药学学报  2017, Vol. 52 Issue (9): 1366-1371   PDF    
新型肿瘤治疗靶点Cdc20的研究进展
乐祥阳, 蒯梦妮, 李乾斌, 陈卓     
中南大学湘雅药学院, 湖南 长沙 410013
摘要: 细胞分裂后期促进复合物(APC)具有调节细胞周期进展的作用,经细胞分裂周期蛋白20(Cdc20)或Cdc20同系物1(Cdh1)活化后形成两种不同的E3泛素连接酶复合物APCCdc20或APCCdh1。Cdc20为促癌因子,Cdh1为抑癌因子,在肿瘤的发生及发展中发挥不同的作用。越来越多的研究表明,Cdc20对肿瘤的发生起促进作用,很多肿瘤中存在Cdc20的高表达。目前Cdc20抑制剂除了apcin外,大部分为非特异性抑制剂,不仅阻断Cdc20与APC的结合,也阻断Cdh1与APC的结合,选择性差。本文就Cdc20在肿瘤发生及发展过程中的作用及其抑制剂进行综述。
关键词: 肿瘤     细胞分裂周期蛋白20     E3泛素连接酶     抑制剂    
Progress in research on the novel cancer therapeutic target Cdc20
LE Xiang-yang, KUAI Meng-ni, LI Qian-bin, CHEN Zhuo     
XiangYa School of Pharmaceutical Sciences, Central South University, Changsha 410013, China
Abstract: The anaphase promoting complex (APC) regulates cell cycle progression by forming two functionally distinct E3 ubiquitin ligase complexes, APCCdc20 activated by cell division cycle protein 20 (Cdc20) and APCCdh1 activated by Cdc20 homologue 1 (Cdh1), respectively. Cdc20 and Cdh1 have different functions in the occurrence and development of the tumor. Cdc20 is a cancer promoter while Cdh1 suppresses tumorigenesis. Emerging evidence has begun to reveal that Cdc20 has positive functions in tumorigenesis, the overexpression of Cdc20 has been observed in many cancers. Currently, Cdc20 inhibitors, mostly non-specific inhibitors except apcin, not only block the combination between Cdc20 and APC, also block the combination between Cdh1 and APC, which leads to a poor selectivity. In this paper, the Cdc20 role in the development and process of cancers and its inhibitors are reviewed.
Key words: cancer     cell division cycle protein 20     E3 ubiquitin ligase     inhibitor    

泛素化在细胞生长、繁殖、凋亡, DNA的损伤、迁移、入侵中发挥至关重要的作用。泛素化是由泛素蛋白酶系统(UPS)介导的一种翻译后修饰, 它能够调节蛋白降解过程[1]。多亚基E3泛素连接酶参与细胞分裂过程, 其中, 细胞分裂后期促进复合物(anaphase promoting complex, APC)是最大的E3泛素连接酶, 由至少14个亚基和1个共活化因子组成, 经细胞分裂周期蛋白20 (Cdc20) 或Cdc20同系物1 (Cdh1) 活化后形成两种不同的E3泛素连接酶复合物APCCdc20或APCCdh1, APCCdc20在有丝分裂中期至后期的过渡期破坏关键细胞周期调控子来调节细胞周期进程(图 1)[2], 而APCCdh1在M期和G1期发挥重要作用[3]

Figure 1 A schematic illustration of cell division cycle protein 20 (Cdc20) participates in the cell division process

Cdc20通常被认为是致癌因子, 促进肿瘤的发生及发展, 抑制Cdc20的活性可调节细胞分裂周期、加速细胞凋亡。同时, 在多种肿瘤细胞甚至肿瘤干细胞中都存在Cdc20的高表达, 为Cdc20作为一个新型抗肿瘤药物研发靶点提供了依据。因此, 本文总结了Cdc20与肿瘤的关系及现有的Cdc20抑制剂。

