Cellular and cell-free studies of catalytic DNA cleavage by ruthenium polypyridyl complexes containing redox-active intercalating ligands.
Cellular and cell-free studies of catalytic DNA cleavage by ruthenium polypyridyl complexes containing redox-active intercalating ligands.
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DOI：
10.1039/c6sc04094b
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发表时间：
2017-05-01
期刊：
影响因子：
8.4
通讯作者：
MacDonnell FM
中科院分区：
文献类型：
--
作者：
Griffith C;Dayoub AS;Jaranatne T;Alatrash N;Mohamedi A;Abayan K;Breitbach ZS;Armstrong DW;MacDonnell FM
黄色焦点显示用IC 50浓度的[（phen）2 Ru（tatpp）Ru（Phen）2] Cl 4处理的H358细胞的细胞核中的时间依赖性DNA双链断裂。钌（ii）多吡啶络合物（RPC），[（phen）2 Ru（tatpp）]2+（32+）和[（phen）2 Ru（tatpp）Ru（phen）2]4+（44+）在温和还原剂即谷胱甘肽（GSH）存在下的无细胞研究中显示出以在降低[O2]时增强的方式切割DNA。由于羟基自由基清除剂减弱切割活性，因此活性氧物质（ROS）参与切割过程。超氧化物歧化酶（SOD）和过氧化氢酶的存在下的裂解实验揭示了一个核心的作用H2 O2作为羟基自由基的直接前体。提出了一种机制，解释了逆[O2]依赖性和活性氧数据，并涉及三种DNA结合的氧化还原异构体32+或44+之间的氧化还原循环。培养的非小细胞肺癌细胞（H358）对32+和44+敏感，IC 50值分别为13和15 μM，当用对映体纯形式的32+和44+处理小鼠时，裸鼠中的异种移植H358肿瘤相对于未处理的肿瘤显示出实质性（约80%）消退（Yadav等人，Mol Cancer Res，2013，12，643）。用15 μM 44+处理的H358细胞的荧光显微镜显示，在处理后短短2小时内，细胞内ROS的产生增加。在用44+处理的2小时内通过免疫荧光检测磷酸化ATM揭示了由于H358细胞核中的ROS损伤和DNA双链断裂（DSB）而启动DNA损伤修复机制，并使用γ H2 AX测定法进行了确认。32+的细胞数据不太清楚，但发生了DNA损伤。值得注意的是，用[Ru（diphenylphen）3]2+（IC 50 1.7 μM）处理的细胞即使在22小时后也没有显示出额外的ROS产生，也没有显示出pATM或γ H2 AX对DNA的损伤。在32+和44+的无细胞切割试验中观察到的低[O2]（4 μM）下增强的DNA切割仅部分反映在缺氧（1.1%O2）下相对于常氧（18%O2）下H358、HCC 2998、HOP-62和Hs 766 t中32+和44+的细胞毒性中。用RPC 32+处理的细胞在缺氧下显示出高达两倍的IC 50增强，而用RPC 44+处理的细胞无论在缺氧还是常氧下都给出相同的IC 50。
Yellow foci show time dependent DNA double strand breaks in the nuclei of H358 cells treated with IC50 concentration of [(phen)2Ru(tatpp)Ru(Phen)2]Cl4. The ruthenium(ii) polypyridyl complexes (RPCs), [(phen)2Ru(tatpp)]2+ (32+) and [(phen)2Ru(tatpp)Ru(phen)2]4+ (44+) are shown to cleave DNA in cell-free studies in the presence of a mild reducing agent, i.e. glutathione (GSH), in a manner that is enhanced upon lowering the [O2]. Reactive oxygen species (ROS) are involved in the cleavage process as hydroxy radical scavengers attenuate the cleavage activity. Cleavage experiments in the presence of superoxide dismutase (SOD) and catalase reveal a central role for H2O2 as the immediate precursor for hydroxy radicals. A mechanism is proposed which explains the inverse [O2] dependence and ROS data and involves redox cycling between three DNA-bound redox isomers of 32+ or 44+. Cultured non-small cell lung cancer cells (H358) are sensitive to 32+ and 44+ with IC50 values of 13 and 15 μM, respectively, and xenograft H358 tumors in nude mice show substantial (∼80%) regression relative to untreated tumors when the mice are treated with enantiopure versions of 32+ and 44+ (Yadav et al. Mol Cancer Res, 2013, 12, 643). Fluorescence microscopy of H358 cells treated with 15 μM 44+ reveals enhanced intracellular ROS production in as little as 2 h post treatment. Detection of phosphorylated ATM via immunofluorescence within 2 h of treatment with 44+ reveals initiation of the DNA damage repair machinery due to the ROS insult and DNA double strand breaks (DSBs) in the nuclei of H358 cells and is confirmed using the γH2AX assay. The cell data for 32+ is less clear but DNA damage occurs. Notably, cells treated with [Ru(diphenylphen)3]2+ (IC50 1.7 μM) show no extra ROS production and no DNA damage by either the pATM or γH2AX even after 22 h. The enhanced DNA cleavage under low [O2] (4 μM) seen in cell-free cleavage assays of 32+ and 44+ is only partially reflected in the cytotoxicity of 32+ and 44+ in H358, HCC2998, HOP-62 and Hs766t under hypoxia (1.1% O2) relative to normoxia (18% O2). Cells treated with RPC 32+ show up to a two-fold enhancement in the IC50 under hypoxia whereas cells treated with RPC 44+ gave the same IC50 whether under hypoxia or normoxia.