新型微管蛋白抑制剂的研发和抗肿瘤机制研究
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摘要
微管是构成细胞骨架的主要成分,存在于所有的真核细胞中,与其他蛋白共同组装成纺锤体、中心粒、鞭毛、神经管等多种结构。正常条件下,微管的聚合和解聚保持着动态平衡,因此细胞分裂高度可控,进展有序。这种不稳定的动力学特性使得微管在维持细胞形态、细胞迁移、细胞有丝分裂、胞内物质的输送及信号传导等细胞关键生物学过程中具有重要调节功能。微管在细胞分裂前期聚合成为纺锤体,而纺锤体在细胞有丝分裂过程中牵引染色体向两极移动进入至两个子细胞中,从而完成细胞增殖。由于微管在细胞有丝分裂过程中承担的重要作用,微管蛋白逐渐成为医药工作者研究与开发抗癌药物的重要靶点之一,而以微管蛋白为靶点的微管蛋白抑制剂也已成为临床证实有效的抗肿瘤药物。该类抑制剂的作用机制是在快速分裂的肿瘤细胞中,通过抑制微管蛋白的聚合或者促进微管蛋白的聚合而干扰细胞的有丝分裂过程,使细胞有丝分裂中断,停滞于M期,从而导致肿瘤细胞发生凋亡,发挥抗肿瘤作用。
根据作用机制的不同,微管蛋白抑制剂可分为两大类：①抑制微管蛋白聚合的微管蛋白解聚剂,如秋水仙碱类和长春碱类化合物；②促进微管蛋白聚合的微管蛋白聚合剂,如紫杉醇及其类似物,埃博霉素及其类似物。根据微管蛋白抑制剂在微管蛋白上的作用位点的不同,微管蛋白抑制剂又可分为3种类型：①作用于秋水仙碱位点的微管蛋白抑制剂；②作用于长春碱位点的微管蛋白抑制剂；③作用于紫杉醇位点的微管蛋白抑制剂。目前,长春碱和紫杉醇位点微管蛋白抑制剂类药物已在肿瘤临床治疗方面占据了重要地位,紫杉醇更是已成为肺癌、乳腺癌和卵巢癌的重要一线治疗药物。然而,和其他抗肿瘤药物一样,难以耐受的副作用及用药后耐药性的出现限制了微管蛋白抑制剂的临床使用。因此,开发新型的具有更强活性、更低毒性、并且对多药耐药肿瘤细胞更有效的新型微管蛋白抑制剂具有很大的应用前景。
HA14-1是本博士论文作者的导师之一Ziwei Huang教授等研发的小分子化合物,是世界上第一例报道的Bcl-2抑制剂,能够强有力地诱导乳腺、结直肠癌、肾癌、宫颈癌、脑胶质瘤、白血病等多种肿瘤细胞系发生凋亡。在后续的研究中,我们发现HA14-1不仅能够通过诱导活性氧(ROS)产生、细胞色素C释放、半胱天冬酶Caspase-9/-3活化这一信号途径而诱导凋亡,在其药物浓度大于10μM时,还可以作用于微管蛋白秋水仙碱位点抑制微管蛋白聚合。我们将HA14-1作为母体药物对其进行结构改构,设计并合成了一系列新型HA14-1类似物(2-amino-4-phenyl-4H-chromene-3-carboxylate analogs),命名为mHA1-19,并对其中抗肿瘤活性较强的mHA1,6,11进行了后续的生物学评价和抗肿瘤机制研究。这些新型化合物表现出更强的抗肿瘤活性和更好的稳定性。肿瘤细胞在接受药物处理后逐渐出现包括细胞变长、不对称及出现长伪足等细胞形态学改变,与HA14-1处理细胞后迅速表现出细胞缩小、核固缩、凋亡小体出现等细胞凋亡的典型改变截然不同。这些现象均提示这一系列HA14-1类似物有着不同于母体药物HA14-1的肿瘤细胞杀伤机制,维持细胞正常形态的微管蛋白极有可能是其主要作用靶点。虽然秋水仙碱类药物因为毒性较大目前还暂未用于肿瘤治疗,但秋水仙碱位点作为一个很有前景的肿瘤药物靶标一直备受关注,目前已有多个化合物进入了临床研究。研究及阐明这一系列mHA类似物的作用靶点及其抗肿瘤机制,将推动新型、高效的秋水仙碱位点抑制剂的设计和开发,具有重要意义。
方法：
1.化合物合成：
该系列化合物总的合成路径为采用苯甲醛、酚类似物和氰乙酸乙酯这三种组分和哌啶进行一步化反应合成。化合物合成后均经过核磁共振氢谱和质谱验证。
(1)mHAl的制备
3-溴-4,5-二甲氧基苯甲醛(0.49g,0.002mol)、3-二甲氨基苯酚(0.27g,0.002mol)、氰乙酸乙酯(0.21mL,0.002mol)和哌啶(0.4mL,0.004mol)溶于15ml无水乙醇,室温下搅拌4小时。加入80ml二氯甲烷稀释后用水清洗,硫酸钠干燥化合物,过滤除去硫酸钠,蒸发干燥溶剂。使用色谱分析法(己烷/二氯甲烷)提纯粗制品,得到0.6g mHAl,收益率63%。
(2)mHA6的制备
采用3-溴-4,5-二甲氧基苯甲醛、1-萘酚和氰乙酸乙酯作为原料,后续步骤同上,得到0.45g mHA6,收益率46%。
(3)mHA11的制备
采用3-氯苯甲醛、3-二甲氨基苯酚和氰乙酸乙酯作为原料,后续步骤同上,得到0.32g mHA11,收益率43%。
2.细胞培养
人白血病细胞HL-60/Bcl-2(pZip-Bcl-2质粒稳定转染)惠赠于Dr. Kapil N. Bhalla(迈阿密大学医学院,佛罗里达州,美国)。细胞培养条件为RPMI1640培养基,10%胎牛血清,2mM谷氨酰胺,100U/ml青霉素,100μg/ml链霉素,5%二氧化碳,37℃。
3.小鼠骨髓细胞收集
小鼠骨髓细胞样本来采样于年龄为3月的C57BL/6雌性小鼠(Charles River Labs)。CO2吸入法处死小鼠,分离取出小鼠的胫骨和股骨,注射器吸取RPMI1640培养基反复冲洗胫骨和股骨,将骨腔内骨髓细胞冲洗出来,采用淋巴细胞分离法获得小鼠骨髓细胞,接种于96孔细胞培养板中,每孔1×105细胞。
4.人正常骨髓细胞样本收集
在通过伦理委员会审查及获得患者知情同意后,取得3例人正常骨髓细胞样本。样本均来自弥漫大B细胞淋巴瘤患者,已通过病理证实为正常骨髓。按骨髓穿刺术操作规范进行操作取得人体骨髓样本,将肝素化的骨髓细胞用RPMI-1640培养基稀释后,采用淋巴细胞分离法获得人体骨髓细胞,接种于96孔细胞培养板中,每孔1X105细胞。
5. CellTiter-Blue法测定细胞活力
HL-60/Bcl-2细胞或小鼠骨髓细胞接种于96孔板,分别接受不同浓度的mHAs药物处理,温箱孵育72小时后,采用CellTiter-Blue细胞活力检测试剂盒按照使用说明测定细胞活力。
6. Cell Count Kit-8(CCK-8)法测定细胞活力
人骨髓细胞接种于96孔板,24小时后分别接受不同浓度的mHAs药物处理,温箱孵育72小时后,采用CCK-8细胞活力检测试剂盒测定细胞活力。
7.细胞集落形成实验
细胞接受不同浓度的mHAs药物及DMSO对照处理24小时后,收集细胞,新鲜培养基稀释后与甲基纤维素半固体培养基混合均匀,接入培养皿使每个培养皿内细胞数目为200,体积为2ml。37℃,5%二氧化碳及饱和湿度的培养箱内孵育10-14天后倒置显微镜下计数细胞克隆数并计算细胞克隆形成率。
8.细胞形态学检测
1μM mHAs处理细胞24小时,倒置显微镜下观察细胞形态改变并拍照。
9.计算机分子模型
采用微管和DAMA-秋水仙碱复合物中的微管晶体结构(PDB:1SA0)作为模板,采用Autodock4程序来预测微管与配体mHAl,6,11的结合模型。
10.秋水仙碱-微管蛋白竞争结合试验
1μM放射性标记的[3H]秋水仙碱,1%DMSO和不同浓度的待测药物均溶于50μl G-PEM缓冲液中,与1μM微管蛋白共同孵育1小时,经DEAE-纤维素柱层析,采用液体闪烁法(Perkin-Elmer)测定滤液中放射性强度。使用GraphPad Prism对数据进行非线性回归分析。
11.微管蛋白聚合实验
按照试剂盒生产公司提供的说明书进行,将微管蛋白溶于G-PEM缓冲液中,得到浓度为3mg/ml的微管蛋白溶液,加入预先加入50μl待测药物溶液的96孔板内,50μl/孔,使用Synergy2酶标仪测定吸光度(360nm/420nm),每分钟一次,共测定1小时。
12.免疫荧光染色
人肺癌细胞CRL5908经1μMmHAl,6,11处理24h后,4%多聚甲醛4℃固定30分钟,0.1%Triton X-100透膜处理15min,2%BSA封闭处理30min,1:2000抗-α-微管蛋白单克隆抗体和7：1000罗丹明-鬼笔环肽标记的抗肌动蛋白抗体避光处理细胞1h,1：200抗鼠IgG二抗避光处理30min,1μg/ml DAPI处理1min。免疫荧光显微镜下观察和拍照。
13.细胞周期测定
1X106HL-60/Bcl-2细胞分别接受1,3,5μM mHAs药物处理24h。收集细胞,PBS清洗2次,重悬于100μlPBS和1ml75%预冷乙醇内,-20℃过夜保存。测定当天离心细胞,移去上清,加入500μl PI染色液(含80μg/mL碘化丙啶,100μg/mL核糖核酸酶A,1%曲拉通),避光孵育至少半小时后流式细胞仪检测,FlowJo7.5软件分析。
14.DNA碎片分析方法测定细胞凋亡
1X106HL-60/Bcl-2细胞分别接受不同药物处理：DMSO对照、阳性对照0.1μM和1μM秋水仙碱,1μM和5μM mHAl,6,11,温箱孵育24h,按照试剂盒生产公司提供的说明书进行提取每个处理组DNA, DNA经2%琼脂糖凝胶电泳,EB显色照相。
结果：
1.mHA系列化合物细胞毒性测定：测定mHAl-19对人白血病细胞HL-60/Bcl-2的细胞毒性并计算其IC50,结果显示该系列化合物中mHA1,6,11具有较强的抗肿瘤活性,选择这3种化合物进行后续的生物学评价和抗肿瘤机制研究。
2.mHA系列化合物构效分析：对该系列化合物进行构效分析,发现C6位点上连接二甲氨基与苯基对化合物活性的影响类似但优于羟基,羟基优于氨基；mHA7,15,17活性均很差,提示C7位点添加任何基团均会降低化合物的活性。
3. mHAl,6,11抗肿瘤活性明显强于其母体药物HA14-1：分别用不同浓度的HA14-1和mHA1,6,11处理人白血病HL-60/Bcl-2细胞,24h或72h后测定细胞活力,结果显示mHAl,6,11的ICso均小于1μM,而HA14-1的ICso为9.394±0.18μM, mHAl,6,11抗肿瘤活性明显强于其母体药物HA14-1。
4. mHAl,6,11对人白血病细胞具有较强细胞毒性,但对正常小鼠骨髓细胞毒性较低：分别对人白血病HL-60/Bcl-2细胞及正常小鼠骨髓细胞进行相同浓度的mHAl,6,11药物处理,72h后分别测定细胞活力。结果显示1μMmHAl,6,11可以杀死约一半的肿瘤细胞,而1μM药物处理对正常小鼠骨髓细胞几乎无影响。
