微管靶点药物Combretastatin A-4及其衍生的抗肿瘤药效学及其机制研究
|
摘要
微管广泛存在于真核细胞的胞质内,是由αβ两种微管蛋白聚合而成的管状聚合物。作为构成细胞骨架的主要成分之一,微管在维持细胞形态,参与细胞的收缩和胞质内物质的运输等方面发挥着重要的作用。尤其是其在细胞分裂前期解聚重组形成纺锤体参与到细胞有丝分裂中的这一特殊的生物学功能使之成为抗肿瘤药物的重要靶点。肿瘤细胞具有快速增殖的能力,若抑制其纺锤体的形成,必将影响细胞有丝分裂的正常进行,使得肿瘤细胞生长停滞于G2/M期。二十世纪初以微管为靶点的紫杉醇类药物在临床上的成功应用激起了人们寻找和改造新型微管类靶点药物的浓厚兴趣。尽管人们对紫杉醇不断地进行结构改造和修饰但其水溶性差,易产生多药耐药以及毒副作用强等问题还是限制了其临床上的广泛运用。寻找新型的微管靶点药物一直是抗肿瘤药物学领域研究的热点之一,直到1987年美国国家癌症研究所Pettit等从南非矮生柳树(Combretum caffrum)中发现了天然活性产物combretastatin A-4(CA-4)。作为经典的微管聚合抑制剂秋水仙碱的类似物,CA-4还具有较强的破坏肿瘤血管的作用。因而与传统的微管类药物相比,CA-4具有抗癌效果强,不易产生耐药等诸多优点,而且由于其结构新颖,便于改造和修饰,颇受药物研究者的青睐。然而随着CA-4临床试验的深入,人们发现CA-4虽然能导致肿瘤细胞的大量坏死,但对残存的肿瘤组织的杀伤力较弱,因而常导致化疗的失败。本论文的研究旨在寻找一些新的CA-4的衍生物以及开发CA-4的临床应用潜能,探索常规的化疗药物或是临床一线用药与CA-4合理的联合用药方案,以期通过以上两方面的研究达到减少用药量,增强药物疗效并减轻化疗药物的毒副作用的最佳治疗效果。同时对限制CA-4抗肿瘤疗效的分子机制做了进一步的研究,希望能为制定该类化合物临床治疗策略提供全新的思路。
本论文将主要分为以下三个部分：(1)对经过结构改造和修饰得到的一系列CA-4衍生物进行抗肿瘤活性的筛选以及相应的药效学评价,并对从中得到的抗肿瘤活性较好,并且毒副作用较低的候选化合物进行初步分子机制的研究。(2)寻找合理的联合配伍用药方案,将不同作用机制的抗肿瘤药物与CA-4合用,以期达到减少药物用量,提高疗效,减轻化疗药物的毒副作用的最佳效果。并进一步探讨两药合用的分子机制,力求为设计临床联合治疗方案提供新的方向。(3)为进一步推动CA-4的临床应用,解决其不能有效抑制化疗后残存的肿瘤细胞增殖的现象,我们对其抗肿瘤机制做了深入的研究,希望通过寻找限制CA-4抗肿瘤疗效的分子机制,采取相应的措施有针对性的进行克服。
第一部分Combretastatin A-4衍生物的抗肿瘤活性及其机制的研究
目的：作为新型的微管聚合抑制剂,CA-4由于其结构简单,抗癌活性显著,因而备受药物化学家的关注。为进一步提高其疗效,降低毒性,我们通过对一系列经过修饰的CA-4衍生物抗癌活性的筛选,旨在寻找具有广谱抗肿瘤活性的新型CA-4类化合物。
方法和结果：XN05是一个新合成的Combretastatin A-4的类似物。首先我们通过MTT法在六株不同组织来源的人肿瘤细胞上(SGC、PC-3、ECA、MCF-7A2780和A549)对XN05的抗肿瘤活性进行细胞水平检测。结果显示其具有较强的抗肿瘤活性,IC5o值均小于2μM,而在两株肝癌细胞BEL-7402和SMMC-7721上的抗肿瘤活性比CA-4更强。值得注意的是,在正常的肝实质细胞HL-7702上XN05的IC50值要明显高于CA-4,提示我们该化合物对正常组织来源的细胞杀伤作用相对较弱。进一步通过离体的微管蛋白聚合实验和免疫荧光实验的结果证实XN05也是以微管为靶点发挥其抗肿瘤活性,并且XN05抑制微管聚合的能力与其母体CA-4类似。引起细胞G2/M期阻滞是微管类药物的重要特征之一,运用流式细胞术在经过XN05处理的两株肝癌细胞中均能观察到明显的G2/M期细胞周期阻滞现象,并且该现象存在浓度和时间依赖性。Western blot结果显示XN05引起的G2/M期阻滞现象也可能与细胞周期关键蛋白cdc2, CDK7以及cyclin B1的表达发生异常有关。随着药物作用时间的延长我们发现处于G2/M期阻滞的细胞最终走向了细胞的凋亡,进一步地肿瘤细胞的生长受到了抑制。
结论：本研究表明经过结构修饰的新型CA-4衍生物XN05具有明确的作用靶点,通过抑制微管的聚合使得细胞阻滞在G2/M期,并最终导致细胞走向凋亡发挥其抗肿瘤活性。此外,我们还发现XN05作用于BEL-7402和SMMC-7721肝癌细胞后引起的不同程度细胞周期阻滞可能与XN05能够对不同的周期蛋白(Cyclins)及周期蛋白依赖性激酶(CDKs)的表达产生影响相关。XN05在体外有着广谱,且优于同系列化合物的抗肿瘤活性,尤其是其在肝癌细胞株中表现出的细胞毒特异性使其非常有希望成为临床治疗的新型抗肿瘤微管类化合物。
第二部分Combretastatin A-4与化疗药物联合用药的抗肿瘤活性评价及机制的研究
目的：随着对CA-4抗肿瘤活性研究的深入,人们发现CA-4还是作用于肿瘤血管的靶向药物。据报道,CA-4在无细胞毒浓度下,作用一定时间后不但能明显地抑制新生血管的生成而且还能破坏即成的血管官腔,造成肿瘤内部大面积的坏死。但是CA-4对肿瘤的增殖抑制只限于其内部,对于肿瘤边缘血管较丰富区域的细胞增殖没有明显的抑制作用,因而常导致临床上治疗的失败。所幸的是,肿瘤边缘血管丰富区域的细胞对传统的放疗化疗方式较敏感,这就提示了我们CA-4具有联合用药的潜力和优势。本实验的研究旨在寻找不同作用机制的传统化疗药物与CA-4合用,克服两药自身临床应用的限制,以期在提高两者疗效的同时降低化疗药物的毒副作用。
方法和结果：通过肿瘤细胞增殖抑制实验结果表明,肿瘤坏死因子超家族成员TRAIL与CA-4合用在人结肠癌SW620和HCT116细胞中具有协同抑制细胞增殖的作用。而DAPI染色和PI单染结合流式细胞术显示这种协同抑制细胞增殖的作用是通过诱导细胞的凋亡来实现的,且两药合用引起的细胞凋亡率比单用组的凋亡率要高6-8倍。进一步根据western blot数据表明这种细胞凋亡的生物学效应可能是通过直接激活caspase酶级联反应而引起的。人结肠癌SW620裸小鼠移植瘤实验也证实TRAIL和CA-4合用后对SW620肿瘤的生长有显著的协同抑制作用,6.25mg/kg CA-4,30mg/kg TRAIL单用组以及合用组在给药20天后的抑瘤率分别为31.2%,59.2%和78.1%。此外肿瘤组织切片的TUNEL染色实验表明给药后确实能引起肿瘤组织中的细胞发生凋亡,并且根据TUNEL染色的荧光强度显示两药合用组引起的细胞凋亡要明显地高于两药单用组。为进一步揭示TRAIL和CA-4合用协同诱导凋亡的潜在机制,我们通过免疫荧光技术以及核质分离试验初步表明转录因子NF--κB的核转位过程受抑制是两药合用产生协同效果的前提。
结论：本实验通过相关抗肿瘤活性的评价,发现肿瘤坏死因子超家族成员TRAIL和CA-4联合用药可协同抑制人结肠癌细胞的增殖。并且采用分子生物学技术初步证实这种联合用药方式可能是通过抑制转录因子NF--κB的核转位,影响了NF-κB对下游相关基因的转录调控,诱导肿瘤细胞发生凋亡来实现的。基于TRAIL对肿瘤细胞的高选择性,而对正常组织细胞的毒性较小,TRAIL和CA-4两药的联合应用将为今后的临床合理用药提供新的思路和方案。
第三部分Combretastatin A-4诱导的自噬及其抗肿瘤作用的机制研究
目的：CA-4具有广谱的抗肿瘤活性,并且在远低于最大耐受剂量的浓度下,CA-4能导致肿瘤血管关闭、坏死进而发挥其抗肿瘤活性。随着临床试验的深入,人们发现CA-4不能完全发挥其药效,究其原因可能是其无法抑制化疗后残存肿瘤组织的增殖,但具体机制并不明确。而本部分实验的研究重点在于探索CA-4的抗肿瘤活性与自噬之间的关系,并希望通过对两者相互作用的分子机制的研究,解决CA-4在临床上运用受限制的原因,有针对性地开发以CA-4为核心的临床用药的配伍方案,以提高CA-4自身抗肿瘤活性,扩大CA-4的临床应用前景。
方法和结果：在本课题的研究中我们利用经典的检测自噬的手段,如吖啶橙染色法,GFP-LC3标示法以及LC3I/II比值法,观察到CA-4能诱导多种肿瘤细胞发生自噬。接着在给予了自噬抑制剂3-MA或者采用siRNA沉默技术降低调节细胞自噬过程中的重要因子Atg5的表达后,流式细胞术检测细胞凋亡率的结果表明CA-4诱导的.自噬属于保护性自噬,抑制自噬的发生可以增强CA-4诱导细胞凋亡的能力。进一步地通过western blot实验表明,在非InTOR依赖性的Bcl2-III型P13K复合物-beclin1信号转导通路中,Bcl-2的磷酸化水平明显上调,而在转入JNK-1siRNA抑制Bcl-2的磷酸化水平后发现CA-4引起的细胞自噬被抑制,而CA-4诱导的细胞凋亡能力明显增强。
结论：我们首次发现作为血管靶向药物CA-4能诱导多种细胞产生自噬,通过采用经典的细胞与分子生物学实验技术与方法初步阐明CA-4引起细胞自噬的分子机制,即Bcl2-PI3K-beclin1信号转导通路可能在CA-4引起的自噬中发挥着重要作用。而在这过程中自噬的发生极大限制了CA-4诱导细胞产生凋亡的能力,引入自噬抑制剂能显著得增强CA-4诱导细胞发生凋亡的能力。本项目的研究初步揭示了CA-4引起肿瘤细胞自噬的机制,及与其抗肿瘤作用之间的关系,而且可为其它可诱导肿瘤细胞自噬的抗肿瘤药物的临床应用提供重要的理论参考。
Introduction
Microtubules, a group of cytoskeletal proteins, are formed by highly dynamic assemblies of tubulin heterodimers, including a-tubulin and β-tubulin. Microtubules and their dynamic assemblies are directly involved in many biological processes, such as mitosis, intracellular transport, maintenance of cell morphology, and signal transduction. Since microtubules play important roles in the regulation of the mitotic progression, disrupting the assembly of microtubules can induce cell cycle arrest in G2/M phase and trigger the signals for programmed cell death. Combretastatin A-4(CA-4), a naturally occurring stilbene derived from the South African tree Combretum caffrum, shows potent cytotoxicity against a broad spectrum of human cancer cell lines. Based on its well-studied structure, a series of newly designed and synthesized compounds of CA-4have been screened for their antitumor activities in order to improve its antitumor activity. Furthermore, due to its limited clinical application, it is necessary to develop effective combination therapies with CA-4and other antitumor agents hoping to maximize the antitumor activity of each agent and reduce their cytotoxicity.
