RU2360692C1 - Agent stimulating apoptosis of human leukaemia cells - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: triterpene glycosides from holothurians are applied to prepare the pharmaceutical composition stimulating apoptosis of human leukaemia cells.

EFFECT: agent described above stimulates apoptosis of human leukaemia cells effectively and does not affect normal cells of human immune system.

3 cl, 14 dwg, 6 ex

Description

The invention relates to medicine and relates to agents having the ability to stimulate apoptosis of human leukemia cells.

Leukemia is a tumor diffusely affecting the hematopoietic tissue of the bone marrow. The incidence of leukemia in different countries of the world varies in a wide range: from 3 to 10 people per 100,000 population. At the same time, men suffer from various forms of leukemia about 1.5 times more often than women. The maximum incidence of chronic leukemia is observed in people over 40-50 years old, and acute - in the age of 10-18 years.

Leukemia is characterized by:

- unlimited growth, uncontrolled cell multiplication - hyperplasia;

- morphological anaplasia - loss of cell ability for differentiation, maturation, immaturity;

- inhibition of normal hematopoiesis due to the rapid proliferation of tumor elements, their "crowding out" of normal hematopoietic growths (metaplasia).

The main treatment for leukemia is chemotherapy [V.I. Makholkin et al. Internal Medicine. M .: Medicine, 1999]. As drugs, both drugs obtained by chemical synthesis and isolated from natural sources are used. According to the mechanism of action on the tumor process, they can be divided into two main groups; a) antimetabolites - substances that disrupt the metabolism of the main biochemical processes that occur in a rapidly dividing cell (tumor), b) cytostatics - substances that directly affect the cell division process.

The former include drugs such as methotrexate, mercaptopurine, thioguanine, cytarabine, fludarabine, hydroxyurea, the second include mitosantrone, vincristine, vinblastine, teniposide, rubomycin, doxorubomycin and others [M. D. Mashkovsky. Medicines, vol. 2, ed. 14. M.: New Wave LLC, 2001, p. 407].

Unfortunately, chemotherapy, as a rule, has a number of very pronounced side effects. Due to poor selectivity, cytostatic drugs affect both sick and healthy blood cells, which leads to the rapid death of a significant number of them and the development of almost complete cytopenia - inhibition of the growth of all blood cells (leukocytes, platelets and red blood cells).

The most dangerous in this case is the development of leukopenia, since white blood cells are one of the main components of the body's natural defense against infection. The degree and duration of leukocytopenia developing after chemotherapy largely determines the number of life-threatening infectious complications.

Thrombocytopenia is also a clinical problem, causing hemorrhagic complications, often fatal, especially in the presence of concomitant infection.

A serious complication of the use of chemotherapy in the treatment of leukemia is the inhibition of the growth of neutrophils - neutropenia, which leads to infectious complications. Given the high likelihood of developing and the potential severity of infectious complications in neutropenia, measures are being taken to limit the entry of infectious agents into the patient’s body from the outside with air, food and water, as well as measures to combat microorganisms that colonize the body. The latter approach includes the prophylactic use of antibiotics and antifungal drugs.

Acute leukemia with timely diagnosis can be cured in 80-90% of cases. Chronic leukemia can be cured in only 20-40% of cases; patients are treated for years, for a long time they need careful medical care and the use of a variety of drugs, due to the relatively fast addiction of tumor cells to the action of one drug.

All this indicates the need to search for new drugs for the treatment of leukemia, based on approaches other than the above.

In the early 70s of the XX century, biologists introduced a new concept into scientific circulation - aloptosis, which describes a cell death system that differs from necrosis by the absence of inflammatory reactions and toxic poisoning of the body, characteristic of the necrotic variant of cell breakdown observed with traditional antitumor drugs [Schutze Osthoff K. et al. Apoptosis signaling by death receptors. Eur. J. Biochem. 1998, vol. 254, p. 439-459; Blatt N. B., Glick G. D. Signaling pathways and effector mechanisms pre-programmed cell death. Bioorg. Med. Chem. 2001, vol. 9, p. 1371-1384].

During apoptosis, cells undergo characteristic morphological changes, including condensation and fragmentation of the nucleus, contraction of the cytoplasm and the formation of so-called apoptotic bodies, which contain fragments of the nucleus surrounded by the cytoplasm and cell membrane. Apoptotic cells are rapidly absorbed by macrophages.

