Human cervical adenocarcinoma HeLa cells from the American Type Culture Collection (Manassas, VA, USA) were cultured in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA). This medium was supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco-BRL, Grand Island, NY, USA). The HeLa cells were maintained in a humidified incubator containing 5% CO₂ at 37°C. The cells were routinely grown in 100 mm plastic tissue culture dishes (Nunc, Roskilde, Denmark) and harvested with a solution of trypsin-EDTA (Gibco-BRL).

Au was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) at 10 mM as a stock solution. The 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 the caspase-9 inhibitor benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethylketone (Z-LEHD-FMK) were obtained from R&D Systems, Inc. (Minneapolis, MN, USA) and were dissolved in DMSO at 10 mM to serve as stock solutions. N-acetyl cysteine (NAC) and L-buthionine sulfoximine (BSO), obtained from Sigma-Aldrich, were dissolved in buffer (20 mM HEPES at pH 7.0) and water at 100 mM as a stock solution, respectively. Cells were pretreated with 15 μM caspase inhibitors, 2 mM NAC or 10 μM BSO for 1 h prior to Au treatment. DMSO (0.01%) was used as a control vehicle and it did not affect cell growth or cell death.

To determine the effect of Au on cell growth, the absorbance of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) was measured in living cells, as described previously (23). Breifly, 5×10³ cells were seeded in 96-well microtiter plates (Nunc). After exposure to the indicated doses of Au with or without 15 μM of each caspase inhibitor, 2 mM NAC or 10 μM BSO for the designated times, MTT solution (20 μl: 2 mg/ml in phosphate buffered saline; PBS) was added to each well of the 96-well plates. The plates were incubated for 3 h at 37°C. Medium was removed from the plates by pipetting and 200 μl DMSO was added to each well for solubilizing the formazan crystals. The optical density was measured at 570 nm using a microplate reader (Synergy™ 2; BioTek Instruments Inc., Winooski, VT, USA).

Sub-G1 cell analysis was determined by propidium iodide (PI; Ex/Em=488/617 nm; Sigma-Aldrich) staining, as described previously (24). In brief, 1×10⁶ cells were incubated in a 60 mm culture dish (Nunc) with the designated doses of Au for 24 h. Cells were washed again with PBS and incubated with PI (10 mg/ml) with simultaneous RNase treatment at 37°C for 30 min. Cellular DNA content was measured using a FACStar flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed using Lysis II and Cellfit software (Becton Dickinson).

To determine apoptotic cell death, cells were stained with annexin V-FITC (Invitrogen Life Technologies, Carlsbad, CA, USA; Ex/Em=488/519 nm), as described previously (24). In brief, 1×10⁶ cells in a 60 mm culture dish (Nunc) were incubated with the designated doses of Au, with or without 15 μM of each caspase inhibitor, 2 mM NAC or 10 μM BSO for 24 h. Cells were washed twice with cold PBS and then resuspended in 500 μl binding buffer (10 mM HEPES/sodium hydroxide pH 7.4, 140 mM sodium chloride and 2.5 mM CaCl₂) at a concentration of 1×10⁶ cells/ml. Annexin V-FITC (5 μl) and PI (1 μg/ml) were then added and the cells were analyzed with a FACStar flow cytometer (Becton Dickinson).

