Katsunori Sasaki

Hedgehog Promotes Neovascularization in Pancreatic Cancers by Regulating Ang-1 and IGF-1 Expression in Bone-Marrow Derived Pro-Angiogenic Cells

Background The hedgehog (Hh) pathway has been implicated in the pathogenesis of cancer including pancreatic ductal adenocarcinoma (PDAC). Recent studies have suggested that the oncogenic function of Hh in PDAC involves signaling in the stromal cells rather than cell autonomous effects on the tumor cells. However, the origin and nature of the stromal cell type(s) that are responsive to Hh signaling remained unknown. Since Hh signaling plays a crucial role during embryonic and postnatal vasculogenesis, we speculated that Hh ligand may act on tumor vasculature specifically focusing on bone marrow (BM)-derived cells. Methodology/Principal Findings Cyclopamine was utilized to inhibit the Hh pathway in human PDAC cell lines and their xenografts. BM transplants, co-culture systems of tumor cells and BM-derived pro-angiogenic cells (BMPCs) were employed to assess the role of tumor-derived Hh in regulating the BM compartment and the contribution of BM-derived cells to angiogenesis in PDAC. Cyclopamine administration attenuated Hh signaling in the stroma rather than in the cancer cells as reflected by decreased expression of full length Gli2 protein and Gli1 mRNA specifically in the compartment. Cyclopamine inhibited the growth of PDAC xenografts in association with regression of the tumor vasculature and reduced homing of BM-derived cells to the tumor. Host-derived Ang-1 and IGF-1 mRNA levels were downregulated by cyclopamine in the tumor xenografts. In vitro co-culture and matrigel plug assays demonstrated that PDAC cell-derived Shh induced Ang-1 and IGF-1 production in BMPCs, resulting in their enhanced migration and capillary morphogenesis activity. Conclusions/Significance We identified the BMPCs as alternative stromal targets of Hh-ligand in PDAC suggesting that the tumor vasculature is an attractive therapeutic target of Hh blockade. Our data is consistent with the emerging concept that BM-derived cells make important contributions to epithelial tumorigenesis.

A flavonoid from Brassica rapa flower as the UV-absorbing nectar guide

The corolla of Brassica rapa has an UV-absorbing zone in its center, known as the nectar guide for attracting pollinating insects. The pigment which plays the role of the nectar guide was isolated from the petals and identified to be isorhamnetin 3,7-O-di-beta-D-glucopyranoside on the basis of MS and NMR spectroscopic data. The D-, L-configurations of the sugar moieties were determined by the fluorometric HPLC method. In plants raised in open field, there was a 13-fold higher content of the compound in the basal parts of the petals compared with the apical parts. This difference in flavonoid content is presumed to contribute to the visual attractiveness of B. rapa flowers to insect pollinators.

Iron overload signature in chrysotile-induced malignant mesothelioma†

Exposure to asbestos is a risk for malignant mesothelioma (MM) in humans. Among the commercially used types of asbestos (chrysotile, crocidolite, and amosite), the carcinogenicity of chrysotile is not fully appreciated. Here, we show that all three asbestos types similarly induced MM in the rat peritoneal cavity and that chrysotile caused the earliest mesothelioma development with a high fraction of sarcomatoid histology. The pathogenesis of chrysotile‐induced mesothelial carcinogenesis was closely associated with iron overload: repeated administration of an iron chelator, nitrilotriacetic acid, which promotes the Fenton reaction, significantly reduced the period required for carcinogenesis; massive iron deposition was found in the peritoneal organs with high serum ferritin; and homozygous deletion of the CDKN2A/2B/ARF tumour suppressor genes, the most frequent genomic alteration in human MM and in iron‐induced rodent carcinogenesis, was observed in 92.6% of the cases studied with array‐based comparative genomic hybridization. The induced rat MM cells revealed high expression of mesoderm‐specific transcription factors, Dlx5 and Hand1, and showed an iron regulatory profile of active iron uptake and utilization. These data indicate that chrysotile is a strong carcinogen when exposed to mesothelia, acting through the induction of local iron overload. Therefore, an intervention to remove local excess iron might be a strategy to prevent MM after asbestos exposure. Copyright

Improved quantification for non-transferrin-bound iron measurement using high-performance liquid chromatography by reducing iron contamination.

