RESUMO
Alzheimer's Disease (AD) is complicated by pro-oxidant intraneuronal Fe(2+) elevation as well as extracellular Zn(2+) accumulation within amyloid plaque. We found that the AD ß-amyloid protein precursor (APP) possesses ferroxidase activity mediated by a conserved H-ferritin-like active site, which is inhibited specifically by Zn(2+). Like ceruloplasmin, APP catalytically oxidizes Fe(2+), loads Fe(3+) into transferrin, and has a major interaction with ferroportin in HEK293T cells (that lack ceruloplasmin) and in human cortical tissue. Ablation of APP in HEK293T cells and primary neurons induces marked iron retention, whereas increasing APP695 promotes iron export. Unlike normal mice, APP(-/-) mice are vulnerable to dietary iron exposure, which causes Fe(2+) accumulation and oxidative stress in cortical neurons. Paralleling iron accumulation, APP ferroxidase activity in AD postmortem neocortex is inhibited by endogenous Zn(2+), which we demonstrate can originate from Zn(2+)-laden amyloid aggregates and correlates with Aß burden. Abnormal exchange of cortical zinc may link amyloid pathology with neuronal iron accumulation in AD.
Assuntos
Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/metabolismo , Ceruloplasmina/antagonistas & inibidores , Zinco/metabolismo , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Animais , Linhagem Celular , Ceruloplasmina/química , Ceruloplasmina/metabolismo , Humanos , Ferro/metabolismo , Camundongos , Alinhamento de SequênciaRESUMO
The strongest genetic risk factor for late-onset Alzheimer's disease (AD) is allelic variation of the APOE gene, with the following risk structure: ε4 > ε3 > ε2. The biochemical basis for this risk profile is unclear. Here, we reveal a new role for the APOE gene product, apolipoprotein E (ApoE) in regulating cellular copper homeostasis, which is perturbed in the AD brain. Exposure of ApoE target replacement (TR) astrocytes (immortalised astrocytes from APOE knock-in mice) to elevated copper concentrations resulted in exacerbated copper accumulation in ApoE4- compared to ApoE2- and ApoE3-TR astrocytes. This effect was also observed in SH-SY5Y neuroblastoma cells treated with conditioned medium from ApoE4-TR astrocytes. Increased intracellular copper levels in the presence of ApoE4 may be explained by reduced levels and delayed trafficking of the copper transport protein, copper-transporting ATPase 1 (ATP7A/Atp7a), potentially leading to impaired cellular copper export. This new role for ApoE in copper regulation lends further biochemical insight into how APOE genotype confers risk for AD and reveals a potential contribution of ApoE4 to the copper dysregulation that is a characteristic pathological feature of the AD brain.
RESUMO
The therapeutic utility of the copper ionophore disulfiram was investigated in a diet-induced obesity mouse model (C57BL/6J background), both through administration in feed (0.05 to 1% (w/w)) and via oral gavage (150 mg/kg) for up to eight weeks. Mice were monitored for body weight, fat deposition (perigonadal fat pads), metabolic changes (e.g., glucose dyshomeostasis) and pathologies (e.g., hepatic steatosis, hyperglycaemia and hypertriglyceridemia) associated with a high-fat diet. Metal-related pharmacological effects across major organs and serums were investigated using inductively coupled plasma mass spectrometry (ICP-MS). Disulfiram treatments (all modes) augmented hepatic copper in mice, markedly moderated body weight and abolished the deleterious systemic changes associated with a high-fat diet. Likewise, another chemically distinct copper ionophore H2(gtsm), administered daily (oral gavage), also augmented hepatic copper and moderated mouse body weight. Postmortem histological examinations of the liver and other major organs, together with serum aminotransferases, supported the reported therapeutic safety of disulfiram. Disulfiram specifically altered systemic copper in mice and altered hepatic copper metabolism, perturbing the incorporation of copper into ceruloplasmin (holo-ceruloplasmin biosynthesis) and subsequently reducing serum copper concentrations. Serum ceruloplasmin represents a biomarker for disulfiram activity. Our results establish copper ionophores as a potential class of antiobesity agents.
