ANTICANCER RESEARCH 33: 3691-3698 (2013)

Fluoxetine-induced Apoptosis in Hepatocellular Carcinoma Cells

A-REUM MUN1,*, SEI-JIN LEE2,*, GI-BEUM KIM1, HYUNG-SUB KANG1, JIN-SHANG KIM1 and SHANG-JIN KIM1

1Department of Pharmacology and Toxicology, College of Veterinary Medicine, Chonbuk National University, Jeonju, Republic of Korea; 2Korea Basic Science Institute Jeonju Center, Jeonju, Republic of Korea

Abstract. Background: In addition to being used to treat cells have extreme chemoresistance and low selectivity to mental disorders, a serious complication of cancer, chemotherapy drugs. In addition, chemotherapy drugs kill antidepressants have been reported to improve cancer patient normal cells as well as tumor cells, leading to significant immunity, inhibit cell growth and have an antitumor effect on adverse effects (3). To overcome the weaknesses of various cancer cell lines. We investigated the apoptotic effect traditional anticancer chemotherapy, alternative or of against the Hep3B human hepatocellular complementary medicine such as combined treatments with carcinoma cell line. Materials and Methods: After treatments therapeutics for other diseases or new agents prepared from of Hep3B cells with fluoxetine, we measured cell viability, natural products is drawing attention as a potential new reactive oxygen species (ROS), mitochondrial membrane approach to anticancer therapy. potential (MMP) and activation of mitogen-activated protein Antidepressants are clinically prescribed to patients for kinases (MAPK). Results: Fluoxetine reduced the viability of management of depression and psychiatric disorders (4), cancer cells, induced loss of MMP and formation of ROS, and many also exhibit substantial benefit in various types reduced expression of extracellular signal-regulated kinase of chronic pain (5). Interestingly, recent studies have 1/2 and increased expression of c-JUN N-terminal kinase documented the anticancer effects of antidepressants in a and p38 MAPK. N-Acetylcysteine, an oxidant-scavenger, and variety of solid tumor types and cancer cell lines (6). 1,2-bis (o-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid Selective serotonin re-uptake inhibitors (SSRI), including (BAPTA-AM), an intracellular Ca2+ chelator, prevented fluoxetine, can inhibit growth of various cancer cell lines fluoxetine-induced modulation of MAPK. Conclusion: from lung, colon, neuroblastoma and breast (6, 7). Fluoxetine appears to exhibit an apoptotic effect against Fluoxetine can improve cancer patient immunity and life Hep3B cells through the loss of MMP, formation of ROS and quality, and extend their life expectancy (8, 9). modulation of MAPK activities. Furthermore, fluoxetine was reported to be a highly effective chemosensitizer (10) that is synergistic with Hepatocellular carcinoma (HCC) is a primary type of anticancer drugs in overcoming multidrug resistance (11). hepatic tumor, an aggressive malignancy with high Fluoxetine has also been reported to have an apoptotic prevalence, and the sixth most common cancer worldwide effect against ovarian cancer cell lines by inducing (1). HCC represents 5% of all cancers with the annual mitochondrial membrane permeability (MMP) changes (12) number of cases exceeding 500,000 worldwide (2). HCC and induce preventive and complex effects against colon cancer development in rats (13). In contrast, several studies have linked fluoxetine with cell proliferation and an increased risk of developing cancer (12), while having no *These Authors contributed equally to this study. effect on cell survival and growth of cancer cells and tumor growth in vivo (14). Correspondence to: Shang-Jin Kim, Department of Pharmacology In the present study, we demonstrated that fluoxetine and Toxicology, College of Veterinary Medicine, Chonbuk National induces apoptosis in Hep3B cells, an HCC cell line. To University, Jeonju-561-756, Republic of Korea. Tel: +82 elucidate the mechanism of fluoxetine-induced apoptosis in 632703927, Fax: +82 632703780, e-mail: [email protected] Hep3B cells, we investigated cell viability, reactive oxygen Key Words: Fluoxetine, hepatocellular carcinoma, apoptosis, species (ROS), MMP, and activation of mitogen-activated mitogen-activated protein kinase, Hep3B cells. protein kinases (MAPK).

