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This article discusses the Israeli press reviews of Konrad Swinarski’s Hamlet, which premiered at the Cameri Theater in Tel Aviv in 1966. Based on material previously unknown in Poland, the analysis makes it possible not only to reconstruct some of the stage actions and effects, but also to follow the reasoning, competences and tastes of Israeli critics. Comparing this material with documents concerning the preparations for the Kraków staging of Hamlet eight years later, the author concludes that the general framework of Swinarski’s conceptualization of the play was already developed in 1966; the director managed to bring some of its aspects to the stage in Tel Aviv. Most importantly, he created a new vision of the characters of Hamlet and Horace, re-examined the image of Claudius, and outlined a historiosophical and political interpretation of Shakespeare’s tragedy. In Israel, the critics usually recognized the ideological message of Swinarski’s staging correctly; however, they questioned its point and denied its value.

HAMLET is a protein‐lipid complex with a specific and broad bactericidal and tumoricidal activity, that lacks cytotoxic activity against healthy cells. In this study, we show that HAMLET also has general immune‐stimulatory effects on primary human monocyte‐derived dendritic cells and macrophages (Mo‐DC and Mo‐M) and murine RAW264.7 macrophages. HAMLET, but not its components alpha‐lactalbumin or oleic acid, induces mature CD14low/–CD83+ Mo‐DC and M1‐like CD14+CD86++ Mo‐M surface phenotypes. Concomitantly, inflammatory mediators, including IL‐2, IL‐6, IL‐10, IL‐12 and MIP‐1α, were released in the supernatant of HAMLET‐stimulated cells, indicating a mainly pro‐inflammatory phenotype. The HAMLET‐induced phenotype was mediated by calcium, NFκB and p38 MAPK signaling in Mo‐DCs and calcium, NFκB and ERK signaling in Mo‐M as inhibitors of these pathways almost completely blocked the induction of mature Mo‐DCs and M1‐like Mo‐M. Compared to unstimulated Mo‐DCs, HAMLET‐stimulated Mo‐DCs were more potent in inducing T cell proliferation and HAMLET‐stimulated macrophages were more efficient in phagocytosis of Streptococcus pneumoniae in vitro. This indicates a functionally activated phenotype of HAMLET‐stimulated DCs and macrophages. Combined, we propose that HAMLET has a two‐fold anti‐bacterial activity; one inducing direct cytotoxic activity, the other indirectly mediating elimination of bacteria by activation of immune cells of the myeloid lineage., HAMLET, a human milk protein‐lipid complex induces a pro‐inflammatory phenotype of macrophages and dendritic cells, resulting in augmented release of inflammatory mediators, stimulation of T‐cell proliferation and phagocytosis of pneumococci. We propose that HAMLET's anti‐bacterial activity is two‐fold; one directly bactericidal and the other mediated by activation of myeloid cells.

Accurately orchestrated course of events normally observed in healing are not followed in diabetic wounds, and bacterial colonization/infection further messes up the process. Novel therapeutic options for treatment of infections caused by multidrug-resistant Staphylococcus aureus are urgently needed. HAMLET (human α-lactalbumin made lethal to tumor cells) has been reported to be able to sensitize bacterial pathogens to traditional antimicrobial agents. The aim was to assess the wound healing activity of curcumin nanoparticles in diabetic wounds infected with methicillin-resistant Staphylococcus aureus (MRSA) sensitized with HAMLET. Fifty male rats were randomized into 5 groups of 10 animals each. In CONTROL group, 0.1-mL sterile saline 0.9% solution was added to the wounds with no infection. In MRSA group, the wounds were infected with MRSA and only treated with 0.1-mL sterile saline 0.9% solution. In MRSA/HAMLET group, infected wounds were treated with HAMLET (100 µg). In MRSA/CNP group, animals with infected wounds were treated with 0.1 mL topical application of 1 mg/mL curcumin nanoparticles. In MRSA/CNP/HAMLET group, animals with infected wounds were treated with topical application of 0.1 mL solution of curcumin nanoparticles (1 mg/mL) and HAMLET (100 µg). All test formulations were applied for 10 days, twice a day, starting from first treatment. Microbiological examination; planimetric, biochemical, histological, and quantitative morphometric studies; immunohistochemical staining for angiogenesis; determination of hydroxyproline levels; and reverse transcription polymerase chain reaction for caspase 3, Bcl-2, and p53 showed that there was significant difference between animals in MRSA/CNP/HAMLET group compared with other groups ( P < .05). Curcumin nanoparticles improved diabetic wounds infected with MRSA sensitized with HAMLET and had the potential to offer more attention to this safer agent for topical use in infected diabetic wounds.

