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1.  Regulation of Hepcidin-25 by Short- and Long-Acting rhEPO May Be Dependent on Ferritin and Predict the Response to rhEPO in Hemodialysis Patients 
Nephron Extra  2014;4(1):55-63.
Background/Aims
We examined whether regulation of hepcidin-25 by short- or long-acting recombinant human erythropoietin (rhEPO) is dependent on ferritin and predicts the response to rhEPO in hemodialysis (HD) patients.
Methods
Two studies with rhEPO were performed in 9 HD patients with a 2-year interval. Serum hepcidin-25 was measured at 0-18 h after intravenous epoetin-β (EPO) or methoxy polyethylene glycol-epoetin-β (PEG-EPO) administration and on days 3-7 after PEG-EPO. Hemoglobin (Hb), serum ferritin, transferrin, C-reactive protein (CRP), and interleukin (IL)-6 were analyzed before hepcidin measurement and 6 months after rhEPO. Based on the serum ferritin levels before hepcidin measurement, the patients in the two studies with EPO or PEG-EPO were combined into low (11; serum ferritin of <15.0 ng/ml) and high ferritin groups (7; serum ferritin of ≥15.0 ng/ml). The response of hepcidin-25 to rhEPO and the effect of rhEPO on anemia were compared between the groups.
Results
The serum hepcidin-25 levels rose at 6-9 h and returned to the baseline at 18 h after EPO. They rose at 6-9 h, returned to the baseline at 18 h, and decreased on day 5-7 after PEG-EPO. Serum hepcidin-25 levels were low (<5.0 ng/ml) in the low ferritin group, but rose at 6-9 h after rhEPO in the high ferritin group. Serum transferrin levels were similar, and CRP and IL-6 were normal in both groups. Hb tended to increase in the low ferritin group, but it significantly decreased in the high ferritin group after rhEPO.
Conclusion
Regulation of hepcidin-25 by rhEPO may be dependent on ferritin, affecting the response to rhEPO in HD patients.
doi:10.1159/000362212
PMCID: PMC4024510  PMID: 24847351
Anemia; Erythropoietin; Ferritin; Hemodialysis; Hepcidin; Iron deficiency

2.  Long-term administration and efficacy of oxaliplatin with no neurotoxicity in a patient with rectal cancer: Association between neurotoxicity and the GSTP1 polymorphism 
Oncology Letters  2014;7(5):1499-1502.
Neurotoxicity is one of the most frequent side-effects of oxaliplatin. Oxaliplatin-induced cumulative and dose-limiting neurotoxicity either results in dose reduction or decreases the patients’ quality of life. However, the symptoms of neurotoxicity often vary among patients. The current study presents the case of a male with rectal cancer, who was administered a cumulative oxaliplatin dose of >5,000 mg/m2 without developing neurotoxicity or allergic reactions. Consequently, this patient continued therapy with modified 5-fluorouracil, leucovorin and oxaliplatin treatment for four years, with stabilization of the disease. This case indicates that if oxaliplatin-containing chemotherapy shows efficacy with no toxicity, the long-term administration of oxaliplatin would be effective and tolerable. Previously, the analysis of genomic polymorphisms in drug target genes has been important for explaining interindividual variations in the efficacy and toxicity of anti-cancer drugs. In the present patient, the glutathione S-transferase P1 (GSTP1) gene polymorphism, which is involved in the detoxification of platinum drugs, was analyzed. The genotype of the present case has been revealed as wild type (Ile/Ile) genotype. In addition, the associations between oxaliplatin-induced neurotoxicity and the GSTP1 polymorphism were also assessed. Certain studies have demonstrated that oxaliplatin-induced neurotoxicity occurs more frequently in patients with the Ile/Ile genotype, while others have demonstrated that those patients with the Val/Val or Ile/Val genotypes are more likely to develop neurotoxicity. Therefore, correlation between the GSTP1 polymorphism and oxaliplatin-induced neurotoxicity remains controversial. Overall, further development of individualized chemotherapy with an analysis of genomic polymorphisms in the drug target genes is required for the prophylaxis oxaliplatin-induced neurotoxicity.
doi:10.3892/ol.2014.1890
PMCID: PMC3997678  PMID: 24765164
oxaliplatin; neurotoxicity; colorectal cancer; GSTP1
3.  Comparative evaluation of the effects of treatment with tocilizumab and TNF-α inhibitors on serum hepcidin, anemia response and disease activity in rheumatoid arthritis patients 
Arthritis Research & Therapy  2013;15(5):R141.
