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Epidermal growth factor (EGF) composed of 53 amino acids
(~ 6 kDa) is a single acid polypeptide and presents
in various body fluids and tissues. The
differentiation of various cells stimulated by EGF via
the EGF receptor has been reported in vivo and in vitro. EGF in
human milk has shown to modulate the growth and
development of fetal intestinal cells.
Therefore, the purpose of this study was to
investigate the role of EGF in human milk on growth and the
protein digestion enzyme - leucine aminopeptidase
(LAP) in human colon adenocarcinoma (Caco-2)
cells. The total cellular RNA and protein, and the
activity and content of LAP were examined after the addition of
different concentrations of EGF. The Caco-2 cells were grown in
80% minimum essential medium (MEM) supplemented
with non-essential amino acids, 20% fetal bovine
serum, 50 U/mL penicillin and 50 mg/mL strep-
tomycin. The cells were incubated at 37℃, in an atmosphere of
95% air -5% CO2. Upon 80% confluency, the cells were
switched to serum-free MEM without antibiotics for 24
h. The cells were then incubated with EGF at 0,
5(close to EGF concentration in human milk), 50 (close to EGF
concen- tration in colostrum) or 250 nM for another 24
h. The cells were trypsinized and frozen for further
analyses. The results showed that the total cellular RNA
of the EGF treatment groups was 1.6- to 3.1-fold higher than the
control group, but no significant differences were observed
among the control, 5 nM and 50 nM EGF treatment groups.
The total cellular RNA of 250 nM EGF treatment group was
significantly elevated compared to the control and 5 nM EGF
treatment groups. Although the cell numbers of the EGF
treatment groups were similar to the control group, the
total cellular protein of 5 nM EGF treatment group was
higher than the control and 250 nM EGF treatment groups. The LAP
activity of the treatment groups became 78~86% of the
control group. The LAP protein (53 kDa) was separated
by 10% SDS-PAGE and analyzed by Western blotting. It
showed that the LAP content of the EGF treatment groups was
86~93% of the control group. In conclusion, 250 nM EGF
treatment increased total cellular RNA and 5 nM EGF
treatment increased total cellular protein content, however,
EGF treatments (5 to 250 nM) slightly decreased the activity and
content of LAP on the cell membranes of Caco-2 cells.
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