化合物B192和EGCG的药效和药理作用机制研究
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摘要
恶性肿瘤严重威胁人类的健康。随着生活环境和生活方式的改变,全球肿瘤的发病率呈现不断升高的趋势。由于对肿瘤的认识尚缺乏全面性以及不同肿瘤的发生发展的复杂性,造成现在肿瘤的治疗还存在着很大的不足之处,需用研发新型抗癌药物。此外,由于肿瘤治疗较为困难,通过控制或者治疗癌前疾病从而实现肿瘤预防的策略越来越受到人们的重视。减缓或抑制癌前疾病发展为肿瘤,可以有效的减少肿瘤的发生率,所以寻找新的能够预防肿瘤发生和发现新的抗肿瘤作用的新化合物具有同等重要的意义。因此,本研究从新型抗癌药物和癌前疾病的治疗药物两个方面进行肿瘤的治疗与预防研究。
采用计算机模拟设计技术,我们针对周期素依赖性激酶(CDK)设计、合成了新型抗肿瘤化合物B192。在对抗肿瘤活性的初步筛选中证实其对多种肿瘤细胞均有较好的抑制作用,并且对肿瘤细胞具有一定的选择性。为了验证其对靶标的作用进行了激酶活性测试,结果显示B192对CDK5/p25和GSK3β激酶活性均有一定的抑制作用,但对CDK2激酶的抑制率较低。进一步以人结肠癌细胞株HCT116为研究模型,生长曲线和克隆形成实验结果显示B192对结肠癌HCT116细胞具有较好的增殖抑制作用,且这种作用呈现时间和剂量依赖性。作用机制研究表明,B192可使p53野生型人结肠癌HCT116细胞、p53敲除型HCT116细胞和p53突变型人结肠癌HT29细胞发生明显的G2/M期阻滞,呈现时间和浓度依赖性,同时还可以引起细胞凋亡,也呈现时间和浓度依赖性。进一步研究表明,在p53野生型HCT116细胞中B192可能通过抑制ERK的活性从而影响细胞的增殖。B192可诱导p53野生型HCT116细胞发生G2/M期阻滞,这种阻滞与细胞内Cyclin B1, Cdc2等G2/M期相关蛋白的变化有关；并且B192对细胞周期的作用并不依赖p53的调节,B192也能诱导p53敲除型HCT116以及p53突变型HT29细胞发生G2/M期阻滞,同时Cdk抑制因子p2l通过p53非依赖方式表达上调。体内实验表明,B192的毒性较小但吸收较差,生物利用度低,抑瘤效果不明显。以上结果说明,我们发现了一个体外具有较好抗肿瘤活性的小分子化合物,但体内结果显示成药性不足,需要通过结构改造得到一个成药性较好的抗肿瘤化合物。
从治疗癌前疾病的角度出发,我们开展了EGCG的抗肝纤维化研究。临床统计结果显示,80%的肝癌由肝纤维化与肝硬化转化而来或与之密切相关,所以抑制肝纤维化的发展可以减少肝癌的发病率。(-)-表没食子儿茶素没食子酸酯(EGCG)是绿茶多酚提取物儿茶素的主要成分。其在儿茶素中含量最高,是典型的黄烷醇类化合物。研究表明EGCG具有抗肿瘤、抗突变等多方面的药理学作用。有研究证实EGCG对四氯化碳所致的肝纤维化模型有治疗作用。但其在胆汁淤积型肝纤维化模型中的作用和机制尚不明确。因此,本研究通过EGCG干预胆管结扎的大鼠模型,考察其抗肝纤维化的作用,同时应用人的肝星状细胞系LX-2,研究EGCG在抗纤维化过程中可能的信号通路,进一步研究其抗肝纤维化的分子作用机制,并考察已经证实的EGCG在肿瘤研究中的直接作用靶蛋白G3BP1是否在肝纤维化的发生发展中起作用,是否是EGCG抗肝纤维化效果的关键靶蛋白。结果显示,在人肝星状细胞系LX-2中,EGCG可以抑制肝纤维化的相关基因的mRNA及蛋白的表达和TGF-β/Smad、NF-κB、p38信号通路中相关蛋白的表达,其主要是通过降低Akt的磷酸化活性,从而影响了TGF-β1诱导的Smad信号通路的表达减少胶原的生成发挥抗肝纤维化的作用。在LX-2细胞中,G3BP1和G3BP2的表达会随着TGF-β1的诱导上调,EGCG处理后下调,但它们的基因下敲或上调均不能影响LX-2细胞中肝纤维化相关基因的表达,这结果说明,G3BP并不是一个抗肝纤维化的靶点,同时在肝纤维化中,EGCG并不是通过直接作用于G3BP而发挥其抗肝纤维化的作用。在胆管结扎的大鼠肝纤维化模型中,EGCG抑制了模型大鼠的肝纤维化的形成和发展,这种抑制主要与抑制肝脏中的炎症反应,并通过抑制肝星状细胞的PI3K/Akt信号通路进而抑制Smad信号通路从而抑制细胞的增殖及表达肝纤维化相关基因造成的。以上结果表明,我们发现了一个EGCG抗肝纤维化的新机制,也为抗肝纤维化和肝癌的预防提供了一个新的潜在的药物。
综上所述,本研究阐明了一个新结构的化合物的抗肿瘤作用机制,虽然其成药性较差,但是为未来合成靶向性和成药性更好的小分子化合物提供了一定的理论基础。本研究还发现了EGCG抗肝纤维化作用的部分新机制,有助于抗肝纤维化药物的研究,为肝癌的预防性治疗提供了新的潜在的化合物。
Cancer is becoming a serious human healthy problem. The incidence of cancer is increasing in world-wide as a result of the change of the environment, as well as increasingly, an adoption of cancer-associated lifestyle choices. There are still many problem of the therapy of tumor, because of the limited knowledge of the development of different of kinds of cancer. In addition, the prevention of the happening of cancer is becoming more and more important for the therapy. Becuae of the difficult of the cure of cancer, it is a meaningful way to develop the therapy of the precancerous disease. The precancerous disease mainly refers to the disease that has a high conversion rate to cancer. If we find a way to inhibit the occur of precancerous disease, the incidence of cancer will decrease significantly. Therefore, screening and identification of new therapeutic agents for precancerous disease are also important. In summary, our study is based on two aspects:the screening of new antiancer small molecule drug and the tactics of prevention of cancer.
