RESUMO
Iron is a crucial element for mammalian cells, considering its intervention in several physiologic processes. Its homeostasis is finely regulated, and its alteration could be responsible for the onset of several disorders. Iron is closely related to inflammation; indeed, during inflammation high levels of interleukin-6 cause an increased production of hepcidin which induces a degradation of ferroportin. Ferroportin degradation leads to decreased iron efflux that culminates in elevated intracellular iron concentration and consequently iron toxicity in cells and tissues. Therefore, iron chelation could be considered a novel and useful therapeutic strategy in order to counteract the inflammation in several autoimmune and inflammatory diseases. Several iron chelators are already known to have anti-inflammatory effects, among them deferiprone, deferoxamine, deferasirox, and Dp44mT are noteworthy. Recently, eltrombopag has been reported to have an important role in reducing inflammation, acting both directly by chelating iron, and indirectly by modulating iron efflux. This review offers an overview of the possible novel biological effects of the iron chelators in inflammation, suggesting them as novel anti-inflammatory molecules.
Assuntos
Sobrecarga de Ferro , Animais , Benzoatos/uso terapêutico , Deferasirox/uso terapêutico , Deferiprona , Desferroxamina/uso terapêutico , Inflamação/complicações , Inflamação/tratamento farmacológico , Ferro/uso terapêutico , Quelantes de Ferro/farmacologia , Quelantes de Ferro/uso terapêutico , Sobrecarga de Ferro/tratamento farmacológico , Sobrecarga de Ferro/etiologia , Mamíferos , Piridonas/uso terapêuticoRESUMO
Iron chelators have long been a target of interest as anticancer agents. Iron is an important cellular resource involved in cell replication, metabolism and growth. Iron metabolism is modulated in cancer cells reflecting their increased replicative demands. Originally, iron chelators were first developed for use in iron overload disorders, however, their potential as anticancer agents has been gaining increasing interest. This is due, in part, to the downstream effects of iron depletion such as the inhibition of proliferation through ribonucleotide reductase activity. Additionally, some chelators form redox active metal complexes with iron resulting in the production of reactive oxygen species and oxidative stress. Newer synthetic iron chelators such as Deferasirox, Triapine and di-2-pyridylketone-4,4,-dimethyl-3-thiosemicrbazone (Dp44mt) have improved pharmacokinetic properties over the older chelator Deferoxamine. This review examines and discusses the various iron chelators that have been trialled for cancer therapy including both preclinical and clinical studies. The successes and shortcomings of each of the chelators and their use in combination therapies are highlighted and future potential in the cancer therapy world is considered.
Assuntos
Antineoplásicos/uso terapêutico , Quelantes de Ferro/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Humanos , Quelantes de Ferro/química , Quelantes de Ferro/metabolismoRESUMO
The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), exhibits pleiotropic activity, inhibiting metastasis of various tumor-types, while being correlated with metastasis in others. Notably, NDRG1 phosphorylation and cleavage are associated with its function, although it is unclear if these modifications occur universally, or selectively, in different cancer cell-types and if it contributes to its pleiotropy. Considering the suggested DNA repair role of nuclear NDRG1, the effects of the above post-translational modifications on its nuclear localization was examined. Herein, the full-length (FL) and truncated (T) NDRG1 isoforms were detected using a C-terminus-directed antibody, while only the FL isoform was identified using an N-terminus-directed antibody. For the first time, we demonstrate that the expression of the NDRG1 FL and T forms occurs in all cancer cell-types examined, as does its phosphorylation (p-NDRG1) at Ser330 and Thr346. The FL isoform localized highly in the nucleus compared to the T isoform. Moreover, p-NDRG1 (Ser330) was also markedly localized in the nucleus, while p-NDRG1 (Thr346) was predominantly cytoplasmic in all cell-types. These results indicate the N-terminus region and phosphorylation at Ser330 could be crucial for NDRG1 nuclear localization and function. PTEN silencing indicated that p-NDRG1 (Thr346) could be regulated differentially in different tumor cell-types, indicating PTEN may be involved in the mechanism(s) underlying the pleiotropic activity of NDRG1. Finally, therapeutics of the di-2-pyridylketone thiosemicarbazone class increased nuclear NDRG1 isoforms (FL and T) detected by the C-terminus-directed antibody in HepG2 cells, while having no significant effect in PC3 cells, indicating differential activity depending on the cell-type.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas de Ciclo Celular/genética , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Metástase Neoplásica , PTEN Fosfo-Hidrolase/genética , Fosforilação/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico/genéticaRESUMO
