AIM:To investigate the relationship between late SV40 factor(LSF)and Notch signaling in the development and progress of hepatocellular carcinoma(HCC).METHODS:Liver cancer tissue specimens from 25 patients were analyze...AIM:To investigate the relationship between late SV40 factor(LSF)and Notch signaling in the development and progress of hepatocellular carcinoma(HCC).METHODS:Liver cancer tissue specimens from 25 patients were analyzed for Notch-1 and LSF expression by immunohistochemistry.The correlation between expression and the biological effects of Notch-1 and LSF were analyzed using genetic and pharmacological strategies in HCC cell lines and human normal cell lines,including hepatic stellate cells(HSC)and human embryonic kidney epithelial cells(HEK).RESULTS:Immunohistochemistry showed that both Notch-1 and LSF were significantly upregulated in HCC samples(76%,19/25,P<0.0001 and 84%,21/25,P<0.0001,respectively)compared with non-cancer samples.Activation of Notch-1 by exogenous transfection of Notch1 intracellular domain increased LSF expression in HSC and HEK cells to levels similar to those seen in HepG2 cells.Furthermore,blocking Notch-1 activation with aγ-secretase inhibitor,DAPT,downregulated LSF expression in HepG2 cells.Additionally,a biological behavior assay showed that forced overexpression of LSF promoted HepG2 cell proliferation and invasion.CONCLUSION:LSF is a key mediator of the Notch signaling pathway,suggesting that it might be a novel therapeutic target for the treatment of HCC.展开更多
目的探讨人工合成的EFEMP1源性抑癌蛋白ZR30与表皮生长因子受体（EGFR）、Notch－1、Akt、基质金属蛋白酶2（MMP－2）的相关性及其对胶质瘤细胞生长、侵袭、血管生成和干性维持的影响，阐明ZR30的抑癌作用机制。方法使用小麦胚芽无细胞...目的探讨人工合成的EFEMP1源性抑癌蛋白ZR30与表皮生长因子受体（EGFR）、Notch－1、Akt、基质金属蛋白酶2（MMP－2）的相关性及其对胶质瘤细胞生长、侵袭、血管生成和干性维持的影响，阐明ZR30的抑癌作用机制。方法使用小麦胚芽无细胞系统合成体外蛋白ZR30。采用明胶酶谱法检测ZR30处理2～3 d后的U87和U251细胞中MMP－2的活性，Western blot检测ZR30处理2～3 d后的胶质瘤亚群细胞U251和U251NS中EGFR、p－Akt、Akt和Notch－1的表达。分别以10和50 ng/ml的ZR30处理U251细胞，以10、50和200 ng/ml的ZR30处理U251NS细胞，四甲基偶氮唑蓝（MTT）法检测ZR30对U251和U251NS细胞增殖的影响。将U251－GFP和U251NS－RFP细胞混合（1∶9）后，注入裸鼠颅内，分别于10和21 d后瘤内注射ZR30或磷酸盐缓冲液（PBS）。提取各组裸鼠右侧大脑的DNA，采用荧光定量聚合酶链反应（CQ－PCR）检测人类基因hSPAG16、鼠基因mSpag16、GFP和RFP的拷贝数。观察各组裸鼠的生存情况，进行生存分析。结果10、50和100 ng/ml ZR30处理2 d后，U87和U251细胞培养液中的活性MMP－2水平均低于对照组。Western blot检测显示，ZR30能抑制U251细胞中EGFR、Notch－1和p－Akt蛋白的表达，以及U251NS细胞中Notch－1和p－Akt蛋白的表达，并可降低U251细胞对表皮生长因子刺激的响应。MTT检测结果显示，ZR30处理U251和U251NS细胞2 d后，能抑制细胞增殖。CQ－PCR结果显示，PBS组、180 ng ZR30组、700 ng ZR30组和1 800 ng ZR30组裸鼠的hSPAG16/mSpag16比值分别为3.67±2.82、1.18±0.97、1.75±1.55和1.38±1.17，180 ng ZR30组、700 ng ZR30组和1 800 ng ZR30组与PBS组比较均降低（均P〈0.05）；GFP/RFP比值分别为1.97±0.80、1.97±0.85、1.48±0.71和1.73±0.77，各组间差异均无统计学意义（均P〉0.05）。U251－GFP与U251NS－RFP细胞混合液移植后10 d治疗，PBS组和ZR30组裸鼠的中位生存时间分别为40.5和59.0 d；移植�展开更多