At present, the research and development of novel small-molecular targeted anti-cancer drug is mainly based on the crystal structures of targeted proteins which are closely related to tumor occurrence, development and maintenance and therational drug design by computer aided design technology. And finally discover the targeted, low toxicity and effective lead compound through oriented synthesis, SAR studies, furtherstructural modification and systemic pharmacological assessment methods. So the study of small-molecular targeted anticancer drugs is one of the most important research frontiers in drug discovery. In the first part of our study, according to the method of scaffold hopping, we designed and synthesized the3-amino-1,4,5,6-tetrahydropyrrolo3,4-c]pyrazole derivatives B192which was based on two small molecule inhibitors in clinical trials. The result showed that B192greatly inhibited cell proliferation in various type of human cancer cells. In the kinase inhibition assay, B192had slight inhibiton effect on CDK5/p25and GSK3β, and little inhibiton effect on the CDK2kinase. The cytotoxicity of B192for human normal liver L02cells was smaller than cancer cell lines. According to the growth curve and colony formation assays, B192inhibited the proliferation of colon cancer cells HCT116in a time-and dose-dependent manner. The flow cytometric analysis revealed that B192induced cell cycle arrest at G2/M phase and resulted in a significant increase of HCT-116cell apoptosis in a time-and dose-dependent manner. It also caused cell cycle arrest at G2/M in HCT116p53-/-and HT29cells. The results of Western Blot showed that B192inhibited the expressions of cdc25C,cdc2,Cylin B1. the expression of p53was not change in HCT116cell and HCT116p53-/-cells. The HCT116p53-/-cell did not express p53, but the levels of p21was significantly increased. The expression of p-Akt, p-ERK1/2and NF-κB were decreased after treated by B192for12h. In vivo experiment showed that B192slightly inhibited the growth of CT26cells by24.13%in tumor weight.Becuse of the solubility of B192, it didn't have a high inhibition effect on the tumor growth in vivo. B192still had a good inhition effect in vitro, it is also provided a theoretical basis for deveolpment a new target small molcecule drugs.
