RU2317304C1 - Peptide possessing apoptotic activity with respect to human cancer cells - Google Patents

Abstract

FIELD: biochemistry, molecular biology.

SUBSTANCE: invention proposes a peptide that represents fragment of human κ-casein and possessing apoptotic activity with respect to breast adenocarcinoma cultured cells. The claimed peptide is isolated from female milk and represents fragment of human κ-casein comprising 74 amino acid residues, molecular mass about 8.6 kDa and found amino acid sequence encoded by the nucleotide sequence SEQ ID NO:1 given in the invention description. Proposed peptide (lacaptin) induces death of cells MCF-7 in its minimal concentration 10 mcg/ml, stops proliferation of cellular culture and causes death of about 10% of cells for 12 h. Increasing concentration of peptide to 100 mcg/ml results to death of 50-70% of breast adenocarcinoma MCF-7 cells for 12 h. Peptide can be used in medicine as abase for development of medicinal formulation of peptide preparation used in therapy of malignant neoplasms.

EFFECT: valuable medicinal properties of peptide.

1 dwg, 2 ex

Description

The invention relates to the field of biochemistry and molecular biology, and in particular to a peptide that is a fragment of human κ-casein having apoptotic activity against human cancer cells in culture.

Apoptosis (or programmed cell death) is a gene-dependent process that is systemic in nature and necessary for the development of a multicellular organism.

Apoptotic cell death is also observed in various pathological conditions of the organism of higher animals. Through programmed cell death, cells are removed whose survival is undesirable for the body, such as mutagenic cells or cells infected with a virus.

Thus, apoptosis is a widespread general biological mechanism responsible for maintaining a constant number of cells, shaping and culling of defective cells.

Apoptotic death is accompanied by characteristic morphological and biochemical changes in the cell, such as shrinkage of the cytoplasm and the destruction of contacts with surrounding cells, the formation of apoptotic bodies containing condensed or morphologically unchanged organelles. Condensation of cells is accompanied by compaction of nuclear contents, aggregation of chromatin along the periphery of the nucleus and its fragmentation, cleavage of nuclear DNA and the destruction of cytoskeletal proteins and lamins.

An important feature of apoptosis is the preservation of cell membrane impermeability. At the same time, the contents of the dying cell are enclosed in the aggregate of apoptotic bodies surrounded by a membrane. The final stage of apoptosis is phagocytosis of apoptotic bodies by neighboring cells or professional phagocytes [Samuilov VD, Aleskin AV, Lagunova EM Biochemistry. 2000.V.65, issue 8. S.1029-1046].

Apoptosis can be induced both by external factors - proapoptotic cytokines, contact with the membrane of activated cytotoxic lymphocytes and natural killers, as well as intracellular events such as depletion of metabolites, dissociation of the oxidative phosphorylation chain, accumulation of unrepairable damage to nuclear DNA, etc.

Currently, the literature describes two main ways of activating apoptosis: mitochondrial and receptor-mediated.

In the mitochondrial activation pathway, critical events in the development of apoptosis are changes in membrane permeability, opening of mitochondrial pores, a decrease in the transmembrane potential of mitochondria, and the formation of reactive oxygen metabolites.

With receptor-mediated apoptosis, the action of physiological inducers is realized through cellular receptors specifically designed to enable the apoptosis program. Common for apoptosis receptors is the functioning in the form of active homotrimers, which allows the simultaneous use of several cytoplasmic signal sequences for communication with multiple conjugated signal proteins [Samuilov VD, Aleskin AV, Lagunova EM Biochemistry. 2000.V.65, issue 8. S.1029-1046].

It is known that during proteolytic cleavage of casein of human milk, regulatory peptides (beta-casomorphins and phosphopeptides) are formed that can act as physiological modulators of metabolism. For example, casein phosphopeptides are carriers of various trace elements, especially calcium, and have cytomodulating effects, stimulating the activity of immunocompetent cells [Meisel H., FitzGerald R.J. Curr. Pharm. Des. 2003. V.9. P.1289-1295]. Casomorphins are involved in ensuring the absorption of nutrients and the subsequent synthesis of hormones, immunopeptides and casokinins provide immune protection and the transfer of information between the endocrine and nervous system.

Lactoferrin, lactoperoxidase and lysozyme are the main antibacterial proteins of milk. Lactoferrin has antiviral activity against viruses such as cytomegalovirus and human immunodeficiency virus [Florisa R., Recio I., Berkhout B. Curr. Pharm. Des. 2003. V.9. P.1257-1275].

