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1.
Tumour Biol ; 42(4): 1010428320914475, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32252611

RESUMO

Hepatocellular carcinoma is a major cause of cancer mortality worldwide. The outcome of hepatocellular carcinoma depends mainly on its early diagnosis. To date, the performance of traditional biomarkers is unsatisfactory. Polo-like kinase 1 is a serine/threonine kinase that plays essential roles in cell cycle progression and deoxyribonucleic acid damage. Moreover, polo-like kinase 1 knockdown decreases the survival of hepatocellular carcinoma cells; therefore, polo-like kinase 1 is an attractive target for anticancer treatments. Nobiletin, a natural polymethoxy flavonoid, exhibits a potential antiproliferative effect against a wide variety of cancers. This study targets to identify a reliable diagnostic biomarker for hepatocellular carcinoma and provide a potential therapeutic target for its treatment. Polo-like kinase 1 levels were analyzed in 44 hepatocellular carcinoma patients, 33 non-hepatocellular carcinoma liver cirrhosis patients and 15 healthy controls using the enzyme-linked immunosorbent assay method. Receiver operating characteristics curve analysis was used to establish a predictive model for polo-like kinase 1 relative to α-fetoprotein in hepatocellular carcinoma diagnosis. Furthermore, in the in vitro study, gene expressions were assessed by quantitative polymerase chain reaction in two human hepatocellular carcinoma cell lines after treatment with doxorubicin and polo-like kinase 1 inhibitor volasertib (Vola) either alone or in combination with nobiletin. Cell viability was also determined using the crystal violet assay.: Serum polo-like kinase 1 levels in hepatocellular carcinoma patients were significantly higher than liver cirrhosis and control groups (p < 0.0001). Polo-like kinase 1 showed a reasonable sensitivity, specificity, positive predictive value, and negative predictive value in hepatocellular carcinoma diagnosis. Moreover, nobiletin improved inhibition of cell growth induced by Vola and doxorubicin. Regarding reverse transcription polymerase chain reaction results, nobiletin suppressed expressions of polo-like kinase 1 and proliferating cell nuclear antigen and elevated expressions of P53, poly (ADPribose) polymerase 1, and caspase-3. Nobiletin/doxorubicin and nobiletin/Vola showed a significant increase in caspase-3 activity indicating cell apoptosis. Polo-like kinase 1 may be a potential biomarker for hepatocellular carcinoma diagnosis and follow-up during treatment with chemotherapies. In addition, nobiletin synergistically potentiates the doxorubicin and Vola-mediated anticancer effect that may be attributed partly to suppression of polo-like kinase 1 and proliferating cell nuclear antigen expression and enhancement of chemotherapy-induced apoptosis.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/metabolismo , Neoplasias Hepáticas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Caspase 3/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Flavonas/farmacologia , Células Hep G2 , Humanos , Cirrose Hepática/patologia , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Pteridinas/farmacologia
2.
Cardiovasc Ther ; 2020: 1926249, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32328171

RESUMO

Isoliquiritigenin (ISL) is a flavonoid isolated mainly from the licorice plant, a traditional Chinese herb. ISL has shown anticancer, anti-inflammatory, antioxidant, and antidiabetic activities. However, the pharmaceutical effects of ISL on atherosclerosis are seldom explored. In this study, we used apolipoprotein E (ApoE) knockout mouse model and angiotensin II- (Ang II-) stimulated vascular smooth muscle cells (VSMCs) to elucidate the pharmacological mechanism of ISL to inhibit atherosclerosis. We found that in ApoE-/- mice ISL could attenuate atherosclerotic lesion, reduce serum lipid levels, and inhibit TRPC5 expression. In vitro, ISL inhibited Ang II-stimulated proliferation of VSMCs and suppressed Ang II-induced TRPC5 and PCNA expressions in a dose-dependent fashion. In conclusion, our findings provide novel insight into the pharmacological effects of ISL on atherosclerosis and suggest that ISL is beneficial for cardiovascular protection.


