RESUMO
Hepatocellular carcinoma (HCC) is one of the most common cancers and a leading cause of cancer mortality. Prognosis of this disease largely depends on its stage. An Enlarged liver, due to dysplasia, may be a critical point in the multi-step progression to HCC. The mechanism underlying hepatomegaly in human and mouse models are poorly understood. We previously reported we observed enlarged liver in hepatitis B virus X protein (HBx) expressing mice (HBx mice). Here we identify the critical role of HBx induced IGF-II in hepatomegaly in mice and abnormal cell growth in human hepatoma cells. We found that HBx induced IGF-II is essential to induce epithelial-mesenchymal transition (EMT) through loss of E-cadherin. In mouse liver, loss of E-cadherin was mediated by post-translational regulation, at least in part, by protease and SUMOylation not by transcriptional regulation. In contrast, in hepatoma cell line (HepG2 cells) Akt signal pathway controls the mRNA expression level of EMT-related transcription factors, especially Twist, in addition to post- translational modification through SUMOylation. Thus, IGF-II-mediated loss of E-cadherin is central in developing hepatomegaly in mice and abnormal cell growth in the hepatoma cell line. HBx induced IGF-II represents a potential biomarker, which is also a therapeutic target in HCC.
Assuntos
Caderinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator de Crescimento Insulin-Like II/metabolismo , Proteína SUMO-1/metabolismo , Transativadores/metabolismo , Animais , Antígenos CD , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/metabolismo , Proteínas Cdh1/metabolismo , Transição Epitelial-Mesenquimal , Células Hep G2 , Hepatócitos/metabolismo , Hepatomegalia/metabolismo , Homozigoto , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Processamento de Proteína Pós-Traducional , Proteínas Virais Reguladoras e AcessóriasRESUMO
BACKGROUND/AIM: The current study investigated the mechanisms underlying the antitumor activity of SB265610, a cysteine-amino acid-cysteine (CXC) chemokines receptor 2 (CXCR2) antagonist. MATERIALS AND METHODS: Cell-cycle progression and regulatory molecules were assessed by flow cytometry, immunoblotting, real-time PCR and immunoprecipitation. Target validation was achieved via RNA interference. RESULTS: G1 arrest induced by SB265610 occurred at concentrations lacking CXCR2 selectivity, persisted upon interleukin 8 (IL8) challenge, and did not affect IL8 downstream target expression. Profiling of G1 regulators revealed cyclin-dependent kinase 2 (CDK2) (Thr160) hypophosphorylation, cyclin D3 gene down-regulation, and p21 post-translational induction. However, only cyclin D3 and CDK2 contributed towards G1 arrest. Furthermore, SB265610 induced a sustained phosphorylation of the p38MAPK. Pharmacological interference with p38MAPK significantly abrogated SB265610-induced G1 arrest and normalized the expression of cyclin D3, with restoration of its exclusive binding to CDK6, but with weak recovery of CDK2 (Thr160) hypo-phosphorylation. CONCLUSION: The present study described the mechanisms for the anti-proliferative activity of SB265610 which may be of value in IL8-rich tumor microenvironments.
Assuntos
Ciclina D3/biossíntese , Quinase 2 Dependente de Ciclina/biossíntese , Compostos de Fenilureia/administração & dosagem , Neoplasias da Próstata/genética , Receptores de Interleucina-8B/biossíntese , Triazóis/administração & dosagem , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Ciclina D3/genética , Quinase 2 Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Fase G1/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-8/genética , Masculino , Fosforilação , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Receptores de Interleucina-8B/genética , Microambiente Tumoral/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
In this study, we have synthesized novel water soluble derivatives of natural compound aloe emodin 4(a-j) by coupling with various amino acid esters and substituted aromatic amines, in an attempt to improve the anticancer activity and to explore the structure-activity relationships. The structures of the compounds were determined by (1) H NMR and mass spectroscopy. Cell growth inhibition assays revealed that the aloe emodin derivatives 4d, 4f, and 4i effectively decreased the growth of HepG2 (human liver cancer cells) and NCI-H460 (human lung cancer cells) and some of the derivatives exhibited comparable antitumor activity against HeLa (Human epithelial carcinoma cells) and PC3 (prostate cancer cells) cell lines compared to that of the parent aloe emodin at low micromolar concentrations.
