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Lipids as Tumoricidal Components of Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET)

UNIQUE AND SHARED EFFECTS ON SIGNALING AND DEATH*
  • James C.S. Ho
    Affiliations
    From the Department of Microbiology, Immunology, and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Sölvegatan 23, S-223 62 Lund, Sweden,
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  • Petter Storm
    Affiliations
    From the Department of Microbiology, Immunology, and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Sölvegatan 23, S-223 62 Lund, Sweden,
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  • Anna Rydström
    Affiliations
    From the Department of Microbiology, Immunology, and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Sölvegatan 23, S-223 62 Lund, Sweden,
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  • Ben Bowen
    Affiliations
    Lawrence Berkeley National Laboratory, Berkeley, California 94720, and
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  • Fredrik Alsin
    Affiliations
    From the Department of Microbiology, Immunology, and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Sölvegatan 23, S-223 62 Lund, Sweden,

    Lawrence Berkeley National Laboratory, Berkeley, California 94720, and
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  • Louise Sullivan
    Affiliations
    Biomedical Sciences Institute, Trinity College Dublin, College Green, Dublin 2, Ireland
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  • Inès Ambite
    Affiliations
    From the Department of Microbiology, Immunology, and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Sölvegatan 23, S-223 62 Lund, Sweden,
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  • K.H. Mok
    Affiliations
    Biomedical Sciences Institute, Trinity College Dublin, College Green, Dublin 2, Ireland
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  • Trent Northen
    Affiliations
    Lawrence Berkeley National Laboratory, Berkeley, California 94720, and
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  • Catharina Svanborg
    Correspondence
    To whom correspondence should be addressed. Tel.: 46-709426549; Fax: 46-46137468;
    Affiliations
    From the Department of Microbiology, Immunology, and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Sölvegatan 23, S-223 62 Lund, Sweden,
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant U54 CA 112970. This study was also supported by a Sharon D. Lund Foundation grant and by the American Cancer Society, the Swedish Cancer Society, the Medical Faculty (Lund University), the Söderberg Foundation, the Segerfalk Foundation, the Anna-Lisa and Sven-Erik Lundgren Foundation for Medical Research, the Knut and Alice Wallenberg Foundation, the Lund City Jubileumsfond, the John and Augusta Persson Foundation for Medical Research, the Maggie Stephens Foundation, the Gunnar Nilsson Cancer Foundation, the Inga-Britt and Arne Lundberg Foundation, and the HJ Forssman Foundation for Medical Research and the Royal Physiographic Society. Support was also obtained from the Danish Council for Independent Research (Medical Sciences).
    This article contains supplemental Figs. S1—S3 and Table S1.
Open AccessPublished:April 29, 2013DOI:https://doi.org/10.1074/jbc.M113.468405
      Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance 13C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 μm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 μm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.
      Background: HAMLET is a broadly tumoricidal complex of partially unfolded α-lactalbumin and oleic acid whose structural and functional contributions to HAMLET remain largely undefined.
      Results: The protonation state and function of the lipid are elucidated.
      Conclusion: HAMLET and oleate possess unique and overlapping effects on tumor cells.
      Significance: Unfolded proteins, like α-lactalbumin, form novel functional entities with deprotonated fatty acids.

      Introduction

      Long-chain fatty acids are essential cellular components, serving as nutrients, membrane constituents, signaling molecules, and precursors for prostaglandins and other crucial bioactive substances (
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      ). Examples of their effects include modifications of enzymatic function, gene expression, synaptic transmission, and metabolism (
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      ). Dysregulated long-chain fatty acid function is associated with numerous medical disorders, including infection, inflammation, atherosclerosis, and cancer (
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      ,
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      Long-chain polyunsaturated fatty acids, endothelial lipase, and atherosclerosis.
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      n-3 polyunsaturated fatty acids and cytokine production in health and disease.
