BACKGROUND Hepatic stellate cell(HSC)activation is key to liver fibrosis.Targeting DNA methylation shows promise.Zebularine,a methylation inhibitor,may suppress HSC activation via the calcineurin(CaN)/NFAT3 pathway.Ma...BACKGROUND Hepatic stellate cell(HSC)activation is key to liver fibrosis.Targeting DNA methylation shows promise.Zebularine,a methylation inhibitor,may suppress HSC activation via the calcineurin(CaN)/NFAT3 pathway.Magnetic resonance imaging(MRI)is a noninvasive tool for evaluating liver fibrosis evaluation tool,but multiparametric MRI for zebularine’s effects in liver fibrosis mouse models has not been studied.AIM To clarify the anti-fibrosis mechanism and MRI-evaluated efficacy of zebularine.METHODS In vitro,transforming growth factor(TGF)-β1-stimulated human HSCs(LX-2)were treated with zebularine.α-smooth muscle actin,fibrotic and anti-fibrotic gene levels,and regulator of calcineurin1(RCAN1)regulation were measured.In vivo,carbon tetrachloride(CCl_(4))-induced liver fibrosis in mice was treated with zebularine,and fibrosis was evaluated using various biochemical,histopathological,and MRI methods.RESULTS Zebularine upregulated RCAN1.4 protein(P<0.01)and inhibited the CaN/NFAT3 pathway(P<0.05).In HSCs,TGF-β1 reduced anti-fibrotic gene massage RNA(mRNA)and increased fibrotic mRNA(P<0.05),whereas zebularine had the opposite effects(P<0.01,P<0.05).CCl4-treated mice exhibited increases in various fibrosis-related indices,all of which were reversed by zebularine treatment(P<0.05).CONCLUSION Zebularine may reduce LX-2 activation and extracellular matrix deposition via RCAN1.4 and CaN/NFAT3 path-ways.Multiparametric MRI can assess its efficacy,suggesting zebularine’s potential as a liver fibrosis treatment.展开更多
Purpose: MCF-7 (ER+, WTP53) and MDA-MB-231 (ER Met, Mutant P53) Caffeic Acid Phenethyl Ester (CAPE) and DNA Methyl Transferase Inhibitor (DNMTi) in breast cancer cell lines of Zebularine (ZEB) single and combined appl...Purpose: MCF-7 (ER+, WTP53) and MDA-MB-231 (ER Met, Mutant P53) Caffeic Acid Phenethyl Ester (CAPE) and DNA Methyl Transferase Inhibitor (DNMTi) in breast cancer cell lines of Zebularine (ZEB) single and combined application of TP53, caspase-9, caspase 8 and caspase-3 genes as a result of the use of single and combined drug methylation profiles are aimed to be evaluated by specific PCR method. Material-Metods: In the MCF-7 and MDA-MB-231 breast cancer cell lines, MTT test and survival analysis were performed as a result of single and combined application of CAPE and Zebularine and Methylation Specific PCR was performed to examine the methylation of caspase-3, caspase-8, caspase-9 and TP53 genes. Results: According to the results of 24-hour drug administration, the IC50 for the MCF-7 cell line was determined as 200 μM, for CAPE 40 μM and for the combined values of 50 μM ZEB + 5 μM CAPE. The effects of caspase-3, caspase-8, caspase-9 and TP53 genes on the methylation level of ZEB, CAPE and ZEB + CAPE drug combination were determined by using bisulfite modified DNAs in MCF-7 and MDA-MB-231 cell lines. Discussion: In the MCF-7 cell line, the 120 μM ZEB viability rate was 51%, and the viability of 80 μM ZEB MDA-MB-231 breast cancer cells decreased by 59.7%. After 20 μM CAPE, viability in MCF-7 cells decreased by 31% in 120 μM CAPE and MDA-MB-231 cells decreased by 41%. The viability with 40 μM CAPE decreased by 19% in MDA-MB-231 cells. It was found that 20 μM CAPE concentration was associated with TP53 methylation in MCF-7 cell lines. The 80 μM ZEB concentration was found to be closely related to the unmethylated status of the TP53 gene. These results obtained with 50 μM ZEB + 5 μM CAPE application were found to be related to the methylated-unmetylated status of the TP53 gene in half (50%). For the caspase-9 gene of MDA-MB-231 cells, 80 μM ZEB concentration was found to be associated with unmetylated status. The effective use of drugs with low concentrations of the drug dose provides a more appropriate approach in terms of treatment.展开更多
The aim of this study is to investigate the anti-proliferative effects of Zebularine on caspase-3 and 9 genes by methylation and protein expression on SKBR3 cells. The SKBR3 cells were treated with Zebularine at diffe...The aim of this study is to investigate the anti-proliferative effects of Zebularine on caspase-3 and 9 genes by methylation and protein expression on SKBR3 cells. The SKBR3 cells were treated with Zebularine at different concentrations (0 - 140 μM) for 24 - 96 hours and the effects on the cell viability were shown. The effects on the cell migration and cell transformation were also demonstrated at the specified dose (IC50 = 40 μM). At the same time, the HRM method and western-blot analysis were used to understand the effects in the apoptotic mechanism. According to the obtained results, it was observed that Zebularine significantly decreased the cell proliferation, cell migration and the cell growth (p < 0.001). As a result of the methylation analyzes performed on SKBR-3 cells at the applied dose, it was observed that Zebularine caused a decrease in both caspase-3 and 9 methylation rates at the 72nd hour. However, both reductions in the methylation levels didn’t cause a significant change (p > 0.05). At the same time, it was detected by the western-blot analyses that there was a time dependent increase in caspase-3 genes while a decrease was detected in the caspase-9 gene in progress of time. The obtained results showed that the methylation changes occurring in the caspase genes weren’t related to the protein levels. According to these results, supportive results are obtained showing that the Zebularine can be used in chemotherapy and that this study is unique since it is the first study in the literature intending to investigate the effects of Zebularine on the SKBR3 cells.展开更多
基金Supported by the Health Research Foundation of Hunan Provincial Health Commission,No.W20243192Natural Science Foundation of Changsha,No.kq2403086+1 种基金National Natural Science Foundation of China,No.81571784Hunan Provincial Health Commission Hunan Provincial High-level Health Talent Major Scientific Research Project,No.R2023022.
