Biosafety Research Institute , College of Veterinary Medicine , Jeonbuk National University , Gobong-ro, Iksan , 54596 , Jeonbuk, Republic of Korea , cbnu.edu
Faculty of Biotechnology and Genetic Engineering , Department of Animal and Fish Biotechnology , Sylhet Agricultural University , Sylhet , 3100 , Bangladesh , sau.ac.bd
Biosafety Research Institute , College of Veterinary Medicine , Jeonbuk National University , Gobong-ro, Iksan , 54596 , Jeonbuk, Republic of Korea , cbnu.edu
Biosafety Research Institute , College of Veterinary Medicine , Jeonbuk National University , Gobong-ro, Iksan , 54596 , Jeonbuk, Republic of Korea , cbnu.edu
Biosafety Research Institute , College of Veterinary Medicine , Jeonbuk National University , Gobong-ro, Iksan , 54596 , Jeonbuk, Republic of Korea , cbnu.edu
Biosafety Research Institute , College of Veterinary Medicine , Jeonbuk National University , Gobong-ro, Iksan , 54596 , Jeonbuk, Republic of Korea , cbnu.edu
Faculty of Biotechnology and Genetic Engineering , Department of Animal and Fish Biotechnology , Sylhet Agricultural University , Sylhet , 3100 , Bangladesh , sau.ac.bd
Biosafety Research Institute , College of Veterinary Medicine , Jeonbuk National University , Gobong-ro, Iksan , 54596 , Jeonbuk, Republic of Korea , cbnu.edu
Biosafety Research Institute , College of Veterinary Medicine , Jeonbuk National University , Gobong-ro, Iksan , 54596 , Jeonbuk, Republic of Korea , cbnu.edu
Biosafety Research Institute , College of Veterinary Medicine , Jeonbuk National University , Gobong-ro, Iksan , 54596 , Jeonbuk, Republic of Korea , cbnu.edu
To overcome TRAIL resistance, we tested the antidepressant drug citalopram (CTL) in combination with TRAIL. The resistance of several types of cancer cells to TRAIL impedes TRAIL-induced cancer cell death. In this study, we investigated the role of, and molecular mechanism by which, the antidepressant CTL-induced cell death in TRAIL-resistant lung cancer cells. We found that CTL increased death receptor 5 (DR5) expression levels by impairing autophagic flux and protecting against lysosomal degradation, thereby increasing the TRAIL-induced apoptosis of TRAIL-resistant A549 lung cancer cells. We also found that CTL impaired autophagic flux and promoted the conversion of light chain 3 (LC3)-I to its lipid-conjugated form, LC3-II, thereby inducing autophagosome accumulation. Our hypothesis that impaired autophagic flux plays an important role in the upregulation of DR5 being confirmed when we determined that DR5 upregulation by CTL was markedly decreased in the presence of rapamycin, an autophagy inducer. Further verification of our theory was achieved through experiments pairing CTL with the early-stage autophagy inhibitor 3-methyladenine (3-MA) and the late-stage autophagy inhibitor chloroquine (CQ). CQ inhibits autophagy by impairing autophagosome–lysosome fusion. Both CTL and CQ markedly increased DR5 expression levels and increase TRAIL-induced apoptosis, whereas 3-MA marginally enhanced TRAIL-induced apoptosis and resulted in minimal DR5 expression. In summary, our findings indicate that CTL impairs autophagic flux, resulting in autophagosome accumulation and augmentation of DR5 to potentiate TRAIL-induced apoptosis, suggesting that CTL may act as a therapeutic agent that sensitizes TRAIL-resistant cancer cells to TRAIL-mediated cancer therapy.
1. Introduction
Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is a cytokine capable of selectively killing cancer cells by binding to cognate death receptors, without causing toxicity to normal cells [1]. Many types of cancer cells are resistant to TRAIL because of inadequate expression of death receptors (DR4/DR5), excessive expression of decoy receptors, and genetic or epigenetic modification of TRAIL receptors [2, 3]. Once bound to its receptors (DR4 and DR5), TRAIL initiates extrinsic and intrinsic apoptotic signals and recruits FADD and Procaspase-8 to form death-inducing signaling complexes (DISCs). DISCs then activate the initiator Caspase-8 and induce apoptotic cell death through the expression of executor Caspases-3/6/7/9 [4, 5]. However, resistance to TRAIL-induced apoptosis remains a significant challenge, especially in aggressive cancers, such as lung cancer. Pharmacological agents that enhance TRAIL receptor expression are currently being considered for inclusion in anticancer combination therapies as a means of overcoming TRAIL resistance [6, 7].
Autophagy is a natural cellular self-regulatory mechanism through which cytosolic components and damaged or misfolded proteins are eliminated via lysosome-mediated degradation. In complete autophagic flux, cellular metabolism and homeostasis are maintained as cytosolic components and are organized into double-membrane vesicles (autophagosomes), which subsequently fuse with lysosomes for degradation and recycling [8]. Autophagosome formation is commonly detected by the conversion of the microtubule-associated protein Light Chain 3 (LC3)-I to its lipid-conjugated form, LC3-II, which is considered a marker of complete autophagosome formation [9]. The ubiquitin-like lysosomal protein p62 (SQSTM1) is involved in LC3-II degradation through autophagosome–lysosome fusion [10]. Accordingly, inhibiting lysosomal fusion by utilizing autophagosomes increases p62 protein levels [11]. The role of autophagy in the anticancer research is complex and controversial. Autophagy suppresses tumors when cancers develop but plays a survival role once cancer progresses [12]. Tumor cells also use autophagy to protect themselves from anticancer treatment [13].
Recent studies have suggested that blocking autophagy renders cancer cells more vulnerable to treatment. This may be a promising strategy for cancer therapy [14, 15]. Various autophagy inhibitors have been studied. 3-MA is an early-stage autophagy inhibitor that blocks autophagosome formation by inhibiting Class III PI3K [16]. Chen et al. showed that silencing autophagy genes (ATGs) and administration of 3-MA inhibit autophagosome accumulation and increases TRAIL-induced apoptosis of A549 lung cancer cells [17]. In contrast, Yang et al. showed that autophagosome accumulation, resulting from the inhibition of lysosomal fusion, increased oridonin-induced apoptosis in A549 cells, whereas 3-MA decreased oridonin-induced apoptotic cell death [18]. Siorkiewicz et al. found that activating autophagosome accumulation and inhibiting lysosome fusion impeded the proliferation of cervical cancer cells and diminished their resistance to the chemotherapeutic drugs cisplatin and paclitaxel [19]. A recent study identified the reason for the resistance of circulating tumor cells to TRAIL, in which death receptor 5 (DR5) was localized to the autophagosome for lysosomal degradation [20]. Therefore, pharmacological agents that induce autophagosome accumulation, but inhibit lysosomal fusion, may be a new means of overcoming TRAIL or any other chemotherapeutic resistance. In the present study, we tested the effectiveness of TRAIL in cancer cells at different stages of autophagy. We used 3-MA and chloroquine (CQ) to block early- and late-stage autophagy, respectively [21].
