Mega-dose sodium ascorbate: a pilot, single-dose, physiological effect, double-blind, randomized, controlled trial.

BACKGROUND: Mega-dose sodium ascorbate (NaAscorbate) appears beneficial in experimental sepsis. However, its physiological effects in patients with septic shock are unknown. METHODS: We conducted a pilot, single-dose, double-blind, randomized controlled trial. We enrolled patients with septic shock within 24 h of diagnosis. We randomly assigned them to receive a single mega-dose of NaAscorbate (30 g over 1 h followed by 30 g over 5 h) or placebo (vehicle). The primary outcome was the total 24 h urine output (UO) from the beginning of the study treatment. Secondary outcomes included the time course of the progressive cumulative UO, vasopressor dose, and sequential organ failure assessment (SOFA) score. RESULTS: We enrolled 30 patients (15 patients in each arm). The mean (95% confidence interval) total 24-h UO was 2056 (1520-2593) ml with placebo and 2948 (2181-3715) ml with NaAscorbate (mean difference 891.5, 95% confidence interval [- 2.1 to 1785.2], P = 0.051). Moreover, the progressive cumulative UO was greater over time on linear mixed modelling with NaAscorbate (P < 0.001). Vasopressor dose and SOFA score changes over time showed faster reductions with NaAscorbate (P < 0.001 and P = 0.042). The sodium level, however, increased more over time with NaAscorbate (P < 0.001). There was no statistical difference in other clinical outcomes. CONCLUSION: In patients with septic shock, mega-dose NaAscorbate did not significantly increase cumulative 24-h UO. However, it induced a significantly greater increase in UO and a greater reduction in vasopressor dose and SOFA score over time. One episode of hypernatremia and one of hemolysis were observed in the NaAscorbate group. These findings support further cautious investigation of this novel intervention. Trial registration Australian New Zealand Clinical Trial Registry (ACTRN12620000651987), Date registered June/5/2020.

Renal arterial infusion of tempol prevents medullary hypoperfusion, hypoxia, and acute kidney injury in ovine Gram-negative sepsis.

AIM: Renal medullary hypoperfusion and hypoxia precede acute kidney injury (AKI) in ovine sepsis. Oxidative/nitrosative stress, inflammation, and impaired nitric oxide generation may contribute to such pathophysiology. We tested whether the antioxidant and anti-inflammatory drug, tempol, may modify these responses. METHODS: Following unilateral nephrectomy, we inserted renal arterial catheters and laser-Doppler/oxygen-sensing probes in the renal cortex and medulla. Noanesthetized sheep were administered intravenous (IV) Escherichia coli and, at sepsis onset, IV tempol (IVT; 30 mg kg-1 h-1 ), renal arterial tempol (RAT; 3 mg kg-1 h-1 ), or vehicle. RESULTS: Septic sheep receiving vehicle developed renal medullary hypoperfusion (76 ± 16% decrease in perfusion), hypoxia (70 ± 13% decrease in oxygenation), and AKI (87 ± 8% decrease in creatinine clearance) with similar changes during IVT. However, RAT preserved medullary perfusion (1072 ± 307 to 1005 ± 271 units), oxygenation (46 ± 8 to 43 ± 6 mmHg), and creatinine clearance (61 ± 10 to 66 ± 20 mL min-1 ). Plasma, renal medullary, and cortical tissue malonaldehyde and medullary 3-nitrotyrosine decreased significantly with sepsis but were unaffected by IVT or RAT. Consistent with decreased oxidative/nitrosative stress markers, cortical and medullary nuclear factor-erythroid-related factor-2 increased significantly and were unaffected by IVT or RAT. However, RAT prevented sepsis-induced overexpression of cortical tissue tumor necrosis factor alpha (TNF-α; 51 ± 16% decrease; p = 0.003) and medullary Thr-495 phosphorylation of endothelial nitric oxide synthase (eNOS; 63 ± 18% decrease; p = 0.015). CONCLUSIONS: In ovine Gram-negative sepsis, renal arterial infusion of tempol prevented renal medullary hypoperfusion and hypoxia and AKI and decreased TNF-α expression and uncoupling of eNOS. However, it did not affect markers of oxidative/nitrosative stress, which were significantly decreased by Gram-negative sepsis.

