A549 and HCC-15 cells originating from lung tumors were obtained from the American Type Culture Collection. Calu-3 cancer cells were purchased from the Korean Cell Line Bank (Korean Cell Line Research Foundation). Roswell Park Memorial Institute (RPMI)-1640 cell culture media (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) fetal bovine serum (Atlas Biologicals, Inc.) and 100 µg/ml penicillin-streptomycin were used for cell culture at 37°C in 5% CO₂.

Sertraline was procured from Cayman Chemical Company. Chloroquine (10 µM) and 3-MA (5 mM) were obtained from Sigma-Aldrich (Merck KGaA), and TRAIL (100 ng/ml) was purchased from AFrontier Co., Ltd.

A549, HCC-15 and Calu-3 cells were seeded into 12-well plates at a density of 1×10⁴ cells and incubated at 37°C for 24 h. Cultured cells were pretreated with sertraline at different concentrations (0, 2.5, 5 and 10 µM) for 18 h, and then treated with recombinant TRAIL (100 ng/ml) for 2 h and 30 min. Some cells were also preincubated with chloroquine (10 µM) or 3-MA (5 mM) for 18 h, and then treated with or without recombinant TRAIL (100 ng/ml) for 2 h 30 min. Cell morphology was observed under an inverted microscope (Nikon Corporation). Crystal violet staining was used for the assessment of cell viability. In this method, viable cells were stained with a crystal violet staining solution (0.5% crystal violet in 30% ethanol and 3% formaldehyde) for 10-15 min at room temperature, washed 3-4 times with phosphate-buffered saline (PBS), and dried. Viability of cells was also assessed by an MTT assay. In brief, 350 µl of 5 mg/ml MTT solution was added to each well and the plate was incubated at 37°C for 2 h and 30 min. The medium was removed and 500 µl DMSO was added to each well. The absorbance was recorded at 570 nm wavelength using a spectrophotometer (Bio-Rad Laboratories, Inc.). All experiments were performed at least three times. Viability was expressed relative to the percentage of the control group, which was set to 100%.

Cell culture supernatants were collected and cytotoxicity was analyzed using an LDH detection kit (cat. no. MK401; Takara Bio, Inc.) according to the manufacturer's protocol. LDH was assessed by measuring absorbance at 490 nm wavelength using a microplate reader (Spectra Max M2; Molecular Devices, LLC).

Cultured A549 cells were washed with 1X cold PBS, harvested with a lysis buffer [25 mM HEPES (pH 7.4), 100 mM ethylenediaminetetraacetic acid (EDTA), 5 mM magnesium chloride (MgCl₂), 0.1 mM dithiothreitol (DTT) and protease inhibitor cocktail], sonicated to obtain cell lysates (4 sec/20 kHz) and centrifuged at 11,200 × g for 10 min at 4°C. The protein concentration was assessed using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). The cell supernatant was collected and the proteins (30 µg) were separated by SDS-PAGE on 10-15% gels. The separated protein bands were transferred to nitrocellulose or PVDF membranes, which were blocked with 5% non-fat dried milk at 25°C for 1 to 2 h. Next, specific primary antibodies in a dilution buffer [1% milk and 1% PBS with 1% Tween-20 (PBST)] for 1 h at 25°C. The following primary antibodies were used for immunoblotting: LC3 (1:1,000; cat. no. 4108), p62 (1:1,000; cat. no. 5114s), cleaved caspase-3 (1:1,000; cat. no. 39665), AMPK (1:1,000; cat. no. 2532), phosphorylated (p)-AMPKα (1:1,000; cat. no. 2535), mTOR (1:1,000; cat. no. 2983) and p-mTOR (1:1,000; cat. no. 5536; all from Cell Signaling Technology, Inc.); cleaved caspase-8 (1:1,000; cat. no. 551242; BD Pharmingen); DR5 (1:10,000; cat. no. ab181846) and DR4 (1:1,000; cat. no. ab8414; both from Abcam); and β-actin (1:1,000; cat. no. A5441; Sigma-Aldrich; Merck KGaA). Then, membranes were probed with horseradish peroxidase-conjugated secondary antibodies (1:5,000; cat. nos. ADI-SAB-100 and ADI-SAB-300; Enzo Life Sciences, Inc.) at 25°C for 1 h. The targeted protein bands were detected with enhanced chemiluminescence reagents (Cytvia). Bands were visualized using a Fusion-FX7 image capturing system (Vilber Lourmat).

