RESUMO
Nicotinamide N-methyl transferase (NNMT) transfers a methyl group from S-adenosyl-L-methionine (SAM) to nicotinamide (NAM), producing 1-methylnicotinamide (1MNA). NNMT has been implicated in several cancer types and recently in metabolism, but its role in autophagy regulation has not yet been investigated. In this study, we determined that NNMT negatively regulated autophagy at the stage of ULK1 activation through protein phosphatase 2A (PP2A) activity. Specifically, NNMT knockdown increased PP2A methylation and subsequently enhanced phosphatase activity. Consequent p-ULK1 (S638) dephosphorylation derepressed ULK1 activity, resulting in autophagy induction. Accordingly, NNMT downregulation rescued tumor cells under nutrient deficiency in vivo, which was alleviated by ULK1 inhibitor treatment. In summary, our results suggest a novel mechanism by which tumor cells protect themselves against nutrient deprivation through NNMT suppression to accelerate autophagy.
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Vaccinia-related kinase 1 (VRK1) and VRK3 are members of the VRK family of serine/threonine kinases and are principally localized in the nucleus. Despite the crucial roles of VRK1/VRK3 in physiology and disease, the molecular and functional interactions of VRK1/VRK3 are poorly understood. Here, we identified over 200 unreported VRK1/VRK3-interacting candidate proteins by affinity purification and LC-MS/MS. The networks of VRK1 and VRK3 interactomes were found to be associated with important biological processes such as the cell cycle, DNA repair, chromatin assembly, and RNA processing. Interactions of interacting proteins with VRK1/VRK3 were confirmed by biochemical assays. We also found that phosphorylations of XRCC5 were regulated by both VRK1/VRK3, and that of CCNB1 was regulated by VRK3. In liver cancer cells and tissues, VRK1/VRK3 were highly upregulated and its depletion affected cell cycle progression in the different phases. VRK3 seemed to affect S phase progression and G2 or M phase entry and exit, whereas VRK1 affects G1/S transition in the liver cancer, which could be explained by different interacting candidate proteins. Thus, this study not only provides a resource for investigating the unidentified functions of VRK1/VRK3, but also an insight into the regulatory roles of VRK1/VRK3 in biological processes.
Assuntos
Proliferação de Células/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/genética , Proteínas Serina-Treonina Quinases/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Ciclina B1/genética , Regulação da Expressão Gênica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Autoantígeno Ku/genética , Neoplasias Hepáticas/patologia , Fosforilação , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Robust mitochondrial respiration provides energy to support physical performance and physiological well-being, whereas mitochondrial malfunction is associated with various pathologies and reduced longevity. In the current study, we tested whether myricetin, a natural flavonol with diverse biological activities, may impact mitochondrial function and longevity. The mice were orally administered myricetin (50 mg/kg/day) for 3 weeks. Myricetin significantly potentiated aerobic capacity in mice, as evidenced by their increased running time and distance. The elevated mitochondrial function was associated with induction of genes for oxidative phosphorylation and mitochondrial biogenesis in metabolically active tissues. Importantly, myricetin treatment led to decreased PGC-1α acetylation through SIRT1 activation. Furthermore, myricetin significantly improved the healthspan and lifespan of wild-type, but not Sir-2.1-deficient, C. elegans. These results demonstrate that myricetin enhances mitochondrial activity, possibly by activating PGC-1α and SIRT1, to improve physical endurance, strongly suggesting myricetin as a mitochondria-activating agent.