Striking a NRF2: The Rusty and Rancid Vulnerabilities Toward Ferroptosis in Alzheimer's Disease.

Significance: The lack of disease-modifying treatments for Alzheimer's disease (AD) that substantially alter the course of the disease highlights the need for new biological models of disease progression and neurodegeneration. Oxidation of macromolecules within the brain, including lipids, proteins, and DNA, is believed to contribute to AD pathophysiology, concomitant with dysregulation of redox-active metals, such as iron. Creating a unified model of pathogenesis and progression underpinned by iron dysregulation and redox dysregulation in AD could lead to new therapeutic targets with disease-modifying potential. Recent Advances: Ferroptosis, which was named in 2012, is a necrotic form of regulated cell death that depends on both iron and lipid peroxidation. While it is distinct from other types of regulated cell death, ferroptosis is regarded as being mechanistically synonymous with oxytosis. The ferroptosis paradigm has great explanatory potential in describing how neurons degenerate and die in AD. At the molecular level, ferroptosis is executed by the lethal accumulation of phospholipid hydroperoxides generated by the iron-dependent peroxidation of polyunsaturated fatty acids, while the major defensive protein against ferroptosis is the selenoenzyme, glutathione peroxidase 4 (GPX4). An expanding network of protective proteins and pathways have also been identified to complement GPX4 in the protection of cells against ferroptosis, with a central role emerging for nuclear factor erythroid 2-related factor 2 (NRF2). Critical Issues: In this review, we provide a critical overview of the utility of ferroptosis and NRF2 dysfunction in understanding the iron- and lipid peroxide-associated neurodegeneration of AD. Future Directions: Finally, we discuss how the ferroptosis paradigm in AD is providing a new spectrum of therapeutic targets. Antioxid. Redox Signal. 39, 141-161.

Perturbed iron biology in the prefrontal cortex of people with schizophrenia.

Despite loss of grey matter volume and emergence of distinct cognitive deficits in young adults diagnosed with schizophrenia, current treatments for schizophrenia do not target disruptions in late maturational reshaping of the prefrontal cortex. Iron, the most abundant transition metal in the brain, is essential to brain development and function, but in excess, it can impair major neurotransmission systems and lead to lipid peroxidation, neuroinflammation and accelerated aging. However, analysis of cortical iron biology in schizophrenia has not been reported in modern literature. Using a combination of inductively coupled plasma-mass spectrometry and western blots, we quantified iron and its major-storage protein, ferritin, in post-mortem prefrontal cortex specimens obtained from three independent, well-characterised brain tissue resources. Compared to matched controls (n = 85), among schizophrenia cases (n = 86) we found elevated tissue iron, unlikely to be confounded by demographic and lifestyle variables, by duration, dose and type of antipsychotic medications used or by copper and zinc levels. We further observed a loss of physiologic age-dependent iron accumulation among people with schizophrenia, in that the iron level among cases was already high in young adulthood. Ferritin, which stores iron in a redox-inactive form, was paradoxically decreased in individuals with the disorder. Such iron-ferritin uncoupling could alter free, chemically reactive, tissue iron in key reasoning and planning areas of the young-adult schizophrenia cortex. Using a prediction model based on iron and ferritin, our data provide a pathophysiologic link between perturbed cortical iron biology and schizophrenia and indicate that achievement of optimal cortical iron homeostasis could offer a new therapeutic target.

Integrin αvß3 Is a Master Regulator of Resistance to TKI-Induced Ferroptosis in HER2-Positive Breast Cancer.

