RU2605321C1 - Fraction of dna-hydrolyse secretory immunoglobulins of class a, with selective apoptotic activity in relation to human cancer cells - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to biochemistry. Purposed a fraction of DNA-hydrolyse secretory immunoglobulins of class A (sIgA), having molecular weight 380 kDa, processing selective apoptotic activity in relation to human cancer cells, extracted from milk of healthy donors by affinity chromatography on protein-A-sepharose, then ion-exchange chromatography on DEAE-cellulose, then by affinity chromatography on DNA-cellulose.

EFFECT: invention enables to inhibit cancer cell growth on the way of apoptosis without inducing death of normal (non-tumorous) cells.

1 cl, 7 dwg, 1 tbl, 3 ex

Description

The invention relates to the field of biochemistry and molecular biology, in particular to a fraction of secretory immunoglobulins of human milk of class A (IgA), which has selective apoptotic activity against human cancer cells, but does not stimulate the death of ordinary human cells.

Apoptosis is a programmed cell death, that is, there are certain mechanisms, as a result of the implementation of which the cell itself ends its existence.

In a multicellular organism, according to the apoptosis mechanism, cells die in the process of embryogenesis, T-cells in the process of differentiation in the thymus, cells infected with viruses, various kinds of altered cells (with insufficient intensity of apoptotic processes, oncological diseases develop) and many others. The main biological purpose of apoptosis is to create organs and tissues with evolutionarily fixed configurations and sizes in the process of embryonic morphogenesis and then maintain these parameters with acceptable tolerances throughout life.

The mechanisms of apoptosis are complex and diverse, they are a complex molecular cascade, the study of which is carried out by a large number of laboratories around the world. The undoubted importance of these studies in the aspect of oncology and gerontology is proved by the success of cancer therapy with some inducers of cancer cell apoptosis.

Immunoglobulins (Ig) of all known classes were found in the milk of women. The bulk of milk immunoglobulins - more than 90% - are class A Ig, represented mainly by their secretory form (sIgA). sIgA are produced by B-lymphocytes of the local mammary gland immune system, coming from specialized formations in the duodenum - Peyer's patches (Ogra PL, Dayton DH (Eds.). Immunology of Breast milk. N. Y: Raven Press, 1979, 284 p.) .

Being the main class of human Ig secrets, sIgA possess a wide range of properties. Several mechanisms of action of sIgA to protect the body against infections are known. So, sIgA molecules can directly participate in the neutralization of toxins, a number of enzymes, and whole virions (Mazanec M.V., et al., Immunol. Today, 1993, 14, 430; Mestecky J., Russell MW, Vet. Quarterly, 1998, 20, S83). In addition, they are able to agglutinate pathogens, as well as inhibit the penetration of foreign antigen through the mucous membrane (Gregory R.L., Filler S.J., Infect. Immun., 1987, 55, 2409).

It is also known that human milk causes the death of mammalian cancer cells in vitro. To date, several components of milk that induce apoptosis of cancer cells are known.

Known lactoferrin, one of the main proteins of milk, possessing not only antiviral activity against viruses such as cytomegalovirus and human immunodeficiency virus (Florisa R. et al., Curr. Pharm. Des., 2003, 9, 1257), but its catalytically active forms possess cytotoxic properties, causing apoptosis of immortalized murine fibroblast cells L929 and human promyelocytes HL-60 (Kanyshkova TG, et al., Eur. J. Biochem. 2003, 270, 3353; Babin EC, et al., Biochemistry, 2004, 69, 1239).

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 R.K., et al. Biochem. Biophys. Res. Commun. 2002. V. 291 (5). P. 1113-1118).

A known protein fragment of κ-casein isolated from human milk with a length of 74 amino acid residues having a molecular weight of about 8.6 kDa and an established amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 described in the description. The known peptide (lactaptin) induces the death of MCF-7 cells at a minimum concentration of 10 μg / ml, while the proliferation of cell culture ceases and about 10% of the cells die within 12 hours (Patent RU 2317304 C1, op. 20.02.2008).

A recombinant analogue of lactaptin with a molecular weight of about 16 kDa is known, consisting of a methionine residue, a fragment of human kappa-casein from 24 to 134 amino acid residues, and a C-terminal histidine tract with the ability to induce cancer cell death (Patent RU 2461566 C1, op. 20.09. 2012).

