与药学上允许的或多种辅料构成的药物组合物。
所述辅料为稀释剂、赋形剂、粘合剂、填充剂、崩裂剂、香味剂、甜味剂中的至少一种。所述药学上允许的一种或多种辅料是指药学领域常规的药物辅料,其中,稀释剂、赋形剂如水等;粘合剂如纤维素衍生物、明胶或聚乙烯吡咯烷酮等;填充剂如淀粉等;崩裂剂如碳酸钙或碳酸氢钠;也可以在药物组合物中加入其他辅助剂如香味剂和/或甜味剂。
本发明的有益效果:
1、本发明提供了激酶抑制剂GSK1838705A在制备抑制食管癌药物中的应用,通过对激酶抑制剂库的激酶抑制剂进行高通量筛选,筛选出的GSK1838705A可抑制食管癌相关成纤维细胞系的增殖,且其抑制作用明显强于对食管癌细胞Eca109、KYSE-150及食管正常细胞Het-1A的增殖抑制。
2、本发明的激酶抑制剂GSK1838705A能够阻滞食管癌相关成纤维细胞系CAFs-1、CAFs-2的细胞周期在G2/M期,并诱导食管癌相关成纤维细胞系中活性氧的产生,并诱导其凋亡,同时激酶抑制剂GSK1838705A能显著抑制食管癌相关成纤维细胞系中α-SMA的表达,能够作为潜在的制备抑制食管癌药物,具有进一步开发的前景,为抑制食管癌药物提供了新的选择。
附图说明
图1是CCK8法检测细胞增殖能力的折线图;
图2是荧光探针法检测细胞活性氧(ROS)生成的荧光图;
图3是流式细胞术检测细胞凋亡的柱状统计图;
图4是流式细胞术检测细胞周期的柱状统计图;
图5是qPCR法检测细胞α-SMA mRNA水平的柱状统计图。
具体实施方式
以下将结合具体实施例对本发明进行详细说明:
激酶抑制剂GSK1838705A在制备抑制食管癌的药物中的应用,具体如下:
实施例一GSK1838705A对细胞增殖能力影响的实验
(1)实验材料:食管癌相关成纤维细胞(CAFs-1、CAFs-2)、食管癌细胞系(KYSE-150、Eca109)、正常食管上皮细胞(Het-1A)。其中,CAFs-1、CAFs-2是分离人源食管癌组织中成纤维细胞所得的原代细胞,命名为食管癌相关成纤维细胞,由发明人经过分离得到,分离及鉴定技术已发表文献“Cancer-associated fibroblasts mediated chemoresistanceby a FOXO1/TGFβ1signaling loop in esophageal squamous cell carcinoma”。食管癌细胞系KYSE-150、Eca109购自上海中科院细胞库,正常食管上皮细胞Het-1A购自美国模式培养物集存库(ATCC)。
(2)实验方法
取对数生长期的CAFs-1、CAFs-2、KYSE-150、Eca109、Het-1A细胞,调整细胞密度,以5000个/孔的细胞密度立即接种到96孔板中,置于37℃,5%CO2饱和湿度培养箱内培养。
激酶抑制剂GSK1838705A设5个浓度,分别为0μM、2.5μM、5μM、7.5μM、10μM。
培养24h待细胞贴壁后,每种细胞都加入以上各个浓度的GSK1838705A,置于37℃,5%CO2饱和湿度培养箱内培养。
24h后弃去全部培养液,每孔加入100μL CCK8工作液(使用前1:10用含10%FBS的完全培养液稀释,CCK8试剂盒购自碧云天生物技术公司,货号C0039),在培养箱内继续孵育2h。
使用酶标仪检测培养液在450nm波长处的吸光度,并计算细胞活度,计算公式为:细胞活度=各浓度吸光度/0μM吸光度。
实验结果:如图1所示,5μM、7.5μM、10μM GSK1838705A明显抑制了CAFs-1、CAFs-2的增殖,且呈浓度依赖性,但GSK1838705A对KYSE-150、Eca109、Het-1A的增殖抑制较弱,且浓度依赖性不强。
实施例二GSK1838705A对细胞活性氧(ROS)生成影响的实验
