Ann-Kristin Mossberg

HAMLET kills tumor cells by an apoptosis-like mechanism—cellular, molecular, and therapeutic aspects

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a protein-lipid complex that induces apoptosis-like death in tumor cells, but leaves fully differentiated cells unaffected. This review summarizes the information on the in vivo effects of HAMLET in patients and tumor models on the tumor cell biology, and on the molecular characteristics of the complex. HAMLET limits the progression of human glioblastomas in a xenograft model and removes skin papillomas in patients. This broad anti-tumor activity includes >40 different lymphomas and carcinomas and apoptosis is independent of p53 or bcl-2. In tumor cells HAMLET enters the cytoplasm, translocates to the perinuclear area, and enters the nuclei where it accumulates. HAMLET binds strongly to histones and disrupts the chromatin organization. In the cytoplasm, HAMLET targets ribosomes and activates caspases. The formation of HAMLET relies on the propensity of alpha-lactalbumin to alter its conformation when the strongly bound Ca2+ ion is released and the protein adopts the apo-conformation that exposes a new fatty acid binding site. Oleic acid (C18:1,9 cis) fits this site with high specificity, and stabilizes the altered protein conformation. The results illustrate how protein folding variants may be beneficial, and how their formation in peripheral tissues may depend on the folding change and the availability of the lipid cofactor. One example is the acid pH in the stomach of the breast-fed child that promotes the formation of HAMLET. This mechanism may contribute to the protective effect of breastfeeding against childhood tumors. We propose that HAMLET should be explored as a novel approach to tumor therapy.

Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET) Kills Human Glioblastoma Cells in Brain Xenografts by an Apoptosis-Like Mechanism and Prolongs Survival

Malignant brain tumors present a major therapeutic challenge because no selective or efficient treatment is available. Here, we demonstrate that intratumoral administration of human α-lactalbumin made lethal to tumor cells (HAMLET) prolongs survival in a human glioblastoma (GBM) xenograft model, by selective induction of tumor cell apoptosis. HAMLET is a protein-lipid complex that is formed from α-lactalbumin when the protein changes its tertiary conformation and binds oleic acid as a cofactor. HAMLET induces apoptosis in a wide range of tumor cells in vitro, but the therapeutic effect in vivo has not been examined. In this study, invasively growing human GBM tumors were established in nude rats (Han:rnu/rnu Rowett, n = 20) by transplantation of human GBM biopsy spheroids. After 7 days, HAMLET was administered by intracerebral convection-enhanced delivery for 24 h into the tumor area; and α-lactalbumin, the native, folded variant of the same protein, was used as a control. HAMLET reduced the intracranial tumor volume and delayed the onset of pressure symptoms in the tumor-bearing rats. After 8 weeks, all α-lactalbumin-treated rats had developed pressure symptoms, but the HAMLET-treated rats remained asymptomatic. Magnetic resonance imaging scans revealed large differences in tumor volume (456 versus 63 mm3). HAMLET caused apoptosis in vivo in the tumor but not in adjacent intact brain tissue or in nontransformed human astrocytes, and no toxic side effects were observed. The results identify HAMLET as a new candidate in cancer therapy and suggest that HAMLET should be additionally explored as a novel approach to controlling GBM progression.

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)

HAMLET (human α‐lactalbumin made lethal to tumor cells) is a complex of human α‐lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis‐like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from α‐lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of α‐lactalbumin is sufficient to induce cell death. We used the bovine α‐lactalbumin Ca2+ site mutant D87A, which is unable to bind Ca2+, and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET‐like complex in the presence of oleic acid. BAMLET (bovine α‐lactalbumin made lethal to tumor cells) and D87A‐BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca2+site, as HAMLET maintained a high affinity for Ca2+ but D87A‐BAMLET was active with no Ca2+ bound. We conclude that partial unfolding of α‐lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca2+‐binding site is not required for conversion of α‐lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca2+site.

Lipids as cofactors in protein folding: Stereo-specific lipid-protein interactions are required to form HAMLET (human alpha-lactalbumin made lethal to tumor cells).

Proteins can adjust their structure and function in response to shifting environments. Functional diversity is created not only by the sequence but by changes in tertiary structure. Here we present evidence that lipid cofactors may enable otherwise unstable protein folding variants to maintain their conformation and to form novel, biologically active complexes. We have identified unsaturated C18 fatty acids in the cis conformation as the cofactors that bind apo α‐lactalbumin and form HAMLET (human α‐lactalbumin made lethal to tumor cells). The complexes were formed on an ion exchange column, were stable in a molten globule‐like conformation, and had attained the novel biological activity. The protein–fatty acid interaction was specific, as saturated C18 fatty acids, or unsaturated C18:1trans conformers were unable to form complexes with apo α‐lactalbumin, as were fatty acids with shorter or longer carbon chains. Unsaturated cis fatty acids other than C18:1:9cis were able to form stable complexes, but these were not active in the apoptosis assay. The results demonstrate that stereo‐specific lipid–protein interactions can stabilize partially unfolded conformations and form molecular complexes with novel biological activity. The results offer a new mechanism for the functional diversity of proteins, by exploiting lipids as essential, tissue‐specific cofactors in this process.

Bladder cancers respond to intravesical instillation of HAMLET (human alpha-lactalbumin made lethal to tumor cells).