Assuntos
Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Longevidade , Mitocôndrias/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Resistência Física/efeitos dos fármacos , Sirtuína 1/metabolismo , Animais , Caenorhabditis elegans , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/efeitos dos fármacos , Biogênese de Organelas , Fosforilação Oxidativa , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Sirtuína 1/genéticaRESUMO
We analyzed the structure of horseradish peroxidase (HRP) under denaturing conditions of 9 M urea or 6 M guanidine hydrochloride (GdnHCl). Far-UV circular dichroism (CD) spectra indicated the existence of native-like secondary structure of holo-HRP in 9 M urea. In addition, slight changes in near-UV and Soret region CD spectra of holo-HRP in 9 M urea suggest that the tertiary structure of holo-HRP and the binding of heme remain partially intact in this condition. A transition in the thermal unfolding transition curve of holo-HRP in 9 M urea indicated the existence of a considerable amount of secondary structure. However, no secondary structure, tertiary structure, or interaction between heme and HRP were observed in holo-HRP in 6 M GdnHCl. Small-angle X-ray scattering indicated that although distal and proximal domains of holo-HRP in 9 M urea might be partially unfolded, the central region that contains the heme might maintain its tertiary structure. Our results suggest that retention of the heme is essential for maintenance of the structure of HRP under highly denaturing conditions.
Assuntos
Heme/química , Peroxidase do Rábano Silvestre/química , Dicroísmo Circular , Guanidina/química , Modelos Moleculares , Desnaturação Proteica , Estrutura Terciária de Proteína , Desdobramento de Proteína , Temperatura , Ureia/química , Difração de Raios XRESUMO
The conventional gene expression profiling approaches have been replaced with DNA microarrays with exhibiting a powerful high-throughput capacity. Most solid surfaces of DNA microarrays contain such a high area density of functional groups to immobilize capture DNAs to the surface that the hybridization of capture DNAs with cDNA can be hindered, resulting in low intensity and reproducibility. Since our previous works showed that the 9-acid dendron was able to increase the hybridization efficiency, we aimed to demonstrate the feasibility of 9-acid dendron-coated glass slides as an advanced microarray platform for gene expression profiling. The 9-acid dendron-coated DNA microarray could reproducibly obtain the expression levels of 2800 human cancer-associated genes in the two liver cancer lines: Hep3B and SK-Hep1. Among the differentially expressed genes, Caveolin-1 (Cav-1) was identified as the most highly up-regulated gene in invasive SK-Hep1 in comparison to non-motile Hep3B. The overexpression of Cav-1 in Hep3B promoted the cell invasion, whereas its knockdown in SK-Hep1 suppressed the invasive feature, which confirms that the overexpression of Cav-1 is closely associated with cell invasion of liver carcinoma. Collectively, the 9-acid dendron-coated surface could successfully detect the transcript levels of cells, demonstrating its feasible potential to identify the candidate genes for further functional studies or diagnosis of diseases.
Assuntos
Antracenos/química , Caveolina 1/genética , Neoplasias Hepáticas/patologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Linhagem Celular Tumoral , Movimento Celular , Humanos , Invasividade Neoplásica/genética , Regulação para CimaRESUMO
RE-1 silencing transcription factor (REST) is a transcriptional repressor that regulates gene expression by binding to repressor element 1. However, despite its critical function in physiology, little is known about its interaction proteins. Here we identified 204 REST-interacting proteins using affinity purification and mass spectrometry. The interactome included proteins associated with mRNA processing/splicing, chromatin organization, and transcription. The interactions of these REST-interacting proteins, which included TRIM28, were confirmed by co-immunoprecipitation and immunocytochemistry, respectively. Gene Ontology (GO) analysis revealed that neuronal differentiation-related GO terms were enriched among target genes that were co-regulated by REST and TRIM28, while the level of CTNND2 was increased by the knockdown of REST and TRIM28. Consistently, the level of CTNND2 increased while those of REST and TRIM28 decreased during neuronal differentiation in the primary neurons, suggesting that CTNND2 expression may be co-regulated by both. Furthermore, neurite outgrowth was increased by depletion of REST or TRIM28, implying that reduction of both REST and TRIM28 could promote neuronal differentiation via induction of CTNND2 expression. In conclusion, our study of REST reveals novel interacting proteins which could be a valuable resource for investigating unidentified functions of REST and also suggested functional links between REST and TRIM28 during neuronal development.
