A folding variant of human α‐lactalbumin induces mitochondrial permeability transition in isolated mitochondria

Abstract

A human milk fraction containing multimeric a-lactalbumin (MAL) is able to kill cells via apoptosis. MAL is a protein complex of a folding variant of a-lactalbumin and lipids. Previous results have shown that upon treatment of transformed cells, MAL localizes to the mitochondria and cytochrome c is released into the cytosol. This is followed by activation of the caspase cascade. In this study, we further investigated the involvement of mitochondria in apoptosis induced by the folding variant of a-lactalbumin. Addition of MAL to isolated rat liver mitochondria induced a loss of the mitochondrial membrane potential (DC m ), mitochondrial swelling and the release of cytochrome c. These changes were Ca 21 -dependent and were prevented by cyclosporin A, an inhibitor of mitochondrial permeability transition. MAL also increased the rate of state 4 respiration in isolated mitochondria by exerting an uncoupling effect. This effect was due to the presence of fatty acids in the MAL complex because it was abolished completely by BSA. BSA delayed, but failed to prevent, mitochondrial swelling as well as dissipation of DC m , indicating that the fatty acid content of MAL facilitated, rather than caused, these effects. Similar results were obtained with HAMLET (human a-lactalbumin made lethal to tumour cells), which is native a-lactalbumin converted in vitro to the apoptosis-inducing folding variant of the protein in complex with oleic acid. Our findings demonstrate that a folding variant of a-lactalbumin induces mitochondrial permeability transition with subsequent cytochrome c release, which in transformed cells may lead to activation of the caspase cascade and apoptotic death.Keywords: a-lactalbumin; cytochrome c; mitochondria; mitochondrial permeability transition; tumour cells.Diverse physiological and pathological stimuli can induce apoptosis by distinct pathways that converge in a common programme of cell suicide. Upon apoptotic triggering, a unique family of cysteine aspartate proteases, caspases, becomes activated [1]. In many models of apoptosis, caspase activation requires the release of apoptogenic factors, such as cytochrome c, from the mitochondrial intermembrane space. Once released into the cytosol, cytochrome c binds to Apaf-1, and together with pro-caspase-9 and dATP, forms the so-called apoptosome complex [2]. This association leads to the activation of pro-caspase-9 which, in turn, initiates the caspase cascade by activating pro-caspase-3. Although cytochrome c release has been observed in many experimental models of apoptosis, the precise mechanism responsible for its movement across the outer mitochondrial membrane remains unclear. Different hypotheses have been proposed [3]. Cytochrome c may be released via specific channels in the outer mitochondrial membrane or the release may be a consequence of mitochondrial swelling leading to the rupture of the outer mitochondrial membrane. Mitochondrial swelling may be due to opening of the mitochondrial permeability transition (MPT) pore, a polyprotein complex fo...

The cytotoxicity of fatty acid/α‐lactalbumin complexes depends on the amount and type of fatty acid

“…HAMLET has also been shown to affect mitochondria [31] and induce apoptosis-like cell death with caspase activation, PS externalization and chromatin condensation irrespective of the p53 and bcl-2 status of the cells [32] and to cause changes in proteasome structure and function [33]. In addition, it has been shown by Aits et al that HAMLET induces autophagy [34], while Rammer et al see activation of the lysosomal death pathway in response to BAMLET treatment, but that the cytotoxicity of BAMLET does not depend on autophagy [18].…”

BAMLET Activates a Lysosomal Cell Death Program in Cancer Cells

“…The nonapoptotic nature of HAMLETinduced cytotoxicity is further emphasized by its ability to kill tumor cells that have acquired resistance to classic apoptosis pathway either by overexpressing the antiapoptotic protein Bcl-2 or by carrying mutations in the p53 tumor suppressor protein (12). Instead, HAMLET has been suggested to act by numerous unrelated mechanisms including (a) direct induction of mitochondrial outer membrane permeabilization (14), (b) histone binding and subsequent DNA damage (15,16), (c) unfolded protein response (17), and (d) autophagic cell death (18).…”

The Alpha-Lactalbumin/Oleic Acid Complex and Its Cytotoxic Activity

“…Apoptotic pathway [2,54,84,87,90,91] Autophagic pathway [81,91] Chomatin structure disorder [67,90,100] Other responses:…”

“…The activation of cytosolic caspases by apogenetic factors released from the inner mitochondrial membrane (e.g. cytochrome c), and subsequent Apaf-1, procaspase-9 and dATP association, resulting in formation of the apoptosome complex, are normal events of the classical apoptotic pathway [85][86][87]. It was later revealed that MAL induced a loss of the mitochondrial membrane potential (ΔΨm) and abnormal mitochondrial permeability transition (MPT) in isolated mitochondria through opening of the MPT pore, resulting in Ca 2+ -dependent release of cytochrome c to the cytosol [87].…”

“…The proposed therapeutic applications of HAMLET and the observed effects are listed in Table 2. One problem in using HAMLET as a potential cancer agent is its interaction with albumin, which through binding of free fatty acids could neutralize the cytotoxic activity of HAMLET or BAMLET [53,87,102]. As albumin is present in nearly all physiologic fluids, it will in many cases be difficult for HAMLET to reach the target cancer cells in an active form.…”