      ). To fulfill these diverse functions, fatty acids engage with cell membranes, and specific fatty acids are taken up from the circulation. A variety of membrane interaction mechanisms have been characterized, but many aspects remain unclear. Fatty acids cross lipid bilayers via flip-flop mechanisms or specific protein-receptor interactions. Receptors identified in caveolae/lipid rafts (
      • Pohl J.
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      ) include fatty acid translocase/Cluster of Differentiation 36 (FAT/CD36), caveolin-1, and interacting cytosolic fatty acid-binding proteins (FABs), which bind anionic phospholipids as well as lipids modified by peroxidation (
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      ,
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      ).
      HAMLET
      The abbreviation used is: HAMLET, human α-lactalbumin made lethal to tumor cells.
      (human alpha-lactalbumin made lethal to tumor cells) is a complex of α-lactalbumin and oleic acid (
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      Apoptosis induced by a human milk protein.
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      Conversion of α-lactalbumin to a protein inducing apoptosis.
      ) and the first member in a new family of complexes formed from partially unfolded proteins with fatty acids as integral constituents. HAMLET displays broad anti-tumor activity in vitro with a high degree of tumor selectivity (
      • Svanborg C.
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      HAMLET kills tumor cells by an apoptosis-like mechanism. Cellular, molecular, and therapeutic aspects.
      ). Studies in patients and animal models have confirmed this selectivity and demonstrated therapeutic efficacy against several tumor types (
      • Fischer W.
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      Human α-lactalbumin made lethal to tumor cells (HAMLET) kills human glioblastoma cells in brain xenografts by an apoptosis-like mechanism and prolongs survival.
      • Gustafsson L.
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      Treatment of Skin Papillomas with Topical α-Lactalbumin-Oleic Acid.
      ,
      • Mossberg A.-K.
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      HAMLET treatment delays bladder cancer development.
      ,
      • Mossberg A.-K.
      • Wullt B.
      • Gustafsson L.
      • Månsson W.
      • Ljunggren E.
      • Svanborg C.
      Bladder cancers respond to intravesical instillation of HAMLET (human α-lactalbumin made lethal to tumor cells).
      • Puthia M.
      • Storm P.
      • Nadeem A.
      • Hsiung S.
      • Svanborg C.
      Prevention and treatment of colon cancer by peroral administration of HAMLET (human α-lactalbumin made lethal to tumour cells).
      ). HAMLET has broad tumoricidal activity unrelated to well defined cellular responses such as apoptosis (
      • Aits S.
      • Gustafsson L.
      • Hallgren O.
      • Brest P.
      • Gustafsson M.
      • Trulsson M.
      • Mossberg A.-K.
      • Simon H.-U.
      • Mograbi B.
      • Svanborg C.
      HAMLET (human α-lactalbumin made lethal to tumor cells) triggers autophagic tumor cell death.
      ,
      • Storm P.
      • Aits S.
      • Puthia M.K.
      • Urbano A.
      • Northen T.
      • Powers S.
      • Bowen B.
      • Chao Y.
      • Reindl W.
      • Lee D.Y.
      • Sullivan N.L.
      • Zhang J.
      • Trulsson M.
      • Yang H.
      • Watson J.D.
      • Svanborg C.
      Conserved features of cancer cells define their sensitivity to HAMLET-induced death. c-Myc and glycolysis.
      • Storm P.
      • Klausen T.K.
      • Trulsson M.
      • Ho CS J.
      • Dosnon M.
      • Westergren T.
      • Chao Y.
      • Rydström A.
      • Yang H.
      • Pedersen S.F.
      • Svanborg C.
      A unifying mechanism for tumor cell death by ion channel activation.
      ). Essential aspects of the conserved death response to HAMLET have been defined, including the dependence on oncogenic transformation (
      • Storm P.
      • Aits S.
      • Puthia M.K.
      • Urbano A.
      • Northen T.
      • Powers S.
      • Bowen B.
      • Chao Y.
      • Reindl W.
      • Lee D.Y.
