Iron accumulation in the brain is associated with the pathogenesis of Parkinson's disease (PD). Misexpression of some iron transport and storage proteins is related to iron dyshomeostasis. Iron regulatory proteins (IRPs) including IRP1 and IRP2 are cytosolic proteins that play important roles in maintaining cellular iron homeostasis. F-box and leucine-rich repeat protein 5 (FBXL5) is involved in the regulation of iron metabolism by degrading IRP2 through the ubiquitin-proteasome system. Nitric oxide (NO) enhances the binding activity of IRP1, but its effect on IRP2 is ambiguous. Therefore, in the present study, we aim to determine whether sodium nitroprusside (SNP), a NO donor, regulates FBXL5 and IRP2 expression in cultured SH-SY5Y cells. MTT assay revealed that treatment of SNP attenuated the cell viability in a dose-dependent manner. Flow cytometry test showed that 100 and 300 μmol/L SNP administration significantly reduced the mitochondrial membrane potential by 45% and 60%, respectively. Moreover, Western blotting analysis demonstrated that 300 μmol/L SNP significantly increased FBXL5 expression by about 39%, whereas the expression of IRP2 was decreased by 46%, correspondingly. These findings provide evidence that SNP could induce mitochondrial dysfunction, enhance FBXL5 expression and decrease IRP2 expression in SH-SY5Y cells.
Cell Line ; Cell Survival ; F-Box Proteins ; metabolism ; Homeostasis ; Humans ; Iron Regulatory Protein 2 ; metabolism ; Nitric Oxide ; metabolism ; Nitroprusside ; pharmacology ; Proteasome Endopeptidase Complex ; Ubiquitin ; metabolism ; Ubiquitin-Protein Ligase Complexes ; metabolism

BACKGROUND AND PURPOSE: Hypoxia, or ischemia, is a common cause of neurological deficits in the elderly. This study elucidated the mechanisms underlying ischemia-induced brain injury that results in neurological sequelae. METHODS: Cerebral ischemia was induced in male Sprague-Dawley rats by transient ligation of the left carotid artery followed by 60 min of hypoxia. A two-dimensional differential proteome analysis was performed using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to compare changes in protein expression on the lesioned side of the cortex relative to that on the contralateral side at 0, 6, and 24 h after ischemia. RESULTS: The expressions of the following five proteins were up-regulated in the ipsilateral cortex at 24 h after ischemia-reperfusion injury compared to the contralateral (i.e., control) side: aconitase 2, neurotensin-related peptide, hypothetical protein XP-212759, 60-kDa heat-shock protein, and aldolase A. The expression of one protein, dynamin-1, was up-regulated only at the 6-h time point. The level of 78-kDa glucose-regulated protein precursor on the lesioned side of the cerebral cortex was found to be high initially, but then down-regulated by 24 h after the induction of ischemia-reperfusion injury. The expressions of several metabolic enzymes and translational factors were also perturbed soon after brain ischemia. CONCLUSIONS: These findings provide insights into the mechanisms underlying the neurodegenerative events that occur following cerebral ischemia.
Aconitate Hydratase ; Aged ; Anoxia ; Brain Injuries ; Brain Ischemia ; Carotid Arteries ; Cerebral Cortex* ; Dynamin I ; Fructose-Bisphosphate Aldolase ; Geriatrics ; Heat-Shock Proteins ; Humans ; Ischemia ; Ligation ; Male ; Mass Spectrometry ; Proteome ; Proteomics ; Rats, Sprague-Dawley ; Reperfusion Injury*

