RU2464974C1 - Means for treatment of malignant ascitic tumours - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and represents application of dopamine as cytotoxic means for treatment of malignant ascetic tumours.

EFFECT: invention ensures extension of arsenal of means for treatment of malignant ascetic tumours and non-toxic for normal cells and in general for entire organism.

3 dwg

Description

The invention relates to medicine and relates to an antitumor agent intended for the treatment of malignant (malignant) ascites.

Malignant (malignant) ascites is a pathological accumulation of fluid in the abdominal cavity that develops as a result of tumor damage to the peritoneum. Ascites causes significant discomfort and leads to various functional disorders in patients in advanced stages of cancer and is a complex clinical problem. Malignant ascites is approximately 10% of all cases of ascites and most often develops in cancer of the breast, ovaries, stomach, pancreas and colon.

Currently, there is a huge arsenal of chemotherapeutic drugs used to treat malignant diseases of various groups: alkylating drugs, cytostatics, antimetabolites, antitumor antibiotics, alkaloids and other biological active substances of plant origin, as well as enzyme and hormonal drugs (Mashkovsky M.D. Medicines M., Medicine, 1984, part 2, pp. 425-475). However, despite the successes achieved in the chemotherapy of malignant tumors, the search for new substances with an antitumor effect continues, since all available antitumor drugs, as a rule, also affect normal tissues, have high toxicity, and cause side effects associated with or not associated with the main mechanism suppression of cell reproduction: nausea, vomiting, loss of appetite, diarrhea, a decrease in the number of leukocytes, platelets and red blood cells in the blood, stomatitis, cardiovascular disorders A temporary loss of hair, and others. In some cases, this limits the dosage or even forces you to pause or stop treatment.

The closest in technical essence to the claimed is the compound jasplakinolide (see Jasplakinolide - An Actin-Specific Reagent that Promotes Actin Polymerization, Methods in Molecular Biology , 2001, Volume 161, Part III, 109-120) - a natural product secreted from sea sponges and having antifungal and anticancer activity. Jasplacinolide binds to a protein that is responsible for cell stability and is included in the cell division mechanism, which is promising for the development of new antitumor drugs.

The objective of the invention is to expand the arsenal of tools suitable for the treatment of malignant ascites tumors and non-toxic to normal cells and the whole organism.

It was experimentally established that to solve this problem, the long-known substance dopamine is promising.

Dopamine - 3,4-dioxiphenylethylamine, oxytyramine, C ₆ H ₃ (OH) ₂ CH ₂ CH ₂ (NH ₂ ), an intermediate product of catecholamine biosynthesis resulting from decarboxylation of dioxiphenylalanine (DOPA). A number of organs and tissues (liver, lungs, intestines, etc.) contain mainly dopamine. Along with adrenaline and norepinephrine, small amounts of dopamine are secreted by the adrenal glands, which indicates a possible independent hormonal function of dopamine. In the central nervous system, dopamine is found mainly in the motor centers, performing the function of a mediator. Dopamine has a number of pharmacological properties characteristic of hormonal and adrenergic substances. However, the possibility of using it as a cytotoxic agent for the treatment of malignant ascites has not been described.

The technical result achieved by the implementation of the invention is the discovery of a new non-obvious property of a known substance - dopamine. The technical result is achieved by the fact that the known dopamine of the general formula C ₆ H ₃ (OH) ₂ CH ₂ CH ₂ (NH ₂ ) is used as an agent for the treatment of malignant ascites.

The possibility of using dopamine for the treatment of malignant ascites has been confirmed on mouse Ehrlich ascites carcinoma. It was shown that a weekly course of daily intraperitoneal injections of dopamine (DA) with a concentration of 10 ^-2 M and 10 ^-1 M reduces, respectively, 10 and 30 times in comparison with the control (saline injection) the total amount and decreases by 27% and 59 % cell diameter in mouse Ehrlich ascites carcinoma.

The invention is illustrated graphic materials, where

figure 1 presents the effect of DA on the morphology of cells of transplantable Ehrlich ascites carcinoma (AKE), detected in a light microscope (at magnification × 50) on the 6th day after the introduction of dopamine into the abdominal cavity, as described in the example, where AB is a view tumor cells from ascites of intact (A, B) and control (B) mice on smears (A, B) and in the histological section (C). G-D is the type of tumor cells after exposure to DA with a concentration of 0.017 M (G), 0.17 M (D) and 10 ^-2 M (E) on smears (G, D) and in the histological section (E). Scale 20 μm (A-B), 15 μm (D) and 10 μm (D, E).

Figure 2 presents morphometric data on the number and size of cells in the ascites of intact, control and experimental mice, where A is the number of tumor cells, B is the diameter of tumor cells, * is a significant difference from control at P <0.05, ** - a significant difference from the control and from experimental values obtained under the influence of DA with a lower concentration at P <0.05.

