CN116173024A - 卡博替尼的用途 - Google Patents
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Abstract
本发明公开了小分子酪氨酸激酶抑制剂卡博替尼的一个新用途，涉及医药技术领域，所述的小分子酪氨酸激酶抑制剂卡博替尼的具体新用途为：卡博替尼或其药学、保健品学或食品学上可接受的盐或酯或衍生物、或它们的混合物可以通过泛素蛋白酶体途径降解突变p53Y220C蛋白；相比较表达其他突变p53或者野生型的肿瘤细胞，携带表达突变p53Y220C的卵巢癌细胞对卡博替尼治疗更加敏感，可用于制备治疗卵巢癌、抑制卵巢癌细胞生长、和/或诱导卵巢癌细胞凋亡的物质。该发现拓展了卡博替尼治疗的应用，为携带突变p53Y220C的卵巢癌患者提供一种新颖的治疗策略。
Description
技术领域
本发明属于生物技术和医学领域，具体涉及一种小分子酪氨酸激酶抑制剂卡博替尼的新用途。
背景技术
相比较传统的放疗以及化疗等方法，靶向治疗具有效果精准，不良反应较轻等优点。卡博替尼是一种多靶点的小分子酪氨酸激酶抑制剂，包括MET、VEGFR1/2/3、ROS1、RET、AXL、NTRK、KIT共九个靶点。卡博替尼被FDA批准用于晚期甲状腺癌的治疗和早期肾细胞癌的二线治疗以及一线治疗。
肿瘤抑制因子p53是细胞中最重要的调节因子之一，各种应激压力(如DNA损伤、致癌信号或营养损耗)会促进p53激活，并诱导细胞周期阻滞、DNA修复、衰老或凋亡、代谢等事件发生，抑制肿瘤的发生发展。然而在超过50％的肿瘤中发生了TP53突变，现有证据表明，突变不仅会消除p53的肿瘤抑制功能，表现为功能缺失(Loss of function,LOF)，而且，突变后的p53还具有显性负活性(dominant-negative，DN)，除此以外突变p53还会获得新的功能，促进肿瘤增殖，转移，耐药，调控肿瘤的微环境等。
卵巢癌作为最为致命的妇科恶性肿瘤之一，现阶段的治疗手段十分有限，因此探究卵巢癌新的治疗方法非常重要。96％的卵巢癌中出现TP53基因突变，p53高突变是卵巢癌非常显著的分子特征之一，p53Y220C是卵巢癌中最常见的错义突变之一(排第一位)。由于其可与p63、p73相互作用，并抑制p63、p73的转录调控，因此，p53Y220C也被认为与R175H、R273H等热点错义突变一样，具有很强的致癌作用。因此，靶向突变p53Y220C是治疗卵巢癌很有吸引力的一个策略。
发明内容
本发明目的是提供一种卡博替尼的新用途，为表达突变p53Y220C卵巢癌患者提供一种新的治疗方向。
本发明的技术方案是：
卡博替尼、或其药学、保健品学或食品学上可接受的盐或酯或衍生物、或它们的混合物用于制备治疗p53突变型卵巢癌、抑制p53突变型卵巢癌的细胞生长、和/或诱导p53突变型卵巢癌细胞凋亡的药物中的用途。
进一步的，所述卡博替尼、或其药学、保健品学或食品学上可接受的盐或酯或衍生物、或它们的混合物用于降解p53突变型卵巢癌中p53突变蛋白。
进一步的，所述p53突变蛋白为p53Y220C突变蛋白。
进一步的，所述p53突变蛋白的降解是通过泛素蛋白酶体降解途径。
进一步的，所述细胞为卵巢癌肿瘤细胞。
进一步的，所述卵巢癌肿瘤细胞为携带突变p53Y220C的肿瘤细胞。
本发明的优点是：本发明所述的卡博替尼可以特异性促进p53Y220C蛋白泛素蛋白酶体途径降解，从而在卵巢癌中达到抗肿瘤作用，为表达突变p53Y220C的卵巢癌患者提供一种新颖的治疗策略。
附图说明
图1为卡博替尼免疫印迹实验结果图，其中，所用卡博替尼浓度为10μM，给药处理时间为48h；
图2为卡博替尼免疫印迹实验结果图，其中，所用卡博替尼浓度为10μM，给药处理时间为48h，MG132/BafilomycinA1浓度为10μM，给药处理时间为8h；