PURPOSE: Zinc is one of the trace minerals in the body and is known to have an anticancer effect by inducing apoptosis in prostate cancer. We aimed to investigate the antiproliferative effects of a zinc-citrate compound in bladder cancer. MATERIALS AND METHODS: A bladder cancer cell line (MBT-2) was treated with a zinc-citrate compound at different time intervals and concentrations. Mitochondrial (m)-aconitase activity was determined by use of the aconitase assay. DNA laddering analysis was performed to investigate apoptosis of MBT-2 cells. The molecular mechanism of apoptosis was investigated by Western blot analysis of p53, p21waf1, Bcl-2, Bcl-xL, and Bax and also by caspase-3 activity analysis. RESULTS: Treatment with the zinc-citrate compound resulted in a time- and dose-dependent decrease in cell number of MBT-2 cells. M-aconitase activity was significantly decreased. DNA laddering analysis indicated apoptosis of MBT-2 cells. The zinc-citrate compound increased the expression of p21waf1 and p53 and reduced the expression of Bcl-2 and Bcl-xL proteins but induced expression of Bax protein. The zinc-citrate compound induced apoptosis of MBT-2 cells by activation of the caspase-3 pathway. CONCLUSIONS: We have shown that a zinc-citrate compound induces apoptotic cell death in a bladder cancer cell line, MBT-2, by caspase-3 activation through up-regulation of apoptotic proteins and down-regulation of antiapoptotic proteins.
Aconitate Hydratase ; Apoptosis ; bcl-2-Associated X Protein ; bcl-X Protein ; Blotting, Western ; Caspase 3 ; Cell Count ; Cell Death ; Cell Line ; DNA ; Down-Regulation ; Minerals ; Prostatic Neoplasms ; Proteins ; Up-Regulation ; Urinary Bladder ; Urinary Bladder Neoplasms ; Zinc

Oxidative damage is thought to be a major cause of the progression of dopamine (DA)rgic neurodegeneration as in Parkinson's disease. We have previously reported that tetrahydrobiopterin (BH4), an endogenous molecule required for DA synthesis, exerts oxidative stress to DA-producing cells and facilitates the production of DA quinone. It is known that aconitase, present in both mitochondrial and cytosolic forms, act as an reactive oxygen species (ROS) sensor, and that their inactivation leads to further generation of ROS. In the present study we investigated whether the BH4-associated vulnerability of DA cells might involve aconitase. In DArgic cell line CATH.a, BH4 treatment caused reduction of activity of both mitochondrial and cytosolic aconitases, and this appeared to be due to direct inactivation of the pre-existing enzyme molecules. Although most of the activity reduced by BH4 was increased upon reactivation reaction under a reducing condition, the restoration was not complete, suggesting that irreversible and covalent modification has occurred. The aconitase inactivation was exacerbated in the presence of DA and attenuated in the presence of tyrosine hydroxylase inhibitor a-methyl-p-tyrosine, suggesting the involvement of DA. The degree of inactivation increased when the cells were treated with the quinone reductase inhibitor dicoumarol and decreased in the presence of quinone reductase inducer sulforaphane. Taken together, BH4 appeared to lead to both reversible and irreversible inactivation of aconitase and that this is facilitated by the presence of DA and accumulation of DA quinone.
Aconitate Hydratase ; Benzoquinones ; Biopterin ; Cell Line ; Cytosol ; Dicumarol ; Dopamine ; NAD(P)H Dehydrogenase (Quinone) ; Oxidative Stress ; Parkinson Disease ; Reactive Oxygen Species ; Thiocyanates ; Tyrosine 3-Monooxygenase

