Ritonavir and ixazomib kill bladder cancer cells by causing ubiquitinated protein accumulation

Abstract

There is no curative treatment for advanced bladder cancer. Causing ubiquitinated protein accumulation and endoplasmic reticulum stress is a novel approach to cancer treatment. The HIV protease inhibitor ritonavir has been reported to suppress heat shock protein 90 and increase the amount of unfolded proteins in the cell. If the proteasome functions normally, however, they are rapidly degraded. We postulated that the novel proteasome inhibitor ixazomib combined with ritonavir would kill bladder cancer cells effectively by inhibiting degradation of these unfolded proteins and thereby causing ubiquitinated proteins to accumulate. The combination of ritonavir and ixazomib induced drastic apoptosis and inhibited the growth of bladder cancer cells synergistically. The combination decreased the expression of cyclin D1 and cyclin-dependent kinase 4, and increased the sub-G₁ fraction significantly. Mechanistically, the combination caused ubiquitinated protein accumulation and endoplasmic reticulum stress. The combination-induced apoptosis was markedly attenuated by the protein synthesis inhibitor cycloheximide, suggesting that the accumulation of ubiquitinated proteins played an important role in the combination's antineoplastic activity. Furthermore, the combination induced histone acetylation cooperatively and the decreased expression of histone deacetylases was thought to be one mechanism of this histone acetylation. The present study provides a theoretical basis for future development of novel ubiquitinated-protein-accumulation-based therapies effective against bladder cancer.

Keywords: Bladder cancer; drug combinations; ixazomib; ritonavir; ubiquitinated proteins.

Figure 1

Combination of ritonavir and ixazomib inhibited bladder cancer cell viability synergistically. (a) MTS assay. Cells were treated with 20–40 μM ritonavir and/or 20–100 nM ixazomib for 48 h. Data are expressed as mean ± SD; n = 6. (b) Photomicrographs after 48 h of treatment. Note that many of the cells treated with the combination are floating. Original magnification, × 100.

Figure 2

Combination of ritonavir and ixazomib perturbed the cell cycle and induced apoptosis in bladder cancer cells. (a) Cell cycle analysis. Cells were treated for 48 h with 40 μM ritonavir and/or 100 nM ixazomib. Ten thousand cells were counted and changes in the cell cycle were evaluated using flow cytometry. Bar graphs show the percentages of the cells in the sub‐G₁ fraction. Data are expressed as mean ± SD from three independent experiments. *P = 0.0495. (b) Western blot analysis for cyclin D1 and cyclin‐dependent kinase (CDK)4. Cells were treated for 48 h with 50 and 100 nM ixazomib with or without 40 μM ritonavir. Actin was used for the loading control. Representative blots are shown. (c) Annexin V assay. Cells were treated for 48 h with 40 μM ritonavir and/or 100 nM ixazomib. Ten thousand cells were counted and apoptotic cells were detected by annexin V assay using flow cytometry. Bar graphs show apoptotic cell percentages. Data are expressed as mean ± SD from three independent experiments. *P = 0.0495. (d) Western blot analysis for cleaved poly(ADP‐ribose) polymerase (PARP), active caspase 3, and NOXA. Cells were treated for 48 h with 50 and 100 nM ixazomib with or without 40 μM ritonavir. Actin was used for the loading control. Representative blots are shown.

Figure 3

Combination of ritonavir and ixazomib caused ubiquitinated protein accumulation and endoplasmic reticulum (ER) stress in bladder cancer cells. (a) Western blot analysis for ER stress markers, ubiquitinated proteins, and an autophagy marker. Cells were treated for 48 h with 50 or 100 nM ixazomib with or without 40 μM ritonavir. Actin was used for the loading control. Representative blots are shown. (b) Western blot analysis for ubiquitinated proteins in detergent‐insoluble fraction. Cells were treated for 48 h with 50 or 100 nM ixazomib with or without 40 μM ritonavir. The detergent‐insoluble fraction was lysed and subjected to Western blotting. Actin was used for the loading control. Representative blots are shown. (c) Western blotting for ubiquitinated proteins. Cells were treated with 40 μM ritonavir and 100 nM ixazomib for 6, 12, 24, and 48 h. Actin was used for the loading control. Representative blots are shown. Ero1‐L, endoplasmic oxidoreductin‐1‐like protein;ERP44, endoplasmic reticulum resident protein 44; GRP78, glucose‐regulated protein 78; HSP70, heat shock protein 70; LC3, light chain 3.

Figure 4

Accumulation of ubiquitinated proteins was important for the anticancer action of ritonavir and ixazomib. (a) Annexin V assay. Cells were treated for 48 h with 40 μM ritonavir and 100 nM ixazomib with or without 5 μg/mL cycloheximide (CHX). Ten thousand cells were counted and apoptotic cells were detected by annexin V assay using flow cytometry. Bar graphs show apoptotic cell percentages. Data are expressed as mean ± SD from three independent experiments. *P = 0.0495. (b) Photomicrographs of UMUC3 cells after 48 h of treatment with the combination of 40 μM ritonavir and 100 nM ixazomib with or without 5 μg/mL CHX. Original magnification, ×100. (c) Western blot analysis for ubiquitinated proteins and light chain 3 (LC3). Cells were treated for 48 h with 40 μM ritonavir and 100 nM ixazomib with or without 5 μg/mL CHX. Actin was used for the loading control. Representative blots are shown. 7‐AAD, 7‐amino‐actinomycin D.

Figure 5

Combination of ritonavir and ixazomib induced histone acetylation in bladder cancer cells. Western blot analysis for histone deacetylase (HDAC)1, 3, and 6 and acetylated histone. Cells were treated for 48 h with 50 or 100 nM ixazomib with or without 40 μM ritonavir. Actin was used for the loading control. Representative blots are shown.