RESUMO
Atherosclerosis is a chronic inflammatory disease of the arterial wall. It has been known that development of atherosclerosis is closely related to activation of tumor necrosis factor α (TNF-α). The objective of this study was to elucidate the effects of TNF-α blockade with brusatol on endothelial activation under pro-atherosclerotic conditions. To this end, we examined the effects of brusatol on TNF-α-induced intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in human aortic endothelial cells (HAECs) using western blot and THP-1 adhesion assays. Brusatol induced a decrease in TNF-α-induced ICAM-1 and VCAM-1 expression through inhibiting TNFR1 expression, leading to suppression of endothelial inflammation independently of the NRF2 (nuclear factor erythroid 2-related factor 2) pathway. The mechanism underlying brusatol-induced TNF receptor 1 (TNFR1) inhibition was investigated with the aid of protein synthesis, co-immunoprecipitation, and cytokine arrays. Notably, brusatol inhibited TNFR1 protein synthesis and suppressed both the canonical nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling pathway and TNF-α-induced cytokine secretion. We further tested the functional effect of brusatol on atherosclerosis development in vivo using two different atherosclerosis mouse models, specifically, acute partial carotid ligation and conventional chronic high-fat diet-fed mouse models. Administration of brusatol led to significant suppression of atherosclerosis development in both mouse models. Our finding that brusatol prevents atherosclerosis via inhibition of TNFR1 protein synthesis supports the potential of downregulation of cell surface TNFR1 as an effective therapeutic approach to inhibit development of atherosclerosis.
Assuntos
Aterosclerose/prevenção & controle , Quassinas/uso terapêutico , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Animais , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complexo de Endopeptidases do Proteassoma/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
It is highly challenging to develop fast and sensitive fluorescent methods for monitoring organic mercury in purely aqueous solutions as well as live cells. Especially, selective fluorescent detection of methylmercury over inorganic mercury ions has not been reported. We developed a fast and sensitive fluorescent detection method for Hg2+ ions as well as methylmercury using an amino acid-based fluorescent probe (1) and SDS micelles. The fluorescent probe in SDS micelles detected sensitively and selectively Hg2+ ions and methylmercury among 16 metal ions in purely aqueous solution by the enhancement of the red emission at 575 nm, and the detection of methylmercury was completed within 1 min. The probe in SDS micelles with EDTA showed highly sensitive and selective turn on detection for methylmercury over Hg2+. The limit of detection was 9.1 nM for Hg2+ (1.8 ppb, R2 = 0.989) and 206 nM for CH3Hg+ (R2 = 0.997). 1 rapidly penetrated live cells and detected intracellular Hg2+ ions as well as CH3Hg+ by the enhancement of both red emissions and green emissions. Subsequent treatment of EDTA into the cell confirmed the selective detection of methylmercury in the cells. The present work indicated that the fluorescent probe with micelle systems provided a fast, sensitive, and selective detection method for monitoring inorganic mercury as well as methyl mercury.