Assuntos
Fármacos Antiobesidade/farmacologia , Cobre/metabolismo , Dissulfiram/farmacologia , Ionóforos/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade/efeitos dos fármacosRESUMO
Mitochondrial dysfunction, ubiquitin-proteasomal system impairment and excitotoxicity occur during the injury and death of neurons in neurodegenerative conditions. The aim of this work was to elucidate the cellular mechanisms that are universally altered by these conditions. Through overlapping expression profiles of rotenone-, lactacystin- and N-methyl-D-aspartate-treated cortical neurons, we have identified three affected biological processes that are commonly affected; oxidative stress, dysfunction of calcium signalling and inhibition of the autophagic-lysosomal pathway. These data provides many opportunities for therapeutic intervention in neurodegenerative conditions, where mitochondrial dysfunction, proteasomal inhibition and excitotoxicity are evident.
Assuntos
Autofagia , Sinalização do Cálcio , Lisossomos/metabolismo , Neurônios/metabolismo , Estresse Oxidativo , Acetilcisteína/análogos & derivados , Acetilcisteína/toxicidade , Animais , Humanos , Análise em Microsséries , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/efeitos dos fármacos , Praguicidas/toxicidade , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/toxicidade , Receptores de N-Metil-D-Aspartato/metabolismo , Rotenona/toxicidade , Ubiquitina/metabolismoRESUMO
BACKGROUND: Therapeutics that target copper for the treatment of prostate cancer are being evaluated in human clinical trials. Elevated intracellular copper is considered to sensitize prostate cancer cells to certain copper-coordination compounds, especially those with ionophoric properties. While there is compelling in vitro evidence that prostate cancer cells accumulate intracellular copper, a corresponding status for copper in patient tissues has not been corroborated. We therefore established whether copper concentrations increase in cancerous prostate tissues, and in sera, in patients throughout disease progression. METHODS: Human prostate tissue samples were obtained from patient prostatectomies (n = 28), and together with patient-matched sera, were analyzed for copper content by inductively coupled plasma mass spectrometry. RESULTS: When grouped together, cancerous prostate tissues exhibiting moderate disease severity (Gleason Score 7) (n = 10) had 1.6-fold more copper than age-matched normal tissues (n = 10) (P < 0.05). Those with more aggressive disease (Gleason Score 9) (n = 8) had 1.8-fold more copper (P < 0.05). In both disease stages however, the copper concentrations between individual samples were rather variable (0.55-3.02 µg/g), with many clearly within the normal range (0.52-1.28 µg/g). Additionally, we found that there was no change in serum copper concentrations in patients with either moderate or aggressive prostate cancer (Gleason Score 7 or 9), compared with reference intervals and to age-matched controls. CONCLUSIONS: The heterogeneous nature of copper concentrations in cancerous prostate tissues, suggest that a small subset of patients may respond to treatments that target elevated intratumoral copper. Therefore, such approaches would likely require personalized treatment strategies.
Assuntos
Biomarcadores Tumorais/metabolismo , Cobre/metabolismo , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Cobre/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Prostatectomia , Neoplasias da Próstata/epidemiologia , Vitória/epidemiologiaRESUMO
Radiolabeled diacetylbis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] is an effective positron-emission tomography imaging agent for myocardial ischemia, hypoxic tumors, and brain disorders with regionalized oxidative stress, such as mitochondrial myopathy, encephalopathy, and lactic acidosis with stroke-like episodes (MELAS) and Parkinson's disease. An excessively elevated reductive state is common to these conditions and has been proposed as an important mechanism affecting cellular retention of Cu from Cu(II)(atsm). However, data from whole-cell models to demonstrate this mechanism have not yet been provided. The present study used a unique cell culture model, mitochondrial xenocybrids, to provide whole-cell mechanistic data on cellular retention of Cu from Cu(II)(atsm). Genetic incompatibility between nuclear and mitochondrial encoded subunits of the mitochondrial electron transport chain (ETC) in xenocybrid cells compromises normal function of the ETC. As a consequence of this impairment to the ETC we show xenocybrid cells upregulate glycolytic ATP production and accumulate NADH. Compared to control cells the xenocybrid cells retained more Cu after being treated with Cu(II)(atsm). By transfecting the cells with a metal-responsive element reporter construct the increase in Cu retention was shown to involve a Cu(II)(atsm)-induced increase in intracellular bioavailable Cu specifically within the xenocybrid cells. Parallel experiments using cells grown under hypoxic conditions confirmed that a compromised ETC and elevated NADH levels contribute to increased cellular retention of Cu from Cu(II)(atsm). Using these cell culture models our data demonstrate that compromised ETC function, due to the absence of O(2) as the terminal electron acceptor or dysfunction of individual components of the ETC, is an important determinant in driving the intracellular dissociation of Cu(II)(atsm) that increases cellular retention of the Cu.