In recent years, antidepressant drugs have gained attention because of their potential therapeutic applications in mental health disorders [22]. These medications, which were originally designed to treat depression and anxiety, have been increasingly explored for their anticancer properties. Among various classes of antidepressants, selective serotonin reuptake inhibitors (SSRIs) have emerged as promising candidates for cancer therapy [23].
Citalopram (CTL), an antidepressant belonging to the SSRI class, has showed anticancer effects against various types of cancer cells [24]. CTL increases ROS formation and cytochrome c release and decreases the expression levels of the antiapoptotic protein Bcl-2 in hepatocellular carcinoma cells (HepG2) [25]. CTL also activates apoptotic signals in human acute myeloid leukemia HL-60 cells, confirming apoptotic cell death indicated by Caspase-3 activation [26]. Resistance to TRAIL-induced apoptosis remains a significant challenge, especially in aggressive cancers such as lung cancer. SSRIs, including CTL, have demonstrated promising anticancer effects across various cancer cell lines, with evidence suggesting that they may enhance sensitivity to TRAIL [23, 27–29]. Therefore, we investigated the role of CTL in TRAIL-resistant lung cancer cell lines. Many types of cancer cells, including lung cancer (A549, HCC-15, and Calu-3) cells, are resistant to the apoptotic effects of TRAIL [30]. In the present study, we observed that combined CTL and TRAIL treatment had a robust killing effect on A549, HCC-15, and Calu-3 cells. After confirming the cell killing effect, we sought to identify the underlying mechanism by which CTL acts as a TRAIL-sensitizing agent in A549 lung cancer cells. We determined that CTL accumulated in autophagosomes because of impaired autophagic flux, thereby inhibiting the breakdown of DR5 by degrading lysosomes and increasing TRAIL-induced apoptosis. We then determined the role of CTL in autophagosome accumulation using the early-stage autophagy inhibitor 3-MA and the late-stage inhibitor CQ. Our observations confirmed that CTL-mediated autophagosome accumulation and enhanced TRAIL-induced apoptosis via DR5 upregulation.
2. Materials and Methods
2.1. Cell Culture Methods
A549 and HCC-15 lung cancer cell lines were acquired from the American Type Culture Collection (Global Bioresource Center, Manassas, VA, USA), while Calu-3 cancer cells were acquired from the Korean Cell Line Bank. These cells were cultured in a CO2 incubator as previously described [30].
2.2. Reagents
CTL, 3-MA, and CQ were obtained from Sigma-Aldrich (St. Louis, MO, USA). TRAIL was purchased from AbFrontier (Seoul, South Korea).
2.3. Cell Viability Assessment
A549, HCC-15, and Calu-3 lung cancer cells were cultured in 12-well plates and incubated at 37°C for 24 h. CTL was applied at various concentrations and time intervals (6, 12, 18, and 24 h) to determine the appropriate subtoxic doses that would not affect cell viability or morphology. Based on these preliminary tests, we selected 50, 100, and 200 μM concentrations of CTL for our experiments. While all time periods showed similar effects, the optimal treatment times found to be 12, 18, and 12 h were chosen for subsequent experiments. Cells were exposed to 100 ng/mL TRAIL for additional 3 h following established protocols [15, 23, 27, 29]. Cell morphology was observed, and images were captured using an inverted microscope (Nikon Corporation, Tokyo, Japan). Crystal violet staining was used to stain the cell biomass. Cells were washed with 1% phosphate-buffered saline (PBS) one or two times, 350–400 μL of crystal violet staining solution was added for 10–20 min at 25°C, and the cells were rinsed with water and dried. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was performed to assess cell viability. In this assay, 350 μL of 5 mg/mL MTT solution was added per well, and the cells were incubated at 37°C for 3 h. The MTT solution was removed, and the cells were rinsed once with PBS before 500 μL of DMSO was added to each well. Finally, the absorbance was measured using a spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All experiments were performed at least thrice. Cell viability was expressed as a percentage relative to the viability of the control group, which was set at 100%.
2.4. Lactate Dehydrogenase (LDH) Assay
Cytotoxicity was assessed in cell supernatants using an LDH cytotoxicity detection kit and performed in accordance with the manufacturer’s protocol (Takara Bio, Inc., Tokyo, Japan). The amount of LDH was determined as previously described [30].
2.5. Protein Isolation and Western Blotting
Cells were washed with PBS, lysed in lysis buffer, and used for protein isolation and blotting, as previously described [23]. DR5 (1:10,000; Abcam, Cambridge, MA, USA), DR4 (1:1000; Abcam), LC3 (1:1000; Sigma-Aldrich), p62 (Sigma-Aldrich), cleaved Caspase-8 (BD Pharmingen, San Jose, CA, USA), cleaved Caspase-3 (Cell Signaling Technology, Danvers, MA, USA), and β-actin (Sigma-Aldrich) were used as received.
2.6. Immunocytochemistry (ICC)
ICC was performed following a previously published protocol [23]. A549 cells were grown on glass coverslips for 24 h in 24-well plates, treated with CTL or CQ for 12 h, further treated with TRAIL for 2 h, washed with PBS, and fixed for 15–20 min in 4% paraformaldehyde. After blocking with 1% bovine serum albumin (BSA) in PBS containing Tween 20, the cells were incubated with primary antibodies against p62 and DR5 for 3 h at 37°C. Subsequently, the cells were incubated with an Alexa-Fluor-488-conjugated donkey anti-rabbit secondary antibody for 2 h at 25°C in the dark. Finally, the cells were stained with DAPI, washed, mounted, and imaged using a Nikon ECLIPSE 80i fluorescence microscope at 400× magnification.
2.7. Transmission Electron Microscopy (TEM)
The TEM protocol was performed as previously described [23]. Trypsinized A549 cells were fixed with 2% glutaraldehyde and 2% paraformaldehyde in 0.05 M sodium cacodylate buffer (pH 7.2) for 2 h at 4°C and postfixed with 2% osmium tetroxide for 1 h at 4°C. After dehydration using a graded ethanol series, the cells were embedded in epoxy resin (Embed 812) and sectioned into 60 nm ultrathin sections using an LKB-III ultramicrotome. The sections were stained with uranyl acetate and lead citrate, and images were acquired at 100,00× magnification using a Hitachi H7650 TEM (Hitachi, Tokyo, Japan) at the Center for University-Wide Research Facilities, Jeonbuk National University (Jeonju, South Korea).
2.8. Small Interfering RNA (siRNA) Transfection
A549 cells were transfected with an siRNA targeting DR5 (Qiagen, Hilden, Germany) (forward, 5′-ACCAGGTGTGATTCAGGTGAA-3′ and reverse, 5′-CCGACTTCACTTGATACTATA-3′) or a scrambled control siRNA (Ambion, Life Technologies, Austin, TX, USA) following the manufacturer’s protocol. The transfection reagent, Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), was used according to the manufacturer’s protocol. Briefly, siRNA (20 μmol/L) and lipofectamine 2000 were diluted in serum-free medium, incubated for 30 min, and then added to the cells. After 6 h, the medium was replaced with complete medium containing 10% FBS. The knockdown efficiency was confirmed using immunoblotting and cell viability assays.
