Vorinostat Induces Reactive Oxygen Species and DNA Damage in Acute Myeloid Leukemia Cells Luca A. Petruccelli., Daphne´ Dupe´ re´ -Richer., Filippa Pettersson, He´ le` ne Retrouvey, Sophia Skoulikas, Wilson H. Miller Jr.* Lady Davis Institute for Medical Research, Segal Cancer Center, Jewish General Hospital, McGill University, Montreal, Canada Abstract Histone deacetylase inhibitors (HDACi) are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML) cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC) reduces vorinostat-induced DNA double strand breaks, G2- M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents. Citation: Petruccelli LA, Dupe´re´-Richer D, Pettersson F, Retrouvey H, Skoulikas S, et al. (2011) Vorinostat Induces Reactive Oxygen Species and DNA Damage in Acute Myeloid Leukemia Cells. PLoS ONE 6(6): e20987. doi:10.1371/journal.pone.0020987 Editor: Venugopalan Cheriyath, Cleveland Clinic, United States of America Received August 26, 2010; Accepted May 17, 2011; Published June 10, 2011 Copyright: 2011 Petruccelli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by Canadian Institutes of Health Research (CIHR) grant # MOP-12863 (www.cihr.ca). Merck provided Vorinostat and financial support (www.merck.com). Wilson H. Miller Jr. is a Chercheur National of the Fonds de la Recherche en Sante´ Que´bec (FRSQ) (www.frsq.gouv.qc.ca). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: This work was financially supported in part by a research grant from Merck. Merck also supplied Vorinostat. This does not affect the authors’ ability to adhere to all of PLoS ONE’s policies on sharing data and materials with regards to this work. * E-mail: wmiller@ldi.jgh.mcgill.ca . These authors contributed equally to this work. Introduction Histone Deacetylases (HDACs) catalyze the removal of acetyl groups from e-lysine residues of histones resulting in chromatin condensation and gene silencing. HDAC overexpression and aberrant recruitment to the promoters of genes implicated in differentiation and tumor suppression has been reported in a number of malignancies [1,2]. HDACs can be divided into four classes: class I (HDAC1, 2, 3, and 8), class IIa (HDAC4, 5, 7, and 9), class IIb (HDAC6 and 10), class III (SIRT1, 2, 3, 4, 5, 6, and 7) and class IV (HDAC11) [3]. Class I, II and IV are zinc-dependent deacetylases, while Class III is NAD+-dependent [3,4]. HDACs can also remove acetyl residues from non-histone proteins, thereby altering their function, providing another mechanism by which HDACs may participate in the malignant phenotype. Targeting HDAC activity using pharmacological small molecule HDAC inhibitors (HDACi) has become an exciting therapeutic strategy. Early studies correlated the anti-tumor effects of HDAC inhibition with restoration of expression of genes involved in differentiation and tumor suppression [5,6]. However, subsequent microarray studies found that HDACi directly or indirectly activate only 7– 10% of all genes, with roughly the same number repressed [7]. This suggested that the anti-tumor activities of HDACi were not limited to their effects on transcription. Indeed, a recent study found 1750 proteins to be acetylated in response to HDACi [8], further suggesting that HDACi can influence diverse cellular pathways. While HDACi were initially associated with differentiation therapy [9,10], recent work with HDACi has expanded into understanding mechanisms of HDACi cytotoxicity. All HDACi have been reported to arrest growth and to activate the extrinsic and/or the intrinsic apoptotic pathways [3]. Additionally, HDACi have been shown to inhibit tumor growth in animal models [11,12]. One mechanism by which HDACi are thought to cause cancer cell apoptosis is through the induction of DNA damage and genomic instability [13]. This has been shown to occur through the generation of reactive oxygen species (ROS), deregulation of repair proteins by acetylation and repression of DNA damage repair protein expression [13,14,15]. It is worth mentioning that normal cells have been reported to be significantly less sensitive than tumor cells to all these effects [16]. Vorinostat (suberoylanilide hydroxamic acid, ZolinzaTM) is a broad spectrum HDACi that inhibits zinc-dependent HDACs (Class I, II and IV). Vorinostat received FDA approval for use in Cutaneous T-cell lymphoma (CTCL) and is in clinical trials, both as a single agent and in combination with other agents in a number of other malignancies [17]. It has also been shown to induce ROS and DNA damage in a number of tumor types [13,18]. Nonetheless, the characterization of vorinostat in acute myeloid leukemia (AML) cells remains incomplete. PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e20987