图3为卡博替尼免疫印迹，免疫共沉淀实验结果图，其中，所用卡博替尼浓度为10μM，给药处理时间为48h；
图4为卡博替尼MTT实验结果图，其中，所用卡博替尼浓度为10μM，给药处理时间为48h。
具体实施方式
经过广泛而深入的研究，发现在卵巢癌，胰腺癌，口咽部鳞状细胞癌患者中，p53Y220C的突变频率较高，尤其是浆液性卵巢癌和口咽部鳞状细胞癌中突变频率最高的突变体。
本发明通过免疫印迹测试了一组携带不同p53突变体或野生型p53的人肿瘤细胞，在给予卡博替尼后，检查p53蛋白的表达情况，发现卡博替尼对p53Y220C突变体具有选择性降解作用。卡博替尼是通过蛋白酶体途径介导p53Y220C的降解，并改变突变p53Y220C的结构，引起K373位点泛素化修饰增加。通过细胞活力以及裸鼠皮下分别接种p53野生型、p53其他突变型以及p53Y220C人源肿瘤细胞证明，卡博替尼体内外均优先阻止p53Y220C型肿瘤细胞的生长。
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合实施例进一步说明本发明的技术方案。但是本发明不限于所列出的实施例，还应包括在本发明所要求的权利范围内其他任何公知的改变。
此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例，也不是单独的或选择性的与其他实施例互相排斥的实施例。
在本发明中所用到的肿瘤或肿瘤细胞是表达人p53野生型或突变型的肿瘤或肿瘤细胞。
所述肿瘤细胞选自下组：人源胰腺癌细胞系BxPC3(p53Y220C)、人源卵巢癌细胞系COV362(p53Y220C)、SKOV3(p53 null)、人源肝癌细胞系HUH7(p53Y220C)、人源非小细胞肺癌细胞株H1975(p53R273H)H1299(p53 null)、人源乳腺癌细胞系MDA-MB-231(p53R280K)、人源骨肉瘤细胞系U20S(p53WT)、人源结直肠癌细胞系HCT116(p53WT)和人肾胚胎干细胞239T。
BxPC3、COV362、SKOV3细胞系的培养方法如下：将细胞接种于含10％小牛血清的RPMI 1640培养基(美国GiBco公司)培养液中，置于37摄氏度、体积分数为5％的CO2培养箱中常规培养，实验前无药培养两周。
HUH7、H1299、HCT116、293T、MDA-MB-231、U20S、H1975细胞系的培养方法如下：将细胞接种于含10％小牛血清的DMEM培养基(美国GiBco公司)培养液中，置于37摄氏度、体积分数为5％的CO2培养箱中常规培养，实验前无药培养两周。
实施例1
卡博替尼引起p53Y220C的选择性下调
通过免疫印迹测试一组携带不同p53状态(野生型、突变p53Y220C、突变p53R175H、突变p53R273H、)的人源肿瘤细胞在给予卡博替尼后，p53的蛋白水平。请参阅图1，图1为卡博替尼免疫印迹实验结果图。如图1所示，卡博替尼可以降低突变p53Y220C的表达水平。但在表达野生型p53的结直肠癌细胞系HCT116(WT)和骨肉瘤细胞系U20S(WT)中，卡博替尼不影响野生型p53的表达，并且卡博替尼对其他的突变p53细胞系如肺癌细胞系H1975(R273H)和乳腺癌细胞系MDA-MB-231(R280K)无明显影响。此外，在不表达p53的细胞系H1299(null)中转染外源性Flag-p53Y220C，经卡博替尼处理后，同样可以降低外源性突变Flag-p53Y220C的表达水平，但外源性野生型p53或其他突变p53的表达水平没有明显影响，证实了卡博替尼可以引起p53Y220C的特异性蛋白表达下调。
实施例2
卡博替尼通过蛋白酶体途径降解p53Y220C蛋白