In order to investigate the influence of iron deficiency on the mRNA expression of iron regulatory protein (IRP(2)) mRNA and ferritins (FN) in intestinal mucosa of rat, the animal model of rat with nutritional iron deficiency was established. According to the measurement of serum iron (sI), serum fertitin (sFn) and Hb, the experiments were divided into 4 groups: control group, recessive iron deficiency group, mild iron deficiency group and moderate iron deficiency group. sI was measured by flame assay and sFN was measured by radioimmunoassay, the expressions of irp(2) mRNA and fn mRNA were detected by RT-PCR. The results showed that (1) with aggravation of iron deficiency, the levels of sI and sFN in experimental groups decreased and had significant difference from that in control group, except sI level in the recessive iron deficiency group; (2) with aggravation of iron deficiency, the expression of irp(2) mRNA in duodenum mucosa elevated, and the expressions of irp(2) mRNA in moderate iron deficiency group and mild iron deficiency group were higher than that in control group (p < 0.01), the expression of irp(2) mRNA in moderate iron deficiency group was higher than that in recessive iron deficiency group (p < 0.05), but the expression of irp(2) mRNA did not showed statistical difference between mild iron deficiency group and moderate iron deficiency group (p > 0.05); (3) with aggragation of iron deficiency, the expression of fn mRNA in dudemum mucosa decreased, the expression levels fn mRNA in control and moderate groups were highest and lowest, respectively, there were significant differences between experimental and control groups (p < 0.05), and between experimental groups (p < 0.05); (4) the expression of irp(2) mRNA and fn mRNA in moderate iron difficiency group showed negative correlation (r = 0.662, p < 0.05). It is concluded that IRP(2) protein serves as an important regulator of iron metabolism in the human body, and regulates iron uptake from the intestine by controlling the expression of fn mRNA at the post transcriptional level.
Anemia, Iron-Deficiency ; metabolism ; Animals ; Duodenum ; metabolism ; Female ; Ferritins ; genetics ; metabolism ; Intestinal Mucosa ; metabolism ; Iron Regulatory Protein 2 ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Wistar

Ascorbic acid has been reported to extend replicative life span of human embryonic fibroblast (HEF). Since the detailed molecular mechanism of this phenomenon has not been investigated, we attempted to elucidate. Continuous treatment of HEF cells with ascorbic acid (at 200 micrometer) from 40 population doubling (PD) increased maximum PD numbers by 18% and lowered SA-beta-gal positive staining, an aging marker, by 2.3 folds, indicating that ascorbic acid extends replicative life span of HEF cells. Ascorbic acid treatment lowered DCFH by about 7 folds and Rho123 by about 70%, suggesting that ascorbic acid dramatically decreased ROS formation. Ascorbic acid also increased aconitase activity, a marker of mitochondrial aging, by 41%, indicating that ascorbic acid treatment restores age-related decline of mitochondrial function. Cell cycle analysis by flow cytometry revealed that ascorbic acid treatment decreased G1 population up to 12%. Further western blot analysis showed that ascorbic acid treatment decreased levels of p53, phospho-p53 at ser 15, and p21, indicating that ascorbic acid relieved senescence-related G1 arrest. Analysis of AP (apurinic/apyrimidinic) sites showed that ascorbic acid treatment decreased AP site formation by 35%. We also tested the effect of hydrogen peroxide treatment, as an additional oxidative stress. Continuous treatment of 20 micrometer of hydrogen peroxide from PD 40 of HEF cells resulted in premature senescence due to increased ROS level, and increased AP sites. Taken together, the results suggest that ascorbic acid extends replicative life span of HEF cells by reducing mitochondrial and DNA damages through lowering cellular ROS.
Aconitate Hydratase ; Aging ; Ascorbic Acid* ; Blotting, Western ; Cell Cycle ; DNA Damage ; DNA* ; Fibroblasts* ; Flow Cytometry ; Humans* ; Hydrogen Peroxide ; Oxidative Stress ; Reactive Oxygen Species

OBJECTIVETo investigate the mRNA expression of transferrin receptor 1 (TfR1) and iron regulatory protein 1 (IRP1) in the full-term placenta from different maternal iron status, and explore the mechanism of placental iron transport and regulation.

METHODSThe mRNA level of TfR1 and IRP1 in full-term placentae was detected by reverse transcription polymerase chain reaction (RT-PCR) in normal group (N), iron deficiency group (ID) and iron deficiency anemia group (IDA).

