Milk Oligosaccharides From Different Cattle Breeds Influence Growth-Related Characteristics of Intestinal Cells.

Sabine Kuntz,Clemens Kunz,Silvia Rudloff

doi:10.3389/fnut.2019.00031

Abstract

Oligosaccharides are present in human milk (HMO) in large amounts and in a high variety: Among other functions they are considered to influence the gut microbiota and gut maturation in infants. Due to the large volume of milk available bovine milk oligosaccharides (BMO) may be an alternative source of functional ingredients to potentially mimic HMO functions. Thus, we investigated direct effects of bovine milk oligosaccharides (BMO) from different cattle breeds on proliferation, differentiation and apoptosis in transformed (HT-29 and Caco-2) and non-transformed human intestinal cells (HIE cells). We observed a profound growth-inhibition effect induced by all BMO isolates in HT-29, Caco-2, and HIE cells in a dose-dependent manner. The effects varied not only between cell lines, i.e., HT-29 and Caco-2 cells were more sensitive than HIE cells, but also between the cattle breeds. Regarding the induction of differentiation, BMO induced differentiation only in HIE cells without affecting apoptosis. Cell cycle analysis via flow cytometry showed that growth inhibition was associated with a G2/M arrest in all cell lines. Expression levels detected by quantitative real-time RT-PCR revealed that this G2/M arrest was associated with changes in mRNA expression levels of cyclin A and B. Cyclin-dependent kinase inhibitors p21cip1 and p27kip1 and the tumor suppressor p53 were only enhanced in HIE cells necessary for arresting cells in the G2/M phase and induction of differentiation. In HT-29 and Caco-2 cells, a loss of p53 expression failed to induce G2/M associated induction of differentiation. The HIE cell specific differentiation induced by BMO was a result of influencing the phosphorylation states of EGFR (epidermal growth factor receptor) and MAP kinase, i.e., ERK1/2 (extracellular signal-regulated kinase 1/2), p38-α, and Akt2 phosphorylation. These results suggest that BMO inhibited intestinal cell proliferation and altered cell cycle dynamics by affecting corresponding regulator genes and mitogen-activated protein kinase signaling. As the development and maturation of digestive and absorptive processes depends on gut differentiation processes, our in vitro experiments show that breed-specific BMO are natural substances influencing various parameter which may be important in vivo in gastrointestinal development. This, however, needs to be proven in future studies.

Highlights

In the last decade, there has been a tremendous research interest in milk oligosaccharides, driven by the advances in chemical sciences, food technology as well as in chemical-enzymatic synthesis to produce single human milk oligosaccharides (HMO) [1, 2]
As we have previously reported on the effects of HMO on proliferation, differentiation and cell signal events [20, 21], the major aim of our current study was to investigate the functional effects of the milk carbohydrate fractions from various cattle breeds using different intestinal cell lines
To address questions related to gut maturation events associated with tissue morphogenetic and cell dynamic changes, we used HT-29 and HIE cells which are intestinal cells with a lower differentiation phenotype, and Caco-2 cells which display characteristics of differentiated epithelial cells [22,23,24,25]

Summary

Introduction

There has been a tremendous research interest in milk oligosaccharides, driven by the advances in chemical sciences, food technology as well as in chemical-enzymatic synthesis to produce single human milk oligosaccharides (HMO) [1, 2] This interest is primarily based upon the progress in HMO and the increasing number of studies investigating their biological functions [3,4,5,6,7]. No study has been carried out proving that the effects of GOS/FOS on, for example, the immune system are comparable to those of HMO Due to this discrepancy in structure between both classes of carbohydrates, prebiotic oligosaccharides and HMO, there is currently a great interest in finding alternatives for HMO

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