It was evidenced that saturated fatty acids (FAs) have a detrimental effect on pancreatic β-cells function and survival, leading to endoplasmic reticulum (ER) calcium release, ER stress, and apoptosis. In the present study, we have tested the effect of three calcium influx inhibitors, i.e., diazoxide, nifedipine, and verapamil, on the apoptosis-inducing effect of saturated stearic acid (SA) in the human pancreatic β-cell lines NES2Y and 1.1B4. We have demonstrated that the application of all three calcium influx inhibitors tested has no inhibitory effect on SA-induced ER stress and apoptosis in both tested cell lines. Moreover, these inhibitors have pro-apoptotic potential per se at higher concentrations. Interestingly, these findings are in contradiction with those obtained with rodent cell lines and islets. Thus our data obtained with human β-cell lines suggest that the prospective usage of calcium channel blockers for prevention and therapy of type 2 diabetes mellitus, developed with the contribution of the saturated FA-induced apoptosis of β-cells, seems rather unlikely.

• Klíčová slova
• 1.1B4, Apoptosis, Calcium influx, Diazoxide, Fatty acids, NES2Y, Nifedipine, Pancreatic β-cells, Type 2 diabetes mellitus, Verapamil,
• Publikační typ
• časopisecké články MeSH

Saturated fatty acids (FAs) induce apoptosis in the human pancreatic NES2Y β-cell line while unsaturated FAs have nearly no detrimental effect. Moreover, unsaturated FAs are capable of inhibiting the pro-apoptotic effect of saturated FAs. Hypoxia is also known to have deleterious effects on β-cells function and viability. In the present study, we have tested the modulatory effect of hypoxia on the effect of FAs on the growth and viability of the human pancreatic NES2Y β-cells. This study represents the first study testing hypoxia effect on effects of FAs in pancreatic β-cells as well as in other cell types. We showed that hypoxia increased the pro-apoptotic effect of saturated stearic acid (SA). Endoplasmic reticulum stress signaling seemed to be involved while redistribution of FA transporters fatty acid translocase/cluster of differentiation 36 (FAT/CD36) and fatty acid-binding protein (FABP) do not seem to be involved in this effect. Hypoxia also strongly decreased the protective effect of unsaturated oleic acid (OA) against the pro-apoptotic effect of SA. Thus, in the presence of hypoxia, OA was unable to save SA-treated β-cells from apoptosis induction. Hypoxia itself had only a weak detrimental effect on NES2Y cells. Our data suggest that hypoxia could represent an important factor in pancreatic β-cell death induced and regulated by FAs and thus in the development of type 2 diabetes mellitus.

• Klíčová slova
• ER stress, NES2Y, apoptosis, caspases, fatty acid transporters, fatty acids, hypoxia, hypoxia-inducible factor 1α, pancreatic β-cells,
• MeSH
• beta-buňky metabolismus   MeSH
• biologické markery MeSH
• buněčné linie MeSH
• hypoxie metabolismus   MeSH
• kaspasy metabolismus   MeSH
• lidé MeSH
• mastné kyseliny metabolismus   MeSH
• proliferace buněk MeSH
• signální transdukce účinky léků   MeSH
• stres endoplazmatického retikula MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biologické markery MeSH
• kaspasy MeSH
• mastné kyseliny MeSH

It has been shown that saturated fatty acids (FAs) have a detrimental effect on pancreatic β-cells function and survival, leading to apoptosis, whereas unsaturated FAs are well tolerated and are even capable of inhibiting the pro-apoptotic effect of saturated FAs. Molecular mechanisms of apoptosis induction and regulation by FAs in β-cells remain unclear; however, mitogen-activated protein (MAP) kinase and endoplasmic reticulum (ER) stress signaling pathways may be involved. In this study, we tested how unsaturated oleic acid (OA) affects the effect of saturated stearic acid (SA) on the p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathways as well as the ER stress signaling pathways during apoptosis induction in the human pancreatic β-cells NES2Y. We demonstrated that OA is able to inhibit all effects of SA. OA alone has only minimal or no effects on tested signaling in NES2Y cells. The point of OA inhibitory intervention in SA-induced apoptotic signaling thus seems to be located upstream of the discussed signaling pathways.

