A gut butyrate-producing bacterium Butyricicoccus pullicaecorum regulates short-chain fatty acid transporter and receptor to reduce the progression of 1,2-dimethylhydrazine-associated colorectal cancer

Abstract

Gut microbes influence tumor development and progression in the intestines and may provide a novel paradigm for the treatment of colorectal cancer (CRC). Gut dysbiosis may be associated with the development and progression of CRC. Identifying the interactions between the colonic tract and gut microbiota may provide novel information relevant to CRC prevention. The present study examined the effects of butyrate-producing Butyricicoccus pullicaecorum (B. pullicaecorum) on mice with 1,2-dimethylhydrazine (DMH)-induced CRC and the microbial metabolite of B. pullicaecorum on CRC cells. Immunohistochemical staining of the mouse colon tissues and reverse transcription PCR of CRC cells were used to determine the protein and mRNA expression levels of the short-chain fatty acid (SCFA) transporter solute carrier family 5 member 8 (SLC5A8) and G-protein-coupled receptor 43 (GPR43). In CRC-bearing mice fed B. pullicaecorum, DMH-induced CRC regressed, body weight increased and serum carcinoembryonic antigen levels decreased. Notably, SLC5A8 and GPR43 were diffusely and moderately to strongly expressed in the neoplastic epithelial cells and underlying muscularis propria in the colons of the mice. In conclusion, administration of B. pullicaecorum or its metabolites improved the clinical outcome of CRC by activating the SCFA transporter and/or receptor. These results indicated that B. pullicaecorum was a probiotic with anti-CRC potential.

Keywords: 1,2-dimethylhydrazine; Butyricicoccus pullicaecorum; butyrate; colorectal cancer.

Figure 1.

Reduction in DMH/DSS-induced CRC formation following B. pullicaecorum administration. (A) Timing of DMH and DSS induction of CRC and B. pullicaecorum administration. Purple arrow, subcutaneous injection of DMH 40 mg/kg body weight. Yellow arrow, sacrifice and colon sampling of H&E staining; blue box, water drinking; red box, DSS drinking; gray triangle, recording body weight; red triangle, blood collection; green triangle, B. pullicaecorum administration. (B) Inner layer of the colon of mice. (C) Histopathological examination of the colon of mice. Red square, higher magnification. Scale bar, 500 µm. (D) The effect of B. pullicaecorum administration on DMH-induced CRC. Scale bar, 100 µm. DMH, 1,2-dimethylhydrazine; DSS, dextran sulfate sodium; CRC, colorectal cancer; CG, control group; BP, B. pullicaecorum; H&E, hematoxylin and eosin; SM, submucosa; MP, muscularis propria; *, the maximal depth of tumor invasion.

Figure 2.

IHC staining of SLC5A8 and GPR43 in the colons of mice. SLC5A8 and GPR43 were examined by IHC staining of the colon of mice. CG: Control group, no DMH/DSS treatment or B. pullicaecorum administration. Scale bar, 100 µm. SLC5A8, solute carrier family 5 member 8; GPR43, G-protein-coupled receptor 43; IHC, immunohistochemistry; CG, control group; DMH, 1,2-dimethylhydrazine; DSS, dextran sulfate sodium; BP, B. pullicaecorum.

Figure 3.

Bleeding images of the anuses of mice. Anuses were imaged at days 29, 32 and 36 following initial B. pullicaecorum administration. BP, B. pullicaecorum; CG, control group; DMH, 1,2-dimethylhydrazine; DSS, dextran sulfate sodium.

Figure 4.

Effects of B. pullicaecorum on mice with DMH-induced CRC. (A) Body weights. Each mouse was weighed once a week. (B) Serum CEA levels. Sera were collected from tail incision of mice on the week for sacrifice. Levels of CEA in sera were measured using a commercial ELISA kit. One-way ANOVA with Fisher's least significant fifference (LSD) post-hoc test was performed to identify significant differences between groups. Data are presented as mean ± SEM. **P<0.01. DMH, 1,2-dimethylhydrazine; CRC, colorectal cancer; CEA, carcinoembryonic antigen; CG, control group; BP, B. pullicaecorum.

Figure 5.

Effects of butyrate on CRC cells. (A) Effect of conditioned medium of B. pullicaecorum cultivation on CRC cell proliferation. Numbers of SW480 and SW620 cells were counted at days 1 and 5 following cell seeding. One-way ANOVA with Fisher's least significant difference (LSD) post-hoc test was performed to identify significant differences between groups. (B) Relative mRNA levels of SLC5A8. (C) Relative mRNA levels of GPR43. Gene quantitation was performed from SW480 cells and SW620 cells treated with 5 mM NaB for 48 h. Differences in gene expressions were compared using Student's t-test. Data are presented as mean ± SEM. *P<0.05. **P<0.01. CRC, colorectal cancer; NaB, sodium butyrate; SLC5A8, solute carrier family 5 member 8; GPR43, G-protein-coupled receptor 43; PY-X, peptone yeast extract broth; HRC, human reference cDNA.