Non-transferrin-bound iron (NTBI) refers to all forms of iron in the plasma that bind to ligands other than transferrin, and is considered to be a marker of iron toxicity. A variety of analytical approaches for measuring NTBI have been reported; however, a clinically relevant level of sensitivity has yet to be achieved. In addition, insufficient values of NTBI in some patients and healthy subjects have led to the assumption that there may be contamination of reagents with background iron. The present study re-evaluated the analytical procedures of the assay with regard to the potential points of iron contamination in each step. NTA and tris carbonatocobaltate (III) solutions were prepared with removal of iron contamination, and then quantification of NTBI was performed. As a result, the sensitivity of the high-performance liquid chromatography (HPLC)-based NTBI method was improved by the successful reduction of background iron contamination. Application of our modified method proved that NTBI was detected even in healthy volunteers, although the concentrations were extremely low; the average NTBI levels were 0.206±0.091 µM (males, n=20) and 0.212±0.095 µM (females, n=16). Thus, our modification of the NTBI assay may be clinically meaningful, and may contribute to the understanding of the clinical significance of relatively low, but elevated concentrations of NTBI in diseases other than typical iron overload.

Hyperferritinemia after adult allogeneic hematopoietic cell transplantation: quantification of iron burden by determining non-transferrin-bound iron.

Iron overload is a common complication in allogeneic hematopoietic cell transplantation (HCT). We studied the prevalence of iron overload using serum ferritin from 122 allogeneic HCT survivors who had survived a median of 1259 (range 134–4261) days. We also quantified iron overload by determining non-transferrin-bound iron (NTBI), which reflects iron overload more directly than ferritin, and compared the results with those of the ferritin assay. Fifty-two patients (43xa0%) showed hyperferritinemia (HF) (serum ferritin >1000xa0ng/mL), and there was a moderate correlation between serum ferritin and the number of transfused red blood cell units (ρxa0=xa00.71). In multivariate analyses, HF was a significant risk factor for liver dysfunction (Pxa0=xa00.0001) and diabetes (Pxa0=xa00.02), and was related to a lesser extent with performance status (Pxa0=xa00.08). There was a significant correlation between serum ferritin and NTBI (ρxa0=xa00.59); however, the association of NTBI with these outcomes was weaker than that of serum ferritin. In conclusion, serum ferritin is a good surrogate marker of iron overload after allogeneic HCT, and reflects organ damage more accurately than NTBI.

Heterogeneous expressions of hepcidin isoforms in hepatoma‐derived cells detected using simultaneous LC‐MS/MS

Hepcidin, a key regulator of iron homeostasis, is known to have three isoforms: hepcidin‐20, ‐22, and ‐25. Hepcidin‐25 is thought to be the major isoform and the only one known to be involved in iron metabolism; the physiological roles of other isoforms are poorly understood. Because of its involvement in the pathophysiology of hereditary hemochromatosis and the anemia of chronic disease, the regulatory mechanisms of hepcidin expression have been extensively investigated, but most studies have been performed only at the transcriptional level. Difficulty in detecting hepcidin has impeded in vitro research. In the present study, we developed a novel method for simultaneous quantification of hepcidin‐20, ‐22, and ‐25 in the media from hepatoma‐derived cell lines. Using this method, we determined the expression patterns of hepcidin isoforms and the patterns of responses to various stimuli in human hepatoma‐derived cultured cells. We found substantial differences among cell lines. In conclusion, a novel method for simultaneous quantification of hepcidin isoforms is presented. Heterogeneous expressions of hepcidin isoforms in human hepatoma‐derived cells were revealed by this method. We believe our method will facilitate quantitative investigation of the role hepcidin plays in iron homeostasis.

Non-transferrin-bound iron assay system utilizing a conventional automated analyzer

BACKGROUNDnIron is an essential metal in the body, but its excessive accumulation causes damage in various organs through free radical production. Iron homeostasis is therefore tightly regulated. However, when iron balance collapses, such as in prolonged transfusion, transferrin (Tf) is fully saturated and non-Tf-bound iron (NTBI) appears in the serum. Monitoring serum NTBI levels is therefore crucial in the assessment of the clinical status of patients with iron overload, since NTBI is associated with cellular and organ damage. Several methods for NTBI determination have been reported, but these are extremely complicated and very few laboratories can quantify NTBI at present.nnnMETHODSnWe established a novel assay system utilizing automated analyzers that are widely used in clinical laboratories for diagnostic testing. In this assay, NTBI is chelated by nitrilotriacetic acid (NTA), after which the iron is reduced and transferred to nitroso-PSAP, a chromogen.nnnRESULTSnThe assay shows excellent linearity, reproducibility, and compatibility with HPLC, one of the most reliable conventional methods for NTBI quantification.nnnCONCLUSIONSnOur novel method for NTBI measurement is high-throughput and may be a useful and powerful tool in the study of the physiological and clinical importance of NTBI.

