Bortezomib inhibits proliferation, migration, and TGF-β1-induced epithelial-mesenchymal transition of RPE cells

Abstract

Purpose: Nuclear factor kappa B (NF-κB) plays an important role in the epithelial-mesenchymal transition (EMT) of RPE cells. We investigated the effects of a proteasome inhibitor, bortezomib, on the EMT in RPE cells. In addition, we assessed the influence of bortezomib on regulation of the NF-κB pathway during this process.

Methods: After treatment with various concentrations of bortezomib, cell viability was analyzed with the water-soluble tetrazolium salt-8 assay, cell-cycle regulation was evaluated with flow cytometry, and cell migration was monitored with in vitro wound healing and Transwell migration assays. To induce fibroblastoid transformation, the RPE cells were treated with recombinant human transforming growth factor (TGF)-β1 (10 ng/ml), and western blot and immunocytochemical analyses were performed to evaluate altered expression of EMT markers after treatment with bortezomib. To verify the effect of bortezomib on shrinkage by myofibroblastic transformation, a contraction assay of the RPE-collagen gel lattice was performed.

Results: Treatment with bortezomib decreased RPE viability in a dose-dependent manner, and flow cytometry revealed that these effects were due to arrest of the G2/M phase cell-cycle. In the in vitro wound healing and Transwell migration assays, treatment with 20 nM bortezomib significantly impeded RPE migration. Treatment with bortezomib also significantly inhibited TGF-β1-induced transdifferentiation of the RPE cells. The effects on proliferation, migration, and the EMT were mediated by regulation of the NF-κB signaling pathway. In addition, bortezomib inhibited contraction of the RPE-collagen gel lattices.

Conclusions: Bortezomib inhibits myofibroblastic transformation of RPE cells by downregulating NF-κB expression and prevents contraction of the RPE-collagen gel matrix. Thus, bortezomib represents a candidate putative therapeutic agent for management of retinal fibrotic diseases.

Figure 1

Effects of bortezomib on proliferation and cell cycle of APRE-19. A: Microscopic observations of a human RPE cell line (ARPE-19) in the presence or absence of 20 nM bortezomib. B: After various concentrations of bortezomib (0, 0.2, 2, and 20 nM) were applied, we evaluated cell viability with the water-soluble tetrazolium salt-8 (WST-8) assay. At a concentration of 20 nM bortezomib, cell viability was statistically significantly reduced (p<0.001). Data represent the mean ± standard deviation (SD) of three replicates. ***p<0.001 with one-way ANOVA, followed by Tukey’s honest significant difference (HSD) post-hoc test. C: Flow-cytometric analysis revealed that bortezomib induced arrest of the G2/M phase at 24 and 48 h. D: After treatment with bortezomib, the proportion of RPE cells in the G2/M phase was statistically significantly higher than that in the control samples (p<0.001). ***p<0.001 with an independent t test, n=3, standard error.

Figure 2

Effects of bortezomib on migration of APRE-19. A: In vitro wound-healing assay revealed that bortezomib, in comparison with vehicle, significantly inhibited wound healing in RPE monolayers. B: Quantification of the remaining wound area. Bortezomib inhibited wound healing after 24 h. ***p<0.001 with an independent t test. C: Transwell migration assays revealed that bortezomib decreased RPE migration. D: Quantification of migrated RPE cells revealed a statistically significant decrease in migration by treatment with 20 nM bortezomib compared to that of control cells. ***p<0.001 with an independent t test, n=3, standard error. E: Filamentous actin staining revealed that bortezomib decreased the number of stress fibers and lamellipodia involved in cell migration. F: Western blot analysis of RAC1 and RHOG revealed that bortezomib downregulated RAC1/RHOG expression. G: Immunocytochemical analysis confirmed that bortezomib downregulated RAC1/RHOG expression.

Figure 3

Regulations of bortezomib on epithelial-mesenchymal transition of APRE-19. A: Western blot analysis after treatment with various concentrations of bortezomib in the presence of transforming growth factor-β1 (TGF-β1, 10 ng/ml). At a concentration of 20 nM, treatment with bortezomib resulted in significant downregulation of mesenchymal factors (N-cadherin, α-smooth muscle actin [SMA], β-catenin, and vimentin) and upregulation of epithelial markers (ZO-1 and occludin). B: Quantification of western blot signals with densitometry. Bortezomib induced statistically significant upregulation of ZO-1 and occludin and downregulation of N-cadherin, α-SMA, β-catenin, and vimentin in the presence of TGF-β1. *p<0.05, **p<0.01, and ***p<0.001 with an independent t test, n=3, standard error, C: Immunocytochemical analysis confirmed the western blot analysis results. Bortezomib (20 nM) statistically significantly increased the expression of ZO-1 and occludin and decreased that of N-cadherin, α-SMA, β-catenin, and vimentin in the presence of TGF-β1.

Figure 4

Inhibitory effect of bortezomib on RPE cell contraction. A: Contraction assay of the RPE–collagen gel complex in the presence or absence of 20 nM bortezomib. Bortezomib efficiently inhibited gel contraction. B: Statistical analysis of the gel area revealed that bortezomib decreased gel contraction after 24 h. ***p<0.001 with an independent t test, n=3, standard error. C: Microscopic observation revealed that cell spreading and cell-to-cell adhesion were impeded in the presence of bortezomib. D: Staining of filamentous actin revealed that the formation of stress fibers was inhibited in the presence of 20 nM bortezomib..

Figure 5

Regulation of NF-kB signaling pathway after treatment of bortezomib. Western blot analysis of the NF-κB signaling pathway in the absence A or presence B of TGF-β1. Bortezomib downregulated nuclear factor kappa B (NF-κB)/phospho-NF-κB expression and upregulated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) regardless of the presence of human transforming growth factor (TGF)-β1. C, D: Immunocytochemical staining at 60 min after the treatment with bortezomib yielded results similar to those for the western blot analysis.