Assuntos
Antraquinonas/química , Antraquinonas/farmacologia , Antineoplásicos/síntese química , Antraquinonas/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células HeLa , Células Hep G2 , Humanos , Relação Estrutura-AtividadeRESUMO
Novel combinations aiming at maximizing the efficacy of bortezomib are highly valued in the clinic. Therefore the current study investigated the outcomes of combining bortezomib with dipyridamole, a well-known antiplatelet. The co-treatment exerted a synergistic lethality in a panel of human leukemia/lymphoma cell lines of different origin. Mechanistically, dipyridamole did not modulate the proteasome inhibitory activity of bortezomib. However, dipyridamole triggered an endoplasmic reticulum (ER) stress, and co-treatment with bortezomib resulted in higher levels of ER stress than either monotherapies. Relieving ER stress with the protein translation inhibitor, cycloheximide suppressed cell death. Moreover, the enhanced ER stress by the co-treatment was associated with an aggravation of reactive oxygen species (ROS) generation and glutathione (GSH) depletion. Replenishing GSH pools significantly scavenged ROS and rescued the cells. Importantly, the cytotoxicity of the co-treatment was executed mainly via the mitochondrial apoptotic pathway with an efficient suppression of the key anti-apoptotic regulators, Mcl-1, Bcl-xl, Bcl-2 and XIAP, driving the independence of the co-treatment-induced apoptosis of a single apoptotic trigger. Furthermore, the intrinsic potential of bortezomib to inhibit important pro-survival pathways was enhanced by dipyridamole in a GSH/ROS-dependent manner. Interestingly, dipyridamole abrogated JAK2 phosphorylation indirectly and selectively in cancer cells, and the co-treatment-induced cytotoxicity was preserved in a model of stromal-mediated chemoresistance. In nude mice, the antitumor activity of the co-treatment surpassed that of bortezomib monotherapy despite that synergy was lacking. In summary, findings of the present study provided a preclinical rationale which warrants further clinical evaluation of bortezomib/dipyridamole novel combination in hematologic malignancies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Hematológicas/tratamento farmacológico , Animais , Bortezomib/farmacologia , Dipiridamol/farmacologia , Células HL-60 , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Humanos , Células Jurkat , Células K562 , Camundongos , Camundongos Nus , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Células U937 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, an event critical for the maintenance of genomic integrity. We show that Plk4 relocalizes from the inner Cep192 ring to the outer Cep152 ring as newly recruited Cep152 assembles around the Cep192-encircled daughter centriole. Crystal-structure analyses revealed that Cep192- and Cep152-derived peptides bind the cryptic polo box (CPB) of Plk4 in opposite orientations and in a mutually exclusive manner. The Cep152 peptide bound to the CPB markedly better than did the Cep192 peptide and effectively 'snatched' the CPB away from a preformed CPB-Cep192 peptide complex. A cancer-associated Cep152 mutation impairing the Plk4 interaction induced defects in procentriole assembly and chromosome segregation. Thus, Plk4 is intricately regulated in time and space through ordered interactions with two distinct scaffolds, Cep192 and Cep152, and a failure in this process may lead to human cancer.
Assuntos
Proteínas de Ciclo Celular/química , Centríolos/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Serina-Treonina Quinases/química , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , Células HEK293 , Células HeLa , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Neoplasias/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de ProteínaRESUMO
Despite advances in treatment, prognosis for many types of carcinoma remains poor. Polo-like kinase 1 (Plk1) has been explored as a target for the development of anticancer drugs. As a mitotic master Ser/Thr kinase, Plk1 is involved in centrosomal maturation, microtubule nucleation, chromosomal segregation, and cytokinesis. Additional functions in interphase and in response to DNA damage have been revealed. The multiple locations of Plk1 correspond to distinct functions, mediated by phosphorylation of multiple substrates. Since it is highly expressed in several carcinomas, and expression of Plk1 is inversely correlated with the survival rate of patients in non-small cell lung, head and neck, and esophageal cancer, Plk1 is recognized as a valid prognostic marker. Connections between Plk1 and p53 or KRAS in carcinoma provide a rationale and several possible routes to the development of therapies. Tumors with both p53-deficiency and high Plk1 expression may be particularly sensitive to Plk1 inhibitors, although some controversial data exist. In KRAS-mutant cancers, on the other hand, Plk1 may be essential for tumor cell survival, but detailed studies as to whether Plk1 inhibitors are more effective in KRAS-mutant cancers must be performed in order to determine whether this is the case. Here, we present evidence for Plk1 as a prognostic marker and potentially effective target for the treatment of patients with carcinoma, to demonstrate the value of Plk1 as a target for the development of cancer treatment, especially for patients with solid tumors. In addition, the effects of Plk1 inhibition in p53- or KRAS-mutated cancer are discussed with respect to clinical implications. Structural specifics of Plk1 are presented, as well as current strategies for discovering new Plk1 inhibitors by targeting the conserved ATP binding site or polo-box domain of Plk1, in order to develop Plk1-specific anticancer drugs.
