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1.
Anticancer Res ; 39(8): 4129-4136, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366497

RESUMO

BACKGROUND/AIM: 5-Aza-2-deoxycytidine (5-Aza-CdR) enhances the sensitivity to 5-fluorouracil (5-FU), but the molecular mechanism is not fully understood. The aim of this study was to investigate the molecular mechanism that enhances the sensitivity to 5-FU treated with 5-Aza-CdR via thymidine phosphorylase (TP). MATERIALS AND METHODS: The sensitivity to drugs was determined on several cancer cell lines by the MTT assay. Protein and mRNA levels were examined by immunoblot and RT-PCR, respectively. Gene silencing, binding of Sp1 to DNA and methylation of DNA was performed by siRNA, ChIP assay and sodium bisulfate genomic sequencing, respectively. RESULTS: Sp1-binding sites in the TP promoter were methylated in epidermoid carcinoma. 5-Aza-CdR demethylated Sp1-binding sites and enhanced sensitivity to 5-FU. CONCLUSION: Demethylation of Sp1-binding sites by 5-Aza-CdR was a key factor enhancing 5-FU sensitivity, which may enable more effective treatments for cancer patients with the combination of 5-Aza-CdR and 5-FU.


Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Metilação de DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Fator de Transcrição Sp1/genética , Timidina Fosforilase/genética , Sítios de Ligação/efeitos dos fármacos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Decitabina/metabolismo , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/genética , Timidina Fosforilase/química
2.
J Agric Food Chem ; 67(26): 7390-7398, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31244202

RESUMO

Wound-induced suberization is an essentially protective healing process for wounded fruit to reduce water loss and microbial infection. It has been demonstrated that abscisic acid (ABA) could promote wound suberization, but the molecular mechanism of ABA regulation remains little known. In this study, the transcript level of Achn030011 (designated as AchnKCS), coding a ß-ketoacyl-coenzyme A synthase (KCS) involved in suberin biosynthesis, was found to be significantly upregulated by ABA in wounded kiwifruit. A bZIP transcription factor (Achn270881), a possible downstream transcription factor in the ABA signaling pathway, was screened and designated as AchnbZIP12 according to its homology with related Arabidopsis transcription factors. A yeast one-hybrid assay demonstrated that AchnbZIP12 could interact with the AchnKCS promoter. Furthermore, significant trans-activation of AchnbZIP12 on AchnKCS was verified. The transcript level of AchnbZIP12 was also upregulated upon treatment with ABA. These results imply that AchnbZIP12 acts as a positive regulator in ABA-mediated AchnKCS transcription during wound suberization of kiwifruit.


Assuntos
Ácido Abscísico/farmacologia , Actinidia/efeitos dos fármacos , Actinidia/fisiologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Reguladores de Crescimento de Planta/farmacologia , Proteínas de Plantas/genética , Actinidia/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Frutas/efeitos dos fármacos , Frutas/genética , Frutas/fisiologia , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos
3.
Anticancer Res ; 39(5): 2395-2403, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31092432

RESUMO

BACKGROUND/AIM: During cancer progression cells undergo epithelial-to-mesenchymal transition (EMT). Although EMT is a complex process, recently, it has been reported that CD146 overexpression in prostate cancer cells is sufficient to induce mesenchymal phenotype. The following study aimed to investigate whether the expression of CD146 is altered by an epigenetic modifier in prostate cancer cells, in vitro. MATERIALS AND METHODS: Three human prostate cancer cell lines were treated with 5-aza-2-deoxycytidine; the expression of CD146 and EMT-related factors was analyzed by RT-PCR and western Blot. The methylation status of the CD146 promoter area was assessed using bisulfite sequencing. RESULTS: Our data showed that, the expression of CD146 was evidently increased in all three studied cell lines in response to a demethylating agent, both at the mRNA and protein level, suggesting epigenetic regulation of the analyzed gene. However, there was no methylation in the studied CpG island in CD146 gene promoter. Moreover, the demethylating agent induced the expression of EMT-related transcription factors (SNAI1, SNAI2, TWIST1 and ZEB1), the pattern of which differed among the cell lines, as well as alterations in cell morphology; altogether accounting for the mesenchymal phenotype. CONCLUSION: The demethylating agent 5-aza-2-deoxycytidine triggers the expression of CD146 in prostate cancer cells independently on the methylation status of the analyzed CpG island fragment in CD146 gene promoter. Moreover, demethylation treatment induces a mesenchymal profile in prostate cancer cells.


