Fluoxetine Induces Apoptosis through Extrinsic/Intrinsic Pathways and Inhibits ERK/NF-κB-Modulated Anti-Apoptotic and Invasive Potential in Hepatocellular Carcinoma Cells In Vitro

The aim of the present study was to verify the effects of fluoxetine on dysregulation of apoptosis and invasive potential in human hepatocellular carcinoma (HCC) SK-Hep1 and Hep3B cells. Cells were treated with different concentrations of fluoxetine for different times. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assays were used for testing the effects of fluoxetine on cell viability. The regulation of apoptosis signaling, and anti-apoptotic, proliferation, and metastasis-associated proteins after fluoxetine treatment were assayed by flow cytometry and Western blotting assay. The detection of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation after fluoxetine treatment was performed by NF-κB reporter gene assay. The results demonstrated that fluoxetine significantly reduced cell viability, cell migration/invasion, NF-κB, extracellular signal-regulated kinases (ERK) activation, and expression of anti-apoptotic (Cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (C-FLIP), Myeloid cell leukemia-1 (MCL-1), X-Linked inhibitor of apoptosis protein (XAIP), and Survivin), proliferation (Cyclin-D1), angiogenesis (vascular endothelial growth factor (VEGF)), and metastasis-associated proteins (matrix metalloproteinase-9 (MMP-9)). Fluoxetine also significantly induced apoptosis, unregulated extrinsic (activation of first apoptosis signal protein and ligand (Fas/FasL), and caspase-8) and intrinsic (loss of mitochondrial membrane potential (ΔΨm) pathways and increased Bcl-2 homologous antagonist killer (BAK) apoptosis signaling. Taken together, these results demonstrated that fluoxetine induced apoptosis through extrinsic/intrinsic pathways and diminished ERK/NF-κB-modulated anti-apoptotic and invasive potential in HCC cells in vitro.


Introduction
Hepatocellular carcinoma (HCC), the most common primary malignancy of the liver, is the second leading cause of cancer-related death worldwide [1]. Dysregulation of apoptosis and high metastatic ability is associated with poor prognosis in patients with HCC [2,3]. Sorafenib and regorafenib are oral multi-kinase inhibitors which promote apoptosis and suppress anti-apoptotic and metastatic activity and are approved for treatment of HCC [4,5]. Therefore, induction of apoptosis and inhibition of anti-apoptotic and metastatic activity may be an available treatment strategy for patients with HCC.
Antidepressants are used to treat depression and improve quality of life in cancer patients [6]. In addition, the anti-cancer effects of antidepressants have been found in several cancer cells [7,8]. Antidepressants modulate inhibition of tumor growth through triggering antitumor immunity, inducing apoptosis, and blocking intracellular signaling transduction [9,10]. Many antidepressants such as desipramine, sertraline, and fluoxetine, have been shown to induce apoptosis in HCC [11][12][13].
Fluoxetine, a selective serotonin reuptake inhibitor (SSRI) antidepressant, is used for the treatment of depression and anxiety disorders [14]. In ovarian cancer-related studies, the apoptotic effect of fluoxetine on human epithelial ovarian cancer cell lines OVCAR-3 and SK-OV-3 growth which was regulated by mitochondria-mediated cell death process was found [15]. In Stepulak et al.'s study, they also suggested that fluoxetine inhibits the extracellular signal-regulated kinase pathway and suppresses growth of colon cancer cells and lung cancer cells [16]. Fluoxetine may also reverse breast cancer cells' resistance and enhance the chemosensitivity to common chemotherapy drugs, such as adriamycin and paclitaxel [17]. Mun et al. reported that fluoxetine induces apoptosis via depletion of the mitochondrial membrane potential (∆Ψ m ), the formation of reactive oxygen species, and the inhibition of mitogen-activated protein kinase (MAPK) activation in HCC Hep3B cells [13]. Whether fluoxetine inhibits anti-apoptotic and metastatic activity in HCC remains ambiguous. Therefore, we investigated the effect of fluoxetine on the dysregulation of apoptosis and invasive potential in HCC.

Fluoxetine Decreased the Cell Viability and NF-κB Activation of Sk-Hep1 and Hep3B Cells
SK-Hep1 and Hep3B cells were treated with 0-40 µM of fluoxetine for 24 and 48 h. Cells were examined for the percentage of viable cells and NF-κB activation by MTT and NF-κB reporter gene assay. The results indicated that fluoxetine significantly decreased the quantity of viable cells ( Figure 1A). The cell viability of SK-Hep1 and Hep3B cells was reduced with an increase of fluoxetine concentration and incubation time. Cell viability was inhibited by 50-60% as compared to the control group (vehicle treatment) at 48 h after 30 µM and 40 µM fluoxetine treatment. Therefore, both 30 µM and 40µM doses of fluoxetine were selected for subsequent experiments. Fluoxetine significantly reduced relative NF-κB activation in SK-Hep1 and Hep3B cells by 20-80% as compared to the control group ( Figure 1B).

