RU2620165C2 - Method for tumour cell death induction - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: to induce tumour cell death in an experiment, HCT116, Jurkat, U937 lines tumour cells are simultaneously subjected to a combined treatment with recombinant DR5-B preparation and 0.1 nM of bortezomib. At that, DR5-B used in a concentration at which the half-maximal effective concentration of DR5-B is 0.330.08± ng/ml for HCT116 cell line, 0.060.01± ng/ml for Jurkat cell line, and± 0.090.02 ng/ml for U937 line.

EFFECT: overcoming of tumour cell lines resistance to TRAIL DR5-B in an experiment.

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Description

Technical field

The invention relates to the field of cell and molecular biology, and specifically to overcoming the resistance of human cell tumor lines to the TRAIL cytokine using the DR5-specific variant of human TRAIL. The method can be used as a promising strategy for the treatment of malignant neoplasia and as a means to study the mechanisms of resistance of tumor cell lines of various origins to the TRAIL cytokine.

State of the art

Cytokine TRAIL (TNF-related apoptosis-inducing ligand, Apo2L, TNFSF10) is one of the members of the family of tumor necrosis factor TNF (tumor necrosis factor). Like other members of the TNF family, TRAIL plays an important role in the observation of tumors, the antiviral response, and is involved in the suppression of autoimmune diseases.

However, the main distinguishing feature of TRAIL is that this cytokine is capable of selectively inducing apoptosis by an external, receptor-dependent mechanism in malignantly transformed cells without cytotoxicity towards normal cells.

TRAIL exhibits apoptotic activity in three-dimensional form, the monomer of this cytokine is a type II transmembrane protein, consisting of 281 amino acid residues. The extracellular domain of TRAIL (amino acids 114 to 281) in the human body can be cleaved by metalloprotease 7 while maintaining apoptosis-inducing activity.

Recombinant preparations of the extracellular domain of TRAIL with a native secondary structure, without affinity sequences and N-terminal amino acids, induce apoptosis in a wide range of tumor cell lines and primary tumors without causing the death of normal cell lines and primary tissues. Recombinant drugs TRAIL showed high antitumor efficacy in experiments on mice with inoculated human tumors. The selectivity of apoptosis-inducing activity is confirmed in clinical studies.

TRAIL has 5 receptors: membrane DR4, DR5, DcR1, DcR2 and soluble osteoprotegerin (OPG). Receptors DR4 and DR5 (death receptors) are capable of signaling apoptosis to a tumor cell. DcR1 and DcR2 receptors (the so-called trap receptors, English decoy receptors) compete with death receptors for binding to TRAIL or activate anti-apoptotic mechanisms in the cell. It is the interaction of TRAIL with trap receptors that is one of the reasons for the resistance of tumor cells to a cytokine. Osteoprotegerin (OPG) is a soluble TRAIL trap receptor that activates the NF-kB protein (RANKL) and is able to interact with ligand molecules in solution. OPG rather plays a role in suppressing osteoclastogenesis, but not in regulating TRAIL-mediated apoptosis, since the interaction of TRAIL-OPG under physiological conditions is rather weak [Almasan A, Ashkenazi A. Apo2L / TRAIL: apoptosis signaling, biology, and potential for cancer therapy / / Cytokine Growth Factor Rev. 2005. Vol. 14, pp. 337-348].

Studies of the biological activity of TRAIL have shown that approximately half of human tumor cell lines and primary tumors are insensitive to TRAIL [Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence DA et al. Safety and antitumor activity of recombinant soluble Apo2 ligand // J. Clin. Invest. 1999. Vol. 104, pp. 155-162]. To date, many methods have been developed to overcome the resistance of tumor lines to wild-type recombinant TRAILs by combining cell lines with TRAILs and chemotherapeutic reagents [Sayers TJ, Murphy WJ. Combining proteasome inhibition with TNF-related apoptosis-inducing ligand (Apo2L / TRAIL) for cancer therapy // Cancer Immunol. Immunother. 2006. Vol. 55, pp. 76-84., Shenoy K, Wu Y, Pervaiz S. LY303511 enhances TRAIL sensitivity of SHEP-1 neuroblastoma cells via hydrogen peroxide-mediated mitogen-activated protein kinase activation and up-regulation of death receptors // Cancer Res. 2009. Vol. 69, pp. 1941-1950 and others.]. However, the wild type TRAIL binds to both death receptors and trap receptors with approximately the same affinity, which can reduce the efficiency of cytopokine induction of apoptosis.

