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16ANTITUMOR AND ANTICARCINOGENIC ACTIVITY OF
THE COMBINED DRUG FROM PROBIOTIC MICROORGANISMS AND PROPOLIS FRACTION ON THE ANIMAL MODEL OF COLON ADENOCARCINOMA
Ogay D. K., Kutliyeva G. D., Miralimova Sh. M., Elova N. A., Enikeeva Z. M.
Republic of Uzbekistan, Tashkent, Institute of Microbiology of AS RUz, Republican
Oncological Scientific Center of Ministry of Health
Abstract. Propolis and its compounds has been the subject of many studies due to their antimicrobial and antiinflammatory activity; however, it is now known that they also possess antitumor properties. It has been studied anticarcinogenic activity of the combined drug from living cells of L. rhamnosus 925 ak, P. avidum 1 and propolis in concentration of 1 and 2.5 % in mice BALB / C with a transplanted tumor strain of colon adenocarcinoma "Akatol". The drug with content of 1 % and 2.5 % of the propolis fraction has both an anticarcinogenic effect and expressed antitumor activity.
Keywords: probiotics, Propionibacterium, lactobacteria, colon adenocarcinoma, propolis, anticancer activity
Propionibacterium is a group of bacteria that renders a high antitumor and anticarcinogenic activity. As far back as in the 60's of the last century, data were obtained displaying a slowing-down of the growth of tumors (including malignant tumors) due to the reinforcement of the organism's defense response. Propionibacterium avidum KP-40 also manifested antitumor activity due to stimulation of nonspecific lymphocytic and macrophage function. It was shown that at infusive administration to humans at presurgical period 10 mg of killed propionibacterial cells after resection of the intestinal carcinoma (54 patients), it was observed a decrease in the percentage of repeated tumor formation [4, 19, 20]. The anticancer property of R. avidum KR-40 at monofactorial treatment of patients with cancer was established by Pulvever et al., in 1994.
A marker of antitumor activity of P.avidum KP-40, according to data of Peters et al., can be neopterin, found in blood and urine of experimental animals and of humans. It was noted that an elevation of the content of neopterin correlates with the stimulation of macrophage activity and the enhancement of antitumor activity [16]. We showed influence of killed cells of the local P. avidum 1 strain on the inhibition of tumor growth (by volume 74.5 %) of the transferred tumor strain of adenocarcinoma of the large intestine - "Akatol" [18].
One of the possible mechanisms of antitumor activity is a synthesis of short-chain fatty acids by microorganisms. Many facts have been obtained that testify the ability of volatile fatty acids to influence on the expression (activity) of genes in enterocytes (the general name for several intestinal epithelial cells) by triggering a cascade of intracellular reactions leading to the programmed death of a tumor (cancer) cell. Specifically, in a number of works [4, 12, 13, 14] it has shown that propionate and acetate synthesized by propionibacterium (P. acidipropionici and P. freudenreichii) are able to induce apoptosis of colorectal carcinoma cells. Oily, propionic, lactic acid act on many cellular regulators participated in the differentiation of large intestine epithelium [12, 22]. Short-chain fatty acids are a main source of energy and regulator of colonocyte functions, maintaining intestinal homeostasis (healthy intestinal mucosa). A protective role of butyric acid in relation to appearance and growth of a colon cancer is described in works of Hung et al [11]. Probiotic cultures, specifically Lactobacillus casei, apart from lactic acid, forms aromatic substances and short-chain fatty acids, so they can be considered as promising antitumor agents. Of great interest are anticancer properties of honey, especially propolis. Propolis in large quantities contains various anticarcinogenic flavonoids and lignans, as well as phenolic aldehydes, terpene compounds, coumarins, amino acids, aromatic acids, phytosterols. Anticarcinogenic effect of propolis, if more specifically - properties of flavonoids (quercetin) - is noted in many scientific studies conducted on animals. Investigations carried out on cell line cultures that cause cancerous tumors have facilitated identification as an inhibitor of the tumor process of the caffeic acid derivative. In a number of animal studies, propolis effectively prevented the development of lung cancer.
In experiments on cell culture propolis and its active components suppressed growth and caused apoptosis of breast, prostate, lung, cervix uteri, colon, pancreatic, liver, oral cavity, brain,
melanoma, and human leukemia cells. In an experiment on the mammary adenocarcinoma cell line [15], it has been established that flavonoids, which are biologically active components of propolis, have anti-cancer properties. It has been studied effect of Egyptian propolis on tumors in mice induced by the cell line of Ehrlich ascites carcinoma. Egyptian propolis possesses a strong inhibitory activity against tumors [5].
Components, showing antiproliferative activity to cancer cell lines have been isolated from Netherlands propolis; two new flavonoids with antiproliferative activity have been isolated from Chinese propolis [12]. Chia-Nan Chen et al., determined that two phenylflavonone (propolin A and propolin B, C) isolated from propolis of Taiwan induce apoptosis in human melanoma cells and significantly inhibit xanthine oxidase activity [5]. As well as substances, extracted from Taiwanese (propolin N, propolin G), suppress the growth of lungs and brain cancer cells [13, 19]. Russo et al studied antioxidant activity of propolis obtained in central Chile. Natural compounds were tested on antiproliferative activity to epidermal cancer cells of the oral cavity (CB), on Caco-2 (adenocarcinoma cells of large intestine) and DU-145 (androgen-sensitive carcinogenic cells of prostate). Obtained results showed that Chilean propolis possesses such biological properties as removal of free radicals and inhibition of tumor cells growth [21]. Natural cytotoxic substances, contained in propolis (specifically attacking cancer cells) - artepillin C and diterpenoid clerodane - allowed to get results respecting to the growth of malignant tumors. Artepillin C, isolated from Brazilian propolis, suppresses the cytotoxic effect and growth of tumor cells in vitro and in vivo [26]. In addition, propolis renders a positive biological effect on the immune system. First of all, this refers to macrophages, as well as the protection of cells from cancer metastases. Investigations have shown that artepellin C activates the immune system and has direct antitumor activity. According to Storcin et al. (2007) anticancer properties of propolis are explained by the high immunostimulating effect, substantially due to an increase in the activity of natural killer cells against tumor cells. Antitumor mechanism can be mediated by preventing oxidative damage and inducing apoptosis [25]. Due to the high level of natural "protective substances", propolis provides a deeper understanding of cancer nature, as well as approach to its treatment.
Aim of this work is to investigate antitumor and anticarcinogenic effects of a combined drug, composing of Propionibacterium avidum 1 (living cells), Lactobacillus rhamnosus 925 ak with different quantitative content of propolis alcohol fraction (1 and 2,5 %) at administration to mice BALB/c.
Materials and methods. Preparation of the drug and its microbiological characteristics. Drugs consist of microorganisms P. avidum 1, L. rhamnosus 925 ak and different concentrations of spirituous fraction of propolis. "Lactopropolis 1" contains at least 107 CFU/ml cultures of L. rhamnosus 925 ak and P. avidum 1 (in 3:1 ratio) and 1 % spirit fraction. Drug contains 3-5 mg of propolis. Accordingly, "Lactopropolis 2" also contains not less than 107 CFU/ml cultures L. rhamnosus 925 ak, P. avidum 1 and 2,5 % spirituous propolis fraction. In this drug, the content of propolis is 10-15 mg. Additional substances: skim cow milk, sucrose, gelatin.
Investigation of anticancerogenic (antitumor) activity of the drug carried out according to methodical recommendations [1]. Drug was examined on a transferred tumor strain of mice — adenocarcinoma of colon (AKATOL). Experimental animals were in a vivarium condition on a normal diet. Animals were divided into 3 groups of 21 mice in each, in all groups the drug was administered 10 days before tumor inoculation. The first group received a drug containing 1 % propolis fraction in 1 vial 3 times a day for 10 days; the second group received a drug containing 5 % propolis fraction in the same regime. The third group of animals was injected 1 ml of physical solution three times for 10 days. 10 days after the start of the drug administration, the animals were transferred with tumors. On the 7th, 14th and 21st days after the tumor was inoculated, 7 animals from each group were killed, take into account alteration of animals weight (without taking into account the tumor mass), size and mass of the tumor, liver weight, and spleen was taken for immunological control.
