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1.
Cancer Sci ; 112(11): 4655-4668, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34533854

RESUMO

Platinum-based regimens are the most widely used chemotherapy regimens, but cancer cells often develop resistance, which impedes therapy outcome for patients. Previous studies have shown that fibroblast growth factor 13 (FGF13) is associated with resistance to platinum drugs in HeLa cells. However, the mechanism and universality of this effect have not been clarified. Here, we found that FGF13 was associated with poor platinum-based chemotherapy outcomes in a variety of cancers, such as lung, endometrial, and cervical cancers, through bioinformatics analysis. We then found that FGF13 simultaneously regulates the expression and distribution of hCTR1 and ATP7A in cancer cells, causes reduced platinum influx, and promotes platinum sequestration and efflux upon cisplatin exposure. We subsequently observed that FGF13-mediated platinum resistance requires the microtubule-stabilizing effect of FGF13. Only overexpression of FGF13 with the -SMIYRQQQ- tubulin-binding domain could induce the platinum resistance effect. This phenomenon was also observed in SK-MES-1 cells, KLE cells, and 5637 cells. Our research reveals the mechanism of FGF13-induced platinum drug resistance and suggests that FGF13 can be a sensibilization target and prognostic biomarker for chemotherapy.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Transportador de Cobre 1/metabolismo , ATPases Transportadoras de Cobre/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fatores de Crescimento de Fibroblastos/fisiologia , Células A549 , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Cisplatino/metabolismo , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Microtúbulos/efeitos dos fármacos , Compostos de Platina/metabolismo , Compostos de Platina/farmacologia , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/metabolismo
2.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064906

RESUMO

Nerve growth factor (NGF) is a protein essential to neurons survival, which interacts with its receptor as a non-covalent dimer. Peptides belonging to NGF N-terminal domain are able to mimic the activity of the whole protein. Such activity is affected by the presence of copper ions. The metal is released in the synaptic cleft where proteins, not yet identified, may bind and transfer to human copper transporter 1 (hCtr1), for copper uptake in neurons. The measurements of the stability constants of copper complexes formed by amyloid beta and hCtr1 peptide fragments suggest that beta-amyloid (Aß) can perform this task. In this work, the stability constant values of copper complex species formed with the dimeric form of N-terminal domain, sequence 1-15 of the protein, were determined by means of potentiometric measurements. At physiological pH, NGF peptides bind one equivalent of copper ion with higher affinity of Aß and lower than hCtr1 peptide fragments. Therefore, in the synaptic cleft, NGF may act as a potential copper chelating molecule, ionophore or chaperone for hCtr1 for metal uptake. Copper dyshomeostasis and mild acidic environment may modify the balance between metal, NGF, and Aß, with consequences on the metal cellular uptake and therefore be among causes of the Alzheimer's disease onset.


Assuntos
Transportador de Cobre 1/metabolismo , Cobre/metabolismo , Fator de Crescimento Neural/metabolismo , Fragmentos de Peptídeos/metabolismo , Sítios de Ligação , Humanos , Ligação Proteica
3.
ChemistryOpen ; 10(4): 486-492, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33908707

RESUMO

The Cu(II)-diacetyl-bis (N4-methylthiosemicarbazone) complex (ATSM-Cu(II)) has been suggested as a promising positron emission tomography (PET) agent for hypoxia imaging. Several in-vivo studies have shown its potential to detect hypoxic tumors. However, its uptake mechanism and its specificity to various cancer cell lines have been less studied. Herein, we tested ATSM-Cu(II) toxicity, uptake, and reduction, using four different cell types: (1) mouse breast cancer cells (DA-3), (2) human embryonic kidney cells (HEK-293), (3) breast cancer cells (MCF-7), and (4) cervical cancer cells (Hela) under normoxic and hypoxic conditions. We showed that ATSM-Cu(II) is toxic to breast cancer cells under normoxic and hypoxic conditions; however, it is not toxic to normal HEK-293 non-cancer cells. We showed that the Cu(I) content in breast cancer cell after treatment with ATSM-Cu(II) under hypoxic conditions is higher than in normal cells, despite that the uptake of ATSM-Cu(II) is a bit higher in normal cells than in breast cancer cells. This study suggests that the redox potential of ATSM-Cu(II) is higher in breast cancer cells than in normal cells; thus, its toxicity to cancer cells is increased.


