Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle-upon-Tyne, NE2 4HH, UK.
Mutagenesis. 2011 Jul;26(4):489-98. doi: 10.1093/mutage/ger006. Epub 2011 Mar 7.
Green tea and its major active constituent, (-)-epigallocatechin-3-gallate (EGCG), are in clinical trials for the prevention and treatment of several diseases such as cancer. DNA topoisomerase (topo) poisons are commonly prescribed anticancer drugs that kill cancer cells by inducing topo-DNA complexes. Using purified topoisomerases, previous in vitro studies have shown that EGCG induces the formation of topo-DNA complexes. Because the activity of a drug on purified topoisomerases does not always represent the activity in a cell, we have used an immunofluorescence technique that allows the visualisation of topo I- and topo II-DNA complexes produced in individual cells to evaluate the activity of EGCG on both enzymes. High levels of topo I- and topo II-DNA complexes were observed in K562 leukaemia cells exposed to EGCG. Similar levels of topo I- and topo II-DNA complexes were visualised in cells treated with gallic acid (GA) (the acid part of the EGCG ester). Pyrogallol (PG) also induced topo-DNA complexes with both enzymes, therefore suggesting that the activity of EGCG and GA is mediated by their PG moieties. Catalase prevented both the cytotoxicity and the formation of topo I- and topo II-DNA complexes induced by EGCG, GA, PG and myricetin (a PG-containing flavonoid recently shown to induce topo I- and topo II-DNA complexes in cells), indicating that hydrogen peroxide mediates these activities. Hydrogen peroxide induced topo I- and topo II (α and β)-DNA complexes in a time- and dose-dependent manner. The formation of topo I- and topo II-DNA complexes in cells exposed to hydrogen peroxide correlated well with the induction of apoptosis, suggesting that the topo-DNA complexes induced at long exposure times by the compounds tested in our study may be apoptotic topo-DNA complexes. Finally, we report results suggesting that PG-containing drugs may selectively kill tumour cells by generating hydrogen peroxide.
绿茶及其主要活性成分（-）-表没食子儿茶素-3-没食子酸酯（EGCG）正在临床试验中，用于预防和治疗多种疾病，如癌症。DNA 拓扑异构酶（topo）毒物是常用的抗癌药物，通过诱导 topo-DNA 复合物杀死癌细胞。以前的体外研究使用纯化的拓扑异构酶表明，EGCG 诱导 topo-DNA 复合物的形成。由于药物在纯化的拓扑异构酶上的活性并不总是代表在细胞中的活性，我们使用免疫荧光技术来评估 EGCG 对两种酶的活性，该技术允许在单个细胞中可视化产生的拓扑异构酶 I 和拓扑异构酶 II-DNA 复合物。在暴露于 EGCG 的 K562 白血病细胞中观察到高水平的拓扑异构酶 I 和拓扑异构酶 II-DNA 复合物。在用没食子酸（GA）（EGCG 酯的酸部分）处理的细胞中观察到类似水平的拓扑异构酶 I 和拓扑异构酶 II-DNA 复合物。焦儿茶酚（PG）也诱导了两种酶的 topo-DNA 复合物，因此表明 EGCG 和 GA 的活性是由它们的 PG 部分介导的。过氧化氢酶可预防 EGCG、GA、PG 和杨梅素（一种最近显示可在细胞中诱导拓扑异构酶 I 和拓扑异构酶 II-DNA 复合物的 PG 含量黄酮类化合物）诱导的细胞毒性和 topo I 和拓扑异构酶 II-DNA 复合物的形成，表明过氧化氢介导这些活性。过氧化氢以时间和剂量依赖的方式诱导拓扑异构酶 I 和拓扑异构酶 II（α 和β）-DNA 复合物。在暴露于过氧化氢的细胞中形成拓扑异构酶 I 和拓扑异构酶 II-DNA 复合物与细胞凋亡的诱导密切相关，这表明在我们的研究中测试的化合物在长时间暴露下诱导的 topo-DNA 复合物可能是凋亡的 topo-DNA 复合物。最后，我们报告的结果表明，含 PG 的药物可能通过生成过氧化氢选择性杀死肿瘤细胞。
