Figure 1.

Therapeutic effect of bortezomib in vitro: (A) BON, LCC-18 and KRJ-I cell lines were treated with increasing concentrations of bortezomib for 24 h, 50 h, and 72 h, respectively, and cell viability was measured by WST-1 proliferation assay. Dose-response curve and histogram of the same data (representative data out of three independent experiments is shown, bar represents means and SD of n = 5 replicates) is shown for a more concise presentation. All cell lines showed a significant antiproliferative response to the treatment (one-way ANOVA: P< 0.0001; and Holm-Sidak's multiple comparisons test; normality was assumed after both, Shapiro-Wilk and Kolmogorov-Smirnov normality test). Dose of relative half-maximal inhibitory effect (IC50) was determined by non-linear regression using Prism 7. Captions: *P< 0.05; **P< 0.0001. (B) In vitro combinatory effects of cisplatin and bortezomib after 50 h of treatment: cell lines were treated with a combination of bortezomib and cisplatin 1:10 (starting from 100 or 50 µM cisplatin + 10 or 5 µM bortezomib, respectively, and subsequent dilutions) versus the corresponding single agents (representative experiment of three independent experiments, bar shows median and interquartile range of n = 5 replicates). Dose response curves were analyzed according to the method of Chou and Talalay. BON cells responded in all measured dilutions (excluding the starting concentrations) with synergistic effects. In LCC-18 cells, bortezomib induced synergism in doses >10 nM, which is lower than the respective IC50. KRJ-I cells, which showed very strong effects even at low concentrations of bortezomib alone, exhibited a very strong response, however, synergism only substantiated for intermediate doses (500 nM cisplatin + 50 nM bortezomib and 5 µM cisplatin + 500 nM bortezomib) (2-way ANOVA with Sidak's multiple comparisons test). *P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001.