Des R Richardson

Publications (133)663.91 Total impact

• Article: Hepcidin, show some self-control! How the hormone of iron metabolism regulates its own expression.

  Darius J R Lane, Michael L-H Huang, Des R Richardson
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  ABSTRACT: Does the hormone of iron metabolism, hepcidin, exhibit 'self-control'? Hepcidin is a small, disulfide-rich peptide synthesized by the liver, which plays a keystone role in regulating systemic iron metabolism in mammals. Hepcidin acts by binding and triggering the lysosomal degradation of the cellular iron exporter ferroportin. Ultimately, decreased ferroportin leads to decreased plasma iron levels. Although various modulators of HAMP (the hepcidin antimicrobial peptide gene) expression are known, no auto-regulatory pathway has been described. In their paper published in the Biochemical Journal in April 2013, Pandur et al. identify an auto-regulatory pathway in which prohepcidin regulates HAMP expression. The authors observe that prohepcidin can bind to the inflammation-regulated STAT3 (signal transducer and activator of transcription 3)-binding site in the HAMP promoter to negatively regulate HAMP expression. Furthermore, the authors find that the prohepcidin-binding partner, α-1 antitrypsin, inhibits prohepcidin's ability to decrease HAMP activity. This is significant as α-1 antitrypsin, similar to hepcidin, is an acute-phase reactant that is up-regulated by inflammation. In conclusion, the discovery of a hepcidin auto-regulatory pathway, first, supports the emerging notion that hepcidin regulation is exquisitely fine-tuned through a process of combinatorial control; and secondly, suggests that hepcidin may play a hand in its own deregulation in diseases of iron metabolism that involve aberrant cytokine signalling (e.g. the anaemia of inflammation).
  Biochemical Journal 06/2013; 452(2):e3-5. · 4.90 Impact Factor
• Article: The Metastasis Suppressor, NDRG1: Mediates its Activity Through Signaling Pathways and Molecular Motors.

  Jing Sun, Daohai Zhang, Dong-Hun Bae, Sumit Sahni, Patric Jansson, Ying Zheng, Qian Zhao, Fei Yue, Minhua Zheng, Zaklina Kovacevic, Des R Richardson
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  ABSTRACT: The metastasis suppressor, N-myc downstream regulated gene 1 (NDRG1), is negatively correlated with tumor progression in multiple neoplasms, being a promising new target for cancer treatment. However, the precise molecular effects of NDRG1 remain unclear. Herein, we summarize recent advances in understanding the impact of NDRG1 on cancer metastasis with emphasis on its interactions with the key oncogenic NF-κB, PI3K/pAKT/mTOR and Ras/Raf/MEK/ERK signaling pathways. Recent studies demonstrating the inhibitory effects of NDRG1 on the epithelial-mesenchymal transition (EMT), a key initial step in metastasis, as well as the WNT/β-catenin pathway are also described. Furthermore, NDRG1 was also demonstrated to regulate molecular motors in cancer cells, leading to inhibition of F-actin stress fiber formation and subsequent reduction of cancer cell migration. Collectively, this review summarizes the underlying molecular mechanisms of the anti-metastatic effects of NDRG1 in cancer cells.
  Carcinogenesis 05/2013; · 5.70 Impact Factor
• Article: Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells.

  Mohammad K Ghalayini, Qihan Dong, Des R Richardson, Stephen J Assinder
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  ABSTRACT: N-myc downstream regulated gene-1 (NDRG1) is a metastasis suppressor that is down-regulated in prostate cancer. NDRG1 phosphorylation is associated with inhibition of metastasis and western blots indicate two bands at ~41 and ~46 kDa. Previous investigations by others suggest the higher band is due to NDRG1 phosphorylation. However, the current study using a dephosphorylation assay and the Phos-tag SDS-PAGE assay, demonstrated the 46 kDa NDRG1 protein band was not due to phosphorylation. Further experiments showed the NDRG1 protein bands were not affected upon glycosidase treatment, despite marked effects of these enzymes on the glycosylated protein, fetuin. Analysis using RT-PCR demonstrated only a single amplicon, and thus, the two bands could not a result from an alternatively spliced NDRG1 transcript. Western blot analysis of prostate cancer cell lysates identified the 41 kDa band to be a truncated form of NDRG1, with mass spectrometry confirming the full and truncated proteins to be NDRG1. Significantly, this truncated protein was not present in normal human prostate epithelial cells. Western blot analysis using anti-NDRG1 raised to its N-terminal sequence failed to detect the truncated protein, suggesting it lacked N-terminus amino acids (residues 1-49). Sequence analysis predicted a pseudotrypsin protease cleavage site between Cys49-Gly50. Such cleavage of NDRG1 in cancer cells may result in loss of NDRG1 tumour suppressive activity.
  Bioscience Reports 05/2013; · 2.38 Impact Factor
• Article: Transferrin iron uptake is stimulated by ascorbate via an intracellular reductive mechanism.