Assuntos
Cateninas/metabolismo , Neurônios/citologia , Mapeamento de Interação de Proteínas/métodos , Proteínas Repressoras/metabolismo , Proteína 28 com Motivo Tripartido/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Ontologia Genética , Células HEK293 , Humanos , Camundongos , Neurônios/metabolismo , Ligação Proteica , Proteínas Repressoras/genética , Proteína 28 com Motivo Tripartido/genética , delta CateninaRESUMO
The role of Sirtuin 6 (SIRT6) as a tumor suppressor or oncogene in liver cancer remains controversial. Thus, we identified the specific role of SIRT6 in the progression of hepatocellular carcinoma (HCC). SIRT6 expression was significantly higher in HCC cell lines and HCC tissues from 138 patients than in an immortalized hepatocyte cell line, THLE-2 and non-tumor tissues, respectively. SIRT6 knockdown by shRNA suppressed the growth of HCC cells and inhibited HCC tumor growth in vivo. In addition, SIRT6 silencing significantly prevented the growth of HCC cell lines by inducing cellular senescence in the p16/Rb- and p53/p21-pathway independent manners. Microarray analysis revealed that the expression of genes involved in nucleosome assembly was apparently altered in SIRT6-depleted Hep3B cells. SIRT6 knockdown promoted G2/M phase arrest and downregulation of genes encoding histone variants associated with nucleosome assembly, which could be attributed to DNA damage. Taken together, our findings suggest that SIRT6 acts as a tumor promoter by preventing DNA damage and cellular senescence, indicating that SIRT6 represents a potential therapeutic target for the treatment of HCC.
Assuntos
Carcinoma Hepatocelular/fisiopatologia , Senescência Celular/fisiologia , Dano ao DNA/fisiologia , Neoplasias Hepáticas/fisiopatologia , Sirtuínas/fisiologia , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Sirtuínas/deficiênciaRESUMO
Ketosteroid isomerase (3-oxosteroid Δ(5)-Δ(4)-isomerase, KSI) from Pseudomonas putida catalyzes allylic rearrangement of the 5,6-double bond of Δ(5)-3-ketosteroid to 4,5-position by stereospecific intramolecular transfer of a proton. The active site of KSI is formed by several hydrophobic residues and three catalytic residues (Tyr14, Asp38, and Asp99). In this study, we investigated the role of a hydrophobic Met112 residue near the active site in the catalysis, steroid binding, and stability of KSI. Replacing Met112 with alanine (yields M112A) or leucine (M112L) decreased the kcat by 20- and 4-fold, respectively. Compared with the wild type (WT), M112A and M112L KSIs showed increased KD values for equilenin, an intermediate analogue; these changes suggest that loss of packing at position 112 might lead to unfavorable steroid binding, thereby resulting in decreased catalytic activity. Furthermore, M112A and M112L mutations reduced melting temperature (Tm) by 6.4°C and 2.5°C, respectively. These changes suggest that favorable packing in the core is important for the maintenance of stability in KSI. The M112K mutation decreased kcat by 2000-fold, compared with the WT. In M112K KSI structure, a new salt bridge was formed between Asp38 and Lys112. This bridge could change the electrostatic potential of Asp38, and thereby contribute to the decreased catalytic activity. The M112K mutation also decreased the stability by reducing Tm by 4.1°C. Our data suggest that the Met112 residue may contribute to the catalytic activity and stability of KSI by providing favorable hydrophobic environments and compact packing in the catalytic core.
