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Yu Y, Rahmanto YS, Richardson DRBp44mT: an orally active iron chelator of the thiosemicarbazone class with potent anti-tumour efficacy. Br J Pharmacol 165(1): 148-166
British Journal of Pharmacology
Abstract
Our previous studies demonstrated that a thiosemicarbazone iron chelator (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone; Dp44mT) possesses potent and selective anti-cancer activity but led to cardiotoxicity at non-optimal doses. In this study, we examined the in vivo anti-tumour efficacy and tolerability of a new-generation 2-benzoylpyridine thiosemicarbazone iron chelator (2-benzoylpyridine-4,4-dimethyl-3-thiosemicarbazone; Bp44mT) administered via the oral or i.v. routes.
BpT chelators were tested in vitro against human lung cancer cells (DMS-53) and in vivo in DMS-53 tumour xenografts in mice. The toxicity of Bp44mT in vivo and its effects on the expression of iron-regulated molecules involved in growth and cell cycle control were investigated.
Administration of Bp44mT by either route resulted in marked dose-dependent inhibition of tumour growth. When administered at 50 mg·kg(-1) via oral gavage three times per week for 23 days, the net xenograft growth was inhibited by 75%, compared with vehicle-treated mice. Toxicological examination showed reversible alterations including slight reduction of RBC count, with a decrease of liver and splenic iron levels, which confirmed iron chelation in vivo. Importantly, in contrast to Dp44mT, the chelator-treated mice did not show cardiac histological abnormalities. There was also no significant weight loss in mice, suggesting oral administration of Bp44mT was well tolerated.
This is the first study to show that Bp44mT can be given orally with potent anti-tumour efficacy. Oral administration of a novel and effective chemotherapeutic agent provides the benefits of convenience for chronic dosing regimens.
... Thiosemicarbazones are an interesting group of compounds. They are known for their numerous pharmacological and biological properties as effective antimicrobial [11], antimalarial [12], and anticancer [13] agents. ...
... Expanded aliphatic region of 1 H-NMR spectrum of mixture of (E)-and (Z)-2'methoxyacetophenone thiosemicarbazone (TSC 1); Figure S4. 13 C{ 1 H}-NMR spectrum of mixture of (E)-and (Z)-2'-methoxyacetophenone thiosemicarbazone (TSC 1); Figure S5. Expanded aromatic region of 13 C{ 1 H}-NMR spectrum of mixture of (E)-and (Z)-2'-methoxyacetophenone thiosemicarbazone (TSC 1); Figure S6. ...
... Expanded aliphatic region of 1 H-NMR spectrum of mixture of (E)-and (Z)-2'-methoxyacetophenone thiosemicarbazone (TSC 4); Figure S15. 13 C{ 1 H}-NMR spectrum of mixture of (E)-and (Z)-2'-aminoacetophenone thiosemicarbazone (TSC 4); Figure S16. Expanded aromatic region of 13 C{ 1 H}-NMR spectrum of mixture of (E)-and (Z)-2'-aminoacetophenone thiosemicarbazone (TSC 4); Figure S17. ...
A set of 12 monosubstituted acetophenone thiosemicarbazone derivatives (TSCs) were synthesized and their inhibitory properties toward tyrosinase activity were tested. Moreover, their ability to inhibit melanogenesis in the B16F10 murine melanoma cell line was studied. In order to investigate the nature of interactions between the enzyme and the inhibitors, molecular docking to the active site was performed. TSCs 5, 6, 8, and 9 revealed a half maximal inhibitory concentration (IC50) below 1 µM. Compound 6 turned out to be the most potent tyrosinase inhibitor. All investigated compounds showed reversible inhibition of competitive or mixed type. The para-substituted TSCs had higher affinity for the enzyme as compared to their ortho- and meta-analogues. All investigated compounds inhibited melanin production in B16F10 cells at the micromolar level. Molecular docking showed that the sulfur atom of the thiourea moiety penetrates the active site and interacts with copper ions. The above outcomes might be helpful in the design of new tyrosinase inhibitors in the food and cosmetic industries.
... Traditionally, thiosemicarbazone anti-tumor activity was associated with inhibition of ribonucleotide reductase, but also more recently with the up-regulation of tumor and metastasis suppressor proteins and the inhibition of oncogenic signaling [54][55][56][57][58]. Additionally, there is a major role for oxidative stress in the anti-tumor activity of some thiosemicarbazone metal complexes, especially those of the di-2-pyridylketone thiosemicarbazone (DpT) class [14,[59][60][61]. These agents include Dp44mT, which showed marked anti-tumor efficacy [14,[62][63][64]. Notably, Dp44mT is a well-characterized tridentate ligand that can form 2:1 ligand to metal complexes with Fe [59] (Fig. 1D). ...
... Dp44mT could penetrate the hydrophobic part of the bilayer (Fig. 10B), move into the surrounding water, and return to the bilayer surface (Fig. 10A). As Dp44mT is highly permeable to tumor cells, these simulation data were in good agreement with the observed ability of this agent to penetrate tumor cell membranes, bind intracellular Fe pools, and then be effluxed out of cells [14,[62][63][64]. ...
Background
Iron (Fe)-induced oxidative stress leads to reactive oxygen species that damage biomembranes, with this mechanism being involved in the activity of some anti-cancer chemotherapeutics.
Methods
Herein, we compared the effect of Fe complexes of the ligand, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), or the potential ligand, Emodin, on lipid peroxidation in cell membrane models (micelles and bicelles). These studies were performed in the presence of hydrogen peroxide (H2O2) and the absence or presence of ascorbate.
Results
In the absence of ascorbate, Fe(II)/Emodin mixtures incubated with H2O2 demonstrated slight pro-oxidant properties on micelles versus Fe(II) alone, while the Fe(III)-Dp44mT complex exhibited marked antioxidant properties. Examining more physiologically relevant phospholipid-containing bicelles, the Fe(II)- and Fe(III)-Dp44mT complexes demonstrated antioxidant activity without ascorbate. Upon adding ascorbate, there was a significant increase in the peroxidation of micelles and bicelles in the presence of unchelated Fe(II) and H2O2. The addition of ascorbate to Fe(III)-Dp44mT substantially increased the peroxidation of micelles and bicelles, with the Fe(III)-Dp44mT complex being reduced by ascorbate to the Fe(II) state, explaining the increased reactivity. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radical anion generation after mixing ascorbate and Emodin, with signal intensity being enhanced by H2O2. This finding suggested Emodin semiquinone radical formation that could play a role in its reactivity via ascorbate-driven redox cycling. Examining cultured melanoma cells in vitro, ascorbate at pharmacological levels enhanced the anti-proliferative activity of Dp44mT and Emodin.
Conclusions and general significance
Ascorbate-driven redox cycling of Dp44mT and Emodin promotes their anti-proliferative activity.
... The DpT derivative di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) was first developed in 2004 as an iron chelator with marked and selective antitumor activity [168], which is also a copper ionophore and efficiently kills breast cancer cells (MCF-7) through intracellular copper release [169,170]. In addition to Dp44mT, the DpT derivative di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone hydrochloride (Dpc) is also one of the most potent compounds with improved anticancer performance and side-effects, whose copper co-administration induces cancer cell death when administered intravenously and orally [171,172]. Dpc was also originally used as an iron chelator for the treatment of pancreatic cancer [173]. Both Dp44mT and Dpc upregulate gene expression of the metastasis suppressor protein, Nmyc downstream regulated gene-1 (Ndrg1), which may enhance the anticancer activity of DpT derivatives [174,175]. ...
Copper, an essential micronutrient, controls multiple fundamental processes throughout all forms of life, such as respiration, cell proliferation and neurotransmitter biosynthesis. High redox activity makes copper a double-edged sword for cell survival, and abnormal copper metabolism is commonly associated with cancer and other diseases. Recently, advances in copper biology have forged new links between researchers from different disciplines to dissect the use of copper targets in cancer treatment. Copper is crucial for maintaining the rapid growth and proliferation of cancer cells, a process known as ‘cuproplasia’. However, excess mitochondrial copper accumulation also triggers ‘cuproptosis’, a new form of programmed cell death, involving the destabilization of Fe-S cluster proteins and the aggregation of DLAT. Accordingly, both activating cuproptosis by copper ionophores and suppressing cuproplasia through copper depletion are noteworthy anticancer options, but this field currently lacks systematic reviews from both biological and chemical perspectives. This review provides a comprehensive overview of the leading known copper biology and copper-targeted cancer therapy, focusing on copper homeostasis, signal transduction, and copper overload/depletion, which should be beneficial for the development of the next-generation of inorganic anticancer drugs. We expect that our review will provide a balanced perspective to the development and comprehension of copper-based cancer therapy.
... These TSCs chelate intracellular iron, disrupting the iron metabolism, affecting the cell cycle progression, and leading to the activation of apoptosis signaling pathways [44]. On the contrary, other TSCs cause a marked decrease in the expression of cyclins [45]. ...
Since the discovery of cisplatin, the search for metal-based compounds with therapeutic potential has been a challenge for the scientific community. In this landscape, thiosemicarbazones and their metal derivatives represent a good starting point for the development of anticancer agents with high selectivity and low toxicity. Here, we focused on the action mechanism of three metal thiosemicarbazones [Ni(tcitr)2], [Pt(tcitr)2], and [Cu(tcitr)2], derived from citronellal. The complexes were already synthesized, characterized, and screened for their antiproliferative activity against different cancer cells and for genotoxic/mutagenic potential. In this work, we deepened the understanding of their molecular action mechanism using an in vitro model of a leukemia cell line (U937) and an approach of transcriptional expression profile analysis. U937 cells showed a significant sensitivity to the tested molecules. To better understand DNA damage induced by our complexes, the modulation of a panel of genes involved in the DNA damage response pathway was evaluated. We analyzed whether our compounds affected cell cycle progression to determine a possible correlation between proliferation inhibition and cell cycle arrest. Our results demonstrate that metal complexes target different cellular processes and could be promising candidates in the design of antiproliferative thiosemicarbazones, although their overall molecular mechanism is still to be understood.
... The use of novel thiosemicarbazone iron chelators as NDRG1 modulators has been explored in both cancer cells and xenograft models [106][107][108]. di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) are iron-binding agents that have been shown to upregulate NDRG1 expression and in turn exhibit anti-metastatic activity [64,92,93]. These compounds deplete cellular iron via hypoxia-inducible factor 1α (HIF-1α) pathways [107], leading to the accumulation of HIF-1α, nuclear translocation, and formation of the HIF-1 complex [109]. ...
N-myc downregulated gene-1 (NDRG1) has been variably reported as a metastasis suppressor, a biomarker of poor outcome, and a facilitator of disease progression in a range of different cancers. NDRG1 is poorly understood in cancer due to its context-dependent and pleiotropic functions. Within breast cancer, NDRG1 is reported to be either a facilitator of, or an inhibitor of tumour progression and metastasis. The wide array of roles played by NDRG1 are dependent on post-translational modifications and subcellular localization, as well as the cellular context, for example, cancer type. We present an update on NDRG1, and its association with hallmarks of cancer such as hypoxia, its interaction with oncogenic proteins such as p53 as well its role in oncogenic and metastasis pathways in breast and other cancers. We further comment on its functional implications as a metastasis suppressor and promoter, its clinical relevance, and discuss its therapeutic targetability in different cancers.
... Clioquinol is a chelator of copper, iron, and zinc. Although Cu simple chelators such as d-penicillamine or trientine have been clinically used for the treatment of Cu overload, novel thiosemicarbazone and clioquinol are lately mostly investigated as potential anticancer agents [229,230]. A novel class of thiosemicarbazone compounds (di-2-pyridylketone thiosemicarbazones) binds copper and have shown great promise in terms of their anticancer activity [231]. ...
Copper is one of the most abundant basic transition metals in the human body. It takes part in oxygen metabolism, collagen synthesis, and skin pigmentation, maintaining the integrity of blood vessels, as well as in iron homeostasis, antioxidant defense, and neurotransmitter synthesis. It may also be involved in cell signaling and may participate in modulation of membrane receptor-ligand interactions, control of kinase and related phosphatase functions, as well as many cellular pathways. Its role is also important in controlling gene expression in the nucleus. In the nervous system in particular, copper is involved in myelination, and by modulating synaptic activity as well as excitotoxic cell death and signaling cascades induced by neurotrophic factors, copper is important for various neuronal functions. Current data suggest that both excess copper levels and copper deficiency can be harmful, and careful homeostatic control is important. This knowledge opens up an important new area for potential therapeutic interventions based on copper supplementation or removal in neurodegenerative diseases including Wilson’s disease (WD), Menkes disease (MD), Alzheimer’s disease (AD), Parkinson’s disease (PD), and others. However, much remains to be discovered, in particular, how to regulate copper homeostasis to prevent neurodegeneration, when to chelate copper, and when to supplement it.
... Even relatively simple substitutions, such as methylation, can result in 1000-fold increased cytotoxicity. 23,[27][28][29] In our previous work differently methylated Triapine derivatives were investigated in detail, revealing that mono-and dimethylation on the terminal NH 2 and/or on the pyridine amine influences the cytotoxicity of the compounds as well as the solution stability and reducibility of their copper complexes. 23 The TSCs with nanomolar anticancer activity and paraptosis-inducing properties were characterized by higher Cu(II) complex solution stability and a slower reduction rate by glutathione (GSH) as compared to TSC ligands possessing weaker cytotoxicity. ...
Effect of exchange of S to O or Se in Triapine and methylation of the hydrazone NH on the p K a , lipophilicity, solution stability and redox properties of Cu( ii ), Fe( ii ) and Fe( iii ) complexes and their anticancer activity.
... Therefore, the effects of metal ion chelation upon cancer cells can be complex, with these effects probably being due to their multi-targeted activity, as metal ions are critical for a variety of key metabolic processes [176]. Nonetheless, thiosemicarbazones and other chelators result in marked anti-oncogenic activity against a broad variety of tumor cells in vitro [177][178][179] and in vivo [151,180,181]. Collectively, the studies described above indicate that the critical catalytic zinc(II) ion in the active site of MMPs is an important molecular target for the design of further chelating agents. ...
The crucial role of extracellular proteases in cancer progression is well-known, especially in relation to the promotion of cell invasion through extracellular matrix remodeling. This also occurs by the ability of extracellular proteases to induce the shedding of transmembrane proteins at the plasma membrane surface or within extracellular vesicles. This process results in the regulation of key signaling pathways by the modulation of kinases, e.g., the epidermal growth factor receptor (EGFR). Considering their regulatory roles in cancer, therapeutics targeting various extracellular proteases have been discovered. These include the metal-binding agents di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), which increase c-MET degradation by multiple mechanisms. Both the direct and indirect inhibition of protease expression and activity can be achieved through metal ion depletion. Considering direct mechanisms, chelators can bind zinc(II) that plays a catalytic role in enzyme activity. In terms of indirect mechanisms, Dp44mT and DpC potently suppress the expression of the kallikrein-related peptidase-a prostate-specific antigen-in prostate cancer cells. The mechanism of this activity involves promotion of the degradation of the androgen receptor. Additional suppressive mechanisms of Dp44mT and DpC on matrix metalloproteases (MMPs) relate to their ability to up-regulate the metastasis suppressors N-myc downstream regulated gene-1 (NDRG1) and NDRG2, which down-regulate MMPs that are crucial for cancer cell invasion.
