Identification of ubiquitin in bovine milk and its growth inhibitory effects on human cancer cell lines

Bovine milk is associated with improved health and reduced risk of several diseases, among them cancer. Milk is a complex mixture of known and unknown components. The components and the mechanisms that contribute to the cancer-preventive effects are largely unknown. We set out to find new peptides in milk and identified ubiquitin (Ub) using matrix-assisted laser desorption ionization-time of flight mass spectrometry and Western blot. Using quantitative Western blot, we estimated the Ub concentration to be about 0.003 μmol/L in milk. We then decided to investigate the effect of treating human colon cancer CaCo-2 cells with Ub, using higher concentrations than in milk. CaCo-2 cells treated with 0.02 to 2.0 μmol/L Ub showed significantly decreased proliferation compared with untreated control cells. A higher growth inhibitory effect than in CaCo-2 cells was found in the neuroblastoma cell line SH-SY5Y treated with 0.02 to 0.2 μmol/L Ub. A bromodeoxyuridine DNA flow cytometric method was used to study cell cycle kinetics in Ub-treated CaCo-2 cells. The data point toward a prolongation of the G₁ phase. The levels of several cell cycle regulatory proteins were affected. Our data point to Ub possibly being one of the components in milk reducing the risk of cancer.