Oxidative Stress Induced by the Deubiquitinase Inhibitor b-AP15 Is Associated with Mitochondrial Impairment

Abstract

Inhibitors of the 20S proteasome such as bortezomib are cytotoxic to tumor cells and have been proven to be valuable for the clinical management of multiple myeloma. The therapeutic efficacy of bortezomib is, however, hampered by the emergence of acquired resistance. Available data suggest that blocking proteasome activity at the level of proteasome-associated deubiquitinases (DUBs) provides a mechanism to overcome resistance to bortezomib and also to other cancer therapies. The small molecule b-AP15 is an inhibitor of proteasome-associated DUB activity that induces both proteotoxic stress and increases in the levels of reactive oxygen species (ROS) in tumor cells. Antioxidants have been shown to decrease apoptosis induction by b-AP15 and we here addressed the question of the mechanism of redox perturbation by this compound. We show that oxidative stress induction by b-AP15 is abrogated in cells deprived of mitochondrial DNA (ρ ⁰ cells). We also show associations between the level of proteotoxic stress, the degree of mitochondrial dysfunction, and the extent of induction of hemeoxygenase-1 (HO-1), a target of the redox-regulated Nrf-2 transcription factor. Decreased expression of COX5b (cytochrome c oxidase subunit 5b) and TOMM34 (translocase of outer mitochondrial membrane 34) was observed in b-AP15-treated cells. These findings suggest a mitochondrial origin of the increased levels of ROS observed in cells exposed to the DUB inhibitor b-AP15.

Figure 1

Induction of mitochondrial dysfunction in HCT116 cells by the deubiquitinase inhibitor b-AP15. (a) HCT116 cells were exposed to 0.5% DMSO or 1 μM b-AP15 for 1, 3, and 6 hours, and extracts were prepared and subjected to immunoblotting using the indicated antibodies. (b) Electron micrographs of HCT116 cells treated with b-AP15 for 1, 6, and 12 h. Scale bar = 0.5 μm. (c) Basal and maximal oxygen consumption rates (OCR) were measured after a 5-hour exposure of HCT116 cells to 1 μM b-AP15 using a Seahorse XF analyzer. Uncoupled respiration was measured after exposure to carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) (mean ± S.D.; ∗∗p < 0.01).

Figure 2

Evidence of oxidative stress in b-AP15-exposed HCT116 cells. (a) HCT116 cells were exposed to 0.5% DMSO or b-AP15 (0.25, 0.5, and 1.0 μM in 0.5% DMSO) for 6 h, and extracts were prepared and subjected to immunoblotting using the indicated antibodies. (b) Increases in the ratio of GSSG/GSH in HCT116 cells exposed to 1 μM b-AP15 for the indicated times (mean ± S.D.; ∗p < 0.05, ∗∗p < 0.01; n = 3). (c) Total levels of GSH were determined in vehicle-treated cells and in cells exposed to 1 μM b-AP15 for 6 h (mean ± S.D.; n = 3; N.S.: not significant at p < 0.05). (d) Glucose-6-phosphate dehydrogenase activity in HCT116 cells exposed to 1 μM b-AP15 for 6 h compared to vehicle-treated cells (mean ± S.D.; ∗p < 0.05; n = 3). (e) Glutathione peroxidase activity in HCT116 cells exposed to 1 μM b-AP15 for 6 h compared to vehicle-treated cells (mean ± S.D.; n = 3). (f) Malondialdehyde levels in HCT116 cells exposed to 1 μM b-AP15 or vehicle for 6 h. Statistical significance was calculated using Student's t-test in (b)–(f).

