Abstract: This study was undertaken to determine the effect of casein glycomacropeptide (CGMP) on the p65 subunit of nuclear factor-κB (NF-κB) of HT-29 human colon cancer cells challenged with lipopolysaccharide (LPS). Furthermore, Western blotting technology was used to detect and compared the expression levels of the key proteins involved in the NF-κB signaling pathway such as IκBα, p-IκBα, E3RSIκB and UBC5 in the control group, milk-derived CGMP group and LPS group to uncover the molecular mechanism of CGMP in regulating the NF-κB signaling pathway. The results showed that all three doses of CGMP (0.001, 0.010 and 0.100 μg/mL) could suppress the degradation of IκBα in some degree, which is involved in the NF-κB signaling pathway of HT-29 cells challenged with LPS and this effect was more significant at the dose of 0.100 μg/mL, which was significantly different when compared with the control group (P < 0.01). Therefore, it was confirmed that CGMP could suppress the degradation of IκBα by repressing the expression of p-IκBα, E3RSIκB and UBC5, thereby inhibiting the activation of the NF-κB signaling pathway. As a result, CGMP can significantly reduce the degradation of IκBα in the NF-κB signal pathway, increase the amount of protein trimer-IκBα-p65-p50 and decrease nuclear translocation of p65 and downstream gene expression by inhibiting the phosphorylation and ubiquitination of IκBα and reducing the expression of p-IκBα and key ubiquitin proteins (E3RSIκB and UBC5). This study illustrates that milk-derived CGMP plays an anti-inflammatory role by regulating the NF-κB signal pathway.

Key words: casein glycomacropeptide, colon cancer cells (HT-29), nuclear factor-κB, IκBα, ubiquitin-protein ligase subunit