Novobiocin And Its Derivative XN4Induce DNA Damage In CML Cells Through Generation Of Reactive Oxygen Species

Purpose To investigate the cytotoxicity of Novobiocin and it’s derivative XN4againstChronic myeloid leukemia (CML) cells, we explored its underlying mechanismsmediating the induction of DNA damage and apoptotic cell death via reactive oxygenspecies (ROS).Methods MTT and CFSE were used to measure the proliferation inhibition ratio ofK562and K562/G01cells. Flow Cytometry (FCM) was used to test the level ofextracellular ROS, DNA damage, cell cycle progression, mitochondria membranepotential (MPP) and apoptosis. qRT-PCR was used to detect the mRNA expression ofNox gene family; Western blot was used to verified the amount of proteins associationwith DNA damage response, cell cycle and cell apoptosis pathway.Result (1) Nov and XN4can significantly inhibited the proliferation of K562withIC50was (232.50±0.22) μmol·L-1and （3.75±0.07）μmol·L-1, respectively, andK562/G01cells with IC50was (237.10±0.13) μmol·L-1and （2.63±0.43）μmol·L-1,respectively. The antitumor activity of XN4was approximately100fold higher thanNov;(2) Both Nov and Nox4could reduce the proliferation division of CML cells.(3)Nov and XN4increased the production of intracellular ROS, followed by induction ofDNA damage and activation of the ATM–p53–r-H2AX pathway andcheckpoint-related signals Chk1/Chk2, which led to increased numbers of cells in theS and G2/M phases of the cell cycle. Furthermore, Nov induced apoptotic cell deaththrough activation of caspase-3and PARP;(4) Nov could increase the mRNA level ofNox5, and XN4could increase the mRNA amount of both Nox4and Nox5genes. Theabove effects were all prevented by the ROS scavenger N-acetylcysteine. ConclusionThese findings therefore suggest that Nov and XN4could increase the expression ofNox5and/or Nox4gene, resulting in inducing DNA damage, cell cycle arrest andapoptosis in CML cells mediated via ROS generation.