The role of DFMO in Helicobacter pylori infection: modulation of ROS response

Background: Gastric cancer remains the third foremost cause of cancer deaths worldwide. The leading cause of gastric cancer, Helicobacter pylori is a thought drive carcinogenesis by inducing chronic inflammation. One pathway implicated in the host immune response involves polyamines, which have been shown by our laboratory to contribute to gastric carcinogenesis. Difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine synthesis reduce gastric inflammation and progression to cancer in gerbils infected with H. pylori. Paradoxically, cell-specific deletion of the Odc gene in macrophages increased inflammation in murine experiments, indicating the likely involvement of alternative mechanisms by which DFMO could be mediating cancer progression, including direct effects on the bacteria. We hypothesize that DFMO modulates the interaction between H. pylori and gastric cancer by affecting the bacteria exposed to oxidative stress generated in an inflammatory environment. Methods: Strains for H. pylori were derived from the parental 7.13 and were either grown on plates passaged 19 times with or without DFMO exposure, or harvested from gerbil stomach tissue after 12 weeks with or without exposure to DFMO in drinking water. RAW 264.7 murine macrophages and H. pylori strains were co-cultured, and mRNA was harvested from cells 6 h post-infection. RT-PCR was used to analyze expression of macrophage activation-associated genes encoding proteins indicative of M1 response (NOS2, TNF-a, IL-1b). For the second focus, we cultured H. pylori 7.13 in liquid media over 24 h with 50 mM DFMO, 50 mM ornithine, or no additional reagent. At each 2 h time-point, we noted the OD600nm and pelleted the bacteria, from which we isolated RNA. To assess oxidative response, we used RT-PCR to compare mRNA expression of genes involved in H. pylori defense mechanisms to ROS/RNS, including sodB and tpx. iTRAQ proteomics analysis demonstrated reduced catalase and TlpB abundance in output strains from gerbils receiving DFMO versus control strains. We performed RT-PCR for katA and tlpB in the parental, in vitro passaged, and gerbil output strains. Lastly, we grew the parental strain and 4 gerbil output strains in Brucella broth, or broth treated with 500 mM or 1 mM H2O2, and plotted growth curves. Results: In co-culture experiments, M1 gene expression levels did not differ between macrophages infected with DFMO-treated or control strains. In our analysis of H. pylori gene expression, we found induction of sodB and tpx at the 2 and 4 h time-points in the samples collected from the DFMO-treated compared to ornithine-treated media or untreated controls. Furthermore, expression of katA and tlpB were lower in DFMO output versus control output strains. Bacterial growth curves demonstrated attenuated growth in H2O2-treated DFMO-exposed H. pylori strains compared to untreated DFMO-exposed controls. Additionally, unexposed control gerbil output strains showed no difference in growth in acutely H2O2-treated strains compared to untreated controls. Conclusions: Our results from this study suggest that chronic exposure of H. pylori to DFMO impedes bacterial response to environmental stress by affecting expression of oxidative defense genes. These data also imply that treatment with DFMO may be contributing to environmental oxidative stress, which leads to an immediate response by the bacteria to prevent ROS-induced DNA damage. Further study may establish the specific role of DMFO in the modulation of H. pylori growth through induction of oxidative stress. The implications are that DFMO has benefits in its effects on H. pylori that add to its chemopreventive potential