  Darius J R Lane, Sherin Chikhani, Vera Richardson, Des R Richardson
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  ABSTRACT: Although ascorbate has long been known to stimulate dietary iron (Fe) absorption and non-transferrin Fe uptake, the role of ascorbate in transferrin Fe uptake is unknown. Transferrin is a serum Fe transport protein supplying almost all cellular Fe under physiological conditions. We sought to examine ascorbate's role in this process, particularly as cultured cells are typically ascorbate-deficient. At typical plasma concentrations, ascorbate significantly increased 59Fe uptake from transferrin by 1.5-2-fold in a range of cells. Moreover, ascorbate enhanced ferritin expression and increased 59Fe accumulation in ferritin. The lack of effect of cycloheximide or the cytosolic aconitase inhibitor, oxalomalate, on ascorbate-mediated 59Fe uptake from transferrin indicate increased ferritin synthesis or cytosolic aconitase activity was not responsible for ascorbate's activity. Experiments with membrane-permeant and -impermeant ascorbate-oxidizing reagents indicate that while extracellular ascorbate is required for stimulation of 59Fe uptake from 59Fe-citrate, only intracellular ascorbate is needed for transferrin 59Fe uptake. Additionally, experiments with l-ascorbate analogs indicate ascorbate's reducing ene-diol moiety is necessary for its stimulatory activity. Importantly, neither N-acetylcysteine nor buthionine sulfoximine, which increase or decrease intracellular glutathione, respectively, affected transferrin-dependent 59Fe uptake. Thus, ascorbate's stimulatory effect is not due to a general increase in cellular reducing capacity. Ascorbate also did not affect expression of transferrin receptor 1 or 125I-transferrin cellular flux. However, transferrin receptors, endocytosis, vacuolar-type ATPase activity and endosomal acidification were required for ascorbate's stimulatory activity. Therefore, ascorbate is a novel modulator of the classical transferrin Fe uptake pathway, acting via an intracellular reductive mechanism.
  Biochimica et Biophysica Acta 02/2013; · 4.66 Impact Factor
• Article: The redox-active, anti-cancer drug Dp44mT inhibits T-cell activation and CD25 through a copper-dependent mechanism.

  Danuta S Kalinowski, Patric J Jansson, Zaklina Kovacevic, Des R Richardson
  Redox report: communications in free radical research 02/2013; · 1.51 Impact Factor
• Article: N-myc Downstream Regulated 1 (NDRG1) Is Regulated by Eukaryotic Initiation Factor 3a (eIF3a) during Cellular Stress Caused by Iron Depletion.

  Darius J R Lane, Federica Saletta, Yohan Suryo Rahmanto, Zaklina Kovacevic, Des R Richardson
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  ABSTRACT: Iron is critical for cellular proliferation and its depletion leads to a suppression of both DNA synthesis and global translation. These observations suggest that iron depletion may trigger a cellular "stress response". A canonical response of cells to stress is the formation of stress granules, which are dynamic cytoplasmic aggregates containing stalled pre-initiation complexes that function as mRNA triage centers. By differentially prioritizing mRNA translation, stress granules allow for the continued and selective translation of stress response proteins. Although the multi-subunit eukaryotic initiation factor 3 (eIF3) is required for translation initiation, its largest subunit, eIF3a, may not be essential for this activity. Instead, eIF3a is a vital constituent of stress granules and appears to act, in part, by differentially regulating specific mRNAs during iron depletion. Considering this, we investigated eIF3a's role in modulating iron-regulated genes/proteins that are critically involved in proliferation and metastasis. In this study, eIF3a was down-regulated and recruited into stress granules by iron depletion as well as by the classical stress-inducers, hypoxia and tunicamycin. Iron depletion also increased expression of the metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), and a known downstream repressed target of eIF3a, namely the cyclin-dependent kinase inhibitor, p27(kip1). To determine if eIF3a regulates NDRG1 expression, eIF3a was inducibly over-expressed or ablated. Importantly, eIF3a positively regulated NDRG1 expression and negatively regulated p27(kip1) expression during iron depletion. This activity of eIF3a could be due to its recruitment to stress granules and/or its ability to differentially regulate mRNA translation during cellular stress. Additionally, eIF3a positively regulated proliferation, but negatively regulated cell motility and invasion, which may be due to the eIF3a-dependent changes in expression of NDRG1 and p27(kip1) observed under these conditions.
  PLoS ONE 01/2013; 8(2):e57273. · 4.09 Impact Factor
• Article: Alkyl Substituted 2'-Benzoylpyridine Thiosemicarbazone Chelators with Potent and Selective Anti-Neoplastic Activity: Novel Ligands that Limit Methemoglobin Formation.