Assuntos
Metilação de DNA/genética , Decitabina/farmacologia , Neoplasias da Próstata/genética , Antígeno CD146/genética , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia
4.
Nat Commun ; 10(1): 2262, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118412

RESUMO

Most biomedical research aimed at understanding gene function uses the Cre-Lox system, which consists of the Cre recombinase-dependent deletion of genes containing LoxP sites. This system enables conditional genetic modifications because the expression and activity of the recombinase Cre/CreERT2 can be regulated in space by tissue-specific promoters and in time by the ligand tamoxifen. Since the precise Cre-Lox recombination event is invisible, methods were developed to report Cre activity and are widely used. However, numerous studies have shown that expression of a given Cre activity reporter cannot be assumed to indicate deletion of other LoxP-flanked genes of interest. Here, we report the generation of an inducible dual reporter-Cre mouse allele, iSuRe-Cre. By significantly increasing Cre activity in reporter-expressing cells, iSuRe-Cre provides certainty that these cells have completely recombined floxed alleles. This genetic tool increases the ease, efficiency, and reliability of conditional mutagenesis and gene function analysis.


Assuntos
Edição de Genes/métodos , Vetores Genéticos/genética , Integrases/genética , Plasmídeos/genética , Animais , Técnicas de Cultura de Células , Clonagem Molecular/métodos , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Recombinação Genética/efeitos dos fármacos , Tamoxifeno/farmacologia
5.
Chemistry ; 25(41): 9691-9700, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31087710

RESUMO

Guanine-rich sequences of DNA are known to readily fold into tetra-stranded helical structures known as G-quadruplexes (G4). Due to their biological relevance, G4s are potential anticancer drug targets and therefore there is significant interest in molecules with high affinity for these structures. Most G4 binders are polyaromatic planar compounds which π-π stack on the G4's guanine tetrad. However, many of these compounds are not very selective since they can also intercalate into duplex DNA. Herein we report a new class of binder based on an octahedral cobalt(III) complex that binds to G4 via a different mode involving hydrogen bonding, electrostatic interactions and π-π stacking. We show that this new compound binds selectivity to G4 over duplex DNA (particularly to the G-rich sequence of the c-myc promoter). This new octahedral complex also has the ability to template the formation of G4 DNA from the unfolded sequence. Finally, we show that upon binding to G4, the complex prevents helicase Pif1-p from unfolding the c-myc G4 structure.


Assuntos
Cobalto/química , Cobalto/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , DNA/química , Quadruplex G/efeitos dos fármacos , Animais , Bovinos , DNA/genética , DNA/metabolismo , DNA Helicases/metabolismo , Genes myc/efeitos dos fármacos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Fenilenodiaminas/química , Fenilenodiaminas/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos
6.
Cancer Sci ; 110(7): 2247-2257, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31099446

RESUMO

Glioblastoma is one of the most devastating human malignancies for which a novel efficient treatment is urgently required. This pre-clinical study shows that eribulin, a specific inhibitor of telomerase reverse transcriptase (TERT)-RNA-dependent RNA polymerase, is an effective anticancer agent against glioblastoma. Eribulin inhibited the growth of 4 TERT promoter mutation-harboring glioblastoma cell lines in vitro at subnanomolar concentrations. In addition, it suppressed the growth of glioblastoma cells transplanted subcutaneously or intracerebrally into mice, and significantly prolonged the survival of mice harboring brain tumors at a clinically equivalent dose. A pharmacokinetics study showed that eribulin quickly penetrated brain tumors and remained at a high concentration even when it was washed away from plasma, kidney or liver 24 hours after intravenous injection. Moreover, a matrix-assisted laser desorption/ionization mass spectrometry imaging analysis revealed that intraperitoneally injected eribulin penetrated the brain tumor and was distributed evenly within the tumor mass at 1 hour after the injection whereas only very low levels of eribulin were detected in surrounding normal brain. Eribulin is an FDA-approved drug for refractory breast cancer and can be safely repositioned for treatment of glioblastoma patients. Thus, our results suggest that eribulin may serve as a novel therapeutic option for glioblastoma. Based on these data, an investigator-initiated registration-directed clinical trial to evaluate the safety and efficacy of eribulin in patients with recurrent GBM (UMIN000030359) has been initiated.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Encéfalo/diagnóstico por imagem , Furanos/administração & dosagem , Glioblastoma/tratamento farmacológico , Cetonas/administração & dosagem , Regiões Promotoras Genéticas/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Reposicionamento de Medicamentos , Feminino , Furanos/farmacologia , Glioblastoma/diagnóstico por imagem , Glioblastoma/genética , Humanos , Injeções Intraperitoneais , Cetonas/farmacologia , Camundongos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Telomerase/genética , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
7.
J Agric Food Chem ; 67(16): 4493-4504, 2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-30938528

