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. 2014 Jul 30;5(14):5453-71.
doi: 10.18632/oncotarget.2113.

Clinically used antirheumatic agent auranofin is a proteasomal deubiquitinase inhibitor and inhibits tumor growth

Ningning Liu  1 , Xiaofen Li  2 , Hongbiao Huang  2 , Chong Zhao  2 , Siyan Liao  2 , Changshan Yang  2 , Shouting Liu  2 , Wenbin Song  2 , Xiaoyu Lu  2 , Xiaoying Lan  2 , Xin Chen  2 , Songgang Yi  2 , Li Xu  3 , Lili Jiang  2 , Canguo Zhao  2 , Xiaoxian Dong  2 , Ping Zhou  2 , Shujue Li  4 , Shunqing Wang  2 , Xianping Shi  2 , Ping Q Dou  5 , Xuejun Wang  6 , Jinbao Liu  2
Affiliations

Clinically used antirheumatic agent auranofin is a proteasomal deubiquitinase inhibitor and inhibits tumor growth

Ningning Liu et al. Oncotarget. .

Abstract

Proteasomes are attractive emerging targets for anti-cancer therapies. Auranofin (Aur), a gold-containing compound clinically used to treat rheumatic arthritis, was recently approved by US Food and Drug Administration for Phase II clinical trial to treat cancer but its anti-cancer mechanism is poorly understood. Here we report that (i) Aur shows proteasome-inhibitory effect that is comparable to that of bortezomib/Velcade (Vel); (ii) different from bortezomib, Aur inhibits proteasome-associated deubiquitinases (DUBs) UCHL5 and USP14 rather than the 20S proteasome; (iii) inhibition of the proteasome-associated DUBs is required for Aur-induced cytotoxicity; and (iv) Aur selectively inhibits tumor growth in vivo and induces cytotoxicity in cancer cells from acute myeloid leukemia patients. This study provides important novel insight into understanding the proteasome-inhibiting property of metal-containing compounds. Although several DUB inhibitors were reported, this study uncovers the first drug already used in clinic that can inhibit proteasome-associated DUBs with promising anti-tumor effects.