BACKGROUND: The cyclin-dependent kinase inhibitor, p21, is well known for its role in cell cycle arrest. Novel anti-cancer agents that deplete iron pools demonstrate marked anti-tumor activity and are also active in regulating p21 expression. These agents induce p21 mRNA levels independently of the tumor suppressor, p53, and differentially regulate p21 protein expression depending on the cell-type. Several chelators, including an analogue of the potent anti-tumor agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), have entered clinical trials, and thus, their molecular mechanism of action is crucial to assess. Hence, this investigation examined how several iron chelators transcriptionally regulate p21. METHODS: Promoter-deletion constructs; luciferase assays; RT-PCR; western analysis; gene silencing; co-immunoprecipitation. RESULTS: The transcriptional regulation of the p21 promoter by iron chelators was demonstrated to be dependent on the chelator and cell-type examined. The potent anti-cancer chelator, Dp44mT, induced p21 promoter activity in SK-MEL-28 melanoma cells, but not in MCF-7 breast cancer cells. Further analysis of the p21 promoter identified a 50-bp region between -104 and -56-bp that was required for Dp44mT-induced activation in SK-MEL-28 cells. This region contained several Sp1-binding sites and mutational analysis of this region revealed the Sp1-3-binding site played a significant role in Dp44mT-induced activation of p21. Further, co-immunoprecipitation demonstrated that Dp44mT induced a marked increase in the interactions between Sp1 and the transcription factors, estrogen receptor-α and c-Jun. CONCLUSIONS AND GENERAL SIGNIFICANCE: Dp44mT-induced p21 promoter activation via the Sp1-3-binding site and increased Sp1/ER-α and Sp1/c-Jun complex formation in SK-MEL-28 cells, suggesting these complexes were involved in p21 promoter activation.
Assuntos
Antineoplásicos/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Quelantes de Ferro/farmacologia , Proteínas de Neoplasias/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Tiossemicarbazonas/farmacologia , Ativação Transcricional/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Células MCF-7 , Melanoma/patologia , Estrutura Molecular , Mutação , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Deleção de Sequência , Fatores de Transcrição/metabolismoRESUMO
N-MYC downstream-regulated gene-1 (NDRG1) is a potent growth and metastasis suppressor that acts through its inhibitory effects on a wide variety of cellular signaling pathways, including the TGF-ß pathway, protein kinase B (AKT)/PI3K pathway, RAS, etc. To investigate the hypothesis that its multiple effects could be regulated by a common upstream effector, the role of NDRG1 on the epidermal growth factor receptor (EGFR) and other members of the ErbB family, namely human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3), was examined. We demonstrate that NDRG1 markedly decreased the expression and activation of EGFR, HER2, and HER3 in response to the epidermal growth factor (EGF) ligand, while also inhibiting formation of the EGFR/HER2 and HER2/HER3 heterodimers. In addition, NDRG1 also decreased activation of the downstream MAPKK in response to EGF. Moreover, novel anti-tumor agents of the di-2-pyridylketone class of thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, which markedly up-regulate NDRG1, were found to inhibit EGFR, HER2, and HER3 expression and phosphorylation in cancer cells. However, the mechanism involved appeared dependent on NDRG1 for di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone, but was independent of this metastasis suppressor for di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone. This observation demonstrates that small structural changes in thiosemicarbazones result in marked alterations in molecular targeting. Collectively, these results reveal a mechanism for the extensive downstream effects on cellular signaling attributed to NDRG1. Furthermore, this study identifies a novel approach for the treatment of tumors resistant to traditional EGFR inhibitors.