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
mental disorders, a serious complication of cancer,
antidepressants have been reported to improve cancer patient
immunity, inhibit cell growth and have an antitumor effect on
various cancer cell lines. We investigated the apoptotic effect
of fluoxetine against the Hep3B human hepatocellular
carcinoma cell line. Materials and Methods: After treatments
of Hep3B cells with fluoxetine, we measured cell viability,
reactive oxygen species (ROS), mitochondrial membrane
potential (MMP) and activation of mitogen-activated protein
kinases (MAPK). Results: Fluoxetine reduced the viability of
cancer cells, induced loss of MMP and formation of ROS,
reduced expression of extracellular signal-regulated kinase
1/2 and increased expression of c-JUN N-terminal kinase
and p38 MAPK. N-Acetylcysteine, an oxidant-scavenger, and
1,2-bis (o-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid
(BAPTA-AM), an intracellular Ca2+ chelator, prevented
fluoxetine-induced modulation of MAPK. Conclusion:
Fluoxetine appears to exhibit an apoptotic effect against
Hep3B cells through the loss of MMP, formation of ROS and
modulation of MAPK activities.
Hepatocellular carcinoma (HCC) is a primary type of
hepatic tumor, an aggressive malignancy with high
prevalence, and the sixth most common cancer worldwide
(1). HCC represents 5% of all cancers with the annual
number of cases exceeding 500,000 worldwide (2). HCC
*These Authors contributed equally to this study.
Correspondence to: Shang-Jin Kim, Department of Pharmacology
and Toxicology, College of Veterinary Medicine, Chonbuk National
University, Jeonju-561-756, Republic of Korea. Tel: +82
632703927, Fax: +82 632703780, e-mail: abbasj@jbnu.ac.kr
Key Words: Fluoxetine, hepatocellular carcinoma, apoptosis,
mitogen-activated protein kinase, Hep3B cells.
cells have extreme chemoresistance and low selectivity to
chemotherapy drugs. In addition, chemotherapy drugs kill
normal cells as well as tumor cells, leading to significant
adverse effects (3). To overcome the weaknesses of
traditional anticancer chemotherapy, alternative or
complementary medicine such as combined treatments with
therapeutics for other diseases or new agents prepared from
natural products is drawing attention as a potential new
approach to anticancer therapy.
Antidepressants are clinically prescribed to patients for
management of depression and psychiatric disorders (4),
and many also exhibit substantial benefit in various types
of chronic pain (5). Interestingly, recent studies have
documented the anticancer effects of antidepressants in a
variety of solid tumor types and cancer cell lines (6).
Selective serotonin re-uptake inhibitors (SSRI), including
fluoxetine, can inhibit growth of various cancer cell lines
from lung, colon, neuroblastoma and breast (6, 7).
Fluoxetine can improve cancer patient immunity and life
quality, and extend their life expectancy (8, 9).
Furthermore, fluoxetine was reported to be a highly
effective chemosensitizer (10) that is synergistic with
anticancer drugs in overcoming multidrug resistance (11).
Fluoxetine has also been reported to have an apoptotic
effect against ovarian cancer cell lines by inducing
mitochondrial membrane permeability (MMP) changes (12)
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
effect on cell survival and growth of cancer cells and tumor
growth in vivo (14).
In the present study, we demonstrated that fluoxetine
induces apoptosis in Hep3B cells, an HCC cell line. To
elucidate the mechanism of fluoxetine-induced apoptosis in
Hep3B cells, we investigated cell viability, reactive oxygen
species (ROS), MMP, and activation of mitogen-activated
protein kinases (MAPK).

0250-7005/2013 $2.00+.40 3691

 ANTICANCER RESEARCH 33: 3691-3698 (2013)
Materials and Methods
Cell culture and reagents. Hep3B cells were obtained from the Korea
Cell Line Bank (KCLB, Seoul, Korea) and grown in Dulbecco’s
modified Eagle’s medium nutrient mixture F-12 HAM (DMEM F-12
HAM) supplemented with 10% fetal bovine serum (Sigma-Aldrich,
St. Louis, MO, USA), 5 mM L-glutamine, 50 U/ml penicillin and 50
μg/ml streptomycin in a humidified 5% CO2-95% air environment at
37˚C. Fluoxetine was purchased from Enzo Life Sciences (Plymouth
Meeting, PA, USA), and 2,7’-dichlorodihydrofluorescin diacetate
(DCFH-DA) was purchased from Molecular Probes (Eugene, OR,
USA). 4’,6-Diamidino-2-phenylindole (DAPI) and 5,5’,6,6’-
tetrachloro-1,1’,3,3’-tetraethyl benzimidazolyl-carbocyanine iodide
(JC-1) were purchased from Enzo Life Sciences.
