ANTIPROLIFERATIVE ACTIVITIES Of EXTRACTS FROM MYCELIAL BIOMASS OF SOME MEDICINAL BASIDIOMYCETES IN HUMAN COLON CANCER CELLS COLO 205.

BACKGROUND: The anticancer effects of phytohormones of cytokinin nature are similar to those of medicinal mushrooms, which are able to synthesize cytokinins in large amounts. AIM: To determine the antiproliferative effect of crude extracts and cytokinin fractions from the mycelial biomass of seven fungi species on colon cancer cells in vitro. MATERIALS AND METHODS: Cytokinin content in mycelial biomass of Ganoderma lucidum, Lentinula edodes, Trametes versicolor, Pleurotus ostreatus, Morchella esculenta, Hericium coralloides, and Fomitopsis officinalis was determined by high performance liquid chromatography mass spectrometry. The antiproliferative effect of the mushroom extracts on the human colon adenocarcinoma Colo 205 cells was assessed by MTT-test. RESULTS: The content of cytokinins (trans-zeatin, zeatin riboside, isopentenyladenosine, isopentenyladenine and zeatin-O-glucoside) was determined in the mycelial biomass of the medicinal macromycetes. Zeatin-type hormones prevailed in all species, though trans-zeatin was the most abundant in H. coralloides and M. esculenta. In P. ostreatus, only zeatin-O-glucoside was detected. The lowest IC50 was found for both the cytokinin fraction (0.21 µg/ml) and the crude extract (0.17 µg/ml) from mycelial biomass of H. coralloides. F. officinalis also demonstrated high antiproliferative effect against Colo 205 cells: IC50 was 0.9 µg/ml for the crude extract and almost twice lower for the cytokinin fraction. In the studied concentration range (0.016-2 µg/ml), the crude extracts from G. lucidum and M. esculenta and the cytokinin fraction from L. edodes did not reach IC50 values. CONCLUSIONS: The present study showed that crude extracts and/or cytokinin fractions of several medicinal Basidiomycetes species are capable to inhibit proliferation of colon cancer cells in vitro. Crude extract cytotoxicity of H. coralloides, P. ostreatus and T. versicolor was higher than that of cytokinin fraction while antiproliferative effect of cytokinin fraction from F. officinalis was higher than that in its crude extract.

Expression of TLR4 and major inflammatory cytokines in patients with bladder cancer of different grade and stage.

BACKGROUND: The bladder cancer is immunogenic, and neoantigens generated by tumor cells trigger a notable immune response in the host. On the other hand, multiple immune escape mechanisms allow for avoiding the recognition by the host immune system. Toll-like receptor type 4 and inflammatory cytokines play major role in the immune response to bladder cancer. AIM: To assess the expression of TLR4 and the genes of major inflammatory cytokines in tumor cells and in unaffected tissue of the bladder. MATERIALS AND METHODS: The pairs of samples from the urinary bladder tumor and unaffected adjacent tissue were obtained from 50 surgically treated patients with bladder cancer. The level of expression of TLR4, TGF-ß1, INF-γ, TNF-α genes was evaluated by real-time polymerase chain reaction. RESULTS: Bladder cancer cells are characterized by lower expression levels of TLR4, TGF-ß1, INF-γ, TNF-α as compared to unaffected tissue. In patients with recurrent cancer, expression of TLR-4 and cytokines does not change both in tumor and in unaffected tissue of the bladder. Expression of TLR4 is identically low both in low- and high-grade cancer. Expression levels of the INF-γ and TNF-α are remarkably low in muscle-invasive cancer compared to the unaffected bladder tissue. The level of TGF-ß1 in bladder cancer is comparable to the unaffected tissue of the bladder, while in the intact and metastatic lymph nodes it is significantly upregulated. CONCLUSION: Bladder cancer tissue differs from the unaffected part of the bladder wall in the level of TLR4, TGF-ß1, INF-γ, TNF-α expression.