5. mHAl,6,11对人正常骨髓细胞几乎无毒性：采集人正常骨髓细胞进行不同浓度的mHAl,6,11药物处理,72h后分别测定细胞活力。结果显示3μM mHAs可杀死几乎全部的恶性HL-60/Bcl-2细胞,而同样处理对人正常骨髓细胞几乎毫无影响。
6.阳性对照药物秋水仙碱对正常小鼠骨髓细胞毒性较大：采用秋水仙碱位点代表药物秋水仙碱作为阳性对照,对人白血病HL-60/Bcl-2细胞及正常小鼠骨髓细胞进行相同浓度药物处理,72h后分别测定细胞活力,结果显示秋水仙碱对肿瘤细胞及正常骨髓细胞均有较强的细胞毒性,尽管其IC50值在人白血病细胞中仅为33.5±3.5nM,但药物浓度在25nM时即可杀伤约30%的正常骨髓细胞。
7.mHA1,6,11可诱导肿瘤细胞丧失克隆形成能力：人白血病HL-60/Bcl-2细胞在接受药物处理后培养10～14d后计数克隆形成数,计算所得IC50值低于细胞毒性试验,提示药物处理不仅能在72h内迅速杀死肿瘤细胞,并可引起部分细胞丧失增殖能力及克隆形成能力。
8.mHA1,6,11引起肿瘤细胞产生特殊形态改变：mHA1,6,11可引起肿瘤细胞产生特殊形态改变。人白血病HL-60/Bcl-2细胞由悬浮细胞典型的球体变为不规则形、细胞变长及出现长伪足；人肺癌CRL5908细胞则由贴壁细胞典型的梭状变为多边形或类圆形。
9.计算机模拟对接研究显示mHA1,6,11可结合于微管蛋白上的秋水仙碱位点：计算机模拟对接研究采用DAMA-秋水仙碱晶体结构作为模板,模拟计算结果显示mHA1,6,11与秋水仙碱均可结合于微管蛋白上的相同位点-秋水仙碱位点,mHA1,6和秋水仙碱结合模式相似而mHA11结合模式略有不同。
10.秋水仙碱-微管蛋白竞争结合试验证实了mHA1,6,11可与秋水仙碱竞争结合位点：秋水仙碱-微管蛋白竞争结合试验显示mHA1,6,11可与放射性标记的秋水仙碱竞争结合位点,抑制其与微管蛋白结合,其作用呈剂量依赖性方式。
11.微管蛋白聚合实验显示mHA1,6,11和秋水仙碱均可抑制微管蛋白聚合,紫杉醇可促进微管蛋白聚合：微管蛋白聚合实验显示紫杉醇可促进微管蛋白聚合,mHA1,6,11和秋水仙碱均可抑制微管蛋白聚合,证实mHA1,6,11具有强效的抗微管聚合作用。
12.免疫荧光染色实验证实mHA1,6,11可降低细胞内微管含量并破坏其网状分布：人肺癌CRL5908细胞接受1μMmHA1,6,11处理24h后行免疫荧光检测,显示胞浆内沿细胞长轴密集分布的微管变为弥散分布,且荧光强度明显降低,提示细胞内微管含量减少。
13.mHAl,6,11可诱导肿瘤细胞发生G2/M细胞周期阻滞：人白血病HL-60/Bcl-2细胞在接受1μM mHA1,6,11药物处理24h后经流式细胞仪检测证实细胞发生特异性G2/M细胞周期阻滞。
14.DNA碎片分析证实mHA1,6,11可诱导肿瘤细胞发生凋亡：mHA1,6,11及秋水仙碱处理人白血病HL-60/Bcl-2细胞24h后行DNA碎片分析,出现明显的凋亡条带,证实秋水仙碱及mHAl,6,11均可诱导人白血病HL-60/Bcl-2细胞发生凋亡。
结论：
1.本研究获得了一系列HA14-1类似物并证实其为具有高效抗肿瘤活性的微管蛋白抑制剂。
2.对该系列化合物的构效分析发现C6位点上连接二甲氨基与苯基类似但均优于羟基,羟基优于氨基；C7位点上不宜连接任何侧链,为日后继续研发新型微管蛋白抑制剂提供了设计思路。
3.本研究结果证明了mHA1,6,11具有较强的肿瘤细胞杀伤作用,其IC50为纳摩尔级别,大大优于其母体药物HA14-1,而且对正常细胞毒性较小,具有进一步研发成药的可能性。
4.计算机模拟对接研究及秋水仙碱-微管蛋白竞争结合实验证实mHA1,6,11均结合于微管蛋白上的秋水仙碱位点并可与秋水仙碱竞争结合位点,为作用于秋水仙碱位点的新型微管蛋白抑制剂。
5.微管蛋白聚合实验进一步证实mHAl,6,11与紫杉醇作用方式相反,与秋水仙碱作用方式一致,为促微管蛋白解聚剂。
6.免疫荧光染色实验显示mHAl,6,11可降低细胞内微管数量及破坏胞浆内正常微管网状结构。
7.流式细胞仪细胞周期检测及DNA碎片分析实验证实mHAl,6,11通过阻滞肿瘤细胞于G2/M期而诱导细胞发生凋亡。
8.本研究结果初步探明了该系列化合物杀伤肿瘤细胞的作用机制：通过作用于微管蛋白,抑制微管蛋白聚合,从而影响纺锤体形成,使肿瘤细胞停滞于G2/M细胞周期,不能完成正常的有丝分裂,从而发生凋亡。
Background and objection:
Microtubules are components of cytoskeleton and are present in virtually all eukaryotic cells. Microtubules form spindle, centriole, flagllum and nerviduct with other proteins in the cells. Under normal condition, there is a balance between microtubule polymerization and depolymerization and the dynamics regulates the mitosis. Microtubules are involved in many cellular processes, including maintenance of cell shape, cell migration, mitosis, intracellular transport and cell signaling. In the eukaryotic cell cycle, microtubules are polymerized and form the mitotic spindle in prophase, which then moves the chromosomes to the opposite sides of the cell, in preparation for cell division into two daughter cells. Because of this important role in cell proliferation, microtubules have been recognized as one of the successful and efficacious drug targets for the development of novel anti-cancer chemotherapeutics. Microtubule-inhibiting agents (MIAs) currently have been used in clinic therapies work through the suppression of the microtubule dynamics by misdirecting the formation of a functional mitotic spindle in fast dividing tumor cells. This arrests the cells in M phase, thereby leading to apoptosis of the tumor cells.
Based on their mechanism of action, Microtubule-inhibiting agents (MIAs) are classified into two broad categories:①microtubule destabilizing agents, including vinca alkaloids and colchicine;②microtubule stabilizing agents, including paclitaxel, epothilone and its analogues. According to their different binding sites on microtubule protein, Microtubule-inhibiting agents (MIAs) are further classified into three groups:colchicine site-binding agents, vinblastine site-binding agents, and paclitaxel site-binding agents. Due to the potent anti-cancer activity, these apoptotic therapeutic agents that target microtubules, including paclitaxel and vinblastine, are among the most commonly prescribed antitumor agents. Paclitaxel is currently recommended as first-line agent in the chemotherapy of lung cancer, breast cancer and ovarian cancer. However, as with other anticancer drugs, intolerable toxicities and the emergence of drug resistance have limited the clinical use of the drugs targeting microtubules. Therefore, a need still exists for discovery and development of novel chemotherapeutic agents that target microtubules, but that show better activicy, lower drug resistance and fewer toxic side effects.
