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We designed and synthesized two novel photocaged peroxide compounds, N5TBHP and N6TBHP, featuring nitrogen-containing fused ring coumarin skeletons. Notably, a tetrahydroquinoline fused coumarin derivative, N6TBHP demonstrated significantly higher photocleavage efficiency under visible light at 455 nm compared to N5TBHP, which contains an indoline fused coumarin. This process effectively releases the oxidative stress inducer tert-butylhydroperoxide (TBHP). Additionally, N6TBHP exhibits high resistance to glutathione (GSH), and its UV spectral analysis suggests enhanced intracellular stability due to reduced reactivity with GSH through self-assembly. Furthermore, N6TBHP can release an optimal amount of TBHP into cells under visible light irradiation with minimal cell damage. These properties position N6TBHP as a promising tool for advancing research in intracellular redox signaling.
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Subretinal hemorrhages result in poor vision and visual field defects. During hemorrhage, several potentially toxic substances are released from iron-based hemoglobin and hemin, inducing cellular damage, the detailed mechanisms of which remain unknown. We examined the effects of excess intracellular iron on retinal pigment epithelial (RPE) cells. A Fe2+ probe, SiRhoNox-1 was used to investigate Fe2+ accumulation after treatment with hemoglobin or hemin in the human RPE cell line ARPE-19. We also evaluated the production of reactive oxygen species (ROS) and lipid peroxidation. Furthermore, the protective effect of-an iron chelator, 2,2'-bipyridyl (BP), and ferrostatin-1 (Fer-1) on the cell damage, was evaluated. Fe2+ accumulation increased in the hemoglobin- or hemin-treated groups, as well as intracellular ROS production and lipid peroxidation. In contrast, BP treatment suppressed RPE cell death, ROS production, and lipid peroxidation. Pretreatment with Fer-1 ameliorated cell death in a concentration-dependent manner and suppressed ROS production and lipid peroxidation. Taken together, these findings indicate that hemoglobin and hemin, as well as subretinal hemorrhage, may induce RPE cell damage and visual dysfunction via intracellular iron accumulation.
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Hemina , Hemoglobinas , Ferro , Epitélio Pigmentado da Retina , Humanos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Cicloexilaminas/farmacologia , Hemina/farmacologia , Hemoglobinas/metabolismo , Ferro/metabolismo , Quelantes de Ferro/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Fenilenodiaminas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologiaRESUMO
The intramolecular Diels-Alder reaction (IMDA) is a powerful method for regioselective and stereoselective construction of functionalised decalin skeletons, and the recent discovery of enzymes that catalyse IMDA cycloaddition in biosynthesis has generated considerable interest. This study focused on the role of the absolute configuration of the C-6 carbon of the substrate polyene in the stereocontrol of the IMDA reaction catalysed by Fsa2 and Phm7, which construct different enantiomeric decalin skeletons. Their enantiomeric precursor polyenes were synthesised and subjected to enzymatic or thermal IMDA reactions to isolate various diastereomeric decalines and determine their absolute configuration. Furthermore, density functional theory calculations were performed to elucidate the stereocontrol mechanism underlying the formation of decalin. The results showed that Fsa2 exhibits the same equisetin-type stereoselectivity for enantiomeric substrates regardless of the 6-methyl group configuration of the substrate, while Phm7 shows two types of stereoselectivity depending on the configuration of the 6-methyl group. We also found a unique stereochemistry-activity relationship in antibacterial activity for decalin diastereomers, including new derivatives. This study provides new insights into the stereoselectivity of DAase, which is important in the synthesis of natural product skeletons.
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Iron metabolism is closely associated with the pathogenesis of obesity. However, the mechanism of the iron-dependent regulation of adipocyte differentiation remains unclear. Here, we show that iron is essential for rewriting of epigenetic marks during adipocyte differentiation. Iron supply through lysosome-mediated ferritinophagy was found to be crucial during the early stage of adipocyte differentiation, and iron deficiency during this period suppressed subsequent terminal differentiation. This was associated with demethylation of both repressive histone marks and DNA in the genomic regions of adipocyte differentiation-associated genes, ãincluding Pparg, which encodes PPARγ, the master regulator of adipocyte differentiation. In addition, we identified several epigenetic demethylases to be responsible for iron-dependent adipocyte differentiation, with the histone demethylase jumonji domain-containing 1A and the DNA demethylase ten-eleven translocation 2 as the major enzymes. The interrelationship between repressive histone marks and DNA methylation was indicated by an integrated genome-wide association analysis, and was also supported by the findings that both histone and DNA demethylation were suppressed by either the inhibition of lysosomal ferritin flux or the knockdown of iron chaperone poly(rC)-binding protein 2. In summary, epigenetic regulations through iron-dependent control of epigenetic enzyme activities play an important role in the organized gene expression mechanisms of adipogenesis.
