A549 lung cancer cells were acquired from the American Type Culture Collection (ATCC). The cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco BRL; Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) fetal bovine serum (Sigma-Aldrich; Merck KGaA) and antibiotics (100 µg/ml penicillin-streptomycin; Sigma-Aldrich; Merck KGaA) at 37°C in a 5% CO₂ incubator.

Amitriptyline was purchased from Cayman Chemical Company, and chloroquine (CQ) (20 µM) was obtained from Sigma-Aldrich; Merck KGaA. Human recombinant TRAIL (100 ng/ml) was purchased from AbFrontier.

Cell viability was assessed with MTT and crystal violet staining assays. The cells were plated in 12-well plates at a density of 1.0×10⁴ cells/well and incubated at 37°C for 24 h. The cells were pretreated with different concentrations of amitriptyline (0, 10, 20 and 40 µM) or CQ for 12 h and then exposed to recombinant TRAIL (100 ng/ml) for 3 h. Cell morphology was observed under an inverted light microscope (magnification, ×100; Nikon Corporation). Cell viability was assessed by adding 50 µl of 5 mg/ml methyl-thiazolyl tetrazolium (MTT) to each well and incubating them at 37°C for 2 h. After incubation, the MTT solution was removed and the cells were treated with 500 µl of dimethyl sulfoxide and the absorbance was measured at 570 nm with a spectrophotometer (Bio-Rad Laboratories). For the crystal violet assay, the cells were stained with a staining solution (0.5% crystal violet in 30% ethanol and 3% formaldehyde) for 10–20 min at room temperature (RT), washed 3–4 times with phosphate-buffered saline (PBS), and then imaged.

Cytotoxicity was analyzed in the collected supernatant and determined by an LDH cytotoxicity detection kit (Takara Bio, Inc.) following the manufacturer's protocol. LDH activity was measured at 490 nm using a microplate reader (Spectra Max M2; Molecular Devices, LLC).

Cells were plated in 6-well plates at 37°C and treated with the indicated doses of amitriptyline (40 µM), CQ (20 µM) and TRAIL (100 ng/ml). Two days later, the culture medium was changed with new medium without amitriptyline, CQ and TRAIL, and the culture continued for 7 days. Colonies were fixed for 20 min at RT in 4% paraformaldehyde, stained with 0.05% (w/v) crystal violet for 10 min at RT, and counted under an inverted light microscope (Nikon Corporation).

Apoptosis was evaluated cells (50 cells/µl) using Annexin V-FITC Assay Kit (Santa Cruz Biotechnology, Inc.), for flow cytometry according to the manufacturer's instructions (Guava EasyCyte HT System; EMD Millipore). The fluorescence was measured at 488 nm of excitation and 525/30 emission using Guava^® InCyte and GuavaSuite Software.

The cells were lysed in lysis buffer [25 mM HEPES (pH 7.4), 100 mM ethylenediaminetetraacetic acid (EDTA), 5 mM MgCl₂, 0.1 mM dithiothreitol (DTT), and a protease inhibitor cocktail], and sonicated to prepare cell lysates. Equal amounts (40 µg) of proteins were separated by 8–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked at 25°C for 1 h, and then incubated with the indicated concentrations of primary antibodies at 25°C for 1 h, and then they were blotted with anti-mouse IgG (Alexa Fluor 647 conjugate) secondary antibodies (product. no. 4410; 1:2,000; Cell Signaling Technology, Inc.) at 25°C for 1 h. The membranes were developed with enhanced chemiluminescence reagents (ECL; GE Healthcare Life Sciences). Primary antibodies used for the immunoblotting included: DR4 (product. code. ab8414; 1:1,000), DR5 (product. code. ab181846; 1:10,000) (both from Abcam), LC3 (product. no. 3868; 1:1,000), p62 (cat. no. 5114; 1:1,000), cleaved caspase-3 (product. no. 9661; 1:500), p-AMPKα (product. no. 2531; 1:1,000) all from Cell Signaling Technology, Inc., cleaved caspase-8 (cat. no. 551242; 1:1,000, BD Pharmingen; BD Biosciences), and β-actin (cat. no. A2228; 1:2,000, Sigma-Aldrich; Merck KGaA). The bands were visualized and captured with a Fusion-FX7 using easy-to-use FusionCapt V16.07 Software (both Vilber Lourmat).

