S. Hudecova

SB203580, a specific inhibitor of p38-MAPK pathway, is a new reversal agent of P-glycoprotein-mediated multidrug resistance

P-glycoprotein (P-gp) is the plasma membrane transport pump responsible for efflux of chemotherapeutic agents from cells and is one of the systems that secures multidrug resistance (MDR) of neoplastic cells. In the present study, drug sensitive L1210 and multidrug resistant L1210/VCR (characterized by overexpression of P-gp) mouse leukemic cell lines were used as an experimental model. We have found that SB203580, a specific inhibitor of p38-MAPK pathway, significantly reduced the degree of the vincristine resistance in L1210/VCR cells. This phenomenon was accompanied by a decrease in the LC(50) value of vincristine from 3.203+/-0.521 to 0.557+/-0.082 microM. The LC(50) value of sensitive cells for vincristine was about 0.011 microM. The effect of SB203580 on L1210/VCR cells was associated with significantly increased intracellular accumulation of [3H]-vincristine in the concentration dependent manner. Prolonged exposure of resistant cells to 30 microM SB203580 did neither significantly influence the gene expression of P-gp, nor change the protein levels of p38-MAPK. Western blot analysis revealed that the MDR phenotype in L1210/VCR cells was associated with increased level and activity of cytosolic p38-MAPK. In resistant cells, the enhanced phosphorylation of both, p38-MAPK and ATF-2 (endogenous substrate for p38-MAPK) was found as well. In conclusion we could remark that SB203580, an inhibitor of p38 kinase pathway, reversed the MDR resistance of L1210/VCR cells. MDR phenotype of these cells is connected with increased levels and activities of p38-MAPK. These findings point to the possible involvement of the p38-MAPK pathway in the modulation of P-gp mediated multidrug resistance in the L1210/VCR mouse leukemic cell line. However, the mechanisms of SB203580 action should be further investigated.

Sulphide signalling potentiates apoptosis through the up‐regulation of IP3 receptor types 1 and 2

To investigate an interaction between the calcium and sulphide signalling pathways, particularly effects of the slow H2S release donor morpholin‐4‐ium‐4‐methoxyphenyl‐(morpholino)‐phosphinodithioate (GYY4137) on the expression of inositol 1,4,5‐trisphosphate receptors (IP3R) with the possible impact on the apoptosis induction in HeLa cells.

Modulation of expression of Na+/Ca2+ exchanger in heart of rat and mouse under stress

Aim:  The Na+/Ca2+ exchanger (NCX) is a major Ca2+ extrusion system in the plasma membrane of cardiomyocytes and an important component participating on the excitation–contraction coupling process in muscle cells. NCX1 isoform is the most abundant in the heart and is known to be changed after development of ischaemia or myocardial infarction. Objective of this study was to investigate the effect of stress factors (immobilization, cold and short‐term hypoxia) on the expression of NCX1, in vivo, in the heart of rat and mouse.

Nuclear all-trans retinoic acid receptors: in vitro effects of selenium.

The present study was undertaken to investigate the effects of selenite (SeIV) and selenate (SeVI) on the all-trans retinoic acid (RA)-nuclear retinoic acid receptor (RAR) complex formation in rat liver. We also present the data on the in vitro effects of SeIV on the RARα and the type I iodothyronine 5′-deiodinase gene expression in the GH4C1 rat pituitary tumor cells. SeIV at 1.0 μmol/L was found to reduce (p<0.05) the RA specific binding to RAR in rat liver. Dithiothreitol (DTT), a protective agent for sulfhydryl groups, was found to be slightly effective in protecting the RAR binding properties when affected by SeIV. SeVI at 0.1 μmol/L reduced (p<0.05) the RA specific binding to RAR in liver, as well. Seleno-l-methionine (Se-II) when compared tol-methionine did not exert any inhibitory effect on the formation of the RA-RAR complex. SeIV (up to 2.5 μmol/L) has no inhibitory effect on GH4C1 cell proliferation as well as the prolactin secretion. SeIV at 1.0 μmol/L significantly decreases the rate of mRNA synthesis and/or degradation of the α form of the RAR and causes the enhancement of the type I iodothyronine 5′-deiodinase gene expression in GH4C1 cells.The results based on in vitro experiments suggest that inorganic selenium may affect the RA specific binding to their cognate receptor molecules, and it may reduce expression of the gene encoding the RARα, with the cell vitality and the cell growth remaining unchanged.

Modulation of the sodium–calcium exchanger in the rat kidney by different sequential stressors

Stress, if exaggerated, modulates a variety of metabolic pathways and results in development of serious health consequences. The cell membrane sodium–calcium exchanger (NCX) is a major calcium extrusion system and is also modulated by stress. It has been shown previously that mRNA, protein levels and activity of the type 1 NCX (NCX1) in the left ventricle of the rat heart are increased by stressors, such as immobilization or hypoxia. In this study we investigated whether exposure to a subsequent different stressor can affect gene expression, protein level and activity of the NCX1 in rat kidney compared to exposure to only one type of stressor. In these experiments, we used immobilization and cold as the model stressors. We found that cold exposure at 4°C for 24 h, when applied after immobilization repeated seven times, completely abolished the immobilization-induced increase in NCX mRNA level and after 7 days cold exposure the increases in NCX1 protein and activity in rat kidney were also abolished. Permanently increased NCX1 expression can result in imbalance of cellular calcium homeostasis and thus contribute to kidney dysfunction. Based on our results, we conclude that exposure to a cold stressor can have a protective effect on the kidney in rats exposed previously to repeated immobilization stress. This might be explained by differential stimulation of sympathetic neural and adrenal medullary responses by these different stressors.

Slow sulfide donor GYY4137 differentiates NG108-15 neuronal cells through different intracellular transporters than dbcAMP

Cellular differentiation is the process, by which a cell changes from one cell type to another, preferentially to the more specialized one. Calcium fluxes play an important role in this action. Differentiated NG108-15 or PC12 cells serve as models for studying neuronal pathways. NG108-15 cell line is a reliable model of cholinergic neuronal cells. These cells differentiate to a neuronal phenotype due to the dibutyryl cAMP (dbcAMP) treatment. We have shown that a slow sulfide donor - GYY4137 - can also act as a differentiating factor in NG108-15 cell line. Calcium is an unavoidable ion required in NG108-15 cell differentiation by both, dbcAMP and GYY4137, since cultivation in EGTA completely prevented differentiation of these cells. In this work we focused primarily on the role of reticular calcium in the process of NG108-15 cell differentiation. We have found that dbcAMP and also GYY4137 decreased reticular calcium concentration by different mechanisms. GYY4137 caused a rapid decrease in type 2 sarco/endoplasmic calcium ATPase (SERCA2) mRNA and protein, which results in lower calcium levels in the endoplasmic reticulum compared to the control, untreated group. The dbcAMP revealed rapid increase in expression of the type 3 IP3 receptor, which participates in a calcium clearance from the endoplasmic reticulum. These results point to the important role of reticular calcium in a NG108-15 cell differentiation.