Liver fibrogenesis is common to various types of chronic liver disease.The possible consequences of fibrogenesis include liver cirrhosis or hepatocelluar carcinoma.(-)-epigallocatechin-3-gallate (EGCG) is one of the most abundant polyphenols present in green tea. EGCG is well known for its anti-tumor activity while other pharmaceutical effects were discovered subsequently. It is reported that EGCG had effect on the liver fibrosis on the model induced by CCl4. Currently, our therapeutic repertoire for the treatment of liver fibrosis and cirrhosis is severly limited. To study the protective effects of (-)-epigallocatechin-3-gallate (EGCG) on hepatic fibrosis induced by bile duct-ligated rats and their mechanisms. The result of our study showed that EGCG significantly reduced the expression of fibrotic marker genes and protein in a human HSC line and inhibited the fibrotic marker protein expression via PI3K/Akt and TGF-β/Smad signaling pathways in LX-2cell line. EGCG reduced the liver fibrosis in BDL rat via blocking PI3K/Akt and TGF-β/Smad signaling pathways. Knock down G3BP1/2and transient high expression G3BP1/2didn't influence the expression of fibrosis-related genes. It is suggested that G3BP was not a new antifibrotic target and the antifibrotic effect of EGCG was not by inhibition of G3BP. In conclusion, we found a new mechanism of the antifibrotic effect of EGCG, and these novel findings suggest that EGCG may be a new drug for the treatment of liver fibrosis.
Our research investigated the anticancer effect of B192, one novel compound designed as a small targeted anticancer drug and discussed the mechanism of the anticancer effect. Though B192has poor water solubility, which is the major limit for ability of becoming a new drug, our work contributed to further design and synthesize better targeted and has better solubility compounds. One the other hand, we focused on the cancer prevention. We confirmed the antifibrotic effect of EGCG on BDL model and discovered new insights in the mechanism of antifibrotic effect.
In summary, our research demonstrated the mechanism of novel compound B192, providing support for further design, synthesis and investigation of small molecule drugs. Our research also revealed that novel antifibrotic mechanism of EGCG, which contributed to the investigation of antifibrotic drugs. Taken together, our research focused on the anticancer small molecule compound and antifibrotic compound EGCG,supporting for further cancer therapy and cancer prevention.
