Role of autophagy and endoplasmic reticulum stress in pro-apoptosis effect of carfilzomib on MCF-7 cells
Objective:To investigate the effect of autophagy and endoplasmic reticulum (ER) stress on MCF-7 cells following carfilzomib treatment.Methods:MCF-7 breast cancer cells cultured in vitro were divided into a control group and an experimental group. The experimental group was treated with carfilzomib at 10, 20, 40, 80, 160, and 320 nmol /L for 0, 3, 6, 12, 24, and 48 h, 3-methyladenine (3-MA, an autophagy inhibitor) at 0.5, 1, 5, 10, 20, 40, 80, and 160 nmol/L for 48 h, and salubrinal (an ER stress inhibitor) at 2.5, 5, 10, 20, 40, 80, 160, and 320 μmol/L for 48 h. MCF-7 cell viability was detected by MTT assay to determine the optimal concentration of the drugs used. Cells were then treated with carfilzomib alone, carfilzomib+3-MA, and carfilzomib+salubrinal. Cell apoptosis was detected by flow cytometry, mRNA level was detected by real-time PCR, and protein expression level was detected by Western blot.Results:Treatment with carfilzomib inhibited the viability of MCF-7 cells, and the inhibitory rate was increased significantly with the increase in the concentration and treatment duration. When cells were treated with carfilzomib at 10 nmol/L for 48 h, the inhibition rate for MCF-7 cells was 20.03±0.34. 3-MA and salubrinal inhibited the viability of MCF-7 cells in a dose-dependent manner. Compared with untreated control cells, carfilzomib induced the apoptosis of MCF-7 cells, with an apoptosis rate of 3.54%. Co-treatment of carfilzomib with either 3-MA or salubrinal resulted in enhanced cell apoptosis (apoptosis rate: 5.04% and 6.95%, respectively) compared with cells treated with carfilzomib alone. Compared with the carfilzomib alone group, cotreatment with 3-MA and salubrinal significantly decreased the mRNA expression levels of Grp-78 and caspase-12, but had no signifciant impact on the mRNA expression level of GADD153. In addition, consistent with mRNA detection results, the expression level of GRP-78 in the carfilzomib+3-MA group and the carfilzomib+salubrinal group was significantly protein reduced. Although salubrinal had an more obvious effect on the protein reduction, there was no significant difference in the expression of GADD153 between the carfilzomib+3-MA group and the carfilzomib+salubrinal group. However, caspase-12 expression was decreased significantly in the carfilzomib+3-MA group compared with the carfilzomib+salubrinal group.Conclusion:Carfilzomib inhibits MCF-7 cell proliferation and induces autophagy partially through the ER stress pathway. Inhibiting autophagy and ER stress can enhance the pro-apoptosis effect of carfilzomib on MCF-7 cells.
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