Thiosemicarbazones reprogram pancreatic cancer bidirectional oncogenic signaling between cancer cells and stellate cells to suppress desmoplasia.

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 ß-catenin and YAP/TAZ signaling in PC cells. Suppression of bidirectional signaling between cell types presents a unique therapeutic opportunity.

Ascorbate-and iron-driven redox activity of Dp44mT and Emodin facilitates peroxidation of micelles and bicelles.

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 the ligand, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), or the potential ligand, Emodin, on Fe-catalyzed 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.

Parkinson's disease: Alterations in iron and redox biology as a key to unlock therapeutic strategies.

A plethora of studies indicate that iron metabolism is dysregulated in Parkinson's disease (PD). The literature reveals well-documented alterations consistent with established dogma, but also intriguing paradoxical observations requiring mechanistic dissection. An important fact is the iron loading in dopaminergic neurons of the substantia nigra pars compacta (SNpc), which are the cells primarily affected in PD. Assessment of these changes reveal increased expression of proteins critical for iron uptake, namely transferrin receptor 1 and the divalent metal transporter 1 (DMT1), and decreased expression of the iron exporter, ferroportin-1 (FPN1). Consistent with this is the activation of iron regulator protein (IRP) RNA-binding activity, which is an important regulator of iron homeostasis, with its activation indicating cytosolic iron deficiency. In fact, IRPs bind to iron-responsive elements (IREs) in the 3ꞌ untranslated region (UTR) of certain mRNAs to stabilize their half-life, while binding to the 5ꞌ UTR prevents translation. Iron loading of dopaminergic neurons in PD may occur through these mechanisms, leading to increased neuronal iron and iron-mediated reactive oxygen species (ROS) generation. The "gold standard" histological marker of PD, Lewy bodies, are mainly composed of α-synuclein, the expression of which is markedly increased in PD. Of note, an atypical IRE exists in the α-synuclein 5ꞌ UTR that may explain its up-regulation by increased iron. This dysregulation could be impacted by the unique autonomous pacemaking of dopaminergic neurons of the SNpc that engages L-type Ca+2 channels, which imparts a bioenergetic energy deficit and mitochondrial redox stress. This dysfunction could then drive alterations in iron trafficking that attempt to rescue energy deficits such as the increased iron uptake to provide iron for key electron transport proteins. Considering the increased iron-loading in PD brains, therapies utilizing limited iron chelation have shown success. Greater therapeutic advancements should be possible once the exact molecular pathways of iron processing are dissected.

The anti-tumor agent, Dp44mT, promotes nuclear translocation of TFEB via inhibition of the AMPK-mTORC1 axis.

Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and its analogues are potent anti-cancer agents through their ability to target lysosomes. Considering this, it was important to understand the mechanisms involved in the Dp44mT-mediated induction of autophagy and the role of 5'-adenosine monophosphate-activated protein kinase (AMPK) as a critical autophagic regulator. As such, this investigation examined AMPK's role in the regulation of the transcription factor EB (TFEB), which transcribes genes involved in autophagy and lysosome biosynthesis. For the first time, this study demonstrated that Dp44mT induces translocation of TFEB to the nucleus. Furthermore, Dp44mT-mediated nuclear translocation of TFEB was AMPK-dependent. Considering that: (1) the mammalian target of rapamycin complex 1 (mTORC1) plays an important role in the regulation of TFEB; and (2) that AMPK is a known regulator of mTORC1, this study also elucidated the mechanisms through which Dp44mT regulates nuclear translocation of TFEB via AMPK. Silencing AMPK led to increased mTOR phosphorylation, that activates mTORC1. Since Dp44mT inhibits mTORC1 in an AMPK-dependent manner through raptor phosphorylation, Dp44mT is demonstrated to regulate TFEB translocation through dual mechanisms: AMPK activation, which inhibits mTOR, and inhibition of mTORC1 via phosphorylation of raptor. Collectively, Dp44mT-mediated activation of AMPK plays a crucial role in lysosomal biogenesis and TFEB function. As Dp44mT potently chelates copper and iron that are crucial for tumor growth, these studies provide insight into the regulatory mechanisms involved in intracellular clearance and energy metabolism that occur upon alterations in metal ion homeostasis.

Antioxidant defense mechanisms and its dysfunctional regulation in the mitochondrial disease, Friedreich's ataxia.

Redox stress is associated with the pathogenesis of a wide variety of disease states. This can be amplified potentially through redox active iron deposits in oxidatively active organelles such as the mitochondrion. There are a number of disease states, including Friedreich's ataxia (FA) and sideroblastic anemia, where iron metabolism is dysregulated and leads to mitochondrial iron accumulation. Considering FA, which is due to the decreased expression of the mitochondrial protein, frataxin, this iron accumulation does not occur within protective storage proteins such as mitochondrial ferritin. Instead, it forms unbound biomineral aggregates composed of high spin iron(III), phosphorous and sulfur, which probably contributes to the observed redox stress. There is also a dysregulated response to the ensuing redox assault, as the master regulator of oxidative stress, nuclear factor erythroid 2-related factor-2 (Nrf2), demonstrates marked down-regulation. The dysfunctional response of Nrf2 in FA is due to multiple mechanisms including: (1) up-regulation of Keap1 that is involved in Nrf2 degradation; (2) activation of the nuclear Nrf2 export/degradation machinery via glycogen synthase kinase-3ß (Gsk3ß) signaling; and (3) inhibited nuclear translocation of Nrf2. More recently, increased microRNA (miRNA) 144 expression has been demonstrated to down-regulate Nrf2 in several disease states, including an animal model of FA. Other miRNAs have also demonstrated to be dysregulated upon frataxin depletion in vivo in humans and animal models of FA. Collectively, frataxin depletion results in multiple, complex responses that lead to detrimental redox effects that could contribute to the mechanisms involved in the pathogenesis of FA.

Treatment of dilated cardiomyopathy in a mouse model of Friedreich's ataxia using N-acetylcysteine and identification of alterations in microRNA expression that could be involved in its pathogenesis.

Deficient expression of the mitochondrial protein, frataxin, leads to a deadly cardiomyopathy. Our laboratory reported the master regulator of oxidative stress, nuclear factor erythroid 2-related factor-2 (Nrf2), demonstrates marked down-regulation after frataxin deletion in the heart. This was due, in part, to a pronounced increase in Keap1. To assess if this can be therapeutically targeted, cells were incubated with N-acetylcysteine (NAC), or buthionine sulfoximine (BSO), which increases or decreases glutathione (GSH), respectively, or the NRF2-inducer, sulforaphane (SFN). While SFN significantly (p < 0.05) induced NRF2, KEAP1 and BACH1, NAC attenuated SFN-induced NRF2, KEAP1 and BACH1. The down-regulation of KEAP1 by NAC was of interest, as Keap1 is markedly increased in the MCK conditional frataxin knockout (MCK KO) mouse model and this could lead to the decreased Nrf2 levels. Considering this, MCK KO mice were treated with i.p. NAC (500- or 1500-mg/kg, 5 days/week for 5-weeks) and demonstrated slightly less (p > 0.05) body weight loss versus the vehicle-treated KO. However, NAC did not rescue the cardiomyopathy. To additionally examine the dys-regulation of Nrf2 upon frataxin deletion, studies assessed the role of microRNA (miRNA) in this process. In MCK KO mice, miR-144 was up-regulated, which down-regulates Nrf2. Furthermore, miRNA screening in MCK KO mice demonstrated 23 miRNAs from 756 screened were significantly (p < 0.05) altered in KOs versus WT littermates. Of these, miR-21*, miR-34c*, and miR-200c, demonstrated marked alterations, with functional clustering analysis showing they regulate genes linked to cardiac hypertrophy, cardiomyopathy, and oxidative stress, respectively.