It is also known that the multimeric forms of milk alpha-lactalbumin possess cytotoxic properties. Alpha-lactalbumin - major Ca ²⁺ - binding protein of human milk with a molecular weight of 14.4 kDa, consisting of 122 amino acids, the main function of which is the interaction with beta-1,4-galactosyltransferase I with the formation of a lactose synthetase complex [Ramakrishnan B., Boeggeman E. Qasba PK, et al. Biochem. Biophys. Res. Commun. 2002. V.291 (5). P.1113-1118]. Multimeric forms of alpha-lactalbumin induced apoptotic death of L1210 murine lymphoblastoid leukemia cells and A549 human lung sarcoma, but did not affect the viability of non-cancerous cells of the primary human kidney fibroblast culture oo (HRTEC) [Hakansson A., Andreasson J., Zhivotovsky B., et. al. Exp. Cell Res. 1999. V.246 (2). P.451-460]. But then it was shown that alpha-lactalbumin is able to induce apoptosis only in the presence of free fatty acids of milk (in particular, oleic acid). Moreover, the apoptotic effect on cells of cancer and embryonic lines may be due to the action of milk fatty acids, and not to alpha-lactalbumin [Svensson M., Hakansson A., Mossberg A.-K., et. al. PNAS. 2000. V. 97 (8). P. 4221-4226].

Closest to the claimed peptide in terms of its functions, the prototype, is the multimeric form of human milk alpha-lactalbumin [Hakansson A., Zhivotovsky V., Orrenius S., et. al. PNAS 1995. V. 92 (17). P.8064-8068]. The disadvantage of the multimeric form of alpha-lactalbumin in human milk is that it does not have the ability to cause apoptotic death of human cancer cells on its own, in the absence of unsaturated fatty acids of milk.

An object of the invention is to identify a new peptide with the ability to induce apoptosis of human cancer cells.

The technical task is achieved by the claimed peptide having apoptotic activity in relation to human cancer cells.

The inventive peptide is isolated from human milk using a series of chromatographies: ion exchange chromatography on DEAE-Toyopearl, ion exchange chromatography on CM-Toyopearl and affinity chromatography on cybacron sepharose. The peptide is a fragment of human κ-casein with a length of 74 amino acid residues, has a molecular weight of about 8.6 kDa, and an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 and shown in FIG.

The study of apoptotic activity of the peptide was carried out on a MCF-7 human breast adenocarcinoma cell culture. The identity of the peptide was confirmed by analysis of protein preparations by electrophoretic separation in the presence of sodium dodecyl sulfate and by high performance reverse phase liquid chromatography.

The peptide structure was determined by analysis of trypsinolysis products by MALDI-TOF MS / MS spectroscopy on a Bruker mass spectrometer.

An analysis of the known level of science and technology showed that the claimed peptide (conventional name lactaptin) is new and differs in functionality from the closest analogues.

Example 1. The selection of the peptide from human milk

Milk provided by donors at various periods of lactation (from 3 weeks to 12 months) was centrifuged at 500 g for 20 minutes, and the milk plasma was stored in aliquots at -70 ° C until use.

The separation of plasma proteins of human milk was performed using ion exchange chromatography on DEAE-Toyopearl. The column (5 × 15 cm) was balanced with 20 mM Tris-HCl buffer, pH 6.8-8.8. After the application of milk plasma (150 ml), the sorbent was washed with the same buffer and elution was performed with a stepwise gradient of concentrations of 30, 100, 250 mM NaCl in the presence of 20 mM Tris-HCl pH 6.8, or 8.8, and then 200 mM NaOH. The pH value in the protein fractions collected by elution with NaOH was adjusted to 7-7.5 using AcOH. The collected fractions were dialyzed for 16-18 hours against 20 mM Tris-HCl buffer, pH 6.8 at 5 ° C.

The fraction of milk proteins eluted by application of milk plasma to DEAE-Toyopearl was applied to a column of CM-Toyopearl 650 M (1.2 × 10 cm). Prior to application, the sorbent was equilibrated with a buffer containing 50 mM Tris-HCl, pH 8.8. Proteins that did not bind to the sorbent were eluted with the same buffer, after which the bound proteins were eluted with a linear gradient of NaCl concentrations from 10 mM to 1 M in the presence of 50 mM Tris-HCl, pH 8.8. The collected fractions were dialyzed for 16-18 hours against a 10-fold excess of buffer containing 10 mM NaCl and 50 mM Tris-HCl, pH 8.8.