Assuntos
Corantes Fluorescentes/química , Compostos de Metilmercúrio/análise , Poluentes Químicos da Água/química , Células A549 , Humanos , Micelas , Conformação Molecular , Imagem Óptica , Soluções , Espectrometria de FluorescênciaRESUMO
The stimuli-responsive conformational transformation of peptides possessing a constrained form triggered by specific biological microenvironment would provide an effective strategy for the development of highly specific peptide therapeutics. Here, we developed a peptide containing a cytotoxic helical KLA sequence with therapeutic specificity through the use of stimuli-responsive conformational transformation. The KLA peptide is modified to form a cyclic structure to allow for constrained helicity that confers low cytotoxicity. The modified KLA peptide is electrostatically complexed to hyaluronic acid to facilitate enhanced endocytosis into the cancer cells. After endocytosis, the peptide is released from the complex into the cellular cytoplasm by hyaluronidases on the surface of the cellular membrane. Specific intracellular stimuli then trigger the release of the strain that suppresses peptide helicity, and the inherent helical conformation of the KLA peptide is restored. Therefore, the stimuli-responsive conformational transformation of a peptide from low to high helicity selectively induces cell death by disruption of the plasma and mitochondrial membrane.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Descoberta de Drogas , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Membranas Mitocondriais/efeitos dos fármacos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Conformação Proteica em alfa-HéliceRESUMO
5-Fluorouracil (5-FU) is an important chemotherapeutic agent for the systemic treatment of colorectal cancer (CRC), but its effectiveness against CRC is limited by increased 5-FU resistance caused by the hypoxic tumor microenvironment. The purpose of our study was to assess the feasibility of using quinacrine (QC) to increase the efficacy of 5-FU against CRC cells under hypoxic conditions. QC reversed the resistance to 5-FU induced by hypoxia in CRC cell lines, as determined using ATP-Glo cell viability assays and clonogenic survival assays. Treatment of cells with 5-FU under hypoxic conditions had no effect on the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a regulator of cellular resistance to oxidative stress, whereas treatment with QC alone or in combination with 5-FU reduced Nrf2 expression in all CRC cell lines tested. Overexpression of Nrf2 effectively prevented the increase in the number of DNA double-strand breaks induced by QC alone or in combination with 5-FU. siRNA-mediated c-Jun N-terminal kinase-1 (JNK1) knockdown inhibited the QC-mediated Nrf2 degradation in CRC cells under hypoxic conditions. The treatment of CRC xenografts in mice with the combination of QC and 5-FU was more effective in suppressing tumor growth than QC or 5-FU alone. QC increases the susceptibility of CRC cells to 5-FU under hypoxic conditions by enhancing JNK1-dependent Nrf2 degradation.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Fluoruracila/farmacologia , Hipóxia/genética , Hipóxia/metabolismo , Fator 2 Relacionado a NF-E2/genética , Quinacrina/farmacologia , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Modelos Animais de Doenças , Fluoruracila/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Modelos Biológicos , Fator 2 Relacionado a NF-E2/metabolismo , Estadiamento de Neoplasias , Proteólise , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Utilizing stimuli-responsive conformational transformation of a cyclic peptide as a gatekeeper for mesoporous nanocarriers has several advantages such as facile introduction of targeting capabilities, low enzymatic degradation during blood circulation and enhanced specific binding to selected cells. In this report, a Asn-Gly-Arg (NGR)-containing dual-functional cyclic peptide gatekeeper on the surface of mesoporous nanocarrier is prepared not only for active targeting of the aminopeptidaseâ N (APN) expressed on cancer cells but also stimuli-responsive intracellular drug release triggered by a glutathione (GSH)-induced conformational transformation of the peptide gatekeeper. The peptide gatekeeper on the surface of nanocarriers exhibits on-off gatekeeping by conformational transformation triggered by intracellular glutathione of the cancer cells. H1299 cells (high APN expression) showed greater uptake of the nanocarrier by endocytosis and higher apoptosis than A549 cells (low APN expression).
Assuntos
Antineoplásicos/farmacologia , Antígenos CD13/metabolismo , Doxorrubicina/farmacologia , Nanocápsulas/química , Peptídeos Cíclicos/química , Dióxido de Silício/química , Antineoplásicos/química , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Preparações de Ação Retardada , Doxorrubicina/química , Liberação Controlada de Fármacos , Glutationa/química , Humanos , Conformação Molecular , Oligopeptídeos/química , Tamanho da Partícula , Porosidade , Propriedades de SuperfícieRESUMO
A fluorescent peptidyl chemosensor for the detection of heavy metal ions in aqueous solution as well as in cells was synthesized on the basis of the peptide receptor for the metal ions using an aggregation-induced emission fluorophore. The peptidyl chemosensor (1) bearing tetraphenylethylene fluorophore showed an exclusively selective turn-on response to Hg(2+) among 16 metal ions in aqueous buffered solution containing NaCl. The peptidyl chemosensor complexed Hg(2+) ions and then aggregated in aqueous buffered solution, resulting in the significant enhancement (OFF-On) of emissions at around 470 nm. The fluorescent sensor showed a highly sensitive response to Hg(2+), and about 1.0 equiv of Hg(2+) was enough for the saturation of the emission intensity change. The detection limit (5.3 nM, R(2) = 0.99) of 1 for Hg(2+) ions was lower than the maximum allowable level of Hg(2+) in drinking water by EPA. Moreover, the peptidyl chemosensor penetrated live cells and detected intracellular Hg(2+) ions by the turn-on response.
