Molecular analysis of cell survival and death pathways in the proteasome inhibitor bortezomib-resistant PC3 prostate cancer cell line.

The ubiquitin-proteasome pathway is an important protein quality control system involved in intracellular homeostasis. To achieve intracellular homeostasis, proteins that are misfolded as a result of translational errors or genetic mutations must be eliminated by the ubiquitin-proteasome pathway. In our previous publications, we determined that 4T1 breast and B16F10 melanoma cancer cells have differential levels of resistance to proteasome inhibitors. Again, in the previous studies, we reported that 4T1 cell cultures, despite being p53-mutant, underwent apoptosis as a result of bortezomib treatment. The first goal of this study was to verify the resistance levels of parental and resistant PC3 prostate cancer cells to bortezomib using WST-1 test. As a result of treatment with different bortezomib concentrations for 48 h, the IC50 value of the parental cells was determined as 32.8 nM and that of the resistant cells was determined as 346 nM. This result showed that the resistant cells were at least 10.5 times more resistant. In addition, to determine whether the resistance gained was reversible or not, the cells were passaged in a medium without bortezomib for one month. The IC50 value determination by WST-1 test showed that the resistant PC3 cells gained an irreversible bortezomib resistance phenotype. The results of the 3D spheroid experiment showed that the 3D spheroid diameter of resistant cells was significantly higher than that of the parental cells. The studies conducted with Western blot showed that ERK1 MAPK T202 phosphorylation and the conversion of autophagy marker LC3-I to LC3-II were significantly increased in parental cells as compared to the resistant cells. Finally, the results showed that while both maternal embryonic leucine zipper kinase (MELK) inhibitor OTSSP167 and Ca2+ chelator BAPTA-AM (also an inhibitor of the expression of antiapoptotic protein GRP78) are promising agents for cancer cells resistant to the proteasome inhibitors, CDK2 inhibitor CVT-313 was found ineffective in both parental and the resistant cells.