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Neoplasias do Colo/metabolismo , Receptores ErbB/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Neoplasias Pancreáticas/metabolismo , Piridinas/uso terapêutico , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-3/antagonistas & inibidores , Tiossemicarbazonas/uso terapêutico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas de Ciclo Celular/agonistas , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/agonistas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/agonistas , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Nus , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Piridinas/farmacologia , Interferência de RNA , Distribuição Aleatória , Receptor ErbB-2/agonistas , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/agonistas , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tiossemicarbazonas/farmacologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The potent and selective anti-tumor agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), localizes in lysosomes and forms cytotoxic copper complexes that generate reactive oxygen species (ROS), resulting in lysosomal membrane permeabilization (LMP) and cell death. Herein, the role of lysosomal membrane stability in the anti-tumor activity of Dp44mT was investigated. Studies were performed using molecules that protect lysosomal membranes against Dp44mT-induced LMP, namely heat shock protein 70 (HSP70) and cholesterol. Up-regulation or silencing of HSP70 expression did not affect Dp44mT-induced LMP in MCF7 cells. In contrast, cholesterol accumulation in lysosomes induced by the well characterized cholesterol transport inhibitor, 3-ß-[2-(diethyl-amino)ethoxy]androst-5-en-17-one (U18666A), inhibited Dp44mT-induced LMP and markedly and significantly (p<0.001) reduced the ability of Dp44mT to inhibit cancer cell proliferation (i.e., increased the IC(50)) by 140-fold. On the other hand, cholesterol extraction using methyl-ß-cyclodextrin enhanced Dp44mT-induced LMP and significantly (p<0.01) increased its anti-proliferative activity. The protective effect of U18666A in increasing lysosomal cholesterol and preventing the cytotoxic activity of Dp44mT was not due to induced autophagy. Instead, U18666A was found to decrease lysosomal turnover, resulting in autophagosome accumulation. Moreover, preincubation with U18666A did not prevent the ability of Dp44mT to induce autophagosome synthesis, indicating that autophagic initiation via Dp44mT occurs independently of LMP. These studies demonstrate the significance of lysosomal membrane stability in relation to the ability of Dp44mT to execute tumor cell death and overcome pro-survival autophagy. Hence, lysosomal-dependent cell death induced by Dp44mT serves as an important anti-tumor strategy. These results are important for comprehensively understanding the mechanism of action of Dp44mT.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Membranas Intracelulares/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Tiossemicarbazonas/farmacologia , Androstenos/farmacologia , Anticolesterolemiantes/farmacologia , Antineoplásicos/metabolismo , Autofagia/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Colesterol/metabolismo , Relação Dose-Resposta a Droga , Feminino , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Concentração Inibidora 50 , Membranas Intracelulares/metabolismo , Membranas Intracelulares/patologia , Lisossomos/metabolismo , Lisossomos/patologia , Células MCF-7 , Permeabilidade , Interferência de RNA , Tiossemicarbazonas/metabolismo , Transfecção , beta-Ciclodextrinas/farmacologiaRESUMO
Essential metals, such as iron and copper, play a critical role in a plethora of cellular processes including cell growth and proliferation. However, concomitantly, excess of these metal ions in the body can have deleterious effects due to their ability to generate cytotoxic reactive oxygen species (ROS). Thus, the human body has evolved a very well-orchestrated metabolic system that keeps tight control on the levels of these metal ions. Considering their very high proliferation rate, cancer cells require a high abundance of these metals compared to their normal counterparts. Interestingly, new anti-cancer agents that take advantage of the sensitivity of cancer cells to metal sequestration and their susceptibility to ROS have been developed. These ligands can avidly bind metal ions to form redox active metal complexes, which lead to generation of cytotoxic ROS. Furthermore, these agents also act as potent metastasis suppressors due to their ability to up-regulate the metastasis suppressor gene, N-myc downstream regulated gene 1. This review discusses the importance of iron and copper in the metabolism and progression of cancer, how they can be exploited to target tumors and the clinical translation of novel anti-cancer chemotherapeutics.
