Casein phosphopeptides : from mineral carriers to modulators of biological activity in intestinal cells

Aims. Calcium introduced with the diet and absorbed in the intestine in addition to the known physiological functions is able to modulate biological activities of intestinal cells such as proliferation, apoptosis and differentiation, all processes involved in the development and / or regression of cancer. Milk and dairy products are known sources of bioavailable calcium for its association with casein and casein phosphopeptides (CPPs). CPPs, derived by in vitro or in vivo casein hydrolysis, apart to bind and solubilise calcium, display the ability to induce calcium uptake in human intestinal tumor cells differentiated in vitro toward an enterocityc phenotype. This CPP aptitude could be correlated with a possible activation of pathways of calcium signalling in intestinal cells either in standard growth condition, or in calcium overload as it might occurs following a meal. Materials and Methods. Undifferentiated and differentiated human intestinal cell line HT-29 was used as in vitro cellular model of tumor or physiologic intestinal epithelium. Cell proliferation rate was estimated by incorporation of bromodeoxyuridine. Cell apoptosis was measured by caspase 3/7 activation and nuclei DAPI staining. Intracellular calcium concentration was quantified by Video-imaging experiments using Fura2. Results. In undifferentiated HT-29 DMEM cells, that mimics the behavior of tumor intestinal cells in their early stages, CPPs increase apoptosis, both at physiological calcium concentration and in calcium overload. These results are also shared by the calcium chelator EGTA, and are presumably due to the ability in binding the extracellular calcium. In differentiated RPMI HT-29 cells, CPPs i) increase proliferation, especially in calcium overload; ii) do not affect apoptosis; iii) protect from calcium overload toxicity. On the contrary, EGTA behaves as a cytotoxic agent, decreasing proliferation and increasing apoptosis. The CPP effect in differentiated RPMI cells is related to their ability of activating cell calcium entry through the interaction with the voltage operated L-type calcium channels. L-type calcium channels activate calcium entry into the cells under depolarization and exert a mitogenic effect. CPPs, as well as other digestion products, such as glucose, amino acids, and oligopeptides, display a depolarizing effect on intestinal cell membranes. Conclusion. Taken together these results demonstrate the ability for CPPs, through the binding with calcium ions and the stimulated entry of these ions in differentiated cells, to modulate biological activity strictly related to normal or cancer phenotype, thus opening the way for a use of CPPs as nutraceutical/functional food.