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.

• Keywords
• 1.1B4, Apoptosis, Calcium influx, Diazoxide, Fatty acids, NES2Y, Nifedipine, Pancreatic β-cells, Type 2 diabetes mellitus, Verapamil,
• Publication type
• Journal Article 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.

• Keywords
• ER stress, NES2Y, apoptosis, caspases, fatty acid transporters, fatty acids, hypoxia, hypoxia-inducible factor 1α, pancreatic β-cells,
• MeSH
• Insulin-Secreting Cells metabolism   MeSH
• Biomarkers MeSH
• Cell Line MeSH
• Hypoxia metabolism   MeSH
• Caspases metabolism   MeSH
• Humans MeSH
• Fatty Acids metabolism   MeSH
• Cell Proliferation MeSH
• Signal Transduction drug effects   MeSH
• Endoplasmic Reticulum Stress MeSH
• Cell Survival MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Biomarkers MeSH
• Caspases MeSH
• Fatty Acids 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.

• Keywords
• NES2Y, apoptosis, endoplasmic reticulum (ER) stress, extracellular signal-regulated kinase (ERK), fatty acids, p38 mitogen-activated protein kinase (MAPK), pancreatic β-cells,
• MeSH
• Apoptosis * MeSH
• Insulin-Secreting Cells cytology   metabolism   pathology   MeSH
• Cell Line MeSH
• Oleic Acid metabolism   MeSH
• Stearic Acids metabolism   MeSH
• Humans MeSH
• MAP Kinase Signaling System * MeSH
• p38 Mitogen-Activated Protein Kinases metabolism   MeSH
• Endoplasmic Reticulum Stress * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Oleic Acid MeSH
• Stearic Acids MeSH
• p38 Mitogen-Activated Protein Kinases 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.

• Keywords
• ERK, NES2Y, apoptosis, fatty acids, p38 MAPK, pancreatic β-cells,
• MeSH
• Enzyme Activation MeSH
• Apoptosis * drug effects   MeSH
• Insulin-Secreting Cells drug effects   metabolism   MeSH
• Cell Line MeSH
• Gene Expression MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Stearic Acids pharmacology   MeSH
• Humans MeSH
• MAP Kinase Signaling System drug effects   MeSH
• Fatty Acids metabolism   pharmacology   MeSH
• p38 Mitogen-Activated Protein Kinases antagonists & inhibitors   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Protein Kinase Inhibitors MeSH
• Stearic Acids MeSH
• Fatty Acids MeSH
• p38 Mitogen-Activated Protein Kinases MeSH
• stearic acid MeSH Browser

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
• Cell Membrane metabolism   MeSH
• Cell Line MeSH
• Ceruloplasmin genetics   metabolism   MeSH
• Cytochrome b Group genetics   metabolism   MeSH
• Gene Expression * MeSH
• Ferroportin MeSH
• Culture Media chemistry   MeSH
• Humans MeSH
• Membrane Proteins * genetics   metabolism   MeSH
• Oxidoreductases genetics   metabolism   MeSH
• Cation Transport Proteins genetics   metabolism   MeSH
• Transferrin metabolism   MeSH
• Iron metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Ceruloplasmin MeSH
• CYBRD1 protein, human MeSH Browser
• Cytochrome b Group MeSH
• Ferroportin MeSH
• HEPH protein, human MeSH Browser
• Culture Media MeSH
• Membrane Proteins * MeSH
• Oxidoreductases MeSH
• Cation Transport Proteins MeSH
• solute carrier family 11- (proton-coupled divalent metal ion transporters), member 2 MeSH Browser
• Transferrin MeSH
• Iron 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
• Fructose-Bisphosphate Aldolase genetics   metabolism   MeSH
• K562 Cells * MeSH
• Iron Deficiencies * MeSH
• Humans MeSH
• Voltage-Dependent Anion Channel 2 genetics   metabolism   MeSH
• Gene Expression Regulation * MeSH
• Up-Regulation MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fructose-Bisphosphate Aldolase MeSH
• Voltage-Dependent Anion Channel 2 MeSH
• VDAC2 protein, human MeSH Browser

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
• Enzyme Activation MeSH
• Apoptosis drug effects   physiology   MeSH
• Cell Division drug effects   physiology   MeSH
• Burkitt Lymphoma MeSH
• Iron Deficiencies * MeSH
• Caspase 3 metabolism   MeSH
• Caspase 6 metabolism   MeSH
• Caspase 7 metabolism   MeSH
• Culture Media MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Cell Survival drug effects   physiology   MeSH
• Iron pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CASP6 protein, human MeSH Browser
• Caspase 3 MeSH
• Caspase 6 MeSH
• Caspase 7 MeSH
• Culture Media MeSH
• Iron 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
• Apoptosis * MeSH
• Humans MeSH
• Mice MeSH
• Tumor Cells, Cultured MeSH
• Tumor Suppressor Protein p53 physiology   MeSH
• bcl-2-Associated X Protein MeSH
• Proto-Oncogene Proteins c-bcl-2 genetics   MeSH
• Proto-Oncogene Proteins genetics   MeSH
• Gene Expression Regulation MeSH
• Iron physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• BAX protein, human MeSH Browser
• Bax protein, mouse MeSH Browser
• Tumor Suppressor Protein p53 MeSH
• bcl-2-Associated X Protein MeSH
• Proto-Oncogene Proteins c-bcl-2 MeSH
• Proto-Oncogene Proteins MeSH
• Iron 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
• Apoptosis * drug effects   MeSH
• Lymphoma, B-Cell MeSH
• Coloring Agents MeSH
• Cell Division MeSH
• Burkitt Lymphoma MeSH
• Doxorubicin pharmacology   MeSH
• Electrophoresis, Agar Gel MeSH
• DNA Fragmentation MeSH
• HeLa Cells MeSH
• Hydroxyurea pharmacology   MeSH
• Culture Media MeSH
• Humans MeSH
• Lymphoma, T-Cell MeSH
• Methotrexate pharmacology   MeSH
• Mice MeSH
• Tumor Cells, Cultured MeSH
• Neoplasms pathology   MeSH
• Propidium MeSH
• Flow Cytometry MeSH
• Rotenone pharmacology   MeSH
• Transferrin MeSH
• Cell Survival MeSH
• Iron administration & dosage   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Coloring Agents MeSH
• Doxorubicin MeSH
• Hydroxyurea MeSH
• Culture Media MeSH
• Methotrexate MeSH
• Propidium MeSH
• Rotenone MeSH
• Transferrin MeSH
• Iron 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
• Leukemia, Erythroblastic, Acute MeSH
• Leukemia, Monocytic, Acute MeSH
• Biological Transport MeSH
• Cell Division * MeSH
• Burkitt Lymphoma MeSH
• Deferoxamine pharmacology   MeSH
• HeLa Cells MeSH
• Kinetics MeSH
• Culture Media MeSH
• Humans MeSH
• Tumor Cells, Cultured MeSH
• Iron-Sulfur Proteins metabolism   MeSH
• Iron-Regulatory Proteins MeSH
• RNA-Binding Proteins metabolism   MeSH
• Transferrin pharmacology   MeSH
• Iron metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Deferoxamine MeSH
• Culture Media MeSH
• Iron-Sulfur Proteins MeSH
• Iron-Regulatory Proteins MeSH
• RNA-Binding Proteins MeSH
• Transferrin MeSH
• Iron MeSH