Selol (Se IV) modulates adhesive molecules in control and TNF-α-stimulated HMEC-1 cells.

Selol, an organic selenitetrigliceride formulation containing selenium at +4 oxidation level, has been suggested as anticancer drug. One of the causes of several diseases including cancer may be inflammation. This study aimed at determining the activity of Selol via measuring its effect on reactive oxygen species (ROS) generation, nuclear factor kappa B (NF-κB) activation, intercellular cell adhesion molecules-1 (ICAM-1), vascular cell adhesive molecule-1 (VCAM-1), and plateled-endothelial cell adhesive molecule-1 (PECAM-1) levels on control and on tumor necrosis factor-α (TNF-α)-stimulated human microvascular endothelial cells (HMEC-1). Cells were treated either with Selol 5% (4 or 8 µgSe/mL) or TNF-α (10 ng/mL) alone or with Selol concomitant with TNF-α. Selol treatment resulted in ROS generation, activation of NF-κB, downregulation of PECAM-1, VCAM-1 and slight upregulation ICAM-1 expression on the cell surface. TNF-α treatment reflected in sharp NF-κB activation, upregulation of both ICAM-1 and VCAM-1 in parallel with the downregulation of PECAM-1 expression on cell surface. Exposure to both compounds upregulated ICAM-1 and VCAM-1, downregulated PECAM-1 level on cell surface in parallel with no changes in level of NF-κB activation as compared with effects mediated by TNF-α alone. These results points to new look at Selol action since it shows a pro-inflammatory activity in parallel with effects on CAMs expression on the cell surface of human microvascular endothelial cells. However, since Selol enhances CAMs expression level when is present concomitantly with TNF-α this fact might suggest that selenium present in the condition of inflammation will make it worse.

Thiram activates NF-kappaB and enhances ICAM-1 expression in human microvascular endothelial HMEC-1 cells.

Thiram (TMTD) is a fungicidal and bactericidal agent used as antiseptic, seed disinfectant and animal repellent. In the light of known properties, thiram is considered to be used as an inhibitor of angiogenesis and/or inflammation. Since angiogenesis requires the growth of vascular endothelial cells we have used microvascular endothelial cell line HMEC-1 to elucidate the effect of thiram on normal and stimulated cells. We cultured HMEC-1 cells in the presence of thiram at low concentration (0.5 µg/mL or 2 µg/mL) (0.2 µM or 0.8 µM) or TNF-α (10 ng/mL) alone, and thiram together with TNF-α. TNF-α was used as a cytokine that triggers changes characteristic for inflammatory state of the cell. We carried out an in vitro study aimed at assessing generation of reactive oxygen species (ROS), activation of NF-κB, and expression of cell adhesion molecules ICAM-1, VCAM-1, PECAM-1. It was found that TMTD produced ROS and activated NF-κB. Activation of NF-κB was concurrent with an increase in ICAM-1 expression on the surface of HMEC-1 cells. ICAM-1 reflects intensity of inflammation in endothelial cell milieu. The expression of VCAM-1 and PECAM-1 on these cells was not changed by thiram. It was also found that stimulation of the HMEC-1 cells with the pro-inflammatory cytokine TNF-α caused activation of ICAM-1 and VCAM-1 expression with concomitant decrease of PECAM-1 cell surface expression above the control levels. Treatment with thiram and TNF-α changed cellular response compared with effects observed after treatment with TNF-α alone, i.e. further increase of ICAM-1 expression and impairment of the TNF-α effect on PECAM-1 and VCAM-1 expression. This study demonstrated that thiram acts as a pro-oxidant, and elicits in endothelial cell environment effects characteristic for inflammation. However, when it is present concurrently with pro-inflammatory cytokine TNF-α interferes with its action.

Modulation of antioxidant defense system by the dithiocarbamate fungicides Maneb and Zineb in Chinese hamster V79 cells and the role of N-acetyl-L-cysteine.

