Duodenal biopsies are considered a suitable source of enterocytes for studies of dietary iron absorption. However, the expression level of molecules involved in iron absorption may vary along the length of duodenum. We aimed to determine whether the expression of molecules involved in the absorption of heme and non-heme iron differs depending on the location in the duodenum. Analysis was performed with samples of duodenal biopsies from 10 individuals with normal iron metabolism. Samples were collected at the following locations: (a) immediately post-bulbar, (b) 1-2 cm below the papilla of Vater and (c) in the distal duodenum. The gene expression was analyzed at the mRNA and protein level using real-time PCR and Western blot analysis. At the mRNA level, significantly different expression of HCP1, DMT1, ferroportin and Zip8 was found at individual positions of duodenum. Position-dependent expression of other molecules, especially of FLVCR1, HMOX1 and HMOX2 was also detected but with no statistical significances. At the protein level, we observed statistically significantly decreasing expression of transporters HCP1, FLVCR1, DMT1, ferroportin, Zip14 and Zip8 with advancing positions of duodenum. Our results are consistent with a gradient of diminishing iron absorption along the duodenum for both heme and non-heme iron.

• Klíčová slova
• heme iron absorption, iron transport, iron uptake in duodenum, non-heme iron absorption,
• MeSH
• duodenum * metabolismus   MeSH
• hem metabolismus   MeSH
• iontový transport MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• železo * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hem MeSH
• messenger RNA MeSH
• železo * MeSH

The importance of iron in the growth and progression of tumors has been widely documented. In this report, we show that tumor-initiating cells (TICs), represented by spheres derived from the MCF7 cell line, exhibit higher intracellular labile iron pool, mitochondrial iron accumulation and are more susceptible to iron chelation. TICs also show activation of the IRP/IRE system, leading to higher iron uptake and decrease in iron storage, suggesting that level of properly assembled cytosolic iron-sulfur clusters (FeS) is reduced. This finding is confirmed by lower enzymatic activity of aconitase and FeS cluster biogenesis enzymes, as well as lower levels of reduced glutathione, implying reduced FeS clusters synthesis/utilization in TICs. Importantly, we have identified specific gene signature related to iron metabolism consisting of genes regulating iron uptake, mitochondrial FeS cluster biogenesis and hypoxic response (ABCB10, ACO1, CYBRD1, EPAS1, GLRX5, HEPH, HFE, IREB2, QSOX1 and TFRC). Principal component analysis based on this signature is able to distinguish TICs from cancer cells in vitro and also Leukemia-initiating cells (LICs) from non-LICs in the mouse model of acute promyelocytic leukemia (APL). Majority of the described changes were also recapitulated in an alternative model represented by MCF7 cells resistant to tamoxifen (TAMR) that exhibit features of TICs. Our findings point to the critical importance of redox balance and iron metabolism-related genes and proteins in the context of cancer and TICs that could be potentially used for cancer diagnostics or therapy.

• Klíčová slova
• FeS cluster, breast cancer, iron metabolism, stem cells, tumor-initiating cells,
• MeSH
• akutní promyelocytární leukemie enzymologie   genetika   MeSH
• analýza hlavních komponent MeSH
• biologický transport MeSH
• buněčné sféroidy MeSH
• chelátory železa farmakologie   MeSH
• chemorezistence MeSH
• fenotyp MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• mitochondrie enzymologie   MeSH
• myši transgenní MeSH
• nádorové kmenové buňky účinky léků   enzymologie   patologie   MeSH
• nádory prostaty farmakoterapie   enzymologie   genetika   patologie   MeSH
• nádory prsu farmakoterapie   enzymologie   genetika   patologie   MeSH
• protinádorové látky farmakologie   MeSH
• regulace genové exprese enzymů MeSH
• regulace genové exprese u nádorů MeSH
• stanovení celkové genové exprese MeSH
• tamoxifen farmakologie   MeSH
• transkriptom * MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• železo metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chelátory železa MeSH
• protinádorové látky MeSH
• tamoxifen MeSH
• železo MeSH

Patients with alcoholic liver disease (ALD) often display disturbed iron indices. Hepcidin, a key regulator of iron metabolism, has been shown to be down-regulated by alcohol in cell lines and animal models. This down-regulation led to increased duodenal iron transport and absorption in animals. In this study, we investigated gene expression of duodenal iron transport molecules and hepcidin in three groups of patients with ALD (with anaemia, with iron overload and without iron overload) and controls. Expression of DMT1, FPN1, DCYTB, HEPH, HFE and TFR1 was measured in duodenal biopsies by using real-time PCR and Western blot. Serum hepcidin levels were measured by using ELISA. Serum hepcidin was decreased in patients with ALD. At the mRNA level, expressions of DMT1, FPN1 and TFR1 genes were significantly increased in ALD. This pattern was even more pronounced in the subgroups of patients without iron overload and with anaemia. Protein expression of FPN1 paralleled the increase at the mRNA level in the group of patients with ALD. Serum ferritin was negatively correlated with DMT1 mRNA. The down-regulation of hepcidin expression leading to up-regulation of iron transporters expression in the duodenum seems to explain iron metabolism disturbances in ALD. Alcohol consumption very probably causes suppression of hepcidin expression in patients with ALD.