The milk protein fraction eluted when applied to a CM-Toyopearl was applied to a cybacron-sepharose column (1 × 10 cm), pre-equilibrated with 50 ml of Tris-HCl, pH 8.8. Unbound proteins were then eluted with 10 volumes of buffer containing 50 mM Tris-HCl, pH 8.8. Proteins bound to cybacron were eluted with a linear gradient of NaCl concentrations from 0 to 1 M in the presence of 50 mM Tris-HCl, pH 8.8.

Peptide identification (determination of amino acid sequence) was performed by MALDI MS / MS spectroscopy on a Bruker mass spectrometer. For this, the fractions containing the claimed peptide after the purification step on cybacron-sepharose were combined, dialyzed against water, lyophilized and dissolved in 0.1% TFA. The solution was applied to a Protein C4 column (4.6 × 20 mm). Elution was carried out by a linear gradient from 0 to 100% buffer water - 0.1% TFA acetonitrile with 0.1% TFA. The column volume was 1.9 cm; the elution rate was 0.2 ml / min.

Example 2. Apoptotic activity of the peptide in relation to human cancer cells in culture

MCF-7 human breast adenocarcinoma cells were grown in IMDM medium with 10% fetal calf serum manufactured by Biolot, containing 40 μg / ml gentamicin.

After the formation of a 30–50% monolayer, the studied peptide was added to the culture medium (the volume was 1/5 of the total volume of the culture medium) and the cell culture growth was monitored during the day.

The effect of the peptide on MCF-7 cells in culture and the cytotoxic effect of the latter were detected using two approaches.

λ=560 нм.The first approach is based on the conversion of the water-soluble MTT dye to the insoluble MTT-formazan by the enzymes of cytoplasm and mitochondria of viable cells. Since nonviable cells (postapoptotic cells, apoptotic bodies) do not have enzymes and cofactors of this transformation are absent, they are not stained with MTT. The resulting MTT-formazan precipitate in viable cells was dissolved in isopropanol and its amount was determined spectrophotometrically by absorption at a wavelength

λ = 560 nm.

The second approach is based on assessing the staining of cytoplasm and nuclei of non-viable cells with trypan blue, followed by counting of such cells in the Goryaev chamber. The approach is based on the fact that the cytoplasmic membrane of viable eukaryotic cells is impervious to this dye.

The minimum concentration of the peptide (lactaptine) in the culture medium, leading to the induction of cell death, is 10 μg / ml. When using such a concentration, the proliferation of the cell culture is stopped and about 10% of the cells die within 12 hours. With an increase in the concentration of the peptide (lactaptin) in the medium up to 100 μg / ml, 50-70% of MCF-7 mammary adenocarcinoma cells die in 12 hours.

Since these methods allow us to evaluate the cytotoxic effect of the peptide on eukaryotic cells, but do not allow us to determine by which mechanism, apoptotic or necrotic, cell death occurred, in addition, using light microscopy, we evaluated the morphological changes in nuclei and cytoplasm in order to discriminate against apoptotic changes (condensation of cells and nuclei) from necrotic (increase in cell volume and nuclear lysis). The results of observations showed that the morphological changes in MCF-7 breast adenocarcinoma cells under the action of lactaptine completely correspond to apoptotic, i.e. there is condensation of cells, compaction of nuclear contents, aggregation of chromatin along the periphery of the nucleus and fragmentation of the nucleus with the formation of apoptotic bodies.

In addition, the death of MCF-7 cells under the action of lactaptine is accompanied by a decrease in the transmembrane potential of mitochondria detected by rhodamine staining. When rhodamine stains living cells, this dye accumulates in the membranes of mitochondria; during apoptotic cell death, mitochondria do not stain with rhodamine. Treatment of cells with lactaptin initiates apoptotic changes in the cell, accompanied by the death of mitochondria, as a result of which MCF-7 apoptotic are not stained with rhodamine.

The proposed peptide, representing a species-specific fraction of human milk protein, specifically a fragment of human κ-casein, which has apoptotic activity against human cancer cells, can be used in medicine as a basis for developing a dosage form of a peptide drug used for the treatment of malignant neoplasms.

Claims (1)

A peptide derived from human milk, having a molecular weight of about 8.6 kDa, having apoptotic activity in relation to the culture of mammary adenocarcinoma cells, having the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1.

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