... Following the DpT series was the evaluation of aromatic substituents, namely 2-benzoylpyridine thiosemicarbazone (BpT) series, which demonstrated enhanced growth inhibition and redox-cycling activity (215). Bp44mT was also effective for inhibiting growth of lung cancer xenografts with no noticeable cardiotoxicity (216). To date though DpC, an analog of Dp44mt is the most potent and well-tolerated compound showing efficacy both in vitro and in vivo in models of pancreatic (217), neuroblastoma (218), and lung (214) cancers. ...
Iron is an essential nutrient that plays a complex role in cancer biology. Iron metabolism must be tightly controlled within cells. Whilst fundamental to many cellular processes and required for cell survival, excess labile iron is toxic to cells. Increased iron metabolism is associated with malignant transformation, cancer progression, drug resistance and immune evasion. Depleting intracellular iron stores, either with the use of iron chelating agents or mimicking endogenous regulation mechanisms, such as microRNAs, present attractive therapeutic opportunities, some of which are currently under clinical investigation. Alternatively, iron overload can result in a form of regulated cell death, ferroptosis, which can be activated in cancer cells presenting an alternative anti-cancer strategy. This review focuses on alterations in iron metabolism that enable cancer cells to meet metabolic demands required during different stages of tumorigenesis in relation to metastasis and immune response. The strength of current evidence is considered, gaps in knowledge are highlighted and controversies relating to the role of iron and therapeutic targeting potential are discussed. The key question we address within this review is whether iron modulation represents a useful approach for treating metastatic disease and whether it could be employed in combination with existing targeted drugs and immune-based therapies to enhance their efficacy.
... Iron overload is known to cause cancer in animal models, 50 and conversely, iron depletion has been reported to suppress tumor growth. 51 The first mechanism by which free iron is believed to trigger malignant transformation is the generation of reactive oxygen species (ROS), which causes peroxidation of membrane fatty acids and subsequent formation of toxic by-products that impair protein synthesis and disrupt DNA, leading to mutations in tumor suppressor genes (such as p53) and DNA repair genes. 52,53 Iron overload may also promote malignant transformation in the liver through the acceleration of fibrosis to cirrhosis by activation of stellate cells and the profibrogenic effects of lipid peroxidation. ...
Due to the recent alarming increase in the incidence of hepatocellular carcinoma (HCC) in thalassemias, the aim of the present report is to review briefly the frequency, the major risk factors and the surveillance of HCC in β-thalassemias. Over the past 33 years, 153 cases of HCC were reported in patients with thalassemia, mainly in Italy, and Greece. Among HCV-infected patients additional factors promoting development of HCC, included: advanced age, male sex, chronic hepatitis B (CHB) coinfection, and iron overload. For early diagnosis of HCC sequential ultrasound screening is recommended especially for thalassemia patients with chronic hepatitis C (CHC), that coincide with (one or more) additional risk factors for HCC. Here we report also the preliminary data of thalassemic patients, above the age of 30 years, followed in 13 different centers. The total number of enrolled patients was 1,313 (males: 612 and 701 females). The prevalence of HCC in thalassemia major patients [characterized by transfusion-dependency (TDT)] and thalassemia intermedia [characterized by nontransfusion dependency (NTDT)] was 1.68 % and 1.98 % ,respectively The lowest age at diagnosis was 36 years in TDT and 47 years in NTDT patients.We hope that our review can be used to develop more refined and prospective analyses of HCC magnitude and risk in patients with thalassemia, and to define specific international guidelines to support clinicians for an early diagnosis and treatment of HCC in thalassemic patients.
... Thiosemicarbazones represent a versatile class of ligands. They possess numerous pharmacological and biological properties which make them effective antimalarial [9] antimicrobial [10], and anticancer [11,12] agents. ...
... Administré à la dose de 50mg/kg, trois fois par semaine à des souris xénogréffées avec la lignée tumorale pulmonaire DMS-53, il s'est révélé être bien toléré, n'a pas causé de dommages cardiaques et a permis d'inhiber la croissance tumorale de 75% par rapport au groupe contrôle. Seule une légère diminution du nombre d'hématies a été observée(Yu et al., 2012).De nombreuses classes de chélateurs de fer synthétiques ont ainsi été développées dans l'objectif d'augmenter l'efficacité, de réduire les toxicités et les effets secondaires, et d'améliorer l'activité par voie orale (Kalinowski et al., 2005). La liste de chélateurs citée cidessus est donc loin d'exhaustive mais elle comporte l'ensemble des molécules qui semblent présenter le meilleur potentiel en vue d'une hypothétique utilisation en oncologie (Figure 21). ...
La dérégulation du métabolisme des cellules tumorales est un hallmark du cancer clairement établi. Pour assurer leur taux de proliferation élevé, les cellules cancéreuses adaptent leur métabolisme, ce qui leur permet de répondre à leurs nouveaux besoins énergétiques. Dans ce contexte, les cellules tumorales présentent des besoins en fer augmentés ainsi que de multiples perturbations du métabolisme du fer, ce qui les rend plus sensibles à la privation en fer. Cette vulnérabilité pourrait ainsi faire l’objet d’un ciblage thérapeutique. Dans les cancers du sein, de nouvelles approches thérapeutiques sont très attendues en particulier pour les cancers triple-négatifs qui développent fréquemment des résistances à la chimiothérapie et qui souffrent d'un manque de cibles thérapeutiques spécifiques. L’activité antitumorale des chélateurs de fer tels que le déférasirox (DFX) évalués en monothérapie a déjà été démontrée dans différents types de cancers mais ne semble pas être suffisamment efficace pour éradiquer les tumeurs. Dans cette étude, nous avons démontré que le DFX agit en synergie avec des molécules de chimiothérapies conventionnelles telles que la doxorubicine, le cisplatine et le carboplatine pour inhiber la prolifération cellulaire et induire l’apoptose et l’autophagie de lignées cellulaires mammaires de sous-type triple-négatif. De plus, la combinaison du DFX avec la doxorubicine et le cyclophosphamide permet de retarder voire d’éviter les récidives dans des xénogreffes de tumeurs mammaires triple-négatives (PDX) sans augmenter les effets secondaires de la chimiothérapie seule ni impacter les réserves en fer globales des souris. Au niveau moléculaire, nous avons montré que la synergie antitumorale du DFX et de la doxorubicine implique une inhibition des voies PI3K et NF-κB. Par ailleurs, étant donné que les patients présentant un cancer triple-négatif avec de faibles réserves en fer tumorales présentent un bon pronostic, nous pensons que la privation en fer au moyen de chélateurs de fer pourrait constituer une approche d’autant plus efficace pour augmenter l’efficacité des chimiothérapies conventionnelles dans le traitement de ces cancers.
... 59 Fe chelation by TSCs often results in the down-regulation of cyclin D1 and subsequent cell cycle arrest at the G 1 /S phase. 60,61 As expected, complex 2 was shown to induce strong dose-dependent downregulation of cyclin D1, whereas the protein expression of cyclin B1, which is responsible for G 2 /M progression, remained unchanged. To further investigate the potential biomolecular targets of Fe-chelating compounds of interest, we studied the phosphorylation of ERK protein, which belongs to the MAPK family. ...
Six morpholine-(iso)thiosemicarbazone hybrids HL1‒HL6 and their copper(II) complexes with good-to-moderate solubility and stability in water were synthesized and characterized. Copper(II) complexes [Cu(L1‒6)Cl] (1‒6) formed weak dimeric associates in the solid state, which did not remain intact in solution as evidenced by ESI-MS. The lead proligands and copper(II) complexes displayed higher antiproliferative activity in cancer cells than Triapine. In addition, complexes 2‒5 were found to specifically inhibit the growth of Gram-positive bacteria S. aureus with MIC50 values at 2 to 5 µg/mL. Insights into the processes controlling intracellular accumulation and mechanism of action were investigated for 2 and 5, including the role of ribonucleotide reductase (RNR) inhibition, endoplasmic reticulum (ER) stress induction and regulation of other cancer signaling pathways. Their ability to moderately inhibit R2 RNR protein in the presence of DTT is likely related to iron chelating properties of the proligands liberated upon reduction.
... Apoptosis is one of the important mechanisms of anti-tumor therapy. Studies have shown that iron chelators can induce leukemia cell apoptosis and may be involved in a variety of apoptosis-related signal transduction pathways, but the mechanism of the induction of apoptosis is not entirely clear [16][17][18][19]. Iron chelators can inhibit the proliferation of tumor cells and induce cell apoptosis; thus, it has great clinical value in the treatment of leukemia. ...
Background
This study aimed to investigate the effect of deferoxamine (DFO) on leukemia in vitro, and to explore the underlying molecular mechanism.
Material/Methods
K562 leukemia cells were treated with various concentrations of DFO (10, 50, and 100 μmol/l) with or without 10 μmol/l ferric chloride for 12 h. Then, total cellular iron was detected. CCK-8 kit and flow cytometry were used for cell viability and apoptosis detection. In addition, expression of apoptosis-related genes was determined by Western blotting and qRT-PCR, respectively.
Results
The results suggested that DFO significantly inhibited K562 cell viability and induced cell apoptosis in a dose-dependent manner. We also found that the protein and mRNA levels of Bax, p53, and Fas dose-dependently increased in DFO-treated K562 cells, while the level of Bcl-2 markedly decreased in a dose-dependent manner. Moreover, the findings showed that ferric chloride eliminated these effects on K562 cells caused by DFO treatment.
Conclusions
Our results indicate that DFO plays a protective role in leukemia via inhibiting leukemia cell viability and inducing cell apoptosis by the regulation of apoptosis-related genes expression.
... Lipocalin-2 was found to be associated with radiation sensitivity in a lung cancer line, and with a fivefold increase in hazard ratio for overall survival if strongly stained in lung tumor tissue in another analysis [166,167]. Unsurprisingly given the poor prognosis of lung cancer, this abundance of preclinical data has encouraged investigators to consider iron pathways as potential therapeutic targets, with already several formulations of iron chelators being studied [168][169][170]. Further understanding in lung iron biology will likely enhance the development of novel antitumor agents. ...
Iron is one of the most abundant transition elements and is indispensable for almost all organisms. While the ability of iron to participate in redox chemistry is an essential requirement for participation in a range of vital enzymatic reactions, this same feature of iron also makes it dangerous in the generation of hydroxyl radicals and superoxide anions. Given the high local oxygen tensions in the lung, the regulation of iron acquisition, utilization, and storage therefore becomes vitally important, perhaps more so than in any other biological system. Iron plays a critical role in the biology of essentially every cell type in the lung, and in particular, changes in iron levels have important ramifications on immune function and the local lung microenvironment. There is substantial evidence that cigarette smoke causes iron dysregulation, with the implication that iron may be the link between smoking and smoking-related lung diseases. A better understanding of the connection between cigarette smoke, iron, and respiratory diseases will help to elucidate pathogenic mechanisms and aid in the identification of novel therapeutic targets.
... Additionally, the activity of cyclin D1 is associated with the p21 protein, which plays a crucial role in triggering various effects on cell cycle regulation. Thus, a decrease in the expression of the p21 CIP1/WAF1 protein may lead to the arrest of the cell cycle in the G1/S phase since this protein can stabilize the cyclin D1-cdk complexes [75,76]. On the other hand, the effect of iron chelation may up-regulate p21 CIP1/WAF1 , which may induce the signaling pathways, thus lead- ing to apoptosis [74,77]. ...
A new class of TSCs containing piperazine (piperazinylogs) of Triapine, was designed to fulfill the di-substitution pattern at the TSCs N4 position, which is a crucial prerequisite for the high activity of the previously obtained TSC compounds–DpC and Dp44mT. We tested the important physicochemical characteristics of the novel compounds L¹-L¹². The studied ligands are neutral at physiological pH, which allows them to permeate cell membranes and bind cellular Fe pools more readily than less lipid-soluble ligands, e.g. DFO. The selectivity and anti-cancer activity of the novel TSCs were examined in a variety of cancer cell types. In general, the novel compounds demonstrated the greatest promise as anti-cancer agents with both a potent and selective anti-proliferative activity. We investigated the mechanism of action more deeply, and revealed that studied compounds inhibit the cell cycle (G1/S phase). Additionally we detected apoptosis, which is dependent on cell line’s specific genetic profile. Accordingly, structure-activity relationship studies suggest that the combination of the piperazine ring with Triapine allows potent and selective anticancer chelators that warrant further in vivo examination to be identified. Significantly, this study proved the importance of the di-substitution pattern of the amine N4 function.
... Di-2-pyridylketone thiosemicarbazone (DpT) is an iron chelator with good anti-tumor activity and selectivity (10)(11)(12), which was first reported by Yuan et al (13). Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT), one of the most effective chelators of the DpT family, has been shown to exhibit a substantial inhibitory effect in transplanted tumors in mice (14) Di-2-pyridylketone-4-cyclohexyl-4-me thyl-3-thiosemicarbazone (DpC) is a second-generation iron chelator. ...
The present study aimed to investigate the antitumor efficacy of di-2-pyridylketone-4-cyclohexyl-4-me thyl-3-thiosemicarbazone (DpC) and di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) on head and neck squamous cell carcinoma (HNSCC) cells. The proliferation and apoptosis of HNSCC cells treated with the iron chelators DpC and Dp44mT were detected. The mechanism of DpC-induced apoptosis on HNSCC cells was investigated. The human HNSCC cell lines FaDu, Cal-27 and SCC-9 were cultured in vitro and exposed to gradient concentrations of DpC and Dp44mT. A Cell Counting Kit-8 assay was used to detect the viability of FaDu, Cal-27, SCC-9 cells. Double staining with annexin V and propidium iodide was performed for the detection of the proportion of apoptotic FaDu, Cal-27 and SCC-9 cells following treatment. The nuclear damage to Cal-27 cells that were treated with DpC was detected by Hoechst staining. Finally, western blot analysis was used to detect the expression of proteins associated with the DNA damage pathway in Cal-27 cells that were treated with DpC. The CCK-8 assay showed that treatment with DpC and Dp44mT was able to markedly inhibit the viability of FaDu, Cal-27 and SCC-9 cells in a concentration-dependent manner. In comparison to Dp44mT, treatment with DpC exhibited a more effective inhibitory effect on the viability of HNSCC cells. The proportion of apoptotic cells detected by flow cytometry increased in a dose-dependent manner in all cell lines following DpC and Dp44mT treatment, with the proportion of apoptotic HNSCC cells induced by DpC treatment being significantly higher compared with Dp44mT (P<0.05). The results of Hoechst staining revealed that the nuclei of Cal-27 cells exhibited morphological changes in response to DpC treatment, including karyopyknosis and nuclear fragmentation. The expression of DNA damage-associated proteins, including phosphorylated (p)-serine-protein kinase ATM, p-serine/threonine-protein kinase Chk1 (p-Chk-1), p-serine/threonine-protein kinase ATR (p-ATR), p-Chk-2, poly (ADP-ribose) polymerase, p-histone H2AX, breast cancer type 1 susceptibility protein, p-tumor protein P53, increased with increasing concentration of DpC in Cal-27 cells. Treatment with DpC and Dp44mT markedly inhibited cell viability and increased the apoptotic rates in human HNSCC cells in a concentration-dependent manner. DpC exhibited a stronger antitumor effect compared with Dp44mT, potentially inducing the apoptosis of HNSCC cells via the upregulation of DNA damage repair-associated proteins.
... [3][4][5][6][7][8][9][10][11][12][13][14] Encouraged by the modest results obtained with traditional metal chelators, a variety of novel thiosemicarbazone-based ligands with antiproliferative activity have been identified (Fig. 1), including 3-AP, 15 Dp44mT, 16,17 Ap44mT, [18][19][20][21] and DpC. [22][23][24] In fact, 3-AP has been investigated in over 20 phase I and phase II clinical trials in a variety of advanced cancers. [25][26][27] Unfortunately, 3-AP has suffered multiple problems, including low efficacy in some tumor-types and serious side effects, which limit its clinical utility. ...