RESUMO
Mitogen- and stress-activated kinase 1 (MSK1) is a nuclear serine/threonine protein kinase that acts downstream of both extracellular signal-regulated kinases and p38 mitogen-activated protein kinase in response to stress or mitogenic extracellular stimuli. Increasing evidence has shown that MSK1 is closely associated with malignant transformation and cancer development. MSK1 should be an effective target for cancer chemoprevention and chemotherapy. However, very few MSK1 inhibitors, especially natural compounds, have been reported. We used virtual screening of a natural products database and the active conformation of the C-terminal kinase domain of MSK1 (PDB id 3KN) as the receptor structure to identify chrysin and its derivative, compound 69407, as inhibitors of MSK1. Compared with chrysin, compound 69407 more strongly inhibited proliferation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ cells with lower cytotoxicity. Western blot data demonstrated that compound 69407 suppressed phosphorylation of the MSK1 downstream effector histone H3 in intact cells. Knocking down the expression of MSK1 effectively reduced the sensitivity of JB6 P+ cells to compound 69407. Moreover, topical treatment with compound 69407 before TPA application significantly reduced papilloma development in terms of number and size in a two-stage mouse skin carcinogenesis model. The reduction in papilloma development was accompanied by the inhibition of histone H3 phosphorylation at Ser10 in tumors extracted from mouse skin. The results indicated that compound 69407 exerts inhibitory effects on skin tumorigenesis by directly binding with MSK1 and attenuates the MSK1/histone H3 signaling pathway, which makes it an ideal chemopreventive agent against skin cancer.
Assuntos
Flavonas/farmacologia , Flavonoides/farmacologia , Fenetilaminas/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Neoplasias Cutâneas/prevenção & controle , Animais , Anticarcinógenos/farmacologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células , Transformação Celular Neoplásica , Histonas/metabolismo , Masculino , Camundongos , Papiloma/prevenção & controle , Fosforilação , Estrutura Terciária de Proteína , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Pele/efeitos dos fármacos , Acetato de Tetradecanoilforbol , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Centrosomes play an important role in various cellular processes, including spindle formation and chromosome segregation. They are composed of two orthogonally arranged centrioles, whose duplication occurs only once per cell cycle. Accurate control of centriole numbers is essential for the maintenance of genomic integrity. Although it is well appreciated that polo-like kinase 4 (Plk4) plays a central role in centriole biogenesis, how it is recruited to centrosomes and whether this step is necessary for centriole biogenesis remain largely elusive. Here we showed that Plk4 localizes to distinct subcentrosomal regions in a temporally and spatially regulated manner, and that Cep192 and Cep152 serve as two distinct scaffolds that recruit Plk4 to centrosomes in a hierarchical order. Interestingly, Cep192 and Cep152 competitively interacted with the cryptic polo box of Plk4 through their homologous N-terminal sequences containing acidic-α-helix and N/Q-rich motifs. Consistent with these observations, the expression of either one of these N-terminal fragments was sufficient to delocalize Plk4 from centrosomes. Furthermore, loss of the Cep192- or Cep152-dependent interaction with Plk4 resulted in impaired centriole duplication that led to delayed cell proliferation. Thus, the spatiotemporal regulation of Plk4 localization by two hierarchical scaffolds, Cep192 and Cep152, is critical for centriole biogenesis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centríolos/fisiologia , Centrossomo/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ciclo Celular/genética , Centríolos/metabolismo , Proteínas Cromossômicas não Histona/genética , Clonagem Molecular , Biologia Computacional , DNA Complementar/genética , Técnica Indireta de Fluorescência para Anticorpo , Immunoblotting , Imunoprecipitação , Lentivirus , Mutagênese , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Unlike other classical protein tyrosine phosphatases (PTPs), PTPRQ (PTP receptor type Q) has dephosphorylating activity towards phosphatidylinositide (PI) substrates. Here, the structure of the catalytic domain of PTPRQ was solved at 1.56â Å resolution. Overall, PTPRQ adopts a tertiary fold typical of other classical PTPs. However, the disordered M6 loop of PTPRQ surrounding the catalytic core and the concomitant absence of interactions of this loop with residues in the PTP loop results in a flat active-site pocket. On the basis of structural and biochemical analyses, it is proposed that this structural feature might facilitate the accommodation of large substrates, making it suitable for the dephosphorylation of PI substrates. Moreover, subsequent kinetic experiments showed that PTPRQ has a strong preferences for PI(3,4,5)P3 over other PI substrates, suggesting that its regulation of cell survival and proliferation reflects downregulation of Akt signalling.
