Background:XIAP-associated factor 1(XAF1)negatively regulates the function of the X-linked inhibitor of apoptosis protein(XIAP),a member of the IAP family that exerts antiapoptotic effects.The extracellular signal-reg...Background:XIAP-associated factor 1(XAF1)negatively regulates the function of the X-linked inhibitor of apoptosis protein(XIAP),a member of the IAP family that exerts antiapoptotic effects.The extracellular signal-regulated kinase(ERK)pathway is thought to increase cell proliferation and to protect cells from apoptosis.The aim of the study was to investigate the correlation between the ERK1/2 signaling pathway and XAF1 in colon cancer.Methods:Four human colon cancer cell lines,HCT1116 and Lovo(wildtype p53),DLD1 and SW1116(mutant p53),were used.Lovo stable transfectants with XAF1 sense and antisense were established.The effects of dominant-negative MEK1(DN-MEK1)and MEK-specific inhibitor U0126 on the ERK signaling pathway and expression of XAF1 and XIAP proteins were determined.The transcription activity of core XAF1 promoter was assessed by dual luciferase reporter assay.Cell proliferation was measured by MTT assay.Apoptosis was determined by Hoechst 33258 staining.Results:U0126 increased the expression of XAF1 in a time-and dose-dependent manner.A similar result was obtained in cells transfected with DN-MEK1 treatment.Conversely,the expression of XIAP was down-regulated.Activity of the putative promoter of the XAF1 gene was significantly increased by U0126 treatment and DN-MEK1 transient transfection.rhEGF-stimulated phosphorylation of ERK appeared to have little or no effect on XAF1 expression.Overexpression of XAF1 was more sensitive to U0126-induced apoptosis,whereas down-regulation of XAF1 by antisense reversed U0126-induced inhibition of cell proliferation.Conclusions:XAF1 expression was up-regulated by inhibition of the ERK1/2 pathway through transcriptional regulation,which required de novo protein synthesis.The results suggest that XAF1 mediates apoptosis induced by the ERK1/2 pathway in colon cancer.展开更多
AIM: To explore epigenetic changes in the gene encoding X chromosome-linked inhibitor of apoptosis-associated factor 1 (XAF1) during esophageal carcinogenesis. METHODS: Methylation status of XAF1 was detected by methy...AIM: To explore epigenetic changes in the gene encoding X chromosome-linked inhibitor of apoptosis-associated factor 1 (XAF1) during esophageal carcinogenesis. METHODS: Methylation status of XAF1 was detected by methylation-specific polymerase chain reaction (MSP) in four esophageal cancer cell lines (KYSE30, KYSE70, BIC1 and partially methylated in TE3 cell lines), nine cases of normal mucosa, 72 cases of primary esophageal cancer and matched adjacent tissue. XAF1 expression was examined by semi-quantitative reverse transcriptional polymerase chain reaction and Western blotting before and after treatment with 5-azadeoxycytidine (5-aza-dc), a demethylating agent. To investigate the correlation of XAF1 expression and methylation status in primary esophageal cancer, immunohistochemistry for XAF1 expression was performed in 32 cases of esophageal cancer and matched adjacent tissue. The association of methylation status and clinicopathological data was analyzed by logistic regression. RESULTS: MSP results were as follows: loss of XAF1 expression was found in three of four esophageal cell lines with promoter region hypermethylation (completely methylated in KYSE30, KYSE70 and BIC1 cell lines and partially in TE3 cells); all nine cases of normal esophageal mucosa were unmethylated; and 54/72 (75.00%) samples from patients with esophageal cancer were methylated, and 25/72 (34.70%) matched adjacent tissues were methylated (75.00% vs 34.70%, 2 = 23.5840, P = 0.000). mRNA level of XAF1 mea- sured with semi-quantitative reverse transcription polymerase chain reaction was detectable only in TE3 cells, and no expression was detected in KYSE30, KYSE70 or BIC1 cells. Protein expression was not observed in KYSE30 cells by Western blotting before treatment with 5-aza-dc. After treatment, mRNA level of XAF1 was detectable in KYSE30, KYSE70 and BIC1 cells. Protein expression was detected in KYSE30 after