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Materials and Methods Measurement of intracellular ROS generation. DCFH is widely used to measure oxidative stress in cells. When the diacetate form of Cell culture and reagents. Hep3B cells were obtained from the Korea DCFH is added to cells, it diffuses across the cell membrane and is Cell Line Bank (KCLB, Seoul, Korea) and grown in Dulbecco’s hydrolyzed by intracellular esterases to liberate DCFH which, upon modified Eagle’s medium nutrient mixture F-12 HAM (DMEM F-12 reaction with oxidizing species, forms its 2-electron oxidation HAM) supplemented with 10% fetal bovine serum (Sigma-Aldrich, product, the highly fluorescent compound 2’-7’-dichlorofluorescein St. Louis, MO, USA), 5 mM L-glutamine, 50 U/ml penicillin and 50 (DCF). The fluorescence intensity can be easily measured and is the basis of the popular cellular assay for oxidative stress (15). After a μg/ml streptomycin in a humidified 5% CO2-95% air environment at 4 37˚C. Fluoxetine was purchased from Enzo Life Sciences (Plymouth 48 h incubation of the Hep3B cells in 12-well plates (1×10 /well), Meeting, PA, USA), and 2,7’-dichlorodihydrofluorescin diacetate the Hep3B cells were treated with fluoxetine (10-100 μM). After a (DCFH-DA) was purchased from Molecular Probes (Eugene, OR, 24 h incubation, Hep3B cells were treated with 10 μM DCFH-DA USA). 4’,6-Diamidino-2-phenylindole (DAPI) and 5,5’,6,6’- for 30 min. Upon incubation with DCFH-DA, the cells were tetrachloro-1,1’,3,3’-tetraethyl benzimidazolyl-carbocyanine iodide observed under a fluorescence microscope (IX-81; Olympus Corp.). (JC-1) were purchased from Enzo Life Sciences. DCFH fluorescence was then determined using a spectrophotometer at excitation and emission wavelengths of 488 nm and 515 nm, Cell viability assay. Hep3B cells were grown on 96-well plates respectively. (5000 cells/well) and cultured for 24 h in DMEM F-12 HAM medium containing 10% fetal bovine serum. After treatment with Western blot analysis of MAPKs. After a 48-h incubation of the fluoxetine (10-100 μM) for 24 h, the culture medium was discarded Hep3B cells in a Petri dish (1×107/well), the Hep3B cells were and cell viability was assessed with the aid of a Cell Counting Kit- treated with fluoxetine (100 μM) with/without N-acetylcysteine 8 (CCK-8; Enzo Life Sciences). Briefly, 100 μl CCK-8 reagent were (NAC) (10 mM) and (acetoxymethyl)-l,Z bis (o-aminophenoxy) added to each well at a 1:10 ratio to cell culture medium. After a 2- ethane N,N,N’,N’-tetra-acetic acid (BAPTA-AM) for 24 h. After h incubation in a humidified atmosphere with 5% CO2 at 37˚C, the incubation, Hep3B cells were washed three times in ice-cold PBS absorbance was read at 450 nm using a spectrophotometer (Spectra and scraped with a cell scraper. The harvested cells were lysed Max M5; Molecular Devices, Sunnyvale, CA, USA). for 30 min at 4˚C in RIPA buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, Nuclear staining with DAPI. Hep3B cells were seeded on coverslips 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM and cultured for 48 h in DMEM F-12 HAM medium containing β-glycerophosphate, 1 mM Na4VO4 and 1 μg/ml leupeptin. The 10% fetal bovine serum. After the treatment with fluoxetine (10-100 sample was then sonicated for five pulses at a 40% using a Sonics μM) for 24 h, cells were washed three times with ice-cold PBS and & Materials Ultrasonic Processor (USA), and then transferred to fixed