Cell viability assay. Hep3B cells were grown on 96-well plates
(5000 cells/well) and cultured for 24 h in DMEM F-12 HAM
medium containing 10% fetal bovine serum. After treatment with
fluoxetine (10-100 μM) for 24 h, the culture medium was discarded
and cell viability was assessed with the aid of a Cell Counting Kit-
8 (CCK-8; Enzo Life Sciences). Briefly, 100 μl CCK-8 reagent were
added to each well at a 1:10 ratio to cell culture medium. After a 2-
h incubation in a humidified atmosphere with 5% CO2 at 37˚C, the
absorbance was read at 450 nm using a spectrophotometer (Spectra
Max M5; Molecular Devices, Sunnyvale, CA, USA).
Nuclear staining with DAPI. Hep3B cells were seeded on coverslips
and cultured for 48 h in DMEM F-12 HAM medium containing
10% fetal bovine serum. After the treatment with fluoxetine (10-100
μM) for 24 h, cells were washed three times with ice-cold PBS and
fixed with 70% ethanol for 1 h. The cells were washed with PBS
and counterstained with DAPI mounting medium for 15 min at
room temperature. Apoptotic nuclei were then visualized under a
fluorescence microscope (IX-81; Olympus Corp.,Tokyo, Japan).
MMP assessment by JC-1 staining. Disruption of the MMP is an
early event in reactive nitrogen species-induced apoptosis.
Mitochondria depolarization is specifically indicated by JC-1, which
is a cationic dye that exhibits potential-dependent accumulation in
mitochondria, indicated by a fluorescence emission shift from green
to red as the mitochondrial membrane becomes more polarized (15).
After a 48-h incubation of the Hep3B cells in 12-well plates
(1×104/well), the Hep3B cells were treated with fluoxetine (10-
100 μM). After 24 h of incubation, Hep3B cells were washed with
PBS and incubated with 10 μg/ml JC-1 for 10 min at 37˚C. The
JC-1 dye accumulates in the mitochondria of healthy cells as
aggregates, which fluoresce red. Upon the collapse of the
mitochondrial potential, JC-1 dye can no longer accumulate in the
mitochondria and remains in the cytoplasm in a monomeric form,
which fluoresces green. Upon incubation with JC-1 dye, the cells
were observed under a fluorescence microscope (IX-81; Olympus
Corp.) and scraped with a cell scraper. The harvested cells were
homogenized immediately, and the extract was transferred to a
1.5 ml microfuge tube. The sample was then sonicated for five
pulses at a 40% using an ultrasonic processor (Sonics & Materials,
Newtown, CT, USA) then microcentrifuged for 10 min at 5,000
rpm. The supernatant was collected and used to measure the MMP.
The fluorescence was measured using a spectrophotometer with
550 nm excitation/600 nm emission for red fluorescence and 485
nm excitation/535 nm emission for green fluorescence.
3692
Measurement of intracellular ROS generation. DCFH is widely used
to measure oxidative stress in cells. When the diacetate form of
DCFH is added to cells, it diffuses across the cell membrane and is
hydrolyzed by intracellular esterases to liberate DCFH which, upon
reaction with oxidizing species, forms its 2-electron oxidation
product, the highly fluorescent compound 2’-7’-dichlorofluorescein
(DCF). The fluorescence intensity can be easily measured and is the
basis of the popular cellular assay for oxidative stress (15). After a
48 h incubation of the Hep3B cells in 12-well plates (1×104/well),
the Hep3B cells were treated with fluoxetine (10-100 μM). After a
24 h incubation, Hep3B cells were treated with 10 μM DCFH-DA
for 30 min. Upon incubation with DCFH-DA, the cells were
observed under a fluorescence microscope (IX-81; Olympus Corp.).
DCFH fluorescence was then determined using a spectrophotometer
at excitation and emission wavelengths of 488 nm and 515 nm,
respectively.