为了确定卡博替尼所导致的p53蛋白的降解是通过蛋白酶体还是溶酶体途径介导的，在本发明中研究者使用蛋白酶体抑制剂MG132以及自噬抑制剂BafilomycinA1与卡博替尼共同处理COV362细胞。请参阅图2，图2为卡博替尼免疫印迹实验结果图。如图2所示，卡博替尼处理后p53蛋白的表达水平降低，MG132能够拮抗卡博替尼诱导的p53Y220C蛋白水平的降解，加入自噬抑制剂后p53蛋白水平无变化，证实了卡博替尼通过蛋白酶体途径降解p53Y220C蛋白。
实施例3
卡博替尼改变突变p53Y220C的结构并引起K373位点泛素化修饰增加
本实例通过分子对接实验和分子动力学模拟实验表明卡博替尼对突变p53Y220C结构影响更大，使得C-terminal结构发生明显的改变。本发明研究者将突变p53Y220C泛素化修饰位点的赖氨酸突变为精氨酸，请参阅图3，图3为卡博替尼免疫印迹，免疫共沉淀实验结果图。如图3所示，通过IP实验发现373位点的赖氨酸突变为精氨酸后可以拮抗卡博替尼诱导的泛素化修饰以及降解，证实了卡博替尼改变突变p53Y220C的结构并引起K373位点泛素化修饰增加。
实施例4
卡博替尼对p53Y220C突变型肿瘤细胞生长具有更强的抑制作用
本实例采用了常规的MTT法。具体方法如下：在SKOV3细胞中构建不同p53突变，分别为SKOV3(control组)、SKOV3(Y220C)、SKOV3(WT)、SKOV3(R273H)；在COV362细胞中敲低p53的表达。将SKOV3细胞以5000个细胞/孔的密度铺96孔板培养过夜。然后，把卡博替尼以不同浓度(1.25μM、2.5μM、5μM、10μM、20μM)加入孔中。48小时后，在每孔中加入MTT(0.5mg/ml)30μl，37℃孵育4小时后吸弃上清。用100μl二甲基亚砜溶解紫色结晶后，置于酶标仪(Bio-Rad公司)，检测570nm处的吸光度。请参阅图4，图4为卡博替尼MTT实验结果图。如图4所示，转染突变p53Y220C的SKOV3细胞对卡博替尼的治疗更加敏感，COV362细胞沉默p53后对卡博替尼治疗敏感性降低，证实了卡博替尼对p53Y220C突变型肿瘤细胞具有更强的抑制作用。
上述实施例的结果表明，本发明发现小分子酪氨酸激酶抑制剂卡博替尼对p53Y220C突变型卵巢癌细胞具有突出的治疗效果。
综上所述，本发明公开了卡博替尼可以选择性降解突变p53Y220C，对野生型p53和其他突变p53的表达没有明显影响。卡博替尼通过与突变p53Y220C相互作用，诱导其构象发生改变，并诱导其通过泛素蛋白酶体途径降解，使表达突变p53Y220C的肿瘤对卡博替尼的治疗更加敏感。本发明发现了卡博替尼的新靶点，能够有效靶向促进p53Y220C卵巢癌细胞p53蛋白的降解，拓展其应用，并同时为表达突变p53Y220C的卵巢癌患者提供一种新颖的治疗策略。
应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。
Claims (6)
1.卡博替尼、或其药学、保健品学或食品学上可接受的盐或酯或衍生物、或它们的混合物用于制备治疗p53突变型卵巢癌、抑制p53突变型卵巢癌的细胞生长、和/或诱导p53突变型卵巢癌细胞凋亡的药物中的用途。
2.根据权利要求1所述的用途，其特征在于：所述卡博替尼、或其药学、保健品学或食品学上可接受的盐或酯或衍生物、或它们的混合物用于降解p53突变型卵巢癌中p53突变蛋白。
3.根据权利要求2所述的用途，其特征在于：所述p53突变蛋白为p53Y220C突变蛋白。
4.根据权利要求2所述的用途，其特征在于：所述p53突变蛋白的降解是通过泛素蛋白酶体降解途径。
5.根据权利要求1所述的用途，其特征在于：所述细胞为卵巢癌肿瘤细胞。
6.根据权利要求5所述的用途，其特征在于：所述卵巢癌肿瘤细胞为携带突变p53Y220C的肿瘤细胞。
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