Assuntos
Cetosteroides/metabolismo , Metionina/genética , Esteroide Isomerases/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Catálise , Domínio Catalítico/genética , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Mutação/genética , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Alinhamento de Sequência , Temperatura de TransiçãoRESUMO
We identified the specific role of vaccinia-related kinase 1 (VRK1) in the progression of hepatocellular carcinoma (HCC) and evaluated its therapeutic and prognostic potential. VRK1 levels were significantly higher in HCC cell lines than a normal hepatic cell line, and were higher in HCC than non-tumor tissue. VRK1 knockdown inhibited the proliferation of SK-Hep1, SH-J1 and Hep3B cells; moreover, depletion of VRK1 suppressed HCC tumor growth in vivo. We also showed that VRK1 knockdown increased the number of G1 arrested cells by decreasing cyclin D1 and p-Rb while upregulating p21 and p27, and that VRK1 depletion downregulated phosphorylation of CREB, a transcription factor regulating CCND1. Additionally, we found that luteolin, a VRK1 inhibitor, suppressed HCC growth in vitro and in vivo, and that the aberrant VRK1 expression correlated with poor prognostic features of HCC. High levels of VRK1 were associated with shorter overall and disease-free survival and higher recurrence rates. Taken together, our findings suggest VRK1 may act as a tumor promoter by controlling the level of cell cycle regulators associated with G1/S transition and could potentially serve as a therapeutic target and/or prognostic biomarker for HCC.
Assuntos
Carcinoma Hepatocelular/enzimologia , Proteínas de Ciclo Celular/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Relação Dose-Resposta a Droga , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Many mitotic kinases have been targeted for the development of anti-cancer drugs, and inhibitors of these kinases have been expected to perform well for cancer therapy. Efforts focused on selecting good targets and finding specific drugs to target are especially needed, largely due to the increased frequency of anti-cancer drugs used in the treatment of lung cancer. Vaccinia-related kinase 1 (VRK1) is a master regulator in lung adenocarcinoma and is considered a key molecule in the adaptive pathway, which mainly controls cell survival. We found that ursolic acid (UA) inhibits the catalytic activity of VRK1 via direct binding to the catalytic domain of VRK1. UA weakens surveillance mechanisms by blocking 53BP1 foci formation induced by VRK1 in lung cancer cells, and possesses synergistic anti-cancer effects with DNA damaging drugs. Taken together, UA can be a good anti-cancer agent for targeted therapy or combination therapy with DNA damaging drugs for lung cancer patients.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Triterpenos/farmacologia , Animais , Antineoplásicos Fitogênicos/química , Sítios de Ligação , Domínio Catalítico , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/química , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Modelos Moleculares , Conformação Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Triterpenos/química , Ensaios Antitumorais Modelo de Xenoenxerto , Ácido UrsólicoRESUMO
Despite a low risk of liver failure and preserved liver function, non-cirrhotic hepatocellular carcinoma (HCC) has a poor prognosis. In the current study, we evaluated an active regulator of SIRT1 (AROS) as a prognostic biomarker in non-cirrhotic HCC. mRNA levels of AROS were measured in tumor and non-tumor tissues obtained from 283 non-cirrhotic HCC patients. AROS expression was exclusively up-regulated in recurrent tissues from the non-cirrhotic HCC patients (P = 0.015) and also in tumor tissues irrespective of tumor stage (P < 0.001) or BCLC stage (P < 0.001). High mRNA levels of AROS were statistically significantly associated with tumor stage (P < 0.001), BCLC stage (P = 0.007), alpha fetoprotein (AFP) level (P = 0.013), microvascular invasion (P = 0.001), tumor size (P = 0.036), and portal vein invasion (P = 0.005). Kaplan-Meir curve analysis demonstrated that HCC patients with higher AROS levels had shorter disease-free survival (DFS) in both the short-term (P < 0.001) and long-term (P = 0.005) compared to those with low AROS. Cox regression analysis demonstrated that AROS is a significant predictor for DFS along with large tumor size, tumor multiplicity, vascular invasion, and poor tumor differentiation, which are the known prognostic factors. In conclusion, AROS is a significant biomarker for tumor aggressiveness in non-cirrhotic hepatocellular carcinoma.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/epidemiologia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/epidemiologia , Neoplasias Hepáticas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Distribuição por Idade , Idoso , Carcinoma Hepatocelular/patologia , Intervalo Livre de Doença , Feminino , Humanos , Cirrose Hepática/epidemiologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Prevalência , Reprodutibilidade dos Testes , República da Coreia/epidemiologia , Fatores de Risco , Sensibilidade e Especificidade , Distribuição por Sexo , Adulto JovemRESUMO
Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ(5)-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ(5)-3-ketosteroid to its conjugated Δ(4)-isomers at a rate that approaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 Oη and C3-O of equilenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 Oη and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1-11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7-2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI.