It was later realized that apoptosis occurs in all body tissues as part of the normal cell cycle. For example, apoptosis occurs during embryogenesis in which individual cells (parts of the body) are “sentenced” to death during the development of the body.

Apoptosis can be divided into three stages. At the first stage, the cell receives an apoptotic signal. A large number of internal and external stimuli of action on the cell can activate an apoptotic sequence of reactions in it. These stimuli include binding to cellular receptors on its surface, removal of critical growth factors, or exposure to various chemical agents. In addition, oxidative stress, irradiation of a cell with UV light or ionizing radiation, heating or a change in osmotic pressure are also factors inducing apoptosis.

At the next stage of apoptosis development, the cell integrates the signals coming from everywhere and can go (or maybe not) to the process of apoptosis, during which enzyme systems are activated, the synthesis of secondary lipid metabolism messengers is activated, gene expression changes and specialized proteases (caspases) are activated. The final decision of the cell to switch to apoptosis depends on many factors. At the final stage of apoptosis, the general signaling mechanism of degradation is simultaneously activated in such a way that the cell immediately acquires the characteristic morphological features inherent in apoptosis.

One of the possible approaches to the destruction of cancer leukemic cells can be forced stimulation (induction) of leukemic cells to apoptosis by exposing them to suitable biologically active natural compounds [S.-Y. Sun, N. et al. Apoptosis as a novel target for cancer. J. Natl. Cancer Inst, 2004, v. 96, No. 9, p. 662-672]. In particular, marine natural compounds and their synthetic analogues and derivatives that are capable of causing apoptosis of cancer cells attract attention.

The presence of triterpene glycosides is a characteristic feature of marine invertebrates belonging to the class Holothurioidea (type Echinodermata).

Holoturia triterpene glycosides exhibit a wide range of biological activities: antifungal, antitumor, hemolytic, cytostatic and immunomodulatory [Stonik, V.A. et al. Handbook of natural toxins and venoms; Tu A., Ed .; Marcel Dekker Inc .: New York, 1988; vol. 3, pp. 107-120]. Holothurium extracts are used as antitumor drugs in alternative medicine in some countries. The use of many preparations from holothuria in traditional eastern medicine has also been reported. The healing properties of food products prepared from commercial holothurians are ascribed to triterpene glycosides [V. Levin Far Eastern trepang; Dalknizhizdat: Vladivostok, 1982].

Holteruria triterpene glycosides have a strong membraneolytic effect on cell and model membranes containing Δ ⁵ - sterols. This effect is the result of the formation of ion channels and pores. The formation of channels and pores in cell membranes is also the cause of hemolytic, antifungal and antitumor activities of these compounds. The antitumor properties of triterpene glycosides from holothuria were first discovered by Nigrelli [Nigrelli, RF Zoologica, 1952, 37, 89-90]. The antitumor activity of glycoside fractions from holothurians collected near the Great Barrier Reef (Australia) and Western Samoa were studied by Kuznetsova and co-workers [Kuznetsova, TA. et al. Sotr. Biochem. Physiol., 1982, 73 C, 41-43]. Glycosides from 19 species of the families Holothuriidae and Stichopodidae inhibited the growth of Sarcoma-37 tumor cells in concentrations from 6.2 to 100 μg / ml in in vitro experiments. It is also known that frondoside A from the holoturia Cucumaria frondosa is used as an immunomodulatory agent [US patent 7163702, 2007.01.16].

There is also evidence in the literature that glycosides isolated from Penlacia quadrangulari holothuria, filinopsids A and E, induce apoptosis in the tissues of murine sarcoma-180 and human microvascular endothelial cells NMEC [Tong, Y et al. J. Int. J. Cancer, 2005, 114, 843-853; Tian, F., et. al. J. Cancer Biology & Therapy, 2005, 4 (8), 874-882], and the triterpene glycoside from the Cucumaria frondosa holothurium, frondoside A, has an antiproliferative effect against cancer cells of the human pancreas AsPC-1 due to the induction of apoptosis [US patent 7144867 , 2006.12.05]. However, the use of filinopsids A and E or frondoside A as a means of inducing apoptosis in human leukemia cells has not been described.

