【作者】 张虹；

【导师】 赵忠熙；

【作者基本信息】 山东大学 ， 药学（专业学位）， 2014， 硕士

【摘要】 过去几十年来,恶性肿瘤已成为了人类健康的最大威胁,其发病率呈逐年上升趋势。据统计,目前已发现的癌症超过200种,全球每年新发癌症1000万例以上,每年逾700万人死于癌症,致死人数约占所有死亡人数的13%。在所有的恶性肿瘤中,乳腺癌是全球女性最高发的癌症,预测到2020年,发展中国家的乳腺癌患者会比目前高出26%。目前临床上乳腺癌的治疗方法主要是手术切除、化学治疗、放射治疗,但存在一定的局限性,特别是化学疗法,毒副反应严重,常伴有耐药现象。该严峻局面促使人们不断寻找新型的抗肿瘤药物,以期发现疗效更好毒性更低的药物。植物、动物和微生物一直是天然药物的重要组成部分,也是人类防病治病的重要资源。因此以天然化合物作为基本骨架来进行结构改造和化学修饰,已成为了新药研发中的重要方法。大蒜属于百合科葱属类植物,既可作为风味调料,也可广泛用于保健药物,其使用历史已有上千年。大蒜中含有丰富的有机硫化物,其种类多达33种,是大蒜的主要活性成分。大量的研究数据表明这些有机硫化物可以通过诱导细胞凋亡而发挥抗肿瘤作用。但是大蒜中的水溶性成分S-烯丙基巯基半胱氨酸(SAMC)目前未有商品化来源；其系统的理化性质研究未见报道；抗乳腺癌的具体机制还有待进一步说明。因此,本论文合成并研究了SAMC的合成及纯化工艺,期望适应大规模生产,并对SAMC进行了初步的药学评价,为制剂开发奠定基础；最后,选择雌激素受体阳性细胞MCF-7和雌激素受体阴性细胞MDA-MB-231,研究SAMC诱导乳腺癌细胞调亡的方式和机制。以L-半胱氨酸盐酸盐、甲氧羰基磺酰氯和烯丙基硫醇为原料,经过两步反应得到S-烯丙基巯基半胱氨酸；采用RP-18柱的柱分离方法纯化得到了纯度大于99%的SAMC。并进一步研究了SAMC的熔点、晶型、平衡溶解度、油水分配系数、稳定性等物化性能,结果揭示SAMC物化性能稳定,适用于临床前和临床制剂开发。使用SRB法研究了SAMC体外细胞毒性作用,结果表明SAMC对乳腺癌细胞MCF-7和MDA-MB-231的细胞毒性呈浓度和时间依赖性,当SAMC作用72h后,MCF-7细胞的IC50值为148μM, MDA-MB-231的IC50值为207μM；利用DAPI染色观察了细胞凋亡形态以及AnnexinV/PI双染法流式细胞仪测定了细胞凋亡率,实验结果表明SAMC可诱导MCF-7和MDA-MB-231细胞凋亡,并伴随着细胞色素C的释放,PARP裂解片段的出现以及Bcl-2家族的改变,即促凋亡蛋白Bax的表达增加,而抗凋亡蛋白Bcl-2的表达下调；针对乳腺癌细胞的周期阻滞作用研究结果显示SAMC可将MCF-7和MDA-MB-231阻滞在G0/G1期,此时周期相关蛋白cyclin D1、cyclin E1的表达增加,并激活了p53基因及其下游基因p21,导致乳腺癌细胞周期阻滞,而PCNA的下调表明SAMC可抑制乳腺癌细胞的增殖：JC-1染色法显示SAMC可使乳腺癌细胞的线粒体膜电位降低；caspase酶活性测定结果表明SAMC可激活caspase-3/7、 caspase-8和caspase-9;划痕实验探索SAMC对乳腺癌细胞迁移能力的影响,发现SAMC可抑制乳腺癌细胞的迁移,并伴随着E-cadherin蛋白的表达增加。上述研究结果显示,SAMC具有较强的抗乳腺癌活性,可通过线粒体途径和死亡受体途径诱导细胞凋亡。更多还原

