【作者】 林雅军；

【导师】 甄永苏；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2008， 博士

【摘要】 恶性肿瘤是人类健康的第二大杀手,在某些地区已上升为头号杀手,世界每年死于恶性肿瘤的人数呈逐年递增的趋势。开发新型抗肿瘤药物将是人类战胜恶性肿瘤的重要手段之一。近年来,随着免疫学和分子生物学的发展,人们对肿瘤形成过程中的受体、基因和信号转导有了比较深入的了解,在此基础上发展的靶向治疗获得了突破性的进展,靶向药物的研究是当前临床研究最活跃的领域之一,其中靶向蛋白酪氨酸激酶的药物已经成为研究的热点。蛋白酪氨酸激酶（PTKs）是一种能选择性地使不同底物的酪氨酸残基磷酸化的一种酶,大致可分为受体酪氨酸激酶、非受体酪氨酸激酶、IR和Janus激酶等。在病理条件下PTKs会持续活化,阻断其对细胞分化、生长和凋亡等的调节功能,诱发肿瘤。据报道受体PTKs家族中超过50%的成员发生与人类恶性肿瘤相关的突变或者过度表达,其中30%的成员与人类实体肿瘤相关。EGFR家族是受体PTKs中重要成员,其包括4个成员,分别是EGFR（ErbB-1,HER-1）、HER-2（ErbB-2）、HER-3（ErbB-3）和HER-4（ErbB-4）。在全世界每年诊断的150万新增乳腺肿瘤患者中,27%—30%与EGFR的过度表达有关,20%—25%与HER-2的过度表达有关。单纯针对EGFR或者HER-2的单克隆抗体或蛋白酪氨酸激酶抑制剂（TKI）存在很大局限性,针对单靶点的药物在肿瘤治疗方面有效率不足30%,虽然与其它药物联用可以在一定程度上提高药物疗效,但是通过受体酪氨酸激酶活性的相互补充,很快产生耐药。因此开发针对EGFR和HER-2双靶点的酪氨酸激酶抑制剂将会提高肿瘤治疗的疗效。蛋白酪氨酸激酶的抑制剂—大黄素能够通过抑制HER-2的自身磷酸化和交互磷酸化,阻断ErbB-2的酪氨酸激酶活性。对HER-2过度表达的乳腺癌细胞采用大黄素处理能够降低酪氨酸的过度磷酸化,提高细胞对化疗药物紫杉醇的敏感性,本文研究同为大黄蒽醌类化合物的大黄酸是否对EGFR和HER-2蛋白酪氨酸激酶活性起到双重抑制作用。大量文献报道大黄中的三种蒽醌类化合物大黄酸、大黄素和芦荟大黄素能够抑制肿瘤细胞增殖、促进肿瘤细胞凋亡、抑制肿瘤血管形成、逆转肿瘤细胞多药耐药等多方面的抗肿瘤活性。但是由于其水溶性低,生物利用度差,目前除应用其泻下作用外,其它药理活性还没有应用于临床。大黄酸与大黄中其它蒽醌类化合物最显著的区别就是大黄酸存在一个游离的羧基,因此我们选择大黄酸的羧基进行结构改造。第一部分以增加水溶性为目标的大黄酸衍生物制备目的:通过对大黄酸进行结构改造,制备出水溶性好的大黄酸衍生物或其盐。方法:方法一,在特定反应条件下,通过大黄酸与赖氨酸反应生成赖氨酰大黄酸,借助赖氨酸存在的游离氨基和羧基增加赖氨酰大黄酸在水中的溶解度。方法二,在一定反应条件下,通过赖氨酸与大黄酸之间的酸碱反应生成稳定的盐,增加大黄酸在水中的溶解度。通过MTT方法检测产物的体外抗肿瘤活性。结果:按照方法一获得了黄色和紫红色两种产物,其中黄色产物不溶于水,紫红色产物水溶性好,对两种产物进行了红外光谱测定,证明两种产物均为大黄酰赖氨酸甲酯,应用MTT方法检测了这两种产物体外抗肿瘤活性,发现二者的体外抗肿瘤活性远远低于大黄酸,而且其产率也比较低,分别为6%和9%,因此没有进行进一步研究。按照方法二获得了水溶性好的紫色产物,经过紫外分光光度法、HPLC法和红外光谱法验证此产物为赖氨大黄酸盐（RHL）,通过MTT方法证实RHL与大黄酸具有同等的抑制肿瘤细胞增殖活性。结论:大黄酰赖氨酸甲酯虽然能够部分改善大黄酸水溶性差的缺点,但是其体外抗肿瘤活性远远低于大黄酸,没有对其进一步研究。RHL不仅解决了大黄酸水溶性难的问题,而且其抗肿瘤活性与大黄酸相当,有进一步开发成抗肿瘤药物的潜力。第二部分赖氨大黄酸体外抗肿瘤活性及分子机制研究目的:观察RHL体外抗肿瘤作用,探讨其分子作用机制。方法:通过MTT检测细胞增殖,Western blot检测受体PTKs通路蛋白表达水平及其磷酸化水平的变化,免疫细胞化学检测EGFR和HER-2蛋白表达水平变化,RT-PCR检测HER-2在基因转录水平上的变化,流式细胞技术（FACS）检测细胞凋亡和细胞周期。结果:RHL抑制不同来源的乳腺癌细胞系（MCF-7、SK-Br-3和MDA-MB-231）、卵巢癌SK-OV-3细胞系和人脐静脉内皮细胞（HUVEC）增殖,IC₅₀分别为95μmol/L、80μmol/L、110μmol/L、85μmol/L和200μmol/L。而且RHL在抑制肿瘤细胞增殖方面与大黄酸相近。对RHL抗肿瘤机制研究中发现:在EGFR高表达和HER-2低表达的MCF-7细胞,其通过抑制EGFR的磷酸化及其下游Raf/MEK/ERK信号通路的磷酸化抑制细胞增殖,并且其与紫杉醇等其它化疗药物联合发挥协同抗肿瘤作用;在EGFR和HER-2高表达的SK-Br-3细胞,其能够抑制EGFR和HER-2蛋白表达和蛋白磷酸化水平,并且能够诱导肿瘤细胞通过p53和p21途径发生细胞凋亡。结论:RHL是新型的靶向EGFR和HER-2双靶点的受体蛋白酪氨酸激酶抑制剂。通过抑制细胞增殖和诱导细胞凋亡途径发挥抗肿瘤作用。第三部分赖氨大黄酸体内抗肿瘤活性及其与紫杉醇的协同作用目的:观察RHL体内抗肿瘤活性和探讨其与紫杉醇协同抗肿瘤作用的机制。方法:建立小鼠肝癌和人类肿瘤裸鼠移植模型,分别采用灌胃、腹腔注射途径给药,观察小鼠对药物的耐受情况和药物的抗肿瘤活性。并通过免疫组化方法观察肿瘤组织EGFR蛋白磷酸化水平变化。结果:RHL单独用药有一定的抑制肿瘤生长作用。采用腹腔注射方法给药优于灌胃给药。其与紫杉醇合用,在小鼠H22肝癌和MCF-7乳腺癌裸鼠移植瘤模型均表现出明显的协同作用。免疫组化研究结果表明RHL能够抑制由紫杉醇引起的EGFR和ERK蛋白磷酸化水平升高。结论:RHL体内应用抑制小鼠H22肝癌和人类乳腺癌裸鼠移植瘤生长,腹腔注射途径给药比灌胃给药有更高抑瘤率。在小鼠H22肝癌和人类乳腺癌裸鼠移植瘤,RHL与Taxol联合有协同抗肿瘤作用。总结:本文通过对大黄酸的改造获得了水溶性好的RHL。RHL抑制乳腺癌细胞和卵巢癌细胞的增殖,并且在抑制肿瘤细胞增殖方面与大黄酸相近。进一步研究发现RHL抑制EGFR和HER-2蛋白磷酸化水平,是靶向EGFR和HER-2双靶点的小分子TKI。其通过抑制EGFR高表达的MCF-7细胞株细胞增殖,和诱导EGFR和HER-2高表达的SK-Br-3细胞株细胞凋亡发挥抗肿瘤活性。其与紫杉醇等化疗药联合应用能增强化疗药的抗肿瘤作用,有望成为肿瘤化疗药或者辅助化疗药。更多还原