Assuntos
Complexos de Coordenação/metabolismo , Imageamento Tridimensional , Mitocôndrias/metabolismo , Semicarbazonas/metabolismo , Ácidos , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico , Complexos de Coordenação/química , Cobre/metabolismo , Meios de Cultura/metabolismo , Transporte de Elétrons , Humanos , Células Híbridas/metabolismo , Espaço Intracelular/metabolismo , Camundongos , Estresse Oxidativo , Ratos , Semicarbazonas/químicaRESUMO
Glutaredoxin1 (GRX1) is a glutathione (GSH)-dependent thiol oxidoreductase. The GRX1/GSH system is important for the protection of proteins from oxidative damage and in the regulation of protein function. Previously we demonstrated that GRX1/GSH regulates the activity of the essential copper-transporting P1B-Type ATPases (ATP7A, ATP7B) in a copper-responsive manner. It has also been established that GRX1 binds copper with high affinity and regulates the redox chemistry of the metallochaperone ATOX1, which delivers copper to the copper-ATPases. In this study, to further define the role of GRX1 in copper homeostasis, we examined the effects of manipulating GRX1 expression on copper homeostasis and cell survival in mouse embryonic fibroblasts and in human neuroblastoma cells (SH-SY5Y). GRX1 knockout led to cellular copper retention (especially when cultured with elevated copper) and reduced copper tolerance, while in GRX1-overexpressing cells challenged with elevated copper, there was a reduction in both intracellular copper levels and copper-induced reactive oxygen species, coupled with enhanced cell proliferation. These effects are consistent with a role for GRX1 in regulating ATP7A-mediated copper export, and further support a new function for GRX1 in neuronal copper homeostasis and in protection from copper-mediated oxidative injury.
Assuntos
Cobre/metabolismo , Glutarredoxinas/fisiologia , Neurônios/enzimologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Cobre/toxicidade , Células HEK293 , Humanos , Camundongos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
ATP7A and ATP7B are copper-transporting P(1B)-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and COMMD1 were previously identified as interacting partners of these Cu-ATPases. In this study, we confirmed that clusterin and COMMD1 interact to down-regulate both ATP7A and ATP7B. Overexpression and knockdown of clusterin/COMMD1 decreased and increased, respectively, endogenous levels of ATP7A and ATP7B, consistent with a role in facilitating Cu-ATPase degradation. We demonstrate that whereas the clusterin/ATP7B interaction was enhanced by oxidative stress or mutation of ATP7B, the COMMD1/ATP7B interaction did not change under oxidative stress conditions, and only increased with ATP7B mutations that led to its misfolding. Clusterin and COMMD1 facilitated the degradation of ATP7B containing the same Wilson disease-causing C-terminal mutations via different degradation pathways, clusterin via the lysosomal pathway and COMMD1 via the proteasomal pathway. Furthermore, endogenous ATP7B existed in a complex with clusterin and COMMD1, but these interactions were neither competitive nor cooperative and occurred independently of each other. Together these data indicate that clusterin and COMMD1 represent alternative and independent systems regulating Cu-ATPase quality control, and consequently contributing to the maintenance of copper homeostasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Clusterina/metabolismo , Proteólise , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenosina Trifosfatases/genética , Animais , Células CHO , Proteínas de Transporte de Cátions/genética , Clusterina/genética , ATPases Transportadoras de Cobre , Cricetinae , Cricetulus , Células HEK293 , Degeneração Hepatolenticular/genética , Degeneração Hepatolenticular/metabolismo , Humanos , Síndrome dos Cabelos Torcidos/genética , Síndrome dos Cabelos Torcidos/metabolismo , Camundongos , Mutação , RatosRESUMO
The copper-transporting P(1B)-type ATPases (Cu-ATPases) ATP7A and ATP7B are key regulators of physiological copper levels. They function to maintain intracellular copper homeostasis by delivering copper to secretory compartments and by trafficking toward the cell periphery to export excess copper. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and toxicity disorders, Menkes and Wilson diseases, respectively. This report describes the interaction between the Cu-ATPases and clusterin and demonstrates a chaperone-like role for clusterin in facilitating their degradation. Clusterin interacted with both ATP7A and ATP7B in mammalian cells. This interaction increased under conditions of oxidative stress and with mutations in ATP7B that led to its misfolding and mislocalization. A Wilson disease patient mutation (G85V) led to enhanced ATP7B turnover, which was further exacerbated when cells overexpressed clusterin. We demonstrated that clusterin-facilitated degradation of mutant ATP7B is likely to involve the lysosomal pathway. The knockdown and overexpression of clusterin increased and decreased, respectively, the Cu-ATPase-mediated copper export capacity of cells. These results highlight a new role for intracellular clusterin in mediating Cu-ATPase quality control and hence in the normal maintenance of copper homeostasis, and in promoting cell survival in the context of disease. Based on our findings, it is possible that variations in clusterin expression and function could contribute to the variable clinical expression of Menkes and Wilson diseases.