Protective Effects of Zerumbone on Colonic Tumorigenesis in Enterotoxigenic Bacteroides fragilis (ETBF)-Colonized AOM/DSS BALB/c Mice

Abstract

Chronic inflammation has been linked to colitis-associated colorectal cancer in humans. The human symbiont enterotoxigenic Bacteroides fragilis (ETBF), a pro-carcinogenic bacterium, has the potential to initiate and/or promote colorectal cancer. Antibiotic treatment of ETBF has shown promise in decreasing colonic polyp formation in murine models of colon cancer. However, there are no reported natural products that have shown efficacy in decreasing polyp burden. In this study, we investigated the chemopreventive effects of oral administration of zerumbone in ETBF-colonized mice with azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced tumorigenesis. Zerumbone significantly reduced the severity of disease activity index (DAI) scores as well as several parameters of colonic inflammation (i.e., colon weight, colon length, cecum weight and spleen weight). In addition, inflammation of the colon and cecum as well as hyperplasia was reduced. Zerumbone treatment significantly inhibited colonic polyp numbers and prevented macroadenoma progression. Taken together, these findings suggest that oral treatment with zerumbone inhibited ETBF-promoted colon carcinogenesis in mice indicating that zerumbone could be employed as a promising protective agent against ETBF-mediated colorectal cancer.

Keywords: colitis; colorectal cancer; enterotoxigenic Bacteroides fragilis; polyps; zerumbone.

Figure 1

Experimental scheme and effects of zerumbone on body weight and the disease activity index in enterotoxigenic Bacteroides fragilis (ETBF)/AOM/DSS mice. BALB/c mice were given a single intraperitoneal injection of AOM (10 mg/kg) and provided drinking water ad libitum containing clindamycin/gentamicin for 5 days (yellow box). ETBF was orally inoculated and the antibiotic cocktail was continued for an additional 7 days (yellow box). Seven days later, BALB/c mice were subjected to two cycles of 1% DSS (5 days per cycle, orange boxes) and distilled water (DW; 16 days per cycle, blue boxes). During the two DSS cycles, BALB/c mice were given zerumbone (30, 60 mg/kg, p.o., three times a week). The total period of the experiment was 9 weeks. (A) Experimental design of the ETBF/AOM/DSS-induced tumorigenesis model. (B) Body weight. (C) Disease activity index (DAI). Body weight and DAI were measured at the last day of ETBF/AOM/DSS-treated experiments. S, sham control; E, ETBF; A, AOM; D, DSS; Z (30), Zerumbone (30 mg/kg) and Z (60), Zerumbone (60 mg/kg). Each dot represents one mouse (n = 6–12 mice per group). Scatter plot. Horizontal bar, median. * p < 0.05, *** p < 0.001. ns, no statistical significance.

Figure 2

Zerumbone reduced ETBF-mediated tumorigenesis in AOM/DSS mice. AOM-treated BALB/c mice were infected with ETBF and subjected to two cycles of DSS (1%) for 9 weeks. During two DSS cycles, BALB/c mice were given zerumbone (30 and 60 mg/kg, p.o., three times a week). (A) Representative gross macroscopic image of the colon. (B) Polyp number. (C) Polyp size distribution. (D) Colon length (mm) and (E) colon weight (mg)/colon length (mm) ratio. Polyp number, polyp size distribution, colon length and colon weight/colon length ratio were measured on the last day of ETBF/AOM/DSS-treated experiments. S, sham control; E, ETBF; A, AOM; D, DSS; Z (30), Zerumbone (30 mg/kg) and Z (60), Zerumbone (60 mg/kg). Each dot represents one mouse. Scatter plot. Horizontal bar, median. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, no statistical significance.

Figure 3

Zerumbone decreases ETBF colonization-promoted high-grade macroadenomas. FFPE colonic tissues of distal colon obtained from AOM-treated BALB/c mice infected with ETBF and subjected to two cycles of DSS (1%) for 9 weeks were excised and stained with H&E. A representative image of microadenoma, low-grade macroadenoma and high-grade macroadenoma is shown. Microadenoma, low-grade macroadenoma and high-grade macroadenoma were counted for two H&E-stained distal colon of sections per mouse. (A) Histology of microadenoma, ×100 magnification. (B) Histology of low-grade macroadenoma, 50× magnification. (C) Histology of high-grade macroadenoma, 50× magnification. (D) Number of microadenoma per mouse. (E) Number of low-grade macroadenoma per mouse. (F) Number of high-grade macroadenoma per mouse. E, ETBF; A, AOM; D, DSS; Z (30), Zerumbone (30 mg/kg) and Z (60), Zerumbone (60 mg/kg). Each dot represents one mouse. Scatter plot. Horizontal bar, median. * p < 0.05. ns, no statistical significance.

Figure 4

Zerumbone decreased large intestinal inflammation and hyperplasia in ETBF-colonized AOM/DSS mice. AOM-treated BALB/c mice were infected with ETBF and subjected to two cycles of DSS (1%) for 9 weeks. During two DSS cycles, BALB/c mice were administered with zerumbone (30 and 60 mg/kg, p.o., three times a week). (A) Histology (H&E) of distal colon tissues, 100× magnification. B. Inflammation score (distal colon). (C) Hyperplasia score (distal colon). (D) Histology (H&E) of cecal tissues, 100× magnification. (E) Inflammation score (cecum). (F) Hyperplasia score (cecum). Each dot represents one mouse. Scatter plot. Horizontal bar, median. S, sham control; E, ETBF; A, AOM; D, DSS and Z (60), Zerumbone (60 mg/kg). * p < 0.05, *** p < 0.001. ns, no statistical significance.

Figure 5

Zerumbone prevented cecum weight loss and spleen enlargement in ETBF-colonized AOM/DSS mice. AOM-treated BALB/c mice were infected with ETBF and subjected to two cycles of DSS (1%) for 9 weeks. During two DSS cycles, BALB/c mice were administered with zerumbone (30 and 60 mg/kg, p.o., three times a week). Mice were euthanized after two cycles of 1% DSS treatment. (A) Representative images of cecum and spleen. (B) Cecum weight. (C) Spleen weight. S, sham control; E, ETBF; A, AOM; D, DSS; Z (30), Zerumbone (30 mg/kg) and Z (60), Zerumbone (60 mg/kg). Each dot represents one mouse. Scatter plot. Horizontal bar, median. * p < 0.05, *** p < 0.001. ns, no statistical significance.

Figure 6

Zerumbone does not affect ETBF colonization in mice. ETBF colonization was assessed by stool plating. Colonization was generally within the range of 10⁷–10⁹ CFU/g stool at 1 (A), 3 (B), 6 (C) and 8 (D) weeks after ETBF infection. S, sham control; E, ETBF; A, AOM; D, DSS; Z (30), Zerumbone (30 mg/kg); Z (60), Zerumbone (60 mg/kg); CFU, colony-forming units and nd, not detected. Each dot represents one mouse. Scatter plot. Horizontal bar, median. ns, no statistical significance.