摘要
AIM:To investigate the inhibitory effect of ubiquitin-proteasome pathway(UPP)on proliferation of esophageal carcinoma cells.METHODS:Esophageal carcinoma cell strain EC9706 was treated with MG-132 to inhibit its UPP specificity.Cell growth suppression was evaluated with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay.DNA synthesis was evaluated by^3H-thymidine(^3H-TdR)incorporation.Morphologic changes of cells were observed under microscope.Activity of telomerase was examined by telomeric repeat amplification protocol(TRAP)of PCRELISA.Cell cycle and apoptosis were detected by flow cytometry(FCM).DNA fragment analysis was used to confirm the presence of apoptosis.Expression of p27^kip1 was detected by immunocytochemical technique.RESULTS:After exposed to MG-132,the growth and value of^3H-TdR incorporation of EC9706 cells were obviously inhibited.Cells became round,small and exfoliative under microscope.TRAP PCR-ELISA showed that light absorption of cells gradually decreased after exposed to 5μmol/L of MG-132 for 24,48,72 and 96 h(P<0.01).The percentage of cells at G0/G1 phase was increased and that at S and G2/M was decreased(P<0.01).The rate of apoptotic cells treated with 5μmol/L of MG-132 for 48 and 96 h was 31.7%and 66.4%,respectively.Agarose electrophoresis showed marked ladders.In addition,the positive signals of p27^kip1 were located in cytoplasm and nuclei in MG-132 group in contrast to cytoplasm staining in control group.CONCLUSION:MG-132 can obviously inhibit proliferation of EC9706 cells and induce apoptosis.The mechanisms include upregulation of p27^kip1 expression,G1 arrest and depression of telomerase activity.The results indicate that inhibiting UPP is a novel strategy for esophageal carcinoma therapy.
抑制作用
泛素
蛋白酶
分芽繁殖
食道癌细胞
肿瘤
EC9706
