RESUMO
5-Aza-2'-deoxycytidine (5-Aza-CdR) is currently acknowledged as a demethylation drug, and causes a certain degree of demethylation in a variety of cancer cells, including pancreatic cancer cells. Emodin, a traditional Chinese medicine (TCM), is an effective monomer extracted from rhubarb and has been reported to exhibit antitumor activity in different manners in pancreatic cancer. In the present study, we examined whether emodin caused demethylation and increased the demethylation of three tumor-suppressor genes P16, RASSF1A and ppENK with a high degree of methylation in pancreatic cancer when combined with 5-Aza-CdR. Our research showed that emodin inhibited the growth of pancreatic cancer Panc-1 cells in a dose- and time-dependent manner. Dot-blot results showed that emodin combined with 5-Aza-CdR significantly suppressed the expression of genome 5mC in PANC-1 cells. In order to verify the effect of methylation, methylation-specific PCR (MSP) and bisulfite genomic sequencing PCR (BSP) combined with TA were selected for the cloning and sequencing. Results of MSP and BSP confirmed that emodin caused faint demethylation, and 5-Aza-CdR had a certain degree of demethylation. When emodin was combined with 5-Aza-CdR, the demethylation was more significant. At the same time, fluorescent quantitative PCR and western blot analysis results confirmed that when emodin was combined with 5-Aza-CdR, the expression levels of P16, RASSF1A and ppENK were increased more significantly compared to either treatment alone. In contrast, the expression levels of DNA methyltransferase 1 (DNMT1) and DNMT3a were more significantly reduced with the combination treatment than the control or either agent alone, further proving that emodin in combination with 5-Aza-CdR enhanced the demethylation effect of 5-Aza-CdR by reducing the expression of methyltransferases. In conclusion, the present study confirmed that emodin in combination with 5-Aza-CdR enhanced the demethylation by 5-Aza-CdR of tumor-suppressor genes p16, RASSF1A and ppENK by reducing the expression of methyltransferases DNMT1 and DNMT3a.
Assuntos
Azacitidina/análogos & derivados , Metilação de DNA/efeitos dos fármacos , Emodina/farmacologia , Encefalinas/genética , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Precursores de Proteínas/genética , Proteínas Supressoras de Tumor/genética , Azacitidina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina , Decitabina , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Regiões Promotoras Genéticas/efeitos dos fármacos , Análise de Sequência de DNA/métodosRESUMO
Emodin, a natural anthraquinone derivative isolated from Rheum palmatum, has been reported to inhibit the growth of pancreatic cancer cells through different modes of action; yet, the detailed mechanism remains unclear. In the present study, we hypothesized that emodin exerts its antitumor effect by participating in the regulation of the DNA methylation level. Our research showed that emodin inhibited the growth of pancreatic cancer PANC-1 cells in a dose- and time-dependent manner. Dot-blot results showed that 40 µM emodin significantly inhibited genomic 5 mC expression in the PANC-1 cells, and mRNA-Seq showed that different concentrations of emodin could alter the gene expression profile in the PANC-1 cells. BSP confirmed that the methylation levels of P16, RASSF1A and ppENK were decreased, while concomitantly the unmethylated status was increased. RT-PCR and western blotting results confirmed that the low expression or absence of expression of mRNA and protein in the PANC-1 cells was re-expressed following treatment with emodin. In conclusion, our study for the first time suggests that emodin inhibits pancreatic cancer cell growth, which may be related to the demethylation of tumor-suppressor genes. The related mechanism may be through the inhibition of methyltransferase expression.
Assuntos
Encefalinas/genética , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Precursores de Proteínas/genética , Proteínas Supressoras de Tumor/genética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina , Metilação de DNA/efeitos dos fármacos , Emodina/administração & dosagem , Encefalinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , NF-kappa B/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Precursores de Proteínas/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
The logical range of laser power density and optimum laser power density were explored for multi-element analysis of pulverized coal flow with laser-induced breakdown spectroscopy in the present paper. The range of laser energy was chosen from 20 to 160 mJ in the experiment. Pulverized coal less than 200 microm in diameter of particles fell freely through feeder outlet and the rate of flow was controlled by screw feeder. Emissions were collected with pulse laser at 1 064 nm focusing on pulverized coal flow and plasma was generated. The intensity and cause of fluctuation of emission spectra at various laser energy levels were studied. A suitable range of laser power density is from 14.4 to 34.4 GW x cm(-2), and the optimum laser power density is 19.5 GW x cm(-2) for the determination of pulverized coal flow with LIBS.