• Klíčová slova
• DCYTB, DMT1, FPN1, HEPH, HFE, TFR1, alcoholic liver disease, gene expression, hepcidin, iron,
• MeSH
• alkoholické nemoci jater metabolismus   MeSH
• cytochromy typu b genetika   metabolismus   MeSH
• dospělí MeSH
• duodenum metabolismus   MeSH
• exprese genu MeSH
• ferroportin MeSH
• hepcidiny fyziologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• membránové proteiny genetika   metabolismus   MeSH
• oxidoreduktasy genetika   metabolismus   MeSH
• proteiny přenášející kationty genetika   metabolismus   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• železo krev   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CYBRD1 protein, human MeSH Prohlížeč
• cytochromy typu b MeSH
• ferroportin MeSH
• hepcidiny MeSH
• HEPH protein, human MeSH Prohlížeč
• membránové proteiny MeSH
• oxidoreduktasy MeSH
• proteiny přenášející kationty MeSH
• solute carrier family 11- (proton-coupled divalent metal ion transporters), member 2 MeSH Prohlížeč
• železo MeSH

It has been shown in previous studies that liver HEP-G2 cells (human hepatocellular carcinoma) lose their ability to express active alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1). Although both are ethanol-inducible enzymes, short-term exposure to ethanol does not cause any changes in expression or activity in cultured HEP-G2 cells. Therefore, we tested the effect of long-term exposure to ethanol on the expression and activity of both ADH and CYP2E1 in these cells. The expression of ADH and CYP2E1 was assessed at the mRNA and/or protein level using real-time PCR and Western blot analysis. Specific colorimetric assays were used for the measurement of ADH and CYP2E1 enzymatic activities. Caco-2 cells (active CYP2E1 and inactive ADH) were used as control cells. Significantly increased protein expression of ADH (about 2.5-fold) as well as CYP2E1 (about 1.6-fold) was found in HEP-G2 cells after long-term (12 mo) exposure to ethanol. The activity of ADH and CYP2E1 was also significantly increased from 12 ± 3 and 6 ± 1 nmol/h/mg of total protein to 191 ± 9 and 57 ± 9 nmol/h/mg of total protein, respectively. We suggest that the loss of activity of ethanol-metabolizing enzymes in cultured HEP-G2 cells is reversible and can be induced by prolonged exposure to ethanol. We are therefore able to reactivate HEP-G2 cells metabolic functions concerning ethanol oxidation just by modification of in vitro culture conditions without necessity of transfection with its side effect - enzyme overexpression.

• MeSH
• alkoholdehydrogenasa biosyntéza   genetika   MeSH
• apoptóza účinky léků   MeSH
• buňky Hep G2 MeSH
• cytochrom P-450 CYP2E1 biosyntéza   genetika   MeSH
• enzymová indukce účinky léků   MeSH
• ethanol farmakologie   MeSH
• hepatocelulární karcinom enzymologie   patologie   MeSH
• játra účinky léků   enzymologie   MeSH
• lidé MeSH
• nádory jater enzymologie   patologie   MeSH
• oxidace-redukce účinky léků   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alkoholdehydrogenasa MeSH
• cytochrom P-450 CYP2E1 MeSH
• ethanol MeSH

Disturbances of iron metabolism are observed in chronic liver diseases. In the present study, we examined gene expression of duodenal iron transport molecules and hepcidin in patients with hereditary hemochromatosis (HHC) (treated and untreated), involving various genotypes (genotypes which represent risk for HHC were examined), and in patients with iron deficiency anaemia (IDA). Gene expressions of DMT1, ferroportin, Dcytb, hephaestin, HFE and TFR1 were measured in duodenal biopsies using real-time PCR and Western blot. Serum hepcidin levels were measured using ELISA. DMT1, ferroportin and TFR1 mRNA levels were significantly increased in post-phlebotomized hemochromatics relative to controls. mRNAs of all tested molecules were significantly increased in patients with IDA compared to controls. The protein expression of ferroportin was increased in both groups of patients but not significantly. Spearman rank correlations showed that DMT1 versus ferroportin, Dcytb versus hephaestin and DMT1 versus TFR1 mRNAs were positively correlated regardless of the underlying cause, similarly to protein levels of ferroportin versus Dcytb and ferroportin versus hephaestin. Serum ferritin was negatively correlated with DMT1 mRNA in investigated groups of patients, except for HHC group. A decrease of serum hepcidin was observed in IDA patients, but this was not statistically significant. Our data showed that although untreated HHC patients do not have increased mRNA levels of iron transport molecules when compared to normal subjects, the expression is relatively increased in relation to body iron stores. On the other hand, post-phlebotomized HHC patients had increased DMT1 and ferroportin mRNA levels possibly due to stimulated erythropoiesis after phlebotomy.

• MeSH
• biologický transport MeSH
• deficit železa * MeSH
• dospělí MeSH
• duodenum metabolismus   MeSH
• fenotyp MeSH
• hemochromatóza krev   genetika   metabolismus   MeSH
• hepcidiny MeSH
• kationické antimikrobiální peptidy krev   metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• proteiny přenášející kationty genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• železo metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HAMP protein, human MeSH Prohlížeč
• hepcidiny MeSH
• kationické antimikrobiální peptidy MeSH
• messenger RNA MeSH
• proteiny přenášející kationty MeSH
• železo MeSH

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