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1.
Nucleic Acids Res ; 51(12): 6120-6142, 2023 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-37158274

RESUMEN

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,  including 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.


Asunto(s)
Estudio de Asociación del Genoma Completo , Hierro , Hierro/metabolismo , Metilación de ADN/genética , Epigénesis Genética , Adipocitos/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo
2.
Bioorg Med Chem ; 111: 117863, 2024 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-39096786

RESUMEN

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.


Asunto(s)
Diseño de Fármacos , Luz , Peróxidos , Especies Reactivas de Oxígeno , Transducción de Señal , Especies Reactivas de Oxígeno/metabolismo , Humanos , Transducción de Señal/efectos de los fármacos , Peróxidos/química , Peróxidos/farmacología , Peróxidos/síntesis química , Estructura Molecular , Relación Estructura-Actividad , terc-Butilhidroperóxido/farmacología , terc-Butilhidroperóxido/química , Cumarinas/química , Cumarinas/farmacología , Cumarinas/síntesis química , Relación Dosis-Respuesta a Droga , Estrés Oxidativo/efectos de los fármacos , Procesos Fotoquímicos
3.
J Pharmacol Sci ; 155(2): 44-51, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38677785

RESUMEN

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.


Asunto(s)
Hemina , Hemoglobinas , Hierro , Epitelio Pigmentado de la Retina , Humanos , Muerte Celular/efectos de los fármacos , Línea Celular , Ciclohexilaminas/farmacología , Hemina/farmacología , Hemoglobinas/metabolismo , Hierro/metabolismo , Quelantes del Hierro/farmacología , Peroxidación de Lípido/efectos de los fármacos , Fenilendiaminas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/patología
4.
J Am Chem Soc ; 144(9): 3793-3803, 2022 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-35133144

RESUMEN

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.


Asunto(s)
Colorantes Fluorescentes , Hemo , Compuestos Ferrosos , Colorantes Fluorescentes/química , Imagen Molecular , Transducción de Señal
5.
J Clin Biochem Nutr ; 71(1): 34-40, 2022 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-35903602

RESUMEN

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.

6.
Org Biomol Chem ; 19(16): 3611-3619, 2021 04 28.
Artículo en Inglés | MEDLINE | ID: mdl-33506853

RESUMEN

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.

7.
FASEB J ; 33(8): 9551-9564, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31145863

RESUMEN

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.


Asunto(s)
Hierro/metabolismo , Músculo Esquelético/metabolismo , Estrés Oxidativo/fisiología , ARN Mensajero/metabolismo , Animales , Western Blotting , Línea Celular , Supervivencia Celular/fisiología , Radical Hidroxilo/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Desarrollo de Músculos/fisiología , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo
8.
Org Biomol Chem ; 18(30): 5843-5849, 2020 08 05.
Artículo en Inglés | MEDLINE | ID: mdl-32756663

RESUMEN

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.

