AIM： To identify the relationship between DNA hyper- methylation and histone modification at a hyperme- thylated, silenced tumor suppressor gene promoter in human gastric cancer cell lines and to elucidate whether al...AIM： To identify the relationship between DNA hyper- methylation and histone modification at a hyperme- thylated, silenced tumor suppressor gene promoter in human gastric cancer cell lines and to elucidate whether alteration of DNA methylation could affect histone modification. METHODS： We used chromatin immunoprecipitation （CHIP） assay to assess the status of histone acetylation and methylation in promoter regions of the p16 and rnutL homolog 1 （MLH1） genes in 2 gastric cancer cell lines, SGC-7901 and MGC-803. We used methylation- specific PCR （MSP） to evaluate the effect of 5-Aza-2＇- deoxycytidine （5-Aza-dC）, trichostatin A （TSA） or their combination treatment on DNA methylation status. We used RT-PCR to determine whether alterations of histone modification status after 5-Aza-dC and TSA treatment are reflected in gene expression. RESULTS： For thep16 and MLH1 genes in two cell lines, silenced loci associated with DNA hypermethylation were characterized by histone H3-K9 hypoacetylation and hypermethylation and histone H3-K4 hypomethylation. Treatment with TSA resulted in moderately increased histone H3-K9 acetylation at the silenced loci with no effect on histone H3-K9 methylation and minimal effects on gene expression. In contrast, treatment with 5-Aza- dC rapidly reduced histone H3-K9 methylation at the silenced loci and resulted in reactivation of the two genes. Combined treatment with 5-Aza-dC and TSA was synergistic in reactivating gene expression at the loci showing DNA hypermethylation. Similarly, histone H3-K4 methylation was not affected alter TSA treatment, andincreased moderately at the silenced loci after 5-Aza-dC treatment. CONCLUSION： Hypermethylation of DNA in promoter CpG islands is related to transcriptional silencing of tumor suppressor genes. Histone H3-K9 methylation in different regions of the promoters studied correlates with DNA methylation status of each gene in gastric cancer cells. However, histone H3-K9 acetylation and H3-K4 methylation inversely correlate 展开更多
Background Histone deacetylase inhibitors （HDACIs） have been reported to induce apoptosis in cancer cells. The effects of trichostatin A （TSA） on gastric cancer cells have not been well characterized. This study w...Background Histone deacetylase inhibitors （HDACIs） have been reported to induce apoptosis in cancer cells. The effects of trichostatin A （TSA） on gastric cancer cells have not been well characterized. This study was aimed to explore the effects and mechanisms of TSA on human gastric cancer SGC-7901 cells. Methods The cells were treated with TSA and analyzed by cell proliferation assay, Western blot, TUNEL assay, flow cytometry by fluorescein isothiocyanate （FITC） conjugated with Annexin V and PI staining, immunofluorescence analysis, analysis of subcellular fractionation, gene chips and real time polymerase chain reaction （PCR）. Results TSA could inhibit cell growth and induced apoptosis in gastric cancer SGC-7901 cells through the regulation of apoptosis-related genes, such as Bcl-2, Bax and survivin. Further study indicated that the pan-caspase inhibitor z-VAD-fmk did not inhibit the apoptosis induced by TSA, and we did not observe the cleavage of poly ADP ribose polymerase （PARP） after TSA treatment too. In addition, apoptosis inducing factor （AIF） and EndoG were found to translocate from mitochondria to nucleus in the immunofluorescence assay and the Western analysis of subcellular fractionation confirmed the result of immunofluorescence assay. Conclusions The apoptosis induced by TSA in gastric cancer SGC-7901 cells involves a caspase-independent pathway.展开更多