Human epidermal growth factor receptor-2 (HER2)-targeting therapies provide clinical benefits for patients with HER2-positive breast cancer. However, the resistance to monotherapies invariably develops and leads to disease relapse and treatment failure. Previous studies have demonstrated a link between the potency of HER2-targeting tyrosine kinase inhibitors (TKIs) and their ability to induce an iron-dependent form of cell death called ferroptosis. The aim of this study was to understand the mechanisms of resistance to TKI-induced ferroptosis and identify novel approaches to overcome treatment resistance. We used mouse and human HER2-positive models of acquired TKI resistance to demonstrate an intimate link between the resistance to TKIs and to ferroptosis and present the first evidence that the cell adhesion receptor αvß3 integrin is a critical mediator of resistance to TKI-induced ferroptosis. Our findings indicate that αvß3 integrin-mediated resistance is associated with the re-wiring of the iron/antioxidant metabolism and persistent activation of AKT signalling. Moreover, using gene manipulation approaches and pharmacological inhibitors, we show that this "αvß3 integrin addiction" can be targeted to reverse TKI resistance. Collectively, these findings provide critical insights into new therapeutic strategies to improve the treatment of advanced HER2-positive breast cancer patients.

Receptor-Independent Anti-Ferroptotic Activity of TrkB Modulators.

Dysregulated brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) signalling is implicated in several neurodegenerative diseases, including Alzheimer's disease. A failure of neurotrophic support may participate in neurodegenerative mechanisms, such as ferroptosis, which has likewise been implicated in this disease class. The current study investigated whether modulators of TrkB signalling affect ferroptosis. Cell viability, C11 BODIPY, and cell-free oxidation assays were used to observe the impact of TrkB modulators, and an immunoblot assay was used to detect TrkB expression. TrkB modulators such as agonist BDNF, antagonist ANA-12, and inhibitor K252a did not affect RSL3-induced ferroptosis sensitivity in primary cortical neurons expressing detectable TrkB receptors. Several other modulators of the TrkB receptor, including agonist 7,8-DHF, activator phenelzine sulphate, and inhibitor GNF-5837, conferred protection against a range of ferroptosis inducers in several immortalised neuronal and non-neuronal cell lines, such as N27 and HT-1080 cells. We found these immortalised cell lines lack detectable TrkB receptor expression, so the anti-ferroptotic activity of these TrkB modulators was most likely due to their inherent radical-trapping antioxidant properties, which should be considered when interpreting their experimental findings. These modulators or their variants could be potential anti-ferroptotic therapeutics for various diseases.

Selective ferroptosis vulnerability due to familial Alzheimer's disease presenilin mutations.

Mutations in presenilin 1 and 2 (PS1 and PS2) cause autosomal dominant familial Alzheimer's disease (FAD). Ferroptosis has been implicated as a mechanism of neurodegeneration in AD since neocortical iron burden predicts Alzheimer's disease (AD) progression. We found that loss of the presenilins dramatically sensitizes multiple cell types to ferroptosis, but not apoptosis. FAD causal mutations of presenilins similarly sensitizes cells to ferroptosis. The presenilins promote the expression of GPX4, the selenoprotein checkpoint enzyme that blocks ferroptosis by quenching the membrane propagation of lethal hydroperoxyl radicals. Presenilin γ-secretase activity cleaves Notch-1 to signal LRP8 expression, which then controls GPX4 expression by regulating the supply of selenium into the cell since LRP8 is the uptake receptor for selenoprotein P. Selenium uptake is thus disrupted by presenilin FAD mutations, suppressing GPX4 expression. Therefore, presenilin mutations may promote neurodegeneration by derepressing ferroptosis, which has implications for disease-modifying therapeutics.

Neuropathological Mechanisms of ß-N-Methylamino-L-Alanine (BMAA) with a Focus on Iron Overload and Ferroptosis.

The incidence of neurodegenerative diseases and cyanobacterial blooms is concomitantly increasing worldwide. The cyanotoxin ß-N-methylamino-L-alanine (BMAA) is produced by most of the Cyanobacteria spp. This cyanotoxin is described as a potential environmental etiology factor for some sporadic neurodegenerative diseases. Climate change and eutrophication significantly increase the frequency and intensity of cyanobacterial bloom in water bodies. This review evaluates different neuropathological mechanisms of BMAA at molecular and cellular levels and compares the related studies to provide some useful recommendations. Additionally, the structure and properties of BMAA as well as its microbial origin, especially by gut bacteria, are also briefly covered. Unlike previous reviews, we hypothesize the possible neurotoxic mechanism of BMAA through iron overload. We also discuss the involvement of BMAA in excitotoxicity, TAR DNA-binding protein 43 (TDP-43) translocation and accumulation, tauopathy, and other protein misincorporation and misfolding.