采用计算机模拟设计技术,我们针对周期素依赖性激酶(CDK)设计、合成了新型抗肿瘤化合物B192。在对抗肿瘤活性的初步筛选中证实其对多种肿瘤细胞均有较好的抑制作用,并且对肿瘤细胞具有一定的选择性。为了验证其对靶标的作用进行了激酶活性测试,结果显示B192对CDK5/p25和GSK3β激酶活性均有一定的抑制作用,但对CDK2激酶的抑制率较低。进一步以人结肠癌细胞株HCT116为研究模型,生长曲线和克隆形成实验结果显示B192对结肠癌HCT116细胞具有较好的增殖抑制作用,且这种作用呈现时间和剂量依赖性。作用机制研究表明,B192可使p53野生型人结肠癌HCT116细胞、p53敲除型HCT116细胞和p53突变型人结肠癌HT29细胞发生明显的G2/M期阻滞,呈现时间和浓度依赖性,同时还可以引起细胞凋亡,也呈现时间和浓度依赖性。进一步研究表明,在p53野生型HCT116细胞中B192可能通过抑制ERK的活性从而影响细胞的增殖。B192可诱导p53野生型HCT116细胞发生G2/M期阻滞,这种阻滞与细胞内Cyclin B1, Cdc2等G2/M期相关蛋白的变化有关；并且B192对细胞周期的作用并不依赖p53的调节,B192也能诱导p53敲除型HCT116以及p53突变型HT29细胞发生G2/M期阻滞,同时Cdk抑制因子p2l通过p53非依赖方式表达上调。体内实验表明,B192的毒性较小但吸收较差,生物利用度低,抑瘤效果不明显。以上结果说明,我们发现了一个体外具有较好抗肿瘤活性的小分子化合物,但体内结果显示成药性不足,需要通过结构改造得到一个成药性较好的抗肿瘤化合物。
从治疗癌前疾病的角度出发,我们开展了EGCG的抗肝纤维化研究。临床统计结果显示,80%的肝癌由肝纤维化与肝硬化转化而来或与之密切相关,所以抑制肝纤维化的发展可以减少肝癌的发病率。(-)-表没食子儿茶素没食子酸酯(EGCG)是绿茶多酚提取物儿茶素的主要成分。其在儿茶素中含量最高,是典型的黄烷醇类化合物。研究表明EGCG具有抗肿瘤、抗突变等多方面的药理学作用。有研究证实EGCG对四氯化碳所致的肝纤维化模型有治疗作用。但其在胆汁淤积型肝纤维化模型中的作用和机制尚不明确。因此,本研究通过EGCG干预胆管结扎的大鼠模型,考察其抗肝纤维化的作用,同时应用人的肝星状细胞系LX-2,研究EGCG在抗纤维化过程中可能的信号通路,进一步研究其抗肝纤维化的分子作用机制,并考察已经证实的EGCG在肿瘤研究中的直接作用靶蛋白G3BP1是否在肝纤维化的发生发展中起作用,是否是EGCG抗肝纤维化效果的关键靶蛋白。结果显示,在人肝星状细胞系LX-2中,EGCG可以抑制肝纤维化的相关基因的mRNA及蛋白的表达和TGF-β/Smad、NF-κB、p38信号通路中相关蛋白的表达,其主要是通过降低Akt的磷酸化活性,从而影响了TGF-β1诱导的Smad信号通路的表达减少胶原的生成发挥抗肝纤维化的作用。在LX-2细胞中,G3BP1和G3BP2的表达会随着TGF-β1的诱导上调,EGCG处理后下调,但它们的基因下敲或上调均不能影响LX-2细胞中肝纤维化相关基因的表达,这结果说明,G3BP并不是一个抗肝纤维化的靶点,同时在肝纤维化中,EGCG并不是通过直接作用于G3BP而发挥其抗肝纤维化的作用。在胆管结扎的大鼠肝纤维化模型中,EGCG抑制了模型大鼠的肝纤维化的形成和发展,这种抑制主要与抑制肝脏中的炎症反应,并通过抑制肝星状细胞的PI3K/Akt信号通路进而抑制Smad信号通路从而抑制细胞的增殖及表达肝纤维化相关基因造成的。以上结果表明,我们发现了一个EGCG抗肝纤维化的新机制,也为抗肝纤维化和肝癌的预防提供了一个新的潜在的药物。
综上所述,本研究阐明了一个新结构的化合物的抗肿瘤作用机制,虽然其成药性较差,但是为未来合成靶向性和成药性更好的小分子化合物提供了一定的理论基础。本研究还发现了EGCG抗肝纤维化作用的部分新机制,有助于抗肝纤维化药物的研究,为肝癌的预防性治疗提供了新的潜在的化合物。
Cancer is becoming a serious human healthy problem. The incidence of cancer is increasing in world-wide as a result of the change of the environment, as well as increasingly, an adoption of cancer-associated lifestyle choices. There are still many problem of the therapy of tumor, because of the limited knowledge of the development of different of kinds of cancer. In addition, the prevention of the happening of cancer is becoming more and more important for the therapy. Becuae of the difficult of the cure of cancer, it is a meaningful way to develop the therapy of the precancerous disease. The precancerous disease mainly refers to the disease that has a high conversion rate to cancer. If we find a way to inhibit the occur of precancerous disease, the incidence of cancer will decrease significantly. Therefore, screening and identification of new therapeutic agents for precancerous disease are also important. In summary, our study is based on two aspects:the screening of new antiancer small molecule drug and the tactics of prevention of cancer.