Assuntos
Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Humanos , Cinética , Modelos Moleculares , Mutação , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Conformação Proteica , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Especificidade por SubstratoRESUMO
Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P(+) cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G1 phase and inhibited the G1/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P(+) cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Quinases Ciclina-Dependentes/antagonistas & inibidores , Flavonoides/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Regulação Alostérica/efeitos dos fármacos , Animais , Sítios de Ligação , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Flavonoides/química , Fase G1/efeitos dos fármacos , Fase G1/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Moleculares , Transplante de Neoplasias , Inibidores de Proteínas Quinases/química , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Fase S/efeitos dos fármacos , Fase S/genética , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologiaRESUMO
Primary cilia are microtubule-based solitary sensing structures on the cell surface that play crucial roles in cell signaling and development. Abnormal ciliary function leads to various human genetic disorders, collectively known as ciliopathies. Outer dense fiber protein 2 (Odf2) was initially isolated as a major component of sperm-tail fibers. Subsequent studies have demonstrated the existence of many splicing variants of Odf2, including Cenexin1 (Odf2 isoform 9), which bears an unusual C-terminal extension. Strikingly, Odf2 localizes along the axoneme of primary cilia, whereas Cenexin1 localizes to basal bodies in cultured mammalian cells. Whether Odf2 and Cenexin1 contribute to primary cilia assembly by carrying out either concerted or distinct functions is unknown. By taking advantage of odf2-/- cells lacking endogenous Odf2 and Cenexin1, but exogenously expressing one or both of these proteins, we showed that Cenexin1, but not Odf2, was necessary and sufficient to induce ciliogenesis. Furthermore, the Cenexin1-dependent primary cilia assembly pathway appeared to function independently of Odf2. Consistently, Cenexin1, but not Odf2, interacted with GTP-loaded Rab8a, localized to the distal/subdistal appendages of basal bodies, and facilitated the recruitment of Chibby, a centriolar component that is important for proper ciliogenesis. Taken together, our results suggest that Cenexin1 plays a critical role in ciliogenesis through its C-terminal extension that confers a unique ability to mediate primary cilia assembly. The presence of multiple splicing variants hints that the function of Odf2 is diversified in such a way that each variant has a distinct role in the complex cellular and developmental processes.
Assuntos
Axonema/genética , Centríolos/genética , Cílios/genética , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Processamento Alternativo , Animais , Axonema/metabolismo , Axonema/ultraestrutura , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Centríolos/metabolismo , Centríolos/ultraestrutura , Cílios/metabolismo , Cílios/ultraestrutura , Células HEK293 , Proteínas de Choque Térmico/metabolismo , Humanos , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais , Transfecção , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
STK295900, a small synthetic molecule belonging to a class of symmetric bibenzimidazoles, exhibits antiproliferative activity against various human cancer cell lines from different origins. Examining the effect of STK295900 in HeLa cells indicates that it induces G(2) phase arrest without invoking DNA damage. Further analysis shows that STK295900 inhibits DNA relaxation that is mediated by topoisomerase 1 (Top 1) and topoisomerase 2 (Top 2) in vitro. In addition, STK295900 also exhibits protective effect against DNA damage induced by camptothecin. However, STK295900 does not affect etoposide-induced DNA damage. Moreover, STK295900 preferentially exerts cytotoxic effect on cancer cell lines while camptothecin, etoposide, and Hoechst 33342 affected both cancer and normal cells. Therefore, STK295900 has a potential to be developed as an anticancer chemotherapeutic agent.