The closest to the claimed fraction of DNA-hydrolyzing sIgA - the prototype is the fraction of DNA-hydrolyzing IgG, with antitumor and DNA-hydrolyzing activity, isolated from the blood of patients with autoimmune pathologies by affinity chromatography on protein G-Sepharose with mol. IgG mass of about 150 kDa (Nevinsky GA. In: Autoimmune Diseases: Symptoms, Diagnosis and Treatment. K.J. Brenner, ed. Nova Science Publishers, Inc. 2010, 1-107).

The main disadvantage of the prototype is the toxic effect of apoptosis of DNA-hydrolyzing blood IgG both on the cancer cell line and on healthy cells.

The objective of the invention is to obtain a fraction of DNA-hydrolyzing sIgA from human milk, causing the death of cancer cells along the path of apoptosis, but not stimulating apoptosis of ordinary human cells.

The problem is achieved by the proposed fraction of DNA-hydrolyzing sIgA from human milk, which has selective apoptotic activity against human cancer cells.

Technical result: a fraction of DNA hydrolyzing sIgA was obtained, which has the ability to selectively inhibit the growth of cancer cells along the path of apoptosis without inducing the death of normal (non-tumor) cells.

The proposed fraction of DNA-hydrolyzing sIgA is capable of inducing apoptosis of cancer cells of the human breast adenocarcinoma line MCF-7 and myeloma RPMI 8226, as well as activating not the death, but the growth and proliferation of bone marrow stem cells.

The inventive fraction of DNA-hydrolyzing sIgA is isolated from the milk of healthy donors by several sequential chromatography: 1) affinity chromatography on protein A-sepharose, which allows you to completely remove non-specifically related components of milk; 2) ion-exchange chromatography on DEAE-cellulose, allowing to completely remove IgG; 3) by affinity chromatography on DNA cellulose, which allows one to obtain a specific fraction of DNA-hydrolyzing sIgA, which has an affinity for DNA.

The homogeneity of the claimed fraction of DNA hydrolyzing sIgA was confirmed by SDS gel electrophoresis, and its unique ability to induce apoptosis of only human cancer cells was confirmed by the inhibition of the fraction of DNA hydrolyzing sIgA milk by the development of MCF-7 and RPMI cancer cells, as well as the activation of bone cell growth the brain of mice of the lines MRL / lpr and (CBAxC57BL) F1.

The invention is based on the revealed ability of a fraction of DNA-hydrolyzing sIgA of human milk to exert an inhibitory dose-dependent effect on the growth of the cell line of mammary adenocarcinoma (MCF-7) and myeloma (RPMI) and to induce the death of cancer cells by the apoptosis mechanism. Moreover, the fraction of DNA-hydrolyzing sIgA milk, in contrast to the induction of death of human cancer cell lines, dose-dependently stimulate not the death, but the growth of normal cells, for example, bone marrow stem cells from the control line of healthy mice (CBAxC57BL) F1.

The invention is illustrated by the following examples.

Example 1. Isolation of a fraction of DNA-hydrolyzing sIgA, analysis of their homogeneity and DNase activity

Samples of the fraction of DNA hydrolyzing sIgA were obtained from milk of 40 female donors according to the methodology (Nevinsky G.A., et al. Appl. Biochem. Biotechnol., 2000, 83, 115). On protein A sepharose containing immobilized protein A, a mixture of sIgA and IgG was isolated under conditions of destruction of nonspecific interactions. Next, IgG and sIgA were effectively separated by ion exchange chromatography on DEAE cellulose.

The chromatographic separation profile of IgG and sIgA is shown in FIG. 1, where: (-) is the optical density profile during elution of mmunoglobulins from the sorbent (A ₂₈₀ ), (---) is the concentration of NaCl. Next, the DEAE cellulose column was pre-equilibrated with 20 mM Tris-HCl pH 7.5. IgG was eluted from the column upon application, and sIgA was a gradient of NaCl concentrations, from 0 to 1 M in 20 mM Tris-HCl pH 7.5. After chromatography, the resulting sIgA preparations were dialyzed against 20 mM Tris-HCl pH 7.5. In FIG. 1 shows that sIgA is quantitatively separated from IgG, the concentration of sIgA was 0.45 mg / ml. Next, the sIgA sample was separated by DNA affinity by affinity chromatography on DNA cellulose.