Introduction
Anemia of inflammation (AI) is a common complication of rheumatoid arthritis (RA) and has a negative impact on RA symptoms and quality of life. Upregulation of hepcidin by inflammatory cytokines has been implicated in AI. In this study, we evaluated and compared the effects of IL-6 and TNF-α blocking therapies on anemia, disease activity, and iron-related parameters including serum hepcidin in RA patients.
Methods
Patients (n = 93) were treated with an anti-IL-6 receptor antibody (tocilizumab) or TNF-α inhibitors for 16 weeks. Major disease activity indicators and iron-related parameters including serum hepcidin-25 were monitored before and 2, 4, 8, and 16 weeks after the initiation of treatment. Effects of tocilizumab and infliximab (anti-TNF-α antibody) on cytokine-induced hepcidin expression in hepatoma cells were analyzed by quantitative real-time PCR.
Results
Anemia at base line was present in 66% of patients. Baseline serum hepcidin-25 levels were correlated positively with serum ferritin, C-reactive protein (CRP), vascular endothelial growth factor (VEGF) levels and Disease Activity Score 28 (DAS28). Significant improvements in anemia and disease activity, and reductions in serum hepcidin-25 levels were observed within 2 weeks in both groups, and these effects were more pronounced in the tocilizumab group than in the TNF-α inhibitors group. Serum hepcidin-25 reduction by the TNF-α inhibitor therapy was accompanied by a decrease in serum IL-6, suggesting that the effect of TNF-α on the induction of hepcidin-25 was indirect. In in vitro experiments, stimulation with the cytokine combination of IL-6+TNF-α induced weaker hepcidin expression than did with IL-6 alone, and this induction was completely suppressed by tocilizumab but not by infliximab.
Conclusions
Hepcidin-mediated iron metabolism may contribute to the pathogenesis of RA-related anemia. In our cohort, tocilizumab was more effective than TNF-α inhibitors for improving anemia and normalizing iron metabolism in RA patients by inhibiting hepcidin production.
doi:10.1186/ar4323
PMCID: PMC3978580  PMID: 24286116
4.  A Low-Molecular-Weight Compound K7174 Represses Hepcidin: Possible Therapeutic Strategy against Anemia of Chronic Disease 
PLoS ONE  2013;8(9):e75568.
Hepcidin is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. The expression of the hepcidin gene, HAMP, is increased in patients with anemia of chronic disease. Previously, the synthetic compound K7174 was identified through chemical screening as a novel inhibitor of the adhesion of monocytes to cytokine-stimulated endothelial cells. K7174 also ameliorated anemia induced by inflammatory cytokines in mice, which suggests a possible involvement of hepcidin regulation. The present study was performed to assess the impact of K7174 on hepcidin expression in a human hematoma cell line and in mice in vivo. We first demonstrated that K7174 treatment in HepG2 cells significantly decreased HAMP expression. Then, we conducted microarray analysis to determine the molecular mechanism by which K7174 inhibits HAMP expression. Transcriptional profiling confirmed the downregulation of HAMP. Surprisingly, we found that K7174 strongly induced GDF15, known as a negative regulator of HAMP expression. Western blotting analysis as well as ELISA confirmed the induction of GDF15 by K7174 treatment. Furthermore, K7174-mediated HAMP suppression was rescued by the silencing of GDF15 expression. Interestingly, we found that K7174 also upregulates CEBPB. Promoter analysis and chromatin immunoprecipitation analysis revealed that CEBPB could contribute to K7174-mediated transcriptional activation of GDF15. Subsequently, we also examined whether K7174 inhibits hepcidin expression in mice. Quantitative RT-PCR analysis with liver samples from K7174-treated mice demonstrated significant upregulation of Gdf15 and downregulation of Hamp expression, as compared to control mice. Furthermore, serum hepcidin concentration was also significantly decreased in K7174-treated mice. In conclusion, K7174 inhibits hepcidin expression partly by inducing GDF15. K-7174 may be a potential therapeutic option to treat anemia of chronic disease.