with 70% ethanol for 1 h. The cells were washed with PBS a 0.5 ml microfuge tube. The sample was heated to 100˚C for 7 and counterstained with DAPI mounting medium for 15 min at min and placed briefly on ice. Then, 30 μL of the supernatant room temperature. Apoptotic nuclei were then visualized under a were loaded onto a sodium dodecyl sulfate-polyacrylamide gel fluorescence microscope (IX-81; Olympus Corp.,Tokyo, Japan). electrophoresis (SDS-PAGE) gel. After electrophoresis, the protein was electrotransferred to a Hybond-ECL polyvinylidene MMP assessment by JC-1 staining. Disruption of the MMP is an fluoride membrane (SurModics Inc. Eden Prairie, MN, USA). The early event in reactive species-induced apoptosis. membrane was blocked with Tris-buffered saline (20 mM Tris and Mitochondria depolarization is specifically indicated by JC-1, which 140 mM NaCl, pH 7.6) containing 0.1% Tween 20 (TBST) and is a cationic dye that exhibits potential-dependent accumulation in 4% milk at room temperature for 1 h. The membrane was then mitochondria, indicated by a fluorescence emission shift from green incubated overnight with a monoclonal rabbit anti-rat antibody to red as the mitochondrial membrane becomes more polarized (15). (Cell Signaling Tech., Danvers, MA, USA) against total or After a 48-h incubation of the Hep3B cells in 12-well plates phosphorylated extracellular signal-regulated kinase 1/2 (1×104/well), the Hep3B cells were treated with fluoxetine (10- (ERK1/2), c-JUN N-terminal kinase (JNK) or p38 MAPK as the 100 μM). After 24 h of incubation, Hep3B cells were washed with primary antibody at a 1:1000 dilution in TBS with 5% milk at 4 PBS and incubated with 10 μg/ml JC-1 for 10 min at 37˚C. The ˚C. The blot was washed three times for 10 min in TBST at room JC-1 dye accumulates in the mitochondria of healthy cells as temperature. The membranes were incubated with horseradish aggregates, which fluoresce red. Upon the collapse of the peroxidase-conjugated secondary antibodies (Cell Signaling mitochondrial potential, JC-1 dye can no longer accumulate in the Tech.) for 60 min. The blot was washed three times for 15 min in mitochondria and remains in the cytoplasm in a monomeric form, TBST (three times for 5 min each), and the bands were detected which fluoresces green. Upon incubation with JC-1 dye, the cells using enhanced chemiluminescence. Representative western blots were observed under a fluorescence microscope (IX-81; Olympus were scanned by a Bio-Rad ChemiDoc XRS and the images Corp.) and scraped with a cell scraper. The harvested cells were quantified in Quantity One 4.5.0 software (Bio-Rad, Hercules, homogenized immediately, and the extract was transferred to a CA, USA). 1.5 ml microfuge tube. The sample was then sonicated for five pulses at a 40% using an ultrasonic processor (Sonics & Materials, Statistical analysis. Results are expressed as mean±standard error Newtown, CT, USA) then microcentrifuged for 10 min at 5,000 of the mean (SEM). The data were analyzed by Student’s t-test or rpm. The supernatant was collected and used to measure the MMP. analysis of variance (ANOVA) with the Bonferroni post-hoc test, The fluorescence was measured using a spectrophotometer with where appropriate, using Prism 5.03 (GraphPad Software Inc., 550 nm excitation/600 nm emission for red fluorescence and 485 San Diego, CA, USA). A p-value<0.05 was considered nm excitation/535 nm emission for green fluorescence. significant.