Assuntos
Compostos de Anilina/farmacologia , Benzimidazóis/farmacologia , Dano ao DNA , DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Inibidores da Topoisomerase/farmacologia , Compostos de Anilina/química , Antineoplásicos Fitogênicos/farmacologia , Benzimidazóis/química , Proteína Quinase CDC2/metabolismo , Camptotecina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Relação Dose-Resposta a Droga , Etoposídeo/farmacologia , Células HCT116 , Células HT29 , Células HeLa , Células Hep G2 , Humanos , Immunoblotting , Células MCF-7 , Estrutura Molecular , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Inibidores da Topoisomerase/químicaRESUMO
Centrosomal proteins play important roles in cell cycle. Among them, the centrosomal protein of 131kDa (CEP131) has been reported as a critical factor for cilia formation which is related with development, signaling, and various diseases, the malfunction of cilia leading to cancer. Specificity protein 1 (SP1), known as a centrosome regulator, is an essential transcription factor regulating the genes involved in multiple cellular processes such as cell cycle, apoptosis, and DNA damages. In this study, we explored the crucial role of SP1 in the regulation of CEP131 gene transcription. A deletion analysis of the CEP131 promoter region revealed dominant promoter elements within the sequence between -400bp and -200bp, which contained consensus binding sites for SP1. Electrophoretic mobility shift assay (EMSA) and chromatin immuno-precipitation (ChIP) assay further confirmed the direct binding of SP1 to the CEP131 promoter. On the other hand, CEP131 transcription could be inhibited by mithramycin (a GC-rich region inhibitor), but exogenous expression of SP1 could increase CEP131 expression as evidenced by a reporter gene assay. In addition, mutation of several SP1 binding sites revealed four SP1 binding sites at -244/-225, -258/-239, -304/-283 and -323/-304 that strongly affect CEP131 expression. Hence, it is suggested that SP1 is a pivotal transcription factor for the regulation of CEP131 expression, consequently leading the control of centrosome functions.
Assuntos
Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica , Proteínas dos Microtúbulos/genética , Fator de Transcrição Sp1/metabolismo , Sítios de Ligação/genética , Centrossomo/efeitos dos fármacos , Centrossomo/metabolismo , Imunoprecipitação da Cromatina , Proteínas do Citoesqueleto , Ensaio de Desvio de Mobilidade Eletroforética , Células HEK293 , Células HeLa , Humanos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Plasmídeos , Plicamicina/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , Deleção de SequênciaRESUMO
The metabolism of glucose and glutamine, primary carbon sources utilized by mitochondria to generate energy and macromolecules for cell growth, is directly regulated by mTORC1. We show that glucose and glutamine, by supplying carbons to the TCA cycle to produce ATP, positively feed back to mTORC1 through an AMPK-, TSC1/2-, and Rag-independent mechanism by regulating mTORC1 assembly and its lysosomal localization. We discovered that the ATP-dependent TTT-RUVBL1/2 complex was disassembled and repressed by energy depletion, resulting in its decreased interaction with mTOR. The TTT-RUVBL complex was necessary for the interaction between mTORC1 and Rag and formation of mTORC1 obligate dimers. In cancer tissues, TTT-RUVBL complex mRNAs were elevated and positively correlated with transcripts encoding proteins of anabolic metabolism and mitochondrial function-all mTORC1-regulated processes. Thus, the TTT-RUVBL1/2 complex responds to the cell's metabolic state, directly regulating the functional assembly of mTORC1 and indirectly controlling the nutrient signal from Rags to mTORC1.