The authors in the patent and other scientific and technical literature found no indication of the use of extracts or individual substances from sea cucumbers as a means of inducing apoptosis in human leukemia cells.

The objective of the invention is the expansion of the arsenal of tools and pharmaceutical compositions capable of selectively stimulating apoptosis of leukemic cells.

The problem was solved by the use of triterpene glycosides (or mixtures thereof) from holothuria as agents inducing apoptosis in human leukemia cells.

Holoturia triterpene glycosides are cucumarioside A ₂ -2 (A ₂ -2) or cucumarioside A ₄ -2 (A ₄ -2), or a mixture thereof, as well as versoposide C (STC) or versoposide D (STD), or a mixture thereof.

These compounds are obtained by isolation and purification from natural raw materials. So, A ₂ -2 and A ₄ -2 were isolated from commercial, edible holothuria Cucumariajaponica, and STC and STD from holoturia Thelenota anax. The process of isolation of triterpene glycosides from holothurians is a standard procedure described, for example, by Avilov et al. [Avilov S.A. et al. The structure of four new triterpene glycosides from holothuria Cucumariajaponica II Chemistry of nature, compound. 1990. No. 6. S.787-792]. The essence of the method is as follows: the animals were crushed and extracted twice with hot ethanol; the combined extracts were evaporated to dryness on a rotary evaporator and the dry residue was chromatographed on a column of Teflon powder or other hydrophobic carrier, equilibrated in water. Inorganic salts and polar impurities were washed off the column with water, and the fraction containing glycosides was washed with 50% ethanol in water. Then the fraction was applied to a column of silica gel; eluent - chloroform-ethanol-water system (100: 100: 17). As a result, two main fractions were isolated: the first contained monosulfated glycosides, and the second contained more polar glycosides. Both fractions were re-chromatographed on silica gel, and then individual glycosides were isolated by high performance column chromatography (HPLC) using reverse phase columns. Usually, after using the column chromatography method, a mixture of glycosides A ₂ -2 and A ₄ -2 or versoposides C and D is obtained, and after using the HPLC method, individual glycosides are obtained.

It is known that cucumariosides A2 and A4, as well as versoposides C and D, have a hemolytic effect [Kalinin, VI et al. J. Nat. Toxins., 1992, 7, 17-30; Kalinin, VI et al. Toxicon, 1996, 34, 475-483], cytotoxic and antitumor activity [Anisimov MM et al. Problems of scientific research and development of the World Ocean: Abstracts. Doc. IV All-Union. Conf. Vladivostok, 1983. P.77-88; Agafonova, IG et al. J. Agric. Food Chem., 2003, 51, 6982-6986], antiviral and antifungal properties [Grishin, Y. et al. In 8th conference of young scientists on organic and bioorganic chemistry; Latvian Academy of Sciences, Institute of Organic Synthesis: Riga, 1991; p. 181; Maltsev I.I. et al. Chemical chemist. Zhurn. 1985, 79, 54-56; RU 2184556 C1, A61K 35/56, A61P 31/12, 07/10/2002], an inhibitory effect on Na ⁺ , K ⁺ -ATPase [Gorshkova, IA et al. Toxicon, 1989, 27, 927-936], with a radioprotective effect [RU 2141833 C1, 11.27.1999 .; Grishin, Yu. I.; et al. Radiobiology, 1990, 30, 556; Kovalevskaya, et al. In VI-th international congress "Actual problems of creation of new medicinal preparations of natural origin"; Saint Petersburg, 2002; pp.406-09], and immunostimulatory properties [Aminin, DL et al. J. Med. Food., 2001, 4, 127-135].

However, the new appointment of cucumariosides A ₂ -2 and A ₄ -2 and versoposides C and D as agents with the ability to stimulate apoptosis of human leukemia cells does not follow with obviousness from their known properties and was discovered by the authors for the first time.

The problem was also solved by the use of cucumarioside A ₂ -2 or cucumarioside A ₄ -2 or a mixture thereof, versoposide C or versoposide D, or a mixture thereof, for the preparation of a pharmaceutical composition that stimulates apoptosis of human leukemia cells.