It was previously shown that apoptosis-inducing signal in many tumor cells is mainly due to the interaction of TRAIL mainly with one of the death receptors. In most types of tumors, especially solid ones, the apoptotic signal is carried out mainly when TRAIL interacts with the DR5 death receptor. In this regard, to overcome the resistance of cell lines to TRAIL, the increase in the specificity of TRAIL with respect to the DR5 receptor is of greatest interest.

Several agonist antibodies to the DR5 receptor and recombinant mutant variants of TRAIL have been developed, with increased specificity for the DR5 receptor. However, agonistic antibodies to the DR5 receptor have a number of significant drawbacks: the lack of trimerization, the need to use additional crosslinking agents for the manifestation of apoptotic activity, and the high cost of production. Therefore, the use of DR5-specific mutant variants has great promise as a strategy for overcoming the resistance of tumor cells to TRAIL.

To date, several DR5-specific mutant variants of TRAIL have been obtained: DR5-8, D269H, rhTRAIL-DR5, DR5-A and DR5-B. Amino acid substitutions in them are shown in table 1.

A strategy for enhancing apoptosis-inducing activity in ovarian carcinoma cells with micromolar concentrations of cisplatin [Duiker EW, de Vries EG, Mahalingam D, Meersma GJ, Boersma-van Ek W et al. Has also been proposed for the receptor-selective variant of TRAIL rhTRAIL-DR5. Enhanced antitumor efficacy of a DR5-specific TRAIL variant over recombinant human TRAIL in a bioluminescent ovarian cancer xenograft model // Clin. Cancer Res. 2009. Vol. 15, pp. 2048-2057]. But all currently existing DR5-selective TRAIL variants, except DR5-B, interact either with the DR4 receptor (D269H) or with DcR2 (DR5-8), and the affinity of these TRAIL variants for receptors, although it is significantly reduced, quite high (see Table 2 from Gasparian ME, Chernyak BV, Dolgikh DA, Yagolovich AV, Popova EN et al. (2009). Generation of new TRAIL mutants DR5A and DR5B with improved selectivity to death receptor 5 // Apoptosis. 2009. Vol. 14, pp. 778-787).

The DR5-specific mutant variant of TRAIL DR5-B is unique in that it does not bind to the death receptor DR4 and receptor trap DcR1 and the fact that its affinity for DcR2 and OPG receptors is reduced by several times more than in other TRAIL variants than 280 and 223 times, respectively (see table 2). Previously, a method for producing a recombinant preparation of a DR5-specific mutant variant of the TRAIL DR5-B cytokine [patent RU No. 2404038 of 11/27/2010] was patented.

DR5-B has been shown to be more potent than wild-type TRAIL on the DR5-dependent TRAIL-sensitive Jurkat T cell leukemia line. In addition, the antitumor activity of the recombinant drug DR5-B on TRAIL-resistant cell lines HeLa and U937 was also well expressed. The sensitivity of cells to DR5-B could be increased while treating the cells with chemotherapeutic reagents. Moreover, it has been shown that many chemotherapeutic agents cause an increase in the expression of the DR5 death receptor on the surface of tumor cells. Thus, DR5-B can be used to overcome the resistance to TRAIL not only of DR5, but also of initially DR4-sensitive cells.

Closest to the claimed is a method of treating tumor cells of the lines Jurkat, HCT116, A549, U937, HT29 and MCF-7 with DR5-B and chemotherapeutic reagents doxorubicin, paclitaxel and bortezomib in an amount of 1-100 nM to overcome the resistance of tumor cells to TRAIL wild cytokine type (BYCHKOV M. Et al., 2013).

However, it remains relevant to search for effective amounts of chemotherapeutic drugs used to induce the death of tumor cells, which, while maintaining antitumor activity, would have minimal toxic effect on normal body tissues.