Assuntos
Aorta/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Chalconas/farmacologia , Placa Aterosclerótica , Canais de Cátion TRPC/antagonistas & inibidores , Animais , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Lipídeos/sangue , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transdução de Sinais , Canais de Cátion TRPC/metabolismo
3.
Medicine (Baltimore) ; 99(16): e19755, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32311975

RESUMO

Although proliferating cell nuclear antigen (PCNA) plays an important role in tumor proliferation and its expression level is closely related to the biological activity of tumor cells, PCNA expression in non-small cell lung cancer (NSCLC) has been seldom reported. In this study, we aimed to investigate the significance of PCNA expression in NSCLC tissues. PCNA expression in NSCLC and adjacent tissues were assessed by immunohistochemistry (IHC), western blotting, and reverse transcription polymerase chain reaction. Single factor analysis was used to study the relationship between the expression of PCNA and clinicopathological features of NSCLC. Multi-factor Cox survival analysis was used to evaluate the relationship between the expression of PCNA and overall survival of postoperative NSCLC patients. The areas under the receiver operating characteristics were calculated to evaluate the value of PCNA expression level in predicting the 3-year survival of NSCLC patients. IHC analysis showed that the positive expression rates of PCNA protein in NSCLC and adjacent tissues were 91.79% (257/280) and 25.83% (31/120), respectively. Western blotting confirmed that PCNA protein level was significantly higher in NSCLC tissues than in the adjacent tissues (P < .05). Reverse transcription polymerase chain reaction showed that the positive rate of PCNA mRNA in NSCLC was 88.93% (249/280), which was significantly higher than that in adjacent tissues 29.17% (35/120) (P < .05). Both PCNA mRNA and protein levels were correlated with tumor differentiation, size, metastasis, and stage in NSCLC. Patients exhibiting higher PCNA protein expression had a significantly shorter disease-specific survival rate than the other patients. PCNA protein level and tumor pathological type, metastasis, differentiation degree, and stage were independent factors affecting the overall survival of postoperative patients. The areas under the receiver operating characteristics of PCNA mRNA for predicting the 3-year survival of NSCLC patients was 0.89 (0.79-0.98), with a sensitivity and specificity of 0.84 and 0.76, respectively. In conclusion, high PCNA protein and mRNA levels may be associated with the occurrence, development, and prognosis of NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/cirurgia , China/epidemiologia , Estudos Transversais , Feminino , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
4.
Zhonghua Zhong Liu Za Zhi ; 42(3): 197-202, 2020 Mar 23.
Artigo em Chinês | MEDLINE | ID: mdl-32252197

RESUMO

Objective: To investigate the effects of metastasis associated gene 1 (MTA1) expression on the proliferation and apoptosis of human esophageal cancer Eca109 cells. Methods: MTA1 siRNA was transfected into human esophageal cancer Eca109 cells, and the control group and blank group were set up. The expression of MTA1 in Eca109 cells with different treatment was detected by real-time fluorescent quantitative PCR (RT-PCR) and western blot. The proliferation of Eca109 cells was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. The cloning formation ability of Eca109 cells was detected by plate cloning assay. The apoptosis of Eca109 cells were detected by flow cytometry. The expression of proliferating cell nuclear antigen (PCNA) and apoptosis-related proteins, including cleaved caspase-3 and total caspase-3 protein in Eca109 cells were detected by western blot. Results: After 48 hours of transfection, RT-PCR result showed that the relative expression levels of MTA1 mRNA in Eca109 cells in the blank group, control group, and siRNA group were 1.00±0.10, 0.98±0.09 and 0.21±0.03, respectively. The expression of MTA1 mRNA in siRNA group was significantly inhibited (P<0.05), while no significant difference of MTA1 mRNA expression between the blank group and the control group has been found (P>0.05). Western blot results were consistent with those of RT-PCR. MTT array results showed that, compared with the blank group and transfection group, the absorbance values of Eca109 cells in siRNA group were dramatically reduced at 48, 72, and 96 h (all P<0.05). There were no significant differences of absorbance values between the blank group and the control group at 48, 72, and 96 h (all P>0.05). The results of the plate colony formation test showed that the number of colony formation in the blank group and control group were 58.64±6.86 and 60.02±7.04, respectively, significantly higher than 18.10±3.16 in siRNA group (P<0.05), while there was no significant difference between the blank group and control group (P>0.05). Flow cytometry results showed that the apoptosis rates in the blank group and control group were (2.13±0.54)% and (2.27±0.61)%, respectively, significantly lower than (32.61±5.28)% in siRNA group (P<0.05), while there was no significant difference between blank group and control group (P>0.05). Western blot results showed that the expression of PCNA protein was down-regulated while cleaved caspase-3 protein expression was upregulated in siRNA group, compared to the control group and blank group (P<0.05). Conclusions: Inhibition of MTA1 expression can inhibit the proliferation of Eca109 cells and induce apoptosis. This process may be related to the down-regulation of PCNA protein expression and activation of caspase-3 protein expression.


