摘要
金刚纂(Euphorbia neriifolia L.)是大戟科植物,采用硅胶柱色谱以及制备HPLC等多种色谱技术,从金刚纂地上部分中分离出5个已知的木脂素成分,并对这5种木脂素的潜在抗肿瘤活性进行体外研究,其中化合物2对食管鳞癌细胞,特别是KYSE-410和KYSE-450细胞,表现出增殖抑制和细胞毒性。机制研究进一步证明化合物2可通过激活caspase-3/9及下调Bcl-2/Bax蛋白表达比例,诱导癌细胞发生显著凋亡。实验结果显示,化合物2在体外对食管鳞癌细胞,尤其是KYSE-410细胞具有显著抑制作用,表明化合物2具有良好的抗肿瘤活性,应对其进行深入研究。
大戟科植物包含约2 000个种属,广泛分布在热带和温带地
从植物中提取的木脂素是中药的重要成分,其中一些已被批准作为治疗药物或药物先导化合
金刚纂药材地上部分采自广西壮族自治区东兰县,由云南省农业科学环境资源研究所王群副研究员进行鉴定。Welch Ultimate XB-C18柱(20 mm × 250 mm,10 μm,上海月旭科技股份有限公司);300 ~ 400目硅胶(青岛海洋化工工厂);MCI凝胶(CHP20P,75 ~ 150 μm,三菱化学工业有限公司);cleaved caspase-3、cleaved caspase-9、caspase 3、caspase 9、Bcl-2、Bax抗体(沈阳万类生物科技有限公司);HRP标记的山羊抗兔IgG 和GAPDH抗体(杭州联科生物技术股份有限公司);TBST(武汉赛维尔生物科技有限公司);Annexin V-FITC/PI细胞凋亡检测试剂盒(南京建成科技有限公司)RPMI-1640培养基(以色列Biological Industries公司);胎牛血清(以色列Biological Industries公司);CDCl3(北京百灵威科技有限公司);其他试剂均为市售分析纯。
金刚纂地上部分2 kg,室温下95%乙醇提取3次(10 L × 3),得到乙醇提取物300 g。残余成分使用水重悬,上清液分别用石油醚、乙酸乙酯和正丁醇萃取。乙酸乙酯部分(92 g)通过MCI凝胶柱以甲醇-水(3∶7 ~ 1∶9)洗脱。90%馏分(12 g)采用硅胶柱色谱(CC),石油醚-乙酸乙酯(30∶1 ~ 1∶1)洗脱,分离得到6个馏分(Fr.1 ~ Fr.6)。Fr.2通过硅胶柱(CHCl3-MeOH,150∶1)和半制备HPLC(CH3CN-H2O,40∶60,3 mL/min)分离,用于制备化合物1(2.1 mg,20.6 min)与化合物2(3.9 mg,19.8 min)。Fr.3c通过硅胶色谱(CHCl3-MeOH,150∶1)分离得到2个组分,Fr.3a和Fr.3b。Fr.3a通过HPLC(CH3CN-H2O,35∶65,3 mL/min)纯化得到化合物4(5.1 mg,40.8 min)。Fr.3c通过Sephadex LH-20(MeOH)凝胶色谱分离得到化合物3(8.9 mg)和化合物5(2.3 mg)。
将人食管鳞癌细胞(TE-13、KYSE-410、KYSE-450)和人正常食管上皮细胞HEEC用含有10%胎牛血清的RPMI-1640培养基,37 ℃,5%CO2环境培养。细胞密度达到70% ~ 90%时,用0.25%胰蛋白酶溶液重悬分离培养,调节密度为每毫升3.0 × 1
细胞凋亡检测步骤按照试剂盒说明书进行。将KYSE-410细胞接种于6孔板中,培养过夜,第2天加入不同浓度的化合物2(终浓度为1,2,4 μg/mL)。孵育24 h后,收集细胞并使用PBS缓冲液轻柔冲洗,然后加入结合缓冲液500 μL重悬细胞,最终得到浓度为每毫升8 × 1
对线粒体凋亡通路相关蛋白,包括Bax、Bcl-2、caspase-3/cleaved caspase-3及caspase-9/cleaved caspase-9进行检测,使用GAPDH作为内参。将KYSE-410细胞接种于6孔板内,培养过夜,并在第2天使用不同浓度的化合物2(终浓度为1,2,4 μg/mL)处理。孵育24 h后,使用冷PBS清洗,并使用含蛋白酶抑制剂的RIPA裂解液在4 ℃裂解30 min,裂解后使用12 000 r/min,4 ℃离心20 min。蛋白质浓度通过BCA定量试剂盒检测。然后进行SDS-PAGE电泳,上样后 80 V恒压电泳,湿转恒流转膜,5%脱脂奶粉封闭2 h后,TBST洗膜3次,每次 10 min,4 ℃孵育一抗过夜;TBST洗膜3次,每次10 min,室温孵育二抗2 h后,TBST 洗膜 3次,每次10 min。采用Tanon 5200图像系统采集发光图像。
通过对金刚纂的乙酸乙酯部分进行分离,从中分离并鉴定出5个木脂素类化合物(

