Human SCLC cell line (Calu-6), adenocarcinoma cell lines (A549, SK-LU-1), and large cell carcinoma cell lines (NCI-H460, NCI-H1299) were obtained from the American Type Culture Collection (Manassas, VA). Normal human pulmonary fibroblast (HPF) cells were obtained from PromoCell GmbH (C-12360, Heidelberg, Germany). These cells were maintained in an incubator containing 5% CO₂ at 37°C. HPF and lung cancer cells were cultured in RPMI-1640 containing 10% fetal bovine serum (Sigma-Aldrich Co., St. Louis, MO) and 1% penicillin-streptomycin (Gibco BRL, Grand Island, NY). Cells were grown in 100 mm plastic cell culture dishes (BD Falcon. Franklin Lakes, NJ) and harvested with trypsin-EDTA (Gibco BRL). HPF cells were used between passages of four to five.

Auranofin was purchased from Sigma-Aldrich Co. and was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich Co.) at 10 mM as a stock solution. Pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), caspase-3 inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK), caspase-8 inhibitor benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone (Z-IETD-FMK), and caspase-9 inhibitor benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethylketone (Z-LEHD-FMK) were obtained from R&D Systems, Inc. (Minneapolis, MN,) and dissolved in 10 mM DMSO as stock solutions. Cells were pretreated with 15 µM of individual caspase inhibitors for 1 h prior to the addition of auranofin. DMSO (0.01%) was used as a control vehicle and did not affect cell growth or cell death.

The effects of auranofin on the proliferation of HPF and lung cancer cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich Co.) assays. Briefly, 3×10⁴ cells were seeded into 96-well microtiter plates (Nunc). After incubation with the indicated doses of auranofin for 24 h, 20 µl of MTT solution [2 mg/ml in phosphate-buffered saline (PBS; GIBCO BRL)] was added to each well. The plates were incubated for 4 h at 37°C. Medium in plates was removed by pipetting, and 100–200 µl of DMSO was added to each well to solubilize formazan crystals. Optical density was measured at 570 nm using a microplate reader (Synergy™ 2, BioTekR Instruments Inc. Winooski, VT).

Necrosis in HPF and lung cancer cells treated with auranofin were evaluated by LDH kit (Sigma-Aldrich Co.) Briefly, 1×10⁶ cells in 60 mm culture dishes (BD Falcon) were incubated with the indicated concentrations of auranofin for 24 h. After treatment, cell culture media were collected and centrifuged for 5 min at 200 × g at room temperature. Supernatants (50 µl) were added to 96-lawell plates along with LDH assay reagent and incubated at room temperature for 30 min. Absorbance values were measured at 490 nm using a microplate reader (Synergy™ 2). LDH release was expressed as the percentage of extracellular LDH activity compared with untreated control cells.

Cell cycle and sub-G1 distributions of cells were determined by propidium iodide (PI, Sigma-Aldrich Co.; Ex/Em=488 nm/617 nm) staining, as previously described (21). Briefly, 1×10⁶ cells in 60 mm culture dishes (BD Falcon) were incubated with the indicated concentrations of auranofin for 24 h. After washing whole cells including floating cells with PBS, cells were fixed in 70% ethanol. These cells were washed with PBS twice and then incubated with PI (10 µg/ml) and RNase (Sigma-Aldrich) at 37°C for 30 min. Proportions of cells in different phases of cell cycle or with sub-G1 DNA content were measured and analyzed with a FAC Star flow cytometer (BD Sciences, Franklin Lakes, NJ, USA).

Apoptosis was identified by staining with annexin V-fluorescein isothiocyanate (FITC, Life Technologies; Ex/Em=488/519 nm), as previously described (22). Briefly, 110⁶ cells in 60 mm culture dishes (BD Falcon) were incubated with the indicated concentrations of auranofin for 24 h with or without individual caspase inhibitors. Cells were washed twice with cold PBS and then suspended in 200 µl of binding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) at a concentration of 5×10⁵ cells/ml at 37°C for 30 min. Annexin V-FITC (2 µl) and PI (1 µg/ml) were added, and cells were analyzed with a FACStar flow cytometer (BD Sciences).

The mitochondrial membrane potential (MMP, ΔΨm) was monitored using a fluorescent dye Rhodamine 123 (Sigma-Aldrich Co.; Ex/Em=485/535 nm), a cell-permeable cationic dye, which preferentially enters into mitochondria of their typical highly negative MMP (∆Ψm). Depolarization of MMP (∆Ψm) results in the loss of Rhodamine 123 from the mitochondria and decreases the intracellular fluorescence of this dye, as previously described (23). In brief, 1×10⁶ cells in 60 mm culture dishes (Nunc) were incubated with the designated doses of auranofin for 24 h with or without 15 µM individual caspase inhibitors. Cells were washed twice with PBS and incubated with Rhodamine 123 (0.1 mg/ml) at a concentration of 5×10⁵ cells/ml at 37°C for 30 min. Rhodamine 123 staining intensities were determined using a FACStar flow cytometer. Rhodamine 123 negative (−) cells indicated MMP (∆Ψm) loss.