doi:10.1371/journal.pone.0075568
PMCID: PMC3785497  PMID: 24086573
5.  Characterization of proteins secreted by pancreatic cancer cells with anticancer drug treatment in vitro 
Oncology Reports  2012;28(6):1968-1976.
Pancreatic cancer is one of the most lethal cancers, with an incidence equaling mortality. It is a heterogeneous group of neoplasms in which pancreatic ductal adenocarcinoma is most common. Pancreatic cancer cannot be cured even if detected early. When treatment is initiated, a suitable method of administration of anticancer drugs must be chosen. Anticancer drugs kill tumor cells. However, side effects including initiation are problematic in anticancer drug therapy. Improved methods for the diagnosis of side effects of pancreatic cancer by using sensitive and specific tumor markers are highly desirable. Therefore, efficient strategies for biomarker discovery are urgently needed. Here, we present an approach based on direct experimental access to proteins released by PANC-1 human pancreatic cancer cells in vitro. A two-dimensional (2-D) map and catalog of this subproteome, herein termed the secretome, were established comprising more than 1,000 proteins observed by ‘2-D difference in-gel electrophoresis analysis using cyanine dye’. We investigated 22 spots that were 1.20-fold upregulated and 31 spots that were 0.66-fold downregulated by gemcitabine chloride treatment. Proteins in these spots were identified by nano-high-performance liquid chromatography electrospray ionization time of flight mass spectrometry/mass spectrometry. Most secretome constituents were nominally cellular proteins. By mass spectrometry screening, 14-3-3 protein sigma (14-3-3 σ), protein S100-A8, protein S100-A9, galectin-7, lactotransferrin (lactoferrin, LF) precursor, serotransferrin (transferrin) precursor, and vitamin D binding protein precursor were identified. Western blotting confirmed the presence of 14-3-3 σ and LF. We found that upregulation of 14-3-3 σ was associated with apoptosis, and downregulation of LF was found to suppress tumorigenesis.
doi:10.3892/or.2012.2020
PMCID: PMC3583485  PMID: 22961650
Panc-1; gemcitabin; secretome; 14-3-3 protein sigma; lactoferrin
6.  Correction: Observation of Live Ticks (Haemaphysalis flava) by Scanning Electron Microscopy under High Vacuum Pressure 
PLoS ONE  2012;7(8):10.1371/annotation/c81bc3a4-63e1-44a6-bf1e-f923087bfa71.
doi:10.1371/annotation/c81bc3a4-63e1-44a6-bf1e-f923087bfa71
PMCID: PMC3414542
7.  Analysis of hepcidin expression: In situ hybridization and quantitative polymerase chain reaction from paraffin sections 
AIM: To establish methods for quantitative polymerase chain reaction (PCR) for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of hepatocellular carcinoma (HCC).
METHODS: Total RNA from paraffin-embedded sections was isolated from 68 paraffin-embedded samples of HCC. Samples came from 54 male and 14 female patients with a mean age of 66.8 ± 7.8 years. Quantitative PCR was performed. Immunohistochemistry and in situ hybridization for hepcidin were also performed.
RESULTS: Quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections of HCC was performed successfully. The expression level of hepcidin mRNA in cancer tissues was significantly higher than that in non-cancer tissues. A method of in situ hybridization for hepcidin was established successfully, and this demonstrated that hepcidin mRNA was expressed in non-cancerous tissue but absent in cancerous tissue.