9.
Exp Eye Res ; 171: 30-36, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29530811

RESUMEN

Excitoneurotoxicity is regarded as one of the mechanisms of the death of retinal ganglion cells induced by retinal central artery occlusion and glaucoma. Oxidative stress is at least in part involved in excitoneurotoxicity. Fenton reaction, which is catalyzed by Fe2+, is known to cause formation of hydroxyl radical, one of reactive oxygen species, suggesting that chelation of iron may be protective against excitoneurotoxicity. In the present study, we histologically evaluated whether zinc-deferoxamine (Zn-DFO) and deferasirox (DFX), common iron-chelating agents, were protective against N-methyl-D-aspartate (NMDA)-induced retinal injury in the rat in vivo. Male Sprague-Dawley rats were subjected to intravitreal NMDA injection (200 nmol/eye). Zn-DFO (1, 3, 10, and 30 mg/kg), Zn (0.1, 0.2 and 0.6 mg/kg) and DFX (20 mg/kg) were intraperitoneally administered. Morphometric evaluations using paraffin-embedded retinal sections, and detection of Fe2+ using SiRhoNox-1, a fluorescent probe of labile Fe2+ in the retinal frozen sections were carried out. Intravitreal NMDA resulted in strong positive signals of SiRhoNox-1 in the ganglion cell layer 24 h after NMDA injection, suggesting that intravitreal NMDA caused Fe2+ accumulation in the retinal ganglion cells. Intravitreal NMDA induced retinal ganglion cell loss 7 days after NMDA injection. Zn-DFO (1, 3, 10, and 30 mg/kg), ZnCl2 (0.2 mg/kg, a corresponding dose of 1 mg/kg Zn-DFO) and DFX (20 mg/kg) prevented the damage of retinal ganglion cells, whereas 0.6 mg/kg ZnCl2, which is a corresponding dose of 3 mg/kg Zn-DFO, did not show any protective effects. Zn-DFO (30 mg/kg) significantly decreased the intensity of the fluorescence of SiRhoNox-1 and the transferrin immunofluorescence 24 h after NMDA injection, the number of TUNEL-positive cells 24 h after NMDA injection, that of 8-OHdG-positive cells, and that of 4-hydroxy-2-nonenal-positive cells 12 and 24 h after NMDA injection. These data suggest that iron-chelating agents protected retinal neurons against excitoneurotoxicity via reduction of iron content and oxidative stress in the rats in vivo. We proposed that treatment with iron-chelating agents would be a new strategy for the retinal diseases caused by excitoneurotoxicity.


Asunto(s)
Agonistas de Aminoácidos Excitadores/toxicidad , Quelantes del Hierro/farmacología , N-Metilaspartato/toxicidad , Estrés Oxidativo/efectos de los fármacos , Degeneración Retiniana/prevención & control , Células Ganglionares de la Retina/efectos de los fármacos , 8-Hidroxi-2'-Desoxicoguanosina , Aldehídos/metabolismo , Animales , Supervivencia Celular/fisiología , Deferasirox/farmacología , Deferoxamina/farmacología , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Colorantes Fluorescentes/metabolismo , Etiquetado Corte-Fin in Situ , Inyecciones Intraperitoneales , Inyecciones Intravítreas , Compuestos de Hierro/metabolismo , Masculino , Microscopía Confocal , Compuestos Organometálicos/farmacología , Ratas , Ratas Sprague-Dawley , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Transferrina/metabolismo
10.
Cell Biol Toxicol ; 34(4): 279-290, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-28871429

RESUMEN

We recently demonstrated the cytotoxic action of a novel phenformin derivative, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on HT-29 cells under a serum- and glucose-deprived condition. In that study, we showed that the ATF6 arm of the ER stress pathway and c-Myc expression were downregulated 12 h after the treatment with 2-Cl-Phen. Through characterization of intracellular events at the early phase of the 2-Cl-Phen treatment before noticeable morphological changes, we found rapid fluctuations in the c-Myc and ATF4 proteins but not in their mRNAs in 2-Cl-Phen-treated HT-29 cells under the serum- and glucose-deprived condition. The 2-Cl-Phen-mediated downregulation of ATF4 protein was not paralleled by the phosphorylation status of PERK and eIF2α. Reduction of c-Myc expression by 2-Cl-Phen was more profound than that of ATF4 expression, and phosphorylated c-Myc was downregulated within 2 h. Pharmacological studies on the expression of c-Myc and ATF4 proteins showed that this decrease was mediated through proteasomal degradation but not by autophagy. Interestingly, treatment with lithium chloride, which is a well-known inhibitor of GSK3ß, partially recovered the expression of ATF4 protein, but its effect on the level of total c-Myc protein was negligible. Treatment with 2-Cl-Phen increased the expression of phosphorylated AMPK, but Compound C, an AMPK inhibitor, did not influence the expression of c-Myc protein in HT-29 cells. Finally, we observed that 2-Cl-Phen partially attenuated the gene expression of integrin subunit α1 (ITGA1), a downstream target of c-Myc. Taken together, these results show that 2-Cl-Phen rapidly downregulated the expression of c-Myc in addition to ER stress responses in a post-translational manner. Further elucidation and improvement of this multi-target-directed compound will provide new insights for developing therapeutic strategies against cancer.