基金Supported by The National Natural Science Foundation of China,No.30470780
文摘AIM:To investigate the relationship between late SV40 factor(LSF)and Notch signaling in the development and progress of hepatocellular carcinoma(HCC).METHODS:Liver cancer tissue specimens from 25 patients were analyzed for Notch-1 and LSF expression by immunohistochemistry.The correlation between expression and the biological effects of Notch-1 and LSF were analyzed using genetic and pharmacological strategies in HCC cell lines and human normal cell lines,including hepatic stellate cells(HSC)and human embryonic kidney epithelial cells(HEK).RESULTS:Immunohistochemistry showed that both Notch-1 and LSF were significantly upregulated in HCC samples(76%,19/25,P<0.0001 and 84%,21/25,P<0.0001,respectively)compared with non-cancer samples.Activation of Notch-1 by exogenous transfection of Notch1 intracellular domain increased LSF expression in HSC and HEK cells to levels similar to those seen in HepG2 cells.Furthermore,blocking Notch-1 activation with aγ-secretase inhibitor,DAPT,downregulated LSF expression in HepG2 cells.Additionally,a biological behavior assay showed that forced overexpression of LSF promoted HepG2 cell proliferation and invasion.CONCLUSION:LSF is a key mediator of the Notch signaling pathway,suggesting that it might be a novel therapeutic target for the treatment of HCC.
文摘目的探讨人工合成的EFEMP1源性抑癌蛋白ZR30与表皮生长因子受体（EGFR）、Notch－1、Akt、基质金属蛋白酶2（MMP－2）的相关性及其对胶质瘤细胞生长、侵袭、血管生成和干性维持的影响，阐明ZR30的抑癌作用机制。方法使用小麦胚芽无细胞系统合成体外蛋白ZR30。采用明胶酶谱法检测ZR30处理2～3 d后的U87和U251细胞中MMP－2的活性，Western blot检测ZR30处理2～3 d后的胶质瘤亚群细胞U251和U251NS中EGFR、p－Akt、Akt和Notch－1的表达。分别以10和50 ng/ml的ZR30处理U251细胞，以10、50和200 ng/ml的ZR30处理U251NS细胞，四甲基偶氮唑蓝（MTT）法检测ZR30对U251和U251NS细胞增殖的影响。将U251－GFP和U251NS－RFP细胞混合（1∶9）后，注入裸鼠颅内，分别于10和21 d后瘤内注射ZR30或磷酸盐缓冲液（PBS）。提取各组裸鼠右侧大脑的DNA，采用荧光定量聚合酶链反应（CQ－PCR）检测人类基因hSPAG16、鼠基因mSpag16、GFP和RFP的拷贝数。观察各组裸鼠的生存情况，进行生存分析。结果10、50和100 ng/ml ZR30处理2 d后，U87和U251细胞培养液中的活性MMP－2水平均低于对照组。Western blot检测显示，ZR30能抑制U251细胞中EGFR、Notch－1和p－Akt蛋白的表达，以及U251NS细胞中Notch－1和p－Akt蛋白的表达，并可降低U251细胞对表皮生长因子刺激的响应。MTT检测结果显示，ZR30处理U251和U251NS细胞2 d后，能抑制细胞增殖。CQ－PCR结果显示，PBS组、180 ng ZR30组、700 ng ZR30组和1 800 ng ZR30组裸鼠的hSPAG16/mSpag16比值分别为3.67±2.82、1.18±0.97、1.75±1.55和1.38±1.17，180 ng ZR30组、700 ng ZR30组和1 800 ng ZR30组与PBS组比较均降低（均P〈0.05）；GFP/RFP比值分别为1.97±0.80、1.97±0.85、1.48±0.71和1.73±0.77，各组间差异均无统计学意义（均P〉0.05）。U251－GFP与U251NS－RFP细胞混合液移植后10 d治疗，PBS组和ZR30组裸鼠的中位生存时间分别为40.5和59.0 d；移植�
