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Oncotarget, 2017, Vol. 8, (No. 2), pp: 3181-3196
Research Paper
The antidepressant fluoxetine induces necrosis by energy
depletion and mitochondrial calcium overload
Emilie Charles1,6,*, Mehdi Hammadi1,6,*, Philippe Kischel2, Vanessa Delcroix1,6,
Nicolas Demaurex3, Cyril Castelbou3, Anne-Marie Vacher1,6, Anne Devin4,6, Thomas
Ducret5,6, Paula Nunes3, Pierre Vacher1,6
1
INSERM U1218, Institut Bergonié, Bordeaux, France
2
Laboratory of Cellular and Molecular Physiology EA4667, Université de Picardie Jules Verne, SFR CAP-SANTE (FED 4231),
Amiens, France
3
Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland
4
Institut de Biochimie et Génétique Cellulaires, UMR 5095, Bordeaux, France
5
INSERM U1045, Centre de Recherche Cardio-Thoracique, Bordeaux, France
6
Université de Bordeaux, Bordeaux, France
*
These authors have contributed equally to this work
Correspondence to: Mehdi Hammadi, email: m.hammadi.2802@gmail.com
Keywords: fluoxetine, CRAC, calcium overload, respiratory chain, cell death
Received: October 19, 2016
Accepted: November 21, 2016
Published: November 29, 2016
ABSTRACT
Selective Serotonin Reuptake Inhibitor antidepressants, such as fluoxetine
(Prozac), have been shown to induce cell death in cancer cells, paving the way for
their potential use as cancer therapy. These compounds are able to increase cytosolic
calcium concentration ([Ca2+]cyt), but the involved mechanisms and their physiological
consequences are still not well understood. Here, we show that fluoxetine induces
an increase in [Ca2+]cyt by emptying the endoplasmic reticulum (ER) through the
translocon, an ER Ca2+ leakage structure. Our data also show that fluoxetine inhibits
oxygen consumption and lowers mitochondrial ATP. This latter is essential for Ca2+
reuptake into the ER, and we postulated therefore that the fluoxetine-induced
decrease in mitochondrial ATP production results in the emptying of the ER, leading to
capacitative calcium entry. Furthermore, Ca2+ quickly accumulated in the mitochondria,
leading to mitochondrial Ca2+ overload and cell death. We found that fluoxetine could
induce an early necrosis in human peripheral blood lymphocytes and Jurkat cells,
and could also induce late apoptosis, especially in the tumor cell line. These results
shed light on fluoxetine-induced cell death and its potential use in cancer treatment.
cancer cells, which are dissociated from their selective
inhibition of serotonin reuptake. Indeed, fluoxetine has
been shown to induce cell death in cancer cells in vitro
[3–11] and to prevent the growth of tumors in vivo [5,
12–14]. Fluoxetine reduces cell viability in various
models of cancer. Moreover, fluoxetine does not decrease
the viability of non-cancer cell lines such as HSF [4]
or primary cells such as peripheral blood mononuclear
cells and B lymphocytes [9], suggesting that fluoxetine
selectively kills tumor cells. Several types of cell death
seem to be involved, with various publications reporting
not only apoptosis [7–10], but also autophagy [10].
However, the precise mechanisms involved in fluoxetineinduced cell death remain largely unresolved at this time.
INTRODUCTION
The antidepressant fluoxetine belongs to the
Selective Serotonin Re-uptake Inhibitor (SSRI) family.
SSRIs enable an increase in serotonin concentration
in the synaptic cleft by preventing serotonin re-uptake
back into the excitatory neuron [1]. SSRIs are effective
antidepressants that possess an advantageous safety
profile, especially concerning overdose. In most countries,
fluoxetine (Prozac®) was the first SSRI that became
available for clinical use [2]. Since then, fluoxetine has
become one of the most widely used antidepressants.
In addition to their neurological effects, SSRIs – and
especially fluoxetine – display toxic properties towards
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Fluoxetine and SSRIs also have reported effects
on cytosolic calcium concentration ([Ca2+]cyt) and on
ion channels, which can be either activated or inhibited.