A series of novel thiosemicarbazone derivatives were synthesized and evaluated for their antiproliferative activity against several selected tumor cell lines of different origins using the MTT assay. The preliminary results indicated that the MGC-803 cell line was remarkably sensitive to all the synthesized compounds. Among this series, compound 5n showed the best inhibitory activity with an IC50 value of 0.93 μM (about 10-fold more potent than 3-AP) against MGC-803. Further mechanism studies revealed that compound 5n could obviously inhibit the proliferation of MGC-803 cells by inducing apoptosis and arresting the cell cycle at the S phase. Compound 5n also showed marked inhibition of cell migration and invasion, without significant cytotoxicity against gastric epithelial immortalized GES-1 cells.
... The authors relied on the resemblance of the tested compounds to Triapine, which is a potent iron chelator that is in clinical trials for anticancer therapy [139]. Today thiosemicarbazones are being extensively studied for their metal-chelating potency and their particularly interesting ability to change the iron metabolism [140][141][142]. Currently, this group of compounds remains one of the most potent anticancer agents [143][144][145]. ...
Background:
Fluorescent compounds had gained strong attention due to their wide and appealing applications. Microscopic techniques and visualization are good examples among others. Introduction of fluorescent dyes into microbiology opens the possibility to observe tissues, organisms or organelle with exceptional sensitivity and resolution. Probes for detection of biologically relevant metals as zinc, iron or copper seems to be particularly important for drug design and pharmaceutical sciences.
Objective:
Quinoline derivatives are well known for their good metal affinity and wide spectrum of biological activity. In this regard, molecular sensors built on this scaffold may be useful not only as analytical but also as therapeutic agents.
Methods:
In the present review, application of quinoline moiety in designing of novel fluorescent probes for zinc is presented and discussed.
Results:
Zinc cations are relevant for vast majority of processes and recently attract a great deal of attention for their role in neurodegenerative diseases. Compounds interacting with Zn2+ may be used for early diagnosis of such disorders, for example the Alzheimer disease.
Conclusion:
Quinoline-based zinc probes may exert some beneficial role in organism acting as theranostic agents. First preliminary drugs for Alzheimer therapy that are based on quinoline moiety are good example of this trend.
... Identifier: NCT02688101) and other thiosemicarbazones, e.g. Dp44mT and Bp44mT, have shown potent antitumor activity in tumor xenografts in mice [16]. However, clinical trials with triapine demonstrated poor activity and side effects such as myelosuppression, hypoxia and methemoglobinemia [13][14][15]. ...
Background:
The thiosemicarbazone CD 02750 (VLX50) was recently reported as a hit compound in a phenotype-based drug screen in primary cultures of patient tumor cells. We synthesized a copper complex of VLX50, denoted VLX60, and characterized its antitumor and mechanistic properties.
Materials and methods:
The cytotoxic effects and mechanistic properties of VLX60 were investigated in monolayer cultures of multiple human cell lines, in tumor cells from patients, in a 3-D spheroid cell culture system and in vivo and were compared with those of VLX50.
Results:
VLX60 showed ≥ 3-fold higher cytotoxic activity than VLX50 in 2-D cultures and, in contrast to VLX50, retained its activity in the presence of additional iron. VLX60 was effective against non-proliferative spheroids and against tumor xenografts in vivo in a murine model. In contrast to VLX50, gene expression analysis demonstrated that genes associated with oxidative stress were considerably enriched in cells exposed to VLX60 as was induction of reactive oxygen. VLX60 compromised the ubiquitin-proteasome system and was more active in BRAF mutated versus BRAF wild-type colon cancer cells.
Conclusions:
The cytotoxic effects of the copper thiosemicarbazone VLX60 differ from those of VLX50 and shows interesting features as a potential antitumor drug, notably against BRAF mutated colorectal cancer.
... (3) DpC exhibits greater activity than Dp44mT in vivo against an aggressive human pancreatic tumor xenograft [26]; (4) DpC demonstrated pronounced in vivo activity after oral and intravenous administration [27], while Dp44mT was not tolerated orally [29]; and (5) while both Dp44mT and DpC display appropriate pharmacokinetics, the markedly greater half-life of DpC (t 1/2 = 10.7 h for DpC vs. 1.7 h for Dp44mT) further underlines its potential [30]. ...
Background:
Neuroblastoma is a relatively common and highly belligerent childhood tumor with poor prognosis by current therapeutic approaches. A novel anti-cancer agent of the di-2-pyridylketone thiosemicarbazone series, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), demonstrates promising anti-tumor activity. Recently, a second-generation analogue, namely di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), has entered multi-center clinical trials for the treatment of advanced and resistant tumors. The current aim was to examine if these novel agents were effective against aggressive neuroblastoma in vitro and in vivo and to assess their mechanism of action.
Methods:
Neuroblastoma cancer cells as well as immortalized normal cells were used to assess the efficacy and selectivity of DpC in vitro. An orthotopic SK-N-LP/Luciferase xenograft model was used in nude mice to assess the efficacy of DpC in vivo. Apoptosis in tumors was confirmed by Annexin V/PI flow cytometry and H&E staining.
Results:
DpC demonstrated more potent cytotoxicity than Dp44mT against neuroblastoma cells in a dose- and time-dependent manner. DpC significantly increased levels of phosphorylated JNK, neuroglobin, cytoglobin, and cleaved caspase 3 and 9, while decreasing IkBα levels in vitro. The contribution of JNK, NF-ĸB, and caspase signaling/activity to the anti-tumor activity of DpC was verified by selective inhibitors of these pathways. After 3 weeks of treatment, tumor growth in mice was significantly (p < 0.05) reduced by DpC (4 mg/kg/day) given intravenously and the agent was well tolerated. Xenograft tissues showed significantly higher expression of neuroglobin, cytoglobin, caspase 3, and tumor necrosis factor-α (TNFα) levels and a slight decrease in interleukin-10 (IL-10).
Conclusions:
DpC was found to be highly potent against neuroblastoma, demonstrating its potential as a novel therapeutic for this disease. The ability of DpC to increase TNFα in tumors could also promote the endogenous immune response to mediate enhanced cancer cell apoptosis.
... The potent anti-tumor agent, di-2-pyridylketone 4,4-dimethyl-3 thiosemicarbazone (Dp44mT; Fig. 1A ; [5,[15][16][17][18][19][20][21][22][23][24][25]), was shown to be lysosomotropic and trapped within lysosomes due to the acidic pH of these vesicles [6]. Within these organelles, Dp44mT forms cytotoxic copper complexes that generate reactive oxygen species (ROS), resulting in lysosomal membrane permeabilization (LMP) that induces tumor cell death [5][6][7]. ...
The potent and selective anti-tumor agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), localizes in lysosomes and forms cytotoxic copper complexes that generate reactive oxygen species (ROS), resulting in lysosomal membrane permeabilization (LMP) and cell death. Herein, the role of lysosomal membrane stability in the anti-tumor activity of Dp44mT was investigated. Studies were performed using molecules that protect lysosomal membranes against Dp44mT-induced LMP, namely heat shock protein 70 (HSP70) and cholesterol. Up-regulation or silencing of HSP70 expression did not affect Dp44mT-induced LMP in MCF7 cells. In contrast, cholesterol accumulation in lysosomes induced by the well characterized cholesterol transport inhibitor, 3-β-[2-(diethyl-amino)ethoxy]androst-5-en-17-one (U18666A), inhibited Dp44mT-induced LMP and markedly and significantly (p<0.001) reduced the ability of Dp44mT to inhibit cancer cell proliferation (i.e., increased the IC50) by 140-fold. On the other hand, cholesterol extraction using methyl-β-cyclodextrin enhanced Dp44mT-induced LMP and significantly (p<0.01) increased its anti-proliferative activity. The protective effect of U18666A in increasing lysosomal cholesterol and preventing the cytotoxic activity of Dp44mT was not due to induced autophagy. Instead, U18666A was found to decrease lysosomal turnover, resulting in autophagosome accumulation. Moreover, preincubation with U18666A did not prevent the ability of Dp44mT to induce autophagosome synthesis, indicating that autophagic initiation via Dp44mT occurs independently of LMP. These studies demonstrate the significance of lysosomal membrane stability in relation to the ability of Dp44mT to execute tumor cell death and overcome pro-survival autophagy. Hence, lysosomal-dependent cell death induced by Dp44mT serves as an important anti-tumor strategy. These results are important for comprehensively understanding the mechanism of action of Dp44mT.
Breast cancer (BCa) is a highly heterogeneous disease, with hormone receptor status being a key factor in patient prognostication and treatment decision‐making. The majority of primary tumours are positive for estrogen receptor alpha (ERα), which plays a key role in tumorigenesis and disease progression, and represents the major target for treatment of BCa. However, around one third of patients with ERα‐positive BCa relapse and progress into the metastatic stage, with 20% of metastatic cases characterised by loss of ERα expression after endocrine treatment, known as ERα‐conversion. It remains unclear whether ERα‐converted cancers are biologically similar to bona fide ERα‐negative disease and which signalling cascades compensate for ERα loss and drive tumour progression. To better understand the biological changes that occur in metastatic BCa upon ERα loss, we performed (phospho)proteomics analysis of 47 malignant pleural effusions derived from 37 breast cancer patients, comparing ERα‐positive, ERα‐converted and ERα‐negative cases. Our data revealed that the loss of ERα‐dependency in this metastatic site leads to only a partial switch to an ERα‐negative molecular phenotype, with preservation of a luminal‐like proteomic landscape. Furthermore, we found evidence for decreased activity of several key kinases, including serum/glucocorticoid regulated kinase 1 (SGK1), in ERα‐converted metastases. Loss of SGK1 substrate phosphorylation may compensate for the loss of ERα‐dependency in advanced disease and exposes a potential therapeutic vulnerability that may be exploited in treating these patients.
To obtain next-generation metal drugs that can overcome the deficiencies of platinum (Pt) drugs and treat cancer more effectively, we proposed to develop a multitargeted palladium (Pd) agent to the tumor microenvironment (TME) based on the specific residue(s) of human serum albumin (HSA). To this end, we optimized a series of Pd(II) 2-benzoylpyridine thiosemicarbazone compounds to obtain a Pd agent (5b) with significant cytotoxicity. The HSA-5b complex structure revealed that 5b bound to the hydrophobic cavity in the HSA IIA subdomain and then His-242 replaced a leaving group (Cl) of 5b, coordinating with the Pd center. The in vivo results showed that the 5b/HSA-5b complex had significant capacity of inhibiting tumor growth, and HSA optimized the therapeutic behavior of 5b. In addition, we confirmed that the 5b/HSA-5b complex inhibited tumor growth through multiple actions on different components of TME: killing cancer cells, inhibiting tumor angiogenesis, and activating T cells.
A novel, potent, and selective antitumor agent, namely (E)-3-phenyl-1-(2-pyridinyl)-2-propen-1-one 4,4-dimethyl-3-thiosemicarbazone (PPP44mT), and its analogues were synthesized and characterized and displayed strikingly distinctive properties. This activity was mediated by the inclusion of a styrene moiety, which through steric and electrochemical mechanisms prevented deleterious oxy-myoglobin or oxy-hemoglobin oxidation relative to other potent thiosemicarbazones, i.e., di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) or di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT). Structure-activity relationship analysis demonstrated specific tuning of PPP44mT electrochemistry further inhibited oxy-myoglobin or oxy-hemoglobin oxidation. Both PPP44mT and its Cu(II) complexes showed conspicuous almost immediate cytotoxicity against SK-N-MC tumor cells (within 3 h). In contrast, [Zn(PPP44mT)2] demonstrated a pronounced delay in activity, taking 48 h before marked antiproliferative efficacy was apparent. As such, [Zn(PPP44mT)2] was designated as a "stealth Zn(II) complex" that overcomes the near immediate cytotoxicity of PPP44mT or its copper complexes. Upon examination of the suppression of oncogenic signaling, [Zn(PPP44mT)2] was superior at inhibiting cyclin D1 expression compared to DpC or Dp44mT.
Standard treatments have shown dismal activity against pancreatic cancer (PC), due in part to the development of a dense stroma (desmoplasia). This perspective discusses the development of the di-2-pyridylketone thiosemicarbazones that overcomes bidirectional oncogenic signaling between PC cells and pancreatic stellate cells (PSCs), which is critical for desmoplasia development. This activity is induced by the up-regulation of the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), which inhibits oncogenic signaling via HGF, IGF-1 and Sonic Hedgehog pathway. More recent studies have deciphered additional pathways including those mediated by Wnt and tenascin C that are secreted by PSCs to activate β-catenin and YAP/TAZ signaling in PC cells. Suppression of bidirectional signaling between cell types presents a unique therapeutic opportunity.
A water-soluble iron complex that produces hydroxyl radical species triggers colorectal cancer cell death via the mitochondrial apoptotic pathway.
Iron is an indispensable requirement for essential biological processes in cancer cells. Due to the greater proliferation of neoplastic cells, their demand for iron is considerably higher relative to normal cells, making them highly susceptible to iron depletion. Understanding this sensitive relationship led to research exploring the effect of iron chelation therapy for cancer treatment. The classical iron-binding ligand, desferrioxamine (DFO), has demonstrated effective anti-proliferative activity against many cancer-types, particularly neuroblastoma tumors, and has the surprising activity of down-regulating the potent oncogene, N-myc, which is a major oncogenic driver in neuroblastoma. Even more remarkable is the ability of DFO to simultaneously up-regulate the potent metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), which plays a plethora of roles in suppressing a variety of oncogenic signaling pathways. However, DFO suffers the disadvantage of demonstrating poor membrane permeability and short plasma half-life, requiring administration by prolonged subcutaneous or intravenous infusions. Considering this, the specifically designed di-2-pyridylketone thiosemicarbazone (DpT) series of metal-binding ligands was developed in our laboratory. The lead agent from the first generation DpT series, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), showed exceptional anti-cancer properties compared to DFO. However, it exhibited cardiotoxicity in mouse models at higher dosages. Therefore, a second generation of agents was developed with the lead compound being di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) that progressed to Phase I clinical trials. Importantly, DpC showed better anti-proliferative activity than Dp44mT and no cardiotoxicity, demonstrating effective anti-cancer activity against neuroblastoma tumors in vivo.
Iron chelators have long been a target of interest as anticancer agents. Iron is an important cellular resource involved in cell replication, metabolism and growth. Iron metabolism is modulated in cancer cells reflecting their increased replicative demands. Originally, iron chelators were first developed for use in iron overload disorders, however, their potential as anticancer agents has been gaining increasing interest. This is due, in part, to the downstream effects of iron depletion such as the inhibition of proliferation through ribonucleotide reductase activity. Additionally, some chelators form redox active metal complexes with iron resulting in the production of reactive oxygen species and oxidative stress. Newer synthetic iron chelators such as Deferasirox, Triapine and di-2-pyridylketone-4,4,-dimethyl-3-thiosemicrbazone (Dp44mt) have improved pharmacokinetic properties over the older chelator Deferoxamine. This review examines and discusses the various iron chelators that have been trialled for cancer therapy including both preclinical and clinical studies. The successes and shortcomings of each of the chelators and their use in combination therapies are highlighted and future potential in the cancer therapy world is considered.