RESUMO

Expression of sodium-iodide symporter (NIS) is stimulated by sterol-regulatory-element-binding transcription factors (SREBFs) in mammary epithelial MCF-7 cells. Because conjugated linoleic acid (CLA) isomers have been shown to inhibit transcriptional activity of SREBFs in the mammary gland, the hypothesis was tested that CLA isomers inhibit NIS expression induced by all- trans retinoic acid (ATRA) in MCF-7 cells through inhibiting SREBF activity. c9t11-CLA and t10c12-CLA decreased ATRA-induced NIS-mRNA expression from 1.00 (ATRA alone) to 0.80 ± 0.12 (200 µM c9t11-CLA, P < 0.05) and 0.62 ± 0.10 (200 µM t10c12-CLA, P < 0.05), NIS-protein expression from 1.00 (ATRA alone) to 0.77 ± 0.08 (200 µM c9t11-CLA, P < 0.05) and 0.63 ± 0.05 (200 µM t10c12-CLA, P < 0.05), and NIS-promoter activity from 1.00 (ATRA alone) to 0.74 ± 0.13 (200 µM c9t11-CLA, P < 0.05) and 0.76 ± 0.13 (200 µM t10c12-CLA, P < 0.05); however, c9t11-CLA and t10c12-CLA increased the mRNA levels of SREBF isoforms and their target genes. In contrast, the mRNA expression of peroxisome-proliferator-activated receptor γ (PPARG) was strongly induced by ATRA alone but decreased by CLA isomers from 1.00 (ATRA alone) to 0.80 ± 0.06 (200 µM c9t11-CLA, P < 0.05) and 0.86 ± 0.06 (200 µM t10c12-CLA, P < 0.05). Overexpression of PPARγ in MCF-7 cells increased basal NIS-promoter activity, and treatment with the PPARγ ligand troglitazone stimulated ATRA-induced NIS-promoter activity. In conclusion, the results suggest that CLA isomers exert their effect on the expression of NIS by decreasing PPARG expression in MCF-7 cells.


Assuntos
Células Epiteliais/efeitos dos fármacos , Ácidos Linoleicos Conjugados/farmacologia , Glândulas Mamárias Humanas/metabolismo , Simportadores/genética , Tretinoína/farmacologia , Células Epiteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Isomerismo , Células MCF-7 , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/efeitos dos fármacos , PPAR gama/genética , PPAR gama/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Iodeto de Sódio/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Simportadores/metabolismo
8.
Prep Biochem Biotechnol ; 49(5): 521-528, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31017522

RESUMO

Staphylococcus aureus, among other staphylococcal species, developed multidrug resistance and causes serious health risks that require complex treatments. Therefore, the development of novel and effective strategies to combat these bacteria has been gaining importance. Since Staphylococcus simulans lysostaphin is a peptidoglycan hydrolase effective against staphylococcal species, the enzyme has a significant potential for biotechnological applications. Despite promising results of lysostaphin as a bacteriocin capable of killing staphylococcal pathogens, it is still not widely used in healthcare settings due to its high production cost. In this study, medium engineering techniques were applied to improve the expression yield of recombinant lysostaphin in E. coli. A new effective inducible araBAD promoter system and different mediums were used to enhance lysostaphin production. Our results showed that the composition of autoinduction media enhanced the amount of lysostaphin production 5-fold with the highest level of active lysostaphin at 30 °C. The production cost of 1000 U of lysostaphin was determined as 4-fold lower than the previously proposed technologies. Therefore, the currently developed bench scale study has a great potential as a large-scale fermentation procedure to produce lysostaphin efficiently.