3692 Mun et al: Antiproliferative Effect of Fluoxetine in Hep3B Cells

Results be 109.6±3.0% for p-p38 MAPK, 108.4±1.2% for p-JNK and 84.7±9.9% for p-ERK. Likewise, the 100 μM fluoxetine- Effect of fluoxetine on the cell viability of Hep3B cells. treated group resulted in values of 59.7±7.1% for p-ERK1/2, Fluoxetine led to a significant reduction in the number of 199.4±10.5% for p-p38 APK, and 143.6±1.7% for p-JNK. cells, as shown in Figure 1A. To characterize the cell death To demonstrate whether ROS and intracellular Ca2+ induced by fluoxetine, we examined the nuclear morphology participate in fluoxetine-induced apoptosis, NAC or BAPTA- of dying cells with a fluorescent DNA-binding dye, DAPI. AM was added to the Hep3B cells growing in the presence of After a 24 h treatment with fluoxetine, cells clearly exhibited fluoxetine. NAC treatment markedly attenuated fluoxetine- condensed and fragmented nuclei, indicative of apoptotic cell induced modulation of MAPK. Using densitometry, 10 mM death (indicated by white arrows). NAC treatment inhibited the fluoxetine-induced increase in In transmitted light images, the concentration-dependent phosphorylation of p38 and JNK (as a percentage of death of the Hep3B cells in the presence of fluoxetine was fluoxetine-induced phophorylation of 100%: 76.4±28.9% and examined. As shown in Figure 1B, treatment with fluoxetine 76.5±18.8%, respectively). Also, NAC treatment inhibited the induced a decrease in cell viability in a dose-dependent fluoxetine-induced decrease in p-ERK. BAPTA-AM at 5 μM manner. For fluoxetine at 10, 30 and 100 μM, the viability significantly attenuated the fluoxetine-induced increase in p- values were 98.3±15.2, 80.4±4.3 and 30.4±3.4%, respectively. p38 and p-JNK MAPK (as a percentage of fluoxetine: 79.4±26.1%, 76.2±19.3%, respectively). BAPTA-AM also Effect of fluoxetine on MMP of Hep3B cells. MMP is an inhibited the fluoxetine-induced decrease in p-ERK (Figure 5). important parameter of mitochondrial function that is used as an indicator of cell health. In order to determine the cause Discussion of reduced mitochondrial activity in the presence of fluoxetine, we analyzed cells using the MMP-sensing dye, In the present study, we demonstrated that fluoxetine had a JC-1. Figure 2A shows the uptake of JC-1 by viable Hep3B potential effect on apoptosis in the specific HCC cell line. cells, as measured by the increase in monomer (green) and We showed that fluoxetine induced cell death, loss of MMP, aggregate (orange-red) fluorescence. Fluoxetine-induced formation of ROS, decrease in anti-apoptotic ERK1/2 mitochondrion depolarization is specifically indicated by a protein and increase in proapoptotic JNK and p38 MAPK in shift in JC-1 fluorescence from red to green in Hep3B cells. Hep3B cells. To corroborate these results, we also detected the Although fluoxetine, used as an anti-depressant for cancer fluorescence of the cationic dye JC-1 by spectrophotometry. patients, has been reported to have an apoptotic effect against As shown in Figure 2B, fluoxetine induced a dramatic ovarian cancer cell lines (12) and colon cancer (13), it has also decrease in the ratio of JC-1 (red/green ratio). Treatment shown to increase risk of promoting cancer (12), but also been with fluoxetine at 30 μM yielded a ratio that was 81.5±7.0% had no effect on cell survival and growth in cancer cells (14). of the control, and fluoxetine at 100 μM yielded a ratio that A recent study showed that fluoxetine, when used alone, had was 56.6±8.4% of the control (p<0.005). little effect on cytotoxicity but had a remarkable effect on breast cancer cells when combined with adriamycin or Effect of fluoxetine on ROS of Hep3B cells. Because paclitaxel (11). In the present study, fluoxetine (10-100 μM) intracellular ROS is considered to be a death signal in induced a dose-dependent antiproliferative effect in Hep3B apoptosis (16), we demonstrated that ROS participates in cells. After fluoxetine treatment at standard therapeutic doses fluoxetine-induced apoptosis. DCFH-DA was used to (40 mg/day, 30 days) as an antidepressant, plasma levels measure ROS levels in fluoxetine-treated Hep3B cells. As reached around 1 μM (9), which is much lower than the shown in Figure 5, the ROS level notably increased in concentrations necessary to induce apoptosis in Hep3B cells, Hep3B cells at 30 μM (125.6±4.6%) and 100 μM as observed in our study. However, the fluoxetine tissue levels (262.3±14.7%) (p<0.005). were up to 20-fold higher than in plasma, as reported for brain tissue, because of its lipophilic properties (17). Indeed, Effect of fluoxetine on the MAPKs expression of Hep3B cells. fluoxetine has been used for the treatment of mental disorders To identify the involvement of ERK1/2, JNK or p38 MAPK, for extended periods of time at doses higher than 100 mg a the levels of phosphorylated as well as of total forms were day, without significant side-effects (18). Fluoxetine has a very determined in Hep3B cells treated with fluoxetine. broad safety range; serious side-effects in humans occur when Phosphorylation of JNK and p38 was clearly increased in administered at doses higher than 75-times the therapeutic fluoxetine-treated Hep3B cells, whereas the phosphorylation doses for depression (19). Therefore, relatively high doses of level of ERK1/2 was inhibited. Using densitometry, the fluoxetine, at concentrations shown to induce apoptosis in percentage variations in the 30 μM fluoxetine-treated group Hep3B cells in our study, could be used alone or as an adjunct compared to the control group (100%) were determined to treatment for cancer, with acceptable side-effects.