The current study falls into three parts:(1) To evaluate the potent antitumor activities of novel CA-4analogues in vitro and investigate the molecular mechanism underlying antitumor action of the novel compounds in carcinoma cells.(2) To explore the effective antitumor therapies by combining CA-4with other conventional chemotherapeutic agents against human carcinoma cells in order to maximize the antitumor activities of each agent and reduce their cytotoxicity.(3) To investigate the potential mechanisms causing the limited clinical application of CA-4and discover possible methods to overcome the limitation.
Part1Potent antitumor activity of the novel Combretastatin A-4analogue and its possible mechanisms
Objective:Combretastatin A-4exhibits potent cytotoxicity against a broad spectrum of human cancer cell lines. The extensively studied structure-activity relationships(S AR) of CA-4leads to numerous chemical modifications on it hoping to overcome its poor water solubility and improve its antitumor activity. Here, we identified some novel CA-4analogues with potent antitumor activities against various human cancer cells.
Methods and results:XN05, as a CA-4derivative, exhibited potent cytotoxicity on different human cancer cells via MTT methods, with the values of IC50less than2μM. Immunofluorescent staining and in vitro microtubule polymerization assay demonstrated that XN05could specifically inhibit the microtubule assembly, thus inducing G2/M phase arrest in two different human liver cancer cells, BEL-7402and SMMC-7721. Flow cytometry results further showed that either extension of treatment time or increasing treatment dosages of XN05could promote apoptosis in human liver cells.
Conclusion:XN05, a novel synthesized compound, possessed potent antitumor activity against human tumor cells via disruption of microtubule. Compared with CA-4, XN05exhibits equivalent or even better antiproliferative activity against human hepatocellular carcinoma cells, indicating that XN05can be a promising compound against human cancer cells. Collectively, such kind of modification indicated a successful structure change for getting stronger antitumor activity.
Part2The effective antitumor therapy by combiningCombretastatin A-4with TRAIL and its underlying mechanism
Objective:As a tumor vascular-targeting agent, CA-4not only aims to prevent the growth of new blood vessels but also targets at well-established tumor tissue endothelia, thus producing a rapid shutdown of tumor blood vessels. Unlike other conventional vascular-targeting agent, CA-4could exert antivascular effects at doses far below its maximum-tolerated dose (MTD) in animals. Such special characteristic made it an excellent candidate for combination therapies with conventional chemotherapeutic agents.
Methods and results:Here, using MTT methods plus flow cytometry assay we demonstrated that the combination of CA-4and TRAIL/Apo-2L exhibited synergistic cytotoxicity and enhanced apoptosis rates in two different human colon cancer cells SW620and HCT116. The synergistic antitumor activity of this combination was also observed in SW620xenografted athymic mice model experiment in vivo. Western blot results indicated that such combination therapy could directly activate the caspase cascades thus leading to the induction of apoptosis in cells. Furthermore, the inhibition of NF-κB translocation may play a role in this combination treatment induced apoptosis according to immunofluorescence staining assay.
Conclusion:In this study, we showed for the first time that the combination treatment of TRAIL/Apo-2L and CA-4had substantial synergistic antitumor activity against human colon cancer cells both in vitro and in vivo. Interestingly, our results demonstrated that CA-4could block the nuclear translocation of NF-κB upon the treatment of TRAIL/Apo-2L. These data together suggested that the combination of TRAIL/Apo-2L and CA-4might be an effective therapeutic strategy to achieve synergistic effect in patients harboring colon tumors.
Part3Autophagy may play a role in the antitumor activity of combretastatin A-4
Objective:Autophagy has been implicated in many physiological and pathological processes and it is widely reported that autophagy is associated with tumor formation and progession. However, its role in cancer is controversy. Here, we try to discover the possible relationship between autophagy and the cancer treatment of CA-4, hoping to find a possible answer to the limited therapeutic effect of CA-4.
Methods and results:By using acridine orange staining, GFP-LC3puncta formation assays and LC3conversion and its turnover assay, we discovered that CA-4could induce autophagy in various cancer cells. Inhibiting autophagy by using its inhibitor or siRNA silencing methods could increase CA-4induced apoptosis in cells according to flow cytometry assay. Finally we identified that JNK-Bcl-2signaling pathway may contribute to CA-4induced autophagy.
Conclusion:In this study, we showed at the first time that CA-4could induce autophagy in a wide variety of cancer cells. This induced autophagy could protect cells from CA-4induced apoptosis, which could be a possible mechanism for the limited clinical therapeutic effect of CA-4. Aiming at the protective effect of CA-4induced autophagy by exploring reasonable combination therapy will be an effective method to solve the problem of its limited clinical use.
本论文将主要分为以下三个部分：(1)对经过结构改造和修饰得到的一系列CA-4衍生物进行抗肿瘤活性的筛选以及相应的药效学评价,并对从中得到的抗肿瘤活性较好,并且毒副作用较低的候选化合物进行初步分子机制的研究。(2)寻找合理的联合配伍用药方案,将不同作用机制的抗肿瘤药物与CA-4合用,以期达到减少药物用量,提高疗效,减轻化疗药物的毒副作用的最佳效果。并进一步探讨两药合用的分子机制,力求为设计临床联合治疗方案提供新的方向。(3)为进一步推动CA-4的临床应用,解决其不能有效抑制化疗后残存的肿瘤细胞增殖的现象,我们对其抗肿瘤机制做了深入的研究,希望通过寻找限制CA-4抗肿瘤疗效的分子机制,采取相应的措施有针对性的进行克服。
第一部分Combretastatin A-4衍生物的抗肿瘤活性及其机制的研究
目的：作为新型的微管聚合抑制剂,CA-4由于其结构简单,抗癌活性显著,因而备受药物化学家的关注。为进一步提高其疗效,降低毒性,我们通过对一系列经过修饰的CA-4衍生物抗癌活性的筛选,旨在寻找具有广谱抗肿瘤活性的新型CA-4类化合物。
方法和结果：XN05是一个新合成的Combretastatin A-4的类似物。首先我们通过MTT法在六株不同组织来源的人肿瘤细胞上(SGC、PC-3、ECA、MCF-7A2780和A549)对XN05的抗肿瘤活性进行细胞水平检测。结果显示其具有较强的抗肿瘤活性,IC5o值均小于2μM,而在两株肝癌细胞BEL-7402和SMMC-7721上的抗肿瘤活性比CA-4更强。值得注意的是,在正常的肝实质细胞HL-7702上XN05的IC50值要明显高于CA-4,提示我们该化合物对正常组织来源的细胞杀伤作用相对较弱。进一步通过离体的微管蛋白聚合实验和免疫荧光实验的结果证实XN05也是以微管为靶点发挥其抗肿瘤活性,并且XN05抑制微管聚合的能力与其母体CA-4类似。引起细胞G2/M期阻滞是微管类药物的重要特征之一,运用流式细胞术在经过XN05处理的两株肝癌细胞中均能观察到明显的G2/M期细胞周期阻滞现象,并且该现象存在浓度和时间依赖性。Western blot结果显示XN05引起的G2/M期阻滞现象也可能与细胞周期关键蛋白cdc2, CDK7以及cyclin B1的表达发生异常有关。随着药物作用时间的延长我们发现处于G2/M期阻滞的细胞最终走向了细胞的凋亡,进一步地肿瘤细胞的生长受到了抑制。
结论：本研究表明经过结构修饰的新型CA-4衍生物XN05具有明确的作用靶点,通过抑制微管的聚合使得细胞阻滞在G2/M期,并最终导致细胞走向凋亡发挥其抗肿瘤活性。此外,我们还发现XN05作用于BEL-7402和SMMC-7721肝癌细胞后引起的不同程度细胞周期阻滞可能与XN05能够对不同的周期蛋白(Cyclins)及周期蛋白依赖性激酶(CDKs)的表达产生影响相关。XN05在体外有着广谱,且优于同系列化合物的抗肿瘤活性,尤其是其在肝癌细胞株中表现出的细胞毒特异性使其非常有希望成为临床治疗的新型抗肿瘤微管类化合物。
第二部分Combretastatin A-4与化疗药物联合用药的抗肿瘤活性评价及机制的研究
目的：随着对CA-4抗肿瘤活性研究的深入,人们发现CA-4还是作用于肿瘤血管的靶向药物。据报道,CA-4在无细胞毒浓度下,作用一定时间后不但能明显地抑制新生血管的生成而且还能破坏即成的血管官腔,造成肿瘤内部大面积的坏死。但是CA-4对肿瘤的增殖抑制只限于其内部,对于肿瘤边缘血管较丰富区域的细胞增殖没有明显的抑制作用,因而常导致临床上治疗的失败。所幸的是,肿瘤边缘血管丰富区域的细胞对传统的放疗化疗方式较敏感,这就提示了我们CA-4具有联合用药的潜力和优势。本实验的研究旨在寻找不同作用机制的传统化疗药物与CA-4合用,克服两药自身临床应用的限制,以期在提高两者疗效的同时降低化疗药物的毒副作用。
方法和结果：通过肿瘤细胞增殖抑制实验结果表明,肿瘤坏死因子超家族成员TRAIL与CA-4合用在人结肠癌SW620和HCT116细胞中具有协同抑制细胞增殖的作用。而DAPI染色和PI单染结合流式细胞术显示这种协同抑制细胞增殖的作用是通过诱导细胞的凋亡来实现的,且两药合用引起的细胞凋亡率比单用组的凋亡率要高6-8倍。进一步根据western blot数据表明这种细胞凋亡的生物学效应可能是通过直接激活caspase酶级联反应而引起的。人结肠癌SW620裸小鼠移植瘤实验也证实TRAIL和CA-4合用后对SW620肿瘤的生长有显著的协同抑制作用,6.25mg/kg CA-4,30mg/kg TRAIL单用组以及合用组在给药20天后的抑瘤率分别为31.2%,59.2%和78.1%。此外肿瘤组织切片的TUNEL染色实验表明给药后确实能引起肿瘤组织中的细胞发生凋亡,并且根据TUNEL染色的荧光强度显示两药合用组引起的细胞凋亡要明显地高于两药单用组。为进一步揭示TRAIL和CA-4合用协同诱导凋亡的潜在机制,我们通过免疫荧光技术以及核质分离试验初步表明转录因子NF--κB的核转位过程受抑制是两药合用产生协同效果的前提。
