Glutathione peroxidase 7 protects against oxidative DNA damage in oesophageal cells
- DunFa Peng1,
- Abbes Belkhiri1,
- TianLing Hu1,
- Rupesh Chaturvedi2,3,
- Mohammad Asim2,3,
- Keith T Wilson2,3,
- Alexander Zaika1,
- Wael El-Rifai1,3
- 1Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA
- 2Division of Gastroenterology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA
- 3Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, USA
- Correspondence to Wael El-Rifai, Vanderbilt University Medical Center, 1255 Light Hall, 2215 Garland Avenue, Nashville, TN 37232, USA;
Contributors DP: study concept and design, experimental design and acquisition of data, analysis and interpretation of data, drafting of the manuscript, statistical analysis, obtained funding, technical and material support; AB: analysis and interpretation of data, experimental troubleshooting, drafting of the manuscript, critical revision of the manuscript; TH: prepared reagents and participated in experiments; RC: assisted in experimental design and acquisition of data; MA: assisted in immunofluorescence experiments; KTW: assisted in analysis and interpretation of data; AZ: assisted in analysis and interpretation of data, experimental troubleshooting; WE-R: study concept and design, study supervision, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript for important intellectual content.
- Revised 18 October 2011
- Accepted 31 October 2011
- Published Online First 9 December 2011
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-H2DCFDA, 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 (H2O2) independent of glutathione. Reconstitution of GPX7 expression in immortalised Barrett's oesophagus cells, BAR-T and CP-A led to resistance to H2O2-induced oxidative stress. Following exposure to acidic bile acids cocktail (pH4), these GPX7-expressing cells demonstrated lower levels of H2O2, 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 H2O2-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 H2O2, 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.
- Barrett's carcinoma
- Barrett's oesophagus
- basic sciences
- bile acids
- cancer immunobiology
- cancer susceptibility
- cancer syndromes
- carcinogen metabolism
- cell signalling
- DNA damage
- gastrointestinal cancer
- gastrointestinal neoplasia
- gastrointestinal pathology
- glutathione peroxidase 7
- molecular carcinogenesis
- oxidative stress
Funding This study was supported by grants from the National Institutes of Health (R01CA106176 (WER) and R01CA138833 (AZ)), Vanderbilt SPORE in Gastrointestinal Cancer (P50 CA95103), Vanderbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt Digestive Disease Research Center (DK058404). The contents of this work are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute or Vanderbilt University.
Competing interests None.
Patient consent Obtained.
Ethics approval Ethics approval was provided by Vanderbilt University Medical Center.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement We will share all reagents and data in this manuscript with the scientific community in accordance with the NIH and Vanderbilt governing policies.