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.

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.

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.

Cancer cell iron metabolism and the development of potent iron chelators as anti-tumour agents.

Cancer contributes to 50% of deaths worldwide and new anti-tumour therapeutics with novel mechanisms of actions are essential to develop. Metabolic inhibitors represent an important class of anti-tumour agents and for many years, agents targeting the nutrient folate were developed for the treatment of cancer. This is because of the critical need of this factor for DNA synthesis. Similarly to folate, Fe is an essential cellular nutrient that is critical for DNA synthesis. However, in contrast to folate, there has been limited effort applied to specifically design and develop Fe chelators for the treatment of cancer. Recently, investigations have led to the generation of novel di-2-pyridylketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) group of ligands that demonstrate marked and selective anti-tumour activity in vitro and also in vivo against a wide spectrum of tumours. Indeed, administration of these compounds to mice did not induce whole body Fe-depletion or disturbances in haematological or biochemical indices due to the very low doses required. The mechanism of action of these ligands includes alterations in expression of molecules involved in cell cycle control and metastasis suppression, as well as the generation of redox-active Fe complexes. This review examines the alterations in Fe metabolism in tumour cells and the systematic development of novel aroylhydrazone and thiosemicarbazone Fe chelators for cancer treatment.