RU2723393C1 - Method of tumour cells radiosensitization - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to molecular biology and can be used to increase efficiency and targeted selectivity of effect on tumour cells using radiation exposure. Method for radiosensitization of tumour cells involves exposing a tumour cell to a substance which is a glycolysis inhibitor and simultaneously blocks cell proliferation, which is represented by glucosamine D hydrochloride in concentration of 10 mM.

EFFECT: invention provides higher radiosensitization and targeted selectivity of tumour cell exposure without affecting non-tumour cells by blocking cell proliferation in the radio-sensitive G1 phase of the cell cycle.

1 cl, 5 dwg

Description

The invention relates to the field of molecular biology, exploring new possibilities for increasing the efficiency and directed selectivity of exposure to tumor cells (death of tumor cells) using radiation exposure.

The present invention also relates to the field of radiosensitization of tumor cells to enhance the damaging effect of ionizing radiation on tumor cells.

The method may find application in the field of treatment of malignant tumors, i.e. chemotherapy treatment and radiation treatment. Characteristic disadvantages of radio and chemotherapy are: toxicity, significant side effects, low tumor specificity. The effectiveness of the action of radiosensitization can be achieved: either by reducing the intrinsic radioprotective capabilities of the cells (reducing the intracellular level of glutathione), or by suppressing the repair from radiation damage (repair inhibitors), or by creating unfavorable culturing conditions for the irradiated objects.

Antimetabolites are often used as radiosensitizers - substances structurally close to physiological metabolites and, as a result, capable of competing with them in various biochemical processes. An important factor in radiosensitization is the regulation of cell proliferation. Cells in different phases of the cell cycle exhibit different radiosensitivity. Cells exhibit less radiosensitivity in the S phase, and greater in the G2 / M phases and G1 phase.

In medical practice, 5-fluorouracil, which affects DNA synthesis and blocks cells in the S phase, is widely used as a radiosensitizer of tumor cells. It has been shown that 5-fluorouracil acts mainly on cells of the proliferative pool: P.Yu. Polyakov et al. Questions of Oncology, 1997, No. 5, Volume 43, p. 487-49 [1].

A known method of radiosensitization of tumor cells using cisplatin after radiation therapy: RF patent 2211716 [2]. Cisplatin refers to platinum-containing drugs that block cells in the S-phase of DNA synthesis. According to the description of the patent, the method allows to reduce the frequency of general and local radiation reactions, radiation injuries of healthy tissues.

The combined use of 5-fluorouracil and platinum-containing preparations (cisplatin, platidiam, plane tree) as a radiosensitizer of tumor cells is known. Here, the radiosensitization of tumor cells is carried out by blocking cells in the S phase of DNA synthesis. Patent RU No. 2088288 [3].

The disadvantage of these methods of radiosensitization, which affect the processes of DNA synthesis and blocks cells in the S-phase, is the low selectivity of action against tumor cells, since dividing cells of normal tissues are also sensitized.

It is also known that the increased dependence of tumor cells on glucose (due to their metabolic features) suggests that glycolysis inhibitors have antitumor activity: R. Dalirfardouei et al. Life Science, 2016; 152: 21-29 [4]. It has been shown that 2-DG significantly enhances radiation damage in cells with a high rate of glucose incorporation and glycolysis: Mesbahi A, et al. Breast. 2017; 33: 97-103 [5]. It is known that 2-deoxy-D-glucose (2-DG) selectively enhances radiation-induced damage to tumor cells without damaging normal cells: Farooque A1 et al., J. Cancer Res Ther. 2009 Sep; 5 Suppl 1: S32-5 [6].

A known method of radiosensitization of tumor cells with a combination of 2DG (as an inhibitor of glycolysis) and ionizing radiation more inhibits tumor growth and survival of mice than the separate effects of these agents: Coleman MC1, Asbury CR, et al. Free Radic Biol Med. 2008 .44 (3): 322-31, [7].

It is also known that 2-DG blocks cell proliferation in the G2 / M phase: Guillano N.Ya. Bondarev et al. Pathological physiology and experimental therapy. 2014.V. 58. No. 4, p. 78-85 [8].

