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: abbasj@jbnu.ac.kr
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
μg/ml streptomycin in a humidified 5% CO2-95% air environment at basis of the popular cellular assay for oxidative stress (15). After a
37˚C. Fluoxetine was purchased from Enzo Life Sciences (Plymouth 48 h incubation of the Hep3B cells in 12-well plates (1×104/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. 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.

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 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
Ca2+ uptake, mitochondria can suffer Ca2+ 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.
Ca2+ 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
accumulation of intracellular Ca2+ 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).

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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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