• Klíčová slova
• NES2Y, apoptosis, endoplasmic reticulum (ER) stress, extracellular signal-regulated kinase (ERK), fatty acids, p38 mitogen-activated protein kinase (MAPK), pancreatic β-cells,
• MeSH
• apoptóza * MeSH
• beta-buňky cytologie   metabolismus   patologie   MeSH
• buněčné linie MeSH
• kyselina olejová metabolismus   MeSH
• kyseliny stearové metabolismus   MeSH
• lidé MeSH
• MAP kinasový signální systém * MeSH
• mitogenem aktivované proteinkinasy p38 metabolismus   MeSH
• stres endoplazmatického retikula * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kyselina olejová MeSH
• kyseliny stearové MeSH
• mitogenem aktivované proteinkinasy p38 MeSH

Saturated stearic acid (SA) induces apoptosis in the human pancreatic β-cells NES2Y. However, the molecular mechanisms involved are unclear. We showed that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway in these cells. Therefore, we tested the role of p38 MAPK signaling pathway activation in apoptosis induction by SA in NES2Y cells. Crosstalk between p38 MAPK pathway activation and accompanying ERK pathway inhibition after SA application was also tested. The inhibition of p38 MAPK expression by siRNA silencing resulted in a decrease in MAPKAPK-2 activation after SA application, but it had no significant effect on cell viability or the level of phosphorylated ERK pathway members. The inhibition of p38 MAPK activity by the specific inhibitor SB202190 resulted in inhibition of MAPKAPK-2 activation and noticeable activation of ERK pathway members after SA treatment but in no significant effect on cell viability. p38 MAPK overexpression by plasmid transfection produced an increase in MAPKAPK-2 activation after SA exposure but no significant influence on cell viability or ERK pathway activation. The activation of p38 MAPK by the specific activator anisomycin resulted in significant activation of MAPKAPK-2. Concerning the effect on cell viability, application of the activator led to apoptosis induction similar to application of SA (PARP cleavage and caspase-7, -8, and -9 activation) and in inhibition of ERK pathway members. We demonstrated that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway and that this activation could be involved in apoptosis induction by SA in the human pancreatic β-cells NES2Y. However, this involvement does not seem to play a key role. Crosstalk between p38 MAPK pathway activation and ERK pathway inhibition in NES2Y cells seems likely. Thus, the ERK pathway inhibition by p38 MAPK activation does not also seem to be essential for SA-induced apoptosis.

• Klíčová slova
• ERK, NES2Y, apoptosis, fatty acids, p38 MAPK, pancreatic β-cells,
• MeSH
• aktivace enzymů MeSH
• apoptóza * účinky léků   MeSH
• beta-buňky účinky léků   metabolismus   MeSH
• buněčné linie MeSH
• exprese genu MeSH
• inhibitory proteinkinas farmakologie   MeSH
• kyseliny stearové farmakologie   MeSH
• lidé MeSH
• MAP kinasový signální systém účinky léků   MeSH
• mastné kyseliny metabolismus   farmakologie   MeSH
• mitogenem aktivované proteinkinasy p38 antagonisté a inhibitory   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inhibitory proteinkinas MeSH
• kyseliny stearové MeSH
• mastné kyseliny MeSH
• mitogenem aktivované proteinkinasy p38 MeSH
• stearic acid MeSH Prohlížeč

We studied the effect of iron deficiency, i.e., 24-h preincubation in iron-free medium, and the effect of high level of non-transferrin iron, i.e., the preincubation in ferric citrate medium containing 500 microM ferric citrate, on the expression of DMT1, Dcytb, ferroportin, hephaestin, and ceruloplasmin in various functional types of human cells. The expression of these proteins potentially involved in non-transferrin iron transport across cell membranes was tested on mRNA level by quantitative real-time PCR as well as on protein level by western blot analysis in Caco-2 (colorectal carcinoma), K562 (erythroleukemia), and HEP-G2 (hepatocellular carcinoma) cells. We found that changes in non-transferrin iron availability, i.e., iron deficiency and high level of non-transferrin iron, affect the expression of tested proteins in a cell type-specific manner. We also demonstrated that changes in the expression on mRNA level do not often correlate with relevant changes on protein level.