Asunto(s)
Biguanidas/farmacología , Glucosa/deficiencia , Factor de Transcripción Activador 4/metabolismo , Adenilato Quinasa/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Biguanidas/química , Proteínas de Ciclo Celular/metabolismo , Medio de Cultivo Libre de Suero , Regulación hacia Abajo/efectos de los fármacos , Factor 2 Eucariótico de Iniciación/metabolismo , Células HT29 , Humanos , Integrina alfa1/genética , Integrina alfa1/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , eIF-2 Quinasa/metabolismo
11.
Lab Invest ; 97(5): 555-566, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28263291

RESUMEN

Renal anemia is a major complication in chronic kidney disease (CKD). Iron supplementation, as well as erythropoiesis-stimulating agents, are widely used for treatment of renal anemia. However, excess iron causes oxidative stress via the Fenton reaction, and iron supplementation might damage remnant renal function including erythropoietin (EPO) production in CKD. EPO gene expression was suppressed in mice following direct iron treatment. Hypoxia-inducible factor-2 alpha (HIF-2α), a positive regulator of the EPO gene, was also diminished in the kidney of mice following iron treatment. Anemia-induced increase in renal EPO and HIF-2α expression was inhibited by iron treatment. In in vitro experiments using EPO-producing HepG2 cells, iron stimulation reduced the expression of the EPO gene, as well as HIF-2α. Moreover, iron treatment augmented oxidative stress, and iron-induced reduction of EPO and HIF-2α expression was restored by tempol, an antioxidant compound. HIF-2α interaction with the Epo promoter was inhibited by iron treatment, and was restored by tempol. These findings suggested that iron supplementation reduced EPO gene expression via an oxidative stress-HIF-2α-dependent signaling pathway.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Eritropoyetina/metabolismo , Hierro/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Eritropoyetina/análisis , Compuestos Férricos/farmacología , Sacarato de Óxido Férrico , Fibroblastos , Ácido Glucárico/farmacología , Células Hep G2 , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Ratones , Regulación hacia Arriba/efectos de los fármacos , Obstrucción Ureteral
12.
Biochem Biophys Res Commun ; 487(4): 789-794, 2017 06 10.
Artículo en Inglés | MEDLINE | ID: mdl-28450115

RESUMEN

Ovarian endometrial cysts cause some kinds of ovarian cancer, and iron is considered as one factor of carcinogenesis. In contrast, hypoxia is associated with progression, angiogenesis, metastasis, and resistance to therapy in cancer. We investigated hypoxia-induced perturbation of iron homeostasis in terms of labile iron, iron deposition, and iron regulatory protein (IRP) in ovarian endometrial cysts. Iron deposition, expression of IRPs, and a protein marker of hypoxia in human ovarian endometrial cysts were analyzed histologically. The concentration of free iron and the pO2 level of the cyst fluid of human ovarian cysts (n = 9) were measured. The expression of IRP2 under hypoxia was investigated in vitro by using Ishikawa cells as a model of endometrial cells. Iron deposition and the expression of IRP2 and Carbonic anhydrase 9 (CA9) were strong in endometrial stromal cells in the human ovarian endometrial cysts. The average concentration of free iron in the cyst fluid was 8.1 ± 2.9 mg/L, and the pO2 was 22.4 ± 5.2 mmHg. A cell-based study using Ishikawa cells revealed that IRP2 expression was decreased by an overload of Fe(II) under normoxia but remained unchanged under hypoxia even in the presence of excess Fe(II). An increase in the expression of IRP2 caused upregulation of intracellular iron as a result of the response to iron deficiency, whereas the protein was degraded under iron-rich conditions. We found that iron-rich regions existed in ovarian endometrial cysts concomitantly with the high level of IRP2 expression, which should generally be decomposed upon an overload of iron. We revealed that an insufficient level of oxygen in the cysts is the main factor for the unusual stabilization of IRP2 against iron-mediated degradation, which provides aberrant uptake of iron in ovarian endometrial stromal cells and can potentially lead to carcinogenesis.


