Anticancer Effects of Auranon in Human Lung Cancer Cells Through Increasing Intracellular ROS Levels and GSH Depletion

Purpose Auranon is known to inhibit thioredoxin reductase (TrxR) and has promising anticancer activity in several cancer types. However, at present, there is no clear explanation for the mechanism underlying the inhibitory effects of Auranon on lung cancer cell growth. In this study, we evaluated the antigrowth effects of Auranon in cells from various lung cancer cell lines with regard to cell death, reactive oxygen species (ROS), and glutathione (GSH) levels. Methods Cell proliferation was assessed using the trypan blue staining cell counting. ROS levels including O 2·- , GSH levels, and MMP ( ∆ Ψm) loss were measured using a ow cytometry. Apoptosis was determined with annexin V-PI staining assay and the change of apoptosis-related protein level was detected by western blotting. TrxR activity was evaluated using a thioredoxin reductase colorimetric assay kit. restored in response to the preincubation with NAC in both Calu-6 and A549 cells. Conclusion Our present ndings demonstrate that Auranon-induced cell death is tightly related to oxidative stress in lung cancer cells. and BSO on cell death, MMP (ΔΨm), the ROS level, and GSH depletion in Auranon-treated Calu-6 and A549 cells


Introduction
Lung cancer is the most frequent malignant tumor and the leading cause of cancer-related mortality and morbidity worldwide (Bray et al. 2018). Lung cancer, which arises from lung epithelial cells, can be histologically divided into two types: small-cell lung cancer (SCLC) and non-small-cell lung cancer To maintain ROS homeostasis, an antioxidant defense system including glutathione (GSH) and thioredoxin (Trx) exists to reduce ROS levels in cells (Trachootham et al. 2009). The Trx system is composed of Trx and a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent Trx reductase (TrxR) (Lu and Holmgren 2014). Trx is a small (12 kDa) reduction-oxidation (redox) protein with a catalytically active dithiol site in a cysteine residue (Cys-Gly-Pro-Cys), which can reduce disul des in target proteins. Oxidized Trx can then be reduced by the TrxR enzyme using NADPH to promote its activities ( Aurano n, as a TrxR inhibitor, is an oral gold ( )-containing phosphine compound that is approved by the United States Food and Drug Administration (FDA) for treatment of rheumatoid arthritis (Chaffman et al. 1984;Onodera et al. 2019). Although it was initially established as an anti-in ammatory drug, recent studies also revealed that Aurano n has potential therapeutic effects for various human diseases including cancer, neurodegenerative disorders, acquired immunode ciency syndrome, and parasitic and bacterial infections (Madeira et al. 2012;Roder and Thomson 2015). The pharmacological effect of Aurano n is due to its high reactivity with cellular nucleophiles such as selenocysteine and cysteine, making Aurano n a potent inhibitor of TrxR (Rigobello et al. 2004; Zhang et al. 2019). In particular, Aurano n exhibits anticarcinogenic activity that is closely related with mitochondrial dysfunction and ROS overproduction in human chronic leukemia and gastric cancer cells (Fiskus et al. 2014;Zou et al. 2015).
Although several studies have demonstrated that Aurano n exhibits potent anticancer activity in human cancer cells, its effect on human lung cancer cells and the underlying mechanisms of action in view of redox state changes still remain to be elucidated. In our previously published reports, we found that Aurano n inhibited cell growth through cell cycle arrest and cell death due to necrosis and caspasedependent apoptosis in lung cancer cells (Cui et al. 2020). In this study, we investigated the antigrowth effects of Aurano n related to the ROS level and GSH depletion in cells from various lung cancer cell lines and evaluated the cellular effects of N-acetyl cysteine (NAC; a well-known antioxidant) and L-buthionine sulfoximine (BSO, an inhibitor of GSH synthesis) on Aurano n-induced cell death in Calu-6 and A549 cells.

Cell culture
Human pulmonary broblast (HPF) cells were obtained from Promo Cell GmbH (Heidelberg, Germany). The human lung adenocarcinoma (A549 and SK-LU-1), large cell carcinoma (NCI-H460 and NCI-H1299), and Calu-6 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA).

Reagents
Aurano n was purchased from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich.) as a 10 mM stock solution. NAC and BSO were also obtained from Sigma-Aldrich. NAC was dissolved in a 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 7.0) buffer, and BSO was dissolved in distilled water. Based on previous studies, cells were pretreated with 2 mM NAC or 10 µM BSO at 37℃ for 1 h followed by treatment with Aurano n at 37℃ for 24 h before assays were performed. DMSO (0.2%) was used as a control vehicle, and it did not affect cell growth or death. All stock solutions were wrapped in foil and kept at 4℃ or − 20℃.