The small compound dubbed HA14-1, identified by one of my supervisor--Dr. Huang ziwei, was the first reported Bcl-2inhibitor which can potently induce apoptosis in a wide variety of human cancers, including breast cancer, colorectal cancer, kidney cancer, cervical cancer, lung cancer, brain glioma and leukemia. In subsequent studies, we found that it not only can induce reactive oxygen species (ROS) generation, cytochrome c release, and Caspase-9/-3activation, but it also can bind microtubules in a manner that is competitive with colchicine when its concentration is greater than10μM. In the present study, we used HA14-1as the initial template compound and developed a new class of novel microtubule-targeting agents (2-amino-4-phenyl-4H-chromene-3-carboxylate analogues) named mHAl-19. mHA1,6and11showed more potent and stable than others, so we choosed them to do subsequent biological assessment. After treatment with these agents, morphological changes including cell elongation, asymmetry, and formation of long pseudopodia were observed which are quite different from the apoptotic morphological changes induced by HA14-1treatment, including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and apoptotic body. This phenomenon indicated that these mHA analogues worked through different pathway from HA14-1to kill cancer cells. Microtubule as the important component of cell shape maintenance was the most probable target. Although the high toxicity of colchicine has prevented its clinical use in cancer therapy, the colchicine-binding site is still a potential drug target that has attracted much attention for development of new agents that bind to this site. Several such compounds have entered clinical trials. Study and find the action mechanism of these mHA analogues is significative to the design and develop of new colchicine site-binding agents.
Methods and materials:
1. Chemical synthesis:
These compounds were synthesized using a one-pot three-component reaction of substituted benzaldehyde, phenol analogs and ethyl cyanoacetate in the presence of piperidine. All of the new compounds described were characterized by1H NMR and mass spectrometry (MS) spectra.
(1) Preparation of mHAl
A mixture of3-Bromo-4,5-dimethoxybenzaldehyde (0.49g,0.002mol),3-(dimethylamino) phenol (0.27g,0.002mol), ethyl cyanoacetate (0.21ml,0.002mol) and piperidine (0.4mL,0.004mol) was suspended in15mL anhydrous ethanol and stirred at room temperature for4h. After diluting with80mLCH2l2and washing with water, the organic layer was dried over Na2SO4. The Na2SO4was then removed by filtration and the solvent was evaporated. The crude product was purified by column chromatography (hexane/CH2Cl2) to give0.6g mHAl at63%yield.
(2) Preparation of mHA6
Starting from3-bromo-4,5-dimethoxybenzaldehyde, naphthalen-1-ol, and ethyl cyanoacetate, we then followed the procedure for the synthesis of mHAl, to give0.45g (46%yield) of mHA6.
(3) Preparation of mHA11
Starting from3-chlorobenzaldehyde,3-(dimethylamino) phenol, and ethyl cyanoacetate, we then followed the procedure for the synthesis of mHAl, to give0.32g (43%yield) of mHA11.
2. Cell culture
The human leukemic HL-60/Bcl-2cell line was obtained from Dr. Kapil N. Bhalla (University of Miami School of Medicine, Miami, FL), which has been stably transfected with pZip-Bcl-2plasmid. Cells were cultured in RPMI1640medium supplemented with10%fetal bovine serum,2mM glutamine,100U/ml penicillin,100μg/mL streptomycin and800μg/mL genticin (G418, Invitrogen, San Diego, CA). Cells were maintained in a humidified5%CO2atmosphere at37℃.
3. Mouse bone marrow cells collection
Mouse bone marrow samples were obtained from3-month-old C57BL/6female mice (Charles River Labs). Mice were sacrificed with CO2the tibias and femurs were dissected. The tibias and femurs were flushed repeatedly with RPMI1640medium through needle. Mouse bone marrow cells were seperated by Ficoll density gradient centrifugation.
4. Isolation and culture of human bone marrow cells
Bone marrow specimens were acquired from Diffuse Large B Cell Lymphoma (DLBCL) patients (n=3) via a protocol approved by our Research Ethics Committee for the use of samples for research, and informed consent was obtained from each patient. All of the patients with normal bone marrow were confirmed by pathology. Heparinized bone marrow samples were diluted with RPMI1640medium and overlaid on5ml separation medium and then centrifuged at3000rpm for30min. The bone marrow cells were washed twice and suspended in RPMI1640medium supplemented with10%fetal bovine serum, added in96-well plate (1×105of cells each well).
5. Measurement of cell viability via CellTiter-Blue assay
HL-60/Bcl-2cells were seeded in96-well plates and treated with various concentrations of these compounds (0.1to3μM). The samples were then incubated at37℃for72h. After incubation, cell viability was measured using a CellTiter-Blue assay kit according to the manufacturer's instructions.
6. Measurement of cell viability via CCK-8assay
The cell viability assays were evaluated by a Cell Counting Kit-8(CCK-8). After overnight culture the bone marrow cells were treated with different concentrations of mHA1, mHA6and mHA11separately, and controls were treated with vehicle (DMSO). After72h treatments, CCK-8solution was added to each well according to the manufacturer's instructions and optical density (OD) was measured at450nm test wavelength using a microplate reader. All experiments were performed in triplicate.
7. Assessment of clonogenicity
After incubation for24h with various drug concentrations or Vehicle (DMSO) control, cells were collected and diluted with fresh medium, then mixed with2%methylcellulose to make the methylcellulose1.3%and fetal bovine serum30%. A1.5ml volume of this mixture, containing200cells, was seeded into culture dish. Dishes were then incubated in incubator for10-14days. Colonies were enumerated with the aid of an inverted microscope.
8. Cell Morphological change
Cells were treated with mHAs at1μM for24h and then examined for morphological changes by inverted fluorescence microscopy and photography.
9. Molecular modeling
The crystal structure of microtubule in a complex with DAMA-colchicine [PDB reference:1SA0(16)] was used to predict the binding models of microtubule bind with designed compounds. The binding modes of designed ligands1,6,11with microtubule were predicted by using the Autodock4program.
10.[3H]Colchicine-tubulin binding assay
One micromolar radiolabeled colchicine [ring C, Methoxy-3H],1%DMSO and various concentrations of test compounds in50μl G-PEM buffer were incubated with1μM tubulin for60min. The binding solutions were filtered through two stacks of DEAE-cellulose filters and washed twice. The radioactivity in the filtrates was determined by liquid scintillation spectrometry. Nonlinear regression was used to analyze the data using GraphPad Prism.
11. Tubulin polymerization assay
Tubulin polymerization assays were conducted using the polymerization assay kit following the manufacturer's instructions. Briefly,50μl of3mg/ml tubulin (>99%pure) proteins in G-PEM buffer was placed in96-well microtiter plates in the presence of test agents. The absorbance at360/420nm was recorded every60s for1h using a Synergy2microplate reader.
12. Immunofluorescence staining
CRL5908cells were treated with1μM mHAl,6, or11for24h. Thereafter, cells were fixed for30min at4℃in4%paraformaldehyde and incubated with0.1%Triton X-100permeabilizing buffer for15minutes. After washing with PBS and blocking with2%BSA in PBS for30minutes, cells were incubated for1h protected from light in1:2000anti-a-tubulin monoclonal antibody and7:1000Rhodamine-Phalloidin-labeled anti-actin antibody in PBS. Cells were then washed with PBS and incubated for30min protected from light with1:200fluorescein isothiocyanate (FITC)-labeled anti-mouse IgG antibody in PBS. Subsequently, all cells were stained with1μg/ml4',6-diamidino-2-phenyl-indole. Samples were examined under a fluorescence microscope and photographed.
13. Cell cycle analysis
HL-60/Bcl-2cells (1×106) were treated with1,3, and5μM of mHA1,6,11, respectively, for24h at37℃. Cells were harvested and washed twice with PBS, then resuspended in100μl of PBS and lml of75%cold ethanol and stored at-20℃overnight. After centrifugation, the supernatant was removed. A500μl PI staining buffer containing80μg/mL of propidium iodide,100μg/mL of RNAse A, and1%Triton was added to the samples. The cells were incubated for at least half an hour (avoid light) and then analyzed by flow cytometry with a FACScalibur system using FlowJo7.5analysis software.
14. DNA fragmentation analysis
HL-60/Bcl-2cells were treated with Vehicle control (DMSO), positive control (colchicine at0.1and1μM), and mHA1,6, or11(at1and5μM), respectively. The plate was incubated for24h, and total DNA was extracted from the cells in each well using an Apoptotic DNA-ladder kit following the manufacture's instructions. The DNA samples were subjected to2%agarose gel electrophoresis and visualized with ethidium bromide staining.
Results:
1. Cytotoxicity of new mHA agents toward leukemic HL-60/Bc;-2cells
Human leukemia HL-60/Bcl-2cells were treated with the series of HA14-1analogs and cell viability assay was performed to get their IC50values. mHAl,6, and11, showed the best anti-tumor activity of the series of compounds and we choose them for further study.
2. Structure-activity analysis
On the basis of the biological result, the structure-activity relationship of these compounds was discussed. We digged up that dimethyl amino in C6is similar with benzene and better than hydroxy(-OH); hydroxy(-OH) is better than amino(-NH2); mHA7,15and17are all very poor which indicated that substitution at C7will decrease its activity.
3. mHAl,6,11were much more potent than their parent HA14-1compound
Human leukemia HL-60/Bcl-2cells were treated with diferent concentrations of HA14-1or mHA1,6,11for24h or72h, respectively. Cell viability assay showed that the IC50values of mHAl,6,11were all less than1μM, while IC50of the parent compound, HA14-1, was9.394±0.18μM. Overall, mHA1,6,11were much more potent than their parent HA14-1compound.
4. mHAl,6,11were very potent to Human leukemia HL-60/Bcl-2cells while they showed low otoxicity against normal mouse bone marrow cells.
Human leukemia HL-60/Bcl-2cells and normal mouse bone marrow cells were treated with diferent concentrations of mHAl,6,11for72h, then cell viability was assessed. The results showed that these compounds had almost no effects on normal mouse bone marrow cells below1μM concentration, while1μM mHAs were able to kill almost half of the malignant HL-60/Bcl-2cells.
5. mHAl,6,11showed almost no cytotoxicity on human bone marrow cells
human bone marrow cells were treated with diferent concentrations of mHAs and DMSO vehicle control for72h, then cell viability was assessed. The results showed that3μM mHAs treatment had almost no effects on normal human bone marrow cells, while3μM mHAs were able to kill almost all of the malignant HL-60/Bcl-2cells.
6. The positive control Colchicine showed high toxicity against normal mouse bone marrow cells
Human leukemia HL-60/Bcl-2cells and normal mouse bone marrow cells were treated with diferent concentrations of colchicine, the colchicine site-binding agent, which was used as the positive control. Cell viability was assessed72h later. The results showed that colchicine was toxic against both malignant and normal cells. Colchicine resulted in the death of30%of normal mouse bone marrow cells at25nM, even though its IC50was only33.5±3.5nM in HL-60/Bcl-2cells.