The protein expression levels were evaluated by western blotting. Briefly, 5×10⁶ cells in 100 mm culture dishes (BD Falcon) were incubated condition with the indicated concentrations of auranofin at 37°C for 24 h with or without pan-caspase inhibitor, (Z-VAD). Cells were washed with PBS and lysed for 30 min in RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Intron Biotechnology, Seongnam Korea). The samples were heated to 100°C for 5 min and placed on ice. Total proteins (30 µg) were resolved using 8–15% SDS-PAGE gels and then transferred to Immobilon-P PVDF membranes (Millipore) by electroblotting. Membranes were probed with anti-PARP (no. 9543, 1:1,000 dilution), anti-cleaved PARP (no. 9541, 1:1,000 dilution), anti-caspase-3 (no. 9662, 1:1,000 dilution), anti-caspase-8 (no. 9746, 1:1,000 dilution), anti-caspase-9 (no. 9502, 1:1,000 dilution), anti-cleaved caspase-3 (no. 9661, 1:1,000 dilution), anti-cleaved caspase-8 (no. 9496, 1:1,000 dilution), anti-cleaved caspase-9 (no. 9501, 1:1,000), anti-Bcl-2 (no. 2872, 1:1,000 dilution), anti-BAX (no. 2774, 1:1,000 dilution) (Cell Signaling Technology); anti-Trx1 (SC-20146, 1:1,000 dilution) and anti-GAPDH (SC-25778, 1:1,000 dilution) (Santa Cruz Biotechnology). Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) at 4°C for 1 h. Blots were developed using an EZ-Western Lumi Pico ECL solution kit (DoGen Bio Co, Seoul, Korea). All band intensities were quantified using the Image J program (Fuji Film, Tokyo, Japan).

The activity of TrxR was assessed using the Thioredoxin Reductase assay kit according to the manufacturer's instructions (Sigma-1Aldrich). In brief, 1×10⁶ cells were incubated in 60 mm culture dish (Nunc) with the indicated dose of auranofin for 24 h. The cells were then washed in PBS and suspended in five volumes of lysis buffer. Protein concentrations were determined using the Bradford method. Supernatant samples containing 30 µg total protein were used for the determination of TrxR activity. These were added to each well in 96-well microtiter plates (Nunc) with 5,5′-dithiobis (2-nitrobenzoic) acid at 25°C for 1 h. The optical density of each well was measured at 412 nm using a microplate reader (Synergy™2).

The results are reported as the mean of at least two or three independent experiments (mean ± SD). Data were analyzed using Instat software (GraphPad Prism5). The Student's t-test or one-way analysis of variance with post-hoc analysis using Tukey's multiple comparison test was used for the parametric data. Statistical significance was defined as P<0.05.

The effect of auranofin, a known inhibitor of TrxR, on the growth of normal lung cell and lung cancer cell types was examined using MTT assays. The growth of normal HPF cells showed dose-dependent inhibition with an IC₅₀ of ~3 µM (Fig. 1A) after a 24-h incubation with auranofin. The growth of Calu-6 cells was also dose-dependently reduced with an IC₅₀ of ~3 µM (Fig. 1B). The growth of A549 and SK-LU-1 cells was marginally reduced by 1–3 µM auranofin and significantly decreased by 4–5 µM auranofin (Fig. 1C and D). Auranofin inhibited the growth of NCI-H460 and NCI-H1299 cells in a dose-dependent manner with IC₅₀ of ~3 µM (Fig. 1E) and 1 µM (Fig. 1F). Furthermore, auranofin significantly decreased the activity of TrxR in Calu-6 and A549 cells (Fig. 1G). Auranofin also downregulated the expression of Trx1 protein in Calu-6 and A549 cells (Fig. S1).

LDH release was measured to determine whether auranofin causes cell necrosis. Treatment increased the release of LDH in the normal HPF cells after a 24 h incubation with 3 µM auranofin (Fig. 2A). Auranofin (3–5 µM) induced significant LDH release in Calu-6, SK-LU-1, and NCI-H460 cells in a dose-dependent manner (Fig. 2B, D and E), at 4–5 µM triggered LDH release in A549 cells (Fig. 2C), and at 1 and 3 µM concentrations increased LDH release in NCI-H1299 cells (Fig. 2F).