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Clusterina/metabolismo , Degeneração Hepatolenticular/metabolismo , Síndrome dos Cabelos Torcidos/metabolismo , Dobramento de Proteína , Adenosina Trifosfatases/genética , Animais , Células CHO , Proteínas de Transporte de Cátions/genética , Clusterina/genética , Cobre/metabolismo , ATPases Transportadoras de Cobre , Cricetinae , Cricetulus , Células HEK293 , Células Hep G2 , Degeneração Hepatolenticular/genética , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Síndrome dos Cabelos Torcidos/genéticaRESUMO
Clioquinol (5-chloro-7-iodo-8-quinolinol) is a copper ionophore that was used primarily during the 1950-1970s as an oral antimicrobial agent. It has been established that clioquinol displays toxicity towards malignant cells, inducing caspase-dependent apoptosis. In the present study we therefore investigated the effect of clioquinol on the XIAP [X-linked IAP (inhibitor of apoptosis protein)], as one of its primary functions is to hinder caspase activity and suppress apoptotic cell death. Clioquinol treatment caused cytoplasmic XIAP to rapidly relocate to the nucleus in multiple human transformed (hyperplasic and carcinoma) prostate lines. Clioquinol also caused the cytoplasmic clearance of other IAP family members (cIAP1 and cIAP2). Copper, and no other relevant bivalent metal (e.g. zinc or iron), was exclusively required for clioquinol to elicit an effect on XIAP. We further demonstrated that clioquinol selectively targets and rapidly destroys transformed prostate lines without harming primary prostate epithelial cells. The toxicity of clioquinol was copper-dependent, positively correlated with the level of extracellular copper and could be abrogated by using the copper chelator TTM (tetrathiomolybdate). Clioquinol forced the profound accumulation of intracellular copper with ensuing toxicity influenced by key regulators of cellular copper homoeostasis. Taken together, our results provide significant insight into clioquinol toxicity and reveal an exciting therapeutic approach for the treatment of prostate cancer.
Assuntos
Antineoplásicos/farmacologia , Clioquinol/farmacologia , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Clioquinol/uso terapêutico , Humanos , Masculino , Próstata/citologia , Próstata/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologiaRESUMO
The copper-transporting P-type ATPases (Cu-ATPases), ATP7A and ATP7B, are essential for the regulation of intracellular copper homeostasis. In this report we describe new roles for glutathione (GSH) and glutaredoxin1 (GRX1) in Cu homeostasis through their regulation of Cu-ATPase activity. GRX1 is a thiol oxidoreductase that catalyzes the reversible reduction of GSH-mixed disulfides to their respective sulfhydryls (deglutathionylation). Here, we demonstrated that glutathionylation of the Cu-ATPases and their interaction with GRX1 were affected by alterations in Cu levels. The data support our hypothesis that the Cu-ATPases serve as substrates for Cu-dependent GRX1-mediated deglutathionylation. This in turn liberates the Cu-ATPase cysteinyl thiol groups for Cu binding and transport. GSH depletion experiments led to reversible inhibition of the Cu-ATPases that correlated with effects on intracellular Cu levels and GRX1 activity. Finally, knockdown of GRX1 expression resulted in an increase in intracellular Cu accumulation. Together, these data directly implicate GSH and GRX1 with important new roles in redox regulation of the Cu-ATPases, through modulation of Cu binding by the Cu-ATPase cysteine motifs.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Adenosina Trifosfatases/genética , Animais , Transporte Biológico/fisiologia , Células CHO , Proteínas de Transporte de Cátions/genética , ATPases Transportadoras de Cobre , Cricetinae , Cricetulus , Técnicas de Silenciamento de Genes , Glutarredoxinas/genética , Glutationa/genética , Células Hep G2 , Humanos , Ligação Proteica/fisiologiaRESUMO
In Alzheimer's disease there is abnormal brain copper distribution, with accumulation of copper in amyloid plaques and a deficiency of copper in neighbouring cells. Excess copper inhibits Abeta (amyloid beta-peptide) production, but the effects of deficiency have not yet been determined. We therefore studied the effects of modulating intracellular copper levels on the processing of APP (amyloid precursor protein) and the production of Abeta. Human fibroblasts genetically disposed to copper accumulation secreted higher levels of sAPP (soluble APP ectodomain)alpha into their medium, whereas fibroblasts genetically manipulated to be profoundly copper deficient secreted predominantly sAPPbeta and produced more amyloidogenic beta-cleaved APP C-termini (C99). The level of Abeta secreted from copper-deficient fibroblasts