结论：本实验通过相关抗肿瘤活性的评价,发现肿瘤坏死因子超家族成员TRAIL和CA-4联合用药可协同抑制人结肠癌细胞的增殖。并且采用分子生物学技术初步证实这种联合用药方式可能是通过抑制转录因子NF--κB的核转位,影响了NF-κB对下游相关基因的转录调控,诱导肿瘤细胞发生凋亡来实现的。基于TRAIL对肿瘤细胞的高选择性,而对正常组织细胞的毒性较小,TRAIL和CA-4两药的联合应用将为今后的临床合理用药提供新的思路和方案。
第三部分Combretastatin A-4诱导的自噬及其抗肿瘤作用的机制研究
目的：CA-4具有广谱的抗肿瘤活性,并且在远低于最大耐受剂量的浓度下,CA-4能导致肿瘤血管关闭、坏死进而发挥其抗肿瘤活性。随着临床试验的深入,人们发现CA-4不能完全发挥其药效,究其原因可能是其无法抑制化疗后残存肿瘤组织的增殖,但具体机制并不明确。而本部分实验的研究重点在于探索CA-4的抗肿瘤活性与自噬之间的关系,并希望通过对两者相互作用的分子机制的研究,解决CA-4在临床上运用受限制的原因,有针对性地开发以CA-4为核心的临床用药的配伍方案,以提高CA-4自身抗肿瘤活性,扩大CA-4的临床应用前景。
方法和结果：在本课题的研究中我们利用经典的检测自噬的手段,如吖啶橙染色法,GFP-LC3标示法以及LC3I/II比值法,观察到CA-4能诱导多种肿瘤细胞发生自噬。接着在给予了自噬抑制剂3-MA或者采用siRNA沉默技术降低调节细胞自噬过程中的重要因子Atg5的表达后,流式细胞术检测细胞凋亡率的结果表明CA-4诱导的.自噬属于保护性自噬,抑制自噬的发生可以增强CA-4诱导细胞凋亡的能力。进一步地通过western blot实验表明,在非InTOR依赖性的Bcl2-III型P13K复合物-beclin1信号转导通路中,Bcl-2的磷酸化水平明显上调,而在转入JNK-1siRNA抑制Bcl-2的磷酸化水平后发现CA-4引起的细胞自噬被抑制,而CA-4诱导的细胞凋亡能力明显增强。
结论：我们首次发现作为血管靶向药物CA-4能诱导多种细胞产生自噬,通过采用经典的细胞与分子生物学实验技术与方法初步阐明CA-4引起细胞自噬的分子机制,即Bcl2-PI3K-beclin1信号转导通路可能在CA-4引起的自噬中发挥着重要作用。而在这过程中自噬的发生极大限制了CA-4诱导细胞产生凋亡的能力,引入自噬抑制剂能显著得增强CA-4诱导细胞发生凋亡的能力。本项目的研究初步揭示了CA-4引起肿瘤细胞自噬的机制,及与其抗肿瘤作用之间的关系,而且可为其它可诱导肿瘤细胞自噬的抗肿瘤药物的临床应用提供重要的理论参考。
Introduction
Microtubules, a group of cytoskeletal proteins, are formed by highly dynamic assemblies of tubulin heterodimers, including a-tubulin and β-tubulin. Microtubules and their dynamic assemblies are directly involved in many biological processes, such as mitosis, intracellular transport, maintenance of cell morphology, and signal transduction. Since microtubules play important roles in the regulation of the mitotic progression, disrupting the assembly of microtubules can induce cell cycle arrest in G2/M phase and trigger the signals for programmed cell death. Combretastatin A-4(CA-4), a naturally occurring stilbene derived from the South African tree Combretum caffrum, shows potent cytotoxicity against a broad spectrum of human cancer cell lines. Based on its well-studied structure, a series of newly designed and synthesized compounds of CA-4have been screened for their antitumor activities in order to improve its antitumor activity. Furthermore, due to its limited clinical application, it is necessary to develop effective combination therapies with CA-4and other antitumor agents hoping to maximize the antitumor activity of each agent and reduce their cytotoxicity.
The current study falls into three parts:(1) To evaluate the potent antitumor activities of novel CA-4analogues in vitro and investigate the molecular mechanism underlying antitumor action of the novel compounds in carcinoma cells.(2) To explore the effective antitumor therapies by combining CA-4with other conventional chemotherapeutic agents against human carcinoma cells in order to maximize the antitumor activities of each agent and reduce their cytotoxicity.(3) To investigate the potential mechanisms causing the limited clinical application of CA-4and discover possible methods to overcome the limitation.
Part1Potent antitumor activity of the novel Combretastatin A-4analogue and its possible mechanisms
Objective:Combretastatin A-4exhibits potent cytotoxicity against a broad spectrum of human cancer cell lines. The extensively studied structure-activity relationships(S AR) of CA-4leads to numerous chemical modifications on it hoping to overcome its poor water solubility and improve its antitumor activity. Here, we identified some novel CA-4analogues with potent antitumor activities against various human cancer cells.
Methods and results:XN05, as a CA-4derivative, exhibited potent cytotoxicity on different human cancer cells via MTT methods, with the values of IC50less than2μM. Immunofluorescent staining and in vitro microtubule polymerization assay demonstrated that XN05could specifically inhibit the microtubule assembly, thus inducing G2/M phase arrest in two different human liver cancer cells, BEL-7402and SMMC-7721. Flow cytometry results further showed that either extension of treatment time or increasing treatment dosages of XN05could promote apoptosis in human liver cells.
Conclusion:XN05, a novel synthesized compound, possessed potent antitumor activity against human tumor cells via disruption of microtubule. Compared with CA-4, XN05exhibits equivalent or even better antiproliferative activity against human hepatocellular carcinoma cells, indicating that XN05can be a promising compound against human cancer cells. Collectively, such kind of modification indicated a successful structure change for getting stronger antitumor activity.
Part2The effective antitumor therapy by combiningCombretastatin A-4with TRAIL and its underlying mechanism
Objective:As a tumor vascular-targeting agent, CA-4not only aims to prevent the growth of new blood vessels but also targets at well-established tumor tissue endothelia, thus producing a rapid shutdown of tumor blood vessels. Unlike other conventional vascular-targeting agent, CA-4could exert antivascular effects at doses far below its maximum-tolerated dose (MTD) in animals. Such special characteristic made it an excellent candidate for combination therapies with conventional chemotherapeutic agents.
Methods and results:Here, using MTT methods plus flow cytometry assay we demonstrated that the combination of CA-4and TRAIL/Apo-2L exhibited synergistic cytotoxicity and enhanced apoptosis rates in two different human colon cancer cells SW620and HCT116. The synergistic antitumor activity of this combination was also observed in SW620xenografted athymic mice model experiment in vivo. Western blot results indicated that such combination therapy could directly activate the caspase cascades thus leading to the induction of apoptosis in cells. Furthermore, the inhibition of NF-κB translocation may play a role in this combination treatment induced apoptosis according to immunofluorescence staining assay.
Conclusion:In this study, we showed for the first time that the combination treatment of TRAIL/Apo-2L and CA-4had substantial synergistic antitumor activity against human colon cancer cells both in vitro and in vivo. Interestingly, our results demonstrated that CA-4could block the nuclear translocation of NF-κB upon the treatment of TRAIL/Apo-2L. These data together suggested that the combination of TRAIL/Apo-2L and CA-4might be an effective therapeutic strategy to achieve synergistic effect in patients harboring colon tumors.
Part3Autophagy may play a role in the antitumor activity of combretastatin A-4
Objective:Autophagy has been implicated in many physiological and pathological processes and it is widely reported that autophagy is associated with tumor formation and progession. However, its role in cancer is controversy. Here, we try to discover the possible relationship between autophagy and the cancer treatment of CA-4, hoping to find a possible answer to the limited therapeutic effect of CA-4.
Methods and results:By using acridine orange staining, GFP-LC3puncta formation assays and LC3conversion and its turnover assay, we discovered that CA-4could induce autophagy in various cancer cells. Inhibiting autophagy by using its inhibitor or siRNA silencing methods could increase CA-4induced apoptosis in cells according to flow cytometry assay. Finally we identified that JNK-Bcl-2signaling pathway may contribute to CA-4induced autophagy.