The cells (~1×10⁶ cells) were grown on glass coverslips, then treated with amitriptyline, washed with 1% PBS, and fixed with 4% paraformaldehyde in PBS at RT for 15 min. They were then washed twice with ice-cold PBS and incubated at RT for 10 min in PBS containing 0.25% Triton X-100. After the incubation, the cells were washed three times with PBS and blocked with 1% BSA in PBST for 30 min. The cells were then incubated with a primary antibody [anti-p62 (1:1,000; product. no. 5114; Cell Signaling Technology, Inc.) and DR4/5 diluted with 1% BSA in PBST] in a 5% CO₂ incubator for 3 h at 37°C. After incubation, the cells were washed three times with PBS. Next, the cells were incubated with a secondary antibody [(Alexa Fluor^® 488-conjugate; donkey polyclonal anti-rabbit, 1:500; cat. no. A-21206; Thermo Fisher Scientific, Inc.), diluted with 1% BSA in PBST] in the dark for 2 h at RT. The solution was removed and the cells were washed 3–4 times with PBS. The cells were treated with DAPI (4′,6-diamidino-2-phenylindole, D9564; Sigma-Aldrich;Merck KGaA) and incubated for 10 min at 25°C. The cells were washed three times, then mounted with fluorescent mounting medium and the images were captured using a fluorescence microscope (Nikon ECLIPSE 80i; magnification, ×400; Nikon Corporation).

Trypsinized cells were fixed with 2% glutaraldehyde (Electron Microscopy Sciences) for 2 h at 4°C in PBS, followed by 2% osmium tetroxide (Electron Microscopy Sciences), and dehydrated with an ethanol series (25, 50, 70, 90 and 100%) for 5 min each. After dehydration, the samples were embedded in epoxy resin (Embed 812; Electron Microscopy Sciences) for 48 h at 60°C according to the manufacturer's instructions. Ultrathin sections (60 nm) were prepared using an LKB III ultratome (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 RT. Images were captured on a Hitachi H7650 electron microscope (magnification ×10,000; Hitachi, Ltd.) installed at the Center for University-Wide Research Facilities (CURF) at Jeonbuk National University (JBNU).

The cell line was transfected with small interfering (si)RNA using Lipofectamine (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Knockdown proficiency was assessed by immunoblotting and cell viability tests. DR4 and DR5 siRNA were purchased from Qiagen China Co., Ltd., each with mixed two target sequences (forward, 5′-TAGCTCAGCTGCAACCATCAA-3′ and reverse, 5′-CAGGCAATCGACATAATATAT-3′ for DR4; forward, 5′-ACCAGGTGTGATTCAGGTGAA-3′ and reverse, 5′-CCGACTTCACTTGATACTATA-3′ for DR5). The synthetic siRNA and scramble siRNA [negative control (NC)] (Qiagen) were transfected using HiPerfect transfection reagent (Qiagen), according to the manufacturer's protocol. Briefly, each sequence of siRNA (20 µmol/ml) and 10 µl Lipofectamine 2000 was diluted in serum-free medium (250 µl) at RT for 5 min, mixed together, and incubated for 30 min at RT. The cells were incubated with siRNA or NC siRNA for 6 h and the medium was then changed with 10% FBS for 24 h, followed by subsequent experimentation.

The DR4 and DR5 mRNA transcripts were measured using quantitative SYBR Green-based real-time qPCR. Total RNA was extracted from the A549 cells with RiboEX (GeneAll Biotechnology). The total RNA extracts were then converted into cDNA by using reverse transcriptase (TOPscript™ One-step RT PCR kit; Enzynomics Co., Ltd.) using a CFX96™ Real-PCR Detection system (Bio-Rad Laboratories, Inc.), following the manufacturer's instructions at 85°C for 5 sec, 37°C for 10 min and 4°C for 15 min. Gene primers (1 µl) and SYBR-Green (Bio-Rad Laboratories, Inc.) contained in a total reaction volume of 20 µl were used to conduct the RT-qPCR . The reaction protocols were as follows: Predenaturation at 95°C for 30 sec, 40 cycles of denaturation at 95°C for 5 sec and annealing at 60°C for 30 sec. GAPDH were used as the respective internal control. The sequences of the primers used were: DR4 forward, 5′-GGGACAGCACGGACCCAGTG-3′ and reverse, 5′-ATCCTTGACCTTGACCATCC-3′; DR5 forward, 5′-GCGGTCCTGCTGTTGGTCTC-3′ and reverse, 5-GCTTCTGTCCACACGCTCAG-3′; and GAPDH as an internal control forward, 5′-TGCACCACCAACTGCTTAG-3′ and reverse, 5′-GGATGCAGGGATGATGTT-3′. All data were evaluated using Bio-Rad CFX manager, version 2.1 analysis software (Bio-Rad Laboratories, Inc.). The collected data from three independent experiments were analyzed using the 2^−ΔΔCq method (29).