Assuntos
Proteínas de Bactérias/biossíntese , Meios de Cultura/metabolismo , Lisostafina/biossíntese , Engenharia Metabólica/métodos , Proteínas Recombinantes/biossíntese , Arabinose/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Meios de Cultura/química , Indução Enzimática/efeitos dos fármacos , Estabilidade Enzimática , Escherichia coli/genética , Fermentação , Lisostafina/isolamento & purificação , Engenharia Metabólica/economia , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Staphylococcus/química , Staphylococcus/metabolismo , Temperatura Ambiente , Fatores de Tempo
9.
Phytomedicine ; 58: 152879, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31005035

RESUMO

BACKGROUND: Anti-angiogenesis is an important strategy of psoriasis treatment, but the side effects of systemic agents remain difficult to overcome. Topical use of indigo naturalis ointment has been proved to improve the skin lesion of psoriasis effectively and safely and one of its major components, tryptanthrin, has been demonstrated to have anti-angiogenic effect. Apelin, which has been reported to act as an angiogenic factor that could stimulate the proliferation and migration of vascular endothelial cells and proved to be elevated in psoriasis patients, is a potential target of anti-angiogenic therapy. PURPOSE: We aim to find out if tryptanthrin works on the apelin pathway and study its anti-angiogenic mechanism. STUDY DESIGN: Human umbilical vein endothelial cells (HUVECs) were used as the in vitro model. METHODS: The effect of tryptanthrin on the expression of apelin and its receptor, APJ, was examined. The mRNA stability, promoter activity, and bioactivity of apelin, were also investigated. Migration and tube formation assay were used to evaluate the relationship between tryptanthrin and apelin. PD98059 and wortmannin were used to study the role of ERK1/2 MAPK and PI3K in apelin signaling pathway. RESULTS: We demonstrated that tryptanthrin could inhibit the expression of apelin, attenuated the stability of apelin mRNA, and significantly inhibited the apelin promoter activity. The addition of apelin-13 restored the suppression of tube formation and migration by tryptanthrin. Both PD98059 and wortmannin could down-regulate the apelin mRNA expression suggesting the important signaling role of ERK1/2 MAPK and PI3K in the gene expression of apelin. CONCLUSION: The anti-angiogenic effect of tryptanthrin was mediated by down-regulating apelin gene expression through suppression of promoter activity and decrease of mRNA stability in human vascular endothelial cells.


Assuntos
Inibidores da Angiogênese/farmacologia , Apelina/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Quinazolinas/farmacologia , RNA Mensageiro/metabolismo , Apelina/metabolismo , Receptores de Apelina/genética , Receptores de Apelina/metabolismo , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Meia-Vida , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Estabilidade de RNA , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Wortmanina/farmacologia
10.
Mol Biotechnol ; 61(6): 427-431, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30941576

RESUMO

Peroxisome proliferator-activated receptor gamma (PPARγ) is involved in the regulation of lipid and glucose homeostasis and inflammation. PPARγ expression level has been widely studied in multiple tissues; however, there are few reports of preceding attempts to produce full-length human PPARγ (hPPARγ) in cellular models, and generally, expression level is not known or measurable. We propose an alternative strategy to express recombinant hPPARγ1, using a transient transfection with an inducible Tet-On 3G system where target and reporter gene were cloned in the same open reading frame. We transiently co-transfected human embryonic kidney 293T (HEK293T) cells with pTRE-ZsGreen1-IRES2-hPPARγ1 and pCMV-TET3G for inducible expression of hPPARγ1. Relative expression of the transcript was evaluated by RT-qPCR 48 h after transfection, obtaining a high expression level of hPPARγ (530-fold change, p < 0.002) in co-transfected HEK293T cells in the presence of doxycycline (1 µg/mL); also a significantly increased production of the reporter protein ZsGreen1 (3.6-fold change, p < 0.05) was determined by fluorescence analysis. These data indicated that HEK293T cells were successfully co-transfected and it could be an alternative model for hPPARγ expression in vitro. Additionally, this model will help to validate the quantification of inducible hPPARγ expression in vivo models for future research.


Assuntos
Clonagem Molecular/métodos , Vetores Genéticos/metabolismo , PPAR gama/genética , Proteínas Recombinantes de Fusão/genética , Doxiciclina/farmacologia , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Vetores Genéticos/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Fases de Leitura Aberta , PPAR gama/biossíntese , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transfecção
11.
Bioengineered ; 10(1): 98-107, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31023186