During mitosis ZEB1 "switches" from being a chromatin-bound epithelial gene repressor, to become a microtubule-associated protein.

Microtubules are polymers of α/ß-tubulin, with microtubule organization being regulated by microtubule-associated proteins (MAPs). Herein, we describe a novel role for the epithelial gene repressor, zinc finger E-box-binding homeobox 1 (ZEB1), that "switches" from a chromatin-associated protein during interphase, to a MAP that associates with α-, ß- and γ-tubulin during mitosis. Additionally, ZEB1 was also demonstrated to associate with γ-tubulin at the microtubule organizing center (MTOC). Using confocal microscopy, ZEB1 localization was predominantly nuclear during interphase, with α/ß-tubulin being primarily cytoplasmic and the association between these proteins being minimal. However, during the stages of mitosis, ZEB1 co-localization with α-, ß-, and γ-tubulin was significantly increased, with the association commonly peaking during metaphase in multiple tumor cell-types. ZEB1 was also observed to accumulate in the cleavage furrow during cytokinesis. The increased interaction between ZEB1 and α-tubulin during mitosis was also confirmed using the proximity ligation assay. In contrast to ZEB1, its paralog ZEB2, was mainly perinuclear and cytoplasmic during interphase, showing some co-localization with α-tubulin during mitosis. Considering the association between ZEB1 with α/ß/γ-tubulin during mitosis, studies investigated ZEB1's role in the cell cycle. Silencing ZEB1 resulted in a G2-M arrest, which could be mediated by the up-regulation of p21Waf1/Cip1 and p27Kip1 that are known downstream targets repressed by ZEB1. However, it cannot be excluded the G2/M arrest observed after ZEB1 silencing is not due to its roles as a MAP. Collectively, ZEB1 plays a role as a MAP during mitosis and could be functionally involved in this process.

Regulation of autophagy and apoptosis by Dp44mT-mediated activation of AMPK in pancreatic cancer cells.

Upon activation, the 5'-adenosine monophosphate-activated protein kinase (AMPK) increases catabolism, while inhibiting anabolism. The anti-cancer agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), activates AMPK in multiple tumor cell-types (Biochim. Biophys Acta 2016;1863:2916-2933). This acts as an initial cell "rescue response" after iron-depletion mediated by Dp44mT. Considering Dp44mT-mediated AMPK activation, the role of AMPK on Dp44mT cytotoxicity was examined. Dp44mT increased the p-AMPK/AMPK ratio in multiple tumor cell-types over short (24 h) and longer (72 h) incubations. Notably, Dp44mT was more effective in inhibiting tumor cell proliferation after AMPK silencing, potentially due to the loss of AMPK-mediated metabolic plasticity that protects cells against Dp44mT cytotoxicity. The silencing of AMPK-increased cellular cholesterol and stabilized lysosomes against Dp44mT-mediated lysosomal membrane permeabilization. This was substantiated by studies demonstrating that the cholesterol-depleting agent, methyl-ß-cyclodextrin (MßCD), restores Dp44mT-mediated lysosomal membrane permeabilization in AMPK silenced cells. The increased levels of cholesterol after AMPK silencing were independent of the ability of AMPK to inhibit the rate-limiting step of cholesterol synthesis via the inactivating phosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) at Ser872. In fact, Dp44mT did not increase phosphorylation of HMGCR at (Ser872), but decreased total HMGCR expression similarly in both the presence or absence of AMPK silencing. Dp44mT was demonstrated to increase autophagic initiation after AMPK silencing via an AMPK- and Beclin-1-independent mechanism. Further, there was increased cleaved caspase 3 and cleaved PARP after incubation of AMPK silenced cells with Dp44mT. Overall, AMPK silencing promotes Dp44mT anti-proliferative activity, suggesting a role for AMPK in rescuing its cytotoxicity by inhibiting autophagy and also apoptosis.

The metastasis suppressor, NDRG1, differentially modulates the endoplasmic reticulum stress response.