Assuntos
Hipóxia/metabolismo , Compostos Organometálicos/metabolismo , Tiossemicarbazonas/metabolismo , Animais , Linhagem Celular Tumoral , Complexos de Coordenação , Radioisótopos de Cobre/química , Radioisótopos de Cobre/metabolismo , Transportador de Cobre 1/metabolismo , Células HEK293 , Humanos , Camundongos , Compostos Organometálicos/química , Compostos Organometálicos/toxicidade , Oxirredução , Tiossemicarbazonas/química , Tiossemicarbazonas/toxicidade
4.
Int J Nanomedicine ; 16: 2071-2085, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33727814

RESUMO

Background: Radiation therapy remains an important treatment modality in cancer therapy, however, resistance is a major problem for treatment failure. Elevated expression of glutathione is known to associate with radiation resistance. We used glutathione overexpressing small cell lung cancer cell lines, SR3A-13 and SR3A-14, established by transfection with γ-glutamylcysteine synthetase (γ-GCS) cDNA, as a model for investigating strategies of overcoming radiation resistance. These radiation-resistant cells exhibit upregulated human copper transporter 1 (hCtr1), which also transports cisplatin. This study was initiated to investigate the effect and the underlying mechanism of iron-platinum nanoparticles (FePt NPs) on radiation sensitization in cancer cells. Materials and Methods: Uptakes of FePt NPs in these cells were studied by plasma optical emission spectrometry and transmission electron microscopy. Effects of the combination of FePt NPs and ionizing radiation were investigated by colony formation assay and animal experiment. Intracellular reactive oxygen species (ROS) were assessed by using fluorescent probes and imaged by a fluorescence-activated-cell-sorting caliber flow cytometer. Oxygen consumption rate (OCR) in mitochondria after FePt NP and IR treatment was investigated by a Seahorse XF24 cell energy metabolism analyzer. Results: These hCtr1-overexpressing cells exhibited elevated resistance to IR and the resistance could be overcome by FePt NPs via enhanced uptake of FePt NPs. Overexpression of hCtr1 was responsible for the increased uptake/transport of FePt NPs as demonstrated by using hCtr1-transfected parental SR3A (SR3A-hCtr1-WT) cells. Increased ROS and drastic mitochondrial damages with substantial reduction of oxygen consumption rate were observed in FePt NPs and IR-treated cells, indicating that structural and functional insults of mitochondria are the lethal mechanism of FePt NPs. Furthermore, FePt NPs also increased the efficacy of radiotherapy in mice bearing SR3A-hCtr1-WT-xenograft tumors. Conclusion: These results suggest that FePt NPs can potentially be a novel strategy to improve radiotherapeutic efficacy in hCtr1-overexpressing cancer cells via enhanced uptake and mitochondria targeting.


Assuntos
Ligas/farmacologia , Transportador de Cobre 1/metabolismo , Ferro/farmacologia , Nanopartículas Metálicas/química , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Platina/farmacologia , Tolerância a Radiação , Aerobiose , Animais , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Nanopartículas Metálicas/ultraestrutura , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Modelos Biológicos , Tolerância a Radiação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Resultado do Tratamento , Raios X
5.
Metallomics ; 12(12): 1995-2008, 2020 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-33146201

RESUMO

Hepatocellular carcinoma (HCC), the most common primary liver cancer, of which ∼800 000 new cases will be diagnosed worldwide this year, portends a five-year survival rate of merely 17% in patients with unresectable disease. This dismal prognosis is due, at least in part, from the late stage of diagnosis and the limited efficacy of systemic therapies. As a result, there is an urgent need to identify risk factors that contribute to HCC initiation and provide targetable vulnerabilities to improve patient survival. While myriad risk factors are known, elevated copper (Cu) levels in HCC patients and the incidence of hepatobiliary malignancies in Wilson disease patients, which exhibit hereditary liver Cu overload, suggests the possibility that metal accumulation promotes malignant transformation. Here we found that expression of the Cu transporter genes ATP7A, ATP7B, SLC31A1, and SLC31A2 was significantly altered in liver cancer samples and were associated with elevated Cu levels in liver cancer tissue and cells. Further analysis of genomic copy number data revealed that alterations in Cu transporter gene loci correlate with poorer survival in HCC patients. Genetic loss of the Cu importer SLC31A1 (CTR1) or pharmacologic suppression of Cu decreased the viability, clonogenic survival, and anchorage-independent growth of human HCC cell lines. Mechanistically, CTR1 knockdown or Cu chelation decreased glycolytic gene expression and downstream metabolite utilization and as a result forestalled tumor cell survival after exposure to hypoxia, which mimics oxygen deprivation elicited by transarterial embolization, a standard-of-care therapy used for patients with unresectable HCC. Taken together, these findings established an association between altered Cu homeostasis and HCC and suggest that limiting Cu bioavailability may provide a new treatment strategy for HCC by restricting the metabolic reprogramming necessary for cancer cell survival.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Quelantes/farmacologia , Cobre/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Molibdênio/farmacologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Transportador de Cobre 1/metabolismo , ATPases Transportadoras de Cobre/metabolismo , Homeostase/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/metabolismo , Proteínas SLC31/metabolismo
6.
Inorg Chem ; 59(23): 16952-16966, 2020 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-33211469