2.9. Statistical Analysis
Data are shown as the mean ± standard deviation (SD) from three independent experiments (n = 3). Significance was determined as described previously [23]. Data were analyzed using one-way analysis of variance followed by Tukey’s multiple comparison test. Statistical analyses were performed using GraphPad Prism Version 5 (GraphPad, San Diego, CA, USA). Differences were considered statistically significant at p < 0.05.
3. Results
3.1. CTL-Enhanced TRAIL-Induced Lung Cancer Cell Death
First, we determined the combined effects of CTL and TRAIL on the inhibition of lung cancer cell proliferation. The combination treatment showed a strong cell-killing effect in all three cell lines examined. Each cell line was preincubated with CTL (200 μM) for 12 h and then cotreated with TRAIL (100 ng/mL) for 3 h. Examination of cell morphologies and their changes by light microscopic observation revealed a dose-dependent severe cell-killing effect of combined treatment, while no significant effect was observed when the cells were treated with CTL or TRAIL alone (Figures 1(a), 1(e), and 1(i)). The density of the dye used in the crystal violet assay gradually decreased, demonstrating that the total cellular biomass decreased because of the severe cell-killing effects of the combined treatment (Figures 1(b), 1(f), and 1(j)). The MTT assay confirmed these results, demonstrating that CTL, in combination with TRAIL, significantly inhibited cell growth in a dose-dependent manner (Figures 1(c), 1(g), and 1(k)). Finally, the LDH assay showed that the combined treatment significantly increased cytotoxicity in lung cancer cells compared to the treatment with only CTL or TRAIL (Figures 1(d), 1(h), and 1(l)). These results suggested that CTL sensitized TRAIL-resistant lung adenocarcinoma cells to TRAIL-mediated cell death.
CTL-enhanced TRAIL-induced cell death in lung cancer cells. A549 (a–d), HCC-15 (e–h), and Calu-3 (i–l) cells were seeded for 24 h and then pretreated with the indicated doses of CTL for 12 h, after which 100 ng/mL TRAIL was added for 2 h 30 min. (a, e, and i) Variations in cell morphology were observed and imaged with a light microscope (magnification, × 100; scale bar, 50 μm). (b, f, and j) The mean density of the crystal violet staining was used to determine the total cellular biomass. (c, g, and k) MTT assays were performed to evaluate cell viability. (d, h, and l) LDH release was measured in collected supernatants to evaluate cytotoxicity. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3). CTL, citalopram; LDH, lactate dehydrogenase; MTT, [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.
CTL-enhanced TRAIL-induced cell death in lung cancer cells. A549 (a–d), HCC-15 (e–h), and Calu-3 (i–l) cells were seeded for 24 h and then pretreated with the indicated doses of CTL for 12 h, after which 100 ng/mL TRAIL was added for 2 h 30 min. (a, e, and i) Variations in cell morphology were observed and imaged with a light microscope (magnification, × 100; scale bar, 50 μm). (b, f, and j) The mean density of the crystal violet staining was used to determine the total cellular biomass. (c, g, and k) MTT assays were performed to evaluate cell viability. (d, h, and l) LDH release was measured in collected supernatants to evaluate cytotoxicity. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3). CTL, citalopram; LDH, lactate dehydrogenase; MTT, [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.
CTL-enhanced TRAIL-induced cell death in lung cancer cells. A549 (a–d), HCC-15 (e–h), and Calu-3 (i–l) cells were seeded for 24 h and then pretreated with the indicated doses of CTL for 12 h, after which 100 ng/mL TRAIL was added for 2 h 30 min. (a, e, and i) Variations in cell morphology were observed and imaged with a light microscope (magnification, × 100; scale bar, 50 μm). (b, f, and j) The mean density of the crystal violet staining was used to determine the total cellular biomass. (c, g, and k) MTT assays were performed to evaluate cell viability. (d, h, and l) LDH release was measured in collected supernatants to evaluate cytotoxicity. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3). CTL, citalopram; LDH, lactate dehydrogenase; MTT, [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.
CTL-enhanced TRAIL-induced cell death in lung cancer cells. A549 (a–d), HCC-15 (e–h), and Calu-3 (i–l) cells were seeded for 24 h and then pretreated with the indicated doses of CTL for 12 h, after which 100 ng/mL TRAIL was added for 2 h 30 min. (a, e, and i) Variations in cell morphology were observed and imaged with a light microscope (magnification, × 100; scale bar, 50 μm). (b, f, and j) The mean density of the crystal violet staining was used to determine the total cellular biomass. (c, g, and k) MTT assays were performed to evaluate cell viability. (d, h, and l) LDH release was measured in collected supernatants to evaluate cytotoxicity. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3). CTL, citalopram; LDH, lactate dehydrogenase; MTT, [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.
CTL-enhanced TRAIL-induced cell death in lung cancer cells. A549 (a–d), HCC-15 (e–h), and Calu-3 (i–l) cells were seeded for 24 h and then pretreated with the indicated doses of CTL for 12 h, after which 100 ng/mL TRAIL was added for 2 h 30 min. (a, e, and i) Variations in cell morphology were observed and imaged with a light microscope (magnification, × 100; scale bar, 50 μm). (b, f, and j) The mean density of the crystal violet staining was used to determine the total cellular biomass. (c, g, and k) MTT assays were performed to evaluate cell viability. (d, h, and l) LDH release was measured in collected supernatants to evaluate cytotoxicity. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3). CTL, citalopram; LDH, lactate dehydrogenase; MTT, [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.
CTL-enhanced TRAIL-induced cell death in lung cancer cells. A549 (a–d), HCC-15 (e–h), and Calu-3 (i–l) cells were seeded for 24 h and then pretreated with the indicated doses of CTL for 12 h, after which 100 ng/mL TRAIL was added for 2 h 30 min. (a, e, and i) Variations in cell morphology were observed and imaged with a light microscope (magnification, × 100; scale bar, 50 μm). (b, f, and j) The mean density of the crystal violet staining was used to determine the total cellular biomass. (c, g, and k) MTT assays were performed to evaluate cell viability. (d, h, and l) LDH release was measured in collected supernatants to evaluate cytotoxicity. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3). CTL, citalopram; LDH, lactate dehydrogenase; MTT, [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.
