CN115813919B - 吲哚-3-丙酮酸或其药用盐在制备治疗乳腺癌药物中的应用 - Google Patents
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Abstract
本发明公开了一种吲哚‑3‑丙酮酸在制备治疗乳腺癌药物中的应用。本发明意外发现,IPyA在生理浓度下(2mM),IPyA对乳腺癌细胞有显著的细胞毒作用。当乳腺癌发展时,IPyA浓度显著下降,且通过补充IPyA使其恢复到接近生理水平,可有效抑制乳腺癌生长,说明IPyA具有极高的安全剂量,而且可有效用于乳腺癌辅助治疗。IPyA在抑制乳腺癌细胞生长的过程中,可通过下调表观遗传调节因子UHRF1的表达,激活AMP依赖的蛋白激酶活性,抑制细胞ATP生成。服用特定益生菌、益生元,调节肠道微生物对Trp的代谢,保持IPyA在体内的生理浓度,可有效预防乳腺癌发生。
Description
技术领域:
本发明属于生物医药领域,具体涉及吲哚-3-丙酮酸或其药用盐在制备治疗乳腺癌药物中的应用。
背景技术:
乳腺癌是女性最常见的癌症,现已跃居全球癌症负担的首位。目前乳腺癌仍是我国女性新发癌症病例数的首位,且年轻化趋势严重,是我国45岁以下女性患癌症死亡的主要原因。尽管除手术外,乳腺癌的治疗可综合运用放疗、化疗、内分泌治疗、靶向治疗等手段,但占15%至20%的三阴性乳腺癌(Triple-negative breast cancer,TNBC)患者,因缺乏治疗靶点无法从疗效更好的靶向药物中获益,所以化疗仍是TNBC辅助治疗的最主要手段。尽管近年肿瘤免疫疗法取得新突破,其中免疫检测点抑制剂(Immune checkpointinhibitors,ICIs)也已进入乳腺癌的临床试验阶段,但临床结果显示ICIs疗法的实际获益人群占患者比例极小,其有效率对大多数肿瘤只有10-30%。可见,从更多途径开发治疗手段,是乳腺癌防治的工作重点,也是“健康中国2030”规划对癌症管理的重要组成部分。
已有大量研究表明,肠道微生物可通过干预宿主代谢而影响宿主健康,如肥胖、高血压、高血脂。在正常情况下,肠道微生物通过一系列特定代谢功能,把宿主饮食中的营养物质代谢为短链脂肪酸、胆汁酸等,维持宿主如免疫功能平衡、血脂/血糖代谢平衡,从而维持宿主生理功能健康;一旦肠道微生态的物种或代谢功能出现异常,上述物质代谢紊乱,多种相关疾病随之发生。越来越多临床实验证实,与肠道微生物相关的益生菌、益生元、代谢产物,以及具有调节肠道微生物物种构成、代谢功能的食品,均可显著改善疾病发展进程。由于肠道微生物对宿主健康有如此深远的影响,肠道微生物及其产物已成为具有巨大潜力的疾病治疗媒介。
至今,已发现肠道微生物所产生的可影响宿主生理功能的代谢物主要包括短链脂肪酸、色氨酸及其衍生物、胆汁酸、胆碱类等。色氨酸(Tryptophan,Trp)是一种人体无法自身合成的必需氨基酸,也是DNA构建模块组成之一。吲哚-3-丙酮酸(Indole-3-pyruvicacid,IPyA)是Trp在肠道微生物代谢过程中的重要代谢产物之一,它天然存在于人体中,已有报道其具有一定的抗炎活性。
发明内容:
针对抗肿瘤药物疗效有限、毒副作用大的技术问题,本发明的目的在于提供吲哚-3-丙酮酸(Indole-3-pyruvic acid,IPyA)或其药用盐在制备治疗乳腺癌药物中的应用。
本发明意外发现,IPyA在生理浓度下(2mM),IPyA对乳腺癌细胞有显著的细胞毒作用。当乳腺癌发展时,IPyA浓度显著下降,且通过补充IPyA使其恢复到接近生理水平,可有效抑制乳腺癌生长,说明IPyA具有极高的安全剂量,而且可有效用于乳腺癌辅助治疗。