The present work compares two literary figures, imagining the internal conflict of each, in connection to a secret. In Hamlet’s case (Hamlet by W. Shakespeare), it is a traumatic impossibility to reveal a secret that leads to his death, while for Gregers (H. Ibsen’s The Wild Duck), the opposite occurs, his inability to keep a secret leads directly to the death of another character. In this work the secret is viewed as the conscious concealment of content, while the motives for keeping or disclosing it at any cost are unconscious.

Frozen storage is necessary to preserve expressed human milk for critically ill and very preterm infants. Milk pasteurization is essential for donor milk given to this special population. Due to these storage and processing conditions, subtle changes occur in milk nutrients. These changes may have clinical implications. Potentially, bioactive complexes of unknown significance could be found in human milk given to preterm infants. One such complex, a cytotoxic α-lactalbumin-oleic acid complex named “HAMLET,” (Human Alpha-Lactalbumin Made Lethal to Tumor cells) is a folding variant of alpha-lactalbumin that is bound to oleic acid. This complex, isolated from human milk casein, has specific toxicity to both carcinogenic cell lines and immature non-transformed cells. Both HAMLET and free oleic acid trigger similar apoptotic mechanisms in tissue and stimulate inflammation via the NF-κB and MAPK p38 signaling pathways. This protein-lipid complex could potentially trigger various inflammatory pathways with unknown consequences, especially in immature intestinal tissues. The very preterm population is dependent on human milk as a medicinal and broadly bioactive nutriment. Therefore, HAMLET’s possible presence and bioactive role in milk should be addressed in neonatal research. Through a pediatric lens, HAMLET’s discovery, formation and bioactive benefits will be reviewed.

The protein-lipid complex alpha1-oleate, derived from HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), is identified as a molecular entity with significant therapeutic potential. Structural characterization of the complex and results of a successful placebo-controlled clinical trial are presented.

HAMLET is a complex of human α-lactalbumin (ALA) and oleic acid and kills several Gram-positive bacteria by a mechanism that bears resemblance to apoptosis in eukaryotic cells. To identify HAMLET's bacterial targets, here we used Streptococcus pneumoniae as a model organism and employed a proteomic approach that identified several potential candidates. Two of these targets were the glycolytic enzymes fructose bisphosphate aldolase (FBPA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Treatment of pneumococci with HAMLET immediately inhibited their ATP and lactate production, suggesting that HAMLET inhibits glycolysis. This observation was supported by experiments with recombinant bacterial enzymes, along with biochemical and bacterial viability assays, indicating that HAMLET's activity is partially inhibited by high glucose-mediated stimulation of glycolysis but enhanced in the presence of the glycolysis inhibitor 2-deoxyglucose. Both HAMLET and ALA bound directly to each glycolytic enzyme in solution and solid-phase assays and effectively inhibited their enzymatic activities. In contrast, oleic acid alone had little to no inhibitory activity. However, ALA alone also exhibited no bactericidal activity and did not block glycolysis in whole cells, suggesting a role for the lipid moiety in the internalization of HAMLET into the bacterial cells to reach its target(s). This was verified by inhibition of enzyme activity in whole cells after HAMLET but not ALA exposure. The results of this study suggest that part of HAMLET's antibacterial activity relates to its ability to target and inhibit glycolytic enzymes, providing an example of a natural antimicrobial agent that specifically targets glycolysis.