Ferroptosis and NRF2: an emerging battlefield in the neurodegeneration of Alzheimer's disease.

Ferroptosis is an iron- and lipid peroxidation-dependent cell death modality and emerging evidence indicates that ferroptosis has great explanatory potential for neuronal loss and associated CNS dysfunction in a range of neurodegenerative diseases (e.g., Alzheimer's, Parkinson's and Huntington's diseases, Motor neuron disease, Friedreich ataxia (FRDA)). Ferroptotic death results from lethal levels of phospholipid hydroperoxides that are generated by iron-dependent peroxidation of polyunsaturated fatty acids (PUFAs), such as arachidonic and adrenic acids, which are conjugated to specific phospholipids (e.g., phosphatidylethanolamines (PEs)). The major cellular protector against ferroptosis is glutathione peroxidase 4 (GPX4), a membrane-associated selenoenzyme that reduces deleterious phospholipid hydroperoxides to their corresponding benign phospholipid alcohols in a glutathione-dependent manner. Other complementary protective systems have also been identified that act to bolster cellular defences against ferroptosis. Many pharmacological modulators of the ferroptosis pathway have been identified, targeting proteins involved in iron homoeostasis and autophagy; the production and detoxification of lipid peroxides, and cyst(e)ine/glutathione metabolism. While a growing number of cell signalling pathways converge to regulate the ferroptosis cascade, an emerging understanding of ferroptosis regulation suggests that the ferroptotic 'tone' of cells can be set by the transcription factor, nuclear factor erythroid 2-related factor 2 (NRF2), which transcriptionally controls many key components of the ferroptosis pathway. In this review, we provide a critical overview of the relationship between ferroptosis and NRF2 signalling. With a focus on the role of ferroptosis in Alzheimer's disease (AD), we discuss how therapeutic modulation of the NRF2 pathway is a viable strategy to explore in the treatment of ferroptosis-driven neurodegeneration.

Systematic Review: Quantitative Susceptibility Mapping (QSM) of Brain Iron Profile in Neurodegenerative Diseases.

Iron has been increasingly implicated in the pathology of neurodegenerative diseases. In the past decade, development of the new magnetic resonance imaging technique, quantitative susceptibility mapping (QSM), has enabled for the more comprehensive investigation of iron distribution in the brain. The aim of this systematic review was to provide a synthesis of the findings from existing QSM studies in neurodegenerative diseases. We identified 80 records by searching MEDLINE, Embase, Scopus, and PsycInfo databases. The disorders investigated in these studies included Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Wilson's disease, Huntington's disease, Friedreich's ataxia, spinocerebellar ataxia, Fabry disease, myotonic dystrophy, pantothenate-kinase-associated neurodegeneration, and mitochondrial membrane protein-associated neurodegeneration. As a general pattern, QSM revealed increased magnetic susceptibility (suggestive of increased iron content) in the brain regions associated with the pathology of each disorder, such as the amygdala and caudate nucleus in Alzheimer's disease, the substantia nigra in Parkinson's disease, motor cortex in amyotrophic lateral sclerosis, basal ganglia in Huntington's disease, and cerebellar dentate nucleus in Friedreich's ataxia. Furthermore, the increased magnetic susceptibility correlated with disease duration and severity of clinical features in some disorders. Although the number of studies is still limited in most of the neurodegenerative diseases, the existing evidence suggests that QSM can be a promising tool in the investigation of neurodegeneration.

Acireductone dioxygenase 1 (ADI1) is regulated by cellular iron by a mechanism involving the iron chaperone, PCBP1, with PCBP2 acting as a potential co-chaperone.

