CN116549440A - 小分子化合物棘壳孢素A在抑制Skp2蛋白及抗前列腺癌中的应用 - Google Patents
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Abstract
本发明公开了天然来源的小分子化合物棘壳孢素A(PrA)在作为Skp2蛋白抑制剂及前列腺癌细胞增殖活性抑制剂中的应用。本发明首次发现棘壳孢素A能够激活Skp2蛋白上游的多聚泛素化,加速Skp2蛋白通过泛素化蛋白酶体途径降解,同时能够调节其下游通路,抑制前列腺癌细胞的DNA复制和合成,从而有效抑制前列腺癌细胞的过度增殖,其将是有效的Skp2蛋白抑制剂与分子靶向治疗领域中有效的前列腺癌抑制剂与杀伤剂。
Description
技术领域
本发明属于医药领域,具体涉及一种小分子化合物棘壳孢素A(PrA)在作为Skp2抑制剂中的应用。
背景技术
棘壳孢素A的分子式为C11H12O4,分子量为208,其在一个吡喃环为基础上由甲基、甲氧基首尾相连所构成的简单化合物()。棘壳孢素A为真菌的次生代谢产物,最早是于1921年由Hiroji等人从洋葱粉根病的植物病原真菌(Pyrenochaetaterrestris)中分离得到。现今已从多种植物真菌与海洋真菌中分离得到该种化合物。该化合物除在植物体上表现出植物毒性和抗病虫害的功效外,还表现出较为良好的抗炎症、抗病毒以及抗肿瘤等效果。现如今棘壳孢素A的应用多用于抗疟药和细胞毒性剂,虽然有肿瘤相关研究但均未有较为清晰的药物作用方式。
S期激酶相关蛋白2(Skp2)是SCFSKP2泛素连接酶复合物的底物识别成分,通过靶向多种肿瘤抑制因子(例如p21、p27和p130)进行泛素化和随后的降解,在肿瘤发生中发挥致癌作用。作为一种充分表征的癌蛋白,在不同的人类癌症中经常观察到Skp2的异常表达和失调。鉴于其在控制肿瘤发生和进展中的关键作用,Skp2已成为抗癌治疗的潜在药理学靶点。
Skp2也被称为FBXLI,编码一个F-box蛋白。Skp2组成SCF Skp2复合物的重要成分并起识别底物蛋白的作用,SCF复合物可以介导涉及细胞周期过程、信号转导和转录目标蛋白的遍在蛋白化和随后的蛋白酶体降解,尤其是识别磷酸化的CDKN1B/p27kip及与G1/S期转换的调节有关。作为SCF-Skp2复合物的底物识别亚单位。Skp2敲除鼠显示细胞的缺陷,包括细胞核增大、中心体重叠和多倍性。在细胞分裂周期阶段,Skp2作用的一个重要底物是p27,这是一个cdk2和cdk1活动的抑制因子,通过降解p27可以促进细胞进入DNA合成期和分裂期。后来,又有几种细胞周期调节蛋白包括cyclin E、p57、p21、p130、cdt1和E2F1相继被报道属于Skp2介导的降解蛋白。此外,Skp2除了诱导p27的降解和增强细胞的增生外,还具有促进肿瘤发生的作用。提出这种上调表达是Skp2促进细胞浸润的一种机制,抑制Skp2的表达也许可以抑制肿瘤的浸润和转移。
Skp2在肿瘤细胞的细胞周期中起着重要的作用,通过药物来抑制Skp2相关蛋白的活性是现如今较为流行的方式。药物抑制分为为三种,第一种为通过抑制Skp2蛋白在细胞周期中的上游等蛋白或基因的表达或者降解,如FAM60A、STAT3;第二种为通过直接作用在Skp2蛋白上,使Skp2蛋白的降解加快,同时Skp2复合体的形成受到阻滞;第三种为通过直接作用在Skp2蛋白,但对Skp2的降解没有影响,通过改变Skp2途径下游蛋白的含量,减少了p27、p21等蛋白的表达,从而改变冲瘤细胞的生长、增殖和转移能力。
发明内容
本发明的目的在于提供一种天然来源的小分子化合物棘壳孢素A在作为Skp2蛋白抑制剂中的应用。
为实现上述目的,本发明采用如下技术方案:
本发明的目的之一是保护小分子化合物棘壳孢素A在作为Skp2蛋白抑制剂中的应用。棘壳孢素A能够通过促进Skp2蛋白的多聚泛素化积累,加速Skp2蛋白通过泛素化蛋白酶体途径降解。