Assuntos
Antineoplásicos , Quelantes , Cobre/metabolismo , Descoberta de Drogas , Ferro/metabolismo , Metais/metabolismo , Animais , Antineoplásicos/uso terapêutico , Quelantes/uso terapêutico , Descoberta de Drogas/métodos , Descoberta de Drogas/tendências , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Oxirredução , Espécies Reativas de Oxigênio/metabolismoRESUMO
Adenosine monophosphate-activated protein kinase (AMPK) is a cellular energy sensor that monitors ATP levels. There is also evidence that AMPK has onco-suppressive properties. Iron plays a crucial role in cellular energy transducing pathways and tumor cell proliferation. Therefore, metals (e.g., iron) could play an important role in the regulation of AMPK-dependent pathways. Hence, this investigation examined the effect of the iron and copper chelator and potent anti-cancer agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), on the AMPK-mediated pathway. These studies demonstrated that Dp44mT, which forms intracellular redox-active complexes with iron and copper, significantly activated AMPK (i.e., p-AMPK/AMPK ratio) in 5 different tumor cell-types. Furthermore, examination of the Dp44mT-metal complexes demonstrated that the effect of Dp44mT on AMPK was due to a dual mechanism: (1) its ability to chelate metal ions; and (2) the generation of reactive oxygen species (ROS). The activation of the AMPK-pathway by Dp44mT was mediated by the upstream kinase, liver kinase B1 (LKB1) that is a known tumor suppressor. Moreover, using AMPKα1-selective silencing, we demonstrated that Dp44mT activated AMPK, resulting in inhibition of acetyl CoA carboxylase 1 (ACC1) and raptor, and activation of Unc-51 like kinase (ULK1). These effects are vital for inhibition of fatty acid synthesis, suppression of protein synthesis and autophagic activation, respectively. Together, this AMPK-mediated repair response aims to rescue the loss of metal ions via chelation and the induction of cytotoxic damage mediated by redox cycling of the Dp44mT-metal ion complex. In conclusion, this study demonstrates for the first time that chelators target the AMPK-dependent pathway.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Antineoplásicos/farmacologia , Células Epiteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Quelantes de Ferro/farmacologia , Tiossemicarbazonas/farmacologia , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Linhagem Celular Tumoral , Metabolismo Energético/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Ácidos Graxos/antagonistas & inibidores , Ácidos Graxos/biossíntese , Fibroblastos , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ferro/metabolismo , Camundongos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Regulatória Associada a mTOR , Transdução de SinaisRESUMO
OBJECTIVE: Inflammation has been closely associated with the development and progression of cancer. Previously, we reported that mast cells play a critical role in tumor growth. The purpose of this study is to investigate the anti-inflammatory effect of an anticancer agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), on an activated human mast cell line, in this case HMC-1 cells. METHODS: We evaluated the effect and specific molecular mechanism of Dp44mT on phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI) using HMC-1 cells. RESULTS: Here, we demonstrated that Dp44mT significantly decreased the protein levels of hypoxia-inducible factor-1α and vascular endothelial growth factor without exposing activated HMC-1 cells to any cytotoxicity. In activated mast cells, Dp44mT mitigated the strong production and mRNA expression of inflammatory cytokines, in this case, interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, and thymic stromal lymphopoietin, through a blockade of caspase-1 and nuclear factor-κB activities. Furthermore, phosphorylations of the mitogen-activated protein kinase family included in inflammatory signaling cascades were significantly inhibited by a Dp44mT treatment. CONCLUSIONS: Overall, our results indicate that the anticancer agent Dp44mT has an anti-inflammatory effect and may be of therapeutic importance for the treatment of mast cell-mediated inflammatory diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Mastócitos/efeitos dos fármacos , Tiossemicarbazonas/farmacologia , Calcimicina/farmacologia , Inibidores de Caspase/farmacologia , Linhagem Celular , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Humanos , Fator 1 Induzível por Hipóxia/farmacologia , Ionóforos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , RNA Mensageiro/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Linfopoietina do Estroma do TimoRESUMO
