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海洋溴酚化合物抗肿瘤活性筛选及作用机制研究
郭传龙
Subtype博士
Thesis Advisor史大永
2019-05-08
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学博士
Degree Discipline海洋生物学
Keyword海洋溴酚化合物 抗肿瘤 Eif4e Parp-1抑制剂
Abstract

肿瘤是威胁人类健康的第二大杀手,其中以肺癌的发病率和死亡率最高。目前针对肺癌的药物大多为靶向药物,如吉非替尼等。虽然针对肺癌的抗肿瘤药物研发取得了一定的进展,但是目前的上市药物普遍存在着耐药性,这限制了这些药物的应用。因此,寻找针对特异性靶点的药物成为抗肿瘤药物研发的重点。

海洋中具有丰富的生物资源,部分海洋来源的天然产物体现出优异的生物学活性。其中,溴酚类化合物是海洋来源独具特色的一类化合物,具有抗氧化、抗菌、抗肿瘤等生物活性。课题组将溴酚与含氮杂环组合,设计合成出一系列的溴酚-吲哚酮化合物。经体外MTT筛选发现,这一系列化合物具有显著的抑制肿瘤细胞增殖作用。其中化合物BOS-102可显著抑制A549细胞增值及克隆形成,诱导A549凋亡和G0/G1期周期阻滞,作用机制研究显示BOS-102可激活活性氧介导的线粒体凋亡途径,并通过PI3K/AktMAPK信号通路发挥作用。

真核细胞翻译起始因子4EeIF4E)在调控真核细胞翻译起始阶段起关键作用。并且eIF4E在肿瘤细胞信号转导途径中处于关键节点位置,使得eIF4E成为抗肿瘤药物研发的热门靶点。课题组根据目前在研的靶向eIF4E的化合物的结构特点,将溴酚基团与氨基-噻唑基团结合,设计合成了一系列的溴酚-噻唑类化合物。我们研究发现化合物EGPI-1可显著抑制肿瘤细胞生长,并可靶向调控eIF4EDuolink PLAwestern blot结果显示化合物EGPI-1可干扰eIF4E/eIF4G相互作用。进一步研究发现,EGPI-1可抑制eIF4E磷酸化并抑制PI3K/Akt/mTOR信号通路。经流式细胞术检测发现EGPI-1可诱导A549凋亡并发生G0/G1期周期阻滞,诱导线粒体损伤及膜电位下降。体内裸鼠移植瘤实验证明EGPI-1在体内同样具有抗肿瘤活性,急性及亚急性毒性实验证明EGPI-1安全性良好。以上研究表明EGPI-1具有开发成新型抗肿瘤药物的潜力。

卵巢癌严重威胁着女性健康,其死亡率高于乳腺癌。近几年随着PARP抑制剂(olaparibrucaparibniraparibtalazoparib)的接连上市,给卵巢癌患者带来了希望。PARP是一种DNA修复酶,在细胞DNA单链损伤修复过程中起关键作用。抑制PARP(主要是PARP-1)可导致细胞发生双链损伤,并可导致BRCA缺陷型细胞的死亡。课题组根据PARP抑制剂上市药物的结构特点,将缩胺基硫脲基团与溴酚基团结合,合成出一系列溴酚-缩胺基硫脲化合物。体外酶活实验证明其中四个化合物可选择性抑制PARP-1活性,其中化合物11PARP-1IC5029.5 nM,对PARP-2IC50>1000 nMMTT检测发现化合物11可显著抑制卵巢癌细胞SK-OV-3增值,诱导SK-OV-3凋亡,ROS积累和G2/M期周期阻滞。进一步实验表明化合物11可诱导DNA双链损伤及H2AX磷酸化,并抑制H2O2所导致的PAR形成。蛋白质组学研究表明化合物11可抑制细胞DNA修复功能。体内裸鼠移植瘤实验表明化合物11可显著抑制裸鼠移植瘤生长,安全性良好;作用机制表明化合物11体内抗肿瘤活性是通过抑制PARP-1实现的。以上实验为开发具有选择性的PARP-1抑制剂提供了有力的实验依据,为研究新型的治疗卵巢癌药物提供了理论依据。

Other Abstract

Tumors are the second biggest threat to human health, and lung cancer is the cancer with highest incidence and mortality. Most of the drugs for lung cancer are targeted drugs, such as Gefitinib. Although anti-tumor drugs for lung cancer have made some progress, drug resistance is currently prevalent in marketed drugs, which limits the application of drugs. Therefore, finding specific targets has become the focus of anti-tumor drug development.