7. Induction of colonogenic cell death by mHA1,6,11.
After exposure various concentrations of mHA1,6, or11for24hours, HL-60/Bcl-2cells were then incubated for10-14days, then the number of colonies were measured. The IC50values for the colony inhibition assay were lower than the values for the cytotoxicity assay, which indicated that treatment with mHAs disrupted proliferation and colony forming ability of some cancer cells without causing actual cell death in72hours.
8. Specific cell morphological changes in response to mHAl,6,11.
mHA1,6and11treatment resulted in specific cell morphological changes. HL-60/Bcl-2cells are suspension cells and typically have a spherical shape. After treatment with mHAs, specific morphological changes including cell elongation, asymmetry, and formation of long pseudopodia were observed. Human lung cancer CRL5908cells changed from spindle shape to multi-angular or nearly-circular shape after treatment with mHAs.
9. The docking study showed that mHAl,6, and11could bind at the colchicine site on the microtubule protein
The crystal structure of microtubule in a complex with DAMA-colchicine was used to predict the binding models of microtubule bind with designed compounds. The results of our docking study showed that mHA1,6, and11could bind at the same site as colchicine on the microtubule protein. mHA1and mHA6adopt a similar binding mode with colchicine, which is slightly different from that adopted by mHA11.
10.[3H]Colchicine-tubulin competition binding assay confirmed that mHAl,6,11could compete with colchicine binding to tubulin.
[3H]Colchicine-tubulin competition binding assay confirmed that mHAl,6,11could inhibit the combination of [3H]colchicine and tubulin in a dose-dependent manner by competing the colchicine binding site.
11. Tubulin polymerization assay showed that mHAl,6,11and colchicine could inhibit the microtubule polymerization while Taxol had opposite effect.
Tubulin polymerization assay showed that mHAl,6,11and colchicine could inhibit the microtubule polymerization while Taxol had opposite effect, suggesting that each of the mHAs possessed strong antitubulin polymerization activity.
12. Immunofluorescent staining study confirmed that mHAl,6,11could decrease the contents of microtubule and destroy the network of microtubules in cells.
Humn lung cancer CRL5908cells were treated with1μM mHA1,6, or11for24hour, then immunofluorescent staining assay were performed. The long microtubule structure along the long axes of the cell in the cytoplasm was disrupted and the microtubule fluorescence intensity was significantly reduced, suggesting the decrease in contents of microtubule.
13. G2/M cell cycle arrest caused by mHAl,6,11.
Human leukemia HL-60/Bcl-2cells were treated with1μM mHA1,6,11, the distribution of cells in different phases of the cell cycle was determined by flow cytometry24hours later. The results showed that mHAs caused a statistically significant increase in the G2-M cell population.
14. DNA fragmentation analysis confirmed that mHAs could induce apoptosis
Human leukemia HL-60/Bcl-2cells were treated with mHAl,6,11and colchicine for24hours, then DNA fragmentation analysis were performed. A clear DNA ladder was observed, indicating that colchicine, and mHAl,6, and11could induce apoptotic cell death in HL-60/Bcl-2cells.
Conclusion:
1. We developed a series of HA14-1analogs and identified them as a new class of microtubule inhibitors with potent anti-cancer growth activity.
2. The structure-activity relationship study of this series of compounds provided references for the design of new microtubule inhibitors in the future.
3. These analogs showed a more stable and more potent anticancer growth activity than did authentic HA14-1, with IC50values are all in nM level. And they all showed lower toxicity towards normal tissues which made them possible to be a drug for further study.
4. The docking study and [3H]Colchicine-tubulin competition binding assay showed that mHA1,6,11are all binding to the colchicine-binding site on microtubule protein and they are all new microtubule inhibitors.
5. Tubulin polymerization assay showed that mHAl,6,11were potent microtubule depolymerizing agent. They worked in a similar manner as colchicine while Taxol was just the opposite.
6. Immunofluorescent staining study indicated that mHA1,6,11decreased the microtubule amount and microtubule network structures in the cells.
7. Flow cytometry analysis and DNA fragmentation analysis showed that mHAl,6,11induced G2/M cell cycle arrest and lead to cell apoptosis.
8. Our study explored the mechanism of this series of HA14-1analogues on malignant cells. They bind at colchicine-binding site on microtubule protein and inhibit microtubule polymerization. The suppression of the microtubule dynamics lead to misdirecting the formation of a functional mitotic spindle, which arrests the cells in G2/M phase, thereby leading to apoptosis of the tumor cell.
根据作用机制的不同,微管蛋白抑制剂可分为两大类：①抑制微管蛋白聚合的微管蛋白解聚剂,如秋水仙碱类和长春碱类化合物；②促进微管蛋白聚合的微管蛋白聚合剂,如紫杉醇及其类似物,埃博霉素及其类似物。根据微管蛋白抑制剂在微管蛋白上的作用位点的不同,微管蛋白抑制剂又可分为3种类型：①作用于秋水仙碱位点的微管蛋白抑制剂；②作用于长春碱位点的微管蛋白抑制剂；③作用于紫杉醇位点的微管蛋白抑制剂。目前,长春碱和紫杉醇位点微管蛋白抑制剂类药物已在肿瘤临床治疗方面占据了重要地位,紫杉醇更是已成为肺癌、乳腺癌和卵巢癌的重要一线治疗药物。然而,和其他抗肿瘤药物一样,难以耐受的副作用及用药后耐药性的出现限制了微管蛋白抑制剂的临床使用。因此,开发新型的具有更强活性、更低毒性、并且对多药耐药肿瘤细胞更有效的新型微管蛋白抑制剂具有很大的应用前景。
HA14-1是本博士论文作者的导师之一Ziwei Huang教授等研发的小分子化合物,是世界上第一例报道的Bcl-2抑制剂,能够强有力地诱导乳腺、结直肠癌、肾癌、宫颈癌、脑胶质瘤、白血病等多种肿瘤细胞系发生凋亡。在后续的研究中,我们发现HA14-1不仅能够通过诱导活性氧(ROS)产生、细胞色素C释放、半胱天冬酶Caspase-9/-3活化这一信号途径而诱导凋亡,在其药物浓度大于10μM时,还可以作用于微管蛋白秋水仙碱位点抑制微管蛋白聚合。我们将HA14-1作为母体药物对其进行结构改构,设计并合成了一系列新型HA14-1类似物(2-amino-4-phenyl-4H-chromene-3-carboxylate analogs),命名为mHA1-19,并对其中抗肿瘤活性较强的mHA1,6,11进行了后续的生物学评价和抗肿瘤机制研究。这些新型化合物表现出更强的抗肿瘤活性和更好的稳定性。肿瘤细胞在接受药物处理后逐渐出现包括细胞变长、不对称及出现长伪足等细胞形态学改变,与HA14-1处理细胞后迅速表现出细胞缩小、核固缩、凋亡小体出现等细胞凋亡的典型改变截然不同。这些现象均提示这一系列HA14-1类似物有着不同于母体药物HA14-1的肿瘤细胞杀伤机制,维持细胞正常形态的微管蛋白极有可能是其主要作用靶点。虽然秋水仙碱类药物因为毒性较大目前还暂未用于肿瘤治疗,但秋水仙碱位点作为一个很有前景的肿瘤药物靶标一直备受关注,目前已有多个化合物进入了临床研究。研究及阐明这一系列mHA类似物的作用靶点及其抗肿瘤机制,将推动新型、高效的秋水仙碱位点抑制剂的设计和开发,具有重要意义。
