CN112159453B - 乙酰华蟾毒精及其在制备治疗肿瘤药物中的应用 - Google Patents

乙酰华蟾毒精及其在制备治疗肿瘤药物中的应用 Download PDF

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CN112159453B
CN112159453B CN202010644562.5A CN202010644562A CN112159453B CN 112159453 B CN112159453 B CN 112159453B CN 202010644562 A CN202010644562 A CN 202010644562A CN 112159453 B CN112159453 B CN 112159453B
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杨乐和
赵承光
戴璇璇
赵承伟
项友群
尹畅甜
胡万乐
周斌
周潮辉
梁广
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Abstract

本发明公开了一种乙酰华蟾毒精在制备抗肿瘤药物中的应用，所述的乙酰华蟾毒精的结构如式(I)所示。该乙酰华蟾毒精可以抑制TNBC细胞BT‑549、MDA‑MB‑231、MDA‑MB‑468的增殖，诱导TNBC细胞的凋亡，抑制TNBC细胞集落的形成，抑制TNBC细胞的迁移，是一种潜在的治疗乳腺癌的活性分子。

Description

乙酰华蟾毒精及其在制备治疗肿瘤药物中的应用
本申请是“乙酰华蟾毒精在制备治疗肿瘤药物中的应用”的分案申请，原申请的申请日为2018年01月22日，申请号为201810059566.X。
技术领域
本发明属于生物医药领域，具体涉及一种乙酰华蟾毒精在制备治疗肿瘤药物中的应用。
背景技术
肿瘤是引起人类死亡的主要原因之一，其发病率和致死率总体呈每年上升的趋势。统计显示，自2010年起，恶性肿瘤己成为城乡居民死亡首要死因。可见肿瘤的预防和治疗十分迫切。药物治疗是肿瘤的主要治疗手段之一。目前，虽然开发出了众多抗肿瘤药物，有效地延长了患者的生命或者提高了患者的生存质量。但肿瘤的药物研究和开发还面临巨大挑战，如抗肿瘤药物多为细胞毒药物，其副作用明显，限制了这些药物的临床应用。近二十年来，肿瘤的靶向疗法得到了飞速的发展，诸如伊马替尼、曲妥珠单抗等靶向肿瘤信号蛋白的药物在临床研究中展现出极具前景的治疗效果以及较低的毒副作用。然而，获得性耐药的出现及肿瘤基因组的多变性，使得靶向疗法同样面临着巨大的挑战。
华蟾毒精，结构如式(II)所示，为蟾蜍科动物如中华大蟾蜍(Bufo bufogargarizans Cantor)或黑眶蟾蜍(Bufo melanostictus Schneider)的干燥分泌物。
Figure BDA0002572542090000011
研究结果表明，华蟾毒精具有一定的抗肿瘤活性，不过其抗肿瘤机理并不明确，同时，其抗肿瘤活性还有待进一步提高。
发明内容
本发明提供了一种乙酰华蟾毒精在制备抗肿瘤药物中的应用，该乙酰华蟾毒精具有更高的疗效。
一种乙酰华蟾毒精在制备抗肿瘤药物中的应用，所述的乙酰华蟾毒精的结构如式(I)所示：
Figure BDA0002572542090000021
作为优选，所述的抗肿瘤药物用于治疗乳腺癌。
作为优选，所述的抗肿瘤药物用于抑制TNBC细胞BT-549、MDA-MB-231、MDA-MB-468的增殖。
作为优选，所述的乙酰华蟾毒精用于抑制STAT3磷酸化。
作为优选，所述的乙酰华蟾毒精用于诱导TNBC细胞的凋亡。
由于肿瘤细胞能够无限增殖形成细胞集落，通过集落形成实验表明，所述的乙酰华蟾毒精可以明显抑制TNBC细胞集落的形成。作为优选，所述的乙酰华蟾毒精用于抑制TNBC细胞集落的形成。
作为优选，所述的乙酰华蟾毒精用于抑制TNBC细胞的迁移；作为进一步的优选，所述的乙酰华蟾毒精用于抑制BT-549人乳腺癌细胞的迁移。
同现有技术相比，本发明的有益效果体现在：
本发明通过对华蟾毒精进行进一步的衍生化，得到的乙酰华蟾毒精对肿瘤细胞具有更好的抑制效果。
附图说明
图1为实施例2华蟾素活性成分对细胞增殖的抑制作用。其中，A为华蟾素三个主要活性成分：华蟾毒精(CBF)，蟾毒灵(BU)，酯蟾蜍配基(RB)对TNBC细胞的细胞增殖的抑制作用；B为华蟾毒精(CBF)对TNBC细胞和正常人乳腺细胞的细胞增殖的抑制作用；C为乙酰华蟾毒精(Ac-CBF)对TNBC细胞和正常人乳腺细胞的细胞增殖的抑制作用。
图2为实施例3中P-STAT3在人正常乳腺细胞株和TNBC细胞株中的蛋白表达结果。
图3为实施例3中乙酰华蟾毒精在TNBC细胞中抑制STAT3磷酸化的结果。
图4为实施例4中乙酰华蟾毒精诱导TNBC细胞凋亡结果。其中，A为流式细胞仪检测乙酰华蟾毒精诱导BT-549细胞凋亡的效果；B为相应细胞凋亡的分布统计图。
图5为实施例5中乙酰华蟾毒精抑制TNBC细胞集落形成的结果。
图6为实施例6中乙酰华蟾毒精抑制TNBC细胞迁移的结果。
具体实施方式
实施例1
向华蟾毒精(15mg，0.0388mmol)和三乙胺(0.39mmol)的二氯乙烷(干燥，1ml)混合溶液中加入乙酰氯(0.39mmol)，反应体系在常温下反应19h。然后反应体系通过硅胶层析柱(DCM/MeOH＝50:1至30:1)进一步纯化得到白色的目标化合物(17mg，产率100％)。