  Christian Stefani, Patric J Jansson, Elaine Gutierrez, Paul V Bernhardt, Des R Richardson, Danuta S Kalinowski
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  ABSTRACT: Thiosemicarbazone chelators, including the 2'-benzoylpyridine thiosemicarbazones (BpT) class, show marked potential as anticancer agents. Importantly, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) has been investigated in >20 phase I and II clinical trials. However, side effects associated with 3-AP administration include methemoglobinemia. Considering this problem, novel BpT analogues were designed bearing hydrophobic, electron-donating substituents at the para position of the phenyl group (RBpT). Their Fe(III/II) redox potentials were all within the range accessible to cellular oxidants and reductants, suggesting they can redox cycle. These RBpT ligands exhibited potent and selective antiproliferative activity, which was comparable or exceeded their BpT counterparts. Major findings include that methemoglobin formation mediated by the lipophilic t-BuBpT series was significantly (p < 0.05-0.001) decreased in comparison to 3-AP in intact red blood cells and were generally comparable to the control. These data indicate the t-BuBpT ligands may minimize methemoglobinemia, which is a marked advantage over 3-AP and other potent thiosemicarbazones.
  Journal of Medicinal Chemistry 12/2012; · 4.80 Impact Factor
• Article: Synthesis and biological evaluation of substituted 2-benzoylpyridine thiosemicarbazones: Novel structure-activity relationships underpinning their anti-proliferative and chelation efficacy.

  Adeline Y Lukmantara, Danuta S Kalinowski, Naresh Kumar, Des R Richardson
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  ABSTRACT: The 2-benzoylpyridine thiosemicarbazone (BpT) chelators demonstrate potent anti-proliferative effects against tumor cells. To understand their structure-activity relationships, BpT analogues incorporating electron-donating substituents on the pyridine and phenyl rings of the BpT scaffold were designed and represent the first attempts to modify the pyridine ring of these thiosemicarbazones. Eight analogues showed significantly (p <0.001) greater anti-proliferative activity than the 'gold-standard' chelator, desferrioxamine. Structure-activity analysis revealed that mono- or di-methoxy substitution at the phenyl ring resulted in lower anti-proliferative activity, while methoxy substitutions at the phenyl ring enhanced iron chelation efficacy. These important findings facilitate the design of thiosemicarbazones with greater anti-tumor activity.
  Bioorganic & medicinal chemistry letters 12/2012; · 2.65 Impact Factor
• Article: Targeting the Metastasis Suppressor, NDRG1, Using Novel Iron Chelators: Regulation of Stress Fiber-Mediated Tumor Cell Migration via Modulation of the ROCK1/pMLC2 Signaling Pathway.

