Effect Of Endoplasmic Reticulum Stress On Breast Cancer MCF-7 Cell Autophagy Induced By Proteasome Inhibitor

Objection:: Breast cancer is a common malignancy, with a high recurrence rate, high transfer rate and high mortality characteristics. Although the surgical treatment of breast cancer has made a good effect, but postoperative radiotherapy and chemotherapy in breast cancer remains an important part of a comprehensive treatment, and the cell tolerance mechanisms of radiotherapy and chemotherapy in breast cancer and the development of new chemotherapy drugs have been important part of breast cancer research. Clinical trials showed that proteasome targeting drugs have good anti-tumoreffect, but studies have shown that chemotherapy drugs like proteasome inhibitor would produce the treatmenttolerancephenomenon in tumor cells, and which is closely related to autophagy. Autophagy is the degradation of proteins and organelles process mediated by lysosome in cells, under normal physiological conditions, autophagy is limited to the scope of the basic level, but the stimulation of hunger and cell environmental changes will be activated autophagy.Although the mechanism of autophagy activation of proteasome inhibitors is still unclear, previous studies have suggested that endoplasmic reticulum stress is closely related to.In this study, we hypothesized that proteasome inhibitor activated autophagy may be partially through endoplasmic reticulum stress pathway and inhibition of autophagy-related upstream endoplasmic reticulum stress signaling pathway can enhance the breast cancer cell killing effect of proteasome inhibition. So in our study,we explore theeffect of endoplasmic reticulum stress on breast cancer MCF-7 cellautophagy induced by proteasome inhibitor.Methods:Breast cancer MCF-7 cells was selected as the research object, select the proteasome inhibitor MG-132, autophagy inhibitor 3-MA and endoplasmic reticulum stressinhibitor Salubrinal for the the experiment. MTT assay was used to explore the optimum concentration of MG-132, 3-MA and the Salubrinal. Experiment were divided into few groups, including control group, MG-132 group, MG-132 + 3-MA group and MG-132 + Salubrinal group. In our study we detected the activity of MCF-7 cellsby MTT assay, using the Western blot method to detect the expression of autophagy-related protein LC3, and apoptosis-related protein Bcl-2, Bax and Caspase-12, and endoplasmic reticulum stress-related protein Grp-78, Caspase-12 avd GADD153. We select flow detector to explore differences between groups on apoptosis of MCF-7 cells and using RT-PCR method to compare the m RNA levels of Grp-78, GADD153 and Caspase-12.Results: 1MG-132 have inhibitory activityin MCF-7 cells,showing a concentrationdependent and time-dependent manner, at a concentration of 2.5μmol/L for 48 h, inhibition rate was 17.4%, and at the concentration of 10μmol/L and 40μmol/L for 48 hours, and inhibition rates were 48.6% and 96.3%, respectively. IC20 value of MG-132 in 48-hour was about 2.5μmol/L. Low concentrations of 3-MA had no significant inhibitory effect on the proliferation of breast cancer MCF-7 cells, at a concentration of 1mmol/L for 48 hours, the inhibitory rate was 6.1%, at the concentration of 10mmol/L and 20mmol/L for 48 hours, inhibition rate reached 46.2% and 92.4%, respectively, had a concentration-dependent manner, IC5 value of 3-MA for 48 hours was 1mmol/L. The low concentrations of Salubrinal had no significant inhibitory effect on the proliferation of MCF-7 cells, at the concentration of 5μmol / L and 10μmol / L for 48 hours, and the inhibition rate was 4.3% and 11.5%, respectively, and at the concentration of 40μmol/L and 80μmol/L for 48 hours, inhibition rate reached 37.9% and 84.8%, respectively, showed a concentration-dependentmanner. IC5 value of Salubrinal for 48 hours was 5μmol/L. 2Chymotrypsin-like proteasome activity assay results showed that proteasomeactivity of MG-132 in MCF-7 cells were inhibited, began at 3h, the inhibition rate was 65%, at 48 h had the strongest inhibition effect, showed a time-dependent manner. Compared withthe results of MTT assay, MG-132 began to show cell inhibitory effect at 3h, at 6h inhibitory effect increased significantly, while at the time of 3h, proteasome inhibition rate was about 65%, showing a significant inhibitory effect, suggesting that proteasome inhibitory activityoccurs beforecell inhibitory activity. 3Autophagy inhibitor 3-MA(1mmol / L) strengthen the inhibitory effect of cell proliferation induced by MG-132(2.5μmol / L), showed a time-dependent manner. Salubrinal(5μmol / L) enhanced MG-132-induced inhibition of MCF-7 cells, and had a time-dependent manner, most visible at 48 h, but not that obvious compared with 3-MA. 4MG-132(2.5μmol / L) could induce apoptosis and cell cycle arrest at G2 phase in MCF-7 cells,apoptosis rate was 10.6% and the percentage of cells in G2 phase was 22.40%. 3-MA(1mmol / L) and Salubrinal(5μmol / L) could enhance the MG-132-induced apoptosis and G2 phase cell cycle arrest, apoptosis rate increased to 23.3% and 24.7%, the percentage of cells in G2 phase increased to 24.80 % and 25.76%. 5MG-132(2.5μmol / L) induced autophagy of MCF-7 cells, autophagy inhibitor 3-MA(1mmol / L) inhibited autophagy and reversed conversion of LC3-I to LC3-II. Endoplasmic reticulum stress inhibitor Salubrinal(5μmol / L) reduced the expression of LC3-I and inhibit LC3-I to LC3-II transformation process, displayed autophagy inhibition effect, but the effect was smaller than 3-MA. 6Compared with MG-132(2.5μmol / L) group, 3-MA(1mmol / L) and Salubrinal(5μmol / L) reduced the protein expression levels of Bcl-2, but protein expressionlevels of Bax and Caspase-3 was increased, displyed good enhancingeffect of MG-132-induced apoptosis in MCF-7 cells. 7MG-132(2.5μmol / L) could induce ER stress of MCF-7 cells, endoplasmic reticulum stress inhibitor Salubrinal(5μmol / L) can inhibit this process in protein and m RNA levels. Similarly, 3-MA(1mmol / L) also performed an inhibitory effect ofendoplasmic reticulum stress induced by MG-132 in MCF-7 cells, but the effect was smaller than Salubrinal.Conclusion: Proteasome inhibitor MG-132 may be activated autophagy partially through endoplasmic reticulum stress pathway; Inhibition of autophagy-related upstream endoplasmic reticulum stress signaling pathway can enhance the cell killing effect of proteasome inhibitor MG-132 in breast cancer MCF-7 cells.