RESUMO

Amyloid beta (Aß) peptides are notorious for their involvement in Alzheimer's disease (AD), by virtue of their propensity to aggregate to form oligomers, fibrils, and eventually plaques in the brain. Nevertheless, they appear to be essential for correct neurophysiology on the synaptic level and may have additional functions including antimicrobial activity, sealing the blood-brain barrier, promotion of recovery from brain injury, and even tumor suppression. Aß peptides are also avid copper chelators, and coincidentally copper is significantly dysregulated in the AD brain. Copper (Cu) is released in significant amounts during calcium signaling at the synaptic membrane. Aß peptides may have a role in maintaining synaptic Cu homeostasis, including as a scavenger for redox-active Cu and as a chaperone for clearing Cu from the synaptic cleft. Here, we employed the Aß1-16 and Aß4-16 peptides as well-established non-aggregating models of major Aß species in healthy and AD brains, and the Ctr1-14 peptide as a model for the extracellular domain of the human cellular copper transporter protein (Ctr1). With these model peptides and a number of spectroscopic techniques, we investigated whether the Cu complexes of Aß peptides could provide Ctr1 with either Cu(II) or Cu(I). We found that Aß1-16 fully and rapidly delivered Cu(II) to Ctr1-14 along the affinity gradient. Such delivery was only partial for the Aß4-16/Ctr1-14 pair, in agreement with the higher complex stability for the former peptide. Moreover, the reaction was very slow and took ca. 40 h to reach equilibrium under the given experimental conditions. In either case of Cu(II) exchange, no intermediate (ternary) species were present in detectable amounts. In contrast, both Aß species released Cu(I) to Ctr1-14 rapidly and in a quantitative fashion, but ternary intermediate species were detected in the analysis of XAS data. The results presented here are the first direct evidence of a Cu(I) and Cu(II) transfer between the human Ctr1 and Aß model peptides. These results are discussed in terms of the fundamental difference between the peptides' Cu(II) complexes (pleiotropic ensemble of open structures of Aß1-16 vs the rigid closed-ring system of amino-terminal Cu/Ni binding Aß4-16) and the similarity of their Cu(I) complexes (both anchored at the tandem His13/His14, bis-His motif). These results indicate that Cu(I) may be more feasible than Cu(II) as the cargo for copper clearance from the synaptic cleft by Aß peptides and its delivery to Ctr1. The arguments in favor of Cu(I) include the fact that cellular Cu export and uptake proteins (ATPase7A/B and Ctr1, respectively) specifically transport Cu(I), the abundance of extracellular ascorbate reducing agent in the brain, and evidence of a potential associative (hand-off) mechanism of Cu(I) transfer that may mirror the mechanisms of intracellular Cu chaperone proteins.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Transportador de Cobre 1/metabolismo , Cobre/metabolismo , Peptídeos beta-Amiloides/química , Cobre/química , Transportador de Cobre 1/química , Humanos , Espectrometria de Fluorescência
7.
Chem Commun (Camb) ; 56(81): 12194-12197, 2020 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-32914794

RESUMO

Employing peptide-based models of copper transporter 1 (CTR1), we show that the trimeric arrangement of its N-terminus tunes its reactivity with Cu, promoting Cu(ii) reduction and stabilizing Cu(i). Hence, the employed multimeric models of CTR1 provide an important contribution to studies on early steps of Cu uptake by cells.


Assuntos
Transportador de Cobre 1/metabolismo , Cobre/metabolismo , Sítios de Ligação , Cobre/química , Transportador de Cobre 1/química , Humanos , Modelos Moleculares , Estrutura Molecular , Oxirredução
8.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 51(5): 643-649, 2020 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-32975078