  Jing Sun, Daohai Zhang, Ying Zheng, Qian Zhao, Minhua Zheng, Zaklina Kovacevic, Des R Richardson
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  ABSTRACT: The iron-regulated metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), is up-regulated by cellular iron depletion mediated by iron chelators and can inhibit cancer cell migration. However, the mechanism of how NDRG1 achieves this effect remains unclear. In this study, we implemented established and newly constructed NDRG1 over-expression and knockdown models using the DU145, HT29 and HCT116 cancer cell lines to investigate the molecular basis by which NDRG1 exerts its inhibitory effect on cell migration. Using these models, we demonstrated that NDRG1 over-expression inhibits cell migration by preventing actin-filament polymerization, stress fiber assembly and formation. In contrast, NDRG1 knockdown had the opposite effect. Moreover, we identified that NDRG1 inhibited an important regulatory pathway mediated by the Rho-associated, coiled-coil containing protein kinase 1 (ROCK1)/phosphorylated myosin light chain 2 (pMLC2) pathway that modulates stress fiber assembly. The phosphorylation of MLC2 is a key process in inducing stress fiber contraction and this was shown to be markedly decreased or increased by NDRG1 over-expression or knockdown, respectively. The mechanism involved in the inhibition of MLC2 phosphorylation by NDRG1 was mediated by a significant (p<0.001) decrease in ROCK1 expression that is a key kinase involved in MLC2 phosphorylation. Considering that NDRG1 is up-regulated after cellular iron depletion, novel thiosemicarbazone iron chelators (e.g., di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone) were demonstrated to inhibit ROCK1/pMLC2-modulated actin-filament polymerization, stress fiber assembly and formation via a mechanism involving NDRG1. These results highlight the role of the ROCK1/pMLC2 pathway in the NDRG1-mediated anti-metastatic signaling network and the therapeutic potential of iron chelators at inhibiting metastasis.
  Molecular pharmacology 11/2012; · 4.53 Impact Factor
• Article: Identification of in vitro metabolites of the novel anti-tumor thiosemicarbazone, DpC, using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry.

  Ján Stariat, Petra Kovaříková, Radim Kučera, Jiří Klimeš, Danuta S Kalinowski, Des R Richardson, Raimo A Ketola
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  ABSTRACT: Di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) is a promising analogue of the dipyridyl thiosemicarbazone class currently under development as a potential anti-cancer drug. In fact, this class of agents shows markedly greater anti-tumor activity and selectivity than the clinically investigated thiosemicarbazone, Triapine®. However, further development of DpC requires detailed data concerning its metabolism. Therefore, we focused on the identification of principal phase I and II metabolites of DpC in vitro. DpC was incubated with human liver microsomes/S9 fractions and the samples were analyzed using ultra-performance liquid chromatography (UPLC(TM)) with electrospray ionization quadrupole-time-of-flight (Q-TOF) mass spectrometry. An Acquity UPLC BEH C(18) column was implemented with 2 mM ammonium acetate and acetonitrile in gradient mode as the mobile phase. The chemical structures of metabolites were proposed based on the accurate mass measurement of the protonated molecules as well as their main product ions. Ten phase I and two phase II metabolites were detected and structurally described. The metabolism of DpC occurred via oxidation of the thiocarbonyl group, hydroxylation and N-demethylation, as well as the combination of these reactions. Conjugates of DpC and the metabolite, M10, with glucuronic acid were also observed as phase II metabolites. Neither sulfate nor glutathione conjugates were detected. This study provides the first information about the chemical structure of the principal metabolites of DpC, which supports the development of this promising anti-cancer drug and provides vital data for further pharmacokinetic and in vivo metabolism studies.
  Analytical and Bioanalytical Chemistry 11/2012; · 3.78 Impact Factor
• Source

  Available from: Prem Ponka

  Article: Identification of nonferritin mitochondrial iron deposits in a mouse model of Friedreich ataxia.