Multidrug resistance (MDR) is a major obstacle in cancer treatment. More than half of human cancers express multidrug-resistant P-glycoprotein (Pgp), which correlates with a poor prognosis. Intriguingly, through an unknown mechanism, some drugs have greater activity in drug-resistant tumor cells than their drug-sensitive counterparts. Herein, we investigate how the novel anti-tumor agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes MDR. Four different cell types were utilized to evaluate the effect of Pgp-potentiated lysosomal targeting of drugs to overcome MDR. To assess the mechanism of how Dp44mT overcomes drug resistance, cellular studies utilized Pgp inhibitors, Pgp silencing, lysosomotropic agents, proliferation assays, immunoblotting, a Pgp-ATPase activity assay, radiolabeled drug uptake/efflux, a rhodamine 123 retention assay, lysosomal membrane permeability assessment, and DCF (2',7'-dichlorofluorescin) redox studies. Anti-tumor activity and selectivity of Dp44mT in Pgp-expressing, MDR cells versus drug-sensitive cells were studied using a BALB/c nu/nu xenograft mouse model. We demonstrate that Dp44mT is transported by the lysosomal Pgp drug pump, causing lysosomal targeting of Dp44mT and resulting in enhanced cytotoxicity in MDR cells. Lysosomal Pgp and pH were shown to be crucial for increasing Dp44mT-mediated lysosomal damage and subsequent cytotoxicity in drug-resistant cells, with Dp44mT being demonstrated to be a Pgp substrate. Indeed, Pgp-dependent lysosomal damage and cytotoxicity of Dp44mT were abrogated by Pgp inhibitors, Pgp silencing, or increasing lysosomal pH using lysosomotropic bases. In vivo, Dp44mT potently targeted chemotherapy-resistant human Pgp-expressing xenografted tumors relative to non-Pgp-expressing tumors in mice. This study highlights a novel Pgp hijacking strategy of the unique dipyridylthiosemicarbazone series of thiosemicarbazones that overcome MDR via utilization of lysosomal Pgp transport activity.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Tiossemicarbazonas/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Feminino , Humanos , Lisossomos/metabolismo , Células MCF-7 , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Fluorescência , Interferência de RNA , Tiossemicarbazonas/metabolismo , Vimblastina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) exhibits significant antitumor activity. However, the mechanism of its pharmacological interaction with human serum albumin (HSA) and DNA remains poorly understood. Here, we aimed to elucidate the interactions of Dp44mT with HSA and DNA using MTT assays, spectroscopic methods, and molecular docking analysis. Our results indicated that addition of HSA at a ratio of 1:1 did not alter the cytotoxicity of Dp44mT, but did affect the cytotoxicity of the Dp44mT-Cu complex. Data from fluorescence quenching and UV-VIS absorbance measurements demonstrated that Dp44mT could bind to HSA with a moderate affinity (Ka = approximately 104 M(-1)). CD spectra revealed that Dp44mT could slightly disrupt the secondary structure of HSA. Dp44mT could also interact with Ct-DNA, but had a moderate binding constant (KEB = approximately 104 M(-1)). Docking studies indicated that the IB site of HSA, but not the IIA and IIIA sites, could be favorable for Dp44mT and that binding of Dp44mT to HSA involved hydrogen bonds and hydrophobic force, consistent with thermodynamic results from spectral investigations. Thus, the moderate binding affinity of Dp44mT with HSA and DNA partially contributed to its antitumor activity and may be preferable in drug design approaches.
Assuntos
DNA/metabolismo , Albumina Sérica/metabolismo , Tiossemicarbazonas/metabolismo , Animais , Sítios de Ligação , Bovinos , Diferenciação Celular/efeitos dos fármacos , Dicroísmo Circular , Complexos de Coordenação/química , Complexos de Coordenação/toxicidade , Cobre/química , DNA/química , Células Hep G2 , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Albumina Sérica/química , Espectrometria de Fluorescência , Termodinâmica , Tiossemicarbazonas/químicaRESUMO
This Editorial refers to.
Assuntos
Antineoplásicos/farmacologia , DNA/metabolismo , Albumina Sérica/metabolismo , Tiossemicarbazonas/farmacologia , Animais , Antineoplásicos/metabolismo , Bovinos , Dicroísmo Circular , Humanos , Neoplasias/tratamento farmacológico , Retratação de Publicação como Assunto , Espectrometria de Fluorescência , Tiossemicarbazonas/metabolismoRESUMO
This response refers to.