Milk contains essential nutrients and functional compounds, such as calcium, fat-soluble vitamins A, D, E, and K, carotenoids, bioactive peptides, and sphingolipids. The bioactive molecules from milk are not expensive and have an added advantage of being derived from food. Therefore, they are more stable and have a broader spectrum than that of other chemicals. Bioactive milk components are useful for treating non-digestive tract disorders, such as cancer, cognitive decline, and hypertension. However, the clinical application of certain breast milk ingredients is limited due to the lack of a large-scale production technology. Once the scaled-up production of lactoferrin became possible, clinical applications were devised and evaluated. Similarly, human alpha-lactalbumin made lethal to tumor cells (HAMLET) can be produced on a large scale as a recombinant protein in microorganisms or in transgenic cattle using suitable separation systems. HAMLET can be used to treat human skin papilloma and cancer. Studies on breast milk that explored the clinical applications of the bioactive components of breast milk have spurred the development of translational medicine and breast milk-derived therapeutics. Some breastmilk derived therapeutic agents are already available to clinicians. Many components of breast milk have shown efficacy in pre-clinical studies and have valid clinical evaluations.

The case for Q1 Hamlet as a pre-Q2 text is coming into focus as the findings of several scholars are reconciled. Additionally, a finding related to the Brudermord text has helped mark that text as a predecessor to both Q1 and Q2/F, with further implications for Q1 as a pre-Q2 text. As this view of Q1 becomes accepted, patterns of Q1 vs. Q2 variance advance the case for Q1 as being the author’s draft of the text that became the censor-approved ‘allowed book’. There is no way to know how most of the Q1 vs. Q2 textual variants progressed from Q1 as printed, to the non-extant allowed book from which the players’ parts were copied, and finally to Q2. However, the 577 Q1 lines that are identifiably concordant but variant to lines in Q2 represent a category of Q1 lines that will be of interest to those planning to edit or stage Q1 Hamlet.

As chaperones, heat shock proteins (HSPs) protect host cells against misfolded proteins that constitute a by-product of protein synthesis. Certain HSPs are also expressed on the surface of tumor cells, possibly to scavenge extracellular unfolded protein ligands and prevent them from becoming cytotoxic. HAMLET-a complex of partially unfolded alpha-lactalbumin and oleic acid-is relying on its N-terminal alpha-helical domain to perturb tumor cell membranes, and the cells die as a consequence of this interaction. Here we show that in parallel, cell surface HSPs bind the beta-sheet domain of alpha-lactalbumin and activate a temporarily protective loop, involving vesicular uptake and lysosomal accumulation. Later, HAMLET destroys lysosomal membrane integrity, and HAMLET release kills the remaining tumor cells. HSPs were identified as HAMLET targets in a proteomic screen and Hsp70-specific antibodies or shRNAs inhibited HAMLET uptake by tumor cells, which showed increased Hsp70 surface expression compared to differentiated cells. The results suggest that HAMLET engages tumor cells by two parallel recognition mechanisms, defined by alpha-helical- or beta-sheet domains of alpha-lactalbumin and resulting in an immediate death response, or a delay due to transient accumulation of the complex in the lysosomes. This dual response pattern was conserved among tumor cells but not seen in normal, differentiated cells. By two different mechanisms, HAMLET thus achieves a remarkably efficient elimination of tumor cells.

Partially unfolded alpha-lactalbumin forms the oleic acid complex HAMLET, with potent tumoricidal activity. Here we define a peptide-based molecular approach for targeting and killing tumor cells, and evidence of its clinical potential (ClinicalTrials.gov NCT03560479). A 39-residue alpha-helical peptide from alpha-lactalbumin is shown to gain lethality for tumor cells by forming oleic acid complexes (alpha1-oleate). Nuclear magnetic resonance measurements and computational simulations reveal a lipid core surrounded by conformationally fluid, alpha-helical peptide motifs. In a single center, placebo controlled, double blinded Phase I/II interventional clinical trial of non-muscle invasive bladder cancer, all primary end points of safety and efficacy of alpha1-oleate treatment are reached, as evaluated in an interim analysis. Intra-vesical instillations of alpha1-oleate triggers massive shedding of tumor cells and the tumor size is reduced but no drug-related side effects are detected (primary endpoints). Shed cells contain alpha1-oleate, treated tumors show evidence of apoptosis and the expression of cancer-related genes is inhibited (secondary endpoints). The results are especially encouraging for bladder cancer, where therapeutic failures and high recurrence rates create a great, unmet medical need., Partially unfolded alpha-lactalbumin forms an oleic acid complex with antitumorigenic properties. Here, the authors define a structurally flexible, peptide-based oleate complex and report a phase I/II clinical trial where this complex is used to treat patients with bladder cancer.