CTL-enhanced TRAIL-induced cell death in lung cancer cells. A549 (a–d), HCC-15 (e–h), and Calu-3 (i–l) cells were seeded for 24 h and then pretreated with the indicated doses of CTL for 12 h, after which 100 ng/mL TRAIL was added for 2 h 30 min. (a, e, and i) Variations in cell morphology were observed and imaged with a light microscope (magnification, × 100; scale bar, 50 μm). (b, f, and j) The mean density of the crystal violet staining was used to determine the total cellular biomass. (c, g, and k) MTT assays were performed to evaluate cell viability. (d, h, and l) LDH release was measured in collected supernatants to evaluate cytotoxicity. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3). CTL, citalopram; LDH, lactate dehydrogenase; MTT, [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.
CTL-enhanced TRAIL-induced cell death in lung cancer cells. A549 (a–d), HCC-15 (e–h), and Calu-3 (i–l) cells were seeded for 24 h and then pretreated with the indicated doses of CTL for 12 h, after which 100 ng/mL TRAIL was added for 2 h 30 min. (a, e, and i) Variations in cell morphology were observed and imaged with a light microscope (magnification, × 100; scale bar, 50 μm). (b, f, and j) The mean density of the crystal violet staining was used to determine the total cellular biomass. (c, g, and k) MTT assays were performed to evaluate cell viability. (d, h, and l) LDH release was measured in collected supernatants to evaluate cytotoxicity. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3). CTL, citalopram; LDH, lactate dehydrogenase; MTT, [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.
CTL-enhanced TRAIL-induced cell death in lung cancer cells. A549 (a–d), HCC-15 (e–h), and Calu-3 (i–l) cells were seeded for 24 h and then pretreated with the indicated doses of CTL for 12 h, after which 100 ng/mL TRAIL was added for 2 h 30 min. (a, e, and i) Variations in cell morphology were observed and imaged with a light microscope (magnification, × 100; scale bar, 50 μm). (b, f, and j) The mean density of the crystal violet staining was used to determine the total cellular biomass. (c, g, and k) MTT assays were performed to evaluate cell viability. (d, h, and l) LDH release was measured in collected supernatants to evaluate cytotoxicity. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3). CTL, citalopram; LDH, lactate dehydrogenase; MTT, [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.
CTL-enhanced TRAIL-induced cell death in lung cancer cells. A549 (a–d), HCC-15 (e–h), and Calu-3 (i–l) cells were seeded for 24 h and then pretreated with the indicated doses of CTL for 12 h, after which 100 ng/mL TRAIL was added for 2 h 30 min. (a, e, and i) Variations in cell morphology were observed and imaged with a light microscope (magnification, × 100; scale bar, 50 μm). (b, f, and j) The mean density of the crystal violet staining was used to determine the total cellular biomass. (c, g, and k) MTT assays were performed to evaluate cell viability. (d, h, and l) LDH release was measured in collected supernatants to evaluate cytotoxicity. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3). CTL, citalopram; LDH, lactate dehydrogenase; MTT, [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.
CTL-enhanced TRAIL-induced cell death in lung cancer cells. A549 (a–d), HCC-15 (e–h), and Calu-3 (i–l) cells were seeded for 24 h and then pretreated with the indicated doses of CTL for 12 h, after which 100 ng/mL TRAIL was added for 2 h 30 min. (a, e, and i) Variations in cell morphology were observed and imaged with a light microscope (magnification, × 100; scale bar, 50 μm). (b, f, and j) The mean density of the crystal violet staining was used to determine the total cellular biomass. (c, g, and k) MTT assays were performed to evaluate cell viability. (d, h, and l) LDH release was measured in collected supernatants to evaluate cytotoxicity. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3). CTL, citalopram; LDH, lactate dehydrogenase; MTT, [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.
CTL-enhanced TRAIL-induced cell death in lung cancer cells. A549 (a–d), HCC-15 (e–h), and Calu-3 (i–l) cells were seeded for 24 h and then pretreated with the indicated doses of CTL for 12 h, after which 100 ng/mL TRAIL was added for 2 h 30 min. (a, e, and i) Variations in cell morphology were observed and imaged with a light microscope (magnification, × 100; scale bar, 50 μm). (b, f, and j) The mean density of the crystal violet staining was used to determine the total cellular biomass. (c, g, and k) MTT assays were performed to evaluate cell viability. (d, h, and l) LDH release was measured in collected supernatants to evaluate cytotoxicity. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3). CTL, citalopram; LDH, lactate dehydrogenase; MTT, [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.
3.2. CTL Increased DR5 Expression Levels to Increase TRAIL-Induced Apoptosis
Then, we investigated the mechanisms underlying the TRAIL-mediated cell-killing effect of the combined treatment. Therefore, we assessed the expression of DR4 and DR5 in A549 lung cancer cells. Western blotting analysis revealed that CTL treatment alone dose-dependently increased DR5 expression levels but had no significant effect on DR4 expression (Figures 2(a), 2(b), and 2(c)). However, the combined treatment with CTL and TRAIL markedly improved the levels of apoptosis marker proteins and cleaved Caspase-8 and Caspase-3 compared with the CTL treatment alone (Figure 2(d)). The ICC results also showed that the combination treatment markedly increased Caspase-8 and Caspase-3 expression levels (Figure 2(e)). These results suggested that CTL-mediated DR5 expression plays a role in TRAIL-induced apoptosis.
CTL-mediated impaired autophagic flux increased DR5 expression levels to enhance TRAIL-mediated apoptosis. A549 cells were cultured and incubated with the indicated doses of CTL for 12 h. (a) Cell lysates were prepared for western blotting, which was performed to evaluate DR4 and DR5 protein expression. (b, c) Density ratio of DR4 (b) and DR5 (c) compared with the controls. (d) Cells were treated with CTL (200 μM) for 12 h and then treated with TRAIL protein (100 ng/mL) for 2 h. Total and cleaved forms of Caspase-8 and Caspase-3 were detected using western blotting. (e) Immunocytochemistry showed that Caspase-8 and Caspase-3 were upregulated in cells treated with a combination of CTL and TRAIL (scale bar, 50 μm). β-actin was used as a loading control. CTL, citalopram; DR4, death receptor 4; DR5, death receptor 5; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand. Results are expressed as the mean ± standard deviation (SD). ∗p < 0.05 and ∗∗∗p < 0.001 vs. the untreated group (n = 3).
CTL-mediated impaired autophagic flux increased DR5 expression levels to enhance TRAIL-mediated apoptosis. A549 cells were cultured and incubated with the indicated doses of CTL for 12 h. (a) Cell lysates were prepared for western blotting, which was performed to evaluate DR4 and DR5 protein expression. (b, c) Density ratio of DR4 (b) and DR5 (c) compared with the controls. (d) Cells were treated with CTL (200 μM) for 12 h and then treated with TRAIL protein (100 ng/mL) for 2 h. Total and cleaved forms of Caspase-8 and Caspase-3 were detected using western blotting. (e) Immunocytochemistry showed that Caspase-8 and Caspase-3 were upregulated in cells treated with a combination of CTL and TRAIL (scale bar, 50 μm). β-actin was used as a loading control. CTL, citalopram; DR4, death receptor 4; DR5, death receptor 5; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand. Results are expressed as the mean ± standard deviation (SD). ∗p < 0.05 and ∗∗∗p < 0.001 vs. the untreated group (n = 3).
