Cytotoxic Lactalbumin-Oleic Acid Complexes in the Human Milk Diet of Preterm Infants.

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.

Bladder cancer therapy without toxicity-A dose-escalation study of alpha1-oleate.

Potent chemotherapeutic agents are required to counteract the aggressive behavior of cancer cells and patients often experience severe side effects, due to tissue toxicity. Our study addresses if a better balance between efficacy and toxicity can be attained using the tumoricidal complex alpha1-oleate, formed by a synthetic, alpha-helical peptide comprising the N-terminal 39 amino acids of alpha-lactalbumin and the fatty acid oleic acid. Bladder cancer was established, by intravesical instillation of MB49 cells on day 0 and the treatment group received five instillations of alpha1-oleate (1.7-17 mM) on days 3 to 11. A dose-dependent reduction in tumor size, bladder size and bladder weight was recorded in the alpha1-oleate treated group, compared to sham-treated mice. Tumor markers Ki-67, Cyclin D1 and VEGF were inhibited in a dose-dependent manner, as was the expression of cancer-related genes. Remarkably, toxicity for healthy tissue was not detected in alpha1-oleate-treated, tumor-bearing mice or healthy mice or rabbits, challenged with increasing doses of the active complex. The results define a dose-dependent therapeutic effect of alpha1-oleate in a murine bladder cancer model.

Divergent Anticancer Activity of Free and Formulated Camel Milk α-Lactalbumin.

Alpha-lactalbumin (α-LA), a small milk calcium-binding globular protein, is known to possess noticeable anticancer activity, which is determined by the ability of this protein to form complexes with oleic acid (OA). To date, in addition to human and bovine α-LA, the ability to form such anti-tumor complexes with OA was described for goat and camel α-LA. Although the mechanisms of the anticancer activity of human and bovine α-LA are already well-studied, little is currently known about the anticancer action of this camel protein. The goal of this study was to fill this gap and to analyze the anticancer and pro-apoptotic activities of camel α-LA in its free form (α-cLA) and as an OA-containing complex (OA-α-cLA) using four human cancer cell lines, including Caco-2 colon cancer cells, PC-3 prostate cancer cells, HepG-2 hepatoma cells, and MCF-7 breast cancer cells as targets. The anti-tumor activities of OA-α-cLA and α-cLA were analyzed using MTT test, annexin/PI staining, cell cycle analysis, nuclear staining, and tyrosine kinase (TK) inhibition methods. We show here that the OA-α-cLA complex does not affect normal cells but has noticeable anti-cancer activity, especially against MCF-7 cells, thus boosting the anticancer activity of α-cLA and improving the selectivity of OA. The OA-α-cLA complex mediated cancer cell death via selective induction of apoptosis and cell-cycle arrest at lower IC50 than that of free α-cLA by more than two folds. However, OA induced apoptosis at higher extent than OA-α-cLA and α-cLA. OA also caused unselective apoptosis-dependent cell death in both normal and cancer cells to a similar degree. The apoptosis and cell-cycle arresting effect of OA-α-cLA may be attributed to the TK inhibition activity of OA. Therefore, OA-α-cLA serves as efficient anticancer complex with two functional components, α-cLA and OA, possessing different activities. This study declared the effectiveness of OA-α-cLA complex as a promising entity with anticancer activity, and these formulated OA-camel protein complexes constitute an auspicious approach for cancer remedy, particularly for breast cancer.

Oleic acid is a key cytotoxic component of HAMLET-like complexes.

HAMLET is a complex of α-lactalbumin (α-LA) with oleic acid (OA) that selectively kills tumor cells and Streptococcus pneumoniae. To assess the contribution of the proteinaceous component to cytotoxicity of HAMLET, OA complexes with proteins structurally and functionally distinct from α-LA were prepared. Similar to HAMLET, the OA complexes with bovine ß-lactoglobulin (bLG) and pike parvalbumin (pPA) (bLG-OA-45 and pPA-OA-45, respectively) induced S. pneumoniae D39 cell death. The activation mechanisms of S. pneumoniae death for these complexes were analogous to those for HAMLET, and the cytotoxicity of the complexes increased with OA content in the preparations. The half-maximal inhibitory concentration for HEp-2 cells linearly decreased with rise in OA content in the preparations, and OA concentration in the preparations causing HEp-2 cell death was close to the cytotoxicity of OA alone. Hence, the cytotoxic action of these complexes against HEp-2 cells is induced mostly by OA. Thermal stabilization of bLG upon association with OA implies that cytotoxicity of bLG-OA-45 complex cannot be ascribed to molten globule-like conformation of the protein component. Overall, the proteinaceous component of HAMLET-like complexes studied is not a prerequisite for their activity; the cytotoxicity of these complexes is mostly due to the action of OA.