RESUMO

Objective: To examine copper transporter 1 (CTR1) expression in pancreatic carcinoma cells, orthotopic xenograft pancreatic tumor model and clinical samples, and verify the effect of copper chelating agent ammonium tetrathiomolybdate (TM) regulate the expression of CTR1 in pancreatic carcinoma cells and the inhibition of pancreatic carcinoma. Methods: The expressions of copper transporter CTR1 and antioxidant protein 1 (ATOX1) in 22 clinical pancreatic ductal carcinoma and paracancer tissues 0.5-1 cm away from the tumor were measured by immunohistochemistry (IHC). PANC-1 cells were used to construct 5 orthotopic xenograft pancreatic tumor of nude mice models. Pancreatic cancer tissues and corresponding normal pancreatic tissues were collected, and the expressions of CTR1 and ATOX1 were detected by IHC and compared with clinical tissues. The proliferation of pancreatic carcinoma cells PANC-1 treated with 10, 30, 50, 100 µmol/L TM for 24 h, 48 h, 72 h was measured by CCK8 assay. The migration abilities of PANC-1 cells treated with 50 µmol/L TM for 24 h, 48 h were detected by scratch test. The expressions of CTR1, vascular endothelial growth factor (VEGF) and CyclinD1 proteins in PANC-1 cells treated with 10, 30, 50, 100 µmol/L TM for 48 h were measured by Western blot. Then the subcutaneous tumor-bearing model of nude mice were established with PANC-1 cells, and the growth of tumor was observed after oral administration of 0.3 mg/d and 1.0 mg/d of TM, respectively. Results: The immunohistochemical results indicated that 19 of the 22 clinical pancreatic ductal cancer tissues of carcinoma patients had high expression of CTR1, and the same high expression of CTR1 was found in the orthotopic transplanted tumor tissues of PANC-1 nude mice. The proliferation inhibition of PANC-1 cells increased with the concentration of TM increased and the treatment time prolonged. The expressions of intracellular CTR1, VEGF and CyclinD1 all decreased with the concentration of TM increased. The cell migration ability decreased after the PANC-1 cells treated with TM. The tumor growth of PANC-1 tumor-bearing nude mice was inhibited after different doses of TM were delivered. The reduction in tumor volume and weight was more pronounced in the high-dose TM group (P<0.05). Conclusion: The expression of CTR1 is abnormally elevated in pancreatic carcinoma, and treatment with copper chelating agent for this target may help to inhibit pancreatic carcinoma.


Assuntos
Compostos de Amônio , Quelantes , Transportador de Cobre 1 , Neoplasias Pancreáticas , Animais , Linhagem Celular Tumoral , Proliferação de Células , Quelantes/farmacologia , Cobre , Transportador de Cobre 1/metabolismo , Transportador de Cobre 1/farmacologia , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/metabolismo
9.
Brain Res Bull ; 164: 339-349, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32795490

RESUMO

BACKGROUND AND PURPOSE: DTNBP1 gene variation and lower dysbindin-1 protein are associated with schizophrenia. Previous evidence suggests that downregulated dysbindin-1 expression results in lower expression of copper transporters ATP7A (intracellular copper transporter) and SLC31A1 (CTR1; extracellular copper transporter), which are required for copper transport across the blood brain barrier. However, whether antipsychotic medications used for schizophrenia treatment may modulate these systems is unclear. EXPERIMENTAL APPROACH: The current study measured behavioral indices of neurological function in dysbindin-1 functional knockout (KO) mice and their wild-type (WT) littermates with or without quetiapine treatment. We assessed serum and brain copper levels, ATP7A and CTR1 mRNA, and copper transporter-expressing cellular population transcripts: TTR (transthyretin; choroid plexus epithelial cells), MBP (myelin basic protein; oligodendrocytes), and GJA1 (gap-junction protein alpha-1; astrocytes) in cortex and hippocampus. KEY RESULTS: Regardless of genotype, quetiapine significantly reduced TTR, MBP, CTR1 mRNA, and serum copper levels. Neurological function of untreated KO mice was abnormal, and ledge instability was rescued with quetiapine. KO mice were hyperactive after 10 min in the open-field assay, which was not affected by treatment. CONCLUSIONS AND IMPLICATIONS: Dysbindin-1 KO results in hyperactivity, altered serum copper, and neurological impairment, the last of which is selectively rescued with quetiapine. Antipsychotic treatment modulates specific cellular populations, affecting myelin, the choroid plexus, and copper transport across the blood brain barrier. Together these results indicate the widespread impact of antipsychotic treatment, and that alteration of dysbindin-1 may be sufficient, but not necessary, for specific schizophrenia pathology.


Assuntos
Encéfalo/metabolismo , Cobre/metabolismo , Disbindina/genética , Esquizofrenia/genética , Animais , Antipsicóticos/farmacologia , Antipsicóticos/uso terapêutico , Encéfalo/efeitos dos fármacos , Transportador de Cobre 1/genética , Transportador de Cobre 1/metabolismo , ATPases Transportadoras de Cobre/genética , ATPases Transportadoras de Cobre/metabolismo , Camundongos , Camundongos Knockout , Fumarato de Quetiapina/farmacologia , Fumarato de Quetiapina/uso terapêutico , Fatores de Risco , Esquizofrenia/tratamento farmacológico , Esquizofrenia/metabolismo
10.
Cancer Res ; 80(19): 4129-4144, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32816860

