CN113876758A - 胭脂素治疗癌症的用途 - Google Patents
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Abstract
本申请公开了胭脂素在制备癌症尤其是肺癌的药物中的应用，还公开了胭脂素与TRAIL联合用于制备癌症尤其是肺癌药物中的应用。
Description
技术领域
本申请涉及胭脂素或胭脂素与TRAIL联合在制备治疗癌症尤其是肺癌的药物中的应用。
背景技术
肺癌是对人类健康和生命威胁最大的恶性肿瘤之一。研究表明85％的患者为非小细胞肺癌，而化疗是治疗非小细胞肺癌的主要方法，但是化疗后肿瘤缓解率为40％至50％，并且化疗常伴随着部分患者出现耐药性以及全身系统副作用，所以当下探索治疗肺癌的新策略至关重要。
胭脂素(Bixin)，又称胭脂树橙、红木素。结构如下：
胭脂素是一种在胭脂树红中发现的脱辅基类胡萝蔔素，既往研究表明该药物可以减少炎症反应和氧化损伤从而预防动脉粥样硬化。
发明内容
本申请旨在提供胭脂素以及胭脂素和TRAIL联合在治疗癌症尤其是肺癌的作用。本申请同时通过胭脂素或胭脂素和TRAIL对人肺癌细胞系A549和H460细胞的作用，证明了胭脂素和TRAIL对抗肺癌细胞生长的潜在机制是通过内质网应激和激活AMPK信号通路诱导细胞凋亡来抑制细胞的增殖，这一研究结果有望为肺癌治疗提供新的治疗策略。
根据本申请的一个方面，本申请提供了胭脂素在制备治疗癌症尤其是肺癌的药物中的应用。
根据本申请的另一方面，本申请提供了胭脂素和TRAIL联合用于制备治疗癌症尤其是肺癌的药物中的应用。
根据一些实施方式，本申请所述的肺癌是由AMPK介导的肺癌。
根据一些实施方式，本申请所述的肺癌是非小细胞肺癌。
本申请所提供的药物包含作为活性成分的胭脂素和药学可接受的载体，或者作为活性成分的胭脂素和TRAIL再加上药学可接受的载体。
在一些实施方式中，胭脂素和TRAIL可以分别与药学可接受的载体制成独立的剂型，也可以将两个活性成分一起与药学可接受的载体制成一个剂型。
本申请所述的药物包括但不限于口服剂型和胃肠外给药剂型。在一些实施方式中，所述药物可以是口服的片剂、胶囊、丸剂、粉剂、缓释制剂、溶液和悬浮液，用于胃肠外注射的无菌溶液、悬浮液或乳液。在其它实施方式中，所述药物为适合单次施予精确剂量的单位剂型。在一些实施方式中，所述活性成分的量在约0.001mg/kg体重/天-约1000mg/kg体重/天的范围内，例如，可以是约0.5mg/kg体重/天-约50mg/kg体重/天。在一些实施方式中，所述活性成分的量为约0.001g/天-约7g/天，例如为约0.002g/天-约6g/天，约0.005g/天-约5g/天，约0.01g/天-约5g/天，约0.02g/天-约5g/天，约0.05g/天-约2.5g/天，约0.1g/天-约1g/天。在其它实施方式中，低于上述范围下限的剂量水平可能已经是足够的或者需要高于上述范围上限的剂量水平。在一些实施方式中，施用所述活性成分，每天一次。在其它实施方式中，施用所述活性成分，每天不只一次。在一些实施方式中，每天施用两次、三次、四次或四次以上的所述活性成分。在一些实施方式中，所述药物施用于的个体为哺乳动物。在其它实施方式中，所述哺乳动物是人。
术语定义
本文所用术语“药学可接受的”是指不影响本申请活性成分的生物活性或性质的物质(如载体)，并且相对无毒，即该物质可施用于个体而不造成不良的生物反应或以不良方式与组合物中包含的任意组分相互作用。
本文所用术语“载体”是指相对无毒的物质，其有助于将本申请的活性成分引入到细胞或组织中。
附图说明
图1示出了胭脂素、TRAIL以及胭脂素与TRAIL共同对肺癌细胞生长的抑制作用；
图2示出了胭脂素、TRAIL以及胭脂素与TRAIL共同对迁移的肺癌细胞比例的降低作用；
图3示出了胭脂素、TRAIL以及胭脂素与TRAIL共同对PERK信号通路的激活作用；