For example, numerous experiments have shown that
fluoxetine induces an increase in [Ca2+]cyt in immune
cells [9, 10, 15–17] and central nervous system cells
[18, 19]. This effect is found both in healthy and cancer
cell models. Reports that fluoxetine induces Ca2+ release
from the Endoplasmic Reticulum (ER) and mimics B-cell
receptor (BCR) ligation [9, 20] suggest that the pathway
could involve PhosphoLipase C (PLC) activation, leading
to the production of Inositol 1,4,5-trisPhosphate (IP3) and
to the activation of the IP3 Receptors (IP3R) located within
the ER membrane. However, other authors have shown
that IP3 is on the contrary not involved in fluoxetineinduced increase in [Ca2+]cyt [16, 17, 20]. Nevertheless,
whether IP3R is involved or not, Ca2+ is released from
an intracellular compartment after a fluoxetine treatment
[20]. It appears that the increase in [Ca2+]cyt induced by
fluoxetine is due to a Ca2+ entry [10, 15–17, 20]. Ca2+ is
a second messenger, which is of utmost importance for
numerous cellular processes including cell death. Hence,
Ca2+ homeostasis is crucial, and it is well known that Ca2+
overload or an alteration in Ca2+ levels within different
cellular compartments can be cytotoxic and may lead
to cell death by necrosis, apoptosis or autophagy [21,
22]. Notably, mitochondria are a central compartment
regarding Ca2+-induced cell death, and fluoxetine is
found mainly accumulated in this organelle [23]. Overall,
further insight is needed in order to elucidate the pathways
involved in the increase in [Ca2+]cyt triggered by fluoxetine.
The purpose of this study was thus to determine the
signaling pathway triggered by fluoxetine, leading to a
[Ca2+]cyt increase in both cancer and healthy immune cells.
For cancer cells, both adherent and non-adherent cell models
were used. Additionally, we studied the relationship between
the Ca2+ pathway and the cell death pathway. We confirmed
that fluoxetine induces an ER-dependent cytosolic Ca2+
increase in adherent and non-adherent cell models. However,
our data shows that this cytosolic Ca2+ increase is due to a
“thapsigargin-like” effect, where Ca2+ leaves the ER via the
translocon and triggers Store-Operated Ca2+ Entry (SOCE).
The initial calcium leak is produced by a direct or indirect
inhibition of SERCA activity, since fluoxetine impairs ATP
production by inhibiting the respiratory chain. The rise in
[Ca2+]cyt resulted in a mitochondrial Ca2+ overload leading to
cell death, mainly by necrosis.
cells and the adherent HeLa cancer cell line, we performed
dose-response experiments using fluoxetine concentrations
ranging between 1 and 100µM, and monitored Fura2AM fluorescence immediately upon fluoxetine addition.
Fluoxetine induces a dose-dependent increase in [Ca2+]cyt
in both PBLs (Figure 1B) and HeLa cells (Figure 1D).
Jurkat cells are also shown for comparison under the same
experimental settings in Figure 1C.
To determine whether this [Ca2+]cyt increase results
from an extracellular influx, from a mobilization of Ca2+
from the intracellular stores or both, experiments were
carried out in the presence or absence of 2mM Ca2+ in
the extracellular medium. As shown in Figure 1E for
Jurkat cells, the addition of fluoxetine induces a sustained
increase in [Ca2+]cyt in the presence of extracellular Ca2+.
In the absence of Ca2+, however, a fluoxetine addition
leads to a smaller change in [Ca2+]cyt, this increase being
transient, returning to the basal level after several minutes.
The importance of extracellular Ca2+ was confirmed by
another experiment in which cells were first placed in a
Ca2+-free medium, and Ca2+ was perfused after fluoxetine
addition (Figure 1F).
RESULTS
Fluoxetine-induced Ca2+ release is independent
of the PLC/IP3/IP3R pathway, RyR and of TCP
Fluoxetine-induced Ca2+ entry involves CRAC
channels
To determine how extracellular Ca2+ is transferred
into cells upon fluoxetine treatment, we specifically
studied Calcium Release-Activated Ca2+ (CRAC)
channels, constituted of Orai1, a plasma membrane
protein, and STIM1, a calcium sensor located in the
ER membrane. When Jurkat cells (Figure 2A and
2B), PBLs (Supplementary Figure S1A and S1B) and
HeLa cells (Supplementary Figure S1C and S1D) were
pre-treated with BTP2 or ML-9 (inhibiting Orai1 and
STIM1, respectively), the addition of fluoxetine in a Ca2+containing medium led to a transient and highly reduced
rise in [Ca2+]cyt (grey traces in Figure 2A, Supplementary
Figure S1A and S1C), quite similar to the effects observed
in the absence of extracellular Ca2+ (red trace in Figure
2A). Furthermore, Jurkat cells expressing shOrai1
exhibited a less intense [Ca2+]cyt increase (Figure 2C and
2D). Similar results were obtained with a non-conducting
pore mutant of Orai1 (E106A), acting as a dominant
negative (Figure 2E and 2F). These results clearly show
that Store-Operated Ca2+ (SOC) channels are involved in
fluoxetine-induced Ca2+ entry.