1 Cdc20与肿瘤 1.1 Cdc20的生物学功能

Cdc20的发现早在40年前, 最初有研究者发现, Cdc20突变可引起有丝分裂异常停止, 使分裂后期染色体分离异常[4]。随后, 有研究表明, Cdc20能调节蛋白−蛋白相互作用[5]。目前, 研究认为Cdc20主要通过活化APC形成一种E3泛素连接酶复合物APCCdc20, 参与其下游底物的降解过程, 调节细胞有丝分裂周期进程、促进细胞凋亡等(图 2)。

Figure 2 A schematic illustration of the pharmacological functions of Cdc20
1.1.1 调节细胞周期

APC经Cdc20活化后形成APCCdc20复合物, 破坏其下游细胞周期调控子securin、cyclin B的泛素化[6], 在有丝分裂中期至后期的过渡期发挥至关重要的作用(图 1)。随着对Cdc20的研究不断深入, 越来越多的APCCdc20底物被发现(表 1)[7-17], 有些底物直接或间接参与细胞分裂过程, Cdc20能调节这些底物的泛素化或降解过程, 从而调节细胞周期进程。因此, 发现更多的APCCdc20底物有助于了解APCCdc20在调节细胞周期进展的分子机制。

Table 1 The functions of the identified substrates for APCCdc20
1.1.2 促进细胞凋亡

除了调节细胞周期进程, Cdc20也被发现参与细胞凋亡过程。细胞凋亡过程与抗凋亡因子和促凋亡因子关系密切, Cdc20通过靶向作用于Mcl-1和Bim调节细胞凋亡。抗有丝分裂药物可以活化纺锤体组装检查点从而抑制APCCdc20的活性[18], 如taxol和nocodazole通过Mad2依赖的途径抑制APCCdc20的活性, 长期使用可促进癌细胞凋亡, 这一策略已经应用于肿瘤治疗方面[19]。也有报道表明, Cdc20通过抑制细胞凋亡引起癌细胞对化学治疗和放射治疗的抗性[20]。随着对Cdc20的不断研究, 其在调节细胞凋亡中的作用将会越来越明确。

1.1.3 Cdc20与肿瘤干细胞

肿瘤干细胞具有高度增殖和分化能力, 具有发展成肿瘤的特性。在越来越多的肿瘤组织中发现了肿瘤干细胞的存在, 有研究认为, 现有的抗肿瘤药物只针对普通肿瘤细胞, 不能杀死肿瘤干细胞, 导致肿瘤化疗耐药和肿瘤治疗失败[21]。因此, 肿瘤干细胞靶向药物研究成为热点。有研究者发现, Cdc20活化APC形成APCCdc20复合物后, 调节其下游多能性相关转录因子Sox2的活性, 促进胶质瘤干细胞的侵袭及更新[17]

1.2 Cdc20在肿瘤中表达情况

Cdc20促进肿瘤的发生及发展, 在很多肿瘤中都发现了Cdc20高表达的现象(表 2), 且Cdc20的表达程度与肿瘤的病理程度相关, Cdc20表达越高, 肿瘤患者的病理分级越高, 预后越差, 5年总体生存率越低。

Table 2 Cdc20 expressions in normal tissues/ tumor tissues from multiple organs. a: The expression levels are shown as number of high expression cases/total number of cases/percentage. ADC: Adenocarcinoma; HCC: Hepatocellular carcinoma; IDC: Invasive duct carcinoma; ILC: Invasive lobular carcinoma; MCADC: Mucinous cystadenocarcinoma; SCADC: Serous cystadenocarcinoma; SCC: Squamous cell carcinoma; TCC: Transitional cell carcinoma

降低Cdc20水平是一条潜在的肿瘤治疗策略。例如, 在胰腺癌患者中, 降低Cdc20的表达水平能有效改善射线对胰腺癌细胞的效果[22, 23]; 在非小细胞肺癌中, 随着Cdc20表达水平增高, 癌细胞胸膜浸润越明显[24], 而下调Cdc20表达水平后, 肺癌细胞生长减缓, 肺癌细胞集落的形成速率减慢[25]。此外, 在乳腺癌[26]、大肠癌[27]、肝癌[28]、前列腺癌[29]、胶质瘤[30]、胃癌[31]、膀胱癌[32]和宫颈癌[33]等肿瘤中也存在Cdc20的高表达, 且其表达程度与肿瘤患者的病理程度、预后关系密切, Cdc20表达水平越高, 风险越大。总而言之, Cdc20的表达水平是一个有效的肿瘤患者预后指标, 并有望成为肿瘤治疗药物研发的新型靶点。