本发明的另一目的是保护小分子化合物棘壳孢素A在作为前列腺癌细胞增殖活性抑制剂中的应用。棘壳孢素A能够通过促进Skp2蛋白降解,进而调节其下游通路,抑制前列腺癌细胞的DNA复制和合成,从而有效抑制前列腺癌细胞的过度增殖。
进一步地,所述前列腺癌细胞为DU145细胞。
更进一步地,小分子化合物棘壳孢素A抑制前列腺癌DU145细胞增殖活性的有效浓度为10 μM-400 μM。
本发明的显著优势在于:
太子参内生真菌来源的小分子化合物棘壳孢素A具有较强的Skp2蛋白的抑制能力和前列腺癌抑制能力,其结构简单,能够稳定分离提取,是一种高效、易于获取的天然来源的抗肿瘤化合物。
本发明首次发现棘壳孢素A能够在DU145细胞中通过增强Skp2蛋白上的多聚泛素化过程降解Skp2,从而来影响细胞的增殖能力,其具体机制是:棘壳孢素A通过增强Skp2蛋白上的多聚泛素化过程,进而明显降低Skp2的半衰期,加快它们的降解速度,从而明显降低DU145细胞内的Skp2的蛋白水平。Skp2蛋白的降解会提高其底物蛋白p27的稳定性,并且会增加p27的表达量,抑癌蛋白p27可以有效控制癌细胞的过度增殖。因此,棘壳孢素A通过增强Skp2蛋白的泛素化可调节其下游通路,从而抑制前列腺癌细胞DU145细胞的DNA复制和合成,有效抑制其过度增殖,以此来达到抑制前列腺癌的作用。
附图说明
图1为实施例1中棘壳孢素A对DU145细胞相关蛋白表达的影响情况对比图。
图2为实施例2中棘壳孢素A影响DU145细胞相关蛋白表达的Western Bolt图(A)及相关蛋白的稳定性曲线图(B)。
图3为实施例3中在蛋白酶体抑制剂作用下棘壳孢素A对DU145细胞相关蛋白表达的影响情况对比图。
图4为实施例4中棘壳孢素A促进Skp2蛋白的多聚泛素化的实验结果图。
图5为实施例5中棘壳孢素A对前列腺癌细胞DU145的增殖与DNA复制的影响情况结果图。
图6为实施例6中棘壳孢素A降低细胞DNA合成能力的实验结果图。
具体实施方式
为了使本发明所述的内容更加便于理解,下面结合具体实施方式对本发明所述的技术方案做进一步的说明,但是本发明不仅限于此。
实施例1 棘壳孢素A造成Skp2蛋白的稳定性降低和加速Skp2蛋白的降解
将棘壳孢素A(PrA)以20 μM的浓度作用于DU145细胞24 h后,测定细胞周期相关蛋白的表达量,实验结果如图1所示。
由图1可见,与对照组相比,经棘壳孢素A处理后Tubulin蛋白表达水平没有明显变化,Skp2蛋白表达水平明显降低,p27蛋白表达水平明显升高,表明棘壳孢素A可通过促进DU145细胞中Skp2蛋白表达水平的降低,促进其下游靶蛋白p27高水平积累,进而诱导细胞的G1/S期阻滞。
实施例2
为进一步确定Skp2蛋白水平降低的原因,通过放线菌酮(CHX)抑制蛋白的降解,检测棘壳孢素A作用下Skp2蛋白以及下游靶蛋白p27的含量变化。其具体操作是以浓度为20 μM的棘壳孢素A作用DU145细胞12 h后,加入CHX 35 μM,分别培养0、2、4、8 h后收集细胞,裂解细胞并收集蛋白进行Western Bolt实验,观察蛋白变化,实验结果见图2。
由图2可见,在棘壳孢素A的作用下,Skp2蛋白的降解明显加剧(A),其可将Skp2蛋白的半衰期由8 h以上分别缩短至4 h左右(B),与此同时,p27蛋白明显积累,半衰期明显延长。
实施例3
细胞内蛋白降解途径主要分为泛素化蛋白酶体途径和溶酶体途径,其中泛素化是蛋白翻译后修饰过程,能够在改变蛋白的水解速率的同时也能高度精准调节蛋白的半衰期变化,是细胞中主要的蛋白调节方式。因此,基于上述结论考虑棘壳孢素A可能通过泛素化蛋白酶体途径加速Skp2蛋白的降解。为了进一步验证这一内容,选取蛋白酶体抑制剂(MG-132)特异性阻断蛋白酶体发挥作用,进而影响泛素化蛋白酶体途径的正常降解。
分别设置对照组、PrA处理组、MG-132处理组、PrA与MG-132联合作用组,培养24小时后收集细胞,裂解细胞并收集蛋白进行Western Bolt实验,观察相关蛋白变化,实验结果见图3。