Conclusion:In this study, we showed at the first time that CA-4could induce autophagy in a wide variety of cancer cells. This induced autophagy could protect cells from CA-4induced apoptosis, which could be a possible mechanism for the limited clinical therapeutic effect of CA-4. Aiming at the protective effect of CA-4induced autophagy by exploring reasonable combination therapy will be an effective method to solve the problem of its limited clinical use.
引文
1. Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nat Rev Cancer 2004;4(4):253-265.
2. Mollinedo F, Gajate C. Microtubules, microtubule-interfering agents and apoptosis. Apoptosis 2003;8(5):413-450.
3. Pohzzi D, Pratesi G, Tortoreto M, et al. A novel taxane with improved tolerability and therapeutic activity in a panel of human tumor xenografts. Cancer Res 1999;59(5):1036-1340.
4. Schiff PB, Horwitz SB. Taxol stabilizes microtubules in mouse fibroblast cells. Proc. Natl.Acad.Sci. USA 1980;77(3):1561-1565.
5. Pellegrini F, Budman DR. Review:tubulin function, action of antitubulin drugs, and new drug development. Cancer Invest.2005;23(3):264-273.
6. Grosios K, Holwell SE, McGown AT, Pettit GR, Bibby MC. In vivo and in vitro evaluation of combretastatin A-4 and its sodium phosphate prodrug. Br J Cancer 1999;81:1318-1327.
7. Nihei Y, Suzuki M, Okamo A, Tsuiji T, et al. Evaluation of antivascular and antimitotic effects of tubulin binding agents in solid tumour therapy. Jpn J Cancer Res 1999;90:1387-1396.
8. Tozer GM, Prise VE, Wilson J, Locke RJ, et al. Combretastatin A-4 phosphate as a tumour vascular targetting agent:early effects in tumours and normal tissues. Cancer Res 1999;59:1626-1634.
9. Pettit GR, Temple C Jr, Narayanan VL, Varma R, et al. Antineo-plastic agents 322. synthesis of combretastatin A 4 prodrugs. Anticancer Drug Des 1995; 10(4):299-309.
10. Ahmed B, Van Eijk LI, Bouma-Ter Steege JC, Van Der Schaft DW, et al. Vascular targeting effect of combretastatin A4 phosphate dominates the inherent angiogenesis inhibitory activity. Int J Cancer 2003;105(1):20-25.
11. Vincent L, Kermani P, Young LM, Cheng J, et al.Combretastatin A4 phosphate induces rapid regression of tumor neovessels and growth throush interference with vascular endothelial-cadherin signaling. J Clin Invest 2005; 115(11):2992-3006.
12. Dark GG, Hill SA, Prise VE, et al. Combretastatin A-4, an agent that displays potent and selective toxicity toward tumor vasculature.Cancer Res 1997;57:1829-1834.
13. Nagaiah G, Remick SC. Combretastatin A4 phosphate:a novel vascular disrupting agent. Future Oncol 2010;6(8):1219-28.
14. Siemann DW, Mercer E, Lepler S, et al. Vascular targeting agents enhance chemotherapeutic agent activities in solid tumor therapy. Int J Cancer 2002;99(1):1-6.
15. Wildiers H, Ahmed B, Guetens G, De Boeck G, et al. Combretastatin A4 phosphate enhances CPT-11 activity independently of the administration sequence. Eur J Cancer 2004;40(2):284-290.
16. Nelkin BD, Ball DW. Combretastatin A4 and doxorubicin combination treatment is effective in a prelinical model of human medulary thyroid carcinoma. Oncol Rep 2001;8(1):157-160.
17. Grosios K, Loadman PM, Swaine DJ, Pettit GR et al. Combination chemotherapy with combretastatin A4 phosphate and 5-fluorouracil in an experimental marine colon adenocarcinoma. Anti Cancer Res 2000;20(1A):229-233.
18. Badn W, Kalliomaki S, Widegren B, Sjogren HO. Low-dose combretastatin A4 phosphate enhances the immune response of tumor hosts to experimenta colon carcinoma. Clin Cancer Res 2006;12(15):4714-4719.
19. Larochelle S, Merrick KA, Terret ME, Wohlbold L, et al. Requirements for Cdk7 in the assembly of Cdkl/cyclin B and activation of Cdk2 revealed by chemical genetics in human cells. Mol Cell 2007;25(6):839-850.
20. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature 2008;451:1069-1075.
21. Yang Z, Klionsky DJ. Eaten alive:a history of macroautophagy. Nat Cell Biol 2010;12:814-822.
22. Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Self-eating and self-killing: crosstalk between autophagy and apoptosis.Nat Rev Mol Cell Biol 2007;8(9):741-752.
23. Kang C, Avery L. To be or not to be, the level of autophagy is the question:dual roles of autophagy in the survival response to starvation. Autophagy 2008;4:82-84.
24. Xie Z, Klionsky DJ. Autophagosome formation:core machinery and adaptations. Nat Cell Biol 2007;9:1102-1109.
25. Levine B, Klionsky D J. Development by selfdigestion:molecular mechanisms and biological functions of autophagy. Dev Cell 2004;6:463-477.
26. Amaravadi RK, Lippincott-Schwartz J, Yin XM, Weiss WA, et al. Principles and current strategies for targeting autophagy for cancer treatment.Clin Cancer Res 2011;17(4):654-666.
27. Jager S, Bucci C, Tanida I, Ueno T, et al. Role for Rab7 in maturation of late autophagic vacuoles. J Cell Sci 2004;117:4837-4848.
28. Cooney MM, Radivoyevitch T, Dowlati A, Overmoyer B, et al. Cardiovascular safety profile of coalbretastatin A4 phosphate in a single dose phase I study in patients with advanced cancer. Clin Cancer Res 2004; 10:96-1013.
29. Stevenson JP, Rosen M, Sun W, Gallagher M, et al. Phase I trial of the antivascular agent combretastatin A4 phosphate on a 5 day schedule to patients with cancer: magnetic resonance imaging evidence for altered tumor blood flow. J Clin Oncol 2003;21(23):4428-4438.
30. Yi-He Ling, Carmen Tornos, Roman Perez-Soler. Phosphorylation of Bcl-2 is a marker of M phase events and not a determinant of apoptosis. J Biol Chem 1998;273:18984-18991.
31. Homesley HD, Filiaci V, Gibbons SK, Long HJ, et al. A randomized phase III trial in advanced endometrial carcinoma of surgery and volume directed radiation followed by cisplatin and doxorubicin with or without paclitaxel:a Gynecologic Oncology Group study. Gynecol Oncol 2009; 112(3):543-552.
32. Jin H, Yang R, Fong S, Totpal K, et al. Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand cooperates with chemotherapy to inhibit orthotopic lung tumor growth and improve survival. Cancer Res 2004;64:4900-4905.
33. Denekamp J.Endothelial cell proliferation as a novel approach to targeting tumour therapy. Br J Cancer 1982;45:136-139.
34. Denekamp J. Review article:angiogenesis, neovascular proliferation and vascular pathophysiology as targets for cancer therapy. Br JRadiol 1993;66:181-196.
35. Tozer GM, Prise VE, Wilson J, Locke RJ, et al. Combretastatin A-4 phosphate as a tumor vascular-targeting agent:early effects in tumors and normal tissues. Cancer Res1999;59:1626-1634.
36. Malcontenti-Wilson C, Muralidharan V, Skinner S, Christophi C, et al. Combretastatin A4 prodrug study of effect on the growth and the microvasculature of colorectal liver metastases in a murine model. Clin Cancer Res 2001;7:1052-1060.
37. Wang S, El-Deiry WS. TRAIL and apoptosis induction by TNF-family death receptors. Oncogene 2003;23:8628-8633.
38. H. Zhu, W.J. Ding, R. Wu, Q.J. Weng, et al. Synergistic anti-cancer activity by the combination of TRAIL/APO-2L and celastrol. Cancer Invest 2010;28:23-32.
39. Koschny R, Walczak H, Ganten TM. The promise of TRAIL-potential and risks of a novel anticancer therapy. J Mol Med 2007;85:923-935.
40. Fisher MJ, Virmani AK, Wu L, et al. Nucleotide substitution in the ectodomain of trail receptor DR4 is associated with lung cancer and head and neck cancer. Clin Cancer Res 2001;7:1688-1697.
41. Shin MS, Kim HS, Lee SH, et al. Mutations of tumor necrosis factor-related apoptosis-inducing ligand receptor 1(TRAIL-R1) and receptor 2 (TRAIL-R2) genes in metastaticbreast cancers. Cancer Res 2001;61:4942-4946.
42. Jeng YM, Hsu HC. Mutation of the DR5/TRAIL receptor 2 gene is infrequent in hepatocellular carcinoma. Cancer Lett 2002; 181:205-208.
43. Kim YS, Schwabe RF, Qian T, et al. TRAIL-mediated apoptosis requires NF-kappaB inhibition and the mitochondrial permeability transition in human hepatoma cells.Hepatology 2002;36:1498-1508.
44. Ehrhardt H, Fulda S, Schmid I, et al. TRAIL induced survival and proliferation in cancer cells resistant towards TRAIL-induced apoptosis mediated by NF-kappaB. Oncogene 2003;22:3842-3852.
45. Okano H, Shiraki K, Inoue H, et al. Cellular FLICE/caspase-8-inhibitory protein as a principal regulator of cell death and survival in human hepatocellular carcinoma. Lab Invest 2003;93:1033-1043.
46. Fulda S, Meyer E, Debatin KM. Inhibition of TRAILinduced apoptosis by Bcl-2 overexpression. Oncogene 2002;21:2283-2294.
47. Chou TC, Talalay P.Quantitative analysis of dose-effect relationships:the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27-55.
48. Nguyen T, Zhang XD, Hersey P. Relative resistance of fresh isolates of melanoma to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Clin Cancer Res 2001;7:966s-973s.
49. Strater J, Hinz U, Walczak H, Mechtersheimer G. Expression of TRAIL and TRAIL receptors in colon carcinoma:TRAIL-R1 is an independent prognostic parameter. Clin Cancer Res 2002;8:3734-3740.
50. Lin CW, Shen SC, Chien CC, Yang LY, et al.12-O-tetradecanoylphorbol-13-acetate-induced invasion/migration of glioblastoma cells through activating PKCalpha/ERK/NF-kappaB-dependent MMP-9 expression. J Cell Physiol 2010; 225(2):472-481.