Disclosure of invention

The claimed invention solves the problem of inducing the death of tumor cells, namely, to a method of overcoming the resistance of tumor cells to wild-type TRAIL cytokine by combining two approaches: inducing apoptosis in tumor cells upon activation of the DR5 receptor using the recombinant drug DR5-B, and enhancing the apoptotic activity of the recombinant DR5-B with chemotherapeutic agents.

The task of overcoming the resistance of tumor lines to TRAIL is achieved by inducing DR5-mediated apoptosis while simultaneously treating tumor cells of the HCT116, Jurkat, U937 lines in an experiment with recombinant DR5-B and bortezomib in an amount of 0.1 nM.

It is preferable to use DR5-B at a concentration at which the half-maximum effective concentration of DR5-B is 0.33 ± 0.08 ng / ml for the HCT116 cell line, 0.06 ± 0.01 ng / ml for the Jurkat cell line and 0, 09 ± 0.02 ng / ml for the U937 line.

The invention was tested on 12 lines of tumor cells: A549 lung adenocarcinoma, Jurkat T-lymphoblastic leukemia, HepG2 hepatocellular carcinoma, HT29 and HCT116 colorectal carcinoma, MCF-7 breast adenocarcinoma, MDA-MB-231 and SKBR-3 leukemia monocytes, monocytes U251 MG, Du145 prostate carcinomas, and SKOV3 ovarian adenocarcinomas. The toxicity of the recombinant drug DR5-B was tested on human umbilical vein endothelial cells (HUVEC) and foreskin fibroblasts (HFF). A549, Du145, HCT116, MDA-MB-231, U251 MG cells were cultured in DMEM supplemented with 10% (by volume) fetal bovine serum and 2 mM glutamine. SKBR-3 and SKOV3 cells were cultured in McCoy 5A supplemented with 10% (v / v) fetal bovine serum. Jurkat, HepG2, HT29, and U937 tumor lines were cultured in RPMI1640 medium supplemented with 5% fetal bovine serum. To cultivate HepG2 cells, amino acids were added to RPMI1640 medium. The MCF-7 breast carcinoma line was cultured in EMEM supplemented with 10 μg / ml recombinant human insulin and 10% (v / v) fetal bovine serum. Normal prepuce fibroblasts (HFFs) were cultured in DMEM supplemented with 10% (v / v) fetal bovine serum. HUVEC endothelial cells were cultured in DMEM / F12 supplemented with 15% fetal bovine serum, 5 ng / ml recombinant human primary fibroblast growth factor (FGF-2), 20 ng / ml recombinant human vascular endothelial growth factor (VEGF), 1% amino acids , 2 mM L-glutamine, 50 U / ml penicillin and 50 ng / ml streptomycin. All cell lines were cultured in 75 ml ³ culture vials (untreated vials were used for Jurkat and U937 cell suspension lines, and treated culture vials were used for adhesive cell lines).

The expression level of TRAIL receptors on the surface of tumor cell lines was determined using flow cytometry. Cells were removed from the culture vials using a 0.05% trypsin-EDTA solution, then washed twice with cold phosphate-buffered saline (PBS) and mixed. Then the cells were poured into 5-ml tubes (35 × 10 ³ cells per sample). FITC conjugated antibodies to TRAIL receptors — DR4, DR5, DcR1, and DcR2 or isotypic control antibodies — were added to the cell suspension and incubated for 1 h at + 4 ° C. Unbound antibodies were washed twice with cold FSB and the cells were transferred to FACS buffer (FSB supplemented with 1 μM propidium iodide). Samples were analyzed on a FACScan flow cytometer (Beckton Dickinson, USA). To analyze the obtained data, we used the WinMDI 2.9 program.