Thiosemicarbazones (TSCs) are a class of Schiff bases usually obtained by the condensation of thiosemicarbazide with a suitable aldehyde or ketone. TSCs have been focus of chemists and biologists due to their wide range of pharmacological effects. One of the promising areas in which these excellent metal chelators are being developed is their use against cancer. TSCs have a wide clinical antitumor spectrum with efficacy in various tumor types such as leukemia, pancreatic cancer, breast cancer, non-small cell lung cancer, cervical cancer, prostate cancer and bladder cancer. To obtain better activity different series of TSCs have been developed by modifying the heteroaromatic system in their molecules. These compounds possessed significant antineoplastic activity when the carbonyl attachment of the side chain was located at a position α to the ring nitrogen atom, whereas attachment of the side chain β or γ to the heterocyclic N atom resulted in inactive antitumor agents. In addition, replacement of the heterocyclic ring N with C also resulted in a biologically inactive compound suggesting that a conjugated N,N,S-tridentate donor set is essential for the biological activities of thiosemicarbazones. Several possible mechanisms have been implemented for the anticancer activity of thiosemicarbazones.
The antioxidant, antimalarial, antibacterial, and antitumor activities of thiosemicarbazones have made this class of compounds important for medicinal chemists. In addition, thiosemicarbazones are among the most potent and well‐known ribonucleotide reductase inhibitors. In this study, 24 new thiosemicarbazone derivatives were synthesized, and the structures and purity of the compounds were determined by IR, 1H NMR, 13C NMR, mass spectroscopy, and elemental analysis. The IC50 values of these 24 compounds were determined with an assay for ribonucleotide reductase inhibition. Compounds 19, 20, and 24 inhibited ribonucleotide reductase enzyme activity at a higher level than metisazone as standard. The cytotoxic effects of these compounds were measured on the MCF7 (human breast adenocarcinoma) and HEK293 (human embryonic kidney) cell lines. Similarly, compounds 19, 20, and 24 had a selective effect on the MCF7 and HEK293 cell lines, killing more cancer cells than cisplatin as standard. The compounds (especially 19, 20, and 24 as the most active ones) were then subjected to docking experiments to identify the probable interactions between the ligands and the enzyme active site. The complex formation was shown qualitatively. The ADME (absorption, distribution, metabolism, and excretion) properties of the compounds were analyzed using in‐silico techniques. A series of new thiosemicarbazone derivatives were synthesized and assayed for their ribonucleotide reductase inhibition activity, with compounds 19, 20, and 24 showing a higher inhibition potency than the standard metisazone. The compounds were subjected to docking experiments to identify the probable interactions between the ligands and the enzyme‐active site. All ligands seemed to fit well into the pocket, giving similar poses.
Background Iron is intimately related to the biology and pathology of gastrointestinal cancer with both iron excess and iron deficiency influencing disease. This thesis examines the biological and clinical effects of iron replacement in gastrointestinal cancer. Methods This thesis reports the effects of iron replacement in colorectal cancers at a biological level using immunohistochemistry, real time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) during clinical treatment for iron deficiency anaemia prior to surgery. Systematic review of the literature also investigates the clinical evidence for such an approach to pre-operative anaemia. The thesis then examines the natural history and impact of anaemia in oesophagogastric cancer before reporting a pilot randomised control study using intravenous iron to treat anaemia in this setting. Results Examining iron administration in colorectal cancer demonstrated that colorectal adenocarcinomas reprogram their iron metabolism to increase the potential labile iron pool. These changes appear to be decoupled from the normal intracellular iron sensing mechanisms. Route of administration of iron to patients did not alter tumour growth or effect iron transport mechanisms. Differential compartmentalisation of iron was noted however. Clinical anaemia in oesophagogastric cancer becomes more severe with time and treatment and was associated with poorer survival outcomes. Furthermore, higher initial haemoglobin, rather than just the absence of anaemia, was associated with better survival outcomes. In a randomised control pilot study for intravenous iron use compared to standard care for anaemia in oesophagogastric cancer, intravenous iron effectively replenished iron stores measured using ferritin and transferrin saturations. Despite chemotherapy and the tumour in situ the intravenous iron group saw an increase in haemoglobin. This was despite a significantly lower starting haemoglobin in the intravenous iron group. Quality of life was also significantly improved in the intravenous iron group. Conclusion This thesis supports the continued clinical use of intravenous iron for anaemia in gastrointestinal cancer, showing no deleterious effects at a biological level despite replenishment of iron stores and increases in haemoglobin at a clinical level. More research is required to investigate compartmentalisation of iron and conclude clinical efficacy in larger adequately powered studies.
Transition metals are essential for the function for numerous metalloproteins and cofactors in living systems. Their acquisition, transport, and storage are tightly regulated and the homeostasis of redox‐active cations such as iron and copper is especially critical to prevent the damaging effects of oxidative stress. Indeed the dyshomeostasis of metals has been observed in the pathophysiology of several diseases, and multiple metal‐binding compounds are currently under investigation as therapeutic candidates. Herein, we focus on the altered metal metabolisms of malignant cells and on metal‐binding strategies for the design of cancer chemotherapeutics. Iron, copper, and zinc chelators with a variety of binding motifs have been studied for their antiproliferative effects in malignant cells, and several compounds have reached clinical trials. Recent pro‐chelation approaches, in which the chelator is activated under specific conditions, are poised to increase therapeutic indexes and avoid unwanted side effects. As additional information emerges on the roles of metals in cancer biology, the design of metal‐binding drug candidates is evolving to improve their selectivity and efficacy and to consider their effects on the immune cells present in the tumor microenvironment. In addition, extensive studies to develop inhibitors of metalloenzymes relevant to cancer growth have led to several new anticancer therapeutics that coordinate the zinc centers in the active site of the enzymes. As illustrated by the examples collected herein, approaches that target either labile or protein‐bound transition metals have significant potential to produce new therapeutic options for cancer treatment.
Tyrosinase plays an essential role in melanogenesis. Excess production of melanin can be a reason of hyperpigmentation skin disorders in mammals and enzymatic browning in plant-derived foods. Catalyzing the rate-limiting step of melanin synthesis, tyrosinase has become the most studied target for melanogenesis inhibition. For over the past ten years a number of synthetic thiosemicarbazone derivatives have been reported to possess strong tyrosinase inhibitory properties with IC50 below 1 µM what locates them among the most potent tyrosinase inhibitors. This review gives an overview of tyrosinase and describes tyrosinase-inhibiting thiosemicarbazones in terms of their structure-activity relationships, kinetics of enzyme inhibition and mechanism of action. Results of the studies on thiosemicarbazones as tyrosinase inhibitors from over 20 research articles have been analyzed, compared and summarized in the present paper. Using thiosemicarbazones as tyrosinase inhibitors is a promising approach in developing anti-melanogenetic agents for skin-whitening cosmetics and anti-browning agents for food.
The group of 19 thiosemicarbazones (TSCs) were synthesized and its inhibitory activity toward mushroom tyrosinase and ability to inhibition of melanogenesis in B16 cells were investigated. Moreover, molecular docking of these compounds to the active site of the enzyme was performed. The obtained results allowed to make the structure-activity relationship (SAR) analysis. Kinetic studies revealed that TSCs 1, 2, 11 and 18 have better inhibitory properties than kojic acid, a reference compound, with the best inhibitory constant (Ki) value of 0.38 µM for TSC 2. According to SAR analysis, the smaller and less branched molecules exhibit higher affinity to the enzyme. Melanin production in B16 cells was inhibited by all investigated compounds at micromolar level. Most of compounds studied in this work can be considered as potent inhibitors of tyrosinase and melanogenesis. They may have broad application in food preservatives and cosmetics. Combined results of molecular docking and SAR analysis can be helpful in designing novel tyrosinase inhibitors of desired properties.
Four metal complexes based on 2-benzoylpyridine N,N-dimethylthiosemicarbazone (Bp44mT) were fully designed. Free ligand and title complexes all demonstrated pronounced activity, which can be indicated by growth inhibition test in vitro. Interestingly, most of the compounds were found to be selective against HepG2 cells but had relative little effect on QSG7701 cells. Particularly, 1 exhibited toxicity on QSG7701 cells ca 12-fold (IC50: the concentration of the compound that produces the cell death of 50%) lower than that of HepG2 cells. The studies of mechanisms of action indicated that 1 induced reactive oxygen species (ROS) generation in a dose-dependent manner via the mitochondria transduction pathway. Protein analyses showed that 1 significantly promoted p21 and p53 gene expression, causing caspase-3 activation.
Ga(III) compounds are very promising candidates for antitumor therapy. Studies have shown that intracellular Reactive Oxygen Species (ROS) is significantly increased after Ga(III) complexes treatment, while these complexes are redox-inactive. To investigate how Ga(III) complexes affects the levels of ROS, we have synthesized three bis-liganded gallium(III)-2-benzoylpyridine-thiosemicarbazones complexes and studied antitumor mechanisms of these complexes. The structures of Ga(III) complexes were identified by X−ray single crystal diffraction. Cytotoxicity studies have shown that ligands and gallium complexes have higher antitumor activity and lower cytotoxicity than normal cells. The most active complex is C3, the antitumor viability of which is better than its related ligands and anticancer agent 3−AP. It shows that Ga(III) complexes not only transmit the iron ions, but also induce intracellular Ca²⁺ to release, and then the standards of ROS in redox-active iron complexes will be increased. Cyt C releases from mitochondria which is lack of membrane potential, then activated Caspase family proteins induce cell apoptosis.
Co(II), Ni(II), and Cu(II) complexes with a Schiff base ligand pyridoxylidenetaurinate (L⁻) were prepared by a template reaction of pyridoxal (3-hydroxy-5-(hydroxymethyl)-2-methylpyridine-4-carbaldehyde), taurine (2-aminoethane-1-sulfonic acid), and the corresponding metal acetate in water–ethanol solution. Composition of the product was [ML2(H2O)2] for all three central metals. Coordination geometry varied from weakly distorted octahedral (Co, Ni) to strongly distorted octahedral (Cu) with trans arrangement of both water molecules and imine N atoms. Each bidentate ligand formed one chelate ring via iminic N and phenolic O donor atoms. The complexes were characterised by elemental analysis, FTIR spectroscopy, and XRD crystal and powder analyses. All complexes crystallize in monoclinic system with space group P21/c. IR spectra of the complexes showed changes typical for Schiff base coordination, comparing with IR spectrum of the ligand.
The aggressiveness of pancreatic cancer urgently requires more efficient treatment options. Because the sigma-2 (σ2) receptor was recently proposed as a promising target for pancreatic cancer therapy, we explored our previously developed multifunctional thiosemicarbazones, designed to synergistically impair cell energy levels, by targeting σ2 and P-gp proteins and chelating Iron. A deconstruction approach was herein applied by removing one function at a time from the potent multifunctional thiosemicarbazones 1 and 2, to investigate the contribution to cytotoxicity of each target involved. The results from in vitro (panel of pancreatic tumor cells) and in vivo experiments (C57BL/6 bearing KP02 tumor), suggest that while the multifunctional activity was not required for the antitumor activity of these thiosemicarbazones, σ2-targeting appeared to allow alternative tumor cell death mechanisms, leading to potent and less toxic off-targets toxicities compared to other thiosemicarbazones devoid of σ2-targeting.
This paper describes the preparation and characterization of polymeric nanoparticles loaded with a potent anti-tumor metal chelator, Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) for delivery to cancer cells. Metal chelators have been increasingly studied for their anti-cancer properties that rely on the high demand of neoplastic cells for iron. Dp44mT has previously shown great antiproliferative characteristics in several cancers including breast cancer and melanoma. To further expand the application of this highly cytotoxic agent for cancer treatment and to enable its specific delivery to malignant cells, here we apply nano-scale particles (NPs) of biodegradable poly(lactic-co-glycolide) (PLGA) for encapsulation of Dp44mT and evaluate its effectiveness in vitro. The results demonstrated that Dp44mT was efficiently encapsulated in PLGA particles. Resulting NPs were uniform in size and shape and had good colloidal stability. Moreover, Dp44mT encapsulation in PLGA enhanced the water solubility of this agent. Lastly, the present formulation showed high level of cytotoxicity in glioma cells. Together, these results show the potential of PLGA NPs as a nano-carrier for Dp44mT with no apparent impact on the anti-tumor activity of this compound.
Metals are vital cellular elements necessary for multiple indispensable biological processes of living organisms, including energy transduction and cell proliferation. Interestingly, alterations in metal levels and also changes in the expression of proteins involved in metal metabolism have been demonstrated in a variety of cancers. Considering this and the important role of metals for cell growth, the development of drugs that sequester metals have become an attractive target for the development of novel anti-cancer agents. Interest in this field has surged with the design and development of new generations of chelators of the thiosemicarbazone class. These ligands have shown potent anti-cancer and anti-metastatic activity in vitro and in vivo. Due to their efficacy and safe toxicological assessment, some of these agents have recently entered multi-center clinical trials as therapeutics for advanced and resistant tumors. This review highlights the role, and changes in homeostasis, of metals in cancer and emphasizes the pre-clinical development and clinical assessment of metal ion-binding agents, namely, thiosemicarbazones, as anti-tumor agents.
Multidrug resistance (MDR) mediated by P-glycoprotein (Pgp) represents a significant impediment to successful cancer treatment. The compound, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), has been shown to induce greater cytotoxicity against resistant cells than their non-resistant counterparts. Herein, the structure-activity relationships of selected thiosemicarbazones are explored and the novel mechanism underlying their ability to overcome resistance is further elucidated. Only thiosemicarbazones with electron-withdrawing substituents at the imine carbon mediated Pgp-dependent potentiated cytotoxicity, which was reversed by Pgp inhibition. Treatment of resistant cells with these thiosemicarbazones resulted in Pgp-dependent lysosomal membrane permeabilization (LMP) that relied on copper (Cu) chelation, reactive oxygen species generation and increased relative lipophilicity. Hence, this study is the first to demonstrate the structural requirements of these thiosemicarbazones necessary to overcome MDR. We also demonstrate the mechanism that enables the targeting of resistant tumors, whereby thiosemicarbazones “hijack” lysosomal Pgp and form redox-active Cu complexes that mediate LMP and potentiate cytotoxicity.
Copper is an essential trace metal required by organisms to perform a number of important biological processes. Copper readily cycles between its reduced Cu(i) and oxidised Cu(ii) states, which makes it redox active in biological systems. This redox-cycling propensity is vital for copper to act as a catalytic co-factor in enzymes. While copper is essential for normal physiology, enhanced copper levels in tumours leads to cancer progression. In particular, the stimulatory effect of copper on angiogenesis has been established in the last several decades. Additionally, it has been demonstrated that copper affects tumour growth and promotes metastasis. Based on the effects of copper on cancer progression, chelators that bind copper have been developed as anti-cancer agents. In fact, a novel class of thiosemicarbazone compounds, namely the di-2-pyridylketone thiosemicarbazones that bind copper, have shown great promise in terms of their anti-cancer activity. These agents have a unique mechanism of action, in which they form redox-active complexes with copper in the lysosomes of cancer cells. Furthermore, these agents are able to overcome P-glycoprotein (P-gp) mediated multi-drug resistance (MDR) and act as potent anti-oncogenic agents through their ability to up-regulate the metastasis suppressor protein, N-myc downstream regulated gene-1 (NDRG1). This review provides an overview of the metabolism and regulation of copper in normal physiology, followed by a discussion of the dysregulation of copper homeostasis in cancer and the effects of copper on cancer progression. Finally, recent advances in our understanding of the mechanisms of action of anti-cancer agents targeting copper are discussed.
