SAN was supplied by Professor Chao-Sheng Li (Institute of Resource, Jilin Academy of Chinese Medicine Sciences, Changchun, China) and dissolved in dimethyl sulfoxide (DMSO). Fetal bovine serum (FBS) and Roswell Park Memorial Institute (RPMI)-1640 culture medium were purchased from Invitrogen (Carlsbad, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), tauroursodeoxycholic acid (TUDCA), N-acetylcysteine (NAC) and Hoechst 33258 were purchased from Sigma (St. Louis, MO, USA). The ROS indicator 5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H₂DCFDA) was purchased from Molecular Probes (Eugene, OR, USA). Enhanced chemiluminescence (ECL) reagents were from Thermo Scientific (Rockford, IL, USA). Anti-CCAAT/enhancer binding protein homologous protein (CHOP), anti-glucose-regulated protein 78 (GRP78), anti-p-protein kinase R (PKR)-like ER kinase (PERK), anti-p-eukaryotic translation initiation factor 2α (eIF2α), anti-activating transcription factor 4 (ATF4), anti-Bax and anti-caspase-3 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-β-actin and horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulin were purchased from Proteintech (Chicago, IL, USA). All reagents and antibodies were purchased from the Changchun Baoxin Biotechnology Company (Changchun, China).

Human lung adenocarcinoma SPC-A1 cells were obtained from the Chinese Academy of Medical Sciences and Peking Union Medical College. Cell lines were cultured at 37°C in 5% (v/v) CO₂ in RPMI-1640 culture medium supplemented with 10% (v/v) FBS. Media were changed at 2-day intervals, and cells were split twice a week by trypsinization. All experiments were performed when cells reached 70% confluency.

The cells were plated in 96-well plates at a density of 1×10⁴ cells/well in 200 µl of complete medium. Each treatment was repeated in 6 separate wells. The cells were treated as indicated for 24 h, after which 20 µl of 5 mg/ml MTT reagent in phosphate-buffered saline (PBS) was added to each well and incubated for 4 h. Formazan crystals were dissolved in 150 µl DMSO. Absorbance was recorded at a wavelength of 490 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Cell viability was calculated as: Viability (%) = absorbance of experimental group/absorbance of control group × 100%. In each experiment, we calculated the mean value of 6 wells/treatment group.

Apoptotic morphological alterations in nuclear chromatin were detected by Hoechst 33258 staining. Briefly, the SPC-A1 cells were cultured in 24-well plates and treated as indicated for 24 h. The cells were washed with ice-cold PBS and fixed with 4% (w/v) paraformaldehyde overnight. The plates were then incubated with 10 µM Hoechst 33258 staining solution for 10 min. The cells were visualized under a fluorescence microscope (IX-71; Olympus).

The Human Unfolded Protein Response RT² Profiler™ PCR Array (SABiosciences, Qiagen, Chicago, IL, USA) profiles the expression of 84 key genes involved in unfolded protein accumulation in the ER. Total cellular RNA was extracted from the cultured cells according to the manufacturer’s instructions. Single-stranded cDNA was obtained by reverse transcription of 1 µg of total RNA using the SABiosciences RT² First Strand kit (SABiosciences). Real-time qPCRs were performed using Applied Biosystems 7300 Fast with SYBR-Green Fluorophore. The reactions were carried out using the RT² SYBR-Green Master Mix. cDNA was used as a template and cycling parameters were 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Fluorescence intensities were analyzed using the manufacturer’s software, and relative quantification was calculated using the 2^™ΔΔCt method. Change in the expression of the 84 genes was shown by heat imaging. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene.

Cells were washed with PBS twice and harvested by scraping into 300 µl of Radio-Immunoprecipitation Assay (RIPA) lysis buffer. Cell lysates were ultrasonicated for 15 sec on ice and then lysed at 4°C for 45 min and centrifuged at 12,000 × g for 10 min. Protein concentrations in the supernatants were determined using the Bio-Rad reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of protein samples (30 µg) were separated by SDS-polyacrylamide gel electrophoresis in duplicate and blotted onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Transfer efficiency was assessed with Ponceau staining. The blots were blocked in Tris-buffered saline containing 5% (w/v) nonfat dry milk and probed with specific primary antibodies overnight at 4°C. The membranes were washed with PBS-Tween-20 and then incubated with a peroxidase-conjugated secondary antibody for 2 h at room temperature. Duplicated membranes were probed for β-actin expression to ensure equal input of cell lysate proteins. The final dilutions and incubation times suggested by the manufacturer were used for each antibody. Immunodetection was performed using the ECL reagents and images were captured using Syngene Bio Imaging (Synoptics, Cambridge, UK).

SPC-A1 cells were seeded onto glass culture slides (BD Biosciences, Bedford, MA, USA) and treated with the indicated drugs. At various time-points, the cells were loaded with 1 µM CM-H₂DCFDA in PBS for 10 min at 37°C in the dark followed by a PBS washing step. DCF-dependent fluorescence was examined by laser-scanning confocal microscopy (FV 1000; Olympus).

