Lotta Gustafsson

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.

Ceramide as a TLR4 agonist; a putative signalling intermediate between sphingolipid receptors for microbial ligands and TLR4

Mucosal Toll‐like receptors (TLRs) respond to pathogens, but remain inert to the indigenous flora, suggesting that the TLRs can receive pathogen‐specific signals. For example, TLR4 signalling is activated in CD14‐negative epithelial cells by P‐fimbriated, uropathogenic Escherichia coli, but not by lipopolysaccharide. The fimbriae use glycosphingolipids as recognition receptors and there is release of ceramide, which is the membrane‐anchoring domain of the receptors. In this study, ceramide was identified as a TLR4 agonist and as a putative signalling intermediate between the glycosphingolipid recognition receptors and TLR4. Exogenous ceramide activated a TLR4‐dependent epithelial cell response, as shown by exposing stably transfected TLR4‐positive or ‐negative human embryonal kidney cells to C2 and C6 ceramide. A similar, TLR4‐dependent response occurred after deliberate release of endogenous long‐chained ceramide with sphingomyelinase. Microbial ligands with glycosphingolipid specificity (P fimbriae or the B subunit of Shiga toxin) were shown to increase the levels of ceramide and to trigger a TLR4‐dependent response in epithelial cells. The results show that ceramide activates TLR4 signalling and suggest that this mechanism might allow pathogens to elicit mucosal TLR4 responses by perturbing sphingolipid receptors for virulence ligands like P fimbriae.

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.

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.

Micro- and macromechanical effects on the wound bed of negative pressure wound therapy using gauze and foam.

Negative pressure wound therapy (NPWT) results in 2 types of tissue deformation, macrodeformation (ie, wound contraction) and microdeformation (ie, the interaction of tissue and dressing on a microscopic level). These effects have been delineated for one type of wound filler, foam, but not for gauze. The mechanical deformation initiates a signaling cascade which ultimately leads to wound healing. The aim of the present study was to examine the effect of gauze and foam on macro- and microdeformation during treatment with negative pressure. An in vivo porcine peripheral wound model was used. NPWT was applied for 72 hours at 0, −75, and −125 mm Hg, using either foam or gauze as wound filler. The mechanical effects of NPWT were examined by measuring the wound surface area reduction and by histologic analysis of the wound bed tissue. Similar degrees of wound contraction (macrodeformation) were seen during NPWT regardless if foam or gauze was used. After negative pressure had been discontinued, the wound stayed contracted. There was no difference in wound contraction between −75 and −125 mm Hg. Biopsies of the wound bed revealed a repeating pattern of wound surface undulations and small tissue blebs (“tissue mushrooms”) were pulled into the pores of the foam dressing and the spaces between the threads in the gauze dressing (microdeformation). This pattern was obvious in wounds treated both with foam and gauze, at atmospheric pressure (0 mm Hg) as well as at subatmospheric pressures (−75 and −125 mm Hg). The degrees of micro- and macrodeformation of the wound bed are similar after NPWT regardless if foam or gauze is used as wound filler.

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.

HAMLET triggers apoptosis but tumor cell death is independent of caspases, Bcl-2 and p53

HAMLET (Human α-lactalbumin Made Lethal to Tumor cells) triggers selective tumor cell death in vitro and limits tumor progression in vivo. Dying cells show features of apoptosis but it is not clear if the apoptotic response explains tumor cell death. This study examined the contribution of apoptosis to cell death in response to HAMLET. Apoptotic changes like caspase activation, phosphatidyl serine externalization, chromatin condensation were detected in HAMLET-treated tumor cells, but caspase inhibition or Bcl-2 over-expression did not prolong cell survival and the caspase response was Bcl-2 independent. HAMLET translocates to the nuclei and binds directly to chromatin, but the death response was unrelated to the p53 status of the tumor cells. p53 deletions or gain of function mutations did not influence the HAMLET sensitivity of tumor cells. Chromatin condensation was partly caspase dependent, but apoptosis-like marginalization of chromatin was also observed. The results show that tumor cell death in response to HAMLET is independent of caspases, p53 and Bcl-2 even though HAMLET activates an apoptotic response. The use of other cell death pathways allows HAMLET to successfully circumvent fundamental anti-apoptotic strategies that are present in many tumor cells.

Changes in Proteasome Structure and Function Caused by HAMLET in Tumor Cells

Background Proteasomes control the level of endogenous unfolded proteins by degrading them in the proteolytic core. Insufficient degradation due to altered protein structure or proteasome inhibition may trigger cell death. This study examined the proteasome response to HAMLET, a partially unfolded protein-lipid complex, which is internalized by tumor cells and triggers cell death. Methodology/Principal Findings HAMLET bound directly to isolated 20S proteasomes in vitro and in tumor cells significant co-localization of HAMLET and 20S proteasomes was detected by confocal microscopy. This interaction was confirmed by co-immunoprecipitation from extracts of HAMLET-treated tumor cells. HAMLET resisted in vitro degradation by proteasomal enzymes and degradation by intact 20S proteasomes was slow compared to fatty acid-free, partially unfolded α-lactalbumin. After a brief activation, HAMLET inhibited proteasome activity in vitro and in parallel a change in proteasome structure occurred, with modifications of catalytic (β1 and β5) and structural subunits (α2, α3, α6 and β3). Proteasome inhibition was confirmed in extracts from HAMLET-treated cells and there were indications of proteasome fragmentation in HAMLET-treated cells. Conclusions/Significance The results suggest that internalized HAMLET is targeted to 20S proteasomes, that the complex resists degradation, inhibits proteasome activity and perturbs proteasome structure. We speculate that perturbations of proteasome structure might contribute to the cytotoxic effects of unfolded protein complexes that invade host cells.