Antidepressant drug enhanced TRAIL receptor-2 expression and sensitized lung cancer cells to TRAIL-induced apoptosis via Inhibition of autophagic flux

Autophagy, an alternative cell death mechanism, is also termed programmed cell death type II. Autophagy in cancer treatment needs to be regulated. In our study, autophagy inhibition by desipramine or the autophagy inhibitor chloroquine (CQ) enhanced tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) receptor-2 [death receptor (DR5)] expression and subsequently TRAIL-induced apoptosis in TRAIL-resistant A549 lung cancer cells. Genetic inhibition of DR5 substantially reduced desipramine-enhanced TRAIL-mediated apoptosis, proving that DR5 was required to increase TRAIL sensitivity in TRAIL-resistant cancer cells. Desipramine treatment upregulated p62 expression and promoted conversion of light chain 3 (LC3)-I to its lipid-conjugated form, LC3-II, indicating that autophagy inhibition occurred at the final stages of autophagic flux. Transmission electron microscopy analysis showed the presence of condensed autophagosomes, which resulted from the late stages of autophagy inhibition by desipramine. TRAIL, in combination with desipramine or CQ, augmented the expression of apoptosis-related proteins cleaved caspase-8 and cleaved caspase-3. Our results contributed to the understanding of the mechanism underlying the synergistic anti-cancer effect of desipramine and TRAIL and presented a novel mechanism of DR5 upregulation. These findings demonstrated that autophagic flux inhibition by desipramine potentiated TRAILinduced apoptosis, suggesting that appropriate regulation of autophagy is required for sensitizing TRAIL-resistant cancer cells to TRAIL-mediated apoptosis.


Introduction
Lung cancer is a common cause of cancer-related deaths [1]. Among the cancers diagnosed in the USA in 2018, lung cancer ranked second in terms of incidence rate [2]. Every year, 1.6 million people die of lung cancer, and 1.8 million people are newly diagnosed. After diagnosis, the 5-year survival rate is 4-17%, which varies based on the stage of the cancer [3]. Various treatment strategies include targeted chemotherapeutic agents, surgery, and radiotherapy for the treatment of advanced non-small cell lung cancer [4]. Newer approaches such as specific combination strategies with potential chemotherapeutic drugs may be a good choice for cancer treatment [5].
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a transmembrane cytokine that has shown the prospect of being used successfully for cancer treatment [6]. It can selectively kill a wide range of tumor cells with no or minimal toxic effects on normal cells [7].
TRAIL-mediated cancer cell killing can occur via both extrinsic and intrinsic apoptotic pathways. TRAIL binds to its receptors, death receptor 4 (DR4) and DR5, prompting apoptotic signals [8]. Subsequently, recruitment of the Fas-associated death domain (FADD) protein and, eventually, procaspase-8 by the FADD protein activates the death-inducing signaling complex (DISC), leading to the activation of caspases-8 and -9 and then the effector caspases-3, -6, and -7. This then results in membrane blebbing, DNA fragmentation, and nuclear shrinkage [9,10].
Although TRAIL is unique for its cancer cell killing capacity, various cancer cells are resistant to TRAIL [11,12]. A large number of tumor cells including human A549 lung cancer cells are resistant to the apoptotic effects of the TRAIL signaling pathway [13,14]. Study has shown that it is possible to overcome TRAIL resistance using effective TRAIL-sensitizing agents [15].
Autophagy, an alternative cell death mechanism, is also named programmed cell death type II [16]. Autophagy eliminates cytosolic components and damaged or misfolded proteins using a lysosome-mediated degradation system, which is promoted under stress conditions such as starvation, hypoxia, growth factor deprivation, and endoplasmic reticulum stress [17].
Autophagic flux is the complete mechanism of autophagy wherein cytosolic components are sequestered into double-membrane vesicles, autophagosomes, which subsequently fuse with lysosomes, initiating the degradation and recycling of these cytosolic components [18]. The formation of autophagosomes is indicated by the conversion of the microtubule-associated protein, light chain 3 (LC3)-I, to its lipid-conjugated form, LC3-II, which is commonly considered a marker of complete autophagosome formation [19]. The autophagosome then combines with lysosomes. p62 (SQSTM1), a ubiquitin-like lysosomal protein, which is incorporated into the autophagosome, degrades LC3-II and additional cargo proteins. Thus, inhibition of autophagy results in increased p62 protein levels [20,21]. Autophagy functions as a mechanism of tumor suppression during tumor formation processes and has been reported to promote tumor cell survival after tumor formation. Thus, autophagy is like a double-edged sword [22]. Many studies have shown that autophagy can play a cell protective role by carrying energy throughout metabolic stress and help avoid cancer cell death [23,24]. Recent studies revealed that pharmacological or genetic inhibition of autophagy enhanced cancer cell death during chemotherapy, indicating that autophagic flux inhibition might be a suitable and promising strategy for cancer treatment [25][26][27]. For example, chloroquine (CQ), or related hydroxychloroquine (HCQ), is an autophagy inhibitor that prevents acidification of lysosomes, inhibits fusion of autophagosomes with lysosomes, and augments the apoptotic effect [28][29][30].
Antidepressants are commonly recommended for the treatment of depression, psychiatric disorders, and chronic pain in cancer patients [31]. For example, desipramine is a tricyclic antidepressant (TCA) that is used as a first-line drug for the treatment of neuropathic pain [32].
As a member of the TCA class of drugs, desipramine has shown cytotoxic effects in many cancer cell lines such as human MG63 osteosarcoma cells [33], human HT29 colon carcinoma cells [34], human PC3 prostate cancer cells [35], C6 glioma cells [36], and mouse Ca3/7 skin squamous cells [37]. However, the anti-cancer effect of desipramine on lung cancer cells has not been reported yet. Therefore, we aimed to investigate the role of desipramine treatment in lung cancer.
In the present study, we demonstrated that inhibition of autophagic flux by desipramine enhanced TRAIL-induced A549 lung cancer cell death and activated DR5. Single treatment with either desipramine or TRAIL did not affect cell death. Although A549 cells are TRAILresistant, desipramine treatment enhanced DR5 expression. Co-treatment of desipramine and TRAIL exhibited a stronger effect on A549 lung adenocarcinoma cells than monotherapy with either desipramine or TRAIL.