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), is a stress response protein that is involved in the inhibition of multiple oncogenic signaling pathways. Initial studies have linked NDRG1 and the endoplasmic reticulum (ER) stress response. Considering this, we extensively examined the mechanism by which NDRG1 regulates the ER stress response in pancreatic and colon cancer cells. We also examined the anti-cancer agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), which induces NDRG1 expression and causes ER stress. The expression of NDRG1 was demonstrated to regulate the three main arms of the ER stress response by: (1) increasing the expression of three major ER chaperones, binding immunoglobulin protein (BiP), calreticulin, and calnexin; (2) suppressing the protein kinase, RNA-activated (PKR)-like ER kinase (PERK); (3) inhibiting the inositol-requiring kinase 1α (IRE1α) arm; and (4) increasing the cleavage of activating transcription factor 6 (ATF6). An important finding was that NDRG1 enhances the anti-proliferative and anti-migratory activity of Dp44mT. This increased efficacy could be related to the following effects in the presence of Dp44mT and NDRG1, namely: markedly increased activation of the PERK target, eukaryotic translation initiation factor 2α (eIF2α); the maintenance of activating transcription factor 4 (ATF4) expression; high cytosolic Ca+2 that increases the sensitivity of cells to apoptosis via activation of the calmodulin-dependent kinase II (CaMKII) signaling cascade; and increased pro-apoptotic C/EBP-homologous protein (CHOP) expression. Collectively, this investigation dissects the molecular mechanisms through which NDRG1 manipulates the ER stress response and its ability to potentiate the activity of the potent anti-cancer agent, Dp44mT.

Regulation and control of nitric oxide (NO) in macrophages: Protecting the "professional killer cell" from its own cytotoxic arsenal via MRP1 and GSTP1.

We recently demonstrated that a novel storage and transport mechanism for nitric oxide (NO) mediated by glutathione-S-transferase P1 (GSTP1) and multidrug resistance protein 1 (MRP1/ABCC1), protects M1-macrophage (M1-MØ) models from large quantities of endogenous NO. This system stores and transports NO as dinitrosyl-dithiol-iron complexes (DNICs) composed of iron, NO and glutathione (GSH). Hence, this gas with contrasting anti- and pro-tumor effects, which has been assumed to be freely diffusible, is a tightly-regulated species in M1-MØs. These control systems prevent NO cytotoxicity and may be responsible for delivering cytotoxic NO as DNICs via MRP1 from M1-MØs, to tumor cell targets.

Cellular iron uptake, trafficking and metabolism: Key molecules and mechanisms and their roles in disease.

Iron is a crucial transition metal for virtually all life. Two major destinations of iron within mammalian cells are the cytosolic iron-storage protein, ferritin, and mitochondria. In mitochondria, iron is utilized in critical anabolic pathways, including: iron-storage in mitochondrial ferritin, heme synthesis, and iron-sulfur cluster (ISC) biogenesis. Although the pathways involved in ISC synthesis in the mitochondria and cytosol have begun to be characterized, many crucial details remain unknown. In this review, we discuss major aspects of the journey of iron from its initial cellular uptake, its modes of trafficking within cells, to an overview of its downstream utilization in the cytoplasm and within mitochondria. The understanding of mitochondrial iron processing and its communication with other organelles/subcellular locations, such as the cytosol, has been elucidated by the analysis of certain diseases e.g., Friedreich's ataxia. Increased knowledge of the molecules and their mechanisms of action in iron processing pathways (e.g., ISC biogenesis) will shape the investigation of iron metabolism in human health and disease.

Glucose modulation induces reactive oxygen species and increases P-glycoprotein-mediated multidrug resistance to chemotherapeutics.