was however regulated and limited by alpha-secretase cleavage. APP can be processed by both alpha- and beta-secretase, as copper-deficient fibroblasts secreted sAPPbeta exclusively, but produced primarily alpha-cleaved APP C-terminal fragments (C83). Copper deficiency also markedly reduced the steady-state level of APP mRNA whereas the APP protein level remained constant, indicating that copper deficiency may accelerate APP translation. Copper deficiency in human neuroblastoma cells significantly increased the level of Abeta secretion, but did not affect the cleavage of APP. Therefore copper deficiency markedly alters APP metabolism and can elevate Abeta secretion by either influencing APP cleavage or by inhibiting its degradation, with the mechanism dependent on cell type. Overall our results suggest that correcting brain copper imbalance represents a relevant therapeutic target for Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Cobre/deficiência , Cobre/farmacologia , Líquido Intracelular , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Células Cultivadas , Cobre/análise , Fibroblastos/química , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Líquido Intracelular/química , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
The Wilson protein (ATP7B) is a copper-translocating P-type ATPase that mediates the excretion of excess copper from hepatocytes into bile. Excess copper causes the protein to traffic from the TGN (trans-Golgi network) to subapical vesicles. Using site-directed mutagenesis, mutations known or predicted to abrogate catalytic activity (copper translocation) were introduced into ATP7B and the effect of these mutations on the intracellular trafficking of the protein was investigated. Mutation of the critical aspartic acid residue in the phosphorylation domain (DKTGTIT) blocked copper-induced redistribution of ATP7B from the TGN, whereas mutation of the phosphatase domain [TGE (Thr-Gly-Glu)] trapped ATP7B at cytosolic vesicular compartments. Our findings demonstrate that ATP7B trafficking is regulated with its copper-translocation cycle, with cytosolic vesicular localization associated with the acyl-phosphate intermediate. In addition, mutation of the six N-terminal metal-binding sites and/or the trans-membrane CPC (Cys-Pro-Cys) motif did not suppress the constitutive vesicular localization of the ATP7B phosphatase domain mutant. These results suggested that copper co-ordination by these sites is not essential for trafficking. Importantly, copper-chelation studies with these mutants clearly demonstrated a requirement for copper in ATP7B trafficking, suggesting the presence of an additional copper-binding site(s) within the protein. The results presented in this report significantly advance our understanding of the regulatory mechanism that links copper-translocation activity with copper-induced intracellular trafficking of ATP7B, which is central to hepatic and hence systemic copper homoeostasis.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Degeneração Hepatolenticular/metabolismo , Oligopeptídeos/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , ATPases Transportadoras de Cobre , Cisteína , Primers do DNA , Ácido Glutâmico/metabolismo , Glicina , Humanos , Modelos Moleculares , Reação em Cadeia da Polimerase , Conformação Proteica , TreoninaRESUMO
Copper homeostasis is tightly regulated in both prokaryotic and eukaryotic cells to ensure sufficient amounts for cuproprotein biosynthesis, while limiting oxidative stress production and toxicity. Over the last century, copper complexes have been developed as antimicrobials and for treating diseases involving copper dyshomeostasis (e.g., Wilson's disease). There now exists a repertoire of copper complexes that can regulate bodily copper through a myriad of mechanisms. Furthermore, many copper complexes are now being appraised for a variety of therapeutic indications (e.g., Alzheimer's disease and amyotrophic lateral sclerosis) that require a range of copper-related pharmacological affects. Cancer therapy is also drawing considerable attention since copper has been recognized as a limiting factor for multiple aspects of cancer progression including growth, angiogenesis, and metastasis. Consequently, 'old copper complexes' (e.g., tetrathiomolybdate and clioquinol) have been repurposed for cancer therapy and have demonstrated anticancer activity in vitro and in preclinical models. Likewise, new tailor-made copper complexes have been designed based on structural and biological features ideal for their anticancer activity. Human clinical trials continue to evaluate the therapeutic efficacy of copper complexes as anticancer agents and considerable progress has been made in understanding their pharmacological requirements. In this chapter, we present a historical perspective on the main copper complexes that are currently being repurposed for cancer therapy and detail several of the more recently developed compounds that have emerged as promising anticancer agents. We further provide an overview of the known mechanisms of action, including molecular targets and we discuss associated clinical trials.
