联邻苯二酚结构的DNA交联剂的设计合成研究及其对黑素肿瘤细胞的选择性杀伤作用
发布时间：2018-12-27 13:54
【摘要】：DNA作为生物体中遗传信息的主要载体在遗传信息的传递中车关重要,以DNA为靶目标的抗癌药物的设计一直是药物研究的热点之一。其中,许多临床上使用的抗肿瘤药物的作用机理涉及到DNA的交联。它们通过与DNA的共价结合作用将DNA双链交联,阻碍DNA双链的打开,使之不能进行遗传信息的复制和传递,从而导致细胞的死亡达到治疗癌症的目的。可诱导的DNA交联剂的引入,增强了药物的靶向性和选择性。 本论文设计合成了十二个含不同连接基团的联邻苯二酚结构的化合物,通过高碘酸钠或者酪氨酸酶的氧化诱导,研究了它们与DNA的交联活性。同时,鉴于恶性黑素瘤细胞中酪氨酸酶的含量高表达,我们建立了一种以联邻苯二酚结构的DNA交联剂选择性的杀伤黑素瘤细胞的方法。具体研究内容涵盖以下几点： 1.首先在体外实验中,我们用琼脂糖凝胶电泳实验检测了化合物与线性DNA交联的能力。在高碘酸钠的氧化诱导下,两个酚羟基在邻位的化合物7a-f都表现出了很强的DNA交联能力,而两个酚羟基在间位的化合物10a-f交联能力很差。在酪氨酸酶的氧化诱导下,所有的化合物均表现出DNA交联能力,两个酚羟基在间位的化合物10a-f比两个酚羟基在邻位的化合物7a-f的交联能力略好。并且,中间连有联苯结构的化合物10f交联活性最好。另外,我们通过3-甲基-2-苯并噻唑酮腙(MBTH)的颜色实验,捕捉到这类化合物在酪氨酸酶氧化诱导后有二醌的中间体的产生,初步提出了交联机理。 2.我们进一步对此类联邻苯二酚化合物的抗癌活性进行了研究。我们用MTT法对药物的细胞毒性进行了研究。细胞毒性实验表明此类联邻苯二酚化合物对酪氨酸酶高表达的恶性黑素瘤细胞的毒性非常明显,而对不含有酪氨酸酶的宫颈癌细胞Hela和正常卵巢细胞CHO毒性很弱。我们通过一系列的分了生物学的方法,包括在细胞内检测邻苯二酚结构的BODIPY荧光探针实验,单细胞凝胶电泳实验,γ-H2AX免疫荧光实验以及通过观测细胞核形态变化,验证了化合物对恶性黑素瘤细胞的选择性杀伤力主要是通过DNA损伤起作用。更重要的是,这些DNA损伤与DNA交联有关。联邻二苯酚的化合物是因为能与恶性黑色素瘤细胞的细胞核中的DNA发生交联,而导致了它具有高选择性的杀伤恶性黑色素瘤细胞的能力。
[Abstract]:As the main carrier of genetic information in organisms, DNA plays an important role in the transmission of genetic information. The design of anticancer drugs targeting DNA has been one of the hot spots in drug research. Among them, many clinical use of anti-tumor drugs related to the mechanism of cross-linking of DNA. By covalent binding with DNA, DNA double strands are linked to each other, which hinders the opening of DNA double strands and makes them unable to replicate and transmit genetic information, thus leading to cell death to achieve the purpose of cancer treatment. The introduction of inducible DNA crosslinker enhanced the targeting and selectivity of the drug. In this paper, twelve dihydroquinone compounds with different connective groups were designed and synthesized, and their crosslinking activities with DNA were studied by oxidation induction of sodium periodate or tyrosinase. At the same time, in view of the high expression of tyrosinase in malignant melanoma cells, we established a selective killing method of melanoma cells with DNA crosslinking agent with dicatechol structure. Specific research covers the following points: 1. Firstly, in vitro, agarose gel electrophoresis was used to test the crosslinking ability of the compounds to linear DNA. Under the oxidation induction of sodium periodate, two phenolic hydroxyl groups exhibited strong DNA crosslinking ability in the ortho compound 7a-f, while the two phenolic hydroxyl groups in the intermediate position compound 10a-f crosslinking ability was very poor. Under the oxidation of tyrosinase, all the compounds showed DNA crosslinking ability. The crosslinking ability of two phenolic hydroxyl compounds in the intermediate position was slightly better than that of two phenolic hydroxyl groups in the ortho position compound 7a-f. Moreover, the crosslinking activity of 10 f compound with intermediate biphenyl structure is the best. In addition, through the color experiment of 3-methyl-2-benzothiazolone Hydrazone (MBTH), we have captured the production of diquinone intermediate after the oxidation of tyrosinase, and put forward the crosslinking mechanism. 2. We further studied the anticancer activity of this kind of catechol compounds. The cytotoxicity of the drug was studied by MTT method. The cytotoxicity test showed that the toxicity of this kind of catechol compounds to malignant melanoma cells with high tyrosinase expression was very obvious, but it was weak to Hela cells without tyrosinase and CHO from normal ovarian cells. We have adopted a series of biological methods, including BODIPY fluorescence probe assay for the detection of catechol structure in cells, single cell gel electrophoresis, 纬-H2AX immunofluorescence assay and nuclear morphological changes. The selective cytotoxicity of the compounds to malignant melanoma cells was confirmed mainly through DNA damage. More importantly, these DNA damage is related to DNA crosslinking. The compound of o-diphenol is capable of crosslinking with DNA in the nucleus of malignant melanoma cells, which leads to its highly selective ability to kill malignant melanoma cells.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R739.5