treatment with 5-aza-dc. Immunohistochemistry was performed on 32 cases of esophageal cancer and adjacent tissue, and demonstrated XAF1 in the nucleus and cytoplasm. XAF1 staining was found in 20/32 samples of adjacent normal tissue but was present in only 8/32 samples of esophageal cancer tissue ( 2= 9.143, P = 0.002). XAF1 expression was decreased in cancer samples compared with adjacent tissues. In 32 cases of esophageal cancer, 24/32 samples were methylated, and 8/32 esophageal cancer tissues were unmethylated. XAF1 staining was found in 6/8 samples of unmethylated esophageal cancer and 2/24 samples of methylated esophageal cancer tissue. XAF1 staining was inversely correlated with XAF1 promoter region methylation (Fisher's exact test, P = 0.004). Regarding methylation status and clinicopathological data, no significant differences were found in sex, age, tumor size, tumor stage, or metastasis with respect to methylation of XAF1 for the 72 tissue samples from patients with esophageal cancer. CONCLUSION: XAF1 is frequently methylated in esophageal cancer, and XAF1 expression is regulated by promoter region hypermethylation.展开更多
目的:通过顺铂诱导人肺腺癌细胞A549的凋亡,研究X染色体连锁凋亡抑制蛋白相关因子1(X-linked inhibitor of apoptosis protein associated factor-1,XAF1)基因在A549细胞中的表达情况,初步探讨该基因在凋亡中的作用。方法:采用4μg/mL...目的:通过顺铂诱导人肺腺癌细胞A549的凋亡,研究X染色体连锁凋亡抑制蛋白相关因子1(X-linked inhibitor of apoptosis protein associated factor-1,XAF1)基因在A549细胞中的表达情况,初步探讨该基因在凋亡中的作用。方法:采用4μg/mL浓度的顺铂处理A549细胞,MTT法检测细胞的增殖抑制率;AnnexinⅤ/7-AAD检测A549细胞凋亡情况;RT-PCR和蛋白质印迹法分别检测A549凋亡后XAF1mRNA和蛋白的表达;Caspase-3试剂盒检测A549细胞凋亡后Caspase-3的活性。结果:用4μg/mL浓度顺铂处理A549细胞2、12和24h后,A549细胞增殖抑制率分别为(3.40±0.65)%、(8.31±0.73)%和(14.72±0.24)%,与对照组(1.03±0.28)%相比均明显增高,P值分别为0.004、0.000和0.000,细胞增殖抑制率随时间增加而增强;A549细胞凋亡率分别为(4.35±0.95)%、(9.69±2.60)%和(22.35±1.24)%,与对照组(1.39±0.21)%相比均明显增加,P值分别为0.006、0.005和0.000,细胞凋亡率随顺铂作用时间延长而增加;实验组XAF1mRNA表达水平分别为(0.199±0.029)、(0.654±0.093)和(1.216±0.101),对照组为(0.091±0.020),XAF1mRNA表达水平随作用时间的延长呈增加趋势,P值分别为0.006、0.001和0.000;实验组XAF1蛋白表达水平分别为(0.322±0.041)、(0.508±0.014)和(0.901±0.014),对照组为(0.124±0.007),XAF1蛋白表达水平随作用时间的延长呈增加趋势,P值分别为0.001、0.000和0.000;Caspase-3活性(A405nm)分别为(34.745±3.781)、(69.524±3.096)和(94.787±5.429),对照组为(21.914±1.962),Caspase-3活性随顺铂作用时间的延长而增加。结论:顺铂诱导A549细胞凋亡可以引起XAF1表达增加,且随细胞凋亡水平的提高而增加,提示XAF1可能参与顺铂诱导人肺腺癌A549细胞的凋亡过程。展开更多
基金Shanghai Medical Key Discipline Construction Foundation(05-Ⅲ-005-017).
文摘Background:XIAP-associated factor 1(XAF1)negatively regulates the function of the X-linked inhibitor of apoptosis protein(XIAP),a member of the IAP family that exerts antiapoptotic effects.The extracellular signal-regulated kinase(ERK)pathway is thought to increase cell proliferation and to protect cells from apoptosis.The aim of the study was to investigate the correlation between the ERK1/2 signaling pathway and XAF1 in colon cancer.Methods:Four human colon cancer cell lines,HCT1116 and Lovo(wildtype p53),DLD1 and SW1116(mutant p53),were used.Lovo stable transfectants with XAF1 sense and antisense were established.The effects of dominant-negative MEK1(DN-MEK1)and MEK-specific inhibitor U0126 on the ERK signaling pathway and expression of XAF1 and XIAP proteins were determined.The transcription activity of core XAF1 promoter was assessed by dual luciferase reporter assay.Cell proliferation was measured by MTT assay.Apoptosis was determined by Hoechst 33258 staining.Results:U0126 increased the expression of XAF1 in a time-and dose-dependent manner.A similar result was obtained in cells transfected with DN-MEK1 treatment.Conversely,the expression of XIAP was down-regulated.Activity of the putative promoter of the XAF1 gene was significantly increased by U0126 treatment and DN-MEK1 transient transfection.rhEGF-stimulated phosphorylation of ERK appeared to have little or no effect on XAF1 expression.Overexpression of XAF1 was more sensitive to U0126-induced apoptosis,whereas down-regulation of XAF1 by antisense reversed U0126-induced inhibition of cell proliferation.Conclusions:XAF1 expression was up-regulated by inhibition of the ERK1/2 pathway through transcriptional regulation,which required de novo protein synthesis.The results suggest that XAF1 mediates apoptosis induced by the ERK1/2 pathway in colon cancer.
