AIM:To study the relationship between the cyclooxy-genase(COX)-2 gene and the proliferation and apopto-sis of esophageal squamous carcinoma EC109 cells.METHODS:The techniques of RNA interference(RNAi)and cell transfec...AIM:To study the relationship between the cyclooxy-genase(COX)-2 gene and the proliferation and apopto-sis of esophageal squamous carcinoma EC109 cells.METHODS:The techniques of RNA interference(RNAi)and cell transfection,as well as the levels of oncogenic-ity in nude mice,were used to study the role of COX-2 in the esophageal squamous carcinoma cell(ESCC)line EC109.Following RNAi and transfection,Western blot-ting analysis was used to determine the expression of the COX-2 protein.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)reduction assay was used to evaluate cell growth,and flow cytometry was used to detect cell apoptosis.RESULTS:Western blotting analysis demonstrated that COX-2 expression was significantly reduced in EC109 cells treated with COX-2-specif ic short interfering RNA(siRNA)but was increased in EC109 cells transfected with COX-2.Furthermore,COX-2 siRNA treatment in-hibited cell proliferation(P < 0.01)and induced apop-tosis in EC109 cells,as determined by an MTT assay and by flow cytometry,respectively.In contrast,trans-fected COX-2 led to increased cell proliferation(P < 0.05)and decreased apoptosis in EC109 cells.In addition,combination treatment of cells with COX-2 siRNA and aspirin had a synergistic effect(P < 0.01).For experi-ments measuring tumorigenicity,xenograft tumors of a greater volume and weight were found in the COX-2 group compared with other groups(P < 0.05).A large dose of aspirin inhibited tumor growth in nude mice ef-fectively(P < 0.05),and the rate of tumor suppression was 51.8% in the high-dose aspirin group.CONCLUSION:COX-2 plays a very critical role in ESCC carcinogenesis,and COX-2 siRNA combined with aspirin has the potential to be an anticancer therapy for the treatment of ESCC.展开更多
AIM: To investigate the mechanism of calcyclin binding protein/Siah-1 interacting protein(Cacy BP/SIP) nuclear translocation in promoting the proliferation of gastric cancer(GC) cells. METHODS: The effect of Cacy BP/S...AIM: To investigate the mechanism of calcyclin binding protein/Siah-1 interacting protein(Cacy BP/SIP) nuclear translocation in promoting the proliferation of gastric cancer(GC) cells. METHODS: The effect of Cacy BP/SIP nuclear translocation on cell cycle was investigated by cell cycle analysis. Western blot analysis was used to assess the change in expression of cell cycle regulatory proteins and proteasome-mediated degradation of p27Kip1. Coimmunoprecipitation(co-IP) analysis was performed to examine the binding of Cacy BP/SIP with Skp1. A Cacy BP/SIP truncation mutant which lacked the Skp1 binding site was constructed and fused to a fluorescent protein. Subsequently, the effect on Skp1 binding with the fusion protein was examined by co-IP, while localization of fluorescent fusion protein observed by confocal laser microscopy, and change in p27Kip1protein expression assessed by Western blot analysis.RESULTS: Cacy BP/SIP nuclear translocation induced by gastrin promoted progression of GC cells from G1 phase. However, while Cacy BP/SIP nuclear translocation was inhibited using si RNA to suppress Cacy BP/SIP expression, cell cycle was clearly inhibited. Cacy BP/SIP nuclear translocation significantly decreased the level of cell cycle inhibitor p27Kip1, increased Cyclin E protein expression whereas the levels of Skp1, Skp2, and CDK2 were not affected. Upon inhibition of Cacy BP/SIP nuclear translocation, there were no changes in protein levels of p27Kip1 and Cyclin E, while p27Kip1 decrease could be prevented by the proteasome inhibitor MG132. Moreover, Cacy BP/SIP was found to bind to Skp1 by immunoprecipitation, an event that was abolished by mutant Cacy BP/SIP, which also failed to stimulate p27Kip1 degradation, even though the mutant could still translocate into the nucleus.CONCLUSION: Cacy BP/SIP nuclear translocation contributes to the proliferation of GC cells, and Cacy BP/SIP exerts this effect, at least in part, by stimulating ubiquitin-mediated degradation of p27Kip1.展开更多