Here, we apply recently developed high-precision K isotope analyses to individual components of the LL4 chondrite Hamlet in order to investigate key processes which occurred during chondrite formation. The K isotopic compositions of all Hamlet chondrules range from −1.36‰ to −0.24‰ δ41K while the matrix and bulk samples show ranges of −0.89‰ to −0.80‰ and −0.86‰ to −1.08‰ δ41K, respectively. This range of δ41K values is significantly less than what was seen by in situ K isotopic analysis of Semarkona and Bishunpur chondrules, a likely effect of the different chondrite petrologic types, analytical artifacts in the SIMS analyses, and chondrule rim effects. Strong evidence for secondary parent-body alteration effects within Hamlet suggests its K fractionation and distribution are dominantly controlled by these processes. Interestingly, the strong correlation between δ41K and chondrule mass suggests that chondrule size played a significant role in the K isotopic distribution within Hamlet. This trend is likely a result of either inherited initial differences in the chondrule K isotopic ratios which were not completely overprinted or mechanisms involved in the metamorphism processes creating variations. This K isotope correlation with chondrule mass could also be suggestive of chondrule-forming nebular processes; nevertheless, it is currently unable to definitively favor any specific model. The K isotopic similarities between Hamlet and bulk ordinary chondrites suggest that all LL chondrites, if not all ordinary chondrites, may have formed via the same processes. Nevertheless, analysis of more pristine chondrules from chondrites of lower metamorphic grade is required to further assess any nebular processes of chondrule formation.

This introductory chapter argues that William Shakespeare's Hamlet can be read as a profound meditation on the nature of human individuality without relying on conceptual frameworks drawn from the eighteenth, nineteenth, twentieth, and twenty-first centuries. Just as historical discourses beyond those of Hamlet itself provide a fuller and more nuanced understanding of the political and dynastic forces shaping life in Shakespeare's Denmark, so what might be called Hamlet's “character” appears in unfamiliar and revealing relief when read against the textual contours of the psychological, rhetorical, and moral-political theorizing that lay at the heart of sixteenth-century humanism. This approach not only better locates Hamlet within Hamlet, but that it offers to rehabilitate a coherent and intensely challenging work of tragedy—albeit one in which Shakespeare steadfastly disregards the rules of Aristotelian and humanist poetics. What sets Hamlet apart from the remainder of the dramatis personae is the degree to which Shakespeare explores through him the insight that the insufficiency of received ethical and political wisdom does not just have public consequences. Transposed onto the person of Hamlet, it calls into question the fundamentals of who and what a human individual might be said to be.

Liprotides are complexes composed of partially denatured proteins and fatty acids in which the fatty acids form a micelle-like core surrounded by a shell of proteins. Liprotides, composed of α-lactalbumin (aLA) and oleic acid (OA), are similar in components and cytotoxicity to the original HAMLET protein-fatty acid complex. Liprotides composed of aLA and OA kill tumor cells by transferring the OA component to, and thus destabilizing, the cell membrane. Here we investigate liprotides' dynamics of transfer of contents between themselves and membranes using the hydrophobic fluorescent probe pyrene. We find that pyrene incorporated into liprotides is exchanged between liprotides within the dead time of a stopped-flow instrument, while the transfer to membranes occurs within 20 s. Transfer kinetics was not affected by the presence of the membrane stabilizing lipid cholesterol. Thus, transfer is a remarkably rapid process which illustrates liprotides' efficacy as transporters of hydrophobic compounds.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a protein-lipid complex derived from human milk that was first described for its tumoricidal activity. Later studies showed that HAMLET also has direct bactericidal activity against select species of bacteria, with highest activity against Streptococcus pneumoniae. Additionally, HAMLET in combination with various antimicrobial agents can make a broad range of antibiotic-resistant bacterial species sensitive to antibiotics. Here, we show that HAMLET has direct antibacterial activity not only against pneumococci but also against Streptococcus pyogenes (group A streptococci [GAS]) and Streptococcus agalactiae (group B streptococci [GBS]). As with pneumococci, HAMLET treatment of GAS and GBS resulted in depolarization of the bacterial membrane, followed by membrane permeabilization and death, which was able to be inhibited by calcium and sodium transport inhibitors. Treatment of clinical antibiotic-resistant isolates of S. pneumoniae, GAS, and GBS with sublethal concentrations of HAMLET in combination with antibiotics decreased the MICs of the antibiotics into the sensitive range. This effect could also be blocked by ion transport inhibitors, suggesting that HAMLET’s bactericidal and combination treatment effects used similar mechanisms. Finally, we show that HAMLET potentiated the effects of erythromycin against erythromycin-resistant bacteria more effectively than penicillin G potentiated killing bacteria resistant to erythromycin. These results show that HAMLET effectively (i) kills three different species of pathogenic streptococci by similar mechanisms and also (ii) potentiates the activities of macrolides and lincosamides more effectively than combination treatment with beta-lactams. These findings suggest a potential therapeutic role for HAMLET in repurposing antibiotics currently causing treatment failures in patients.