CONCLUSION: We have established novel methods for quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of HCC.
doi:10.3748/wjg.v18.i28.3727
PMCID: PMC3406426  PMID: 22851866
Hepcidin; Expression; In situ hybridization; Immunohistochemistry; Real-time polymerase chain reaction
8.  Hepcidin Expression in Iron Overload Diseases Is Variably Modulated by Circulating Factors 
PLoS ONE  2012;7(5):e36425.
Hepcidin is a regulatory hormone that plays a major role in controlling body iron homeostasis. Circulating factors (holotransferrin, cytokines, erythroid regulators) might variably contribute to hepcidin modulation in different pathological conditions. There are few studies analysing the relationship between hepcidin transcript and related protein expression profiles in humans. Our aims were: a. to measure hepcidin expression at either hepatic, serum and urinary level in three paradigmatic iron overload conditions (hemochromatosis, thalassemia and dysmetabolic iron overload syndrome) and in controls; b. to measure mRNA hepcidin expression in two different hepatic cell lines (HepG2 and Huh-7) exposed to patients and controls sera to assess whether circulating factors could influence hepcidin transcription in different pathological conditions. Our findings suggest that hepcidin assays reflect hepatic hepcidin production, but also indicate that correlation is not ideal, likely due to methodological limits and to several post-trascriptional events. In vitro study showed that THAL sera down-regulated, HFE-HH and C-NAFLD sera up-regulated hepcidin synthesis. HAMP mRNA expression in Huh-7 cells exposed to sera form C-Donors, HFE-HH and THAL reproduced, at lower level, the results observed in HepG2, suggesting the important but not critical role of HFE in hepcidin regulation.
doi:10.1371/journal.pone.0036425
PMCID: PMC3346721  PMID: 22586470
9.  Observation of Live Ticks (Haemaphysalis flava) by Scanning Electron Microscopy under High Vacuum Pressure 
PLoS ONE  2012;7(3):e32676.
Scanning electron microscopes (SEM), which image sample surfaces by scanning with an electron beam, are widely used for steric observations of resting samples in basic and applied biology. Various conventional methods exist for SEM sample preparation. However, conventional SEM is not a good tool to observe living organisms because of the associated exposure to high vacuum pressure and electron beam radiation. Here we attempted SEM observations of live ticks. During 1.5×10−3 Pa vacuum pressure and electron beam irradiation with accelerated voltages (2–5 kV), many ticks remained alive and moved their legs. After 30-min observation, we removed the ticks from the SEM stage; they could walk actively under atmospheric pressure. When we tested 20 ticks (8 female adults and 12 nymphs), they survived for two days after SEM observation. These results indicate the resistance of ticks against SEM observation. Our second survival test showed that the electron beam, not vacuum conditions, results in tick death. Moreover, we describe the reaction of their legs to electron beam exposure. These findings open the new possibility of SEM observation of living organisms and showed the resistance of living ticks to vacuum condition in SEM. These data also indicate, for the first time, the usefulness of tick as a model system for biology under extreme condition.
doi:10.1371/journal.pone.0032676
PMCID: PMC3303806  PMID: 22431980
10.  Protein Profiling of Blood Samples from Patients with Hereditary Leiomyomatosis and Renal Cell Cancer by Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry 
Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an extremely rare syndrome with autosomal dominant inheritance. HLRCC is characterized by a predisposition to leiomyomas of the skin and the uterus as well as renal cell carcinoma. The disease-related gene has been identified as fumarate hydratase (fumarase, FH), which encodes an enzyme involved in the mitochondrial tricarboxylic acid cycle. Protein profiling may give some insight into the molecular pathways of HLRCC. Therefore, we performed protein profiling of blood samples from HLRCC patients, their family members, and healthy volunteers, using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) coupled with IMAC-Cu chips. For hierarchical clustering analysis, we used the 45 peaks that revealed significant differences in single-marker analysis over the range from 1500 to 15,000 m/z. Heat map analysis based on the results of clustering distinguished the HLRCC kindred from non-HLRCC subjects with a sensitivity of 94% and a specificity of 90%. SELDI-TOF MS profiling of blood samples can be applied to identify patients with HLRCC and to assess specific molecular mechanisms involved in this condition.
doi:10.3390/ijms131114518
PMCID: PMC3509594  PMID: 23203078
Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC); fumarate hydratase (FH); surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS); metastasis
11.  A novel clinical entity, IgG4-related disease (IgG4RD): general concept and details 
Modern Rheumatology  2011;22(1):1-14.