Oxidative stress plays a role in the development of drug resistance in cancer cells. Cancer cells must constantly and rapidly adapt to changes in the tumor microenvironment, due to alterations in the availability of nutrients, such as glucose, oxygen and key transition metals (e.g., iron and copper). This nutrient flux is typically a consequence of rapid growth, poor vascularization and necrosis. It has been demonstrated that stress factors, such as hypoxia and glucose deprivation up-regulate master transcription factors, namely hypoxia inducible factor-1α (HIF-1α), which transcriptionally regulate the multi-drug resistance (MDR), transmembrane drug efflux transporter, P-glycoprotein (Pgp). Interestingly, in addition to the established role of plasma membrane Pgp in MDR, a new paradigm of intracellular resistance has emerged that is premised on the ability of lysosomal Pgp to transport cytotoxic agents into this organelle. This mechanism is enabled by the topological inversion of Pgp via endocytosis resulting in the transporter actively pumping agents into the lysosome. In this way, classical Pgp substrates, such as doxorubicin (DOX), can be actively transported into this organelle. Within the lysosome, DOX becomes protonated upon acidification of the lysosomal lumen, causing its accumulation. This mechanism efficiently traps DOX, preventing its cytotoxic interaction with nuclear DNA. This review discusses these effects and highlights a novel mechanism by which redox-active and protonatable Pgp substrates can utilize lysosomal Pgp to gain access to this compartment, resulting in catastrophic lysosomal membrane permeabilization and cell death. Hence, a key MDR mechanism that utilizes Pgp (the “gun”) to sequester protonatable drug substrates safely within lysosomes can be “turned on” MDR cancer cells to destroy them from within.
The focus of this research is on the study of a series of copper (II) benzoylpyridine thiosemicarbazone
complexes. Of the six benzoylpyridine thiosemicarbazone ligands used in this study, two
are reported for the first time; 2-benzoylpyridine tert-butyl thiosemicarbazone (BZP-tBTSC), and
2-benzoylpyridine benzyl thiosemicarbazone (BZP-BzTSC). Once characterized by NMR, melting
point, and MS, these mono-anionic tridentate ligands were then reacted with Cu2+ to form the new
square planar metal complexes [Cu(BZP-tBTSC)Cl] and [Cu(BZP-BzTSC)Cl]. All of the copper complexes
display marked inhibition of human topoisomerase IIα. The [Cu(BZP-tBTSC)Cl] complex
shows marked activity against human breast cancer cell lines.
Human hepatocellular carcinoma (HCC) is known to have a poor prognosis. Sorafenib, a molecular targeted drug, is most commonly used for HCC treatment. However, its effect on HCC is limited in clinical use and therefore new strategies regarding sorafenib treatment are required. Iron overload is known to be associated with progression of chronic hepatitis and increased risk of HCC. We previously reported that iron depletion inhibited cancer cell proliferation and conversely induced angiogenesis. Indeed iron depletion therapy including iron chelator needs to be combined with anti-angiogenic drug for its anti-cancer effect. Since sorafenib has an anti-angiogenic effect by its inhibitory targeting VEGFR, we hypothesized that sorafenib could complement the anti-cancer effect of iron depletion. We retrospectively analyzed the relationship between the efficacy of sorafenib and serum iron-related markers in clinical HCC patients. In clinical cases, overall survival was prolonged in total iron binding capacity (TIBC) high- and ferritin low-patients. This result suggested that the low iron-pooled patients, who could have a potential of more angiogenic properties in/around HCC tumors, could be adequate for sorafenib treatment. We determined the effect of sorafenib (Nexavar®) and/or deferasirox (EXJADE®) on cancer cell viability, and on cell signaling of human hepatocarcinoma HepG2 and HLE cells. Both iron depletion by deferasirox and sorafenib revealed insufficient cytotoxic effect by each monotherapy, however, on the basis of increased angiogenesis by iron depletion, the addition of deferasirox enhanced anti-proliferative effect of sorafenib. Deferasirox was confirmed to increase vascular endothelial growth factor (VEGF) secretion into cellular supernatants by ELISA analysis. In in vivo study sorafenib combined with deferasirox also enhanced sorafenib-induced apoptosis. These results suggested that sorafenib combined with deferasirox could be a novel combination chemotherapy for HCC.
A phase II trial of a single 5-day course of deferoxamine in 9 patients with neuroblastomas was completed. Within 2 days of completion of treatment responses were observed in 7 of 9 patients and there was no drug toxicity. These responses were a decrease in bone marrow infiltration and, in one patient, a measurable reduction in her tumor mass. We conclude that deferoxamine given as an 8-h i.v. infusion daily for 5 days at 150 mg/kg/day has antitumor activity.
Mouse NIH 3T3 cells were transformed to multidrug resistance with high-molecular-weight DNA from multidrug-resistant human
KB carcinoma cells. The patterns of cross resistance to colchicine, vinblastine, and doxorubicin hydrochloride (Adriamycin;
Adria Laboratories Inc.) of the human donor cell line and mouse recipients were similar. The multidrug-resistant human donor
cell line contains amplified sequences of the mdr1 gene which are expressed at high levels. Both primary and secondary NIH
3T3 transformants contained and expressed these amplified human mdr1 sequences. Amplification and expression of the human
mdr1 sequences and amplification of cotransferred human Alu sequences in the mouse cells correlated with the degree of multidrug
resistance. These data suggest that the mdr1 gene is likely to be responsible for multidrug resistance in cultured cells.
Numerous studies have suggested that iron (Fe) chelators such as desferrioxamine (DFO) may be useful antitumor agents (Blatt and Stitely, Cancer Res 47:1749, 1987; Becton and Bryles, Cancer Res 48:7189, 1988). Recent work with several analogues of the lipophilic Fe chelator, pyridoxal isonicotinoyl hydrazone (PIH), indicate that some of these ligands are considerably more efficient than DFO both in terms of their Fe chelation efficacy and at preventing 3H-thymidine incorporation by neuroblastoma (NB) cells (Richardson and Ponka, J Lab Clin Med 124:660, 1994). Considering this fact, the present study was designed to test the antiproliferative effect of a wide range of PIH analogues to identify the most active compounds. A total of 36 ligands have been examined that were synthesized by condensation of three types of aromatic aldehydes (pyridoxal, salicylaldehyde, and 2-hydroxy-1-naphthyladehyde) with a range of acid hydrazides. The effects of these chelators were assessed using the human NB cell line, SK-N-MC. Although PIH was far more effective than DFO at preventing Fe uptake from transferrin, it was less effective than DFO at preventing cellular proliferation (DFO ID50 = 22 mumol/L; PIH ID50 = 75 mumol/L). In contrast, 14 PIH analogues were far more efficient than DFO at preventing proliferation (ID50 = 1 to 7 mumol/L) and may have potential as antitumor agents. The most effective compounds were those hydrazones derived from 2-hydroxy-1-naphthylaldehyde. Most of the PIH analogues were considerably more effective than DFO at both preventing 59Fe uptake from 59Fe-transferrin and in mobilizing 59Fe from prelabeled NB cells. In addition, a linear relationship between Fe chelation efficacy and antiproliferative activity was found only for hydrazones derived from salicylaldehyde. Apart from gallium (Ga) nitrate having an antiproliferative effect by itself, this metal potentiated the antiproliferative effect of PIH but not that of DFO. Spectrophotometric studies showed that PIH could chelate Ga, and it can be suggested that, like the PIH-Fe complex that donates Fe to reticulocytes (Ponka et al, Biochim Biophys Acta 718:151, 1982), the PIH-Ga complex may efficiently bestow Ga to NB cells. The results suggest that analogues of PIH deserve further vigorous investigation because they may be useful therapeutic agents for the treatment of cancer.
Iron (Fe) is known to be necessary for cellular proliferation. Previous studies have suggested that neuroblastoma cells appear to be relatively sensitive to growth inhibition by a specific Fe chelator, deferrioxamine (DFO), in vitro. Also, DFO has been recently used for the treatment of neuroblastoma patients. In this paper we demonstrate that neuroblastoma cell proliferation in vitro is extremely sensitive to inhibition by DFO as compared to another cell line with almost identical growth kinetics. Neuroblastoma cells treated with DFO adapt appropriately to Fe chelation as measured by marked upregulation of transferrin receptor mRNA, increased functional transferrin receptor, and decreased cellular ferritin concentration. Further studies that quantitated cellular incorporation of 59Fe from added transferrin-59Fe in the presence of DFO indicated that neuroblastoma cells were more sensitive to inhibition of Fe incorporation by the chelator as compared to the other cell line. Neuroblastoma cells treated with DFO showed a consistent arrest in the G1 phase of the cell cycle. For cells taken from the "resting" state this block occurred before the vast majority of cells had entered S or G2-M phases of the cell cycle. Further evidence that neuroblastoma cells were arrested before the G1-S interface was provided when cells inhibited by DFO and released into aphidicolin exhibit arrest at the G1-S interface, whereas release from aphidicolin into DFO resulted in entry into S phase. Also, DFO-treated cells exhibited a decrease in both p34cdc2 immunoreactive protein as well as kinase activity. The results of these latter studies strongly indicate evidence for a Fe requirement for malignant cell proliferation before the onset of DNA synthesis. Our results also provide a basis for further studies that will better define a therapeutic approach to patients with neuroblastoma utilizing DFO treatment.
Cyclin D1 plays an important role in regulating the progression of cells through the G1 phase of the cell cycle. This gene is frequently overexpressed in human colon cancer. To address the role of cyclin D1 in growth control and tumorigenesis in this disease, we have overexpressed an antisense cyclin D1 cDNA construct in the human colon cancer cell line SW480E8, which expresses high levels of cyclin D1. The integration and expression of the antisense construct was verified by Southern and Northern blot analyses, respectively, and resulted in decreased expression of the cyclin D1 protein. This was associated with decreased levels of the Rb and p27Kip1 proteins. In addition, the hypophosphorylated form of Rb was increased in these cells. The SW480E8 antisense cyclin D1 cells displayed an increased doubling time, a decrease in saturation density, decreased plating efficiency and anchorage-independent growth, and a loss of tumorigenicity in nude mice. These findings provide direct evidence that increased expression of cyclin D1 in colon tumor cells contributes to their abnormal growth and tumorigenicity. The ability to revert the transformed phenotype of these cells with antisense cyclin D1 suggests that cyclin D1 or its associated cyclin-dependent kinase 4 may be useful targets in the therapy of colon cancer.
Neuroblastoma (NB) is a high risk tumor of childhood, and raised serum ferritin is an adverse prognostic factor. The hypothesis that iron chelation therapy impacts tumor status and patient prognosis through changes in iron metabolism has been systematically evaluated here in a xenograft model of human NB. One of two iron chelators was given in seven different regimens to nude mice xenografted s.c. with either IMR-32, an established cell line, or JBN-1, heterotransplanted directly from a patient. Nude mice (a total of 160 in 24 cohorts) were given: desferrioxamine (DFO) by s.c. bolus or reservoir; 1,2-dimethyl-3-hydroxypyridin-4-one (L1), i.p. or orally; or saline. Measurements of mean Hb and liver iron levels were compared with corresponding saline cohorts per regimen as well as for pooled cohorts per agent for both cell lines. For IMR-32 xenografts, significant differences in Hb were achieved with L1 (10.9 g/dl pooled versus 13.7 g/dl controls) and in liver iron with DFO and L1 (235 microg/g and 306 microg/g, respectively, versus 520 microg/g). For JBN-1, the pattern was similar. With L1, H6 was 10.2 g/dl and controls were 11.7 g/dl (individual DFO cohorts were also significant); liver iron with DFO was 303 microg/g, liver iron with L1 was 270 microg/g, and controls were 387 microg/g. Additional therapy prior to tumor injection (67 mice and 10 cohorts) did not increase the depletion. Despite documentation of iron depletion, no reductions in tumor engraftment, latency, or tumor size at end point were achieved in the chelator-treated mice, compared with controls populations. Accordingly, inclusion of these iron chelators in clinical trials for NB appears unwarranted.
Deferiprone is an orally active iron-chelating agent that is being evaluated as a treatment for iron overload in thalassemia major. Studies in an animal model showed that prolonged treatment is associated with a decline in the effectiveness of deferiprone and exacerbation of hepatic fibrosis.
Hepatic iron stores were determined yearly by chemical analysis of liver-biopsy specimens, magnetic susceptometry, or both. Three hepatopathologists who were unaware of the patients' clinical status, the time at which the specimens were obtained, and the iron content of the specimens examined 72 biopsy specimens from 19 patients treated with deferiprone for more than one year. For comparison, 48 liver-biopsy specimens obtained from 20 patients treated with parenteral deferoxamine for more than one year were similarly reviewed.
Of the 19 patients treated with deferiprone, 18 had received the drug continuously for a mean (+/-SE) of 4.6+/-0.3 years. At the final analysis, 7 of the 18 had hepatic iron concentrations of at least 80 micromol per gram of liver, wet weight (the value above which there is an increased risk of cardiac disease and early death in patients with thalassemia major). Of 19 patients in whom multiple biopsies were performed over a period of more than one year, 14 could be evaluated for progression of hepatic fibrosis; of the 20 deferoxamine-treated patients, 12 could be evaluated for progression. Five deferiprone-treated patients had progression of fibrosis, as compared with none of those given deferoxamine (P=0.04). By the life-table method, we estimated that the median time to progression of fibrosis was 3.2 years in deferiprone-treated patients. After adjustment for the initial hepatic iron concentration, the estimated odds of progression of fibrosis increased by a factor of 5.8 (95 percent confidence interval, 1.1 to 29.6) with each additional year of deferiprone treatment.
Deferiprone does not adequately control body iron burden in patients with thalassemia and may worsen hepatic fibrosis.
The widely prevailing view that the cyclin-dependent kinase inhibitors (CKIs) are solely negative regulators of cyclin-dependent kinases (CDKs) is challenged here by observations that normal up-regulation of cyclin D- CDK4 in mitogen-stimulated fibroblasts depends redundantly upon p21(Cip1) and p27(Kip1). Primary mouse embryonic fibroblasts that lack genes encoding both p21 and p27 fail to assemble detectable amounts of cyclin D-CDK complexes, express cyclin D proteins at much reduced levels, and are unable to efficiently direct cyclin D proteins to the cell nucleus. Restoration of CKI function reverses all three defects and thereby restores cyclin D activity to normal physiological levels. In the absence of both CKIs, the severe reduction in cyclin D-dependent kinase activity was well tolerated and had no overt effects on the cell cycle.
Patients with thalassemia major require lifelong chelation therapy to prevent iron-induced organ damage. The orally active chelator deferiprone has been proposed as an alternative for patients unable or unwilling to use deferoxamine. One report has concluded that deferiprone may worsen hepatic fibrosis in patients with thalassemia, whereas others have found no detrimental effect. A panel of 3 pathologists evaluated 112 coded liver biopsies obtained from 56 patients before and after deferiprone therapy. Fibrosis was scored with the Laennec and Ishak systems. The mean interval between liver biopsies was 3.1 years (range, 1.2-4.9 years). In 11 patients seronegative for hepatitis C, fibrosis scores before and after therapy were 1.12 +/- 1.07 and 0.97 +/- 0.84 (P =.42) with the use of the Ishak system, and 0.71 +/- 0.65 and 0.70 +/- 0.53 (P =.91) with the Laennec system. Among 45 patients seropositive for hepatitis C, fibrosis scores before and after therapy were 1.91 +/- 1.13 and 2.04 +/- 1.30 (P =.43) with the use of the Ishak system and 1.26 +/- 0.73 and 1.35 +/- 0.90 (P =.41) with the Laennec system. When the data set was limited to biopsies that each contained 6 or more portal tracts (31 patients), analysis still showed no significant change in fibrosis with time. With the use of the Laennec system, the fibrosis score did not increase by more than one level in any patients without hepatitis C; it increased by more than one level in 1 patient with hepatitis C; and it did not decrease by more than one level in any of the 56 patients. This analysis of the largest collection of liver biopsies reported to date in patients receiving deferiprone demonstrates no evidence of deferiprone-induced progression of hepatic fibrosis during long-term therapy.