RESUMO

Progranulin has multiple functions in several physiological and pathological processes, including embryonic development, wound repair, tumorigenesis, inflammation and neurodegeneration. To investigate the transcriptional regulation of the PGRN gene, a luciferase knock-in reporter system was established in HEK293 cells by integrating luciferase gene in the genome controlled by the endogenous PGRN promoter using CRISPR/Cas9. PCR results demonstrated the site-specific integration of the exogenous luciferase gene into the genome. To validate the novel luciferase knock-in system, a CRISPR/Cas9 transcription activation/repression system for the PGRN gene was constructed and applied to the knock-in system. In addition, phorbol ester (phorbol 12-myristate, 13-acetate), previously reported as activating the expression of PGRN, was applied to the system. The results indicated that luciferase activity was directly correlated with the activity of the PGRN endogenous promoter. This novel system will be a useful tool for investigating the transcriptional regulation of PGRN, and it has great potential in screening the drugs targeting PGRN.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Efeito Fundador , Técnicas de Introdução de Genes/métodos , Luciferases/genética , Progranulinas/genética , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Genoma Humano , Células HEK293 , Humanos , Luciferases/metabolismo , Progranulinas/agonistas , Progranulinas/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Genética/efeitos dos fármacos
12.
Cell Prolif ; 52(4): e12617, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31012173

RESUMO

OBJECTIVES: The roles and related mechanisms of six2 in regulating non-small cell lung cancer (NSCLC) cells progression are unclear. This work aimed to explore the roles of six2 in NSCLC cell stemness. MATERIALS AND METHODS: Kaplan-Meier plotter analysis was used to examine the correlation between six2 expression and the survival of NSCLC patients. Quantitative reverse transcription PCR and Western blot were performed to detect six2 expression in clinical samples. Moreover, transwell migration, tumour spheroid formation and in vivo tumour formation assays were used to examine the effects of six2 on NSCLC cell progression. Additionally, methylation analysis was carried out to measure E-cadherin methylation level in different cells. Finally, cell viability assay was performed to explore the effects of six2 on chemotherapeutic sensitivity of NSCLC cells. RESULTS: Lung cancer patients with a higher six2 expression level displayed a shorter overall survival. Six2 expression was higher in lung cancer tissues than in normal adjacent tissues. Additionally, six2 knockdown suppressed NSCLC cell stemness. Mechanistically, six2 overexpression inhibited epithelial marker E-cadherin expression via stimulating its promoter methylation. And E-cadherin knockdown rescued six2 knockdown-induced decrease of NSCLC cancer cell stemness. Notably, six2 knockdown enhanced cisplatin sensitivity in parental NSCLC cells and attenuated cisplatin resistance in cisplatin-resistant NSCLC cells. CONCLUSIONS: Our results suggest that six2 facilitates NSCLC cell stemness and attenuates chemotherapeutic sensitivity via suppressing E-cadherin expression.


Assuntos
Antígenos CD/genética , Caderinas/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Epigênese Genética/genética , Proteínas de Homeodomínio/genética , Neoplasias Pulmonares/genética , Proteínas do Tecido Nervoso/genética , Transcrição Genética/genética , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cisplatino/farmacologia , Progressão da Doença , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Masculino , Metilação/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Transcrição Genética/efeitos dos fármacos
13.
Pharmazie ; 74(3): 163-167, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30961683

RESUMO

The natural phytoalexin resveratrol (RES) has exhibited excellent anti-tumor effects on a variety of tumors including malignant melanoma. However, its specific mechanism of anti-melanoma needs to be further explored. It has been reported, that the expression of tumor suppressor gene RUNX3 was lost or substantially decreased in melanoma. Whether RES exerts its anti-tumor effect by regulating the expression of RUNX3 gene in melanoma is worthy of study. In the present study, we found the RUNX3 promoter is hypermethylated and the expression of RUNX3 mRNA and protein are absent in melanoma cells B16F10. After intervention with RES, promoter hypermethylation of RUNX3 in B16F10 cells could be significantly decreased and mRNA and protein expression of it was upregulated in a dose-dependent manner. We further investigated the effects of RES on B16F10 xenograft models. The intervention of RES and treatment of melanoma positive drug dacarbazine (DTIC) both could significantly inhibit tumor growth in xenograft mice, but only RES could upregulate the expression of RUNX3 mRNA and protein in peripheral blood and tumor tissues. Therefore, the upregulation of mRNA and protein expression of RUNX3 resulting from promoter demethylation might be one of the mechanisms of RES inhibiting melanoma. This research has revealed a novel mechanism for RES against melanoma from the epigenetic perspective, which is helpful to improve the understanding of the anti-tumor mechanism of RES and provide new insights for the treatment of melanoma.


Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core/genética , Desmetilação do DNA/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/genética , Resveratrol/farmacologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Feminino , Crescimento/efeitos dos fármacos , Xenoenxertos , Masculino , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/efeitos dos fármacos
14.
Chemosphere ; 227: 323-328, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30999172

RESUMO

In the present study, we investigated the association between methylation of DNA damage response-related genes such as cyclin-dependent kinase inhibitor (CDKN)2A, Ras association (RalGDS/AF-6) domain family member (RASSF)1A, O6-methylguanine DNA methyltransferase (MGMT), Kirsten rat sarcoma viral oncogene homolog (KRAS), and spleen-associated tyrosine kinase (SYK) and DNA damage in hepatocytes of rats following subchronic exposure to vinyl chloride (VC). Sixty-four healthy rats were randomly divided into three VC exposure groups (5, 25, and 125 mg/kg) and an untreated negative control group (n = 16 each). VC was administered by intraperitoneal injection every other day for a total of three times a week. Eight randomly selected rats from each group were sacrificed at the end of 6 and 12 weeks, and liver tissue was harvested for the comet assay and for assessment of DNA methylation level and mRNA expression of related genes by PCR. Overall methylation levels in the genome of hepatocytes in VC-exposed rats were higher than those in the control group at 6 and 12 weeks (P < 0.05), although no differences were observed with regarding to dose (P > 0.05). After 12 weeks of exposure, differences in the methylation of RASSF1A and MGMT promoter regions were observed between the high-dose group and other groups (P < 0.05), whereas no differences were observed for the KRAS, SYK, and CDKN2A promoters (P > 0.05). These results suggest that DNA damage and increased genome-wide methylation are biomarkers for VC exposure and that RASSF1A and MGMT promoter methylation is related to the carcinogenic mechanism of VC.


Assuntos
Dano ao DNA/genética , Metilação de DNA , Hepatócitos/efeitos dos fármacos , Cloreto de Vinil/toxicidade , Animais , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Metilases de Modificação do DNA/genética , Relação Dose-Resposta a Droga , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Fígado/patologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Ratos , Proteínas Supressoras de Tumor/genética
15.
Oncol Rep ; 41(6): 3464-3474, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31002343

RESUMO

The EF­hand calcium binding protein tescalcin (TESC) is highly expressed in various human and mouse cancer tissues and is therefore considered a potential oncogene. However, the underlying mechanism that governs TESC expression remains unclear. Emerging evidence suggests that TESC expression is under epigenetic regulation. In the present study, the relationship between the epigenetic modification and gene expression of TESC in gastric cancer was investigated. To evaluate the relationship between the methylation and expression of TESC in gastric cancer, the methylation status of CpG sites in the TESC promoter was analyzed using microarray with the Illumina Human Methylation27 BeadChip (HumanMethylation27_270596_v.1.2), gene profiles from the NCBI Dataset that revealed demethylated status were acquired, and real­time methylation­specific PCR (MSP) in gastric cancer cells was conducted. In the present study, it was demonstrated that the hypermethylation of TESC led to the downregulation of TESC mRNA/protein expression. In addition, 5­aza­2c­deoxycytidine (5'­aza­dC) restored TESC expression in the tested gastric cancer cells except for SNU­620 cells. ChIP assay further revealed that the methylation of the TESC promoter was associated with methyl­CpG binding domain protein (MBD)1, histone deacetylase (HDAC)2, and Oct­1 and that treatment with 5'­aza­dC facilitated the dissociation of MBD1, HDAC2, and Oct­1 from the promoter of TESC. Moreover, silencing of TESC increased MBD1 expression and decreased the H3K4me2/3 level, thereby causing transcriptional repression and suppression of cell survival in NCI­N87 cells; conversely, overexpression of TESC downregulated MBD1 expression and upregulated the H3K4me2 level associated with active transcription in SNU­638 cells. These results indicated that the differential expression of TESC via the modification status of the promoter and histone methylation controled cell survival in gastric cancer cells. Overall, the present study provided a novel therapeutic strategy for gastric cancer.