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ROS are constantly generated and eliminated in biological systems and play important roles in a variety of normal biochemical functions and abnormal pathological processes (20). Mitochondrial damage causes ROS to be released from mitochondria, since mitochondria are the major source of ROS. On the other hand, overproduction of ROS causes an imbalance of pro- and antioxidative processes, thus creating a phenomenon known as oxidative stress that results in mitochondria damage, loss of MMP and the execution of apoptosis (21). In the present study, a high level of intracellular ROS production was observed in fluoxetine- treated Hep3B cells. In addition, our results showed that fluoxetine caused a decrease in MMP in Hep3B cells. Reduction of MMP is among the very first intracellular events preceding the execution phase of apoptosis via the mitochondria-mediated death pathway (22). The evidence suggests that ROS are important signaling molecules regulating MAPK activity (23). Several studies have demonstrated that most anticancer agents and ionizing radiation that can induce ROS generation can also concurrently activate MAPK pathways in multiple cell types (24, 25). In addition, recent research has shown that phosphorylation of MAPK plays an important role in the apoptotic cascade of various cancer cell lines (26). We showed that fluoxetine causes an increase in phosphorylation of JNK and p38 MAPK but a decrease in phosphorylation of ERK1/2 in Hep3B cells. It is universally accepted that activation of ERK1/2 enhances cell proliferation (27), but activation of JNK and/or p38 MAPK induces cell death and apoptosis (28, 29). Figure 1. The effects of fluoxetine on cell viability of Hep3B cells. A: Furthermore, activation of p38 kinase has also been implicated Cell morphology was assessed by optical microscopy and fluorescent in the mechanism of apoptosis triggered by chemotherapeutic photographic assessment of apoptosis in Hep3B cells stained with 4’,6- agents (30). It was reported that fluoxetine inhibited the Diamidino-2-phenylindole (×200). B: The data are reported as the growth of lung (A549) and colon (HT29) cancer cells as a mean±SEM (n=8) for each group. *p<0.05, **p<0.01, ***p<0.005: vs. control, Bonferroni’s post-hoc test. result of inhibition of ERK1/2 activation (6). In several cell types, mitochondria also serve as a very efficient Ca2+ buffer, taking up substantial amounts of cytosolic Ca2+ at the expense of MMP. As a consequence of 2+ 2+ Ca uptake, mitochondria can suffer Ca overload, ERK1/2 protein and increase in proapoptotic JNK and p38 triggering the opening of the permeability transition pore MAPK in Hep3B cells. (PTP), which is associated with apoptosis via the In view of the above arguments and the new data mitochondrial pathway, and necrosis due to mitochondrial presented herein, we propose that fluoxetine-induced damage (31). Opening of PTP has been shown to be apoptosis results from the production of intracellular ROS, promoted by thiol oxidation and inhibited by antioxidants, the accumulation of intracellular Ca2+, the loss of MMP, a lending support for a role of ROS in pore opening (32). decrease in antiapoptotic ERK1/2 protein and an increase in Furthermore, it has been demonstrated that mitochondrial proapoptotic JNK and p38 MAPK proffers in Hep3B cells. 2+ Ca uptake can lead to free radical production (33). In order Therefore, fluoxetine may be a potential antiproliferative to determine that the production of intracellular ROS or the agent against HCC, although further research must be carried 2+ accumulation of intracellular Ca is essential for fluoxetine- out to fully investigate this possibility. induced apoptosis, we further investigated whether NAC, an oxidant-scavenger, and BAPTA-AM, an intracellular Ca2+ Acknowledgements chelator, can protect against fluoxetine-induced modulation of MAPK. Our results showed that NAC and BAPTA-AM This research was supported by research funds of the Korean both inhibit the fluoxetine-induced decrease in antiapoptotic Ministry of Science (2010- 0021909 and 2011-0013872).