Changes in expression of TLR-4, TGF-ß, INF-γ, TNF-α in cultured T24/83 cells of invasive bladder cancer treated with cisplatin and/or polyphenolic adjuvant melanin.

BACKGROUND: Toll-like receptor 4 (TLR4) is known to be involved in carcinogenesis and cancer progression. Changes in TLR4 expression are associated with changes in the expression of key cellular cytokines (transforming growth factor-ß (TGF-ß), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ)), which affect cancer progression and metastasis. AIM: To study changes in the expression of TLR4, TGF-ß, TNF-α, IFN-γ genes, the level of apoptosis and cell cycle distribution in human invasive urothelial carcinoma T24/83 cells under the treatment with polyphenolic adjuvant compound of fungal origin melanin, cytotoxic drug cisplatin, and combination of both. MATERIALS AND METHODS: T24/83 cells were incubated with cisplatin (0.05 mM), melanin (5 µg/ml), or their combination. The expression level of TLR-4, TGF-ß, INF-γ, TNF-α was evaluated by the real time polymerase chain reaction. The flow cytometry was used to study cell cycle distribution, proliferative activity and level of apoptosis. Morphological analysis of the Т24/83 cells was performed as well. RESULTS: Melanin, cisplatin, and their combination downregulate TLR4 expression (2.67; 1.28; and 2.73-fold decrease, respectively) and TNF-α expression (6.5; 1.4; and 1.7-fold decrease, respectively). Melanin did not affect TGF-ß expression while cisplatin caused 13-fold downregulation of TGF-ß. The combined use of cisplatin and melanin decreased TGF-ß expression by 6.5 times. The upregulation of IFN-γ by melanin, cisplatin, and their combination was demonstrated (4.3; 6.7; and 2-fold increase, respectively). All treatment modalities increased the level of apoptosis in T24/83 cells. Melanin treatment increased significantly the proportion of fibroblast-like cells in T24/83 culture with decreased cell adhesion to the substrate. CONCLUSIONS: Melanin, cisplatin, and combination of both agents affect significantly TLR4, TNF-α, TGF-ß, INF-γ expression, cell cycle distribution and morphology in T24/83 cells suggesting their transition to less aggressive phenotype.

STATUS OF GLUTATHIONE-DEPENDENT LINK OF BLOOD ANTIOXIDANT SYSTEM UNDER APPLICATION OF FENUGREEK SEED POWDER IN ANIMALS WITH EXPERIMENTAL OBESITY.

The content of reduced glutathione (GSH) and activity of glutathione-dependent enzymes: glutathione peroxidase (GP) and glutathione-S-transferase (GT) in blood serum of rats in the diet supplemented of fine powder seeds Trigonella foenum graecum L. on the background of experimental obesity. It was established that the development of obesity in rats was accompanied by violation of homeostasis in the glutathione-dependent system of blood serum. It has been shown that animals on high-calorie diet-along 14 weeks had a decrease (1,2) in GSH concentration, a decline (1,7) in GP activity and the increase (1,7) of GT activity compared with control values. Addition to high-calorie diet 2% fine powder seeds Trigonella foenum graccum L. reduced the body weight gain by 21%, carried positive modulating effect on the content of GSH and GP, GT activity in animals with experimental obesity.

Prx II and CKBB proteins interaction under physiologic al and thermal stress conditions in A549 and HeLa cells.

Peroxiredoxins (Prxs) are versatile enzymes that demonstrate various cell functions as peroxidases, protein chaperones, functions of signal modulators and binding partners. It is well established that Prxs can interact with multiple proteins in cells, such as ASK1, Cdk5-p35, JNK, MIF, PDGF, TK R4 and others. In this study, we attempted to evaluate a possible association between ubiquitous Prx II and ATP/ADP buffering enzyme - brain-type creatine kinase (CK BB). Our co-immunoprecipitation (Co-IP) results from the A549 and HeLa cell lysates with overexpressed HA-Prx II and Flag-CK BB have demonstrated strong association between two proteins under non-stressed conditions. This protein interaction was enhanced by the heat treatment with further HA-Prx II precipitation to the immobilized Flag-CK BB depending on the temperature increase. Temperature induced oligomerization of Prx II may contribute to the formation of Prx II conglomerates, which in turn, can associate with CK BB and increase signal intensities on the blotted membranes. Thus, such association and oligomerization of Prx II could take part in recovery and protection of the CK BB enzyme activity from inactivation during heat-induced stress.