Assuntos
Equilenina/metabolismo , Pseudomonas putida/enzimologia , Esteroide Isomerases/química , Esteroide Isomerases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Equilenina/química , Ligação de Hidrogênio , Modelos Moleculares , Mutação , Ligação Proteica , Espectroscopia de Prótons por Ressonância Magnética , Pseudomonas putida/genética , Esteroide Isomerases/metabolismo , Especificidade por SubstratoRESUMO
To date, many anticancer drugs have been developed by directly or indirectly targeting microtubules, which are involved in cell division. Although this approach has yielded many anticancer drugs, these drugs produce undesirable side effects. An alternative strategy is needed, and targeting mitotic exit may be one alternative approach. Localization of phosphorylated barrier-to-autointegration factor (BAF) to the chromosomal core region is essential for nuclear envelope compartment relocalization. In this study, we isolated brazilin from Caesalpinia sappan Leguminosae and demonstrated that it inhibited BAF phosphorylation in vitro and in vivo. Moreover, we demonstrated direct binding between brazilin and BAF. The inhibition of BAF phosphorylation induced abnormal nuclear envelope reassembly and cell death, indicating that perturbation of nuclear envelope reassembly could be a novel approach to anticancer therapy. We propose that brazilin isolated from C. sappan may be a new anticancer drug candidate that induces cell death by inhibiting vaccinia-related kinase 1-mediated BAF phosphorylation.
Assuntos
Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Benzopiranos/isolamento & purificação , Benzopiranos/farmacologia , Caesalpinia/química , Proteínas de Ligação a DNA/metabolismo , Membrana Nuclear/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Animais , Antineoplásicos/metabolismo , Benzopiranos/metabolismo , Morte Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Membrana Nuclear/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Telófase/efeitos dos fármacosRESUMO
Uncontrolled proliferation, a major feature of cancer cells, is often triggered by the malfunction of cell cycle regulators such as protein kinases. Recently, cell cycle-related protein kinases have become attractive targets for anti-cancer therapy, because they play fundamental roles in cellular proliferation. However, the protein kinase-targeted drugs that have been developed so far do not show impressive clinical results and also display severe side effects; therefore, there is undoubtedly a need to investigate new drugs targeting other protein kinases that are critical in cell cycle progression. Vaccinia-related kinase 1 (VRK1) is a mitotic kinase that functions in cell cycle regulation by phosphorylating cell cycle-related substrates such as barrier-to-autointegration factor (BAF), histone H3, and the cAMP response element (CRE)-binding protein (CREB). In our study, we identified luteolin as the inhibitor of VRK1 by screening a small-molecule natural compound library. Here, we evaluated the efficacy of luteolin as a VRK1-targeted inhibitor for developing an effective anti-cancer strategy. We confirmed that luteolin significantly reduces VRK1-mediated phosphorylation of the cell cycle-related substrates BAF and histone H3, and directly interacts with the catalytic domain of VRK1. In addition, luteolin regulates cell cycle progression by modulating VRK1 activity, leading to the suppression of cancer cell proliferation and the induction of apoptosis. Therefore, our study suggests that luteolin-induced VRK1 inhibition may contribute to establish a novel cell cycle-targeted strategy for anti-cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Luteolina/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Antineoplásicos/química , Domínio Catalítico/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Luteolina/química , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/químicaRESUMO
Sirtuins are NAD(+) -dependent deacetylases that regulate a range of cellular processes. Although diverse functions of sirtuins have been proposed, those functions of SIRT6 and SIRT7 that are mediated by their interacting proteins remain elusive. In the present study, we identified SIRT6- and SIRT7-interacting proteins, and compared their interactomes to investigate functional links. Our interactomes revealed 136 interacting proteins for SIRT6 and 233 for SIRT7 while confirming seven and 111 proteins identified previously for SIRT6 and SIRT7, respectively. Comparison of SIRT6 and SIRT7 interactomes under the same experimental conditions disclosed 111 shared proteins, implying related functional links. The interaction networks of interactomes indicated biological processes associated with DNA repair, chromatin assembly, and aging. Interactions of two highly acetylated proteins, nucleophosmin (NPM1) and nucleolin, with SIRT6 and SIRT7 were confirmed by co-immunoprecipitation. NPM1 was found to be deacetylated by both SIRT6 and SIRT7. In senescent cells, the acetylation level of NPM1 was increased in conjunction with decreased levels of SIRT6 and SIRT7, suggesting that the acetylation of NPM1 could be regulated by SIRT6 and SIRT7 in the aging process. Our comparative interactomic study of SIRT6 and SIRT7 implies important functional links to aging by their associations with interacting proteins. All MS data have been deposited in the ProteomeXchange with identifiers PXD000159 and PXD000850 (http://proteomecentral.proteomexchange.org/dataset/PXD000159, http://proteomecentral.proteomexchange.org/dataset/PXD000850).
