Enterococcus faecalis is a human colonic commensal that induces colon inflammation and cancer in several mouse models. We have shown that E. faecalis-infected macrophages produce 4-hydroxy-2-nonenal (4-HNE), a lipid peroxidation byproduct of ω-6 polyunsaturated fatty acids, and that this reactive aldehyde is genotoxic and mediates bystander effects (BSE).
To further investigate mechanisms for 4-HNE-mediated BSE, we treated primary murine colonic epithelial cells (YAMC) with 4-HNE and studied Wnt/β-catenin signaling. Western blotting showed activation of β-catenin after 1 hr exposure of YAMC cells to 1 μM 4-HNE. Immunofluorescence staining confirmed nuclear translocation of activated β-catenin 5 hrs following treatment. These findings suggested that 4-HNE as an endogenous carcinogen that potentially contributes to colorectal carcinogenesis. Because glutathione S-transferase alpha 4 (Gsta4) is a detoxifying enzyme for this reactive aldehyde, we determined whether exposure of YAMC cells to 4-HNE increased the production of Gsta4. Indeed, Gsta4 was significantly increased at 24-72 hrs following 4-HNE treatment, suggesting upregulation of Gsta4 in response to 4-HNE-induced genotoxicity. In addition, we measured Gsta4 in 6 human colon cancer cell lines and compared these results to normal human colonic epithelial cells (FHC). Our findings showed that Gsta4 was strongly produced by all cancer cell lines compared to FHC. Finally, we colonized interleukin (IL)-10 knockout mice with E. faecalis for short- (2 weeks), intermediate- (3 months), and long-term (9 months) periods of time. Gsta4 levels in serum significantly increased for Il10−/− mice colonized with E. faecalis at 2 weeks compared to no change for colonized wildtype mice. Immunohistochemical staining for Gsta4 was apparent in tissue macrophages from colon biopsies for colonized mice, implying macrophage activation. Gsta4-positivity significantly increased in colon macrophages after 3 and 9 months of colonization in areas of increased inflammation. Of note, although Gsta4-positive staining was not seen in epithelial cells in areas of inflammation, intense staining was noted in neoplastic epithelial cells. These results suggest that activation of Gsta4 may be an early event in macrophage activation and associated with neoplasia.
In summary, in the IL-10 knockout model, E. faecalis-infected macrophages produce 4-HNE and this lipid peroxidation breakdown product induces Wnt/β-catenin signaling and Gsta4 production. These findings implicate Gsta4 as a potential surrogate biomarker for colon cancer and should permit the development of new chemopreventive strategies.
Citation Format: Xingmin Wang, Yonghong Yang, Mark Huycke, Department of Veterans Affairs Medical Center. Glutathione S-transferase alpha 4 is a potential biomarker for Enterococcus faecalis-induced inflammation and colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1344. doi:10.1158/1538-7445.AM2013-1344