Asunto(s)
Endometrio/metabolismo , Proteína 2 Reguladora de Hierro/metabolismo , Quistes Ováricos/metabolismo , Femenino , Humanos , Hipoxia/metabolismo , Hierro/metabolismo , Proteína 2 Reguladora de Hierro/biosíntesis , Células Tumorales Cultivadas
13.
Chem Res Toxicol ; 30(2): 699-704, 2017 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-27943678

RESUMEN

Tirapazamine (TPZ) is an anticancer drug with highly selective cytotoxicity toward hypoxic cells. TPZ is converted to a radical intermediate under hypoxic conditions, and this intermediate interacts with intracellular macromolecules, including DNA. TPZ has been reported to indirectly induce DNA double-strand breaks (DSBs) through the formation of various intermediate DNA lesions under hypoxic conditions. Although the topoisomerase II-DNA complex has been identified as one of these intermediates, other lesions have not yet been defined. In order to obtain a deeper understanding of the mechanisms responsible for the selective cytotoxicity of TPZ toward hypoxic cells, its cellular sensitivity was systematically examined with genetically isogenic DNA-repair-deficient mutant DT40 cell lines. Our results showed that tdp1-/-, tdp2-/-, parp1-/-, and aptx1-/- cells displayed hypersensitivity to TPZ only under hypoxic conditions. These results strongly suggest that the accumulation of the topoisomerase I-trapped DNA complex, topoisomerase II-trapped DNA complex, and abortive ligation products with 5'-AMP are the potential causes of TPZ-induced hypoxic cell death. Furthermore, our genetic analysis revealed that under normoxic conditions (as well as hypoxic conditions), TPZ exhibited significant cytotoxicity toward cell lines deficient in homologous recombination, nonhomologous end joining, base excision repair, and translesion synthesis. Ascorbic acid, a radical scavenger, suppressed TPZ-induced cytotoxicity toward normoxic cells. These results suggest the involvement of oxidative DNA damage and DSBs produced by reactive oxygen species generated from superoxide, a byproduct of the oxidation of TPZ radical intermediates in normoxic cells. Collectively, our results demonstrate that TPZ induces oxidative DNA damage under normoxic and hypoxic conditions and selectively introduces abortive topoisomerase-DNA complexes and unligatable DNA ends under hypoxic conditions.


Asunto(s)
Antineoplásicos/toxicidad , Daño del ADN , ADN/efectos de los fármacos , Triazinas/toxicidad , Animales , Línea Celular , Pollos , Ensayo Cometa , Especies Reactivas de Oxígeno/metabolismo , Tirapazamina
14.
J Clin Biochem Nutr ; 60(1): 39-48, 2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-28163381

RESUMEN

Owing to its distinctive electrochemical properties with interconvertible multiple oxidation states, iron plays a significant role in various physiologically important functions such as respiration, oxygen transport, energy production, and enzymatic reactions. This redox activity can also potentially produce cellular damage and death, and numerous diseases are related to iron overload resulting from the dysfunction of the iron regulatory system. In this case, "free iron" or "labile iron," which refers to iron ion weakly bound or not bound to proteins, causes aberrant production of reactive oxygen species. With the aim of elucidating the variation of labile iron involved in pathological processes, some chemical tools that can qualitatively and/or quantitatively monitor iron have been utilized to investigate the distribution, accumulation, and flux of biological iron species. Since iron ions show unique reactivity depending on its redox state, i.e., Fe2+ or Fe3+ (or transiently higher oxidative states), methods for the separate detection of iron species with different redox states are preferred to understand its physiological and pathological roles more in detail. The scope of this review article covers from classical chromogenic to newly emerging chemical tools for the detection of Fe ions. In particular, chemical tools applicable to biological studies will be presented.