图4：4A示出了胭脂素、TRAIL以及二者的联合可增加细胞内ROS水平；4B示出了胭脂素、TRAIL以及二者的联合可增强NADPH活性；4C示出了胭脂素、TRAIL以及二者的联合可显著上调Caspase-3的活性；4D示出了胭脂素、TRAIL以及二者的联合可显著上调Caspase-9的活性。
图5：5A和5B示出单独用TRAIL或胭脂素处理可上调磷酸化AMPK，DR4，Caspase-9，Caspase-3和Bax的表达，并下调Bcl-2的表达，二者的组合进一步增强或抑制了上述蛋白质的表达；
图6示出了AMPK在胭脂素和TRAIL共同诱导的细胞凋亡中的作用；
图7：7A示出TRAIL和AMPK siRNA联合治疗会降低胭脂素和TRAIL的抗肿瘤能力；7B示出了在肿瘤重量上显示出相同的趋势；7C示出不同治疗组之间小鼠的体重没有显著差异；7D示出胭脂素和TRAIL不会对小鼠的重要器官产生明显的毒性；
图8示出胭脂素和TRAIL联合用药诱导AMPK、DR4、Caspase-9、Caspase-3、Bax和Bcl2的抑制作用被AMPK抑制剂所逆转。
具体实施方式
以下通过实施例结合附图详细阐述本申请的技术方案，但本申请并不受限于此。
实施例
生物活性测试试验
材料和方法
细胞培养与处理
从美国典型培养物保藏中心(ATCC，USA)购买肺癌细胞系A549和H460，以及BEAS-2B的人正常肺上皮细胞。常规培养的所有细胞均在含有10％胎牛血清(FBS)(GIBCO)，1％青霉素/链霉素的Dulbecco改良的Eagle's培养基(DMEM，Gibco，Waltham，MA，USA)中进行培养。用于人类肺癌治疗的胭脂素(纯度>98％，美国，Sigma)溶于DMSO中，并保存在-20℃进行后续研究，然后在DMEM培养基中稀释以进行实验治疗。
细胞活力分析
为了计算胭脂素(Bixin/Bix)、TRAIL和这两种药物在不同细胞系中的抑制作用，将约1×103个细胞/孔种植在96孔板中。24小时后，用不同浓度的药物处理癌细胞，并在37℃下孵育24小时。最终通过MTT测定法评估细胞活力。
成克隆实验
用指定浓度的Bixin和TRAIL在生长培养基中处理约65％的人肺癌细胞24小时。孵育后，将细胞收集在单独的试管中。从每个试管中，分别在6孔板中铺600个细胞/孔，并使其生长两周。14天后，将细胞用冷磷酸盐缓冲盐水(PBS)洗涤一次，使用冷却的甲醇固定细胞15分钟。然后在室温下，将细胞在25％甲醇中的0.5％结晶紫溶液(ChemCatch，美国)染色5分钟。将细胞用水洗涤3次并风干以使用显微镜计数。
划痕实验
先用马克笔在6孔板背后均匀划横线，再将人肺癌细胞按1×105个细胞/孔种植于6孔板中，待细胞长满后，使用枪头垂直于背后的直线划痕。然后用PBS洗涤以去除细胞碎片，并继续培养24小时。
氧自由基(ROS)检测
将细胞用不同浓度的药物处理后，用胰蛋白酶将细胞消化，然后在PBS中的5μMDCFH-DA中于37℃孵育2小时。用荧光酶标仪在488nm激发和522nm发射下评估样品的DCFH荧光强度(Biotech Instruments，USA)。
Caspase-3和Caspase-9活性
使用Caspase-9/-3活性测定试剂盒(美国Clontech)检测其活性。将人肺癌细胞在冰冷的裂解缓冲液中裂解10分钟，然后以10,000×g离心5分钟。然后将具有特定肽底物的Caspases底物溶液添加到上清液中，并在37℃下培养2小时，然后在405nm处进行ELISA读数器测定。
蛋白质印迹分析
在不同条件下处理后，收集细胞并除去培养基。然后将细胞用冰冷的PBS洗涤3次，并在新鲜蛋白酶抑制剂混合物的存在下在冰冷的裂解缓冲液中裂解。在体内实验中，从异种移植裸鼠获得冷冻的肿瘤组织样品，用1ml裂解缓冲液裂解约100mg肿瘤组织。将细胞裂解液在4℃以15,000g离心20分钟，收集上清液。使用BSA蛋白测定试剂盒(美国，Thermo)评估蛋白浓度。然后将蛋白质提取物通过10％SDS-PAGE分离胶，并转移至聚偏二氟乙烯膜(PVDF)。用5％脱脂脂肪干奶(溶于含0.1％Tween-20的Tris缓冲盐溶液(TBS)中)封闭1小时。4℃过夜孵育一抗，第二天回收抗体，室温孵育2抗1小时。