The optical absorption profile obtained by eluting sIgA with cellulose DNA is shown in FIG. 2, where: ( ─ ) is the optical density profile for elution of mmunoglobulins from the sorbent (A ₂₈₀ ), (-) is the concentration of NaCl. Chromatography of human milk sIgA on DNA cellulose yielded fraction (1) that did not have an affinity for DNA, and fraction (2) that was eluted with a NaCl concentration gradient from 0 to 3 M, which had an affinity for DNA, DNA hydrolyzing activity, and the ability to suppress cancer cell growth, sIgA concentration was 0.07 mg / ml.

An electrophoretic analysis of the homogeneity of the fraction of DNA hydrolyzing sIgA was performed using Lemmley SDS electrophoresis (Laemmli UK, Nature, 1970, 227, 680). The concentrating gel contained 4% acrylamide (AA: bisAA ratio = 32: 1), 125 mM Tris-HCl pH 6.8, 0.1% sodium dodecyl sulfate (SDS); separating gel - 12%, 15% or gradient (4.5-18%) acrylamide, 375 mM Tris-HCl pH 8.8, 0.1% SDS. Protein samples (5-10 μg) were incubated in a buffer containing 50 mM Tris-HCl pH 6.8, 1% SDS, 15% glycerol, 0.025%) bromphenol blue at 100 ° C for 2 min, after which the solution was applied to gel (0.6 × 100 × 150 mm). The homogeneity analysis of the fraction of DNA-hydrolyzing sIgA was carried out both under non-dissociating and dissociating conditions, in the latter case 0.1% mercaptoethanol was added to the buffer. Electrophoresis was carried out for 4-5 hours at 25 ° C in a buffer: 25 mM Tris-glycine, pH 8.3, 0.1% SDS at 7-15 mA. Proteins were stained with AgNO ₃ : the gel was fixed for 20 min in a 20% TCA solution, then washed with water overnight. After that, the gel was kept for 30 min in a solution of dithiothreitol (DTT) (4-5 mg / ml DTT per 300 ml of H ₂ O) and 30 min in a 0.1% solution of AgNO ₃ (150 ml) in the dark. Then the gel was washed with water 4 times for 10 minutes. The gel was developed with a 2% Na ₂ CO ₃ solution containing 0.02% formaldehyde until protein staining. Staining was stopped by adding 2% solution of CH ₃ COOH (100 ml) for 10 minutes and washed 2-3 times with water for 10 min.

An analysis of the homogeneity of the sIgA DNA hydrolyzing fraction using SDS gel electrophoresis before and after treatment with 10 mM DTT is shown in FIG. 3. As can be seen from the data presented in FIG. 3, until the disulfide bonds are restored to the sample, the main protein band sIgA with mol. mass of 380 kDa and a small band corresponding to the sum of the light chain sIgA (L, 25 kDa) and the connecting chain (J-chain, 15 kDa) - L + J. After restoration of the antibodies, four protein bands are found corresponding to the light (L), heavy chains (H), secretory component of these antibodies (S) and the connecting chain (J).

From the data of FIG. Figure 3 shows that the samples of the fraction of DNA hydrolyzing sIgA are electrophoretically homogeneous and correspond in molecular weight to sIgA. In addition, the affiliation of these Igs to class A secretory antibodies is shown by enzyme-linked immunosorbent assay using specific monoclonal mouse antibodies against human sIgA according to (Buneva V.N., et al. Appl. Biochem. Biotechnol., 1998, 75, 63). According to previous studies (Buneva V.N., et al. Appl. Biochem. Biotechnol., 1998, 75, 63), catalytic activity is a property of the obtained sIgA preparations that do not contain enzyme impurities with nuclease and other possible activities.

Relative DNA hydrolyzing activity was determined for each of 40 samples of the fraction of DNA hydrolyzing sIgA according to the results of three experiments.

