Lynda Addo

The three isoforms of hepcidin in human serum and their processing determined by liquid chromatography-tandem mass spectrometry (LC-tandem MS)

Hepcidin, the iron regulatory hormone, has three isoforms; -20, -22 and -25. While hepcidin-25 has been studied extensively, the physiological significance of other isoforms remains poorly understood. Using a quantitative method based on liquid chromatography-tandem mass spectrometry (LC-tandem MS) developed by our group, we quantified hepcidin isoforms in human serum to elucidate their characteristics, and investigated the role of hepatocytes in isoform processing. Hepcidin isoforms in serum obtained from 40 healthy volunteers were quantified. Synthetic hepcidin peptides were added to healthy serum, and to HepG2 culture media, and hepcidin isoform concentrations determined. All three hepcidin isoforms were detected in human serum; however, hepcidin-25 concentrations were highest. The three hepcidin isoforms showed a strong positive correlation with each other and with serum ferritin. Additionally, while hepcidin-20 was strongly correlated with serum creatinine, the other isoforms were not. Hepcidin-20 and -25 levels were also increased in chronic kidney disease (CKD) serum. Hepcidin-22 rapidly degraded into hepcidin-20, whereas hepcidin-25 remained relatively stable. Finally, hepcidin-22 degradation into hepcidin-20 was accelerated in the presence of HepG2. This method has enabled us to reveal fundamental characteristics of the three hepcidin isoforms in serum and may be a powerful tool for quantifying hepcidin isoform expression and processing.

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

BACKGROUND Iron 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. METHODS We 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. RESULTS The assay shows excellent linearity, reproducibility, and compatibility with HPLC, one of the most reliable conventional methods for NTBI quantification. CONCLUSIONS Our 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.

Successful Control of Disseminated Intravascular Coagulation by Recombinant Thrombomodulin during Arsenic Trioxide Treatment in Relapsed Patient with Acute Promyelocytic Leukemia

Disseminated intravascular coagulation (DIC) frequently occurs in patients with acute promyelocytic leukemia (APL). With the induction of therapy in APL using all-trans retinoic acid (ATRA), DIC can be controlled in most cases as ATRA usually shows immediate improvement of the APL. However, arsenic trioxide (ATO) which has been used for the treatment of relapse in APL patients has shown to take time to suppress APL cells, therefore the control of DIC in APL with ATO treatment is a major problem. Recently, the recombinant soluble thrombomodulin fragment has received a lot of attention as the novel drug for the treatment of DIC with high efficacy. Here, we present a relapsed patient with APL in whom DIC was successfully and safely controlled by rTM during treatment with ATO.

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)

Upregulation of iron regulatory hormone hepcidin by interferon α.

Interferon (IFN) activates various immune systems in vivo and is administered to patients with diseases such as viral hepatitis B, C, and malignant tumors. Iron dysregulation has been reported during treatment with IFN; however, it remains unclear whether IFN itself affects iron metabolism. We therefore determined the effect of IFN on iron metabolism.

Hepatic nerve growth factor induced by iron overload triggers defenestration in liver sinusoidal endothelial cells

The fenestrations of liver sinusoidal endothelial cells (LSECs) play important roles in the exchange of macromolecules, solutes, and fluid between blood and surrounding liver tissues in response to hepatotoxic drugs, toxins, and oxidative stress. As excess iron is a hepatotoxin, LSECs may be affected by excess iron. In this study, we found a novel link between LSEC defenestration and hepatic nerve growth factor (NGF) in iron-overloaded mice. By Western blotting, NGF was highly expressed, whereas VEGF and HGF were not, and hepatic NGF mRNA levels were increased according to digital PCR. Immunohistochemically, NGF staining was localized in hepatocytes, while TrkA, an NGF receptor, was localized in LSECs. Scanning electron microscopy revealed LSEC defenestration in mice overloaded with iron as well as mice treated with recombinant NGF. Treatment with conditioned medium from iron-overloaded primary hepatocytes reduced primary LSEC fenestrations, while treatment with an anti-NGF neutralizing antibody or TrkA inhibitor, K252a, reversed this effect. However, iron-loaded medium itself did not reduce fenestration. In conclusion, iron accumulation induces NGF expression in hepatocytes, which in turn leads to LSEC defenestration via TrkA. This novel link between iron and NGF may aid our understanding of the development of chronic liver disease.

Iron-induced epigenetic abnormalities of mouse bone marrow through aberrant activation of aconitase and isocitrate dehydrogenase

Iron overload remains a concern in myelodysplastic syndrome (MDS) patients. Iron chelation therapy (ICT) thus plays an integral role in the management of these patients. Moreover, ICT has been shown to prolong leukemia-free survival in MDS patients; however, the mechanisms responsible for this effect are unclear. Iron is a key molecule for regulating cytosolic aconitase 1 (ACO1). Additionally, the mutation of isocitrate dehydrogenase (IDH), the enzyme downstream of ACO1 in the TCA cycle, is associated with epigenetic abnormalities secondary to 2-hydroxyglutarate (2-HG) and DNA methylation. However, epigenetic abnormalities observed in many MDS patients occur without IDH mutation. We hypothesized that iron itself activates the ACO1-IDH pathway, which may increase 2-HG and DNA methylation, and eventually contribute to leukemogenesis without IDH mutation. Using whole RNA sequencing of bone marrow cells in iron-overloaded mice, we observed that the enzymes, phosphoglucomutase 1, glycogen debranching enzyme, and isocitrate dehydrogenase 1 (Idh1), which are involved in glycogen and glucose metabolism, were increased. Digital PCR further showed that Idh1 and Aco1, enzymes involved in the TCA cycle, were also elevated. Additionally, enzymatic activities of TCA cycle and methylated DNA were increased. Iron chelation reversed these phenomena. In conclusion, iron activation of glucose metabolism causes an increase of 2-HG and DNA methylation.