The toxicity of bovine α-lactalbumin made lethal to tumor cells is highly dependent on oleic acid and induces killing in cancer cell lines and noncancer-derived primary cells.

A complex between α-lactalbumin and oleic acid (C18:1, 9 cis) has been reported to be cytotoxic to cancer cells. We have prepared such complexes and tested their activity against both cancer cell lines and noncancer-derived primary cells. Unexpectedly, some primary cell types were more sensitive to treatment than cancer cell lines. We found the complex to be cytotoxic to all of the tested cells, with a 46-fold difference between the most sensitive and the least sensitive cell type. Oleic acid by itself exhibited a remarkably similar activity. The cell-killing mechanisms of the complex and of oleic acid alone were examined by flow cytometry, testing for apoptosis- and necrosis- inducing activity. The T-cell leukemia-derived Jurkat cells primarily underwent cell death resembling apoptosis, whereas the monocytic leukemia-derived THP1 cells adopted a more necrotic-like cell death. Erythrocytes were sensitive to lysis by the complex and oleic acid. We conclude that oleic acid is cytotoxic by itself and that, in contrast to the literature, a complex of α-lactalbumin and oleic acid has cytotoxic activity against primary cells, as well as cancer cells.

Pro-inflammatory effects of commercial alpha-lactalbumin on RAW 264.7 macrophages is due to endotoxin contamination.

This study investigated the effects of alpha-lactalbumin (alpha-LA) on cellular signaling molecules associated with inflammatory responses in RAW 264.7 macrophages. The results indicated that commercial alpha-LA could increase prostaglandin E(2) (PGE(2)) and the expression of COX-2 via increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK) and jun N-terminal kinase (JNK), and increase nitric oxide (NO) and the expression of iNOS via the activation of ERK1/2 and JNK. Furthermore, commercial alpha-LA could increase nuclear translocation of p65 nuclear factor-kappa B (p65 NF-kappaB) through stimulation on inhibitor kappa B-alpha (IkappaB-alpha) degradation. Since endotoxin also has these effects, we assayed the content of endotoxin in the commercial alpha-LA. We found to our surprise that endotoxin was there and that alpha-LA-induced NO and PGE(2) production could be suppressed by polymyxin B, a specific inhibitor of endotoxin. Thus, the pro-inflammatory effects of commercial alpha-LA might be caused by endotoxin contamination through activation and expression of iNOS and COX-2 which were upregulated by MAPKs or nuclear translocation of p65 NF-kappaB in RAW 264.7 cells. It is therefore crucial to assess the possibility of endotoxin contamination within any biological product being studied for immune augmenting activities before a meaning result can be obtained.

Structure-function characteristics of the biomaterials based on milk-derived proteins.

There is an impetus on development of implantable biomaterials with the characteristics of improved biodegradability, bio-absorbability and wound healing activities. The milk proteins have valuable nutritional and biological properties, which lead to the promotion of quality health. In this study, whey protein isolate or WPI (highly aggregated) and its component lactalbumin (less aggregated) were melt blended with polycaprolactone (PCL) and then compression moulded into thin sheets ( approximately 1mm thickness). The effects of structural morphologies of the proteins on the mechanical, morphological, in vitro enzymatic degradation, and cytotoxicity and cell proliferation characteristics of the biomaterials were examined. In general, the tensile strength and modulus of the biomaterials decreased with increasing protein content. Compared to WPI, lactalbumin showed a better compatibility with the PCL matrix as observed in the mechanical properties and scanning electron microscopic morphology. The biomaterials exhibited a good retention of the mechanical characteristics after digestion in a physiologically simulated fluid containing trypsin enzyme. However, lactalbumin containing biomaterials showed a better retention of the tensile properties compared to WPI containing biomaterials. The cell culture studies indicated that the biomaterials have no cytotoxic effects, moreover they enhanced the proliferation of L929 cells compared to the pure PCL. Finally, this study indicated that the PCL based biomaterials with a protein content of 20wt% may be applied in fabrication of implantable devices for soft tissue engineering, where it requires a reasonably low to moderate mechanical strength (e.g., approximately 10MPa tensile strength), and improved biodegradability, biocompatibility and tissue healing activities.