BACKGROUND AND PURPOSE: Cancer cells develop resistance to stress induced by chemotherapy. In tumours, a considerable glucose gradient exists, resulting in stress. Notably, hypoxia-inducible factor-1 (HIF-1) is a redox-sensitive transcription factor that regulates P-glycoprotein (Pgp), a crucial drug-efflux transporter involved in multidrug resistance (MDR). Here, we investigated how glucose levels regulate Pgp-mediated drug transport and resistance. EXPERIMENTAL APPROACH: Human tumour cells (KB31, KBV1, A549 and DMS-53) were incubated under glucose starvation to hyperglycaemic conditions. Flow cytometry assessed reactive oxygen species (ROS) generation and Pgp activity. HIF-1α, NF-κB and Pgp expression were assessed by reverse transcriptase-PCR and Western blotting. Fluorescence microscopy examined p65 distribution and a luciferase-reporter assay assessed HIF-1 promoter-binding activity. The effect of glucose-induced stress on Pgp-mediated drug resistance was examined after incubating cells with the chemotherapeutic and Pgp substrate, doxorubicin (DOX), and performing MTT assays validated by viable cell counts. KEY RESULTS: Changes in glucose levels markedly enhanced cellular ROS and conferred Pgp-mediated drug resistance. Low and high glucose levels increased (i) ROS generation via NADPH oxidase 4 and mitochondrial membrane destabilization; (ii) HIF-1 activity; (iii) nuclear translocation of the NF-κB p65 subunit; and (iv) HIF-1α mRNA and protein levels. Increased HIF-1α could also be due to decreased prolyl hydroxylase protein under these conditions. The HIF-1α target, Pgp, was up-regulated at low and high glucose levels, which led to lower cellular accumulation of Pgp substrate, rhodamine123, and greater resistance to DOX. CONCLUSIONS AND IMPLICATIONS: As tumour cells become glucose-deprived or exposed to high glucose levels, this increases stress, leading to a more aggressive MDR phenotype via up-regulation of Pgp.

Glutathione S-transferase and MRP1 form an integrated system involved in the storage and transport of dinitrosyl-dithiolato iron complexes in cells.

Nitrogen monoxide (NO) is vital for many essential biological processes as a messenger and effector molecule. The physiological importance of NO is the result of its high affinity for iron in the active sites of proteins such as guanylate cyclase. Indeed, NO possesses a rich coordination chemistry with iron and the formation of dinitrosyl-dithiolato iron complexes (DNICs) is well documented. In mammals, NO generated by cytotoxic activated macrophages has been reported to play a role as a cytotoxic effector against tumor cells by binding and releasing intracellular iron. Studies from our laboratory have shown that two proteins traditionally involved in drug resistance, namely multidrug-resistance protein 1 and glutathione S-transferase, play critical roles in intracellular NO transport and storage through their interaction with DNICs (R.N. Watts et al., Proc. Natl. Acad. Sci. USA 103:7670-7675, 2006; H. Lok et al., J. Biol. Chem. 287:607-618, 2012). Notably, DNICs are present at high concentrations in cells and are biologically available. These complexes have a markedly longer half-life than free NO, making them an ideal "common currency" for this messenger molecule. Considering the many critical roles NO plays in health and disease, a better understanding of its intracellular trafficking mechanisms will be vital for the development of new therapeutics.

The lure of a LYR: The logistics of iron sulfur cluster delivery.

How are nascent iron-sulfur (Fe-S) clusters directed to specific recipient proteins? In this issue of Cell Metabolism, Maio et al. (2014) show that the mitochondrial Fe-S cochaperone protein HSC20 guides nascent Fe-S clusters based on a highly conserved motif, LYR, that exists in target proteins in different molecular contexts.

Fixing frataxin: 'ironing out' the metabolic defect in Friedreich's ataxia.