For example, in FIG. 4 shows an electrophoretic analysis of DNA hydrolyzing activity of 10 samples of the fraction of DNA hydrolyzing sIgA from milk in a 1.5% agarose gel, where: dor. K - DNA in the absence of AT; dor. 1-10 - AT from milk 10 milk donors. The arrows indicate the position on the gel of supercoiled (sc), ring relaxed (co) and linear (lin) forms of DNA.

In FIG. Figure 4 shows that for all samples of the fraction of DNA-hydrolyzing sIgA compared to the control (DNA incubated in the absence of Ig), an effective transition of DNA from a superscored form to a ring relaxed and linear form occurs. The relative DNase activity of the fraction of DNA hydrolyzing sIgA was evaluated by the degree of DNA loss in the band corresponding to the supercoiled form. The complete disappearance of this form of DNA was taken as 100% DNase activity. DNA hydrolyzing activity was determined by a decrease in the amount of supercoiled DNA. The relative DNase activity values for 40 samples of the fraction of DNA-hydrolyzing sIgA are significantly different: the relative activity of the preparations varies from 30 to 90%.

The table shows 6 of 40 samples of the fraction of DNA hydrolyzing sIgA with relative activity from 27 to 60%, which were used to analyze their effect on cancer cells and bone marrow stem cells.

Example 2. Investigation of the apoptotic effect of the fraction of DNA hydrolyzing sIgA on human cancer cells of the MCF-7 and RPMI lines

To determine the cytotoxic properties of the fraction of DNA-hydrolyzing sIgA, we studied the effect of 6 different samples on the growth of MCF-7 human breast adenocarcinoma cells.

MCF-7 cells are estrogen-dependent, caspase 3 mutant; therefore, cell apoptosis follows the mitochondrial pathway and is accompanied by oligonucleosomal fragmentation of nuclear DNA. Cells were incubated for three days in a medium containing preparations of catalytically active antibodies in various concentrations. Living cells were stained with 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT).

This method (MTT test) consists in the formation of colored formazan crystals only in the mitochondria of living cells. Four controls were set: 1) the cells grew in an Ig-free medium for the same period of time; 2) cells were incubated with a KCl solution in which Ig was diluted; 3) in a medium without cells, MTT was added after three days, and 4) the antibody solution was incubated for 2 hours with MTT, then this MTT was added to cells growing without Ig.

For the first, second, and fourth controls, the number of living cells was estimated to be close to 100 ± 10%. In the case of the third control, reliably tested staining of MTT solutions did not occur. It was found that the fraction of DNA-hydrolyzing sIgA has a pronounced cytotoxic effect, which manifests itself after the first 12 hours of incubation.

The study of the effect of the concentration of the fraction of DNA hydrolyzing sIgA on the growth and proliferation of MCF-7 cells showed that with an increase in the concentration of the fraction of DNA hydrolyzing sIgA, the MTT index decreases. All samples of the fraction of DNA hydrolyzing sIgA inhibited the growth of cancer cells.

To determine the apoptotic cells used a standard kit for fluorescence analysis of annexin-V. Cells treated with the sIgA DNA hydrolyzing fraction showed binding to annexin V and morphological changes typical of apoptosis.

A study was conducted of the effect of five samples of the fraction of DNA-hydrolyzing sIgA milk of healthy donors on cancer cells of the RPMI line (myeloma). In FIG. Figure 5 shows the effect of the concentration of the fraction of DNA-hydrolyzing sIgA milk on the growth and proliferation of RPMI cells. The averaged data of three experiments are presented; the determination error did not exceed 17%.

The MTT test data for RPMI cells, which were incubated with six samples of the fraction of DNA hydrolyzing sIgA milk (table) at various concentrations for three days, showed that the fraction of DNA hydrolyzing sIgA milk of healthy women in labor has a pronounced cytotoxic effect on cancer cells of this lines.

Another typical indicator of cell apoptosis is DNA fragmentation to oligonucleosome size fragments. After incubation of cancer cells of the RPMI line with two preparations of the fraction of DNA hydrolyzing sIgA milk, an electrophoretic analysis of their DNA was performed. From the data presented in FIG. 6, it can be seen that the DNA of cells incubated with preparations of the sIgA DNA hydrolyzing fraction is expensively fragmented (doors 2 and 3), in contrast to the DNA of cells incubated in the absence of the sIgA DNA hydrolyzing fraction (doors 1). Therefore, the fraction of DNA hydrolyzing sIgA induces apoptosis of cancer cells of this line.

