Mechanisms of apoptosis induced by a protein complex isolated from human milk : with focus on the role of mitochondria

Abstract

Human milk contains a protein complex comprising of a folding variant of human alpha-lactalbumin and oleic acid (C18:1). Both, a milk fraction containing this complex (MAL, for multimeric (alpha- lactalbumin)., as well as native alpha-lactalbumin converted in vitro into the folding variant (HAMLET, for human alpha-lactalbumin Made lethal to tumour cells), kills various transformed cells via apoptosis-a highly regulated and conserved form of cell suicide.

Upon treatment of transformed cells, MAL/HAMLET binds to the cell surface, enters the cell and exhibits a granular pattern in the cytoplasm. Subsequently, the complex translocates to and accumulates in the nucleus. MAL/HAMLET-treated Jurkat or A549 cells showed typical biochemical characteristics of apoptotic cells, such as caspase activation and cleavage of specific caspase substrates. The caspase activation did not depend on death receptor ligation. However, mitochondrial cytochrome c was released, an event required for activation of pro-caspase-9 in the presence of Apaf-1 and dATP. Active caspase-9, in turn, cleaves and activates executioner caspases. Mitochondria play a critical role in apoptotosis by releasing apoptogenic proteins, including cytochrome c to the cytosol. At the time of investigation, cytochrome c and AIF (apoptosis inducing factor) were the only proteins known to be released from mitochondria during apoptosis.

We showed that another intermembrane space protein, adenylate kinase (AK)-2, was released together with cytochrome c, while AK-3-a matrix protein remained inside mitochondria during apoptosis induced by different stimuli, including MAL. This suggested a general release of intermembrane space proteins, rather than a specific release of cytochrome c and AIF during apoptotic cell death.

Co-localization of MAL/HAMLET with the mitochondrial dye MitoTracker Red indicated that MAL/HAMLET may cause the release of intermembrane Apace proteins in treated cells by direct targeting of mitochondria. Addition of MAL/HAMLET to Ca2+-loaded isolated mitochondria induced loss of delta-psim, swelling and release of proteins from the intermembrane space, including cytochrome c and AK-2. These effects were prevented by Cyclosporin A, an inhibitor of mitochondrial permeability transition (MPT), indicating that MAL/HAMLET induced cytochrome c release via opening of MPT pores. Moreover, in the absence of Ca2+-loading MAL/HAMLET increased the rate of state 4 respiration and induced a sub-maximal drop of AT. in isolated mitochondria, suggesting that this complex possesses uncoupling properties. Tits uncoupling effect of MAL/HAMLET was due to the presence of fatty acids in the complex since it was completely abolished by bovine serum albumin (BSA). In contrast, BSA failed to prevent MPT, but slightly delayed it, indicating that the fatty acids in MAL/HAMLET rather facilitated than were responsible for MPT induction.

Treatment of cells with HAMLET caused a rapid decrease in AT- that was accompanied by cytochrome c release and a decreased ability of mitochondria to accumulate Ca2+. MPT inhibitors did not block the loss in delta-psim in HAMLET-treated cells. However, when the inhibitors were added in combination with BSA, they markedly prevented the drop in AT. and restored the ability of mitochondria to accumulate Ca2+, suggesting again that the mitochondrial changes in HAMLET-treated cells were a consequence of both MPT induction and uncoupling.

Thus, our data demonstrate that a protein complex containing a folding variant of human alpha-lactalbumin is directly toxic to mitochondria resulting in MPT pore opening, release of cytochrome c, and activation of the caspase cascade; taken together, these events leads to apoptotic death of transformed cells.