【Abstract】 Malignant tumor has been the biggest threat for human health since the morbidity rate has been ever increasing in last few decades. It has been reported that there were more than200types of cancers has been found, and more than10million new cases were diagnosed each year. Over700,000people die from cancers each year, which is about13%of total morbidity. Breast cancer is the top cancer in women and the breast cancer patients will increase26%by2020in the developing country. The common treatment methods for breast cancer at present include surgery, chemotherapy and radiotherapy. All the existing treatment methods for breast cancer have their own limitations; for example, the toxic and side effects of chemotherapy are usually serious and often accompanied by drug resistance. Therefore, the searching for new antitumor drugs with better efficacy and less toxicity has been the focus for past decades. Plants, animals and microorganisms have always been the important sources of medicines for treatment tumors and other diseases. Additional structural modifications based on natural compounds have become an important strategy for development of novel drugs.Garlic (Allium sativum), a member of the lily family, has been used as the flavors as well as healthy supplements for thousands of years. Garlic’s bioactivities are thought to be attributed to its pharmacological characteristics of many sulfur-containing compounds; more than33organosulfur compounds in garlic are now discovered. A large amount of data indicates that garlic’s anticarcinogenic activities are partially due to cancer cell apoptosis induced by organosulfur compounds. Among the garlic organosulfur compounds, the water-soluble constituent S-allylmercaptocysteine (SAMC) is not commercially available and its physical and chemical properties as well as anti-breast cancer mechanism have not been well studied. In this work, the synthetic routes and purification procedures of SAMC were studied in order to establish a starting point for the industrial scale production; preliminary pharmaceutical evaluation of SAMC was performed for further pharmaceutical development; and finally, the antitumor mechanism of SAMC in breast cancer cell lines MCF-7(ER-positive) and MDA-MB-231(ER-negative) was investigated.SAMC was synthesized in a two-step reaction by using L-cysteine hydrochloride, methoxycarbonyl chloride and allyl mercaptan as raw materials. The newly synthesized SAMC was purified by a PR-18column and the purity of the purified SAMC was determined to be above99%. The melting point, the equilibrium solubility, oil-water partition coefficients and stability of the purified SAMC were determined and the results indicate that the purified SAMC was suitable for further pharmaceutical development. SRB analysis indicated that SAMC exhibited an effective cell growth inhibition of human breast cancer cell lines MCF-7and MDA-MB-231in a dose and time-dependent manner. The IC50values of SAMC were found to be148μM and207μM for MCF-7and MDA-MB-231cells at72h, respectively. DAPI staining and Annexin V/PI analysis suggested that SAMC induced apoptosis along with the release of cytochrome-c, the cleavage of PARP and the expression change of Bcl-2family such as the increase of the pro-apoptotic protein Bax and decrease of the anti-apoptotic protein Bcl-2. The cell cycle arrest effect of SAMC was analysed by flow cytometry using PI, the results indicated that the cell cycles of MCF-7and MDA-MB-231were arrested in the G0/G1phase by SAMC, and the expression of cell cycle proteins, such as cyclin D1, cyclin E1were increased; p53and p21that is the downstream gene of p53were activated and responsible for the arrest effect. The down-regulation of PCNA suggested that SAMC inhibited the proliferation of cells. The mitochondrial membrane potential was decreased with the treatment of SAMC by JC-1staining. Caspase activity analysis indicated that SAMC activated the caspase-3/7, caspase-8and caspase-9. The wound scratch assay found that SAMC inhibited the migration of breast cancer cells and the expression of E-cadherin was increased.All the results indicated that SAMC inhibited cell proliferation by delaying the cell cycle at G0/G1phase, and induced the cell apoptosis through mitochondrial (intrinsic) and death receptor (extrinsic) pathways.更多还原