Oxidative stress is one of the major factors leading to Maneb- and Zineb-induced disorders. The aim of this in vitro study was to examine (i) the potency of Maneb and Zineb to induce changes in antioxidant enzyme activities in Chinese hamster fibroblasts V79 cells and (ii) the role of N-acetyl-L-cysteine (NAC) in the preventing their action. Maneb increased mitochondrial superoxide dismutase (SOD2) activity but failed to affect the activity of cytoplasmic superoxide dismutase (SOD1), whereas Zineb did not change the activity of any of superoxide dismutases. The activity of catalase (CAT) was reduced only by Zineb. The activity of both glutathione peroxidases (non-Se-GPx, Se-GPx) and glutathione reductase (GR) was decreased after exposure to these agents. After NAC pre-treatment Maneb increased the activity of GR, whereas the activity of non-Se-GPx was decreased as compared to that in NAC-treated cells. On the other hand, the activity of both SODs and CAT was decreased. Zineb decreased the activity of both GPxs and SOD2 with a concomitant increase in CAT activity comparing to NAC-treated cells. The results obtained thus suggest that Zineb acts by another mechanism, than Maneb does, and that one of the mechanisms of NAC protection against Maneb or Zineb-induced effects in V79 cells is its impact on enzymatic defense. Activity of GR, SOD2, and GPxs are the most affected enzymes.

Protective effect of N-acetyl-L-cysteine against maneb induced oxidative and apoptotic injury in Chinese hamster V79 cells.

The role of antioxidant N-acetyl-L-cysteine (NAC) in protection against cellular changes triggered by maneb during in vitro exposure was investigated in cultured Chinese hamster V79 cells. We observed high apoptotic activity and high oxidative stress induced by exposure to maneb evidenced by a statistically significant increase in lipid peroxidation (measured as TBARS--thiobarbituric acid reactive substances) as well as a decrease of glutathione (GSH) and glutathione disulfide (GSSG) ratio (GSH/GSSG). Maneb did not exhibit any effect on protein oxidation (measured by protein carbonyls content). NAC suppressed cellular changes induced by maneb in V79 cells. NAC pre-treatment prevented TBARS production and significantly decreased the number of apoptotic cells. However, protective effect of NAC on GSH and GSSG levels has been shown only in cells exposed to lower concentration of maneb (100 µM).

Protective effect of N-acetyl-L-cysteine against disulfiram-induced oxidative stress and apoptosis in V79 cells.

This work investigated the effect of N-acetyl-L-cysteine (NAC) on disulfiram (DSF) induced oxidative stress in Chinese hamster fibroblast cells (V79). An increase in oxidative stress induced by DSF was observed up to a 200 µM concentration. It was evidenced by a statistically significant increase of both GSH(t) and GSSG levels, as well as elevated protein carbonyl (PC) content. There was no increase in lipid peroxidation (measured as TBARS). DSF increased CAT activity, but did not change SOD1 and SOD2 activities. Analysis of GSH related enzymes showed that DSF significantly increased GR activity, did not change Se-dependent GPx, but statistically significantly decreased non-Se-dependent GPx activity. DSF showed also pro-apoptotic activity. NAC alone did not produce any significant changes, besides an increase of GSH(t) level, in any of the variables measured. However, pre-treatment of cells with NAC ameliorated DSF-induced changes. NAC pre-treatment restored the viability of DSF-treated cells evaluated by Trypan blue exclusion assay and MTT test, GSSG level, and protein carbonyl content to the control values as well as it reduced pro-apoptotic activity of DSF. The increase of CAT and GR activity was not reversed. Activity of both GPx was significantly increased compared to their values after DSF treatment. In conclusion, oxidative properties are at least partially attributable to the cellular effects of disulfiram and mechanisms induced by NAC pre-treatment may lower or even abolish the observed effects. These observations illustrate the importance of the initial cellular redox state in terms of cell response to disulfiram exposure.

[Biological role of phosphatidylcholine-specific phospholipase C in mammalian cells]. / Biologiczna rola fosfolipazy C zaleznej od fosfatydylocholiny w komórkach ssaków.

Phosphatidylcholine-specific phospholipase C (PC-PLC) catalyzes the hydrolysis of the ester linkage between glycerol and phosphate in phosphocholine (PC) and other phosphatides, such as sphingomylin (SM) and phosphatidylethanolamine (PE). PC-PLC activity has been described in many organisms, from bacteria to mammals. In mammalian cells the enzyme has been found in erythrocytes, lymphocytes, muscular tissue, adipose tissue, and the nervous system. Hydrolysis of PC by PC-PLC results in the production of phosphocholine and diacylglycerol (DAG), a well-characterized lipid second-messenger molecule. The PC-degradation pathway by PC-PLC is activated by many factors, including cytokines, growth factors, mitogens, and calcium ions. Degradation of PC has been implicated in intracellular signal transduction involved in the regulation of cell metabolism, growth, differentiation, and induction of apoptosis. In this review the structure and biological function of mammalian PC-PLC are discussed.