Cell viability was determined using the MTT test. 24 hours before the determination of viability, the adhesion cells were adsorbed into the wells of a 96 well-treated culture plate at a rate of 10 ⁴ cells per well. Then, the cells were washed from the medium and dilutions of recombinant TRAIL or DR5-B preparations, as well as chemotherapeutic reagents in the corresponding culture medium without embryonic bovine serum, were added to them. After that, the cells were incubated for 24 hours. Suspension cells U937 and Jurkat were besieged at 900 rpm for 5 minutes, suspended in serum-free medium and applied to an untreated culture plate at a rate of 5 × 10 ⁴ cells per well. TRAIL preparations and chemotherapeutic agents diluted in serum-free culture medium were added to the cells. After 24 hours, MTT was added to the cells (final concentration 0.5 mg / ml) and incubated for 4 hours. After incubation, the contents of the plates were precipitated for 5 minutes at 3000 rpm. The supernatants were removed and the crystals of formazan formed in DMSO formed during MTT reduction were dissolved. Cell viability was determined by the optical density of the contents of the wells, which were detected using a Bio-Rad 680 plate spectrophotometer (Bio-Rad, USA) at a wavelength of 540 nm with background subtraction at 655 nm. The apoptotic nature of cell death was confirmed by studying the morphology of cell nuclei when stained with Hoechst 33342 dyes and propidium iodide. Fluorescence microscopy was performed using a Nikon Eclipse T100f microscope.

Statistical data processing was performed using Microsoft Office Excel 2013. All experiments were repeated at least three times. The half-maximum effective concentrations (EC ₅₀ ) of the TRAIL and DR5-B preparations were calculated using the GraphPad Prism 6 program.

The technical result of the claimed invention is the development of a new method for inducing the death of tumor cells. The method combines two approaches: the induction in tumor cells of DR5-mediated apoptosis using the DR5-specific mutant variant of the TRAIL cytokine, and the increase in the apoptosis-inducing activity of DR5-B using chemotherapeutic reagents. The first approach overcomes the resistance of DR5-dependent tumor lines to TRAIL, and the second approach promotes the sensitization of DR4-dependent cells to DR5-mediated apoptosis. This method can be used to overcome the resistance of tumor cell lines to TRAIL in cell and preclinical models, as well as to study the mechanisms of resistance of cells to TRAIL.

The method can be used as a promising treatment strategy for malignant neoplasia. The prospects of the method lies in the fact that it allows the use of these chemotherapeutic reagents in reduced dosages, which, in turn, leads to a decrease in their toxic effects on normal cells and body tissues and a synergistic interaction of drugs during the induction of cell death of malignant neoplasias. In the future, to create a combined preparation. The concentrations of the recombinant drug DR5-specific variant of TRAIL DR5-B and the chemotherapeutic reagent are selected depending on the type of tumor, and their ratio may vary depending on the sensitivity of cells to the drugs.

Brief Description of the Drawings

The invention is illustrated by the drawings:

Figure 1. Expression of death receptors and TRAIL traps on the surface of tumor cells. Cells were incubated with FITC-conjugated antibodies to TRAIL membrane receptors and the level of expression of the receptors was determined using flow cytometry. Values are presented as mean ± standard deviation (n = 3).

Figure 2. Cytotoxicity of TRAIL and DR5-B on various tumor cells. Cells were incubated with TRAIL or DR5-B for 24 hours and viability was determined using the MTT test. Values are presented as mean ± standard deviation (n = 3).

Figure 3. Effect of doxorubicin on TRAIL or DR5-B mediated tumor cell death. Cells were incubated for 24 hours with the indicated amounts of TRAIL or DR5-B and doxorubicin (Jurkat - 0.1 nM, U937 - 1 nM, MCF-7, HT29, HCT116, SKBR-3, A549 and MDA-MB-231 - 100 nM, Du145 and SKOV3 - 500 nM, HepG2 - 1 μM and U251 MG - 2 μM) and cell viability was evaluated using the MTT test. Values are presented as mean ± standard deviation (n = 3).

Figure 4. The effect of paclitaxel on TRAIL or DR5-B induced cell death. These cancer cells were incubated with the indicated amounts of TRAIL and paclitaxel variants (Jurkat - 0.1 nM, U937 - 1 nM, HCT116 - 5 nM, HT29, SKBR-3 and SKOV3 - 10 nM, MDA-MB-231 - 50 nM, MCF -7 and A549 - 100 nM, Du145 - 500 nM, HepG2 - 1 μM and U251 MG - 10 μM) for 24 hours and cell viability was determined using the MTT test. Values are presented as mean ± standard deviation, (n = 3).