A known method of radiosensitization of tumor cells, comprising treating the cells with doxorubicin (an antibiotic with antitumor activity), and 2DG - a glycolysis inhibitor that blocks cell proliferation in the G2 / M phase, in combination with ionizing radiation. The work shows an increase in the action of radiation in 1.14 and 1.16 times: Islamian JP., Asian Ras J Cancer Prev. 2015; 16 (18): 8431-8. [nine].

Thus, the above methods are based on the same mechanism of action on cells: blocking proliferation in the radiosensitive G2 / M phase of the cell cycle and the use of glycolysis inhibitors. However, the effectiveness of radiosensitization is insignificant and is within the statistical error in the dosimetric assessment of the radiation dose and can be explained by the variability in the sensitivity of tumor cell lines. Perhaps this is due to the fact that both ionizing radiation and 2DG block proliferation in the G2 / M phases of the cell cycle, and that the cytostatic effect of 2DG is offset by the large cytostatic effect of gamma radiation.

The technical effect of the claimed invention is to increase radiosensitization and directed selectivity of action on tumor cells without affecting non-tumor cells.

The task is to find a method that provides radiosensitization due to directed inhibition of tumor cell metabolism and block cell proliferation in the cell cycle phase, which does not coincide with radiation blocking of gamma radiation proliferation.

The technical effect is achieved by the fact that the proposed method of radiosensitization of tumor cells, which consists in exposing the tumor cell to a substance that is an inhibitor of glycolysis and at the same time blocking cell proliferation, where it is new that glucosamine D hydrochloride is used in a substance that blocks cell proliferation , which blocks cell proliferation in the radiosensitive G1 phase of the cell cycle, which does not coincide with radiation blocking proliferation of gamma radiation.

It is known that glucosamine D, as well as 2DG, inhibits glycolysis, reducing the activity of hexokinase. D-glucosamine is known to be toxic to several malignant cell lines and in vivo tumors at concentrations having little effect on normal cells: Zhang L, Liu W-S, Han B-Q, et al. Antitumor activities of D-glucosamine and its derivatives. Journal of Zhejiang University SCIENCE B. 2006; 7: 608-614 [10]. Studies of the molecular mechanisms of the antitumor activity of low glucosamine D concentrations have revealed changes in the activity of genes responsible for the survival and proliferation of tumor cells, in particular, the suppression of the STAT3 gene that stimulates the proliferation and survival of tumor cells: Chesnokov V1, Sun C, Itakura K. Glucosamine suppresses proliferation of human prostate carcinoma DU145 cells through inhibition of STAT3 signaling. Cancer Cell Int. 2009 Sep 10; 9:25 [11].

However, the effect of non-toxic concentrations of glucosamine D hydrochloride on the increase in the radiosensitization of tumor cells and the targeted selectivity of its effect (glucosamine) on tumor cells is not known.

The inventors experimentally discovered a targeted increase in the selective radiosensitization of tumor cells without damaging non-tumor cells with non-toxic concentrations of the glycolysis inhibitor glucosamine D hydrochloride.

The essence of the invention is based on the fact that glucosamine D hydrochloride, which inhibits glycolysis, is the main energy pathway for ATP production by tumor cells, which affects the energy-dependent repair processes of radiation-induced damage and leads to apoptotic death of tumor cells. Since non-tumor cells receive ATP through oxidative forforylation, treatment with glycolysis inhibitors does not practically change the level of radiation-induced cell death. As a result, it becomes possible to increase the effect of ionizing radiation without increasing the radiation dose.

To clarify the invention, the following graphic materials.

In FIG. Figure 1 presents the results of experiments to study the effect of 24-hour cell treatment with glucosamine D on the cell progression in a cycle (distribution of cells by phase of the cell cycle).

In FIG. Figure 2 shows histograms of one of three experiments evaluating the effect of gamma rays emitted during cobalt 60 decay on changes in the distribution of cells by DNA content (by the phases of the cell cycle) in the studied cell lines 24 hours after irradiation.

In FIG. Figure 3 presents the results of a cytometric analysis of the composition of the cell population 48 hours after irradiation in tumor cells HeLa G63, Hep G2 and endothelial cells ECV 304

In FIG. Figure 4 presents the results of experiments to assess the level of cells with DNA content <2s (apoptotic dying cells) after separate and combined treatment of 10mM glucosamine D and 7Gy cells with Co60 gamma rays.

