α-Difluoromethylornithine reduces gastric carcinogenesis by causing mutations in Helicobacter pylori cagY

Abstract

Infection by Helicobacter pylori is the primary cause of gastric adenocarcinoma. The most potent H. pylori virulence factor is cytotoxin-associated gene A (CagA), which is translocated by a type 4 secretion system (T4SS) into gastric epithelial cells and activates oncogenic signaling pathways. The gene cagY encodes for a key component of the T4SS and can undergo gene rearrangements. We have shown that the cancer chemopreventive agent α-difluoromethylornithine (DFMO), known to inhibit the enzyme ornithine decarboxylase, reduces H. pylori-mediated gastric cancer incidence in Mongolian gerbils. In the present study, we questioned whether DFMO might directly affect H. pylori pathogenicity. We show that H. pylori output strains isolated from gerbils treated with DFMO exhibit reduced ability to translocate CagA in gastric epithelial cells. Further, we frequently detected genomic modifications in the middle repeat region of the cagY gene of output strains from DFMO-treated animals, which were associated with alterations in the CagY protein. Gerbils did not develop carcinoma when infected with a DFMO output strain containing rearranged cagY or the parental strain in which the wild-type cagY was replaced by cagY with DFMO-induced rearrangements. Lastly, we demonstrate that in vitro treatment of H. pylori by DFMO induces oxidative DNA damage, expression of the DNA repair enzyme MutS2, and mutations in cagY, demonstrating that DFMO directly affects genomic stability. Deletion of mutS2 abrogated the ability of DFMO to induce cagY rearrangements directly. In conclusion, DFMO-induced oxidative stress in H. pylori leads to genomic alterations and attenuates virulence.

Keywords: Helicobacter pylori; chemoprevention; difluoromethylornithine; gastric cancer; polyamines.

Fig. 1.

Mongolian gerbils infected for 12 wk with H. pylori strain 7.13 and treated with 1% DMFO in the drinking water. (A) H&E staining of gastric tissues, showing invasive adenocarcinoma without treatment and low-grade dysplasia with DFMO treatment. (Scale bars: 100 μm.) (B) Frequency of invasive adenocarcinoma in infected gerbils; all of the uninfected gerbils had normal histology (seven gerbils per group in control and DFMO-treated). (C) Inflammation scores in the gastric tissues of infected gerbils with or without DFMO treatment. NS, not significant. (D) Western blot for phosphorylated CagA (pCagA) in AGS cells cocultured with gerbil output strains for 4 h. Data shown are representative of output strains from 16 gerbils in the control group and 15 gerbils in the DFMO-treated group. (E) Densitometric analysis of CagA translocation. The translocation index was calculated by the ratio of phospho-CagA to total CagA (tCagA), standardized to β-actin. (F) NF-κB activation in AGS cells cocultured with gerbil output strains for 3 h. (G) CXCL8 mRNA expression by real-time PCR in AGS cells cocultured with gerbil output strains for 6 h. In C and E–G, each symbol represents an individual output strain from a different infected gerbil, error bars represent SEM, and statistical analysis were performed using unpaired t test. For cancer incidence (B), significance was calculated using Fisher’s exact test.

Fig. 2.

cagY rearrangements in gerbil output strains. (A) cagY RFLP profile of H. pylori 7.13 parental and output strains from infected gerbils. Arrows indicate strains with rearrangements in cagY compared with the parental strain (DFMO-2, DFMO-4, DFMO-5, and DFMO-8). (B) Schematic representation of the cagY sequencing by SMRT. Repeat domains and identity with VirB10 are shown. The orange and blue boxes represent the repeat motifs designated 2A and 2B. The dashed boxes indicate insertion of a motif. (C) Western blot analysis for CagY in output strains from gerbils in the control and DFMO-treated group. (D) cagY RFLP profiles of DFMO output strains and parental strain 7.13 complemented with a rearranged cagY. (E) Western blot analysis for CagY in output strains from gerbils in DFMO-4, DFMO-8, and complemented strains. (F) Protein levels of CXCL8 quantified by ELISA from supernatants of AGS cells cocultured for 24 h with the parental strain 7.13, DFMO output strains, or parental strain 7.13 complemented with rearranged cagY. Error bars represent SEM. ANOVA with Newman–Keuls multiple comparisons test was used. (G) Western blot for pCagA in AGS cells cocultured for 4 h with parental strain 7.13, DFMO output strains, or parental strain 7.13 complemented with rearranged cagY.