Ewing sarcoma is the second most common bone tumor in childhood. Despite aggressive chemotherapy and radiotherapy strategies, the prognosis of patients with metastatic disease remains poor. We have recently reported that Ewing tumor cell proliferation was strongly inhibited by IFN-beta and to a lesser degree by IFN-alpha. Moreover, under IFN-beta treatment, some cell lines undergo apoptosis. Since the possibility of using IFNs for Ewing tumor treatments may be of interest, we have evaluated the efficacy of Hu-IFNs in a nude mice model of Ewing tumor xenografts. The results reported here show that human type I IFNs, Hu-IFN-alpha and Hu-IFN-beta impaired tumor xenograft take and displayed an anti-growth effect toward established xenografts. Furthermore, we have also shown that combined therapy with Hu-IFNs and ifosfamide (IFO), an alkylating agent widely used in high-dose chemotherapy of Ewing tumors, results in a strong antitumor effect. Pathological analysis showed that Hu-IFN-alpha/IFO and Hu-IFN-beta/IFO were characterized by a dramatic decrease in the mitotic index and marked necrosis, as well as extensive fibrosis associated with numerous calcifications. To our knowledge, this is the first demonstration of a potential antitumor effect of human type I IFNs and IFO on Ewing tumors, providing a rational foundation for a promising therapeutic approach to Ewing sarcoma.
Abramoff, M.D., Magelhaes, P.J., Ram, S.J. "Image Processing with ImageJ". Biophotonics International, volume 11, issue 7, pp. 36-42, 2004.
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic-helix-loop-helix-PAS transcription factor consisting of HIF-1 alpha and HIF-1 beta subunits. HIF-1 alpha expression and HIF-1 transcriptional activity increase exponentially as cellular O2 concentration is decreased. Several dozen target genes that are transactivated by HIF-1 have been identified, including those encoding erythropoietin, glucose transporters, glycolytic enzymes, and vascular endothelial growth factor. The products of these genes either increase O2 delivery or allow metabolic adaptation to reduced O2 availability. HIF-1 is required for cardiac and vascular development and embryonic survival. In fetal and postnatal life, HIF-1 is required for a variety of physiological responses to chronic hypoxia. HIF-1 expression is increased in tumor cells by multiple mechanisms and may mediate adaptation to hypoxia that is critical for tumor progression. HIF-1 thus appears to function as a master regulator of O2 homeostasis that plays essential roles in cellular and systemic physiology, development, and pathophysiology.
Although hypoxia (lack of oxygen in body tissues) is perhaps the most physiological inducer of the wild-type p53 gene, the mechanism of this induction is unknown. Cells may detect low oxygen levels through a haem-containing sensor protein. The hypoxic state can be mimicked by using cobalt chloride and the iron chelator desferrioxamine: like hypoxia, cobalt chloride and desferrioxamine activate hypoxia-inducible factor 1alpha (HIF-1alpha), which stimulates the transcription of several genes that are associated with hypoxia. Here we show that these treatments induce accumulation of wild-type p53 through HIF-1alpha-dependent stabilization of p53 protein. Induction of p53 does not occur in either a mutant hepatoma cell line that is unable to induce HIF-1alpha or embryonic stem cells derived from mice lacking HIF-1beta. HIF-1alpha is found in p53 immunoprecipitates from MCF7 cells that express wild-type p53 and are either hypoxic or have been exposed to desferrioxamine. Similarly, anti-haemagglutinin immunoprecipitates from lysates of normoxic PC3M cells that had been co-transfected with haemagglutinin-tagged HIF-1alpha and wild-type p53 also contain p53. Transfection of normoxic MCF7 cells with HIF-1alpha stimulates a co-transfected p53-dependent reporter plasmid and increases the amount of endogenous p53. Our results suggest that hypoxic induction of transcriptionally active wild-type p53 is achieved as a result of the stabilization of p53 by its association with HIF-1alpha.
Iron chelation therapy was initially designed to alleviate the toxic effects of excess iron evident in iron-overload diseases. However, some iron chelator-metal complexes have also gained interest due to their high redox activity and toxicological properties that have potential for cancer chemotherapy. This communication addresses the conflicting results published recently on the ability of the iron chelator, Dp44mT, to induce hydroxyl radical formation upon complexation with iron (B.B. Hasinoff and D. Patel, J Inorg. Biochem.103 (2009), 1093-1101). This previous study used EPR spin-trapping to show that Dp44mT-iron complexes were not able to generate hydroxyl radicals. Here, we demonstrate the opposite by using the same technique under very similar conditions to show the Dp44mT-iron complex is indeed redox-active and induces hydroxyl radical formation. This was studied directly in an iron(II)/H(2)O(2) reaction system or using a reducing iron(III)/ascorbate system implementing several different buffers at pH 7.4. The demonstration by EPR that the Dp44mT-iron complex is redox-active confirms our previous studies using cyclic voltammetry, ascorbate oxidation, benzoate hydroxylation and a plasmid DNA strand-break assay. We discuss the relevance of the redox activity to the biological effects of Dp44mT.
Iron is a critical nutrient for DNA synthesis and cellular proliferation. Targeting iron in cancer cells using specific chelators is a potential new strategy for the development of novel anticancer agents. One such chelator, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), possesses potent and selective anticancer activity (J Med Chem 50:3716-3729, 2007). To elucidate the mechanisms of its potent antitumor activity, Bp4eT was labeled with (14)C. Its efficacy was then compared with the (14)C-labeled iron chelator pyridoxal isonicotinoyl hydrazone (PIH), which exhibits low anticancer activity. The ability of these ligands to permeate the cell membrane and their cellular retention was examined under various conditions using SK-N-MC neuroepithelioma cells. The rate of [(14)C]PIH uptake into cells was significantly (p < 0.001) lower than that of [(14)C]Bp4eT at 37°C, indicating that the increased hydrophilicity of [(14)C]PIH reduced membrane permeability. In contrast, the efflux of [(14)C]PIH was significantly (p < 0.05) higher than that of [(14)C]Bp4eT, leading to increased cellular retention of [(14)C]Bp4eT. In addition, the uptake and release of the (14)C-labeled chelators was not reduced by metabolic inhibitors, indicating that these processes were energy-independent. No significant differences were evident in the uptake of [(14)C]Bp4eT at 37 or 4°C, demonstrating a temperature-independent mechanism. Furthermore, adjusting the pH of the culture medium to model the tumor microenvironment did not affect [(14)C]Bp4eT membrane transport. It can be concluded that [(14)C]Bp4eT more effectively permeated the cell membrane and evaded rapid efflux in contrast to [(14)C]PIH. This property, in part, accounts for the more potent anticancer activity of Bp4eT relative to PIH.
Iron chelators have potential therapeutic use for the treatment of cancer. Studies with a range of chelators indicate that Fe chelation alone is not enough to generate compounds with pronounced antitumor efficacy. Structure-activity relationships demonstrated that chelators containing hard electron donors such as oxygen typically lead to ligands that bind FeIII with high affinity that do not have pronounced antitumor efficacy. Such compounds aremore suitable for the treatment of iron-overload disease, e.g., β-thalassemia major. In contrast, ligands with soft donors such as sulfur and nitrogen lead to compounds that can redox cycle and induce a "double punch", namely, marked chelation and redox activity. Such compounds include the thiosemicarbazone chelators such as 3-AP and the ApT, BpT, and DpT series. Detailed investigations of the thiosemicarbazone group of ligands have demonstrated that they are highly effective chelators that, besides RR, also target a range of other molecules including NDRG1 and top2α, all of which contribute to their anticancer effects.
Melanotransferrin (MTf) is a transferrin homologue that binds iron (Fe) through a high affinity Fe-binding site. MTf has been implicated in diverse processes, e.g., iron metabolism, plasminogen activation, eosinophil differentiation and cancer cell migration, proliferation and tumourigenesis. Our previous studies using a knockout mouse demonstrated that MTf does not have an essential function in Fe metabolism (E.O. Sekyere, L.L. Dunn, Y.S. Rahmanto, D.R. Richardson, Role of melanotransferrin in iron metabolism: studies using targeted gene disruption in vivo, Blood 107 (2006) 2599-2601). However, it does play a role in melanoma cell proliferation and tumourigenesis. In this investigation, we report generation and characterization of transgenic mice bearing the MTf gene (MTf(Tg)) produced via lentiviral delivery. In MTf(Tg) mice, MTf mRNA and protein were hyper-expressed in tissues compared to control mice. These animals exhibited no gross morphological, histological, nor Fe status changes. The MTf(Tg) mice were also born in accordance with classical Mendelian ratios. However, hyper-expression of MTf leads to a mild, but significant decrease in erythrocyte count. This animal provides a novel MTf hyper-expression transgenic model for further investigating the biological function(s) of MTf.
Iron chelators have been reported to induce apoptosis and cell cycle arrest in cancer cells. Recent studies suggest broad and selective antitumor activity of the new iron chelator, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT; Whitnall et al., Proc Natl Acad Sci USA 2006;103:14901-14906). However, little is known concerning its effects on hematological malignancies. Using acute leukemia cells, the effect of Dp44mT on apoptosis, cell cycle, caspase-3 activation, and mitochondrial trans-membrane potential has been examined by flow cytometry. Dp44mT acted to induce a G(1)/S arrest in NB4 promyelocytic leukemia cells at low concentrations (0.5-2.5 microM), being far more effective than the clinically used chelator, desferrioxamine (DFO). Moreover, Dp44mT induced apoptosis of NB4 cells in a dose- and time-dependent manner with markedly less effect on nonproliferating cells. The apoptosis-inducing activity of Dp44mT was significantly more effective than DFO. Furthermore, this study also showed that Dp44mT had broad activity, inducing apoptosis in several types of acute leukemia and also multiple myeloma cell lines. Additional studies examining the cytotoxic mechanisms of Dp44mT showed that a reduction in the mitochondrial trans-membrane potential and caspase-3 activation could be involved in the mechanism of apoptosis. Our results suggest that Dp44mT possesses potential as an effective cytotoxic agent for the chemotherapeutic treatment of acute leukemia.
The liver is fundamentally important in drug metabolism. In oncology, the astute clinician must not only understand the meaning and limitations of commonly ordered liver biochemical tests, but also be aware of which anticancer agents might induce liver dysfunction, and of the strategies for appropriate dosing of patients with pre-existing liver dysfunction. In part I of our Review, we highlighted both the importance and inadequacies of identifying serum biochemical liver abnormalities in oncology; we also discussed a lack of routine formal investigation of liver function. We summarised chemotherapy-related hepatotoxicity and other causes of liver toxic effects in patients with cancer. Here in part II, we discuss trials that have specifically assessed chemotherapy dosing strategies in the setting of overt biochemical liver dysfunction and we note their recommendations. Furthermore, we review other assessments of liver metabolic and excretory function, particularly in the setting of chemotherapy drug handling. We discuss the potential use of these metabolic probes in practice.
The liver has a key role in the metabolism (ie, inactivation or activation) of many commonly used anticancer agents-cytotoxics or new biological agents. Therefore, assessment of liver function is a fundamental part of initial work-up and management of patients with cancer. An understanding of the meaning of conventional serum biochemical testing of liver function and status, what variables they are measuring, and usefulness for chemotherapy dosing is essential. Emerging awareness of the drawbacks of conventional serum biochemical testing and further understanding of the intricacies of liver function is leading to the development of alternative strategies for appropriate chemotherapy regimens and dosing. We present an overview of assessment of liver function and chemotherapy dosing. We consider the use of serum liver biochemical testing to predict liver function, potential causes of biochemical abnormalities in patients with cancer, and chemotherapy drugs that are associated with hepatotoxicity. Part II will overview the current knowledge surrounding chemotherapy dosing in the setting of liver dysfunction; as well as alternative tests of hepatic metabolic function that are beginning to be used as strategies for appropriate individualised chemotherapy administration.
Plasma concentrations of desferrioxamine and ferrioxamine were measured following bolus injections of desferrioxamine and during 24 h infusions of the drug. [59Fe]ferrioxamine clearance and urinary iron excretion were also measured.
Higher plasma ferrioxamine concentrations are found in iron loaded subjects and higher desferrioxamine concentrations in subjects with normal iron loads. There is a correlation between the circulating concentration of ferrioxamine during an infusion and the 48 h urinary iron excretion. The data suggests that the amount of iron chelated in vivo is related to an increase in the size of an intermediate chelatable pool rather than the total amount of the iron load. The well-recognized delay in urinary iron excretion appears to be related to active tubular reabsorption of ferrioxamine.
The effects of two different cell cycle inhibitors on the proliferation of human lymphoblastoid cells have been analyzed by flow cytometric techniques. Mimosine, a plant amino acid, reversibly blocks the cell cycle at a point which occurs roughly 2 h before the arrest mediated by aphidicolin, an inhibitor of DNA polymerase alpha activity, which defines the G1/S phase boundary. The levels of thymidine kinase mRNA, which increase at the onset of S phase, are higher in cells blocked with aphidicolin than in cells treated with mimosine whereas the opposite results are obtained in the case of p53 mRNA levels, which are known to be maximal in the late G1 phase. These results indicate that mimosine inhibits cell cycle traverse in the late G1 phase prior to the onset of DNA synthesis and identifies a previously undefined reversible cell cycle arrest point.
The role of the transferrin homologue, melanotransferrin (p97), in iron metabolism has been studied using the human melanoma cell line, SK-MEL-28, which expresses this antigen in high concentrations. The mechanisms of iron and transferrin uptake were investigated using human transferrin labelled with iodine-125 and iron-59. Internalised and membrane-bound iron and transferrin were separated using the proteinase, pronase. The uptake of iron from transferrin occurred by at least two processes. The first process was saturable and consistent with receptor-mediated endocytosis, involving internalisation of transferrin bound to specific binding sites. Uptake of iron also occurred by a second process which was non-saturable up to 0.06 mg/ml (0.75 microM) and was of higher efficiency than the saturable process. This process of iron uptake may be the dominant one at physiological serum transferrin concentrations. A membrane-bound, pronase-sensitive, temperature-dependent, iron-binding component was also identified. The number of binding sites was estimated to be approx. 340,000 per cell (assuming 2 atoms of iron per site) and it is suggested that this binding component may be melanotransferrin.
The basic components of erythropoiesis are stem cell precursors, their stimulation by erythropoietin, and an adequate supply of iron from which to make hemoglobin. This article discusses the effects of each of these and the simple tests by which they may be evaluated clinically.
We examined the short-term efficacy and toxicity of high doses of intravenous deferoxamine (DFO) in children with recurrent neuroblastoma. Ten children (3 2/12-20 years, median 6 5/12 years) had measurable recurrent disease following 1-3 prior treatment regimens. DFO (120-240 mg/kg/d) was planned as a continuous i.v. infusion for five days every other week. Serum ferritins at the start of this therapy ranged from 133-->5000 ng/ml (median 611 ng/ml). Of eight patients begun at a dose of 120-150 mg/kg/d, a single patient experienced visual disturbances which resolved after DFO was discontinued. Two patients begun at 240 mg/kg/d (with serum ferritins levels of 505 and 717 ng/ml) both experienced dose-limiting toxicity including lethargy, dizziness, blurred vision and leg cramps. Although decreases in serum ferritin levels of a least 10% were noted in 4 patients, there were no partial or complete response. DFO given at a dose of 150 mg/kg/d i.v. according to this schedule appears to be ineffective as a single agent against neuroblastoma. Starting doses of 240 mg/kg/d have unacceptable short-term toxicity.