背景与目的:腺病毒感染依赖于靶细胞表面柯萨奇-腺病毒受体(Coxsackie and adenovirus receptor,CAR)的表达,肿瘤细胞CAR表达的缺失和下调是造成腺病毒为载体基因治疗效果不佳的根本原因。本研究通过观察组氨酸去乙酰化酶(histone deace...背景与目的:腺病毒感染依赖于靶细胞表面柯萨奇-腺病毒受体(Coxsackie and adenovirus receptor,CAR)的表达,肿瘤细胞CAR表达的缺失和下调是造成腺病毒为载体基因治疗效果不佳的根本原因。本研究通过观察组氨酸去乙酰化酶(histone deacetylase,HDAC)抑制剂TrichostatinA(TSA)对卵巢癌细胞株A2780CAR表达水平的影响,探讨HDAC抑制剂在腺病毒载体基因治疗中应用的可能性。方法:分别在TSA作用A2780细胞前后检测CARmRNA和蛋白水平的表达,同时采用流式细胞术测定病毒转染效率,MTT实验评价腺病毒携带的胸苷激酶(ADV/TK)的体外抗瘤效应。结果:TSA处理后,A2780细胞内CARmRNA和蛋白表达明显增高。通过流式细胞仪分析GFP/ADV转染后GFP的表达发现,未处理组细胞转染率为(1.24±0.14)%;5nmol/L和100nmol/LTSA处理48h后,细胞的转染效率分别为(7.58±0.32)%和(7.94±0.28)%。MTT结果表明,ADV/TK介导的体外杀伤作用在5nmol/L和100nmol/LTSA处理组较未处理组增强了4～10倍。结论:TSA可以增强针对卵巢癌细胞的腺病毒基因转染效率,为增强卵巢癌基因治疗效果提供可能的方法。展开更多
The secondary laticifer, a specific tissue in the secondary phloem of rubber tree, is differentiated from the vascular cambia. The number of the secondary laticifer in the trunk bark of rubber tree is positively corre...The secondary laticifer, a specific tissue in the secondary phloem of rubber tree, is differentiated from the vascular cambia. The number of the secondary laticifer in the trunk bark of rubber tree is positively correlated with rubber yield. Although jasmonates have been demonstrated to be crucial in the regulation of secondary laticifer differentiation, the mechanism for the jasmonate-induced secondary laticifer differentiation remains to be elucidated.By using an experimental morphological technique, the present study revealed that trichostatin A(TSA), an inhibitor of histone deacetylation, could induce the secondary laticifer differentiation in a concentrationdependent manner. The results suggest that histone acetylation is essential for the secondary laticifer differentiation in rubber tree.展开更多
Background Histone deacetylase （HDAC） inhibitors are a group of small chemical molecules that inhibit histone deacetylase. At cell level, HDAC inhibitors have multiple biological effects such as cell cycle arrest, a...Background Histone deacetylase （HDAC） inhibitors are a group of small chemical molecules that inhibit histone deacetylase. At cell level, HDAC inhibitors have multiple biological effects such as cell cycle arrest, apoptosis, cell differentiation and auotophagy. At molecular level, HDAC inhibitors cause histone and nonhistone acetylation and induce gene expression. HDAC inhibitors are widely used in cancer therapy because of its function of inducing apoptosis. However, the mechanisms of apoptosis effect are not fully understood. TSA is a classical HDAC inhibitor and widely used in epigenetic and anti-cancer research. In this study, we selected Trichostatin A （TSA） to investigate the mechanisms of HDAC inhibitors apoptotic effect on cancer cells. Methods Cervical cancer cell lines such as Hela, Caski and normal human keratinocyte line HaCaT were treated with various concentrations of TSA. Crystal violent assay and 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay were performed to determine cell number. PARP cleavage and FITC-AnexinV were performed to determine apoptosis. DNA-methyltransferase （DNMT）I, DNMT3A and DNMT3B were determined by regular PCR, qPCR and Western Blotting. Small interfering RNA （SiRNAi） was used to knock down DNMT3B. Results HDAC inhibitors only induce cervical cancer cell apoptosis. At 1 Iumol/L of TSA, 86% of Hela cell and 76% of Caski went apoptosis. For normal cells, HDAC inhibitors have no cytotoxic effect at therapeutic dosage, （90.0＋8.4）% of normal cell survive after treated with 1 IJmol/L of TSA. We compared 1 pmol/L group with untreated control with t-test. There was no significance between 1 pmol/L group and untreated control for normal cell （P 〉0.05）. HDAC inhibitors decreased DNMT3B in cancer cell but not in normal cell. Manually knock-down of DNMT3B induced Hela and Caski cell apoptosis. More than 99% of Hela and Caski cell went apoptosis after deprived of DNMT3B. Conclusions DNMT3B was essential to cervical cancer cell su展开更多