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Figures

Figure 1
Figure 1. Auranofin (Aur) induces cell apoptosis in human HepG2 and MCF-7 cells
(A) Cytotoxic effects of Aur on HepG2 and MCF-7 cells. HepG2 and MCF-7 cells were exposed to Aur in various concentrations for 24 or 48 h, and then were subjected to MTS assay. Data from three biological repeats are presented. Mean±SD (n=3). (B, C) Cell death induction by Aur in HepG2 and MCF-7. HepG2 and MCF-7 cells were treated with different doses of Aur for 12 or 24 h, then apoptotic cells were detected by Annexin V-FITC / Propidium iodide (PI) double staining, and the stained cells were either recorded using an inverted fluorescence microscope (Axio Obsever Z1, Zeiss, Germany) or detected by flow cytometry (FACScan, Becton-Dickinson). Representative images of the 24 h time point are shown in (B). Cell death data at 12 and 24 h are summarized in (C). Mean±SD (n=3). *P<0.05, compared with DMSO (DM) treatment. (D) PARP cleavage and caspase activation induced by Aur. HepG2 cells (left) and MCF-7 cells (right) were treated with Aur at the indicated doses for 18 h and then pro-caspases and PARP were detected by Western blot. GAPDH was used as a loading control.
Figure 2
Figure 2. Aur inhibits the proteasome function
(A) Accumulation of ubiquitinated proteins (Ub-prs). HepG2 and MCF-7 cells were exposed to Aur (0.5, 1.0, 2.0 μM) for 3 h and 6 h. Ub-prs were detected using antibodies against all Ub, K48-linked, or K63-linked polyubiquitin. GAPDH was used as a loading control. The western blot images were representatives from at least three independent experiments. (B) Accumulation of endogenous proteasome substrates. p21 and c-Jun proteins were detected after treatment with Aur (0.5, 1.0, 2.0 μM) or bortezomib/Velcade (Vel, 100 nM) for 9 h in both HepG2 and MCF-7 cells. (C, D) Accumulation of GFPu, a surrogate proteasome substrate. GFPu-HEK293 cells, a clonal HEK293 cell line stably transfected with GFPu (a surrogate UPS substrate created by carboxyl fusion of an enhanced green fluorescence protein with degron CL1), were treated with Aur (0.5, 1.0, 2.0 μM) for 6 h and then GFPu and Ub-prs were detected by western blot (C). Fluorescent GFPu images in the GFPu-HEK293 cells treated with Aur (2.0 μM) or Vel (50 nM) were shown in (D). (E) Comparison of the accumulation of K48-linked Ub-prs induced by Aur and Vel. K562 cells were treated with the indicated doses of Vel and Aur (0.5 μM) for 9 h and then K48-linked Ub-prs were detected by western blot analysis.
Figure 3
Figure 3. Aur inhibits the 19S proteasome DUB activity rather than 20S proteasome peptidases
(A) Computational molecular docking of Au+ with UCHL5 of 19S proteasomes. The hydrolysed form of chloro (triethylphosphine) gold or Aur, (triethylphosphine) gold cation (L2, left), and its binding mode at the active site of UCHL5 were shown (right). (B) Effect of Aur on DUB activities in cell lysate. Cell lysate was treated with Aur (2μM) or NEM (N-ethylmaleimide, 2 mM), then the DUB activity at different times was recorded by using the Ub-AMC substrate. The experiment was repeated three times, yielding the similar results. (C) Inhibition of the DUB activity in 26S proteasomes. Purified 26S proteasomes were treated with increasing doses of Aur, then DUB activity was kinetically detected as in (B). (D) NAC rescues Aur-mediated DUB inhibition. Purified 26S proteasomes were treated with Aur (2 μM), Aur+NAC (100 μM), or NEM (2 mM) for 15 min, then DUB activity was detected. (E) Ubiquitin chain disassembly assay. K48-linked ubiquitin tetramers were disassembled by the 26S proteasomes after treatment with Aur (2.0, 40 μM). (F) Active-site–directed labeling of proteasomal DUBs. Purified 26S proteasomes were treated with Aur (2.0, 40 μM) for 10 min and then labeled with HA-UbVS and fractionated via SDS-PAGE. The covalently bound HA-UbVS was detected by western blot for the HA tag. (G) The effect of 26S proteasome disassembly by siRNA-mediated knockdown of RPN11 on Aur induced Ub-prs accumulation. HepG2 cells were transfected with specific siRNA against RPN11 for 48 h, and then treated with Aur (0.5, 1.0, 2.0 μM) for 6 h. Scrambled siRNA was used as control. K48-linked polyubiquitin and RPN11 protein was detected by western blot analyses. GAPDH was used as a loading control. (H) GFPu accumulation with RPN11 siRNA silencing and Aur treatment. GFPu-HEK293 cells were transfected with control siRNA or RPN11 siRNA for 48 h, and then treated with 0.5 μM Aur for 6 h. GFPu and RPN11 protein was detected by western blot analyses.
Figure 4
Figure 4. Proteasome inhibition is required for Aur to induce apoptosis
(A) The time course of proteasome inhibition, caspase activation and apoptosis induction by Aur treament. HepG2 and MCF-7 cancer cells were treated with Aur (0.5 μM), then the cells were collected at the indicated time points for western blot analyses for ubiquitinated proteins including total ubiquitin conjugates, K48- and K63-linked polyubiquitins, as well as apoptosis-related proteins (caspases and PARP) in the whole cell lysate. C-Cas: cleaved caspases. GAPDH was used as a loading control. (B) An illustration of the binding of Aur with N-acetyl-L-cysteine (NAC) to inactivate Aur. In phosphate buffered saline (PBS), NAC binds with Aur, forming a new product. (C) NAC completely reversed Aur-induced proteasome inhibition and apoptosis. HepG2 and MCF-7 cells were treated with Aur, NAC, or Aur+NAC (A+N) for 18 h. Western blot analyses for the indicated proteins were performed. (D, E) NAC completely blocked Aur from inducing apoptosis. HepG2 and MCF-7 cells were treated as in (C) for 24 h, apoptotic cells were detected with Annexin V-PI staining followed by either flow cytometry (Mean±SD, n=3) or fluorescence microscopy. Flow cytometry data were summarized in (D), *P<0.05, versus Aur-treated alone. The phase contrast and fluorescent images were shown in (E). Red stain indicates PI-positive; green stain indicates Annexin V-positive. Scale bar=50 μm.
