ABSTRACT
To explore the effect and mechanisms of demethylation drug zebularine on esophageal cancer cells apoptosis, ECA109 cells and KYSE170 cells were treated with zebularine at different concentrations (25, 50, 100, 200, and 400 μmol·L-1). The cell viability was measured by CCK-8. Flow cytometry was used to detect the cell apoptosis rate, Western blot was performed to determine the expression of apoptosis protein (Bcl-2, Bax, cleaved-caspase-3, and cleaved-PARP) and Wnt signal pathway molecules (β-catenin, cyclin D1, and c-Myc), real-time quantitative PCR was used to detect the expression level of negative regulatory genes of Wnt signaling pathway, methylation specific PCR (MSP) was used to detect the methylation status of secreted frizzled related protein 2 (SFRP2) and dickkopf 3 (Dkk3) genes. After knockdown of SFRP2 and Dkk3, the effect of zebularine on apoptosis was detected. The studies showed that zebularine could inhibit the activity of ECA109 and KYSE170 cells in a dose-dependent and time-dependent manner; zebularine could induce cell apoptosis, down-regulate the expression of Bcl-2 protein, up-regulate the expression of Bax, cleaved-caspase-3, and cleaved-PARP protein, and inhibit the expression of β-catenin, cyclin D1, and c-Myc protein (P < 0.05); the mRNA expression levels of Dkk3 and SFRP2 were significantly up-regulated by zebularine, while the methylation levels of SFRP2 and Dkk3 promoters were decreased; knockdown of SFRP2 and Dkk3 could reduce the apoptosis induced by zebularine. In summary, zebularine could reduce the methylation level of SFRP2 and Dkk3 gene promoter, promote the expression of SFRP2 and Dkk3 gene, and then induce the apoptosis of esophageal cancer cells by inhibiting Wnt/β-catenin signaling pathway.
ABSTRACT
Abstract?AlM: To investigate the effect of zebularine ( Zeb ) loaded Poly ( ethylene glycol ) - block - poly ( ε -caprolactone) methyl ether ( MePEG-PCL) nanoparticles ( NPs) on the viability, attachment, and apoptosis of in vitro cultured lens epithelial cells ( LECs) .?METHODS: In vitro cultured infant human lens tissue HLE B-3 immortalized cells were distributed randomly divided into six groups. Each group was administered with free Zeb 50μmol/L ( ZebF1 group ) , 100μmol/L ( ZebF2 group) , Zeb -loaded MePEG-PCL NPs 50μmol/L ( ZebNP1 group) , Zeb -loaded MePEG-PCL NPs 100μmol/L ( ZebNP2 group) , MePEG-PCL empty NPs( NPs group) or blank medium (group C) respectively. A tetrazolium dye assay ( MTT) test and modified MTT test were performed to determine cell viability and cell attachment. DNA ladder was used to detect the cell apoptosis.?RESULTS: Determined by MTT colorimetric method:Cell proliferation rate of LECs were suppressed by all Zeb administration groups in a concentration-time dependent manner (P ZebNP1>ZebF2 (P<0. 05).?CONCLUSlON: Zeb loaded MePEG-PCL NPs had better effect on suppressing the viability and attachment of in vitro cultured LECs than the free Zeb groups , as well as enhancing the apoptosis.