EXPRESSION OF REG1Α, GAST AND TGFB1 GENES IN CONDITIONS OF STRESSINDUCED GASTRIC MUCOUS LESIONS DEVELOPMENT AND HEALING IN RATS.

Analysis of Reg1a gene expression in rat gastric mucosa under development and healing of stress-induced lesions was carried out. Increased expression of Reg1a was observed after 1 hour of stressor impact - 2,1 times, and achieved the maximum level expression after 3 hours of stress exposure - 3,5 times, that occurred on the background of lipid peroxidation intensification and antioxidant system dysfunction. The sharp decrease in 1,6 and 2 times of Reg1a gene expression was shown in 12 and 24 hours respectively after termination of the stressor action. Analysis of Gast gene expression did not confirm that gastrin stimulated Reg1a expression in gastric mucosa under water immersion restraint stress. The positive correlation between Reg1a and Tgfb1 genes expression was determined in the dynamics of stress-induced gastric lesions' development and healing, which may indicate the involvement of Tgfb1 to acceleration of lesion's healing.

TP53 codon 72 polymorphic variants (Rs1042522) frequency in the Ukrainian population. / ChASTOTA POLIMORFNYKh VARIANTIV 72 KODONU GENA TP53 (RS1042522) SERED NASELENNIa UKRAINY.

OBJECTIVE: To estimate frequencies of polymorphic variants of TP53 codon 72 in the Ukrainian population. MATERIALS AND METHODS: We determined the allele frequencies for 148 healthy people. Genotyping was performed by allele specific polymerase chain reaction. RESULTS: We identified 31 individuals (20.9 %) with Arg/Arg genotype, Arg/Pro genotype was identified for 116 indi viduals (78.4 %), whereas genotype Pro/Pro was rare and was found in one person only (0.7 %). Genotype distribu tions were not within Hardy Weinberg equilibrium (χ2 = 59,7, p < 0.0001). CONCLUSIONS: Arg and Pro allele frequencies in the population of Ukraine are 60 and 40 % respectively, which is sig nificantly differ from the frequencies described in the literature for Poland, the Czech Republic, the USA and Brazil.

INDICATORS OF HUMORAL IMMUNITY UNDER CHEMICAL BURNS OF ESOPHAGUS IN RATS.

It is well known that the immune system has been actively involved in the regeneration and healing processes of post burn wounds. However, unanswered questions remain concerning the role of humoral immunity in the healing mechanisms and development of burn wound complications. We have developed an experimental model of chemical esophageal burn (CEB) which corresponds to esophageal burn in 1-8 years old children. We studied the features of humoral immunity upon CEB in rats. A decrease in IgG levels and an increase in levels of medium- and low- molecular circulating immune complexes (CIC) on the first day of esophageal burns were observed. On the 21st day of burn, we observed an increase in the IgG concentration and a tendency to accumulation of medium- and low-molecular CIC. The studied indicators can be used to differentiate CEB development and create a timeline of burn wounds.

[THE CONTENT OF LIPIDS AND PRODUCTS OF THEIR PEROXIDATION OF RAT THYMOCYTES IN EXPERIMENTAL ULCEROGENESIS].