CTL-mediated impaired autophagic flux increased DR5 expression levels to enhance TRAIL-mediated apoptosis. A549 cells were cultured and incubated with the indicated doses of CTL for 12 h. (a) Cell lysates were prepared for western blotting, which was performed to evaluate DR4 and DR5 protein expression. (b, c) Density ratio of DR4 (b) and DR5 (c) compared with the controls. (d) Cells were treated with CTL (200 μM) for 12 h and then treated with TRAIL protein (100 ng/mL) for 2 h. Total and cleaved forms of Caspase-8 and Caspase-3 were detected using western blotting. (e) Immunocytochemistry showed that Caspase-8 and Caspase-3 were upregulated in cells treated with a combination of CTL and TRAIL (scale bar, 50 μm). β-actin was used as a loading control. CTL, citalopram; DR4, death receptor 4; DR5, death receptor 5; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand. Results are expressed as the mean ± standard deviation (SD). ∗p < 0.05 and ∗∗∗p < 0.001 vs. the untreated group (n = 3).
CTL-mediated impaired autophagic flux increased DR5 expression levels to enhance TRAIL-mediated apoptosis. A549 cells were cultured and incubated with the indicated doses of CTL for 12 h. (a) Cell lysates were prepared for western blotting, which was performed to evaluate DR4 and DR5 protein expression. (b, c) Density ratio of DR4 (b) and DR5 (c) compared with the controls. (d) Cells were treated with CTL (200 μM) for 12 h and then treated with TRAIL protein (100 ng/mL) for 2 h. Total and cleaved forms of Caspase-8 and Caspase-3 were detected using western blotting. (e) Immunocytochemistry showed that Caspase-8 and Caspase-3 were upregulated in cells treated with a combination of CTL and TRAIL (scale bar, 50 μm). β-actin was used as a loading control. CTL, citalopram; DR4, death receptor 4; DR5, death receptor 5; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand. Results are expressed as the mean ± standard deviation (SD). ∗p < 0.05 and ∗∗∗p < 0.001 vs. the untreated group (n = 3).
CTL-mediated impaired autophagic flux increased DR5 expression levels to enhance TRAIL-mediated apoptosis. A549 cells were cultured and incubated with the indicated doses of CTL for 12 h. (a) Cell lysates were prepared for western blotting, which was performed to evaluate DR4 and DR5 protein expression. (b, c) Density ratio of DR4 (b) and DR5 (c) compared with the controls. (d) Cells were treated with CTL (200 μM) for 12 h and then treated with TRAIL protein (100 ng/mL) for 2 h. Total and cleaved forms of Caspase-8 and Caspase-3 were detected using western blotting. (e) Immunocytochemistry showed that Caspase-8 and Caspase-3 were upregulated in cells treated with a combination of CTL and TRAIL (scale bar, 50 μm). β-actin was used as a loading control. CTL, citalopram; DR4, death receptor 4; DR5, death receptor 5; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand. Results are expressed as the mean ± standard deviation (SD). ∗p < 0.05 and ∗∗∗p < 0.001 vs. the untreated group (n = 3).
3.3. Silencing DR5 Expression Alleviated the TRAIL-Induced Cell-Killing Effect
To confirm the role of CTL-mediated DR5 expression in TRAIL-induced apoptosis, a DR5-specific siRNA was used to silence DR5 expression. The cytotoxic effects of CTL combined with TRAIL were markedly reduced after siRNA transfection. Morphological observation of the cells, MTT assays, and crystal violet staining confirmed that cell death was reduced after DR5 siRNA transfection. However, as expected, the combined effect of CTL and TRAIL on cell viability was similar in negative control siRNA-transfected cells (Figures 3(a), 3(b), and 3(c)). The silencing efficacy of the DR5 siRNA was assessed using western blotting, which showed that DR5 siRNA transfection almost completely blocked DR5 expression (Figures 3(d) and 3(e)). In summary, these results confirmed that the upregulation of DR5 by CTL played an important role in TRAIL-induced cell death.
Silencing DR5 expression alleviated the TRAIL-induced cell-killing effect. A549 cells were transfected with a DR5-specific siRNA or a negative control (NC) siRNA (40 nM) for 24 h. Cells were treated with CTL (200 μM) for 12 h and then treated to TRAIL (100 ng/mL) for 2 h 30 min. (a) Cell images were captured, and morphological variations were observed using a light microscope (× 100). (b) The total cellular biomass density was determined using crystal violet staining. (c) Cell viability, as a percentage of the control, was assessed using MTT assays and presented as a bar diagram. (d) Cell lysates were prepared for the western blotting analysis to determine DR5 expression levels. (e) Ratio of DR5 to control density. Every result reflects the average of three independent experiments. The results are expressed as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. the untreated group (n = 3).
Silencing DR5 expression alleviated the TRAIL-induced cell-killing effect. A549 cells were transfected with a DR5-specific siRNA or a negative control (NC) siRNA (40 nM) for 24 h. Cells were treated with CTL (200 μM) for 12 h and then treated to TRAIL (100 ng/mL) for 2 h 30 min. (a) Cell images were captured, and morphological variations were observed using a light microscope (× 100). (b) The total cellular biomass density was determined using crystal violet staining. (c) Cell viability, as a percentage of the control, was assessed using MTT assays and presented as a bar diagram. (d) Cell lysates were prepared for the western blotting analysis to determine DR5 expression levels. (e) Ratio of DR5 to control density. Every result reflects the average of three independent experiments. The results are expressed as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. the untreated group (n = 3).
Silencing DR5 expression alleviated the TRAIL-induced cell-killing effect. A549 cells were transfected with a DR5-specific siRNA or a negative control (NC) siRNA (40 nM) for 24 h. Cells were treated with CTL (200 μM) for 12 h and then treated to TRAIL (100 ng/mL) for 2 h 30 min. (a) Cell images were captured, and morphological variations were observed using a light microscope (× 100). (b) The total cellular biomass density was determined using crystal violet staining. (c) Cell viability, as a percentage of the control, was assessed using MTT assays and presented as a bar diagram. (d) Cell lysates were prepared for the western blotting analysis to determine DR5 expression levels. (e) Ratio of DR5 to control density. Every result reflects the average of three independent experiments. The results are expressed as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. the untreated group (n = 3).
Silencing DR5 expression alleviated the TRAIL-induced cell-killing effect. A549 cells were transfected with a DR5-specific siRNA or a negative control (NC) siRNA (40 nM) for 24 h. Cells were treated with CTL (200 μM) for 12 h and then treated to TRAIL (100 ng/mL) for 2 h 30 min. (a) Cell images were captured, and morphological variations were observed using a light microscope (× 100). (b) The total cellular biomass density was determined using crystal violet staining. (c) Cell viability, as a percentage of the control, was assessed using MTT assays and presented as a bar diagram. (d) Cell lysates were prepared for the western blotting analysis to determine DR5 expression levels. (e) Ratio of DR5 to control density. Every result reflects the average of three independent experiments. The results are expressed as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. the untreated group (n = 3).