因此,本发明提供了吲哚-3-丙酮酸或其药用盐在制备治疗乳腺癌药物中的应用。
优选,所述的治疗乳腺癌药物是治疗三阴性乳腺癌或管腔A型乳腺癌的药物。
优选,所述的治疗乳腺癌药物含有药学上可接受的辅料。
优选,所述的治疗乳腺癌药物是将吲哚-3-丙酮酸或其药用盐用医学可接受的各种制剂辅料制备成口服给药剂型。
优选,所述的治疗乳腺癌药物是将吲哚-3-丙酮酸或其药用盐用医学可接受的各种制剂辅料制备成静脉给药剂型。
优选,所述的治疗乳腺癌药物是通过吲哚-3-丙酮酸或其药用盐直接抑制肿瘤细胞表观遗传调节因子UHRF1表达,激活AMP依赖的蛋白激酶活性,抑制细胞ATP生成的药物。
优选,所述的治疗乳腺癌药物是杀伤鼠源乳腺癌细胞系4T1,人源乳腺癌细胞系MDA-MB-231、MCF-7的药物。
本发明的第二个目的是提供一种治疗乳腺癌药物,其含有吲哚-3-丙酮酸或其药用盐作为活性成分。
目前临床上大部分抗肿瘤治疗为外源性化合物或者重组单克隆抗体,在取得一定疗效的同时常引起一系列毒副作用。因此,从人体内源性代谢物中寻找控制肿瘤细胞生长的有效物质是一个具有巨大潜力的方向。吲哚-3-丙酮酸(Indole-3-pyruvic acid,IPyA)是一种天然存在于人体、动物的色氨酸(Tryptophan,Trp)代谢物,其生理浓度约为2mM。IPyA是Trp在肠道微生物代谢过程中的重要代谢产物之一,其在人体、动物体内的天然积累与肠道微生物状态密切相关。
本发明意外发现,IPyA在生理浓度下(2mM),IPyA对乳腺癌细胞有显著的细胞毒作用。当乳腺癌发展时,IPyA浓度显著下降,且通过补充IPyA使其恢复到接近生理水平,可有效抑制乳腺癌生长,说明IPyA具有极高的安全剂量,而且可有效用于乳腺癌辅助治疗。IPyA在抑制乳腺癌细胞生长的过程中,可通过下调表观遗传调节因子UHRF1(ubiquitin-likewith PHD and ring finger domains 1)的表达,激活AMP依赖的蛋白激酶(Adenosine 5‘-monoph osphate(AMP)-activated protein kinase,AMPK)活性,抑制细胞ATP生成。服用特定益生菌、益生元,调节肠道微生物对Trp的代谢,保持IPyA在体内的生理浓度,可有效预防乳腺癌发生。
附图说明
图1是吲哚-3-丙酮酸(IPyA)对乳腺癌细胞活力的影响。
图2是吲哚-3-丙酮酸(IPyA)对乳腺癌细胞克隆形成能力的影响。
图3是哚-3-丙酮酸(IPyA)对乳腺癌细胞相关蛋白表达的影响。
图4是吲哚-3-丙酮酸(IPyA)对乳腺癌细胞总ATP生成的影响。
图5是吲哚-3-丙酮酸(IPyA)对乳腺癌细胞氧化磷酸化途径ATP生成的影响。
图6是吲哚-3-丙酮酸(IPyA)对乳腺癌细胞糖酵解途径ATP生成的影响。
图7是吲哚-3-丙酮酸(IPyA,灌胃给药)对乳腺癌荷瘤动物肿瘤生长的影响。
图8是吲哚-3-丙酮酸(IPyA,腹腔注射)对乳腺癌荷瘤动物肿瘤生长的影响。
具体实施方式
以下实施例是对本发明的进一步解释和说明,而不是对本发明的限制。
本发明以下实施例使用细胞系及其培养方法如下。
鼠源乳腺癌细胞系4T1,人源乳腺癌细胞系MDA-MB-231、MCF-7购自于中国科学院典型培养物委员会细胞库。4T1培养于含10%胎牛血清(Biological Industries公司)、1%青霉素/链霉素(Gibco公司)的RPMI培养基(Gibco公司),MDA-MB-231和MCF-7培养于含10%胎牛血清(Biological Industries公司)、1%青霉素/链霉素(Gibco公司)的DMEM培养基(Gibco公司)。上述含有特定添加物的培养基以下均称为完全培养基。所有细胞系均在5%CO2、37℃恒温培养箱中培养。