Assuntos
Metais Pesados/análise , Peptídeos/química , Fluorescência , Concentração de Íons de Hidrogênio , Limite de Detecção , Soluções , Espectrometria de Fluorescência , ÁguaRESUMO
Aneuploidy is the most common characteristic of human cancer cells. It also causes genomic instability, which is involved in the initiation of cancer development. Various lines of evidence indicate that nicotinamide adenine dinucleotide(P)H quinone oxidoreductase 1 (NQO1) plays an important role in cancer prevention, but the molecular mechanisms underlying this effect have not yet been fully elucidated. Here, we report that ionizing radiation (IR) induces substantial aneuploidy and centrosome amplification in NQO1-deficient cancer cells, suggesting that NQO1 plays a crucial role in preventing aneuploidy. NQO1 deficiency markedly increased the protein stability of Aurora-A in irradiated cancer cells. Small interfering RNA targeting Aurora-A effectively attenuated IR-induced centrosome amplification concerned with aneuploidy in NQO1-deficient cancer cells. Furthermore, we found that NQO1 specifically binds to Aurora-A via competing with the microtubule-binding protein, TPX2 (targeting protein for Xklp2), and contributes to the degradation of Aurora-A. Our results collectively demonstrate that NQO1 plays a key role in suppressing IR-induced centrosome amplification and aneuploidy through a direct interaction with Aurora-A.
Assuntos
Aneuploidia , Aurora Quinase A/metabolismo , Neoplasias da Mama/patologia , Centrossomo , Radioisótopos de Césio , Raios gama , NAD(P)H Desidrogenase (Quinona)/metabolismo , Apoptose/efeitos da radiação , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/radioterapia , Ciclo Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Feminino , Humanos , Imunoprecipitação , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/genética , RNA Interferente Pequeno/genética , Células Tumorais CultivadasRESUMO
Lead is one of the most toxic substances. However, there are few ratiometric fluorescent probes for sensing Pb2+ in aqueous solution as well as living cells because specific ligands for Pb2+ ions have not been well characterized. Considering the interactions between Pb2+ and peptides, we developed ratiometric fluorescent probes for Pb2+ based on the peptide receptor in two steps. First, we synthesized fluorescent probes (1-3) based on the tetrapeptide receptor (ECEE-NH2) containing hard and soft ligands by conjugation with diverse fluorophores that showed excimer emission when they aggregated. After investigation of fluorescent responses to metal ions, benzothiazolyl-cyanovinylene was evaluated as an appropriate fluorophore for ratiometric detection of Pb2+. Next, we modified the peptide receptor to decrease the number of hard ligands and/or to replace Cys with disulfide bond and methylated Cys for improving selectivity and cell permeability. From this process, we developed two fluorescent probes (3 and 8) among the probes (1-8) that exhibited remarkable ratiometric sensing properties for Pb2+ including high water solubility (≤2% DMF), visible light excitation, high sensitivity, selectivity for Pb2+, low detection limits (<10 nM), and fast response (<6 min). The binding mode study revealed that specific Pb2+-peptide interactions of the probes caused nanosized aggregates in which the fluorophores of the probes came close each other, exhibiting excimer emission. In particular, 8 based on tetrapeptide bearing a disulfide bond and two carboxyl groups with a good permeability successfully quantified intracellular uptake of Pb2+ in live cells through ratiometric fluorescent signals. The ratiometric sensing system based on specific metal-peptide interactions and excimer emission process could provide a valuable tool to quantify Pb2+ in live cells and pure aqueous solutions.