The expression of proteins was evaluated using western blot analysis, as previously described (25). In brief, 1×10⁶ cells were incubated in a 60 mm culture dish (Nunc) with the designated doses of Au for 24 h. The cells were then washed in PBS and suspended in five volumes of lysis buffer [20 mM HEPES, pH 7.9, 20% glycerol, 200 mM potassium chloride, 0.5 mM EDTA, 0.5% NP40, 0.5 mM DTT and 1% protease inhibitor cocktail (Sigma-Aldrich)]. The concentration of supernatant proteins was determined using the Bradford method. Supernatant samples containing 30 mg total protein were resolved by 7.5 or 12.5% SDS-PAGE gels depending on the sizes of target proteins, transferred onto Immobilon-P polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) by electroblotting and then probed with anti-poly ADP ribose polymerase, monoclonal rabbit anti-B-cell lymphoma 2 (Bcl-2), polyclonal rabbit anti-BCL2-associated X protein (Bax) purchased from Cell signaling Technology, Inc. (Danvers, MA, USA), polyclonal rabbit anti-Trx1, monoclonal mouse anti-TrxR1, polyclonal rabbit anti-Trx2, polyclonal goat anti-TrxR2 and monoclonal goat anti-β-actin antibodies (Santa Cruz Biotechnology, Inc.). Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and blots were developed using an ECL kit (Amersham, Arlington Heights, IL, USA).

To measure MMP levels, a rhodamine 123 fluorescent dye (Sigma-Aldrich; Ex/Em=485/535 nm) was used as described previously (24,26). In brief, 1×10⁶ cells in a 60 mm culture dish (Nunc) were incubated with the designated doses of Au for 24 h. Cells were washed twice with PBS and incubated with the rhodamine 123 (0.1 mg/ml) at 37°C for 30 min. Rhodamine 123 staining intensity was determined using a FACStar flow cytometer. Rhodamine 123 negative cells indicated a MMP loss in cells.

Necrosis in the cells treated with Au was evaluated using an LDH kit (Sigma-Aldrich). In brief, 1×10⁶ cells were incubated in a 60 mm culture dish (Nunc) with the indicated doses of Au, with or without 15 μM caspase inhibitors, 2 mM NAC or 10 μM BSO for 24 h. Following treatment, the culture media were collected and centrifuged for 5 min at 211 × g. Media supernatant (50 μl) was added to a fresh 96-well plate with LDH assay reagent and incubated at room temperature for 30 min. The absorbance values were measured at 490 nm using a microplate reader. LDH release was expressed as the percentage of extracellular LDH activity compared with the control cells.

The level of intracellular O₂^•− was specifically detected using dihydroethidium (DHE; Ex/Em=510/580 nm; Invitrogen Life Technologies). In brief, 1×10⁶ cells were incubated in a 60 mm culture dish (Nunc) with the designated doses of Au, with or without 15 μM caspase inhibitors, 2 mM NAC or 10 μM BSO for 24 h. Cells were then washed in PBS and incubated with 20 μM DHE at 37°C for 30 min. DHE fluorescence was detected using a FACStar flow cytometer (Becton Dickinson). Levels of mitochondrial O₂^•− are expressed as mean fluorescence intensity, which was calculated using CellQuest software (Becton Dickinson).

Levels of cellular GSH were analyzed using a 5-chloromethylfluorescein diacetate dye (CMFDA; Ex/Em=522/595 nm; Invitrogen Life Technologies), as previously described (27). In brief, 1×10⁶ cells were incubated in a 60 mm culture dish (Nunc) with the designated doses of Au, with or without 15 μM caspase inhibitors, 2 mM NAC or 10 μM BSO for 24 h. After washing with PBS, the cells were incubated with 5 μM CMFDA at 37°C for 30 min. The intensity of 5-chloromethylfluorescein (CMF) fluorescence was determined using a FACStar flow cytometer. The percentage of negative CMF cells indicated GSH depletion of cells.

The activity of TrxR was assessed using the Thioredoxin Reductase assay kit according to the manufacturer’s instructions (Sigma-Aldrich). In brief, 1×10⁶ cells were incubated in a 60 mm culture dish (Nunc) with the indicated doses of Au for 24 h. The cells were then washed in PBS and suspended in five volumes of lysis buffer (R&D systems, Inc.). 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, BioTek^® Instruments Inc.).

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

Initially, the effect of Au on the growth inhibition of HeLa cells was examined using MTT assays. Following exposure to Au for 24, 48 and 72 h, the growth of HeLa cells dose and time-dependently decreased with an IC₅₀ of ~2, 1.5 and 1 μM at 24, 48 and 72 h, respectively (Fig. 1A).