Pharmaceutical compositions that stimulate apoptosis of human leukemia cells are prepared by mixing an effective amount of triterpene glycosides from holothuria with traditional pharmaceutically or pharmacologically acceptable excipients. The expression "pharmaceutically or pharmacologically acceptable" means molecular components and compositions that do not cause negative, allergic or other undesirable reactions when administered to an animal or person. The nature of the filler depends on the route of administration. For example, if oral administration is desired, a solid excipient may be selected, while liquid saline may be used for intravenous administration.

The technical result provided by the invention is the ability of compounds A ₂ -2 and A ₄ -2, as well as STC and STD or mixtures thereof to induce aloptosis of human leukemia cells, as well as the possibility of using these substances for the preparation of pharmaceutical compositions that stimulate apoptosis of leukemia cells person.

Studies have shown that triterpene glycosides of holothuria do not significantly affect normal cells of the human immune system (neutrophils). This reduces the risk of neutropenia that accompanies leukemia treatment with traditional anti-cancer drugs.

The invention expands the arsenal of tools and pharmaceutical compositions that stimulate apoptosis of human leukemia cells.

Figure 1 presents the dose-dependent cytotoxic effect of compounds A ₂ -2 and A ₄ -2 in relation to human leukemia cells HL-60, determined by MTT after 6 hours of incubation of cells with substances. Each point on the graph corresponds to the average number of living cells, expressed as a percentage ± SD (standard deviation from the mean) obtained from three independent experiments, three samples for each substance concentration in each experiment.

Figure 2 presents the dose-dependent cytotoxic effect of STC and STD glycosides in relation to human TNR-1 leukemia cells, determined by MTT after 24 hours of incubation of cells with substances. Each point on the graph corresponds to the average number of living cells, expressed as a percentage ± SD (standard deviation from the mean) obtained from three independent experiments, three samples for each substance concentration in each experiment.

Figure 3 presents the time-dependent induction of apoptosis by cucumariosides A ₂ -2 and A ₄ -2, taken at a concentration of 5 μM, in HL-60 leukemia cells, determined by flow cytometry. Each point on the graph corresponds to the average number of apoptotic cells in percent ± SD (standard deviation from the mean) obtained from three independent experiments.

Figure 4 presents the time-dependent induction of apoptosis with STC and STD glycosides taken at a concentration of 0.3 μM in HL-60 leukemia cells, determined by flow cytometry. Each point on the graph corresponds to the average number of apoptotic cells in percent ± SD (standard deviation from the mean) obtained from three independent experiments.

Figure 5 presents the dose-dependent induction of apoptosis by cucumariosides A ₂ -2 and

And ₄ -2 in HL-60 leukemia cells, determined by flow cytometry after 6 hours of incubation of substances with cells. Each point on the graph corresponds to the average number of apoptotic cells in percent ± SD (standard deviation from the mean) obtained from three independent experiments.

Figure 6 shows the effect of apoptosis induction by cucumariosides A ₂ -2 and

A ₄ -2, taken in concentrations of 0.5 and 1 μM, after 24 hours of incubation with HL-60 leukemia cells, obtained by flow cytometry staining of apoptotic cells with Annexin V-FITC and PI fluorescent dyes. The effect is presented as the number of apoptotic cells, expressed as a percentage (right lower quadrant - early apoptosis; right upper quadrant - late apoptosis). One of three independent experiments is presented.

Figure 7 shows the effect of apoptosis induction STC and STD taken at concentrations of 0.05-1.0 μM after 24 hours of incubation with HL-60 leukemia cells obtained by flow cytometry. The effect is presented as the number of apoptotic cells, expressed as a percentage (right lower quadrant - early apoptosis; right upper quadrant - late apoptosis). One of three independent experiments is presented.

On Fig presents the effect of the induction of apoptosis by cucumariosides A ₂ -2 and A ₄ -2, taken at a concentration of 5 μm, in various types of leukemic cells, determined by flow cytometry after 6 hours of incubation of substances with cells. Each “column” corresponds to the average number of apoptotic cells in percent ± SD (standard deviation from the mean) obtained from three independent experiments.

Figure 9 shows the effect of apoptosis induction STC and STD taken at a concentration of 0.3 μM in various types of leukemic cells, determined by flow cytometry after 24 hours of incubation of substances with cells. Each “column” corresponds to the average number of apoptotic cells in percent ± SD (standard deviation from the mean) obtained from three independent experiments.