Western blot analysis of MAPKs. After a 48-h incubation of the
Hep3B cells in a Petri dish (1×107/well), the Hep3B cells were
treated with fluoxetine (100 μM) with/without N-acetylcysteine
(NAC) (10 mM) and (acetoxymethyl)-l,Z bis (o-aminophenoxy)
ethane N,N,N’,N’-tetra-acetic acid (BAPTA-AM) for 24 h. After
incubation, Hep3B cells were washed three times in ice-cold PBS
and scraped with a cell scraper. The harvested cells were lysed
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,
1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM
β-glycerophosphate, 1 mM Na4VO4 and 1 μg/ml leupeptin. The
sample was then sonicated for five pulses at a 40% using a Sonics
& Materials Ultrasonic Processor (USA), and then transferred to
a 0.5 ml microfuge tube. The sample was heated to 100˚C for 7
min and placed briefly on ice. Then, 30 μL of the supernatant
were loaded onto a sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) gel. After electrophoresis, the
protein was electrotransferred to a Hybond-ECL polyvinylidene
fluoride membrane (SurModics Inc. Eden Prairie, MN, USA). The
membrane was blocked with Tris-buffered saline (20 mM Tris and
140 mM NaCl, pH 7.6) containing 0.1% Tween 20 (TBST) and
4% milk at room temperature for 1 h. The membrane was then
incubated overnight with a monoclonal rabbit anti-rat antibody
(Cell Signaling Tech., Danvers, MA, USA) against total or
phosphorylated extracellular signal-regulated kinase 1/2
(ERK1/2), c-JUN N-terminal kinase (JNK) or p38 MAPK as the
primary antibody at a 1:1000 dilution in TBS with 5% milk at 4
˚C. The blot was washed three times for 10 min in TBST at room
temperature. The membranes were incubated with horseradish
peroxidase-conjugated secondary antibodies (Cell Signaling
Tech.) for 60 min. The blot was washed three times for 15 min in
TBST (three times for 5 min each), and the bands were detected
using enhanced chemiluminescence. Representative western blots
were scanned by a Bio-Rad ChemiDoc XRS and the images
quantified in Quantity One 4.5.0 software (Bio-Rad, Hercules,
CA, USA).
Statistical analysis. Results are expressed as mean±standard error
of the mean (SEM). The data were analyzed by Student’s t-test or
analysis of variance (ANOVA) with the Bonferroni post-hoc test,
where appropriate, using Prism 5.03 (GraphPad Software Inc.,
San Diego, CA, USA). A p-value<0.05 was considered
significant.
 Mun et al: Antiproliferative Effect of Fluoxetine in Hep3B Cells
Results
Effect of fluoxetine on the cell viability of Hep3B cells.
Fluoxetine led to a significant reduction in the number of
cells, as shown in Figure 1A. To characterize the cell death
induced by fluoxetine, we examined the nuclear morphology
of dying cells with a fluorescent DNA-binding dye, DAPI.
After a 24 h treatment with fluoxetine, cells clearly exhibited
condensed and fragmented nuclei, indicative of apoptotic cell
death (indicated by white arrows).
In transmitted light images, the concentration-dependent
death of the Hep3B cells in the presence of fluoxetine was
examined. As shown in Figure 1B, treatment with fluoxetine
induced a decrease in cell viability in a dose-dependent
manner. For fluoxetine at 10, 30 and 100 μM, the viability
values were 98.3±15.2, 80.4±4.3 and 30.4±3.4%, respectively.
Effect of fluoxetine on MMP of Hep3B cells. MMP is an
important parameter of mitochondrial function that is used
as an indicator of cell health. In order to determine the cause
of reduced mitochondrial activity in the presence of
fluoxetine, we analyzed cells using the MMP-sensing dye,
JC-1. Figure 2A shows the uptake of JC-1 by viable Hep3B
cells, as measured by the increase in monomer (green) and
aggregate (orange-red) fluorescence. Fluoxetine-induced
mitochondrion depolarization is specifically indicated by a
shift in JC-1 fluorescence from red to green in Hep3B cells.
To corroborate these results, we also detected the
fluorescence of the cationic dye JC-1 by spectrophotometry.
As shown in Figure 2B, fluoxetine induced a dramatic
decrease in the ratio of JC-1 (red/green ratio). Treatment
with fluoxetine at 30 μM yielded a ratio that was 81.5±7.0%
of the control, and fluoxetine at 100 μM yielded a ratio that
was 56.6±8.4% of the control (p<0.005).