The data are expressed as the mean ± standard deviation (SD) from three independent experiments. The significance of the differences between the treatments was analyzed using one-way analysis of variance (ANOVA), followed by the Tukey-Kramer post hoc test. Statistical analyses were executed using GraphPad Prism 7 (GraphPad Software, Inc). P<0.05 was considered to indicate a statistically significant difference.

To explore the synergistic effect of amitriptyline with TRAIL on the inhibition of lung cancer cell viability, the A549 lung adenocarcinoma cell line was selected. The results revealed a strong synergistic effect on this cell line. The cells were pretreated with 40 µM amitriptyline for 12 h, followed by co-treatment with 100 ng/ml of TRAIL for 3 h. The cell morphologies were examined under a light microscope. Co-treatment with TRAIL increased the number of cells undergoing apoptotic death (Fig. 1A). The MTT assay revealed that the combined treatment triggered significant growth inhibition in a dose-dependent manner (Fig. 1B). The LDH levels after combined treatment demonstrated that amitriptyline induced apoptosis in a dose-dependent manner; however, the individual use of amitriptyline or TRAIL alone failed to show similar effects (Fig. 1C). Additionally, the colony-forming capacity of A549 cancer cells after combination treatment of amitriptyline and TRAIL was examined. Amitriptyline alone treatment not shown any inhibition effects (data now shown), but combine treatment with TRAIL gradually reduced the colony formation in a dose-dependent manner (Fig. 1D). These results indicated that amitriptyline significantly sensitized TRAIL-resistant A549 lung adenocarcinoma cells to TRAIL-mediated apoptosis.

To evaluate the principal mechanism underlying the apoptosis of A549 cells prompted by the combination of amitriptyline and TRAIL, the augmented expression of DRs associated with TRAIL-induced apoptosis was explored. An important reason for TRAIL resistance in numerous cancer cell lines is associated with the decreased expression of TRAIL receptors DR4 and DR5 or upregulation of the decoy receptors DcR1 and DcR2 (30). Western blot analysis demonstrated that amitriptyline increased DR4 and DR5 expression levels in a dose-dependent manner and time-dependent manner (Fig. 2A). When assessed via mRNA expression, amitriptyline treatment increased the transcription of DR5, but not DR4, (Fig. 2B). These results indicated that amitriptyline may increase DR5 expression through transcriptional or post-transcriptional regulation and concurrently amitriptyline stabilized DR4 protein expression by inhibiting its degradation through post-translational regulation. Moreover, immunocytochemistry results demonstrated the significant expression of DR4 and DR5 in amitriptyline-treated cells compared to non-treated cells (Fig. 2C). The apoptosis-indicating proteins cleaved caspase-8 and cleaved caspase-3 were activated after treatment with amitriptyline and TRAIL compared to treatments with each individually (Fig. 2D). Furthermore, the apoptosis percentage by Annexin V assay was measured, which indicated that amitriptyline and TRAIL in combination enhanced apoptotic cell death (Fig. 2E and F). Collectively, these findings indicated that DR4 and DR5 upregulated by amitriptyline induced TRAIL-mediated apoptosis in TRAIL-resistant A549 lung cancer cells.