Recognition of the central role of iron in the generation of toxic, oxygen-derived species through the Haber-Weiss reaction, the ability of desferrioxamine (DFX) to prevent the damage associated with free radical generation in reperfusion injury, and its inhibitory effect on cell proliferation by inactivation of the iron dependent enzyme ribonucleotide reductase, resulted in an increasing number of studies exploring the novel therapeutic applications of iron chelating drugs: (a) Animal models of reperfusion injury have shown that DFX is able to decrease post-anoxic damage to the brain and heart as manifested in decreased infarct size and improved functional recovery. Iron chelators may be particularly useful in improving the preservation of organs intended for transplantation such as the heart, lung or kidney. (b) Anthracycline cardiotoxicity is aggravated by iron and inhibited by iron chelators. Because the mechanism of its antineoplastic effect differs from its cardiotoxic effect, it is possible to inhibit anthracycline cardiotoxicity without interfering with therapeutic efficacy. In vivo and in vitro animal studies have yielded encouraging results but much additional experimental work is still required before iron chelating therapy may be advocated for use in patients on anthracycline therapy. (c) Cell proliferation can be inhibited by iron chelators through the reversible inhibition of ribonucleotide reductase, a rate-limiting enzyme in DNA synthesis. This may be exploited for the treatment of malignant disease, and preliminary studies have already shown that DFX in combination with multidrug chemotherapy is effective in controlling neuroblastoma and other tumours. However, the contribution of DF to the overall clinical effect is unclear. Prospective controlled clinical studies are required in order to establish whether the antiproliferative, or cell synchronizing properties of DFX may be of practical usefulness in the control of malignant disease. (d) Control of protozoal infection: Experimental in vivo and in vitro models have shown that malarial infection may be inhibited by iron chelating therapy. This useful effect of DFX and other iron chelators is most probably related to ribonucleotide reductase inhibition. Clinical studies of asymptomatic P. falciparum malaria and of cerebral malaria have shown both an accelerated rate of parasite clearance and earlier recovery from coma. These observations lend new meaning to the term 'nutritional immunity' and open new channels for exploring the possibility of controlling infection by means of selective intracellular iron deprivation. Experimental models for studying the effect of iron chelators on other intracellular pathogens such as Toxoplasma gondii, Chlamydia psittaci, or Mycobacterium tuberculosis should be established.(ABSTRACT TRUNCATED AT 400 WORDS)
Charles J. Sherr Howard Hughes Medical Institute Department of Tumor Cell Biology St. Jude Children’s Research Hospital 332 North Lauderdale Memphis, Tennessee 38104 Recent advances in our understanding of the cell division cycle are now tying the functions of Gl phase regulators to diverse processes involving signal transduction, differ- entiation, senescence, apoptosis, and malignant transfor- mation. What determines the rate of Gl phase progression, and how do cells integrate mitogenic and antiproliferative signals with the cell cycle machinery? Lessons From Budding Yeast In Saccharomyces cerevisiae, a single 34 kDa cyclin- dependent kinase (cdk) (p34cDCZB/cdc2, also known as cdkl) serves as a master controller of the cell cycle, assembling sequentially into active holoenzyme complexes with Gl, S phase, or mitotic cyclins temporally to direct distinct transitions (reviewed by Nasmyth, 1993; Reed, 1992). In the presence of appropriate nutrients, Gl cells that reach a critical size initiate DNA replication, form buds, and dupli- cate their spindle bodies in preparation for subsequent division. Gl cyclins (Clnl, Cln2, and Cln3) are required for these processes (Richardson et al., 1989) (see Figure I), and their overexpression contracts Gl phase and de- creases cell size. Cln3-Cdc28 is present throughout Gl, and its kinase activity appears necessary for the subse- quent transcriptional activation of the CLN7 and CLN2 genes (Tyers et al., 1993). In turn, the induced Clnl and Cln2 proteins associate with Cdc28, whose kinase activity further stimulates CLN7 and CLN2 transcription. CLN7 and CLN2 gene expression is controlled by a heterodimeric transcription factor composed of Swi4 and Swi6, and Cln- CdcPm Schwab and Nasmyth, 1993). The kinase activities of Clb-Cdc28 complexes are held in check by an inhibitory protein (p40sfc’) (Mendenhall, 1993) that accumulates early in Gl and is degraded shortly before S phase (Schwab et al., 1994). Phosphorylation of ~40~‘~’ by Clnl,Cln2-Cdc28 might trigger its ubiquitin- mediated degradation, thereby enabling the Cln-regulated kinases to control S phase entry indirectly. Haploid Gl phase cells can also undergo cell cycle ar- rest and mate to form diploids. Conjugation is provoked by pheromones (a and a factors), secreted by cells of oppo- site mating types, that trigger a receptor-mediated sig- naling pathway (serpentine receptor-heterotrimeric G
Deregulation of cell proliferation is a hallmark of neoplastic transformation. Alteration in growth control pathways must translate into changes in the cell-cycle regulatory machinery, but the mechanism by which this occurs is largely unknown. Compared with normal human fibroblasts, cells transformed with a variety of viral oncoproteins show striking changes in the subunit composition of the cyclin-dependent kinases (CDKs). In normal cells, CDKs exist predominantly in multiple quaternary complexes, each containing a CDK, cyclin, proliferating cell nuclear antigen and the p21 protein. However, in many transformed cells, proliferating cell nuclear antigen and p21 are lost from these multiprotein enzymes. Here we have investigated the significance of this phenomenon by molecular cloning of p21 and in vitro reconstitution of the quaternary cell-cycle kinase complexes. We find that p21 inhibits the activity of each member of the cyclin/CDK family. Furthermore, overexpression of p21 inhibits the proliferation of mammalian cells. Our results indicate that p21 may be a universal inhibitor of cyclin kinases.
In view of the high relapse rate following chemotherapy for patients with advanced neuroblastoma (NB) and primitive neuroectodermal tumors (PNET), we designed a novel chemotherapy program which incorporated the iron chelator deferoxamine. The purpose of the deferoxamine was to sensitize the cells to standard chemotherapy. The D-CECaT regimen contained (in mg/m2): deferoxamine 4500 during days 1-5; cyclophosphamide 600 mg over days 6 and 7; etoposide 300 mg over days 7 and 8; carboplatin 100 mg over days 7 and 8; and thiotepa 30 mg over days 6-8. Between October 1989 and May 1992 we entered 23 advanced NB and two PNET patients. Sepsis occurred in four courses, nausea and vomiting in 30 courses, and 50 courses required blood and platelets. Responses observed in previously untreated patients with stage III NB: six out of six CR (17+ to 41+ months), with stage IV NB, nine out of 11 CR (14+ to 28+ months), two out of 11 VGPR (22+ months), with stage IV PNET two out of two CR (1+ to 35+ months). With previously treated and failed stage IV NG, two out of six VGPR for 19+ and 20 months, and four out of six PR 1, 8, 9 and 11 months. Median survival for 19 new patients was 22+ months (6 to 41+ months; two patients in CR died at 7 months during adjuvant autologous marrow transplant). In conclusion, D-CECaT is an effective initial cytoreductive regimen for advanced stage NB/PNET patients. Additional patients and studies are required to determine its use as an alternative to autologous bone marrow transplantation.
As an essential nutrient and a potential toxin, iron poses an exquisite regulatory problem in biology and medicine. At the cellular level, the basic molecular framework for the regulation of iron uptake, storage, and utilization has been defined. Two cytoplasmic RNA-binding proteins, iron-regulatory protein-1 (IRP-1) and IRP-2, respond to changes in cellular iron availability and coordinate the expression of mRNAs that harbor IRP-binding sites, iron-responsive elements (IREs). Nitric oxide (NO) and oxidative stress in the form of H2O2 also signal to IRPs and thereby influence cellular iron metabolism. The recent discovery of two IRE-regulated mRNAs encoding enzymes of the mitochondrial citric acid cycle may represent the beginnings of elucidating regulatory coupling between iron and energy metabolism. In addition to providing insights into the regulation of iron metabolism and its connections with other cellular pathways, the IRE/IRP system has emerged as a prime example for the understanding of translational regulation and mRNA stability control. Finally, IRP-1 has highlighted an unexpected role for iron sulfur clusters as post-translational regulatory switches.
The growth and tumorigenicity of murine lung cancer cells transfected with an antisense cyclin D1 construct were evaluated in studies pertaining to mouse lung carcinogenesis. This antisense construct inhibited the expression of cyclin D in these cells, significantly reducing both their in vitro proliferation and tumorigenicity in nude mice relative to control cells. These data may have implications regarding the treatment of human neoplasms of aerodigestive tract origin that either overexpress the cyclin D oncogene or exhibit mutations that influence cell cycle progression via cyclin D-dependent mechanisms.
Iron-chelating therapy with deferoxamine in patients with thalassemia major has dramatically altered the prognosis of this previously fatal disease. The successes achieved with deferoxamine, as well as the limitations of this treatment, have stimulated the design of alternative strategies of iron-chelating therapy, including orally active iron chelators. The development of the most promising of these, deferiprone, has progressed rapidly over the last 5 years; data from several trials have provided direct and supportive evidence for its short-term efficacy. At the same time, the toxicity of this agent mandates a careful evaluation of the balance between risk and benefit of deferiprone in patients with thalassemia, in most of whom long-term deferoxamine is safe and efficacious therapy.
We have recently screened 36 analogues of the lipophilic iron (Fe) chelator, pyridoxal isonicotinoyl hydrazone (PIH), for their antiproliferative effect (Richardson et al, Blood 86:4295, 1995). Of these compounds, 1 chelator derived from salicylaldehyde benzoyl hydrazone (206) and 4 ligands derived from 2-hydroxy-1-naphthylaldehyde benzoyl hydrazone (308, 309, 311, and 315) showed pronounced antiproliferative activity, being far more effective than desferrioxamine (DFO). The present study was designed to investigate in detail the mechanism of action of these PIH analogues in a variety of neoplastic cell lines. This investigation showed that the analogues were far more active than DFO at inhibiting cellular proliferation and 3H-thymidine, 3H-leucine, and 3H-uridine incorporation. Additional experiments showed that, in contrast to DFO, the 5 analogues were potent at preventing 59Fe uptake from transferrin (Tf) and increasing 59Fe release from cells at concentrations as low as 10 micromol/L. Examination of the distribution of 59Fe in neoplastic cells using native polyacrylamide gel electrophoresis (PAGE)/59Fe-autoradiography showed that most of the 59Fe taken up from Tf was incorporated into ferritin, although 3 other previously unrecognized components (bands A, B, and C) were also identified. Band C comigrated with 59Fe-citrate and was chelated on incubation of neuroblastoma cells with DFO, PIH, or the PIH analogues, with this compartment being the main intracellular target of these ligands. Further work showed that the effects of the chelators at inducing characteristics consistent with apoptosis or necrosis were cell line-specific, and while DFO increased the percentage of cells in the G0/G1 phases in all cell types, the effect of analogue 311 on the cell cycle was variable depending on the cell line. This study provides further evidence for the potential use of these Fe chelators as anticancer agents.
The enzyme RR catalyzes the conversion of ribonucleoside diphosphates to their deoxyribonucleotide counterparts. RR is critical for the generation of the cytosine, adenine, and guanine deoxyribonucleotide 5'-triphosphate building blocks of DNA, which are present in cells as exceedingly small intracellular pools. Therefore, interference with the function of RR might well result in an agent with significant antineoplastic activity, particularly against rapidly proliferating tumor cells. HUr is the only inhibitor of RR in clinical usage; this agent, however, is a relatively poor inhibitor of the enzyme and has a short serum half-life. Consequently, HUr is a relatively weak anticancer agent. In an effort to develop a more potent inhibitor of RR with utility as an anticancer agent, we have synthesized 3-AP and demonstrated (a) potent inhibition of L1210 leukemia cells in vitro, (b) curative capacity for mice bearing the L1210 leukemia, (c) marked inhibition of RR, and (d) sensitivity of HUr-resistant cells to 3-AP. These findings collectively demonstrate the clinical potential of 3-AP as an antineoplastic agent.
Some chelators of the pyridoxal isonicotinoyl hydrazone class have antiproliferative activity that is far greater than desferrioxamine (DFO). In this study, DFO was compared with one of the most active chelators (311) on the expression of molecules that play key roles in cell-cycle control. This was vital for understanding the role of iron (Fe) in cell-cycle progression and for designing chelators to treat cancer. Incubating cells with DFO, and especially 311, resulted in a decrease in the hyperphosphorylated form of the retinoblastoma susceptibility gene product (pRb). Chelators also decreased cyclins D1, D2, and D3, which bind with cyclin-dependent kinase 4 (cdk4) to phosphorylate pRb. The levels of cdk2 also decreased after incubation with DFO, and especially 311, which may be important for explaining the decrease in hyperphosphorylated pRb. Cyclins A and B1 were also decreased after incubation with 311 and, to a lesser extent, DFO. In contrast, cyclin E levels increased. These effects were prevented by presaturating the chelators with Fe. In contrast to DFO and 311, the ribonucleotide reductase inhibitor hydroxyurea increased the expression of all cyclins. Hence, the effect of chelators on cyclin expression was not due to their ability to inhibit ribonucleotide reductase. Although chelators induced a marked increase in WAF1 and GADD45 mRNA transcripts, there was no appreciable increase in their protein levels. Failure to translate these cell-cycle inhibitors may contribute to dysregulation of the cell cycle after exposure to chelators. (Blood. 2001;98:842-850)
Transferrin receptor expression is controlled by the amount of iron required by the cell to maintain its metabolism and therefore tumor cells in a highly proliferative state have a high density of transferrin receptors. In this study, phosphorothioated antisense TfR oligonucleotides (TfR-ODna) targeted to the sequences of TfR mRNA including the AUG initiation codon and the control sense chain (TfR-ODns) were synthesized. The rate of cellular DNA synthesis was determined by [3H]-thymidine incorporation. Administering TfR-ODna to three morphologically distinct breast cancer cell lines (MCF-7, T47D, and MDA-MB-231) and a normal breast cell line (MCF-12A) caused specific inhibition of tumor cell growth. The IC50 (50% inhibition of DNA synthesis) of the TfR-ODna for the MCF-7, T47D and MDA-MB-231 cells were 0.5, 0.5, and 1.0 microM, respectively, whereas the MCF-12A normal breast cells were about 30 times (IC50 of 30 microM) less sensitive to TfR-ODna than the breast cancer cells. The cytotoxicity of the antisense TfR-ODna was 10 to 60 times greater than that of the sense chain (TfR-ODns). TfR mRNA and protein synthesis were demonstrated by RT-PCR and immunohistochemical staining, respectively. Approximately 50% inhibition of the expression of TfR mRNA was observed when breast cancer cells were treated with 1 microM antisense TfR ODNa for 72 hrs but 1 microM antisense only caused 14% inhibition in normal breast cells. The decreased cytotoxicity and inhibition of TfR gene expression when the tumor cells were treated with the same concentration (1 microM) of TfR-ODns demonstrated the specificity of the TfR-ODna for blocking the target TfR gene. The combined cytotoxicities to human breast tumor MCF-7 cells of the antisense TfR-ODna and the iron chelator deferoxamine (DFO) or the ribonucleotide reductase inhibitor hydroxyurea were observed in this study. IC50s (50% inhibition of DNA synthesis) for DFO and hydroxyurea individually were 0.3 microM and 250 microM, respectively. The CalcuSyn program was used to determine the combined effects among the agents and synergism (Combined Indexes (CI) < 1) were found with the following two combinations: TfR-ODna (0.007 microM to 0.15 microM) with DFO (0.15 microM to 5 microM) and TfR-ODna (0.007 microM to 0.15 microM) with hydroxyurea (50 microM to 800 microM). However, inhibition by TfR-ODns was not synergistic with either DFO or hydroxyurea. The synergistic effects on inhibition of DNA synthesis between TfR-ODna and DFO or hydroxyurea suggest that inhibition of breast cancer cell growth by TfR-ODna is produced by depletion of iron pools that are required for DNA synthesis in tumor cells. The fact that TfR-ODna specifically decreases cell viability and proliferation, and reduces TfR mRNA and protein expression in human breast carcinoma cells without affecting normal breast cells, suggests that the antisense oligonucleotide to the transferrin receptor may be a novel therapeutic approach in breast cancer.
