Individual and Combined Effects of Nucleotides and Human Milk Oligosaccharides on Proliferation, Apoptosis and Necrosis in a Human Fetal Intestinal Cell Line

Abstract

Nucleotides (NT) and human milk oligosaccharides (HMO) individually affect epithelial cell growth, but their combined effects had not been studied. Herein, the impact of NT and HMO on cell proliferation, apoptosis, necrosis and cell cycle in the fetal epithelial cell line (FHs-74 Int) was determined. Cells were incubated with media containing 2.5% FBS and no epidermal growth factor (Control); fucosyllactose (FL) mix [85% 2'FL/15% 3'FL], sialyllactose (SL) mix [40% 6'SL/10% 3'SL/50% sialic acid (SA)] or LNnT at 125, 250, 500 or 1000 μg/mL with and without 250 μg/mL NT (43% CMP, 18.5% UMP, 16.4% AMP, and 22.0% GMP) for 24 or 72 h. NT alone significantly increased proliferation, but did not affect cell cycle or apoptosis/necrosis. All HMO treatments at 1000 μg/mL significantly decreased proliferation and some were also inhibitory at 250 or 500 μg/mL. When NT and HMO were simultaneously added, NT ameliorated the anti-proliferative effect of HMO. FL significantly increased cells in S phase and SL and LNnT treatments significantly increased cells in G2/M and S phases, which concomitantly decreased cells in G0/G1. HMO with NT significantly decreased the percent of cells in the G2/M phase compared to HMO alone. Higher HMO doses significantly increased the percentage of apoptotic and necrotic cells compared to control. In conclusion, HMO reduced cell proliferation and this effect is partially ameliorated by NT. It appears that HMO initially induced apoptosis/necrosis, which was later evidenced by G2/M cell cycle arrest and decreased proliferation.

Metabolism of Caprine Milk Carbohydrates by Probiotic Bacteria and Caco-2:HT29–MTX Epithelial Co-Cultures and Their Impact on Intestinal Barrier Integrity

“…A previous study by Kuntz et al [ 57 ] reported that proliferation rates of HT29 and Caco-2 cells was inhibited by neutral oligosaccharides (2.3 and 0.2 mg/mL respectively) and acidic oligosaccharide (0.3 and 0.7 mg/mL respectively) isolated from human milk [ 57 ]. In contrast, 1 mg/mL lactose had no effect on the proliferation of primary human foetal intestinal cells [ 58 ], but proliferation of the same cell line was increased after exposure to whey produced from bovine colostrum [ 59 ] and complete human milk [ 60 ]. Thus, it could be suggested that treatment of IECs with purified preparations of neutral or acidic oligosaccharides inhibits cellular proliferation to a greater extent than IECs exposed to treatments comprised of a combination of carbohydrates such as the CF.…”

“…Thus, it could be suggested that treatment of IECs with purified preparations of neutral or acidic oligosaccharides inhibits cellular proliferation to a greater extent than IECs exposed to treatments comprised of a combination of carbohydrates such as the CF. How this relates to the mechanism of action was not determined in this study, but it is known that neutral and acidic oligosaccharides inhibit proliferation rates of IECs through alterations in epidermal growth factor receptor (EGFR) signalling and cell cycle regulators [ 58 , 61 ], and human milk increases proliferation through a unique tyrosine kinase pathway [ 60 ].…”

Effect of a Semi-Purified Oligosaccharide-Enriched Fraction from Caprine Milk on Barrier Integrity and Mucin Production of Co-Culture Models of the Small and Large Intestinal Epithelium

“…Also, proliferation rates of pre-confluent HT29 cell cultures were reduced after incubation with neutral and acidic oligosaccharides (0.2 and 0.4 mg/mL respectively) from human milk, although Caco-2 brush border-expressing cultures were unaffected at these concentrations [ 77 ]. However, proliferation of primary human foetal intestinal cells when incubated (72 h) with 1 mg/mL lactose and tri-galactose was unchanged [ 78 ]. In contrast, proliferation of the same cell line, was increased after 48 h exposure to whey produced from bovine colostrum at concentrations ranging from 0.3% to 10% v / v [ 79 ] and also complete human milk at concentrations ranging from 1% to 10% v / v after 24 h exposure [ 80 ].…”

“…Thus, it could be hypothesised that neutral or acidic oligosaccharides, as purified preparations, are able to elicit a greater response in inhibiting cellular proliferation than samples comprised of combinations of acidic and neutral oligosaccharides in conjunction with other saccharides components. Whether this could also be related to their mechanism of action requires further study; previous reports indicate that neutral and acidic oligosaccharides induce growth inhibition in intestinal epithelial cell cultures through changes in EGFR signalling and gene expression of cell cycle regulators [ 59 , 78 ]. In contrast, complete human milk increases cell proliferation but not through the EGFR signalling but via a unique tyrosine kinase pathway [ 80 ].…”

Select human milk oligosaccharides directly modulate peripheral blood mononuclear cells isolated from 10-d-old pigs

“…Indeed, both 2′-FL ( P = 0·07) and the combination of fucosylated HMO (FL mix; P = 0·10) tended to inhibit proliferation. Whether HMO affect immune cell proliferation through the regulation of cyclin expression, as has been demonstrated in epithelial cells ( 34 , 42 ) , or through a more novel mechanism, such as fucose modification of Notch receptors or ligands ( 43 ) , is unknown and should be tested. In terms of cytokine production, stimulation of PBMC with iHMO doubled IL-10 production by PBMC.…”