Assuntos
Mapas de Interação de Proteínas , Sirtuínas/metabolismo , Acetilação , Envelhecimento , Células HEK293 , Humanos , Imunoprecipitação , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Nucleofosmina , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Proteômica/métodos , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/metabolismo , Sirtuínas/análise , Espectrometria de Massas em Tandem/métodos , NucleolinaRESUMO
PURPOSE: Although transcatheter arterial chemoembolization (TACE) is the most common treatment option in patients with hepatocellular carcinoma (HCC), its clinical benefits remain still controversial. Since TACE induces hypoxic necrosis in tumors, hypoxia-inducible factor 1α (HIF-1α) could critically affect biology in residual tumors after TACE treatment and subsequent prognosis. However, HIF-1α and its prognostic relevance in TACE have rarely been examined in human specimens. In the current study, we investigated the prognosis and expression of genes regulated by HIF-1α in HCC patients receiving preoperative TACE for the first time. METHODS: In total, 35 patients with HCC (10 patients undergoing preoperative TACE) were retrospectively studied. The prognostic significance of TACE was analyzed using Kaplan-Meier and Cox regression models. Protein levels of HIF-1α and mRNA levels of HIF-1α-associated genes were examined using immunohistochemistry (IHC) and real-time RT-PCR, respectively. RESULTS: Preoperative TACE was significantly associated with increased 2-year recurrence rate (80 vs. 36 %, P = 0.00402) and shorter disease-free survival (DFS) time (11.9 vs. 35.7 months, P = 0.0182). TACE was an independent prognostic factor for recurrence (P = 0.007) and poor DFS (P = 0.010) in a multivariate analysis. Immunohistochemical staining revealed in vivo activation of HIF-1α in human specimens treated with TACE. Notably, protein levels of HIF-1α were significantly increased in TACE tissues demonstrated by IHC. Transcriptional targets of HIF-1α showed mRNA expression patterns consistent with activation of HIF-1α in TACE tissues. CONCLUSIONS: Our findings collectively demonstrate that preoperative TACE confers poor prognosis in HCC patients through activation of HIF-1α.