AIM:To gain molecular insights into the action of the histone deacetylase inhibitor(HDACI) trichostatin-A(TSA) in pancreatic cancer(PC) cells.METHODS:Three PC cell lines,BxPC-3,AsPC-1 and CAPAN-1,were treated with var...AIM:To gain molecular insights into the action of the histone deacetylase inhibitor(HDACI) trichostatin-A(TSA) in pancreatic cancer(PC) cells.METHODS:Three PC cell lines,BxPC-3,AsPC-1 and CAPAN-1,were treated with various concentrations of TSA for def ined periods of time.DNA synthesis was assessed by measuring the incorporation of 5-bromo-2'deoxyuridine.Gene expression at the level of mRNA was quantif ied by real-time polymerase chain reaction.Expression and phosphorylation of proteins was monitored by immunoblotting,applying an infrared imaging technology.To study the role of p38 MAP kinase,the specif ic enzyme inhibitor SB202190 and an inactive control substance,SB202474,were employed.RESULTS:TSA most eff iciently inhibited BrdU incorporation in BxPC-3 cells,while CAPAN-1 cells displayed the lowest and AsPC-1 cells an intermediate sensitivity.The biological response of the cell lines correlated with the increase of histone H3 acetylation after TSA application.In BxPC-3 cells(which are wild-type for KRAS),TSA strongly inhibited phosphorylation of ERK 1/2 and AKT.In contrast,activities of ERK and AKT in AsPC-1 and CAPAN-1 cells(both expressing oncogenic KRAS) were not or were only modestly affected by TSA treatment.In all three cell lines,but most pronounced in BxPC-3 cells,TSA exposure induced an activation of the MAP kinase p38.Inhibition of p38 by SB202190 slightly but signif icantly diminished the antiproliferative effect of TSA in BxPC-3 cells.Interestingly,only BxPC-3 cells responded to TSA treatment by a signif icant increase of the mRNA levels of bax,a pro-apoptotic member of the BCL gene family.Finally,in BxPC-3 and AsPC-1 cells,but not in the cell line CAPAN-1,signif icantly higher levels of the cell cycle inhibitor protein p21Waf1 were observed after TSA application.CONCLUSION:The biological effect of TSA in PC cells correlates with the increase of acetyl-H3,p21Waf1,phospho-p38 and bax levels,and the decrease of phosphoERK 1/2 and phospho-AKT.展开更多
基金National Natural Science Foundation of China,No.30271477,No.30572162
文摘AIM： To identify the relationship between DNA hyper- methylation and histone modification at a hyperme- thylated, silenced tumor suppressor gene promoter in human gastric cancer cell lines and to elucidate whether alteration of DNA methylation could affect histone modification. METHODS： We used chromatin immunoprecipitation （CHIP） assay to assess the status of histone acetylation and methylation in promoter regions of the p16 and rnutL homolog 1 （MLH1） genes in 2 gastric cancer cell lines, SGC-7901 and MGC-803. We used methylation- specific PCR （MSP） to evaluate the effect of 5-Aza-2＇- deoxycytidine （5-Aza-dC）, trichostatin A （TSA） or their combination treatment on DNA methylation status. We used RT-PCR to determine whether alterations of histone modification status after 5-Aza-dC and TSA treatment are reflected in gene expression. RESULTS： For thep16 and MLH1 genes in two cell lines, silenced loci associated with DNA hypermethylation were characterized by histone H3-K9 hypoacetylation and hypermethylation and histone H3-K4 hypomethylation. Treatment with TSA resulted in moderately increased histone H3-K9 acetylation at the silenced loci with no effect on histone H3-K9 methylation and minimal effects on gene expression. In contrast, treatment