Human mesothelial cells (HM69 and HM72) and human mesothelioma cells (ADA, CON, Hmeso, Mill, Phi, REN and ROB) were obtained from Queen's Medical Center (Honolulu, HI, USA). These cells were cultured in Ham's F-12 media containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (both from Gibco BRL, Grand Island, NY, USA). Mesothelial and mesothelioma cells were maintained in incubator containing 5% CO₂ at 37°C. Cells were grown in 100 mm plastic cell culture dishes (BD Falcon, Franklin Lakes, NJ, USA) and harvested with a trypsin-EDTA (Gibco BRL).

Auranofin purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich Chemical Co., St. Louis, MO, USA) at 10 mM as a stock solution. The pan-caspase inhibitor (Z-VAD-FMK; benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) was obtained from R&D Systems, Inc. (Minneapolis, MN, USA) and were dissolved in DMSO at 10 mM. NecroX-2 and necrostatin-1 from Enzo Life Science (Plymouth Meeting, PA, USA) were dissolved in DMSO at 1 and 50 mM, respectively. NAC and BSO obtained from Sigma-Aldrich Chemical Co. were dissolved in 20 mM HEPES (pH 7.0) and water at 100 mM, respectively. Cells were pretreated with 15 μM Z-VAD, 1 μM NecroX-2, 50 μM necrostatin-1, 2 mM NAC or 10 μM BSO for 1 h prior to auranofin treatment.

The protein expression levels were evaluated by western blot analysis. In brief, 1×10⁶ cells in 60 mm culture dish (BD Falcon) were incubated with or without 3 μM auranofin for 24 h. Then cells were washed with phosphate-buffered saline (PBS) and added in 4 volumes of protein extract buffer (Life Technologies, Carlsbad, CA, USA). The concentrations of protein were determined using the Bradford method. A total of 30 μg total proteins were resolved by 4–20% SDS-PAGE gels, and then transferred to Immobilon-P PVDF membranes (Millipore, Billerica, MA, USA) by electroblotting. Then membranes were probed with anti-PARP and anti-c-PARP (Cell signaling Technology, Danvers, MA, USA) and anti-TrxR1, anti-GAPDH and anti-β-actin (Santa Cruz Biotechnology). Membrane was incubated with fluorescence-conjugated secondary antibodies. Bands were visualized by using a LI-COR Odyssey Imager (LI-COR Biosciences, Lincoln, NE, USA).

The effect of auranofin on proliferation in mesothelioma cells was determined by CellTiter 96^® AQueous Non-Radioactive Cell Proliferation Assay kit (Promega, Madison, WI, USA). In brief, 5×10³ cells in 96-well microtiter plate (BD Falcon) were incubated with the indicated concentrations of auranofin with or without NAC or BSO for 24 h. Then, 20 μl of 3-(4,5-dimethylthazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosulfate (PMS) mixture was added to each well in 96-well plates. The plates were incubated for 3 h at 37°C. The optical density was measured at 490 nm using a microplate reader (VersaMax plate reader; Molecular Devices, Sunnyvale, CA, USA).

Sub-G1 analysis was determined by propidium iodide (PI; Sigma-Aldrich Chemical Co.; Ex/Em=488/617 nm) staining. Briefly, 1×10⁶ cells in 60 mm culture dish (BD Falcon) were incubated with the indicated concentrations of auranofin with or without Z-VAD, NecroX-2 or necrostatin-1 for 24 h. Cells were washed with PBS and then incubated with 10 μg/ml PI with RNase at 37°C for 30 min. Sub-G1 DNA content cells were analyzed with an Accuri C6 flow cytometer (BD Sciences, Franklin Lakes, NJ, USA).

Apoptosis was detected by staining cells with Annexin V-fluorescein isothiocyanate (FITC; Life Technologies; Ex/Em=488/519 nm) and PI (Sigma-Aldrich Chemical Co.). Briefly, 1×10⁶ cells in 60 mm culture dish (BD Falcon) were incubated with the indicated concentrations of auranofin with or without Z-VAD, NecroX-2, necrostatin-1, NAC or BSO for 24 h. Then cells were washed twice with cold PBS and added 500 μl of binding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) at a concentration of 1×10⁶ cells/ml. Five microliters of Annexin V-FITC and PI were added to these cells, which were analyzed with Accuri C6 flow cytometer (BD Sciences).

Necrosis in cells was evaluated by LDH kit (Sigma-Aldrich Chemical Co.) Briefly, 1×10⁶ cells in 60 mm culture dish (BD Falcon) were incubated with the indicated concentration of auranofin with or without NAC or BSO for 24 h. After treatment, the cell culture media were collected and centrifuged for 5 min at 1,500 rpm. A total of 50 μl of the media supernatant was added to a fresh 96-well plate (SPL Life Sciences, Pocheon, Gyeonggi-do, Korea) with LDH assay reagent and then incubated at room temperature for 30 min. The absorbance values were measured at 490 nm using a microplate reader (Synergy™ 2; BioTek^® Instruments Inc., Winooski, VT, USA). LDH release was expressed as the percentage of extracellular LDH activity compared with the control cells.