Fluoxetine induces an increase in the cytosolic
Ca2+ concentration resulting from Ca2+ release as
well as Ca2+ entry
The involvement of CRAC channels highly suggests
that fluoxetine leads to a depletion of intracellular Ca2+
stores. In line with this, depletion of ER calcium with
thapsigargin (TG) abolished fluoxetine-induced Ca2+
signals in HeLa cells (Supplementary Figure S2A).
However, when cells were pre-treated with fluoxetine, TG-
In Jurkat cells, fluoxetine can induce a dosedependent increase in [Ca2+]cyt (Figure 1A). In order to
determine the effects of fluoxetine on [Ca2+]cyt in PBLs
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induced Ca2+ signals were also abolished (Supplementary
Figure S2B). Several publications have shown that
fluoxetine indeed induces Ca2+ release [9, 20], potentially
in a PLC and IP3-independent manner [15, 17, 20]. To
more thoroughly characterize the precise ER receptors
or pumps responsible for the [Ca2+]cyt increase, we used
Figure 1: Dose-dependent effects of fluoxetine on [Ca2+]cyt. A. Jurkat cells loaded with FuraPE3-AM Ca2+ probe were placed in
a 2mM Ca2+ containing medium. Fluoxetine injection (5 to 100µM) is indicated by the arrow. Ratios were normalized to baseline. Results
correspond to 3 independent experiments with 12 averaged wells per experiment. B, C and D. Quantification of Ca2+ responses induced
by fluoxetine in a medium containing 2mM Ca2+ in PBL cells (B), Jurkat cells (C) and HeLa cells (D). The reported values represent the
area under the curve (AUC), expressed in arbitrary units (A.U.). E. Jurkat cells were loaded with FuraPE3-AM, and fluorescence ratios
were recorded in the presence (2mM, black trace) or absence (0mM, red trace) of external Ca2+. F. Jurkat cells loaded with Fura2AM were
incubated first with fluoxetine (100µM) in a Ca2+-free medium. External Ca2+ was only added a few minutes later.
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Figure 2: Fluoxetine-induced Ca2+ entry involves CRAC channels. A. Jurkat cells were loaded with FuraPE3-AM, and
fluorescence ratios were recorded in the presence (2mM, black trace) or in the absence (0mM, red trace) of external Ca2+. Fluoxetine
injection (100µM) is indicated by the arrow. Inhibitors of Orai1 channels (BTP2, 10µM, dark grey trace) and Stim proteins (ML9, 10µM,
light grey trace) were added in the presence of 2mM Ca2+. B. Quantification of results obtained in A: histogram bars represent the area
under the curve (AUC), expressed in arbitrary units (A.U.), for experiments without external calcium (0mM Ca2+), with external calcium
(2mM Ca2+), with BTP2 and with ML9. C. and E. Fluorescence ratios of Jurkat cells loaded with FuraPE3-AM, either transfected with a
non-targeting shRNA (shNT) or a shRNA targeting Orai1 (shOrai1, C), or with a control GFP expression plasmid (GFP) and a plasmid
allowing the expression of a non-conducting pore - dominant negative Orai1 (Orai1 E106A, E). Fluoxetine injection (100µM) is indicated
by the arrow. D and F. Quantification of results obtained in C and E, respectively: histogram bars represent the area under the curve (AUC),
expressed in arbitrary units (A.U.).
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transferase, leaving the translocon closed [26]). When
PBL cells were pretreated 30 minutes with anisomycin, in
the absence of extracellular Ca2+, the increase in [Ca2+]cyt
was reduced by 35% after fluoxetine addition in PBLs
(Figure 4A and 4B). Interestingly, in Jurkat cells, pretreatment with anisomycin (30 minutes) reduced the levels
of fluoxetine-induced Ca2+ release by 85% (Figure 4C and
4D), confirming the role of the translocon in fluoxetineinduced [Ca2+]cyt rise.
various pharmacological inhibitors and modified cell
lines. First, we have ruled out the implication of the PLC/
IP3 pathway in our cellular model by monitoring IP3
localization in cells after the addition of fluoxetine. For this
purpose, we used a fusion protein made of the Pleckstrin
Homology (PH) domain of PLCδ and eGFP, which is
able to bind PIP2 and IP3 and hence allows the monitoring
of IP3 localization after PIP2 cleavage, regardless of the
PLC isoform (Figure 3A). As a positive control we used
TRAIL, a death ligand able to induce the PLC/IP3 pathway,
leading to an increase in [Ca2+]cyt. The results show that
TRAIL induces relocation of the IP3 fluorescence from
the membrane to the cytosol, revealing PIP2 cleavage
and IP3 release (Figure 3B). In contrast, fluoxetine does
not elicit such a relocation of the fluorescence (Figure
3C). Thus, we conclude that fluoxetine does not activate
the PLC/IP3 pathway in order to trigger the increase in
[Ca2+]cyt. Using PLCγ1-/- Jurkat cells, we also show that
PLCγ1 deficiency does not suppress fluoxetine-induced
intracellular Ca2+ mobilization (Figure 3D). Moreover,
upon treatment with two inhibitors of IP3Rs (Figure 3E
and 3F), namely 2-APB (red trace) and xestospongin C
(green trace), no significant difference concerning Ca2+
mobilization was revealed upon fluoxetine application.