Effects of desipramine treatment on TRAIL-induced death of lung cancer cells
To study the combined effect of desipramine and TRAIL on the inhibition of lung cancer cell viability, we used the A549 lung cancer cell line. The results demonstrated a strong combined effect on this cell line. Cells were preincubated with desipramine (30 µM) for 12 h and then co-treated with TRAIL (100 ng/mL) for 3 h. Cell morphologies and their changes were captured under a light microscope. Desipramine, in combination with TRAIL, increased apoptotic cell death ( Figures 1A, B, E, F, I and J). MTT assay demonstrated that the combined treatment significantly inhibited growth in a dose-dependent manner ( Figure 1C, G and K). The trypan blue exclusion assay showed that the combined treatment, compared to single treatment, robustly decreased the number of viable cells to a greater extent ( Figure 1D, H and L). These results suggested that desipramine sensitized TRAIL-resistant A549 lung adenocarcinoma cells to TRAIL-mediated apoptotic cell death.

Combined desipramine and TRAIL treatment effectively inhibited the formation of A549 cell colonies and enhances TRAIL-mediated apoptosis
We further examined the combined effects of desipramine and TRAIL on the colony-forming capacity of A549 cancer cells. When A549 cells were cultured with desipramine (30 µM) for 3 days, colony formation was completely inhibited; thus, the desipramine dose was reduced to 15 µM. Single TRAIL or desipramine treatment slightly reduced colony formation, whereas combined TRAIL-drug treatment significantly reduced colony formation and size ( Collectively, these results confirmed that desipramine increased TRAIL-mediated apoptosis in TRAIL-resistant lung adenocarcinoma A549 cells.

Effects of TRAIL receptor-2 (DR5) on TRAIL-induced apoptosis
To understand the underlying molecular mechanism of apoptosis of A549 cells induced by combined treatment with desipramine and TRAIL, we investigated whether the expression of death receptors was related to TRAIL-induced apoptosis. TRAIL resistance in several cancer cells was found to be associated with downregulated expression of the TRAIL receptors DR4 and DR5 or upregulated expression of the decoy receptors DcR1 and DcR2. Western blot analysis showed that desipramine treatment enhanced DR5 expression in a dose-and time-dependent manner but had no effect on DR4 expression ( Figure 3A). Desipramine treatment also increased DR5 transcript levels ( Figure 3B). Furthermore, ICC revealed substantially greater appearance of DR5 in desipramine-treated cells than in non-treated cells ( Figure 3C).
Finally, the apoptosis marker proteins, cleaved caspase-8 and cleaved caspase-3, were more markedly expressed after combined desipramine and TRAIL treatment than after single treatment ( Figure 3D). Therefore, DR5 potentiation by desipramine is essential for TRAILinduced apoptosis.