Fluoxetine Induced Apoptosis and Reduced Expression of Anti-Apoptotic Proteins in SK-Hep1 Cells
Detection of cell cycle and caspase-3 activation, Annexin V/PI-double staining, and western blotting were used to investigate the effect of fluoxetine on dysregulation of apoptosis in SK-Hep1 cells. In Figure 2A,B indicated fluoxetine significantly induced accumulation of sub-G 1 and caspase-3 activation by 25-50% and 18-48%. The results of dot plots ( Figure 2C) indicated that 30 µM and 40 µM of fluoxetine induced apoptosis of cells, with an increase in the percentage of early apoptotic cells (2-4%) and late apoptotic cells (10-30%). Fluoxetine significantly induced early-stage and late-stage apoptosis in a dose-dependent manner. Expression of anti-apoptotic proteins (C-FLIP, MCL-1, XIAP, and Survivin) was reduced with fluoxetine treatment by 22-92% as compared to the control group ( Figure 2D). (C) A NF-κB reporter gene assay was performed on SK-Hep1/NF-κB-luc2 and Hep3B/NF-κB-luc2 cells at 48 h. * p < 0.05 and ** p < 0.01, significant difference between fluoxetine-treated groups and the control as analyzed by Student's t test.

Fluoxetine Promoted Extrinsic and Intrinsic Apoptotic Signaling Transduction in SK-Hep1 and Hep3B Cells
To investigate apoptosis signaling induced by fluoxetine, we performed various apoptosis determination methods as follows. The results shown in Figure 3A-C revealed that fluoxetine promoted the activation of Fas, FasL, and caspase-8. Loss of mitochondria membrane potential (∆Ψ m ) is required for intrinsic apoptosis. Figure 3D indicated fluoxetine significantly triggered loss of ∆Ψ m . Additionally, we found extrinsic and intrinsic apoptosis mechanisms were both activated by fluoxetine in Hep3B cells as well ( Figure 3E,F). Protein levels of Fas, FasL, and BAK were significantly enhanced by fluoxetine treatment in SK-Hep1 cells ( Figure 3G). (C) evaluation of early and late apoptosis events by Annexin V/PI-double staining; (D) expression of anti-apoptotic proteins (C-FLIP, MCL-1, XIAP, and Survivin) are presented with Western blotting assay. Quantification data were averaged over three repeated experiments. * p < 0.05 and ** p < 0.01, significant difference between the control and fluoxetine-treated groups.

Fluoxetine Suppressed Cell Migration/Invasion and Reduced ERK Activation and Expression of Metastasis-Associated and Proliferative Proteins in SK-Hep1 and Hep3B Cells
Transwell cell migration and invasion assays were used for measuring cell migration and invasion in SK-Hep1 and Hep3B cells after exposure to fluoxetine. The results indicated that fluoxetine significantly inhibited cell migration and invasion by 80-90% and 70-80%, respectively, as compared to the control group ( Figure 4A,B). Furthermore, fluoxetine may also decrease the number of migration and invasion Hep3B cells ( Figure 4C,D). As shown in Figure 4E, fluoxetine significantly reduced levels of metastasis-associated (MMP-9 and VEGF) and proliferative proteins (Cyclin-D1). Moreover, we also investigated effect of fluoxetine on MAPK/ERK activation with western blotting. The results indicated that fluoxetine markedly suppressed the protein level of pERK ( Figure 4E).  and BAK on SK-Hep1 cells were investigated with Western blotting assay. Quantification data were normalized by β-actin expression and averaged over three repeated experiments. * p < 0.05, ** p < 0.01, significant difference between control and fluoxetine-treated groups.

Fluoxetine Not Only Induced Apoptosis, but Suppressed Tumor Progression in SK-Hep1 and Hep3B Cells
To explain the mechanism of fluoxetine on Sk-Hep1 and Hep3B cells, we propose the mechanism shown in Figure 5 based on our experimental results. Fluoxetine may trigger extrinsic (through death receptor) and intrinsic (through mitochondria) apoptosis mechanisms. Fluoxetine also reduced the production of anti-apoptosis proteins such as C-FLIP, MCL-1 and XIAP. Moreover, tumor invasion and migration ability were both suppressed by fluoxetine. Tumor invasion-related protein MMP-9 was significantly inhibited by fluoxetine. ERK-mediated tumor progression signaling was also blocked by fluoxetine. In sum, fluoxetine may enhance apoptosis effects and suppress tumor progression of HCC.