由图3可见,与对照组相比,PrA处理组中Skp2蛋白的表达水平明显降低,p27蛋白的表达水平明显升高;MG-132处理组中Skp2蛋白和p27蛋白表达水平均有升高;而与单加PrA组相比,PrA与MG-132联合作用组中Skp2蛋白的降解趋势受到抑制,蛋白表达水平有明显回增。
实施例4 棘壳孢素A促进Skp2蛋白的多聚泛素化降解
为验证棘壳孢素A是否通过泛素化蛋白酶体途径介导Skp2的降解,在HEK 293T细胞模型中构建泛素化模型,HEK 293T细胞模型共分为4个实验组,分别为①空白对照组、②阴性对照组、③阳性对照组、④实验组,每组细胞加入相应转染质粒后,正常培养36 h,加入化合物处理8 h后根据Western Blotting蛋白收集方法收集细胞并根据以下步骤采用镍磁珠亲和提取方法提取组氨酸标记(His-tag)的泛素化蛋白,结果如图4所示:
1.取每组培养好的细胞,用适量PBS洗涤1次,用500 μL结合缓冲液(6 Mguanidine-HCl, 0.1 M Na2HPO4/NaH2PO4,10 mM imidazole,pH=7.4)重悬,超声破碎细胞,置于冰上10 min,12000rpm离心15 min后去上清液即为粗提蛋白样品。
2.磁珠预处理:将磁珠置于涡旋混匀器上充分混匀,然后取适量悬液于离心管中,进行磁性分离,弃上清。加入与分离出珠子相同体积的结合缓冲液,上下翻转或吹打数次,磁性分离去上清,重复洗涤2次。
3.目标蛋白与磁珠结合:
1 mL磁珠至少可结合5 mg蛋白,6 cm皿取150 μL(保证珠子体积不至于过少,结合充分)。将第2步裂解好的粗提蛋白样品加入到有磁珠的离心管中,涡旋震荡15 s,充分混匀,将混匀后的样品放置在旋转混匀器上4℃培养4 h使得磁珠与蛋白充分结合,然后进行磁性分离,弃上清。
4.磁珠洗涤:
加入1 mL洗涤缓冲液(20 mM Sodium Phosphate,500 mM NaCl,50 mMImidazole,pH=7.4)到装有磁珠的离心管中,翻转或吹打数次,使磁珠悬浮,磁性分离,上清收集备检,重复1次。加1 mL洗涤缓冲液使磁珠重新悬浮,将磁珠移到新离心管(防止原离心管壁其他蛋白非特异性吸附),磁性分离,弃上清。
5.目标蛋白洗脱:
加入200 μL蛋白洗脱缓冲液(20 mM Sodium Phosphate,500 mM NaCl,500 mMImidazole,pH=7.4)于上步磁珠中,翻转吹打数次,磁性分离,收集洗脱液到新离心管,即为纯化蛋白样品。
由图4可见,对比不加棘壳孢素A的阳性对照组(③),加入10 μM棘壳孢素A处理(④)能够促进Skp2多聚泛素化,从而加速Skp2降解。
实施例5 棘壳孢素A抑制前列腺癌细胞DU145的增殖与DNA复制
通过CCK8实验测定6个不同浓度的棘壳孢素A处理24 h对DU145细胞的抑制效果,其具体实验流程如下,实验结果如图5所示:
1. 按8000个/孔的细胞浓度在96孔细胞培养板内接种DU145细胞,每孔细胞悬液200 μL,设置空白实验组、阴性对照组、实验组,每组设置3~4个复孔,将种盘后的细胞转移到37℃、5% CO2培养箱中培养24 h;
2. 24h后取出细胞,用真空泵沿孔壁轻柔吸弃原有旧培养基,禁止碰触孔底,以防吸弃孔底细胞,并加入含有相应浓度的棘壳孢素A(7 μM、14 μM、28 μM、36 μM、48 μM、60 μM),并继续培养48h;
3. 培养后进行CCK8孵育;将CCK8母液充分混匀,按1:9比例用完全培养基稀释,混匀备用;
4. 将待处理的细胞从CO2培养箱中取出,用真空泵沿孔壁轻柔吸弃原有旧培养基(禁止碰触孔底,以防吸弃孔底细胞);
5. 将上述混匀的含有CCK8的完全培养基按每孔100 μL沿壁轻轻加入孔中;
6. 将细胞培养皿转移到37 ℃、5% CO2培养箱中培养2h;
7. 孵育完全后将96孔细胞培养板放进酶标仪中测OD450吸光值;
8. 将各数据放入GraphPad Prism 7.0软件,运用软件自带的IC50拟合功能计算IC50值,并绘制抑制曲线。