Figure 5. Effect of bortezomib on TRAIL or DR5-B-induced death of tumor cells. Cells were incubated for 24 hours with TRAIL or DR5-B and bortezomib (HCT116, Jurkat, U937 - 0.1 nM, MDA-MB-231 - 25 nM, SKBR-3, A549 and Du145 - 50 nM, MCF-7, HT29 and SKOV3 - 100 nM, HepG2 and U251 MG - 500 nM) and cell viability was determined using MTT. Values are presented as mean ± standard deviation (n = 3).

Figure 6. DR5-B does not show cytotoxic activity to normal human cells. (A) Analysis of expression of TRAIL cytokine membrane receptors on the surface of human foreskin fibroblasts (HFF) and human umbilical vein endothelial cells (HUVEC). The effect of TRAIL and DR5-B on the viability of HFF (B) and HUVEC (C). Cells were incubated with different concentrations of TRAIL and DR5-B for 24 hours and their viability was determined using the MTT test. Values are presented as mean ± standard deviation (n = 3).

The implementation of the invention

The invention is illustrated by examples:

Example 1. The recombinant drug DR5-B exhibits greater cytotoxicity compared with the recombinant drug TRAIL wild on cells with different levels of expression of TRAIL receptors.

The expression level of TRAIL receptors on the surface of Jurkat, U937, MCF-7, HT29, HCT116, SKBR-3, A549 and MDA-MB-231, Du145, SKOV3, HepG2 and U251 MG tumor cells was studied using flow cytometry. Inhibition cells with antibodies to TRAIL receptors labeled with FITC or with antibodies of isotypic control and expression of receptors on the cell membrane was determined using a FACScan cytometer (Fig. 1).

Cell lines express different numbers of TRAIL receptors, but no direct correlation between the sensitivity of cancer cells to TRAIL and the number of death receptors has been recorded. In all studied cell lines, the level of expression of trap receptors was relatively low compared to death receptors (2-11% DcR1 and 6-19% DcR2 of total receptor expression). The exception was A549 (DcR2 30%) and MDA-MB-231 (DcR1 35.5%) cells, in which the number of trap receptors was quite high (Fig. 1).

Next, we analyzed the effect of different concentrations of the recombinant preparations TRAIL and DR5-B on the viability of the cell lines Jurkat, U937, MCF-7, HT29, HCT116, SKBR-3, A549 and MDA-MB-231, Du145, SKOV3, HepG2 and U251 MG (Fig. . 2). Tumor line cells were incubated with TRAIL preparations for 24 hours and their viability was determined using the MTT test. The analysis of the research results showed that:

1. Half of the 12 analyzed lines are insensitive to wild-type TRAIL (cell death less than 20%).

2. On nine of the twelve lines (MCF-7, HT29, HCT116, HepG2, Jurkat, U937, A549, SKBR-3 and U251 MG), DR5-B caused cell death more efficiently than wild-type TRAIL (Fig. 2 table 3).

3. The cytotoxicity of the TRAIL and DR5-B preparations on three cell lines that mainly (but not completely) conduct the signal for apoptosis through the DR4 receptor (Du145, SKOV3, and MDA-MB-231) did not differ.

The results of the example show that the recombinant DR5-B preparation is superior to the wild-type cytokine preparation in the efficiency of apoptosis induction.

Example 2. The chemotherapy drugs doxorubicin, taxol and bortezomib to a greater extent enhance DR5-B mediated apoptosis, compared with TRAIL-induced.

Analysis of the effect on the viability of the cell lines Jurkat, U937, MCF-7, HT29, HCT116, SKBR-3, A549 and MDA-MB-231, Du145, SKOV3, HepG2 and U251 MG of the combined effects of recombinant drugs TRAIL and DR5-B and chemotherapeutic reagents doxorubicin, bortezomib and taxol was carried out as follows. The concentration of chemotherapeutic agents for joint incubation was selected based on their cytotoxicity to cells. Cell death by chemotherapy did not exceed 30% of the cells. After selecting the concentrations, the cells were incubated with doxorubicin (Fig. 3), paclitaxel (Fig. 4) and bortezomib (Fig. 5) for 24 hours, after which their viability was determined using the MTT test.