方法：
1.化合物合成：
该系列化合物总的合成路径为采用苯甲醛、酚类似物和氰乙酸乙酯这三种组分和哌啶进行一步化反应合成。化合物合成后均经过核磁共振氢谱和质谱验证。
(1)mHAl的制备
3-溴-4,5-二甲氧基苯甲醛(0.49g,0.002mol)、3-二甲氨基苯酚(0.27g,0.002mol)、氰乙酸乙酯(0.21mL,0.002mol)和哌啶(0.4mL,0.004mol)溶于15ml无水乙醇,室温下搅拌4小时。加入80ml二氯甲烷稀释后用水清洗,硫酸钠干燥化合物,过滤除去硫酸钠,蒸发干燥溶剂。使用色谱分析法(己烷/二氯甲烷)提纯粗制品,得到0.6g mHAl,收益率63%。
(2)mHA6的制备
采用3-溴-4,5-二甲氧基苯甲醛、1-萘酚和氰乙酸乙酯作为原料,后续步骤同上,得到0.45g mHA6,收益率46%。
(3)mHA11的制备
采用3-氯苯甲醛、3-二甲氨基苯酚和氰乙酸乙酯作为原料,后续步骤同上,得到0.32g mHA11,收益率43%。
2.细胞培养
人白血病细胞HL-60/Bcl-2(pZip-Bcl-2质粒稳定转染)惠赠于Dr. Kapil N. Bhalla(迈阿密大学医学院,佛罗里达州,美国)。细胞培养条件为RPMI1640培养基,10%胎牛血清,2mM谷氨酰胺,100U/ml青霉素,100μg/ml链霉素,5%二氧化碳,37℃。
3.小鼠骨髓细胞收集
小鼠骨髓细胞样本来采样于年龄为3月的C57BL/6雌性小鼠(Charles River Labs)。CO2吸入法处死小鼠,分离取出小鼠的胫骨和股骨,注射器吸取RPMI1640培养基反复冲洗胫骨和股骨,将骨腔内骨髓细胞冲洗出来,采用淋巴细胞分离法获得小鼠骨髓细胞,接种于96孔细胞培养板中,每孔1×105细胞。
4.人正常骨髓细胞样本收集
在通过伦理委员会审查及获得患者知情同意后,取得3例人正常骨髓细胞样本。样本均来自弥漫大B细胞淋巴瘤患者,已通过病理证实为正常骨髓。按骨髓穿刺术操作规范进行操作取得人体骨髓样本,将肝素化的骨髓细胞用RPMI-1640培养基稀释后,采用淋巴细胞分离法获得人体骨髓细胞,接种于96孔细胞培养板中,每孔1X105细胞。
5. CellTiter-Blue法测定细胞活力
HL-60/Bcl-2细胞或小鼠骨髓细胞接种于96孔板,分别接受不同浓度的mHAs药物处理,温箱孵育72小时后,采用CellTiter-Blue细胞活力检测试剂盒按照使用说明测定细胞活力。
6. Cell Count Kit-8(CCK-8)法测定细胞活力
人骨髓细胞接种于96孔板,24小时后分别接受不同浓度的mHAs药物处理,温箱孵育72小时后,采用CCK-8细胞活力检测试剂盒测定细胞活力。
7.细胞集落形成实验
细胞接受不同浓度的mHAs药物及DMSO对照处理24小时后,收集细胞,新鲜培养基稀释后与甲基纤维素半固体培养基混合均匀,接入培养皿使每个培养皿内细胞数目为200,体积为2ml。37℃,5%二氧化碳及饱和湿度的培养箱内孵育10-14天后倒置显微镜下计数细胞克隆数并计算细胞克隆形成率。
8.细胞形态学检测
1μM mHAs处理细胞24小时,倒置显微镜下观察细胞形态改变并拍照。
9.计算机分子模型
采用微管和DAMA-秋水仙碱复合物中的微管晶体结构(PDB:1SA0)作为模板,采用Autodock4程序来预测微管与配体mHAl,6,11的结合模型。
10.秋水仙碱-微管蛋白竞争结合试验
1μM放射性标记的[3H]秋水仙碱,1%DMSO和不同浓度的待测药物均溶于50μl G-PEM缓冲液中,与1μM微管蛋白共同孵育1小时,经DEAE-纤维素柱层析,采用液体闪烁法(Perkin-Elmer)测定滤液中放射性强度。使用GraphPad Prism对数据进行非线性回归分析。
11.微管蛋白聚合实验
按照试剂盒生产公司提供的说明书进行,将微管蛋白溶于G-PEM缓冲液中,得到浓度为3mg/ml的微管蛋白溶液,加入预先加入50μl待测药物溶液的96孔板内,50μl/孔,使用Synergy2酶标仪测定吸光度(360nm/420nm),每分钟一次,共测定1小时。
12.免疫荧光染色
人肺癌细胞CRL5908经1μMmHAl,6,11处理24h后,4%多聚甲醛4℃固定30分钟,0.1%Triton X-100透膜处理15min,2%BSA封闭处理30min,1:2000抗-α-微管蛋白单克隆抗体和7：1000罗丹明-鬼笔环肽标记的抗肌动蛋白抗体避光处理细胞1h,1：200抗鼠IgG二抗避光处理30min,1μg/ml DAPI处理1min。免疫荧光显微镜下观察和拍照。
13.细胞周期测定
1X106HL-60/Bcl-2细胞分别接受1,3,5μM mHAs药物处理24h。收集细胞,PBS清洗2次,重悬于100μlPBS和1ml75%预冷乙醇内,-20℃过夜保存。测定当天离心细胞,移去上清,加入500μl PI染色液(含80μg/mL碘化丙啶,100μg/mL核糖核酸酶A,1%曲拉通),避光孵育至少半小时后流式细胞仪检测,FlowJo7.5软件分析。
14.DNA碎片分析方法测定细胞凋亡
1X106HL-60/Bcl-2细胞分别接受不同药物处理：DMSO对照、阳性对照0.1μM和1μM秋水仙碱,1μM和5μM mHAl,6,11,温箱孵育24h,按照试剂盒生产公司提供的说明书进行提取每个处理组DNA, DNA经2%琼脂糖凝胶电泳,EB显色照相。
结果：
1.mHA系列化合物细胞毒性测定：测定mHAl-19对人白血病细胞HL-60/Bcl-2的细胞毒性并计算其IC50,结果显示该系列化合物中mHA1,6,11具有较强的抗肿瘤活性,选择这3种化合物进行后续的生物学评价和抗肿瘤机制研究。
2.mHA系列化合物构效分析：对该系列化合物进行构效分析,发现C6位点上连接二甲氨基与苯基对化合物活性的影响类似但优于羟基,羟基优于氨基；mHA7,15,17活性均很差,提示C7位点添加任何基团均会降低化合物的活性。
3. mHAl,6,11抗肿瘤活性明显强于其母体药物HA14-1：分别用不同浓度的HA14-1和mHA1,6,11处理人白血病HL-60/Bcl-2细胞,24h或72h后测定细胞活力,结果显示mHAl,6,11的ICso均小于1μM,而HA14-1的ICso为9.394±0.18μM, mHAl,6,11抗肿瘤活性明显强于其母体药物HA14-1。
4. mHAl,6,11对人白血病细胞具有较强细胞毒性,但对正常小鼠骨髓细胞毒性较低：分别对人白血病HL-60/Bcl-2细胞及正常小鼠骨髓细胞进行相同浓度的mHAl,6,11药物处理,72h后分别测定细胞活力。结果显示1μMmHAl,6,11可以杀死约一半的肿瘤细胞,而1μM药物处理对正常小鼠骨髓细胞几乎无影响。
5. mHAl,6,11对人正常骨髓细胞几乎无毒性：采集人正常骨髓细胞进行不同浓度的mHAl,6,11药物处理,72h后分别测定细胞活力。结果显示3μM mHAs可杀死几乎全部的恶性HL-60/Bcl-2细胞,而同样处理对人正常骨髓细胞几乎毫无影响。
6.阳性对照药物秋水仙碱对正常小鼠骨髓细胞毒性较大：采用秋水仙碱位点代表药物秋水仙碱作为阳性对照,对人白血病HL-60/Bcl-2细胞及正常小鼠骨髓细胞进行相同浓度药物处理,72h后分别测定细胞活力,结果显示秋水仙碱对肿瘤细胞及正常骨髓细胞均有较强的细胞毒性,尽管其IC50值在人白血病细胞中仅为33.5±3.5nM,但药物浓度在25nM时即可杀伤约30%的正常骨髓细胞。
7.mHA1,6,11可诱导肿瘤细胞丧失克隆形成能力：人白血病HL-60/Bcl-2细胞在接受药物处理后培养10～14d后计数克隆形成数,计算所得IC50值低于细胞毒性试验,提示药物处理不仅能在72h内迅速杀死肿瘤细胞,并可引起部分细胞丧失增殖能力及克隆形成能力。
8.mHA1,6,11引起肿瘤细胞产生特殊形态改变：mHA1,6,11可引起肿瘤细胞产生特殊形态改变。人白血病HL-60/Bcl-2细胞由悬浮细胞典型的球体变为不规则形、细胞变长及出现长伪足；人肺癌CRL5908细胞则由贴壁细胞典型的梭状变为多边形或类圆形。
9.计算机模拟对接研究显示mHA1,6,11可结合于微管蛋白上的秋水仙碱位点：计算机模拟对接研究采用DAMA-秋水仙碱晶体结构作为模板,模拟计算结果显示mHA1,6,11与秋水仙碱均可结合于微管蛋白上的相同位点-秋水仙碱位点,mHA1,6和秋水仙碱结合模式相似而mHA11结合模式略有不同。
10.秋水仙碱-微管蛋白竞争结合试验证实了mHA1,6,11可与秋水仙碱竞争结合位点：秋水仙碱-微管蛋白竞争结合试验显示mHA1,6,11可与放射性标记的秋水仙碱竞争结合位点,抑制其与微管蛋白结合,其作用呈剂量依赖性方式。
11.微管蛋白聚合实验显示mHA1,6,11和秋水仙碱均可抑制微管蛋白聚合,紫杉醇可促进微管蛋白聚合：微管蛋白聚合实验显示紫杉醇可促进微管蛋白聚合,mHA1,6,11和秋水仙碱均可抑制微管蛋白聚合,证实mHA1,6,11具有强效的抗微管聚合作用。
12.免疫荧光染色实验证实mHA1,6,11可降低细胞内微管含量并破坏其网状分布：人肺癌CRL5908细胞接受1μMmHA1,6,11处理24h后行免疫荧光检测,显示胞浆内沿细胞长轴密集分布的微管变为弥散分布,且荧光强度明显降低,提示细胞内微管含量减少。
13.mHAl,6,11可诱导肿瘤细胞发生G2/M细胞周期阻滞：人白血病HL-60/Bcl-2细胞在接受1μM mHA1,6,11药物处理24h后经流式细胞仪检测证实细胞发生特异性G2/M细胞周期阻滞。
14.DNA碎片分析证实mHA1,6,11可诱导肿瘤细胞发生凋亡：mHA1,6,11及秋水仙碱处理人白血病HL-60/Bcl-2细胞24h后行DNA碎片分析,出现明显的凋亡条带,证实秋水仙碱及mHAl,6,11均可诱导人白血病HL-60/Bcl-2细胞发生凋亡。
结论：
1.本研究获得了一系列HA14-1类似物并证实其为具有高效抗肿瘤活性的微管蛋白抑制剂。
2.对该系列化合物的构效分析发现C6位点上连接二甲氨基与苯基类似但均优于羟基,羟基优于氨基；C7位点上不宜连接任何侧链,为日后继续研发新型微管蛋白抑制剂提供了设计思路。
3.本研究结果证明了mHA1,6,11具有较强的肿瘤细胞杀伤作用,其IC50为纳摩尔级别,大大优于其母体药物HA14-1,而且对正常细胞毒性较小,具有进一步研发成药的可能性。
4.计算机模拟对接研究及秋水仙碱-微管蛋白竞争结合实验证实mHA1,6,11均结合于微管蛋白上的秋水仙碱位点并可与秋水仙碱竞争结合位点,为作用于秋水仙碱位点的新型微管蛋白抑制剂。
5.微管蛋白聚合实验进一步证实mHAl,6,11与紫杉醇作用方式相反,与秋水仙碱作用方式一致,为促微管蛋白解聚剂。
6.免疫荧光染色实验显示mHAl,6,11可降低细胞内微管数量及破坏胞浆内正常微管网状结构。
7.流式细胞仪细胞周期检测及DNA碎片分析实验证实mHAl,6,11通过阻滞肿瘤细胞于G2/M期而诱导细胞发生凋亡。
8.本研究结果初步探明了该系列化合物杀伤肿瘤细胞的作用机制：通过作用于微管蛋白,抑制微管蛋白聚合,从而影响纺锤体形成,使肿瘤细胞停滞于G2/M细胞周期,不能完成正常的有丝分裂,从而发生凋亡。
Background and objection:
Microtubules are components of cytoskeleton and are present in virtually all eukaryotic cells. Microtubules form spindle, centriole, flagllum and nerviduct with other proteins in the cells. Under normal condition, there is a balance between microtubule polymerization and depolymerization and the dynamics regulates the mitosis. Microtubules are involved in many cellular processes, including maintenance of cell shape, cell migration, mitosis, intracellular transport and cell signaling. In the eukaryotic cell cycle, microtubules are polymerized and form the mitotic spindle in prophase, which then moves the chromosomes to the opposite sides of the cell, in preparation for cell division into two daughter cells. Because of this important role in cell proliferation, microtubules have been recognized as one of the successful and efficacious drug targets for the development of novel anti-cancer chemotherapeutics. Microtubule-inhibiting agents (MIAs) currently have been used in clinic therapies work through the suppression of the microtubule dynamics by misdirecting the formation of a functional mitotic spindle in fast dividing tumor cells. This arrests the cells in M phase, thereby leading to apoptosis of the tumor cells.
