CN115089571A - 菊苣酸在制备三阴乳腺癌治疗药物中的应用 - Google Patents
菊苣酸在制备三阴乳腺癌治疗药物中的应用 Download PDFInfo
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Abstract
本发明公开了菊苣酸在制备三阴乳腺癌治疗药物中的应用，属于医药技术领域。本发明一方面通过体外细胞实验，证实菊苣酸可以抑制MDA‑MB‑231细胞的增殖和迁移，另一方面通过MDA‑MB‑231异种移植，验证了菊苣酸可以抑制体内三阴乳腺癌肿瘤的生长。本发明所述的菊苣酸可作为活性成分，应用于抗肿瘤的药物中，开拓了菊苣酸的新用途，为治疗三阴乳腺癌提供了新选择。
Description
技术领域
本发明属于医药技术领域，具体涉及菊苣酸在制备三阴乳腺癌治疗药物中的应用。
背景技术
菊苣酸(cichoric acid,CA)，又称二咖啡酰酒石酸，是一种纯天然化合物，主要是从菊科植物(菊苣、紫锥菊、莴苣、蒲公英等)中提取分离得到的咖啡酸类成分。研究表明菊苣酸具有抗炎、抗氧化、抗肿瘤、抗病毒、提高机体免疫等多种药理作用。近年来，菊苣酸在抗肿瘤方面的效果也日益显现，Sun等(Sun,X.,Zhang,X.,Zhai,H.,Zhang,D.,&Ma,S,2019.Chicoric acid(CA)induces autophagy in gastric cancer through promotingendoplasmic reticulum(ER)stress regulated by AMPK.Biomed Pharmacother,118,109144)发现菊苣酸可显著降低胃癌细胞活力，诱导胃癌细胞凋亡和自噬；另有报道菊苣酸可抑制结肠癌细胞生长，推测是端粒酶活性降低和诱导细胞凋亡所致(Tsai,Y.L.,Chiu,C.C.,Yi-Fu Chen,J.,Chan,K.C.,&Lin,S.D,2012.Cytotoxic effects of Echinaceapurpurea flower extracts and cichoric acid on human colon cancer cellsthrough induction of apoptosis.J Ethnopharmacol,143(3),914-919.)。
三阴乳腺癌(Triple-negative breast cancer,TNBC)是一种具有高度异质性和临床侵袭性的疾病，占乳腺癌的15％～20％，在所有乳腺癌亚型中死亡率最高。TNBC是一种IHC分类的乳腺癌亚型，即雌激素受体(ER)、孕激素受体(PR)，人表皮生长因子受体2(HER2)阴性的乳腺癌。与激素受体阳性乳腺癌(通常采用内分泌治疗)或HER2阳性乳腺癌(可采用抗体治疗(如曲妥珠单抗))相比，TNBC没有有效的靶向治疗，常规化疗仍然是TNBC患者的标准治疗方式。因此，迫切需要寻找靶向治疗三阴乳腺癌新药物，这将大大有利于TNBC患者的临床治疗(Liao,C.,Zhang,Y.,Fan,C.,Herring,L.E.,Liu,J.,Locasale,J.W.,...Zhang,Q,2020.Identification of BBOX1 as a Therapeutic Target in Triple-NegativeBreast Cancer.Cancer Discov,10(11),1706-1721.)。
现有的文献报道主要集中在菊苣酸的抗炎、抗氧化方面，但尚未有菊苣酸用于治疗三阴乳腺癌的报道。
发明内容
本发明的目的是提供菊苣酸在制备三阴乳腺癌治疗药物中的应用。
