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

白介素8(IL-8)最初被认为是一种中性粒细胞趋化因子，具有很强的血管生成活性，在此，我们证明了IL-8在人肝细胞癌细胞中有结构性表达，在肝癌细胞系HepG2中也有大量表达。为了研究人IL-8启动子在肝细胞癌中的表达所必需的启动子区域，我们利用含有人IL-8启动子缺失的CAT报告构建物进行了瞬时转染实验。我们发现IL-8启动子-137bp至-132bp的5‘-AGGAAG-3’序列是多瘤病毒增强子A结合蛋白3(PEAS)位点，与激活蛋白1(AP-1)具有协同作用。在PEA3或AP-1位点引入突变使构成启动子活性丧失，并且在PEA3和AP-1位点的突变在HepG2细胞中显示最低的IL-8启动子活性。此外，凝胶迁移率改变分析显示，PEA3和AP-1复合体在HepG2细胞中形成。在免疫组织化学分析中，PEA8和IL-8蛋白在有明显血管生成的肝细胞癌中共存。此外，我们还利用顺式双链寡核苷酸(ODN)，针对PEA3结合位点，将PEA3分子捕获到肝癌细胞核中。PEA3诱骗寡核苷酸可显著抑制裸鼠移植的HepG2细胞的血管生成，降低IL-8mRNA的表达。这是首次报道在肝癌中PEA3与AP-1协同在促进组成性IL-8表达方面发挥关键作用，从而诱导肝癌肿瘤血管生成。因此，转录因子PEA3有可能成为抗肝癌血管生成治疗的新靶点。

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.