Isolectins of phytohemagglutinin are able to induce apoptosis in HEp-2 carcinoma cells in vitro.

AIM: To study the effects of total phytohemagglutinin (PHA) and its isolectins on cell death and apoptosis in human HEp-2 carcinoma cells and to analyze the possible molecular mechanisms of lectin induced apoptosis. MATERIALS AND METHODS: The commercial preparation of the kidney beans (Phaseolus vulgaris) lectins and HEp-2 cells were used. Apoptosis index was determined using acridine orange and ethidium bromide staining. The expression levels of apoptosis mediator cleaved caspase-3 and proapoptotic Bax protein were studied by Western blot analysis. The gene expression levels were analyzed by qPCR. RESULTS: PHA and its isolectins induced apoptosis in HEp-2 cells accompanied by the increased expression of caspase-3 cleaved form, with PHA-E being the most effective. The treatment of HEp-2 cells with PHA or its isolectins resulted in a marked increase of Bax on both mRNA and protein levels. CONCLUSIONS: PHA and its isolectins were shown to induce the apoptosis in human HEp-2 carcinoma cells via increasing proapoptotic protein Bax and activating caspases-3.

[Proapoptotic properties of total phytohemagglutinine and its individual isolectins in human cell culture 4BL].

Phytohemagglutinine (PHA) is widely investigated lectin with mitogenic properties. Recently it was shown that PHA is not only cell proliferation inducer, but also has a toxic or cytostatic effect. However concentration dependence and molecular mechanisms of this effect are not enough investigated. To study proapoptotic properties of total phytohemagglutinine and its isolectins in human cell culture of not tumor origin 4BL we observed a change in the frequency of apoptotic cells in the tested cell culture under the influence of the total phytohemagglutinine and erythroagglutinin by the method of specific color luminescent dye. The activation of caspases-3 and -8 and induction of protein Bax expression under the influence of lectins were detected by Western blot analysis. It was revealed that erythroagglutinin induced apoptosis with the highest efficiency compared with leukoagglutinin and total phytohemagglutinine. The induction of apoptosis in human cell culture of not tumor origin 4BL is probably caused by activating caspase-dependent and mitochondrial signalling.

[Influence of omega-3 polyunsaturated fatty acids on oxidative stress and cytochrome P450 2E1 expression in rat liver].

Polyunsaturated fatty acids (PUFAs), omega-3 ones in particular, form phospholipid layer of biological membranes, which provides normal functioning of membrane-associated complexes of enzymes and transmembrane transport. Free omega-3 PUFAs regulate the transcription of many genes, and thereby have an effect on the level of metabolic processes, particularly control of lipid and carbohydrate metabolism in the liver. Cytochrome P450 2El (1.14.14.1) causes the transformation of lipophilic exogenous and endogenous substances, as well as involvement in homeostasis, both at the cellular and systemic levels. The aim is to study changes in expression of cytochrome P450 2E1, and to assess the antioxidant system and the level of peroxidation processes in the liver of experimental animals under the chronic action omega-3 PUFAs. During experiment more than two-fold increase in the content of cytochrome P450 2El was observed in the liver of rats which additionally received omega-3 PUFAs for 4 weeks in the standard daily diet. At the same time, such changes in the enzyme expression did not lead to an imbalance of pro- and antioxidant processes in the liver.

[The influence of omega-3 polyunsaturated fatty acids on the expression of enzymes of the prooxidant and antioxidant systems in the rat liver].

Omega-3 polyunsaturated fatty acids (PUFAs) are involved in the regulation of many physiological processes by modulating the activity of transcription factors. However, the properties of the compounds to influence the pro - antioxidant processes in liver are poorly investigated. We aimed to study the changes in expression of cytochrome P450 2E1 (CYP2E1) with a strong prooxidant activity, and heme oxygenase 1 (HO-1), which is characterized by anti-apoptotic and anti-inflammatory properties, and evaluation of the activity of antioxidant enzymes in the liver of experimental animals under the action of omega-3 PUFAs. It was shown a significant increase in cytochrome P450 2E1 and heme oxygenase 1 expression in the liver of rats treated with the omega-3 PUFAs (per os, daily for 4 weeks). At the same time, there were no changes in the activity of antioxidant enzymes - superoxide dismutase and catalase. Probably an increase of the level of cytochrome P450 2E1 leads to induction of expression of enzymes with cytoprotective properties, such as heme oxygenase-1, by ROS-dependent activation of certain signaling pathways. Such CYP2E1-dependent activation of expression of HO-1 is one of the possible mechanisms of manifestation of antioxidant, antiapoptotic and anti-inflammatory properties of omega-3 PUFAs.

[Stress-induced changes in the content of cytochrome P450 2E1 in the liver of mice with chronic psychoemotional overexertion].

In this work we investigated changes in the cytochrome P450 2E1 (CYP2E1) expression level in the liver of mice that have been exposed to psycho-emotional stress (PES). It was shown twofold relative to control reduction of the enzyme, which does not normalize after termination of the stressor. The changes in level of Cyp2e1 mRNA is not observed at any time point of the experiment. At the same time it was found a significant decrease in the expression level of hsp90--one of the factors determines of the level of CYP2E1 degradation in the cell. Also it was shown, the oxidative stress develops in the liver of experimental animals, and this is indicated by a 3-fold increase in the malondialdehyde level on the background of a 5-fold decrease in catalase activity. A decrease in the level of expression of cytochrome P450 2E1 in the liver of mice that have been exposed to psycho-emotional stress is due to the intensification of the peroxide process and the development of oxidative stress. Reduction of protein content of CYP2E1 is apparently not related to the hsp90-dependent processes of its degradation in the cell.

[Optimization of animal model for investigation of pathogenesis of type 2 diabetes].

Diabetes mellitus is one of the three most common modem diseases. A number of animal models is used in investigations of the mechanisms of development of the disease. Most of these models replicate the symptoms of type 1 diabetes mellitus. The development of type 2 diabetes is caused by the insulin resistance, hyperglycemia, structural and functional disorders of the pancreatic cells. Investigation of pathogenesis of type 2 diabetes is complicated by the lack of adequate models of this disease. In this work, based on existing hyperglycemia model, we propose the model of metabolic syndrome as a precursor of type 2 diabetes. The development of metabolic syndrome symptoms was caused by 28 days long intramuscular injection of protamine sulfate to guinea pigs at a dose of 15 mg/kg along with keeping of animals on a high glucose diet. Increased blood glucose and cholesterol levels, reduction of glycogen in liver, the structural and functional damage and reduce in the number of functionally active beta-cells in the pancreas of the experimental animals were observed. The results confirm the development of the metabolic syndrome symptoms in experimental animals, which makes it possible to use such methodical approach in creation of promising type 2 diabetes model.

[Influence of long-term combined gamma-radiation and ethanol on cytochrome P450 2E1 expression in the mice liver].

The effect of continuous single and combined influence of exogenous stress factors (ethanol and low intensity gamma-radiation) on cytochrome P450 2E1 (1.14.14.1) expression in mice liver was studied. The chronic ethanol intake induced Cyp2E1 protein expression but not increased Cyp2e1 mRNA level. The first week of continuous combined influence of ethanol and gamma-irradiation was characterized by the increase of Cyp2E1 protein level, that was back to normal on second week. A small decrease of this index was observed at the end of the fifth week. The changes of Cyp2E1 protein amount accompanied the decrease of Cyp2e1 mRNA level.

[Cytochrome P450 2E1 expression in mice liver after exposure to continuous and acute gamma-radiation].

The effect of continuous low-intensity and acute high-intensity ionizing radiation in low and high doses on expression of cytochrome P450 2El (1.14.14.1) in mice liver was studied. It was shown, that changes of Cyp2e1 amount depended on the dose and intensity of ionizing radiation. Low doses of continuous gamma-radiation caused reliable decrease of Cyp2e1 expression on protein mRNA levels. Low doses of acute gamma-radiation lead to an increase of Cyp2el amount, while high doses--to a corresponding decrease. We showed, that low and high doses of acute gamma-radiation caused a significant decrease of Cyp2e1 mRNA level. We suppose that the detected changes of Cyp2el expression are associated with peroxidation process and development of oxidative stress.

Molecular chaperone, HSP60, and cytochrome P450 2E1 co-expression in dilated cardiomyopathy.

Physiological stresses (heat, hemodynamics, genetic mutations, oxidative injury and myocardial ischemia) produce pathological states in which protein damage and misfolded protein structures are a common denominator. The specialized proteins family of antistress proteins - molecular chaperons (HSPs) - are responsible for correct protein folding, dissociating protein aggregates and transport of newly synthesized polypeptides to the target organelles for final packaging, degradation or repair. They are inducible at different cell processes such as cell division, apoptosis, signal transduction, cell differentiation and hormonal stimulation. HSPs are involved in numerous diseases including cardiovascular pathologies, revealing changes of expression and cell localization. We studied the possible changes in expression level of abundant mitochondrial chaperon Hsp60 and main human cytochrome P450 monooxygenase (2E1 isoform) at dilated cardiomyopathy (DCM) progression at the end stage of heart failure using Western blot analysis. The ischemic and normal humans' hearts were studied as control samples. We observed the decrease of Hsp60 level in cytoplasmic fraction of DCM- and ischemia-affected hearts' left ventricular and significant increase of Hsp60 in mitochondrial fractions of all hearts investigated. At the same time we detected the increase of P450 2E1 expression level in ischemic and dilated hearts' cytoplasmic fractions in comparison with normal myocardium and no detectable changes in microsomal fractions of hearts investigated which could be linked with increased level of oxidative injury for DCM heart muscle. In addition, all the changes described are accompanied by significant decrease of ATPase activity of myosin purified from DCM-affected heart in comparison with normal and ischemic myocardia as well. The data obtained allow us to propose a working hypothesis of functional link between antistress (HSPs) and antioxidative (cytochromes) systems at DCM progression.