Discussion
Initiation of the extrinsic apoptotic pathway is mediated by cell death receptor Fas/Fas ligand (FasL) interaction [18]. Resistance to apoptosis is associated with upregulation of anti-apoptotic proteins and downregulation of pro-apoptotic proteins in HCC [2]. Reduction of Fas expression was found in HCC with poor differentiation [19]. Overexpression of anti-apoptotic proteins, including MCL-1, C-FLIP, XIAP, and Survivin, is implicated with a poor prognosis in patients with HCC [20,21]. MCL-1 and C-FLIP mediate resistance to apoptosis through suppression of intrinsic and extrinsic apoptotic signaling transduction. Both XIAP and Survivin, diminish apoptosis induced by anti-cancer agents through a decrease of caspase-3 activation [20,22,23]. Pro-apoptotic protein BAK-mediated dissipation of the mitochondrial membrane potential (∆Ψ m ) is blocked by MCL-1 [24]. Furthermore, Fluoxetine has been demonstrated to induce cell death through induction of the mitochondrial apoptosis pathway in HCC Hep3B cells [13]. In our data, we found that fluoxetine significantly induced apoptosis, enhanced expression of apoptotic proteins (Fas, FasL, and BAK), and triggered extrinsic and intrinsic apoptotic signaling (increases of Fas, FasL, Caspase-8 activation and induced the loss of ∆Ψm). In addition, expression of anti-apoptotic proteins (C-FLIP, XIAP, MCL-1, and Survivin) was significantly reduced by fluoxetine treatment.
Mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) is the critical activator for tumor progression through upregulation of downstream kinases and transcription factors [36]. High expression of phosphorylated ERK (pERK) as the prognostic factor is linked to aggressive tumor behavior and poor overall survival in HCC [37]. Fluoxetine has also been shown to trigger apoptosis by inhibiting phosphorylation of ERK in Hep3B cells [13]. Previous studies indicated that PD98059, an ERK inhibitor, diminishes NF-κB activation and sorafenib, regorafenib, anti-HCC drugs, inhibit NF-κB-modulated tumor progression via suppression of ERK activation in HCC in vitro and in vivo [4,32,38]. Tine et al., reported that 5 µM fluoxetine did not affect ERK phosphorylation, but induced cell invasion, NF-κB activation, and expression of metastasis-associated proteins (MMP-2 and -9) in HCC HepG2 cells [39]. However, 30 µM of fluoxetine may induce cytotoxicity and ERK dephosphorylation in HCC Hep3B cells [13]. In a previous study, we demonstrated that NF-κB modulated invasive potential was diminished with 40 µM fluoxetine treatment in non-small cell lung cancer in vitro [35]. Notably, in this study, we indicated that 10-40 µM fluoxetine significantly reduced NF-κB activation, and 30-40 µM fluoxetine markedly suppressed protein levels of pERK, Cyclin-D1, VEGF, MMP-9, and cell migration/invasion in SK-Hep1 cells.
In conclusion, fluoxetine not only induces apoptosis through extrinsic/intrinsic pathways but also inhibits ERK/NF-κB-modulated anti-apoptotic and metastatic activity in HCC SK-Hep1 and Hep3B cells in vitro. We suggest fluoxetine as a potential inhibitor of ERK/NF-κB signaling which may provide therapeutic benefits for the treatment of HCC.

Cell Culture
SK-Hep1 and Hep3B, human hepatocellular carcinoma, were both obtained from Professor Jing-Gung Chung's laboratory and routinely tested for mycoplasma contamination [39]. Cells were grown in a humidified incubator with 5% CO 2 at 37 • C and cultured in DMEM medium. DMEM was supplemented with 10% heat inactivated fetal bovine serum (FBS), 2 mM L-glutamine and antibiotics (100 units/mL penicillin, 100 µg/mL streptomycin).

NF-κB Reporter Gene Assay
SK-Hep1/NF-κB-luc2 or Hep3B/NF-κB-luc2 cells were seeded into 96-well plates at 3 × 10 4 /well overnight, and then treated with different concentrations (0, 10, 20, 30 and 40 µM) of fluoxetine for 48 h. After treatments, the cell medium in each well was replaced with 100 µL of 500 µM D-luciferin (Promega, Madison, WI, USA) and incubated for 15 min in the dark. The effect of fluoxetine on relative NF-κB activity was evaluated using an IVIS imaging system and corrected with cell viability as described [41].

Cell Cycle Analysis
SK-Hep1 cells (5 × 10 5 ) were seeded into 6-well plates and treated with different concentrations (0, 30, and 40 µM) of fluoxetine for 48 h. Cells were trypsinized and collected. Cells were fixed in 70% ethanol for 24 h at −20 • C, rehydrated with ice-cold PBS, and then stained with 40 µg/mL propidium iodide (PI) reagent (Biovision) with 100 µg/mL RNase in the dark for 30 min. Cell cycle analysis was then performed with flow cytometry and analyzed with FlowJo software [43].

