BAMLET Activates a Lysosomal Cell Death Program in Cancer Cells

Abstract

A complex of human α-lactalbumin and oleic acid (HAMLET) was originally isolated from human milk as a potent anticancer agent. It kills a wide range of transformed cells of various origins while leaving nontransformed healthy cells largely unaffected both in vitro and in vivo. Importantly, purified α-lactalbumins from other mammals form complexes with oleic acid that show biological activities similar to that of HAMLET. The mechanism by which these protein-lipid complexes kill tumor cells is, however, largely unknown. Here, we show that complex of bovine α-lactalbumin and oleic acid (BAMLET), the bovine counterpart of HAMLET, kills tumor cells via a mechanism involving lysosomal membrane permeabilization. BAMLET shows potent cytotoxic activity against eight cancer cell lines tested, whereas nontransformed NIH-3T3 murine embryonic fibroblasts are relatively resistant. BAMLET accumulates rapidly and specifically in the endolysosomal compartment of tumor cells and induces an early leakage of lysosomal cathepsins into the cytosol followed by the activation of the proapoptotic protein Bax. Ectopic expression of three proteins known to stabilize the lysosomal compartment, i.e. heat shock protein 70 (Hsp70), Hsp70-2, and lens epithelium-derived growth factor, confer significant protection against BAMLET-induced cell death, whereas the antiapoptotic protein Bcl-2, caspase inhibition, and autophagy inhibition fail to do so. These data indicate that BAMLET triggers lysosomal cell death pathway in cancer cells, thereby clarifying the ability of α-lactalbumin:oleate complexes to kill highly apoptosis-resistant tumor cells. Mol Cancer Ther; 9(1); 24-32. ©2010 AACR.

The cytotoxicity of fatty acid/α‐lactalbumin complexes depends on the amount and type of fatty acid

“…HAMLET has also been shown to affect mitochondria [31] and induce apoptosis-like cell death with caspase activation, PS externalization and chromatin condensation irrespective of the p53 and bcl-2 status of the cells [32] and to cause changes in proteasome structure and function [33]. In addition, it has been shown by Aits et al that HAMLET induces autophagy [34], while Rammer et al see activation of the lysosomal death pathway in response to BAMLET treatment, but that the cytotoxicity of BAMLET does not depend on autophagy [18]. However, apart from these quite specific effects of HAMLET, there is also evidence of a more crude effect connected to membrane interactions and alterations.…”

“…Furthermore, complexes of peptide fragments of α-LA have been formed by mixing the protein with OA in ethanol dispersed in a buffer [15] and BAMLET has also been made by mixing α-LA with the more soluble sodium salt of OA, sodium oleate [16]. The -LA:OA complexes are cytotoxic to a wide range of cancer cells [17][18][19] as well as some types of primary cells [17]. The structure of the H/BAMLET has been a subject of multiple studies, often with conflicting results.…”

The Alpha-Lactalbumin/Oleic Acid Complex and Its Cytotoxic Activity

“…These include OA-conditioned anion exchange chromatography using either a DEAE-Trisacryl M in the conventional method [3,5,6,19] or a DEAE-Sepharose column in an alternative method [53]. Direct mixing under heat denaturation [30,36,52], heat denaturation of -LA and OA-conditioned anion exchange chromatography [62], direct mixing in solution at room temperature with alternated pH conditions [45,48,49,63], direct mixing followed by anion exchange chromatography [61] and alkaline conditions [64] have also been used.…”

“…Initial gas chromatography and mass spectrometry data suggested that the average number of oleic acid molecules bound in the HAMLET complex was 0.9, with some batch variation [6]. The assumption of a 1:1 protein/fatty acid ratio was also reported in the preparation of BAMLET by chromatography [61]. By gas chromatography and mass spectrometry a later study estimated an -LA to OA ratio of 1:5.4 in a HAMLET complex prepared by the OA-preconditioned anion exchange chromatography method [38].…”

Milk proteins-derived bioactive peptides in dairy products: molecular, biological and methodological aspects

“…In an acidic environment, α-lactalbumin forms the HAMLET (human α-lactalbumin made lethal to tumor cells) complex with oleic acid which inhibits the proliferation of various tumors by an apoptosislike mechanism [Svanborg et al 2003]. The BAM-LET complex, the bovine equivalent of HAMLET, has been recently found to exert a strong cytotoxic effect on eight lines of tumor cells by increasing the permeability of the lysosomal membrane [Rammer et al 2010].…”