Suppression of DR5 altered the results of desipramine-induced TRAIL-mediated apoptosis
We applied DR5-specific siRNA to block DR5 expression, thereby restoring cancer cell viability. This finding provided evidence that DR5 plays an important role in augmenting the effect of desipramine on TRAIL-induced apoptosis. Cells, transfected with DR5-specific siRNA or negative control (NC) siRNA for 24 h and treated with desipramine for 12 h, were incubated with TRAIL for an additional 3 h for cell viability evaluation and an additional 2 h for western blot analysis. After siRNA transfection, the ability of desipramine, in combination with TRAIL, to induce cell death was reduced. However, the combined effect of desipramine and TRAIL on cell viability was similar in NC siRNA-transfected cells ( Figures 4A, B, C).
Western blot results showed that DR5-specific siRNA-transfected cells, compared to the nontransfected cells, showed blocked DR5 expression ( Figure 4D). The upregulation of DR5 by desipramine demonstrated the important role of DR5 in attenuating TRAIL resistance.

Effects of desipramine treatment on autophagic flux
To identify the function of desipramine in autophagic flux, we checked the levels of the autophagy markers LC3-II and p62. Immunoblotting assay revealed that desipramine inhibited autophagy at the final stages of autophagic flux. Desipramine converted LC3-I to LC3-II, indicating the formation of complete autophagosomes, and increased p62 levels by inhibiting its degradation in lysosomes or proteasomes ( Figure 5A). The genetic autophagy inhibitor atg5 did not alter the expression of p62 and LC3-II. Therefore, atg5-independent autophagosome accumulation occurred in desipramine-treated cells ( Figure 5B). TEM exposed the condensed accumulation of autophagic vacuoles. This was absent in the control, thereby establishing that desipramine inhibited autophagic flux ( Figure 5C). ICC analysis also revealed that autophagic flux inhibition by desipramine increased the expression level of p62 in a dose-dependent manner ( Figure 5D). These experiments indicated that desipramine inhibited autophagic flux by inhibiting autophagosome-lysosome fusion in lung cancer cells.

Autophagic flux inhibition upregulated DR5
To investigate the role of autophagic flux inhibition in DR5 expression, we used the autophagy inhibitor CQ. CQ inhibited autophagic flux and thus upregulated DR5 expression. Cell culture plates were pretreated with CQ (20 μM) or indicated doses of desipramine for 12 h.
Immunoblotting assay showed that desipramine and CQ increased the levels of LC3-II.
Moreover, desipramine alone enhanced p62 expression in a dose-dependent manner. These findings suggested that desipramine inhibited autophagic flux to induce apoptosis ( Figure 6A). Furthermore, DR5 expression was upregulated in desipramine-and CQ-treated cells ( Figure   6B). Finally, we checked the levels of apoptosis-related proteins cleaved caspase-8 and cleaved caspase-3. Desipramine or CQ, in combination with TRAIL, increased the levels of cleaved caspase-8 and cleaved caspase-3 ( Figure 6C). Overall, these findings proved that autophagy inhibition enhanced TRAIL-mediated apoptosis by upregulating DR5 expression.

Autophagy inhibition by desipramine augmented TRAIL-induced cell death
To investigate the role of desipramine in autophagy inhibition and subsequent TRAIL-mediated cell death, a functionally active autophagy inhibitor CQ was applied. Cells were pretreated with CQ or desipramine at specified doses for 12 h and finally incubated with TRAIL for 3 h.
Morphological analysis of cells by light microscopy and crystal violet assay revealed that A549 cells treated with either TRAIL or desipramine showed slight cell death, whereas cells treated with a combination of desipramine or CQ and TRAIL showed significantly improved TRAILmediated cell death (Figures 7A, B). MTT and trypan blue staining assays showed that cells treated with desipramine or CQ, in combination with TRAIL, demonstrated decreased cell viability and augmented cell death ( Figure 7C). Collectively, these findings showed that desipramine enhanced TRAIL-induced apoptosis by inhibiting autophagic flux.