Based on their mechanism of action, Microtubule-inhibiting agents (MIAs) are classified into two broad categories:①microtubule destabilizing agents, including vinca alkaloids and colchicine;②microtubule stabilizing agents, including paclitaxel, epothilone and its analogues. According to their different binding sites on microtubule protein, Microtubule-inhibiting agents (MIAs) are further classified into three groups:colchicine site-binding agents, vinblastine site-binding agents, and paclitaxel site-binding agents. Due to the potent anti-cancer activity, these apoptotic therapeutic agents that target microtubules, including paclitaxel and vinblastine, are among the most commonly prescribed antitumor agents. Paclitaxel is currently recommended as first-line agent in the chemotherapy of lung cancer, breast cancer and ovarian cancer. However, as with other anticancer drugs, intolerable toxicities and the emergence of drug resistance have limited the clinical use of the drugs targeting microtubules. Therefore, a need still exists for discovery and development of novel chemotherapeutic agents that target microtubules, but that show better activicy, lower drug resistance and fewer toxic side effects.
The small compound dubbed HA14-1, identified by one of my supervisor--Dr. Huang ziwei, was the first reported Bcl-2inhibitor which can potently induce apoptosis in a wide variety of human cancers, including breast cancer, colorectal cancer, kidney cancer, cervical cancer, lung cancer, brain glioma and leukemia. In subsequent studies, we found that it not only can induce reactive oxygen species (ROS) generation, cytochrome c release, and Caspase-9/-3activation, but it also can bind microtubules in a manner that is competitive with colchicine when its concentration is greater than10μM. In the present study, we used HA14-1as the initial template compound and developed a new class of novel microtubule-targeting agents (2-amino-4-phenyl-4H-chromene-3-carboxylate analogues) named mHAl-19. mHA1,6and11showed more potent and stable than others, so we choosed them to do subsequent biological assessment. After treatment with these agents, morphological changes including cell elongation, asymmetry, and formation of long pseudopodia were observed which are quite different from the apoptotic morphological changes induced by HA14-1treatment, including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and apoptotic body. This phenomenon indicated that these mHA analogues worked through different pathway from HA14-1to kill cancer cells. Microtubule as the important component of cell shape maintenance was the most probable target. Although the high toxicity of colchicine has prevented its clinical use in cancer therapy, the colchicine-binding site is still a potential drug target that has attracted much attention for development of new agents that bind to this site. Several such compounds have entered clinical trials. Study and find the action mechanism of these mHA analogues is significative to the design and develop of new colchicine site-binding agents.
Methods and materials:
1. Chemical synthesis:
These compounds were synthesized using a one-pot three-component reaction of substituted benzaldehyde, phenol analogs and ethyl cyanoacetate in the presence of piperidine. All of the new compounds described were characterized by1H NMR and mass spectrometry (MS) spectra.
(1) Preparation of mHAl
A mixture of3-Bromo-4,5-dimethoxybenzaldehyde (0.49g,0.002mol),3-(dimethylamino) phenol (0.27g,0.002mol), ethyl cyanoacetate (0.21ml,0.002mol) and piperidine (0.4mL,0.004mol) was suspended in15mL anhydrous ethanol and stirred at room temperature for4h. After diluting with80mLCH2l2and washing with water, the organic layer was dried over Na2SO4. The Na2SO4was then removed by filtration and the solvent was evaporated. The crude product was purified by column chromatography (hexane/CH2Cl2) to give0.6g mHAl at63%yield.
(2) Preparation of mHA6
Starting from3-bromo-4,5-dimethoxybenzaldehyde, naphthalen-1-ol, and ethyl cyanoacetate, we then followed the procedure for the synthesis of mHAl, to give0.45g (46%yield) of mHA6.
(3) Preparation of mHA11
Starting from3-chlorobenzaldehyde,3-(dimethylamino) phenol, and ethyl cyanoacetate, we then followed the procedure for the synthesis of mHAl, to give0.32g (43%yield) of mHA11.
2. Cell culture
The human leukemic HL-60/Bcl-2cell line was obtained from Dr. Kapil N. Bhalla (University of Miami School of Medicine, Miami, FL), which has been stably transfected with pZip-Bcl-2plasmid. Cells were cultured in RPMI1640medium supplemented with10%fetal bovine serum,2mM glutamine,100U/ml penicillin,100μg/mL streptomycin and800μg/mL genticin (G418, Invitrogen, San Diego, CA). Cells were maintained in a humidified5%CO2atmosphere at37℃.
3. Mouse bone marrow cells collection
Mouse bone marrow samples were obtained from3-month-old C57BL/6female mice (Charles River Labs). Mice were sacrificed with CO2the tibias and femurs were dissected. The tibias and femurs were flushed repeatedly with RPMI1640medium through needle. Mouse bone marrow cells were seperated by Ficoll density gradient centrifugation.
4. Isolation and culture of human bone marrow cells
Bone marrow specimens were acquired from Diffuse Large B Cell Lymphoma (DLBCL) patients (n=3) via a protocol approved by our Research Ethics Committee for the use of samples for research, and informed consent was obtained from each patient. All of the patients with normal bone marrow were confirmed by pathology. Heparinized bone marrow samples were diluted with RPMI1640medium and overlaid on5ml separation medium and then centrifuged at3000rpm for30min. The bone marrow cells were washed twice and suspended in RPMI1640medium supplemented with10%fetal bovine serum, added in96-well plate (1×105of cells each well).
5. Measurement of cell viability via CellTiter-Blue assay
HL-60/Bcl-2cells were seeded in96-well plates and treated with various concentrations of these compounds (0.1to3μM). The samples were then incubated at37℃for72h. After incubation, cell viability was measured using a CellTiter-Blue assay kit according to the manufacturer's instructions.
6. Measurement of cell viability via CCK-8assay
The cell viability assays were evaluated by a Cell Counting Kit-8(CCK-8). After overnight culture the bone marrow cells were treated with different concentrations of mHA1, mHA6and mHA11separately, and controls were treated with vehicle (DMSO). After72h treatments, CCK-8solution was added to each well according to the manufacturer's instructions and optical density (OD) was measured at450nm test wavelength using a microplate reader. All experiments were performed in triplicate.
7. Assessment of clonogenicity
After incubation for24h with various drug concentrations or Vehicle (DMSO) control, cells were collected and diluted with fresh medium, then mixed with2%methylcellulose to make the methylcellulose1.3%and fetal bovine serum30%. A1.5ml volume of this mixture, containing200cells, was seeded into culture dish. Dishes were then incubated in incubator for10-14days. Colonies were enumerated with the aid of an inverted microscope.
8. Cell Morphological change
Cells were treated with mHAs at1μM for24h and then examined for morphological changes by inverted fluorescence microscopy and photography.
9. Molecular modeling
The crystal structure of microtubule in a complex with DAMA-colchicine [PDB reference:1SA0(16)] was used to predict the binding models of microtubule bind with designed compounds. The binding modes of designed ligands1,6,11with microtubule were predicted by using the Autodock4program.
10.[3H]Colchicine-tubulin binding assay
One micromolar radiolabeled colchicine [ring C, Methoxy-3H],1%DMSO and various concentrations of test compounds in50μl G-PEM buffer were incubated with1μM tubulin for60min. The binding solutions were filtered through two stacks of DEAE-cellulose filters and washed twice. The radioactivity in the filtrates was determined by liquid scintillation spectrometry. Nonlinear regression was used to analyze the data using GraphPad Prism.
11. Tubulin polymerization assay
Tubulin polymerization assays were conducted using the polymerization assay kit following the manufacturer's instructions. Briefly,50μl of3mg/ml tubulin (>99%pure) proteins in G-PEM buffer was placed in96-well microtiter plates in the presence of test agents. The absorbance at360/420nm was recorded every60s for1h using a Synergy2microplate reader.
12. Immunofluorescence staining
CRL5908cells were treated with1μM mHAl,6, or11for24h. Thereafter, cells were fixed for30min at4℃in4%paraformaldehyde and incubated with0.1%Triton X-100permeabilizing buffer for15minutes. After washing with PBS and blocking with2%BSA in PBS for30minutes, cells were incubated for1h protected from light in1:2000anti-a-tubulin monoclonal antibody and7:1000Rhodamine-Phalloidin-labeled anti-actin antibody in PBS. Cells were then washed with PBS and incubated for30min protected from light with1:200fluorescein isothiocyanate (FITC)-labeled anti-mouse IgG antibody in PBS. Subsequently, all cells were stained with1μg/ml4',6-diamidino-2-phenyl-indole. Samples were examined under a fluorescence microscope and photographed.