Some natural proteins can be complexed with oleic acid (OA) to form an active protein–lipid formulation that can induce tumor-selective apoptosis. The first explored protein was human milk α-lactalbumin (α-LA), called HAMLET when composed with OA in antitumor form. Several groups have prepared active protein–lipid complexes using a variety of approaches, all of which depend on target protein destabilization or direct OA–protein incubation to alter pH to acid or alkaline condition. In addition to performing vital roles in inflammatory processes and immune responses, fatty acids can disturb different metabolic pathways and cellular signals. Therefore, the tumoricidal action of these complexes is related to OA rather than the protein that keeps OA in solution and acts as a vehicle for transferring OA molecules to tumor cells. However, other studies have suggested that the antitumor efficacy of these complexes was exerted by both protein and OA together. The potential is not limited to the anti-tumor activity of protein–lipid complexes but extends to other functions such as bactericidal activity. The protein shell enhances the solubility and stability of the bound fatty acid. These protein–lipid complexes are promising candidates for fighting various cancer types and managing bacterial and viral infections.

Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), has surpassed HIV/AIDS as the leading cause of death from a single infectious agent. The increasing occurrence of drug-resistant strains has become a major challenge for health care systems and, in some cases, has rendered TB untreatable. However, the development of new TB drugs has been plagued with high failure rates and costs. Alternative strategies to increase the efficacy of current TB treatment regimens include host-directed therapies or agents that make M. tuberculosis more susceptible to existing TB drugs. In this study, we show that HAMLET, an α-lactalbumin-oleic acid complex derived from human milk, has bactericidal activity against M. tuberculosis HAMLET consists of a micellar oleic acid core surrounded by a shell of partially denatured α-lactalbumin molecules and unloads oleic acid into cells upon contact with lipid membranes. At sublethal concentrations, HAMLET potentiated a remarkably broad array of TB drugs and antibiotics against M. tuberculosis For example, the minimal inhibitory concentrations of rifampin, bedaquiline, delamanid, and clarithromycin were decreased by 8- to 16-fold. HAMLET also killed M. tuberculosis and enhanced the efficacy of TB drugs inside macrophages, a natural habitat of M. tuberculosis Previous studies showed that HAMLET is stable after oral delivery in mice and nontoxic in humans and that it is possible to package hydrophobic compounds in the oleic acid core of HAMLET to increase their solubility and metabolic stability. The potential of HAMLET and other liprotides as drug delivery and sensitization agents in TB chemotherapy is discussed here.