【Abstract】 Malignancy is the second cause of death, and in some areas it has been the first one. The mortality of malignancy increases yearly in the world. The important way for human to overcome malignancy is to explore new anti-tumor drugs. With the development of immunology and molecular biology, a number of receptors, genes and signal transduction pathways involved in tumor generation had been reported. Based on this discovery, targeted therapy of tumor has been made a breakthrough. The development of targeted drugs is being one of the most attractive fields in tumor research and therapy. The drugs targeting protein tyrosine kinase had already been the focus point. A tyrosine kinase is an enzyme that can transfer a phosphate group from ATP to a tyrosine residue in a protein, including receptor PTKs, non-receptor PTKs, IR and Janus. In the abnormal condition, PTKs are constructively activated, consequently blocking the regulation of cell differentiation, growth and apoptosis, and inducing tumor progression.More than 50% of the known receptor PTKs have been found to be either mutated or overexpressed in association with human malignancies and 30% of them have been implicated in human solid toumors. EGFR family, an important group of PTKs, includes four members: EGFR （ErbB-1）, HER-2 （ErbB-2）, HER-3 （ErbB-3） and HER-4 （ErbB-4）. There are one million and five hundred thousand new cases of breast cancer around the world every year, 27%—30% of them with EGFR overexpression and 20%—25% of them with HER-2 overexpression. Therapeutic use of protein tyrosine kinase inhibitors （TKIs） and monoclonal antibody targeting EGFR or HER-2 have some limitations. The efficacy of an anti-tumor drug that targets only a single point is less than 30%. The anti-tumor activity can be improved by the combination of chemotherapeutic agents; however, resistance occours soon through the mutual compensation of receptor PTKs. So, dual receptor tyrosine kinase inhibitors targeting both EGFR and HER-2 may increase the response rate. Emodin, a tyrosine kinase inhibitor, suppresses HER-2 atuo-phosphorylation and trans-phosphorylation and blocks tyrosine kinase activity of ErbB-2. Emodin can inhibit the growth of HER-2 -overexpressing tumors in mice and also sensitize these tumors to taxol. In this article, we studied if rhein analog showed dual inhibition to EGFR and HER-2.Rhein, emodin and aloe-emodin are the main anthraquinone derivatives of rhubarb. It was reported that the anthraquinone derivatives of rhubarb could inhibit tumor cell proliferation, induce tumor cell apoptosis, block blood vessel formation and even reverse multi-drug resistance. However, all of them have poor water solubility and poor bioavailability. Their therapeutic applications are limited in cathartic. The most significant difference between rhein and other anthraquinone derivatives is that rhein has a free carboxyl group. Therefore structural modification can be directed to the free carboxyl group.PartⅠPreparation of rhein analog with high solubility in waterPurpose: To prepare rhein derivatives easily dissolving in water.Methods: One is the preparation of lysyl-rhein. The carboxyl group of rhein reacts with the amino group of lysine in special condition to form lysyl-rhein, which improves its solubility in water depending on the free amino group and the free carboxyl group of lysine. Another is the preparation of rhein lysinate （RHL）. The carboxyl group of rhein reacts with the amino group of lysine in room temperature to form RHL, which easily dissolves in water. By MTT assay, we observed the anti-tumor activities of these new products in vitro.Results: We obtained two new products by the first method. One was yellow and did not dissolve in water. Another was purple and easy to dissolve. These two compounds were proved to be rheinyl lysine methyl ester, by infrared spectrum analysis. The anti-tumor efficacy of these two compounds was far less than that of rhein and the yield of them was very low, only 6% and 9% respectively. No further study was conducted. By second mthod we obtained a purple compound with good solubility. It was proved to be RHL by violet spectrophotometry, HPLC, and infrared spectrum analysis. By MTT assay, RHL had the same anti-tumor activity as rhein （in DMSO solution）.Conclusions: Although rheinyl lysine methyl ester could partly improve the solubility, the anti-tumor activity was far less than rhein. No further study was conducted. RHL not only easily dissolved in water but also showed the same anti-tumor activity as rhein. That warrants further development of RHL.PartⅡThe anti-tumor activity of rhein lysinate and its molecular mechanism in tumor treatment in vitroPurpose: To observe the anti-tumor activity of RHL and investigate its molecular mechanism.Methods: By MTT assay investigating tumor cell proliferation; by Western blot investigating the phosphorylation of protein and the expression level of proteins in PTKs signaling pathway; by immunocytochemistry observing the expression level of EGFR and HER-2; by RT-PCR observing HER-2 change in transcriptional level; by FACS observing cell apoptosis and cell cycle arrest.Results: RHL inhibited the proliferation of different breast cancer cells （MCF-7, SK-Br-3 and MDA-MB-231）, ovarian cancer cells （SK-OV-3） and