文摘BACKGROUND Hepatic stellate cell(HSC)activation is key to liver fibrosis.Targeting DNA methylation shows promise.Zebularine,a methylation inhibitor,may suppress HSC activation via the calcineurin(CaN)/NFAT3 pathway.Magnetic resonance imaging(MRI)is a noninvasive tool for evaluating liver fibrosis evaluation tool,but multiparametric MRI for zebularine’s effects in liver fibrosis mouse models has not been studied.AIM To clarify the anti-fibrosis mechanism and MRI-evaluated efficacy of zebularine.METHODS In vitro,transforming growth factor(TGF)-β1-stimulated human HSCs(LX-2)were treated with zebularine.α-smooth muscle actin,fibrotic and anti-fibrotic gene levels,and regulator of calcineurin1(RCAN1)regulation were measured.In vivo,carbon tetrachloride(CCl_(4))-induced liver fibrosis in mice was treated with zebularine,and fibrosis was evaluated using various biochemical,histopathological,and MRI methods.RESULTS Zebularine upregulated RCAN1.4 protein(P<0.01)and inhibited the CaN/NFAT3 pathway(P<0.05).In HSCs,TGF-β1 reduced anti-fibrotic gene massage RNA(mRNA)and increased fibrotic mRNA(P<0.05),whereas zebularine had the opposite effects(P<0.01,P<0.05).CCl4-treated mice exhibited increases in various fibrosis-related indices,all of which were reversed by zebularine treatment(P<0.05).CONCLUSION Zebularine may reduce LX-2 activation and extracellular matrix deposition via RCAN1.4 and CaN/NFAT3 path-ways.Multiparametric MRI can assess its efficacy,suggesting zebularine’s potential as a liver fibrosis treatment.
文摘Purpose: MCF-7 (ER+, WTP53) and MDA-MB-231 (ER Met, Mutant P53) Caffeic Acid Phenethyl Ester (CAPE) and DNA Methyl Transferase Inhibitor (DNMTi) in breast cancer cell lines of Zebularine (ZEB) single and combined application of TP53, caspase-9, caspase 8 and caspase-3 genes as a result of the use of single and combined drug methylation profiles are aimed to be evaluated by specific PCR method. Material-Metods: In the MCF-7 and MDA-MB-231 breast cancer cell lines, MTT test and survival analysis were performed as a result of single and combined application of CAPE and Zebularine and Methylation Specific PCR was performed to examine the methylation of caspase-3, caspase-8, caspase-9 and TP53 genes. Results: According to the results of 24-hour drug administration, the IC50 for the MCF-7 cell line was determined as 200 μM, for CAPE 40 μM and for the combined values of 50 μM ZEB + 5 μM CAPE. The effects of caspase-3, caspase-8, caspase-9 and TP53 genes on the methylation level of ZEB, CAPE and ZEB + CAPE drug combination were determined by using bisulfite modified DNAs in MCF-7 and MDA-MB-231 cell lines. Discussion: In the MCF-7 cell line, the 120 μM ZEB viability rate was 51%, and the viability of 80 μM ZEB MDA-MB-231 breast cancer cells decreased by 59.7%. After 20 μM CAPE, viability in MCF-7 cells decreased by 31% in 120 μM CAPE and MDA-MB-231 cells decreased by 41%. The viability with 40 μM CAPE decreased by 19% in MDA-MB-231 cells. It was found that 20 μM CAPE concentration was associated with TP53 methylation in MCF-7 cell lines. The 80 μM ZEB concentration was found to be closely related to the unmethylated status of the TP53 gene. These results obtained with 50 μM ZEB + 5 μM CAPE application were found to be related to the methylated-unmetylated status of the TP53 gene in half (50%). For the caspase-9 gene of MDA-MB-231 cells, 80 μM ZEB concentration was found to be associated with unmetylated status. The effective use of drugs with low concentrations of the drug dose provides a more appropriate approach in terms of treatment.
文摘The aim of this study is to investigate the anti-proliferative effects of Zebularine on caspase-3 and 9 genes by methylation and protein expression on SKBR3 cells. The SKBR3 cells were treated with Zebularine at different concentrations (0 - 140 μM) for 24 - 96 hours and the effects on the cell viability were shown. The effects on the cell migration and cell transformation were also demonstrated at the specified dose (IC50 = 40 μM). At the same time, the HRM method and western-blot analysis were used to understand the effects in the apoptotic mechanism. According to the obtained results, it was observed that Zebularine significantly decreased the cell proliferation, cell migration and the cell growth (p < 0.001). As a result of the methylation analyzes performed on SKBR-3 cells at the applied dose, it was observed that Zebularine caused a decrease in both caspase-3 and 9 methylation rates at the 72nd hour. However, both reductions in the methylation levels didn’t cause a significant change (p > 0.05). At the same time, it was detected by the western-blot analyses that there was a time dependent increase in caspase-3 genes while a decrease was detected in the caspase-9 gene in progress of time. The obtained results showed that the methylation changes occurring in the caspase genes weren’t related to the protein levels. According to these results, supportive results are obtained showing that the Zebularine can be used in chemotherapy and that this study is unique since it is the first study in the literature intending to investigate the effects of Zebularine on the SKBR3 cells.