      • Sullivan N.L.
      • Zhang J.
      • Trulsson M.
      • Yang H.
      • Watson J.D.
      • Svanborg C.
      Conserved features of cancer cells define their sensitivity to HAMLET-induced death. c-Myc and glycolysis.
      ), proteasome inhibition (
      • Gustafsson L.
      • Aits S.
      • Onnerfjord P.
      • Trulsson M.
      • Storm P.
      • Svanborg C.
      Changes in proteasome structure and function caused by HAMLET in tumor cells.
      ), and nucleosome-histone binding (
      • Düringer C.
      • Hamiche A.
      • Gustafsson L.
      • Kimura H.
      • Svanborg C.
      HAMLET interacts with histones and chromatin in tumor cell nuclei.
      ). We have recently identified ion channel activation as a new, unifying mechanism of HAMLET tumoricidal effect (
      • Storm P.
      • Klausen T.K.
      • Trulsson M.
      • Ho CS J.
      • Dosnon M.
      • Westergren T.
      • Chao Y.
      • Rydström A.
      • Yang H.
      • Pedersen S.F.
      • Svanborg C.
      A unifying mechanism for tumor cell death by ion channel activation.
      ). Rapid ion fluxes triggered by HAMLET were shown to initiate changes in morphology, viability, gene expression, and MAPK signaling, and especially Na+ or K+ fluxes were essential for these responses to occur.
      In a screen for suitable fatty acids cofactors, C18:1, cis-monounsaturated fatty acids were identified as optimal for HAMLET formation (
      • Svensson M.
      • Mossberg A.-K.
      • Pettersson J.
      • Linse S.
      • Svanborg C.
      Lipids as cofactors in protein folding. Stereo-specific lipid-protein interactions are required to form HAMLET (human α-lactalbumin made lethal to tumor cells).
      ), suggesting that these fatty acids may share specific structural features required both for HAMLET formation and to engage targets involved in tumor cell death. In contrast, trans-poly/monounsaturated or fully saturated fatty acids failed to form a HAMLET-like complex under near identical experimental conditions (
      • Svensson M.
      • Mossberg A.-K.
      • Pettersson J.
      • Linse S.
      • Svanborg C.
      Lipids as cofactors in protein folding. Stereo-specific lipid-protein interactions are required to form HAMLET (human α-lactalbumin made lethal to tumor cells).
      • Lisková K.
      • Kelly A.L.
      • O'Brien N.
      • Brodkorb A.
      Effect of denaturation of α-lactalbumin on the formation of BAMLET (bovine α-lactalbumin made lethal to tumor cells).
      ,
      • Permyakov S.E.
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      • Khasanova L.M.
      • Fadeev R.S.
      • Zhadan A.P.
      • Roche-Hakansson H.
      • Håkansson A.P.
      • Akatov V.S.
      • Permyakov E.A.
      Oleic acid is a key cytotoxic component of HAMLET-like complexes.
      • Vukojević V.
      • Bowen A.M.
      • Wilhelm K.
      • Ming Y.
      • Ce Z.
      • Schleucher J.
      • Hore P.J.
      • Terenius L.
      • Morozova-Roche L.A.
      Lipoprotein complex of equine lysozyme with oleic acid (ELOA) interactions with the plasma membrane of live cells.
      ). The contribution of the C18:1 cis fatty acids to the tumoricidal activity of HAMLET has been debated, however. Studies of complexes with high lipid content have recently suggested that unfolded proteins may function solely as “lipid carriers” and that the tumoricidal response is triggered by the long-chain fatty acids alone (
      • Permyakov S.E.
      • Knyazeva E.L.
      • Khasanova L.M.
      • Fadeev R.S.
      • Zhadan A.P.
      • Roche-Hakansson H.
      • Håkansson A.P.
      • Akatov V.S.
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      Oleic acid is a key cytotoxic component of HAMLET-like complexes.
      ,
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      • Frare E.
      • Canton M.