Assuntos
Apoptose , Neoplasias Esofágicas/patologia , Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/fisiologia , Proteínas Repressoras/fisiologia , Transativadores/metabolismo , Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Esofágicas/metabolismo , Histona Desacetilases/genética , Humanos , Antígeno Nuclear de Célula em Proliferação , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transativadores/genética , Transfecção
5.
Life Sci ; 250: 117561, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32198052

RESUMO

AIMS: Pyruvate kinase M2 (PKM2), a unique isoform of the pyruvate kinases, not only acts as a crucial metabolic enzyme when it locates in the cytoplasm, but also plays important roles in tumor formation and growth when it accumulates in the nuclei. Our aim was to investigate the potential role of PKM2 in liver regeneration in mice insulted with carbon tetrachloride (CCl4). MATERIAL AND METHODS: The liver regeneration model was established by intraperitoneal injection of CCl4 for 48 h in male BALB/c mice. The expression of PKM2, phospho-STAT3, STAT3, proliferating cell nuclear antigen (PCNA) and Cyclin D1 were evaluated by western blot. The distribution of PKM2 was verified by immunofluorescence staining. The degree of injured region was assessed by hematoxylin and eosin (HE) staining. The proliferation of liver cells was tested by Immunohistochemistry. KEY FINDINGS: The nuclear accumulation of PKM2 increased in the liver treated with CCl4, but treatment with ML-265 significantly suppressed CCl4-induced nuclear accumulation of PKM2. In addition, treatment with ML-265 suppressed the level of cyclin D1 and proliferating cell nuclear antigen (PCNA), reduced the count of Ki67-positive hepatocytes, and expanded the damaged region in histological examination. Meanwhile, treatment with ML-265 suppressed the phosphorylation of nuclear signal transducer and activator of transcription 3 (STAT3). Inhibition of STAT3 by stattic made the same effects as ML-265. SIGNIFICANCE: These data uncovered the role of nuclear PKM2 in liver regeneration and the pro-proliferation effects of nuclear PKM2 may be through targeting its downstream transcription factor STAT3.


Assuntos
Núcleo Celular/metabolismo , Regeneração Hepática , Piruvato Quinase/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Tetracloreto de Carbono , Proliferação de Células , Citoplasma/metabolismo , Hepatócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/metabolismo , Hepatopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(11): 5791-5800, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123106

RESUMO

Targeted degradation approaches such as proteolysis targeting chimeras (PROTACs) offer new ways to address disease through tackling challenging targets and with greater potency, efficacy, and specificity over traditional approaches. However, identification of high-affinity ligands to serve as PROTAC starting points remains challenging. As a complementary approach, we describe a class of molecules termed biological PROTACs (bioPROTACs)-engineered intracellular proteins consisting of a target-binding domain directly fused to an E3 ubiquitin ligase. Using GFP-tagged proteins as model substrates, we show that there is considerable flexibility in both the choice of substrate binders (binding positions, scaffold-class) and the E3 ligases. We then identified a highly effective bioPROTAC against an oncology target, proliferating cell nuclear antigen (PCNA) to elicit rapid and robust PCNA degradation and associated effects on DNA synthesis and cell cycle progression. Overall, bioPROTACs are powerful tools for interrogating degradation approaches, target biology, and potentially for making therapeutic impacts.


Assuntos
Antígeno Nuclear de Célula em Proliferação/metabolismo , Engenharia de Proteínas/métodos , Proteólise , Ubiquitina-Proteína Ligases/genética , Sítios de Ligação , Células HEK293 , Humanos , Terapia de Alvo Molecular/métodos , Antígeno Nuclear de Célula em Proliferação/química , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
7.
Nat Commun ; 11(1): 1109, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111820

RESUMO

In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. We present the high-resolution cryo-EM structure of the human processive Pol δ-DNA-PCNA complex in the absence and presence of FEN1. Pol δ is anchored to one of the three PCNA monomers through the C-terminal domain of the catalytic subunit. The catalytic core sits on top of PCNA in an open configuration while the regulatory subunits project laterally. This arrangement allows PCNA to thread and stabilize the DNA exiting the catalytic cleft and recruit FEN1 to one unoccupied monomer in a toolbelt fashion. Alternative holoenzyme conformations reveal important functional interactions that maintain PCNA orientation during synthesis. This work sheds light on the structural basis of Pol δ's activity in replicating the human genome.


