Background: Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency that affects premature neonates. Despite recent advances in neonatal medicine, the morbidity and mortality from NEC have not changed appreciably over the last three decades. Risk factors for NEC include very low birth weight (<1,500 g), formula feeding, hypoxia, and bacterial colonization of the gut. Breast milk is highly protective against NEC. P-glycoprotein (Pgp) is an efflux ATP-binding cassette transmembrane transporter that protects against intestinal inflammation by pumping out a broad range of toxins and xenobiotics from enterocytes. Pgp is encoded by the multidrug resistant gene (MDR1). Pgp is found in the liver, brain, kidney, and intestine. Previous studies from our lab have shown that breast-fed, but not formula-fed neonatal rats have increased expression of Pgp in the small intestine, and that rat breast milk induces Pgp expression in rat enterocyte cell lines in vitro.

Purpose: The purpose of this study is to determine the kinetics of (human) breast milk-induced expression of Pgp in Caco-2 and HT-29 enterocytes. Methods: The human-derived cell lines, Caco-2 and HT-29, were treated with various concentrations (0-20%) of the water-soluble fraction of human breast milk for 0-24 h. Pgp induction was determined by Western blotting with the anti-Pgp antibody, C219. Chinese hamster ovary cell line, CHCR5, which constitutively expresses Pgp, was used as a positive control. To determine which component of breast milk induces Pgp, low molecular weight components (Mr<10 kD) of human breast milk were separated by gel filtration using Sephadex G-50 column.

Results: Human breast milk induced Pgp in Caco-2 and HT-29 cells in a dose-dependent manner at concentrations ranging from 0.5-20%. Expression of Pgp increased between 2-6 h of treatment and remained elevated for 24 h. Pgp was induced in Caco-2 cells by the high molecular weight fraction of breast milk.

Conclusion: The high molecular weight water-soluble fraction of breast milk induces Pgp in Caco-2 and HT-29 cells in a dose- and time-dependent manner. Induction of Pgp by human breast milk may contribute to the intestinal protection against NEC.

Dose-response Induction of Pgp in Caco-2 Cells