Fig. 3.

Mongolian gerbils infected for 12 wk with H. pylori strain 7.13, DFMO output strain (DFMO-4), or 7.13 parental strain complemented with rearranged cagY (7.13[DFMO-4]). (A) Inflammation score in the gastric tissues of gerbils infected with 7.13, DFMO-4, or 7.13 [DFMO-4]. Error bars represent SEM. ANOVA with Newman–Keuls multiple comparisons test was used for A. (B) Frequency of diagnoses in gerbils. The adjacent numbers correspond to the number of gerbils in each section of the bar. ^§§§P < 0.001 for DFMO-4 and 7.13 [DFMO-4]-infected gerbils versus 7.13-infected, comparing cancer frequency; ^¶¶¶P < 0.001 for DFMO-4 and 7.13 [DFMO-4]-infected gerbils versus 7.13-infected, comparing cancer + dysplasia frequency. For diagnosis comparisons significance was calculated using Fisher’s exact test. (C) H&E staining of gastric tissues, showing invasive adenocarcinoma in a 7.13-infected gerbil, normal histology in a DFMO-4-infected animal, and gastritis in a 7.13 [DFMO-4]-infected gerbil. None of the seven uninfected gerbils developed dysplasia or adenocarcinoma. (Scale bars: 100 μm.)

Fig. 4.

H. pylori serially passaged on DFMO-supplemented plates. (A) cagY RFLP profile of H. pylori 7.13 serially passaged on regular plates (–) or plates supplemented with DFMO (D) or ornithine (O). RFLP profiles are shown for passage 1 (p1), 5 (p5), or 20 (p20). For all of the RFLP studies, four individual colonies per condition were analyzed. (B) NF-κB activation in AGS cells cocultured for 3 h with H. pylori serially passaged 20 times on control, DFMO, or ornithine-containing plates. Western blot (C) and densitometric (D) analysis for pCagA in AGS cells cocultured with gerbil output strains for 4 h. ANOVA with Newman–Keuls multiple comparisons test was used for B and D.

Fig. 5.

Effect of DFMO on sodB and katA mRNA expression in H. pylori. H. pylori strain 7.13 was treated with DFMO or ornithine for 2, 4, or 8 h in liquid culture. sodB (A) and katA (B) mRNA expression, determined by real-time PCR, is expressed as fold increase compared with the untreated control at each time point. ^§§P < 0.01 versus untreated control. ^§§§P < 0.001 versus untreated control. ^¶P < 0.05 versus DFMO-treated. ^¶¶P < 0.01 versus DFMO-treated. ^¶¶¶P < 0.001 versus DFMO-treated. ANOVA with Newman–Keuls multiple comparisons test was used for A and B.

Fig. 6.

Oxidative DNA damage in H. pylori. Confocal microscopy (A) and flow cytometry plots (B) for 8-oxoguanine in H. pylori treated with DFMO or ornithine for 24 h, or exposed to oxygen for 4 h. (Scale bars: 5 μm.) (C) Quantification of 8-oxoguanine–positive bacteria by flow cytometry. ANOVA with Newman–Keuls multiple comparisons test was used.

Fig. 7.

Oxidative DNA damage and cagY RFLP in H. pylori ΔmutS2. (A) mutS2 mRNA expression by real-time PCR, expressed as fold increase compared with the untreated control at each time point. ^§§P < 0.01 versus untreated control. ^§§§P < 0.001 versus untreated control. ^¶¶P < 0.01 versus DFMO-treated. ^¶¶¶P < 0.001 versus DFMO-treated. (B) Percentage of 8-oxoguanine–positive bacteria assessed by flow cytometry in H. pylori WT and ΔmutS2 after DFMO or ornithine treatment for 24 h. (C) cagY RFLP profile of H. pylori WT and ΔmutS2 after five passages in DFMO-supplemented plates. ANOVA with Newman–Keuls multiple comparisons test was used for A and B.