Assuntos
DNA Polimerase III/química , DNA Polimerase III/metabolismo , Motivos de Aminoácidos , Domínio Catalítico , Microscopia Crioeletrônica , DNA/metabolismo , DNA Polimerase III/genética , Replicação do DNA , Endonucleases Flap/química , Endonucleases Flap/metabolismo , Holoenzimas , Humanos , Modelos Moleculares , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Subunidades Proteicas , Relação Estrutura-Atividade
8.
Life Sci ; 248: 117445, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32081664

RESUMO

AIMS: Atherosclerosis (AS) is a common cardiovascular disease with complicated pathogenesis. Long non-coding RNAs (lncRNAs) have been reported to be associated with AS progression. We aimed to explore the role and underlying mechanism of HOXA transcript at the distal tip (HOTTIP) in AS. MATERIALS AND METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of HOTTIP, miR-490-3p and high mobility group B 1 (HMGB1) in AS patients' sera and oxidized low-density lipoprotein (ox-LDL) induced human aortic vascular smooth muscle cells (HA-VSMCs). Cell Counting Kit-8 (CCK-8) assay and transwell assay were conducted to evaluate the proliferation and migration of HA-VSMCs, respectively. Western blot assay was carried out to determine the levels of proliferating cell nuclear antigen (PCNA), matrix metalloprotein 2 (MMP2), MMP9 and HMGB1. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were conducted to verify the targeting association between HOTTIP and miR-490-3p, as well as miR-490-3p and HMGB1. KEY FINDINGS: HOTTIP and HMGB1 were upregulated and miR-490-3p was downregulated in the sera of AS patients and ox-LDL-stimulated HA-VSMCs. HOTTIP knockdown suppressed ox-LDL induced proliferation and migration in HA-VSMCs. MiR-490-3p was identified as a target of HOTTIP and HOTTIP overexpression abolished the inhibition on cell proliferation and migration mediated by miR-490-3p in ox-LDL-induced HA-VSMCs. Moreover, miR-490-3p inhibition promoted cell proliferation and migration by directly targeting HMGB1 in ox-LDL-induced HA-VSMCs. Besides, HOTTIP knockdown repressed the activation of PI3K-AKT signaling pathway. SIGNIFICANCE: HOTTIP knockdown suppressed cell proliferation and migration by regulating miR-490-3p/HMGB1 axis and PI3K-AKT pathway in ox-LDL-induced HA-VSMCs.


Assuntos
Aterosclerose/genética , Proteína HMGB1/genética , MicroRNAs/genética , Miócitos de Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Longo não Codificante/genética , Aorta/metabolismo , Aorta/patologia , Aterosclerose/sangue , Aterosclerose/patologia , Estudos de Casos e Controles , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica , Proteína HMGB1/metabolismo , Humanos , Lipoproteínas LDL/farmacologia , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
9.
Nucleic Acids Res ; 48(6): 3042-3052, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32009145

RESUMO

Ubiquitylation of the eukaryotic sliding clamp, PCNA, activates a pathway of DNA damage bypass that facilitates the replication of damaged DNA. In its monoubiquitylated form, PCNA recruits a set of damage-tolerant DNA polymerases for translesion synthesis. Alternatively, modification by K63-linked polyubiquitylation triggers a recombinogenic process involving template switching. Despite the identification of proteins interacting preferentially with polyubiquitylated PCNA, the molecular function of the chain and the relevance of its K63-linkage are poorly understood. Using genetically engineered mimics of polyubiquitylated PCNA, we have now examined the properties of the ubiquitin chain required for damage bypass in budding yeast. By varying key parameters such as the geometry of the junction, cleavability and capacity for branching, we demonstrate that either the structure of the ubiquitin-ubiquitin junction or its dynamic assembly or disassembly at the site of action exert a critical impact on damage bypass, even though known effectors of polyubiquitylated PCNA are not strictly linkage-selective. Moreover, we found that a single K63-junction supports substantial template switching activity, irrespective of its attachment site on PCNA. Our findings provide insight into the interrelationship between the two branches of damage bypass and suggest the existence of a yet unidentified, highly linkage-selective receptor of polyubiquitylated PCNA.


Assuntos
Dano ao DNA/genética , Proteínas de Ligação a DNA/genética , Antígeno Nuclear de Célula em Proliferação/genética , Ubiquitinação/genética , Reparo do DNA/genética , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Poliubiquitina/genética , Mapas de Interação de Proteínas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/genética
10.
PLoS One ; 15(2): e0229000, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32092106

RESUMO

Site-specific conjugation of ubiquitin onto a range of DNA repair proteins regulates their critical functions in the DNA damage response. Biochemical and structural characterization of these functions are limited by an absence of tools for the purification of DNA repair proteins in purely the ubiquitinated form. To overcome this barrier, we designed a ubiquitin fusion protein that is N-terminally biotinylated and can be conjugated by E3 RING ligases onto various substrates. Biotin affinity purification of modified proteins, followed by cleavage of the affinity tag leads to release of natively-mono-ubiquitinated substrates. As proof-of-principle, we applied this method to several substrates of mono-ubiquitination in the Fanconi anemia (FA)-BRCA pathway of DNA interstrand crosslink repair. These include the FANCI:FANCD2 complex, the PCNA trimer and BRCA1 modified nucleosomes. This method provides a simple approach to study the role of mono-ubiquitination in DNA repair or any other mono-ubiquitination signaling pathways.