Figure 3

HeLa Rho⁰ (ρ ⁰) cells show a decreased oxidative stress response to b-AP15. (a) HeLa cells were exposed to 0.5% DMSO or 1 μM b-AP15 for 1, 3, and 6 hours, and extracts were prepared and subjected to immunoblotting using the indicated antibodies. All cultures received 0.5% DMSO. (b) HeLa cells were exposed to 0.5% DMSO or b-AP15 (0.25, 0.5, and 1.0 μM in 0.5% DMSO) for 6 h, and extracts were prepared and subjected to immunoblotting using the indicated antibodies. (c) HeLa cells were exposed to EtBr and uridine to generate mitochondrial DNA depleted cells (HeLa ρ ⁰). The ratio of mtDNA to nDNA was compared in HeLa parental and ρ ⁰ cells using RT-PCR (∗∗∗p < 0.001). (d) Electron micrographs of mitochondria in HeLa parental and ρ ⁰ cells. Scale bar = 0.5 μm. (e) Basal oxygen consumption rates (OCR) of HeLa parental and ρ ⁰ cells (n = 3; mean ± S.D.; ∗∗∗∗p < 0.0001). (f) HeLa ρ ⁰ cells were treated with 100 nM bortezomib (BZ) or 1μM b-AP15 for 5 h followed by western blot analysis for K48-linked polyubiquitin chains, Nrf-2, HO-1, MT-COXII, and β-actin. Note the impaired induction of Nrf-2 and HO-1 by UPS inhibitors in ρ ⁰ cells. (g) The ratio of GSSG/GSH was determined in parental HeLa and ρ ⁰ cells exposed to 1 μM b-AP15 or vehicle for 6 h (n = 3; mean ± S.D.; ∗∗p < 0.01). (h) HCT116 cells were exposed to 0.5% DMSO, 1 μM b-AP15, 5 μM CB113, 5 μM CB826, 5 μM CB916, and 1.5 μM auranofin (AUF) for 6 h, and extracts were prepared and subjected to immunoblotting using the indicated antibodies.

Figure 4

Increased levels of proteotoxic stress are associated with decreased mitochondrial function and increased induction of HO-1. (a) HCT116 cells were exposed to 0.5% DMSO, 1 μM b-AP15, and 10 μM CpdA for 6 h, as indicated. Extracts were prepared and subjected to immunoblotting using the indicated antibodies. Note the increased levels of polyubiquitinated proteins, Hsp70, and HO-1 in cells exposed to b-AP15 and the ER translocation inhibitor CpdA. (b, c) HCT116 cells were treated with b-AP15 (1 μM) and/or CpdA (10 μM) for 5 hours and oxygen consumption rates were measured using a Seahorse XF analyzer (n = 3 in each group). A: DMSO or compounds; B: oligomycin; C: FCCP; D: antimycin and rotenone. (b) Measurement of OCR in real time after exposure to different compounds; (c) left: basal OCR after 300 min of treatment with compounds (mean ± S.D.; ∗∗∗p < 0.0001; n = 3); right: uncoupled OCR after addition of FCCP (mean ± S.D.; ∗∗∗p < 0.0001; ∗p < 0.05; n = 3).

Figure 5

Proteomic analysis of mitochondrial proteins. (a) Volcano plot showing log₂(fold change) versus p values for proteins from isolated mitochondria prepared from HCT116 cells treated with DMSO or 1 μM b-AP15 for 6 h. (b) Top candidates with significant changes from (a) (p ≤ 0.05, log₂ ≥ 0.4 or log₂ < −0.4). (c) Volcano plot showing log₂(fold change) versus p values for proteins from isolated mitochondria prepared from HCT116 cells treated with DMSO or 1 μM b-AP15 and 10 μM CpdA for 6 h. (d) Top candidates with significant changes from (c) (p ≤ 0.05, log₂ ≥ 0.4 or log₂ < −0.4). (e, f) Upper part: HCT116 cells were exposed to 0.5% DMSO, 1 μM b-AP15 in the presence or absence of 10 μM CpdA for 1, 3, and 6 h, as indicated. Extracts were prepared and subjected to immunoblotting using antibodies to Hsp60 and β-actin. (e, f) Lower part: mitochondria were purified from cells treated with 1 μM b-AP15 in the presence or absence of 10 μM CpdA and analyzed by shotgun proteomics. Data was normalized to control samples (treated with 0.5% DMSO).