15.
J Clin Biochem Nutr ; 61(3): 176-182, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-29203958

RESUMEN

Congenital diaphragmatic hernia (CDH) is a life-threatening neonatal disease that leads to lung hypoplasia and pulmonary hypertension. We recently found that maternal prenatal administration of Saireito (TJ-114) ameliorates fetal CDH in a nitrofen-induced rat model. Here, we studied the role of iron and oxidative stress in neonates of this model and in lung fibroblasts IMR90-SV in association with nitrofen and Saireito. We observed increased immunostaining of 8-hydroxy-2'-deoxyguanosine in the lungs of neonates with CDH, which was ameliorated by maternal Saireito intake. Pulmonary transferrin receptor expression was significantly decreased in both CDH and CDH after Saireito in comparison to normal controls, indicating functional lung immaturity, whereas catalytic Fe(II) and pulmonary DMT1/ferroportin expression remained constant among the three groups. Saireito revealed a dose-dependent scavenging capacity with electron spin resonance spin trapping in vitro against hydroxyl radicals but not against superoxide. Finally, nitrofen revealed dose-dependent cytotoxicity to IMR90-SV cells, accompanied by an increase in oxidative stress, as seen by 5(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate and catalytic Fe(II). Saireito ameliorated all of these in IMR90-SV cells. In conclusion, catalytic Fe(II)-dependent oxidative stress by nitrofen may be the pathogenic cause of CDH, and the antioxidative activity of Saireito is at least partially responsible for improving nitrofen-induced CDH.

16.
Cancer Sci ; 107(3): 250-7, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26679080

RESUMEN

Multi-wall carbon nanotubes (MWCNT) are a form of flexible fibrous nanomaterial with high electrical and thermal conductivity. However, 50-nm MWCNT in diameter causes malignant mesothelioma (MM) in rodents and, thus, the International Agency of Research on Cancer has designated them as a possible human carcinogen. Little is known about the molecular mechanism through which MWCNT causes MM. To elucidate the carcinogenic mechanisms of MWCNT in mesothelial cells, we used a variety of lysates to comprehensively identify proteins specifically adsorbed on pristine MWCNT of different diameters (50 nm, NT50; 100 nm, NT100; 150 nm, NT150; and 15 nm/tangled, NTtngl) using mass spectrometry. We identified >400 proteins, which included hemoglobin, histone, transferrin and various proteins associated with oxidative stress, among which we selected hemoglobin and transferrin for coating MWCNT to further evaluate cytotoxicity, wound healing, intracellular catalytic ferrous iron and oxidative stress in rat peritoneal mesothelial cells (RPMC). Cytotoxicity to RPMC was observed with pristine NT50 but not with NTtngl. Coating NT50 with hemoglobin or transferrin significantly aggravated cytotoxicity to RPMC, with an increase in cellular catalytic ferrous iron and DNA damage also observed. Knockdown of transferrin receptor with ferristatin II decreased not only NT50 uptake but also cellular catalytic ferrous iron. Our results suggest that adsorption of hemoglobin and transferrin on the surface of NT50 play a role in causing mesothelial iron overload, contributing to oxidative damage and possibly subsequent carcinogenesis in mesothelial cells. Uptake of NT50 at least partially depends on transferrin receptor 1. Modifications of NT50 surface may decrease this human risk.


Asunto(s)
Carcinógenos/toxicidad , Hemoglobinas/fisiología , Mesotelioma/metabolismo , Nanotubos de Carbono/toxicidad , Transferrina/fisiología , Adsorción , Animales , Carcinógenos/química , Línea Celular , Epitelio/efectos de los fármacos , Epitelio/patología , Femenino , Hemoglobinas/química , Masculino , Mesotelioma/inducido químicamente , Nanotubos de Carbono/química , Tamaño de la Partícula , Ratas Endogámicas F344 , Receptores de Transferrina/metabolismo , Transferrina/química
17.
Biochem Biophys Res Commun ; 476(4): 600-606, 2016 08 05.
Artículo en Inglés | MEDLINE | ID: mdl-27262439