Cytotoxic aggregates of alpha-lactalbumin induced by unsaturated fatty acid induce apoptosis in tumor cells.

The effects of three fatty acids on cytotoxic aggregate formation of Ca(2+)-depleted bovine alpha-lactalbumin (apo-BLA) have been studied by UV absorbance spectroscopy and transmission electron microscopy. The experimental results demonstrate that two unsaturated fatty acids, oleic acid and linoleic acid, and one saturated fatty acid, stearic acid, induce the intermediate of apo-BLA at pH 4.0-4.5 to form amorphous aggregates in time- and concentration-dependent manners. These aggregates are dissolved under physiological conditions at 37 degrees C and further characterized by fluorescence spectroscopy, circular dichroism and time-of-flight mass spectrometry. Our data here indicate that the structural characteristics of these aggregates are similar to those of HAMLET/BAMLET (human/bovine alpha-lactalbumin made lethal to tumor cells), a complex of the partially unfolded alpha-lactalbumin with oleic acid. Cell viability experiments indicate the aggregates of apo-BLA induced by oleic acid and linoleic acid show significant dose-dependent cytotoxicity to human lung tumor cells of A549 but those induced by stearic acid have no toxicity to tumor cells. Furthermore, the cytotoxic aggregates of apo-BLA induced by both unsaturated fatty acids induce apoptosis of human lung cancer cell line A549, suggesting that such cytotoxic aggregates of apo-BLA could be potential antitumor drugs. The present study provides insight into the mechanism of fatty acid-dependent oligomerization and cytotoxicity of alpha-lactalbumin, and will be helpful in the understanding of the molecular mechanism of HAMLET/BAMLET formation.

Who is Mr. HAMLET? Interaction of human alpha-lactalbumin with monomeric oleic acid.

A specific state of the human milk Ca(2+) binding protein alpha-lactalbumin (hLA) complexed with oleic acid (OA) prepared using an OA-pretreated ion-exchange column (HAMLET) triggers several cell death pathways in various tumor cells. The possibility of preparing a hLA-OA complex with structural and cytotoxic properties similar to those of the HAMLET but under solution conditions has been explored. The complex was formed by titration of hLA by OA at pH 8.3 up to OA critical micelle concentration. We have shown that complex formation strongly depends on calcium, ionic strength, and temperature; the optimal conditions were established. The spectrofluorimetrically estimated number of OA molecules irreversibly bound per hLA molecule (after dialysis of the OA-loaded preparation against water followed by lyophilization) depends upon temperature: 2.9 at 17 degrees C (native apo-hLA; resulting complex referred to as LA-OA-17 state) and 9 at 45 degrees C (thermally unfolded apo-hLA; LA-OA-45). Intrinsic tryptophan fluorescence measurements revealed substantially decreased thermal stability of Ca(2+)-free forms of HAMLET, LA-OA-45, and OA-saturated protein. The irreversibly bound OA does not affect the Ca(2+) association constant of the protein. Phase plot analysis of fluorimetric and CD data indicates that the OA binding process involves several hLA intermediates. The effective pseudoequilibrium OA association constants for Ca(2+)-free hLA were estimated. The far-UV CD spectra of Ca(2+)-free hLA show that all OA-bound forms of the protein are characterized by elevated content of alpha-helical structure. The various hLA-OA complexes possess similar cytotoxic activities against human epidermoid larynx carcinoma cells. Overall, the LA-OA-45 complex possesses physicochemical, structural, and cytotoxic properties closely resembling those of HAMLET. The fact that the HAMLET-like complex can be formed in aqueous solution makes the process of its preparation more transparent and controllable, opening up opportunities for formation of active complexes with specific properties.