(1)实验材料:CAFs-1、CAFs-2、KYSE-150、Eca109、Het-1A
(2)实验方法
取对数生长期的CAFs-1、CAFs-2、KYSE-150、Eca109、Het-1A细胞,调整细胞密度,以2×105个/孔的细胞密度立即接种到6孔板中,置于37℃,5%CO2饱和湿度培养箱内培养。
激酶抑制剂GSK1838705A设3个浓度,分别为0μM、6μM、10μM。
培养24h待细胞贴壁后,每种细胞都加入以上各个浓度的GSK1838705A,置于37℃,5%CO2饱和湿度培养箱内培养。
24h后弃去全部培养液,每孔加入1mL DCFH-DA活性氧检测工作液(使用前1:1000用无血清培养液稀释,使终浓度为10μM,活性氧检测试剂盒购自碧云天生物技术公司,货号S0033),在培养箱内继续孵育20min。
弃去多余的DCFH-DA,用无血清细胞培养液洗涤细胞三次,荧光显微镜下分析细胞内活性氧生成情况。
实验结果:如图2所示,10μM GSK1838705A明显促进了CAFs-1、CAFs-2中ROS的生成,但对KYSE-150、Eca109、Het-1A中ROS生成影响不显著。
实施例三GSK1838705A对细胞凋亡影响的实验
(1)实验材料:CAFs-1、CAFs-2、KYSE-150、Eca109、Het-1A。
(2)实验方法
取对数生长期的CAFs-1、CAFs-2、KYSE-150、Eca109、Het-1A细胞,调整细胞密度,以2×105个/孔的细胞密度立即接种到6孔板中,置于37℃,5%CO2饱和湿度培养箱内培养。
GSK1838705A设3个浓度,为0μM、6μM、10μM。
培养24h待细胞贴壁后,每种细胞都加入以上各个浓度的GSK1838705A,每个浓度设3个复孔,置于37℃,5%CO2饱和湿度培养箱内培养。
24h后收集细胞,用PBS轻轻重悬细胞并计数,
离心,弃去PBS,加入结合液,调整细胞浓度为1×106个/mL,
取100μL重悬的细胞,加入5μL Annexin V-PE,轻轻混匀;再加入5μL 7-AAD,轻轻混匀,室温避光孵育15min,随即进行流式细胞仪检测。
实验结果:如图3所示,10μM GSK1838705A明显促进了CAFs-1、CAFs-2的凋亡,但对KYSE-150、Eca109、Het-1A的凋亡影响不显著。
实施例四GSK1838705A对细胞周期影响的实验
(1)实验材料:CAFs-1、CAFs-2、KYSE-150、Eca109、Het-1A。
(2)实验方法
取对数生长期的CAFs-1、CAFs-2、KYSE-150、Eca109、Het-1A细胞,调整细胞密度,以2×105个/孔的细胞密度立即接种到6孔板中,置于37℃,5%CO2饱和湿度培养箱内培养。
GSK1838705A设3个浓度,为0μM、6μM、10μM。
培养24h待细胞贴壁后,每种细胞都加入以上各个浓度的GSK1838705A,每个浓度设3个复孔,置于37℃,5%CO2饱和湿度培养箱内培养。
24h后收集细胞,用PBS轻轻重悬细胞清洗细胞两次,
离心,弃去PBS,逐滴加入预冷的75%乙醇,置于-20℃冰箱固定过夜,
1500rpm离心5min,弃去固定液,加入PBS清洗细胞两次,
离心,弃去PBS,加入含有RNA酶的细胞周期染色液PI,轻轻混匀,室温避光孵育15min,
随即进行流式细胞仪检测,PI为红色荧光。
实验结果:如图4所示,10μM GSK1838705A明显阻滞CAFs-1、CAFs-2于G2/M期,但对KYSE-150、Eca109、Het-1A的细胞周期影响不显著。
实施例五GSK1838705A对CAFs-1、CAFs-2成纤维细胞活化标志物α-SMA mRNA影响的实验