Assuntos
Antineoplásicos/uso terapêutico , Cobre/uso terapêutico , Neoplasias/tratamento farmacológico , Compostos Organometálicos/uso terapêutico , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Antineoplásicos/metabolismo , Complexos de Coordenação , Cobre/efeitos adversos , Cobre/química , Cobre/metabolismo , Desenho de Fármacos , Reposicionamento de Medicamentos , Humanos , Estrutura Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Compostos Organometálicos/efeitos adversos , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Relação Estrutura-AtividadeRESUMO
Cellular senescence is characterised by the irreversible arrest of proliferation, a pro-inflammatory secretory phenotype and evasion of programmed cell death mechanisms. We report that senescence alters cellular iron acquisition and storage and also impedes iron-mediated cell death pathways. Senescent cells, regardless of stimuli (irradiation, replicative or oncogenic), accumulate vast amounts of intracellular iron (up to 30-fold) with concomitant changes in the levels of iron homeostasis proteins. For instance, ferritin (iron storage) levels provided a robust biomarker of cellular senescence, for associated iron accumulation and for resistance to iron-induced toxicity. Cellular senescence preceded iron accumulation and was not perturbed by sustained iron chelation (deferiprone). Iron accumulation in senescent cells was driven by impaired ferritinophagy, a lysosomal process that promotes ferritin degradation and ferroptosis. Lysosomal dysfunction in senescent cells was confirmed through several markers, including the build-up of microtubule-associated protein light chain 3 (LC3-II) in autophagosomes. Impaired ferritin degradation explains the iron accumulation phenotype of senescent cells, whereby iron is effectively trapped in ferritin creating a perceived cellular deficiency. Accordingly, senescent cells were highly resistant to ferroptosis. Promoting ferritin degradation by using the autophagy activator rapamycin averted the iron accumulation phenotype of senescent cells, preventing the increase of TfR1, ferritin and intracellular iron, but failed to re-sensitize these cells to ferroptosis. Finally, the enrichment of senescent cells in mouse ageing hepatic tissue was found to accompany iron accumulation, an elevation in ferritin and mirrored our observations using cultured senescent cells.
Assuntos
Apoptose/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Ferro/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Ferritinas/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HEK293 , Humanos , Ferro/análise , Ferro/metabolismo , Lisossomos/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3RESUMO
Cellular senescence is characterized by irreversible growth arrest incurred through either replicative exhaustion or by pro-oncogenic cellular stressors (radioactivity, oxidative stress, oncogenic activation). The enrichment of senescent cells in tissues with age has been associated with tissue dyshomeostasis and age-related pathologies including cancers, neurodegenerative disorders (e.g. Alzheimer's, Parkinson's, etc.) and metabolic disorders (e.g. diabetes). We identified copper accumulation as being a universal feature of senescent cells [mouse embryonic fibroblasts (MEF), human prostate epithelial cells and human diploid fibroblasts] in vitro. Elevated copper in senescent MEFs was accompanied by elevated levels of high-affinity copper uptake protein 1 (Ctr1), diminished levels of copper-transporting ATPase 1 (Atp7a) (copper export) and enhanced antioxidant defence reflected by elevated levels of glutathione (GSH), superoxide dismutase 1 (SOD1) and glutaredoxin 1 (Grx1). The levels of intracellular copper were further increased in senescent MEFs cultured in copper supplemented medium and in senescent Mottled Brindled (Mobr) MEFs lacking functional Atp7a. Finally, we demonstrated that the restoration/preservation of autophagic-lysosomal degradation in senescent MEFs following rapamycin treatment correlated with attenuation of copper accumulation in these cells despite a further decrease in Atp7a levels. This study for the first time establishes a link between Atp7a and the autophagic-lysosomal pathway, and a requirement for both to effect efficient copper export. Such a connection between cellular autophagy and copper homeostasis is significant, as both have emerged as important facets of age-associated degenerative disease.