Assuntos
Dicroísmo Circular , Simulação de Acoplamento Molecular , Animais , Bovinos , DNA/química , Humanos , Albumina Sérica/química , Espectrometria de Fluorescência , Termodinâmica , TiossemicarbazonasRESUMO
Autophagy functions as a survival mechanism during cellular stress and contributes to resistance against anticancer agents. The selective antitumor and antimetastatic chelator di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) causes lysosomal membrane permeabilization and cell death. Considering the integral role of lysosomes in autophagy and cell death, it was important to assess the effect of Dp44mT on autophagy to further understand its mechanism of action. Notably, Dp44mT affected autophagy by two mechanisms. First, concurrent with its antiproliferative activity, Dp44mT increased the expression of the classical autophagic marker LC3-II as a result of induced autophagosome synthesis. Second, this effect was supplemented by a reduction in autophagosome degradation as shown by the accumulation of the autophagic substrate and receptor p62. Conversely, the classical iron chelator desferrioxamine induced autophagosome accumulation only by inhibiting autophagosome degradation. The formation of redox-active iron or copper Dp44mT complexes was critical for its dual effect on autophagy. The cytoprotective antioxidant N-acetylcysteine inhibited Dp44mT-induced autophagosome synthesis and p62 accumulation. Importantly, Dp44mT inhibited autophagosome degradation via lysosomal disruption. This effect prevented the fusion of lysosomes with autophagosomes to form autolysosomes, which is crucial for the completion of the autophagic process. The antiproliferative activity of Dp44mT was suppressed by Beclin1 and ATG5 silencing, indicating the role of persistent autophagosome synthesis in Dp44mT-induced cell death. These studies demonstrate that Dp44mT can overcome the prosurvival activity of autophagy in cancer cells by utilizing this process to potentiate cell death.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Lisossomos/metabolismo , Neoplasias/tratamento farmacológico , Fagossomos/metabolismo , Tiossemicarbazonas/farmacologia , Acetilcisteína/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cobre/metabolismo , Desferroxamina/farmacologia , Feminino , Sequestradores de Radicais Livres/farmacologia , Humanos , Ferro/metabolismo , Lisossomos/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Oxirredução/efeitos dos fármacos , Fagossomos/patologia , Sideróforos/farmacologiaRESUMO
N-myc downstream regulated gene 1 (NDRG1) is a potent metastasis suppressor with an undefined role in the stress response. Autophagy is a pro-survival pathway and can be regulated via the protein kinase-like endoplasmic reticulum kinase (PERK)/eIF2α-mediated endoplasmic reticulum (ER) stress pathway. Hence, we investigated the role of NDRG1 in stress-induced autophagy as a mechanism of inhibiting metastasis via the induction of apoptosis. As thiosemicarbazone chelators induce stress and up-regulate NDRG1 to inhibit metastasis, we studied their effects on the ER stress response and autophagy. This was important to assess, as little is understood regarding the role of the stress induced by iron depletion and its role in autophagy. We observed that the chelator, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), which forms redox-active iron and copper complexes, effectively induced ER stress as shown by activation of the PERK/eIF2α pathway. Dp44mT also increased the expression of the autophagic marker, LC3-II, and this was dependent on activation of the PERK/eIF2α axis, as silencing PERK prevented LC3-II accumulation. The effect of Dp44mT on LC3-II expression was at least partially due to iron-depletion, as this effect was also demonstrated with the classical iron chelator, desferrioxamine (DFO), and was not observed for the DFO-iron complex. NDRG1 overexpression also inhibited basal autophagic initiation and the ER stress-mediated autophagic pathway via suppression of the PERK/eIF2α axis. Moreover, NDRG1-mediated suppression of the pro-survival autophagic pathway probably plays a role in its anti-metastatic effects by inducing apoptosis. In fact, multiple pro-apoptotic markers were increased, whereas anti-apoptotic Bcl-2 was decreased upon NDRG1 overexpression. This study demonstrates the role of NDRG1 as an autophagic inhibitor that is important for understanding its mechanism of action.