We studied if bladder cancers respond to HAMLET (human α‐lactalbumin made lethal to tumor cells) to establish if intravesical HAMLET application might be used to selectively remove cancer cells in vivo. Patients with nonmuscle invasive transitional cell carcinomas were included. Nine patients received 5 daily intravesical instillations of HAMLET (25 mg/ml) during the week before scheduled surgery. HAMLET stimulated a rapid increase in the shedding of tumor cells into the urine, daily, during the 5 days of instillation. The effect was specific for HAMLET, as intravesical instillation of NaCl, PBS or native α‐lactalbumin did not increase cell shedding. Most of the shed cells were dead and an apoptotic response was detected in 6 of 9 patients, using the TUNEL assay. At surgery, morphological changes in the exophytic tumors were documented by endoscopic photography and a reduction in tumor size or change in tumor character was detected in 8 of 9 patients. TUNEL staining was positive in biopsies from the remaining tumor in 4 patients but adjacent healthy tissue showed no evidence of apoptosis and no toxic response. The results suggest that HAMLET exerts a direct and selective effect on bladder cancer tissue in vivo and that local HAMLET administration might be of value in the future treatment of bladder cancers.

Apoptosis-Like Death in Bacteria Induced by HAMLET, a Human Milk Lipid-Protein Complex

Background Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. Methodology/Principal Findings We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. Conclusions/Significance Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) triggers autophagic tumor cell death.

HAMLET, a complex of partially unfolded α‐lactalbumin and oleic acid, kills a wide range of tumor cells. Here we propose that HAMLET causes macroautophagy in tumor cells and that this contributes to their death. Cell death was accompanied by mitochondrial damage and a reduction in the level of active mTOR and HAMLET triggered extensive cytoplasmic vacuolization and the formation of double‐membrane‐enclosed vesicles typical of macroautophagy. In addition, HAMLET caused a change from uniform (LC3‐I) to granular (LC3‐II) staining in LC3‐GFP‐transfected cells reflecting LC3 translocation during macroautophagy, and this was blocked by the macroautophagy inhibitor 3‐methyladenine. HAMLET also caused accumulation of LC3‐II detected by Western blot when lysosomal degradation was inhibited suggesting that HAMLET caused an increase in autophagic flux. To determine if macroautophagy contributed to cell death, we used RNA interference against Beclin‐1 and Atg5. Suppression of Beclin‐1 and Atg5 improved the survival of HAMLET‐treated tumor cells and inhibited the increase in granular LC3‐GFP staining. The results show that HAMLET triggers macroautophagy in tumor cells and suggest that macroautophagy contributes to HAMLET‐induced tumor cell death.

Apoptosis and tumor cell death in response to HAMLET (human alpha-lactalbumin made lethal to tumor cells).

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a molecular complex derived from human milk that kills tumor cells by a process resembling programmed cell death. The complex consists of partially unfolded alpha-lactalbumin and oleic acid, and both the protein and the fatty acid are required for cell death. HAMLET has broad antitumor activity in vitro, and its therapeutic effect has been confirmed in vivo in a human glioblastoma rat xenograft model, in patients with skin papillomas and in patients with bladder cancer. The mechanisms of tumor cell death remain unclear, however. Immediately after the encounter with tumor cells, HAMLET invades the cells and causes mitochondrial membrane depolarization, cytochrome c release, phosphatidyl serine exposure, and a low caspase response. A fraction of the cells undergoes morphological changes characteristic of apoptosis, but caspase inhibition does not rescue the cells and Bcl-2 overexpression or altered p53 status does not influence the sensitivity of tumor cells to HAMLET. HAMLET also creates a state of unfolded protein overload and activates 20S proteasomes, which contributes to cell death. In parallel, HAMLET translocates to tumor cell nuclei, where high-affinity interactions with histones cause chromatin disruption, loss of transcription, and nuclear condensation. The dying cells also show morphological changes compatible with macroautophagy, and recent studies indicate that macroautophagy is involved in the cell death response to HAMLET. The results suggest that HAMLET, like a hydra with many heads, may interact with several crucial cellular organelles, thereby activating several forms of cell death, in parallel. This complexity might underlie the rapid death response of tumor cells and the broad antitumor activity of HAMLET.

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

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex consisting of partially unfolded protein and fatty acid and was first identified in casein fractions of human breast milk. The complex can be produced from its pure components through a modified chromatographic procedure where preapplied oleic acid binds with partially unfolded alpha-lactalbumin on the stationary phase in situ. Because native alpha-lactalbumin itself cannot trigger cell death, HAMLETs remarkable tumor-selective cytotoxicity has been strongly correlated with the conformational change of the protein upon forming the complex, but whether a recovery to the native state subsequently occurs upon entering the tumor cell is yet unclear. To this end, we utilize a recombinant variant of human alpha-lactalbumin in which all eight cysteine residues are substituted for alanines (rHLA(all-Ala)), rendering the protein nonnative and biologically inactive under all conditions. The HAMLET analogue formed from the complex of rHLA(all-Ala) and oleic acid (rHLA(all-Ala)-OA) exhibited equivalent strong tumoricidal activity against lymphoma and carcinoma cell lines and was shown to accumulate within the nuclei of tumor cells, thus reproducing the cellular trafficking pattern of HAMLET. In contrast, the fatty acid-free rHLA(all-Ala) protein associated with the tumor cell surface but was not internalized and lacked any cytotoxic activity. Structurally, whereas HAMLET exhibited some residual native character in terms of NMR chemical shift dispersion, rHLA(all-Ala)-OA showed significant differences to HAMLET and, in fact, was found to be devoid of any tertiary packing. The results identify alpha-lactalbumin as a protein with strikingly different functions in the native and partially unfolded states. We posit that partial unfolding offers another significant route of functional diversification for proteins within the cell.

HAMLET interacts with lipid membranes and perturbs their structure and integrity.

Background Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure. Methodology/Principal Findings We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLAall-Ala). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles. Conclusions/Significance The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death.