Discussion
Depression is the most common symptom in cancer patients, and it suppresses their anti-cancer immunity. The main purpose of our study was to investigate the function of desipramine and co-treatment of desipramine and TRAIL in A549 lung cancer cells. We found that desipramine inhibited autophagic flux, resulting in DR5 upregulation, which finally enhanced TRAILinduced apoptosis of A549 cells.
TRAIL is a transmembrane cytokine and shows promising anti-cancer activities in tumor cells without cytotoxic effects. Since it is a safe and potent biological agent, there is scope for its use in cancer therapy in humans [38,39]. However, the observed TRAIL resistance in certain cancer cells remains unclear. Autophagy is an alternative cell-death mechanism, and it plays an important role in recycling cellular components. Complete autophagic flux is a mechanism by which cellular components are recruited to lysosomes for degradation [40,41]. Anti-malarial drugs such as CQ function as autophagy inhibitors and have been shown to impair autophagy in clinical trials for cancer therapy [42]. Recent studies suggested that autophagy inhibition sensitized cancer cells to apoptosis and a complete autophagic flux promoted cancer cell survival [43,44].
A549 lung cancer cells are resistant to TRAIL [13,45]. In the present study, we detected that desipramine or TRAIL alone was not capable of inducing cell death in A549 lung cancer cells.
Notably, the combined treatment of desipramine and TRAIL strongly promoted cell death in A549 cells. Moreover, the combined treatment robustly inhibited colony formation and reduced size in A549 cells (Figures 1, 2). Desipramine, which upregulated DR5 expression, exerted this apoptotic effect, owing to the combined effect of TRAIL and desipramine ( Figure 3). This experiment proposed that desipramine, in combination with TRAIL, played a role as an anticancer agent that could be used to sensitize lung cancer cells to TRAIL-induced apoptosis.
Treatment with desipramine alone increased LC3-II and p62 levels in A549 cells in a dosedependent manner.
Our findings showed that the inhibition of DR5 expression by DR5-specific siRNA abundantly increased cell viability and thus inhibited the effects of desipramine on TRAIL-mediated apoptosis. These results indicated that DR5 upregulation was required for the combined effect of desipramine and TRAIL. Moreover, these findings, for the first time, revealed that desipramine enhanced DR5 expression via autophagy inhibition (Figure 4). It was also revealed that desipramine increased autophagosome formation, as indicated by LC3-II accumulation, and inhibited lysosomal fusion with autophagosomes, thereby resulting in increased p62 levels.
This confirmed the inhibition of autophagic flux ( Figure 5). Furthermore, the combined treatment of TRAIL and desipramine or CQ, compared with single treatment regimens, increased cell death to a greater extent. The inhibition of autophagy by desipramine and the lysosomal inhibitor CQ increased DR5 expression level and improved TRAIL-mediated caspase-dependent apoptotic cell death. Our findings were confirmed by the amplified levels of the intracellular apoptosis-related proteins-activated caspase-3 and activated caspase-8 ( Figures 6, 7).
In conclusion, we reported that desipramine treatment enhanced the function of TRAIL by upregulating DR5 expression. Moreover, the combined treatment of desipramine and TRAIL

Cell viability assay: Cell viability was assessed by methyl thiazolyltetrazolium (MTT)
and crystal violet staining assays. Cells were plated in 12-well plates at a density of 1.0×10 4 cells/well and incubated at 37°C for 24 h. Cells were pretreated with different concentrations of desipramine for 12 h and then exposed to recombinant TRAIL (100 ng/mL) for 3 h. In addition, the cells were pretreated with CQ (20 μM) for 1 h and then treated with desipramine.
Cell morphology was observed under an inverted microscope (Nikon, Tokyo, Japan). Cell viability was assessed by adding 5 mg/mL MTT (500 µL) to each well and incubating the plates at 37°C for 2 h. After incubation, the MTT solution was removed, and the wells were treated with dimethyl sulfoxide (500 µL). The absorbance was measured at 570 nm using a spectrophotometer (Bio-Rad, Hercules, CA, USA). For the crystal violet assay, cells were stained with a staining solution (0.5% crystal violet in 30% ethanol and 3% formaldehyde) at room temperature (25C) for 10-20 min, washed 3-4 times with phosphate-buffered saline (PBS), and then imaged.

Trypan blue exclusion assay:
The number of live cells was counted using microscopy and a hemocytometer after staining the cells with trypan blue (Sigma-Aldrich). The results were calculated as percentages and compared to those of the vehicle-treated controls.

Colony-formation assay:
Cells were plated in 12-well plates and treated with the indicated doses of TRAIL and desipramine. After 2 days, the medium was replaced with new medium without TRAIL and desipramine and further cultured for a week. Colonies were fixed in 100% methanol for 20 min and stained with 0.05% (w/v) crystal violet for 5 min. After washing, colonies were counted under an inverted microscope (Nikon, Japan).  Gene primers (1 µL), with SYBR Green (Bio-Rad Laboratories) and a total reaction volume of 20 µL, were used for qRT-PCR. The sequences of the primers used were DR5 (forward: 5'-GCGGTCCTGCTGTTGGTCTC-3', reverse: 5'-GCTTCTGTCCACACGCTCAG-3') and GAPDH, which was used as an internal control (forward: 5'-TGCACCACCAACTGCTTAG-3', reverse: 5'-GGATGCAGGGATGATGTT-3'). All data were evaluated using Bio-Rad CFX manager version 2.1 analysis software (Bio-Rad Laboratories).

Statistical analysis:
The data are expressed as the mean ± standard deviation (SD). The significance of the differences between treatments was determined using one-way analysis of variance (ANOVA), followed by the Tukey-Kramer test. Statistical analyses were performed using the GraphPad Prism 5 software (GraphPad Software, Inc.). A p-value <0.05 was Acknowledgements: Not applicable.

Conflicts of Interest:
The authors declare no conflict of interest.