  Megan Whitnall, Yohan Suryo Rahmanto, Michael L-H Huang, Federica Saletta, Hiu Chuen Lok, Lucía Gutiérrez, Francisco J Lázaro, Adam J Fleming, Tim G St Pierre, Marc R Mikhael, Prem Ponka, Des R Richardson
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  ABSTRACT: There is no effective treatment for the cardiomyopathy of the most common autosomal recessive ataxia, Friedreich ataxia (FA). This disease is due to decreased expression of the mitochondrial protein, frataxin, which leads to alterations in mitochondrial iron (Fe) metabolism. The identification of potentially toxic mitochondrial Fe deposits in FA suggests Fe plays a role in its pathogenesis. Studies using the muscle creatine kinase (MCK) conditional frataxin knockout mouse that mirrors the disease have demonstrated frataxin deletion alters cardiac Fe metabolism. Indeed, there are pronounced changes in Fe trafficking away from the cytosol to the mitochondrion, leading to a cytosolic Fe deficiency. Considering Fe deficiency can induce apoptosis and cell death, we examined the effect of dietary Fe supplementation, which led to body Fe loading and limited the cardiac hypertrophy in MCK mutants. Furthermore, this study indicates a unique effect of heart and skeletal muscle-specific frataxin deletion on systemic Fe metabolism. Namely, frataxin deletion induces a signaling mechanism to increase systemic Fe levels and Fe loading in tissues where frataxin expression is intact (i.e., liver, kidney, and spleen). Examining the mutant heart, native size-exclusion chromatography, transmission electron microscopy, Mössbauer spectroscopy, and magnetic susceptibility measurements demonstrated that in the absence of frataxin, mitochondria contained biomineral Fe aggregates, which were distinctly different from isolated mammalian ferritin molecules. These mitochondrial aggregates of Fe, phosphorus, and sulfur, probably contribute to the oxidative stress and pathology observed in the absence of frataxin.
  Proceedings of the National Academy of Sciences 11/2012; · 9.68 Impact Factor
• Article: The Iron Chelator, Deferasirox, as a Novel Strategy for Cancer Treatment: Oral Activity Against Human Lung Tumor Xenografts and Molecular Mechanism of Action.

  Goldie Y L Lui, Peyman Obeidy, Samuel J Ford, Chris Tselepis, Danae M Sharp, Patric J Jansson, Danuta S Kalinowski, Zaklina Kovacevic, David B Lovejoy, Des R Richardson
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  ABSTRACT: Deferasirox is an orally-effective iron chelator currently used for the treatment of iron (Fe) overload disease and has been implemented as an alternative to the "gold standard" chelator, desferrioxamine (DFO). Earlier studies demonstrated that DFO has anti-cancer activity due to its ability to deplete cancer cells of Fe. In this investigation, we examined the in vitro and in vivo activity of deferasirox against cells from human solid tumors. To date, there have been no studies to investigate the effect of deferasirox on these types of tumors in vivo. Deferasirox demonstrated similar activity at inhibiting proliferation of DMS-53 lung carcinoma and SK-N-MC neuroepithelioma cell lines when compared to DFO. Further, deferasirox was generally similar or slightly more effective than DFO at mobilizing cellular (59)Fe and inhibiting iron uptake from Tf depending on the cell-type. However, deferasirox potently inhibited DMS-53 xenograft growth in nude mice when given by oral gavage, with no marked alterations in normal tissue histology. To understand the anti-tumor activity of deferasirox, we investigated its effect on the expression of molecules that play key roles in metastasis, cell cycle control and apoptosis. We demonstrated that deferasirox increased expression of the metastasis suppressor protein, N-myc downstream regulated gene-1 (NDRG1), and up-regulated the cyclin-dependent kinase inhibitor p21(CIP1/WAF1), while decreasing cyclin D1 levels. Moreover, this agent increased the expression of apoptosis markers, including cleaved caspase-3 and cleaved poly [ADP-ribose] polymerase 1. Collectively, we demonstrate that deferasirox is an orally-effective anti-tumor agent against solid tumors.
  Molecular pharmacology 10/2012; · 4.53 Impact Factor
• Article: Development of a sensitive HPLC method to measure in vitro permeability of E- and Z-isomeric forms of thiosemicarbazones in Caco-2 monolayers.

  Zufan Debebe, Sergei Nekhai, Meseret Ashenafi, David B Lovejoy, Danuta S Kalinowski, Victor R Gordeuk, W Malcolm Byrnes, Des R Richardson, Pradeep K Karla
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  ABSTRACT: In the current study, we developed a HPLC method to quantitatively measure the permeability of the BpT-based chelators, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and 2-benzoylpyridine 4-allyl-3-thiosemicarbazone (Bp4aT), across human colorectal adenocarcinoma (Caco-2) monolayers as a model of gut absorption. In aqueous solution, Bp4eT and Bp4aT formed inter-convertible Z and E isomers that were resolved by HPLC. Peak area was linear with respect to chelator concentration. Acceptable within-day and between-day precision (<22%) and accuracy (85-115% of true values) were obtained over a range of 1.0-100μM for Bp4eT and 1.5-300μM for Bp4aT. Limits of detection were 0.3μM and 1μM for Bp4eT and Bp4aT, respectively, while corresponding limits of quantification were 1μM and 5μM. Both chelators showed significant ability to chelate iron in THP-1 cells using a calcein-based assay and no apparent cytotoxicity was observed within 24h. Ratios of the apical to basolateral and basolateral to apical transport for Bp4eT were 1.10 and 0.89 at 100μM and 300μM respectively, indicating equal bi-directional movement of the compounds. Similarly, ratios were 0.77 and 0.92 for Bp4aT, respectively. This study demonstrates that Bp4eT and Bp4aT can be efficiently transported through Caco-2 cells and can potentially be formulated for oral delivery.
  Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 08/2012; 906:25-32. · 2.78 Impact Factor
• Article: Novel second-generation di-2-pyridylketone thiosemicarbazones show synergism with standard chemotherapeutics and demonstrate potent activity against lung cancer xenografts after oral and intravenous administration in vivo.