Intracellular ROS such as H₂O₂, ^•OH and ONOO^• were detected by an oxidation-sensitive fluorescent probe dye, 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA, Life Technologies; Ex/Em=495/529 nm). As H₂DCFDA is poorly selective for O₂^•−, dihydroethidium (DHE, Life Technologies; Ex/Em=518/605 nm), which is highly selective for O₂^•−, was used for its detection. Briefly, 1×10⁶ cells in 60 mm culture dish (BD Falcon) were incubated with the indicated concentrations of auranofin with or without NAC or BSO for 24 h. The cells were washed in PBS and incubated with 20 μM H₂DCFDA and DHE at 37°C for 30 min. DCF and DHE fluorescences were detected by using Accuri C6 flow cytometer (BD Sciences).

Cellular GSH levels were analyzed using a 5-chloromethylfluorescein diacetate dye (CMFDA, Ex/Em=522/595 nm; Life Technologies). In brief, 1×10⁶ cells were incubated in a 60 mm culture dishes (BD Falcon) with 3 μM auranofin with or without NAC or BSO for 24 h. Cells were then washed with PBS and incubated with 5 μM CMFDA at 37°C for 30 min. CMF fluorescence intensity was determined using Accuri C6 flow cytometer (BD Sciences). Negative CMF staining (GSH-depletion) of cells is expressed as the percentage of (−) CMF cells.

The results represent the mean of at least three independent experiments (mean ± SD). Data were analyzed using Instat software (GraphPad Prism4, San Diego, CA, USA). The Student's t-test or one-way analysis of variance (ANOVA) with post hoc analysis using Tukey's multiple comparison test was used for parametric data. P<0.05 was considered to indicate a statistically significant difference.

Firstly, we observed the protein expression levels of TrxR1 in human mesothelial and mesothelioma cells. As a result, there was no difference of TrxR1 expression in either mesothelial or mesothelioma cells (Fig. 1A). Instead, the levels of TrxR1 in ADA, CON and Hmeso were lower than those in other mesothelioma cells (Fig. 1A). Treatment with auranofin attenuated the proliferation of mesothelioma cells in a dose-dependent manner (Fig. 1B). ADA and CON cells, which showed lower TrxR1 levels, were more sensitive to auranofin than Mill and Phi cells (Fig. 1B). Auranofin completely reduced the level of TrxR1 in ADA cells and this agent also decreased that of Phi cells (Fig. 1B). In addition, auranofin increased LDH release, sub-G1 cells and Annexin V positive cells in ADA and Phi cells (Fig. 1C–E). LDH release was high in Phi cells whereas sub-G1 cells and Annexin V positive cells were high in ADA cells. It also induced a cleavage in PARP protein in ADA and Phi cells (Fig. 1F).

It was investigated whether auranofin induces apoptosis and/or necrosis in ADA cells. When auranofin-treated ADA cells were co-incubated with Z-VAD, a pan-caspase inhibitor, Z-VAD did not change the percentages of sub-G1 and Annexin V positive cells in these cells (Fig. 2A and B). In contrast, NecroX-1, necrosis inhibitor, decreased the numbers of sub-G1 and Annexin V positive cells in auranofin-treated ADA cells and necrostatin-1, necroptosis inhibitor, reduced the numbers of decreased sub-G1 and Annexin V positive cells in these cells as well (Fig. 2C and D).

Auranofin is an inhibitor of TrxR1. Therefore, it can induce cell death through an oxidative stress. We pre-treated ADA and Phi cells with 2 mM NAC for 1 h prior to the treatment of auranofin. NAC significantly recovered the reduced cell proliferation caused by auranofin in ADA and Phi cells (Fig. 3A). NAC also inhibited auranofin-induced LDH release in both cells (Fig. 3B). NAC significantly prevented cell death in auranofin-treated ADA and Phi cells, and the prevention was dramatic in ADA cells (Fig. 3C). As expected, auranofin increased ROS levels including O₂^•− in ADA and Phi cells at 24 h, and NAC decreased the levels in these cells (Fig. 4A and B). Auranofin also induced GSH depletion in ADA and Phi cells (Fig. 4C and D). NAC completely blocked the GSH depletion caused by auranofin in ADA and Phi cells (Fig. 4C and D).

There are two main antioxidant systems, Trx and GSH in cells. For this reason, inhibition of GSH synthesis might be a novel strategy to disturb the redox status and finally lead to cell death. As expected, BSO intensified the inhibition of cell proliferation in Phi cells, which were relatively resistant to auranofin compared with ADA cells (Fig. 3A). BSO did not additionally increase the LDH release in auranofin-treated ADA and Phi cells (Fig. 3B). However, BSO significantly increased apoptotic cell death in auranofin-treated ADA and Phi cells (Fig. 3C). BSO also augmented the increased ROS levels including O₂^•− in auranofin-treated cells and the augmentation was strong in Phi cells (Fig. 4A and B). Moreover, BSO significantly increased the numbers of GSH-depleted cells in auranofin-treated Phi cells (Fig. 4D).