RESUMO

Therapeutic checkpoint antibodies blocking programmed death receptor 1/programmed death ligand 1 (PD-L1) signaling have radically improved clinical outcomes in cancer. However, the regulation of PD-L1 expression on tumor cells is still poorly understood. Here we show that intratumoral copper levels influence PD-L1 expression in cancer cells. Deep analysis of the The Cancer Genome Atlas database and tissue microarrays showed strong correlation between the major copper influx transporter copper transporter 1 (CTR-1) and PD-L1 expression across many cancers but not in corresponding normal tissues. Copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells and RNA sequencing revealed that copper regulates key signaling pathways mediating PD-L1-driven cancer immune evasion. Conversely, copper chelators inhibited phosphorylation of STAT3 and EGFR and promoted ubiquitin-mediated degradation of PD-L1. Copper-chelating drugs also significantly increased the number of tumor-infiltrating CD8+ T and natural killer cells, slowed tumor growth, and improved mouse survival. Overall, this study reveals an important role for copper in regulating PD-L1 and suggests that anticancer immunotherapy might be enhanced by pharmacologically reducing intratumor copper levels. SIGNIFICANCE: These findings characterize the role of copper in modulating PD-L1 expression and contributing to cancer immune evasion, highlighting the potential for repurposing copper chelators as enhancers of antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4129/F1.large.jpg.


Assuntos
Antígeno B7-H1/metabolismo , Neoplasias Encefálicas/imunologia , Cobre/metabolismo , Neuroblastoma/imunologia , Evasão Tumoral/fisiologia , Animais , Antígeno B7-H1/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Quelantes/farmacologia , Transportador de Cobre 1/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Imunoterapia/métodos , Células Matadoras Naturais , Linfócitos do Interstício Tumoral/patologia , Camundongos Endogâmicos BALB C , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Trietilenofosforamida/farmacologia , Evasão Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Aquat Toxicol ; 226: 105561, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32688145

RESUMO

In the aquatic environment, metals are present as mixtures, therefore studies on mixture toxicity are crucial to thoroughly understand their toxic effects on aquatic organisms. Common carp (Cyprinus carpio) were used to assess the effects of short-term Cu(II) and Cd(II) mixtures, using a fixed concentration of one of the metals, representing 25 % of its individual 96h-LC50 (concentration lethal for 50 % of the population) combined with a variable concentration of the other metal corresponding to 10, 25 or 50 % of its 96h-LC50, and vice versa. Our results showed a fast Cu and Cd bioaccumulation, with the percentage of increase in the order gill > liver > carcass. An inhibitory effect of Cu on Cd uptake was observed; higher Cu concentrations at fixed Cd levels resulted in a decreased accumulation of Cd. The presence of the two metal ions resulted in losses of total Na, K and Ca. Fish tried to compensate for the Na loss through the induction of the genes coding for Na+/K+-ATPase and H+-ATPase. Additionally, a counterintuitive induction of the gene encoding the high affinity copper transporter (CTR1) occurred, while a downregulation was expected to prevent further metal ion uptake. An induction of defensive mechanisms, both metal ion binding protein and anti-oxidant defences, was observed. Despite the metal accumulation and electrolyte loss, the low mortality suggest that common carp is able to cope with these metal levels, at least during a one-week exposure.


Assuntos
Bioacumulação/efeitos dos fármacos , Cádmio/toxicidade , Carpas/metabolismo , Cobre/toxicidade , Homeostase/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Cádmio/metabolismo , Carpas/genética , Cobre/metabolismo , Transportador de Cobre 1/genética , Transportador de Cobre 1/metabolismo , Eletrólitos/metabolismo , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Transporte de Íons , Dose Letal Mediana , Potássio/metabolismo , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Poluentes Químicos da Água/metabolismo
12.
Biochim Biophys Acta Mol Basis Dis ; 1866(10): 165843, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32454166

RESUMO

Age related macular degeneration (AMD) is a multifactorial disease with genetic, biochemical and environmental risk factors. We observed a significant increase in copper levels in choroid-RPE from donor eyeballs with AMD. Adult retinal pigment epithelial cells (ARPE19 cells) exposed to copper in-vitro showed a 2-fold increase in copper influx transporter CTR1 and copper uptake at 50 µM concentration. Further there was 2-fold increase in cytochrome C oxidase activity and a 2-fold increase in the mRNA expression of NRF 2 with copper treatment. There was a significant increase in mitochondrial biogenesis markers PGC1ß and TFAM which was confirmed by mitochondrial mass and copy number. On the contrary, in AMD choroid-RPE, the CTR1 mRNA was found to be significantly down-regulated compared to its respective controls. SCO1 and PGC1ß mRNA showed an increase in choroid-RPE. Our study proposes copper to play an important role in mitochondrial biogenesis in RPE cells.