实施例1吲哚-3-丙酮酸(IPyA)对乳腺癌细胞活力的影响
4T1,MDA-MB-231,MCF-7细胞接种于96孔细胞培养板中(1×104cells/mL,100μL/well),贴壁过夜后,加入IPyA(0~4mM)处理48小时;然后把含药培养基换为含10%CCK-8(Promega,Wisconsin公司)的RPMI培养基(Gibco公司),继续培养2~3小时,在490nm下检测各孔吸光度(OD),按如下公式计算细胞相对活力,cell viability(%)=(OD给药组孔-OD校正孔)/(OD对照组孔-OD校正孔)%。
IPyA对4T1,MDA-MB-231,MCF-7相对活力的影响如图1所示。在0.5mM IPyA浓度下,上述各细胞系的相对活力均仅在50%,而在2mM IPyA浓度下,上述细胞系的相对活力几乎为0%,说明IPyA在生理浓度下对乳腺癌细胞有显著杀伤作用。
实施例2吲哚-3-丙酮酸(IPyA)对乳腺癌细胞克隆形成能力的影响
上述乳腺癌细胞接种于24孔板中(1×103cells/well),贴壁过夜后,加入含IPyA(1,2,4mM)的完全培养基培养一段时间(以不添加IPyA的作为对照,即图中的0),每3天更换一次含药完全培养基。待孔中大多数单个克隆中细胞数大于70个为止,弃上清,PBS洗涤细胞1次。每孔加入1mL 4%多聚甲醛,室温固定细胞20min,PBS洗涤细胞1次。加适量结晶紫染色液染20min,流水洗干净染色液,空气干燥。将细胞板倒置并叠加一张带网格的透明胶片,用肉眼直接计数克隆,或在显微镜(低倍镜)计数大于10个细胞的克隆数。最后计算克隆形成率。克隆形成率(colony formation,%)=克隆数/接种总细胞数(%).
IPyA对4T1,MDA-MB-231,MCF-7克隆形成能力的影响如图2所示。在2mM IPyA浓度下,上述各细胞系的克隆形成率基本低于50%,说明IPyA在生理浓度下对乳腺癌细胞克隆形成有显著抑制作用。
实施例3吲哚-3-丙酮酸(IPyA)对乳腺癌细胞相关蛋白表达的影响
4T1细胞接种于100mm细胞培养皿中(170000cell/dish),加入含IPyA(0.125,0.25,0.5mM)的完全培养基培养48h(以不添加IPyA的作为对照,即图中的0),弃培养基,预冷PBS洗涤2次,加入含有磷酸酶抑制剂和蛋白酶抑制剂的蛋白裂解液,按说明书相应步骤提取总蛋白。总蛋白经微量分光光度法进行浓度定量后,各样品取一致量的总蛋白与上样缓冲液混合,变性(100℃,10min)。所得样品按照常规蛋白印迹方法(Western blotting)比较UHRF1、AMPK、p-AMPK表达差异。
IPyA对4T1细胞中UHRF1、AMPK、p-AMPK表达的影响如图3所示。在0.5mM IPyA浓度下,4T1细胞的UHRF1蛋白表达量显著下降,AMPK总蛋白表达无显著变化,但其磷酸化程度显著提高,说明IPyA可通过逆转UHRF1对AMPK的负调节作用,激活AMPK,抑制乳腺癌细胞生长。
实施例4吲哚-3-丙酮酸(IPyA)对乳腺癌细胞ATP生成的影响
参照实施例3条件,把IPyA(0.125,0.25,0.5mM)作用于4T1细胞48小时,然后用含0.25%EDTA的胰蛋白酶消化各组细胞,调节浓度,接种至Seahorse XF 96孔微孔板中(20000cells/well),贴壁过夜后,参照Seahorse XF Real-Time ATP Rate Assay Kit(Agilent,公司),测定各组细胞ATP生成量。