It was hypothesized that DR4 and DR5 played important roles in amitriptyline-induced TRAIL-mediated apoptosis. In support of this hypothesis, DR4 and DR5-specific siRNA were applied to silence DR4 and DR5 expression, respectively. The silencing of DR4 and DR5 expression with specific siRNA restored cell viability. These data provided evidence that DR4 and DR5 play an important role in enhancing the effect of amitriptyline on TRAIL-induced apoptosis. Cells were transfected with DR4 and DR5-specific siRNAs or a NC siRNA for 24 h and the cells were treated with amitriptyline for 12 h, followed by incubation with TRAIL for an additional 3 h to assess cell viability or for 2 h for western blot analysis. The cell death induction capacity of amitriptyline combined with TRAIL significantly decreased after siRNA transfection. The combined effect of amitriptyline and TRAIL, however, on viability was similar in the NC siRNA-transfected cells (Figs. 3A and B, and 4A and B). Moreover, the colony formation-inhibiting capacity of amitriptyline combined with TRAIL-treated cells considerably decreased after siRNA transfection. The colony formation-inhibiting capacity of amitriptyline combined with TRAIL was similar in the NC control siRNA-transfected cells (Figs. 3C and 4C). Western blot analysis revealed that the expression of DR4 and DR5 was blocked after siRNA transfection compared to the non-transfected cells (Figs. 3D and 4D). These experimental findings confirmed that the upregulation of DR4 and DR5 is required in attenuating TRAIL resistance.

To investigate the role of amitriptyline in autophagy flux, the well-known autophagy markers LC3-II and p62 were analyzed. Western blot analysis revealed the conversion of LC3I to LC3-II, indicating the formation of complete autophagosomes. However, p62 is a cargo adaptor protein that depends on lysosomes or proteasomes for degradation (31). The expression of LC3-II and p62 was increased following amitriptyline treatment, indicating the blocking of autophagy flux by inhibiting autophagosome-lysosome fusion in the late stage of autophagy (Fig. 5A). The immunocytochemistry images also demonstrated the increased expression of p62 in a dose-dependent manner (Fig. 5B). Transmission electron microscopy revealed the higher accumulation of autophagic vacuoles compared to the control, confirming autophagy flux inhibition by amitriptyline (Fig. 5C). These results indicated that amitriptyline blocked autophagy flux at the final stage of autophagy.

The role of autophagy blocking in death receptor expression was investigated using an autophagy inhibitor. Blocking autophagy flux with a final stage autophagy inhibitor CQ upregulated both DR4 and DR5 expression, leading to an increase in apoptosis. The cells were treated with or without 20 µM CQ and the indicated doses of amitriptyline for 12 h. Western blot analysis revealed that amitriptyline and CQ increased the levels of LC3-II. Moreover, amitriptyline alone increased p62 levels in a dose-dependent manner. These results revealed that amitriptyline blocked autophagy flux to induce apoptosis (Fig. 6A). Furthermore, amitriptyline and the autophagy inhibitor CQ increased DR4 and DR5 expression (Fig. 6B). After 12 h treatment with CQ and amitriptyline, along with an additional 2 h TRAIL treatment, the expression of apoptosis-associated proteins cleaved caspase-8 and cleaved caspase-3 were observed. Cell lysates analyzed by western blotting demonstrated that treatment with CQ and TRAIL also activated caspase-8 and caspase-3 (Fig. 6C). The immunocytochemistry results also revealed that the CQ and TRAIL co-treatment expressed cleaved caspase-8 and cleaved caspase-3 compared to treatment with CQ or TRAIL alone (Fig. 6D). Additionally, to investigate the role of autophagy in TRAIL-mediated cell death, the cells were preincubated with CQ or amitriptyline with the indicated doses for 12 h, and then additionally incubated with TRAIL for 3 h. The cell morphology analyzed by light microscopy demonstrated slight cell death of the A549 cells treated with either TRAIL or amitriptyline alone. TRAIL-mediated cell death, however, was strongly increased by the combination of amitriptyline or CQ with TRAIL (Fig. 7A). In addition, A549 cells treated with either TRAIL, amitriptyline or CQ alone slightly reduced colony formation capacity; but, TRAIL in combination with amitriptyline or CQ strongly inhibited the colony formation capacity of A549 cells (Fig. 7B). The MTT assay showed reduced viability and significantly increased cell death in cells treated with amitriptyline or CQ plus TRAIL (Fig. 7C). The LDH assay also showed that CQ or amitriptyline combined with TRAIL increased apoptotic cell death (Fig. 7D). Overall, these results indicated that blocking autophagy-induced DR4 and DR5 upregulation aggravated TRAIL-mediated apoptosis.