The iron-containing protein, acireductone dioxygenase 1 (ADI1), is a dioxygenase important for polyamine synthesis and proliferation. Using differential proteomics, the studies herein demonstrated that ADI1 was significantly down-regulated by cellular iron depletion. This is important, since ADI1 contains a non-heme, iron-binding site critical for its activity. Examination of multiple human cell-types demonstrated a significant decrease in ADI1 mRNA and protein after incubation with iron chelators. The decrease in ADI1 after iron depletion was reversible upon incubation of cells with the iron salt, ferric ammonium citrate (FAC). A significant decrease in ADI1 mRNA levels was observed after 14 h of iron depletion. In contrast, the chelator-mediated reduction in ADI1 protein occurred earlier after 10 h of iron depletion, suggesting additional post-transcriptional regulation. The proteasome inhibitor, MG-132, prevented the iron chelator-mediated decrease in ADI1 expression, while the lysosomotropic agent, chloroquine, had no effect. These results suggest an iron-dependent, proteasome-mediated, degradation mechanism. Poly r(C)-binding protein (PCBPs) 1 and 2 act as iron delivery chaperones to other iron-containing dioxygenases and were shown herein for the first time to be regulated by iron levels. Silencing of PCBP1, but not PCBP2, led to loss of ADI1 expression. Confocal microscopy co-localization studies and proximity ligation assays both demonstrated decreased interaction of ADI1 with PCBP1 and PCBP2 under conditions of iron depletion using DFO. These data indicate PCBP1 and PCBP2 interact with ADI1, but only PCBP1 plays a role in ADI1 expression. In fact, PCBP2 appeared to play an accessory role, being involved as a potential co-chaperone.

Ascorbate and Tumor Cell Iron Metabolism: The Evolving Story and Its Link to Pathology.

Significance: Vitamin C or ascorbate (Asc) is a water-soluble vitamin and an antioxidant that is involved in many crucial biological functions. Asc's ability to reduce metals makes it an essential enzyme cofactor. Recent Advances: The ability of Asc to act as a reductant also plays an important part in its overall role in iron metabolism, where Asc induces both nontransferrin-bound iron and transferrin-bound iron uptake at physiological concentrations (∼50 µM). Moreover, Asc has emerged to play an important role in multiple diseases and its effects at pharmacological doses could be important for their treatment. Critical Issues: Asc's role as a regulator of cellular iron metabolism, along with its cytotoxic effects and different roles at pharmacological concentrations, makes it a candidate as an anticancer agent. Ever since the controversy regarding the studies from the Mayo Clinic was finally explained, there has been a renewed interest in using Asc as a therapeutic approach toward cancer due to its minimal side effects. Numerous studies have been able to demonstrate the anticancer activity of Asc through selective oxidative stress toward cancer cells via H2O2 generation at pharmacological concentrations. Studies have demonstrated that Asc's cytotoxic mechanism at concentrations (>1 mM) has been associated with decreased cellular iron uptake. Future Directions: Recent studies have also suggested other mechanisms, such as Asc's effects on autophagy, polyamine metabolism, and the cell cycle. Clearly, more has yet to be discovered about Asc's mechanism of action to facilitate safe and effective treatment options for cancer and other diseases.

Exploiting Cancer Metal Metabolism using Anti-Cancer Metal- Binding Agents.

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 has 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 anticancer 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 antitumor agents.

The old and new biochemistry of polyamines.

Polyamines are ubiquitous positively charged amines found in all organisms. These molecules play a crucial role in many biological functions including cell growth, gene regulation and differentiation. The three major polyamines produced in all mammalian cells are putrescine, spermidine and spermine. The intracellular levels of these polyamines depend on the interplay of the biosynthetic and catabolic enzymes of the polyamine and methionine salvage pathway, as well as the involvement of polyamine transporters. Polyamine levels are observed to be high in cancer cells, which contributes to malignant transformation, cell proliferation and poor patient prognosis. Considering the critical roles of polyamines in cancer cell proliferation, numerous anti-polyaminergic compounds have been developed as anti-tumor agents, which seek to suppress polyamine levels by specifically inhibiting polyamine biosynthesis, activating polyamine catabolism, or blocking polyamine transporters. However, in terms of the development of effective anti-cancer therapeutics targeting the polyamine system, these efforts have unfortunately resulted in little success. Recently, several studies using the iron chelators, O-trensox and ICL670A (Deferasirox), have demonstrated a decline in both iron and polyamine levels. Since iron levels are also high in cancer cells, and like polyamines, are required for proliferation, these latter findings suggest a biochemically integrated link between iron and polyamine metabolism.