Figure 10 presents the Tunnel test (TUNEL) for the induction of apoptosis in HL-60 leukemia cells treated with cucumariosides A ₂ -2 and A ₄ -2 at a concentration of 5 μM for 6 hours. One of three independent experiments is presented.

11 shows the effect of cucumariosides A ₂ -2 and A ₄ -2, taken at a concentration of 1 μM, on the cell cycle of HL-60 leukemia cells, determined by flow cytometry after 24 hours of incubation of cells with substances. The effect is presented as the number of cells in a given phase of the cell cycle, with the number of cells in the Sub G ₁ phase expressed as a percentage. One of three independent experiments is presented.

12 shows the effect of STC and STD taken at a concentration of 0.3 μM on the cell cycle of HL-60 leukemia cells, determined by flow cytometry after 24 hours of incubation of substances with cells. The effect is presented as the number of cells in a given phase of the cell cycle, and the number of cells in the Sub G ₁ phase is expressed as a percentage. One of three independent experiments is presented.

On Fig presents the effect of A ₂ -2 and A ₄ -2 at a concentration of 1 μM on the intracellular level of pro-caspase-3 in HL-60 cells. One of three independent experiments is presented.

On Fig presents the effect of STC and STD taken at a concentration of 0.3 and 1 μM, respectively, on the intracellular levels of procaspase-3, -8, 9, 12, as well as PARP and BID proteins in HL-60 cells. One of three independent experiments is presented.

The preparation of pharmaceutical compositions is illustrated by the following examples.

Example 1. Cucumarioside A ₂ -2 in an effective amount is mixed with an appropriate amount of a mixture of starch and gypsum and pressed into a tablet.

Example 2. The pharmaceutical composition is prepared as described in example 1, but cucumarioside A ₄ -2 is used as triterpene glycoside, and a mixture of sorbitol and calcium stearate is used as filler.

Example 3. The pharmaceutical composition is prepared as described in example 1, but as triterpene glycoside use versoposide C.

Example 4. The pharmaceutical composition is prepared as described in example 2, but as a triterpene glycoside use versoposide D.

Example 5. A mixture of glycosides, cucumariosides A ₂ -2 and A ₄ -2 in effective concentrations is dissolved in an appropriate amount of saline.

Example 6. The pharmaceutical composition is prepared as described in example 5, but as a mixture of glycosides using a mixture of versoposide C and versoposide D.

The study of biological activity

I. Materials and methods.

1. Accepted abbreviations.

A ₂ -2 - cucumarioside A ₂ -2; A ₄ -2. - cucumarioside A ₄ -2; STC - versoposide C; STD - versoposide D; mM is millimol / liter; µM - micromol / liter; µl - microliter; SD is the standard deviation from the mean; MTT - a method for determining the cytotoxicity of substances; Annexin V-FITC - fluorescent dye; PI - propidium iodide, fluorescent dye; TUNEL - a method for determining apoptosis; Sub-Gi is one of the phases of the cell cycle characteristic of cells in apoptosis; PARP and BID - proteins that are directly involved in the process of apoptosis in the cell; PBS - phosphate buffered saline; FBS - serum of bovine embryos; DMSO - dimethyl sulfoxide.

2. The cultivation of cells.

HL-60, THP-1, NB4 or K562 cells (ATCC, Rockville, MD) were cultured in RPMI-1640 medium containing 10% FBS, 100 μg / ml penicillin and 100 μg / ml streptomycin, in an atmosphere containing 5% CO ₂ , at 37 ° C, and were reseeded every 2 days.

3. Preparation of solutions of substances.

Basic (stock) glycoside solutions with a substance concentration of 20 mM were prepared in dimethyl sulfoxide (DMSO) (Fisher, USA), from which solutions of the desired concentration were obtained by dilution in a culture medium. The content of DMSO in dilute solutions did not exceed 0.5% in all experiments.