• MeSH
• buněčná membrána metabolismus   MeSH
• buněčné linie MeSH
• ceruloplasmin genetika   metabolismus   MeSH
• cytochromy typu b genetika   metabolismus   MeSH
• exprese genu * MeSH
• ferroportin MeSH
• kultivační média chemie   MeSH
• lidé MeSH
• membránové proteiny * genetika   metabolismus   MeSH
• oxidoreduktasy genetika   metabolismus   MeSH
• proteiny přenášející kationty genetika   metabolismus   MeSH
• transferin metabolismus   MeSH
• železo metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ceruloplasmin MeSH
• CYBRD1 protein, human MeSH Prohlížeč
• cytochromy typu b MeSH
• ferroportin MeSH
• HEPH protein, human MeSH Prohlížeč
• kultivační média MeSH
• membránové proteiny * MeSH
• oxidoreduktasy MeSH
• proteiny přenášející kationty MeSH
• solute carrier family 11- (proton-coupled divalent metal ion transporters), member 2 MeSH Prohlížeč
• transferin MeSH
• železo MeSH

We have shown previously that iron deprivation significantly stimulates the uptake of non-transferrin ferric iron from ferric citrate by erythroleukemia K562 cells and that this stimulation depends on protein synthesis. However, we have not detected increased expression of any known iron transport protein (Kovar J. et al. (2006) Blood Cells Mol Dis 37:95-99). Therefore, in order to identify membrane proteins of K562 cells with increased expression under iron deprivation, we employed the isolation of membrane proteins by two-phase partitioning system, protein separation by high-resolution 2D electrophoresis, computer differential analysis, and tandem mass spectrometry. Employing these techniques we identified two proteins with statistically significant upregulation, i.e., aldolase A (ALDA) and voltage-dependent anion channel 2 (VDAC2). The upregulation of aldolase A and VDAC2 in K562 cells under iron deprivation was also confirmed by western blot analysis. This is the first time when the control of aldolase A and VDAC2 levels by iron status of the cell is demonstrated.

• MeSH
• aldolasa genetika   metabolismus   MeSH
• buňky K562 * MeSH
• deficit železa * MeSH
• lidé MeSH
• napětím ovládaný aniontový kanál 2 genetika   metabolismus   MeSH
• regulace genové exprese * MeSH
• upregulace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aldolasa MeSH
• napětím ovládaný aniontový kanál 2 MeSH
• VDAC2 protein, human MeSH Prohlížeč

We tested the effect of iron deprivation on cell death induction in human Raji cells pre-adapted to differing availability of extracellular iron. Iron deprivation was achieved by incubation in a defined iron-free medium. Original Raji cells have previously been adapted to long-term culture in a defined medium with 5 microg/ml of iron-saturated human transferrin as a source of iron. Raji/lowFe cells were derived from original Raji cells by subsequent adaptation to culture in the medium with 50 microm ferric citrate as a source of iron. Raji/lowFe-re cells were derived from Raji/lowFe cells by re-adaptation to the transferrin-containing (5 microg/ml) medium. Iron deprivation induced cell death in both Raji cells and Raji/lowFe-re cells; that is, cells pre-adapted to a near optimum source of extracellular iron (5 microg/ml of transferrin). However, Raji/lowFe cells preadapted to a limited source of extracellular iron (50 microm ferric citrate) became resistant to the induction of cell death by iron deprivation. We demonstrated that cell death induction by iron deprivation in Raji cells correlates with the activation of executioner caspase-3 and the cleavage of caspase-3 substrate, poly-ADP ribose polymerase. Two other executioner caspases, caspase-7 and caspase-6, were not activated. Taken together, we suggest that in human Raji cells, iron deprivation induces apoptotic cell death related to caspase-3 activation. However, the sensitivity of the cells to death induction by iron deprivation can be reversibly changed by extracellular iron availability. The cells pre-adapted to a limited source of extracellular iron became resistant.