Finally, similar to B cells [20], other experiments using
U73122, a pharmacological inhibitor of PLCβ [24], did
not reveal significant difference in the [Ca2+]cyt profile
after fluoxetine treatment (Figure 3G and 3H). Together,
these results demonstrate that the PLC/IP3/IP3R pathway
is likely not involved in the fluoxetine-induced calcium
release from the ER. Other receptors that could potentially
have a role in the increase in [Ca2+]cyt are the Ryanodine
receptors (RyR). Using dantrolene (an inhibitor of RyR1
and RyR3 isoforms), we ruled out the potential implication
of these receptors in fluoxetine-induced increase in
[Ca2+]cyt (Figure 3G and 3H). Finally, we ensured that Ca2+
was not released from lysosomes by using the Two Pore
Channel (TPC) inhibitor, Ned19. Cells treated with Ned19
did not show significant modification of the fluoxetineinduced Ca2+ increase (Figure 3G and 3H).
Fluoxetine-induced increase in [Ca2+]cyt leads to
calcium accumulation in mitochondria
To decipher the downstream events of the [Ca2+]cyt
increase after a fluoxetine addition, we took a closer look
at the mitochondria, in order to determine if Ca2+ could be
stored in this compartment. For that purpose, we performed
confocal imaging experiments with HeLa cells, using the
mitochondrial marker and Ca2+ sensing probe Rhod2, in
association with the Mitotracker Green mitochondrial
probe. The addition of fluoxetine led to an increase in
red fluorescence, with the overlap with the Mitotracker
Green signal yielding a yellow color, demonstrating the
increase in mitochondrial Ca2+ concentration ([Ca2+]mt,
Figure 5A, CTL). For the sake of comparison, we also
used TG, a compound that is well known for inhibiting
SERCA pumps, raising thus [Ca2+]cyt by ER depletion and
Ca2+ entry through SOCE (Figure 5A, Tg). Tg induced
a larger and faster, but more transient, Ca2+ entry in the
mitochondria of HeLa cells (Figure 5B). Interestingly, we
could not observe any increase in [Ca2+]mt when cells were
pretreated with the translocon inhibitor, anisomycin (Figure
5A and 5B). Similar results were obtained on PBLs cells
(Figure 5C and 5D), demonstrating that a reduction of only
35% of the fluoxetine-induced cytosolic calcium increase
(Figure 4A and 4B) was sufficient to completely abolish
the fluoxetine-induced mitochondrial Ca2+ increase in these
cells (Figure 5D). Next, to identify whether the Ca2+ uptake
was mediated by the Mitochondrial Ca2+ Uniporter (MCU),
we used Ru360, a specific inhibitor of the uniporter. We
show that in the presence of Ru360, the fluoxetine-induced
increase in [Ca2+]mt was abolished in HeLa cells (Figure 5A
and 5B), as well as in PBL (Figure 5C and 5D), suggesting
the implication of the MCU in the fluoxetine-induced
increase in [Ca2+]mt.
Fluoxetine induces ER calcium depletion
through the translocon
Since IP3R and RyR receptors, among others, are
not likely to be involved in the ER Ca2+ release upon
fluoxetine addition, we searched for another candidate.