Assuntos
Azacitidina/farmacologia , Proteínas de Ligação ao Cálcio/genética , Metilação de DNA/genética , Neoplasias Gástricas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Código das Histonas/genética , Histona Desacetilase 2/genética , Humanos , Análise em Microsséries , Fator 1 de Transcrição de Octâmero/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Neoplasias Gástricas/patologia , Fatores de Transcrição/genética
16.
Biomed Res Int ; 2019: 5689613, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30931327

RESUMO

Sinapic acid (SA) modulates the nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling pathway in chondrocytes. In order to test the hypothesis that SA is protective against the development of osteoarthritis (OA), primary mouse chondrocytes were treated in vitro with SA and the promoter transactivation activity of heme oxygenase 1 (HO-1), nuclear translocation of Nrf2, and protein expression of HO-1 were assayed. To test the hypothesis in vivo, a destabilization of the medial meniscus (DMM) model was used to induce OA in the knees of mice and SA was delivered orally to the experimental group. The chondrocytes were harvested for further analysis. The expression of HO-1 was similarly upregulated in cartilage from both the experimental mice and human chondrocytes from osteoarthritic knees. SA was found to enhance the promoter transactivation activity of heme oxygenase 1 (HO-1) and increase the expression of Nrf2 and HO-1 in primary chondrocytes. Histopathologic scores showed that the damage induced by the DMM model was significantly lower in the SA treatment group. The addition of a HO-1 inhibitor with SA did not show additional benefit over SA alone in terms of cartilage degradation or histopathologic scores. The expression of TNF-α, IL-1ß, IL-6, MMP-1, MMP-3, MMP-13, ADAMTS4, and ADAMTS5 was significantly reduced both in vitro and in vivo by the presence of SA. Protein expressions of HO-1 and Nrf2 were substantially increased in knee cartilage of mice that received oral SA. Our results suggest that SA should be further explored as a preventative treatment for OA.


Assuntos
Ácidos Cumáricos/administração & dosagem , Heme Oxigenase-1/genética , Fator 2 Relacionado a NF-E2/genética , Osteoartrite do Joelho/tratamento farmacológico , Administração Oral , Idoso , Animais , Cartilagem/efeitos dos fármacos , Cartilagem/patologia , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Meniscos Tibiais/efeitos dos fármacos , Meniscos Tibiais/fisiopatologia , Camundongos , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/patologia , Regiões Promotoras Genéticas/efeitos dos fármacos
17.
Ann Hematol ; 98(6): 1383-1392, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30877373

RESUMO

Poly (ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that participates in the DNA repair of malignant cells, with various consequences on their survival. We have recently shown that PARP1 mRNA levels in the bone marrow of patients with myelodysplastic syndrome (MDS) are correlated to prognosis. To evaluate PARP1 as a biomarker of response to 5-azacytidine in patients with MDS, we measured PARP1 mRNA levels by a quantitative real-time PCR in diagnostic bone marrow samples of 77 patients with MDS treated with 5-azacytidine. Patients with higher PARP1 mRNA levels had a better response to 5-azacytidine per the IWG criteria (p = 0.006) and a longer median survival after 5-azacytidine initiation (p = 0.033). Multivariate analysis revealed that PARP1 mRNA level was the only factor affecting response to treatment and survival after treatment with 5-azacytidine. A next-generation sequencing for 40 genes of interest in MDS and quantification of the methylation levels of the PARP1 promoter were also carried out in a subset of samples (16 and 18 samples respectively). It is the first time that a single, easily measurable biomarker shows a clear correlation with response to treatment and survival in a patient population consisting of previously untreated patients with MDS homogeneously treated with 5-azacytidine. The fact that PARP1 is also a treatment target in several malignancies underscores the importance of our finding for the potential use of PARP1 inhibitors in MDS.


Assuntos
Antimetabólitos/uso terapêutico , Azacitidina/uso terapêutico , Medula Óssea/química , Síndromes Mielodisplásicas/tratamento farmacológico , Poli(ADP-Ribose) Polimerase-1/biossíntese , RNA Mensageiro/análise , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos/efeitos adversos , Azacitidina/efeitos adversos , Biomarcadores , Dano ao DNA , Metilação de DNA/efeitos dos fármacos , Reparo do DNA , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Prognóstico , Regiões Promotoras Genéticas/efeitos dos fármacos , Modelos de Riscos Proporcionais , Regulação para Cima/efeitos dos fármacos
18.
J Agric Food Chem ; 67(17): 4808-4816, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30888162

RESUMO

Cellular senescence is the state of irreversible cell cycle arrest that provides a blockade during oncogenic transformation and tumor development. Avenanthramide A (AVN A) is an active ingredient exclusively extracted from oats, which possesses antioxidant, anti-inflammatory, and anticancer activities. However, the underlying mechanism(s) of AVN A in the prevention of cancer progression remains unclear. In the current study, we revealed that AVN A notably attenuated tumor formation in an azoxymethane/dextran sulfate sodium (AOM/DSS) mouse model. AVN A treatment triggered cellular senescence in human colon cancer cells, evidenced by enlarging cellular size, upregulating ß-galactosidase activity, γ-H2AX positive staining, and G1 phase arrest. Moreover, AVN A treatment significantly increased the expression of miR-129-3p, which markedly repressed the E3 ubiquitin ligase Pirh2 and two other targets, IGF2BP3 and CDK6. The Pirh2 silencing by miR-129-3p led to a significant increase in protein levels of p53 and its downstream target p21, which subsequently induced cell senescence. Taken together, our data indicate that miR-129-3p/Pirh2/p53 is a critical signaling pathway in AVN A induced cellular senescence and AVN A could be a potential chemopreventive strategy for cancer treatment.