51. Darzynkiewicz Z, Zhao H. Detection of DNA strand breaks in apoptotic cells by flow-and image-cytometry. Methods Mol Biol 2010;682:91-101.
52. Horak P, Pils D, Haller G, et al. Contribution of epigenetic silencing of tumor necrosis factor-related apoptosis inducing ligand receptor 1 (DR4) to TRAIL resistance and ovarian cancer. Mol Cancer Res 2005;3:335-343.
53. Larribere L, Khaled M, Tartare-Deckert S, et al. PI3K mediates protection against TRAIL-induced apoptosis in primary human melanocytes. Cell Death Differ 2004; 11:1084-91.
54. Weldon CB, Parker AP, Patten D, et al. Sensitization of apoptotically resistant breast carcinoma cells to TNF and TRAIL by inhibition of p38 mitogen-activated protein kinase signaling. Int J Oncol 2004;24:1473-80.
55. Singh TR, Shankar S, Chen X, Asim M, Srivastava RK. Synergistic interactions of chemotherapeutic drugs and tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand on apoptosis and on regression of breast carcinoma in vivo. Cancer Res 2003;63:5390-5400.
56. Baldwin, A. S., Jr. Series introduction:the transcription factor NF-κB and human disease. J Clin Investig 2001; 107:3-6.
57. Baeuerle, P. A. and Baltimore, D. NF-κB:ten years after. Cell 1996;87:13-20.
58. Baeuerle, P.A.and Henkel, T. Function and activation of NF-κB in the immunesystem. Annu Rev Immunol 1994;12:141-179.
59. Verma IM, Stevenson JK, Schwarz EM, Van Antwerp D, et al. Rcl/NF-κB/IκB family:intimate tales of association and dissociation. Genes Dev 1995;9:2723-2735.
60. Hayakawa M, Miyashita H, Sakamoto I, Kitagawa M, et al. Evidence that reactive oxygen species do not mediate NF-kappaB activation. EMBO J 2003;22:3356-3366.
61. Xufeng Chen, Karthikeyan Kandasamy, Rakesh K. Srivastava. Differential Roles of RelA (p65) and c-Rel Subunits of Nuclear Factor κB in Tumor Necrosis Factor-related Apoptosis-inducing Ligand Signaling. Cancer Res 2003;63:1059-1066.
62. Mizushima N. Autophagy:process and function. Genes Dev 2007;21:2861-2873.
63. Levine B, Klionsky DJ. Development by self-digestion:molecular mechanisms and biological functions of autophagy. Dev Cell 2004;6:463-477.
64. Uttenweiler A, Mayer A. Microautophagy in the yeast Saccharomyces cerevisiae. Methods Mol Biol 2008;445:245-259.
65. Hayashi-Nishino M, et al. A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat Cell Biol 2009; 11:1433-1437.
66. Nakai A, Yamaguchi O, Takeda T, Higuchi Y, et al. The role of antophagy in cardiomyocytes in the basal state and in re-sponse to hemodynamic stress. Nat Med 2007;13(5):619-624.
67. Sun Y, Liu JH, Jin L, Lin SM, Yang Y, Sui YX, Shi H. Over-expression of the Beclinl gene upregulates chemosensitivity to anti-cancer drugs by enhancing therapy-induced apoptosis in cervix squamous carcinoma CaSki cells. Cancer Lett 2010;294(2):204-10.
68. Liu YL, Yang PM, Shun CT, Wu MS, et al. Autophagy potentiates the anti-cancer effects of the histone deacetylase inhibitors in hepatocellular carcinoma. Autophagy 2010;6(8):1057-65.
69. Mizushima N. Methods for monitoring autophagy. Int J Biochem Cell Biol 2004;36:2491-2502.
70. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, et al. LC3, a mammalian homologue of yeast Apg8p, is localized inautophagosome membranes after processing. EMBO J 2000; 19:5720-5728.
71. Pyo JO, Jang MH, Kwon YK, Lee HJ, et al. Essential roles of Atg5 and FADD in autophagic cell death:dissection of autophagic cell death into vacuole formationand cell death. J Biol Chem 2005;280:20722-20729.
72. Mizushima N, Levine B,Cuervo AM, et al. Autophagy fights disease through cellular self-digestion. Nature 2008;451(7182):1069-1075.
73. Anderson HL, Yap JT, Miller MP, Robbins A, et al. Assessment of pharmacodynamic vascular response in a phase I trial of combretastatin A4 phosphate. J Clin Oncol.2003;21(15):2823-30.
74. Cuervo, A. M. Autophagy:in sickness and in health. Trends Cell Biol 2004;14:70-77.
75. Ajabnoor GM, Crook T, Coley HM. Paclitaxel resistance is associated with switch from apoptotic to autophagic cell death in MCF-7 breast cancer cells.Cell Death Dis 2012;26(3):e260.
76. Liu YL, Yang PM, Shun CT, Wu MS, et al. Autophagy potentiates the anti-cancer effects of the histone deacetylase inhibitors in hepatocellular carcinoma.Autophagy 2010;6(8):1057-65.
77. Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene 2004;23:2891-2906.
78. Petiot A, Ogier-Denis E, Blommaart EF, Meijer AJ et al. Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. JBiol Chem 2000; 275:992-998.
79. Amaravadi RK, Yu D, Lum JJ, et al. Autophagy inhibition enhances therapy induced apoptosis in a Myc-induced model of lymphoma.J Clin Invest 2007;117(2):326-336.
80. Yongjie Wei,Sophie Pattingre, Sangita Sinha, Michael Bassik and Beth Levine. JNK1-Mediated Phosphorylation of Bcl-2Regulates Starvation-Induced Autophagy. Molecular Cell 2008;30:678-688.
1. Lowe J, Li H, Downing KH et al. Refined structure of αβ-tubulin at 3.5 A resolution. J Mol Biol 2001;13(5):1045-1057.
2. Zhou J, Joshi HC. Exquisite modulation of micro tubule dynamics is required for chromosome congression during mitosis. Mol Biol Cell 2001; 12:173A.
3. Wittmann T, Bokoch GM, Waterman-Storer CM. Regulation of microtubule destabilizing activity of Op18/stathmin downstream of Racl. J Biol Chem 2004;279(7):6196-6203.
4. Alli E, Bash-Babula J, Yang JM, et al. Effect of stathmin on the sensitivity to antimicrotubule drugs in human breast cancer. Cancer Res 2002;62(23):6864-6869.
5. Quasthof S, Hartung HP. Chemotherapy-induced peripheral neuropathy.J Neurol 2002;249(1):9-17.
6. Ke Y, Ye K, Grossniklaus HE, Archer DR, Joshi HC, Kapp JA. Noscapine inhibits tumor growth with little toxicity to normal tissues or inhibition of immune responses. Cancer Immunol Immunother 2000;49:217-225.
7. Ye K, Ke Y, Keshava N, Shanks J, Kapp JA, Tekmal RR, Petros J, Joshi HC. Opium alkaloidnoscapine is an antitumor agent that arrests metaphase and induces apoptosis in dividing cells. Proc Natl Acad Sci USA 1998;95:1601-1606.
8. Heidemann S. Microtubules, leukemia, and cough syrup. Blood 2006; 107:2216-2217.
9. Nogales E, Wolf SG, Khan IA, Luduena RF, Downing KH. Structure of tubulin at 6.5 A and location of the taxol-binding site. Nature 1995;375:424-427.
10. Morris PG, Fornier MN. Microtubule active agents:Beyond the taxane frontier. Clin Cancer Res 2008;14:7167-7172.
11. Seve P, Dumontet C. Is class III beta-tubulin a predictive factor in patients receiving tubulinbinding agents? Lancet Oncol 2008;9:168-175.
12. Goodin S, Kane MP, Rubin EH. Epothilones:Mechanism of action and biologic activity. J Clin Oncol 2004;22:2015-2025.
13. Thomas ES, Gomez HL, Li RK, Chung HC, et al. Ixabepilone plus capecitabine for metastatic breast cancer progressing after anthracycline and taxane treatment.J Clin Oncol 2007;25:5210-5217.
14. Mooberry SL, Tien G, Hernandez AH, et al. Laulimalide and isolaulimalide, new paclitaxel-like microtubule stabilizing agent. Cancer Res 1999;59 (3):653-660.
15. Wilmes A, Bargh K, Kelly C, Northcote PT, Miller JH. Peloruside A synergizes with othermicrotubule stabilizing agents in cultured cancer cell lines. Mol Pharmacol 2007;4:269-280.
16. Hamel E, Day BW, Miller JH, Jung MK, et al. Synergistic effects of peloruside A and laulimalide with taxoid site drugs, but not with each other, on tubulin assembly. Mol Pharmacol 2006;70:1555-1564.
17. Jordan MA. Mechanism of action of antitumor drugs that interact with microtubules and tubulin. Curr Med Chem Anti-Cane Agents 2002;2(1):1-17.
18. Lobert S, Correia JJ. Energetics of vines, alkaloid interactions with tubulin. Methods Enzymol 2000;323:77-103.
19. Iwasaki S, Kobayashi H, Furukawa J, Namikoshi M, et al. Studies on macrocyclic lactone antibiotics.Ⅶ. Structure of a phytotoxin "rhizoxin" produced by Rhizopus chinensis. J Antibiot 1984;37:354-362.
20. Gupta S, Bhattacharyya B. Antimicrotubular drugs binding to vinca domain of tubulin. Mol Cell Biochem 2003;253:41-47.
21. Bai RL, Pettit GR, Hamel E. Binding of dolastatin 10 to tubulin at a distinct site for peptideantimitotic agents near the exchangeable nucleotide and vinca alkaloid sites. J Biol Chem 1990;265:17141-17149.
22. Luduena RF, Roach MC, Prasad V, Lacey E. Effect of phomopsin A on the alkylation of tubulin. Biochem Pharmacol 1990;39:1603-1608.
23. Giannakakou P, Gussio R, Nogales E, et al. A common pharmacophore for epothilone and taxanes:molecular basis for drug resistance conferred by tubulin mutations in human cancer cells. Proc Nat Acad Sci USA 2000;97(6):2904-2909.
24. Krishnamurthy G, Cheng W, Lo MC, et al. Biophysical characterization of the interactions of HTI-286 with tubulin heterodimer and microtubules. Biochemistry 2003;42(46):13484-13495.