(1)实验材料:CAFs-1、CAFs-2。
(2)实验方法
取对数生长期的CAFs-1、CAFs-2细胞,调整细胞密度,以2×105个/孔的细胞密度立即接种到6孔板中,置于37℃,5%CO2饱和湿度培养箱内培养。
GSK1838705A设3个浓度,为0μM、6μM、10μM。
培养24h待细胞贴壁后,每种细胞都加入以上各个浓度的GSK1838705A,置于37℃,5%CO2饱和湿度培养箱内培养。
24h后弃去全部培养液,mRNA提取试剂盒(试剂盒购自Axygen,货号AP-MN-MS-RNA-250)提取各组总mRNA,
并用逆转录试剂盒(试剂盒购自Takara,货号RR036A)逆转录为cDNA,
设计α-SMA引物,上游引物序列为:AAAAGACAGCTACGTGGGTGA(5’-3’),下游引物序列为:GCCATGTTCTATCGGGTACTTC(5’-3’),引物由上海生工生物工程股份有限公司合成,
制备q-PCR反应:
a.稀释cDNA:将上述cDNA产物加入40uL DEPC水稀释。
b.配制体系:SYBR Green 5uL,cDNA 1uL,上下游引物各0.5uL,DEPC水3uL。每组均设3个复孔。
c.上机:95℃,2min预温,95℃,10min预变性,95℃,15s,60℃,1min(采集荧光),共40个循环。熔解曲线:95℃,15s,60℃,1min(采集荧光),95℃,30s,60℃,15s。
d.下机:实验结束后进行数据分析。
实验结果:如图5所示,GSK1838705A明显降低了CAFs-1、CAFs-2中α-SMA的mRNA水平。
综上,可以看出,激酶抑制剂GSK1838705A能够诱导食管癌肿瘤微环境中的食管癌相关成纤维细胞CAFs-1、CAFs-2中活性氧的产生,促进CAFs-1、CAFs-2细胞的凋亡,同时还能够显著抑制CAFs-1、CAFs-2中α-SMA mRNA的表达,阻滞CAFs-1、CAFs-2细胞周期在G2/M期,由此可以有效改善食管癌耐治疗的抵抗性,进而保证治疗效果,在一定程度上降低肿瘤复发或转移的几率。
以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。本发明未详细描述的技术、形状、构造部分均为公知技术。
Claims (5)
2.根据权利要求1所述的应用,其特征在于,所述抑制食管癌的药物具体是抑制食管癌相关成纤维细胞系的增殖的药物。
3.根据权利要求2所述的激酶抑制剂GSK1838705A在制备抑制食管癌的药物中的应用,其特征在于,所述激酶抑制剂GSK1838705A阻滞食管癌相关成纤维细胞系的细胞周期在G2/M期。
4.根据权利要求1-3任一权利要求所述的激酶抑制剂GSK1838705A在制备抑制食管癌的药物中的应用,其特征在于,所述抑制食管癌的药物是以激酶抑制剂GSK1838705A为唯一活性成分,或包括激酶抑制剂GSK1838705A的药物组合物。
5.根据权利要求4所述的激酶抑制剂GSK1838705A在制备抑制食管癌的药物中的应用,其特征在于,所述包括激酶抑制剂GSK1838705A的药物组合物是指激酶抑制剂GSK1838705A与药学上允许的或多种辅料构成的药物组合物。
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CN101827848A (zh) * | 2007-08-08 | 2010-09-08 | 葛兰素史密丝克莱恩有限责任公司 | 作为IGF-1R抑制剂用于治疗癌症的2-[(2-{苯基氨基}-1H-吡咯并[2,3-d]嘧啶-4-基)氨基]苯甲酰胺衍生物 |
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