The metabolically active and redox-active mitochondrion appears to play a major role in the cellular metabolism of the transition metal, iron. Frataxin, a mitochondrial matrix protein, has been identified as playing a key role in the iron metabolism of this organelle due to its iron-binding properties and is known to be essential for iron-sulphur cluster formation. However, the precise function of frataxin remains elusive. The decrease in frataxin expression, as seen in the inherited disorder Friedreich's ataxia, markedly alters cellular and mitochondrial iron metabolism in both the mitochondrion and the cell. The resulting dysregulation of iron trafficking damages affects tissues leading to neuro- and cardiodegeneration. This disease underscores the importance of iron homeostasis in the redox-active environment of the mitochondrion and the molecular players involved. Unravelling the mechanisms of altered iron metabolism in Friedreich's ataxia will help elucidate a biochemical function for frataxin. Consequently, this will enable the development of more effective and rationally designed treatments. This review will focus on the emerging function of frataxin in relation to the observed alterations in mitochondrial iron metabolism in Friedreich's ataxia. Tissue-specific alterations due to frataxin loss will also be discussed, as well as current and emerging therapeutic strategies.

Dp44mT targets the AKT, TGF-ß and ERK pathways via the metastasis suppressor NDRG1 in normal prostate epithelial cells and prostate cancer cells.

BACKGROUND: Effective treatment of prostate cancer should be based on targeting interactions between tumour cell signalling pathways and key converging downstream effectors. Here, we determined how the tumourigenic phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), tumour-suppressive phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and transforming growth factor-ß (TGF-ß) pathways are integrated via the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1). Moreover, we assessed how the novel anti-tumour agent, Dp44mT, may target these integrated pathways by increasing NDRG1 expression. METHODS: Protein expression in Dp44mT-treated normal human prostate epithelial cells and prostate cancer cells (PC-3, DU145) was assessed by western blotting. The role of NDRG1 was examined by transfection using an NDRG1 overexpression vector or shRNA. RESULTS: Dp44mT increased levels of tumour-suppressive PTEN, and decreased phosphorylation of ERK1/2 and SMAD2L, which are regulated by oncogenic Ras/MAPK signalling. Importantly, the effects of Dp44mT on NDRG1 and p-SMAD2L expression were more marked in prostate cancer cells than normal prostate epithelial cells. This may partly explain the anti-tumour selectivity of these agents. Silencing NDRG1 expression increased phosphorylation of tumourigenic AKT, ERK1/2 and SMAD2L and decreased PTEN levels, whereas NDRG1 overexpression induced the opposite effect. Furthermore, NDRG1 silencing significantly reduced the ability of Dp44mT to suppress p-SMAD2L and p-ERK1/2 levels. CONCLUSION: NDRG1 has an important role in mediating the tumour-suppressive effects of Dp44mT in prostate cancer via selective targeting of the PI3K/AKT, TGF-ß and ERK pathways.

Endoplasmic reticulum protein 29 regulates epithelial cell integrity during the mesenchymal-epithelial transition in breast cancer cells.

The epithelial-mesenchymal transition (EMT) correlates with disruption of cell-cell adhesion, loss of cell polarity and development of epithelial cell malignancy. Identifying novel molecules that inhibit EMT has profound potential for developing mechanism-based therapeutics. We previously demonstrated that the endoplasmic reticulum protein 29 (ERp29) is a novel factor that can drive mesenchymal-epithelial transition (MET) and induce cell growth arrest in MDA-MB-231 cells. Here, we show that ERp29 is an important molecule in establishing epithelial cell integrity during the MET. We demonstrate that ERp29 regulates MET in a cell context-dependent manner. ERp29 overexpression induced a complete MET in mesenchymal MDA-MB-231 cells through downregulating the expression of transcriptional repressors (for example, Slug, Snai1, ZEB2 and Twist) of E-cadherin. In contrast, overexpression of ERp29 induces incomplete MET in basal-like BT549 cells in which the expression of EMT-related markers (for example, vimentin; cytokeratin 19 (CK19) and E-cadherin) and the transcriptional repressors of E-cadherin were not altered. However, ERp29 overexpression in both cell-types resulted in loss of filamentous stress fibers, formation of cortical actin and restoration of an epithelial phenotype. Mechanistic studies revealed that overexpression of ERp29 in both cell-types upregulated the expression of TJ proteins (zonula-occludens-1 (ZO-1) and occludin) and the core apical-basal polarity proteins (Par3 and Scribble) at the membrane to enhance cell-cell contact and cell polarization. Knockdown of ERp29 in the epithelial MCF-7 cells decreased the expression of these proteins, leading to the disruption of cell-cell adhesion. Taken together, ERp29 is a novel molecule that regulates MET and epithelial cell integrity in breast cancer cells.