with 5-Aza- dC rapidly reduced histone H3-K9 methylation at the silenced loci and resulted in reactivation of the two genes. Combined treatment with 5-Aza-dC and TSA was synergistic in reactivating gene expression at the loci showing DNA hypermethylation. Similarly, histone H3-K4 methylation was not affected alter TSA treatment, andincreased moderately at the silenced loci after 5-Aza-dC treatment. CONCLUSION： Hypermethylation of DNA in promoter CpG islands is related to transcriptional silencing of tumor suppressor genes. Histone H3-K9 methylation in different regions of the promoters studied correlates with DNA methylation status of each gene in gastric cancer cells. However, histone H3-K9 acetylation and H3-K4 methylation inversely correlate
文摘Background Histone deacetylase inhibitors （HDACIs） have been reported to induce apoptosis in cancer cells. The effects of trichostatin A （TSA） on gastric cancer cells have not been well characterized. This study was aimed to explore the effects and mechanisms of TSA on human gastric cancer SGC-7901 cells. Methods The cells were treated with TSA and analyzed by cell proliferation assay, Western blot, TUNEL assay, flow cytometry by fluorescein isothiocyanate （FITC） conjugated with Annexin V and PI staining, immunofluorescence analysis, analysis of subcellular fractionation, gene chips and real time polymerase chain reaction （PCR）. Results TSA could inhibit cell growth and induced apoptosis in gastric cancer SGC-7901 cells through the regulation of apoptosis-related genes, such as Bcl-2, Bax and survivin. Further study indicated that the pan-caspase inhibitor z-VAD-fmk did not inhibit the apoptosis induced by TSA, and we did not observe the cleavage of poly ADP ribose polymerase （PARP） after TSA treatment too. In addition, apoptosis inducing factor （AIF） and EndoG were found to translocate from mitochondria to nucleus in the immunofluorescence assay and the Western analysis of subcellular fractionation confirmed the result of immunofluorescence assay. Conclusions The apoptosis induced by TSA in gastric cancer SGC-7901 cells involves a caspase-independent pathway.
文摘背景与目的:腺病毒感染依赖于靶细胞表面柯萨奇-腺病毒受体(Coxsackie and adenovirus receptor,CAR)的表达,肿瘤细胞CAR表达的缺失和下调是造成腺病毒为载体基因治疗效果不佳的根本原因。本研究通过观察组氨酸去乙酰化酶(histone deacetylase,HDAC)抑制剂TrichostatinA(TSA)对卵巢癌细胞株A2780CAR表达水平的影响,探讨HDAC抑制剂在腺病毒载体基因治疗中应用的可能性。方法:分别在TSA作用A2780细胞前后检测CARmRNA和蛋白水平的表达,同时采用流式细胞术测定病毒转染效率,MTT实验评价腺病毒携带的胸苷激酶(ADV/TK)的体外抗瘤效应。结果:TSA处理后,A2780细胞内CARmRNA和蛋白表达明显增高。通过流式细胞仪分析GFP/ADV转染后GFP的表达发现,未处理组细胞转染率为(1.24±0.14)%;5nmol/L和100nmol/LTSA处理48h后,细胞的转染效率分别为(7.58±0.32)%和(7.94±0.28)%。MTT结果表明,ADV/TK介导的体外杀伤作用在5nmol/L和100nmol/LTSA处理组较未处理组增强了4～10倍。结论:TSA可以增强针对卵巢癌细胞的腺病毒基因转染效率,为增强卵巢癌基因治疗效果提供可能的方法。
基金supported by the National Natural Science Foundation of China (31300504)Fundamental Research Funds for Rubber Research Institute,Chinese Academy of Tropical Agricultural Sciences (1630022016006)the Earmarked Fund for China Agriculture Research System (CARS-34-GW1)
文摘The secondary laticifer, a specific tissue in the secondary phloem of rubber tree, is differentiated from the vascular cambia. The number of the secondary laticifer in the trunk bark of rubber tree is positively correlated with rubber yield. Although jasmonates have been demonstrated to be crucial in the regulation of secondary laticifer differentiation, the mechanism for the jasmonate-induced secondary laticifer differentiation remains to be elucidated.By using an experimental morphological technique, the present study revealed that trichostatin A(TSA), an inhibitor of histone deacetylation, could induce the secondary laticifer differentiation in a concentrationdependent manner. The results suggest that histone acetylation is essential for the secondary laticifer differentiation in rubber tree.