Assuntos
Metabolismo Energético , Lisossomos/metabolismo , Proteínas/metabolismo , Estresse Fisiológico , ATPases Associadas a Diversas Atividades Celulares , Trifosfato de Adenosina/metabolismo , Adenilato Quinase/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Ciclo do Ácido Cítrico , DNA Helicases/genética , DNA Helicases/metabolismo , Feminino , Glucose/deficiência , Glutamina/deficiência , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais , Estatísticas não Paramétricas , Serina-Treonina Quinases TOR , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Nuclear factor-κB (NF-κB) ligand (RANKL) was shown to induce osteoclast differentiation by increasing the expression of c-Fos, NFATc1 and TRAP. Salubrinal treatment to bone marrow macrophage (BMM) cells, however, significantly blocked NFATc1 expression and osteoclast differentiation by RANKL. Overexpression of NFATc1 further confirmed that NFATc1 is a key factor affected by salubrinal in osteoclast differentiation by RANKL. Unexpectedly, NFATc1 and c-Fos mRNA expressions were not affected by salubrinal, implicating that NFATc1 expression is regulated at a translational stage. In support of this, salubrinal increased the phosphorylation of a translation factor eIF2α, decreasing the global protein synthesis including NFATc1. In contrast, a phosphorylation mutant plasmid pLenti-eIF2α-S51A restored RANKL-induced NFATc1 expression and osteoclast differentiation even in the presence of salubrinal. Furthermore, knockdown of ATF4 significantly reduced salubrinal-induced osteoblast differentiation as evidenced by decreased calcium accumulation and lowered expressions of the osteoblast differentiation markers, alkaline phosphatase and RANKL in MC3T3-E1 osteoblast cells. Salubrinal treatment to co-cultured BMM and MC3T3-E1 cells also showed reduction of osteoclast differentiation. Finally, salubrinal efficiently blocked osteoporosis in mice model treated with RANKL as evidenced by elevated bone mineral density (BMD) and other osteoporosis factors. Collectively, our data indicate that salubrinal could affect the differentiation of both osteoblast and osteoclast, and be developed as an excellent anti-osteoporosis drug. In addition, modulation of ATF4 and NFATc1 expressions through eIF2α phosphorylation could be a valuable target for the treatment of osteoporosis.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Cinamatos/farmacologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Osteoblastos/citologia , Osteoclastos/citologia , Tioureia/análogos & derivados , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Cálcio/metabolismo , Células Cultivadas , Cinamatos/uso terapêutico , Técnicas de Cocultura , Modelos Animais de Doenças , Fêmur/diagnóstico por imagem , Camundongos , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Osteoporose/patologia , Fosforilação , Ligante RANK/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Radiografia , Transdução de Sinais , Tioureia/farmacologia , Tioureia/uso terapêutico , Tíbia/diagnóstico por imagemRESUMO
RNase H (retroviral ribonuclease H) cleaves the phosphate backbone of the RNA template within an RNA/DNA hybrid to complete the synthesis of double-stranded viral DNA. In the present study we have determined the complete structure of the RNase H domain from XMRV (xenotropic murine leukaemia virus-related virus) RT (reverse transcriptase). The basic protrusion motif of the XMRV RNase H domain is folded as a short helix and an adjacent highly bent loop. Structural superposition and subsequent mutagenesis experiments suggest that the basic protrusion motif plays a role in direct binding to the major groove in RNA/DNA hybrid, as well as in establishing the co-ordination among modules in RT necessary for proper function.
Assuntos
Ribonuclease H/química , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Sequência Conservada , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismoRESUMO
The serine phosphatase SerB653 plays a crucial role in the infection of Porphyromonas gingivalis, which contributes to the pathogenesis of periodontitis, an inflammatory disease of teeth-supporting tissues. Because functional loss of SerB653 eliminates the virulence of P. gingivalis, SerB653 inhibitors are considered potential periodontitis therapeutic or preventive agents. To identify SerB653 inhibitors with potent anti-periodontitis activity, we conducted a high-throughput screen of a representative 6800-compound subset of a synthetic chemical library of the Korea Chemical Bank (KCB) for compounds with activity against SerB653. The primary screening yielded 150 hits, and subsequent confirmatory studies identified eight compounds, mainly within a single cluster of 3-acyl-2-phenylamino-1,4-dihydroquinolin-4-one derivatives, that showed greater than 50% inhibition of SerB653 activity at a concentration of 50µM. A second screening with a focused library identified 10 compounds with IC(50) values less than 10µM. In antibacterial tests, three of these compounds showed a minimum inhibitory concentration against P. gingivalis growth of 5-50nM.