Figure 1 Chemical structures of compounds 1-5
MTT检测法是一种快速方便高通量筛选方法,可用于评估药物在细胞增殖的作用和细胞毒
化合物2对KYSE-410人食管鳞癌细胞系具有高细胞毒性。由于肿瘤细胞凋亡是影响肿瘤生存主要原因,首先测试化合物2对细胞凋亡的影响。使用化合物2处理KYSE-410细胞系,孵育24 h后,采用流式细胞术检测KYSE-410细胞早期和晚期凋亡情况(

Figure 2 Compound 2 induced KYSE-410 cells apoptosis
A-D:The cells were treated with compound 2 for 24 h at 1, 2 and 4 μg/mL to evaluate the early and late apoptosis respectively; E:Represent histograms for apoptosis rate in KYSE-40 ()
线粒体途径的主要特征是线粒体参与凋亡通路,异常激活的caspase相关蛋白在诱导线粒体凋亡中起到重要作

Figure 3 Compound 2 induced KYSE-410 cells apoptosis through mitochondrial pathway. KYSE-410 cells were treated with compound 2 for 24 h at 1, 2 and 4 μg/mL to detect the expression of related proteins by Western blot ()
A:Expression of Bax, Bcl-2, caspase-3/cleaved caspase-3 and caspase-9/cleaved caspase-9 were detected by Western blot;B:Represent histograms for the fold change of cleaved caspas 3/caspase 3 in KYSE-40 treated with compound 2;C:Represent histograms for the fold change of cleaved caspas 9/caspase 9 in KYSE-40 treated with compound 2;D:Represent histograms for the fold change of Bcl-2 and Bax in KYSE-40 treated with compound 2
Bcl-2蛋白家族通过线粒体应激在细胞凋亡中发挥重要作用。Bax和Bcl-2均属于Bcl-2蛋白家族。Bcl-2是细胞凋亡抑制因子,而Bax则发挥促细胞凋亡功能。Bcl-2和Bax蛋白的表达水平对凋亡有关键影
肿瘤干细胞被认为在肿瘤的形成、恶化和化疗药物耐药中发挥重要作

Figure 4 Cells pretreated with the compound 2 exhibited a good inhibition of colony formation capacity in KYSE-410 cells ()
A:Colony formation of KYSE-410 cells pretreated with compound 2 for 48 h at 1, 2 and 4 μg/mL, respectively;B:Quantitation of cell colony numbers
本研究对金刚纂地上部分中分离得到的5个已知木脂素类化合物进行活性研究,发现化合物2对食管鳞癌细胞具有较好的细胞毒活性,并显著抑制细胞集落形成。此外,化合物2可通过调控线粒体凋亡通路促进KYSE-410细胞的凋亡,值得进一步深入研究。
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