Glutathione peroxidase 7 protects against oxidative DNA damage in oesophageal cells

Abstract

Objective: Exposure of the oesophageal mucosa to gastric acid and bile acids leads to the accumulation of reactive oxygen species (ROS), a known risk factor for Barrett's oesophagus and progression to oesophageal adenocarcinoma (OAC). This study investigated the functions of glutathione peroxidase 7 (GPX7), frequently silenced in OAC, and its capacity in regulating ROS and its associated oxidative DNA damage.

Design: Using in-vitro cell models, experiments were performed that included glutathione peroxidase (GPX) activity, Amplex UltraRed, CM-H(2)DCFDA, Annexin V, 8-oxoguanine, phospho-H2A.X, quantitative real-time PCR and western blot assays.

Results: Enzymatic assays demonstrated limited GPX activity of the recombinant GPX7 protein. GPX7 exhibited a strong capacity to neutralise hydrogen peroxide (H(2)O(2)) independent of glutathione. Reconstitution of GPX7 expression in immortalised Barrett's oesophagus cells, BAR-T and CP-A led to resistance to H(2)O(2)-induced oxidative stress. Following exposure to acidic bile acids cocktail (pH4), these GPX7-expressing cells demonstrated lower levels of H(2)O(2), intracellular ROS, oxidative DNA damage and double-strand breaks, compared with controls (p<0.01). In addition, these cells demonstrated lower levels of ROS signalling, indicated by reduced phospho-JNK (Thr183/Tyr185) and phospho-p38 (Thr180/Tyr182), and demonstrated lower levels of apoptosis following the exposure to acidic bile acids or H(2)O(2)-induced oxidative stress. The knockdown of endogenous GPX7 in immortalised oesophageal squamous epithelial cells (HET1A) confirmed the protective functions of GPX7 against pH4 bile acids by showing an increase in the levels of H(2)O(2), intracellular ROS, oxidative DNA damage, double-strand breaks, apoptosis, and ROS-dependent signalling (p<0.01).

Conclusion: The dysfunction of GPX7 in oesophageal cells increases the levels of ROS and oxidative DNA damage, which are common risk factors for Barrett's oesophagus and OAC.

Figure 1. GPX7 neutralizes H₂O₂ independent of glutathione

A) Glutathione peroxidase activity assay was performed using a recombinant GPX7 protein. The change of the absorbance at 340 nm (Δ340 nm) was used to evaluate the relative glutathione peroxidase activity compared to a glutathione peroxidase positive control. B) Amplex UltraRed peroxidase assay was performed using recombinant GPX7 protein. An inverse relationship between the level of GPX7 and H₂O₂ was observed (r=0.92, p<0.0001). C) GPX7 expression was significantly downregulated in Barrett’s dysplasia (BD) and adenocarcinomas (EAC), in comparison with normal oesophagus (NS).

Figure 2. GPX7 protects oesophageal epithelial cells from H₂O₂ induced oxidative stress

A) BAR-T cells were reconstituted with a GPX7 expression adenoviral system. The levels of mRNA and protein in the control (Ad-CTRL) and GPX7 expressing cells (Ad-GPX7) are shown. B) HET1A cells were used for knockdown of GPX7 with a GPX7 shRNA lentiviral system. The levels of mRNA and protein in control (Sc shRNA) and GPX7 knockdown cells (GPX7 shRNA) are shown. BAR-T cells and HET1A cells were treated with different concentrations of H₂O₂ for 16h, and then cell viability was measured using ATPGlo assay. The results were normalized to the values in cells treated with PBS. The results demonstrate an increase in cell viability in GPX7 expressing BAR-T cells (C) and a decrease in cell viability in GPX7 knockdown HET1A cells (D). E–F) Western blot analysis for cleaved caspase 3 in BAR-T (E) and HET1A cells (F). ImageJ software was used to determine the bands density of cleaved and total caspase 3. A histogram presenting the ratio of cleaved caspase 3 to total caspase 3 is displayed below the western blot images of BAR-T and HET1A respectively. **, P<0.01.