Assuntos
Inibidores Enzimáticos/farmacologia , Periodontite/microbiologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Porphyromonas gingivalis/efeitos dos fármacos , Porphyromonas gingivalis/enzimologia , Quinolinas/farmacologia , Infecções por Bacteroidaceae/tratamento farmacológico , Infecções por Bacteroidaceae/enzimologia , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Periodontite/tratamento farmacológico , Monoéster Fosfórico Hidrolases/metabolismo , Porphyromonas gingivalis/crescimento & desenvolvimento , Quinolinas/química , Relação Estrutura-AtividadeRESUMO
Patulin is a fungal mycotoxin of Aspergilus and Penicillium that is commonly found in rotting fruits and exerts its potential toxic effect mainly by reactive oxygen species (ROS) generation. However, the effect of patulin on cancer cells as well as its intracellular mechanism has been controversial and not clearly defined yet. In this study, patulin was found to induce G1/S accumulation and cell growth arrest accompanied by caspase-3 activation, PARP cleavage and ATF3 expression in human colon cancer cell line HCT116. Ser/Thr phosphorylation of a transcription factor, EGR-1, was increased while its expression did not change upon patulin treatment to the cells. Knockdown of ATF3 and EGR-1 using their respective siRNAs showed EGR-1 dependent ATF3 expression. Moreover, treatment of the cells with antioxidants N-acetylcysteine (NAC) and glutathione (GSH) revealed that patulin induced ATF3 expression and apoptosis were dependent on ROS generation. ATF3 expression was also increased by patulin in other colorectal cancer cell types, Caco2 and SW620. Collectively, our data present a new anti-cancer molecular mechanism of patulin, suggesting EGR-1 and ATF3 as critical targets for the development of anti-cancer chemotherapeutics. In this regard, patulin could be a candidate for the treatment of colorectal cancers.
Assuntos
Fator 3 Ativador da Transcrição/agonistas , Apoptose/genética , Neoplasias Colorretais/patologia , Patulina/farmacologia , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Acetilcisteína/farmacologia , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Antioxidantes/farmacologia , Células CACO-2 , Caspase 3/genética , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Glutationa/farmacologia , Células HCT116 , Humanos , Micotoxinas/farmacologia , Fosforilação , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor IGF Tipo 1/genéticaAssuntos
Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Homeostase/fisiologia , Modelos Biológicos , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Centrômero/metabolismo , Histonas , Fosforilação , Ligação Proteica , Quinase 1 Polo-LikeRESUMO
Bipolar spindle formation is pivotal for accurate segregation of mitotic chromosomes during cell division. A growing body of evidence suggests that, in addition to centrosome- and chromatin-based microtubule (MT) nucleation, MT-based MT nucleation plays an important role for proper bipolar spindle formation in various eukaryotic organisms. Although a recently discovered Augmin complex appears to play a central role in this event, how Augmin is regulated remains unknown. Here we provide evidence that a mammalian polo-like kinase 1 (Plk1) localizes to mitotic spindles and promotes MT-based MT nucleation by directly regulating Augmin. Mechanistically, we demonstrated that Cdc2-dependent phosphorylation on a γ-tubulin ring complex (γ-TuRC) recruitment protein, Nedd1/GCP-WD, at the previously uncharacterized S460 residue induces the Nedd1-Plk1 interaction. This step appeared to be critical to allow Plk1 to phosphorylate the Hice1 subunit of the Augmin complex to promote the Augmin-MT interaction and MT-based MT nucleation from within the spindle. Loss of either the Nedd1 S460 function or the Plk1-dependent Hice1 phosphorylation impaired both the Augmin-MT interaction and γ-tubulin recruitment to the spindles, thus resulting in improper bipolar spindle formation that ultimately leads to mitotic arrest and apoptotic cell death. Thus, via the formation of the Nedd1-Plk1 complex and subsequent Augmin phosphorylation, Plk1 regulates spindle MT-based MT nucleation to accomplish normal bipolar spindle formation and mitotic progression.