Assuntos
Avidina/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Proteínas de Grupos de Complementação da Anemia de Fanconi , Antígeno Nuclear de Célula em Proliferação , Ubiquitina-Proteína Ligases , Ubiquitina , Animais , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/isolamento & purificação , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/isolamento & purificação , Humanos , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/isolamento & purificação , Células Sf9 , Spodoptera , Ubiquitina/química , Ubiquitina/isolamento & purificação , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/isolamento & purificação , Proteínas Ubiquitinadas/química , Proteínas Ubiquitinadas/isolamento & purificação
11.
Ecotoxicol Environ Saf ; 192: 110256, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32014724

RESUMO

The modulatory role of the Spirulina platensis (SP) against furan-induced (FU) hepatic and renal damage was assessed in this study. For achieving this, sixty rats were distributed into six groups: control group, SP-administered group (300 mg/kg b.wt orally for 28 days), a FU-intoxicated group (16 mg/kg b.wt, orally, daily for 28 days), protective co-treated group SP/F (administered SP 300 mg/kg b.wt, one week before, and concurrently with FU intoxication), therapeutic co-treated group FU/SP (administered SP 300 mg/kg b.wt, one week after FU intoxication for 28 days) and protective/therapeutic co-treated group SP/FU/SP (administered SP one week before and after, concurrently with FU intoxication). Subsequently, the biochemical responses and the histology of hepatic and renal tissues in treated rats were assessed. The results indicated that FU intoxication induced a significant hepato- and nephropathy represented by the elevation in the values of tissue injury biomarkers and reduction in protein levels. Histologically, a wide range of morphological, cytotoxic, inflammatory, and vascular alterations as well as downregulation in the immunoexpression of the proliferating cell nuclear antigen (PCNA) and the proliferation-associated nuclear antigen (Ki-67) were induced by FU intoxication. Oral SP administration, particularly in the protective/therapeutic co-treated group, markedly supressed the serum levels of the tissue injury biomarkers, diminished the inflammatory response, restored the cytotoxic alterations, upregulated the immunoexpression of PCNA and Ki-67, and restored the perturbed morphology of the hepatic and renal tissues. In conclusion, the obtained data demonstrated that SP co-administration elicits both protective and therapeutic potential against the FU-induced hepato- and nephropathy.


Assuntos
Proliferação de Células/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/terapia , Furanos/toxicidade , Nefropatias/terapia , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Spirulina , Animais , Biomarcadores/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Antígeno Ki-67/metabolismo , Rim/metabolismo , Rim/patologia , Rim/ultraestrutura , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Nefropatias/patologia , Fígado/metabolismo , Fígado/patologia , Fígado/ultraestrutura , Masculino , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos
12.
Nat Commun ; 11(1): 304, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949141

RESUMO

Biological processes in development and disease are controlled by the abundance, localization and modification of cellular proteins. We have developed versatile tools based on recombinant E3 ubiquitin ligases that are controlled by light or drug induced heterodimerization for nanobody or DARPin targeted depletion of endogenous proteins in cells and organisms. We use this rapid, tunable and reversible protein depletion for functional studies of essential proteins like PCNA in DNA repair and to investigate the role of CED-3 in apoptosis during Caenorhabditis elegans development. These independent tools can be combined for spatial and temporal depletion of different sets of proteins, can help to distinguish immediate cellular responses from long-term adaptation effects and can facilitate the exploration of complex networks.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Técnicas Citológicas , Luz , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/efeitos da radiação , Animais , Apoptose , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/efeitos da radiação , Caspases/efeitos dos fármacos , Caspases/metabolismo , Caspases/efeitos da radiação , Engenharia Celular/métodos , Dano ao DNA , DNA Ligase Dependente de ATP , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Lamina Tipo A/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética
13.
World Neurosurg ; 136: e460-e468, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31953094

