[Juglone induces proliferation inhibition and apoptosis of cervical cancer cells via promoting c-Myc ubiquitination]

Abstract

Objective: To observe the expression of c-Myc protein in cervical cancer HeLa cells and explore the effect of juglone on the proliferation and apoptosis of HeLa cells by affecting c-Myc ubiquitination.

Methods: HeLa cells treated with different concentrations (0, 10, 20, or 50 μmol/L) of juglone or with 20 μmol/L juglone for different time lengths were examined for expression of c-Myc protein with Western blotting. The half-life of c-Myc protein was determined using cycloheximide (CHX) and c-Myc protein degradation was detected using coimmunoprecipitation. We also assessed the effects of 20 μmol/L juglone combined with 0, 1.0 or 2.0 μmol/L MG132 (a proteasome inhibitor) on c-Myc expression. The effects of 20 μmol/L juglone on the proliferation and apoptosis of HeLa cells with RNA interference-mediated knockdown of c-Myc were evaluated with MTT assay and flow cytometry.

Results: Treatment with juglone significantly lowered c-Myc protein expression in HeLa cells in a concentration-and time-dependent manner (P < 0.05). Juglone obviously shortened the half-life of c-Myc protein, and the addition of MG132 significantly up-regulated the expression level of c-Myc protein (P < 0.05). Juglone treatment also promoted ubiquitination of c-Myc protein in HeLa cells. Compared with the cells transfected with a negative control construct, the cells transfected with si-c-Myc showed significantly decreased proliferation inhibition and a lowered cell rate with early apoptosis after juglone treatment (P < 0.05).

Conclusion: Juglone inhibits proliferation and promotes apoptosis of HeLa cells by affecting the ubiquitination of c-Myc protein.

Keywords: HeLa cells; c-Myc; cervical cancer; juglone; ubiquitination.

图 1

胡桃醌对HeLa细胞c-Myc蛋白表达的影响 Effect of juglone on expression of c-Myc protein in HeLa cells. A: Western blots of c-Myc protein in HeLa cells treated with different concentrations of juglone. B: Western blots of c-Myc in HeLa cells treated with 20 μmol/L juglone for different time lengths. C: Quantitative analysis of c-Myc protein expression. **P < 0.01 vs untreated (0 μmol/LJuglone) group.

图 2

胡桃醌对c-Myc蛋白半衰期的影响 Effect of juglone on half-life of c-Myc protein. A, B: Effect of juglone on expression of c-Myc protein at different time points. C: Quantitative analysis of protein expression. *P < 0.05, **P < 0.01 vs 0 h group; ^#P < 0.05, ^##P < 0.01 vs CHX+20 μmol/L group.

图 3

蛋白酶体抑制剂对胡桃醌处理后c-Myc蛋白的影响 Effects of proteasome inhibitor on c-Myc protein expression in HeLa cells after juglone treatment. A: Western blots of c-Myc protein expressions in different groups. B: Quantitative analysis of the protein expression. *P < 0.05 vs jugloen single treated group; ^#P < 0.05, ^##P < 0.01 vs untreated group.

图 4

胡桃醌促进c-Myc蛋白泛素化降解 Ubiquitination of c-Myc protein in juglone-treated HeLa cells.

图 5

胡桃醌对敲及不敲除c-Myc基因HeLa细胞后增殖及凋亡的影响 Effects of juglone on proliferation and apoptosis of HeLa cells with and without c-Myc gene knockout. A, B: estern blotting after c-Myc gene knockout and quantitative analysis of the protein expression. C, D: Effects of juglone on proliferation and apoptosis of cells with and without c-Myc gene knockout. E: Comparison of early apoptosis rate. *P < 0.05; **P < 0.01 vs siNAgroup; ^#P < 0.05 vs siNAplus jugone group.