The translocon is an ER structure that enables the
transfer of secretory proteins and lumenal domains of
membrane proteins from the cytoplasm to the ER lumen
[25]. However, it has also been shown that the translocon
can mediate a Ca2+ leakage from the ER stores into the
cytoplasm [26]. To determine whether this mechanism of
calcium release could be involved in fluoxetine-induced
[Ca2+]cyt increase, we used a pharmacological inhibitor
of the translocon, anisomycin (an inhibitor of peptidylwww.impactjournals.com/oncotarget
Fluoxetine reduces oxygen consumption and
leads to a drop in ATP content
Since our results demonstrate that fluoxetine induces
a mitochondrial Ca2+ overload, we next sought to determine
if fluoxetine exerted other effects on mitochondria, for
instance by addressing the potential involvement of the
respiratory chain. We thus monitored oxygen consumption
rate in different cell lines, and we showed that fluoxetine
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Figure 3: Fluoxetine does not induce hydrolysis of the membrane bound PIP2. A. Confocal microscopy images of HeLa
cells, transfected with a construct enabling transient expression of a fusion protein [Pleckstrin Homology (PH) domain of PLC] / eGFP
fluorescent protein, allowing to locate membrane PIP2, as well as cytosolic IP3. Fluorescence was measured in zones of interest delimited
by rectangles, corresponding to membrane (rectangle “1”) and cytosolic regions (rectangle “2”), respectively. B and C. Time course
of fluorescence emission, at the membrane level (black trace) and cytoplasm (red or blue trace): B - To induce IP3 translocation from
membrane to cytoplasm, rhTRAIL (100ng/mL) was injected when indicated by arrow. C - Fluoxetine (100μM) does not induce IP3
translocation. D. Jurkat cells were loaded with FuraPE3-AM Ca2+ probe. Cells were either PLCγ deficient (PLC def) or reconstituted with
a functional PLCγ (PLC rec). E. Jurkat cells were loaded with FuraPE3-AM, and fluorescence ratios were recorded in the presence of
2mM of external Ca2+ (black trace). Ratios were also recorded in the presence of the IP3Rs inhibitors xestospongin C (500nM) and 2-APB
(44µM, green and red traces, respectively). F. Quantification of results obtained in E: histogram bars represent the area under the curve
(AUC), expressed in arbitrary units (A.U.), for control (CTL), xestospongin C and 2-APB experiments. G. Jurkat cells were loaded with
FuraPE3-AM, and fluorescence ratios were recorded in the presence of 10µM dantrolene (inhibitor of RyR1 and RyR3 isoforms, green
trace), 10µM U73122 (inhibitor of PLCβ, purple trace) and 5μM Ned19 (TPC inhibitor, blue trace). H. Quantification of results obtained
in G: histogram bars represent the area under the curve (AUC), expressed in arbitrary units (A.U.), for control (CTL), dantrolene, U73122
and Ned19 experiments.
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actually inhibits oxygen consumption in PBLs (Figure
6A), as well as in Jurkat cells (Figure 6B), and HeLa
cells (Figure 6C), suggesting that fluoxetine inhibits the
respiratory chain. When the ATPase activity was stimulated
with dinitrophenol (DNP, Figure 6A, B and 6C), fluoxetine
was still able to exert an inhibitory effect. Since respiration
is coupled to ATP production, we monitored the AMPactivated protein kinase (AMPk) activity by assessing the
ratio p-AMPk / AMPk in Jurkat cells treated either with
fluoxetine or with AICAR (an activator of AMP kinase).
The AMPk system acts as a sensor of cellular energy status,
and is activated by increases in the cellular AMP:ATP ratio
caused by metabolic stresses that either interfere with ATP
production or accelerate ATP consumption [27]. Figure 6D
shows that fluoxetine is able to increase the ratio p-AMPk /
AMPk, suggesting again that fluoxetine may be able to
impair ATP production. We thus monitored ATP production
via ATP imaging in single living cells using a Förster
resonance energy transfer (FRET) - based fluorescent
ATP probe, named ATeam [28]. We found that fluoxetine
was able to reproducibly lower the steady-state levels of
mitochondrial ATP (Figure 6E and 6F). Although the
absolute decrease in ATP content in HeLa cells, which are
highly glycolytic cells, was only on the order of 10%, it is
noteworthy that this fluoxetine-induced decrease was equal
to one third of the decrease observed using the same method
with the potent ATP-synthase inhibitor oligomycin (Figure
6E and 6F). Thus, these observations are consistent with a
model where fluoxetine partially decreases ATP synthesis
by reducing respiratory chain activity. Additionally, we
monitored the oligomycin and fluoxetine induced [Ca2+]cyt
changes (Figure 6G and 6H). When oligomycin was first
added, a general [Ca2+]cyt increase occurred due to ATP
synthase inhibition, and fluoxetine was not able to further
enhance [Ca2+]cyt increase (Figure 6G). When fluoxetine
was first added, the classical [Ca2+]cyt increase occurred, and
Figure 4: Fluoxetine induces ER Ca2+ depletion through the translocon. Measurements of [Ca2+]cyt in PBLs A. and in the
Jurkat cell line C. cells were loaded with FuraPE3-AM, and fluorescence ratios were recorded either in control cells (CTL) or in cells pretreated with anisomycin (30 min., 50µM), in absence of external Ca2+. B and D. Quantification of results obtained in A and C, respectively:
histogram bars represent the area under the curve (AUC), expressed in arbitrary units (A.U.).