由图5可见,棘壳孢素A对DU145细胞表现出浓度依赖型抑制效果,其半数抑制浓度(IC50)为19.72 μM。高浓度的棘壳孢素A(50 μM)则几乎完全杀死所有DU145细胞而非单纯抑制其增殖,说明棘壳孢素A可能同时通过增殖抑制与诱导细胞死亡的多重途径杀伤DU145细胞。
实施例6
棘壳孢素A可能降低了DU145细胞的DNA合成能力,从而影响其细胞增殖活性。而细胞增殖标记物BrdU是胸腺嘧啶的衍生物,常用于标记新合成的DNA(细胞周期S期),这种标记可以稳定存在,并随着DNA复制进入子代细胞中,因此可以用于检测判断细胞的DNA合成能力。为了确定棘壳孢素A是否会影响DU145细胞DNA的合成能力,设计BrdU标记实验来验证棘壳孢素A对DU145细胞DNA合成能力的影响,其具体实验流程如下,实验结果如图6所示:
1. 在12孔细胞培养板中放入处理干净的细胞爬片与每孔5×105个DU145细胞共培养12 h;
2. 12 h后将原有完全培养基吸弃,用1 mL 1×PBS洗2遍,吸弃;
3. 每孔加入1 mL基础培养基进行同步化处理36 h;
4. 36 h后用1×PBS洗2遍,然后换成完全培养基,并分别加入10、20 μM的棘壳孢素A,培养12 h;
5. 培养12 h后,进行BrdU标记(终浓度50 μM),放入37 ℃、5% CO2培养箱孵育2h;
6. 将-20℃冰冻甲醇用于固定细胞,沿孔壁缓慢加入1 mL,使液体完全覆盖细胞,固定15 min;
7. 然后吸去固定液后,开盖,生物安全柜自然风干;
8. 固定好的细胞爬片置于12孔板中,用自封袋封好,4 ℃保存备用(可保存1周,用于后续BrdU染色);
9. 取出对应的细胞爬片,一分为二,一份置于12孔板4℃保存备用,另一份放入湿盒中,做好标记,每片爬片加入适量体积的2 M HCl,恒温培养箱37℃孵育65 min;
10. 加入1×PBS洗3次,吸弃PBS(操作时注意区分正反面);
11. 滴加适量的PBS-BSA(含5%BSA)室温条件下封闭1 h,然后吸净封闭液;
12. 用PBST 1:100稀释抗BrdU抗体,加入适量稀释后的抗体,室温孵育1 h,避光;
13. 加入1×PBS洗3次,当洗第3次时配制二抗稀释液,用PBST 1:300稀释;
14. 加入二抗,室温孵育45 min,避光;
15. 加入1×PBS洗1次,吸干,晾干,取浓度为1 mg/mL的DAPI母液,用PBS 1:1000稀释,室温孵育5 min左右,避光;
16. PBS再洗1次,轻轻用吸水纸吸净PBS,晾干,滴加一滴50%甘油封片(封片时注意不要产生气泡,正面朝下);
17. 镜检并拍照。
由图6可见,棘壳孢素A作用后DU145细胞的BrdU阳性细胞率与对照组相比显著降低,且随着棘壳孢素A浓度的增加,BrdU阳性细胞率从对照组的58.1%分别降低至24.8%和11.6%,表明棘壳孢素A可以有效降低DU145细胞的DNA合成能力并具有浓度依赖性。
综上所述,棘壳孢素A能够通过促进Skp2蛋白上泛素蛋白的多聚泛素化积累,加速Skp2蛋白的泛素化蛋白酶体途径的降解,进而增加Skp2蛋白下游靶蛋白p27蛋白的积累,从而改变DU145细胞中相关蛋白的含量,进而诱导细胞周期阻滞与抑制细胞增殖。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (4)
1.小分子化合物棘壳孢素A在作为Skp2抑制剂中的应用,其特征在于:棘壳孢素A能够通过促进Skp2蛋白的多聚泛素化积累,加速Skp2蛋白通过泛素化蛋白酶体途径降解。
2.小分子化合物棘壳孢素A在作为前列腺癌细胞增殖活性抑制剂中的应用,其特征在于:棘壳孢素A能够通过促进Skp2蛋白降解,进而调节其下游通路,抑制前列腺癌细胞的DNA复制和合成,从而有效抑制前列腺癌细胞的过度增殖。
3.如权利要求2所述的应用,其特征在于:所述前列腺癌细胞为DU145细胞。
4. 如权利要求3所述的应用,其特征在于:棘壳孢素A的有效抑制浓度为10 μM-400 μM。
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