RESUMEN

Iron is an essential nutrient for every type of life on earth. However, excess iron is cytotoxic and can lead to an increased cancer risk in humans. Catalytic ferrous iron [Fe(II)] is an initiator of the Fenton reaction, which causes oxidative stress by generating hydroxyl radicals. Recently, it became possible to localize catalytic Fe(II) in situ with a turn-on fluorescent probe, RhoNox-1. Here, we screened each organ/cell of rats to globally evaluate the distribution of catalytic Fe(II) and found that eosinophils showed the highest abundance. In various cells, lysosomes were the major organelle, sharing ∼40-80% of RhoNox-1 fluorescence. We then used an ovalbumin-induced allergic peritonitis model to study the dynamics of catalytic Fe(II). Peritoneal lavage revealed that the total iron contents per cell were significantly decreased, whereas an increase in the number of inflammatory cells (macrophages, neutrophils, eosinophils and lymphocytes) resulted in an increased total iron content of the peritoneal inflammatory cells. Notably, macrophages, eosinophils and neutrophils exhibited significantly increased catalytic Fe(II) with increased DMT1 expression and decreased ferritin expression, though catalytic Fe(II) was significantly decreased in the peritoneal lavage fluid. In conclusion, catalytic Fe(II) in situ more directly reflects cellular activity and the accompanying pathology than total iron does.


Asunto(s)
Hierro/metabolismo , Peritonitis/metabolismo , Animales , Líquido Ascítico/metabolismo , Catálisis , Línea Celular , Modelos Animales de Enfermedad , Eosinófilos/metabolismo , Espacio Extracelular/metabolismo , Colorantes Fluorescentes , Células HL-60 , Humanos , Espacio Intracelular/metabolismo , Lisosomas/metabolismo , Macrófagos/metabolismo , Masculino , Ovalbúmina/inmunología , Ovalbúmina/toxicidad , Peritonitis/etiología , Ratas , Ratas Endogámicas F344
18.
Mol Cell Biochem ; 419(1-2): 29-40, 2016 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-27392906

RESUMEN

Recently, we developed a variety of phenformin derivatives as selective antitumor agents. Based on previous findings, this study evaluated a promising compound, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on the basis of stress responses in the human colon cancer cell line HT-29 under a serum- and glucose-deprived condition. 2-Cl-Phen triggered morphological changes such as shrinkage and plasma membrane disintegration, as well as a decrease in mitochondrial activity and an increase in LDH leakage. To understand intracellular issues relating to 2-Cl-Phen, this study focused on the expression levels of ER stress-inducible genes and several oncogenic genes. Serum and glucose deprivation significantly induced a variety of ER stress-inducible genes, but a 12-h treatment of 2-Cl-Phen down-regulated expression of several ER stress-related genes, with the exception of GADD153. Interestingly, the expression levels of ATF6α, GRP78, MANF, and CRELD2 mRNA were almost completely decreased by 2-Cl-Phen. This study also observed that a 24-h treatment of 2-Cl-Phen attenuated the expression levels of GRP78, GADD153, and c-Myc protein. The decrease in c-Myc protein occurred before the fluctuation of GRP78 protein, while the expression of c-Myc mRNA showed little change with cotreatment of serum and glucose deprivation with 2-Cl-Phen. To further understand the 2-Cl-Phen-induced down-regulation of ATF6-related genes, this study investigated the stability of ATF6α and GRP78 proteins using NanoLuc-tagged constructs. The expression levels of NanoLuc-tagged ATF6α and GRP78 were significantly down-regulated by 2-Cl-Phen in the presence or absence of the translation inhibitor cycloheximide. Taken together, our novel phenformin derivative 2-Cl-Phen has the unique characteristic of diminishing tumor adaptive responses, especially the expression of ATF6-related genes, as well as that of c-Myc protein, in a transcriptional and posttranscriptional manner under a serum- and glucose-deprived condition. Further characterization of cytotoxic mechanisms related to phenformin derivatives may give new insights into developing additional promising anticancer agents.