Percentage of inhibition of tumor growth (% of ITG) by weight was determined by formula: % ITG = (Bk-Bo) / Bk x 100, where Bk is the average tumor mass in the animals of the control group, BO is the average tumor mass in the animals of testing group.
% of ITG on volume: ITG % = (Vk - Vo) / Vk x 100, where Vk — is average tumor volume in the animals of control group, VO — is the average tumor volume in the animals of the test group, calculated by formula: V = a-b-c where a, b and c are the length, width and height of tumor node in mm (the average tumor volume in the group V is taken into account).
It was also calculated efficacy index (IE), which is determined by ratio BO - the average tumor mass in the animals of testing group to Bk - average tumor mass in animals of control group: IE = BO/Bk. IE shows, how many times mass of tumors in the experimental group decreases compared to the control group.
Statistical processing of obtained data was carried out by Student-Fisher method in the modification of M. D. Mashkovskiy on 95 % significance level.
Drug of liver and spleen homogenates for immunological investigations. After sacrifice, spleen and liver of animals were cut out. Spleen was placed into a homogenizer, added 2 ml of physical solution and homogenized until a homogeneous liquid will obtained. Liquid (spleen homogenate) was collected in a washed with a solution of heparin (12,5 ml physiological solution+0,5 ml heparin) tube. Separately, liver of the killed mouse was placed into a homogenizer, 2 ml of physiological solution was added and triturated 2-3 min. Resulting liquid (liver homogenate) was collected in a test tube and used for further inoculation.
Determination of immunological parameters. For determination of activity of natural killer cells to liver homogenate in a volume of 1,0-1,5 ml, 0,5 ml of heparin and 12,5 ml of physiological solution were added. Isolation of lymphocytes from spleen homogenate was carried out according to the methodical recommendation of Zalyalieva M.V. "Methods for estimation of subpopulations of peripheral blood lymphocytes in human" (2004). In our experiments we used CD16 antigen, expressing on normal killers, neutrophils, macrophages. It was used a method of indirect rosette formation. Principle of the method is based on the ability of antigen-antibody complex, in which erythrocytes (indicator particles) are loaded with monoclonal antibodies by means of chlorine chromium and mixed with lymphocytes.
Sensibilized erythrocytes bind to cells, expressing on the surface of the appropriate antigens. As a result, rosettes are formed, consisting of a centrally located lymphocyte surrounded by red blood cells. Reaction was recorded using a light microscope, counting a number of dead and living cells in each socket per 100 cells. Dead cells are dyed pink with ambiguous boundary, and living lymphocytes do not stain. Results are expressed as a percentage by formula: % SD + cells = (AB)/(100-B) x 100 %, where A are the dead cells in the experiment (arithmetic mean of 3 parallel sockets). B - % of dead cells in the control (arithmetic mean of 3 parallel sockets).
Results and discussion. In animals of control group, all tumors were transferred to the end of the 7th day of experiment, average tumor mass attained 273,8 mg and its volume was 0,149 cm3 (Table 1). Body weight of animals increased in comparison with the initial mass by 36,2 %. On the 14th day of experiment there was also a 100 % vaccination, average volume of tumors reached 10,344 cm , their average mass was 2,259 g. Weight of animals is increased by 16,06 %. In comparison with the group, sacrificed on 7th day, the weight of liver increased by 29,7 %. On the 21st day of experiment there was also a 100 % vaccination, average size of tumors reached 13,930 cm3, their average mass was 3,766 g. Weight of animals decreased by 19,37 %. Compared with the group, sacrificed on 7th day, weight of liver was less by 18,3 %.