  David B Lovejoy, Danae M Sharp, Nicole Seebacher, Peyman Obeidy, Thomas Prichard, Christian Stefani, Maram T Basha, Philip C Sharpe, Patric J Jansson, Danuta S Kalinowski, Paul V Bernhardt, Des R Richardson
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  ABSTRACT: We developed a series of second-generation di-2-pyridyl ketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) ligands to improve the efficacy and safety profile of these potential antitumor agents. Two novel DpT analogues, Dp4e4mT and DpC, exhibited pronounced and selective activity against human lung cancer xenografts in vivo via the intravenous and oral routes. Importantly, these analogues did not induce the cardiotoxicity observed at high nonoptimal doses of the first-generation DpT analogue, Dp44mT. The Cu(II) complexes of these ligands exhibited potent antiproliferative activity having redox potentials in a range accessible to biological reductants. The activity of the copper complexes of Dp4e4mT and DpC against lung cancer cells was synergistic in combination with gemcitabine or cisplatin. It was demonstrated by EPR spectroscopy that dimeric copper compounds of the type [CuLCl](2), identified crystallographically, dissociate in solution to give monomeric 1:1 Cu:ligand complexes. These monomers represent the biologically active form of the complex.
  Journal of Medicinal Chemistry 08/2012; 55(16):7230-44. · 4.80 Impact Factor
• Source

  Available from: Maciej Serda

  Article: Synthesis and characterization of quinoline-based thiosemicarbazones and correlation of cellular iron-binding efficacy to anti-tumor efficacy.

  Maciej Serda, Danuta S Kalinowski, Anna Mrozek-Wilczkiewicz, Robert Musiol, Agnieszka Szurko, Alicja Ratuszna, Namfon Pantarat, Zaklina Kovacevic, Angelica M Merlot, Des R Richardson, Jaroslaw Polanski
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  ABSTRACT: Iron chelators have emerged as a potential anti-cancer treatment strategy. In this study, a series of novel thiosemicarbazone iron chelators containing a quinoline scaffold were synthesized and characterized. A number of analogs show markedly greater anti-cancer activity than the 'gold-standard' iron chelator, desferrioxamine. The anti-proliferative activity and iron chelation efficacy of several of these ligands (especially compound 1b), indicates that further investigation of this class of thiosemicarbazones is worthwhile.
  Bioorganic & medicinal chemistry letters 07/2012; 22(17):5527-31. · 2.65 Impact Factor
• Article: The ins and outs of mitochondrial iron-loading: the metabolic defect in Friedreich’s ataxia

  Des R. Richardson, Michael L-H. Huang, Megan Whitnall, Erika M. Becker, Prem Ponka, Yohan Suryo Rahmanto
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  ABSTRACT: Friedreich’s ataxia is a cardio- and neurodegenerative disease due to decreased expression of the mitochondrial protein, frataxin. This defect results in mitochondrial iron-overload, and in this review, we discuss the mechanisms that lead to this iron accumulation. Using a conditional knockout mouse model where frataxin is deleted in the heart, it has been shown that this mutation leads to transferrin receptor-1 upregulation, resulting in increased iron uptake from transferrin. There is also marked downregulation of ferritin that is required for iron storage and decreased expression of the iron exporter, ferroportin1, leading to decreased cellular iron efflux. The increased mitochondrial iron uptake is facilitated by upregulation of the mitochondrial iron transporter, mitoferrin2. This stimulation of iron uptake probably attempts to rescue the deficit in mitochondrial iron metabolism that is due to downregulation of mitochondrial iron utilization, namely, heme and iron–sulfur cluster (ISC) synthesis and also iron storage (mitochondrial ferritin). The resultant decrease in heme and ISC synthesis means heme and ISCs are not exiting the mitochondrion for cytosolic use. Hence, increased mitochondrial iron uptake coupled with decreased utilization and release leads to mitochondrial iron-loading. More generally, disturbance of mitochondrial iron utilization in other diseases probably also results in similar compensatory alterations. KeywordsIron-Iron metabolism-Transferrin receptor 1-Heme-Iron–sulfur cluster
  Journal of Molecular Medicine 04/2012; 88(4):323-329. · 4.67 Impact Factor
• Article: Sustained expression of heme oxygenase-1 alters iron homeostasis in nonerythroid cells.