Figure 3. GPX7 decreases the H₂O₂ levels and intracellular ROS level in oesophageal epithelial cells upon pH 4 bile acids exposure

A–B) BAR-T cells and HET1A cells, as in Figure 2, were exposed to pH 4 bile acids (PH4 BA) for 10, 30, and 60 min. The media from BAR-T (A) and HET1A (B) cells were then subjected to Amplex UltraRed hydrogen peroxide assay to measure the H₂O₂ level. H₂O₂ concentrations were retrieved by comparing to standard curve. C–D) To measure intracellular ROS, BAR-T (C) cells and HET1A (D) cells were treated with pH 4 bile acids for 30 min, and then subjected to flow cytometry analysis of CM-H₂DCFDA. In (C) (BAR-T) and (D) (HET1A), left panels show representative flow cytometry histograms whereas the right panels depict the quantitative data from flow cytometry. * P<0.05, ** P<0.01.

Figure 4. GPX7 decreased pH 4 bile acids-induced oxidative DNA damage

A–B) Immunofluorescence staining of 8-oxoguanine (green) in BAR-T cells (A) and HET1A cells (B) following the exposure to pH 4 bile acids (PH4 BA). The quantification of immunostaining results using ImageJ is shown in the right panels. * P<0.05. The reconstitution of GPX7 in BAR-T led to a significant reduction in fluorescence intensity per cell for 8-oxoguanine (A) whereas the knockdown of GPX7 in HET1A cells led to a significant increase in the fluorescence intensity per cell for 8-oxoguanine (B).

Figure 5. GPX7 reduces pH 4 bile acid-induced double strand breaks

A–B) Immunofluorescence staining of phospho-H2A.X (green) in BAR-T cells (A) and HET1A cells (B) following the exposure to pH 4 bile acids (PH4 BA). The quantification of immunostaining results from ImageJ is shown in the right panels. * P<0.05. The reconstitution of GPX7 in BAR-T led to a significant reduction in percentage of cells positive for phospho-H2A.X (A) whereas the knockdown of GPX7 in HET1A cells led to a significant increase in the percentage of cells positive for phospho-H2A.X (B).

Figure 6. GPX7 suppresses ROS signaling on exposure to pH 4 bile acids

BAR-T cells and HET1A cells were exposed to pH 4 bile acids (PH4 BA) for 10, 30, and 60 minutes. Western blotting analysis demonstrated lower levels of phospho-JNK and phospho-p38 in BAR-T cells following reconstitution of GPX7 (Ad-GPX7) as compared to control (AD-CTRL) (left panel), whereas HET1A cells demonstrated higher levels of phospho-JNK and phospho-p38 following shRNA knockdown of GPX7 as compared to control sc shRNA (right panel).

Figure 7. GPX7 protects oesophageal epithelial cells from acidic bile acids-induced apoptosis

BAR-T (A–C) and HET1A (D–F) cells were exposed to pH 4 bile acids (PH4 BA) for 10 and 30 minutes. A) ATPGlo cell viability assay demonstrated a significant increase in the percentage of viable cells following reconstitution of GPX7 (Ad-GPX7) as compared to control (Ad-CTRL) at the 30 min time point. B) Flow cytometry analysis of Annexin V apoptosis assay showed lower percentage of apoptotic cells following reconstitution of GPX7 (Ad-GPX7) as compared to control (Ad-CTRL) at the 30 min time point. C) Western blot analysis of cleaved caspase 3. The ratio of cleaved caspase 3 to total caspase 3 was quantified using ImageJ and is shown in the right of the western blotting images. **, P<0.01. The results demonstrate lower levels of cleaved caspase 3 in cells with reconstitution of GPX7, following exposure to PH4 BA. D–F) The knockdown of GPX7 in HET1A as compared to control sc shRNA cells, led to reduction in cell viability (D), increase in Annexin V positive cells (E) and an increase in the level of cleaved caspase 3 (F), following exposure to PH4 BA.

Figure 8. GPX7 expression was upregulated after acidic bile acids exposure in HET1A and EPC2 cells

HET1A and EPC2 cells were exposed to pH4 bile acids for 30 min, then cells were washed in PBS and cultured in regular medium for the indicated time points. Quantitative real time RT PCR and western blot analysis were used to determine mRNA (A & C) and protein levels (B & D) in HET1A and EPC2.