In FIG. 5 (a, b) presents a morphological analysis of the composition of the cell population 48 hours after irradiation in HeLa G63 tumor cells (5, a); in endothelial cells ECV 304 (5, b).

Examples of specific implementation of the method

The method was tested on cervical epithelioid carcinoma cells of the HeLa G 63 line, Hep G2 hepatocarcinomas and human endothelial cells of the ECV 304 line. These cells were treated with a glycosylation inhibitor glucosamine D at non-toxic concentrations of 3 and 10 mM hydrochloride and then irradiated with gamma rays of cobalt-60 b at a dose of 7Gy and after 24 and / or 48 hours, a cytometric and morphological analysis of the composition of the cell population was performed.

Cell lines are cultured in the Eagle medium (Biolot company) with the addition of 10% cattle serum (Biolot company) and 100 units / ml penicillin-streptomycin (Gibco BRL) at 37 ° C. Cells were grown in penicillin vials with coverslips, which allowed for simultaneous morphological and cytometric analysis of the same cell population.

As a radiosensitizer, glucosamine hydrochloride (glucosamine HCl) is used, which is non-toxic, stable, and highly hydrophilic.

The irradiation of cells with Co 60 gamma rays was carried out at the Researcher PNPI named after B.P. Konstantinova. Dose rate 7Gy / min.

From the presented results (Fig. 1), 24-hour treatment of cells with glucosamine D leads to blocking the progression of the cycle and accumulation of cells in the G1 phase of the cell cycle, regardless of the type of cell.

Subsequent irradiation with gamma rays was carried out on a cell population synchronized in G1 phase, which allows to reduce variations in the cellular response due to different radiosensitivity of the phases of the cell cycle.

In FIG. Figure 2 shows histograms of one of three experiments evaluating the effect of gamma rays emitted during cobalt 60 decay on changes in the distribution of cells by DNA content 24 hours after irradiation. The histograms also show the average values of this distribution for 3 independent experiments for Hep G2 cells, and for 5 independent experiments for HeLa G63 and ECV 304 cells. From the histograms it is seen that irradiation of cells with 60 Go gamma rays at a dose of 7 Gy led to the accumulation of cells in the G2 / M (4c = 75-88%) cell cycle, regardless of the type of cell. At the same time, the proportion of cells with a DNA content of less than 2 s (apoptotically dying) increased slightly during cell fixation 24 hours after irradiation, which may indicate that this time is insufficient for the implementation of the radiation-induced apoptosis program. Obviously, the selective focus on tumor cells (HeLa, and Hep G2) is absent during separate treatment with glucosamine (in a non-toxic concentration) and gamma radiation during fixation 24 hours after treatment. With an increase in the time interval to 48 hours, the implementation of radiation-induced injuries in HeLa G63 tumor cells and in ECV 304 endothelial cells proceeds differently. On carcinoma cells, we see fragmentation of nuclear material characteristic of apoptotic death, and micronucleus formation is recorded in ECV 304 cells , which, as a rule, are formed due to the loss of genetic material during mitosis (Fig. 3 and Fig. 5a, 5b).

In FIG. Figure 4 presents the results of experiments to assess the level of cells with DNA content <2s (apoptotic dying cells) after separate and combined treatment of cells with glucosamine and 7Gy Co60 gamma rays. It can be seen from the diagrams that the sensitivity of the cells of the studied lines to ionizing radiation does not differ much, it is within confidence intervals. To this glucosamine concentration (10 mM), the sensitivity of carcinoma cells (both HeLa and Hep G2) is higher than the sensitivity of ECV 304 endothelial cells, but not as significantly as with combined exposure. The combined effects of glucosamine and gamma rays show significant differences between carcinoma cells and endotheliocytes. Preradiation incubation of HeLa G63 cells with glucosamine led to a significant increase in the proportion of cells with DNA content <2s (7 times compared with irradiation without glucosamine and 3 times compared with glucosamine without irradiation).

For Hep G2 cells, pre-radiation incubation with glucosamine led to an increase in the proportion of cells with DNA content <2s doubled (24.6 + 3.8%) compared with glucosamine (10.3 + 1.9%) and tripled compared with irradiation (7.6 + 0.5%).