[Phosphoinositide-specific phospholipase C in mammalian cells: structure, properties, and function]. / Fosfolipaza C zalezna od fosfatydyloinozytoluw komórkach ssaków - budowa, wlasciwosci i funkcja.

Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) to yield diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). Phospholipase C activities have been described in several organisms, including bacteria, yeast, plants, and mammals. In mammalian cells, PLC (PLC-beta, PLC-gamma, PLC-delta, PLC-epsilon, PLC-zeta, and PLC-eta isoforms) has been implicated in intracellular signal transduction, vesicle transport, endocytosis, exocytosis, ion channel function, mitosis, cytoskeletal reorganization, and neuronal signal transduction. Mammalian phospholipase C is regulated by many factors, including calcium ions, receptor tyrosine kinases, and small G-proteins of the Ras and Rho families. In this review the structure and biological function of PLC are discussed.

[Phospholipase D in mammalian cells: structure, properties, physiological and pathological role]. / Fosfolipaza D w komórkach ssaków - budowa, wlasciwosci, rola fizjologiczna i patologiczna.

Phospholipase D (PLD) catalyzes the hydrolysis of the phosphodiester bond of glycerophospholipid phosphatidylcholine to generate phosphatidic acid (PA) and choline. Phosphatidic acid is widely considered to be the intracellular lipid mediator of many biological functions. PA is a precursor of many other bioactive lipids, including diacylglycerol (DAG) and lysophosphatidic acid (LPA). Phospholipase D activities have been described in multiple organisms, including bacteria, yeast, plants, and mammals. In mammalian cells, PLD (PLD1 and PLD2 isoenzymes) has been implicated in intracellular signal transduction, vesicle transport, endocytosis, exocytosis, cell migration, mitosis, and cytoskeletal reorganization. Mammalian phospholipase D is regulated by many factors, including phosphatidylinositol-4,5-bisphosphate (PIP2), protein kinase C (PKC), and small G-proteins of the Rho, Ral, and ARF families. In this review we discuss the relationships of PLD1 and PLD2, their structure, biological function, and implications in pathological states.

[Phospholipases A in mammalian cells: structure, properties, physiological and pathological role]. / Fosfolipazy A w komórkach ssaków--budowa, wlasciwosci, rola fizjologiczna i patologiczna.

Phospholipases A are a diverse group of enzymes, which catalyzes the hydrolysis of the ester bond at the sn-1 and sn-2 positions of glycerophospholipids, forming free fatty acids and lysophospholipids. In normal conditions, these enzymes regulate the turnover of free fatty acids in membrane phospholipids, affecting membrane stability, fluidity, and transport processes. Phospholipase activity is also responsible for the generation of intracellular messengers, including arachidonic acid metabolites. Phospholipases are regulated by many factors including selective phosphorylation, intracellular calcium, and pH. In this review we discuss the relationships of phospholipases A1 and A2, their structure, biological function, and implications in various human diseases.

Defective Brca2 influences topoisomerase I activity in mammalian cells.

The Chinese hamster cell mutant V-C8 is defective in the Brca2 gene (Kraakman-van der Zwet et al., 2002, Cell Biol.; 22: 669). Here we report that V-C8 cells were 10-fold more sensitive to camptothecin, an inhibitor of topoisomerase I, than the parental V79 cells. The level of the relaxation activity of topoisomerase I in nuclear extracts was also lower (4-fold) in V-C8 than V79 cells, in spite of the fact that the level of the topoisomerase I protein was the same in these cells. The survival of V-C8 cells in the presence of camptothecin, the sensitivity of V-C8 topoisomerase I to camptothecin, and the level of the relaxation activity in V-C8 nuclear extract were almost completely restored by transfection of V-C8 cells with the murine Brca2 gene or by the transfer of human chromosome 13 providing the BRCA2 gene. These results indicate that the observed changes in the topoisomerase I activity in V-C8 are due to the defective function of the Brca2 gene.