RESUMO

BACKGROUND: Allicin can suppress liver and cardiac fibrosis; thus, we hypothesized that it might prevent scar tissue from extensive epidural fibrosis after laminectomy. METHODS: Human epidural scar fibroblasts were isolated from surgical specimens and treated with allicin at a gradient of concentrations. Morphology, viability, migration rate, cell cycle, and apoptosis rate were measured by fluorescence microscope, Cell Counting Kit-8 assay, scratch assay, and flow cytometry. Western blot was used to measure the expression level of proliferation-related proteins. RESULTS: After treatment by allicin, cell viability (P = 0.042) and migration rate (P = 0.010 in scratch assay and P = 0.025 in transwell assay) decreased significantly in a dose-dependent manner. The percentage of G1 phase cells significantly decreased (P = 0.017), whereas the percentage of S phase cells (P = 0.096) and G2 phase cells (P = 0.038) significantly increased in a dose-dependent manner. Similarly, the percentage of apoptotic cells increased significantly in a dose-dependent manner (P = 0.036). Compared with the control group, the expression level of proliferating cell nuclear antigen protein (P = 0.081) and Bcl-2 protein (P = 0.029) significantly decreased, whereas the BAX protein level significantly increased (P = 0.017). CONCLUSIONS: Allicin can suppress human epidural scar fibroblast migration, induce cell apoptosis, and block cell proliferation at S phase and G2 phase.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cicatriz/patologia , Espaço Epidural/patologia , Fibroblastos/efeitos dos fármacos , Ácidos Sulfínicos/farmacologia , Biomarcadores/metabolismo , Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fibroblastos/citologia , Fibrose/patologia , Humanos , Vértebras Lombares , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína X Associada a bcl-2/metabolismo
14.
Cell Prolif ; 53(1): e12710, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31663659

RESUMO

OBJECTIVE: Clinical trials have demonstrated the efficacy of indigo naturalis, a traditional Chinese medicine ingredient, against psoriasis, a skin disease characterized by keratinocyte hyperproliferation and inflammation. The present study investigates the efficacy of tryptanthrin, a bioactive compound in indigo naturalis, against non-melanoma skin cancer (NMSC) and the signalling events involved. METHODS: Efficacy of tryptanthrin against NMSC was assessed using DMBA/PMA-induced skin carcinogenesis model in Swiss albino mice. Immunostaining for PCNA and ki-67 was used to mark proliferating cells in tissues. Haematoxylin and eosin staining and toluidine staining were employed to assess inflammation, and TUNEL assay was used to detect apoptosis in tissues. The signalling events were evaluated using Western blot, imunohistochemistry and immunofluorescence staining. MTT assay and clonogenic assay were performed to assess the viability and proliferation of cancer cells, in vitro. RESULTS: In mice, topical application of tryptanthrin suppressed skin carcinogenesis. It attenuated inflammation, impeded the proliferation of hair follicle (HF) cells and suppressed the activation of ß-catenin, a major driver of HF cell proliferation. Additionally tryptanthrin suppressed the activation of ERK1/2 and p38, both of which promote ß-catenin activation and lowered the expression of c-Myc and cyclin-D1. Tryptanthrin suppressed the proliferation of the human NMSC cell line, A431 and abrogated EGF-induced activation of ß-catenin and subsequent cytoskeletal rearrangement. CONCLUSION: The study demonstrates with molecular evidence that tryptanthrin is an effective suppressor of NMSC.


Assuntos
Quinazolinas/farmacologia , Neoplasias Cutâneas/prevenção & controle , Animais , Ensaios de Seleção de Medicamentos Antitumorais , Antígeno Ki-67/metabolismo , Camundongos , Proteínas de Neoplasias/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol/toxicidade , Ácido gama-Aminobutírico/análogos & derivados , Ácido gama-Aminobutírico/toxicidade
15.
Neoplasma ; 67(1): 15-26, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31607135

RESUMO

Glycogen synthase kinase beta (GSK3ß) is considered as a promising target for lung cancer treatment and its inhibitor lithium chloride (LiCl) is widely regarded as having potent anti-proliferative and apoptosis-modulating activities. Proliferating cell nuclear antigen (PCNA), as an auxiliary protein for DNA polymerase delta, which regulates DNA replication and repair, has been reported to play an important role in regulating apoptosis. Here, we showed that GSK3ß interacted with PCNA in H1299 lung adenocarcinoma cells using GST pull-down and co-immunoprecipitation experiments. We discovered that their interaction can be enhanced within the first 3 h after UVC irradiation and decreased gradually with time. Overexpression of PCNA protein decreased GSK3ß Ser9 phosphorylation, whereas knockdown of PCNA using small interfering RNA (siRNA) increased Ser9 phosphorylated GSK3ß, which was attenuated by phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 after UVC irradiation, indicating the involvement of the PI3K-AKT pathway. Functional analyses suggested that downregulation of PCNA sensitized H1299 cells to LiCl-induced apoptosis. Thus, our results unraveled a novel regulatory of GSK3ß by PCNA and provided a promising direction for treatment of lung cancer.