Assuntos
Autofagia/genética , Senescência Celular/genética , ATPases Transportadoras de Cobre/genética , Cobre/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Transportador de Cobre 1 , ATPases Transportadoras de Cobre/metabolismo , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Glutarredoxinas/genética , Glutationa/genética , Homeostase , Humanos , Lisossomos/metabolismo , Masculino , Camundongos , Próstata/metabolismo , Superóxido Dismutase-1/genéticaRESUMO
Ceruloplasmin (Cp) is a multicopper ferroxidase that is considered to be an important source of copper in milk for normal neonatal development. We investigated the expression, subcellular localization and secretion of Cp in PMC42-LA cell culture models representative of resting, lactating and suckled human mammary epithelia. Both secreted Cp (sCp) and plasma membrane associated glycosylphosphatidylinositol-linked Cp (GPI-Cp) were expressed in PMC42-LA cells. In all three epithelial models (resting, lactating and suckled), the expression and secretion of copper-bound, ferroxidase active, Cp (holo-Cp) was dependent on media copper concentration. In low copper (bathocuproinedisulphonic acid/d-penicillamine treated models) there was greater than a 2-fold decrease in holo-Cp expression and secretion, which was mirrored by a 2-fold increase in the expression and secretion of copper-free Cp protein (apo-Cp). Cell surface biotinylation studies revealed that the state of PMC42-LA cell differentiation (functionality), and the level of extracellular copper, had no significant effect on the level of plasma membrane bound GPI-Cp. Quantitative real time PCR analyses determined that there was no significant (P > 0.05) difference in Cp mRNA levels across all copper conditions investigated (0, 5, 50 µM). However, there was a significant (P < 0.05) increase (â¼2-fold) in Cp mRNA in both the lactating and suckled models in comparison to the resting model. Furthermore, the Cp mRNA increase in response to PMC42-LA differentiation corresponded with more secreted Cp protein, both apo and holo forms, indicating a link between function and Cp requirement. Our results provide significant insight on the regulation of Cp expression and secretion in lactation and copper incorporation into milk.
Assuntos
Ceruloplasmina/metabolismo , Cobre/farmacologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônios/farmacologia , Lactação/metabolismo , Glândulas Mamárias Animais/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ceruloplasmina/genética , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feminino , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/efeitos dos fármacos , CamundongosRESUMO
Copper-ionophores that elevate intracellular bioavailable copper display significant therapeutic utility against prostate cancer cells in vitro and in TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice. However, the pharmacological basis for their anticancer activity remains unclear, despite impending clinical trails. Herein we show that intracellular copper levels in prostate cancer, evaluated in vitro and across disease progression in TRAMP mice, were not correlative with copper-ionophore activity and mirrored the normal levels observed in patient prostatectomy tissues (Gleason Score 7 & 9). TRAMP adenocarcinoma cells harbored markedly elevated oxidative stress and diminished glutathione (GSH)-mediated antioxidant capacity, which together conferred selective sensitivity to prooxidant ionophoric copper. Copper-ionophore treatments [CuII(gtsm), disulfiram & clioquinol] generated toxic levels of reactive oxygen species (ROS) in TRAMP adenocarcinoma cells, but not in normal mouse prostate epithelial cells (PrECs). Our results provide a basis for the pharmacological activity of copper-ionophores and suggest they are amendable for treatment of patients with prostate cancer. Additionally, recent in vitro and mouse xenograft studies have suggested an increased copper requirement by prostate cancer cells. We demonstrated that prostate adenocarcinoma development in TRAMP mice requires a functional supply of copper and is significantly impeded by altered systemic copper distribution. The presence of a mutant copper-transporting Atp7b protein (tx mutation: A4066G/Met1356Val) in TRAMP mice changed copper-integration into serum and caused a remarkable reduction in prostate cancer burden (64% reduction) and disease severity (grade), abrogating adenocarcinoma development. Implications for current clinical trials are discussed.
