Oxidative DNA Damage and Cisplatin Neurotoxicity Is Exacerbated by Inhibition of OGG1 Glycosylase Activity and APE1 Endonuclease Activity in Sensory Neurons

Abstract

Cisplatin can induce peripheral neuropathy, which is a common complication of anti-cancer treatment and negatively impacts cancer survivors during and after completion of treatment; therefore, the mechanisms by which cisplatin alters sensory neuronal function to elicit neuropathy are the subject of much investigation. Our previous work suggests that the DNA repair activity of APE1/Ref-1, the rate-limiting enzyme of the base excision repair (BER) pathway, is critical for neuroprotection against cisplatin. A specific role for 8-oxoguanine DNA glycosylase-1 (OGG1), the glycosylase that removes the most common oxidative DNA lesion, and putative coordination of OGG1 with APE1/Ref-1 in sensory neurons, has not been investigated. We investigated whether inhibiting OGG1 glycosylase activity with the small molecule inhibitor, TH5487, and/or APE1/Ref-1 endonuclease activity with APE Repair Inhibitor III would alter the neurotoxic effects of cisplatin in sensory neuronal cultures. Sensory neuron function was assessed by calcitonin gene-related peptide (CGRP) release, as a marker of sensitivity and by neurite outgrowth. Cisplatin altered neuropeptide release in an inverse U-shaped fashion, with low concentrations enhancing and higher concentrations diminishing CGRP release. Pretreatment with BER inhibitors exacerbated the functional effects of cisplatin and enhanced 8oxo-dG and adduct lesions in the presence of cisplatin. Our studies demonstrate that inhibition of OGG1 and APE1 endonuclease activity enhances oxidative DNA damage and exacerbates neurotoxicity, thus limiting oxidative DNA damage in sensory neurons that might alleviate cisplatin-induced neuropathy.

Keywords: DNA damage; base excision repair; chemotherapy-induced peripheral neuropathy; cisplatin; neurite outgrowth; neuropeptide; oxidative stress; sensory neuron.

Figure 1

CGRP release from neuronal cultures is altered following exposure to cisplatin. (A). Columns represent the mean ± SEM of CGRP release stimulated by a 10 min exposure to 30 nM capsaicin following a 24 h exposure to increasing concentrations of cisplatin (30 μM). * p < 0.0001 comparing stimulated release in CIS to no treatment control, two-way ANOVA with Tukey’s posttest. (B). Each column represents the mean ± SEM of the total CGRP content following exposure to cisplatin treatments. * p < 0.05 comparing content in CIS (100 μM) to no treatment control, one-way ANOVA with Dunnett’s posttest.

Figure 2

CGRP release from neuronal cultures is altered following exposure to cisplatin and the changes in stimulated CGRP release following exposure to base excision repair inhibitors. (A). Columns represent the mean ± SEM of CGRP release stimulated by a 10 min exposure to 30 nM capsaicin following a 24 h exposure to cisplatin (30 μM). * p < 0.0001 comparing stimulated release in CIS to VEH controls; † p < 0.001 comparing CIS effects in the presence and absence of base excision repair inhibitors, two-way ANOVA with Tukey’s posttest. (B). Each column represents the mean ± SEM of the total CGRP content following the indicated treatments.

Figure 3

Effects of cisplatin and base excision repair inhibitors on 8-oxodG levels in cells adjacent to sensory neurons. (A), Representative immunostaining for 8-oxodG (green) in sensory neuron cultures. The original magnification was ×20. Scale bar represents 200 µm. (B), Quantitative analysis of the integrated density of 8-oxodG staining within PGP9.5+ regions of the image field, acquired as relative fluorescence units using Cytation5 software. * p < 0.0001 comparing TH and ARI3 to VEH; † p < 0.0001 comparing TH/CIS and ARI3/CIS to inhibitors alone, two-way ANOVA with Tukey’s posttest.

Figure 4

Effects of cisplatin and base excision repair inhibitors on cisplatin adduct levels in cells adjacent to sensory neurons. (A) Representative immunostaining for cisplatin adducts (green) in sensory neuron cultures. The original magnification was ×20. Scale bar represents 200 µm. (B) Quantitative analysis of the integrated density of adduct staining within PGP9.5+ regions of the image field, acquired as relative fluorescence units using Cytation5 software. † p < 0.0001 comparing TH/CIS and ARI3/CIS to TH/VEH and ARI3/VEH, respectively, * p < 0.0001 comparing VEH/CIS to ARI3/CIS; two-way ANOVA with Tukey’s posttest.

Figure 5

Effects of cisplatin and base excision repair inhibitors on neurite outgrowth. (A) Representative immunostaining for PGP9.5 (green) in sensory neuron cultures. The original magnification was ×20. Scale bar represents 200 µm. (B) Quantitative analysis of the axonal area of PGP9.5 staining, acquired using Cytation5 software. * p < 0.01 comparing the indicated groups to the Vehicle-treated control, two-way ANOVA with Tukey’s posttest.

Figure 6

A summary of the proposed roles of oxidative DNA damage, OGG1 and APE1 in sensory neurons. Figure created with Biorender.com, accessed on 29 December 2021.