MDA-MB-231细胞是三阴乳腺癌细胞中极具侵袭性的一类细胞，其特点是紧密连接蛋白(claudin)低，具有干细胞样和间充质样特征。本发明一方面通过体外细胞实验，证实菊苣酸可以抑制MDA-MB-231细胞的增殖和迁移，另一方面通过MDA-MB-231异种移植，验证了菊苣酸可以抑制体内三阴乳腺癌肿瘤的生长，优选的剂量为66mg/kg。进一步在对优选的给药剂量(66mg/kg)组肿瘤切片进行H&E、Ki-67、TUNEL染色，结果表明，菊苣酸可以抑制三阴乳腺癌增殖并促进其凋亡，进而发挥治疗三阴乳腺癌的疗效。
本发明所述的菊苣酸可作为活性成分，应用于抗肿瘤的药物中，开拓了菊苣酸的新用途，为治疗三阴乳腺癌提供了新选择。
附图说明
图1为菊苣酸(CA)抑制MDA-MB-231细胞生长和迁移的结果。其中：a为0μM、10μM、20μM菊苣酸给药48h后MDA-MB-231细胞集落形成图片；b为0μM、10μM、20μM菊苣酸给药48h后MDA-MB-231细胞集落形成率；c为0μM、12.5μM、25μM菊苣酸给药0h和24h后MDA-MB-231细胞迁移图片；d为0μM、12.5μM、25μM菊苣酸给药24h后MDA-MB-231细胞伤口愈合率。每个实验均进行三次以上独立重复实验，数据以mean±SEM表示，**p<0.01、****p<0.0001vs对照组。
图2为菊苣酸(CA)抑制体内三阴乳腺癌肿瘤生长的结果。其中：a为治疗期间各组BALB/c裸鼠肿瘤生长曲线；b为治疗结束后各组BALB/c裸鼠肿瘤重量；c为Model组和CA(66mg/kg)组肿瘤组织H&E染色(黑色箭头指示蓝色细胞核)、Ki-67染色(黑色箭头指示的棕色细胞为Ki-67阳性细胞)及TUNEL染色(白色箭头指示的绿色荧光细胞为TUNEL阳性细胞)具有代表性的显微照片(200×)。数据以mean±SEM表示，n＝6/组。*p<0.05、**p<0.01vs对照组。
具体实施方式
下面结合附图和具体实施例对本发明作进一步详细说明，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
以下实施例采用的材料如下：
1、药品
菊苣酸购自成都曼斯特生物科技有限公司(成都，中国)。
2、细胞和动物
MDA-MB-231细胞购自中国科学院上海生物化学与细胞生物学研究所。
清洁级雌性BALB/c裸鼠，体重16-18g，购自杭州子源实验动物科技有限公司，合格证号：SCXK(浙)2019-0004。
实施例1
菊苣酸对于MDA-MB-231细胞增殖和迁移的影响
菊苣酸溶液的配制：称取一定量菊苣酸粉末，溶于DMSO中配制成200mM的母液，给药前用DMEM培养基稀释至各浓度。
1、集落实验
将MDA-MB-231细胞以1000个/孔的密度接种于六孔板中，在含10％胎牛血清的DMEM培养基中培养24h，然后用菊苣酸(10μM、20μM)处理48h。48h后将含药液的培养基弃去，并重新添加含10％胎牛血清的DMEM继续培养在5％CO2、37℃条件下，培养7天形成细胞集落。PBS洗后，在室温下用4％多聚甲醛固定细胞集落，然后用结晶紫染液染色30min后拍照。使用Image J对细胞集落计数，细胞集落形成率(％)＝给药组细胞集落数/对照组集落数*100％
2、划痕实验
将MDA-MB-231细胞(5×105个/孔)置于6孔板中。过夜孵育后，用无菌的1mL移液枪尖端在每孔中产生一个划痕，在尼康倒置荧光显微镜(Nikon Ts2R)下拍摄(0h)照片。分别用菊苣酸(12.5μM和25μM)处理细胞，培养24h后再拍照。
伤口愈合率(wound healing proportion,WHP)(％)＝(0h划痕长度-24h划痕长度)/0h划痕长度*100％。