Effect of fluoxetine on ROS of Hep3B cells. Because
intracellular ROS is considered to be a death signal in
apoptosis (16), we demonstrated that ROS participates in
fluoxetine-induced apoptosis. DCFH-DA was used to
measure ROS levels in fluoxetine-treated Hep3B cells. As
shown in Figure 5, the ROS level notably increased in
Hep3B cells at 30 μM (125.6±4.6%) and 100 μM
(262.3±14.7%) (p<0.005).
Effect of fluoxetine on the MAPKs expression of Hep3B cells.
To identify the involvement of ERK1/2, JNK or p38 MAPK,
the levels of phosphorylated as well as of total forms were
determined in Hep3B cells treated with fluoxetine.
Phosphorylation of JNK and p38 was clearly increased in
fluoxetine-treated Hep3B cells, whereas the phosphorylation
level of ERK1/2 was inhibited. Using densitometry, the
percentage variations in the 30 μM fluoxetine-treated group
compared to the control group (100%) were determined to
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-
treated group resulted in values of 59.7±7.1% for p-ERK1/2,
199.4±10.5% for p-p38 APK, and 143.6±1.7% for p-JNK.
To demonstrate whether ROS and intracellular Ca2+
participate in fluoxetine-induced apoptosis, NAC or BAPTA-
AM was added to the Hep3B cells growing in the presence of
fluoxetine. NAC treatment markedly attenuated fluoxetine-
induced modulation of MAPK. Using densitometry, 10 mM
NAC treatment inhibited the fluoxetine-induced increase in
phosphorylation of p38 and JNK (as a percentage of
fluoxetine-induced phophorylation of 100%: 76.4±28.9% and
76.5±18.8%, respectively). Also, NAC treatment inhibited the
fluoxetine-induced decrease in p-ERK. BAPTA-AM at 5 μM
significantly attenuated the fluoxetine-induced increase in p-
p38 and p-JNK MAPK (as a percentage of fluoxetine:
79.4±26.1%, 76.2±19.3%, respectively). BAPTA-AM also
inhibited the fluoxetine-induced decrease in p-ERK (Figure 5).
Discussion
In the present study, we demonstrated that fluoxetine had a
potential effect on apoptosis in the specific HCC cell line.
We showed that fluoxetine induced cell death, loss of MMP,
formation of ROS, decrease in anti-apoptotic ERK1/2
protein and increase in proapoptotic JNK and p38 MAPK in
Hep3B cells.
Although fluoxetine, used as an anti-depressant for cancer
patients, has been reported to have an apoptotic effect against
ovarian cancer cell lines (12) and colon cancer (13), it has also
shown to increase risk of promoting cancer (12), but also been
had no effect on cell survival and growth in cancer cells (14).
A recent study showed that fluoxetine, when used alone, had
little effect on cytotoxicity but had a remarkable effect on
breast cancer cells when combined with adriamycin or
paclitaxel (11). In the present study, fluoxetine (10-100 μM)
induced a dose-dependent antiproliferative effect in Hep3B
cells. After fluoxetine treatment at standard therapeutic doses
(40 mg/day, 30 days) as an antidepressant, plasma levels
reached around 1 μM (9), which is much lower than the
concentrations necessary to induce apoptosis in Hep3B cells,
as observed in our study. However, the fluoxetine tissue levels
were up to 20-fold higher than in plasma, as reported for brain
tissue, because of its lipophilic properties (17). Indeed,
fluoxetine has been used for the treatment of mental disorders
for extended periods of time at doses higher than 100 mg a
day, without significant side-effects (18). Fluoxetine has a very
broad safety range; serious side-effects in humans occur when
administered at doses higher than 75-times the therapeutic
doses for depression (19). Therefore, relatively high doses of
fluoxetine, at concentrations shown to induce apoptosis in
Hep3B cells in our study, could be used alone or as an adjunct
treatment for cancer, with acceptable side-effects.
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 ANTICANCER RESEARCH 33: 3691-3698 (2013)

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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 Mun et al: Antiproliferative Effect of Fluoxetine in Hep3B Cells
Figure 2. The effects of fluoxetine on mitochondrial membrane
permeability (MMP) in Hep3B cells. (A): Fluorescent photographic
assessment of apoptosis in Hep3B cells stained with JC-1 (×200). (B:)
MMP levels in Hep3B cells were detected as described in the Materials
and Methods section. The data are reported as mean±SEM (n=8) for
each group. *p<0.05, **p<0.01, ***p<0.005: vs. control, Bonferroni’s
post-hoc test.
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Received July 14, 2013
Revised July 22, 2013
Accepted July 23, 2013
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