Fluoxetine-Induced Apoptosis in Hepatocellular Carcinoma Cells
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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 fluoxetine 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). 0250-7005/2013 $2.00+.40 3691 ANTICANCER RESEARCH 33: 3691-3698 (2013) 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 nitrogen 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.