RESUMO
Rationale: Overexpression of NAD(P)H:quinone oxidoreductase 1 (NQO1) is associated with tumor cell proliferation and growth in several human cancer types. However, the molecular mechanisms underlying the activity of NQO1 in cell cycle progression are currently unclear. Here, we report a novel function of NQO1 in modulation of the cell cycle regulator, cyclin-dependent kinase subunit-1 (CKS1), at the G2/M phase through effects on the stability of cFos. Methods: The roles of the NQO1/c-Fos/CKS1 signaling pathway in cell cycle progression were analyzed in cancer cells using synchronization of the cell cycle and flow cytometry. The mechanisms underlying NQO1/c-Fos/CKS1-mediated regulation of cell cycle progression in cancer cells were studied using siRNA approaches, overexpression systems, reporter assays, co-immunoprecipitation, pull-down assays, microarray analysis, and CDK1 kinase assays. In addition, publicly available data sets and immunohistochemistry were used to investigate the correlation between NQO1 expression levels and clinicopathological features in cancer patients. Results: Our results suggest that NQO1 directly interacts with the unstructured DNA-binding domain of c-Fos, which has been implicated in cancer proliferation, differentiation, and development as well as patient survival, and inhibits its proteasome-mediated degradation, thereby inducing CKS1 expression and regulation of cell cycle progression at the G2/M phase. Notably, a NQO1 deficiency in human cancer cell lines led to suppression of c-Fos-mediated CKS1 expression and cell cycle progression. Consistent with this, high NQO1 expression was correlated with increased CKS1 and poor prognosis in cancer patients. Conclusions: Collectively, our results support a novel regulatory role of NQO1 in the mechanism of cell cycle progression at the G2/M phase in cancer through effects on cFos/CKS1 signaling.
Assuntos
Ciclo Celular , NAD(P)H Desidrogenase (Quinona) , Neoplasias , Humanos , Divisão Celular , Linhagem Celular Tumoral , Fase G2 , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Neoplasias/genéticaRESUMO
Histone deacetylase (HDAC) plays an important role in cancer onset and progression. Therefore, inhibition of HDAC offers potential as an effective cancer treatment regimen. CG200745, (E)-N(1)-(3-(dimethylamino)propyl)-N(8)-hydroxy-2-((naphthalene-1-loxy)methyl)oct-2-enediamide, is a novel HDAC inhibitor presently undergoing a phase I clinical trial. Enhancement of p53 acetylation by HDAC inhibitors induces cell cycle arrest, differentiation, and apoptosis in cancer cells. The purpose of the present study was to investigate the role of p53 acetylation in the cancer cell death caused by CG200745. CG200745-induced clonogenic cell death was 2-fold greater in RKO cells expressing wild-type p53 than in p53-deficient RC10.1 cells. CG200745 treatment was also cytotoxic to PC-3 human prostate cancer cells, which express wild-type p53. CG200745 increased acetylation of p53 lysine residues K320, K373, and K382. CG200745 induced the accumulation of p53, promoted p53-dependent transactivation, and enhanced the expression of MDM2 and p21(Waf1/Cip1) proteins, which are encoded by p53 target genes. An examination of CG200745 effects on p53 acetylation using cells transfected with various p53 mutants showed that cells expressing p53 K382R mutants were significantly resistant to CG200745-induced clonogenic cell death compared with wild-type p53 cells. Moreover, p53 transactivation in response to CG200745 was suppressed in all cells carrying mutant forms of p53, especially K382R. Taken together, these results suggest that acetylation of p53 at K382 plays an important role in CG200745-induced p53 transactivation and clonogenic cell death.
Assuntos
Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Naftalenos/farmacologia , Neoplasias da Próstata/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Meia-Vida , Histonas/metabolismo , Humanos , Lisina , Masculino , Mutação , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Fatores de Tempo , Ativação Transcricional/efeitos dos fármacos , Transfecção , Ensaio Tumoral de Célula-Tronco , Proteína Supressora de Tumor p53/genéticaRESUMO
We developed a novel method for harvesting endothelial cells from blood vessels of freshly obtained cancer and adjacent normal tissue of human breast, and compared the response of the cancer-derived endothelial cells (CECs) and normal tissue-derived endothelial cells (NECs) to ionizing radiation. In brief, when tissues were embedded in Matrigel and cultured in endothelial cell culture medium (ECM) containing growth factors, endothelial cells grew out of the tissues. The endothelial cells were harvested and cultured as monolayer cells in plates coated with gelatin, and the cells of 2nd-5th passages were used for experiments. Both CECs and NECs expressed almost the same levels of surface markers CD31, CD105 and TEM-8 (tumor endothelial marker-8), which are known to be expressed in angiogenic endothelial cells, i.e., mitotically active endothelial cells. Furthermore, both CECs and NECs were able to migrate into experimental wound in the monolayer culture, and also to form capillary-like tubes on Matrigel-coated plates. However, the radiation-induced suppressions of migration and capillary-like tube formations were greater for CECs than NECs from the same patients. In addition, in vitro clonogenic survival assays demonstrated that CECs were far more radiosensitive than NECs. In summary, we have developed a simple and efficient new method for isolating endothelial cells from cancer and normal tissue, and demonstrated for the first time that endothelial cells of human breast cancer are significantly more radiosensitive than their normal counterparts from the same patients.