4. Determination of cytotoxicity.

3- (4,5-ethylthiazol-2-yl) -2,5-diphenyltetrazole bromide (MTT) (Sigma, St. Louis, MO, USA) was used to determine the cytotoxicity of the substances. HL-60 or THP-1 cells were seeded in a 96-well plate (1 × 10 ⁴ cells per well) and then treated with the indicated concentrations of cucumariosides A ₂ -2 or A ₄ -2. Then, an MTT solution (5 mg / ml) was added to each well and the cells were incubated at 37 ° C and 5% CO ₂ for 4 hours. The cell suspension was centrifuged at 1000 rpm for 5 min and the cells in each well were then extracted with 100 μl of dimethyl sulfoxide. The optical density of the solutions in each well was then determined on a flatbed spectrophotometer reader at 550 nm. The percentage of living cells was then calculated by the formula (OD _exp / OD _etrl ) × 100,

where OD _exp is the optical density of the experimental well, OD _ctrl is the optical density of the control well.

5. Determination of apoptosis by flow cytometry.

Cells (1 × 10 ⁶ ) were treated with glycosides at the indicated concentrations, incubated for a specified period of time, then washed with phosphate buffer (PBS) by centrifugation at 1000 rpm for 5 min. The cells were then treated to detect early and late apoptosis with the fluorescent dyes of Annexia V-FITC and propidium iodide (PI) in accordance with the manufacturer's recommendations. Cells (1 × 10 ⁵ -5 × 10 ⁵ ) were resuspended in 500 μl of binding buffer (Annexin V-FITC Apoptosis Detection kit. Medical & Biological Laboratory), 5 μl of Annexin V-FITC and 5 μl of PI were added and incubated for 15 min in darkness at room temperature. Then the cells (1 × 10 ⁴ ) were analyzed on a flow cytometer manufactured in the USA (Beckton Dickinson, Franklin Lakes, NJ) to detect early and late apoptosis.

6. Analysis by the TUNEL method.

HL-60 cells (1 × 10 ⁴ cells per well) were seeded in a 96-well plate and treated with A ₂ -2 or A ₄ -2 cucumariosides at a concentration of 5 μM for 6 hours. Then the cells were washed by centrifugation and fixed in 100 μl per well of freshly prepared 4% formaldehyde in PBS (pH 7.4) for 10 min. The resulting slides were incubated for 5 min with 0.1% Triton X-100, washed twice with PBS and dried. The TUNEL reagent kit (Roche, Basel, Switzerland) was used to detect apoptosis, in accordance with the recommendations of the manufacturer. Cells were incubated for 60 min at 37 ° C with a reaction mixture (50 μl per slide) containing fluorescein-12-dUTP in the presence of terminal deoxynucleotidyl transferase (TdT), with the green fluorescent label attached to free 3'-OH groups fragmented DNA. Then the samples were washed three times with PBS, treated with 500 μl of a buffer containing PI for 15 min, and analyzed using a fluorescence microscope (Carl Zeiss, LSM 510) at an excitation wavelength of 450-500 nm and a detection wavelength of 515-565 nm .

7. Cell cycle analysis.

Cells (1 × 10 ⁶ ) were treated with glycosides at the indicated concentrations, incubated for 24 hours, then washed with phosphate buffer (PBS) by centrifugation at 1000 rpm for 5 min. After this, the cells were suspended in PBS and fixed with cold 70% ethanol for 3 hours. Fixed cells were stained with 50 μg / ml PI (propidium iodide) containing 50 μg / ml RNase A at 37 ° C for 30 minutes. Cell DNA (10,000 cells per experimental group) was then analyzed using a cell cytometer.

8. Western blot analysis.

HL-60 cells (1 × 10 ⁷ cells in a Petri dish, 10 cm in diameter) were incubated with appropriate solutions (1 μM for glycosides A ₂ -2 or A ₄ -2; 0.3 or 1 μM for glycosides STC or STD) in 10 ml RPMI-1640 medium containing 10% FBS for 24 hours. Then the cells were washed with cold PBS (2 × 10 ml), suspended in a lysis buffer containing 20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1.0% Triton X-100, 50 mM NaF, 2.5 mM Na ₃ VO ₄ and protease inhibitors (1 mM PMSF, 20 u / ml aprotinin). Lysates (25 μg) were subjected to electrophoresis on 10% dodecyl sulfate-polyacrylamide gel, transferred to a nitrocellulose membrane, which was blocked for 1 hour at 25 ° C in blocking buffer (10 mM Tris-HCl, 0.15 M NaCl, 0.1% sodium azide and 5% skim milk) and incubated overnight at 4 ° C with the corresponding primary antibodies in dilution (1: 1000). The membranes were then incubated with secondary anti-IgG antibodies in a 1: 5000 dilution in blocking buffer. Signals of protein-antibody complexes were recorded by chemiluminescent luminescence.