A549 cells (1×10⁵ cells/well) were cultured on glass coverslips at 37°C for 24 h and treated with sertraline for 18 h, followed by washing with 1% PBS and fixing with 4% paraformaldehyde in PBS at room temperature for 15 min. Cells were washed twice with ice-cold PBS and incubated with PBS containing 0.25% Triton X-100 at room temperature for 10 min. After incubation, cells were washed three times with PBS and blocked with 1% bovine serum albumin (BSA; GenDEPOT) in PBST for 30 min at 4̊C. Cells were probed with primary antibodies [anti-p62 (1:100; cat. no. 5114s; Cell Signaling Technology, Inc.) and anti-DR5 (1:100; cat. no. ab181846; Abcam) diluted in PBST containing 1% BSA] in a 5% CO₂ incubator at 37°C for 3 h, followed by washing three times with PBS. These cells were then incubated with the Alexa Fluor^® 488-conjugated donkey polyclonal anti-rabbit secondary antibody (1:1,000; cat. no. A-21206; Thermo Fisher Scientific, Inc.) for 2 h at 25°C in a dark condition. The solution was washed off and cells were further washed 3-4 times with PBS and stained with DAPI for 10 min at room temperature (25°C). Cells were washed three times and mounted with a fluorescent mounting medium. Images were captured using a fluorescence microscope (Nikon ECLIPSE 80i; Nikon Corporation) at ×400 magnification.

Adherent A549 cells (1×10⁶ cells/well) were detached using trypsin and then fixed with 2% glutaraldehyde (Electron Microscopy Sciences) and 2% paraformaldehyde in 0.05 M sodium cacodylate buffer (pH 7.2; Electron Microscopy Sciences) for 2 h at 4°C. Next, cells were treated with 2% osmium tetroxide (Electron Microscopy Sciences) for 1 h at 4°C and dehydrated with graded ethanol (25, 50, 70, 90 and 100%) for 5 min each. After dehydration, samples were embedded in epoxy resin (Embed 812; Electron Microscopy Sciences) for 48 h at 60°C, according to the manufacturer's instructions. Ultra-thin sections (60 nm) were prepared using an LKB-III ultramicrotome (Leica Microsystems GmbH) and stained with 0.5% uranyl acetate (Electron Microscopy Sciences) for 20 min and 0.1% lead citrate (Electron Microscopy Sciences) for 7 min at room temperature. Images were captured at ×10,000 magnification on a Hitachi H7650 electron microscope (Hitachi, Ltd.) installed at the Center for University-Wide Research Facilities at Jeonbuk National University (Republic of Korea).

Small interfering RNA (siRNA) targeting DR5 and scramble control siRNA were purchased from Ambion (Thermo Fisher Scientific, Inc.). The transfection reagent Lipofectamine^® 2000 was obtained from Invitrogen (Thermo Fisher Scientific, Inc.). A549 lung cancer cells (1×10⁵ cells/well) were transfected with 40 nM DR5 siRNA (siRNA ID 104279; Sequence 5′-UUU AGC CAC CUU UAU CUC AUU GUC C-3′; Ambion; Thermo Fisher Scientific, Inc.) using Lipofectamine 2000, according to the manufacturer's protocol. The cells were incubated with siRNA for 6 h and the medium was then changed to RPMI-1640 with 10% FBS for 24 h. Cells were then treated with sertraline, or sertraline in combination with TRAIL. At 24-h post transfection, knockdown efficiency was assessed by the cell viability assay and immunoblotting at the protein level.

Data are expressed as the mean ± SD. The significance of the differences between treatments were analyzed using one-way ANOVA followed by Tukey's test. Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Effects of sertraline on TRAIL-mediated apoptosis were evaluated following the treatment of lung adenocarcinoma cells. Human lung cancer cell lines (A549, HCC-15 and Calu-3) were preincubated with the indicated doses of sertraline for 18 h and then treated with TRAIL for 2 h and 30 min. Cells were imaged and variations in morphology related to apoptosis, such as cell shrinkage, were investigated under a light microscope. Cells treated with sertraline or TRAIL alone had slight changes in viability and showed no obvious morphological changes compared with control cells (Fig. 1). Thus, indicating that A549, HCC-15 and Calu-3 cells were highly resistant to TRAIL-mediated apoptosis. However, treatment with different concentrations of sertraline along with TRAIL significantly increased the number of apoptotic cells (Fig. 1A, B, E, F, I and J). The result of the MTT assay showed that combination treatment with sertraline and TRAIL reduced cell viability, which indicated that the number of apoptotic cells increased, for all cell lines tested (Fig. 1C, G and K). Sertraline or TRAIL alone failed to cause any significant increase in levels of LDH. Whereas, sertraline in combination with TRAIL significantly increased the level of LDH in all cell lines, indicative of apoptosis induction in a sertraline dose-dependent manner (Fig. 1D, H and L). These results demonstrated that sertraline could sensitize TRAIL-resistant human lung adenocarcinoma A549, HCC-15 and Calu-3 cells to TRAIL-mediated apoptosis.

Next, the molecular function of sertraline in TRAIL-mediated apoptosis of A549 lung cancer cells was investigated (Fig. 2). A549 cells were treated with indicated doses of sertraline for 18 h, and the harvested cell lysates were analyzed by western blotting to determine DR4 and DR5 expression. Sertraline upregulated DR5 expression in a dose-dependent manner in A549 cells; however, the expression of DR4 was unaltered (Fig. 2A). A549 cells were preincubated with the indicated doses of sertraline for 18 h and then exposed to TRAIL for 2 h. Intracellular apoptosis-regulatory proteins, cleaved caspase-8 and cleaved caspase-3, were activated in cells subjected to a combined treatment of sertraline and TRAIL compared with cells treated with sertraline alone (Fig. 2B). Furthermore, ICC results revealed the increased expression of DR5 in sertraline-treated cells compared with that in non-treated cells (Fig. 2C). These findings indicated that sertraline could induce TRAIL-mediated apoptosis in lung adenocarcinoma cells by upregulating DR5 expression.