Silencing DR5 expression alleviated the TRAIL-induced cell-killing effect. A549 cells were transfected with a DR5-specific siRNA or a negative control (NC) siRNA (40 nM) for 24 h. Cells were treated with CTL (200 μM) for 12 h and then treated to TRAIL (100 ng/mL) for 2 h 30 min. (a) Cell images were captured, and morphological variations were observed using a light microscope (× 100). (b) The total cellular biomass density was determined using crystal violet staining. (c) Cell viability, as a percentage of the control, was assessed using MTT assays and presented as a bar diagram. (d) Cell lysates were prepared for the western blotting analysis to determine DR5 expression levels. (e) Ratio of DR5 to control density. Every result reflects the average of three independent experiments. The results are expressed as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. the untreated group (n = 3).
3.4. CTL Impaired Autophagic Flux and Protected DR5 From Degradation
To explore the role of CTL in autophagic flux, we detected two autophagy markers, LC3-II and p62, using western blotting. The CTL treatment impaired autophagic flux by inhibiting autophagosomes and lysosomes, as indicated by the formation of complete autophagosomes and increased p62 levels (Figure 4(a)). TEM results revealed that CTL-treated cells showed autophagic vacuoles with condensed cellular components and that untreated cells, as well as cells treated with the autophagy-inducer rapamycin, showed normal cellular degradation through autophagy (Figure 4(b)). Similar to CTL, CQ prevented lysosomal acidification, inhibited autophagosome fusion, and affected DR5 expression. Both CQ and CTL increased DR5 expression levels; however, in the presence of rapamycin, CQ- and CTL-mediated DR5 expression levels were markedly decreased. Changes in p62 and LC3 expression levels confirmed that DR5 was degraded via autophagy (Figures 4(c), 4(d), and 4(e)). In summary, these experiments indicated that CTL inhibited autophagosome–lysosome fusion and protected DR5 from degradation in A459 lung cancer cells.
CTL-mediated autophagic flux was impaired, which protected DR5 from degradation. A549 cells were treated with the indicated doses of CTL for 12 h. (a) LC3 conversion and p62 protein expression levels were assessed by the western blotting analysis. (b) Cells were treated with CTL (200 μM) or RAPA (20 nM) for 12 h. Transmission electron microscopy was used to visualize the accumulation of autophagosomes (scale bar, 0.5 μm). DR5, LC3, and p62 protein expression were evaluated after treatment with CTL, CQ, CTL with RAPA, or CQ with RAPA. (c) Relative protein expression levels of DR5 and β-actin (relative densitometry analysis). (d) Relative protein expression levels of LC3 and β-actin (relative densitometry analysis). (e) Relative protein expression levels of p62 and β-actin (relative densitometry analysis). The results are expressed as the mean ± SD. ∗∗∗p < 0.001 vs. the untreated group (n = 3).
CTL-mediated autophagic flux was impaired, which protected DR5 from degradation. A549 cells were treated with the indicated doses of CTL for 12 h. (a) LC3 conversion and p62 protein expression levels were assessed by the western blotting analysis. (b) Cells were treated with CTL (200 μM) or RAPA (20 nM) for 12 h. Transmission electron microscopy was used to visualize the accumulation of autophagosomes (scale bar, 0.5 μm). DR5, LC3, and p62 protein expression were evaluated after treatment with CTL, CQ, CTL with RAPA, or CQ with RAPA. (c) Relative protein expression levels of DR5 and β-actin (relative densitometry analysis). (d) Relative protein expression levels of LC3 and β-actin (relative densitometry analysis). (e) Relative protein expression levels of p62 and β-actin (relative densitometry analysis). The results are expressed as the mean ± SD. ∗∗∗p < 0.001 vs. the untreated group (n = 3).
CTL-mediated autophagic flux was impaired, which protected DR5 from degradation. A549 cells were treated with the indicated doses of CTL for 12 h. (a) LC3 conversion and p62 protein expression levels were assessed by the western blotting analysis. (b) Cells were treated with CTL (200 μM) or RAPA (20 nM) for 12 h. Transmission electron microscopy was used to visualize the accumulation of autophagosomes (scale bar, 0.5 μm). DR5, LC3, and p62 protein expression were evaluated after treatment with CTL, CQ, CTL with RAPA, or CQ with RAPA. (c) Relative protein expression levels of DR5 and β-actin (relative densitometry analysis). (d) Relative protein expression levels of LC3 and β-actin (relative densitometry analysis). (e) Relative protein expression levels of p62 and β-actin (relative densitometry analysis). The results are expressed as the mean ± SD. ∗∗∗p < 0.001 vs. the untreated group (n = 3).
CTL-mediated autophagic flux was impaired, which protected DR5 from degradation. A549 cells were treated with the indicated doses of CTL for 12 h. (a) LC3 conversion and p62 protein expression levels were assessed by the western blotting analysis. (b) Cells were treated with CTL (200 μM) or RAPA (20 nM) for 12 h. Transmission electron microscopy was used to visualize the accumulation of autophagosomes (scale bar, 0.5 μm). DR5, LC3, and p62 protein expression were evaluated after treatment with CTL, CQ, CTL with RAPA, or CQ with RAPA. (c) Relative protein expression levels of DR5 and β-actin (relative densitometry analysis). (d) Relative protein expression levels of LC3 and β-actin (relative densitometry analysis). (e) Relative protein expression levels of p62 and β-actin (relative densitometry analysis). The results are expressed as the mean ± SD. ∗∗∗p < 0.001 vs. the untreated group (n = 3).
CTL-mediated autophagic flux was impaired, which protected DR5 from degradation. A549 cells were treated with the indicated doses of CTL for 12 h. (a) LC3 conversion and p62 protein expression levels were assessed by the western blotting analysis. (b) Cells were treated with CTL (200 μM) or RAPA (20 nM) for 12 h. Transmission electron microscopy was used to visualize the accumulation of autophagosomes (scale bar, 0.5 μm). DR5, LC3, and p62 protein expression were evaluated after treatment with CTL, CQ, CTL with RAPA, or CQ with RAPA. (c) Relative protein expression levels of DR5 and β-actin (relative densitometry analysis). (d) Relative protein expression levels of LC3 and β-actin (relative densitometry analysis). (e) Relative protein expression levels of p62 and β-actin (relative densitometry analysis). The results are expressed as the mean ± SD. ∗∗∗p < 0.001 vs. the untreated group (n = 3).
