Structure and Function of ABCG2-Rich Extracellular Vesicles Mediating Multidrug Resistance

Multidrug resistance (MDR) is a major impediment to curative cancer chemotherapy. The ATP-Binding Cassette transporters ABCG2, ABCB1 and ABCC2 form a unique defense network against multiple structurally and functionally distinct chemotherapeutics, thereby resulting in MDR. Thus, deciphering novel mechanisms of MDR and their overcoming is a major goal of cancer research. Recently we have shown that overexpression of ABCG2 in the membrane of novel extracellular vesicles (EVs) in breast cancer cells results in mitoxantrone resistance due to its dramatic sequestration in EVs. However, nothing is known about EVs structure, biogenesis and their ability to concentrate multiple antitumor agents. To this end, we here found that EVs are structural and functional homologues of bile canaliculi, are apically localized, sealed structures reinforced by an actin-based cytoskeleton and secluded from the extracellular milieu by the tight junction proteins occludin and ZO-1. Apart from ABCG2, ABCB1 and ABCC2 were also selectively targeted to the membrane of EVs. Moreover, Ezrin-Radixin-Moesin protein complex selectively localized to the border of the EVs membrane, suggesting a key role for the tethering of MDR pumps to the actin cytoskeleton. The ability of EVs to concentrate and sequester different antitumor drugs was also explored. Taking advantage of the endogenous fluorescence of anticancer drugs, we found that EVs-forming breast cancer cells display high level resistance to topotecan, imidazoacridinones and methotrexate via efficient intravesicular drug concentration hence sequestering them away from their cellular targets. Thus, we identified a new modality of anticancer drug compartmentalization and resistance in which multiple chemotherapeutics are actively pumped from the cytoplasm and highly concentrated within the lumen of EVs via a network of MDR transporters differentially targeted to the EVs membrane. We propose a composite model for the structure and function of MDR pump-rich EVs in cancer cells and their ability to confer multiple anticancer drug resistance

L-DOPA Neurotoxicity Is Mediated by Up-Regulation of DMT1−IRE Expression

2008-2009 > Academic research: refereed > Publication in refereed journalpublished_fina

Mathematical modeling of the dynamic storage of iron in ferritin

<p>Abstract</p> <p>Background</p> <p>Iron is essential for the maintenance of basic cellular processes. In the regulation of its cellular levels, ferritin acts as the main intracellular iron storage protein. In this work we present a mathematical model for the dynamics of iron storage in ferritin during the process of intestinal iron absorption. A set of differential equations were established considering kinetic expressions for the main reactions and mass balances for ferritin, iron and a discrete population of ferritin species defined by their respective iron content.</p> <p>Results</p> <p>Simulation results showing the evolution of ferritin iron content following a pulse of iron were compared with experimental data for ferritin iron distribution obtained with purified ferritin incubated <it>in vitro </it>with different iron levels. Distinctive features observed experimentally were successfully captured by the model, namely the distribution pattern of iron into ferritin protein nanocages with different iron content and the role of ferritin as a controller of the cytosolic labile iron pool (cLIP). Ferritin stabilizes the cLIP for a wide range of total intracellular iron concentrations, but the model predicts an exponential increment of the cLIP at an iron content > 2,500 Fe/ferritin protein cage, when the storage capacity of ferritin is exceeded.</p> <p>Conclusions</p> <p>The results presented support the role of ferritin as an iron buffer in a cellular system. Moreover, the model predicts desirable characteristics for a buffer protein such as effective removal of excess iron, which keeps intracellular cLIP levels approximately constant even when large perturbations are introduced, and a freely available source of iron under iron starvation. In addition, the simulated dynamics of the iron removal process are extremely fast, with ferritin acting as a first defense against dangerous iron fluctuations and providing the time required by the cell to activate slower transcriptional regulation mechanisms and adapt to iron stress conditions. In summary, the model captures the complexity of the iron-ferritin equilibrium, and can be used for further theoretical exploration of the role of ferritin in the regulation of intracellular labile iron levels and, in particular, as a relevant regulator of transepithelial iron transport during the process of intestinal iron absorption.</p

1B/(−)IRE DMT1 Expression during Brain Ischemia Contributes to Cell Death Mediated by NF-κB/RelA Acetylation at Lys310