RESULTS(1) The expression of TfR1 mRNA in N group was 0.4813 +/- 0.1891, in ID group was 0. 6647 +/- 0.2788, and in IDA group was 0.9767 +/- 0.2858. There was significant difference between IDA group and N group or ID group (t = 0.002, P < 0.01 or t = 0.028, P < 0.05), and was no difference between ID group and N group (t = 0.117, P > 0.05). (2) The expression of IRP1 mRNA in N group was 0.2616 +/- 0.0785, in ID group was 0.3696 +/- 0.1801, and in IDA group was 0.3971 +/- 0.0902 and was no difference among the three groups (F = 1.845, P = 0.179).

CONCLUSIONSThe expression of TfR1 mRNA is increased when maternal iron deficiency progressed while there is no change in the expression of IRP1 mRNA in the placentae of TfR1 mRNA indicated that IRP1 takes part in the regulation of placenta iron transport.

Anemia, Iron-Deficiency ; genetics ; Antigens, CD ; metabolism ; Female ; Humans ; Iron Regulatory Protein 1 ; metabolism ; Placenta ; metabolism ; Pregnancy ; RNA, Messenger ; metabolism ; Receptors, Transferrin ; metabolism

AIMTo investigate the possible role of manganese in the regulation of mitochondrial aconitase (mACON) activity human prostate carcinoma cell line PC-3 cells.

METHODSThe mACON enzymatic activities of human prostate carcinoma cell line PC-3 cells were determined using a reduced nicotinamide adenine dinucleotide-coupled assay. Immunoblot and transient gene expression assays were used to study gene expression of the mACON. The putative response element for gene expression was identified using reporter assays with site-directed mutagenesis and electrophoretic mobility-shift assays.

RESULTSIn vitro study revealed that manganese chloride (MnCl2) treatment for 16 h inhibited the enzymatic activity of mACON, which induced the inhibition of citrate utility and cell proliferation of PC-3 cells. Although results from transient gene expression assays showed that MnCl2 treatment upregulated gene translation by approximately 5-fold through the iron response element pathway, immunoblot and reporter assays showed that MnCl2 treatments inhibited protein and gene expression of mACON. This effect was reversed by co-treatment with ferric ammonium citrate. Additional reporter assays with site-directed mutagenesis and electrophoretic mobility-shift assays suggested that a putative metal response element in the promoter of the mACON gene was involved in the regulation of MnCl2 on the gene expression of mACON.

CONCLUSIONThese findings suggest that manganese acts as an antagonist of iron, disrupting the enzymatic activity and gene expression of mACON and citrate metabolism in the prostate.

Aconitate Hydratase ; antagonists & inhibitors ; genetics ; Actins ; genetics ; Adenosine Triphosphate ; metabolism ; Cell Line, Tumor ; Chlorides ; pharmacology ; Citrates ; metabolism ; DNA Primers ; Gene Expression Regulation, Enzymologic ; drug effects ; Gene Expression Regulation, Neoplastic ; drug effects ; Genes, Reporter ; Humans ; Iron ; metabolism ; Male ; Manganese Compounds ; pharmacology ; Mitochondria ; enzymology ; Prostatic Neoplasms ; enzymology