IgG4-related disease (IgG4RD) is a novel clinical disease entity characterized by elevated serum IgG4 concentration and tumefaction or tissue infiltration by IgG4-positive plasma cells. IgG4RD may be present in a certain proportion of patients with a wide variety of diseases, including Mikulicz’s disease, autoimmune pancreatitis, hypophysitis, Riedel thyroiditis, interstitial pneumonitis, interstitial nephritis, prostatitis, lymphadenopathy, retroperitoneal fibrosis, inflammatory aortic aneurysm, and inflammatory pseudotumor. Although IgG4RD forms a distinct, clinically independent disease category and is attracting strong attention as a new clinical entity, many questions and problems still remain to be elucidated, including its pathogenesis, the establishment of diagnostic criteria, and the role of IgG4. Here we describe the concept of IgG4RD and up-to-date information on this emerging disease entity.
doi:10.1007/s10165-011-0508-6
PMCID: PMC3278618  PMID: 21881964
IgG4-related diseases; Mikulicz’s disease; Sjögren’s syndrome; Autoimmune pancreatitis; Castleman’s disease
12.  Ultraviolet B-induced expression of amphiregulin and growth differentiation factor 15 in human lens epithelial cells 
Molecular Vision  2011;17:159-169.
Purpose
Epidemiological and experimental studies have revealed that exposure to ultraviolet B (UVB) light can induce cataractogenesis. The objective of this study was to determine gene expression changes in human lens epithelial cells in response to UVB exposure and identify factors that can be involved in UVB-induced cataractogenesis.
Methods
SV40 T-antigen-transformed human lens epithelial cells (SRA01/04) were irradiated at various UVB-energy levels (10–80 mJ/cm2) and checked for viability. An irradiation condition of 30 mJ/cm2 was adopted for transcriptome analysis. Total RNAs isolated from UVB-exposed and unexposed cells at 12 h and 24 h after UVB exposure were examined for global gene expression changes using Affymetrix Human Gene 1.0 ST array. mRNA levels of specific genes were examined by RT–PCR and real-time PCR, and protein levels in the conditioned media were assayed by ELISA. To examine mRNA expression in human lens, primary cultured human lens epithelial (HLE) cells were prepared from surgically removed lens epithelium, and used for UVB-irradiation and expression analysis. Effects of certain gene products on SRA01/04 cell metabolism were examined using commercially available recombinant proteins.
Results
Expression of most the genes analyzed was essentially unchanged (between 0.5 and 2.0 fold) in UVB-irradiated cells compared to non-irradiated cells at both 12 and 24 h after UVB exposure. Sixty one and 44 genes were upregulated more than twofold by UVB exposure at 12 h and 24 h, respectively. Emphasis was placed on genes encoding extracellular proteins, especially growth factors and cytokines. A total of 18 secreted protein genes were upregulated more than twofold at either or both time points. Amphiregulin (AREG) and growth differentiation factor 15 (GDF15) were chosen because of their higher upregulation and novelty, and their upregulation was confirmed in SRA01/04 cells using RT–PCR and real-time PCR analysis. AREG and GDF15 protein levels in conditioned media significantly increased at all UVB-energy points at 24 h, while they were scarcely detectable at 12 h. AREG and GDF15 mRNA levels were also significantly upregulated in UVB-irradiated primary cultured HLE cells compared with the corresponding control culture. AREG significantly stimulated 3H-thymidine and 3H-leucine uptake in SRA01/04 cells as did a positive control epidermal growth factor (EGF). Recombinant GDF15 did not stimulate 3H-thymidine incorporation at any concentration tested, but significantly stimulated 3H-leucine uptake. RT–PCR analysis demonstrated that primary cultured HLE and SRA01/04 cells expressed not only epidermal growth factor receptor (EGFR) mRNA but also transforming growth factor β receptors (TGFBR1 and TGFBR2) mRNAs.