Assuntos
Carcinoma Hepatocelular/genética , Expressão Gênica/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Hepáticas/genética , Carcinoma Hepatocelular/patologia , Quimioembolização Terapêutica/métodos , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica/métodos , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Prognóstico , RNA Mensageiro/genética , Estudos RetrospectivosRESUMO
We analyzed the enzymatic profiles of on-chip DNA ligation as we controlled the lateral spacing of surface-immobilized DNA substrates using dendron molecules with different sizes at the nanoscale. Enzymatic on-chip DNA ligation was performed on the dendron-coated surface within 20 min with no need for post-ligation gel electrophoresis. The enzymatic DNA repair was assessed by the fluorescence intensity at the repaired DNA duplex after thermally dissociating the unligated Cy3-labeled DNA from the DNA duplex, in which the Cy3-labeled DNA was hybridized prior to the on-chip DNA ligation. The rate of the nick-sealing reaction on the 27-acid dendron surface was 3-fold higher than that on the 9-acid dendron surface, suggesting that the wider lateral spacing determined by the larger dendron molecule could facilitate the access of DNA ligase to the nick site. The performance of on-chip DNA ligation was dropped to 10% and 3% when the nick was replaced by one- and two-nucleotide-long gaps, respectively. The 5' terminal phosphorylation of DNA strands by polynucleotide kinase and the on-chip DNA cleavage by endonucleases were also quantitatively monitored throughout the on-chip DNA ligation on the dendron-coated surface. A better understanding of the enzymatic kinetics of on-chip DNA ligation will contribute to a more reliable performance of various on-chip DNA ligation-based assays.
Assuntos
DNA Ligases/metabolismo , DNA/química , DNA/metabolismo , Dendrímeros/química , Técnicas Genéticas/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Carbocianinas/química , Cinética , Análise de Sequência com Séries de Oligonucleotídeos/métodosRESUMO
We independently controlled surface topography and wettability of polystyrene (PS) films by CF4 and oxygen plasma treatments, respectively, to evaluate the adhesion and proliferation of human fetal osteoblastic (hFOB) cells on the films. Among the CF4 plasma-treated PS films with the average surface roughness ranging from 0.9 to 70 nm, the highest adhesion of hFOB cells was observed on a PS film with roughness of ~11 nm. When this film was additionally treated by oxygen plasma to provide a hydrophilic surface with a contact angle less than 10°, the proliferation of bone-forming cell was further enhanced. Thus, the plasma-based independent modification of PS film into an optimum nanotexture for human osteoblast cells could be appplied to materials used in bone tissue engineering.
Assuntos
Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Poliestirenos/farmacologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Microscopia de Força Atômica , Osteoblastos/metabolismo , Oxigênio/farmacologia , Espectroscopia Fotoeletrônica , Gases em Plasma , Molhabilidade/efeitos dos fármacosRESUMO
BACKGROUND: The effectiveness of molecular targeted agents is modest in hepatocellular carcinoma (HCC). Efficacy of molecular targeted therapies has been better in cancer patients with high expression of actionable molecules defined as cognate target molecules. However, patient stratification based on the actionable molecules dictating the effectiveness of targeted drugs has remained understudied in HCC. EXPERIMENTAL DESIGN & RESULTS: Paired tumor and non-tumoral tissues derived from a total of 130 HCC patients were studied. Real-time RT-PCR was used to analyze the mRNA expression of actionable molecules in the tissues. mRNA levels of EGFR, VEGFR2, PDGFRß, FGFR1, and mTOR were up-regulated in tumors compared to non-tumors in 35.4, 42.3, 61.5, 24.6, and 50.0% of patients, respectively. Up-regulation of EGFR was observed at early stage and tended to gradually decrease toward late stages (BCLC stage A: 41.9%; B: 30.8%; C: 17.6%). Frequency of VEGFR2 expression in tumors at stage C was lower than that in the other stages (BCLC stage A: 45.9%; B: 41.0%; C: 29.4%). PDGFRß and mTOR were observed to be up-regulated in more than 50% of tumors in all the stages whereas FGFR1 was up-regulated in only about 20% of HCC irrespective of stages. A cluster analysis of actionable gene expression revealed that HCC can be categorized into different subtypes that predict the effectiveness of molecular targeted agents and combination therapies in clinical trials. Analysis of in vitro sensitivity to sorafenib demonstrated that HCC cells with up-regulation of PDGFRß and c-Raf mRNA are more susceptible to sorafenib treatment in a dose and time-dependent manner than cells with low expression of the genes. CONCLUSIONS: mRNA expression analysis of actionable molecules could provide the rationale for new companion diagnostics-based therapeutic strategies in the treatment of HCC.