Assuntos
Adenocarcinoma de Pulmão/patologia , Apoptose , Glicogênio Sintase Quinase 3 beta/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Adenocarcinoma de Pulmão/metabolismo , Linhagem Celular Tumoral , Cromonas/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno
16.
J Fish Biol ; 96(1): 102-110, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31674006

RESUMO

The eye of the fish has a lifelong persistent neurogenesis unlike eye of mammals, so it's highly interesting to study retinal neurogenesis and its genetic control to give complete knowledge about the cause of this property in fish in comparison to mammals. We performed fluorescent in situ hybridisation for loach Misgurnus anguillicaudatus bmi1, msi1 and sox2 genes, which are used as an indicator of the sites of multipotent stem cells. Proliferating cell nuclear antigen (PCNA), bromodeoxyuridine (BRDU) and KI67 markers were used as indicators of proliferating cells and glial fibrillary acidic protein (GFAP) immunofluorescence was used for detection of the glial property of cells, as well as, immunohistochemistry detected the role of peroxisome proliferator-activated receptor (PPAR)α and γ in retinal neurogenesis. Our results determined that the lens and the retina of loach M. anguillicaudatus contain proliferative and pluripotent stem cells that have both glial and neuroepithelial properties, which add new cells continuously throughout life even without injury-induced proliferation. The PPARα has an essential function in providing energy supply for retinal neurogenesis more than PPARγ.


Assuntos
Proliferação de Células , Cipriniformes/fisiologia , Cristalino/citologia , Retina/citologia , Células-Tronco/fisiologia , Animais , Biomarcadores , Bromodesoxiuridina/metabolismo , Cipriniformes/genética , Regulação da Expressão Gênica , Proteína Glial Fibrilar Ácida , Antígeno Ki-67/metabolismo , Cristalino/fisiologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Retina/fisiologia
17.
Gen Comp Endocrinol ; 285: 113230, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31348955

RESUMO

During the ovarian ontogeny in birds, five fundamental events can be recognized: migration and colonization of the primordial germ cells, differentiation and proliferation of oogonies, an organization of germinal nests, beginning of the meiotic process and folliculogenesis. The knowledge of these events is fundamental for the interpretation of the processes involved in the differentiation of female gametes. However, there are only references for some model species such as Gallus gallus domesticus and Coturnix coturnix. In a previous study, the histological structure of embryonic ovaries of Columba livia was revealed. Therefore, the objective of this work is to characterize the processes of meiosis and folliculogenesis C. livia from the analysis of the expression of the GnRH receptor, the 3ßHSD enzyme and the cell proliferation protein PCNA in embryonic and postnatal ovaries. Therefore, the expression of GnRHR, 3ßHSD, and PCNA was revealed in histological testicular and ovarian preparations in embryos (stages 41-43) and neonates (2, 5, 7, 10 and 75 days post-hatching). The present study demonstrates that the fate of germline cells is dictated by their location during gonadal development. Thus, the germline cells located in the cortex of the left gonad enter meiosis, while those in the right gonad and those in the medulla of the left ovary fail to go into meiosis. This indicates that somatic signals, instead of an autonomous cellular mechanism, regulate the entry of the germline cells into meiosis in the C. livia embryo. Future studies will be focused on the analysis of proteins associated with meiotic events and folliculogenesis in embryonic and neonatal ovaries of C. livia, to evaluate the regulation of meiosis in vitro.


Assuntos
3-Hidroxiesteroide Desidrogenases/metabolismo , Columbidae/metabolismo , Meiose , Folículo Ovariano/crescimento & desenvolvimento , Receptores LHRH/metabolismo , Animais , Proliferação de Células , Columbidae/embriologia , Embrião não Mamífero/citologia , Feminino , Células Germinativas/metabolismo , Imuno-Histoquímica , Oócitos/citologia , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo
18.
Biochim Biophys Acta Mol Cell Res ; 1867(4): 118628, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31884068

RESUMO

One neurotoxic mechanism of amyloid-beta peptide (Aß), the major component of senile plaques in the brains of Alzheimer's disease (AD) patients, is to trigger cell cycle reentry in fully differentiated neurons. However, the detailed underlying mechanisms remain unclear. Using Aß25-35-treated primary rat cortical neurons as the experimental system, in the present study we tested whether Aß-induced inhibitor of differentiation-1 (Id1)/hypoxia-inducible factor-1alpha (HIF-1α) and cyclin-dependent kinase-5 (CDK5) contribute to cell cycle reentry in fully differentiated post-mitotic neurons. We found that Id1-induced HIF-1α mediated Aß25-35-dependent expression of the cell cycle markers cyclin D1 and proliferating cell nuclear antigen (PCNA), both colocalized with microtubule-associated protein-2 (MAP-2) + cells, indicative of cell cycle reentry in the mature neurons. Aß25-35 also enhanced p35 cleavage to p25 without affecting CDK5 expression. The CDK5 inhibitor roscovitine and the siRNA targeting CDK5 both suppressed Aß25-35-dependent HIF-1α expression and cell cycle reentry. Intriguingly, Aß25-35-induced Id1 repressed p25 production while CDK5/p25 reciprocally inhibited Id1 expression, despite the observation that both Id1 and CDK5/p25 acted upstream of HIF-1α. These results demonstrated that both Id1/HIF-1 and CDK5/HIF-1 contribute to Aß-induced cell cycle reentry in post-mitotic neurons; furthermore, Id1 and CDK5/p25 reciprocally suppress expression of each other.


