Helicobacter pylori infection-induced H3Ser10 phosphorylation in stepwise gastric carcinogenesis and its clinical implications

Abstract

Background: Our previous works have demonstrated that Helicobacter pylori (Hp) infection can alter histone H3 serine 10 phosphorylation status in gastric epithelial cells. However, whether Helicobacter pylori-induced histone H3 serine 10 phosphorylation participates in gastric carcinogenesis is unknown. We investigate the expression of histone H3 serine 10 phosphorylation in various stages of gastric disease and explore its clinical implication.

Materials and methods: Stomach biopsy samples from 129 patients were collected and stained with histone H3 serine 10 phosphorylation, Ki67, and Helicobacter pylori by immunohistochemistry staining, expressed as labeling index. They were categorized into nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia, low-grade intraepithelial neoplasia, high-grade intraepithelial neoplasia, and intestinal-type gastric cancer groups. Helicobacter pylori infection was determined by either ¹³ C-urea breath test or immunohistochemistry staining.

Results: In Helicobacter pylori-negative patients, labeling index of histone H3 serine 10 phosphorylation was gradually increased in nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia groups, peaked at low-grade intraepithelial neoplasia, and declined in high-grade intraepithelial neoplasia and gastric cancer groups. In Helicobacter pylori-infected patients, labeling index of histone H3 serine 10 phosphorylation followed the similar pattern as above, with increased expression over the corresponding Helicobacter pylori-negative controls except in nonatrophic gastritis patient whose labeling index was decreased when compared with Helicobacter pylori-negative control. Labeling index of Ki67 in Helicobacter pylori-negative groups was higher in gastric cancer than chronic atrophic gastritis and low-grade intraepithelial neoplasia groups, and higher in intestinal metaplasia group compared with chronic atrophic gastritis group. In Helicobacter pylori-positive groups, Ki67 labeling index was increased stepwise from nonatrophic gastritis to gastric cancer except slightly decrease in chronic atrophic gastritis group. In addition, we noted that histone H3 serine 10 phosphorylation staining is accompanied with its location changes from gastric gland bottom expanded to whole gland as disease stage progress.

Conclusions: These results indicate that stepwise gastric carcinogenesis is associated with altered histone H3 serine 10 phosphorylation, Helicobacter pylori infection enhances histone H3 serine 10 phosphorylation expression in these processes; it is also accompanied with histone H3 serine 10 phosphorylation location change from gland bottom staining expand to whole gland expression. The results suggest that epigenetic dysregulation may play important roles in Helicobacter pylori-induced gastric cancer.

Keywords: Helicobacter pylori; H3 serine 10 phosphorylation; epigenetic; gastric cancer; histone modification.

Figure 1

Representative photographs of p‐H3Ser10 and Ki67 immunostaining in gastric tissues. A, Negative control in nonatrophic gastritis (NAG) patients in which primary antibody has been omitted (control); B, Helicobacter pylori‐negative (Hp−) in NAG patient; C, H. pylori‐positive (Hp+) in NAG patient; D, Ki67 in H. pylori‐positive NAG patient; E‐L, p‐H3Ser10 staining, E and I, chronic atrophic gastritis (CAG); F and J, intestinal metaplasia (IM); G and K, high‐grade intraepithelial neoplasia (HGIN); H and L, intestinal‐type gastric cancer (GC). H3Ser10‐stained cells are located mainly in the bottom and body of gastric gland in NAG (E and I). Almost all cells show nuclear immunoreactivity in IM, GC, and H. pylori‐positive HGIN (F, H, J, K, and L). Arrows indicate representative immunostaining cells. Original magnifications ×100 (A‐L)

Figure 2

Labeling index of p‐H3Ser10 and Ki67 in various stages of gastric disease. Labeling index (LI) of p‐H3Ser10 (A and B and E)‐ and Ki67 (C and D and F)‐stained cells was generated by visualizing and recording the image data under microscope; it was calculated as mentioned in method and expressed as mean ± SD in each group. The average level of p‐H3Ser10 and Ki67 LI among various stages of gastric disease was compared by ANOVA. X‐axes indicate different stages of disease; Y‐axes indicate LI of p‐H3Ser10 and Ki67. Dots represent LI of either p‐H3Ser10 or Ki67 in each group; the short bars between dots indicate mean value of each group. One asterisk represents value that is statistically significant (P < .05); two asterisks indicate value that is highly statistically significant (P < .01). Nonatrophic gastritis, NAG; chronic atrophic gastritis, CAG; intestinal metaplasia, IM; low‐grade intraepithelial neoplasia, LGIN; high‐grade intraepithelial neoplasia, HGIN; intestinal‐type gastric cancer, GC; Helicobacter pylori infection status, H. pylori‐positive; H. pylori noninfection status, H. pylori‐negative; H. pylori infection or noninfection status, H. pylori (+/−)

Figure 3

Location changes in p‐H3Ser10‐ and Ki67‐stained cells in stepwise gastric disease. Location of p‐H3Ser10 (A and B)‐ and Ki67 (C and D)‐positive cells in gastric gland was visualized and recorded under microscope; the percentage of stained cell in each location of gastric gland in every section was evaluated to generate mean value. X‐axes indicate different stages of disease; Y‐axes indicate the percentages of p‐H3Ser10‐ or Ki67‐positive cell's location. Nonatrophic gastritis, NAG; chronic atrophic gastritis, CAG; intestinal metaplasia, IM; low‐grade intraepithelial neoplasia, LGIN; high‐grade intraepithelial neoplasia, HGIN; intestinal‐type gastric cancer, GC; Helicobacter pylori infection status, H. pylori‐positive; H. pylori noninfection status, H. pylori‐negative