Figure 5
Figure 5. Phenol-containing antioxidant Tertiary butylhydroquinone (Tbhq) could scavenge Aur-induced ROS generation but could not rescue Aur-induced proteasome inhibition and apoptosis.
(A, B) HepG2 cells were treated with Aur (0.5 μM), Tbhq (20 μM) or their combination for 12 h. ROS was detected by flow cytometry. Relative level of ROS was shown. Mean±SD (n=3). *P<0.05, compared with other treatments. (C) HepG2 cells were treated with increasing doses of Tbhq in the absence or presence of Aur (0.5 μM) for 24 h. Ubiquitinated proteins and PARP were detected by western blot analyses (upper). Cell death was detected by Annexin V/PI staining with flow cytometry. Mean±SD (n=3). *P<0.05, compared with Aur treatment alone. Cell viability was detected by MTS assay. Mean±SD (n=3). *P<0.05, compared with each treatment alone. (D) MCF-7 cells were treated with Aur (0.5 μM), Tbhq (5 μM) or their combination for 12 h. ROS was detected and shown as in (A). Mean±SD (n=3). *P<0.05, compared with Aur treatment alone, (E, F, G) MCF-7 cells were treated as in (D) for 24 h. Cell death and cell viability were detected as in (B). Cell death images and summary were shown in (E, F), and cell viability was shown in (G). Mean±SD (n=3). *P<0.05, compared with Aur control for cell death; compared with vehicle control for cell viability.
Figure 6
Figure 6. Aur interferes with multiple apoptosis-related signal pathways in cancer cells
(A) CHOP and caspase 12 (Cas-12) protein expression. HepG2 and MCF-7 cells were exposed to Aur (0.5, 1.0, 2.0 μM) for 18 h. Western blot was performed for the detection of the ER stress-related proteins CHOP and Cas-12. (B) Changes in cytoplasmic IκBα and nuclear NF-κB p65 protein levels. HepG2 and MCF-7 cells were treated with Aur (0.5, 1.0, 2.0 μM) for 12 h. Cytoplasmic and nuclear proteins were extracted for western blot analyses for IκBα and NF-κB p65, respectively. GAPDH and histone 3 were used as cytoplasmic and nuclear protein loading control, respectively. (C) Mitochondrial membrane potential (ΔΨm) depolarization. As treated in (B), loss of ΔΨm was detected by flow cytometry. Representative flow images were shown (upper) and the quantitative data were summarized (lower). Mean±SD (n=3). *P<0.05, versus control. (D) HepG2 and MCF-7 cells were co-treated with Aur (0.5 μM) and z-VAD-FMK (50μM) for 18 h. Ub-prs and PARP proteins were assessed by western blotting. GAPDH was used as a loading control. (E) HepG2 and MCF-7 were treated as in (D) for 24 h, then apoptotic cells were detected with PI/annexin V staining, followed by either imaging under an inverted fluorescent microscope or detecting by flow cytometry. Representative phase contrast and fluorescent images were shown in (E, left). Red indicates PI-positive; green indicates annexin V-positive. Scale bar=50 μm. Cell death data by flow cytometry were shown in (E, right). Mean±SD (n = 3). #P<0.05, versus DM control; *P<0.05, versus Aur treatment alone.
Figure 7
Figure 7. Aur inhibits tumor growth and the proteasome of tumor xenografts in mice without affecting mouse body weight
BALB/c nude mice bearing HepG2 and MCF-7 tumors were treated with vehicle or Aur (6 mg/kg/day, i.p.) for 15 and 21 days, respectively. Tumor size was recorded every other day. Tumor images and tumor weight (A), tumor size (B) and body weight (C) and were shown. *P<0.05, compared with the control. (D) Representative micrographs of immunohistochemistry staining for total (Ub-prs), K48-linked (K48-), or K63-linked (K63-) ubiquitinated proteins and the indicated proteasome substrate proteins (c-Jun and p21) in nude mouse tumor tissues. All the immunostaining was repeated in three mouse tumor tissues and the images shown were collected at a magnification of 200×.
Figure 8
Figure 8. Aur inhibits the proteasome and specifically induces cytotoxicity in cancer cells from acute myeloid leukemia (AML) patients
(A) Cancer cells from 6 AML patients (Pt) and peripheral blood mononuclear cells from 6 healthy volunteers (Nm) were treated with Aur at the indicated doses or with Vel (50 nM) for 24 h and the cell viability was detected by the MTS assay. The scatter plot of the IC50 values in each group was shown (A, left). *P<0.05, versus patients. Cell viability with Vel treatment in each group was shown (A, right). Mean±SD (n=3). *P<0.05, versus AML patients. (B, C) Cancer cells from 3 AML patients or the peripheral mononuclear cells from 3 normal human individuals were incubated with Aur at the indicated doses or with Vel (50 nM) for 12 h. Cell death was analyzed by flow cytometry. The typical images from flow cytometry were shown in (B, C, left) and cell death were summarized in (B, C, right). Mean±SD (n=3). (D) As treated in (B, C), cancer cells from AML patients were treated with Aur or Vel for 15 h, then cells were stained with Annexin V/PI and imaged under a fluorescent microscope. The phase contrast and fluorescent images were taken and merged. Scale bar=50 μm. (E) AML cancer cells and human peripheral mononuclear cells were treated with Aur or Vel for 6 h followed by detecting ubiquitinated proteins with western blot analyses. Western blot images of cells from 3 individuals of each group are shown. At the top of the panel, 1, 2, 3, 4, and 5 denote control, Aur (0.25, 0.5, 1.0 μM), and Vel (50 nM), respectively.
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