Implications of apurinic/apyrimidinic endonuclease in reactive oxygen signaling response after cisplatin treatment of dorsal root ganglion neurons

Abstract

Peripheral neuropathy is one of the major side effects of the anticancer drug cisplatin. Although previous work suggests that this neuropathy correlates with formation of DNA adducts in sensory neurons, growing evidence suggests that cisplatin also increases the generation of reactive oxygen species (ROS), which could cause DNA damage. Apurinic/apyrimidinic endonuclease/redox factor-1 (Ape1/Ref-1) is a multifunctional protein involved in DNA base excision repair of oxidative DNA damage and in redox regulation of a number of transcription factors. Therefore, we asked whether altering Ape1 functions would influence cisplatin-induced neurotoxicity. Sensory neurons in culture were exposed to cisplatin for 24 hours and several end points of toxicity were measured, including production of ROS, cell death, apoptosis, and release of the immunoreactive calcitonin gene-related peptide (iCGRP). Reducing expression of Ape1 in neuronal cultures using small interfering RNA (siRNA) enhances cisplatin-induced cell killing, apoptosis, ROS generation, and cisplatin-induced reduction in iCGRP release. Overexpressing wild-type Ape1 attenuates all the toxic effects of cisplatin in cells containing normal endogenous levels of Ape1 and in cells with reduced Ape1 levels after Ape1siRNA treatment. Overexpressing the redox deficient/repair competent C65-Ape1 provides partial rescue, whereas the repair-deficient Ape1 (N226A + R177A) does not protect neurons from cisplatin toxicity. We also observe an increase in phosphorylation of p53 after a decrease in Ape1 levels in sensory neuronal cultures. These results strongly support the notion that Ape1 is a potential translational target such that protecting Ape1 levels and particularly its DNA repair function could reduce peripheral neuropathy in patients undergoing cisplatin treatment.

Figure 1. Effect of cisplatin treatments on sensory neuronal cells

A. Sensory neuronal cell cultures were incubated with different concentrations of cisplatin for 24 hrs. Cytotoxicity was determined using the trypan blue exclusion assay as described. Each point represents the mean ± SE for three independent harvests of cells. Asterisks indicates a statistically significant (*p<0.05) difference from control. B. ROS generation in sensory neuronal cell cultures following cisplatin treatment. After incubation with cisplatin for 24 hrs, sensory neuronal cell cultures were loaded with 10 μM carboxy-H2DCFDA and incubated for 1 hour. Carboxy-H2DCFDA is an indicator for reactive oxygen species (ROS) and only fluoresces when hydrolyzed by esterases and oxidation occurs within the reaction system. The top portion of the figure shows the fluorescence of the oxidized form of carboxy-H2DCFDA as analyzed in a representative flow cytometry experiment. As a positive control for oxidative stress, cells were treated for 1 hr with 100 μM TBHP. The panel at the bottom represents the summary of three experiments expressed as the amount of change in fluorescent cells following cisplatin treatment. Each point represents the mean ± SE for three independent harvests of cells. Statistically different points from controls are indicated with an asterisk (*p<0.05). C. Ape1 protein expression levels following cisplatin treatment. The panel on the top shows a representative Western blot of the expression of Ape1 after sensory neuronal cell cultures were incubated with different cisplatin concentrations for 24 hrs. The panel at the bottom represents the summary of Ape1 expression following cisplatin treatment normalized to the amount of actin detected by densitometry. Each point represents the mean ± SE for three independent harvests of cells. Statistically different points from controls are indicated with an asterisk (*p<0.05).