基金This article was supported by the grants from the National Natural Science Foundation of China，the “985” Project of the Peking University Health Science Center and the Scientific Research Foundation for the Returned Overseas Chinese Scholars,State Education Ministry
文摘Background Histone deacetylase （HDAC） inhibitors are a group of small chemical molecules that inhibit histone deacetylase. At cell level, HDAC inhibitors have multiple biological effects such as cell cycle arrest, apoptosis, cell differentiation and auotophagy. At molecular level, HDAC inhibitors cause histone and nonhistone acetylation and induce gene expression. HDAC inhibitors are widely used in cancer therapy because of its function of inducing apoptosis. However, the mechanisms of apoptosis effect are not fully understood. TSA is a classical HDAC inhibitor and widely used in epigenetic and anti-cancer research. In this study, we selected Trichostatin A （TSA） to investigate the mechanisms of HDAC inhibitors apoptotic effect on cancer cells. Methods Cervical cancer cell lines such as Hela, Caski and normal human keratinocyte line HaCaT were treated with various concentrations of TSA. Crystal violent assay and 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay were performed to determine cell number. PARP cleavage and FITC-AnexinV were performed to determine apoptosis. DNA-methyltransferase （DNMT）I, DNMT3A and DNMT3B were determined by regular PCR, qPCR and Western Blotting. Small interfering RNA （SiRNAi） was used to knock down DNMT3B. Results HDAC inhibitors only induce cervical cancer cell apoptosis. At 1 Iumol/L of TSA, 86% of Hela cell and 76% of Caski went apoptosis. For normal cells, HDAC inhibitors have no cytotoxic effect at therapeutic dosage, （90.0＋8.4）% of normal cell survive after treated with 1 IJmol/L of TSA. We compared 1 pmol/L group with untreated control with t-test. There was no significance between 1 pmol/L group and untreated control for normal cell （P 〉0.05）. HDAC inhibitors decreased DNMT3B in cancer cell but not in normal cell. Manually knock-down of DNMT3B induced Hela and Caski cell apoptosis. More than 99% of Hela and Caski cell went apoptosis after deprived of DNMT3B. Conclusions DNMT3B was essential to cervical cancer cell su
文摘AIM:To gain molecular insights into the action of the histone deacetylase inhibitor(HDACI) trichostatin-A(TSA) in pancreatic cancer(PC) cells.METHODS:Three PC cell lines,BxPC-3,AsPC-1 and CAPAN-1,were treated with various concentrations of TSA for def ined periods of time.DNA synthesis was assessed by measuring the incorporation of 5-bromo-2'deoxyuridine.Gene expression at the level of mRNA was quantif ied by real-time polymerase chain reaction.Expression and phosphorylation of proteins was monitored by immunoblotting,applying an infrared imaging technology.To study the role of p38 MAP kinase,the specif ic enzyme inhibitor SB202190 and an inactive control substance,SB202474,were employed.RESULTS:TSA most eff iciently inhibited BrdU incorporation in BxPC-3 cells,while CAPAN-1 cells displayed the lowest and AsPC-1 cells an intermediate sensitivity.The biological response of the cell lines correlated with the increase of histone H3 acetylation after TSA application.In BxPC-3 cells(which are wild-type for KRAS),TSA strongly inhibited phosphorylation of ERK 1/2 and AKT.In contrast,activities of ERK and AKT in AsPC-1 and CAPAN-1 cells(both expressing oncogenic KRAS) were not or were only modestly affected by TSA treatment.In all three cell lines,but most pronounced in BxPC-3 cells,TSA exposure induced an activation of the MAP kinase p38.Inhibition of p38 by SB202190 slightly but signif icantly diminished the antiproliferative effect of TSA in BxPC-3 cells.Interestingly,only BxPC-3 cells responded to TSA treatment by a signif icant increase of the mRNA levels of bax,a pro-apoptotic member of the BCL gene family.Finally,in BxPC-3 and AsPC-1 cells,but not in the cell line CAPAN-1,signif icantly higher levels of the cell cycle inhibitor protein p21Waf1 were observed after TSA application.CONCLUSION:The biological effect of TSA in PC cells correlates with the increase of acetyl-H3,p21Waf1,phospho-p38 and bax levels,and the decrease of phosphoERK 1/2 and phospho-AKT.