Results are expressed as the mean ± standard deviation (SD) of repeated experiments, as indicated in the figure legends. Data are representative of three independent experiments performed in triplicate. Statistical analysis of the data was performed using one-way ANOVA. The Tukey’s post hoc test was used to determine the significance for all pairwise comparisons of interest. Differences were considered statistically significant at P<0.05.

To measure the effects of SAN on the growth of lung cancer cells, we treated SPC-A1 cells with different doses (0.5, 1, 2 and 4 µM) of SAN for 24 h and detected cell viability using the MTT assay. The results revealed that treatment with SAN inhibited cell growth in a dose-dependent manner (Fig. 1A). To investigate whether inhibition of cell growth by SAN is related to cell apoptosis, we monitored apoptosis using Hoechst 33258 staining. After treatment with 0.5, 1, 2 and 4 µM SAN for 24 h, the cells showed obvious features of apoptosis: chromatin condensation and nuclear fragmentation (Fig. 1B). We monitored the expression levels of the pro-apoptotic proteins Bax and cleaved caspase-3 to confirm SAN-induced apoptosis. As shown in Fig. 1C, SAN treatment upregulated the expression of Bax and cleaved caspase-3, suggesting that inhibition of growth by SAN involves apoptosis. Overall, these results indicate an anticancer role of SAN in SPC-A1 cells.

Classically, chemotherapeutic agent-induced apoptosis was thought to be associated with ROS production (24,26,22). In the present study, we aimed to determine whether ROS were involved in SAN-induced SPC-A1 cell death. After treatment with 1 and 2 µM SAN for 24 h, ROS production was detected using CM-H₂DCFDA staining and laser-scanning confocal microscopy. The results showed that ROS were significantly elevated in a dose-dependent manner (Fig. 2A). ROS production is closely related to the induction of apoptosis. Thus, we investigated whether ROS affect SAN-induced growth inhibition and apoptosis. Pretreatment of SPC-A1 cells with the ROS scavenger (NAC, 100 µM for 1 h) significantly reversed growth inhibition (Fig. 2B) and apoptosis (Fig. 2C) induced by SAN (2 µM for 24 h). Therefore, these results suggest that SAN-mediated growth inhibition and apoptosis partially rely on the generation of ROS.

ROS trigger oxidative stress, which may lead to ER stress through the accumulation of unfolded proteins and/or misfolded proteins. ER stress triggers the unfolded protein response (UPR), which involves a great variety of molecules. We monitored changes in the expression of genes associated with the UPR pathway using the PCR array assay. The results showed that 32 genes were upregulated more than 2-fold after treatment with SAN (2 µM for 24 h) in the SPC-A1 cells, including glucose-regulated protein 78 (GRP78, also called HSPA5, position D4), inositol requiring kinase 1α (IRE1α, also called ERN1, position C2), activating transcription factor 6α (ATF6α, position A3), activating transcription factor 4 (ATF4, position A2), growth arrest and DNA-damage-inducible gene 34 (GADD34, also called PPP1R15A, position E11) and CAAT/enhancer binding protein homologous protein (CHOP, also called DDIT3, position B2) (Fig. 3A). We next confirmed ER stress by measuring protein expression by western blotting. Consistently, the expression of GRP78, p-protein kinase R (PKR)-like ER kinase (p-PERK), p-eukaryotic translation initiation factor 2α (p-elF2α), ATF4 and CHOP were significantly increased in a dose-dependent manner (Fig. 3B). Therefore, SAN can induce ER stress in SPC-A1 cells.

ER stress is thought to play an important role in the cell pathophysiology process associated with cell death (27). To confirm the effects of ER stress on SAN-induced growth inhibition, we blocked ER stress using 50 µM TUDCA for 1 h, followed by treatment with 2 µM SAN for 24 h, and then detected cell viability using the MTT assay. Results indicated that TUDCA partly reversed SAN-induced cell growth inhibition (Fig. 3C). In addition, the Hoechst 33258 staining assay showed similar results, with TUDCA inhibiting SAN-induced apoptosis (Fig. 3D). These data suggest that ER stress is associated with SAN-induced cell growth inhibition and apoptosis in SPC-A1 cells.

To investigate the relationship between ER stress and ROS, we aimed to determine whether ER stress is involved in the regulation of ROS production. Notably, pretreatment with 50 µM TUDCA for 1 h, followed by treatment with 2 µM SAN for 24 h, partially reversed SAN-induced ROS production (Fig. 4A). Next, the expression of the ER stress-related proteins GRP78 and CHOP were monitored after blocking ROS with NAC. After pretreatment of SPC-A1 cells with 100 µM NAC for 1 h, followed by treatment with 2 µM SAN for 24 h, SAN-induced expression of GRP78 and CHOP was markedly attenuated (Fig. 4B). These data suggest that blocking ER stress with TUDCA can reduce ROS production, whereas eliminating ROS by NAC can attenuate the extent of ER stress. Therefore, ER stress and ROS production promote each other and form a vicious cycle.