• MeSH
• aktivace enzymů MeSH
• apoptóza účinky léků   fyziologie   MeSH
• buněčné dělení účinky léků   fyziologie   MeSH
• Burkittův lymfom MeSH
• deficit železa * MeSH
• kaspasa 3 metabolismus   MeSH
• kaspasa 6 metabolismus   MeSH
• kaspasa 7 metabolismus   MeSH
• kultivační média MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• viabilita buněk účinky léků   fyziologie   MeSH
• železo farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CASP6 protein, human MeSH Prohlížeč
• kaspasa 3 MeSH
• kaspasa 6 MeSH
• kaspasa 7 MeSH
• kultivační média MeSH
• železo MeSH

Iron deprivation induces apoptosis in some sensitive cultured tumour cells, while other cells are resistant. In order to elucidate the mechanisms involved in apoptosis induction by iron deprivation, we studied the expression of p53 and the expression of selected p53-regulated genes. To discriminate between changes coupled only with iron deprivation and changes involved in apoptosis induction by iron deprivation, we compared the expression of the genes in sensitive (human Raji, mouse 38C13) versus resistant (human HeLa, mouse EL4) cells under iron deprivation. Iron deprivation was achieved by incubation in a defined iron-free medium. The level of p53 mRNA decreased significantly under iron deprivation in sensitive cells, but it did not change in resistant cells. On the contrary, the level of the p53 protein under iron deprivation was slightly increased in sensitive cells while it was not changed in resistant cells. The activity of p53 was assessed by the expression of selected p53-regulated targets, i.e. p21(WAF1/CIP1) gene, mdm2, bcl-2 and bax. We did not detect any relevant change in mRNA levels as well as in protein levels of these genes under iron deprivation with the exception of p21(WAF1/CIP1). We detected a significant increase in the level of p21 mRNA in both (sensitive and resistant) mouse cell lines tested, however, we did not find any change in both (sensitive and resistant) human cell lines. Moreover, the p21(WAF1/CIP1) protein was accumulated in mouse-sensitive 38C13 cells under iron deprivation while all other cell lines tested, including human-sensitive cell line Raji, did not show any accumulation of p21(WAF1/CIP1) protein. It seems that the p21(WAF1/CIP1) mRNA, as well as protein accumulation, is not specifically coupled with apoptosis induction by iron deprivation and that it is rather cell-line specific. Taken together, we suggest that iron deprivation induces apoptosis at least in some cell types independently of the p53 pathway.

• MeSH
• apoptóza * MeSH
• lidé MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• nádorový supresorový protein p53 fyziologie   MeSH
• protein X asociovaný s bcl-2 MeSH
• protoonkogenní proteiny c-bcl-2 genetika   MeSH
• protoonkogenní proteiny genetika   MeSH
• regulace genové exprese MeSH
• železo fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• BAX protein, human MeSH Prohlížeč
• Bax protein, mouse MeSH Prohlížeč
• nádorový supresorový protein p53 MeSH
• protein X asociovaný s bcl-2 MeSH
• protoonkogenní proteiny c-bcl-2 MeSH
• protoonkogenní proteiny MeSH
• železo MeSH

We studied the sensitivity of tumor cells to the induction of apoptosis by iron deprivation. Iron deprivation was achieved by the employment of a defined iron-deficient culture medium. Mouse 38C13 cells and human Raji cells die within 48 and 96 h of incubation in iron-deficient medium, respectively. On the contrary, mouse EL4 cells and human HeLa cells are completely resistant to the induction of death under the same experimental arrangement. Deoxyribonucleic acid fragmentation analysis by agarose gel electrophoresis as well as flow cytometric analysis after propidium iodide staining detected in 38C13 and Raji cells, but not in EL4 and HeLa cells, changes characteristic to apoptosis. The 38C13 cells, sensitive to iron deprivation, also displayed a similar degree of sensitivity to apoptosis induction by thiol deprivation (achieved by 2-mercaptoethanol withdrawal from the culture medium) as well as by rotenone (50 nM), hydroxyurea (50 microM), methotrexate (20 nM), and doxorubicin (100 nM). Raji cells shared with 38C13 cells a sensitivity to rotenone, methotrexate, doxorubicin, and, to a certain degree, to hydroxyurea. However, Raji cells were completely resistant to thiol deprivation. EI4 and HeLa cells, resistant to iron deprivation, also displayed a greater degree of resistance to most of the other apoptotic stimuli than did their sensitive counterparts. We conclude that some tumor cells in vitro are sensitive to apoptosis induction by iron deprivation, while other tumor cells are resistant. All the tumors found to be sensitive to iron deprivation in this study (four cell lines) are of hematopoietic origin. The mechanism of resistance to apoptosis induction by iron deprivation differs from the mechanism of resistance to thiol deprivation.