As growth inhibition of Calu-6 and A549 cells by auranofin could be explained by an arrest during cell cycle progression, distribution of the cells in different stages of the cell cycle were examined after a 24 h incubation with auranofin. DNA flow cytometric analysis indicated that 2 and 3 µM auranofin induced a G2/M phase arrest of the cell cycle in Calu-6 cells and that 1 µM auranofin did not affect cell cycle distributions. In addition, auranofin did not show specific cell cycle arrest in A549 cells (Fig. 3A and B). Moreover, auranofin significantly increased the percentages of sub-G1 cells in Calu-6 and A549 cells at 24 h (Fig. 3B).

Whether auranofin induces apoptosis in cells was assessed using an annexin V-staining assay. The number of annexin V-positive normal HPF and Calu-6 cells significantly increased in a dose-dependent manner after treatment with 1–5 µM auranofin (Fig. 4A and B). At 4–5 µM, the number of annexin V-positive A549 cells was greatly increased (Fig. 4C). Similarly, treatment with 1–5 µM auranofin increased the number of annexin V-positive SK-LU-1 cells (Fig. 4D). The number of annexin V-positive NCI-H460 and NCI-H1299 cells were increased after incubation in 3–5 and 1–3 µM concentrations of auranofin, respectively (Fig. 4E and F).

Since apoptosis is closely related to the collapse of MMP (∆Ψm), loss of MMP (∆Ψm) in auranofin-treated cells was assessed using Rhodamine 123 dye. Loss of MMP (∆Ψm) in the normal HPF cells was dose-dependently induced by auranofin at concentrations of 2–5 µM (Fig. 5A). Similar loss of MMP (∆Ψm) was observed after treatment of Calu-6 and SK-LU-1 cells (Fig. 5B and D), A549 cells (Fig. 5C), and NCI-H460 cells (Fig. 5E) with auranofin at 3–5, 4–5, and 2–5 µM, respectively. Concentrations of 1–3 µM auranofin did not show this effect in A549 cells (Fig. 5C). Furthermore, auranofin at concentrations of 0.1–0.5 µM significantly increased loss of MMP (∆Ψm) in NCI-H1299 cells (Fig. 5F).

As auranofin increased the number of annexin V-positive cells, levels of apoptosis-related proteins were evaluated by western blot analysis. Intact forms of PARP decreased in auranofin-treated Calu-6 and A549 cells whereas the cleavage forms of PARP increased in these cells (Fig. 6A and C). In addition, the levels of cleaved caspase-3 were dose-dependently upregulated in auranofin-treated Calu-6 and A549 cells (Fig. 6A and C). Auranofin also decreased the levels of Bcl-2 and increased the levels of BAX in Calu-6 and A549 cells (Fig. 6A and C). The ratios of cleaved PARP/total PARP and cleaved caspase-3/total caspase-3 were increased in auranofin-treated Calu-6 and A549 cells (Fig. 6B and D). All blots presented together were probed from the same membrane.

To determine which caspases were involved in auranofin-induced apoptosis, cells were pretreated with various caspase inhibitors before treatment with auranofin. Z-VAD (a pan-caspase inhibitor) significantly decreased the number of annexin V-positive Calu-6 cells treated with 3 µM auranofin (Fig. 7A). Furthermore, all of the tested caspase inhibitors (Z-DVED for caspase-3, Z-IETD for caspase-8, and Z-LEHD for caspase-9) significantly reduced the death of Calu-6 cells following auranofin treatment (Fig. 7A). In addition, all caspase inhibitors significantly protected against the loss of MMP (∆Ψm) in Calu-6 cells caused by auranofin (Fig. 7B). Likewise, all tested caspase inhibitors slightly decreased apoptotic A549 cell death following incubation with 5 µM auranofin (Fig. 7C). However, these decreases were not statistically significant. Caspase inhibitors marginally reduced the loss of MMP (∆Ψm) in auranofin-treated A549 cells (Fig. 7D). The expression of apoptosis-related proteins showed an increase in the intact form of PARP in auranofin-treated Calu-6 cells in the presence of Z-VAD and a decrease in the cleavage form of PARP in those cells (Fig. 7E). Furthermore, Z-VAD reduced cleavage forms of caspase-3, −8, and −9 in auranofin-treated Calu-6 cells (Fig. 7E). Finally, the expression of Bcl-2 in auranofin-treated cells was clearly upregulated in the presence of Z-VAD, and the levels of BAX in those cells were downregulated (Fig. 7E). The ratio of cleaved PARP/total PARP, cleaved caspase-3/total caspase-3, cleaved caspase-8/total caspase-8 and cleaved caspase-9/total caspase-9 were increased in auranofin-treated Calu-6 cells (Fig. 7F). However, these were decreased in auranofin and Z-VAD treated Calu-6 cells (Fig. 7F). All blots presented together were probed from the same membrane.