To explore the outcomes of sertraline exposure on autophagic flux, LC3-II and p62 expression levels were assessed by western blotting (Fig. 3). The expression levels of LC3-II and p62 increased after sertraline treatment, indicating the inhibition of autophagic flux (Fig. 3A). Treatment with sertraline alone or in combination with TRAIL upregulated LC3-II and p62 expression compared with TRAIL treatment alone (Fig. 3B). ICC results also showed that sertraline increased the expression of p62 (Fig. 3C). The AMPK pathway is involved in stabilizing cellular energy through the control of autophagic flux via downregulation of mTOR expression (29). Sertraline inhibited phosphorylation of AMPK, resulting in the inhibition of autophagic flux (Fig. 3D). TEM revealed the inhibition of autophagic flux, as confirmed by the accumulation of autophagic vacuoles containing intracellular material in the treatment group (Fig. 3E). These findings indicated that the inhibition of AMPK phosphorylation by sertraline may result in the suppression of autophagic flux in lung cancer cells.

The well-known autophagy inhibitors chloroquine and 3-MA were used to examine the effects of sertraline-induced TRAIL-mediated apoptosis in A549 cells (Fig. 4). A549 cells were incubated with chloroquine, 3-MA and sertraline at the indicated concentrations for 18 h. Then, cells were exposed to TRAIL for an additional 2 h and 30 min. Cells were photographed to investigate any morphological changes under light microscope. Cell viability was analyzed by crystal violet staining and an MTT assay. The number of dead A549 cells increased slightly after treatment with either TRAIL or sertraline alone. However, the combination of TRAIL and chloroquine or 3-MA notably increased cell death. Based on cellular morphology, it was observed that TRAIL with sertraline, chloroquine or 3-MA enhanced cell death compared with sertraline or TRAIL alone (Fig. 4A and B). The MTT assay showed that both autophagy inhibitors, chloroquine and 3-MA, in combination with TRAIL significantly decreased the viability of A549 cells compared with control cells (Fig. 4C). Additionally, the combination of TRAIL and chloroquine or 3-MA also enhanced LDH release compared with control cells (Fig. 4D). These results indicated that sertraline enhanced the TRAIL-mediated apoptosis of lung cancer cells by inhibiting autophagic flux.

To further explore the mechanism underlying sertraline-induced TRAIL-mediated apoptosis, autophagic flux was inhibited using autophagy inhibitors, chloroquine and 3-MA. Inhibition of autophagic flux with chloroquine and 3-MA resulted in the upregulation of DR5 expression, thereby increasing apoptosis (Fig. 5B). Cells were treated with the indicated concentration of sertraline and two autophagy inhibitors [chloroquine (10 µM) and 3-MA (5 mM)] for 18 h. Cell lysates were collected for western blotting and it was found that sertraline and chloroquine treatment notably increased the expression levels of p62 and LC3-II; 3-MA could also slightly increase p62 and LC3-II expression (Fig. 5A). Moreover, DR5 expression in the chloroquine or 3-MA groups was similar to that observed with sertraline treatment alone (Fig. 5B). The expression of key apoptosis indicators, cleaved caspase-8 and cleaved caspase-3, were also evaluated in the lysates of the cells treated with chloroquine, 3-MA and sertraline with the indicated doses for 18 h, and TRAIL for 2 h. Autophagy inhibitors chloroquine and 3-MA in the presence of TRAIL could trigger the expression of cleaved caspase-8 and cleaved caspase-3 (Fig. 5C). Taken together, these findings suggested that the inhibition of autophagy may result in the upregulation of DR5 expression and sertraline-induced TRAIL-mediated apoptosis.

Silencing DR5 expression using DR5 siRNA significantly altered the effect on cell viability (Fig. 6). This experiment showed that DR5 plays a key role in sertraline-induced TRAIL-mediated apoptosis of cancer cells. After 24 h of transfection with DR5 siRNA or scramble control siRNA, cells were treated with sertraline for 18 h and then with TRAIL (100 ng/ml) for 2 h and 30 min. Cells were then subjected to viability and western blot analyses. As a result, it was found that DR5 siRNA-transfected cells blocked cell death despite co-treatment with sertraline and TRAIL. The viability of cells treated with scramble siRNA was similar to that of the cells exposed to sertraline and TRAIL co-treatment (Fig. 6A-C). Western blot analysis showed that DR5 expression was inhibited in DR5 siRNA-transfected cells compared with that in non-transfected cells. These experimental findings indicated that sertraline-mediated upregulation of DR5 plays an important role in weakening TRAIL resistance. Overall, these findings demonstrated that the induction of TRAIL-mediated apoptosis with sertraline via AMPK-mediated autophagic flux inhibition is a possible therapeutic strategy to target the TRAIL-DR5 apoptotic pathway.