如图1中a、b所示，10μM和20μM的菊苣酸对MDA-MB-231细胞集落形成具有显著抑制作用，说明菊苣酸可以抑制MDA-MB-231细胞的增殖；如图1中c、d所示，12.5μM和25μM的菊苣酸对MDA-MB-231细胞迁移也具有显著抑制作用。
实施例2
菊苣酸对体内三阴乳腺癌肿瘤的影响
菊苣酸溶液的配制：称取菊苣酸115.99mg，加入175μL DMSO至其完全溶解，再加入175μL吐温20混匀，待其完全溶解得到331.40mg/mL的菊苣酸母液；再用20％羟丙基-β-环糊精(生理盐水配置)分别稀释至0.6mg/mL、1.6mg/mL、6.6mg/mL的溶液，按10μL/g/d给裸鼠腹腔注射。每次给药均现配现用。
1、MDA-MB-231异种移植实验
所有动物实验均经过中国药科大学伦理委员会批准，5周龄雌性BALB/c裸鼠24只，体重16-18g，购自杭州子源实验动物科技有限公司，采用无菌食物和水，单独通风的笼子饲养，MDA-MB-231细胞(5×106个)悬浮于200μL生理盐水中，皮下注射右前肢，平均肿瘤体积达100mm3后，将小鼠随机分为4组，每组6只，分别进行Model(2.5％DMSO+2.5％吐温20+95％生理盐水)、菊苣酸(6mg/kg/d)、菊苣酸(16mg/kg/d)、菊苣酸(66mg/kg/d)腹腔注射(i.p)处理，每隔1d测量肿瘤体积和体重。肿瘤计算公式为：1/2×长×宽2，在治疗的最后一天，处死小鼠，解剖异种移植肿瘤。抑瘤率(％)＝(治疗结束后对照组肿瘤的平均体积－治疗结束后实验组肿瘤的平均体积)/治疗结束后对照组肿瘤的平均体积×100％。
2、肿瘤H&E、TUNEL染色
肿瘤组织在4％多聚甲醛中固定24h，脱水，并包埋在石蜡中。4μm厚的切片分别用苏木精-伊红(H&E)和TUNEL染色进行形态学观察。在正置荧光显微镜(Olympus BX53；放大倍数：×200)下观察和拍摄图像。
3、肿瘤Ki-67染色
石蜡包埋的组织切片(4μm厚)脱蜡并重新水化，在4℃下与Ki-67特异性抗体孵育过夜，在室温下与HRP(辣根过氧化物酶)结合的二级抗体孵育2h，在苏木精中染色3分钟，并在正置荧光显微镜(Olympus BX53；放大倍数：×200)下观察和拍摄图像。
如表1和图2中a、b所示，菊苣酸(6mg/kg、16mg/kg、66mg/kg)对MDA-MB-231异种移植肿瘤均有一定的抑制作用，抑制作用最明显的是66mg/kg。
表1不同剂量菊苣酸(CA)体内抑制三阴乳腺癌结果
如图2中c所示，在对优选的给药剂量(66mg/kg)组肿瘤切片进行H&E、Ki-67、TUNEL染色，从Ki-67结果可以看出，菊苣酸(66mg/kg)组对比模型组，Ki-67蛋白表达量显著降低，证明菊苣酸可以显著抑制体内肿瘤的增殖。此外，H&E染色结果显示模型组肿瘤细胞核密度高，有明显的团块生长趋势。菊苣酸治疗组(66mg/kg)的肿瘤切片出现细胞核浓缩、细胞质空泡化等与细胞凋亡相关的形态学变化。TUNEL染色结果显示，菊苣酸(66mg/kg)组对比模型组绿色荧光细胞数明显增加，进一步说明菊苣酸可以诱导体内三阴乳腺癌肿瘤细胞凋亡。
Claims (5)
1.菊苣酸在制备三阴乳腺癌治疗药物中的应用。
2.根据权利要求1所述的应用，其特征在于：所述菊苣酸的给药剂量为6-66 mg/kg。
3.根据权利要求1所述的应用，其特征在于：所述菊苣酸的给药剂量为66 mg/kg。
4.一种三阴乳腺癌治疗药物，其特征在于：所述药物的活性成分为菊苣酸。
5.根据权利要求4所述的三阴乳腺癌治疗药物，其特征在于：所述药物还包括药学上可接受的载体。
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