文殊兰抗肿瘤活性单体生物碱的分离筛选及其抑制有丝分裂的分子机制研究

Because of the unique advantages of Crinum asiaticum L.var. sinicum Bak, it has become a traditional Chinese medicine with promising development foreground. Reports showed that Crinum asiaticum L.var. sinicum Bak had definite antitumor effects. Our previous study showed that Crinum asiaticum L.var. sinicum Bak alkaloids(CA) had strong antitumor effects, which were effective fraction of Crinum asiaticum L.var. sinicum Bak. But up to now, there were no systematic research on extraction, isolation, identification, antitumor activity screening and the molecular antitumor mechanism of CA. For further development of Crinum asiaticum L.var. sinicum Bak, the systematic study on extraction and isolation is urgently needed to find new kinds of active compounds.Therefore, this task mainly focus on the above contents. In previous study, we have seperated 3 compouds from CA. Our privious study also showed that CA could induce G2/M phase arrest of tumor cells. So we can put forward hypothesis that blocking tumor cell cycle progression and preventing its passing the mitotic checkpoint may be the key mechanism of CA. After the research of screening and identification of monomer active alkaloid(CMA), the topic will study the effect of CMA on microtubule dynamics of tumor cells and comfirm the molecular target of CMA by methods of RT-PCR, Western blot, LCSM and FCM. From the above research, we will verify the validity of the hypothesis, and in the end elucidate the antineoplastic effective substances of CA and its molecular mechanism.

文殊兰以其独有的优势已成为一种极具开发价值的中药。有研究表明，文殊兰具有一定的抗肿瘤作用。本课题前期研究表明，文殊兰生物碱（CA）具有极强的抗肿瘤活性，是文殊兰的抗肿瘤有效部位。但目前尚无针对CA的系统分离鉴定、抗肿瘤活性筛选以及其分子机制研究。为进一步开发文殊兰，急需进行分离纯化研究，找出新型活性化合物。因此，本课题拟开展CA的系统分离和抗肿瘤活性筛选研究，前期已分离鉴定了3个化合物。前期研究还发现，CA可使肿瘤细胞阻滞于G2/M 期，由此我们提出假说：阻滞肿瘤细胞周期进程、使其不能跨越有丝分裂检查点可能是CA抗肿瘤的关键机制。本课题拟在筛选鉴定文殊兰活性单体生物碱基础上，进一步采用RT-PCR、Western blot、LCSM、FCM等方法研究其对细胞微管动力学的影响及作用靶点。通过研究验证上述假说的正确性，阐明CA抗肿瘤药效物质基础及其分子作用机制。

文殊兰以其独有的优势已成为一种极具开发价值的中药。文殊兰具有一定的抗肿瘤作用。 通过乙醇加热回流等方法提取分离纯化得出5种文殊兰单体成分，分别为（1）对羟基苯甲酸；（2）石蒜碱（3）N-反式-阿魏酰基酪胺、（4）甘草素、（5）朱顶红星碱；并确定石蒜碱为文殊兰抗肿瘤的主要药效单体成分，其敏感细胞株为人乳腺癌MCF-7细胞。石蒜碱对人乳腺癌MCF-7细胞具有生长抑制作用，其IC50为4.829μmol/L。石蒜碱可将MCF-7细胞阻滞在G2/M期，其作用机制为：石蒜碱可使乳腺癌细胞的微管蛋白的聚合状态发生改变，通过“非依赖p53途径”通路，显著上调MCF-7细胞中p21蛋白表达量，抑制Cdc2(Cdk1)/CyclinB复合体活性，从而对人乳腺癌MCF-7细胞周期起到阻滞作用。石蒜碱直接通过抑制BRCA1的磷酸化，上调BRCA1的E3泛素连接酶活性，导致BRCA1抑制中心体微管成核的功能减弱，进而影响微管功能，使着丝点-微管稳定性降低，纺锤体检测点信号增强，导致纺锤体检测点检测到动点间缺乏合适张力，阻止有丝分裂的继续进行。石蒜碱是从线粒体途径和内质网途径两条通路来诱导MCF-7细胞凋亡的，并且确定了线粒体途径占主导的作用机制。石蒜碱诱导MCF-7细胞凋亡的机制是一方面通过引起MCF-7细胞中的Ca2+平衡紊乱，进而诱发MCF-7细胞内质网应激。细胞内钙离子浓度的改变促使了Bip/GRP78蛋白高表达，使之与未折叠蛋白结合以缓解内质网应激。与此同时细胞通过XBP1循环激活作用的多次重复来处理内质网应激反应。但由于内质网应激水平较高，最终分别启动凋亡信号Caspase-12和CHOP/GADD153来诱导MCF-7细胞的凋亡。另一方面通过降低Bcl-2蛋白的表达，增加Bax蛋白的表达，从而促进MPTP孔开放，降低线粒体膜电位，导致线粒体膜内相对高渗，线粒体基质膨胀，外膜破裂，并伴随细胞内酸化，同时，加速了Cytochrome C的释放。在ATP和dATP共同作用下，Cytochrome C首先与胞浆中的凋亡蛋白酶活化因子Apaf-1结合，聚合成为复合体，这种多聚复合物充分募集(caspascs)，启动Caspase-9形成全酶，然后裂解，导致procaspases-9自动活化，活化的Caspases-9分裂，继续使活化下游的Caspases-3，最终诱导MCF-7细胞凋亡。