2 Cdc20抑制剂

Cdc20促进肿瘤的发生及发展, 在很多肿瘤中都存在Cdc20的高表达, 因此, 使用小分子抑制剂降低Cdc20水平是治疗肿瘤的一条策略。尽管有些蛋白酶抑制剂可以限制APC的活性, 但往往是以一种非特异性的方式, 作用于多种蛋白, 破坏下游许多抑癌因子, 引起发热、贫血、腹泻和恶心等不良反应, 而靶向作用于Cdc20的特异性抑制剂可以避免直接抑制APC的活性, 从而减轻毒副作用[34]。遗憾的是, 目前特异性Cdc20抑制剂研究非常少。以下将总结一些特异性和非特异性Cdc20抑制剂(表 3)[35-39], 为特异性Cdc20抑制剂的研发提供思路。

Table 3 The structures and functions of Cdc20 inhibitors
2.1 特异性Cdc20抑制剂

目前, 特异性Cdc20抑制剂的研究尚处于起步阶段。有研究组在蟾蜍卵提取物中构建APCCdc20−底物模型来筛选Cdc20抑制剂:依照文献[40]方法制备处于分裂间期的蟾蜍卵提取物(不加入钙离子载体A23187活化), 保存于−80 ℃条件下, 加入难降解性cyclin蛋白(MBP-△90) 诱导处于分裂间期的蟾蜍卵提取物进入有丝分裂分裂期, MBP-△90由麦芽糖结合蛋白MBP与缺失N端90位氨基酸的cyclin蛋白融合而成。为消除分裂间期残留蛋白的影响, 加入Cdh1蛋白和细胞周期依赖性蛋白激酶抑制剂roscovitine促进其降解, roscovitine能减缓Cdk1对Cdh1蛋白去磷酸化的抑制作用。用待筛选化合物处理蟾蜍卵提取物后加入同位素标记的cyclin蛋白N端碎片(cyc B1-NT), 根据cyc B1-NT的剩余量可以计算所筛选化合物的活性[35]

对所筛选化合物构效关系研究(图 3)发现, 用吡啶环替换apcin中嘧啶环时活性降低(apcin-P); 而用吗啉环替换(apcin-M)活性消失; 但用氨基替换apcin中的硝基咪唑基团时, 活性影响很小(apcin-A)[35]。在实验过程中, 用apcin处理的蟾蜍卵提取物实验组中Cdc20的含量减少, 而加入体外制备的Cdc20后, cyclin蛋白水解受到的抑制作用解除, 这说明apcin通过靶向作用于Cdc20, 从而抑制cyclin蛋白水解。随后, Cdc20的晶体结构也被发现(图 3), apcin与Cdc20的对接结果显示, apcin嘧啶环中的氮原子、链上的2个氮氢与Cdc20中的177位天冬氨酸有3个氢键结合作用, apcin中的三氯甲基埋入由200位缬氨酸、209位酪氨酸和467位亮氨酸组成的疏水口袋中, 而apcin中的硝基咪唑基团则暴露在溶剂中。这些研究都表明, apcin是通过靶向作用于Cdc20, 阻断Cdc20与APC之间的连接, 从而调节其下游底物的活性, 进而发挥其生物功能。

Figure 3 The structures of apcin derivatives (A) and crystal structure of the apcin Cdc20 complex (B)

目前, 本课题组也在进行Cdc20特异性抑制剂的研究。对Cdc20晶体结构分析后, 以apcin为先导化合物, 保留已有的氢键作用, 对apcin中的嘧啶环、三氯甲基和甲硝唑基团进行改造, 并使用计算机辅助药物设计进行验证, 以期发现活性更好的Cdc20特异性抑制剂。