Figure 3 shows the effect of DA on the ultrastructure of AKE cells detected in a transmission electron microscope (at magnification × 10,000), where A-B is the type of tumor cells from ascites of intact (A, B) and control (C) mice in ultrathin sections. The insets (a, c) show images of cell areas showing partial resorption of the contents of the adenogranule (a) and vacuolar cell dystrophy (c). GE - the type of tumor cells after exposure to DA concentration of 10 ^-1 M. Designations: MB - microvilli, C - cytosol, I - core, M - mitochondria, LC - fat drop (adenogranule), (arrow indicates the beginning of resorption of the contents of the granule ) KS - cortical layer, MU - moire pattern. Y — angularity, CoC — annular lamella, PV — phagocytic vacuole. The scale bar in Fig. 3A, a, B, D, d, D, E = 1 μm, in Fig. 3c = 0.1 μm.

Example

In the experiments, 140 male outbred mice of the SHK line were used, about 3 months old, weighing 28-36 g, 20 of them for passaging the tumor. Work with animals was carried out in accordance with internationally recognized rules and ethical standards. The object of the study was the ELD strain of transplantable Ehrlich ascites carcinoma (AKE). Tumor inoculation was performed by injection into the abdominal cavity of the mouse 10 ⁶ AKE cells in 0.1 ml of physiological saline (RF) for infusion. The size of the inoculum was chosen in preliminary experiments, taking into account the previously described methodology for working with AKE (E.V. Inzhevatkin. Workshop on experimental oncology as an example of Ehrlich ascites carcinoma: Method. Development. Krasnoyarsk. 2004. 10 pp.) And the results of previous experiments conducted by us in vitro on cell cultures (Moshkov D.A., Abramova M.B. et al. // Bull. experimental biology and medicine. 2010. V.149, No. 3, S.335-339).

Dopamine (DA) was injected intraperitoneally in mice with inoculated AKE in a volume of 1 ml 18 hours after tumor inoculation and then, DA was injected for 6 days 2 times a day with an interval between injections of 5 hours. For this, a pharmaceutical preparation DA from ampoules containing a 4% (or approximately 0.2 M) solution with metabisulfite (Pedrosa R., Soares-da-Siva P. et al. // Br. J. Pharmacol. 2002. Vol. 137, No. 8. P.1305-1313, OJSC “Biochemist”, Russian Federation and Orion, Pharma, Finland), diluted with pharmacy physiological saline (RF) for injection to working concentrations of 0.02 M, 10 ^-1 M, 10 ^-2 M or undiluted. Control mice were injected simultaneously with DA injection of equal volume RF. Some of the mice with the inoculated tumor were left intact until the end of the experiment. At the end of the experiments, the mice were sacrificed by the cervical dislocation method, ascites fluid was removed into penicillin vials; Samples were taken to count the number of cells using the Goryaev’s camera, and the remaining ascites were used for morphological analysis of cells in smears, in histological and ultrathin sections. Statistical processing of the results was carried out in Microsoft Excel (Microsoft Office 2003). The significance of the data differences was determined using the Student t-test. Quantitative data are represented by arithmetic mean ± error of the mean at a significance level of the confidence interval of 0.05.

Counting cells in ascites revealed (Fig. 2) that in the intact and control groups of mice, the number of malignant cells in ascites was 10 times higher than in the experimental groups receiving DA injections at a concentration of 0.02 M, 10 ^-2 M, and more than 25 times more than in mice that received injections of DA at a concentration of 10 ^-1 M, or undiluted. Compared to intact and control mice, the size of cells isolated from ascites in experimental mice, when measured on smears and in histological sections, turned out to be a quarter and a half smaller, depending on DA concentrations. It is known that the change in the volume of AKE cells is directly due to the degree of polymerization of the cytosolic actin and the formation of the F-actin cytoskeleton (Pedersen SF, Hoffmann EK et al. // Comp Biochem Physiol A Mol Integr Physiol. 2001. Vol.130, N.3. P. 385-399). Cytological (histological, ultrastructural) studies (Figs. 1, 3) confirmed that dopamine causes actin polymerization and cytoskeleton disruption in malignant cells.

Analysis of the histological structure of AKE cells detected on total preparations (smears) and in thick sections shows that the tumor cells have a rounded shape and approximately the same size (figure 1). Tumor cells of intact and control animals on smears have clear contours, their cytoplasm is dense without noticeable vacuoles or inclusions, control preparations were characterized by a slightly larger proportion of damaged cells than intact cells, the reason for which was not clarified. In slices, they have somewhat blurry contours, different densities and more variable sizes; all this depends on the cut level of individual cells. With all other conditions being equal, the cells of mouse AKE exposed to DA, both on smears and in sections, visually have a noticeably smaller diameter, and it is more variable within one frame.

According to the results of electron microscopic studies (figa, B), the ultrastructure of ascites cells taken from intact and control mice is identical to the previously described ultrastructure of cells of the classic "wild" strain of AKE ( Roth J., Li WP, Knibbs RN et al. // Proc Natl Acad Sci USA . 1994. Vol. 91, N.24. P.11353-11357).