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cell), a molecular complex of human α-lactalbumin and oleic acid, is known to have selective cytotoxic activity against certain types of tumors. This cytotoxicity is known to stem from water-insoluble oleic acid. In this study, we manufactured an alternative complex using liposome as an oleic acid delivery vesicle. We named this nanolipoplex LIMLET (LIposome Made LEthal to Tumor cell). The LIMLET vesicle contained approximately 90,200 oleic acid molecules inserted into its lipophilic phospholipid bilayer and had a nominal mean diameter of 127nm. Using a WST-1 assay, its cytotoxicity against two cancer cell lines, MDA-MB-231 (human breast cancer) and A549 (human lung cancer), were tested. The results were compared with that of a normal cell line, Vero (from monkey kidney). We found that (1) LIMLET showed distinctive cytotoxicity against A549 and MDA-MB-231 cells, whereas bare liposomes (containing no oleic acid) had no toxicity, even at high concentrations, and (2) LIMLET demonstrated selective, concentration-dependent toxicity against the cancer cells: the LD50 values of MDA-MB-231 and A549 cells were 1.3 and 2.2nM LIMLET, respectively, whereas the LD50 of Vero was 5.7nM. The strength of the tumoricidal effect appeared to stem from the number of oleic acid molecules present. Our result suggests that LIMLET, like HAMLET, is an interesting nanolipoplex that can potentially be developed into tumor treatments.

Interaction of a HAMLET-like La–OA cytotoxic complex (human α-lactalbumin-oleic acid) and its constituents with the excitable plasmalemma of giant Chara corallina cells was investigated. The voltage–clamp technique was used to study Ca2+ and Cl− transient currents in the plasmalemma of intact cells. The action of the complex and OA on the target cell membrane has a dose-dependent character. It was found that the La–OA complex has an inhibiting effect on Ca2+ current across the plasmalemma, while α-lactalbumin alone does not affect the electrophysiological characteristics of the cellular membrane. However, oleic acid blocks Ca2+ current across the plasmalemma. This is accompanied by the induction of a non-selective conductivity in the cellular membrane, a decrease in the resting potential and plasma membrane resistance of algal cells. We propose that the cytotoxicity of La–OA and other HAMLET-like complexes is determined by oleic acid acting as a blocker of potential-dependent Ca2+ channels in the plasma membrane of target cells. The presented results show that the study model of green algae C. corallina cells plasmalemma is a convenient tool for the investigation of ion channels in many animal cells.

Oleic acid (OA) complexes of human alpha-lactalbumin (α-LA) and several other proteins are effective in the killing of a variety of tumor cells. While debate on whether the key component of the complexes is the OA or protein continues, studies probing the mechanism of action of the complexes at the tumor cell surface or in the cell interior assume the action of a molecule in the form of an undissociated complex. Recent evidence however suggests that OA complexes of protein are stripped of bound OA on interaction with artificial or natural membranes before entering the cell. Properties of BAMLET stripped of its OA by exposure to erythrocytes (ET-BAMLET) were investigated in the study. ET-BAMLET resembled α-LA in its inability to induce hemolysis of erythrocytes and behaviour in a gel filtration column. Spectroscopy techniques-fluorescence, far- and near UV CD as well as calorimetry and proteolysis however suggest the molecule to be different both from native α-LA and the apo form. Remarkably, unlike native α-LA and apo-α-LA, ET-BAMLET binds OA and turns hemolytic by simple mixing with the fatty acid around neutral pH. Since BAMLET/HAMLET incubated cells take up large amounts of OA, the study suggests the possibility of ET-BAMLET combining with OA and reforming the complex inside the cells.

Human α-lactalbumin made lethal to tumor cells (HAMLET) is a tumoricidal complex consisting of human α-lactalbumin and multiple oleic acids (OAs). OA has been shown to play a key role in the activity of HAMLET and its related complexes, generally known as protein–fatty acid (PFA) complexes. In contrast to what is known about the fate of the protein component of such complexes, information about what happens to OA during their action is still lacking. We monitored the membrane, OA and protein components of bovine α-lactalbumin complexed with OA (BLAOA; a HAMLET-like substance) and how they associate with each other. Using ultracentrifugation, we found that the OA and lipid components follow each other closely. We then firmly identify a transfer of OA from BLAOA to both artificial and erythrocyte membranes, indicating that natural cells respond similarly to BLAOA treatment as artificial membranes. Uncomplexed OA is unable to similarly affect membranes at the conditions tested, even at elevated concentrations. Thus, BLAOA can spontaneously transfer OA to a lipid membrane. After the interaction with the membrane, the protein is likely to have lost most or all of its OA. We suggest a mechanism for passive import of mainly uncomplexed protein into cells, using existing models for OA's effect on membranes. Our results are consistent with a membrane destabilization mediated predominantly by OA insertion being a significant contribution to PFA cytotoxicity. publishedVersion

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted.