1H NMR(400MHz，CDCl3)δ7.92(s,1H),7.17(d,J＝10Hz,1H),6.21(d,J＝10Hz,1H),4.33(t,J＝9Hz,1H)，3.66(m,1H),3.10(m,1H),2.76(m,1H)，2.21(s,3H),2.01(s,3H),1.80-1.27(m,18H),1.04(s,6H).3C NMR(400MHz,CDCl3)δ170.2,169.2,162.6,149.3,147.8,116.9,115.2,74.8,75.5,73.6,61.4,50.4,45.2,42.1,40.1,39.3,38.0,35.8,34.9,32.6,28.8,28.3,27.6,21.0,20.8,20.5,18.4,13.5.；ESI-HR MS:calcd.for C28H37O7 +(M+H)+485.2461,found 485.2432.
反应方程式如下：
Figure BDA0002572542090000031
实施例2MTT试验测定乙酰华蟾毒精对肿瘤细胞增殖的抑制作用
分别对华蟾素三个主要活性成分：蟾毒灵(Bufalin，BU)、华蟾毒精(Cinobufagin，CBF)、酯蟾蜍配基(Resibufogenin，RB)和实施例1合成的乙酰华蟾毒精进行MTT实验。所用的细胞为TNBC细胞株BT-549、MDA-MB-231、MDA-MB-468和人正常乳腺细胞株MCF-10A，结果如图1所示。蟾毒灵、华蟾毒精、酯蟾蜍配基对BT-549TNBC细胞IC50分别是62.98nM、57.33nM和734.6nM，其中华蟾毒精的抗肿瘤效果最强，接着检测发现华蟾毒精对三株TNBC细胞BT-549、MDA-B-231和MDA-MB-468的IC50分别是57.33nM、99.6nM和87.42nM，而对正常人乳腺细胞MCF-10a的IC50则大于200nM。目标化合物乙酰华蟾毒精对三株胃癌细胞的IC50分别是17.57μM、28.31μM和27.57μM，均明显低于先导物华蟾毒精。此外，乙酰华蟾毒精对人正常乳腺细胞的增殖影响较小，表明类似物对肿瘤细胞具有明显的选择性。
实验方法：细胞生存率用MTT实验测定。细胞以8000每孔的密度接种于96孔板中，24h后换新鲜培养基。将不同浓度(6.25，12.5，25，50，100，200，400，800nm)的药物加入每孔中，终体积为100μL，每个浓度设置3个复孔，继续培养24小时后，加入20μL 5mg/mL的溴化噻唑蓝四氮唑(MTT)，随后在37℃培养箱中继续孵育4h，去掉培养基加入150μL DMSO。在490nm波长读数据，每个实验重复3次，计算IC50值。
实施例3乙酰华蟾蜍素的抗肿瘤作用的机制
采用WB实验检测STAT3在人正常乳腺细胞MCF-10A和TNBC细胞BT-549、MDA-MB-231、MDA-MB-468中的表达，结果如图2所示。结果显示：P-STAT3在三株TNBC细胞中的蛋白表达相比于人正常乳腺细胞都是显著升高。
用不同浓度的乙酰华蟾毒精(终浓度10、20和30nM)处理TNBC细胞BT-549，12小时后，用免疫印迹检测总细胞蛋白质提取物(60mg)中各种靶蛋白水平，GAPDH作为内参。结果如图3所示，结果显示TNBC细胞中乙酰华蟾毒精都浓度依赖性的抑制STAT3的磷酸化，30nM能明显抑制STAT3的磷酸化(图3)。因此，乙酰华蟾毒精是通过抑制STAT3发挥抗肿瘤作用。
实施例4乙酰华蟾毒精诱导TNBC细胞的凋亡试验
采用Annexin V-FITC/PI双染法流式细胞仪检测乙酰华蟾毒精(10、20和30nM)诱导TNBC细胞凋亡，结果见图4，由图4可知，乙酰华蟾毒精作用24h后能够剂量依赖性的诱导BT-549细胞产生凋亡。
实施例5乙酰华蟾毒精能够显著抑制TNBC细胞集落形成
采用不同浓度的乙酰华蟾毒精(10、20和30nM)作用BT-549和MDA-MB-231TNBC细胞24h后，更换新鲜培养基继续培养一周以形成单个细胞集落。结果如图5所示，表明乙酰华蟾毒精能剂量依赖性地抑制这两株TNBC细胞集落形成。
实施例6乙酰华蟾毒精能够显著抑制TNBC细胞迁移
为了评价乙酰华蟾毒精对TNBCBT-549及MDA-MB-231细胞迁移能力的影响，细胞划痕后分别经不同浓度乙酰华蟾毒精(5、10和15nM)处理18h，0h和24h两个时间节点拍照，结果如图6所示，表明BT-549及MDA-MB-231细胞的迁移能力与对照组相比，均有下降的趋势，且BT-549细胞株效果好于MDA-MB-231细胞株，说明乙酰华蟾毒精具有抑制人TNBC细胞系BT-549及MDA-MB-231迁移的作用。

Claims (2)

1.一种乙酰华蟾毒精在制备抗肿瘤药物中的应用，其特征在于，所述的乙酰华蟾毒精的结构如式（I）所示：
Figure DEST_PATH_IMAGE002
（I）
所述的乙酰华蟾毒精用于诱导TNBC细胞的凋亡。
2.根据权利要求1所述的乙酰华蟾毒精在制备抗肿瘤药物中的应用，其特征在于，所述的乙酰华蟾毒精还用于抑制TNBC细胞集落的形成，抑制TNBC细胞的迁移。
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