The results showed that doxorubicin, paclitaxel, and bortezomib enhance both TRAIL and DR5-B induced cell death, but for most cells this effect was more pronounced for DR5-B (Fig. 3-5). When combined with chemotherapeutic agents and DR5-B, cell death of all lines exceeds 60%, while in the case of TRAIL, less than half of the combinations (16 of 36) showed this result (Table 4).

When combined with doxorubicin or paclitaxel with TRAIL, in no line was more than 80% cell death observed, whereas in the case of DR5-B this barrier is overcome in five lines. Of the three chemotherapy drugs tested, bortezomib most effectively enhances cell death caused by both TRAIL drugs, but this sensitizing effect is more pronounced with DR5-B. Thus, DR5-B together with bortezomib causes the death of more than 70% of cells in all cell lines, and for TRAIL-sensitive lines HCT116 and MDA-MB-231, as well as for TRAIL-resistant cell lines MCF-7, HepG2 and SKOV3 death of more than 90% of cells. At the same time, wild-type TRAIL together with bortezomib caused the death of 70% of cells in only three lines, and only TRAIL-sensitive HCT116 cells died by 90% (Table 4).

The half-maximum effective concentrations (EC ₅₀ ) of DR5-B on nine cell lines are approximately 2-10 times lower compared to TRAIL (Table 3).

All three chemotherapy drugs tested (doxorubicin, paclitaxel and bortezomib) significantly reduced the EC ₅₀ values of both forms of TRAIL (Table 5), but DR5-B, when combined with chemotherapy, remains 1.5-10 times more effective than TRAIL for most cell lines . The EC ₅₀ for DR5-B, together with doxorubicin and paclitaxel, was less than 0.35 ng / ml and 0.25 ng / ml, respectively, for all cell lines. Such EC ₅₀ values for wild-type TRAIL were achieved only in four lines with doxorubicin and in six lines with paclitaxel. The most effective cytotoxicity of both forms of TRAIL is enhanced by bortezomib. When co-incubated with bortezomib, the EC ₅₀ values for DR5-B decreased several times (less than 0.1 ng / ml in ten lines), whereas for wild-type TRAIL this only happened on the SKOV3 line.

The results show that doxorubicin, taxol, and bortezomib are more likely to enhance DR5-B induced cell death than with TRAIL-induced apoptosis.

Example 3. The recombinant drug DR5-B does not show toxicity to normal cells.

The level of expression of TRAIL receptors on the surface of HUVEC endothelial cells and human foreskin fibroblasts HFF was studied using flow cytometry. Cells were incubated with antibodies to TRAIL receptors labeled with FITC or with antibodies of isotypic control and expression of receptors on the cell membrane was determined using a FACScan cytometer (Fig. 6 (A)). The results of the study showed that normal cells, in contrast to tumor cells, express smaller amounts of both TRAIL death receptors and cytokine trap receptors.

The effect of TRAIL and DR5-B preparations on the viability of HFF and HUVEC was analyzed using the MTT test after incubation of cells with recombinant wild-type and DR5-B recombinant drugs (Fig. 6 (B) and (C)). Cells were incubated with different concentrations of TRAIL and DR5-B for 24 hours and their viability was determined using the MTT test. The results of the study showed that the recombinant preparations of TRAIL and its DR5-specific mutant variant DR5-B are non-toxic to normal cells at concentrations of up to 10 μg / ml, which is 100 times higher than the maximum toxic concentration of drugs on 12 tumor cell lines of various nature.

Example 3 shows that the recombinant preparation of the DR5-specific variant of TRAIL DR5-B is non-toxic to normal cells and can be studied in preclinical models as a selective and highly active antitumor agent.

Claims (2)

1. A method for inducing the death of tumor cells of the HCT116, Jurkat, U937 lines in an experiment, which consists in the simultaneous combined treatment of tumor cells with recombinant DR5-B and bortezomib in an amount of 0.1 nM. 2. The method of claim 1, wherein DR5-B is used at a concentration at which the half-maximum effective concentration of DR5-B is 0.33 ± 0.08 ng / ml for the HCT116 cell line, 0.06 ± 0 , 01 ng / ml for the Jurkat cell line and 0.09 ± 0.02 ng / ml for the U937 line.

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