Assuntos
Cobre/metabolismo , Células Epiteliais/metabolismo , Degeneração Macular/metabolismo , Mitocôndrias/metabolismo , Biogênese de Organelas , Epitélio Pigmentado da Retina/metabolismo , Pigmentos da Retina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular , Corioide/metabolismo , Cobre/farmacologia , Transportador de Cobre 1/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Degeneração Macular/patologia , Masculino , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/patologia , Pigmentos da Retina/genética , Fatores de Transcrição/metabolismo
13.
J Cell Mol Med ; 24(9): 5274-5289, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32207235

RESUMO

Chemoresistance is the main obstacle of treatment in patients with osteosarcoma. RNA-binding protein PTBP1 has been identified as an oncogene in various cancers. However, the role of PTBP1 in osteosarcoma, especially in chemoresistant osteosarcoma, and the underlying mechanism remain unclear. In this study, we aimed to explore the functions of PTBP1 in chemoresistance of osteosarcoma. We found that PTBP1 was significantly increased in chemotherapeutically insensitive osteosarcoma tissues and cisplatin-resistant osteosarcoma cell lines (MG-63CISR and U-2OSCISR ) as compared to chemotherapy-sensitive osteosarcoma tissues and cell lines. Knock-down of PTBP1 can enhance the anti-proliferation and apoptosis-induced effects of cisplatin in MG-63CISR and U-2OSCISR cells. Moreover, PTBP1 knock-down significantly up-regulated the expression of the copper transporter SLC31A1, as indicated by transcriptome sequencing. Through RNA immunoprecipitation, dual-luciferase reporter assay and RNA stability detection, we confirmed that PTBP1 binds to SLC31A1 mRNA and regulates the expression level of SLC31A1 by affecting mRNA stability. Additionally, SLC31A1 silencing abrogated the chemosensitizing effect of PTBP1 knock-down in MG-63CISR and U-2OSCISR cells. Using a nude mouse xenograft model, we further confirmed that PTBP1 knock-down enhanced chemoresistant osteosarcoma responsiveness to cisplatin treatment in vivo. Collectively, the present study suggests that PTBP1 is a crucial determinant of chemoresistance in osteosarcoma.


Assuntos
Cisplatino/uso terapêutico , Transportador de Cobre 1/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Adolescente , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Criança , Cisplatino/farmacologia , Transportador de Cobre 1/metabolismo , Regulação para Baixo/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Inativação Gênica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Masculino , Camundongos Nus , Osteossarcoma/patologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Adulto Jovem
14.
Hear Res ; 388: 107893, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-32006874

RESUMO

BACKGROUND: Antitumor agents based on platinum have gained a well-established place in the treatment of several forms of cancer. Their efficiency is hampered by serious toxic effects against healthy tissues as well. Ototoxicity is a serious side effect leading to hearing impairment and represents an important issue affecting the patients' quality of life. The currently used platinum chemotherapeutics exert different toxicity towards cochlear cells. The aim of our study was to answer some questions regarding the differential uptake and cellular pharmacodynamics of Cisplatin (CDDP), Carboplatin (CBDCA) and Oxaliplatin (L-OHP) in the HEI-OC1 cochlear cell line. METHODS: We studied the expression of copper transporters CTR1, ATP7A and ATP7B which are presumably involved in the uptake, cellular transport and efflux of platinum compounds by immunofluorescence microscopy and flow-cytometry. The cellular uptake of the compounds was evaluated through the determination of intracellular platinum concentration by atomic absorption spectroscopy. The effects of the treatment of HEI-OC1 cells with platinum compounds were also evaluated: cytotoxicity with the Cell Titer Blue viability test, formation of reactive oxygen species with 2',7' -dichlorofluorescein diacetate, genotoxicity with the comet assay and apoptosis with the cleaved PARP ELISA test. RESULTS: CTR1, ATP7A and ATP7B were all expressed by HEI-OC1 cells. The treatment with the platinum compounds led to a modulation of their expression, manifested in a differential platinum uptake. Treatment with Cisplatin led to the highest intracellular concentration of platinum compared to Oxaliplatin and Carboplatin at the same dose. Treatment with CuSO4 reduced platinum uptake of all the compounds, significantly in the case of Cisplatin and Carboplatin. CDDP was the most cytotoxic against HEI-OC1 cells, with an IC50 = 65.79  µM, compared to 611.7 µM for L-OHP and 882.9 µM for CBDCA, at the same molar concentration. The production of ROS was the most intense after CDDP, followed by L-OHP and CBDCA. In the comet assay, at the 100 µM concentration, L-OHP and CBDCA induced DNA adducts while CDDP induced adducts as well as DNA strand breaks. CBDCA and L-OHP lead to a significant increase of cleaved PARP at 24h (p < 0.001), suggesting an important apoptotic process induced by these compounds at the used concentrations. CONCLUSIONS: The results obtained in the current study suggest that the modulation of copper transporters locally may represent a new strategy against platinum drugs ototoxicity.