II. The results of the study of the biological activity of cucumariosides A ₃ -2 and A ₄ -2, as well as versoposides C and D and their discussion.

All studied glycosides (or mixtures thereof) already in non-cytotoxic concentrations showed apoptosis-stimulating activity on several lines of human leukemia cells, which is confirmed by data on cytotoxicity studies, the study of apoptosis by flow cytometry and TUNEL, as well as data on the effects on the cell cycle and experimental data Western blotting. It was found that cucumariosides A ₂ -2 and A ₄ -2 or versoposides C and D induce apoptosis of human leukemia cells at optimal concentrations of 0.3-5 μM. The optimal time for exposure to tumor cells is 3-24 hours. Apoptotic changes (early and late apoptosis) in the cells were measured using fluorescent dyes of Annexia V-FITC and propidium iodide (PI). At the same time, a significant increase in the number of cells in the phase of the cell cycle Sub-G _{1 was} also observed, which is typical for cells in a state of apoptosis. In cells treated with cucumariosides A ₂ -2 and A ₄ -2 or versoposides C and D, DNA fragmentation characteristic of apoptosis was also recorded. The studied glycosides induced apoptosis in various leukemia cell lines, including HL-60 (acute promyelocytic leukemia), NB4 (acute promyelocytic leukemia), THP-1 (acute monocytic leukemia), K562 (chronic myelogenous leukemia).

Cytotoxic activity.

A ₂ -2 and A ₄ -2, as well as STC and STD, showed cytotoxic properties on HL-60 and THP-1 human leukemia cells. The cytotoxic effect was evaluated by the MTT method.

So, we studied the dose-dependent cytotoxic effect of A ₂ -2 and A ₄ -2 (see figure 1) and STC and STD (see figure 2) in relation to human leukemia cells HL-60 and THP-1, respectively . The data obtained indicate that A ₂ -2 and A ₄ -2 show high cytotoxic activity against human leukemia cells HL-60 after 6 hours of incubation with cells, within a concentration of 1-5 μM, with A ₂ -2 showing stronger effect than A ₄ -2. In a pair of STC and STD glycosides (see FIG. 2), STC has a stronger cytotoxic effect. So, with an STC concentration of 2.5 μM, only about 20% of THP-1 cells survive, and when exposed to cells of the same concentration of STD, about 70% of THP-1 cells survive. This is also evidenced by IC ₅₀ STC and STD in relation to THP-1 cells: 1.7 and 3 μM, respectively.

Assessment of apoptosis using flow cytometry

In-depth study of the cause of cytoxic changes caused by A ₂ -2

and A ₄ -2, as well as STC and STD in leukemic cells, were performed by flow cytometry. In this method, apoptotic changes in cells that occur under the influence of glycosides lead to the release of phosphatidylserine onto the external surface of leukemic cells, to which the protein Annexin-V FITC, covalently crosslinked with a fluorescent dye, selectively binds. The appearance among cells treated with glycosides of a population of cells bearing a green fluorescent label associated with the protein Annexin-V FITC, is evidence of the entry of cells into the phase of early apoptosis. After some time (6-24 hours), the cells undergo deeper changes, allowing the fluorescent dye propidium iodide to penetrate the cell, which leads to the staining of the components of the nucleus in red. As can be seen from the results shown in figures 3-9, A ₂ -2 and A ₄ -2, like STC and STD, induce apoptosis in various types of human leukemic cells. The percentage of apoptotic cells depends on the concentration of glycosides (Figs. 5-7), on the duration of exposure of the glycosides to the cells (Figs. 3, 4), on the type of leukemic cells (Figs. 8, 9), as well as on the chemical structure of the glycosides themselves (Figs. 3-9). Here, as well as in experiments on the cytotoxic effect, A ₂ -2 is more active than A ₄ -2, and STC is more active than STD, which is especially clearly seen in Figs. 8 and 9. From the same results ( Figs. 8 and 9) that the glycosides tested stimulate apoptosis in HL-60, THP-1, NB-4, and K562 human leukemia cell lines. The concentrations at which glycosides stimulate apoptosis in human leukemia cells are in the range of 0.3–5 μM, which depends not only on the structure of the glycoside, but also on the time of incubation of cells with substances. Thus, the studied glycosides A ₂ -2, A ₄ -2, STC and STD induce apoptosis in human leukemia cells already in non-cytotoxic concentrations. So, from the results presented in figures 5 and 6, it follows that the percentage of apoptosis caused by cucumariosides A ₂ -2