3694 Mun et al: Antiproliferative Effect of Fluoxetine in Hep3B Cells

Figure 3. The effects of fluoxetine on the generation of intracellular Figure 2. The effects of fluoxetine on mitochondrial membrane reactive oxygen species (ROS) in Hep3B cells. The involvement of ROS permeability (MMP) in Hep3B cells. (A): Fluorescent photographic in fluoxetine-induced cell death was determined by measuring the levels assessment of apoptosis in Hep3B cells stained with JC-1 (×200). (B:) of ROS in Hep3B cells. The cells were incubated with 10 mM 2,7’- MMP levels in Hep3B cells were detected as described in the Materials dichlorodihydrofluorescin diacetate as described in the Materials and and Methods section. The data are reported as mean±SEM (n=8) for Methods section. The fluorescence intensity was evaluated using each group. *p<0.05, **p<0.01, ***p<0.005: vs. control, Bonferroni’s fluorescent microscopy (3B cel(A) and spectrophotometry (B). The data post-hoc test. are reported as mean±SEM (n=8) for each group. ***p<0.005: vs. control, Bonferroni’s post-hoc test.

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Figure 4. The effects of fluoxetine on mitogen-activated protein kinases (MAPK) expression in Hep3B cells. The amount of MAPK was measured by western blot analysis. This image shows typical changes in MAPK levels after exposure to fluoxetine (A). The blots were quantified by scanning densitometry. The data are reported as a mean±SEM (n=4) for each group (B). ***p<0.005: vs. control, Bonferroni’s post-hoc test.

Figure 5. The effects of fluoxetine with/without N-acetylcysteine (NAC) and (acetoxymethyl)-l,Z bis (o-aminophenoxy) ethane N,N,N’,N’-tetra-acetic acid (BAPTA-AM) on mitogen-activated protein kinases (MAPK) expression in Hep3B cells. The amount of MAPK was measured by western blot analysis. This image shows typical changes in MAPK levels after exposure to fluoxetine (A). The blots were quantified by scanning densitometry. The data are reported as a mean±SEM (n=4) for each group (B). ***p<0.005: vs. control; ###p<0.005: vs. control, Bonferroni’s post-hoc test.

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