IPyA对4T1细胞ATP生成的影响如图4~图6所示。随着IPyA浓度提高,4T1细胞的ATP生成量逐渐下降(图4),其中氧化磷酸化途径(Oxidative phosphorylation)生成的ATP(mitoATP)占比更显著下降(图5),而糖酵解途径(Glycolysis)生成的ATP(glyATP)占比显著提高(图6),说明IPyA在逆转UHRF1对AMPK的负调节的过程中,抑制了细胞ATP生成,负反馈性地激活AMPK,从而达到抑制乳腺癌细胞生长的目的。
实施例5吲哚-3-丙酮酸(IPyA,口服给药)对乳腺癌荷瘤动物肿瘤生长的影响
取雌性Balb/c小鼠(6~8周龄),在右侧第四对乳腺脂肪垫处皮下注射4T1细胞(8×104cells/小鼠)。肿瘤体积增长至约30mm3时,把荷瘤小鼠按肿瘤体积大分层随机分配到模型组(Model组),低剂量组(IPyA组,60mg/kg),高剂量组(IPyA组,120mg/kg),每组8只。从分组日起,给药组给予IPyA(灌胃,每天1次),模型组给予等体积生理盐水,连续给药28天。给药期间,每周2次检测肿瘤大小和体重变化。第28天,剖取各组小鼠肿瘤,称量肿瘤重量,拍照。
IPyA(灌胃给药)对乳腺癌荷瘤动物肿瘤生长的影响如图7所示。IPyA(灌胃给药)可明显减缓4T1乳腺癌肿瘤生长速度,最终降低肿瘤重量。
实施例6吲哚-3-丙酮酸(IPyA,腹腔注射)对乳腺癌荷瘤动物肿瘤生长的影响
取雌性Balb/c小鼠(6~8周龄),在右侧第四对乳腺脂肪垫处皮下注射4T1细胞(8×104cells/小鼠)。肿瘤体积增长至约30mm3时,把荷瘤小鼠按肿瘤体积大分层随机分配到模型组(Model组),给药组(IPyA组,60mg/kg),每组5只。从分组日起,给药组给予IPyA(腹腔注射,每周2次),模型组给予等体积生理盐水,连续给药28天。给药期间,每周2次检测肿瘤大小和体重变化。第28天,剖取各组小鼠肿瘤,称量肿瘤重量,拍照。
IPyA(腹腔注射)对乳腺癌荷瘤动物肿瘤生长的影响如图8所示。IPyA(腹腔注射)也可明显减缓4T1乳腺癌肿瘤生长速度,最终降低肿瘤重量。
Claims (7)
1.吲哚-3-丙酮酸或其药用盐在制备治疗乳腺癌药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述的治疗乳腺癌药物是治疗三阴性乳腺癌或管腔A型乳腺癌的药物。
3.根据权利要求1所述的应用,其特征在于,所述的治疗乳腺癌药物含有药学上可接受的辅料。
4.根据权利要求1、2或3所述的应用,其特征在于,所述的治疗乳腺癌药物是将吲哚-3-丙酮酸或其药用盐用医学可接受的各种制剂辅料制备成口服给药剂型。
5.根据权利要求1、2或3所述的应用,其特征在于,所述的治疗乳腺癌药物是将吲哚-3-丙酮酸或其药用盐用医学可接受的各种制剂辅料制备成静脉给药剂型。
6.根据权利要求1、2或3所述的应用,其特征在于,所述的治疗乳腺癌药物是杀伤鼠源乳腺癌细胞系4T1,人源乳腺癌细胞系MDA-MB-231、MCF-7的药物。
7.根据权利要求1、2或3所述的应用,其特征在于,所述的治疗乳腺癌药物是通过吲哚-3-丙酮酸或其药用盐直接抑制肿瘤细胞表观遗传调节因子UHRF1表达,激活AMP依赖的蛋白激酶活性,抑制细胞ATP生成的药物。
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