Alanyl-glutamine and glutamine supplementation improves 5-fluorouracil-induced intestinal epithelium damage in vitro

Abstract

In this study, we have examined the role of glutamine derivatives in reducing 5-fluorouracil (5-FU)-induced epithelial damage in an undifferentiated crypt intestinal cell line, IEC-6. In this model, we have investigated proliferation indirectly by detecting the enzyme-derived formazan dye from the tetrazolium salt WST-1 in viable cells at 24 and 48 h after 5-FU treatment. Migration was measured at 12 and 24 h after razor scraping of the cell monolayer. Cell death was measured by quantifying the percentage of apoptotic and necrotic figures by flow cytometry at 12 and 24 h following 5-FU challenge. Neither glutamine nor alanyl-glutamine prevented 5-FU-induced apoptosis and necrosis in IEC-6 cells at 12 and 24 h after 5-FU challenge. However, glutamine and alanyl-glutamine enhanced migration and proliferation when compared with 5-FU-treated controls (P < 0.05). These new findings support our earlier study on the benefit of oral glutamine in enhancing epithelial recovery after 5-FU challenge.

Fig. 1

(a) Diagram showing a representative digital image of the scraping area with superimposed grid (each square = 0.1 mm²), overlapping the column of farthest cell migration. IEC-6 adherent cells were tracked by traced red dots for counting. The traced red dots within the column were changed to black on a white background and counted digitally by Image Pro Plus software. A yellow line was drawn to depict the migration distance from the scraping point. (b) Dose-response and time-effect graphs of 5-fluorouracil (5-FU) at 6 and 24 h, showing inhibition of cell migration in vitro. IEC-6 cell monolayers were scraped to induce migration, and incubated for 1 h with 5-FU immediately after standard medium replacement, diluted in standard media at doses of 0.3, 1, 3, 10, or 30 mM. The bars represent means ± standard error (SE) for the number of migrating cells per square millimeter of scraped area. *Statistically significant difference (P < 0.05) compared to migrated cells in standard medium without 5-FU treatment (SM), determined by analysis of variance (ANOVA) and Bonferroni’s test

Fig. 2

(a) Dose-response and time-effect graphs of glutamine (A) and alanyl-glutamine (B) at 12 and 24 h, in enhancing cell migration in vitro. IEC-cell monolayers were scraped to induce migration, and incubated with glutamine and alanyl-glutamine (1, 5, 10, and 50 mM) diluted in glutamine-free media, immediately after standard medium replacement. The bars represent means ± SE for the number of migrating cells per square millimeter of scraped area. *Statistically significant difference (P <0.05) compared to migrated cells in medium without glutamine (WG), determined by ANOVA and Bonferroni’s test

Fig. 3

(a) Protective effect of glutamine (G) and alanyl-glutamine (AG) (10 mM) in cell migration following 24 h of 5-FU challenge, in enhancing cell migration in vitro. IEC-6 cell monolayers were scraped to induce migration, and incubated with glutamine and alanyl-glutamine diluted in glutamine-free media, immediately after standard medium replacement. The bars represent means ± SE for the number of migrating cells per square millimeter of scraped area. *P <0.05 compared to migrated cells in glutamine-free media without treatment (WG), ^#P <0.05, compared to migrated cells in standard media pretreated with 1 mM of 5-FU, by ANOVA and Bonferroni’s test. (B) Representative migration columns of adherent IEC-6 cells from the experimental groups 24 h following the 5-FU challenge. The control group is shown at the top, representing migrating cells not challenged with 5-FU embedded in standard media

Fig. 4

Effect of glutamine (G) and alanyl-glutamine (AG) on cell proliferation assay by detected absorbance using an ELISA microplate reader at 450 nm in 96-well seeded IEC-6 cells, following 1 h of 5-fluorouracil (5-FU) exposure (1 mM). 5-FU was diluted in DMEM Gln-free media. After 24 and 48 h, wells were incubated for 4 h with 10 μl of the tetrazolium salt and the absorbance was measured. *P <0.05 compared to untreated control in glutamine-free media (WG) at 24 and 48 h, ^#P <0.05, compared to standard media pretreated with 1 mM of 5-FU at 24 and 48 h, by Student’s t test and ANOVA and Bonferroni’s test, respectively

Fig. 5

Apoptosis and necrosis induced by 1 h 5-fluorouracil (5-FU) pretreatment (at 1 mM, diluted in glutamine-free media) were not inhibited by alanyl-glutamine (AG) and glutamine (G) at 12 (a) and 24 h (b) in IEC-6 cells. Cells were stained with FITC-conjugated annexin V and propidium iodide and analyzed by flow cytometry. Results are shown as density plots with propidium iodide versus annexin V-FITC. Viable cells have low annexin-V-FITC and low propidium iodide staining (lower left quadrant), apoptotic cells have high annexin V-FITC and low propidium iodide staining (lower right quadrant), and necrotic cells have high propidium iodide and annexin V-FITC staining (upper right quadrant)

Fig. 6

Effect of glutamine (G) and alanyl-glutamine (AG) on apoptosis (a) and necrosis (b) by flow cytometry in IEC-6 cells, following 1 h of 5-fluorouracil (5-FU) exposure (1 mM). 5-FU was diluted in DMEM Gln-free media. *P <0.05 compared to media enriched with 10 mM of AG, pretreated with 1 mM of 5-FU at 12 and 24 h, ^#P <0.05, compared to media enriched with 10 mM of G, pretreated with 1 mM of 5-FU at 12 and 24 h, + P <0.05 compared to media enriched with 10 mM of G at 12 h, by Student’s t test