Nutritional Immunity: Butyrate and Colon Carcinogenesis

Abstract

Early-onset of colorectal cancer (EOCRC) is increasing amongst younger populations. A main contributing factor is modern-day diets. Clinical studies indicate that high fibre nutrition regimens may be protective against the EOCRC. An abundant metabolite of fibre is butyrate, which has been observed to mitigate the loss of intestinal mucus and E-cadherin in human colorectal adenocarcinoma cell lines. E-cadherin facilitates intercellular signalling, which is essential for the suppression of tumorigenesis and metastasis progression. Bacteroides fragilis toxin (BFT) is a metalloprotease virulence factor of enterotoxigenic B. fragilis (ETBF) that induces the cleavage and degradation of E-cadherin. ETBF is commonly found colonised in the intestinal mucus of patients who were susceptible to developing colorectal neoplasia and pre-cancerous lesions. Associations between butyrate’s potential level of protection against BFT is yet to be explored in current literature. Thus, in this study, ETBF was used as a bacterial model to test the hypothesis that butyrate acts to upregulate E-cadherin and/or mucus production, which protects against B. fragilis-mediated cellular responses in colonic adenocarcinoma cells.

The cellular effects of butyrate are conventionally investigated in the undifferentiated HT29 cell line. However, the mucus secreting HT29-MTX cells are postulated to be more physiologically representative of the colonic epithelia. Both HT29 and HT29-MTX cell lines were used to model BFT-mediated loss of E-cadherin. These cells were treated with 5 mM of butyrate and/or B. fragilis for 24-hours. Trypan blue exclusion was applied to quantify cell proliferation and viability. RT-qPCR was utilized to quantify CDH1 and MUC2 gene expression, which encodes for E-cadherin and mucin 2 (intestinal mucus) respectively. Alcian blue staining was implemented to assess acidic mucin expression. Immunofluorescent microscopy was used to evaluate E-cadherin and mucin 2 protein levels.

Findings of this study indicated 2-8 mM of butyrate had no significant effect on B. fragilis growth or viability. In uninfected HT29 cells, butyrate increased CDH1 (3.30-fold, P<0.05) and plasma membrane-associated E-cadherin expression. This effect was not concentration dependent. Butyrate (5 mM) also increased CDH1 (1.99-fold, P<0.01) and E-cadherin protein levels in ETBF-infected HT29 cells. Similarly, butyrate increased CDH1 expression in uninfected (7.32-fold, P<0.01) and infected (6.11-fold, P<0.05) HT29-MTX cells. Butyrate also upregulated acidic mucin expression in mucin granulae and reduced MUC2 expression. Further investigation is needed to determine the effect of butyrate on E-cadherin and mucin 2 protein expression in HT29-MTX cells. This study provides insight into further investigation of butyrate as a potential chemo-preventative therapy against BFT-mediated colorectal carcinogenesis.