Assuntos
Ciclo Celular , Quinase 5 Dependente de Ciclina/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteína 1 Inibidora de Diferenciação/metabolismo , Neurônios/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Células Cultivadas , Córtex Cerebral/citologia , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Quinase 5 Dependente de Ciclina/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteína 1 Inibidora de Diferenciação/genética , Mitose , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Roscovitina/farmacologia
19.
Biomed Pharmacother ; 121: 109681, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31810125

RESUMO

OBJECTIVES: Cynaroside (CYN) is the predominant derivative of luteolin in aerial parts of Bidens tripartita which has been used in folk medicine as a diaphoretic, diuretic, antiseptic and anti-inflammatory agent. In our study, alginate (ALG), which is an anionic polymer with bioadhesive properties, was used as a CYN carrier, and multiple hydrogel formulations were created. Additionally, the present study evaluated the in vivo anti-inflammatory and anti-allergic activities of all preparations. METHODS: Novel gel formulations as topical carriers for CYN obtained from B. tripartita were developed and characterized. The bioadhesive properties of the designed preparations were also evaluated in an ex vivo model using the skin of hairless mice. In vitro CYN release from all formulations was examined and analysed by HPLC. Histopathological evaluation of mouse skin sections stained with H&E after carrageenan and oxazolone administration was also carried out. In addition, the influence of CYN on cell proliferation was examined by the PCNA staining method. RESULTS: The results showed that 10 % CYN inhibited the release of anti-inflammatory mediators, and both tested concentrations, which included 5 % and 10 % (2 mg and 20 mg CYN per site, respectively), reduced oxazolone-induced ear swelling. Histopathological examination of the samples revealed a marked reduction in paw skin and ear tissue inflammation and in inflammatory infiltrates. The influence of CYN on cell proliferation was examined by the PCNA staining method, and the staining and distribution of PCNA-immunoreactive (PCNA-IR) cells were observed. After the application of the 5 % and 10 % hydrogels, the investigated samples showed decreased nuclear immunoreactivity to PCNA, which was similar to that of the control. Moreover, after application of the placebo formulation, fewer PCNA-IR cells were also observed. CONCLUSION: The obtained data suggest that the topical application of CYN significantly reduces the number of T cells, mast cells and histiocytes in mouse skin with inflammation or atopic dermatitis.


Assuntos
Antialérgicos/farmacologia , Anti-Inflamatórios/farmacologia , Composição de Medicamentos , Glucosídeos/farmacologia , Hidrogéis/química , Luteolina/farmacologia , Animais , Antialérgicos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Modelos Animais de Doenças , Liberação Controlada de Fármacos , Edema/tratamento farmacológico , Masculino , Camundongos Endogâmicos C57BL , Oxazolona , Antígeno Nuclear de Célula em Proliferação/metabolismo
20.
Proc Natl Acad Sci U S A ; 117(2): 1081-1089, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31879348

RESUMO

The tethering together of sister chromatids by the cohesin complex ensures their accurate alignment and segregation during cell division. In vertebrates, sister chromatid cohesion requires the activity of the ESCO2 acetyltransferase, which modifies the Smc3 subunit of cohesin. It was shown recently that ESCO2 promotes cohesion through interaction with the MCM replicative helicase. However, ESCO2 does not significantly colocalize with the MCM complex, suggesting there are additional interactions important for ESCO2 function. Here we show that ESCO2 is recruited to replication factories, sites of DNA replication, through interaction with PCNA. We show that ESCO2 contains multiple PCNA-interaction motifs in its N terminus, each of which is essential to its ability to establish cohesion. We propose that multiple PCNA-interaction motifs embedded in a largely flexible and disordered region of the protein underlie the unique ability of ESCO2 to establish cohesion between sister chromatids precisely as they are born during DNA replication.


Assuntos
Acetiltransferases/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Replicação do DNA/fisiologia , Animais , Proteínas de Ciclo Celular/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , DNA Helicases/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vertebrados/genética
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