13. Cell cycle analysis
HL-60/Bcl-2cells (1×106) were treated with1,3, and5μM of mHA1,6,11, respectively, for24h at37℃. Cells were harvested and washed twice with PBS, then resuspended in100μl of PBS and lml of75%cold ethanol and stored at-20℃overnight. After centrifugation, the supernatant was removed. A500μl PI staining buffer containing80μg/mL of propidium iodide,100μg/mL of RNAse A, and1%Triton was added to the samples. The cells were incubated for at least half an hour (avoid light) and then analyzed by flow cytometry with a FACScalibur system using FlowJo7.5analysis software.
14. DNA fragmentation analysis
HL-60/Bcl-2cells were treated with Vehicle control (DMSO), positive control (colchicine at0.1and1μM), and mHA1,6, or11(at1and5μM), respectively. The plate was incubated for24h, and total DNA was extracted from the cells in each well using an Apoptotic DNA-ladder kit following the manufacture's instructions. The DNA samples were subjected to2%agarose gel electrophoresis and visualized with ethidium bromide staining.
Results:
1. Cytotoxicity of new mHA agents toward leukemic HL-60/Bc;-2cells
Human leukemia HL-60/Bcl-2cells were treated with the series of HA14-1analogs and cell viability assay was performed to get their IC50values. mHAl,6, and11, showed the best anti-tumor activity of the series of compounds and we choose them for further study.
2. Structure-activity analysis
On the basis of the biological result, the structure-activity relationship of these compounds was discussed. We digged up that dimethyl amino in C6is similar with benzene and better than hydroxy(-OH); hydroxy(-OH) is better than amino(-NH2); mHA7,15and17are all very poor which indicated that substitution at C7will decrease its activity.
3. mHAl,6,11were much more potent than their parent HA14-1compound
Human leukemia HL-60/Bcl-2cells were treated with diferent concentrations of HA14-1or mHA1,6,11for24h or72h, respectively. Cell viability assay showed that the IC50values of mHAl,6,11were all less than1μM, while IC50of the parent compound, HA14-1, was9.394±0.18μM. Overall, mHA1,6,11were much more potent than their parent HA14-1compound.
4. mHAl,6,11were very potent to Human leukemia HL-60/Bcl-2cells while they showed low otoxicity against normal mouse bone marrow cells.
Human leukemia HL-60/Bcl-2cells and normal mouse bone marrow cells were treated with diferent concentrations of mHAl,6,11for72h, then cell viability was assessed. The results showed that these compounds had almost no effects on normal mouse bone marrow cells below1μM concentration, while1μM mHAs were able to kill almost half of the malignant HL-60/Bcl-2cells.
5. mHAl,6,11showed almost no cytotoxicity on human bone marrow cells
human bone marrow cells were treated with diferent concentrations of mHAs and DMSO vehicle control for72h, then cell viability was assessed. The results showed that3μM mHAs treatment had almost no effects on normal human bone marrow cells, while3μM mHAs were able to kill almost all of the malignant HL-60/Bcl-2cells.
6. The positive control Colchicine showed high toxicity against normal mouse bone marrow cells
Human leukemia HL-60/Bcl-2cells and normal mouse bone marrow cells were treated with diferent concentrations of colchicine, the colchicine site-binding agent, which was used as the positive control. Cell viability was assessed72h later. The results showed that colchicine was toxic against both malignant and normal cells. Colchicine resulted in the death of30%of normal mouse bone marrow cells at25nM, even though its IC50was only33.5±3.5nM in HL-60/Bcl-2cells.
7. Induction of colonogenic cell death by mHA1,6,11.
After exposure various concentrations of mHA1,6, or11for24hours, HL-60/Bcl-2cells were then incubated for10-14days, then the number of colonies were measured. The IC50values for the colony inhibition assay were lower than the values for the cytotoxicity assay, which indicated that treatment with mHAs disrupted proliferation and colony forming ability of some cancer cells without causing actual cell death in72hours.
8. Specific cell morphological changes in response to mHAl,6,11.
mHA1,6and11treatment resulted in specific cell morphological changes. HL-60/Bcl-2cells are suspension cells and typically have a spherical shape. After treatment with mHAs, specific morphological changes including cell elongation, asymmetry, and formation of long pseudopodia were observed. Human lung cancer CRL5908cells changed from spindle shape to multi-angular or nearly-circular shape after treatment with mHAs.
9. The docking study showed that mHAl,6, and11could bind at the colchicine site on the microtubule protein
The crystal structure of microtubule in a complex with DAMA-colchicine was used to predict the binding models of microtubule bind with designed compounds. The results of our docking study showed that mHA1,6, and11could bind at the same site as colchicine on the microtubule protein. mHA1and mHA6adopt a similar binding mode with colchicine, which is slightly different from that adopted by mHA11.
10.[3H]Colchicine-tubulin competition binding assay confirmed that mHAl,6,11could compete with colchicine binding to tubulin.
[3H]Colchicine-tubulin competition binding assay confirmed that mHAl,6,11could inhibit the combination of [3H]colchicine and tubulin in a dose-dependent manner by competing the colchicine binding site.
11. Tubulin polymerization assay showed that mHAl,6,11and colchicine could inhibit the microtubule polymerization while Taxol had opposite effect.
Tubulin polymerization assay showed that mHAl,6,11and colchicine could inhibit the microtubule polymerization while Taxol had opposite effect, suggesting that each of the mHAs possessed strong antitubulin polymerization activity.
12. Immunofluorescent staining study confirmed that mHAl,6,11could decrease the contents of microtubule and destroy the network of microtubules in cells.
Humn lung cancer CRL5908cells were treated with1μM mHA1,6, or11for24hour, then immunofluorescent staining assay were performed. The long microtubule structure along the long axes of the cell in the cytoplasm was disrupted and the microtubule fluorescence intensity was significantly reduced, suggesting the decrease in contents of microtubule.
13. G2/M cell cycle arrest caused by mHAl,6,11.
Human leukemia HL-60/Bcl-2cells were treated with1μM mHA1,6,11, the distribution of cells in different phases of the cell cycle was determined by flow cytometry24hours later. The results showed that mHAs caused a statistically significant increase in the G2-M cell population.
14. DNA fragmentation analysis confirmed that mHAs could induce apoptosis
Human leukemia HL-60/Bcl-2cells were treated with mHAl,6,11and colchicine for24hours, then DNA fragmentation analysis were performed. A clear DNA ladder was observed, indicating that colchicine, and mHAl,6, and11could induce apoptotic cell death in HL-60/Bcl-2cells.
Conclusion:
1. We developed a series of HA14-1analogs and identified them as a new class of microtubule inhibitors with potent anti-cancer growth activity.
2. The structure-activity relationship study of this series of compounds provided references for the design of new microtubule inhibitors in the future.
3. These analogs showed a more stable and more potent anticancer growth activity than did authentic HA14-1, with IC50values are all in nM level. And they all showed lower toxicity towards normal tissues which made them possible to be a drug for further study.
4. The docking study and [3H]Colchicine-tubulin competition binding assay showed that mHA1,6,11are all binding to the colchicine-binding site on microtubule protein and they are all new microtubule inhibitors.
5. Tubulin polymerization assay showed that mHAl,6,11were potent microtubule depolymerizing agent. They worked in a similar manner as colchicine while Taxol was just the opposite.
6. Immunofluorescent staining study indicated that mHA1,6,11decreased the microtubule amount and microtubule network structures in the cells.
7. Flow cytometry analysis and DNA fragmentation analysis showed that mHAl,6,11induced G2/M cell cycle arrest and lead to cell apoptosis.
8. Our study explored the mechanism of this series of HA14-1analogues on malignant cells. They bind at colchicine-binding site on microtubule protein and inhibit microtubule polymerization. The suppression of the microtubule dynamics lead to misdirecting the formation of a functional mitotic spindle, which arrests the cells in G2/M phase, thereby leading to apoptosis of the tumor cell.
引文
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[3]Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nat Rev Cancer 2004;4:253-65.
[4]Risinger AL, Giles FJ, Mooberry SL. Microtubule dynamics as a target in oncology. Cancer Treat Rev 2009;35:255-61.
[5]Mollinedo F, Gajate C. Microtubules, microtubule-interfering agents and apoptosis. Apoptosis 2003;8:413-50.
[6]Zhou J, Giannakakou P. Targeting microtubules for cancer chemotherapy. Curr Med Chem Anticancer Agents 2005;5:65-71.
[7]Dumontet C, Jordan MA. Microtubule-binding agents:a dynamic field of cancer therapeutics. Nat Revs Drug Discov 2010;9:790-803.
[8]Carlson RO. New tubulin targeting agents currently in clinical development. Expert Opin Investig Drugs 2008; 17:707-22.
[9]Perez EA. Microtubule inhibitors:Differentiating tubulin-inhibiting agents based on mechanisms of action, clinical activity, and resistance. Mol Cancer Ther 2009;8:2086-95.
[10]Morris PG, MN. F. Microtubule active agents:beyond the taxane frontier. Clin Cancer Res 2008; 14:7167-72.
[11]Gascoigne KE, Taylor SS. How do anti-mitotic drugs kill cancer cells? J Cell Sci 2009;122:2579-85.
[12]Wang JL, Liu D, Zhang ZJ, Shan S, Han X, Srinivasula SM,et al. Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells. Proc Natl Acad Sci U S A 2000;97:7124-9.
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[14]Risinger AL, Westbrook CD, Encinas A, Mulbaier M, Schultes CM, Wawro S, et al. ELR510444, a novel microtubule disruptor with multiple mechanisms of action. J Pharmacol Exp Ther 2011;336:652-60.
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[2]Hall A. The cytoskeleton and cancer. Cancer Metastasis Rev 2009;28:5-14.
[3]Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nat Rev Cancer 2004;4:253-65.
[4]Risinger AL, Giles FJ, Mooberry SL. Microtubule dynamics as a target in oncology. Cancer Treat Rev 2009;35:255-61.
