Morgan Andrew G M, Babu Dinesh, Michail Karim, Siraki Arno G
Faculty of Pharmacy and Pharmaceutical sciences, University of Alberta, Edmonton, Canada.
Faculty of Pharmacy and Pharmaceutical sciences, University of Alberta, Edmonton, Canada; Faculty of Pharmacy, Alexandria University, Alexandria, Egypt.
Toxicol Lett. 2017 Oct 5;280:48-56. doi: 10.1016/j.toxlet.2017.07.894. Epub 2017 Jul 25.
Several lines of evidence have pointed towards the potential therapeutic benefit of NSAIDs in cancer therapy. In this study, we have investigated the acute bio-activation of NSAIDs and their metabolites via myeloperoxidase (MPO), a highly-expressed peroxidase enzyme in acute myeloid leukemia. As bio-activation involves the formation of reactive metabolites, we hypothesized that NSAIDs which produced reactive metabolites would be correlated with leukemia cell toxicity. We tested the enzymatic peroxidation of three NSAIDs, namely diclofenac, indomethacin, and naproxen in comparison with their hepatic metabolites, 4'- hydroxydiclofenac (4'-OHD), 5-hydroxydiclofenac (5-OHD), O-desmethyl-N-deschlorobenzoylindomethacin (DMBI), O-desmethylindomethacin (DMI) and O-desmethylnaproxen (ODN). Firstly, we used purified peroxidases in kinetic UV-vis kinetic spectrophotometry, and electron paramagnetic resonance (EPR) experiments to determine oxidation of ascorbic acid and glutathione (GSH), respectively. We then used HL-60 cells, as a model of acute myelogenous leukemia to carry out trypan blue exclusion, cellular ATP analysis, mitochondrial membrane potential (MMP) and cytofluorometric GSH assays. Our results present evidence that diclofenac, 4'-OHD, 5-OHD, DMBI and DMI demonstrated significant cytotoxic effect in the leukemic cells through oxidation by intracellular MPO. In the same vein, only diclofenac and its two metabolites caused a significant drop in the MMP and cellular ATP level; however, the cell death induced by indomethacin metabolites reflected a subtle effect on MMP or GSH content. Interestingly, only diclofenac and 4'-OHD (and not 5- OHD) caused a significant drop in HL-60 cells' GSH content. Among diclofenac compounds, only 4'-OHD also generated GS radical and caused a significant increase in ascorbate co-oxidation rate. Lastly, even though ODN also generated GS radical and potently cooxidized ascorbate, it showed no significant cytotoxicity. These results provide evidence of a correlation between acute cytotoxicity and MPO-bioactivated NSAIDs, though this was not correlated for all compounds (e.g., ODN). Further studies are required to determine both the MPO-dependent and MPO-independent mechanisms of cytotoxicity.
多条证据表明非甾体抗炎药（NSAIDs）在癌症治疗中具有潜在的治疗益处。在本研究中，我们研究了NSAIDs及其代谢产物通过髓过氧化物酶（MPO）的急性生物活化作用，MPO是急性髓系白血病中一种高表达的过氧化物酶。由于生物活化涉及活性代谢产物的形成，我们假设产生活性代谢产物的NSAIDs与白血病细胞毒性相关。我们测试了三种NSAIDs（双氯芬酸、吲哚美辛和萘普生）及其肝脏代谢产物4'-羟基双氯芬酸（4'-OHD）、5-羟基双氯芬酸（5-OHD）、O-去甲基-N-去氯苯甲酰吲哚美辛（DMBI）、O-去甲基吲哚美辛（DMI）和O-去甲基萘普生（ODN）的酶促过氧化作用。首先，我们在动力学紫外可见分光光度法和电子顺磁共振（EPR）实验中使用纯化的过氧化物酶，分别测定抗坏血酸和谷胱甘肽（GSH）的氧化情况。然后，我们使用HL-60细胞作为急性髓性白血病模型，进行台盼蓝排斥试验、细胞ATP分析、线粒体膜电位（MMP）和细胞荧光GSH测定。我们的结果表明，双氯芬酸、4'-OHD、5-OHD、DMBI和DMI通过细胞内MPO氧化在白血病细胞中表现出显著的细胞毒性作用。同样，只有双氯芬酸及其两种代谢产物导致MMP和细胞ATP水平显著下降；然而，吲哚美辛代谢产物诱导的细胞死亡对MMP或GSH含量的影响较小。有趣的是，只有双氯芬酸和4'-OHD（而不是5-OHD）导致HL-60细胞的GSH含量显著下降。在双氯芬酸化合物中，只有4'-OHD还产生了GS自由基并导致抗坏血酸共氧化速率显著增加。最后，尽管ODN也产生了GS自由基并有力地共氧化了抗坏血酸，但它没有表现出显著的细胞毒性。这些结果提供了急性细胞毒性与MPO生物活化的NSAIDs之间存在相关性的证据，尽管并非所有化合物（如ODN）都如此。需要进一步研究以确定细胞毒性的MPO依赖性和MPO非依赖性机制。