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oligomycin was nonetheless able to raise slightly [Ca2+]cyt,
most probably because of its more potent effect on ATP
production (Figure 6H).
(DISC) upon binding to its receptor, that culminate in
the induction of apoptosis [29]; ii) hydrogen peroxide
(H2O2), one of the most biologically relevant member of
the Reactive Oxygen Species (ROS [30]) generated in both
intracellular and extracellular space in normal conditions,
and also by phagocytic cells at sites of inflammation. High
levels of ROS can cause necrosis, while lower levels can
cause apoptosis [31]. H2O2 was indeed highly effective
in inducing necrosis, especially at 24 and 48 hours posttreatment, in PBLs (Figure 7B and 7C) and Jurkat cells
(Figure 7E and 7F). The 4h time frame was indeed not
Fluoxetine-induced Ca2+ signaling leads to late
apoptosis and necrosis
We next sought to determine which mechanism
was responsible for fluoxetine-induced cell death. Cell
death was also induced by i) CD95L, a death ligand
known to initiate the death-inducing signaling complex
Figure 5: Fluoxetine leads to Ca2+ accumulation into mitochondria. A, C. Confocal microscopy images obtained from HeLa
cells (A) and PBL cells (C) loaded with the mitochondrial Ca2+ probe Rhod2-AM, as well as the mitochondrial probe MitoTracker® green.
The increase in mitochondrial Ca2+ load induced an increase in the red staining after fluoxetine or TG application, but not after pretreatment
with an inhibitor of the translocon (anisomycin, 50µM) or an inhibitor of the MCU (RU360, 10µM). B. Rhod-2 fluorescence intensity of
representative HeLa cells (as shown in A) over time before and after addition of Tg or fluoxetine (either in control cells or in cells pretreated with anisomycin or RU360). D. Rhod-2 fluorescence intensity of representative PBL cells (as shown in C) over time before and after
addition of Tg or fluoxetine (either in control cells or in cells pre-treated with anisomycin or RU360).
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Figure 6: Fluoxetine blocks oxygen consumption and leads to a drop in ATP content. A, B and C. Oxygen consumption
rates were measured using an Oroboros oxygraph-2k, either without (CTL) or in the presence of fluoxetine or dinitrophenol (DNP, 50 to
200µM), in PBLs (A), Jurkat cells (B) or HeLa cells (C). All values were normalized vs. 5mM pyruvate. D. Western blotting showing the
ratio AMPk / p-AMPk in Jurkat cells in control conditions (CTL, first lane) and in treated conditions, either with fluoxetine (50µM, second,
third and fourth lanes, at 10, 30 and 60 minutes post-treatment, respectively) or with AICAR (an activator of AMP kinase, 0.65mM, fifth
lane). E and F. Monitoring of ATP production via ATP imaging in single living cells using a Förster resonance energy transfer (FRET) based fluorescent ATP probe (ATeam). Loss of ATP is visualized by a decrease in FRET (E), and quantified by the “Δ loss” value. The “Δ
loss” values, obtained either with fluoxetine or the ATP synthase inhibitor oligomycin, calculated as the difference between initial and final
normalized FRET signals levels after 10 min, are reported in F. G. Variations of [Ca2+]cyt induced by fluoxetine (50μM) with oligomycin
pre-treatment. H. Variations of [Ca2+]cyt induced by 0.4µM oligomycin with fluoxetine pre-treatment. [Ca2+]cyt was recorded in a whole cell
population. Experiments shown in G and H. were performed in a Ca2+-free medium.
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Figure 7: Fluoxetine-induced Ca2+ signaling leads to late apoptosis and/or necrosis. Cell death was quantified in PBLs A, B
and C. and in Jurkat cells D, E and F. using FACS. Analyses were conducted at 4h post-treatment (A and D), 24h post treatment (B and E)
and 48h post treatment (C and F). Cell death was assessed by Annexin V (AV) and Propidium Iodide (PI) staining. Cells were cultured in
normal conditions or treated with 20µM QVD, a broad spectrum caspase inhibitor. Cell death was induced with fluoxetine (40µM), but also
with CD95L (2.5ng.ml-1 for Jurkat cells, 100ng.ml-1 for PBLs) and hydrogen peroxide (H2O2, 50µM). HPT: hours post-treatment.