Cell proliferation assay
Viable and dead cell numbers were determined using the trypan blue staining method. In brief, 1 × 10 6 cells per well were seeded into 60 mm culture dishes (BD Falcon) for cell counting. After exposure to the indicated concentrations of Aurano n for 24 h, the cells then underwent trypan blue cell counting. For all experimental conditions, three replicates were used, and the experiment was performed at least twice.  TrxR activity assay TrxR activity was determined using a uorescent thioredoxin reductase colorimetric assay kit (Cayman Chemical, Ann Arbor, MI, USA). Brie y, 1 × 10 6 cells in 60 mm culture dishes (BD Falcon) were pretreated with 2 mM NAC or 10 µM BSO for 1 h and then treated with the indicated concentrations of Aurano n for 24 h. Cells were homogenized in a cold radioimmunoprecipitation assay (RIPA) buffer (CureBio, Seoul, Korea) and centrifuged at 13,500 × g for 20 min at 4℃. The supernatant was collected after centrifugation, and protein concentrations were then determined using the Bradford method with a Bio-Rad assay kit (Bio-Rad Laboratories., Hercules, CA, USA). Equal amounts of total protein (30 µg) were added to each well in a Nunc 96-well plate (Thermo Fisher Scienti c, Waltham, MA, USA) with a master mix containing 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) and NADPH. The uorescence intensity of each well was measured at 410 nm using a Synergy™ 2 spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). TrxR activity was calculated using a formula provided by the protocol.

Statistical analysis
The results are reported as the mean of at least two or three independent experiments (mean ± SD). Data were analyzed using Instat (GraphPad, San Diego, CA, USA). The Student's t-test or one-way analysis of variance with a post-hoc analysis using Tukey's multiple comparison test was used for the parametric data. Statistical signi cance was de ned as p < 0.05.

Results
Effects of Aurano n on cell growth and death in lung cancer cells in SK-LU-1 cells (Fig. 1D), and 1-3 µM in NCI-H1299 cells (Fig. 1F). These results indicate that normal lung cells and cells from different lung cancer cell lines have differential sensitivity to Aurano n.

Effects of Aurano n on intracellular ROS levels in lung cancer cells
To assess intracellular ROS levels in Aurano n-treated normal lung cells and cells from lung cancer cell lines at 24 h, two uorescent probe dyes (H 2 DCFDA and DHE) were used to detect non-speci c ROS and O 2 •− levels, respectively. We found that Aurano n affected ROS (DCF) and O 2 •− (DHE) levels depending on the treatment dose and cell type (Figs. 2 and 3). Among the tested concentrations of Aurano n, treatment with 4 µM signi cantly increased the ROS level in HPF cells (#5), but 5 µM Aurano n did not ( Fig. 2A), whereas treatment with 3 µM Aurano n led to a maximum ROS level in Calu-6 cells, yet 4-5 µM Aurano n decreased the ROS level (Fig. 2B). In A549 and NCI-H460 cells, we found that all tested concentrations of Aurano n increased ROS levels, but these levels were not raised in a dose-dependent manner ( Fig. 2C and 2E). ROS levels in 4 µM Aurano n-treated A549 and 3 µM Aurano n-treated NCI-H460 cells were the highest (Figs. 2C and 2D). Treatment with 4 µM Aurano n led to a maximum level of ROS in SK-LU-1 cells, whereas 5 µM Aurano n did not affect the ROS level (Fig. 2D). In NCI-H1299 cells, all tested concentrations of Aurano n generally decreased ROS levels, and the levels of 2-3 µM Aurano n-treated cells were remarkably reduced compared to untreated cells (Fig. 2F). The level of red uorescence derived from DHE, which re ects O 2 •− accumulation, gradually increased in Aurano n-  Fig. 5B and 5F). Next, we investigated whether ROS levels in Aurano n-treated Calu-6 and A549 cells were changed by NAC or BSO. As expected, Aurano n signi cantly increased the level of O 2 •− in Calu-6 and A549 cells at 24 h, and the level of O 2 •− in Aurano ntreated Calu-6 and A549 cells was decreased by NAC ( Fig. 5C and 5G). BSO dramatically augmented the increased O 2 •− level in Aurano n-treated Calu-6 cells (Fig. 5C), but it failed to enhance the level of O 2 •− in Aurano n-treated A549 cells (Fig. 5G). Regarding GSH depletion, Aurano n treatment alone led to an increase in the number of GSH-depleted Calu-6 and A549 cells. Treatment with NAC reduced the number of GSH-depleted cells induced by Aurano n in Calu-6 and A549 cells, whereas BSO intensi ed the GSH depletion caused by Aurano n in these cells (Figs. 5D and 5H).