Asunto(s)
Neoplasias del Colon/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucosa , Proteínas de Neoplasias/biosíntesis , Fenformina , Línea Celular Tumoral , Neoplasias del Colon/patología , Chaperón BiP del Retículo Endoplásmico , Humanos , Fenformina/análogos & derivados , Fenformina/farmacología
19.
Org Biomol Chem ; 14(6): 2090-111, 2016 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-26779679

RESUMEN

Triostin A is a biosynthetic precursor of echinomycin which is one of the most potent hypoxia inducible factor 1 (HIF-1) inhibitors. An improved solution-phase synthesis of triostin A on a preparative scale has been achieved in 17.5% total yield in 13 steps. New analogues of triostin A with various aromatic chromophores, oxidized intra-peptide disulfide bridges and diastereoisomeric cyclic depsipeptide cores were also successfully synthesized. All analogues had a significant inhibitory effect on HIF-1 transcriptional activation in hypoxia and cytotoxicity on MCF-7 cells, with the exception of the derivatives containing a naphthalene chromophore or a thiosulfonate bridge. For the first time, triostin A, echinomycin and the thiosulfinate analogue of triostin A have been revealed to inhibit not only DNA binding of HIF-1 but also HIF-1α protein accumulation in MCF-7 cells. Furthermore, the thiosulfinate analogue and triostin A exhibited a hypoxia-selective cytotoxicity on MCF-7 cells. The improved solution-phase synthetic procedure described herein will contribute to the development of diverse bicyclic depsipeptide drug candidates with the potential to act as novel anti-cancer agents targeting hypoxic tumor microenvironments.


Asunto(s)
Antineoplásicos/farmacología , Hipoxia de la Célula/efectos de los fármacos , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , ADN de Neoplasias/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HEK293 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Células MCF-7 , Modelos Moleculares , Estructura Molecular , Unión Proteica/efectos de los fármacos , Quinoxalinas/síntesis química , Quinoxalinas/química , Quinoxalinas/farmacología , Soluciones , Relación Estructura-Actividad
20.
Graefes Arch Clin Exp Ophthalmol ; 254(12): 2373-2385, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27572140

RESUMEN

PURPOSE: Retinal hypoxia plays a crucial role in ocular neovascular diseases, such as diabetic retinopathy, retinopathy of prematurity, and retinal vascular occlusion. Fluorescein angiography is useful for identifying the hypoxia extent by detecting non-perfusion areas or neovascularization, but its ability to detect early stages of hypoxia is limited. Recently, in vivo fluorescent probes for detecting hypoxia have been developed; however, these have not been extensively applied in ophthalmology. We evaluated whether a novel donor-excited photo-induced electron transfer (d-PeT) system based on an activatable hypoxia-selective near-infrared fluorescent (NIRF) probe (GPU-327) responds to both mild and severe hypoxia in various ocular ischemic diseases animal models. METHODS: The ocular fundus examination offers unique opportunities for direct observation of the retina through the transparent cornea and lens. After injection of GPU-327 in various ocular hypoxic diseases of mouse and rabbit models, NIRF imaging in the ocular fundus can be performed noninvasively and easily by using commercially available fundus cameras. To investigate the safety of GPU-327, electroretinograms were also recorded after GPU-327 and PBS injection. RESULT: Fluorescence of GPU-327 increased under mild hypoxic conditions in vitro. GPU-327 also yielded excellent signal-to-noise ratio without washing out in vivo experiments. By using near-infrared region, GPU-327 enables imaging of deeper ischemia, such as choroidal circulation. Additionally, from an electroretinogram, GPU-327 did not cause neurotoxicity. CONCLUSIONS: GPU-327 identified hypoxic area both in vivo and in vitro.


Asunto(s)
Coroides/irrigación sanguínea , Endotelio Vascular/patología , Hipoxia/complicaciones , Enfermedades de la Retina/diagnóstico , Espectrometría de Fluorescencia/métodos , Espectroscopía Infrarroja Corta/métodos , Animales , Células Cultivadas , Coroides/patología , Modelos Animales de Enfermedad , Electrorretinografía , Angiografía con Fluoresceína , Colorantes Fluorescentes/farmacología , Fondo de Ojo , Humanos , Hipoxia/diagnóstico , Ratones , Ratones Endogámicos C57BL , Conejos , Reproducibilidad de los Resultados , Enfermedades de la Retina/etiología
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