[Abstract]:As the main carrier of genetic information in organisms, DNA plays an important role in the transmission of genetic information. The design of anticancer drugs targeting DNA has been one of the hot spots in drug research. Among them, many clinical use of anti-tumor drugs related to the mechanism of cross-linking of DNA. By covalent binding with DNA, DNA double strands are linked to each other, which hinders the opening of DNA double strands and makes them unable to replicate and transmit genetic information, thus leading to cell death to achieve the purpose of cancer treatment. The introduction of inducible DNA crosslinker enhanced the targeting and selectivity of the drug. In this paper, twelve dihydroquinone compounds with different connective groups were designed and synthesized, and their crosslinking activities with DNA were studied by oxidation induction of sodium periodate or tyrosinase. At the same time, in view of the high expression of tyrosinase in malignant melanoma cells, we established a selective killing method of melanoma cells with DNA crosslinking agent with dicatechol structure. Specific research covers the following points: 1. Firstly, in vitro, agarose gel electrophoresis was used to test the crosslinking ability of the compounds to linear DNA. Under the oxidation induction of sodium periodate, two phenolic hydroxyl groups exhibited strong DNA crosslinking ability in the ortho compound 7a-f, while the two phenolic hydroxyl groups in the intermediate position compound 10a-f crosslinking ability was very poor. Under the oxidation of tyrosinase, all the compounds showed DNA crosslinking ability. The crosslinking ability of two phenolic hydroxyl compounds in the intermediate position was slightly better than that of two phenolic hydroxyl groups in the ortho position compound 7a-f. Moreover, the crosslinking activity of 10 f compound with intermediate biphenyl structure is the best. In addition, through the color experiment of 3-methyl-2-benzothiazolone Hydrazone (MBTH), we have captured the production of diquinone intermediate after the oxidation of tyrosinase, and put forward the crosslinking mechanism. 2. We further studied the anticancer activity of this kind of catechol compounds. The cytotoxicity of the drug was studied by MTT method. The cytotoxicity test showed that the toxicity of this kind of catechol compounds to malignant melanoma cells with high tyrosinase expression was very obvious, but it was weak to Hela cells without tyrosinase and CHO from normal ovarian cells. We have adopted a series of biological methods, including BODIPY fluorescence probe assay for the detection of catechol structure in cells, single cell gel electrophoresis, 纬-H2AX immunofluorescence assay and nuclear morphological changes. The selective cytotoxicity of the compounds to malignant melanoma cells was confirmed mainly through DNA damage. More importantly, these DNA damage is related to DNA crosslinking. The compound of o-diphenol is capable of crosslinking with DNA in the nucleus of malignant melanoma cells, which leads to its highly selective ability to kill malignant melanoma cells.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R739.5
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