Apoptosis induced by a human milk protein complex. Cellular and structural studies in tumour cells and bacteria

Sammanfattning: Human milk contains a vast array of bioactive molecules, with nutritional and protective functions. This thesis describes the effects of a human milk protein complex, MAL, on tumour cells and bacteria. During our studies on the anti-adhesive properties of human milk we observed that a milk fraction killed tumour cells. The morphology of the cells indicated apoptotic cell death and this was verified by DNA fragmentation analysis (Proc Natl Acad Sci U.S.A 1995; 92: 8064-8068). The effect of MAL was selective for tumour cells and immature embryonic cells while mature differentiated cells were resistant. MAL bound to the surface of both sensitive and resistant cells but was internalised only in sensitive cells. MAL was shown to co-localise with mitochondria and this interaction caused a disruption of the mitochondrial membrane potential, which caused release of cytochrome c and caspase activation (Exp Cell Res 1999; 249: 260-268). The caspase activation was functional as shown by the cleavage of intracellular substrates. In parallel MAL was also transported via active nuclear transport to the nuclei of sensitive cells. MAL exerted a direct effect on isolated nuclei causing the induction of apoptotic DNA fragmentation without involvement of mitochondrial activation pathways (Exp Cell Res 1999; 2246: 451-460). MAL was purified from the casein fraction of human milk and was shown by N-terminal amino acid sequencing to contain alpha-lactalbumin. Native alpha-lactalbumin had no bactericidal or tumouricidal activity, suggesting that the alpha-lactalbumin of the active fraction had different structural properties. By spectroscopic techniques MAL was shown to have a more flexible tertiary structure with exposure of hydrophobic surfaces like the molten globule form of alpha-lactalbumin (J Biol Chem 1999; 274: 6388-6396). Native alpha-lactalbumin was inactive, but could be converted to the active form by a folding change combined with a fatty acid to stabilise this fold. These studies verified that alpha-lactalbumin was the active component and that the conformational change was required for the apoptosis-inducing activity. MAL was also shown to be bactericidal against S. pneumoniae, but had little effect against gram-negative and other gram-positive bacteria. In S. pneumoniae MAL was shown to induce identical DNA fragmentation as seen in tumour cells, suggesting apoptosis-like features of bacterial death. The bacterial death did not involve cytochrome c and caspase-activation but the bacteria had sequence homologies in three open reading frames of the pneumococcal genome with a caspase-independent effector molecule in eukaryotic cells, apoptosis inducing factor (AIF). These sequences are currently investigated.