HUVEC. The IC₅₀ was 95μmol/L, 80μmol/L, 110μmol/L, 85μmol/L and 200μmol/L, respectively. It had the same anti-prliferation with rhein. In MCF-7 cells with EGFR overexpression and without HER-2 overexpression, RHL inhibited the phosphorylation of EGFR, the phosphorylation of downstream Raf/MEK/ERK signaling pathway and showed synergistic effect with chemotherapeutic drugs such as taxol. In SK-Br-3 cells with both EGFR and HER-2 overexpression, RHL inhibited the phosphorylation of EGFR and HER-2 and induced cell apoptosis through p53 and p21 pathway.Conclusions: RHL was a new dual target protein tyrosine kinase inhibitor aiming directly at EGFR and HER-2. RHL not only inhibited tumor cell proliferation but also induced tumor cell apoptosis.PartⅢThe anti-tumor activity of rhein lysinate and its synergism with taxol in tumor treatment in vivoPurpose: To observe anti-tumor activity of RHL and explore its synergistic effect with taxol. Methods: Establishing the model of mouse H22 liver cancer and human MCF-7 breast cancer xenografts in athymic mice, administering RHL by intragastric administration and intraperitoneal injection, observing the tolerance in mice and the therapeutic effect of RHL, and observing the phosphorylation of EGFR in tumor tissue by immunohistochemistry method.Results: RHL inhibited tumor growth. The efficacy of RHL administered by intraperitoneal injection was better than by intragastric administration. RHL in combination with taxol showed the synergistic effect in inhibiting tumor growth in mouse H22 liver cancer and MCF-7 breast cancer xenografts in athymic mice. The result of immunohistochemistry demonstrated that RHL partly abrogated the increase of the phosphorylation of EGFR and ERK induced by taxol.Conclusions: RHL inhibited tumor growth of mouse liver cancer H22 and human breast cancer MCF-7 xenografts in athymic mice. The efficacy of RHL administered by intraperitoneal injection is better than that by intragastric administration. It had a synergistic anti-tumor activity with taxol.In summary, we obtained a new compound - RHL. That is easily dissolved in water. RHL inhibited the proliferation of breast cancer and ovarian cancer cells and had the same potency with rhein. Our results indicated that RHL, as a new dual tyrosine kinase inhibitor, was capable of repressing the tyrosine kinase activity of EGFR and HER-2, accordingly inhibiting the proliferation of EGFR overexpressing MCF-7 breast cancer cells and inducing cell apoptosis of EGFR and HER-2 overexpressing SK-Br-3 breast cancer cells. Moreover, it sensitized tumor cells to chemotherapeutic drugs such as taxol. These results may have important therapeutic implications in cancer chemotherapy.更多还原