Abstract: To further elucidate the anti-inflammatory activity of casein glycomacropeptide (CGMP) and its efficacy in enhancing recovery from intestinal inflammation, the effect of CGMP on the NF-κB p65 subunit in colon carcinoma cells HT-29 treated with lipopolysaccharide (LPS) and its role in regulating the NF-κB signaling pathway were determined. Furthermore, plasmids were constructed to express CGMP and NF-κB?essential?modulator (NEMO) and then used to co-transfect HT-29 cells. Co-immunoprecipitation was used to identify the interaction between the expressed proteins. Finally, we detected the pro-apoptotic effect of CGMP on HT-29 cells by measuring the change in mitochondrial transmembrane potential. The experimental results indicated that after 30 h transfection of HT-29 cells, the co-expression and interaction of CGMP and NEMO were confirmed. CGMP affected the NF-κB signaling pathway through its interaction with NEMO with a concomitant increase in apoptosis of HT-29 cells in a time-dependent manner. This effect of CGMP was maximum at 0.1 μg/mL. In conclusion, the interaction of CGMP and NEMO can indirectly affect IKK (IκB kinase) and therefore the NF-κB signaling pathway through IκB. The study revealed another way CGMP could regulate the NF-κB signaling pathway, and fully indicated that there are multiple ways CGMP could execute anti-inflammatory effect.

Key words: casein glycomacropeptide (CGMP), colon carcinoma cells HT-29, NF-κB signaling pathway, NF-κB essential modulator (NEMO), apoptosis