3.5. CTL Impaired Autophagic Flux and Enhanced TRAIL-Mediated Apoptosis
We investigated the role of autophagy inhibition by early- (3-MA) and late-stage (CQ) autophagy inhibitors on DR5 expression and TRAIL-induced apoptosis. Cell culture samples were pretreated with 3-MA (2 mM), CQ (20 μM), or the indicated doses of CTL for 12 h. The immunoblotting assay results showed that CTL and CQ increased DR5 expression levels (Figures 5(a) and 5(b)) through autophagosome accumulation, as confirmed by highly elevated LC3-II and p62 levels, and that 3-MA inhibited autophagosome accumulation and marginally increased DR5 expression levels. These tests confirmed the role of autophagosome accumulation in DR5 expression (Figures 5(c), 5(d), and 5(e)). We also checked the expression levels of apoptosis-related proteins, cleaved Caspase-8, and cleaved Caspase-3. When combined with TRAIL, CTL, CQ, and 3-MA increased cleaved Caspase-8 levels. 3-MA only marginally increased the level of cleaved Caspase-3 compared to the effects of CTL and CQ (Figure 5(f)). In summary, these findings demonstrated that CTL-impaired autophagy upregulated DR5 expression, thereby mediating autophagosome accumulation and enhancing TRAIL-mediated apoptosis.
CTL-mediated impaired autophagic flux enhanced TRAIL-mediated apoptosis. Cells were incubated with CTL (200 μM), CQ (20 μM), or 3-MA (2 mM) for 12 h. (a) DR5 expression was assessed by western blotting. (b) Ratio of DR5 and control density, (c) p62 and LC3 expression was assessed by western blotting. (d, e) Ratio of LC3 and p62 and control densities. (f) Cells were incubated with CTL (200 μM), CQ (20 μM), or 3-MA (5 mM) for 12 h and then exposed or not to TRAIL (100 ng/mL) for 2 h. Western blotting results show the expression levels of cleaved Caspase-8 and cleaved Caspase-3. 3-MA, 3-methyladenine. The results are expressed as the mean ± SD. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3).
CTL-mediated impaired autophagic flux enhanced TRAIL-mediated apoptosis. Cells were incubated with CTL (200 μM), CQ (20 μM), or 3-MA (2 mM) for 12 h. (a) DR5 expression was assessed by western blotting. (b) Ratio of DR5 and control density, (c) p62 and LC3 expression was assessed by western blotting. (d, e) Ratio of LC3 and p62 and control densities. (f) Cells were incubated with CTL (200 μM), CQ (20 μM), or 3-MA (5 mM) for 12 h and then exposed or not to TRAIL (100 ng/mL) for 2 h. Western blotting results show the expression levels of cleaved Caspase-8 and cleaved Caspase-3. 3-MA, 3-methyladenine. The results are expressed as the mean ± SD. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3).
CTL-mediated impaired autophagic flux enhanced TRAIL-mediated apoptosis. Cells were incubated with CTL (200 μM), CQ (20 μM), or 3-MA (2 mM) for 12 h. (a) DR5 expression was assessed by western blotting. (b) Ratio of DR5 and control density, (c) p62 and LC3 expression was assessed by western blotting. (d, e) Ratio of LC3 and p62 and control densities. (f) Cells were incubated with CTL (200 μM), CQ (20 μM), or 3-MA (5 mM) for 12 h and then exposed or not to TRAIL (100 ng/mL) for 2 h. Western blotting results show the expression levels of cleaved Caspase-8 and cleaved Caspase-3. 3-MA, 3-methyladenine. The results are expressed as the mean ± SD. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3).
CTL-mediated impaired autophagic flux enhanced TRAIL-mediated apoptosis. Cells were incubated with CTL (200 μM), CQ (20 μM), or 3-MA (2 mM) for 12 h. (a) DR5 expression was assessed by western blotting. (b) Ratio of DR5 and control density, (c) p62 and LC3 expression was assessed by western blotting. (d, e) Ratio of LC3 and p62 and control densities. (f) Cells were incubated with CTL (200 μM), CQ (20 μM), or 3-MA (5 mM) for 12 h and then exposed or not to TRAIL (100 ng/mL) for 2 h. Western blotting results show the expression levels of cleaved Caspase-8 and cleaved Caspase-3. 3-MA, 3-methyladenine. The results are expressed as the mean ± SD. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3).
CTL-mediated impaired autophagic flux enhanced TRAIL-mediated apoptosis. Cells were incubated with CTL (200 μM), CQ (20 μM), or 3-MA (2 mM) for 12 h. (a) DR5 expression was assessed by western blotting. (b) Ratio of DR5 and control density, (c) p62 and LC3 expression was assessed by western blotting. (d, e) Ratio of LC3 and p62 and control densities. (f) Cells were incubated with CTL (200 μM), CQ (20 μM), or 3-MA (5 mM) for 12 h and then exposed or not to TRAIL (100 ng/mL) for 2 h. Western blotting results show the expression levels of cleaved Caspase-8 and cleaved Caspase-3. 3-MA, 3-methyladenine. The results are expressed as the mean ± SD. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3).
CTL-mediated impaired autophagic flux enhanced TRAIL-mediated apoptosis. Cells were incubated with CTL (200 μM), CQ (20 μM), or 3-MA (2 mM) for 12 h. (a) DR5 expression was assessed by western blotting. (b) Ratio of DR5 and control density, (c) p62 and LC3 expression was assessed by western blotting. (d, e) Ratio of LC3 and p62 and control densities. (f) Cells were incubated with CTL (200 μM), CQ (20 μM), or 3-MA (5 mM) for 12 h and then exposed or not to TRAIL (100 ng/mL) for 2 h. Western blotting results show the expression levels of cleaved Caspase-8 and cleaved Caspase-3. 3-MA, 3-methyladenine. The results are expressed as the mean ± SD. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. the untreated group (n = 3).
3.6. CTL-Mediated Autophagosome Accumulation Influenced the TRAIL-Mediated Cell-Killing Effect in A549 Cells
A549 cells were incubated with 3-MA, CQ, or CTL at the indicated concentrations for 12 h and were then treated with or without TRAIL for an additional 2 h 30 min. Morphological changes in the cells were observed under a light microscope. Total cellular biomass was observed using crystal violet staining, and A549 cells were imaged (Figures 6(a) and 6(b)). Cellular death was strongly triggered by CTL or CQ in combination with TRAIL; however, the combination of 3-MA and TRAIL demonstrated only a slight cell-killing effect. According to the MTT assay, the combination of 3-MA and TRAIL resulted in a slight decrease in cell viability, whereas the treatment with CTL or CQ resulted in a significant decrease in the viability of A549 cells compared to control cells (Figure 6(c)). Moreover, the combination of TRAIL with either CTL or CQ enhanced the release of LDH during cell death compared to the combined application of 3-MA and TRAIL (Figure 6(d)). These results indicated that late-stage autophagic flux inhibition exerted a greater influence on TRAIL-induced cell death than early-stage autophagic flux inhibition, and that CTL-mediated autophagosome accumulation influenced the TRAIL-mediated cell-killing effect on A549 cells.