      • Polverino de Laureto P.
      • Fontana A.
      The oleic acid complexes of proteolytic fragments of α-lactalbumin display apoptotic activity.
      ).
      The effect of lipids on host cells is influenced by the protonation state. For example, experiments using anionic inhibitors suggested that the deprotonated form of long-chain fatty acids may be the most relevant for cellular uptake (
      • Hajri T.
      • Abumrad N.A.
      Fatty acid transport across membranes. Relevance to nutrition and metabolic pathology.
      ). The protonation state of oleic acid in HAMLET is unclear due to the phase behavior of oleic acid, which increases the apparent pKa to between 8.0 and 8.5 (
      • Cistola D.P.
      • Hamilton J.A.
      • Jackson D.
      • Small D.M.
      Ionization and phase behavior of fatty acids in water. Application of the Gibbs phase rule.
      ) or 9.85 (
      • Kanicky J.R.
      • Shah D.O.
      Effect of degree, type, and position of unsaturation on the pKa of long-chain fatty acids.
      ). Oleic acid is expected to be deprotonated in HAMLET, however, as the complex is formed by ion exchange chromatography at basic pH. Oleate has been used successfully to form HAMLET-like complexes (
      • Lišková K.
      • Auty M.A.E.
      • Chaurin V.
      • Min S.
      • Mok K.H.
      • O'Brien N.
      • Kelly A.L.
      • Brodkorb A.
      Cytotoxic complexes of sodium oleate with β-lactoglobulin.
      ), but structural or biological differences between oleic acid and oleate as cofactors in HAMLET have not been examined.
      To address this question, we first show by 13C NMR that oleate and oleic acid produce structurally homologous HAMLET complexes. Using a variety of cellular assays, we subsequently identify dose-dependent effects of oleate on tumor cells, partially overlapping with those of HAMLET. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.

      DISCUSSION

      Oleic acid is a key constituent of the HAMLET complex, but the extent to which the lipid contributes to tumor cell death has remained unclear as have the mechanisms involved. HAMLET is optimally formed from 18-carbon, monounsaturated, cis-fatty acids and partially unfolded α-lactalbumin, in contrast to unsaturated trans- or saturated fatty acids, which fail to form complexes with the protein. In this study we show that the deprotonated form of oleic acid (oleate) is an active lipid cofactor in HAMLET formation and that when presented in the context of partially unfolded α-lactalbumin, oleate acts directly on the cell death machinery through membrane sensing, uptake, and metabolic processing. The lipid per se did not reproduce the cell death response to HAMLET or the metabolic paralysis, however, confirming that the protein and lipid are both required for the full-fledged tumoricidal response to HAMLET. At higher oleate concentrations, a larger part of the HAMLET profile was reproduced, but major differences in cellular response profiles were still present. Oleate recognition by tumor cells is thus quantitatively and qualitatively altered in the context of partially unfolded α-lactalbumin.
      A number of HAMLET-related complexes have been prepared using different α-lactalbumin variants (
      • Pettersson J.
      • Mossberg A.K.
      • Svanborg C.
      α-Lactalbumin species variation, HAMLET formation, and tumor cell death.
      ,
      • Pettersson-Kastberg J.
      • Aits S.
      • Gustafsson L.
      • Mossberg A.
      • Storm P.
      • Trulsson M.
      • Persson F.
      • Mok K.H.
      • Svanborg C.
      Can misfolded proteins be beneficial? The HAMLET case.
      ), aggregation states (
      • Lisková K.
      • Kelly A.L.
      • O'Brien N.
      • Brodkorb A.
      Effect of denaturation of α-lactalbumin on the formation of BAMLET (bovine α-lactalbumin made lethal to tumor cells).
      ), related proteins like equine lysozyme (
      • Wilhelm K.
      • Darinskas A.
      • Noppe W.
      • Duchardt E.
      • Mok K.H.
      • Vukojević V.
      • Schleucher J.