Iron and Alzheimer's Disease: An Update on Emerging Mechanisms.

Iron is a crucial transition metal for life and is the most abundant transition metal in the brain. However, iron's biological utility as an effective redox cycling metal also endows it with the potential to catalyze production of noxious free radicals. This "Janus-faced" nature of iron demands a tight regulation of cellular its metabolism. This regulation is crucial in the CNS, where iron plays myriad keystone roles in CNS processes, including mitochondrial energy transduction, enzyme catalysis, mitochondrial function, myelination, neurotransmitter anabolism and catabolism. Aberrations in brain iron homeostasis can elevate levels of this redox-active metal, leading to mislocalization of the metal and catastrophic oxidative damage to sensitive cellular and subcellular structures. Iron dyshomeostasis has been strongly linked to the pathogenesis of Alzheimer's disease (AD), as well as other major neurodegenerative diseases. Despite the growing societal burden of AD, no disease-modifying therapy exists, necessitating continued investment into both drug-development and the fundamental science investigating the disease-causing mechanisms. Targeting iron dyshomeostasis in the brain represents a rational approach to treat the underlying disease. Here we provide an update on known and emerging iron-associated mechanisms involved in AD. We conclude with an overview of evidence suggesting that, in addition to apoptosis, neuronal loss in AD involves "ferroptosis", a newly discovered iron- and lipid-peroxidation-dependent form of regulated necrosis. The ferroptosis field is rapidly progressing and may provide key insights for future drug-development with disease-modifying potential in AD.

Coupling of the polyamine and iron metabolism pathways in the regulation of proliferation: Mechanistic links to alterations in key polyamine biosynthetic and catabolic enzymes.

Many biological processes result from the coupling of metabolic pathways. Considering this, proliferation depends on adequate iron and polyamines, and although iron-depletion impairs proliferation, the metabolic link between iron and polyamine metabolism has never been thoroughly investigated. This is important to decipher, as many disease states demonstrate co-dysregulation of iron and polyamine metabolism. Herein, for the first time, we demonstrate that cellular iron levels robustly regulate 13 polyamine pathway proteins. Seven of these were regulated in a conserved manner by iron-depletion across different cell-types, with four proteins being down-regulated (i.e., acireductone dioxygenase 1 [ADI1], methionine adenosyltransferase 2α [MAT2α], Antizyme and polyamine oxidase [PAOX]) and three proteins being up-regulated (i.e., S-adenosyl methionine decarboxylase [AMD1], Antizyme inhibitor 1 [AZIN1] and spermidine/spermine-N1-acetyltransferase 1 [SAT1]). Depletion of iron also markedly decreased polyamine pools (i.e., spermidine and/or spermine, but not putrescine). Accordingly, iron-depletion also decreased S-adenosylmethionine that is essential for spermidine/spermine biosynthesis. Iron-depletion additionally reduced 3H-spermidine uptake in direct agreement with the lowered levels of the polyamine importer, SLC22A16. Regarding mechanism, the "reprogramming" of polyamine metabolism by iron-depletion is consistent with the down-regulation of ADI1 and MAT2α, and the up-regulation of SAT1. Moreover, changes in ADI1 (biosynthetic) and SAT1 (catabolic) partially depended on the iron-regulated changes in c-Myc and/or p53. The ability of iron chelators to inhibit proliferation was rescuable by putrescine and spermidine, and under some conditions by spermine. Collectively, iron and polyamine metabolism are intimately coupled, which has significant ramifications for understanding the integrated role of iron and polyamine metabolism in proliferation.