CTL enhanced TRAIL-mediated apoptosis by inhibiting autophagic flux. Cells were preincubated with or without CQ (10 μM), 3-MA (2 mM), and CTL (200 μM) for 12 h. Cells were then exposed with or without TRAIL (100 ng/mL) for 2 h 30 min. (a) Morphological variations in cells were observed with a light microscope (magnification, × 100; scale bar, 50 μm). (b) Crystal violet staining was performed to observe the density of cellular biomass. (c) Cell viability, as a percentage of control, was assessed using MTT assays, and the results are presented as a bar diagram. (d) LDH release was measured using the collected supernatants. ∗∗∗p < 0.001 vs. the untreated (control) group (n = 3).
CTL enhanced TRAIL-mediated apoptosis by inhibiting autophagic flux. Cells were preincubated with or without CQ (10 μM), 3-MA (2 mM), and CTL (200 μM) for 12 h. Cells were then exposed with or without TRAIL (100 ng/mL) for 2 h 30 min. (a) Morphological variations in cells were observed with a light microscope (magnification, × 100; scale bar, 50 μm). (b) Crystal violet staining was performed to observe the density of cellular biomass. (c) Cell viability, as a percentage of control, was assessed using MTT assays, and the results are presented as a bar diagram. (d) LDH release was measured using the collected supernatants. ∗∗∗p < 0.001 vs. the untreated (control) group (n = 3).
CTL enhanced TRAIL-mediated apoptosis by inhibiting autophagic flux. Cells were preincubated with or without CQ (10 μM), 3-MA (2 mM), and CTL (200 μM) for 12 h. Cells were then exposed with or without TRAIL (100 ng/mL) for 2 h 30 min. (a) Morphological variations in cells were observed with a light microscope (magnification, × 100; scale bar, 50 μm). (b) Crystal violet staining was performed to observe the density of cellular biomass. (c) Cell viability, as a percentage of control, was assessed using MTT assays, and the results are presented as a bar diagram. (d) LDH release was measured using the collected supernatants. ∗∗∗p < 0.001 vs. the untreated (control) group (n = 3).
CTL enhanced TRAIL-mediated apoptosis by inhibiting autophagic flux. Cells were preincubated with or without CQ (10 μM), 3-MA (2 mM), and CTL (200 μM) for 12 h. Cells were then exposed with or without TRAIL (100 ng/mL) for 2 h 30 min. (a) Morphological variations in cells were observed with a light microscope (magnification, × 100; scale bar, 50 μm). (b) Crystal violet staining was performed to observe the density of cellular biomass. (c) Cell viability, as a percentage of control, was assessed using MTT assays, and the results are presented as a bar diagram. (d) LDH release was measured using the collected supernatants. ∗∗∗p < 0.001 vs. the untreated (control) group (n = 3).
4. Discussion
In this study, we investigated the effects of CTL and CTL combined with TRAIL on A549 non–small lung cancer cells, including the role of autophagic flux in mediating these effects. CTL increased DR5 expression levels and protected against its degradation by impairing autophagic flux, thereby increasing TRAIL-induced apoptosis.
TRAIL binds to death receptors and initiates the caspase cascade, which induces cancer cell death [4]. Similar to many other cancer cells, A549 lung cancer cells are resistant to TRAIL [23]. CTL is often prescribed to treat depression [31]. Our findings revealed that CTL sensitized lung cancer cells and enhanced TRAIL-induced apoptosis. We observed that neither CTL nor TRAIL alone exhibited a cytotoxic effect on A549, HCC-15, or Calu-3 lung cancer cell lines; however, CTL administered with TRAIL resulted in a significant increase in cell death. CTL upregulates DR5 expression, which activates the TRAIL-induced apoptotic caspase cascade. Using a DR5-specific siRNA to inhibit DR5 expression increased cell viability, but CTL failed to show an effect on TRAIL-mediated apoptosis. These results confirmed that CTL plays a role in the upregulation of DR5 and is a prerequisite for the manifestation of the beneficial effects of combined CTL and TRAIL treatment.
Autophagy plays a cytoprotective role against various types of cellular stress and is involved in cell death [32]. Autophagosomes are typically formed when LC3-1 is cleaved and conjugated to phosphatidylethanolamine to become LC3-II [9]. p62 is degraded in autophagosomes via direct binding to LC3-II. Total p62 and LC3-II expression levels increased when lysosomes inhibited autophagosome degradation. Western blotting showed that CTL treatment markedly increased p62 and LC3-II expression levels, which in turn, inhibited autophagic degradation and disrupted autophagosome–lysosome fusion [11]. TEM revealed highly condensed autophagic vacuoles in CTL-treated cells. These experiments provide strong evidence that CTL induces autophagosome accumulation and impairs autophagosome–lysosome fusion, ultimately blocking autophagic flux. As autophagy blockade restored DR5 expression, TRAIL-induced apoptosis was enhanced.
A previous study using an in vitro model showed that circulating tumor cells develop TRAIL resistance through autophagic degradation of DR5 [20]. Our experiments suggest a possible reason for this finding. Similar to normal cells, tumor cells develop via autophagy [33, 34]. Our experiments revealed that the CTL treatment significantly increased DR5 protein levels but had the opposite effect when administered in the presence of rapamycin. After the treatment with rapamycin, the LC3 and p62 levels suggested that DR5 was removed by autophagy. This suggests that lung cancer cells develop resistance to TRAIL as part of the autophagic degradation of DR5.
We confirmed that CTL induces autophagosome accumulation and inhibits fusion between autophagosomes and lysosomes, ultimately preventing DR5 degradation. The application of CQ, which also inhibits autophagosome–lysosome fusion, has an effect similar to that of CTL; that is, it increases DR expression levels and markedly increases TRAIL-mediated apoptotic cell death. The application of 3-MA, which inhibits autophagosome accumulation [35], marginally increased DR5 expression levels and slightly increased TRAIL-mediated apoptosis compared to the TRAIL treatment alone. These findings suggested that the autophagic flux inhibition is more prominent at the late stage of TRAIL-induced apoptosis than at the early stage [36].
Our findings suggested that the antidepressant CTL may be a valuable addition to TRAIL therapy for overcoming drug resistance in lung cancer. CTL may enhance the effectiveness of TRAIL in inducing cancer cell death by inhibiting late-stage autophagic flux and preventing DR5 degradation. However, further evaluations and experiments are needed to establish the therapeutic potential of this antidepressant. Future research should focus on testing these effects in animal models to confirm our findings and potentially lead to clinical trials evaluating the combined use of CTL and TRAIL for lung cancer treatment. In addition, exploring the effects of CTL on autophagy-related resistance in other cancer types may broaden its therapeutic applications.
K.M.A.Z., A.N.M., J.-W.S., and S.-Y.P. designed and performed the study, analyzed the data, and wrote the manuscript. All the authors have read and approved the final version of the manuscript.
Funding
This study was supported by the National Research Foundation (NRF) of the Ministry of Education (2019R1A6A1A03033084) and the Ministry of Agriculture, Food, and Rural Affairs (322087051HD020).
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