Example 3. The effect of the fraction of DNA-hydrolyzing sIgA milk on the proliferation of stem cells of the bone marrow of control (healthy) mice of lines (CBAxC57BL) F1

The studies were performed using bone marrow stem cells (CCMs) of control healthy mice of the (CBAxC57BL) F1 line. The HSC differentiation profile was evaluated by the number of granulocyte-macrophage (GM-CFU), burst-forming (PFU-E) and mixed colony-forming units (CFU-HEMM). GM-CFU are the precursors of the myeloid branch of hematopoiesis, PFU-E - the erythroid, and mixed colonies contain the predecessors of the granulocytic, erythroid, megakaryocytic and monocytic branches of bone marrow stem cell differentiation.

In FIG. Figure 7 shows the differentiation profiles of granulocytic CCM (A) and erythroid CCM (B) of the mouse line (CBAxC57BL) F1, after incubation with five samples of the fraction of DNA hydrolyzing sIgA, (Nos. 1-4, 6 tables.) Milk in various concentrations, where white columns - 0.05 μM, bars with a dash - 0.1 μM, black columns - 0.2 μM (average values of three experiments are given). From FIG. Figure 7 shows that the fraction of DNA-hydrolyzing sIgA milk exhibits a dose-dependent effect and that at none of the concentrations of the fraction of DNA-hydrolyzing sIgA there is a statistically significant decrease in the number of colonies of these cells compared to the control, and at some concentrations they significantly stimulate the growth of CCM in control mice (CBAxC57BL ) F1 line of mice. As shown above, all preparations of the fraction of DNA hydrolyzing sIgA had a strong cytotoxic effect, inhibiting the growth of cancer cells of carcinoma and especially myeloma.

Thus, it was found that the fraction of DNA-hydrolyzing sIgA of human milk inhibits the growth of the cell line of mammary adenocarcinoma (MCF-7) and myeloma (RPMI) and induce the death of cancer cells by the apoptosis mechanism, while they either have almost no effect or stimulate growth healthy mouse (CBAxC57BL) F1 bone marrow stem cell colonies.

Thus, a fraction of DNA-hydrolyzing sIgA from milk of healthy donors has been proposed, which has unique properties compared to antibodies from the blood of healthy donors and patients with autoimmune diseases, capable of inhibiting the growth of cancer cells without inducing the death of normal (non-tumor) cells.

It is known that human immunoglobulins, unlike many proteins, oligopeptides and enzymes that are rapidly cleaved by proteases, exist in the blood for a long time.

Using the proposed fraction of DNA-hydrolyzing sIgA from breast milk will allow:

- increase the treatment efficiency by 5 times with catalytically active sIgA of human milk of malignant tumors in the range of a single dose of 25 mg / kg of mass for tumor regression by 60% (see Fig. 6, 0.5-0.9 μM, molar mass of sIgA is equal to 380 kDa) compared with the recombinant analogue of the protein fragment of casein (Patent 2461566 C1, op. September 20, 2012, example 3): to achieve the same effect, 120 mg / kg of body weight is required (3-day course at a dose of 40 mg / kg of body weight);

- use the proposed fraction of DNA-hydrolyzing sIgA of human milk not only for the treatment of malignant neoplasms, but also as an immunocorrector, since the detected stimulation of bone marrow cell growth can lead to an improvement in the state of the immune and hematopoietic systems of the human body;

- reduce the number and frequency of drugs administered, since antibodies in the blood may not undergo degradation and elimination from the body for up to several months;

- expand the range of antitumor drugs - proteins of natural origin.

Claims (1)

A fraction of DNA-hydrolyzing secretory immunoglobulins of class A (sIgA), having a molecular mass of 380 kDa, having selective apoptotic activity against human cancer cells, isolated from healthy donor milk by affinity chromatography on protein A-sepharose, then ion-exchange chromatography on DEAE-cellulose , further by affinity chromatography on DNA cellulose.

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