Identification of the HT-29 cell line as a model for investigating MCT1 transporters in sigmoid colon adenocarcinoma.

MCT1 transporters play a crucial role in the symbiotic relationship between humans and their colonic microbiome by facilitating the transport of bacteria-derived short chain fatty acids. Expression of colonic MCT1 transporters, localized in surface epithelial cells, is regulated by luminal butyrate levels. However, MCT1 also transports lactate and can be used by cancer cells to facilitate anaerobic glycolysis. Using immunolocalization techniques, this study investigated whether changes in MCT1 during cancer varied between different colonic regions. Whilst MCT1 abundance did not significantly change in transverse colon adenocarcinoma (P = 0.363, N = 6, paired T-Test), there was an increase in MCT1 in sigmoid colon adenocarcinoma (P = 0.010, N = 21, paired T-test). Using RT-PCR and western blotting, three human intestinal cell lines were tested for their suitability as a MCT1 cancer cell model. Experiments with Caco-2 cells confirmed that they modelled normal cells, with MCT1 only expressed after exposure to butyrate. In contrast, MCT1 was expressed in the absence of butyrate in both HCT-8 and HT-29 cell lines, with consistently high levels of MCT1 protein being present in HT-29 cells. Furthermore, butyrate treatment of HT-29 cells significantly decreased both MCT1 protein abundance (P < 0.001, N = 4, unpaired T-test) and glycosylation of its' chaperone protein, CD147 (P < 0.001, N = 4, unpaired T-test). These data suggest that (i) MCT1 transporter abundance increases in sigmoid colon adenocarcinoma, and (ii) HT-29 cells are an appropriate cell model with which to investigate MCT1 function in this disease.

Investigation of facilitative urea transporters in the human gastrointestinal tract.

The symbiotic relationship between humans and their intestinal microbiome is supported by urea nitrogen salvaging. Previous studies have shown that colonic UT-B urea transporters play a significant role in this important physiological process. This current study investigated UT-A and UT-B urea transporter expression along the human gastrointestinal tract. Initial end-point PCR experiments determined that UT-A RNA was predominantly expressed in the small intestine, while UT-B RNA was expressed in stomach, small intestine, and colon. Using western blotting experiments, a strong 40-60 kDa UT-B signal was found to be abundant in both ileum and colon. Importantly, this signal was deglycosylated by PNGaseF enzyme treatment to a core protein of 30 kDa in both tissues. Further immunolocalization studies revealed UT-B transporter proteins were present at the apical membrane of the villi in the ileum, but predominantly at the basolateral membrane of the colonic surface epithelial cells. Finally, a blind scoring immunolocalization study suggested that there was no significant difference in UT-B abundance throughout the colon (NS, ANOVA, N = 5-21). In conclusion, this current study suggested UT-B to be the main human intestinal urea transporter. Intriguingly, these data suggested that the same UT-B isoform was present in all intestinal epithelial cells, but that the precise cellular location varied.