Fig. 6

The combined effects of FKB and proteasome inhibitors MG132 and Bortezomib on cell viabilities, apoptosis and expression of Skp2 and p27/Kip1. a, C4-2B, DU145 and PC3 cells were treated with 0.1%DMSO, 8.8 μM FKB, 5 μM MG132, 5 nM Bortezomib or their combinations for 24 h. MTT assay was performed to measure cell viabilities. Bars are mean ± SD of three independent experiments. “*” denotes P < 0.05 and “**” denotes P < 0.01. b, left panels: protein expression of Skp2, p27/Kip1, p21/WAF1, and cleaved PARP was measured after DU145 cells were treated with 0.01% DMSO, 8.8 μM FKB, 5 μM MG132), or their combinations for 24 h. Right panels: DU145 and PC3 cells were treated with 8.8 μM FKB, 20 μM flavokawain A (FKA), 5 nM Bortezomib, or their combinations. Skp2 protein levels were examined. c, the caspase 3/7 activities were measured by ELISA kit after DU145 cells were treated with 0.1% DMSO, 8.8 μM FKB, 5 μM MG132, and their combination for 24 h. Bars are mean ± SD of three independent experiments. d, schematic presentation of mechanisms for the combined effects of FKB and proteasome inhibitors. FKB degrades Skp2 via inhibition of Ubc12/Cullin1 neddylation, whereas proteasome inhibitors down-regulation of Skp2 expression through transcriptional suppression. The combination results in enhanced up-regulation of p21/WAF1, p27/Kip1, and cleaved PARP, leading to greater growth inhibition and apoptosis