Assuntos
Autofagia , Proteínas de Ciclo Celular/biossíntese , Estresse do Retículo Endoplasmático , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Neoplasias/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Cobre/metabolismo , Desferroxamina/farmacologia , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ferro/metabolismo , Quelantes de Ferro/farmacologia , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Neoplasias/genética , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tiossemicarbazonas/farmacologia , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
N-myc down-regulated gene 1 (NDRG1) is a known metastasis suppressor in multiple cancers, being also involved in embryogenesis and development, cell growth and differentiation, lipid biosynthesis and myelination, stress responses and immunity. In addition to its primary role as a metastasis suppressor, NDRG1 can also influence other stages of carcinogenesis, namely angiogenesis and primary tumour growth. NDRG1 is regulated by multiple effectors in normal and neoplastic cells, including N-myc, histone acetylation, hypoxia, cellular iron levels and intracellular calcium. Further, studies have found that NDRG1 is up-regulated in neoplastic cells after treatment with novel iron chelators, which are a promising therapy for effective cancer management. Although the pathways by which NDRG1 exerts its functions in cancers have been documented, the relationship between the molecular structure of this protein and its functions remains unclear. In fact, recent studies suggest that, in certain cancers, NDRG1 is post-translationally modified, possibly by the activity of endogenous trypsins, leading to a subsequent alteration in its metastasis suppressor activity. This review describes the role of this important metastasis suppressor and discusses interesting unresolved issues regarding this protein.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neoplasias/terapia , Proteínas Supressoras de Tumor/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/química , Diferenciação Celular , Desenvolvimento Embrionário , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/química , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Tripsina/fisiologiaRESUMO
The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, is over expressed in renal cell carcinoma (RCC). However, the cell biology functions of RCC are not well understood. The present study aimed to verify the ability of CDKN3 to promote the proliferation and migration of RCC through in vitro experiments. Subsequently, the clinical prognostic effects were analyzed using The Cancer Genome Atlas (TCGA; https://www.cancer.gov/) and Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/). The chelators, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), an analogue of the anti-tumor agent, were screened through bioinformatics analysis. The expression of CDKN3 is positively correlated with the IC50 of Dp44mT. In two RCC cell lines, 786-0 and Caki-1, we conducted small interfering RNA (siRNA) knockdown of CDKN3 and overexpression of CDKN3 by transfection plasmid. Subsequently, we administered Dp44mT to examine the resulting alterations in cell proliferation, migration, and apoptosis, thereby elucidating the role of CDKN3 and Dp44mT in these processes. The results of the experiment revealed a positive association between CDKN3 expression and the proliferation of RCC cell lines. Down-regulating CDKN3 significantly increased the apoptosis rate and inhibited cell migration in 786-0 and Caki-1 cells. Furthermore, bioinformatics analysis revealed a high expression of CDKN3 in RCC and a negative association between CDKN3 expression and survival. Gene set enrichment analysis (GSEA) revealed a significant association between high CDKN3 expression and the cell cycle pathway. Furthermore, we identified Dp44mT as a drug highly correlated with CDKN3 through the database. Subsequent addition of Dp44mT resulted in similar findings to those observed upon CDKN3 knockdown. Our findings have important implications for the diagnosis and treatment of CDKN3 in RCC. Additionally, Dp44mT is likely to be a promising candidate for future clinical applications.
Assuntos
Carcinoma de Células Renais , Movimento Celular , Proliferação de Células , Proteínas Inibidoras de Quinase Dependente de Ciclina , Neoplasias Renais , Humanos , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Renais/patologia , Neoplasias Renais/metabolismo , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Linhagem Celular Tumoral , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Tiossemicarbazonas/farmacologia , RNA Interferente Pequeno/metabolismo , Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fosfatases de Especificidade DuplaRESUMO
BACKGROUND: Iron (Fe)-induced oxidative stress leads to reactive oxygen species that damage biomembranes, with this mechanism being involved in the activity of some anti-cancer chemotherapeutics. METHODS: Herein, we compared the effect of the ligand, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), or the potential ligand, Emodin, on Fe-catalyzed lipid peroxidation in cell membrane models (micelles and bicelles). These studies were performed in the presence of hydrogen peroxide (H2O2) and the absence or presence of ascorbate. RESULTS: In the absence of ascorbate, Fe(II)/Emodin mixtures incubated with H2O2 demonstrated slight pro-oxidant properties on micelles versus Fe(II) alone, while the Fe(III)-Dp44mT complex exhibited marked antioxidant properties. Examining more physiologically relevant phospholipid-containing bicelles, the Fe(II)- and Fe(III)-Dp44mT complexes demonstrated antioxidant activity without ascorbate. Upon adding ascorbate, there was a significant increase in the peroxidation of micelles and bicelles in the presence of unchelated Fe(II) and H2O2. The addition of ascorbate to Fe(III)-Dp44mT substantially increased the peroxidation of micelles and bicelles, with the Fe(III)-Dp44mT complex being reduced by ascorbate to the Fe(II) state, explaining the increased reactivity. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radical anion generation after mixing ascorbate and Emodin, with signal intensity being enhanced by H2O2. This finding suggested Emodin semiquinone radical formation that could play a role in its reactivity via ascorbate-driven redox cycling. Examining cultured melanoma cells in vitro, ascorbate at pharmacological levels enhanced the anti-proliferative activity of Dp44mT and Emodin. CONCLUSIONS AND GENERAL SIGNIFICANCE: Ascorbate-driven redox cycling of Dp44mT and Emodin promotes their anti-proliferative activity.