and A ₄ -2, taken at a concentration of 3 μM, and at an incubation time with cells of 6 hours (see FIG. 5), it is equal to the percentage of apoptosis caused by the same glycosides, but at a concentration of 1 μM and an incubation time of 24 hours ( see Fig.6). In general, the results presented in Figs. 3-9 demonstrate that A ₂ -2 and A ₄ -2, like STC and STD, are capable of inducing 15-90% apoptosis in various types of human leukemic cells within concentrations of 0.3-5 µM, with an exposure time of 3-24 hours and that the antileukemic properties of the studied glycosides, at least in part, can be explained by the induction of apoptosis.

TUNEL observation of apoptosis

Another evidence of the induction of apoptosis by the studied glycosides was obtained by the TUNEL method on the example of A ₂ -2 and A ₄ -2. (see figure 10). In this method, the fragmented DNA that appears in the cell during apoptotic transformations is stained with the FITC dye and gives a green fluorescent color, and the genomic DNA of the control, untreated, non-apoptotic cells is stained only with propidium iodide (PI) in red. HL-60 leukemia cells (FIG. 10), treated with A ₂ -2 and A ₄ -2 at a concentration of 5 μM for 6 hours, showed, in contrast to control cells, the inclusion of a green fluorescent label, indicating the presence of fragmented DNA and is a sign of apoptosis.

Cell Cytometry

An analysis of the cell cycle of HL-60 cells treated for 24 hours with solutions of A ₂ -2 and A ₄ -2 (Fig. 11) at a concentration of 1 μM and STC and STD at a concentration of 0.3 μM (Fig. 12) showed a significant increase in population cells in the phase of the cell cycle sub-G ₁ compared with control cells, which is an undoubted sign of the onset of apoptosis. And in this experiment, A ₂ -2 was more active than A ₄ -2, and STC was much more active than STD. So, the effect of A ₂ -2 led to the transition of 20.1% of the cells into the sub-G ₁ phase, while A ₄ -2 caused the transition of a smaller number of cells, 17.6% (Fig. 11). The difference in a similar effect on the glycoside STC and STD cells is much more significant: 36.7 and 2.8%, respectively (Fig. 12).

Evidence of stimulation of apoptosis by Western immuno-blotting.

In the late stages of apoptosis in cells, a set of intracellular proteins, enzymes, caspases are activated, which cause splitting of intracellular biopolymers. In normal cells, these proteins are inactive form, in the form of precursors - procaspases. In experiments on Western immuno-blotting performed on HL-60 leukemia cells, it was found that the studied glycosides A ₂ -2 and A ₄ -2 decrease the intracellular level of pro-caspase-3 protein (Fig. 13), while STC and STD lower the level of proteins procaspase-3, -8, -9, 12 in the cells (Fig. 14). This is due to the transformation of procaspases into an active form after treatment of leukemia cells with the studied glycosides. This experiment is another clear evidence of the stimulation of apoptosis by the studied glycosides and suggests that apoptosis induced by A ₂ -2 and A ₄ -2, as well as STC and STD in leukemic cells HL-60 and THP-1, is caspase-dependent.

Claims (3)

1. The use of triterpene glycosides from holothuria as a means of stimulating apoptosis of human leukemia cells. 2. The use according to claim 1, where the specified triterpene glycosides from holothuria are cucumarioside A ₂ -2, or cucumarioside A ₄ -2, or a mixture thereof, or versoposide C, or versoposide D, or a mixture thereof. 3. The use of triterpene glycosides from holothuria, as defined in any of claims 1 and 2, for the preparation of a pharmaceutical composition that stimulates apoptosis of human leukemia cells.

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