Deferasirox (ICL670A) effectively inhibits oesophageal cancer growth in vitro and in vivo.

BACKGROUND AND PURPOSE: Growing evidence implicates iron in the aetiology of gastrointestinal cancer. Furthermore, studies demonstrate that iron chelators possess potent anti-tumour activity, although whether iron chelators show activity against oesophageal cancer is not known. EXPERIMENTAL APPROACH: The effect of the iron chelators, deferoxamine (DFO) and deferasirox, on cellular iron metabolism, viability and proliferation was assessed in two oesophageal adenocarcinoma cell lines, OE33 and OE19, and the squamous oesophageal cell line, OE21. A murine xenograft model was employed to assess the effect of deferasirox on oesophageal tumour burden. The ability of chelators to overcome chemoresistance and to enhance the efficacy of standard chemotherapeutic agents (cisplatin, fluorouracil and epirubicin) was also assessed. KEY RESULTS: Deferasirox and DFO effectively inhibited cellular iron acquisition and promoted intracellular iron mobilization. The resulting reduction in cellular iron levels was reflected by increased transferrin receptor 1 expression and reduced cellular viability and proliferation. Treating oesophageal tumour cell lines with an iron chelator in addition to a standard chemotherapeutic agent resulted in a reduction in cellular viability and proliferation compared with the chemotherapeutic agent alone. Both DFO and deferasirox were able to overcome cisplatin resistance. Furthermore, in human xenograft models, deferasirox was able to significantly suppress tumour growth, which was associated with decreased tumour iron levels. CONCLUSIONS AND IMPLICATIONS: The clinically established iron chelators, DFO and deferasirox, effectively deplete iron from oesophageal tumour cells, resulting in growth suppression. These data provide a platform for assessing the utility of these chelators in the treatment of oesophageal cancer patients.

Iron chelators for the treatment of cancer.

The study of iron chelators as anti-tumor agents is still in its infancy. Iron is important for cellular proliferation and this is demonstrated by observations that iron-depletion results in cell cycle arrest and also apoptosis. In addition, many iron chelators are known to inhibit ribonucleotide reductase, the iron-containing enzyme that is the rate-limiting step for DNA synthesis. Desferrioxamine is a well known chelator used for the treatment of iron-overload disease, but it has also been shown to possess anti-cancer activity. Another class of chelators, namely the thiosemicarbazones, have been shown to possess anti-cancer activity since the 1950's, although their mechanism(s) of action have only recently been more comprehensively elucidated. In fact, the redox activity of thiosemicarbazone iron complexes is thought to be important in mediating their potent cytotoxicity. Moreover, unlike typical iron chelators which simply act to deplete tumors of iron, several thiosemicarbazones (i.e., Bp44mT and Dp44mT) do not induce this effect, their anti-cancer efficacy being due to other mechanisms e.g., redox activity. Other reports have also shown that some thiosemicarbazones inhibit topoisomerase IIα, demonstrating that this class of agents have multiple molecular targets and act by various mechanisms. The most well characterized thiosemicarbazone iron chelator in terms of its assessment in humans is 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP). Observations from these clinical trials highlight the less than optimal activity of this ligand and several side effects related to its use, including myelo-suppression, hypoxia and methemoglobinemia. The mechanisms responsible for these latter effects must be elucidated and the design of the ligand altered to minimize these problems and increase efficacy. This review discusses the development of chelators as unique agents for cancer treatment.

Bp44mT: an orally active iron chelator of the thiosemicarbazone class with potent anti-tumour efficacy.

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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. CONCLUSIONS AND IMPLICATIONS: 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.