Effects of NAC and BSO on apoptosis-related protein levels in Calu-6 and A549 cells
To investigate the relationship between ROS accumulation and cell apoptosis, PARP degradation and cleavage of caspase-3 as apoptotic markers were measured by western blot analysis in Aurano n-treated Calu-6 and A549 cells under different redox states using NAC or BSO. As predicted, compared with the control group, activated caspase-3 and cleaved PARP were strongly expressed by treatment with Aurano n in Calu-6 and A549 cells. By contrast, the expression levels of caspase-3 and PARP were notably reduced in Aurano n-treated Calu-6 and A549 cells (Figs. 6A-6D). Furthermore, pretreatment with NAC and subsequent treatment with Aurano n increased the expression levels of intact caspase-3 and PARP compared to treatment with Aurano n alone in Calu-6 and A549 cells, whereas the cleavage of caspase-3 and degradation of PARP by Aurano n were prevented in the presence of NAC in these cells (Figs. 6A and 6C). In cells pretreated with BSO and then co-treated with Aurano n, the expression levels of pro-caspase-3 and PARP were further decreased compared to Aurano n treatment in the absence of BSO in Calu-6 and A549 cells (Figs. 6B and 6D). Activated caspase-3 in cells pretreated with BSO and cotreated with Aurano n was detected similarly to the changes observed in Aurano n-treated Calu-6 cells (Fig. 6B). In A549 cells treated with BSO and Aurano n, despite a diminishing level of pro-caspase-3, cleaved active caspase-3 fragments were not detected (Fig. 6D). In addition, cleaved PARP was observed in the combined treatment with BSO and Aurano n compared to controls in Calu-6 and A549 cells ( Fig. 6B and 6D). These results suggest that apoptosis is a mechanism of Aurano n-mediated cell death, and ROS accumulation contributes to Aurano n-induced apoptosis in lung cancer cells.
Effects of NAC and BSO on TrxR activity in Aurano n-treated Calu-6 and A549 cells Inhibition of TrxR activity by Aurano n leads to increased intracellular oxidative stress and induced cell death. As expected, after exposure to Aurano n for 24 h, we found that TrxR activity was dosedependently decreased with concentrations ≥ IC 50  However, Aurano n susceptibility of A549 and SK-LU-1 cells was higher than that of normal lung cells. This nding implies that Aurano n can be an effective antitumor agent for lung cancer, but before using it in a clinical trial, we must rst consider the differential susceptibility of Aurano n depending on the type of lung cancer cell. . Therefore, regulation of the Trx system can be a prime target for cancer therapy. Our previous results indicated that Aurano n dose-dependently reduced the activity of TrxR in Calu-6 and A549 cells, and, moreover, NAC prevented the observed decrease of TrxR activity by Aurano n in Calu-6 and A549 cells. By contrast, BSO bolstered the decrease in TrxR activity by Aurano n in Calu-6 cells but did not affect TrxR activity in Aurano n-treated A549 cells. These results also suggest that the TrxR inhibitor Aurano n may be an effective antitumor drug for patients with lung cancer.
In the present study, we mainly focused on the potential of Aurano n as an anticancer drug in lung cancer, and conclude that Aurano n-induced growth inhibition and death of lung cancer cells, which were accompanied by increasing ROS levels and GSH depletion. In addition, the changes of ROS and GSH levels by NAC or BSO affected cell growth inhibition and death in Aurano n-treated lung cancer cells. Our present ndings provide useful information toward better understanding of the antitumor effects of Aurano n in lung cancer cells in relation to ROS and GSH levels.

Con icts of interest
The authors do not have any con icts of interest to declare.

Availability of data and materials
Data collected during the present study are available from the corresponding author upon reasonable request.

Ethics declarations
The material in this paper has not been published and is not under active consideration by another journal. The research was conducted in accordance with the declaration of Helsinki.      Effects of NAC and BSO on apoptosis-related protein levels in Aurano n-treated Calu-6 and A549 cells.
Exponentially growing Calu-6 cells or A549 cells were incubated in the presence of the indicated concentrations of Aurano n for 24 h following 1 h preincubation of 2 mM NAC or 10 μM BSO. Whole-cell extracts were prepared, and equal amounts of lysates were then resolved through SDS-PAGE, transferred onto PVDF membranes, and immunoblotted with indicated antibodies against PARP, cleaved PARP, procaspase-3, cleaved-caspase-3, and GAPDH (A, B, C, and D, respectively). GAPDH detection was used to con rm equal protein loading.

Figure 7
Effects of NAC and BSO on TrxR activity in Aurano n-treated Calu-6 and A549 cells. Exponentially growing Calu-6 cells or A549 cells were incubated in the presence of the indicated concentrations of Aurano n for 24 h following 1 h preincubation of 2 mM NAC or 10 μM BSO. TrxR activity was measured using the insulin reduction assay. The graphs indicate inhibition of TrxR activity by Aurano n or combination treatment with NAC or BSO in Calu-6 (A and B) and A549 (C and D) cells. * p < 0.05 compared with Aurano n-untreated control cells.