OBJECTIVE: Human seminal plasma has diverse biological activities including cytotoxic effect. It contains high concentrations of zinc and citric acid. Zinc inhibits several carcinoma cell growths through induction of cell cycle arrest and apoptosis. We tried to investigate the effects of zinc-citrate compound (CIZAR(R)) on normal human ovarian epithelial (NOSE) cells and human epithelial ovarian cancer cells, OVCAR-3. METHODS: To investigate the potential effect of CIZAR(R) on cell growth and survival, cells were treated with different dose and exposed to different time. Mitochondrial(m)-aconitase activity was determined in cell extracts using aconitase assay. The flow cytometric assay, DNA laddering, telomerase activity and morphological analysis were done to investigate apoptosis of OVCAR-3 cells. Molecular mechanism of apoptosis was investigated by p53, Bcl-XL, Bcl-2, Bax protein, and caspase activity. RESULTS: Treatment of OVCAR-3 cells with CIZAR(R) resulted in a time- and dose-dependent decrease in cell number in comparison with NOSE cells. M-aconitase activity was significantly decreased in OVCAR-3 cells but relatively constant in NOSE cells. The flow cytometric assay, DNA laddering and morphological analysis indicated apoptosis of OVCAR-3 cells. CIZAR(R) did not affect p53 but increased the expression of p21waf1 upon the indicated times and induced reduction of telomerase activity. CIZAR(R) reduced expression of Bcl-2 and Bcl-xL proteins but induced expression of Bax protein. CIZAR(R) induced apoptosis of OVCAR-3 cells by activation of caspase-3 pathway. CONCLUSION: These results show that CIZAR(R) prevent the proliferation of OVCAR-3 cells by inactivation of m-aconitase activity and induce apoptosis by induction of apoptotic genes and repression of antiapoptotic genes without adverse effect on normal ovarian epithelial cells. These results will offer new window in prevention and treatment of epithelial ovarian cancer.
Aconitate Hydratase ; Apoptosis ; bcl-2-Associated X Protein ; bcl-X Protein ; Caspase 3 ; Cell Count ; Cell Cycle Checkpoints ; Cell Extracts ; Citric Acid ; DNA ; Epithelial Cells ; Humans* ; Nose ; Ovarian Neoplasms* ; Repression, Psychology ; Semen ; Telomerase ; Zinc

To explore the mechanism of iron metabolism and its regulation as well as the roles of IRP(2) in ion metabolism of HL-60 cells, HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, which was treated with ferric chloride (FeCl(3)) or deferoxamine (DFO). The cells were harvested at 12, 24 and 48 hours of proliferation, and total RNA was isolated; cDNA was synthesized by reverse transcription (RT), and relative expression levels of IRP(2) mRNA, Fn mRNA and TfR mRNA were determined by RT-PCR. The results showed at follows: (1) the level of IRP(2) mRNA remained constant in all cells, whether or not treated with DFO or FeCl(3). However, the expression of IRP(2) mRNA decreased when the time of cell culture was prolonged. There was no significant difference between groups (F(B-S) = 1.199, P > 0.05), but there was significant difference among the different time culture (F(W-S) = 43.418, P < 0.01). (2) Cells which treated neither with DFO nor ferri chloride showed significant difference from the control (F(W-S) = 7.184, F(B-S) = 113.926; P < 0.01). The level of TfR mRNA increased in the cells treated with DFO. Surprisingly, when cells treated with FeCl(3), there was not decline of TfR mRNA expression, but it increased lightly at 12 hours and peaked at 24 hours and declined drastically at 48 hours. (3) The level of Fn mRNA in the cells treated with FeCl(3) was approximately 2-fold as the control cells. In contrast with the control cells, there was significant difference (P < 0.05). The level of Fn mRNA of the cells treated with DFO had little change. As compared with the control cells, no significant difference was seen (P > 0.05). (4) There was not any significant correlation between IRP(2) mRNA and TfR mRNA or Fn mRNA in HL-60 cells (r = -0.005; r = 0.074; P > 0.05). It is concluded that (1) IRP(2) may regulate the iron metabolism in HL-60 cells by altering amounts of the IRP(2) 3.7- or 6.4-kb mRNA at the transcriptional level, or by IRP(2) degradation at the post transcriptional level. (2) Both of Fn mRNA and TfR mRNA participated, more or less, in the iron metabolism in HL-60 cells.
Ferritins ; genetics ; Gene Expression Regulation, Neoplastic ; HL-60 Cells ; Humans ; Iron Regulatory Protein 2 ; genetics ; RNA, Messenger ; genetics ; metabolism ; Receptors, Transferrin ; genetics ; Reverse Transcriptase Polymerase Chain Reaction