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enough to see any hydrogen peroxide-induced necrosis on
Jurkat cells (Figure 7D), whereas this compound was the
most effective in inducing early necrosis in PBLs (Figure
7A). Fluoxetine was quite as effective in inducing necrosis
in PBLs, with the largest effect seen at 48h (Figure 7C),
and a less pronounced effect at 4h (Figure 7A) and 24h
(Figure 7B). Jurkat cells followed apparently the same
pattern, but the results were influenced by the application of
QVD, a broad spectrum caspase inhibitor [32], suggesting
that part of the cells positive for both annexin V (AV+) and
propidium iodide (PI+) were late apoptotic cells (Figure 7E
and 7F). CD95L also induced late apoptosis, especially in
Jurkat cells (Figure 7E and 7F), but was less effective in
PBLs (Figure 7A, B and 7C). No necrosis was evidenced
with CD95L since the QVD inhibitor indeed always
abolished detection of AV+/PI+ cells. Early apoptosis could
also be induced by CD95L in PBLs or by fluoxetine in
Jurkat cells, but levels of apoptosis were far lower than
necrosis / late apoptotic levels in the same conditions. This
apoptosis was efficiently blocked by QVD, (Figure 7A,
B, C and 7F). H2O2 was clearly not a potent inducer of
apoptosis. These results clearly show that late apoptosis
and necrosis were the main mechanisms by which cell
death occurred, in PBLs and Jurkat cells treated with
fluoxetine (especially at 48h). The original Annexin V / PI
plots are shown in Supplementary Figure S3.
increasing the [Ca2+]cyt in HeLa cells (Supplementary
Figure S2B). This effect is not in agreement with what
was found in human bladder cells [20], and the mechanism
underlying these cell-type dependent differences remains
to be determined. These results led us hypothesize that
fluoxetine might have a “TG-like” effect on the ER. Upon
TG application, this latter releases ER Ca2+ passively
through leak channels, including the translocon [36]. In
agreement with this hypothesis, the translocon blocker
anisomycin either partially or strongly reduced fluoxetineinduced Ca2+ signals in PBL and Jurkat cells. How does
fluoxetine induce a passive Ca2+ leak from the ER? One
possibility is that it influences SERCA function, at least
indirectly. Of note, the energy used for ER Ca2+ reuptake
by SERCAs preferentially comes from ATP produced in
mitochondria [37]. Since fluoxetine induced a decrease
in respiration and a drop in mitochondrial ATP, it is
conceivable that this could contribute to an inhibition
of the Ca2+ reuptake back into the ER. Fluoxetine could
be, in this case, considered as an indirect inhibitor of
SERCAs. In line with this hypothesis, it has already been
shown that the UCP3-mediated uncoupling of oxidative
phosphorylation from ATP synthesis can modulate SERCA
activity by decreasing mitochondrial ATP production [38].
Previous studies have shown that calcium leaking via
the translocon triggers cell death, especially in epithelial
cancer cells [39]. Our results demonstrate indeed that the
fluoxetine-induced [Ca2+]cytincrease involving translocon
correlates with cell death in PBLs and Jurkat cells. It
is noteworthy that the Jurkat cell line was found to be
sensitive to this fluoxetine-induced cell death, contrary
to what Gordon’s team reported [9]. Unlike TG, that
is able to cause cell death mainly by apoptosis (with
necrosis being involved to a lesser extent [40]), we found
that fluoxetine-induced cell death was mainly caused
by necrosis (and / or late apoptosis, especially in Jurkat
cells). We hypothesized that these effects could be due to a
mitochondrial Ca2+ overload, as recently shown in glioma
cells [41]. It has already been demonstrated that fluoxetine
preferentially accumulates into mitochondria [23], and
most importantly, we show that fluoxetine induced Ca2+
accumulation into the mitochondria. At lower levels, Ca2+
can actually promote ATP synthesis, by stimulating the
activity of various metabolic mitochondrial enzymes [42].
