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Molecular mechanism of constitutive expression of interleukin 8 in the cancer progress and the development of the angiogenesis control therapy

白细胞介素8组成型表达在癌症进展中的分子机制及血管生成控制治疗的发展

基本信息

批准号：
13670315
负责人：
KITAJIMA Isao
金额：
$ 2.24万
依托单位：
TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670315/
关键词：
Hpatoma Colon Cancer Angiogenesis IL-8 PEA3 promoter Decoy Gene therapy 浸潤アポトーシス HepG2細胞 AP1 NF-кB

项目摘要

Interleukin-8 (IL-8), originally identified as a neufrophil chemotactic factor, exhibits a potent angiogenic activity, Here, we demonstrated the IL-8 mRNA is constitutively expressed in human hepatocellular cartinoma cells (HCCs) and also abundantly expressed in the hepatoma cell line, HepG2. However, IL-8 mRNA was not detected in uninvolved liver tissue surrounding HCCs.In order to investigate the promoter regions essential for constitutive IL-8 gene expression in HCCs, we performed transient transfection experiments utilizing CAT reporter constructs containing deletions of the human IL-8 promoter. We found that the sequence 5'-AGGAAG-3' at -137 bp to -132bp of the IL-8 promoter was shown to be a polyomavirus enhancer A binding protein 3(PEAS) site which can cooperate with the activator protein 1(AP-1). The introduction of a mutation in either the PEA3 or AP-1 site abolished the constitutive promoter activity, and mutation at both the PEA3 and AP-1 sites showed the lowest IL-8 promoter activity in HepG2 cells. Moreover, electrophoretic mobility shift assay demonstrated constitutive formation of PEA3 and AP-1 complexes in HepG2 cells. In immunohistochemistiy analysis, PEA8 and IL-8 proteins coexisted in HCC tumors with marked angiogenesis.Further, we applied cis-element double strand oligodeoxynucleotides (ODNs), "decoy" ODNs against PEA3 binding sites in order to trap PEA3 molecules in the nuclei of HCCs. Transfection of PEA3 decoy ODNs showed rapid regression of transplanted HepG2 cells in nude mice, which significantly inhibited angiogenesis with decreased IL-8 mRNA expression.This is the first report that in HCCs PEA3 cooperates with AP-1 to play crucial a role in promoting constitutive IL-8 expression, which can induce tumor angiogenesis in hepatomas. Therefore, it is possible that transcription factor PEA3 is a new target of anti-angiogenic cancer therapy for hepatoma.

白细胞介素-8 （IL-8）最初被认为是一种神经粒细胞趋化因子，具有强大的血管生成活性。在这里，我们证明了IL-8 mRNA在人肝细胞癌细胞（hcc）中组成性表达，并且在肝癌细胞系HepG2中也大量表达。然而，在hcc周围未受累的肝组织中未检测到IL-8 mRNA。为了研究hcc中构成IL-8基因表达所必需的启动子区域，我们利用含有人类IL-8启动子缺失的CAT报告基因构建物进行了瞬时转染实验。我们发现IL-8启动子-137 ~ -132bp的5′-AGGAAG-3′序列是多瘤病毒增强子a结合蛋白3（PEAS）位点，可与激活蛋白1（AP-1）协同工作。在PEA3或AP-1位点引入突变使组成启动子活性消失，PEA3和AP-1位点的突变显示HepG2细胞中IL-8启动子活性最低。此外，电泳迁移率转移实验显示PEA3和AP-1复合物在HepG2细胞中形成。免疫组化分析显示，PEA8和IL-8蛋白在血管生成明显的HCC肿瘤中共存。此外，我们利用顺式双链寡脱氧核苷酸（ODNs）作为“诱饵”，针对PEA3结合位点，将PEA3分子诱捕到hcc细胞核中。转染PEA3诱饵ODNs后，裸鼠移植HepG2细胞快速消退，IL-8 mRNA表达降低，明显抑制血管生成。这是首次报道在hcc中PEA3与AP-1协同在促进构成性IL-8表达中发挥重要作用，从而在肝癌中诱导肿瘤血管生成。因此，转录因子PEA3可能是肝癌抗血管生成肿瘤治疗的新靶点。