Assuntos
Antineoplásicos/toxicidade , Carboplatina/toxicidade , Cisplatino/toxicidade , Cóclea/efeitos dos fármacos , Transportador de Cobre 1/metabolismo , ATPases Transportadoras de Cobre/metabolismo , Cobre/metabolismo , Oxaliplatina/toxicidade , Animais , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Carboplatina/metabolismo , Linhagem Celular , Cisplatino/metabolismo , Cóclea/metabolismo , Cóclea/patologia , Relação Dose-Resposta a Droga , Camundongos , Ototoxicidade , Oxaliplatina/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo
15.
World J Biol Psychiatry ; 21(1): 13-28, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30230404

RESUMO

Objectives: Several schizophrenia brain regions exhibit decreased dysbindin. Dysbindin modulates copper transport crucial for myelination, monoamine metabolism and cellular homeostasis. Schizophrenia patients (SZP) exhibit increased plasma copper, while copper-decreasing agents produce schizophrenia-like behavioural and pathological abnormalities. Therefore, we sought to determine dysbindin and copper transporter protein expression and copper content in SZP.Methods: We studied the copper-rich substantia nigra (SN) using Western blot and inductively-coupled plasma mass spectrometry. We characterised specific protein domains of copper transporters ATP7A, CTR1, ATP7B and dysbindin isoforms 1 A and 1B/C in SZP (n = 15) and matched controls (n = 11), and SN copper content in SZP (n = 14) and matched controls (n = 11). As a preliminary investigation, we compared medicated (ON; n = 11) versus unmedicated SZP (OFF; n = 4).Results: SZP exhibited increased C terminus, but not N terminus, ATP7A. SZP expressed less transmembrane CTR1 and dysbindin 1B/C than controls. ON exhibited increased C terminus ATP7A protein versus controls. OFF exhibited less N terminus ATP7A protein than controls and ON, suggesting medication-induced rescue of the ATP7A N terminus. SZP exhibited less SN copper content than controls.Conclusions: These results provide the first evidence of disrupted copper transport in schizophrenia SN that appears to result in a copper-deficient state. Furthermore, copper homeostasis may be modulated by specific dysbindin isoforms and antipsychotic treatment.


Assuntos
Encéfalo/patologia , Transportador de Cobre 1/metabolismo , ATPases Transportadoras de Cobre/metabolismo , Cobre/metabolismo , Disbindina/metabolismo , Esquizofrenia/genética , Estudos de Casos e Controles , Cobre/deficiência , Humanos , Espectrometria de Massas , Substância Negra/metabolismo
16.
Curr Genet ; 66(3): 531-548, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31784768

RESUMO

While it is known that ScRad9 DNA damage checkpoint protein is recruited to damaged DNA by recognizing specific histone modifications, here we report a different way of Rad9 recruitment on chromatin under non DNA damaging conditions. We found Rad9 to bind directly with the copper-modulated transcriptional activator Mac1, suppressing both its DNA binding and transactivation functions. Rad9 was recruited to active Mac1-target promoters (CTR1, FRE1) and along CTR1 coding region following the association pattern of RNA polymerase (Pol) II. Hir1 histone chaperone also interacted directly with Rad9 and was partly required for its localization throughout CTR1 gene. Moreover, Mac1-dependent transcriptional initiation was necessary and sufficient for Rad9 recruitment to the heterologous ACT1 coding region. In addition to Rad9, Rad53 kinase also localized to CTR1 coding region in a Rad9-dependent manner. Our data provide an example of a yeast DNA-binding transcriptional activator that interacts directly with a DNA damage checkpoint protein in vivo and is functionally restrained by this protein, suggesting a new role for Rad9 in connecting factors of the transcription machinery with the DNA repair pathway under unchallenged conditions.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Dano ao DNA , Reparo do DNA , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Transportador de Cobre 1/genética , Transportador de Cobre 1/metabolismo , FMN Redutase/genética , FMN Redutase/metabolismo , Proteínas Nucleares/genética , Fosforilação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
17.
Nat Commun ; 10(1): 5080, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31704944