Assuntos
Neoplasias da Mama/irrigação sanguínea , Mama/irrigação sanguínea , Células Endoteliais/efeitos da radiação , Tolerância a Radiação , Biomarcadores/metabolismo , Movimento Celular/efeitos da radiação , Separação Celular/métodos , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Relação Dose-Resposta à Radiação , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Humanos , Neovascularização Fisiológica/efeitos da radiação , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
Although diverse cell penetrating motifs not only from naturally occurring proteins but also from synthetic peptides have been discovered and developed, the selectivity of cargo delivery connected to these motifs into the desired target cells is generally low. Here, we demonstrate the selective cytotoxicity tuning of an anticancer KLA peptide with a cell penetrating motif activatable by matrix metalloproteinase-2 (MMP2). The anionic masking sequence introduced at the end of the KLA peptide through an MMP2-cleavable linker is selectively cleaved by MMP2 and the cationic cell penetrating motif is activated. Upon treatment of the peptide to H1299 cells (high MMP2 level), it is selectively internalized into the cells by MMP2, which consequently induces membrane disruption and cell death. In contrast, the peptide shows negligible cytotoxicity toward A549 cancer cells with low MMP2 levels. Furthermore, the selective therapeutic efficacy of the peptide induced by MMP2 is also corroborated using in vivo study.
RESUMO
Three-dimensional (3D) cancer cell culture systems have been developed to aid the study of molecular mechanisms in cancer development, identify therapeutic targets, and test drug candidates. In this study, we developed a strategy for mimicking the hypoxic tumor microenvironment in a 3D cancer cell culture system using multi-layer, nanofibrous poly(ε-caprolactone) (PCL) scaffold (pNFS)-based cancer cell cultures. We found that human colon cancer cells infiltrated pNFS within 3 days and could be cultured three-dimensionally within the NFS. When incubated in four stacks of 30 µm-thick pNFS for 3 days, colon cancer cells in layer three showed partially reduced entry into the S phase, whereas those in layer four, located farthest from the media, showed a marked reduction in S-phase entry. As a consequence, cells in layer four exhibited hypoxia-induced disorganization of F-actin on day 3, and those in layers three and four showed an increase in the expression of the hypoxia-induced transcription factor HIF-1α and its target genes, Glut1, CA9, VEGF, and LDHA. Consistent with these results, doxorubicin- and ionizing radiation-induced cell death was reduced in colon cancer cells cultured in layers three and four. These results suggest that pNFS-based multi-layer colon cancer cell cultures mimic the hypoxic tumor microenvironment and are useful for bioassays.
RESUMO
We present a reaction-based fluorescent probe (1) for Hg2+ and CH3Hg+, based on the displacement reaction of the arylboronic acid with the mercury species. 1 showed promising sensing properties for Hg2+ and CH3Hg+, such as high selectivity and sensitivity, turn-on response, fast response to Hg2+ (<2 min) and CH3Hg+ (<5 min), low detection limits and operation in purely aqueous solutions.