HAMLET (human α-lactalbumin made lethal to tumour cells) is a complex of α-lactalbumin (aLA) and oleic acid (OA) which kills transformed cells, while leaving fully differentiated cells largely unaffected. Other protein-lipid complexes show similar anti-cancer potential. We call such complexes liprotides. The cellular impact of liprotides, while intensely investigated, remains unresolved. To address this, we report on the cell-killing mechanisms of liprotides prepared by incubating aLA with OA for 1 h at 20 or 80 °C (lip20 and lip80, respectively). The liprotides showed similar cytotoxicity against MCF7 cells, though lip80 acts more slowly, possibly due to intermolecular disulphide bonds formed during preparation. Liprotides are known to increase the fluidity of a membrane and transfer OA to vesicles, prompting us to focus on the effect of liprotides on the cell membrane. Extracellular Ca2+ influx is important for activation of the plasma membrane repair system, and we found that removal of Ca2+ from the medium enhanced the liprotides’ killing effect. Liprotide cytotoxicity was also increased by knockdown of Annexin A6 (ANXA6), a protein involved in plasma membrane repair. We conclude that MCF7 cells counteract liprotide-induced membrane permeabilization by activating their plasma membrane repair system, which is triggered by extracellular Ca2+ and involves ANXA6.

HAMLET (Human Alpha-Lactalbumin Made LEthal to Tumor cells), a complex of oleic acid (OA) with partially unfolded human α-lactalbumin, shows remarkable toxicity towards a spectrum of tumor cells as well as few differentiated cells including mammalian erythrocytes. Human erythrocytes, for this reason, have been used as convenient model cells to study toxic properties of the OA complexes. The toxicity of HAMLET-like complexes, prepared using immunoglobulin gamma (IgG) isolated from the sera of rabbits immunized with human erythrocytes as well as those unimmunized, towards the red cells was investigated. The OA complex of the IgG prepared by the heat-treatment procedure comprised of protein monomers and oligomers with bound OA. The IgG in the complexes retained most secondary but only partial tertiary structure and complex formation with OA did not abolish the ability of anti-erythrocyte IgG to bind to the erythrocytes. Anti-erythrocyte IgG-OA complexes were remarkably more hemolytic than those prepared using non-specific IgG, while complexes prepared using affinity purified anti-erythrocyte IgG were most effective in hemolyzing the cells. The work suggests that antibodies that exhibit affinity towards target cells may be useful in the preparation of selective and highly toxic OA complexes for the cells.

SummaryMembers of the SWI/SNF chromatin-remodeling complex are among the most frequently mutated genes in human cancer, but how they suppress tumorigenesis is currently unclear. Here, we use Drosophila neuroblasts to demonstrate that the SWI/SNF component Osa (ARID1) prevents tumorigenesis by ensuring correct lineage progression in stem cell lineages. We show that Osa induces a transcriptional program in the transit-amplifying population that initiates temporal patterning, limits self-renewal, and prevents dedifferentiation. We identify the Prdm protein Hamlet as a key component of this program. Hamlet is directly induced by Osa and regulates the progression of progenitors through distinct transcriptional states to limit the number of transit-amplifying divisions. Our data provide a mechanistic explanation for the widespread tumor suppressor activity of SWI/SNF. Because the Hamlet homologs Evi1 and Prdm16 are frequently mutated in cancer, this mechanism could well be conserved in human stem cell lineages.PaperClip

α-Lactalbumin (α-la) is a major whey protein found in milk. Previous data suggested that α-la has antiproliferative effects in human adenocarcinoma cell lines such as Caco-2 and HT-29. However, the cell death inducing α-la was not a naturally occurring monomer but either a multimeric variant or an α-la:oleic acid complex (HAMLET/BAMLET). Proteolysis showed that both human and bovine α-la are susceptible to digestion. ELISA assays assessing cell death with the native undigested α-la fractions showed that undigested protein fractions did have a significant cell death effect on CaCo-2 cells. Bovine α-la was also more effective than human α-la. A reduction in activity corresponded with lower concentrations of the protein and partial digestion and fragmentation of the protein using trypsin and pepsin. This suggests that the tertiary structure is vital for the apoptotic effect.