As shown in Fig. 1B, Au did not affect the proportion of sub-G1 cells in the HeLa cells at 24 h. However, when HeLa cells were stained with annexin V-FITC to evaluate the induction of apoptosis, the percentages of annexin V-staining cells increased in a dose-dependent manner in Au-treated HeLa cells (Fig. 1C). This agent decreased the expression of PARP-1 and Bcl-2, whereas it increased the expression of Bax (Fig. 1D). In addition, Au triggered the loss of MMP and increased the release of LDH in HeLa cells at 24 h in a dose-dependent manner (Fig. 1E and F). These results indicated that Au induced necrosis as well as apoptosis in the HeLa cells.

Alterations in the levels of intracellular O₂^•− and GSH in Au-treated HeLa cells were also examined. As shown in Fig. 2A, Au significantly increased the levels of intracellular O₂^•− (DHE) in HeLa cells at 24 h. Au, a known inhibitor of TrxR, also decreased the activities of TrxR in HeLa cell lysates and cells (Fig. 2B). In addition, examination of the expression of Trx system-related proteins demonstrated that the levels of TrxR1 and Trx2 were downregulated by Au (Fig. 2B). Additionally, Au markedly increased the percentage of GSH-depleted cells in HeLa cells at 24 h (Fig. 2C).

To determine which caspases were involved in the death of Au-treated HeLa cells, the effects of caspase inhibitors in those cells were assessed. For this experiment, 2 μM Au was selected as a suitable dose to differentiate the level of cell death in the presence or absence of each of the following caspase inhibitors: pan-caspase inhibitor (Z-VAD), caspase-3 inhibitor (Z-DEVD), caspase-8 inhibitor (Z-IELD) and the caspase-9 inhibitor (Z-LEHD). The concentration of each caspase inhibitor, 15 μM, was selected as the optimal dose for the present study as it did not significantly affect cell death in the control cells (28). Among the caspase inhibitors, Z-VAD significantly attenuated apoptosis induced by Au in HeLa cells at 24 h (Fig. 3A). Although Z-DEVD and Z-IETD also partially prevented apoptotic cell death in Au-treated HeLa cells, Z-LEHD did not have an effect (Fig. 3A). Furthermore, all caspase inhibitors, particularly Z-VAD, decreased the release of LDH triggered by Au in HeLa cells (Fig. 3B). Therefore, a variety of caspases appeared to be involved in apoptotic and necrotic cell death in Au-treated HeLa cells.

In association with the levels of O₂^•− and GSH, Z-VAD and Z-DEVD significantly reduced levels of O₂^•− (DHE) in HeLa cells treated with Au and prevented the depletion of GSH in those cells (Fig. 3C and D). While Z-IETD prevented depletion of GSH in Au-treated HeLa cells, it did not affect levels of O₂^•− in those cells (Fig 3C and D). Z-LEHD did not result in any alterations in the levels of O₂^•− or GSH (Fig. 3C and D).

The effects of NAC and BSO on cell growth, cell death and levels of ROS and GSH were then assessed in 2 μM Au-treated HeLa cells at 24 h. As shown in Fig. 4A, NAC significantly recovered the inhibition of cell growth induced by Au. NAC also prevented apoptotic cell death and release of LDH in Au-treated HeLa cells (Fig. 4B and C). However, as an inhibitor of GSH synthesis, BSO enhanced the inhibition of cell growth, apoptosis and the release of LDH induced by Au in HeLa cells (Fig. 4A–C). In the assessment of whether NAC and BSO affect the levels of O₂^•− and GSH in Au-treated HeLa cells, NAC attenuated the levels of O₂^•− and the depletion of GSH in these cells (Fig. 4D and E). By contrast, BSO significantly enhanced the levels of O₂^•− and the depletion of GSH induced by Au in HeLa cells (Fig. 4D and E).