2.2 非特异性Cdc20抑制剂 2.2.1 TAME和pro-TAME

研究组[36]发现另外一个小分子化合物TAME也能抑制蟾蜍卵提取物中cyclin蛋白水解, 然而, TAME是通过与APC结合, 阻断Cdc20或Cdh1活化APC, 从而抑制APC的活性。尽管TAME可以阻断Cdc20活化APC, 对APCCdc20的底物进行调节, 但它同时也可以阻断Cdh1活化APC, 而Cdh1是一个抑癌因子, APC经Cdh1活化后形成APCCdh1复合物, 抑制肿瘤的形成及发展(图 4)。由于TAME的渗透性不佳, Zeng等[36]发现pro-TAME能水解生成TAME, pro-TAME也能阻断Cdc20或Cdh1与APC的结合, 抑制APC的活性, 而且pro-TAME能诱导细胞凋亡, 与其他抑制剂联合使用时能大大提高细胞死亡率。

Figure 4 The difference between specific Cdc20 inhibitors and non-specific Cdc20 inhibitors
2.2.2 NAHA、CFM-4和BCHHD

在乳腺癌小鼠模型中, NAHA能减小肿瘤的体积, 减轻肿瘤的重量, 降低Cdc20水平, 减缓细胞生长速率, 抑制癌细胞集落的形成。有研究者报道, NAHA能通过抑制尿激酶型纤溶酶原激活物减缓癌细胞的入侵和迁移[37]。这些都表明, NAHA以一种不明机制的方式抑制乳腺癌细胞中Cdc20的表达。CARP-1能把Cdc20或Cdh1与APC2结合起来, 调节细胞的生长和凋亡, 而化合物CFM-4能阻断这种结合, 延长细胞周期并诱导凋亡[38]。Nasr等[39]发现在产生耐药性的胰腺癌细胞中, 化合物BCHHD 7c能抑制Cdc20的表达。

2.2.3 天然产物成分

金雀异黄素是一种异黄酮类植物雌激素, 对恶性肿瘤具有多效作用, 它能抑制酪氨酸激酶活性, 作用于多种信号通路抑制细胞生长、迁移和入侵, 诱导凋亡。在胶质瘤、横纹肌肉瘤、肝癌和乳腺癌等肿瘤细胞中, 金雀异黄素能下调包括Cdc20在内的很多关键调控因子的表达, 表现出抗肿瘤性[41]。从药用真菌中提取的一种灵芝醇ganodermanontriol (GDNT), 能下调Cdc20水平, 抑制乳腺癌细胞的生长及癌细胞集落形成[42]。从茄科植物中提取出的withaferin A对胰腺癌、乳腺癌和大肠癌等多种肿瘤细胞都有抗肿瘤活性, 而近期有研究者发现withaferin A是通过促进Cdc20和Mad2的降解和抑制纺锤体组装检查点功能来发挥抗肿瘤活性[43]

3 结语

越来越多的研究表明, Cdc20作为一个促癌因子, 参与很多肿瘤的发生和发展过程, Cdc20抑制剂是肿瘤治疗的一种新策略。Apcin是一种特异性Cdc20抑制剂, 与Cdc20靶向结合, 抑制APC的活性, 而其他抑制剂为非特异性抑制剂, 选择性较低。金雀异黄素、GDNT和withaferin A等植物成分具有无毒的特点, 是一种安全的肿瘤治疗途径。对Cdc20在肿瘤中作用的不断研究, 将为特异性Cdc20抑制剂的研发提供新的思路, 一方面, 研究者要不断发现APCCdc20的下游蛋白, 更深入、全面地了解Cdc20在肿瘤中的作用; 另一方面, 也要探索Cdc20在肿瘤的发生及发展中除了调节细胞周期和细胞凋亡以外的药理作用。为满足进一步的研发及临床需要, 应致力于特异性更高、活性更好的Cdc20抑制剂研究。

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