HAMLET is a complex of human α-lactalbumin (hLA) with oleic acid (OA) that kills various tumor cells and strains of Streptococcus pneumoniae. More potent protein-OA complexes were previously reported for bovine α-lactalbumin (bLA) and β-lactoglobulin (bLG), and pike parvalbumin (pPA), and here we explore their structural features. The concentration dependencies of the tryptophan fluorescence of hLA, bLA, and bLG complexes with OA reveal their disintegration at protein concentrations below the micromolar level. Chemical cross-linking experiments provide evidence that association with OA shifts the distribution of oligomeric forms of hLA, bLA, bLG, and pPA toward higher-order oligomers. This effect is confirmed for bLA and bLG using the dynamic light scattering method, while pPA is shown to associate with OA vesicles. Like hLA binding, OA binding increases the affinity of bLG for small unilamellar dipalmitoylphosphatidylcholine vesicles, while pPA efficiently binds to the vesicles irrespective of OA binding. The association of OA with bLG and pPA increases their α-helix and cross-β-sheet content and resistance to enzymatic proteolysis, which is indicative of OA-induced protein structuring. The lack of excess heat sorption during melting of bLG and pPA in complex with OA and the presence of a cooperative thermal transition at the level of their secondary structure suggest that the OA-bound forms of bLG and pPA lack a fixed tertiary structure but exhibit a continuous thermal transition. Overall, despite marked differences, the HAMLET-like complexes that were studied exhibit a common feature: a tendency toward protein oligomerization. Because OA-induced oligomerization has been reported for other proteins, this phenomenon is inherent to many proteins.

A complex formed by human α-lactalbumin (α-LA) and oleic acid (OA), named HAMLET, has been shown to have an apoptotic activity leading to the selective death of tumor cells. In numerous publications it has been reported that in the complex α-LA is monomeric and adopts a partly folded or “molten globule” state, leading to the idea that partly folded proteins can have “beneficial effects”. The protein/OA molar ratio initially has been reported to be 1:1, while recent data have indicated that the OA-complex is given by an oligomeric protein capable of binding numerous OA molecules per protein monomer. Proteolytic fragments of α-LA, as well as other proteins unrelated to α-LA, can form OA-complexes with biological activities similar to those of HAMLET, thus indicating that a generic protein can form a cytotoxic complex under suitable experimental conditions. Moreover, even the selective tumoricidal activity of HAMLET-like complexes has been questioned. There is recent evidence that the biological activity of long chain unsaturated fatty acids, including OA, can be ascribed to their effect of perturbing the structure of biological membranes and consequently the function of membrane-bound proteins. In general, it has been observed that the cytotoxic effects exerted by HAMLET-like complexes are similar to those reported for OA alone. Overall, these findings can be interpreted by considering that the protein moiety does not have a toxic effect on its own, but merely acts as a solubilising agent for the inherently toxic fatty acid.

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 (13)C 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.

Although HAMLET (human α-lactalbumin made lethal to tumor cells), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat α-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50 °C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin, and β2-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane.

Extracellular application of 5–30 μM oleic acid (C18:1:9cis) produced a significant decrease in the amplitude of Ca2+ currents and Ca2+-activated chloride currents in plasma membrane of green alga Chara corallina. Introduction of 10 μM oleic acid intracellularly suppressed the development of the Ca2+ current. This inhibition of the Ca2+ current was reversible, and the Ca2+ conductivity of the cell membrane was restored after the removal of oleic acid. There were no changes in the non-specific leakage current, which indicates the intactness of the lipid component of the membrane. An increase of the oleic acid concentration up to 50–100 μM in the extracellular solution induced almost complete and irreversible suppression of inward currents and caused a substantial increase in the non-specific leakage current and conductivity of plasma membrane, suggesting its disruption. We suggest that the mechanism of cytotoxicity of oleic acid may be related to transport across plasma membrane of oleic acid and its complexes with α-lactalbumin (HAMLET and La-OA).