1.
Düringer, Caroline, et al.
(författare)
HAMLET; a novel tool to identify apoptotic pathways in tumor cells.
2005
Ingår i: Application of apoptosis to cancer treatment.. - Berlin/Heidelberg : Springer-Verlag. - 9781402033032 ; , s. 223-245
Bokkapitel (övrigt vetenskapligt/konstnärligt) abstract
Tumor cells often carry mutations in genes that control cell survival, and become resistant to signals that trigger cell death. Yet, some cell death pathways remain intact in tumor cells. If identified, these pathways might be exploited to selectively remove tumor cells. HAMLET (human α-lactalbumin made lethal to tumor cells) is a protein-lipid complex derived from human milk that activates cell death programs in tumor cells but not in healthy differentiated cells. We use HAMLET as a tool to identify apoptosis and apoptosis-like cell death mechanisms in tumor cells and to understand if these mechanisms differ between tumor and healthy cells. HAMLET interacts with the cell surface, translocates into the cytoplasm and accumulates in cell nuclei, where it disrupts the chromatin. Recent in vivo studies have shown that HAMLET maintains the tumoricidal activity in glioblastoma, papilloma and bladder cancer models, with no significant side effects. The results suggest that HAMLET should be explored as a new therapeutic agent with selectivity for the tumor and with little toxicity for adjacent healthy tissue. Such therapies are a much-needed complement to conventional treatments, to reduce the side effects and improve the selectivity.
2.
Baumann, Anne, et al.
(författare)
HAMLET Forms Annular Oligomers When Deposited with Phospholipid Monolayers
2012
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 418:1-2, s. 90-102
Tidskriftsartikel (refereegranskat) abstract
Recently, the anticancer activity of human a-lactalbumin made lethal to tumor cells (HAMLET) has been linked to its increased membrane affinity in vitro, at neutral pH, and ability to cause leakage relative to the inactive native bovine alpha-lactalbumin (BLA) protein. In this study, atomic force microscopy resolved membrane distortions and annular oligomers (AOs) produced by HAMLET when deposited at neutral pH on mica together with a negatively charged lipid monolayer. BLA, BAMLET (HAMLET's bovine counterpart) and membrane-binding Peptide C, corresponding to BLA residues 75-100, also form AO-like structures under these conditions but at higher subphase concentrations than HAMLET. The N-terminal Peptide A, which binds to membranes at acidic but not at neutral pH, did not form AOs. This suggests a correlation between the capacity of the proteins/peptides to integrate into the membrane at neutral pH as observed by liposome content leakage and circular dichroism experiments and the formation of AOs, albeit at higher concentrations. Formation of AOs, which might be important to HAMLET's tumor toxic action, appears related to the increased tendency of the protein to populate intermediately folded states compared to the native protein, the formation of which is promoted by, but not uniquely dependent on, the oleic acid molecules associated with HAMLET. (C) 2012 Elsevier Ltd. All rights reserved.
3.
Brest, Patrick, et al.
(författare)
MiR-129-5p is required for histone deacetylase inhibitor-induced cell death in thyroid cancer cells
2011
Ingår i: Endocrine-Related Cancer. - 1479-6821. ; 18:6, s. 711-719
Tidskriftsartikel (refereegranskat) abstract
The molecular mechanism responsible for the antitumor activity of histone deacetylase inhibitors (HDACi) remains elusive. As HDACi have been described to alter miRNA expression, the aim of this study was to characterize HDACi-induced miRNAs and to determine their functional importance in the induction of cell death alone or in combination with other cancer drugs. Two HDACi, trichostatin A and vorinostat, induced miR-129-5p overexpression, histone acetylation and cell death in BCPAP, TPC-1, 8505C, and CAL62 cell lines and in primary cultures of papillary thyroid cancer (PTC) cells. In addition, miR-129-5p alone was sufficient to induce cell death and knockdown experiments showed that expression of this miRNA was required for HDACi-induced cell death. Moreover, miR-129-5p accentuated the anti-proliferative effects of other cancer drugs such as etoposide or human a-lactalbumin made lethal for tumor cells (HAMLET). Taken together, our data show that miR-129-5p is involved in the antitumor activity of HDACi and highlight a miRNA-driven cell death mechanism. Endocrine-Related Cancer (2011) 18 711-719
4.
Brisuda, Antonín, et al.
(författare)
Bladder cancer therapy using a conformationally fluid tumoricidal peptide complex
2021
Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 12
Tidskriftsartikel (refereegranskat) abstract
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.
5.
Casbarra, A, et al.
(författare)
Conformational analysis of HAMLET, the folding variant of human alpha-lactalbumin associated with apoptosis
2004
Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 13:5, s. 1322-1330
Tidskriftsartikel (refereegranskat) abstract
A combination of hydrogen/deuterium (H/D) exchange and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the conformation in solution of HAMLET, the folding variant of human alpha-lactalbumin, complexed to oleic acid, that induces apoptosis in tumor and immature cells. Although near- and far-UV CD and fluorescence spectroscopy were not able to discriminate between HAMLET and apo-alpha-lactalbumin, H/D exchange experiments clearly showed that they correspond to two distinct conformational states, with HAMLET incorporating a greater number of deuterium atoms than the apo and holo forms. Complementary proteolysis experiments revealed that HAMLET and apo are both accessible to proteases in the P-domain but showed substantial differences in accessibility to proteases at specific sites. The overall results indicated that the conformational changes associated with the release of Ca2+ are not sufficient to induce the HAMLET conformation. Metal depletion might represent the first event to produce a partial unfolding in the beta-domain of a-lactalbumin, but some more unfolding is needed to generate the active conformation HAMLET, very likely allowing the protein to bind the C18:1 fatty acid moiety. On the basis of these data, a putative binding site of the oleic acid, which stabilizes the HAMLET conformation, is proposed.