The molecular mechanisms responsible for increasing iron and neurodegeneration in brain ischemia are an interesting area of research which could open new therapeutic approaches. Previous evidence has shown that activation of nuclear factor kappa B (NF-κB) through RelA acetylation on Lys310 is the prerequisite for p50/RelA-mediated apoptosis in cellular and animal models of brain ischemia. We hypothesized that the increase of iron through a NF-κB-regulated 1B isoform of the divalent metal transporter-1 (1B/DMT1) might contribute to post-ischemic neuronal damage. Both in mice subjected to transient middle cerebral artery occlusion (MCAO) and in neuronally differentiated SK-N-SH cells exposed to oxygen-glucose-deprivation (OGD), 1A/DMT1 was only barely expressed while the 1B/DMT1 without iron-response-element (−IRE) protein and mRNA were early up-regulated. Either OGD or over-expression of 1B/(−)IRE DMT1 isoform significantly increased iron uptake, as detected by total reflection X-ray fluorescence, and iron-dependent cell death. Iron chelation by deferoxamine treatment or (−)IRE DMT1 RNA silencing displayed significant neuroprotection against OGD which concomitantly decreased intracellular iron levels. We found evidence that 1B/(−)IRE DMT1 was a target gene for RelA activation and acetylation on Lys310 residue during ischemia. Chromatin immunoprecipitation analysis of the 1B/DMT1 promoter showed there was increased interaction with RelA and acetylation of H3 histone during OGD exposure of cortical neurons. Over-expression of wild-type RelA increased 1B/DMT1 promoter-luciferase activity, the (−)IRE DMT1 protein, as well as neuronal death. Expression of the acetylation-resistant RelA-K310R construct, which carried a mutation from lysine 310 to arginine, but not the acetyl-mimic mutant RelA-K310Q, down-regulated the 1B/DMT1 promoter, consequently offering neuroprotection. Our data showed that 1B/(−)IRE DMT1 expression and intracellular iron influx are early downstream responses to NF-κB/RelA activation and acetylation during brain ischemia and contribute to the pathogenesis of stroke-induced neuronal damage

Iron-Responsive Olfactory Uptake of Manganese Improves Motor Function Deficits Associated with Iron Deficiency

Iron-responsive manganese uptake is increased in iron-deficient rats, suggesting that toxicity related to manganese exposure could be modified by iron status. To explore possible interactions, the distribution of intranasally-instilled manganese in control and iron-deficient rat brain was characterized by quantitative image analysis using T1-weighted magnetic resonance imaging (MRI). Manganese accumulation in the brain of iron-deficient rats was doubled after intranasal administration of MnCl2 for 1- or 3-week. Enhanced manganese level was observed in specific brain regions of iron-deficient rats, including the striatum, hippocampus, and prefrontal cortex. Iron-deficient rats spent reduced time on a standard accelerating rotarod bar before falling and with lower peak speed compared to controls; unexpectedly, these measures of motor function significantly improved in iron-deficient rats intranasally-instilled with MnCl2. Although tissue dopamine concentrations were similar in the striatum, dopamine transporter (DAT) and dopamine receptor D1 (D1R) levels were reduced and dopamine receptor D2 (D2R) levels were increased in manganese-instilled rats, suggesting that manganese-induced changes in post-synaptic dopaminergic signaling contribute to the compensatory effect. Enhanced olfactory manganese uptake during iron deficiency appears to be a programmed “rescue response” with beneficial influence on motor impairment due to low iron status

Snx3 Regulates Recycling of the Transferrin Receptor and Iron Assimilation

Sorting of endocytic ligands and receptors is critical for diverse cellular processes. The physiological significance of endosomal sorting proteins in vertebrates, however, remains largely unknown. Here we report that sorting nexin 3 (Snx3) facilitates the recycling of transferrin receptor (Tfrc) and thus is required for the proper delivery of iron to erythroid progenitors. Snx3 is highly expressed in vertebrate hematopoietic tissues. Silencing of Snx3 results in anemia and hemoglobin defects in vertebrates due to impaired transferrin (Tf)-mediated iron uptake and its accumulation in early endosomes. This impaired iron assimilation can be complemented with non-Tf iron chelates. We show that Snx3 and Vps35, a component of the retromer, interact with Tfrc to sort it to the recycling endosomes. Our findings uncover a role of Snx3 in regulating Tfrc recycling, iron homeostasis, and erythropoiesis. Thus, the identification of Snx3 provides a genetic tool for exploring erythropoiesis and disorders of iron metabolism.National Institutes of Health (U.S.) (P01 HL032262

Associations of iron metabolism genes with blood manganese levels: a population-based study with validation data from animal models

<p>Abstract</p> <p>Background</p> <p>Given mounting evidence for adverse effects from excess manganese exposure, it is critical to understand host factors, such as genetics, that affect manganese metabolism.</p> <p>Methods</p> <p>Archived blood samples, collected from 332 Mexican women at delivery, were analyzed for manganese. We evaluated associations of manganese with functional variants in three candidate iron metabolism genes: <it>HFE </it>[hemochromatosis], <it>TF </it>[transferrin], and <it>ALAD </it>[δ-aminolevulinic acid dehydratase]. We used a knockout mouse model to parallel our significant results as a novel method of validating the observed associations between genotype and blood manganese in our epidemiologic data.</p> <p>Results</p> <p>Percentage of participants carrying at least one copy of <it>HFE C282Y</it>, <it>HFE H63D</it>, <it>TF P570S</it>, and <it>ALAD K59N </it>variant alleles was 2.4%, 17.7%, 20.1%, and 6.4%, respectively. Percentage carrying at least one copy of either <it>C282Y </it>or <it>H63D </it>allele in <it>HFE </it>gene was 19.6%. Geometric mean (geometric standard deviation) manganese concentrations were 17.0 (1.5) μg/l. Women with any <it>HFE </it>variant allele had 12% lower blood manganese concentrations than women with no variant alleles (β = -0.12 [95% CI = -0.23 to -0.01]). <it>TF </it>and <it>ALAD </it>variants were not significant predictors of blood manganese. In animal models, <it>Hfe</it>^-/-mice displayed a significant reduction in blood manganese compared with <it>Hfe</it>^+/+mice, replicating the altered manganese metabolism found in our human research.</p> <p>Conclusions</p> <p>Our study suggests that genetic variants in iron metabolism genes may contribute to variability in manganese exposure by affecting manganese absorption, distribution, or excretion. Genetic background may be critical to consider in studies that rely on environmental manganese measurements.</p