      • Morozova-Roche L.A.
      Protein oligomerization induced by oleic acid at the solid-liquid interface. Equine lysozyme cytotoxic complexes.
      ), bovine β-lactoglobulin, or the α isoform of pike parvalbumin (
      • Permyakov S.E.
      • Knyazeva E.L.
      • Khasanova L.M.
      • Fadeev R.S.
      • Zhadan A.P.
      • Roche-Hakansson H.
      • Håkansson A.P.
      • Akatov V.S.
      • Permyakov E.A.
      Oleic acid is a key cytotoxic component of HAMLET-like complexes.
      ). Some of these complexes, which contain a high number of oleic acid molecules (
      • Lisková K.
      • Kelly A.L.
      • O'Brien N.
      • Brodkorb A.
      Effect of denaturation of α-lactalbumin on the formation of BAMLET (bovine α-lactalbumin made lethal to tumor cells).
      ), have been proposed to permeabilize cells by virtue of their fatty acid content alone, giving rise to suggestions that the protein acts merely as a fatty acid delivery vehicle (
      • Baumann A.
      • Gjerde A.U.
      • Ying M.
      • Svanborg C.
      • Holmsen H.
      • Glomm W.R.
      • Martinez A.
      • Halskau O.
      HAMLET forms annular oligomers when deposited with phospholipid monolayers.
      ,
      • Mossberg A.-K.
      • Puchades M.
      • Halskau Ø.
      • Baumann A.
      • Lanekoff I.
      • Chao Y.
      • Martinez A.
      • Svanborg C.
      • Karlsson R.
      HAMLET interacts with lipid membranes and perturbs their structure and integrity.
      • Zherelova O.M.
      • Kataev A.A.
      • Grishchenko V.M.
      • Knyazeva E.L.
      • Permyakov S.E.
      • Permyakov E.A.
      Interaction of antitumor α-lactalbumin-oleic acid complexes with artificial and natural membranes.
      ). It may be speculated that upon interaction of tumor cell membranes with several HAMLET molecules, membrane patches with higher lipid concentrations might be formed, resulting in membrane disruption and necrosis. The present study clearly demonstrates that HAMLET and oleate-HAMLET complexes carry a limited number of lipid moieties and that distinct cellular responses including ion fluxes and specific death pathways are activated, arguing against nonspecific membrane disruptions as a mechanism of tumor cell death. Although we show that the lipid is active in the context of the partially unfolded protein, we found no evidence of independent cytotoxic activity at concentrations equimolar to those present in HAMLET. At higher concentrations, the lipid was cytotoxic on its own, however. Importantly, cellular response profiles at either lipid concentration were not those involved in necrosis, as would have been expected if HAMLET merely permeabilizes the cell membrane due to its lipid content.
      In this study, HAMLET and oleate were shown to trigger overlapping but also fundamentally different responses in tumor cells. At equimolar concentrations, oleate alone was shown to activate Na+ fluxes but not the full ion channel repertoire seen in HAMLET-treated cells. This limited effect may explain the low transcriptional response to oleate (175 μm) and its lack of cytotoxicity. At higher oleate concentrations, effects on ion fluxes were stronger, compatible with the transcriptional changes, and the cytotoxic response and overlapping effects on transcription included cancer-related pathways, MAPKs genes, and innate immune response genes. Ion fluxes were not identical to HAMLET, however, as oleate (500 μm) caused partial activation of Na+ and Ca2+ compatible with the differences in transcriptomic profiles. The results emphasize that the HAMLET complex is functionally defined in part by oleate but possesses unique additional properties not reproduced by the lipid.
      Cancer metabolism is optimized for cell growth and survival under conditions of metabolic stress (
      • Hsu P.P.
      • Sabatini D.M.
      Cancer cell metabolism. Warburg and beyond.
      ). In addition to the shift in glycolysis known as the Warburg effect, there is evidence that fatty acid oxidation contributes to the metabolic changes accompanying oncogene overexpression (
      • Menendez J.A.
