AIM:To investigate cytokeratin 8(CK8)overexpression during hepatitis C virus(HCV)infection and its pathogenesis,and the effect of ectopic CK8 expression on hepatoma cell lines.METHODS:We successfully established an in...AIM:To investigate cytokeratin 8(CK8)overexpression during hepatitis C virus(HCV)infection and its pathogenesis,and the effect of ectopic CK8 expression on hepatoma cell lines.METHODS:We successfully established an in vitro HCV cell culture system(HCVcc)to investigate the different expression profiles of CK8 in Huh-7-HCV and Huh-7.5-HCV cells.The expression of CK8 at the mRNA level was determined by real-time polymerase chain reaction(RT-PCR).The expression of CK8 at the protein level was evaluated by Western blotting.We then constructed a eukaryotic expression combination vector containing the coding sequence of human full length CK8 gene.CK8cDNA was amplified by reverse transcription-PCR and inserted into pEGFP-C1 and the positive clone pEGFPCK8 was obtained.After confirming the sequence,the recombinant plasmid was transfected into SMMC7721cells with lipofectamine2000 and CK8 expression was detected using inverted fluorescence microscopy,RTPCR and Western blotting.Besides,we identified biological function of CK8 on SMMC7721 cells,including cell proliferation,cell cycle and apoptosis detection.RESULTS:RT-PCR showed that the expression level of CK8 in Huh-7-HCV and Huh-7.5-HCV cells was 2.88and 2.95 times higher than in control cells.Western blot showed that CK8 expression in Huh-7-HCV and Huh-7.5-HCV cells was 2.53 and 3.26 times higher than that in control cells,respectively.We found that CK8at mRNA and protein levels were both significantly increased in HCVcc.CK8 was up-regulated in SMMC7721cells.CK8 expression at the mRNA level was significantly upregulated in SMMC7721/pEGFP-CK8 cells.CK8expression in SMMC7721/pEGFP-CK8 cells was 2.69times higher than in SMMC7721 cells,and was 2.64times higher than in SMMC7721/pEGFP-C1 cells.CK8expression at the protein level in SMMC7721/pEGFPCK8 cells was 2.46 times higher than in SMMC7721cells,and was 2.29 times higher than in SMMC7721/pEGFP-C1 cells.Further analysis demonstrated that forced expression of CK8 slowed cell growth and induced apoptosis of SMMC7721 cells.CONCLU展开更多
Gastrin and cyclooxygenase-2(COX-2) playimportant roles in the carcinogenesis and progression ofgastric cancer.However,it remains unknown whether the combination of cholecystokinin-2(CCK-2) receptor antagonist plus CO...Gastrin and cyclooxygenase-2(COX-2) playimportant roles in the carcinogenesis and progression ofgastric cancer.However,it remains unknown whether the combination of cholecystokinin-2(CCK-2) receptor antagonist plus COX-2 inhibitor exerts synergistic anti-tumor effects on human gastric cancer.Here,we demonstrated that the combination of AG-041R(a CCK-2 receptor antagonist) plus NS-398(a selective COX-2 inhibitor) treatment had synergistic effects on proliferation inhibition,apoptosis induction,down-regulation of Bcl-2 and up-regulation of Bax expression in MKN-45 cells.These results indicate that simultaneous targeting of CCK-2 receptor and COX-2 may inhibit gastric cancer development more effectively than targeting either molecule alone.(C)2008 Elsevier Ireland Ltd.All rights reserved.展开更多
基金Supported by National Natural Science Foundation of China,No.81170393
文摘AIM:To investigate cytokeratin 8(CK8)overexpression during hepatitis C virus(HCV)infection and its pathogenesis,and the effect of ectopic CK8 expression on hepatoma cell lines.METHODS:We successfully established an in vitro HCV cell culture system(HCVcc)to investigate the different expression profiles of CK8 in Huh-7-HCV and Huh-7.5-HCV cells.The expression of CK8 at the mRNA level was determined by real-time polymerase chain reaction(RT-PCR).The expression of CK8 at the protein level was evaluated by Western blotting.We then constructed a eukaryotic expression combination vector containing the coding sequence of human full length CK8 gene.CK8cDNA was amplified by reverse transcription-PCR and inserted into pEGFP-C1 and the positive clone pEGFPCK8 was obtained.After confirming the sequence,the recombinant plasmid was transfected into SMMC7721cells with lipofectamine2000 and CK8 expression was detected using inverted fluorescence microscopy,RTPCR and Western blotting.Besides,we identified biological function of CK8 on SMMC7721 cells,including cell proliferation,cell cycle and apoptosis detection.RESULTS:RT-PCR showed that the expression level of CK8 in Huh-7-HCV and Huh-7.5-HCV cells was 2.88and 2.95 times higher than in control cells.Western blot showed that CK8 expression in Huh-7-HCV and Huh-7.5-HCV cells was 2.53 and 3.26 times higher than that in control cells,respectively.We found that CK8at mRNA and protein levels were both significantly increased in HCVcc.CK8 was up-regulated in SMMC7721cells.CK8 expression at the mRNA level was significantly upregulated in SMMC7721/pEGFP-CK8 cells.CK8expression in SMMC7721/pEGFP-CK8 cells was 2.69times higher than in SMMC7721 cells,and was 2.64times higher than in SMMC7721/pEGFP-C1 cells.CK8expression at the protein level in SMMC7721/pEGFPCK8 cells was 2.46 times higher than in SMMC7721cells,and was 2.29 times higher than in SMMC7721/pEGFP-C1 cells.Further analysis demonstrated that forced expression of CK8 slowed cell growth and induced apoptosis of SMMC7721 cells.CONCLU
文摘Gastrin and cyclooxygenase-2(COX-2) playimportant roles in the carcinogenesis and progression ofgastric cancer.However,it remains unknown whether the combination of cholecystokinin-2(CCK-2) receptor antagonist plus COX-2 inhibitor exerts synergistic anti-tumor effects on human gastric cancer.Here,we demonstrated that the combination of AG-041R(a CCK-2 receptor antagonist) plus NS-398(a selective COX-2 inhibitor) treatment had synergistic effects on proliferation inhibition,apoptosis induction,down-regulation of Bcl-2 and up-regulation of Bax expression in MKN-45 cells.These results indicate that simultaneous targeting of CCK-2 receptor and COX-2 may inhibit gastric cancer development more effectively than targeting either molecule alone.(C)2008 Elsevier Ireland Ltd.All rights reserved.