In the first research group, where the drug contained 1 % of the propolis fraction, after 7th days of the grafting there was a decrease in the vaccination by 57 %, and a decrease in tumor growth, tumors were less in mass and in volume than tumors in the control group, in the 4th animals tumors did not appear. There was a significant increase in the body weight of animals to the 7th day in relation to the initial mass (to 37,7 %), but average weight of liver at 19,6 was lower than in the control animals (Table 2). % of ITG 76,32 on weight and 79,87 % on volume, IE 4,23 (on weight) and 4,96 (on volume). The group, sacrificed in 14 days after the grafting, also it was observed decrease in grafting by 43 %, and tumor growth was more pronounced (by 7,43 %) than in 7th days. Three animals had no tumors to the 14th day. Body mass of animals increased compared to initial body mass (on 13,2 %) and the average weight of liver increased up to 8,8 % compared to control animals - tumor carriers. % of ITG was 83,75 % by weight and 88,04 % by volume. IE was 6,14 (by weight) and 8,36 (by volume). Body mass of animals by the 21st day in relation to the initial mass increased to 19,5 %, average weight of liver to 30,84 % in comparison with control animals - tumor carriers. On the 21st day, the highest reduction in grafting was observed at 71 %, % of ITG was 67,87 % (by weight) and 54,56 % (by volume), thus their IE on mass was 3,11 and by volume 2,20, i.e. on average tumors were 2.2 times less than in the control.
Table 1. Parameters of the control group of mice BALB/c after grafting by ACATOL strains
Time after Animals Quantity without tumors Body mass Average tumor volume, cm3 Liver Tumor
grafting quantity At the beginning At the end mass, g mass, mg
7 day 7 0 20,5 27,9 0,149 1,31 273,8±44
14 day 7 0 21,60 27,92 10,344 1,70 2259±56
21 day 7 0 25,50 23,0 13,910 1,07 3766±980
In the second research group using drug containing 2,5 % propolis, in 7 days after grafting, a decrease in the grafting by 43 % and it was observed a decrease of tumor growth. In this group, by the 7th day, the tumors were less in mass and in volume than tumors in the control group, tumors did not appear in 3 animals. Slightly increased body weight of animals relative to the initial mass (by 1,6 %),
average liver mass was 6,87 % lower than in control animals. % of ITG was 84,98 % by weight and 99,32 % by volume. The IE was 6,68 by weight and 14,9 by volume.
90 j= 80 I 70
M
60 50
° 40
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30
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20 10
so OS
7 days
I on mass I on volume
14 days
21 days
Fig. 1. Investigation of anticarcinogenic activity of "Lactopropolis 1" drug at a dose of 1 % on the 7Ü
14th and 21st day after grafting
0
120
% 100
80
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20
on mass
on volume
7 days
14 days
21 days
th th
Fig. 2. Study of anticarcinogenic activity of "Lactopropolis 2" drug at a dose of 2,5 % on the 7th 14
and 21st day after grafting
0
Table 2. Antitumor activity of biodrug in research groups on mice BALB/c with grafted tumor strain ACATOL
Time after grafting Dose of administered drug ml/20g Animals quantity in a group Quantity and % of alive without tumor Body mass, g Average tumor volume at the end, Liver mass Tumor mass, mg
At the beginning At the end
7 d 1 % 7 4 (54,4) 16,4 26,7 0,030 1,05 64,83±12
2,5 % 7 3(43 %) 24,8 25,2 0,001 1,22 41,0+ 6,5
14 d 1 % 7 3 (43 %) 23,5 26,6 1,237 1,85 367±145
2,5 % 7 1(14 %) 19,5 22,25 2,400 1,13 1000±73,5
21 d 1 % 7 5(71 %) 21,5 25,7 6,330 1,40 1240±345
2,5 % 7 6 (86 %) 27,00 26,07 4,15 1,13 926±103
At sacrifice of animals the third group in 14 days after grafting, only one animal had no tumor (a 14 % reduction in grafting), and a smaller (by 29,25 %) decrease in tumor growth than in the 7th day. The body mass of animals with respect to the initial mass increased up to 16.,7 % and liver mass to 33,5 % was less in comparison with control animals with tumor carriers. In this group, to the 14th day, % of % ITG was 55,73 % by weight and 78.02 % by volume; in this connection, IE was lower and was 2,26 (by weight) and 4.31 by volume. For 21st day in the third research group it was noted the maximum reduction in grafting (86 %). Body mass of animals to the 21st day in relation to the initial mass decreased insignificantly - to 3,4 %, and the average weight of the liver was increased to 5,6 % in comparison with the control animals with tumor carriers. The most representative are data on the percent inhibition of tumor growth. So, in the first group, to the 7th day of grafting with the drug with 1 % propolis content, inhibition rate was 76,32 % by weight and 79,87 % by volume, IE 4,23 (by weight) and 4.96 (by volume). In the 1st group, by the 14th day, % of ITG was 83.75 % by weight and 88.04 % by volume. IE was 6.14 (by weight) and 8.36 (by volume). On the 21st day, the highest reduction in grafting was observed at 71 %, % % ITG was 67,87 % (by weight) and 54.56 % (by volume), thus their IE by mass was 3,11 and by volume 2, 20, i.e. on average, tumors were 2,2 times less than in control (Figure 1). In the second group of the drug with using 2,5 % propolis after 7 days of grafting, % of % ITG was 84,98 % by weight and 99,32 % by volume. IE was 6,68 by weight and 14.9 by volume. In this group, by the 14th day, % of % ITG was 55,73 % by mass and 78,02 % by volume, in this connection, IE was lower and amounted to 2,26 (by weight) and 4,31 by volume (Figure 2).
Immunological investigations with using monoclonal antibodies CD16 for determination expression of receptors on cells of natural killer by the indirect rosette method have shown that combined drug with different propolis content of 1 % and 2,5 % differently causes expression: 1,7 times immediately in 7 days and is maintained almost on the same level for 21 days; with an increase in the concentration of propolis up to 2,5 %, immunological parameters decrease in the first week (5) and gradually increase to 2 and 3 weeks (9 and 10, respectively).
Table 3. Expression of CD16 receptors
№ Animals group Time of readings removal
7 days 14 days 21 days
1. Control 7,3 7,5 7
2. Drug with 1 % of propolis 12,3 12 11
3. Drug with 2,5 % of propolis 5 9 10
Conclusions. Thus, the preparation containing 1 % of the propolis fraction after 10 days of preventive use has both anti-carcinogenic effect (reduces tumor incidence by 57 %, 43 % and 71 % on the 7, 14 and 21 days after tumor transplantation respectively) and pronounced antitumor effect, especially on days 7 and 14. The use of the drug with 2.5 % propolis at all times after its 10-day administration contributes to both a reduction in tumor incidence (by day 7 by 43 %, by day 14 by 14 % and by 21 days-by 86 %), and by a pronounced antitumor effect, especially on days 7 and 21. Also, the drug at this dose at all times after administration contributes to an increase in the body weight of the experimental animals.
Therefore, the preparations "Lactopropolis 1" and "Lactopropolis 2 (with 1 % content, 2.5 % propolis content) can be recommended for use, since they have anticarcinogenic and antitumor effect. Combined drugs with oral administration cause the activation of the immune process due to the development of tumor cell activity of natural killer cells.
Thus, we can state that the problem of using domestic biologics in oncology deserves the most serious attention. The variety of their positive clinical and immunological effects, the absence of contraindications should promote wider introduction of these drugs into practical oncology for the correction of immunodeficient conditions, elimination of side effects of radiation and chemotherapy, increasing the overall and disease-free survival of patients with oncopathology, improving their quality of life. One can expect that the search for effective anticarcinogenic and antitumor drugs will lead to the creation of new biologics that will occupy a worthy place in the oncological clinic.
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