6.
Cirl, Christine, et al.
(författare)
Subversion of Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins
2008
Ingår i: Nature Medicine. - : Springer Science and Business Media LLC. - 1546-170X .- 1078-8956. ; 14:4, s. 399-406
Tidskriftsartikel (refereegranskat) abstract
Pathogenic microbes have evolved sophisticated molecular strategies to subvert host defenses. Here we show that virulent bacteria interfere directly with Toll-like receptor (TLR) function by secreting inhibitory homologs of the Toll/interleukin-1 receptor (TIR) domain. Genes encoding TIR domain containing–proteins (Tcps) were identified in Escherichia coli CFT073 (TcpC) and Brucella melitensis (TcpB). We found that TcpC is common in the most virulent uropathogenic E. coli strains and promotes bacterial survival and kidney pathology in vivo. In silico analysis predicted significant tertiary structure homology to the TIR domain of human TLR1, and we show that the Tcps impede TLR signaling through the myeloid differentiation factor 88 (MyD88) adaptor protein, owing to direct binding of Tcps to MyD88. Tcps represent a new class of virulence factors that act by inhibiting TLR- and MyD88-specific signaling, thus suppressing innate immunity and increasing virulence.
7.
8.
Farooq, Shukkur Muhammed, et al.
(författare)
Therapeutic Effect of Blocking CXCR2 on Neutrophil Recruitment and Dextran Sodium Sulfate-Induced Colitis
2009
Ingår i: Journal of Pharmacology and Experimental Therapeutics. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 1521-0103 .- 0022-3565. ; 329:1, s. 123-129
Tidskriftsartikel (refereegranskat) abstract
Dextran sodium sulfate (DSS)-induced colitis in mice is characterized by polymorphonuclear neutrophil (PMN) infiltration into the colonic mucosa and lumen. The mechanism by which this occurs is unclear. To begin to understand the mechanism, we determined the role of the PMN chemokine receptor, CXCR2, in DSS-induced colitis by using CXCR2(-/-) mice or by neutralizing CXCR2. DSS was administered through drinking water to CXCR2(-/-) and BALB/c mice for 5 days followed by regular water for 1 day. In the neutralization study, mice were injected with control serum or goat anti-CXCR2 antiserum. BALB/c mice receiving DSS and control serum-injected mice receiving DSS lost weight and showed considerable clinical illness. Histological observation revealed submucosal edema, PMN infiltration into the submucosa and mucosa, extensive crypt damage with abscesses, and ulceration. In contrast, both the CXCR2(-/-) and anti-CXCR2 antiserum-treated mice gained weight and had significantly lower symptom scores. Histology of these mice showed submucosal edema but relatively intact crypt architecture and very few ulcers. Significantly fewer PMNs were found in the mucosa in anti-CXCR2 antiserum compared with control serum-injected inflamed mice, but no significant difference in eosinophil infiltration was observed between the groups. Our experiments identify a role for CXCR2 in DSS-induced colitis and suggest that antagonizing CXCR2 provides some therapeutic efficacy, possibly by impeding PMN recruitment into the mucosa. Antagonizing CXCR2 may form the basis for therapeutic drugs directed at controlling colitis.
9.
Fast, Jonas, et al.
(författare)
Stability of HAMLET--A kinetically trapped {alpha}-lactalbumin oleic acid complex.
2005
Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 14:2, s. 329-340
Tidskriftsartikel (refereegranskat) abstract
The stability toward thermal and urea denaturation was measured for HAMLET (human -lactalbumin made lethal to tumor cells) and -lactalbumin, using circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Under all conditions examined, HAMLET appears to have the same or lower stability than -lactalbumin. The largest difference is seen for thermal denaturation of the calcium free (apo) forms, where the temperature at the transition midpoint is 15°C lower for apo HAMLET than for apo -lactalbumin. The difference becomes progressively smaller as the calcium concentration increases. Denaturation of HAMLET was found to be irreversible. Samples of HAMLET that have been renatured after denaturation have lost the specific biological activity toward tumor cells. Three lines of evidence indicate that HAMLET is a kinetic trap: (1) It has lower stability than -lactalbumin, although it is a complex of -lactalbumin and oleic acid; (2) its denaturation is irreversible and HAMLET is lost after denaturation; (3) formation of HAMLET requires a specific conversion protocol.
10.
Fischer, Walter, et al.
(författare)
Human alpha-lactalbumin made lethal to tumor cells (HAMLET) kills human glioblastoma cells in brain xenografts by an apoptosis-like mechanism and prolongs survival.
2004
Ingår i: Cancer Research. - 1538-7445. ; 64:6, s. 2105-2112
Tidskriftsartikel (refereegranskat) abstract
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.