The work is dedicated to the research of the content of lipids and products of their peroxidation in rats thymocytes in experimental ulceration. It was found significant increase of the content of lipid peroxidation products diene conjugates (DC), malondialdehyde (MDA), schiff base (SB) in experimental models of gastric ulcers (ethanol and stress). It was established that under ethanol gastric the contents of DC increases by 1.8 times, MDA by 2.1 and SB by 1.3 times relative to control values. Under stress model it was observed an increase in the number of DC by 2 times, MDA by 1.9 and SB by 1.3 times relative to control. When ethanol and stress ulcers cholesterol increased by 1.7 and 1.5 times, triacylglycerol by 2 and 2.3 times and fatty acids by 2.2 and 1.9 times, respectively, relative to controls. Phosphatidylethanolamine content decreases by 1.5 and 1.3 times compared to control. Also, the stress model, it was observed reduction of phosphatidylinositol by 1.3 times and increased lizofosfatydylholinu by 1.7 times compared to control. Therefore, our studies indicate quantitative changes of lipid content (neutral- and phospholipids) in rats' thymocytes under experimental (ethanol and stress) ulceration. The reason of this changes may be activation of lipid peroxidation, as shown by the increase of lipid peroxidation products' (DK, MDA, SB) content.

Analysis of creatine kinase activity with evaluation of protein expression under the effect of heat and hydrogen peroxide.

Protein oxidation has detrimental effects on the brain functioning, which involves inhibition of the crucial enzyme, brain type creatine kinase (CKBB), responsible for the CK/phosphocreatine shuttle system. Here we demonstrate a susceptibility of CKBB to several ordinary stressors. In our study enzymatic activity of purified recombinant brain-type creatine kinase was evaluated. We assayed 30 nMconcentration of CKBB under normal and stress conditions. In the direction of phosphocreatine formation hydrogen peroxide and heat treatments altered CKBB activity down to 26 and 14%, respectively. Also, examination of immunoblotted membrane patterns by SDS-PAGE electrophoresis and western blot analysis showed a decrease in expression levels of intrinsic CKBB enzyme in HeLa andA549 cells. Hence, our results clearly show that cytosolic CKBB is extremely sensitive to oxidative stress and heat induced inactivation. Therefore, due to its susceptibility, this enzyme may be defined as a potential target in brain damage.

THE SEARCH OF COMPOUNDS WITH ANTIAGGREGATION ACTIVITY AMONG S-ESTERS OF THIOSULFONIC ACIDS.

According to the current understanding, the hyperactivation of platelets may lead to increased intravascular coagulation and thrombosis. Today a relevant issue is the search for new anti-thrombotic agents that are able to modulate the activity of platelet receptors, thus, influence the processes of activation and aggregation of platelets. The aim of this study was to investigate the effects of newly synthesized thiosulfonate derivatives on platelet aggregation. The activity of the compounds was tested in vitro using platelet-rich plasma. As a result of the screening test, structural formulas of four agents with high antiaggregative activity were established. These compounds inhibited ADP- and collagen-induced platelet aggregation in a dose-dependent manner. Two of these compounds were shown to be more effective inhibitors of aggregation induced by ADP (IC50 - 8-10 µM), as well as collagen (IC50 - 1.5-2.0 µM).

[CARBOHYDRATE METABOLISM IN TYPE 1 DIABETIC RATS UNDER THE CONDITIONS OF THE KIDNEY BEAN PODS AQUEOUS EXTRACT APPLICATION].

The influence of the aqueous pods extract of kidney bean (Phaseolus vulgaris) on indicators of carbohydrate metabolism under the condition of experimental type 1 diabetes in rats was studied. It was shown that long-term oral administration of the extract at a dose of 200 mg/kg to rats leads to the decreasing of blood glucose and glycosylated hemoglobin in the background of chronic hypoinsulinemia conditions. The use of studied extract led to an increase of glycogen synthase activity in rat muscle cells and hexokinase activity in rat liver cells under the conditions of type 1 diabetes. It was estimated that administration of the aqueous extract to control rats and animals with studied model of diabetes increases GLUT-4 protein content in muscle tissue. Thus, the mechanisms of P. vulgaris hypoglycemic action can be related with the ability of the particular phytoconstituents directly effect on key intracellular elements of insulin target tissues carbohydrate metabolism under the conditions of type 1 diabetes.

