2-Hydroxy-3′,5,5′-trimethoxychalcone (named DK-139) is a synthetic chalcone derivative that has anti-inflammatory, anti-tumor, and endoplasmic reticulum-mediated apoptosis activities. However, the mode of action of DK-139 on reactive oxygen species (ROS)-induced apoptosis remains unknown. In this study, we found that DK-139 activated DNA damage responses, as was revealed by the accumulation of the tumor suppressor p53 and the phosphorylation of histone H2AX at Ser139 (called γ-H2AX), which are hallmarks of DNA damage responses. The occurrence of DK-139-induced DNA damage was confirmed through single-cell gel electrophoresis (comet tail assay). Interestingly, using p53-null HCT116 cells revealed that p53 was not involved in DK-139-induced apoptosis. Instead, we found that DK-139 increased the production of ROS, which led to the processing of caspase-2, BH3 interacting-domain death agonist (BID), caspase-9, and caspase-7. Pretreatment with the ROS scavenger N-acetyl cysteine reduced the frequency of DK-139-induced γ-H2AX formation, demonstrating that DK-139 triggered DNA damage through ROS production. In addition, NAC pretreatment prevented DK-139-induced processing of caspase-2, BID, caspase-9, caspase-7, and poly(ADP-ribose) polymerase. These results suggest that DK-139 triggers apoptosis through ROS-mediated DNA damage and activation of the caspase-2 cascade in A549 human lung cancer cells.

2-羟基-3'，5,5'-三甲氧基查耳酮（命名为DK-139）是具有抗炎，抗肿瘤和内质网介导的细胞凋亡活性的合成查尔酮衍生物。但是，DK-139对活性氧（ROS）诱导的细胞凋亡的作用方式仍然未知。在这项研究中，我们发现DK-139激活了DNA损伤反应，这是由肿瘤抑制因子p53的积累和组蛋白H2AX在Ser139处的磷酸化（称为γ-H2AX）所揭示的，这是DNA损伤反应的标志。通过单细胞凝胶电泳（彗尾试验）证实了DK-139诱导的DNA损伤的发生。有趣的是，使用无p53的HCT116细胞表明，p53不参与DK-139诱导的细胞凋亡。相反，我们发现DK-139增加了ROS的产生，导致处理了caspase-2，BH3相互作用域死亡激动剂（BID），caspase-9和caspase-7。用ROS清除剂N-乙酰半胱氨酸进行预处理可以降低DK-139诱导的γ-H2AX形成的频率，这表明DK-139通过ROS产生触发了DNA损伤。另外，NAC预处理阻止了DK-139诱导的caspase-2，BID，caspase-9，caspase-7和聚（ADP-核糖）聚合酶的加工。这些结果表明DK-139通过ROS介导的DNA损伤和A549人肺癌细胞中caspase-2级联的激活来触发细胞凋亡。另外，NAC预处理阻止了DK-139诱导的caspase-2，BID，caspase-9，caspase-7和聚（ADP-核糖）聚合酶的加工。这些结果表明DK-139通过ROS介导的DNA损伤和A549人肺癌细胞中caspase-2级联的激活来触发细胞凋亡。另外，NAC预处理阻止了DK-139诱导的caspase-2，BID，caspase-9，caspase-7和聚（ADP-核糖）聚合酶的加工。这些结果表明，DK-139通过ROS介导的DNA损伤和A549人肺癌细胞中caspase-2级联的激活来触发细胞凋亡。