基金Supported by The National Natural Science Foundation of China,No.81071974The National 863 High Technology Research and Development Plan of China,No.2007AA02Z4Z4
文摘AIM:To study the relationship between the cyclooxy-genase(COX)-2 gene and the proliferation and apopto-sis of esophageal squamous carcinoma EC109 cells.METHODS:The techniques of RNA interference(RNAi)and cell transfection,as well as the levels of oncogenic-ity in nude mice,were used to study the role of COX-2 in the esophageal squamous carcinoma cell(ESCC)line EC109.Following RNAi and transfection,Western blot-ting analysis was used to determine the expression of the COX-2 protein.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)reduction assay was used to evaluate cell growth,and flow cytometry was used to detect cell apoptosis.RESULTS:Western blotting analysis demonstrated that COX-2 expression was significantly reduced in EC109 cells treated with COX-2-specif ic short interfering RNA(siRNA)but was increased in EC109 cells transfected with COX-2.Furthermore,COX-2 siRNA treatment in-hibited cell proliferation(P < 0.01)and induced apop-tosis in EC109 cells,as determined by an MTT assay and by flow cytometry,respectively.In contrast,trans-fected COX-2 led to increased cell proliferation(P < 0.05)and decreased apoptosis in EC109 cells.In addition,combination treatment of cells with COX-2 siRNA and aspirin had a synergistic effect(P < 0.01).For experi-ments measuring tumorigenicity,xenograft tumors of a greater volume and weight were found in the COX-2 group compared with other groups(P < 0.05).A large dose of aspirin inhibited tumor growth in nude mice ef-fectively(P < 0.05),and the rate of tumor suppression was 51.8% in the high-dose aspirin group.CONCLUSION:COX-2 plays a very critical role in ESCC carcinogenesis,and COX-2 siRNA combined with aspirin has the potential to be an anticancer therapy for the treatment of ESCC.
基金Supported by the National Natural Science Foundation of China,No.81072040the Specialized Research Fund for the Doctoral Program of Ningxia Medical University
文摘AIM: To investigate the mechanism of calcyclin binding protein/Siah-1 interacting protein(Cacy BP/SIP) nuclear translocation in promoting the proliferation of gastric cancer(GC) cells. METHODS: The effect of Cacy BP/SIP nuclear translocation on cell cycle was investigated by cell cycle analysis. Western blot analysis was used to assess the change in expression of cell cycle regulatory proteins and proteasome-mediated degradation of p27Kip1. Coimmunoprecipitation(co-IP) analysis was performed to examine the binding of Cacy BP/SIP with Skp1. A Cacy BP/SIP truncation mutant which lacked the Skp1 binding site was constructed and fused to a fluorescent protein. Subsequently, the effect on Skp1 binding with the fusion protein was examined by co-IP, while localization of fluorescent fusion protein observed by confocal laser microscopy, and change in p27Kip1protein expression assessed by Western blot analysis.RESULTS: Cacy BP/SIP nuclear translocation induced by gastrin promoted progression of GC cells from G1 phase. However, while Cacy BP/SIP nuclear translocation was inhibited using si RNA to suppress Cacy BP/SIP expression, cell cycle was clearly inhibited. Cacy BP/SIP nuclear translocation significantly decreased the level of cell cycle inhibitor p27Kip1, increased Cyclin E protein expression whereas the levels of Skp1, Skp2, and CDK2 were not affected. Upon inhibition of Cacy BP/SIP nuclear translocation, there were no changes in protein levels of p27Kip1 and Cyclin E, while p27Kip1 decrease could be prevented by the proteasome inhibitor MG132. Moreover, Cacy BP/SIP was found to bind to Skp1 by immunoprecipitation, an event that was abolished by mutant Cacy BP/SIP, which also failed to stimulate p27Kip1 degradation, even though the mutant could still translocate into the nucleus.CONCLUSION: Cacy BP/SIP nuclear translocation contributes to the proliferation of GC cells, and Cacy BP/SIP exerts this effect, at least in part, by stimulating ubiquitin-mediated degradation of p27Kip1.