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Estudo de Associação Genômica Ampla , Ferro , Ferro/metabolismo , Metilação de DNA/genética , Epigênese Genética , Adipócitos/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismoRESUMO
We developed a caged hydroperoxide, BhcTBHP, releasing prooxidant TBHP under blue light irradiation. MitoTBHP with triphenylphosphonium at position 7 triggered selective oxidative stress and membrane depolarization in mitochondria upon photoirradiation. This study presents a powerful tool for studying redox signaling and oxidative stress in living cells.
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Estresse Oxidativo , Peróxidos , Peróxidos/farmacologia , Espécies Reativas de Oxigênio , Oxirredução , Peróxido de Hidrogênio , terc-Butil Hidroperóxido/farmacologiaRESUMO
Haloperidol is a widely used antipsychotic agent that exerts antipsychotic effects through a strong antagonism of dopamine D2 receptors. In addition, haloperidol is classified as a sigma-1 receptor (S1R) antagonist that prevents endogenous oxidative stress in cultured cells. However, pharmacological activities of haloperidol against oxidative stress remain unclear. Oxytosis/ferroptosis are iron-dependent nonapoptotic oxidative cell deaths that are regarded as two names for the same cell death pathway and the potential physiological relevance of oxytosis/ferroptosis in multiple diseases is suggested. In the present study, the effects of haloperidol on oxytosis/ferroptosis were investigated in S1R-knockdown mouse hippocampal HT22 cells. The results indicate that haloperidol is a strong inhibitor of oxytosis/ferroptosis independent of S1R. Imaging of HT22 cells with a newly developed fluorescent probe showed that haloperidol was localized to late endosomes and lysosomes and reduced the accumulation of lysosomal ferrous ions, resulting in reduced production of intracellular reactive oxygen species and inhibition of cell death. These results indicate that haloperidol is useful not only as an antipsychotic agent but also as a neuroprotective agent against endogenous oxidative stress via distinct mechanisms. Furthermore, lysosome-targeting ferroptosis inhibitors could be useful for the treatment of various diseases, including cancers, ischemia-reperfusion injury, and neurodegenerative disorders, which have been associated with ferroptosis.
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Antipsicóticos , Ferroptose , Fármacos Neuroprotetores , Animais , Antipsicóticos/farmacologia , Dopamina , Corantes Fluorescentes , Haloperidol/farmacologia , Íons , Ferro/metabolismo , Lisossomos/metabolismo , Camundongos , Fármacos Neuroprotetores/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Dopamina D2 , Receptores sigma , Receptor Sigma-1RESUMO
Artesunate, an antimalarial drug, induces ferroptosis, but the mechanism is still unclear. In the present study, we investigated how Artesunate induces ferroptosis in ovarian serous carcinoma. Experiments were performed using the ovarian serous carcinoma cell lines CaOV3 and SKOV3ip1, and the sensitivity of CaOV3 to Artesunate was higher than that of SKOV3ip1. Ferroptosis inhibitors inhibited Artesunate-induced intracellular lipid peroxi-dation and cell death. However, unlike class 1 ferroptosis inducer erastin, Artesunate had no effect on intracellular glutathione-SH levels. We found that Artesunate-induced changes in lysosomal Fe|2+ were parallel to the induction of ferroptosis. Therefore, ferritin, which oxidizes and binds intracellular Fe|2+, may have an inhibitory effect on ferroptosis. Knockdown of nuclear coactivator 4, a key molecule of ferritinophagy (ferritin-specific autophagy), suppressed Artesunate-induced cell death. Knockdown of ferritin heavy chain by siRNA greatly enhanced the sensitivity to Artesunate, and overexpression of ferritin heavy chain greatly reduced the sensitivity of ovarian cancer cell lines to Artesunate. These results can explain the differential sensitivity of CaOV3 and SKOV3ip1 to Artesunate. In conclusion, enhancement of ferritinophagy is an important step involved in the mechanism of Artesunate-induced ferroptosis, and ferritin heavy chain levels may contribute to the regulation of sensitivity in Artesunate-induced ferroptosis in ovarian serous carcinoma cells.