Conclusions
These results indicate that AREG and GDF15 produced in response to UVB exposure can affect the growth and protein synthesis of lens epithelial cells, suggesting that they have autocrine and paracrine roles related to pathological changes of lens tissue during long-term UVB exposure.
PMCID: PMC3021579  PMID: 21245963
13.  Increased serum hepcidin-25 level and increased tumor expression of hepcidin mRNA are associated with metastasis of renal cell carcinoma 
BMC Cancer  2009;9:270.
Background
Hepcidin has an important role in iron metabolism. We investigated whether hepcidin was involved in renal cell carcinoma (RCC).
Methods
We measured serum hepcidin-25 levels in 32 patients by liquid chromatograpy (LC)-mass spectrometry (MS)/MS, and assessed hepcidin mRNA expression in paired tumor and non-tumor tissue samples from the surgical specimens of 53 consecutive patients with RCC by real-time reverse transcription polymerase chain reaction.
Results
The serum hepcidin-25 level was higher in patients with metastatic RCC than nonmetastatic RCC (P < 0.0001), and was positively correlated with the serum interleukin-6 and C-reactive protein levels (P < 0.001). Expression of hepcidin mRNA was lower in tumor tissues than in non-tumor tissues (P < 0.0001). The serum hepcidin-25 level was not correlated with the expression of hepcidin mRNA in the corresponding tumor tissue specimens from 32 patients. Hepcidin mRNA expression in tumor tissue was correlated with metastatic potential, but not with histological differentiation or tumor stage. Kaplan-Meier analysis showed that over expression of hepcidin mRNA was related to shorter overall survival in RCC patients. Univariate analysis (Cox proportional hazards model) showed that the hepcidin mRNA level was an independent prognostic factor for overall survival.
Conclusion
Our findings suggest that a high serum hepcidin-25 level may indicate the progression of RCC, and that upregulation of hepcidin mRNA expression in tumor tissue may be related to increased metastatic potential.
doi:10.1186/1471-2407-9-270
PMCID: PMC2729778  PMID: 19656379
14.  Expression of hepcidin mRNA is uniformly suppressed in hepatocellular carcinoma 
BMC Cancer  2008;8:167.
Background
The present study evaluated the expression of hepcidin mRNA in hepatocellular carcinoma (HCC).
Methods
Samples of cancerous and non-cancerous liver tissue were taken from 40 patients with HCC who underwent hepatectomy. Expression of hepcidin mRNA was evaluated by real-time PCR, and compared in tumors differing in their degree of differentiation, number of tumors, and vessel invasion. Correlations between hepcidin expression and the interval until HCC recurrence, and the serum concentration of hepcidin were evaluated, together with the expression of mRNAs for other iron metabolism molecules, ferroportin and transferrin receptor 2 (Trf2).
Results
Hepcidin mRNA expression in non-cancerous and cancerous tissues was 1891.8 (32.3–23187.4) and 53.4 (1.9–3185.8), respectively (P < 0.0001). There were no significant differences in hepcidin expression among tumors differing in their degree of differentiation, number of tumors, or vessel invasion. There was no significant correlation between hepcidin expression and the interval until HCC recurrence. The serum concentration of hepcidin-25 was not correlated with hepcidin-mRNA expression. Finally, there were no significant differences in the expression of mRNA for ferroportin and Trf2 between cancerous and non-cancerous tissues.
Conclusion
Expression of hepcidin mRNA is strikingly suppressed in cancerous, but not in non-cancerous tissues, in patients with HCC, irrespective of ferroportin or Trf2 expression. Uniform suppression of hepcidin may be linked to the development of HCC.
doi:10.1186/1471-2407-8-167
PMCID: PMC2430575  PMID: 18541040

Results 1-14 (14)