Unlike HeLa G63 and Hep G2 cells, pre-radiation incubation of ECV 304 cells (non-tumor cells) with glucosamine did not significantly change the distribution of cell phases. The population of cells with DNA content <2 s differed from the untreated control (1.4 + 0.9%) and 5.2 + 0.4% (irradiation with glucosamine), but practically did not differ from separate treatment with glucosamine (5.0 + 3.2%) and irradiation (4.4 + 1.9 %).

It has been proved that glucosamine-induced radiosensitization has a selective focus on hepatocarcinoma tumor cell lines (Hep G2) and cervical carcinoma (HeLa G63), and does not affect the sensitivity of non-tumor ECV 304 cells. Since the manifestation of the effect is not detected immediately, but after 48 hours and later, it is obvious that glucosamine D inhibits the energy (ATP) dependent post-radiation processes of DNA repair, selectively enhances the radiation damage of only tumor cells.

The data presented are confirmed by experiments on cells of the cervical epithelioid carcinoma HeLa G 63 line, hepatocarcinomas Hep G2 line and human endothelial cells line ECV 304. It was shown that treatment of cells with non-toxic concentrations of glycosylation inhibitor glucosamine D hydrochloride disrupts the progression of the cycle by synchronizing cells to cell cycle in the G1 phase, which leads to an increase in apoptotic death of tumor cells. A significant increase in apoptosis is recorded in HeLa G 63 and Hep G2 cells when combined with glucosamine and gamma rays compared to the separate exposure of each of them, in contrast to ECV 304 cells.

Literature

1. P.Yu. Polyakov et al. Non-traditional approaches to radiation treatment of cancer patients. Questions of Oncology, 1997, No. 5, Volume 43, p. 487-49.

2. RF patent No. 2211716.

3. Patent RU No. 2088288.

4. Dalirfardouei R., Rfrimi G., Jamialahmadi K. Molecular mechanisms and biomedical applications of glucosamine as a potential multifunctional therapeutic agent. Life Science, 2016; 152: 21-29.

5. Rashidi MR, Mesbahi A, Mohammadzadeh M et al. Targeted superparamagnetic nanoparticles coated with 2-deoxy-d-gloucose and doxorubicin more sensitize breast cancer cells to ionizing radiation Breast. 2017; 33: 97-103.

6. Farooque A1, Afrin F, Adhikari JS, Dwarakanath BS. Protection of normal cells and tissues during radio- and chemosensitization of tumors by 2-deoxy-D-glucose. J Cancer Res Ther. 2009 Sep; 5 Suppl 1: S32-5.

7. Coleman MC1, Asbury CR, Daniels D, et al. 2-deoxy-D-glucose causes cytotoxicity, oxidative stress, and radiosensitization in pancreatic cancer. Free Radic Biol Med. 2008.44 (3): 322-31.

8. Gigliano N.Ya., Bondarev G.N., Konevega L.V., Noskin L.A., Zhurishkina U.V., Alchinova I.B. Possible mechanisms for the selective action of glycolysis inhibitors on endotheliocytes and human carcinoma cells. Pathological physiology and experimental therapy. 2014.V. 58. No. 4, p. 78-85.

9. Islamian JP, Aghaee F, Farajollahi A, et al. Combined Treatment with 2-Deoxy-D-Glucose and Doxorubicin Enhances the in Vitro Efficiency of Breast Cancer Radiotherapy. Asian Ras J Cancer Prev. 2015; 16 (18): 8431-8.

10. Zhang L, Liu W-S, Han B-Q, et al. Antitumor activities of D-glucosamine and its derivatives. Journal of Zhejiang University SCIENCE B. 2006; 7: 608-614.

11. Chesnokov V1, Sun C, Itakura K. Glucosamine suppresses proliferation of human prostate carcinoma DU145 cells through inhibition of STAT3 signaling. Cancer Cell Int. 2009, Sep 10; 9:25 a.m.

Claims (1)

The method of radiosensitization of tumor cells, which consists in exposing the tumor cell to a substance that is an inhibitor of glycolysis and at the same time blocking cell proliferation, characterized in that glucosamine D hydrochloride at a concentration of 10 mM is used as a substance that blocks cell proliferation.

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