At present, the research and development of novel small-molecular targeted anti-cancer drug is mainly based on the crystal structures of targeted proteins which are closely related to tumor occurrence, development and maintenance and therational drug design by computer aided design technology. And finally discover the targeted, low toxicity and effective lead compound through oriented synthesis, SAR studies, furtherstructural modification and systemic pharmacological assessment methods. So the study of small-molecular targeted anticancer drugs is one of the most important research frontiers in drug discovery. In the first part of our study, according to the method of scaffold hopping, we designed and synthesized the3-amino-1,4,5,6-tetrahydropyrrolo3,4-c]pyrazole derivatives B192which was based on two small molecule inhibitors in clinical trials. The result showed that B192greatly inhibited cell proliferation in various type of human cancer cells. In the kinase inhibition assay, B192had slight inhibiton effect on CDK5/p25and GSK3β, and little inhibiton effect on the CDK2kinase. The cytotoxicity of B192for human normal liver L02cells was smaller than cancer cell lines. According to the growth curve and colony formation assays, B192inhibited the proliferation of colon cancer cells HCT116in a time-and dose-dependent manner. The flow cytometric analysis revealed that B192induced cell cycle arrest at G2/M phase and resulted in a significant increase of HCT-116cell apoptosis in a time-and dose-dependent manner. It also caused cell cycle arrest at G2/M in HCT116p53-/-and HT29cells. The results of Western Blot showed that B192inhibited the expressions of cdc25C,cdc2,Cylin B1. the expression of p53was not change in HCT116cell and HCT116p53-/-cells. The HCT116p53-/-cell did not express p53, but the levels of p21was significantly increased. The expression of p-Akt, p-ERK1/2and NF-κB were decreased after treated by B192for12h. In vivo experiment showed that B192slightly inhibited the growth of CT26cells by24.13%in tumor weight.Becuse of the solubility of B192, it didn't have a high inhibition effect on the tumor growth in vivo. B192still had a good inhition effect in vitro, it is also provided a theoretical basis for deveolpment a new target small molcecule drugs.
Liver fibrogenesis is common to various types of chronic liver disease.The possible consequences of fibrogenesis include liver cirrhosis or hepatocelluar carcinoma.(-)-epigallocatechin-3-gallate (EGCG) is one of the most abundant polyphenols present in green tea. EGCG is well known for its anti-tumor activity while other pharmaceutical effects were discovered subsequently. It is reported that EGCG had effect on the liver fibrosis on the model induced by CCl4. Currently, our therapeutic repertoire for the treatment of liver fibrosis and cirrhosis is severly limited. To study the protective effects of (-)-epigallocatechin-3-gallate (EGCG) on hepatic fibrosis induced by bile duct-ligated rats and their mechanisms. The result of our study showed that EGCG significantly reduced the expression of fibrotic marker genes and protein in a human HSC line and inhibited the fibrotic marker protein expression via PI3K/Akt and TGF-β/Smad signaling pathways in LX-2cell line. EGCG reduced the liver fibrosis in BDL rat via blocking PI3K/Akt and TGF-β/Smad signaling pathways. Knock down G3BP1/2and transient high expression G3BP1/2didn't influence the expression of fibrosis-related genes. It is suggested that G3BP was not a new antifibrotic target and the antifibrotic effect of EGCG was not by inhibition of G3BP. In conclusion, we found a new mechanism of the antifibrotic effect of EGCG, and these novel findings suggest that EGCG may be a new drug for the treatment of liver fibrosis.
Our research investigated the anticancer effect of B192, one novel compound designed as a small targeted anticancer drug and discussed the mechanism of the anticancer effect. Though B192has poor water solubility, which is the major limit for ability of becoming a new drug, our work contributed to further design and synthesize better targeted and has better solubility compounds. One the other hand, we focused on the cancer prevention. We confirmed the antifibrotic effect of EGCG on BDL model and discovered new insights in the mechanism of antifibrotic effect.
In summary, our research demonstrated the mechanism of novel compound B192, providing support for further design, synthesis and investigation of small molecule drugs. Our research also revealed that novel antifibrotic mechanism of EGCG, which contributed to the investigation of antifibrotic drugs. Taken together, our research focused on the anticancer small molecule compound and antifibrotic compound EGCG,supporting for further cancer therapy and cancer prevention.
引文
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