Assuntos
Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , MicroRNAs/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , ortoaminobenzoatos/administração & dosagem , Animais , Ciclo Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/fisiopatologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genética
19.
J Biosci ; 44(1)2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30837363

RESUMO

Wilms tumor 1 (WT1) has long been overexpressed in acute myeloid leukemia and has a prognostic value in its diagnosis. Lately, the formation of G-quadruplexes in oncogenic promoters like (WT1) has been widely investigated since stabilization of these structures leads to transcriptional inhibition of the oncogene. Daunorubicin and mitoxantrone considered as crucial components of almost all standard acute myeloid leukemia induction regimens. Herein we have proposed a probable molecular mechanism of action through which the drugs may stabilize (WT1) promoter G-quadruplexes. Differential pulse voltammetry, circular dichroism, and polyacrylamide gel electrophoresis, electrophoretic mobility shifts assay, polymerase chain reaction (PCR) stop assays, and quantitative RT-PCR were performed in order to better understanding the nature of interactions between the drugs and G-quadruplexes. Data revealed that both drugs had potential to stabilize G-quadruplexes and down-regulate WT1 transcription but daunorubicin exposed more silencing impact. The results illustrated the therapeutic association of these two commercial FDA-approved drugs in (WT1) transcriptional down-regulation. Since (WT1) has known as a transcriptional regulator of at least 137 target genes, so the new data are significant for the development of new approaches to regulating WT1 and other target genes by employing special drugs in cancer treatment.


Assuntos
Daunorrubicina/farmacologia , Leucemia Mieloide Aguda/genética , Mitoxantrona/farmacologia , Proteínas WT1/genética , Linhagem Celular Tumoral , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Ensaio de Desvio de Mobilidade Eletroforética , Quadruplex G/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Regiões Promotoras Genéticas/efeitos dos fármacos
20.
Clin Epigenetics ; 11(1): 46, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30867047

RESUMO

BACKGROUND: Both SOX2 promoter methylation and air pollution have been associated with lung cancer risk. However, little has been done to assess SOX2 promoter methylation in individuals living in air pollution areas. The aim of this study was to investigate SOX2 promoter methylation in non-smoking Taiwanese adults living in areas with different levels of air pollution especially particulate matter with diameter < 2.5 µm (PM2.5). METHODS: A total of 1142 individuals aged 30-70 years were recruited. Data on SOX2 methylation, residence, age, and exposure to second-hand smoke (SHS) among others were extracted from the Taiwan Biobank dataset (2008-2015). After excluding former and current smokers, alongside those with incomplete information, a total of 461 non-smokers comprising 176 men and 285 women were included in the study. Participants' residences were grouped under northern and central/southern areas because air pollution (PM2.5) is lower in northern compared to central and southern areas. RESULTS: The methylation levels in men (0.16310 ± 0.01230) and women (0.15740 ± 0.01240) were significantly different (P < .0001). In both sexes, the SOX2 promoter region was shown to be significantly hypermethylated in central and southern areas compared with the northern areas. The regression coefficient (ß) was 0.00331 (P = 0.0257) in men and 0.00514 (P < .0001) in women. CONCLUSION: SOX2 was significantly hypermethylated in both men and women residing in central and southern areas. The consistency in the results for both sexes shows that SOX2 promoter methylation could serve as a potential biomarker for industrial air pollution exposure. Moreover, it might reflect predisposition to cancer. Hence, healthy non-smokers at precancerous stages who have not been clinically diagnosed could be identified.


Assuntos
Metilação de DNA , Fatores de Transcrição SOXB1/genética , Poluição por Fumaça de Tabaco/análise , Adulto , Idoso , Poluição do Ar/efeitos adversos , Poluição do Ar/análise , Metilação de DNA/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/efeitos dos fármacos , Análise de Regressão , Taiwan , Poluição por Fumaça de Tabaco/efeitos adversos
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