The ocean is rich in biological resources, and some marine-derived natural products exhibit excellent biological activity. Among them, bromophenol compounds are unique compounds of marine origin, and have biological activities such as anti-oxidation, antibacterial and anti-tumor. Our team combined bromophenol with nitrogen-containing heterocycles into a series of bromophenol-fluorenone compounds. In vitro MTT screening revealed that this series of compounds has a significant inhibitory effect on tumor cell proliferation. Among them, the compound BOS-102 significantly inhibited the proliferation and clonal formation of A549 cells, induced apoptosis and G0/G1 phase arrest in A549 cells. The mechanism study showed that BOS-102 can activate the mitochondrial apoptotic pathway mediated by reactive oxygen species. Further research revealed that BOS-102 deactivated the PI3K/Akt pathway and activated MAPK signaling pathways.

The eukaryotic translation initiation factor 4E (eIF4E) plays a key role in regulating the initiation of eukaryotic translation. And eIF4E is at a critical node in the tumor signaling pathway, making eIF4E a hot target for anti-tumor drug development. The current research on targeting eIF4E mainly targets the following aspects: interference with eIF4E/4IF4G interaction; inhibition of PI3K/Akt/mTOR-4EBP1 signaling pathway and inhibition of Ras/ERK/MNK-eIF4E signaling pathway. Based on the structural characteristics of the current eIF4E-targeted compounds, we combined with the bromophenol and the amino-thiazole to synthesize a series of compounds. Our study found that the compound EGPI-1 significantly inhibited tumor cell growth and targeted regulation of eIF4E. Duolink PLA and western blot results showed that compound EGPI-1 distrupt eIF4E/eIF4G interaction. Further studies revealed that EGPI-1 inhibitd eIF4E phosphorylation and inhibitd PI3K/Akt/mTOR signaling pathway. Flow cytometry analysis showed that EGPI-1 induced apoptosis and G0/G1 phase arrest of A549 cells; induced mitochondrial damage and decreased mitochondrial membrane potential. In vivo nude mice xenograft study demonstrated that EGPI-1 has antitumor activity in vivo and that EGPI-1 was safe in animal experiments. The above studies indicated that EGPI-1 has the potential to be developed into a novel anti-tumor drug.

Ovarian cancer is a serious threat to women's health and its mortality rate is higher than breast cancer. In recent years, with the successive listing of PARP inhibitors (olaparib, rucaparib, niraparib, talazoparib), hopes have been given to patients with ovarian cancer. PARP is a DNA repair enzyme that plays a key role in the repair of cellular DNA single-strand damage. Inhibition of PARP (primarily PARP-1) can cause double-stranded damage to cells and can lead to death of BRCA-deficient cells. Based on the structural characteristics of the listed drugs, the research group combined a thiol group with a bromophenol group to synthesize a series of bromophenol-amino thiourea compounds. In vitro enzymatic activity demonstrated that four of the compounds selectively inhibited PARP-1 activity, with compound 11 having an IC50 of 29.5 nM for PARP-1 and an IC50 of >1000 nM for PARP-2. MTT assay showed that compound 11 significantly inhibited the proliferation of ovarian cancer cell SK-OV-3, induced apoptosis, ROS accumulation and G2/M phase arrest in SK-OV-3 cells. Further experiments showed that compound 11 can induce DNA double-strand damage and H2AX phosphorylation, and inhibit PAR formation caused by H2O2. Proteomics studies showed that compound 11 inhibits cellular DNA repair. In vivo nude mice xenograft experiments showed that compound 11 significantly inhibit the growth of xenografts in mice, and the mechanism study indicated that the antitumor activity of compound 11 in vivo was through inhibiting PARP-1. Animal experiments indicated that compound 11 was safe. The above experiments provide a powerful experimental basis for the development of selective PARP-1 inhibitors, and provide a theoretical basis for the study of new drugs for the treatment of ovarian cancer.

MOST Discipline Catalogue理学 ; 理学::海洋科学
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/156801
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
郭传龙. 海洋溴酚化合物抗肿瘤活性筛选及作用机制研究[D]. 中国科学院海洋研究所. 中国科学院大学,2019.
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