  Cheng Li, Maria E Lönn, Xiangcong Xu, Ghassan J Maghzal, David M Frazer, Shane R Thomas, Barry Halliwell, Des R Richardson, Gregory J Anderson, Roland Stocker
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  ABSTRACT: Heme oxygenases initiate the catabolism of heme, releasing carbon monoxide, iron, and biliverdin. Sustained induction of heme oxygenase-1 (HO-1) in nonerythroid cells plays a key role in many pathological processes, yet the effect of long-term HO-1 expression on cellular iron metabolism in the absence of exogenous heme is poorly understood. Here we report that in a model nonerythroid cell, both transient and stable HO-1 expression increased heme oxygenase activity, but total cellular heme content was decreased only with transient enzyme expression. Sustained HO-1 activity increased the expression of both the mitochondrial iron importer mitoferrin-2 and the rate-limiting enzyme in heme synthesis, aminolevulinate synthase-1, and it augmented the mitochondrial content of heme. Also, the expression of transferrin receptor-1 and the activities of iron-regulatory proteins 1 and 2 decreased, whereas total labile iron and the regulatory activity of the heme-binding transcription factor Bach1 were unaltered. In addition, stable, but not transient, HO-1 expression decreased the activities of aconitase, as well as increasing proteasomal degradation of ferritin. Together, our results reveal a novel and coordinated adaptive response of nonerythroid cells to sustained HO-1 induction that has an impact on cellular iron homeostasis.
  Free radical biology & medicine 04/2012; 53(2):366-74. · 5.42 Impact Factor
• Article: Methemoglobin formation by triapine, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), and other anticancer thiosemicarbazones: identification of novel thiosemicarbazones and therapeutics that prevent this effect.

  Patricia Quach, Elaine Gutierrez, Maram Talal Basha, Danuta S Kalinowski, Philip C Sharpe, David B Lovejoy, Paul V Bernhardt, Patric J Jansson, Des R Richardson
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  ABSTRACT: Thiosemicarbazones are a group of compounds that have received comprehensive investigation as anticancer agents. The antitumor activity of the thiosemicarbazone, 3-amino-2-pyridinecarboxaldehyde thiosemicarbazone (3-AP; triapine), has been extensively assessed in more than 20 phase I and II clinical trials. These studies have demonstrated that 3-AP induces methemoglobin (metHb) formation and hypoxia in patients, limiting its usefulness. Considering this problem, we assessed the mechanism of metHb formation by 3-AP compared with that of more recently developed thiosemicarbazones, including di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT). This was investigated using intact red blood cells (RBCs), RBC lysates, purified oxyhemoglobin, and a mouse model. The chelation of cellular labile iron with the formation of a redox-active thiosemicarbazone-iron complex was found to be crucial for oxyhemoglobin oxidation. This observation was substantiated using a thiosemicarbazone that cannot ligate iron and also by using the chelator, desferrioxamine, that forms a redox-inactive iron complex. Of significance, cellular copper chelation was not important for metHb generation in contrast to its role in preventing tumor cell proliferation. Administration of Dp44mT to mice catalyzed metHb and cardiac metmyoglobin formation. However, ascorbic acid administered together with the drug in vivo significantly decreased metHb levels, providing a potential therapeutic intervention. Moreover, we demonstrated that the structure of the thiosemicarbazone is of importance in terms of metHb generation, because the DpT analog, di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), does not induce metHb generation in vivo. Hence, DpC represents a next-generation thiosemicarbazone that possesses markedly superior properties. This investigation is important for developing more effective thiosemicarbazone treatment regimens.
  Molecular pharmacology 04/2012; 82(1):105-14. · 4.53 Impact Factor
• Article: The Iron-Regulated Metastasis Suppressor NDRG1 Targets NEDD4L, PTEN, and SMAD4 and Inhibits the PI3K and Ras Signaling Pathways.