IEC-6 intestinal cell death induced by bovine milk alpha-lactalbumin.

Potent inhibition of cell proliferation was found for commercial preparations of bovine alpha-lactalbumin on cultured intestinal cell line IEC-6 albeit lot-dependent. The inhibition was irreversible and a single exposure to the culture medium containing alpha-lactalbumin of an active lot for a period as short as 30 min was enough to provoke cell death, possibly through apoptosis. The oligomer fraction from size exclusion chromatography was significantly robust, while the monomer fraction remained totally inert, in inducing cell death. Incubation at 37 degrees C for 5 d with 30% trifluoroethanol in acetate, pH 5.5, in a slowly rotating test tube rendered the monomer fraction cytotoxic. Again, the resulting inhibitory activity was found in the oligomer fraction from size exclusion chromatography, with emergence of subtle peaks at 22- and 30-kDa. Furthermore, the occurrence of SDS-stable 30-kDa as well as 20-kDa bands on electrophoresis was a common feature for alpha-lactalbumin with the activity inducing cell death. Thus, a certain dimeric state can be implicated in the cytotoxicity of bovine alpha-lactalbumin.

Involvement of SDS-stable higher M(r) forms of bovine normal milk alpha-lactalbumin in inducing intestinal IEC-6 cell death.

Monomeric 14-kDa bovine alpha-lactalbumin was purified with a preparation of lower molecular weight whey protein concentrate from Holstein cow normal milk followed by size exclusion chromatography. The protein showed a stimulatory rather than an inhibitory effect on the proliferation of a cultured IEC-6 cell line from the rat small intestine. But incubation in 30% trifluoroethanol/acetate buffer (pH 5.5) at 37 degrees C for 5 d in a slowly rotating test tube rendered it highly cytotoxic with concomitant appearance of SDS-stable 20- and 30-kDa forms of alpha-lactalbumin on electrophoresis. Furthermore, alpha-lactalbumin obtained by a one-step purification procedure by affinity chromatography on an anti-alpha-lactalbumin antibody column from the lower molecular weight whey protein concentrate, which had been found to contain several SDS-stable higher M(r) forms of alpha-lactalbumin, exhibited potent inhibitory activity on IEC-6 cell growth. These results indicate the involvement of SDS-stable higher M(r) forms of bovine normal milk alpha-lactalbumin in inducing cell death on the intestinal IEC-6 cell line.

No need to be HAMLET or BAMLET to interact with histones: binding of monomeric alpha-lactalbumin to histones and basic poly-amino acids.

The ability of a specific complex of human alpha-lactalbumin with oleic acid (HAMLET) to induce cell death with selectivity for tumor and undifferentiated cells was shown recently to be mediated by interaction of HAMLET with histone proteins irreversibly disrupting chromatin structure [Duringer, C., et al. (2003) J. Biol. Chem. 278, 42131-42135]. Here we show that monomeric alpha-lactalbumin (alpha-LA) in the absence of fatty acids is also able to bind efficiently to the primary target of HAMLET, histone HIII, regardless of Ca(2+) content. Thus, the modification of alpha-LA by oleic acid is not required for binding to histones. We suggest that interaction of negatively charged alpha-LA with the basic histone stabilizes apo-alpha-LA and destabilizes the Ca(2+)-bound protein due to compensation for excess negative charge of alpha-LA's Ca(2+)-binding loop by positively charged residues of the histone. Spectrofluorimetric curves of titration of alpha-LA by histone H3 were well approximated by a scheme of cooperative binding of four alpha-LA molecules per molecule of histone, with an equilibrium dissociation constant of 1.0 microM. Such a stoichiometry of binding implies that the binding process is not site-specific with respect to histone and likely is driven by just electrostatic interactions. Co-incubation of positively charged poly-amino acids (poly-Lys and poly-Arg) with alpha-LA resulted in effects which were similar to those caused by histone HIII, confirming the electrostatic nature of the alpha-LA-histone interaction. In all cases that were studied, the binding was accompanied by aggregation. The data indicate that alpha-lactalbumin can be used as a basis for the design of antitumor agents, acting through disorganization of chromatin structure due to interaction between alpha-LA and histone proteins.