Role of APE1 in differentiated neuroblastoma SH-SY5Y cells in response to oxidative stress: Use of APE1 small molecule inhibitors to delineate APE1 functions

Abstract

Oxidative DNA damage has been implicated in a number of central nervous system pathologies. The base excision repair (BER) pathway is one of the most important cellular protection mechanisms that respond to oxidative DNA damage. Human apurinic (apyrimidinic) endonuclease/redox effector factor (APE1/Ref-1 or APE1) is an essential enzyme in the BER pathway and is expressed in both mitotic and post-mitotic cells in humans. In neurons, a reduction of APE1 expression increases chemotherapy-induced cytotoxicity, while overexpression of APE1 protects cells against the cytotoxicity. However, given the multiple functions of APE1, knockdown of total APE1 is not completely informative of whether it is the redox or DNA repair activity, or interactions with other proteins. Therefore, the use of selective small molecules that can block each function independent of the other is of great benefit in ascertaining APE1 function in post-mitotic cells. In this study, we chose differentiated SH-SY5Y cells as our post-mitotic cell line model to investigate whether a druginduced decrease in APE1 DNA repair or redox activity contributes to the growth and survival of post-mitotic cells under oxidative DNA damaging conditions. Here, we demonstrate that overexpression of WT-APE1 or C65-APE1 (repair competent) results in significant increase in cell viability after exposure to H 2 O 2 . However, the 177/226-APE1 (repair deficient) did not show a protective effect. This phenomenon was further confirmed by the use of methoxyamine (MX), which blocks the repair activity of APE1 that results in enhanced cell killing and apoptosis in differentiated SH-SY5Y cells and in neuronal cultures after oxidative DNA damaging treatments. Blocking APE1 Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conflict of interest:No conflicts of interest were disclosed. NIH Public Access

Identification and Characterization of New Chemical Entities Targeting Apurinic/Apyrimidinic Endonuclease 1 for the Prevention of Chemotherapy-Induced Peripheral Neuropathy

“…We demonstrated that reducing the activity of the DNA BER pathway by reducing APE1 expression increased the neurotoxicity produced by anticancer treatment, whereas augmenting APE1 activity lessened the neurotoxicity (Vasko et al, 2005(Vasko et al, , 2011Jiang et al, 2008Jiang et al, , 2009. In addition, we demonstrated that APE1's DNA repair function, not its redox signaling function, is crucial for sensory neuron survival and function (Vasko et al, 2011).…”

APE1/Ref‐1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single‐cellRNAsequencing

“…Apurinic/apyrimidinic endonuclease 1/redox factor‐1 (APE1/Ref‐1; henceforth referred to as APE1) is a multifunctional protein that is involved in repairing DNA damage via its endonuclease activity in base excision repair (Fung and Demple, 2005; Izumi et al ., 2005; Jiang et al ., 2009; Kelley et al ., 2014), and using its redox protein–protein signaling function to control the activity of numerous transcription factors such as STAT3, NFκB, AP‐1, p53, and hypoxia‐inducible factor 1α (HIF1α), among others (Cardoso et al ., 2012; Fishel et al ., 2015; Gaiddon et al ., 1999; Jiang et al ., 2010; Kelley et al ., 2012; Lando et al ., 2000; Logsdon et al ., 2016). It also contributes to the removal of damaged bases within RNA (Poletto et al ., 2016; Vascotto et al ., 2014).…”

APE1, the DNA base excision repair protein, regulates the removal of platinum adducts in sensory neuronal cultures by NER

“…In previous work from our laboratory, however, we demonstrated that augmenting the base excision repair (BER) pathway by increasing the expression of apurinic/apyrimidinic endonuclease (APE1) is neuroprotective against cisplatin-induced toxicity in isolated sensory neurons [18]. We and others also have shown that cisplatin increases production of reactive oxygen species (ROS) and that oxidative stress may contribute to cisplatin-induced toxicity [18–21].…”