Assuntos
Emodina , Tiossemicarbazonas , Ácido Ascórbico/química , Emodina/farmacologia , Compostos Ferrosos , Peróxido de Hidrogênio , Ferro/metabolismo , Ligantes , Micelas , Oxirredução , Espécies Reativas de Oxigênio , Tiossemicarbazonas/farmacologiaRESUMO
BACKGROUND: The di-2-pyridylketone thiosemicarbazones, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), demonstrate potent and selective anti-tumor activity. In fact, DpC entered Phase I clinical trials for advanced and resistant tumors. METHODS: This investigation examined the activity of these thiosemicarbazones in five tumor cell-types compared to nine clinically used chemotherapeutics and also in combination with these drugs. RESULTS: Dp44mT and especially DpC demonstrated potent anti-proliferative activity that was significantly greater than a range of standard anti-cancer therapeutics. As most anti-cancer drugs are given in combination, further studies were performed to examine the synergistic activity of DpC or Dp44mT with these chemotherapeutics. Combination experiments revealed broad synergy between Dp44mT or DpC upon addition of these drugs, with a sequential protocol of treating first with standard chemotherapies followed by incubation with the thiosemicarbazones being optimal. However, combining DpC and Dp44mT resulted in a pronounced antagonistic drug interaction. To dissect the mechanism of this latter effect, custom-prepared 14C-DpC was implemented and examined for its uptake by cells. The avid uptake of 14C-DpC by tumor cells observed at 37 °C was suppressed at 4 °C and by the metabolic inhibitor, sodium fluoride, suggesting a temperature- and energy-dependent mechanism. Furthermore, competition studies using an excess of unlabeled Dp44mT or DpC inhibited 14C-DpC or 14C-Dp44mT uptake, respectively, suggesting these ligands utilize the same carrier/receptor, antagonizing the internalization of each other. CONCLUSIONS AND GENERAL SIGNIFICANCE: These studies demonstrate the potent and broad anti-proliferative activity of Dp44mT and particularly DpC, and are important for establishing optimized combinations with standard chemotherapies.
Assuntos
Antineoplásicos , Tiossemicarbazonas , Antineoplásicos/farmacologia , Transporte Biológico , Linhagem Celular Tumoral , Temperatura , Tiossemicarbazonas/farmacologiaRESUMO
Selective targeting of elevated copper (Cu) in cancer cells by chelators to induce tumor-toxic reactive oxygen species (ROS) may be a promising approach in the treatment of glioblastoma multiforme (GBM). Previously, the Cu chelator di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) attracted much interest due to its potent anti-tumor activity mediated by the formation of a highly redox-active Cu-Dp44mT complex. However, its translational potential was limited by the development of toxicity in murine models of cancer reflecting poor selectivity. Here, we overcame the limitations of Dp44mT by incorporating it in new biomimetic nanoparticles (NPs) optimized for GBM therapy. Biomimetic design elements enhancing selectivity included angiopeptide-2 functionalized red blood cell membrane (Ang-M) camouflaging of the NPs carrier. Co-loading Dp44mT with regadenoson (Reg), that transiently opens the blood-brain-barrier (BBB), yielded biomimetic Ang-MNPs@(Dp44mT/Reg) NPs that actively targeted and traversed the BBB delivering Dp44mT specifically to GBM cells. To further improve selectivity, we innovatively pre-loaded GBM tumors with Cu. Oral dosing of U87MG-Luc tumor bearing mice with diacetyl-bis(4-methylthiosemicarbazonato)-copperII (Cu(II)-ATSM), significantly enhanced Cu-level in GBM tumor. Subsequent treatment of mice bearing Cu-enriched orthotopic U87MG-Luc GBM with Ang-MNPs@(Dp44mT/Reg) substantially prevented orthotopic GBM growth and led to maximal increases in median survival time. These results highlighted the importance of both angiopeptide-2 functionalization and tumor Cu-loading required for greater selective cytotoxicity. Targeting Ang-MNPs@(Dp44mT/Reg) NPs also down-regulated antiapoptotic Bcl-2, but up-regulated pro-apoptotic Bax and cleaved-caspase-3, demonstrating the involvement of the apoptotic pathway in GBM suppression. Notably, Ang-MNPs@(Dp44mT/Reg) showed negligible systemic drug toxicity in mice, further indicating therapeutic potential that could be adapted for other central nervous system disorders.