[5]Mollinedo F, Gajate C. Microtubules, microtubule-interfering agents and apoptosis. Apoptosis 2003;8:413-50.
[6]Zhou J, Giannakakou P. Targeting microtubules for cancer chemotherapy. Curr Med Chem Anticancer Agents 2005;5:65-71.
[7]Dumontet C, Jordan MA. Microtubule-binding agents:a dynamic field of cancer therapeutics. Nat Revs Drug Discov 2010;9:790-803.
[8]Carlson RO. New tubulin targeting agents currently in clinical development. Expert Opin Investig Drugs 2008; 17:707-22.
[9]Perez EA. Microtubule inhibitors:Differentiating tubulin-inhibiting agents based on mechanisms of action, clinical activity, and resistance. Mol Cancer Ther 2009;8:2086-95.
[10]Morris PG, MN. F. Microtubule active agents:beyond the taxane frontier. Clin Cancer Res 2008; 14:7167-72.
[11]Gascoigne KE, Taylor SS. How do anti-mitotic drugs kill cancer cells? J Cell Sci 2009;122:2579-85.
[12]Wang JL, Liu D, Zhang ZJ, Shan S, Han X, Srinivasula SM,et al. Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells. Proc Natl Acad Sci U S A 2000;97:7124-9.
[13]An J, Chen Y, Huang Z. Critical Upstream Signals of Cytochrome c Release Induced by a Novel Bcl-2 Inhibitor. J Biol Chem 2004;279:19133-40.
[14]Risinger AL, Westbrook CD, Encinas A, Mulbaier M, Schultes CM, Wawro S, et al. ELR510444, a novel microtubule disruptor with multiple mechanisms of action. J Pharmacol Exp Ther 2011;336:652-60.
[15]LaVallee TM, Burke PA, Swartz GM, Hamel E, Agoston GE, Shah J, et al. Significant antitumor activity in vivo following treatment with the microtubule agent ENMD-1198. Mol Cancer Ther 2008;7:1472-82.
[16]Ravelli RB, Gigant B, Curmi PA, Jourdain I, Lachkar S, Sobel A, et al. Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain. Nature 2004;428:198-202.
[17]Kitazumi I, Tsukahara M. Regulation of DNA fragmentation:the role of caspases and phosphorylation. FEBS J 2011;278:427-41.
[18]An J, Nie A, Chervin A, Ducoff HS, Huang Z. Overcoming the radioresistance of prostate cancer cells with a novel Bcl-2 inhibitor. Oncogene 2007;26:652-61.
[19]Manero F, Gautier F, Gallenne T, Cauquil N, Gree D, Cartron PF, et al. The small organic compound HA 14-1 prevents Bcl-2 interaction with Bax to sensitize malignant glioma cells to induction of cell death. Cancer Res 2006;66:2757-64.
[20]Sinicrope FA, Penington RC, Tang XM. Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis is inhibited by Bcl-2 but restored by the small molecule Bcl-2 inhibitor, HA 14-1, in human colon cancer cells. Clin Cancer Res 2004; 10:8284-92.
[21]Campas C, Cosialls AM, Barragan M, Iglesias-Serret D, Santidrian AF, Coll-Mulet L, et al. Bcl-2 inhibitors induce apoptosis in chronic lymphocytic leukemia cells. Exp hematol 2006;34:1663-9.
[22]Hermanson D, Addo SN, Bajer AA, Marchant JS, Das SG, Srinivasan B, et al. Dual mechanisms of sHA 14-1 in inducing cell death through endoplasmic reticulum and mitochondria. Mol Pharmacol 2009;76:667-78.
[23]Yue QX, Liu X, Guo DA. Microtubule-binding natural products for cancer therapy. Planta Med 2010;76:1037-43.
[24]Fojo AT, Menefee M. Microtubule targeting agents:basic mechanisms of multidrug resistance (MDR). Semin Oncol 2005;32:S3-8.
[25]Loganzo F, Hari M, Annable T, Tan X, Morilla DB, Musto S, et al. Cells resistant to HTI-286 do not overexpress P-glycoprotein but have reduced drug accumulation and a point mutation in alpha-tubulin. Mol Cancer Ther 2004;3:1319-27.
[26]Kanakkanthara A, Wilmes A, O'Brate A, Escuin D, Chan A, Gjyrezi A, et al. Peloruside-and laulimalide-resistant human ovarian carcinoma cells have betaⅠ-tubulin mutations and altered expression of betaⅡ-and betaⅢ-tubulin isotypes. Mol Cancer Ther 2011;10:1419-29.
[27]Wilmes A, O'Sullivan D, Chan A, Chandrahasen C, Paterson I, Northcote PT, et al. Synergistic interactions between peloruside A and other microtubule-stabilizing and destabilizing agents in cultured human ovarian carcinoma cells and murine T cells. Cancer Chemother Pharmacol 2011;68:117-26.
[28]Owonikoko TK, Ramalingam SS, Kanterewicz B, Balius TE, Belani CP, Hershberger PA. Vorinostat increases carboplatin and paclitaxel activity in non-small-cell lung cancer cells. Int J Cancer.2010;126:743-55.
[29]Yardley DA, Raefsky E, Castillo R, Lahiry A, Locicero R, Thompson D, et al. Phase II Study of Neoadjuvant Weekly nab-Paclitaxel and Carboplatin, With Bevacizumab and Trastuzumab, As Treatment For Women With Locally Advanced HER2(+) Breast Cancer. Clin Breast Cancer 2011;11:297-305.
[30]Banerjee S, Wang ZW, Mohammad M, et al. Efficacy of selected natural products as therapeutic agents against cancer. J Nat Prod 2008; 71:492-496.
[31]Butler MS. Natural products to drugs:natural product- derived compounds in clinical trials. Nat Prod Rep 2008; 25:475-516.
[32]Bennouna, J., Delord, J.-P., Campone, M., and Nguyen, L. Vinflunine:A New Microtubule Inhibitor Agent, Clinical Cancer Research 2008; 14:1625-1632.
[33]Kingston, D. G. I. Tubulin-Interactive Natural Products as Anticancer Agents, Journal of Natural Products 2009; 72,507-515.
[34]Direas V, Limentani S, Romieu G, et al. Phase II multicenter study of larotaxel (XRP9881), a novel taxoid, in patients with metastatic breast cancer who previously received taxane-based therapy. Ann Oncol 2008; 19:1255-1260.
[35]Vulfovich, M., and Rocha-Lima, C. Novel advances in pancreatic cancer treatment, Expert Review of Anticancer Therapy 2008; 8,993-1002.
[36]Aghajanian, C., Burris, H. A., Jones, S., Spriggs, D. R., Cohen, M. B., Peck, R., Sabbatini, P., Hensley, M. L., Greco, F. A., Dupont, J., and O'Connor, O. A. Phase I Study of the Novel Epothilone Analog Ixabepilone (BMS-247550) in Patients With Advanced Solid Tumors and Lymphomas, Journal of Clinical Oncology 2007; 25,1082-1088.
[37]Hunt, J. T. Discovery of Ixabepilone, Molecular Cancer Therapeutics 2009; 8, 275-281.
[38]Qi WY, Meng ZY, Dou GF. Clinical research progress of epothilone analogues. J Int Pharm Res(国际药学研究杂志)2009；36,336-339.
[39]Chang CE, Zhang YT, Fu DX, et al. Ixabepilone, a new antineoplastic drug for breast cancer. Chin J New Drugs(中国新药杂志)2008；17：1629-1633.
[40]Zhu Y, Fu DX. Advances in the study of microtubule stabilizing agents. Chin J New Drugs(中国新药杂志)2009；18：1105-1109.
[41]Cigler, T., and Vahdat, L. T. Eribulin mesylate for the treatment of breast cancer, Expert Opinion on Pharmacotherapy 2010; 11,1587-1593.
[42]Jackson, K. L. et al. The halichondrins and E7389. Chem 2009; 109, 3044-3079.
[43]Cortes, J., Vahdat, L., Blum, J. L., Twelves, C., Campone, M., Roche, H., Bachelot, T., Awada, A., Paridaens, R., Goncalves, A., Shuster, D. E., Wanders, J., Fang, F., Gurnani, R., Richmond, E., Cole, P. E., Ashworth, S., and Allison, M. A. Phase Ⅱ Study of the Halichondrin B Analog Eribulin Mesylate in Patients With Locally Advanced or Metastatic Breast Cancer Previously Treated With an Anthracycline, a Taxane, and Capecitabine, Journal of Clinical Oncology 2010; 28,3922-3928.
[44]Twelves, C., Cortes, J., Vahdat, L., Wanders, J., Akerele, C., and Kaufman, P. Phase Ⅲ Trials of Eribulin Mesylate (E7389) in Extensively Pretreated Patients With Locally Recurrent or Metastatic Breast Cancer, Clinical Breast Cancer 2010; 10,160-163.
[45]Beslija, S., Bonneterre, J., Burstein, H. J., Cocquyt, V., Gnant, M., Heinemann, V., Jassem, J.,Ko"stler, W. J.,Krainer,M., Menard, S., Petit, T., Petruzelka, L., Possinger, K., Schmid, P., Stadtmauer, E., Stockler, M., Van Belle, S., Vogel, C., Wilcken, N., Wiltschke, C., Zielinski, C. C., Zwierzina, H., and for the Central European Cooperative Oncology, G. Third consensus on medical treatment of metastatic breast cancer, Annals of Oncology 2009; 20, 1771-1785.
[46]Dufresne, A., Pivot, X., Tournigand, C., Facchini, T., Altweegg, T., Chaigneau, L., and Gramont, A. Impact of chemotherapy beyond the first line in patients with metastatic breast cancer, Breast Cancer Research and Treatment 2008; 107,275-279.
[47]Cai, S. X. Small Molecule Vascular Disrupting Agents:Potential New Drugs for Cancer Treatment, Recent Patents on Anti-Cancer Drug Discovery 2007; 2, 79-101.
[48]Lippert Iii, J. W. Vascular disrupting agents, Bioorganic & Medicinal Chemistry 2007; 15,605-615.
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