Investigation of the effects of proteasome inhibition in breast cancer cell lines by proteomic methods

Inhibition of the 26S proteasome, which plays important roles in many intracellular processes, is extremely important in terms of cancer treatment. Also, the proteomics techniques, making the analysis of almost all proteins possible encoded by the genome under certain conditions in a specific cell, has gained considerable value recently. In our studies, firstly 4T1 breast cancer cells were treated with the proteasome inhibitor bortezomib for 24 hours, and the proteins were then separated by two-dimensional (2D) gel electrophoresis and analyzed by label-free nLC-MS/MS proteomic techniques. The analyzes showed that especially anti-apoptotic GRP78 protein was overexpressed. To increase the apoptotic effects of the proteasome inhibitors as well as to determine the cell death mechanism, 4T1 cells were then treated with bortezomib and the GRP78 inhibitor BAPTA-AM combination for 24 hours. Following the treatment, label-free nLC-MS/MS analyzes showed that the expression of 1266 proteins varied significantly among the experimental groups. Cdc5L and DBNL proteins were among these proteins. The increased expression levels of Cdc5L and DBNL were confirmed by Western blot method after treatment with bortezomib and BAPTA-AM combination. The increase in DBNL expression was also confirmed by the Dot blot method. IC50 values of Cdc5L inhibitor CVT-313, DBNL inhibitor OTSSP167 and BAPTA-AM in 4T1 cells were found as 20,1 nM, 43 nM and 19,9 μM, respectively. WST-1 analyzes showed that bortezomib + CVT-313 and bortezomib + OTSSP167 combinations for 24 hours can have highly cytotoxic effects on 4T1 cells. The three-dimensional (3D) spheroid cell model, carried out with 4T1 cells and followed for 14 days; also proved that bortezomib + OTSSP167 combined therapy was more effective than monotherapies, bortezomib + CVT-313 as well as bortezomib + BAPTA-AM combinations. Bortezomib + OTSSP167 combined therapy has also been shown to inhibit the 3D spheroid formation in MDA-MB-231 human breast cancer cells. Since altogether the results presented here show that OTSSP167 is effective at nanomolar range, it is believed that the combination of bortezomib and OTSSP167 may offer a novel approach in breast cancer treatment and is therefore worthwhile to be tested in vivo and clinically.