• MeSH
• apoptóza * účinky léků   MeSH
• B-buněčný lymfom MeSH
• barvicí látky MeSH
• buněčné dělení MeSH
• Burkittův lymfom MeSH
• doxorubicin farmakologie   MeSH
• elektroforéza v agarovém gelu MeSH
• fragmentace DNA MeSH
• HeLa buňky MeSH
• hydroxymočovina farmakologie   MeSH
• kultivační média MeSH
• lidé MeSH
• lymfom T-buněčný MeSH
• methotrexát farmakologie   MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• nádory patologie   MeSH
• propidium MeSH
• průtoková cytometrie MeSH
• rotenon farmakologie   MeSH
• transferin MeSH
• viabilita buněk MeSH
• železo aplikace a dávkování   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• barvicí látky MeSH
• doxorubicin MeSH
• hydroxymočovina MeSH
• kultivační média MeSH
• methotrexát MeSH
• propidium MeSH
• rotenon MeSH
• transferin MeSH
• železo MeSH

We studied the factors that determine the differing growth requirements of low-iron-tolerant (LIT) versus high-iron-dependent (HID) cells for extracellular nontransferrin iron. The growth of LIT cells HeLa and THP-1, when transferred from transferrin (5 micrograms/ml) medium into low-iron (5 microM ferric citrate) medium, was not significantly affected while HID cells Jiyoye and K562 showed nearly no growth. HeLa and THP-1 cells, as well as Jiyoye and K562 cells, do not produce transferrin in sufficient amounts to support their growth in low-iron medium. Surprisingly, similar rates of iron uptake in low-iron medium (0.033 and 0.032 nmol Fe/min and 10(6) cells) were found for LIT cells HeLa and HID cells K562. Furthermore, the intracellular iron level (4.64 nmol/10(6) cells) of HeLa cells grown in low-iron medium was much higher than iron levels (0.15 or 0.20 nmol/10(6) cells) of HeLa or K562 cells grown in transferrin medium. We demonstrated that the activity (ratio activated/total) of the iron regulatory protein (IRP) in HID cells Jiyoye and K562 increased more than twofold (from 0.32 to 0.79 and from 0.47 to 1.12, respectively) within 48 h after their transfer into low-iron medium. In the case of LIT cells HeLa and THP-1, IRP activity stayed at similar or slightly decreased levels (0.86-0.73 and 0.58-0.55, respectively). Addition of iron chelator deferoxamine (50 microM, i.e., about half-maximal growth-inhibitory dose) resulted in significantly increased activity of IRP also in HeLa and THP-1 cells. We hypothesize that the relatively higher bioavailability of nontransferrin iron in LIT cells, over that in HID cells, determines the differing responses observed under low-iron conditions.

• MeSH
• akutní erytroblastická leukemie MeSH
• akutní monocytární leukemie MeSH
• biologický transport MeSH
• buněčné dělení * MeSH
• Burkittův lymfom MeSH
• deferoxamin farmakologie   MeSH
• HeLa buňky MeSH
• kinetika MeSH
• kultivační média MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• proteiny obsahující železo a síru metabolismus   MeSH
• proteiny regulující obsah železa MeSH
• proteiny vázající RNA metabolismus   MeSH
• transferin farmakologie   MeSH
• železo metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• deferoxamin MeSH
• kultivační média MeSH
• proteiny obsahující železo a síru MeSH
• proteiny regulující obsah železa MeSH
• proteiny vázající RNA MeSH
• transferin MeSH
• železo MeSH