RESUMO

Hyperaccumulators typically refer to plants that absorb and tolerate elevated amounts of heavy metals. Due to their unique metal trafficking abilities, hyperaccumulators are promising candidates for bioremediation applications. However, compared to bacteria-based bioremediation systems, plant life cycle is long and growing conditions are difficult to maintain hindering their adoption. Herein, we combine the robust growth and engineerability of bacteria with the unique waste management mechanisms of plants by using a more tractable platform-the common baker's yeast-to create plant-like hyperaccumulators. Through overexpression of metal transporters and engineering metal trafficking pathways, engineered yeast strains are able to sequester metals at concentrations 10-100 times more than established hyperaccumulator thresholds for chromium, arsenic, and cadmium. Strains are further engineered to be selective for either cadmium or strontium removal, specifically for radioactive Sr90. Overall, this work presents a systematic approach for transforming yeast into metal hyperaccumulators that are as effective as their plant counterparts.


Assuntos
Proteínas de Transporte/genética , Engenharia Metabólica/métodos , Metais Pesados/metabolismo , Saccharomyces cerevisiae/genética , Antiporters/genética , Antiporters/metabolismo , Arsênio/metabolismo , Biodegradação Ambiental , Cádmio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Cromo/metabolismo , Proteínas de Transporte de Cobre/genética , Proteínas de Transporte de Cobre/metabolismo , Transportador de Cobre 1/genética , Transportador de Cobre 1/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas SLC31/genética , Proteínas SLC31/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estrôncio/metabolismo , Radioisótopos de Estrôncio/metabolismo
18.
FASEB J ; 33(12): 14325-14336, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31661638

RESUMO

Cisplatin (CP) is one of the most effective chemotherapeutics in the treatment of human cancers. However, the beneficial effects of CP are limited by the toxic effects, especially nephrotoxicity. Fluorofenidone (AKFPD) is a promising multifunctional antifibrosis pyridinone drug discovered by our group. But there is no evidence of its protective effects against acute kidney injury (AKI). Therefore, we investigated the protective effects of AKFPD on CP-induced AKI in vivo and in vitro. Compared with the model group, treatment with AKFPD effectively ameliorated kidney damages. In order to elucidate the mechanisms, we discovered that AKFPD treatment notably alleviated generation of reactive oxygen species, reduced the phosphorylation levels of MAPKs (ERK1 and 2, JNKs, and p38), suppressed inflammatory response, inhibited apoptosis, and abated the expression of CP transporters (organic cation transporter 2 and copper transport protein 1) compared with the model group. Moreover, because renal ischemia reperfusion injury (IRI)-induced AKI and LPS-induced AKI are the major models representative of renal transplantation-correlated AKI and sepsis-related AKI, which are also the main causes of AKI, we have also proved the effectiveness of AKFPD on these models. In conclusion, these findings suggest that AKFPD is a potent drug for CP-, IRI-, and LPS-caused AKI and elucidate the underlying mechanism.-Jiang, Y., Quan, J., Chen, Y., Liao, X., Dai, Q., Lu, R., Yu, Y., Hu, G., Li, Q., Meng, J., Xie, Y., Peng, Z., Tao, L. Fluorofenidone protects against acute kidney injury.


Assuntos
Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/prevenção & controle , Cisplatino/toxicidade , Piridonas/farmacologia , Animais , Antineoplásicos/toxicidade , Linhagem Celular , Transportador de Cobre 1/genética , Transportador de Cobre 1/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/prevenção & controle , Rim/citologia , Rim/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Transportador 2 de Cátion Orgânico/genética , Transportador 2 de Cátion Orgânico/metabolismo , Peroxidase/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio , Traumatismo por Reperfusão
19.
Chem Commun (Camb) ; 55(74): 11107-11110, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31461100

RESUMO

Cu(i) binds to the N-terminal metal binding domain (MBD) of hCTR1 and induces its conformational change, which promotes the interaction of the MBD with cell membranes. The membrane interaction was confirmed in living cells. This process could be the first step to initiate the cellular uptake of copper ions by hCTR1.


Assuntos
Membrana Celular/metabolismo , Transportador de Cobre 1/metabolismo , Cobre/metabolismo , Lipossomos/metabolismo , Fragmentos de Peptídeos/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Linhagem Celular Tumoral , Humanos , Micelas , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos , Dodecilsulfato de Sódio/metabolismo
20.
Proc Natl Acad Sci U S A ; 116(37): 18285-18294, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31451653

RESUMO

Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes.


Assuntos
Cobre/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Glutationa/metabolismo , Técnicas de Sonda Molecular , Oncogenes/fisiologia , Transportador de Cobre 1/metabolismo , Fluoresceína , Células HEK293 , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Neoplasias/metabolismo , Oxirredução , Estresse Oxidativo , Rodaminas , Transdução de Sinais
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