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      ,
      • Nomura D.K.
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      • Niessen S.
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      ). We detected two major alterations in cellular metabolism, one entailing a global metabolic paralysis, which was seen exclusively in HAMLET-treated cells, and the second, a shift in lipid metabolites, citric acid cycle constituents, and amino acids, seen both in HAMLET and oleate-treated cells but not in oleic acid-treated cells. HAMLET and oleate both increased fatty acid metabolism, consistent with lipid recognition and uptake by tumor cells, but oleic acid had no significant effect. The inertia of cells to the protonated acid was surprising but may indicate that the de-protonated fatty acid is the more active cellular agonist, as indicated by previous, cellular uptake studies (
      • Mashek D.G.
      • Coleman R.A.
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      ).
      Critical starting metabolites in the citric acid cycle were less abundant after HAMLET treatment, consistent with a loss of pyruvate and inhibition of glycolysis. Previously, HK1 and the glycolytic machinery have been shown to influence HAMLET sensitivity, and direct binding of HAMLET to the kinase has been detected (
      • Storm P.
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      • Urbano A.
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      • Sullivan N.L.
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      • Trulsson M.
      • Yang H.
      • Watson J.D.
      • Svanborg C.
      Conserved features of cancer cells define their sensitivity to HAMLET-induced death. c-Myc and glycolysis.
      ). In parallel, an increased rate of amino acid catabolism and a buildup of metabolites toward the end of the cycle were observed, further suggesting that HAMLET may force tumor cells to use glucose-independent metabolic pathways and amino acid catabolism as an energy source potentially compensating for the inhibition of glycolysis. Oleate alone seemed to direct metabolism toward amino acid catabolism, suggesting that the deprotonated lipid deregulates the TCA cycle but does not trigger death. Amino acid metabolism is important for cancer cell survival, and the use of amino acid metabolites for energy production has been proposed to offer a glucose-independent solution to ATP generation in tumor cells (
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      ).
      This study resolves for the first time the question of the lipid contribution to the tumoricidal activity of HAMLET. The results confirm that the lipid is a necessary constituent to achieve the tumoricidal effect of the complex (
      • Svensson M.
      • Håkansson A.
      • Mossberg A.K.
      • Linse S.
      • Svanborg C.
      Conversion of α-lactalbumin to a protein inducing apoptosis.
      ). This was previously inferred from the finding that partially unfolded α-lactalbumin protein alone does not trigger tumor cell death (
      • Svensson M.
      • Fast J.
      • Mossberg A.-K.
      • Düringer C.
      • Gustafsson L.
      • Hallgren O.
      • Brooks C.L.
      • Berliner L.
      • Linse S.
      • Svanborg C.
      α-Lactalbumin unfolding is not sufficient to cause apoptosis, but is required for the conversion to HAMLET (human α-lactalbumin made lethal to tumor cells).
      ,
      • Pettersson-Kastberg J.
      • Mossberg A.K.
      • Trulsson M.
      • Yong Y.J.
      • Min S.
      • Lim Y.
      • O'Brien J.E.
      • Svanborg C.
      • Mok K.H.
      α-Lactalbumin, engineered to be nonnative and inactive, kills tumor cells when in complex with oleic acid. A new biological function resulting from partial unfolding.
      ). On the other hand, oleic acid alone was largely inactive, presenting somewhat of a paradox. In this study the deprotonated lipid is shown to be a fully functional cellular agonist both alone and in the context of α-lactalbumin. The oleate effect was concentration-dependent, as higher oleate concentrations reproduced more of the cellular response to HAMLET. However, important qualitative differences were observed between the HAMLET complex and oleate alone at equimolar or higher concentrations. Although the effects on gene expression were overlapping, significant sets of genes were uniquely responsive to oleate or HAMLET, respectively. We conclude that partially unfolded α-lactalbumin is a suitable partner for oleate to offer tumor cells the “kiss of death.”

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