An investigation of the mechanisms underlying the proteasome inhibitor bortezomib resistance in PC3 prostate cancer cell line

Abstract

The phenomenon of acquired resistance to chemotherapeutic agents is a long-standing conundrum in cancer treatment. To help delineate drug resistance mechanisms and pave the way for the development of novel strategies, we generated a PC3 prostate cancer cell line resistant to proteasome inhibitor bortezomib for the first time. The resistant cells were found to have an IC₅₀ value of 359.6 nM, whereas the IC₅₀ value of parental cells was 82.6 nM after 24 h of treatment with varying doses of bortezomib. The resistant cells were also partly cross-resistant to the novel proteasome inhibitor carfilzomib; however, they were not resistant to widely used chemotherapeutic agent vincristine sulfate, indicating that enhanced cellular drug efflux via the multidrug resistance (MDR) transporters is not the molecular basis of the resistance. Since both bortezomib and carfilzomib target and inhibit the chymotrypsin-related activity residing in the β5 subunit of the proteasome (PSMB5), we next examined its expression and found surprisingly no significant alteration in the expression profile of the mature form. However, a significant increase in the accumulation of the precursor form of PSMB5 in response to 100 nM bortezomib was observed in the parental cells without a significant accumulation in the resistant cells. The results presented here thus suggest that the molecular mechanisms causing resistance to proteasome inhibitors need to be examined in-depth to overcome the resistance to ubiquitin-proteasome pathway inhibitors in cancer treatment.

Keywords: Bortezomib; Cancer; Metastasis; PC3; Proteasome.

Fig. 1

The IC₅₀ values of bortezomib in a parental PC3 and b resistant PC3 cells. The cells were treated with the indicated concentrations of bortezomib in “Materials and methods” section. The cell survival was determined by WST-1 assay (n = 3–5)

Fig. 2

Effect of proteasome inhibitor carfilzomib (50 nM) and bortezomib (100 nM) on parental PC3 and resistant PC3 cells. a The cell proliferation was determined by the iCELLigence system. b The statistical analysis of cell index values for each treatment. The results are presented as mean ± SD. The groups were compared with two-way ANOVA with Bonferroni multiple comparison post-test. ** represents a p value < 0.01; *** represents a p-value < 0.001

Fig. 3

Effect of carboplatin (1 µM) and vincristine sulfate (1 µM) on parental PC3 and resistant PC3 cells. a The cell proliferation was determined by the iCELLigence system. b The statistical analysis of cell index values for each treatment. The results are presented as mean ± SD. The groups were compared with two-way ANOVA with Bonferroni multiple comparison post-test. ** represents a p-value < 0.01; *** represents a p-value < 0.001

Fig. 4

Effect of flavonoids EGCG (10 µM) and quercetin (10 µM) on parental PC3 and resistant PC3 cells. a The cell proliferation was determined by the iCELLigence system. b The statistical analysis of cell index values for each treatment. The results are presented as mean ± SD. The groups were compared with two-way ANOVA with Bonferroni multiple comparison post-test. * represents a p-value < 0.05; ** represents a p-value < 0.01; *** represents a p-value < 0.001

Fig. 5

Western blot analysis of polyubiquitin conjugates, PSMB5 and cyclin D1 in parental and resistant PC3 cells. Cells were treated with 10 nM and 100 nM bortezomib for 24 h. Afterwards, 35 µg protein was separated on 12% SDS-PAGE followed by Western blot analysis as described in detail in “Materials and methods” section. PC3-P, parental PC3 cells; PC3-R, resistant PC3 cells. The result is representative of two experiments, each run in duplicate