Assuntos
Ácidos Borônicos/análise , Mercúrio/análise , Compostos de Metilmercúrio/análise , Espectrometria de Fluorescência/métodos , Limite de DetecçãoRESUMO
NAD(P)H:quinone oxidoreductase (NQO1) has been reported to play an important role in cell death caused by beta-lapachone (beta-lap), 3,4-dihydro-22,2-dimethyl-2H-naphthol[1,22b]pyran-5,6-dione. This study investigated whether cisplatin (cis-diamminedichloroplatinum) sensitizes cancer cells to beta-lap by upregulating NQO1. The cytotoxicity of cisplatin and beta-lap alone or in combination against FSaII fibrosarcoma cells of C3H mice in vitro was determined with a clonogenic survival assay and assessment of gamma-H2AX foci formation, a hallmark of DNA double-strand breaks. The cellular sensitivity to beta-lap progressively increased during the 24 h after cisplatin treatment. The expression and enzymatic activity of NQO1 also increased during the 24 h after cisplatin treatment, and dicoumarol, an inhibitor of NQO1, was found to nullify the cisplatin-induced increase in beta-lap sensitivity. The role of NQO1 in the cell death caused by beta-lap alone or in combination with cisplatin was further elucidated using NQO1-positive and NQO1-negative MDA-MB-231 human breast cancer cells. Cisplatin increased the sensitivity of the NQO1-positive but not the NQO1-negative MDA-MB-231 cells to beta-lap treatment. Combined treatment with cisplatin and beta-lap suppressed the growth of FSaII tumors in the legs of C3H mice in a manner greater than additive. It is concluded that cisplatin markedly increases the sensitivity of cancer to beta-lap in vitro and in vivo by upregulating NQO1.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Cisplatino/farmacologia , Naftoquinonas/farmacologia , Regulação para Cima/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C3H , NAD(P)H Desidrogenase (Quinona) , NADPH Desidrogenase/metabolismoRESUMO
PURPOSE: To determine the efficacy of a caffeine derivative 1-methylxanthine (1-MTX) in increasing radiosensitivity of cancer cells and elucidate the underlying mechanisms in vitro. MATERIALS AND METHODS: RKO human colorectal cancer cells carrying wild type protein 53 kDa (p53) were incubated with 3 mM 1-MTX for 30 min, exposed to 4 Gy ionizing radiation, and further incubated with 1-MTX for three days. The clonogenic cell death was determined, and the cell cycle distribution and apoptosis were studied with flow cytometry at different times after irradiation. The DNA double strand break (DNA DSB) was examined using phosphorylated Histone2A (gamma-H2AX) foci formation, and the expression/activity of checkpoint 2 kinase (Chk2), cell division cycle 25 (Cdc25) phosphatase and cyclin B1/Cdc2 kinase were also investigated using western blotting and in vitro kinase assays. RESULTS: The treatment with 3 mM 1-MTX increased the radiation-induced clonogenic and apoptotic cell death. The radiation-induced phosphorylation of Chk2 and Cdc25c and the radiation-induced increase in the cyclin B1/Cdc2 kinas activity were little affected by 1-MTX. The radiation-induced G2/M arrest was only slightly shortened and the expression of radiation-induced gamma-H2AX was markedly prolonged by 1-MTX. CONCLUSIONS: 1-MTX significantly increased the radiosensitivity of RKO human colorectal cancer cells carrying wild type p53 mainly by inhibiting the repair of radiation-induced DNA DSB without causing significant alteration in radiation-induced G2/M arrest. Such a radiosensitization occurred at 1-MTX concentrations almost non-toxic to the target tumor cells.