Figure 2. Effect of Ape1 siRNA knockdown on sensory neuronal cells following cisplatin treatment

A. Sensory neuronal cells were incubated with 100 nM Ape1siRNA on day four in culture and the Ape1siRNAs were removed on day six. Ape1 expression was analyzed on day eleven by western blot. SCsiRNA (100 nM) was used as negative control. B. Ape1siRNA knockdown and SCsiRNA transfected sensory neuronal cells were incubated with cisplatin (0, 50, 100μM) for 24 hrs. The ordinate represents the percent of cell survival measured by trypan blue exclusion. Each point represents the mean ± SE for three independent harvests of cells. Statistically different points from controls are indicated with an asterisk (*p<0.05). C. ROS generation. The fluorescence of the oxidized form of carboxy-H2DCFDA was analyzed by flow cytometry in SCsiRNA and Ape1siRNA knockdown cells. These data are representative of four individual experiments. Statistically different points from controls are indicated with an asterisk (*p<0.05). D. Ape1 protein expression levels in SCsiRNA and Ape1siRNA knockdown DRG cells following cisplatin 50μM treatment at indicated timepoints (left) as analyzed by Western blot. The summary of Ape1 expression was normalized to the amount of actin detected by densitometry (right). Each point represents the mean ± SE for three independent harvests of cells. Statistically different points from controls are indicated with an asterisk (*p<0.05).

Figure 3. Effect of Ape1 overexpression on sensory neuronal cell viability after cisplatin treatment

A. Sensory neuronal cells were incubated with 100 nM Ape1siRNA on day four in culture and the Ape1siRNAs were removed on day six. The cells were then infected with one of three adenoviral constructs; vector control, WT-Ape1or C65-Ape1 on day eight for 24 hrs. The level of adenovirus infection was measured on day eleven by fluorescence microscope. Ape1 expression was analyzed on day eleven by western blot. B. Survival of sensory neuronal cells following cisplatin treatment with or without Ape1 knockdown and the addition of transgene Ape1 constructs. The ordinate represents percent of cells surviving at 24 hrs after various doses of cisplatin treatment as measured by trypan blue exclusion in DRG cells, without (left) or with (right) Ape1 knockdown. Each point represents the mean ± SE for three independent harvests of cells. Statistically different points from controls are indicated with an asterisk (*p<0.05).

Figure 4. Effect of Ape1 levels on the apoptosis levels of sensory neuronal cells following cisplatin treatment

A. Sensory neuronal cell cultures were incubated with Ape1siRNA or SCsiRNA control oligos and were then infected with either WT-Ape1 or C65-Ape1. The cells were then treated with cisplatin (100uM) for 24hrs. The level of cells undergoing apoptosis were detected by Annexin-V and 7ADD staining and FACS analysis as discussed in materials and methods. B. Quantification of the data presented in A. The percentage of cells undergoing apoptosis (c, lower right quadrant) plus late apoptosis (b, upper right quadrant) are shown. Statistically different points from controls are indicated with an asterisk (*p<0.05).

Figure 5. Effect of altered Ape1 levels on cisplatin-induced iCGRP release from sensory neuronal cells

A. Sensory neuronal cells were treated for 24 hrs with increasing amounts of cisplatin. There was a decrease in the amount of iCGRP released in the capsaicin-evoked release of iCGRP. Each column represents the mean +/- SE of the percent of total content of iCGRP after exposure to 30 nM capsaicin for nine wells of neurons. B. Sensory neuronal cells were treated with cisplatin, Ape1-siRNA or SC-siRNA and/or virus as indicated. Compared to untreated controls, SCsiRNA and Ape1siRNA treated cells had a statistically significant (*p < 0.05) decrease in release after exposed to cisplatin. C. Sensory neuronal cells were treated with cisplatin at 10 uM and either SCsiRNA or Ape1-siRNA and then either adenoviral vector control or adenovirus with either WT-Ape1, C65-Ape1 or N226A+R177A Ape1. There was a significantly significant increase in the release in the C65A-Ape1 and the WT-Ape1 transgenes compared to the vector or the N226A+R177A-Ape1 (p < 0.05).

Figure 6. Effect of altering Ape1 levels on the p53 signaling and dsb pathway following cisplatin treatment of sensory neuronal cells

A. A representative western blot analysis of the protein levels of p53 and Gadd45a and the phosphorylation of p53^ser15 and phosphorylation levels of H2AX. Sensory neuronal cell cultures were incubated with Ape1siRNA or SCsiRNA control oligos and were then treated with 50 μM cisplatin for 48 hrs. B. The protein levels of three independent experiments following normalization to actin. Each point represents the mean ± SE for at least three independent harvests of cells. Differences were considered significant at *p<0.05.