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Labile heme (LH) is a complex of Fe(II) and protoporphyrin IX, an essential signaling molecule in various biological systems. Most of the subcellular dynamics of LH remain unclear because of the lack of efficient chemical tools for detecting LH in cells. Here, we report an activity-based fluorescence probe that can monitor the fluctuations of LH in biological events. H-FluNox is a selective fluorescent probe that senses LH using biomimetic N-oxide deoxygenation to trigger fluorescence. The selectivity of H-FluNox to LH is >100-fold against Fe(II), enabling the discrimination of LH from the labile Fe(II) pool in living cells. The probe can detect the acute release of LH upon NO stimulation and the accumulation of LH by inhibiting the heme exporter. In addition, imaging studies using the probe revealed a partial heme-export activity of the ATP-binding cassette subfamily G member 2 (ABCG2), potential LH pooling ability of G-quadruplex, and involvement of LH in ferroptosis. The successful use of H-FluNox in identifying fluctuations of LH in living cells offers opportunities for studying the physiology and pathophysiology of LH in living systems.
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Corantes Fluorescentes , Heme , Compostos Ferrosos , Corantes Fluorescentes/química , Imagem Molecular , Transdução de SinaisRESUMO
BACKGROUND: Cisplatin is widely used as an antitumor drug for the treatment of solid tumors. However, its use has been limited owing to nephrotoxicity, a major side effect. The mechanism of cisplatin-induced nephrotoxicity (CIN) has long been investigated in order to develop preventive/therapeutic drugs. Ferroptosis is a newly identified form of non-apoptotic regulated cell death induced by iron-mediated lipid peroxidation and is involved in the pathophysiology of various diseases. In this study, we examined the role of ferroptosis in CIN. METHODS: We evaluated the role of ferroptosis in CIN by in vivo experiments in a mouse model. RESULTS: Cisplatin increased the protein expressions of transferrin receptor-1 and ferritin, and iron content in the kidney of mice. In addition, treatment with cisplatin augmented renal ferrous iron and hydroxyl radical levels with co-localization. Mice administered cisplatin demonstrated kidney injury, with renal dysfunction and increased inflammatory cytokine expression; these changes were ameliorated by Ferrostatin-1 (Fer-1), an inhibitor of ferroptosis. The expression of the ferroptosis markers, COX2 and 4-hydroxynonenal (4-HNE), increased with cisplatin administration, and decreased with the administration of Fer-1. By contrast, cisplatin-induced apoptosis and necroptosis were inhibited by treatment with Fer-1. Moreover, deferoxamine, an iron chelator, also inhibited CIN, with a decrease in the expression of COX-2 and 4-HNE. CONCLUSION: Ferroptosis is involved in the pathogenesis of CIN and might be used as a new preventive target for CIN.
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Cisplatino/toxicidade , Ferroptose , Animais , Ferritinas , Ferro/metabolismo , Peroxidação de Lipídeos , CamundongosRESUMO
To develop antitumor drugs capable of targeting energy metabolism in the tumor microenvironment, we produced a series of potent new biguanide derivatives via structural modification of the arylbiguanide scaffold. We then conducted biological screening using hypoxia inducible factor (HIF)-1- and unfolded protein response (UPR)-dependent reporter assays and selective cytotoxicity assay under low glucose conditions. Homologation studies of aryl-(CH2)n-biguanides (n = 0-6) yielded highly potent derivatives with an appropriate alkylene linker length (n = 5, 6). The o-chlorophenyl derivative 7l (n = 5) indicated the most potent inhibitory effects on HIF-1- and UPR-mediated transcriptional activation (IC50; 1.0 ± 0.1 µM, 7.5 ± 0.1 µM, respectively) and exhibited selective cytotoxicity toward HT29 cells under low glucose condition (IC50; 1.9 ± 0.1 µM). Additionally, the protein expression of HIF-1α induced by hypoxia and of GRP78 and GRP94 induced by glucose starvation was markedly suppressed by the biguanides, thereby inhibiting angiogenesis. Metabolic flux and fluorescence-activated cell sorting analyses of tumor cells revealed that the biguanides strongly inhibited oxidative phosphorylation and activated compensative glycolysis in the presence of glucose, whereas both were strongly suppressed in the absence of glucose, resulting in cellular energy depletion and apoptosis. These findings suggest that the pleiotropic effects of these biguanides may contribute to more selective and effective killing of cancer cells due to the suppression of various stress adaptation systems in the tumor microenvironment.
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Antineoplásicos , Biguanidas , Metabolismo Energético/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Estresse Fisiológico/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Células A549 , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Biguanidas/síntese química , Biguanidas/química , Biguanidas/farmacologia , Galinhas , Células HEK293 , Células HT29 , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismoRESUMO
Bismuth-rhodamine compounds stand out for their unique excitable photosensitizing properties and concomitant fluorescence; however, further knowledge of the structure-property relationship is required to expand the scope of their practical application. With this aim, this study describes the first examples of asymmetric bismuth-incorporated rhodamines, BiRNH and BiRAc, including their synthesis, photophysical properties, and photosensitizing abilities. Upon red light excitation, BiRNH exhibits detectable emission and photosensitizing properties, while the N-acetylated derivative BiRAc shows a hypsochromic shift in the absorption wavelength and attenuation of emission and photosensitizing ability. These significantly different photophysical properties enabled us to design an activatable fluorogenic photosensitizer, BiRGlu, which bears a γ-glutamyl group instead of the acetyl group in BiRAc. The γ-glutamyl group can be cleaved by γ-glutamyl transpeptidase (GGT) to produce BiRNH, which acts as a red-light-excitable fluorophore and photosensitizer. A cell study revealed that the phototoxicity and fluorescence of BiRGlu could be simultaneously and selectively activated in the cells with high GGT activity. Thus, we established that BiRNH could be envisaged as a versatile scaffold for activatable fluorogenic photosensitizers.
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Visualizing biological events and states to resolve biological questions is challenging. Tissue clearing permits three-dimensional multicolor imaging. Here, we describe a pH-adjustable tissue clearing solution, Seebest (SEE Biological Events and States in Tissues), which preserves lipid ultrastructures at an electron microscopy level. Adoption of polyethylenimine was required for a wide pH range adjustment of the tissue clearing solution. The combination of polyethylenimine and urea had a good tissue clearing ability for multiple tissues within several hours. Blood vessels stained with lipophilic carbocyanine dyes were deeply visible using the solution. Adjusting the pH of the solution was important to maximize the fluorescent intensity and suppress dye leakage during tissue clearing. The spatial distribution of doxorubicin and oxidative stress were observable using the solution. Moreover, spatial distribution of liposomes in the liver was visualized. Hence, the Seebest solution provides pH-adjustable, rapid, sufficient tissue clearing, while preserving lipid ultrastructures, which is suitable for drug delivery system evaluations.
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Asbestos is still a social burden worldwide as a carcinogen causing malignant mesothelioma. Whereas recent studies suggest that local iron reduction is a preventive strategy against carcinogenesis, little is known regarding the cellular and molecular mechanisms surrounding excess iron. Here by differentially using high-risk and low-risk asbestos fibers (crocidolite and anthophyllite, respectively), we identified asbestos-induced mutagenic milieu for mesothelial cells. Rat and cell experiments revealed that phagocytosis of asbestos by macrophages results in their distinctive necrotic death; initially lysosome-depenent cell death and later ferroptosis, which increase intra- and extra-cellular catalytic Fe(II). DNA damage in mesothelial cells, as assessed by 8-hydroxy-2'-deoxyguanosine and γ-H2AX, increased after crocidolite exposure during regeneration accompanied by ß-catenin activation. Conversely, ß-catenin overexpression in mesothelial cells induced higher intracellular catalytic Fe(II) with increased G2/M cell-cycle fraction, when p16INK4A genomic loci localized more peripherally in the nucleus. Mesothelial cells after challenge of H2O2 under ß-catenin overexpression presented low p16INK4A expression with a high incidence of deletion in p16INK4A locus. Thus, crocidolite generated catalytic Fe(II)-rich mutagenic environment for mesothelial cells by necrotizing macrophages with lysosomal cell death and ferroptosis. These results suggest novel molecular strategies to prevent mesothelial carcinogenesis after asbestos exposure.
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Amianto , Ferroptose , Mesotelioma , Animais , Epitélio , Compostos Ferrosos , Peróxido de Hidrogênio , Macrófagos , Mutagênicos , Ratos , beta Catenina/genéticaRESUMO
High-throughput methods for monitoring subcellular labile Fe(II) are important for conducting studies on iron homeostasis and for the discovery of potential drug candidates for the treatment of iron deficiency or overload. Herein, a highly sensitive and robust fluorescent probe for the detection of intracellular labile Fe(II) is described. The probe was designed through the rational optimization of the reactivity and responsiveness for an Fe(II)-induced fluorogenic reaction based on deoxygenation of an N-oxide, which was developed in-house. The probe is ready to use for a 96-well-plate-based high-content imaging of labile Fe(II) in living cells. Using this simple method, we were able to conduct high-throughput screening of a chemical library containing 3399 compounds. The compound lomofungin was identified as a potential drug candidate for the intracellular enhancement of labile Fe(II) via a novel mechanism in which the ferritin protein was downregulated.
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Corantes Fluorescentes , Ensaios de Triagem em Larga Escala , Homeostase , FerroRESUMO
Iron deposits are often observed in the brains of patients with neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. This study outlines the development of F-Nox-1 as the first example of a 19F-MRI probe that can selectively detect Fe(ii) in aqueous solutions. The use of tetrafluoro-p-phenylenediamine (TFPDA) as a 19F signal emitter with an Fe(ii)-selective chemical switch, based on our previously reported N-oxide chemistry, yielded a readout of a symmetry-dependent 19F signal change in response to Fe(ii). The addition of Fe(ii) ions to F-Nox-1 triggered a 19F signal change, both in the chemical shift and signal intensity, and the response was highly selective to Fe(ii) over other biologically relevant metal ions. The probe could also detect Fe(ii) in serum containing various biological contaminants by 19F magnetic resonance imaging (19F-MRI). Imaging of soluble Fe(ii) species, which is the major component of water-soluble iron species, by 19F-MRI will potentially enable the direct monitoring of the elevation of Fe(ii) levels prior to the formation of iron deposits, which is a potential risk factor for neurodegenerative diseases.
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By carrying out structural modifications based on the bicyclic peptide structure of echinomycin, we successfully synthesized various powerful antitumor derivatives. The ring conformation in the obtained compounds was restricted by cross-linking with an unnatural bond. The prepared derivatives were demonstrated to strongly suppress the hypoxia inducible factor (HIF)-1 transcriptional activation and hypoxia induction of HIF-1 protein expression. Particularly, alkene-bridged derivative 12 exhibited remarkably potent cytotoxicity (IC50 = 0.22 nM on the MCF-7 cell line) and HIF-1 inhibition (IC50 = 0.09 nM), which considerably exceeded those of echinomycin. Conformational analyses and molecular modeling studies revealed that the biological activities were enhanced following restriction of the conformation by cross-linking through a metabolically stable and rigid bridge bond. In addition, we proposed a new globular conformation stabilized by intramolecular π stacking that can contribute to the biological effects of bicyclic depsipeptides. The developments presented in the current study serve as a useful guide to expand the chemical space of peptides in drug discovery.
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Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Depsipeptídeos/síntese química , Depsipeptídeos/farmacologia , Desenho de Fármacos , Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Células A549 , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Células HEK293 , Células HT29 , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Células MCF-7 , Relação Estrutura-AtividadeRESUMO
Skeletal muscle atrophy is caused by disruption in the homeostatic balance of muscle degeneration and regeneration under various pathophysiological conditions. We have previously reported that iron accumulation induces skeletal muscle atrophy via a ubiquitin ligase-dependent pathway. However, the potential effect of iron accumulation on muscle regeneration remains unclear. To examine the effect of iron accumulation on myogenesis, we used a mouse model with cardiotoxin (CTX)-induced muscle regeneration in vivo and C2C12 mouse myoblast cells in vitro. In mice with iron overload, the skeletal muscles exhibited increased oxidative stress and decreased expression of satellite cell markers. Following CTX-induced muscle injury, these mice also displayed delayed muscle regeneration with a decrease in the size of regenerating myofibers, reduced expression of myoblast differentiation markers, and decreased phosphorylation of MAPK signaling pathways. In vitro, iron overload also suppressed the differentiation of C2C12 myoblast cells but the suppression could be reversed by superoxide scavenging using tempol. Excess iron inhibits myogenesis via oxidative stress, leading to an imbalance in skeletal muscle homeostasis.-Ikeda, Y., Satoh, A., Horinouchi, Y., Hamano, H., Watanabe, H., Imao, M., Imanishi, M., Zamami, Y., Takechi, K., Izawa-Ishizawa, Y., Miyamoto, L., Hirayama, T., Nagasawa, H., Ishizawa, K., Aihara, K.-I., Tsuchiya, K., Tamaki, T. Iron accumulation causes impaired myogenesis correlated with MAPK signaling pathway inhibition by oxidative stress.