Assuntos
Neoplasias/radioterapia , Radiossensibilizantes/farmacologia , Xantinas/farmacologia , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Dano ao DNA , Fase G2/efeitos da radiação , Histonas/análise , Humanos , Proteína Supressora de Tumor p53/análise , Fosfatases cdc25/fisiologiaRESUMO
NAD(P)H:quinone oxidoreductase (NQO1) mediates cell death caused by the novel anti-cancer drug beta-lapachone (beta-lap). Therefore, beta-lap sensitivity of cells is positively related to the level of cellular NQO1. Heat shock up-regulates NQO1 expression in cancer cells, thereby enhancing the clonogenic cell death caused by beta-lap. The mechanisms by which heat shock elevates NQO1 expression were investigated in the present study using human A549 lung cancer cells and human MDA-MB-231 breast cancer cells. When MDA-MB-231(NQO1+) cells stably transfected with NQO1 were heated at 42 degrees C for 1 h the expression of NQO1 and the sensitivity of the cells to beta-lap progressively increased during the 24-48 h post-heating period. Heating increased NQO1 transcription by cis-acting elements such as xenobiotic response element and antioxidant response element located in the NQO1 gene promoter region. The turnover of NQO1 protein in heated cells was much slower than in unheated cells. NQO1 and heat shock protein 70 (Hsp70) co-precipitated and co-localised in cells before and after heating, demonstrating the close association of these two proteins in the cells. These results suggest that NQO1 is stabilised by the Hsp70 molecular chaperone. It is concluded that the prolonged increase in NQO1 expression after heat shock is due to increased NQO1 transcription, and also increased Hsp70-mediated NQO1 stabilisation.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proteínas de Choque Térmico HSP70/fisiologia , Resposta ao Choque Térmico , NAD(P)H Desidrogenase (Quinona)/biossíntese , Naftoquinonas/farmacologia , Morte Celular , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hipertermia InduzidaRESUMO
Hypoxia leads to cancer progression and promotes the metastatic potential of cancer cells. MicroRNAs (miRNAs) are small non-coding RNA that have emerged as key players involved in cancer development and progression. Hypoxia alters a set of hypoxia-mediated miRNAs expression during tumor development and it may function as oncogenes or tumor-suppressors. However, the roles and molecular mechanisms of hypoxia-regulatory miRNAs in colorectal cancer (CRC) progression remain poorly understood. Here we firstly identified miR-590-5p as hypoxia-sensitive miRNAs which was upregulated in colon cancer cells under hypoxia. Hypoxia-induced miR-590-5p suppressed the expression of RECK, in turn, promoting cell invasiveness and migratory abilities via activation of matrix metalloproteinases (MMPs) and filopodia protrusion in vitro. Inhibition of miR-590-5p suppressed tumor growth and metastasis in mouse xenograft and CRC liver metastasis models via inhibition of MMPs activity. Clinical analysis revealed higher miR-590-5p expression in CRC, compared to normal specimens. Furthermore, miR-590-5p expression was significantly increased in liver metastasis as compared to their matched primary CRC. Taken together, our findings provide the first evidence that miR-590-5p may have potential as a therapeutic target for CRC patients with metastasis.
Assuntos
Neoplasias Colorretais/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Metaloproteinases da Matriz/genética , MicroRNAs/genética , Regiões 3' não Traduzidas/genética , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Progressão da Doença , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Células HCT116 , Humanos , Hipóxia , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos Nus , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Transplante HeterólogoRESUMO
HIF-1 (hypoxia-inducible factor-1) regulates the expression of ~100 genes involved in angiogenesis, metastasis, tumor growth, chemoresistance and radioresistance, underscoring the growing interest in targeting HIF-1 for cancer control. In the present study, we investigated the molecular mechanisms underlying brusatol-induced HIF-1α degradation and cell death in colorectal cancer under hypoxia (0.5% O2). Under hypoxia, pretreatment of cancer cells with brusatol increased HIF-1α degradation and cancer cell death in a dose-dependent manner. This effect was mediated by activation of prolyl hydroxylases (PHDs), as evidenced by the block of brusatol-induced HIF-1α degradation and cancer cell death by both pharmacological inhibition and siRNA-mediated knockdown of PHDs. In addition, a ferrous iron chelator (2,2'-bypyridyl) blocked brusatol-induced degradation of HIF-1α and cancer cell death in hypoxia by inhibiting PHD activation. We further found that brusatol inhibited c-Myc expression, and showed that overexpression of c-Myc prevented brusatol-induced degradation of HIF-1α and cancer cell death by increasing mitochondrial ROS production and subsequent ROS-mediated transition of ferrous iron to ferric iron. Consistent with these results, treatment of tumor-bearing mice with brusatol significantly suppressed tumor growth by promoting PHD-mediated HIF-1α degradation. Collectively, our results suggest that brusatol-mediated inhibition of c-Myc/ROS signaling pathway increases HIF-1α degradation by promoting PHD activity and induces cell death in colorectal cancer under hypoxia.