Measurement of Caspase-3 and -8 Activation
SK-Hep1 or Hep3B cells were seeded into 6-well plates at 5 × 10 5 cells/well overnight and then treated with different concentrations (0, 30, and 40 µM) of fluoxetine for 48 h. Cells were stained with CaspGlow Fluorescein Active Caspase-3 or -8 Staining kit from BioVision (Milpitas, CA, USA) adhering to the manufacturer's instructions. Briefly, cells were stained with active-caspase-3 (1 µL active caspase-3 FITC antibody in 300 µL PBS) or active-caspase-8 staining solution (1 µL caspase-8 PE antibody in 300 µL PBS) for 30 min in a humidified incubator with 5% CO 2 at 37 • C, respectively. Cells were washed once and harvested with 200 µL wash buffer and centrifugation and then resuspended in 500 µl wash buffer for immediate FACS analysis [43]. Data were analyzed using FlowJo software.

Assessment of Fas-L and Fas Activation
Sk-Hep1 cells (5 × 10 5 ) were seeded into 6-well plates at cells/well and treated with 0, 30 and 40 µM fluoxetine for 48 h. For detection of cell surface antigens, collected cells were treated with PE-conjugated anti-CD178 (FAS-L) antibody (BioLegend, Inc., San Diego, CA, USA) or FITC-conjugated anti-CD95 (Fas) antibody and incubated on ice for 15 min in the dark. Then, they were washed twice with 500 µL cell staining buffer and resuspended in 500 µL cell staining buffer for flow cytometry. Each assay included at least 10,000 gated events. The histograms obtained were analyzed using FlowJo software [45].

Western Blotting
3 × 10 6 SK-Hep1 cells were placed in a 10-cm dish overnight and then treated with different concentrations (0, 30, and 40 µM) of fluoxetine for 48 h. Cells were collected by centrifugation and lysised with in a lysis buffer (50mM Tris-HCl (pH 8.0), 120mM NaCl, 0.5% Nonide P-40) for 30 min and the total protein was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). 40-60 µg of total protein from each treatment was separated by 10-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene difluoride (PVDF) membrane (FluoroTrans ® Pall Corporation, Port Washington, NY, USA) by electroblotting and blocked with 5% (w/v) non-fat milk in Tris buffered saline (TBS) containing 0.05% Tween-20. Blots were probed by using primary antibodies and β-actin, followed by secondary antibody and Immobilon Western Chemiluminescent HRP Substrate (Pierce, Rockford, IL, USA) for enhanced chemiluminescence as described previously [32,46]. The immuno-detected proteins from each sample were then revealed by the MultiGel-21 imaging system (TOP BIO CO., Taipei, Taiwan) and their band intensities were quantified using Image J (version 1.50, National Institutes of Health, Bethesda, MD, USA). Quantification data were normalized by β-actin expression and averaged over three repeated experiments.

Invasion and Migration Assay
Sk-Hep1 or Hep3B cells (3 × 10 6 cells in a 10-cm dish) were pretreated without or with 30 and 40 µM of fluoxetine for 48 h and harvested for Transwell assay. The invasion or migration of Sk-Hep1 and Hep3B cells was investigated using a 8 µm pore size Transwell assay (BD Biosciences, Franklin Lakes, NJ, USA) as described previously [27]. The upper chamber of the 24-Transwell plate was covered with (invasion) or without (migration) 0.5% Matrigel. 1 × 10 6 cells were placed in the upper chamber of a 24-Transwell plate in DMEM medium containing 1% FBS. The cells invade or migrate from the upper chamber containing 1% FBS to the lower chamber, containing 10% FBS in DMEM media for incubation for 48 h. At the end of incubation, on the underside of the membrane, invaded cells were fixed with 4% formaldehyde in PBS and stained with 3% crystal violate, and air-dried for 15 min. Then, the cell number was photographed under a light a microscope (Nikon ECLIPSE Ti-U) at ×100 and quantified by ImageJ software [27].

Statistical Analysis
The results are presented as means ± S.D. from at least three experiments and the differences between fluoxetine-treated and control groups were measured by Student's t-test. If p-values were <0.05, differences were considered statistically significant. Excel 2017 (Microsoft, Redmond, WA, USA) was used for statistical analyses in this study.

Conclusions
In this study, we suggested that fluoxetine induced apoptosis through extrinsic/intrinsic pathways. Moreover, fluoxetine may also decline ERK/NF-κB-modulated anti-apoptotic and invasive potential in two types of HCC cells (SK-Hep1 and Hep3B) in vitro.