Although with a few exceptions, most new anticancer agents are initially developed for intravenous use, oral treatment with anticancer agents is, if feasible, to be preferred, as this route of administration is convenient to patients, reduces administration costs and facilitates the use of more chronic treatment regimens. Recent studies have identified various physiological barriers limiting the oral absorption of anticancer drugs. Presently, several strategies are explored to alter the low and variable oral bioavailability of several important anticancer agents by taking advantage of an intentional interaction between anticancer agents and drugs that modulate active intestinal drug transporters or (intestinal) enzymes.
We previously demonstrated that 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) and other aroylhydrazone chelators possess potent antineoplastic activity because of their ability to bind iron (Fe). From these studies, we identified structural components of the hydrazones that provide antineoplastic activity, namely the salicylaldehyde and 2-hydroxy-1-naphthylaldehyde moieties. A related group of chelators known as the thiosemicarbazones also show pronounced antitumor activity because of their ability to inhibit ribonucleotide reductase. Considering this, we designed a new series of "hybrid ligands" by condensation of the aldehydes described above with a range of thiosemicarbazides. The parent compound of these ligands is 2-hydroxy-1-naphthylaldehyde thiosemicarbazone (NT). Of 8 NT analogues, 3 chelators, namely NT, N4mT (2-hydroxy-1-naphthylaldehyde-4-methyl-3-thiosemicarbazone), and N44mT (2-hydroxy-1-naphthylaldehyde-4,4-dimethyl-3-thiosemicarbazone), showed high antiproliferative activity against SK-N-MC neuroepithelioma cells (50% inhibitory concentration [IC(50)] = 0.5-1.5 microM). Indeed, their activity was significantly (P <.0001) greater than that of desferrioxamine (DFO) (IC(50) = 22 microM). We demonstrate that 311, a 311 analogue (311m), and several NT-series chelators have significantly (P <.001) greater antiproliferative activity against tumor cells than against a range of normal cell types. For example, the IC(50) values of NT and N4mT in SK-N-MC neuroepithelioma cells were 0.5 microM, whereas for fibroblasts the IC(50) values were greater than 25 microM. Further, the effect of one of the most potent chelators (311m) on preventing the growth of bone marrow stem cell cultures was far less than that of doxorubicin and similar to that of cisplatin. These studies support the further development of these chelators as antiproliferative agents.
Iron is suspected to be involved in the induction and/or progression of various human tumors. More particularly, we have previously shown that iron may be involved in the pathogenesis of Kaposi's sarcoma (KS). We have also shown that the iron chelator desferrioxamine (DFO) has a potent anti-KS activity in vitro, suggesting that it may represent a potential therapeutic approach for the treatment of KS. The present study was designed to investigate the effect of DFO on the growth of human KS xenografts in immunodeficient mice. Unexpectedly, we found that mice treated with DFO (400 mg/kg, 3 times weekly) (n = 30) exhibited a marked enhancement of tumor growth compared with control mice (n = 33) (230 +/- 134 mm(2) versus 143 +/- 70 mm p < 0.01). No enhancement of tumor growth was seen in mice treated with iron-saturated DFO. At least 2 findings suggest that this paradoxic pro-KS activity occurred independently of mice iron stores. First, treatment with DFO had only a marginal effect on ferritin and hematocrit levels. Second, induction of effective iron depletion by an iron-poor diet (6.7 mg iron/kg diet) (n = 23) did not have a deleterious effect on the growth of the KS xenografts. The lesions obtained from the DFO-treated animals exhibited a significantly decreased apoptotic index (p < 0.05), indicating that some antiapoptotic mechanism induced by DFO may be operating in vivo to favour tumor growth. In conclusion, our data show that DFO has a stimulatory effect on KS growth in immunodeficient mice, suggesting that this drug is not indicated in patients with KS.
Iron chelation therapy is the only therapeutic approach that leads to enhanced iron excretion in beta-thalassaemia major and other transfusion-dependent patients. Although desferrioxamine has been used in such treatment over the last three decades, it is not an ideal drug due to its poor oral availability. Consequently extensive research effort has been directed towards the identification of non-toxic, orally active iron chelators. An ideal candidate must possess a range of critical physicochemical and biological properties, such as high selectivity and affinity for iron(III), tightly controlled distribution and metabolic profiles and low toxicity. Unfortunately, hexadentate ligands are generally associated with poor oral bioavailability, whereas many tridentate and bidentate molecules are orally active. The tridentate triazoles have been investigated for clinical potential; they are readily absorbed from the gastrointestinal tract and promote iron excretion with high efficacy. In similar fashion, several bidentate hydroxypyridinones have been demonstrated to possess potential as oral chelating agents.
Iron (Fe) chelators are potential antitumor agents. Cellular Fe depletion results in a G1/S arrest but the precise molecular mechanisms involved remain unclear. Recent studies have shown that this process is complex with multiple cell cycle molecules being involved. We previously showed that Fe chelators such as 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) were far more potent antitumor agents than the clinically used ligand, desferrioxamine (DFO). To further characterize the effects of chelators on cell cycle arrest, we compared their activity with the DNA-damaging agents actinomycin D (Act D) and cisplatin (CP). These latter two compounds increase the expression of p53 and its target genes such as the universal cyclin-dependent kinase inhibitor, p21(CIP1/WAF1). Incubation of normal and neoplastic cells with all agents resulted in increased nuclear p53, the effect being pronounced for Act D and CP. As expected, both Act D and CP also markedly increased nuclear p21(CIP1/WAF1) protein levels, while DFO and 311 caused a significant (P<0.0004) decrease. This latter effect was surprising, as these chelators markedly increased mRNA levels of this molecule. Immunofluorescence studies showed that Act D and CP caused nuclear localization of p21(CIP1/WAF1). In contrast, the chelators prevented translation of p21(CIP1/WAF1). This did not appear to be due to a general effect of the chelators on preventing translation, as transferrin receptor 1 was markedly up-regulated 15- to 21-fold by DFO and 311. Combination of 311 with Act D or CP prevented translation of p21(CIP1/WAF1) and its nuclear localization observed with these DNA-damaging agents. Significantly, the effect of chelation on reducing nuclear p21(CIP1/WAF1) was reversed by the Fe donor ferric ammonium citrate, indicating that p21(CIP1/WAF1) translation was dependent on intracellular Fe levels. This study demonstrates that while Fe chelators markedly up-regulate the mRNA levels of p21(CIP1/WAF1) they paradoxically inhibit translation.
Whereas 40% of human breast carcinomas harbor mutations in the tumor suppressor protein p53, the use of tests demonstrating the presence of p53 mutations as a prognostic marker in breast cancer has not altered clinical management. Therefore, the search for new markers, especially among cell cycle-regulatory molecules, is a high priority, both in terms of prognostication and for identification of novel targets. p21 regulates the outcome of the p53 response to DNA damage, as might occur after administration of a chemotherapeutic agent, and we have shown that attenuation of p21 using an antisense oligodeoxynucleotide (ODN) inhibits cell proliferation in vitro and decreases growth of Met-1 mammary carcinomas in mice. In the current study, we extend this work to human cells and tissue. Three of eight human breast tumors that we obtained from a tissue bank show markedly increased p21 levels, variably staining in the nucleus and cytosol. All corresponding normal tissues were p21 negative. In the three p21-positive tumors, the phosphatidylinositol 3'-kinase-relevant signaling proteins p85 and PTEN were also increased. To investigate whether p21 is a feasible target for attenuation in human breast cancer, we investigated two human carcinoma cell lines. When transfected with antisense p21 ODN, both MCF7 and T47D breast cancer cells exhibit dose-dependent attenuation of p21 levels, associated with apoptosis in the absence of an additional apoptotic stimulus. Because p21 regulates the cellular repair response to damaged DNA, our work suggests that attenuation of p21 using our antisense p21 ODN may be effective in modulating the progression of breast cancer in either the presence or absence of combination chemotherapy and sets the stage for future clinical trials.
The cyclin kinase inhibitor p21, originally described as a universal inhibitor of cyclin-dependent kinases, has since been shown to have additional functions other than CDK inhibition. It is likely that a key role of p21 is to keep cells alive after DNA damage and subsequent p53 induction, in order for the cell to effect repairs. Thus, the increase in p21 seen in some cancers may impart these cells with a survival advantage. Here we discuss how this antiapoptotic aspect of p21 makes it an attractive target for cancer therapy; attenuation of p21 in malignant cells may subvert the normal repair process induced by DNA-damaging chemotherapeutic agents and thus make such drugs more effective.
The study was conducted to investigate the effects of a novel therapeutic approach, i.e. the combination of chemotherapy and immunotherapy, against a human prostate carcinoma xenograft. A topoisomerase I inhibitor, topotecan, and CpG-containing oligodeoxynucleotides (CpG-ODN) were combined. Athymic mice bearing the PC-3 human prostate carcinoma were treated with the maximum tolerated dose (MTD) of topotecan (3 weekly treatments) and with repeated treatments of CpG-ODN (40 and 20 microg/mouse); tumour growth and lethal toxicity were monitored. Topotecan effect on CpG-ODN-induced production of interleukin (IL) 12, interferon (IFN)-gamma and tumour necrosis factor-alpha was also assessed. Since topotecan pretreatment differentially influenced CpG-ODN-induced production of IL-12 and IFN-gamma, the antitumour effects of the two therapies were investigated in a sequential (full topotecan regimen followed by CpG-ODN) or in an alternating sequence (starting with CpG-ODN). Topotecan inhibited PC-3 tumour growth, inducing 95% tumour volume inhibition. All combined treatments resulted in a significant delay in tumour growth, compared to the effects in topotecan-treated mice (P<0.01, by analysis of tumour growth curves). The combination regimens were well tolerated, except for the alternating sequence of 40 microg CpG-ODN and topotecan, which resulted in three out of eight toxic deaths. This alternating sequence was highly toxic even when another cytotoxic drug (doxorubicin) was used in healthy mice. In conclusion, the combination of topotecan and CpG-ODN increased antitumour effects over chemotherapy alone in the growth of a human prostate carcinoma xenograft. Administration sequence was critical to the combination toxicity: the complete regimen of the cytotoxic drug followed by repeated administrations of the immunomodulator seemed the most promising for further investigations.
Aroylhydrazone and thiosemicarbazone iron (Fe) chelators have potent antitumor activity. The aim of the current study was to examine the antitumor effects and mechanisms of action of a novel series of Fe chelators, the di-2-pyridyl thiosemicarbazones. Of 7 new chelators synthesized, 4 showed pronounced antiproliferative effects. The most active chelator was Dp44mT, which had marked and selective antitumor activity-for example, an IC(50) of 0.03 microM in neuroepithelioma cells compared with more than 25 microM in mortal fibroblasts. Indeed, this antiproliferative activity was the greatest yet observed for an Fe chelator. Efficacy was greater than it was for the cytotoxic ligand 311 and comparable to that of the antitumor agent doxorubicin. Strikingly, Dp44mT significantly (P <.01) decreased tumor weight in mice to 47% of the weight in the control after only 5 days, whereas there was no marked change in animal weight or hematologic indices. Terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) staining demonstrated apoptosis in tumors taken from mice treated with Dp44mT. This chelator caused a marked increase of caspase-3 activity in murine Madison-109 (M109) cells. Caspase activation was at least partially mediated by the release of mitochondrial holo-cytochrome c (h-cytc) after incubation with Dp44mT. In conclusion, Dp44mT is a novel, highly effective antitumor agent in vitro and in vivo that induces apoptosis.
Iron (Fe) is critical for proliferation, but its precise role in cell cycle progression remains unclear. In this study, we examined the mechanisms involved by assessing the effects of Fe chelators on the expression of molecules that play key roles in this process. In initial studies, gene arrays were used to assess gene expression after incubating cells with 2 Fe chelators, namely, desferrioxamine (DFO) and 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), or the DNA-damaging agent, actinomycin D. From the genes assessed, only the N-myc downstream-regulated gene 1 (Ndrg1) was specifically up-regulated by Fe chelation. Although the function of Ndrg1 is unclear, previous studies showed it markedly slows tumor growth and acts as a potent metastasis suppressor. Incubation of cells with chelators markedly increased Ndrg1 mRNA and protein expression, but this was not found with their Fe complexes or when the Fe-binding site had been inactivated. Increased Ndrg1 expression following Fe chelation was related to the permeability and antiproliferative activity of chelators and could be reversed by Fe repletion. Moreover, Ndrg1 up-regulation after chelation occurred at the transcriptional level and was mediated by hypoxia inducible factor-1alpha (HIF-1alpha)-dependent and -independent mechanisms. Our investigation suggests Ndrg1 is a novel link between Fe metabolism and the control of proliferation.
beta-Thalassaemia major is an inherited blood disorder which is complicated by repeated blood transfusion and excessive gastrointestinal iron (Fe) absorption, which leads to toxic Fe overload. Current treatment using the chelator, desferrioxamine (DFO), is expensive and cumbersome since the drug requires long subcutaneous infusions and it is not orally active. A novel chelator, 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone (PCTH), was recently designed and shown to have high Fe chelation efficacy in vitro. The aim of this investigation was to examine the Fe chelation efficacy of PCTH in vitro implementing primary cultures of cardiomyocytes and in vivo using mice. We showed that PCTH was significantly (P<0.005) more effective than DFO at mobilising (59)Fe from prelabelled cardiomyocytes. Moreover, PCTH prevented the incorporation of (59)Fe into ferritin during Fe uptake from (59)Fe-labelled transferrin. These effects were important to assess as cardiac complications caused by Fe deposition are a major cause of death in beta-thalassaemia major patients. Further studies showed that PCTH was orally active and well tolerated by mice at doses ranging from 50 to 200 mg/kg, twice daily (bd), for 2 days. A dose-dependent increase in faecal (59)Fe excretion was observed in the PCTH-treated group. This level of Fe excretion at 200 mg/kg was similar to the same dose of the orally effective chelators, pyridoxal isonicotinoyl hydrazone (PIH) and deferiprone (L1). Effective Fe chelation in the liver by PCTH was shown via its ability to reduce ferritin-(59)Fe accumulation. Mice treated for 3 weeks with PCTH at doses of 50 and 100 mg/kg/bd showed no overt signs of toxicity as determined by weight loss and a range of biochemical and haematological indices. In subchronic Fe excretion studies over 3 weeks, PIH and PCTH at 75 mg/kg/bd for 5 days/week increased faecal (59)Fe excretion to 140% and 145% of the vehicle control, respectively. This study showed that PCTH was well tolerated at 100 mg/kg/bd and induced considerable Fe excretion by the oral route, suggesting its potential as a candidate to replace DFO.