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

Abstract

Peripheral neuropathy is one of the major side effects of treatment with the anticancer drug, cisplatin. One proposed mechanism for this neurotoxicity is the formation of platinum adducts in sensory neurons that could contribute to DNA damage. Although this damage is largely repaired by nuclear excision repair (NER), our previous findings suggest that augmenting the base excision repair pathway (BER) by overexpressing the repair protein APE1 protects sensory neurons from cisplatin-induced neurotoxicity. The question remains whether APE1 contributes to the ability of the NER pathway to repair platinum-damage in neuronal cells. To examine this, we manipulated APE1 expression in sensory neuronal cultures and measured Pt-removal after exposure to cisplatin. When neuronal cultures were treated with increasing concentrations of cisplatin for two or three hours, there was a concentration-dependent increase in Pt-damage that peaked at four hours and returned to near baseline levels after 24 hours. In cultures where APE1 expression was reduced by ~80% using siRNA directed at APE1, there was a significant inhibition of Pt-removal over eight hours which was reversed by overexpressing APE1 using a lentiviral construct for human wtAPE1. Reduction in APE1 expression also altered the expression of the NER proteins RPA70 and XPA in sensory neuronal cultures. Overexpressing a mutant APE1 (C65 APE1), which only has DNA repair activity, but not its other significant redox-signaling function, mimicked the effects of wtAPE1. Overexpressing DNA repair activity mutant APE1 (226+177APE1), with only redox activity was ineffective suggesting it is the DNA repair function of APE1 and not its redox-signaling, that restores the Pt-damage removal. Together, these data provide the first evidence that a critical BER enzyme, APE1, helps regulate the NER pathway in the repair of cisplatin damage in sensory neurons.

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

“…We previously demonstrated that E3330 protects against both cisplatin-and oxaliplatin-induced neurotoxicity (Kelley et al, 2014). Therefore, we wanted to determine whether APX2009 has a similar protective effect after oxaliplatin treatment, which would also support our hypothesis that repair of oxidative DNA damage participates in the regulation of the platinum crosslink removal as shown with E3330 (Kelley et al, 2014;Kim et al, 2015). As shown in Fig.…”

“…In our previous studies using an experimental model of isolated sensory neurons in culture, we established a causal relationship between cancer therapy-induced neurotoxicity and DNA damage and repair and, more specifically, BER and APE1 (Vasko et al, 2005(Vasko et al, , 2011Jiang et al, 2008;Kelley et al, 2014;Kim et al, 2015;Georgiadis et al, 2016). 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.…”

“…Cisplatin and oxaliplatin both produce significant levels of reactive oxygen species in cells, with cisplatin producing higher levels (Preston et al, 2009;Kelley et al, 2014;Kim et al, 2015). However, the DNA crosslinks produced by these two agents differ: cisplatin produces Pt-1-2-d(GpG) intrastrand DNA crosslinks, whereas oxaliplatin creates predominantly Pt-1-3-d(ApG) interstrand DNA crosslinks (Kelley et al, 2014).…”

“…Although the neuroprotective effects of APX2009 are evident, we also wanted to investigate whether these E3330 analogs were capable of tumor cell killing similar to what we have observed with E3330 (Vasko et al, 2011;Kelley et al, 2014;Kim et al, 2015). A three-dimensional coculture model of pancreatic cancer established in our laboratories was used as an ex vivo system that included both low-passage, patient-derived tumor cells and cancer-associated fibroblasts (Arpin et al, 2016).…”

“…Therapies including platinum agents and ionizing radiation cause DNA damage in sensory neurons, and augmenting the base excision repair (BER) pathway reverses the toxic effects of these chemotherapies (Vasko et al, 2005(Vasko et al, , 2011Jiang et al, 2008;Kelley et al, 2014;Kim et al, 2015). Reducing the expression of a critical enzyme in the BER pathway, apurinic/apyrimidinic endonuclease (APE)-1 (also called , in sensory neurons amplifies the toxicity to sensory neurons exposed to anticancer therapies; by contrast, overexpressing APE1 and an APE1 DNA repair-proficient, redox-deficient mutant protein attenuates the neurotoxicity (Vasko et al, 2005(Vasko et al, , 2011Jiang et al, 2008;Kelley et al, 2014).…”