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Ferro/metabolismo , Músculo Esquelético/metabolismo , Estresse Oxidativo/fisiologia , RNA Mensageiro/metabolismo , Animais , Western Blotting , Linhagem Celular , Sobrevivência Celular/fisiologia , Radical Hidroxila/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular/fisiologia , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
After intracranial hemorrhage (ICH), iron is released from the hematoma and induces secondary brain injury. However, the detail effect of iron on blood-brain barrier (BBB) function is still unknown. We investigated whether hemoglobin (Hb), ferrous ammonium sulfate (FAS) or hemin which contains iron have the detrimental effect on both human brain microvascular endothelial cells and pericytes by cellular function analysis in vitro. We developed an iron (Fe2+)-detectable probe, Si-RhoNox-1, to investigate intracellular Fe2+ accumulation (Fe2+intra). After FAS treatment, there was the correlation between Fe2+intra and cell death. Moreover, Hb or hemin treatment induced cell death, increased reactive oxygen species and promoted Fe2+intra in both cells. These changes were inhibited by the Fe2+ chelator, 2,2'-bipyridil (BP). Furthermore, hemin induced endothelial barrier dysfunction via disruption of junction integrity. Based on in vitro studies, we used a hemin-injection ICH mice model in vivo. Hemin injection (10 mM/10 µL, i.c.) induced deleterious effects including BBB hyper-permeability, neuronal deficits, neuronal damage, altered proteins expression, and Fe2+intra in BBB composed cells. Lastly, BP (40 mg/kg, i.p.) administration attenuated neuronal deficits at 3 days after surgery. Collectively, Hb or hemin damaged BBB composed cells via Fe2+intra. Therefore, the regulation of the Fe2+ movement in BBB might be effective for treatment of ICH.
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Barreira Hematoencefálica/metabolismo , Lesões Encefálicas/etiologia , Lesões Encefálicas/metabolismo , Células Endoteliais/metabolismo , Espaço Intracelular/metabolismo , Hemorragias Intracranianas/complicações , Ferro/metabolismo , Pericitos/metabolismo , 2,2'-Dipiridil/metabolismo , 2,2'-Dipiridil/farmacologia , Animais , Apoptose/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Endotélio Vascular/citologia , Compostos Ferrosos/metabolismo , Compostos Ferrosos/farmacologia , Hematoma/metabolismo , Hematoma/fisiopatologia , Hemina/metabolismo , Hemina/farmacologia , Hemoglobinas/metabolismo , Hemoglobinas/farmacologia , Humanos , Masculino , Camundongos , Compostos de Amônio Quaternário/metabolismo , Compostos de Amônio Quaternário/farmacologiaRESUMO
Iron is involved in numerous physiologically essential processes in our body. However, excessive iron is a pathogenic factor in neurodegenerative diseases, causing aberrant oxidative stress. Divalent metal transporter 1 (DMT1) acts as a primary transporter of Fe(ii) ions. The intracellular delivery of DMT1 toward the cellular membrane via the trans-Golgi network during the endocytotic process is partially regulated by a retromer-mediated protein-sorting system comprising vacuolar protein-sorting proteins (VPSs). Thus, together with DMT1, the Golgi-apparatus acts as a hub organelle in the delivery system for intracellular Fe(ii) ions. Dysfunction of the VPS-relevant protein sorting system can induce the abnormal delivery of DMT1 toward lysosomes concomitantly with Fe(ii) ions. To explore this issue, we developed a fluorescent probe, Gol-SiRhoNox, for the Golgi-specific detection of Fe(ii) ions by integrating our original N-oxide-based Fe(ii)-specific chemical switch, a new Golgi-localizable chemical motif, and polarity-sensitive fluorogenic scaffold. Our synchronous imaging study using Gol-SiRhoNox and LysoRhoNox, a previously developed fluorescent probe for lysosomal Fe(ii), revealed that the intracellular distribution balance of Fe(ii) ions between the Golgi apparatus and lysosomes is normally Golgi-dominant, whereas the lysosome-specific elevation of Fe(ii) ions was observed in cells with induced dysfunction of VPS35, a member of the retromer complex. Treatment of cells with dysfunctional VPS35 with R55, a molecular chaperone, resulted in the restoration of the subcellular distribution of Fe(ii) ions to the Golgi-dominant state. These results indicate that the impairment of the DMT1 traffic machinery affects subcellular iron homeostasis, promoting Fe(ii) leakage at the Golgi and lysosomal accumulation of Fe(ii) through missorting of DMT1.