Hepcidin production in response to iron is controlled by monocyte-derived humoral factors

Hepcidin, which is mainly produced by the liver, is the key regulator in iron homeostasis. Hepcidin expression is up-regulated by iron loading in vivo, but the mechanism underlying this process is not completely understood. In the present study, we investigated the mechanism, following the hypothesis that hepcidin production in response to iron loading is regulated by extra-hepatic iron sensors. We measured serum hepcidin concentrations and iron indices in Wistar rats treated with saccharated ferric oxide (SFO). Human hepatoma-derived HepG2 cells were stimulated using SFO-administered rat sera, and co-cultured with rat spleen cells, human monocyte-derived THP-1 cells, or human monocytes with diferric transferrin (holo-Tf), and hepcidin concentrations in the conditioned media were measured. SFO elevated rat serum hepcidin concentrations. SFO-treated rat sera increased hepcidin production from HepG2 cells, and this induction correlated with serum hepcidin levels, but not with iron indices. Holo-Tf up-regulated hepcidin concentrations in media from HepG2 cells co-cultured with rat spleen cells, THP-1 cells, or human monocytes with or without cell-to-cell contacts, while holo-Tf did not up-regulate hepcidin from HepG2 cells alone. Our results suggest the existence of humoral factors capable of inducing hepcidin production that are secreted by extra-hepatic cells, such as reticuloendothelial monocytes, in response to iron.

Iron facilitator LS081 reduces hypoxia‐inducible factor‐1α protein and functions as anticancer agent in hepatocellular carcinoma

Hypoxia inducible factor‐1α (HIF‐1α) has a central role in cellular oxygen‐sensing, and its overexpression in many types of cancer is considered important in tumor progression. Thus, targeting HIF‐1α production and activity has been of great therapeutic interest. In normoxic conditions, HIF‐1α is hydroxylated by oxygen‐dependent prolyl‐hydroxylases, which require ferrous iron for its activity. The tumor suppressor protein von Hippel Lindau binds to the hydroxylated HIF‐1α, which is then ubiquitinated and degraded by proteasomes. We focused on the physiological degradation machinery of HIF‐1α mediated by prolyl hydroxylases. Previously, we identified a small molecule, LS081, that is capable of stimulating iron uptake into cells. In the present study, we aimed to inhibit the expression of HIF‐1α protein and growth of hepatocellular carcinoma by using the iron‐facilitating activity of LS081. In the human hepatocellular carcinoma cell lines Hep3B and HepG2, a combination of LS081 and ferric ammonium citrate (LS081/FeAC) inhibited HIF‐1α protein expression but did not inhibit HIF‐1α mRNA expression. A mutated HIF‐1α protein, which has proline residues that were replaced with alanine and transfected into HEK293 cells, was not affected by the combination of LS081 and FeAC. Furthermore, the iron‐facilitating activity of LS081 resulted in Hep3B and HepG2 growth inhibition in vitro and in vivo. These results indicate that the iron‐facilitating activity of LS081 inhibits HIF‐1α expression through prolyl‐hydroxylation of HIF‐1α and might have a therapeutic effect in the treatment of hepatocellular carcinoma. (Cancer Sci 2012; 103: 767–774)

Iron overload patients with unknown etiology from national survey in Japan

Transfusion is believed to be the main cause of iron overload in Japan. A nationwide survey on post-transfusional iron overload subsequently led to the establishment of guidelines for iron chelation therapy in this country. To date, however, detailed clinical information on the entire iron overload population in Japan has not been fully investigated. In the present study, we obtained and studied detailed clinical information on the iron overload patient population in Japan. Of 1109 iron overload cases, 93.1% were considered to have occurred post-transfusion. There were, however, 76 cases of iron overload of unknown origin, which suggest that many clinicians in Japan may encounter some difficulty in correctly diagnosing and treating iron overload. Further clinical data were obtained for 32 cases of iron overload of unknown origin; median of serum ferritin was 1860.5xa0ng/mL. As occurs in post-transfusional iron overload, liver dysfunction was found to be as high as 95.7% when serum ferritin levels exceeded 1000xa0ng/mL in these patients. Gene mutation analysis of the iron metabolism-related genes in 27 cases of iron overload with unknown etiology revealed mutations in the gene coding hemojuvelin, transferrin receptor 2, and ferroportin; this indicates that although rare, hereditary hemochromatosis does occur in Japan.