[PROTEIN CONTENT CHANGES IN RATS' LIVER AND BRAIN UNDER CHRONIC ALCOHOL INTOXICATION].

An electrophoretic investigation of quantitative protein content in rat's' tissyes under chronic alcoholic intoxication has been done. It was shown the significant increase of protein content with molecular mass 55, 48-50, 43-45, 39-41 KDa and the decrease-of protein fractions with molecular mass 46-48, 34-35, 27-30, 16-18 KDa accordingly in liver and brain homogenates of experimental animals.

[FUNCTIONING PROTEASES IN THE ESOPHAGUS MUCOSA AFTER CHEMICAL BURNS].

The main result of esophagus burn is the formation of scars, that caused by excessive synthesis of collagen and changes the balance of metalloproteinases and their tissue inhibitors. It was studied the activity of proteolytic enzymes, participation of MMP (metalloproteinase) and their tissue inhibitors (TIMP) in alkali burns of the esophagus 1st and 2nd degrees. We have shown a significant increase of TIMP level in homogenate after alkali burns of the esophagus (an average of 31-56% depend on of burn degree). We observed a reduced activity of serine proteinase after 1st degree burns on 15th, 21st day 35 and 18% respectively, after burns 2nd degree on 15th, 21st day 54 and 50%. The decrease of activity MMP after 1st degree burns on 15th and 21st day 30, 19%, respectively, in conditions of chemical burns 2nd degree on 15th and 21st day 30, 37%. These data may indicate the development of scarring after burn simulation of 2nd degree. Further investigation of the MMP and TIMP in the process of wound healing can be useful in creating effective approaches to prevent formation of post scarring of the esophagus.

[State of antioxidant system of rat thymocytes in experimental ulcerogenesis].

Features of free radical processes and their impact on the implementation of immunocompetent cells of their functions under conditions of peptic ulcer are insufficiently studied today. Reduced activity of catalase 1.7 and 3.4 times and that of glutathione peroxidise ~ 2.0 times, accordingly, were observed in both models of gastric ulceration (stress ulcer and ethanol one). Enzymatic activity of superoxide dismutase decreased 1.5 times and activity of glutathione transferase increased 1.8 times in the stress model in contrast to the ethanol model of stomach ulcer. Obtained results indicate the exhaustion of antioxidant system in rats' thymocytes under experi- mental ulcerogenesis. These data confirm complex negative effect of ulcer on the organism.

Combined influence of teichoic acids from Staphylococcus aureus and heterometallik Cu/Cd ethylenediamine complex on peritoneal macrophages and tumor cells.

We investigated the effects of teichoic acid (TA) from Staphylococcus aureus Wood 46 on tumor growth and metastasis of the experimental Lewis lung carcinoma (LLC) in mice. Intranasal administration of TA alone aggravated both tumor growth and metastasis, whereas combined administration of TA with a synthetic bimetallic (copper : cadmium) ethylene diamine complex PO244 resulted in pronounced antitumor and antimetastatic effects. The group of animals subjected to the combined treatment with TA and PO244 manifested the highest degree of lymphocyte infiltration into the tumor tissue, compared to the control group and those exposed to TA or PO244 alone. Moreover, the combined treatment negatively affected the adhesive properties of peritoneal macrophages in the LLC bearing mice. Co-cultivation of the isolated macrophages with primary LLC cultures revealed significant (p < 0.05) cytotoxic and cytostatic effects, detected as an increased level of apoptosis and a reduced fraction of replicating cells.

The phospho-c-Jun (Ser63) content in mucosa cells under experimental development of gastric cancer.