  Zaklina Kovacevic, Sherin Chikhani, Goldie Y L Lui, Sutharshani Sivagurunathan, Des R Richardson
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  ABSTRACT: Abstract Aims: The metastasis suppressor gene, N-myc downstream regulated gene-1 (NDRG1), is negatively correlated with tumor progression in multiple neoplasms, including pancreatic cancer. Moreover, NDRG1 is an iron-regulated gene that is markedly upregulated by cellular iron-depletion using novel antitumor agents such as the chelator, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), in pancreatic cancer cells. However, the exact function(s) of NDRG1 remain to be established and are important to elucidate. Results: In the current study, using gene-array analysis along with NDRG1 overexpression and silencing, we identified the molecular targets of NDRG1 in three pancreatic cancer cell lines. We demonstrate that NDRG1 upregulates neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) and GLI-similar-3 (GLIS3). Further studies examining the downstream effects of NEDD4L led to the discovery that NDRG1 affects the transforming growth factor-β (TGF-β) pathway, leading to the upregulation of two key tumor suppressor proteins, namely phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and mothers against decapentaplegic homolog-4 (SMAD4). Moreover, NDRG1 inhibited the phosphatidylinositol 3-kinase (PI3K) and Ras oncogenic pathways. Innovation: This study provides significant insights into the mechanisms underlying the antitumor activity of NDRG1. For the first time, a role for NDRG1 is established in regulating the key signaling pathways involved in oncogenesis (TGF-β, PI3K, and Ras pathways). Conclusion: The identified target genes of NDRG1 and their effect on the TGF-β signaling pathway reveal its molecular function in pancreatic cancer and a novel therapeutic avenue. Antioxid. Redox Signal. 00, 000-000.
  Antioxidants & Redox Signaling 04/2012; · 8.20 Impact Factor
• Article: The iron chelators Dp44mT and DFO inhibit TGF-β-induced epithelial-mesenchymal transition via up-regulation of N-Myc downstream-regulated gene 1 (NDRG1).

  Zhiqiang Chen, Daohai Zhang, Fei Yue, Minhua Zheng, Zaklina Kovacevic, Des R Richardson
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  ABSTRACT: The epithelial-mesenchymal transition (EMT) is a key step for cancer cell migration, invasion, and metastasis. Transforming growth factor-β (TGF-β) regulates the EMT and the metastasis suppressor gene, N-myc downstream-regulated gene-1 (NDRG1), could play a role in regulating the TGF-β pathway. NDRG1 expression is markedly increased after chelator-mediated iron depletion via hypoxia-inducible factor 1α-dependent and independent pathways (Le, N. T. and Richardson, D. R. (2004) Blood 104, 2967-2975). Moreover, novel iron chelators show marked and selective anti-tumor activity and are a potential new class of anti-metabolites. Considering this, the current study investigated the relationship between NDRG1 and the EMT to examine if iron chelators can inhibit the EMT via NDRG1 up-regulation. We demonstrated that TGF-β induces the EMT in HT29 and DU145 cells. Further, the chelators, desferrioxamine (DFO) and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), inhibited the TGF-β-induced EMT by maintaining E-cadherin and β-catenin, at the cell membrane. We then established stable clones with NDRG1 overexpression and knock-down in HT29 and DU145 cells. These data showed that NDRG1 overexpression maintained membrane E-cadherin and β-catenin and inhibited TGF-β-stimulated cell migration and invasion. Conversely, NDRG1 knock-down caused morphological changes from an epithelial- to fibroblastic-like phenotype and also increased migration and invasion, demonstrating NDRG1 knockdown induced the EMT and enhanced TGF-β effects. We also investigated the mechanisms involved and showed the TGF-β/SMAD and Wnt pathways were implicated in NDRG1 regulation of E-cadherin and β-catenin expression and translocation. This study demonstrates that chelators inhibit the TGF-β-induced EMT via a process consistent with NDRG1 up-regulation and elucidates the mechanism of their activity.
  Journal of Biological Chemistry 03/2012; 287(21):17016-28. · 4.77 Impact Factor