Study on aldo-keto reductases as potential targets of antitumor agents : its involvement in the drug resistance of cancer cells

一种醛酮还原酶AKR1B10已被证明可诱导人非小细胞肺癌。本研究旨在研究AKR1B10基因表达的调控机制，以开发药物代谢酶靶向抗肿瘤药物。首先，我们采用RT-PCR技术检测了AKR1B10在人非小细胞肺癌细胞系A549和H23中的表达。AKR1B10在A549细胞中有显著的表达，而在H23细胞中几乎没有表达，这表明由于AKR1B10的表达差异显著，这两种细胞系可以作为研究AKR1B10基因调控的有用研究工具。为了确定AKR1B10基因的调控区域，我们从A549细胞中分离出5'-侧翼区域进行分析。在-720 ~ -588之间存在一个微卫星，两个等位基因之间存在CCTT重复序列。然而，在荧光素酶报告基因实验中，该微卫星并未参与基础AKR表达的调控。在5'侧区有几个潜在的调控共识序列。其中，有一个共识序列是抗氧化反应元件（ARE），它与基因表达的氧化还原调控有关。然后，利用甲氧基醌增强are介导的表达，在H23细胞中进行了AKR1B10诱导试验，结果表明，甲氧基醌可以提高AKR1B10的表达。由于转录因子Nrf2结合ARE并增强基因表达，因此在荧光素酶报告基因试验中共同引入了表达Nrf2的质粒。Nrf2的共表达显著提高了AKR1B10基因的转录活性，提示ARE和Nrf2可能参与了AKR1B10基因的调控。此外，通过RT-PCR，活性氧过氧化氢可以诱导H23细胞中AKR1B10的表达。这些结果表明，AKR1B10的表达受氧化应激的调控，而氧化应激与癌变密切相关。这些发现有望为阐明醛酮还原酶在癌细胞癌变和耐药中的作用，以及开发靶向醛酮还原酶的抗肿瘤药物提供基础信息。少

An aldo-keto reductase, AKR1B10 has been shown to be induced in the human non-small cell lung carcinoma. In this study, we studied the regulatory mechanisms of gene expression of AKR1B10 for the purpose of developing the drug metabolizing enzyme-targeting antitumor agents. First, we examined the AKR1B10 expression in human non-small cell lung carcinoma cell lines, A549 and H23 with RT-PCR technique. While significant AKR1B10 expression was observed in A549 cells, very little expression was detected in H23 cells, showing that these two cell lines can be useful research tools to study the gene regulation of AKR1B10 because of the remarkable contrast in the AKR1B10 expression. In order to identify the regulatory region upon AKR1B10 gene, the 5'-flanking region was isolated from A549 cells and analyzed. There is a microsatellite raging from -720 to -588 with CCTT repeats between the two alleles. However, this microsatellite was shown not to be involved in the regulation of basal AKR expres … More sion in luciferase reporter assay. There are several potential regulatory consensus sequences within the 5'-flanking region. Among them, there is a consensus sequence of antioxidant response element (ARE), which is related to the redox regulation of gene expression. Then, ethoxyquin, a reagent that enhances the ARE-mediated expression, was test for the AKR1B10 induction in H23 cells, and was shown to increase the AKR1B10 expression. Since transcription factor Nrf2 binds to ARE and enhances the gene expression, an Nrf2 expressing plasmid was co-introduced in the luciferase reporter assay. Co-expression of Nrf2 significantly increased the transcriptional activity of AKR1B10 gene, suggesting that ARE and Nrf2 can be involved in the AKR1B10 gene regulation. In addition, hydrogen peroxide, an active oxygen species, could induce the AKR1B10 expression in H23 cells with RT-PCR. These results suggest that AKR1B10 expression is regulated by oxidative stress that is tightly related to carcinogenesis. These findings are expected to be fundamental information to clarify the implication of aldo-keto reductases in the carcinogenesis and drug resistance of cancer cells, and to develop the aldo-keto reductase-targeting antitumor agents. Less