Neoplastic transformation of rat liver epithelial cells is enhanced by non-transferrin-bound iron

<p>Abstract</p> <p>Background</p> <p>Iron overload is associated with liver toxicity, cirrhosis, and hepatocellular carcinoma in humans. While most iron circulates in blood as transferrin-bound iron, non-transferrin-bound iron (NTBI) also becomes elevated and contributes to toxicity in the setting of iron overload. The mechanism for iron-related carcinogenesis is not well understood, in part due to a shortage of suitable experimental models. The primary aim of this study was to investigate NTBI-related hepatic carcinogenesis using T51B rat liver epithelial cells, a non-neoplastic cell line previously developed for carcinogenicity and tumor promotion studies.</p> <p>Methods</p> <p>T51B cells were loaded with iron by repeated addition of ferric ammonium citrate (FAC) to the culture medium. Iron internalization was documented by chemical assay, ferritin induction, and loss of calcein fluorescence. Proliferative effects were determined by cell count, toxicity was determined by MTT assay, and neoplastic transformation was assessed by measuring colony formation in soft agar. Cyclin levels were measured by western blot.</p> <p>Results</p> <p>T51B cells readily internalized NTBI given as FAC. Within 1 week of treatment at 200 μM, there were significant but well-tolerated toxic effects including a decrease in cell proliferation (30% decrease, p < 0.01). FAC alone induced little or no colony formation in soft agar. In contrast, FAC addition to cells previously initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in a concentration dependent increase in colony formation. This was first detected at 12 weeks of FAC treatment and increased at longer times. At 16 weeks, colony formation increased more than 10 fold in cells treated with 200 μM FAC (p < 0.001). The iron chelator desferoxamine reduced both iron uptake and colony formation. Cells cultured with 200 μM FAC showed decreased cyclin D1, decreased cyclin A, and increased cyclin B1.</p> <p>Conclusion</p> <p>These results establish NTBI as a tumor promoter in T51B rat liver epithelial cells. Changes in cyclin proteins suggest cell cycle disregulation contributes to tumor promotion by NTBI in this liver cell model.</p

Natural history of SLC11 genes in vertebrates: tales from the fish world

<p>Abstract</p> <p>Background</p> <p>The <it>SLC11A1/Nramp1 </it>and <it>SLC11A2/Nramp2 </it>genes belong to the <it>SLC11/Nramp </it>family of transmembrane divalent metal transporters, with <it>SLC11A1 </it>being associated with resistance to pathogens and <it>SLC11A2 </it>involved in intestinal iron uptake and transferrin-bound iron transport. Both members of the <it>SLC11 </it>gene family have been clearly identified in tetrapods; however <it>SLC11A1 </it>has never been documented in teleost fish and is believed to have been lost in this lineage during early vertebrate evolution. In the present work we characterized the <it>SLC11 </it>genes in teleosts and evaluated if the roles attributed to mammalian <it>SLC11 </it>genes are assured by other fish specific <it>SLC11 </it>gene members.</p> <p>Results</p> <p>Two different <it>SLC11 </it>genes were isolated in the European sea bass (<it>Dicentrarchus. labrax</it>), and named <it>slc11a2-α </it>and <it>slc11a2-β</it>, since both were found to be evolutionary closer to tetrapods <it>SLC11A2</it>, through phylogenetic analysis and comparative genomics. Induction of <it>slc11a2-α </it>and <it>slc11a2-β </it>in sea bass, upon iron modulation or exposure to <it>Photobacterium damselae </it>spp. <it>piscicida</it>, was evaluated in <it>in vivo </it>or <it>in vitro </it>experimental models. Overall, <it>slc11a2-α </it>was found to respond only to iron deficiency in the intestine, whereas <it>slc11a2-β </it>was found to respond to iron overload and bacterial infection in several tissues and also in the leukocytes.</p> <p>Conclusions</p> <p>Our data suggests that despite the absence of <it>slc11a1</it>, its functions have been undertaken by one of the <it>slc11a2 </it>duplicated paralogs in teleost fish in a case of synfunctionalization, being involved in both iron metabolism and response to bacterial infection. This study provides, to our knowledge, the first example of this type of sub-functionalization in iron metabolism genes, illustrating how conserving the various functions of the SLC11 gene family is of crucial evolutionary importance.</p