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. 2004 Oct 1;10(19):2779-84.
doi: 10.3748/wjg.v10.i19.2779.

Inhibitory effect of ubiquitin-proteasome pathway on proliferation of esophageal carcinoma cells

Wei-Guo Zhang  1 , Jie-Ping Yu, Qing-Ming Wu, Qiang Tong, Sheng-Bao Li, Xiao-Hu Wang, Guo-Jian Xie
Affiliations

Inhibitory effect of ubiquitin-proteasome pathway on proliferation of esophageal carcinoma cells

Wei-Guo Zhang et al. World J Gastroenterol. .

Abstract

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 (3)H-thymidine ((3)H-TdR) incorporation. Morphologic changes of cells were observed under microscope. Activity of telomerase was examined by telomeric repeat amplification protocol (TRAP) of PCR-ELISA. 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 (3)H-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 5 micromol/L of MG-132 for 24, 48, 72 and 96 h (P<0.01). The percentage of cells at G(0)/G(1) phase was increased and that at S and G(2)/M was decreased (P<0.01). The rate of apoptotic cells treated with 5 micromol/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, G(1) arrest and depression of telomerase activity. The results indicate that inhibiting UPP is a novel strategy for esophageal carcinoma therapy.

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Figures

Figure 1
Figure 1
MTT assay of EC9706 cells after exposed to MG-132.
Figure 2
Figure 2
Morphologic changes of EC9706 cells observed un-der microscope after treated with 5 μmol/L of MG-132. A: control group; B: EC9706 cells treated with MG-132 for 24 h; C: EC9706 cells treated with MG-132 for 48 h; D: EC9706 cells treated with MG-132 for 72 h; E: EC9706 cells treated with MG-132 for 96 h (× 200).
Figure 3
Figure 3
Cell cycle and apoptosis of EC9706 cells exposed to 5 μmol/L of MG-132. A: control group. Apoptotic sub-G1 peak was not found; B: EC9706 cells exposed to MG-132 for 48 h. The ratio of apoptotic cells was 31.7%; C: EC9706 cells ex-posed to MG-132 for 96 h. The ratio of apoptotic cells was 66.4%.
Figure 4
Figure 4
Results of DNA agarose electrophoresis. Lane1: con-trol group; Lane 2: EC9706 cells exposed to 5 μmol/L of MG-132 for 48 h; Lane 3: EC9706 cells exposed to 5 μmol/L of MG-132 for 96 h; Lane 4: 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000 bp ladder markers.
Figure 5
Figure 5
Results of immunohistochemical staining of EC9706 cells. A: control group. Immunohistochemical staining of p27kip1 protein located in cytoplasm of EC9706 cells; B: MG-132 group.
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