25. Chen SM, Meng LH, Ding J. New microtubule-inhibiting anticancer agents. Expert Opin Investig Drugs 2010; 19:329-343.
26. Cormier A, Marchand M, Ravelli RB, Knossow M, Gigant B. Structural insight into theinhibition of tubulin by vinca domain peptide ligands. EMBO Rep 2008;9:1101-1106.
27. Boukari H, Sackett DL, Schuck P, Nossal RJ. Single-walled tubulin ring polymers. Biopolymers 2007;86:424-436.
28. Risinger AL, Giles FJ, Mooberry SL. Microtubule dynamics as a target in oncology. Cancer Treat Rev 2009;35:255-261.
29. Bhattacharyya B, Panda D, Gupta S, Banerjee M. Anti-mitotic activity of colchicine and thestructural basis for its interaction with tubulin. Med Res Rev 2008;28:155-183.
30. Pettit GR, Singh SB, Hamel E, Lin CM, et al. Isolation and structure of the strong cell growth and tubulin inhibitor combretastatin A-4. Experientia 1989;45:209-211.
31. Hamel E. Antimitotic natural products and their interactions with tubulin. Med Res Rev 1996; 16:207-231.
32. Mann J. Natural products in cancer chemotherapy:Past, present and future. Nat Rev Cancer 2002;2:143-148.
33. Iyer S, Chaplin DJ, Rosenthal DS, Boulares AH, et al. Induction of apoptosis in proliferating human endothelial cells by the tumor-specific antiangiogenesis agent combretastatin A-4. Cancer Res 1998;58:4510-4514.
34. Ahmed B, Van Eijk LI, Bouma-Ter Steege JC, Van Der Schaft DW, et al. Vascular targeting effect of combretastatin A-4 phosphate dominates the inherent angiogenesis inhibitory activity. Int J Cancer 2003; 105:20-25.
35. Siemann DW, Mercer E, Lepler S, Rojiani AM. Vascular targeting agents enhance chemotherapeutic agent activities in solid tumor therapy. Int J Cancer 2002;99:1-6.
36. Mabjeesh NJ, Escuin D, LaVallee TM, Pribluda VS, et al.2-ME inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF. Cancer Cell 2003;3:363-375.
37. D'Amato RJ, Lin CM, Flynn E, Folkman J, Hamel E.2-Methoxyestradiol, an endogenousmammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site. Proc Natl Acad Sci USA 1994;91:3964-3968.
38. Desbene S, Giorgi-Renault S. Drugs that inhibit tubulin polymerization:The particular case of podophyllotoxin and analogues. Curr Med Chem Anticancer Agents 2002;2:71-90.
39. Peyrot V, Leynadier D, Sarrazin M, Briand C, et al. Mechanism of binding of the new antimitotic drug MDL 27048 to the colchicine site of tubulin:Equilibrium studies. Biochemistry 1992;31:11125-11132.
40. Smith DB, Ewen C, Mackintosh J, Fox BW, et al. A phase I and pharmacokinetic study of amphethinile. Br J Cancer 1988;57:623-627.
41. Edward DR, Berg WB, Sprigss DR, et al.MDL-27048, a novel syntheticmicrotubule inhibitor, exerts curative antitumor activity in vivo, show efficacy toward multi drug-resistant tumor cells, and lacks neurotoxicity. Clin Cancer Res 2004;s6:563.
42. Segal MS, Glodstein MM, Attinger EO. The use of noscapine (narcotine) as an antitussive agent. Dis Chest 1957;32:305-309.
43. Ye K, Ke Y, Keshava N, Shanks J, et al. Opium alkaloid noscapine is an antitumor agent that arrests metaphase and induces apoptosis in dividing cells. Proc Natl Acad Sci USA 1998;95:1601-1606.
44. Landen JW, Hau V, Wang M, Davis T, et al. Noscapine crosses the blood-brain barrier and inhibits glioblastoma growth. Clin Cancer Res 2004; 10:5187-5201.
45. Landen JW, Lang R, McMahon SJ, Rusan NM, et al. Noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma. Cancer Res 2002;62:4109-4114.
46. Zhou J, Gupta K, Yao J, Ye K, et al. Paclitaxel-resistant human ovarian cancer cells undergo c-Jun NH2-terminal kinase-mediated apoptosis in response to noscapine.J Biol Chem 2002;277:39777-39785.
47. Aneja R, Asress S, Dhiman N, Awasthi A, et al. Nontoxic melanoma therapy by a novel tubulin-binding agent. Int J Cancer 2010;126:256-265.
48. Karna P, Rida PC, Pannu V, Gupta KK, et al. A novel microtubule-modulating noscapinoid triggers apoptosis by inducing spindle multipolarity via centrosome amplification and declustering. Cell Death Differ 2010; 18:632-644.
49. Aneja R, Zhou J, Zhou B, Chandra R, Joshi HC. Treatment of hormone-refractory breast cancer:Apoptosis and regression of human tumors implanted in mice. Mol Cancer Ther 2006;5:2366-2377.
50. Aneja R, Miyagi T, Karna P, Ezell T, et al. A novel microtubule-modulating agent induces mitochondrially driven caspase-dependent apoptosis via mitotic checkpoint activation in human prostate cancer cells. Eur J Cancer 2010;46:1668-1678.
2. Mollinedo F, Gajate C. Microtubules, microtubule-interfering agents and apoptosis. Apoptosis 2003;8(5):413-450.
3. Pohzzi D, Pratesi G, Tortoreto M, et al. A novel taxane with improved tolerability and therapeutic activity in a panel of human tumor xenografts. Cancer Res 1999;59(5):1036-1340.
4. Schiff PB, Horwitz SB. Taxol stabilizes microtubules in mouse fibroblast cells. Proc. Natl.Acad.Sci. USA 1980;77(3):1561-1565.
5. Pellegrini F, Budman DR. Review:tubulin function, action of antitubulin drugs, and new drug development. Cancer Invest.2005;23(3):264-273.
6. Grosios K, Holwell SE, McGown AT, Pettit GR, Bibby MC. In vivo and in vitro evaluation of combretastatin A-4 and its sodium phosphate prodrug. Br J Cancer 1999;81:1318-1327.
7. Nihei Y, Suzuki M, Okamo A, Tsuiji T, et al. Evaluation of antivascular and antimitotic effects of tubulin binding agents in solid tumour therapy. Jpn J Cancer Res 1999;90:1387-1396.
8. Tozer GM, Prise VE, Wilson J, Locke RJ, et al. Combretastatin A-4 phosphate as a tumour vascular targetting agent:early effects in tumours and normal tissues. Cancer Res 1999;59:1626-1634.
9. Pettit GR, Temple C Jr, Narayanan VL, Varma R, et al. Antineo-plastic agents 322. synthesis of combretastatin A 4 prodrugs. Anticancer Drug Des 1995; 10(4):299-309.
10. Ahmed B, Van Eijk LI, Bouma-Ter Steege JC, Van Der Schaft DW, et al. Vascular targeting effect of combretastatin A4 phosphate dominates the inherent angiogenesis inhibitory activity. Int J Cancer 2003;105(1):20-25.
11. Vincent L, Kermani P, Young LM, Cheng J, et al.Combretastatin A4 phosphate induces rapid regression of tumor neovessels and growth throush interference with vascular endothelial-cadherin signaling. J Clin Invest 2005; 115(11):2992-3006.
12. Dark GG, Hill SA, Prise VE, et al. Combretastatin A-4, an agent that displays potent and selective toxicity toward tumor vasculature.Cancer Res 1997;57:1829-1834.
13. Nagaiah G, Remick SC. Combretastatin A4 phosphate:a novel vascular disrupting agent. Future Oncol 2010;6(8):1219-28.
14. Siemann DW, Mercer E, Lepler S, et al. Vascular targeting agents enhance chemotherapeutic agent activities in solid tumor therapy. Int J Cancer 2002;99(1):1-6.
15. Wildiers H, Ahmed B, Guetens G, De Boeck G, et al. Combretastatin A4 phosphate enhances CPT-11 activity independently of the administration sequence. Eur J Cancer 2004;40(2):284-290.
16. Nelkin BD, Ball DW. Combretastatin A4 and doxorubicin combination treatment is effective in a prelinical model of human medulary thyroid carcinoma. Oncol Rep 2001;8(1):157-160.
17. Grosios K, Loadman PM, Swaine DJ, Pettit GR et al. Combination chemotherapy with combretastatin A4 phosphate and 5-fluorouracil in an experimental marine colon adenocarcinoma. Anti Cancer Res 2000;20(1A):229-233.
18. Badn W, Kalliomaki S, Widegren B, Sjogren HO. Low-dose combretastatin A4 phosphate enhances the immune response of tumor hosts to experimenta colon carcinoma. Clin Cancer Res 2006;12(15):4714-4719.
19. Larochelle S, Merrick KA, Terret ME, Wohlbold L, et al. Requirements for Cdk7 in the assembly of Cdkl/cyclin B and activation of Cdk2 revealed by chemical genetics in human cells. Mol Cell 2007;25(6):839-850.
20. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature 2008;451:1069-1075.
21. Yang Z, Klionsky DJ. Eaten alive:a history of macroautophagy. Nat Cell Biol 2010;12:814-822.
22. Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Self-eating and self-killing: crosstalk between autophagy and apoptosis.Nat Rev Mol Cell Biol 2007;8(9):741-752.
23. Kang C, Avery L. To be or not to be, the level of autophagy is the question:dual roles of autophagy in the survival response to starvation. Autophagy 2008;4:82-84.
24. Xie Z, Klionsky DJ. Autophagosome formation:core machinery and adaptations. Nat Cell Biol 2007;9:1102-1109.
25. Levine B, Klionsky D J. Development by selfdigestion:molecular mechanisms and biological functions of autophagy. Dev Cell 2004;6:463-477.
26. Amaravadi RK, Lippincott-Schwartz J, Yin XM, Weiss WA, et al. Principles and current strategies for targeting autophagy for cancer treatment.Clin Cancer Res 2011;17(4):654-666.
27. Jager S, Bucci C, Tanida I, Ueno T, et al. Role for Rab7 in maturation of late autophagic vacuoles. J Cell Sci 2004;117:4837-4848.
28. Cooney MM, Radivoyevitch T, Dowlati A, Overmoyer B, et al. Cardiovascular safety profile of coalbretastatin A4 phosphate in a single dose phase I study in patients with advanced cancer. Clin Cancer Res 2004; 10:96-1013.
29. Stevenson JP, Rosen M, Sun W, Gallagher M, et al. Phase I trial of the antivascular agent combretastatin A4 phosphate on a 5 day schedule to patients with cancer: magnetic resonance imaging evidence for altered tumor blood flow. J Clin Oncol 2003;21(23):4428-4438.
30. Yi-He Ling, Carmen Tornos, Roman Perez-Soler. Phosphorylation of Bcl-2 is a marker of M phase events and not a determinant of apoptosis. J Biol Chem 1998;273:18984-18991.
31. Homesley HD, Filiaci V, Gibbons SK, Long HJ, et al. A randomized phase III trial in advanced endometrial carcinoma of surgery and volume directed radiation followed by cisplatin and doxorubicin with or without paclitaxel:a Gynecologic Oncology Group study. Gynecol Oncol 2009; 112(3):543-552.
32. Jin H, Yang R, Fong S, Totpal K, et al. Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand cooperates with chemotherapy to inhibit orthotopic lung tumor growth and improve survival. Cancer Res 2004;64:4900-4905.
33. Denekamp J.Endothelial cell proliferation as a novel approach to targeting tumour therapy. Br J Cancer 1982;45:136-139.
34. Denekamp J. Review article:angiogenesis, neovascular proliferation and vascular pathophysiology as targets for cancer therapy. Br JRadiol 1993;66:181-196.
35. Tozer GM, Prise VE, Wilson J, Locke RJ, et al. Combretastatin A-4 phosphate as a tumor vascular-targeting agent:early effects in tumors and normal tissues. Cancer Res1999;59:1626-1634.
36. Malcontenti-Wilson C, Muralidharan V, Skinner S, Christophi C, et al. Combretastatin A4 prodrug study of effect on the growth and the microvasculature of colorectal liver metastases in a murine model. Clin Cancer Res 2001;7:1052-1060.
37. Wang S, El-Deiry WS. TRAIL and apoptosis induction by TNF-family death receptors. Oncogene 2003;23:8628-8633.
38. H. Zhu, W.J. Ding, R. Wu, Q.J. Weng, et al. Synergistic anti-cancer activity by the combination of TRAIL/APO-2L and celastrol. Cancer Invest 2010;28:23-32.
39. Koschny R, Walczak H, Ganten TM. The promise of TRAIL-potential and risks of a novel anticancer therapy. J Mol Med 2007;85:923-935.
40. Fisher MJ, Virmani AK, Wu L, et al. Nucleotide substitution in the ectodomain of trail receptor DR4 is associated with lung cancer and head and neck cancer. Clin Cancer Res 2001;7:1688-1697.
41. Shin MS, Kim HS, Lee SH, et al. Mutations of tumor necrosis factor-related apoptosis-inducing ligand receptor 1(TRAIL-R1) and receptor 2 (TRAIL-R2) genes in metastaticbreast cancers. Cancer Res 2001;61:4942-4946.
42. Jeng YM, Hsu HC. Mutation of the DR5/TRAIL receptor 2 gene is infrequent in hepatocellular carcinoma. Cancer Lett 2002; 181:205-208.
43. Kim YS, Schwabe RF, Qian T, et al. TRAIL-mediated apoptosis requires NF-kappaB inhibition and the mitochondrial permeability transition in human hepatoma cells.Hepatology 2002;36:1498-1508.
44. Ehrhardt H, Fulda S, Schmid I, et al. TRAIL induced survival and proliferation in cancer cells resistant towards TRAIL-induced apoptosis mediated by NF-kappaB. Oncogene 2003;22:3842-3852.
45. Okano H, Shiraki K, Inoue H, et al. Cellular FLICE/caspase-8-inhibitory protein as a principal regulator of cell death and survival in human hepatocellular carcinoma. Lab Invest 2003;93:1033-1043.
46. Fulda S, Meyer E, Debatin KM. Inhibition of TRAILinduced apoptosis by Bcl-2 overexpression. Oncogene 2002;21:2283-2294.
47. Chou TC, Talalay P.Quantitative analysis of dose-effect relationships:the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27-55.
48. Nguyen T, Zhang XD, Hersey P. Relative resistance of fresh isolates of melanoma to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Clin Cancer Res 2001;7:966s-973s.
49. Strater J, Hinz U, Walczak H, Mechtersheimer G. Expression of TRAIL and TRAIL receptors in colon carcinoma:TRAIL-R1 is an independent prognostic parameter. Clin Cancer Res 2002;8:3734-3740.
50. Lin CW, Shen SC, Chien CC, Yang LY, et al.12-O-tetradecanoylphorbol-13-acetate-induced invasion/migration of glioblastoma cells through activating PKCalpha/ERK/NF-kappaB-dependent MMP-9 expression. J Cell Physiol 2010; 225(2):472-481.
51. Darzynkiewicz Z, Zhao H. Detection of DNA strand breaks in apoptotic cells by flow-and image-cytometry. Methods Mol Biol 2010;682:91-101.
52. Horak P, Pils D, Haller G, et al. Contribution of epigenetic silencing of tumor necrosis factor-related apoptosis inducing ligand receptor 1 (DR4) to TRAIL resistance and ovarian cancer. Mol Cancer Res 2005;3:335-343.
53. Larribere L, Khaled M, Tartare-Deckert S, et al. PI3K mediates protection against TRAIL-induced apoptosis in primary human melanocytes. Cell Death Differ 2004; 11:1084-91.
54. Weldon CB, Parker AP, Patten D, et al. Sensitization of apoptotically resistant breast carcinoma cells to TNF and TRAIL by inhibition of p38 mitogen-activated protein kinase signaling. Int J Oncol 2004;24:1473-80.
55. Singh TR, Shankar S, Chen X, Asim M, Srivastava RK. Synergistic interactions of chemotherapeutic drugs and tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand on apoptosis and on regression of breast carcinoma in vivo. Cancer Res 2003;63:5390-5400.
56. Baldwin, A. S., Jr. Series introduction:the transcription factor NF-κB and human disease. J Clin Investig 2001; 107:3-6.
57. Baeuerle, P. A. and Baltimore, D. NF-κB:ten years after. Cell 1996;87:13-20.
58. Baeuerle, P.A.and Henkel, T. Function and activation of NF-κB in the immunesystem. Annu Rev Immunol 1994;12:141-179.
59. Verma IM, Stevenson JK, Schwarz EM, Van Antwerp D, et al. Rcl/NF-κB/IκB family:intimate tales of association and dissociation. Genes Dev 1995;9:2723-2735.
60. Hayakawa M, Miyashita H, Sakamoto I, Kitagawa M, et al. Evidence that reactive oxygen species do not mediate NF-kappaB activation. EMBO J 2003;22:3356-3366.
61. Xufeng Chen, Karthikeyan Kandasamy, Rakesh K. Srivastava. Differential Roles of RelA (p65) and c-Rel Subunits of Nuclear Factor κB in Tumor Necrosis Factor-related Apoptosis-inducing Ligand Signaling. Cancer Res 2003;63:1059-1066.
62. Mizushima N. Autophagy:process and function. Genes Dev 2007;21:2861-2873.
63. Levine B, Klionsky DJ. Development by self-digestion:molecular mechanisms and biological functions of autophagy. Dev Cell 2004;6:463-477.
64. Uttenweiler A, Mayer A. Microautophagy in the yeast Saccharomyces cerevisiae. Methods Mol Biol 2008;445:245-259.
65. Hayashi-Nishino M, et al. A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat Cell Biol 2009; 11:1433-1437.
66. Nakai A, Yamaguchi O, Takeda T, Higuchi Y, et al. The role of antophagy in cardiomyocytes in the basal state and in re-sponse to hemodynamic stress. Nat Med 2007;13(5):619-624.
67. Sun Y, Liu JH, Jin L, Lin SM, Yang Y, Sui YX, Shi H. Over-expression of the Beclinl gene upregulates chemosensitivity to anti-cancer drugs by enhancing therapy-induced apoptosis in cervix squamous carcinoma CaSki cells. Cancer Lett 2010;294(2):204-10.
68. Liu YL, Yang PM, Shun CT, Wu MS, et al. Autophagy potentiates the anti-cancer effects of the histone deacetylase inhibitors in hepatocellular carcinoma. Autophagy 2010;6(8):1057-65.
69. Mizushima N. Methods for monitoring autophagy. Int J Biochem Cell Biol 2004;36:2491-2502.
70. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, et al. LC3, a mammalian homologue of yeast Apg8p, is localized inautophagosome membranes after processing. EMBO J 2000; 19:5720-5728.
71. Pyo JO, Jang MH, Kwon YK, Lee HJ, et al. Essential roles of Atg5 and FADD in autophagic cell death:dissection of autophagic cell death into vacuole formationand cell death. J Biol Chem 2005;280:20722-20729.
72. Mizushima N, Levine B,Cuervo AM, et al. Autophagy fights disease through cellular self-digestion. Nature 2008;451(7182):1069-1075.
73. Anderson HL, Yap JT, Miller MP, Robbins A, et al. Assessment of pharmacodynamic vascular response in a phase I trial of combretastatin A4 phosphate. J Clin Oncol.2003;21(15):2823-30.
74. Cuervo, A. M. Autophagy:in sickness and in health. Trends Cell Biol 2004;14:70-77.
75. Ajabnoor GM, Crook T, Coley HM. Paclitaxel resistance is associated with switch from apoptotic to autophagic cell death in MCF-7 breast cancer cells.Cell Death Dis 2012;26(3):e260.
76. Liu YL, Yang PM, Shun CT, Wu MS, et al. Autophagy potentiates the anti-cancer effects of the histone deacetylase inhibitors in hepatocellular carcinoma.Autophagy 2010;6(8):1057-65.
77. Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene 2004;23:2891-2906.
78. Petiot A, Ogier-Denis E, Blommaart EF, Meijer AJ et al. Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. JBiol Chem 2000; 275:992-998.
79. Amaravadi RK, Yu D, Lum JJ, et al. Autophagy inhibition enhances therapy induced apoptosis in a Myc-induced model of lymphoma.J Clin Invest 2007;117(2):326-336.
80. Yongjie Wei,Sophie Pattingre, Sangita Sinha, Michael Bassik and Beth Levine. JNK1-Mediated Phosphorylation of Bcl-2Regulates Starvation-Induced Autophagy. Molecular Cell 2008;30:678-688.
1. Lowe J, Li H, Downing KH et al. Refined structure of αβ-tubulin at 3.5 A resolution. J Mol Biol 2001;13(5):1045-1057.
2. Zhou J, Joshi HC. Exquisite modulation of micro tubule dynamics is required for chromosome congression during mitosis. Mol Biol Cell 2001; 12:173A.
3. Wittmann T, Bokoch GM, Waterman-Storer CM. Regulation of microtubule destabilizing activity of Op18/stathmin downstream of Racl. J Biol Chem 2004;279(7):6196-6203.
4. Alli E, Bash-Babula J, Yang JM, et al. Effect of stathmin on the sensitivity to antimicrotubule drugs in human breast cancer. Cancer Res 2002;62(23):6864-6869.
5. Quasthof S, Hartung HP. Chemotherapy-induced peripheral neuropathy.J Neurol 2002;249(1):9-17.
6. Ke Y, Ye K, Grossniklaus HE, Archer DR, Joshi HC, Kapp JA. Noscapine inhibits tumor growth with little toxicity to normal tissues or inhibition of immune responses. Cancer Immunol Immunother 2000;49:217-225.
7. Ye K, Ke Y, Keshava N, Shanks J, Kapp JA, Tekmal RR, Petros J, Joshi HC. Opium alkaloidnoscapine is an antitumor agent that arrests metaphase and induces apoptosis in dividing cells. Proc Natl Acad Sci USA 1998;95:1601-1606.
8. Heidemann S. Microtubules, leukemia, and cough syrup. Blood 2006; 107:2216-2217.
9. Nogales E, Wolf SG, Khan IA, Luduena RF, Downing KH. Structure of tubulin at 6.5 A and location of the taxol-binding site. Nature 1995;375:424-427.
10. Morris PG, Fornier MN. Microtubule active agents:Beyond the taxane frontier. Clin Cancer Res 2008;14:7167-7172.
11. Seve P, Dumontet C. Is class III beta-tubulin a predictive factor in patients receiving tubulinbinding agents? Lancet Oncol 2008;9:168-175.
12. Goodin S, Kane MP, Rubin EH. Epothilones:Mechanism of action and biologic activity. J Clin Oncol 2004;22:2015-2025.
13. Thomas ES, Gomez HL, Li RK, Chung HC, et al. Ixabepilone plus capecitabine for metastatic breast cancer progressing after anthracycline and taxane treatment.J Clin Oncol 2007;25:5210-5217.
14. Mooberry SL, Tien G, Hernandez AH, et al. Laulimalide and isolaulimalide, new paclitaxel-like microtubule stabilizing agent. Cancer Res 1999;59 (3):653-660.
15. Wilmes A, Bargh K, Kelly C, Northcote PT, Miller JH. Peloruside A synergizes with othermicrotubule stabilizing agents in cultured cancer cell lines. Mol Pharmacol 2007;4:269-280.
16. Hamel E, Day BW, Miller JH, Jung MK, et al. Synergistic effects of peloruside A and laulimalide with taxoid site drugs, but not with each other, on tubulin assembly. Mol Pharmacol 2006;70:1555-1564.
17. Jordan MA. Mechanism of action of antitumor drugs that interact with microtubules and tubulin. Curr Med Chem Anti-Cane Agents 2002;2(1):1-17.
18. Lobert S, Correia JJ. Energetics of vines, alkaloid interactions with tubulin. Methods Enzymol 2000;323:77-103.
19. Iwasaki S, Kobayashi H, Furukawa J, Namikoshi M, et al. Studies on macrocyclic lactone antibiotics.Ⅶ. Structure of a phytotoxin "rhizoxin" produced by Rhizopus chinensis. J Antibiot 1984;37:354-362.
20. Gupta S, Bhattacharyya B. Antimicrotubular drugs binding to vinca domain of tubulin. Mol Cell Biochem 2003;253:41-47.
21. Bai RL, Pettit GR, Hamel E. Binding of dolastatin 10 to tubulin at a distinct site for peptideantimitotic agents near the exchangeable nucleotide and vinca alkaloid sites. J Biol Chem 1990;265:17141-17149.
22. Luduena RF, Roach MC, Prasad V, Lacey E. Effect of phomopsin A on the alkylation of tubulin. Biochem Pharmacol 1990;39:1603-1608.
23. Giannakakou P, Gussio R, Nogales E, et al. A common pharmacophore for epothilone and taxanes:molecular basis for drug resistance conferred by tubulin mutations in human cancer cells. Proc Nat Acad Sci USA 2000;97(6):2904-2909.
24. Krishnamurthy G, Cheng W, Lo MC, et al. Biophysical characterization of the interactions of HTI-286 with tubulin heterodimer and microtubules. Biochemistry 2003;42(46):13484-13495.
25. Chen SM, Meng LH, Ding J. New microtubule-inhibiting anticancer agents. Expert Opin Investig Drugs 2010; 19:329-343.
26. Cormier A, Marchand M, Ravelli RB, Knossow M, Gigant B. Structural insight into theinhibition of tubulin by vinca domain peptide ligands. EMBO Rep 2008;9:1101-1106.
27. Boukari H, Sackett DL, Schuck P, Nossal RJ. Single-walled tubulin ring polymers. Biopolymers 2007;86:424-436.
28. Risinger AL, Giles FJ, Mooberry SL. Microtubule dynamics as a target in oncology. Cancer Treat Rev 2009;35:255-261.
29. Bhattacharyya B, Panda D, Gupta S, Banerjee M. Anti-mitotic activity of colchicine and thestructural basis for its interaction with tubulin. Med Res Rev 2008;28:155-183.
30. Pettit GR, Singh SB, Hamel E, Lin CM, et al. Isolation and structure of the strong cell growth and tubulin inhibitor combretastatin A-4. Experientia 1989;45:209-211.
31. Hamel E. Antimitotic natural products and their interactions with tubulin. Med Res Rev 1996; 16:207-231.
32. Mann J. Natural products in cancer chemotherapy:Past, present and future. Nat Rev Cancer 2002;2:143-148.
33. Iyer S, Chaplin DJ, Rosenthal DS, Boulares AH, et al. Induction of apoptosis in proliferating human endothelial cells by the tumor-specific antiangiogenesis agent combretastatin A-4. Cancer Res 1998;58:4510-4514.
34. Ahmed B, Van Eijk LI, Bouma-Ter Steege JC, Van Der Schaft DW, et al. Vascular targeting effect of combretastatin A-4 phosphate dominates the inherent angiogenesis inhibitory activity. Int J Cancer 2003; 105:20-25.
35. Siemann DW, Mercer E, Lepler S, Rojiani AM. Vascular targeting agents enhance chemotherapeutic agent activities in solid tumor therapy. Int J Cancer 2002;99:1-6.
36. Mabjeesh NJ, Escuin D, LaVallee TM, Pribluda VS, et al.2-ME inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF. Cancer Cell 2003;3:363-375.
37. D'Amato RJ, Lin CM, Flynn E, Folkman J, Hamel E.2-Methoxyestradiol, an endogenousmammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site. Proc Natl Acad Sci USA 1994;91:3964-3968.
38. Desbene S, Giorgi-Renault S. Drugs that inhibit tubulin polymerization:The particular case of podophyllotoxin and analogues. Curr Med Chem Anticancer Agents 2002;2:71-90.
39. Peyrot V, Leynadier D, Sarrazin M, Briand C, et al. Mechanism of binding of the new antimitotic drug MDL 27048 to the colchicine site of tubulin:Equilibrium studies. Biochemistry 1992;31:11125-11132.
40. Smith DB, Ewen C, Mackintosh J, Fox BW, et al. A phase I and pharmacokinetic study of amphethinile. Br J Cancer 1988;57:623-627.
41. Edward DR, Berg WB, Sprigss DR, et al.MDL-27048, a novel syntheticmicrotubule inhibitor, exerts curative antitumor activity in vivo, show efficacy toward multi drug-resistant tumor cells, and lacks neurotoxicity. Clin Cancer Res 2004;s6:563.
42. Segal MS, Glodstein MM, Attinger EO. The use of noscapine (narcotine) as an antitussive agent. Dis Chest 1957;32:305-309.
43. Ye K, Ke Y, Keshava N, Shanks J, et al. Opium alkaloid noscapine is an antitumor agent that arrests metaphase and induces apoptosis in dividing cells. Proc Natl Acad Sci USA 1998;95:1601-1606.
44. Landen JW, Hau V, Wang M, Davis T, et al. Noscapine crosses the blood-brain barrier and inhibits glioblastoma growth. Clin Cancer Res 2004; 10:5187-5201.
45. Landen JW, Lang R, McMahon SJ, Rusan NM, et al. Noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma. Cancer Res 2002;62:4109-4114.
46. Zhou J, Gupta K, Yao J, Ye K, et al. Paclitaxel-resistant human ovarian cancer cells undergo c-Jun NH2-terminal kinase-mediated apoptosis in response to noscapine.J Biol Chem 2002;277:39777-39785.
47. Aneja R, Asress S, Dhiman N, Awasthi A, et al. Nontoxic melanoma therapy by a novel tubulin-binding agent. Int J Cancer 2010;126:256-265.
48. Karna P, Rida PC, Pannu V, Gupta KK, et al. A novel microtubule-modulating noscapinoid triggers apoptosis by inducing spindle multipolarity via centrosome amplification and declustering. Cell Death Differ 2010; 18:632-644.
49. Aneja R, Zhou J, Zhou B, Chandra R, Joshi HC. Treatment of hormone-refractory breast cancer:Apoptosis and regression of human tumors implanted in mice. Mol Cancer Ther 2006;5:2366-2377.
50. Aneja R, Miyagi T, Karna P, Ezell T, et al. A novel microtubule-modulating agent induces mitochondrially driven caspase-dependent apoptosis via mitotic checkpoint activation in human prostate cancer cells. Eur J Cancer 2010;46:1668-1678.