Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221-0006, USA.
Chem Res Toxicol. 2012 Nov 19;25(11):2542-52. doi: 10.1021/tx300337j. Epub 2012 Oct 22.
Agents that chemically modify DNA form a backbone of many cancer treatments. A key problem for DNA-modifying agents is lack of specificity. To address this issue, we designed novel molecular scaffolds, termed An-Hq and An-Hq(2), which are activated by a hallmark of some cancers: elevated concentrations of reactive oxygen species. Elevated reactive oxygen species are linked to oncogenesis and are found to increase in several aggressive cancers. The agents are quinones that, upon oxidation, form highly electrophilic species. In vitro studies identified the mode of addition to DNA. The aniline portion of An-Hq serves to enhance nucleophilic addition to the ethyl phenyl ether instead of forming common Michael additions. Structural characterization showed that the agents add to 2'-deoxyguanosine at the N2,N3-positions. The product formed is a bulky hydroxy-N2,3-benzetheno-2'-deoxyguanosine adduct. In addition, the oxidatively activated agents added to 2'-deoxyadenosine and 2'-deoxycytidine but not thymidine or 2'-deoxyinosine. These findings are confirmed by primer extension analysis of a 392 base pair DNA. The full-length primer extension product was reduced by 69.0 ± 0.6% upon oxidative activation of An-Hq(2) as compared to controls. Little sequence dependence was observed with 76% of guanine, adenine, and cytosine residues showing an increase in extension stops between 2- and 4-fold above controls. Benzetheno-nucleobase addition to double-stranded DNA was confirmed by LC/MS of a self-complementary oligonucletide. Experiments were carried out to confirm in vivo DNA damage. Because of the lesion identified in vitro, we reasoned that nucleotide excision repair should be involved in reversing the effects of these oxidatively activated agents and enhance toxicity in Drosophila melanogaster. Using an RNAi-based approach, Ercc1 was silenced, and survival was monitored after injection of an agent. As expected, bulky cross-linking DNA-modifying agents, cisplatin and chlorambucil, showed statistically significant enhanced toxicity in Drosophila with silenced Ercc1. In addition, 5-fluorouracil, which does not produce bulky lesions, showed no selective toxicity. An-Hq and An-Hq(2) showed statistically significant toxicity in Drosophila with silenced Ercc1. Examination of cytotoxicity shows renal carcinoma cell lines as a target of these agents with a median IC(50) of 1.8 μM. Taken together, these data show that the designed oxidatively activated agents form distinct, bulky DNA modifications that prove difficult for cancer cells possessing an elevated reactive oxygen species phenotype to overcome. The modification produced is relatively unique among anticancer agents.
能够化学修饰 DNA 的试剂构成了许多癌症治疗方法的基础。DNA 修饰试剂的一个关键问题是缺乏特异性。为了解决这个问题，我们设计了新型分子支架，称为 An-Hq 和 An-Hq(2)，它们被一些癌症的一个标志激活：活性氧浓度升高。活性氧与致癌有关，并且在几种侵袭性癌症中发现其增加。这些试剂是醌，在氧化时形成高亲电物种。体外研究确定了与 DNA 的加成模式。An-Hq 的苯胺部分有助于增强对乙基苯醚的亲核加成，而不是形成常见的迈克尔加成。结构表征表明，这些试剂在 N2，N3-位置添加到 2'-脱氧鸟苷。形成的产物是一个大体积的羟基-N2，3-苯并噻吩-2'-脱氧鸟苷加合物。此外，氧化激活的试剂添加到 2'-脱氧腺苷和 2'-脱氧胞嘧啶，但不添加胸苷或 2'-脱氧肌苷。这些发现通过对 392 个碱基对 DNA 的引物延伸分析得到证实。与对照相比，氧化激活 An-Hq(2)后，全长引物延伸产物减少了 69.0±0.6%。观察到很少的序列依赖性，76%的鸟嘌呤、腺嘌呤和胞嘧啶残基的延伸停止增加了 2-4 倍以上。通过自互补寡核苷酸的 LC/MS 证实了双链 DNA 上的苯并噻吩-核苷加合物。进行了体内 DNA 损伤的实验以确认。由于体外鉴定的损伤，我们推断核苷酸切除修复应该参与逆转这些氧化激活试剂的作用，并增强果蝇黑色素体中的毒性。使用基于 RNAi 的方法，沉默了 Ercc1，并在注射试剂后监测了存活情况。正如预期的那样，具有大体积交联的 DNA 修饰试剂顺铂和氯氨苯丁酸在沉默 Ercc1 的果蝇中表现出统计学上显著增强的毒性。此外，不产生大体积损伤的 5-氟尿嘧啶没有表现出选择性毒性。An-Hq 和 An-Hq(2)在沉默 Ercc1 的果蝇中表现出统计学上显著的毒性。细胞毒性检查显示肾癌细胞系是这些试剂的靶标，其半数最大抑制浓度（IC50）为 1.8 μM。总之，这些数据表明，设计的氧化激活试剂形成了独特的、大体积的 DNA 修饰，这使得具有升高的活性氧表型的癌细胞难以克服。产生的修饰在抗癌剂中相对独特。