Tumor stressors induce two mechanisms of intracellular P-glycoprotein-mediated resistance that are overcome by lysosomal-targeted thiosemicarbazones.

Multidrug resistance (MDR) is a major obstacle in cancer treatment due to the ability of tumor cells to efflux chemotherapeutics via drug transporters (e.g. P-glycoprotein (Pgp; ABCB1)). Although the mechanism of Pgp-mediated drug efflux is known at the plasma membrane, the functional role of intracellular Pgp is unclear. Moreover, there has been intense focus on the tumor micro-environment as a target for cancer treatment. This investigation aimed to dissect the effects of tumor micro-environmental stress on subcellular Pgp expression, localization, and its role in MDR. These studies demonstrated that tumor micro-environment stressors (i.e. nutrient starvation, low glucose levels, reactive oxygen species, and hypoxia) induce Pgp-mediated drug resistance. This occurred by two mechanisms, where stressors induced 1) rapid Pgp internalization and redistribution via intracellular trafficking (within 1 h) and 2) hypoxia-inducible factor-1α expression after longer incubations (4-24 h), which up-regulated Pgp and was accompanied by lysosomal biogenesis. These two mechanisms increased lysosomal Pgp and facilitated lysosomal accumulation of the Pgp substrate, doxorubicin, resulting in resistance. This was consistent with lysosomal Pgp being capable of transporting substrates into lysosomes. Hence, tumor micro-environmental stressors result in: 1) Pgp redistribution to lysosomes; 2) increased Pgp expression; 3) lysosomal biogenesis; and 4) potentiation of Pgp substrate transport into lysosomes. In contrast to doxorubicin, when stress stimuli increased lysosomal accumulation of the cytotoxic Pgp substrate, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), this resulted in the agent overcoming resistance. Overall, this investigation describes a novel approach to overcoming resistance in the stressful tumor micro-environment.

Transcriptional regulation of the cyclin-dependent kinase inhibitor, p21CIP1/WAF1, by the chelator, Dp44mT.

BACKGROUND: The cyclin-dependent kinase inhibitor, p21, is well known for its role in cell cycle arrest. Novel anti-cancer agents that deplete iron pools demonstrate marked anti-tumor activity and are also active in regulating p21 expression. These agents induce p21 mRNA levels independently of the tumor suppressor, p53, and differentially regulate p21 protein expression depending on the cell-type. Several chelators, including an analogue of the potent anti-tumor agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), have entered clinical trials, and thus, their molecular mechanism of action is crucial to assess. Hence, this investigation examined how several iron chelators transcriptionally regulate p21. METHODS: Promoter-deletion constructs; luciferase assays; RT-PCR; western analysis; gene silencing; co-immunoprecipitation. RESULTS: The transcriptional regulation of the p21 promoter by iron chelators was demonstrated to be dependent on the chelator and cell-type examined. The potent anti-cancer chelator, Dp44mT, induced p21 promoter activity in SK-MEL-28 melanoma cells, but not in MCF-7 breast cancer cells. Further analysis of the p21 promoter identified a 50-bp region between -104 and -56-bp that was required for Dp44mT-induced activation in SK-MEL-28 cells. This region contained several Sp1-binding sites and mutational analysis of this region revealed the Sp1-3-binding site played a significant role in Dp44mT-induced activation of p21. Further, co-immunoprecipitation demonstrated that Dp44mT induced a marked increase in the interactions between Sp1 and the transcription factors, estrogen receptor-α and c-Jun. CONCLUSIONS AND GENERAL SIGNIFICANCE: Dp44mT-induced p21 promoter activation via the Sp1-3-binding site and increased Sp1/ER-α and Sp1/c-Jun complex formation in SK-MEL-28 cells, suggesting these complexes were involved in p21 promoter activation.

Letter to the Editor: "Analysis of the Interaction of Dp44mT with Human Serum Albumin and Calf Thymus DNA Using Molecular Docking and Spectroscopic Techniques".

n/a.

Copper and conquer: copper complexes of di-2-pyridylketone thiosemicarbazones as novel anti-cancer therapeutics.

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.