小干扰RNA的构建
针对人AMPK的siRNA序列和对照siRNA来自Santa Cruz Biotech(美国)。将3×105A549细胞/孔种植于6孔板上，然后按照制造商的说明在每孔中用Lipofectamine 2000(美国Invitrogen，美国)用100pmol siRNA双链体转染。然后将细胞注入动物体内实验，以探讨AMPK在肺癌进展中的作用。
无胸腺裸鼠模型构建
从上海实验动物中心(中国上海)购买了40只6-8周大的雄性Athymic裸鼠，并将其饲养在温度和湿度可控的无菌条件下(25±2℃，50±10％湿度)，所有动物实验都是根据1996年美国国家卫生研究院发布的《实验动物的护理和使用指南》进行的，以最大程度地减少动物的痛苦。所有过程均符合吉林大学第一医院机构动物护理和使用委员会的要求。简而言之，将5×105个A549细胞皮下注射到裸鼠的背侧。通过每两天计算两个最大垂直肿瘤直径来测量肿瘤体积。将所有荷瘤裸鼠随机分为4组：(1)对照组，不做任何处理；(2)AMPKsiRNA组；(3)Bixin(20mg/kg)和TRAIL(100μg/小鼠)组合组；(4)Bixin和TRAIL与AMPKsiRNA(100μl)的组合每天使用28天。将Bixin溶于DMSO，然后在蒸馏水中稀释。小鼠口服Bixin。对照组给予DMSO稀释于水中。每周测量三次体重和肿瘤大小。用式1/2(L1×L2×H)评价肿瘤体积，L1代表长径，L2代表短径，H是肿瘤的高度。最后，将小鼠处死。取出心脏，肾脏和肝脏以及肿瘤组织样品，通过蛋白质印迹进行病理分析和分子机理研究。
病理分析
取出心脏，肾脏和肝脏，并在4％中性福尔马林液体中固定。脱水后，进行苏木精伊红(HE)染色，随机选择五个视野进行评估。
实验结果
1.胭脂素增强了TRAIL诱导的人类肺癌细胞株活力的抑制。
既往研究表明，TRAIL因其选择性诱导各种癌细胞系而不是正常细胞的凋亡而成为癌症治疗的候选药物。通过光学显微镜或CCK-8试验研究了胭脂素对包括A549和H460细胞在内的肺癌细胞系的生长抑制能力。如图1A所示，单独用不同浓度的TRAIL或胭脂素处理的细胞显示出细胞附着减少和明显的细胞收缩，这在人正常的肺上皮细胞中没有发现的。但是，TRAIL和胭脂素的联合治疗增强了这种对细胞生长的抑制作用。此外，CCK-8分析表明，用TRAIL和胭脂素处理的肺癌细胞的细胞活力要比单一处理的细胞低。正常细胞BEAS-2B在用胭脂素和/或TRAIL处理后，各组之间无显著差异(图1B，C，D)。
2.胭脂素和TRAIL联合抑制人肺癌细胞系中的集落形成和迁移
为确定胭脂素和TRAIL联合治疗在细胞集落形成和迁移中的作用。在A549和H460细胞中进行集落形成测定和划痕实验测定。如图2A所示单独用胭脂素或TRAIL处理的肺癌细胞株的集落形成水平以剂量依赖的方式明显减弱。值得注意的是，与单一药物治疗相比，联合治疗促进了肺癌细胞克隆能力的降低(图2A，B，C)。划痕实验表明，单独使用TRAIL或胭脂素的治疗可适度降低迁移的肺癌细胞的比例。但是，联合处理增强了这种效应(图2D，E，F)。
3.胭脂素和TRAIL联合治疗人肺癌细胞的分子机制与PERK信号通路相关
内质网应激(ERs)在癌细胞的增殖和细胞活力中发挥着至关重要的作用。因此，我们假设ERs通过促进PERK信号通路参与抑制胭脂素和/或TRAIL处理的肺癌细胞的迁移和侵袭。p-PERK，GRP78，CHOP，p-eIF2α和ATF4在不同浓度的胭脂素和/或TRAIL刺激的A549和H460细胞中的表达。数据显示单独使用TRAIL或胭脂素进行处理可增加PERK磷酸化，GRP78，CHOP，eIF2α磷酸化和ATF4表达。如图3A和3B所示，通过与胭脂素(40μM)和TRAIL(200ng/ml)的联合处理可以增强该信号通路的激活。
