Immunomodulatory effects of live and UV-killed Bacillus subtilis natto on inflammatory response in human colorectal adenocarcinoma cell line in vitro

Abstract

Background and objectives: Colorectal cancer (CRC) is a heterogeneous disease of the colon or rectum arising from adenoma precursors and serrated polyps. Recently, probiotics have been proposed as an effective and potential therapeutic approach for CRC prevention and treatment. Probiotics have been shown to alleviate inflammation by restoring the integrity of the mucosal barrier and impeding cancer progression.

Materials and methods: In this study, we aimed to investigate the immunomodulatory effects of live and UV-killed Bacillus subtilis natto on the inflammatory response in CRC. Caco-2 cells were exposed to various concentrations of live and UV- killed B. subtilis natto, and cell viability was assessed using MTT assay. Gene expression analysis of IL-10, TGF-β, TLR2 and TLR4 was performed using RT-qPCR.

Results: Our findings showed that both live and UV-killed B. subtilis natto caused significant reduction in inflammatory response by decreasing the gene expression of TLR2 and TLR4, and enhancing the gene expression of IL-10 and TGF-β in Caco-2 cells as compared to control group.

Conclusion: The results of this study suggest that live and UV-killed B. subtilis natto may hold potential as a therapeutic supplement for modulating inflammation in CRC.

Keywords: Bacillus subtilis; Colorectal neoplasms; Gene expression; Immunomodulation; Probiotics.

Fig. 1

Cell viability by MTT assay to determine the effects of live and UV-killed B. subtilis natto on Caco-2 cells. Different MOIs (MOI 1, 10, 100) of live and UV-killed B. subtilis natto were added to Caco-2 cell monolayer and incubated for 24 h. The data are expressed as the mean ± SEM of three independent experiments. The results are presented as the percentage of cell survival compared to control samples. MOI, multiplicity of infection; Bsn, B. subtilis natto.

Fig. 2

Effects of live (MOI 100) and UV-killed (MOI 100) B. subtilis natto (Bsn) on the expression levels of anti-inflammatory genes IL-10 (A) and TGF-β (B) in Caco-2 cells by RT-qPCR. Expression data are normalized using β-actin as a reference gene. Caco-2 cells monolayers were treated with LPS for 24 h as a positive control. Data are shown as mean ± SEM from three independent experiments. **P < 0.01, ***P < 0.001 compared to the untreated control were considered statistically significant.

Fig. 3

Effects of live (MOI 100) and UV-killed (MOI 100) B. subtilis natto (Bsn) on TLR2 (A) and TLR4 (B) gene expression in Caco-2 cells after 24 h. Gene expression data are normalized using β-actin as the reference gene. Caco-2 cell monolayers were treated with LPS for 24 h as a positive control. Data are presented as the mean ± SEM of three independent experiments. *P ˂ 0.05, **P ˂ 0.01, ***P ˂ 0.001 was considered statistically significant compared to the untreated control.