AIM: The aim of the study was to determine the phosphorylated c-Jun content and reduced and oxidized glutathione (GSH/GSSG) ratio in gastric mucosa cells during the process of gastric cancer development in rats. MATERIALS AND METHODS: Gastric carcinoge-nesis was initiated in 80 white male rats by 10-week replacement of drinking water with 0.01% solution of N-methyl-N´-nitro-N-nitrosoguanidine, at the same time they were redefined on diet containing 5% NaCl. Then the animals were fed with standard vivarium diet till the end of 24(th) week. The gastric mucosa cells were examined at the end of 4(th), 6(th), 8(th), 10(th), 12(th), 18(th), and 24(th) weeks. Sandwich ELISA method was used to determine the content of phospho-c-Jun. The contents of GSH and GSSG were analyzed by spectrofluorymetric method with the use of orthophthalic aldehyde. RESULTS: At the end of 6(th), 8(th), 10(th) weeks of MNNG and NaCl treatment the gastric mucosa cells were characterized by 4-, 6.3-, 1.9-fold higher content of phospho-c-Jun compared to the control, respectively, and 12, 18 and 24 weeks there was registered a stable increase of phospho-c-Jun content on the average at 3.6-fold compared to control values. Starting from 6(th) week of gastric cancer development an average decrease of GSH/GSSG was 3.4-fold compared with the control. CONCLUSION: During gastric carcinogenesis there was registered the decrease of GSH/GSSG ratio and increased level of phosphorylated c-Jun what points on MAP-kinase cascade activation in prooxidant conditions.

[Effects of 2-(2-hydroxyphenoxy) acetyl-L-prolinate sodium on antioxidant defense system of the gastric mucosa of rats under conditions of stress action].

We investigated the preventive effect of low molecular weight organic compound 2-(2-hydroxyphenoxy) acetyl-L-prolinat sodium on erosive and ulcerative lesions in the gastric mucosa caused by stress. It was established that prophylactic administration of this substance to rats at a dose of 1 mg/kg reduced the lesion area by 56.7%. 2-(2-hydroxyphenoxy) acetyl-L-prolinat sodium efficiently restored the impaired pro-/antioxidative balance in the gastric mucosa of rats under stress: the content of diene conjugates was reduced by 1.24 times, TBA-active products reduced by 1.31 times, Schiff's bases reduced by 1.28 times. This effect was accompanied by increased activities of the superoxide dismutase, catalase and of glutathione system.

[The activity of interferon-dependent 2',5'-oligoadenylatesynthetase in rat thymus lymphoid cells under the action of ethanol and acetic zinc administration].

The activity of interferon-induced 2',5'-oligoadenylatesynthetase (2',5'-OA-synthetase) in thymocytes of rats simultaneously treated with ethanol and acetic zinc preparation was studied. The 2',5'-OA-synthetase activity was decreased by 75% after daily ethanol administration for 14 days. The changes were less pronounced at the late terms of the experiment. In vitro stimulation of enzyme activity by inducer cycloferon was more pronounced at the late terms of ethanol action. Combined administration of ethanol and acetic zinc increased the 2',5'-OA-synthetase activity (by 81% at 21th day and by 30% at 28th day) in comparison to the group of animals that consumed ethanol only. The zinc preparation augmented the stimulation of enzyme activity induced by cycloferon, the effect was most prominent at the late terms of the ethanol action (at 28th day). Probably, ethanol depresses the synthesis of interferon or disturbs the functioning of 2',5'-oligoadenylate cascade. The acetic zinc amplifies the interferon synthesis.

[Effects of natural antioxidants on the content of lipids, proteins and carbohydrates in rat's gastric mucosal cells under experimental ulceration].

Effects of natural antioxidants on the contents of lipids, proteins and carbohydrates in rat's gastric mucosal cells under experimental ethanolinduced ulceration have been studied. A significant decrease in the contents of neutral and phospholipids was found under experimental ulceration and their increase was detected under addition of squalene and ammivit. Under ulceration we found a significant decrease of protein fractions with molecular weight within the range of 11-138 kDa, galactose and fucose contents. Following addition of squalene and ammivit, a normalization of these parameters was observed.