The three isoforms of Hepcidin measured in human serum by liquid chromatographytandem mass spectrometry

H the iron regulatory hormone, is known to have three isoforms (-20, -22, -25 amino acids). Research into hepcidin-25 has been extensive whereas the other isoforms have not received much attention. Because hepcidin dysregulation is evident in iron overload disorders, quantifying this peptide will prove vital in understanding its role in the development of such diseases. However, hepcidin assay methods still remain widely unavailable. Reports on the measurement of this peptide suggest that whereas hepcidin-25 and hepcidin-20 can be found in serum and urine, hepcidin -22 is only found in urine and not in serum. We sought to measure the three hepcidin isoforms in human serum using a method we previously established, based on LC-tandem/ MS and to elucidate their characteristics. Using our LC-tandem/MS method, we identified the hepcidin isoforms in sera from 40 randomly selected healthy volunteers. All three hepcidin isoforms were detected in human sera. Hepcidin-25 levels were highest; however, hepcidin isoform concentrations varied among the individual samples. All three hepcidin isoforms correlated positively with serum ferritin, consistent with the role of hepcidin in iron metabolism. Also, all three isoforms correlated positively with each other. Hepcidin-20 showed a relatively positive correlation with serum creatinine. We found high concentrations of hepcidin-20 in the sera of chronic renal dysfunction patients. In conclusion, we found all three hepcidin isoforms in human sera. Although further studies on the other hepcidin isoforms are required, a simultaneous quantification of all the hepcidin isoforms may provide researchers with novel information and also serve as novel biomarkers.Cancer is a class of diseases, classified by the organ of origin and characterized by uncontrollable cell growth. Our focus is on prostate cancer (PC) which starts in the prostate and is generally a walnut-sized gland located below the bladder. Approximately 4500 males in South Africa are diadnosed annually, thus making it the second most common cause of cancer death in men. Current diagnostic methods include digital rectal examinations (DRE), prostate specific antigen test (PSA), biopsy and ultrasound however, they are invasive and lack specificity and sensitivity. Therefore, the development of a noninvasive, specific and sensitive early detection method is required. Biomarkers are biological indicators i.e. DNA, proteins and miRNA, which have recently been identified as targets for the early detection of disease. MiRNAs are small, naturally occurring, non-coding RNA molecules directly involved in regulating gene expression at the post-transcriptional level. They offer great potential as biomarkers for cancer detection due to their remarkable stability in blood and characteristic expression in different diseases. The aim of this study is therefore to identify miRNAs as specific biomarkers for the early detection of PC. The identification of specific miRNAs and their targets will be done using various bioinformatics techniques including programming and statistical analyses. Once identified, these miRNAs will be experimentally validated to generate expression profiles using molecular techniques. Furthermore, newly identified, experimentally validated miRNAs will be used in combination with nanotechnology to develop a diagnostic kit for the early detection of PC. IWBBIO 2013. Proceedings Granada, 18-20 March, 2013 161

Abstract 149: Antitumor effect of itraconazole on hematological malignant cells by suppression of Hedgehog signal transduction via inhibition of lipid raft formation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL [Introduction and aim] Itraconazole (ITCZ) is one of the antifungal agents that inhibits fungal cell membrane ergosterol synthesis. ITCZ has been widely used for the treatment of fungal infections with high efficiency. Recently, anti-tumor effect of ITCZ was reported for solid tumors (Kim J et al. Cancer Cell 2010;17:388-399). During the treatment of hematological malignancies, ITCZ has been quite often used for invasive fungal infections; however, there has been no report for the effect of ITCZ on hematological malignancies. Therefore, we aimed to investigate the anti-tumor effect of ITCZ on hematological malignancies in the present study. [Methods] DAUDI, Jurkat, K562, Karpas299 cell lines were used as a model of hematological malignancies in the present study. At first, MTT assays were performed by treating the cells to determine if ITCZ possessed anti-tumor effect on the cell growth. Then, comprehensive analysis of intracellular signal transductions was performed utilizing reporter assay system. To evaluate the involvement of lipid raft formation by ITCZ, Gli1 expression concerning the Hedgehog signal transduction was determined by real-time PCR and western blotting, and fluorescent staining of the cells using lipid raft marker was also performed. [Results] ITCZ inhibited the cell growth by 40 to 60% determined by MTT assays in all four cell lines tested in the present study. Comprehensive analysis of intracellular signal transductions utilizing reporter assay system in Karpas299 cells showed the strong decrease of Gli1 transcription, implying the inhibition of Hedgehog signal transduction. Concerning Hedgehog signal pathway, Gli1 protein has been reported to be overexpressed in tumor cells and considered to be involved in the tumor growth, and Gli1 has also been reported to localize in lipid raft. Therefore, we hypothesized that ITCZ might influenced on the lipid raft of cell membrane and then Gli1 expression was suppressed, leading to the weakened Hedgehog signal transduction and cell growth inhibition. Fluorescent staining of Jurkat cells using lipid raft marker showed that the presence of ITCZ apparently inhibited the formation of lipid raft on cell membrane. This finding was quite similar compared to that observed when lipid raft inhibitor was added to cell culture medium. [Conclusions] ITCZ has an inhibitory effect on the cell growth in the cultured cell lines derived from hematological malignancies as same as the previous finding observed in solid tumors. Our results further indicated that ITCZ inhibited the formation of lipid raft on cell membrane, and then Hedgehog signal transduction should be weakened, finally leading to the cell growth inhibition. Taken together, the tumor inhibition by ITCZ should be favorable effect and may enhance the efficacy of the treatment of various hematological malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 149. doi:1538-7445.AM2012-149