Assuntos
Adenocarcinoma/metabolismo , Antineoplásicos/farmacologia , Cobre/metabolismo , Ionóforos/farmacologia , Neoplasias da Próstata/metabolismo , Animais , ATPases Transportadoras de Cobre/genética , Masculino , Camundongos , Camundongos TransgênicosRESUMO
The Wilson protein (ATP7B) is a copper-transporting CPx-type ATPase defective in the copper toxicity disorder Wilson disease. In hepatocytes, ATP7B delivers copper to apo-ceruloplasmin and mediates the excretion of excess copper into bile. These distinct functions require the protein to localize at two different subcellular compartments. At the trans-Golgi network, ATP7B transports copper for incorporation into apo-ceruloplasmin. When intracellular copper levels are increased, ATP7B traffics to post-Golgi vesicles in close proximity to the canalicular membrane to facilitate biliary copper excretion. In the present study, we investigated the role of the six N-terminal MBSs (metal-binding sites) in the trafficking process. Using site-directed mutagenesis, we mutated or deleted various combinations of the MBSs and assessed the effect of these changes on the localization and trafficking of ATP7B. Results show that the MBSs required for trafficking are the same as those previously found essential for the copper transport function. Either MBS 5 or MBS 6 alone was sufficient to support the redistribution of ATP7B to vesicular compartments. The first three N-terminal motifs were not required for copper-dependent intracellular trafficking and could not functionally replace sites 4-6 when placed in the same sequence position. Furthermore, the N-terminal region encompassing MBSs 1-5 (amino acids 64-540) was not essential for trafficking, with only one MBS close to the membrane channel, necessary and sufficient to support trafficking. Our findings were similar to those obtained for the closely related ATP7A protein, suggesting similar mechanisms for trafficking between copper-transporting CPx-type ATPases.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Espaço Intracelular/metabolismo , Peptídeos/fisiologia , Adenosina Trifosfatases/biossíntese , Adenosina Trifosfatases/imunologia , Adenosina Trifosfatases/fisiologia , Processamento Alternativo/genética , Animais , Sítios de Ligação/genética , Células CHO/enzimologia , Proteínas de Transporte de Cátions/biossíntese , Proteínas de Transporte de Cátions/imunologia , Proteínas de Transporte de Cátions/fisiologia , ATPases Transportadoras de Cobre , Cricetinae , Cricetulus , Membranas Intracelulares/metabolismo , Isoenzimas/biossíntese , Isoenzimas/genética , Mutagênese Sítio-Dirigida/genética , Peptídeos/genética , Estrutura Terciária de Proteína/fisiologia , Deleção de Sequência/genética , Deleção de Sequência/fisiologiaRESUMO
Copper is an essential micronutrient involved in fundamental life processes that are conserved throughout all forms of life. The ability of copper to catalyze oxidation-reduction (redox) reactions, which can inadvertently lead to the production of reactive oxygen species (ROS), necessitates the tight homeostatic regulation of copper within the body. Many cancer types exhibit increased intratumoral copper and/or altered systemic copper distribution. The realization that copper serves as a limiting factor for multiple aspects of tumor progression, including growth, angiogenesis and metastasis, has prompted the development of copper-specific chelators as therapies to inhibit these processes. Another therapeutic approach utilizes specific ionophores that deliver copper to cells to increase intracellular copper levels. The therapeutic window between normal and cancerous cells when intracellular copper is forcibly increased, is the premise for the development of copper-ionophores endowed with anticancer properties. Also under investigation is the use of copper to replace platinum in coordination complexes currently used as mainstream chemotherapies. In comparison to platinum-based drugs, these promising copper coordination complexes may be more potent anticancer agents, with reduced toxicity toward normal cells and they may potentially circumvent the chemoresistance associated with recurrent platinum treatment. In addition, cancerous cells can adapt their copper homeostatic mechanisms to acquire resistance to conventional platinum-based drugs and certain copper coordination complexes can re-sensitize cancer cells to these drugs. This review will outline the biological importance of copper and copper homeostasis in mammalian cells, followed by a discussion of our current understanding of copper dysregulation in cancer, and the recent therapeutic advances using copper coordination complexes as anticancer agents.