4.胭脂素和TRAIL的联合治疗会影响ROS的产生以及Caspase-3和Caspase-9的活性。
我们使用DCFH-DA荧光探针评估胭脂素和TRAIL是否通过ROS产生诱导A549和H460细胞凋亡。单一药物处理可增加细胞内ROS水平并以剂量依赖性方式增加，两者的结合效果增强(图4A)。此外，在本部分中还对被认为是ROS的主要来源之一的NADPH进行了测量。因此，在NADPH活性中也观察到了相似的趋势，联合治疗增强了NADPH活性(图4B)。对于胭脂素和/或TRAIL处理，A549和H460细胞中的Caspase-3和Caspase-9活性显著上调(图4C和4D)。
5.胭脂素和TRAIL共同治疗可增强AMPK和人肺癌细胞凋亡相关蛋白的表达
AMPK具有促进或抑制肿瘤的双重功能，某些AMPK激活剂可以抑制肿瘤的生长。为了评估胭脂素和TRAIL联合治疗的抗肿瘤作用是否由AMPK介导，通过以下方法研究了磷酸化的AMPK，DR4，Caspase-9，Caspase-3，抗凋亡成员Bcl-2和促凋亡信号Bax。结果表明，单独用TRAIL或胭脂素处理可上调磷酸化AMPK，DR4，Caspase-9，Caspase-3和Bax的表达，并下调Bcl-2的表达。组合疗法进一步增强或抑制了上述蛋白质的表达(图5A和5B)。
6.AMPK激活是胭脂素和TRAIL共同诱导人肺癌细胞凋亡的必要条件
为了进一步探讨AMPK在胭脂素和TRAIL共同诱导的细胞凋亡中的作用，CompoundC(或称为Dorsomorphin，其为AMPK抑制剂)与胭脂素和TRAIL一起作用于A549和H460细胞。如图6A-6B所示，Compound C的预处理显著减弱了胭脂素和TRAIL诱导的磷酸化AMPK，DR4，Caspase-9，Caspase-3以及Bax的上调和Bcl-2的下调。另外，用Compound C处理细胞后，再次测量细胞活力，凋亡细胞的比率，Caspase-3活性和ROS产生。与胭脂素和TRAIL治疗组相比，Compound C预处理后细胞活力显著提高(图6C)。同时，当AMPK被抑制时，凋亡细胞的比例和Caspase-3活性显著降低(图6D和6F)。ROS含量显示出相同的趋势(图6E)。
7.胭脂素和TRAIL共同治疗可通过激活AMPK从而抑制A549肿瘤异种移植小鼠体内的肿瘤发展。
在体内实验中，将A549细胞皮下孵育到无胸腺裸鼠中。显然，与对照组相比，胭脂素和TRAIL联合治疗可以显著抑制具有A549肿瘤异种移植物的小鼠的肿瘤生长。但是，用胭脂素、TRAIL和AMPK siRNA联合治疗会降低胭脂素和TRAIL的抗肿瘤能力(图7A)。在肿瘤重量上显示出相同的趋势(图7B)。不同治疗组之间小鼠的体重没有显著差异(图7C)。此外，对心脏，肾脏和肝脏的H&E染色表明，在不注射A549的情况下，胭脂素和TRAIL联合治疗不会对小鼠的重要器官产生明显的毒性(图7D)。
8.通过体内实验的蛋白质印迹分析了AMPK和凋亡相关信号。如图8所示，胭脂素和TRAIL共同处理可诱导AMPK，DR4，Caspase-9，Caspase-3，Bax和Bcl2的抑制作用被AMPK抑制剂所逆转。
Claims (8)
1.胭脂素在制备治疗癌症的药物中的应用。
2.根据权利要求1所述的应用，其中所述癌症为肺癌。
3.根据权利要求2所述的应用，其中所述肺癌是由AMPK介导的肺癌。
4.根据权利要求2或3所述的应用，其中所述肺癌是非小细胞肺癌。
5.胭脂素和TRAIL联合用于制备治疗癌症的药物中的应用。
6.根据权利要求5所述的应用，其中所述癌症是肺癌。
7.根据权利要求6所述的应用，其中所述肺癌是由AMPK介导的肺癌。
8.根据权利要求6或7所述的应用，其中所述肺癌是非小细胞肺癌。
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