As can be seen from FIGS. 3A, B, the surface of rounded or markedly angular tumor cells of intact mice around the entire perimeter is covered with partially deformed narrow and short microvilli. Immediately beneath the plasma membrane is a narrow cortical layer composed of parallel actin filaments. The cortical layer is uneven, in the form of islands, between them the cytosol goes directly to the membrane. Occasionally, small phagocytotic vacuoles are found under the membrane. The rest of the cytoplasm is occupied by numerous mitochondria of the orthodox conformation, with longitudinal or transverse cristae and a light matrix, very rare fragments of single cisterns of the rough reticulum and countless free-lying ribosomes. One of the identifying signs of AKE cells is the rounded osmiophil adenogranules (lipid droplets), the rudiment of secreted mammary epithelium, from which this type of cancer cell originated. Their full or partially filled or empty form (Fig. 3, insert a) indicates the inclusion of granules in the metabolism of carcinoma cells. In the nuclei of intact AKE cells, chromatin is highly condensed, the nucleoli are very large. The ultrastructure of the ascites cells of the control and intact mice is identical. The main feature of both of them is that they do not contain a noticeable F-actin cytoskeleton. This is clearly seen in living cells according to ultrastructural characteristics, but partially damaged cells, in which the matrix is clearly visible due to the loss of part of the ribosomes. The cytoplasm of such cells undergoes strong vacuole dystrophy and actin filaments of the cytoskeleton are present only in the cortical layer (Fig. 3B, insert c).

Cancer cells subjected to periodic exposure to DA had a different look. Microvilli are damaged, become significantly wider and longer, or acquire a mushroom shape with a pronounced thin stalk and a wide round head, large vacuoles are often located inside them, indicating an increased phagocytic activity of cells (Fig. 3G, D). The cortical layer becomes much thicker than in the control. The size and osmiophilicity of adenogranules decreases, almost all of them have traces of resorption of the contents, mitochondria acquire a condensed conformation, with swollen cristae and a dark matrix, the ribosomes for the most part disappear, and the cytosol as a whole is substantially clarified. It is clearly seen that the entire thickness of the cytoplasm from the plasma membrane to the nucleus is filled with a dense network of actin filaments (Fig. 3 insert g, D). In addition, in the cytoplasm of cells, the formation of moiré patterns is observed (Fig. 3E), the same as in cultured cells after the chronic action of DA (Parnyshkova E.Yu., Lavrovskaya V.P. et al. // Morphology. 2011. T. 140, No. 6. 00-00), caused by uneven condensation of the cytoplasm due to the mass polymerization of G-actin under the influence of DA. Cell nuclei do not change from the action of DA, but apoptotic bodies are often found instead of nuclei, indicating increased cell death. Significant changes in the surface structure (G), filling the entire space between the plasma membrane and the nuclear membrane with a network of actin filaments (g), the emergence of mushroom-like microvillas (D) are observed.

Thus, it was found that DA in vivo exerts a cytotoxic effect on AKE cells, similar to the previously identified effect on cultured fibroblast-like and cancer cells in vitro. The ultrastructural features of AKE cells exposed to DA indicate that the mechanism of its cytotoxicity consists in interaction with cytosolic G-actin, judging by the similarity of the effects of DA on AKE, on cultured cells of various types and on model systems, including isolated G -actin (Moshkov D.A., Pavlik L.L., Shubina BC, Parnyshkova E.Yu. et al. // Biophysics. 2010. V. 55, No. 5, S.850-856).

In particular, a noticeable deformation of microvilli and an expansion of the cortical layer, formed mainly by filamentous actin, the appearance in the cytosol of an extensive network formed by filaments, which we identify as actin filaments by their diameter and localization in places previously occupied exclusively by G-actin, as well as discovered for the first time a significant reduction in cell size under the influence of DA.

Animal experiments have shown that the oncotherapeutically effective concentration of DA for AKE inoculated in animals is an order of magnitude higher than that considered lethal for AKE cells in culture, possibly due to the volumetric nature of AKE and the two-dimensionality of cell cultures grown in plates. Further observation of a group of animals that received dopamine only for 6 days (since the study did not set the goal of curing the animals) at a concentration of 10 ^-1 M or undiluted showed that they lived 2 months longer than control animals.

All the above shows that dopamine, which is a natural substance synthesized in the body, can be considered as a prototype of a new class of compounds (Takeuchi H., Ara G. // Cancer Chemother. Pharmacol. 1998. Vol.42, N.6. P. 491-496), possessing anticancer activity, acting on the basis of a direct effect on G-actin as a therapeutic target of cells, the pathologically high content of which is characteristic of cancer cells, in particular, ascites tumor cells.

Claims (1)

The use of dopamine as a cytotoxic agent for the treatment of malignant ascites tumors.

Images