基金Supported by Grants from the National Basic Research Program(973 Program), No. 2012CB934002, 2010CB912802National Key Scientific Instrument Special Programme of China, No.2011YQ03013405National Science Foundation of China,No. 81121004, 81071953 and 81161120432
文摘AIM: To explore epigenetic changes in the gene encoding X chromosome-linked inhibitor of apoptosis-associated factor 1 (XAF1) during esophageal carcinogenesis. METHODS: Methylation status of XAF1 was detected by methylation-specific polymerase chain reaction (MSP) in four esophageal cancer cell lines (KYSE30, KYSE70, BIC1 and partially methylated in TE3 cell lines), nine cases of normal mucosa, 72 cases of primary esophageal cancer and matched adjacent tissue. XAF1 expression was examined by semi-quantitative reverse transcriptional polymerase chain reaction and Western blotting before and after treatment with 5-azadeoxycytidine (5-aza-dc), a demethylating agent. To investigate the correlation of XAF1 expression and methylation status in primary esophageal cancer, immunohistochemistry for XAF1 expression was performed in 32 cases of esophageal cancer and matched adjacent tissue. The association of methylation status and clinicopathological data was analyzed by logistic regression. RESULTS: MSP results were as follows: loss of XAF1 expression was found in three of four esophageal cell lines with promoter region hypermethylation (completely methylated in KYSE30, KYSE70 and BIC1 cell lines and partially in TE3 cells); all nine cases of normal esophageal mucosa were unmethylated; and 54/72 (75.00%) samples from patients with esophageal cancer were methylated, and 25/72 (34.70%) matched adjacent tissues were methylated (75.00% vs 34.70%, 2 = 23.5840, P = 0.000). mRNA level of XAF1 mea- sured with semi-quantitative reverse transcription polymerase chain reaction was detectable only in TE3 cells, and no expression was detected in KYSE30, KYSE70 or BIC1 cells. Protein expression was not observed in KYSE30 cells by Western blotting before treatment with 5-aza-dc. After treatment, mRNA level of XAF1 was detectable in KYSE30, KYSE70 and BIC1 cells. Protein expression was detected in KYSE30 after treatment with 5-aza-dc. Immunohistochemistry was performed on 32 cases of esophageal cancer and adjacent tissue, and demonstrated XAF1 in the nucleus and cytoplasm. XAF1 staining was found in 20/32 samples of adjacent normal tissue but was present in only 8/32 samples of esophageal cancer tissue ( 2= 9.143, P = 0.002). XAF1 expression was decreased in cancer samples compared with adjacent tissues. In 32 cases of esophageal cancer, 24/32 samples were methylated, and 8/32 esophageal cancer tissues were unmethylated. XAF1 staining was found in 6/8 samples of unmethylated esophageal cancer and 2/24 samples of methylated esophageal cancer tissue. XAF1 staining was inversely correlated with XAF1 promoter region methylation (Fisher's exact test, P = 0.004). Regarding methylation status and clinicopathological data, no significant differences were found in sex, age, tumor size, tumor stage, or metastasis with respect to methylation of XAF1 for the 72 tissue samples from patients with esophageal cancer. CONCLUSION: XAF1 is frequently methylated in esophageal cancer, and XAF1 expression is regulated by promoter region hypermethylation.
文摘目的:通过顺铂诱导人肺腺癌细胞A549的凋亡,研究X染色体连锁凋亡抑制蛋白相关因子1(X-linked inhibitor of apoptosis protein associated factor-1,XAF1)基因在A549细胞中的表达情况,初步探讨该基因在凋亡中的作用。方法:采用4μg/mL浓度的顺铂处理A549细胞,MTT法检测细胞的增殖抑制率;AnnexinⅤ/7-AAD检测A549细胞凋亡情况;RT-PCR和蛋白质印迹法分别检测A549凋亡后XAF1mRNA和蛋白的表达;Caspase-3试剂盒检测A549细胞凋亡后Caspase-3的活性。结果:用4μg/mL浓度顺铂处理A549细胞2、12和24h后,A549细胞增殖抑制率分别为(3.40±0.65)%、(8.31±0.73)%和(14.72±0.24)%,与对照组(1.03±0.28)%相比均明显增高,P值分别为0.004、0.000和0.000,细胞增殖抑制率随时间增加而增强;A549细胞凋亡率分别为(4.35±0.95)%、(9.69±2.60)%和(22.35±1.24)%,与对照组(1.39±0.21)%相比均明显增加,P值分别为0.006、0.005和0.000,细胞凋亡率随顺铂作用时间延长而增加;实验组XAF1mRNA表达水平分别为(0.199±0.029)、(0.654±0.093)和(1.216±0.101),对照组为(0.091±0.020),XAF1mRNA表达水平随作用时间的延长呈增加趋势,P值分别为0.006、0.001和0.000;实验组XAF1蛋白表达水平分别为(0.322±0.041)、(0.508±0.014)和(0.901±0.014),对照组为(0.124±0.007),XAF1蛋白表达水平随作用时间的延长呈增加趋势,P值分别为0.001、0.000和0.000;Caspase-3活性(A405nm)分别为(34.745±3.781)、(69.524±3.096)和(94.787±5.429),对照组为(21.914±1.962),Caspase-3活性随顺铂作用时间的延长而增加。结论:顺铂诱导A549细胞凋亡可以引起XAF1表达增加,且随细胞凋亡水平的提高而增加,提示XAF1可能参与顺铂诱导人肺腺癌A549细胞的凋亡过程。