However, upon continuous Ca2+ overload, two mechanisms
are engaged to eliminate Ca2+ excess from mitochondria:
the NCLX (the mitochondria-specific sodium / calcium
exchanger) and / or the PTP (Permeability Transition
Pore) [43, 44]. The opening of this latter can cause the
inner membrane of the mitochondria to depolarise [45],
oxydative phosphorylations to stop (as evidenced herein),
matrix swelling and membrane breaks, causing Ca2+ as
well as proapoptotic molecules (such as cytochrome
c) to leak out. Continuous mitochondrial permeability
transition can lead to cell death by apoptosis or necrosis,
depending on remaining cellular ATP. Intracellular ATP
DISCUSSION
Although it is now widely accepted that SSRIs (such
as fluoxetine) are able to induce a rise in intracellular Ca2+
and cell death, the exact underlying mechanisms remain
obscure. Several groups agreed that fluoxetine releases
Ca2+ from intracellular stores (including the recent work
of Gobin and collaborators in human T lymphocytes
[33]), in addition to stimulating extracellular Ca2+ entry
[9, 20, 34, 35]]. In the present study, we confirm that
fluoxetine indeed induces an increase in the cytosolic
Ca2+ concentration, resulting from an intracellular ER
Ca2+ release, followed by extracellular Ca2+ entry. Since
this mechanism is reminiscent of capacitative channel
activation, we further assessed the implication of Stim1
and Orai1. We found that these typical actors of store
operated Ca2+ entry were involved in fluoxetine-induced
[Ca2+]cyt increase, in contrast to the recent finding in human
T lymphocytes [33]. By using a variety of pharmacological
tools and fluorescent assays, we show that the canonical
PLC/IP3/IP3R pathway is not involved, in good agreement
with other studies [15, 17, 20, 34]]. Our results suggest
thus that fluoxetine effects are most likely not induced by
G protein–coupled receptors signaling, nor through RyR or
TPC. We were able to confirm reduced fluoxetine-induced
Ca2+ signals following pre-treatment with agents depleting
ER calcium, such as TG [9, 20] or ATP [34], as shown
in Supplementary Figure S2A. However, it is noteworthy
that fluoxetine, in turn, was also able to prevent TG from
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present study to decipher the molecular mechanisms are
admittedly high, but in line with most in vitro published
studies, in which apoptotic effects of fluoxetine are seen
from 0.1 to 100μM. Plasma concentrations found in patients
treated with fluoxetine are usually in the low micromolar
range: plasma concentrations at equilibrium in depressivetreated patients are rather comprised between 0.5 and
1.6μM, with high inter-individual variability [51]. After
a 30-days treatment with a 40mg daily intake (neurologic
disorders being treated within the 20 to 80mg range),
plasma concentrations reach 1μM [52]. This concentration
could conceivably retain significant apoptotic effects in
vivo. Due to the relative safety of fluoxetine, dose escalation
would also be an option: a study in which fluoxetine
intakes ranged from 280 to 1260mg showed that plasma
concentrations could reach 0.75 to 4.5μM (a study of 234
cases with fluoxetine overdose suggest that 1500mg intake
is the upper limit [53]). Future investigation on the effects
of fluoxetine concentrations within therapeutic range, as
well as differential effects between normal and cancer
cells (as already suggested by others [4, 9]), will both be
necessary to assess whether and how fluoxetine may bring
added value to existing cancer treatments in the clinic.
concentration appear indeed to be a key switch, pointing
either to apoptosis or necrosis [46]. If the proportion of
damaged mitochondria remains small, but sufficient to
activate apoptosis, there will be enough ATP from intact
mitochondria for apoptosis to occur [47]. If the proportion
of damaged mitochondria is high, it is likely that signals
emitted from damaged mitochondria will spread to all
mitochondria [48], and subsequent ATP depletion will
hinder assembly of the apoptotic machinery, ultimately
leading to necrosis [46, 49]. Fluoxetine, at the concentration
we used, may therefore induce directly necrosis and/or
late apoptosis on most cells, and also apoptosis on a more
resistant subpopulation of the cells. All results, as well
as the proposed mechanism, are summarized in Figure
8. On the other hand, depletion of ER Ca2+ can also lead
to ER stress, another pathway able to trigger cell death
[50], especially when overstimulation occurs. Whether
fluoxetine induces ER stress and whether this contributes
to fluoxetine toxicity would require further study.
The cytotoxic fluoxetine effects are highly interesting
for potential use in cancer therapies, particularly in light of
beneficial psychological effects it may have on patients
facing a life-threatening disease. The doses used in the
Figure 8: Proposed mechanism for the fluoxetine-induced increase in [Ca2+]cyt and cell death. Fluoxetine, known to enter
mitochondria, is likely able to inhibit directly the respiratory chain, which is necessary for mitochondrial ATP production, and thus for
pumping calcium into the ER via SERCAs. Calcium leaks from the ER via the translocon, leading to ER calcium depletion, which in
turn activates the store-operated calcium channels Orai1 via the reticular STIM1 protein. Once released from the endoplasmic reticulum,
calcium quickly accumulates into the mitochondria, leading to mitochondrial calcium overload and cell death, mainly by necrosis.
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which CaCl2 was omitted and 100µM EGTA was added
in order to chelate residual Ca2+ ions. This medium
was added to the cells just before recording to avoid
intracellular calcium stores leaking.
MATERIALS AND METHODS
Cell lines and PBLs
Jurkat cells and Jurkat variants (deficient for
signaling proteins PLCγ1-/-) were obtained from Dr.
Patrick Legembre (ER440-OSS CLCC Eugène Marquis,
Université de Rennes 1). HeLa cells were bought from
DSMZ. Peripheral blood mononuclear cells PBMC
from healthy donors were isolated by Ficoll gradient
centrifugation. Blood pouches were obtained from the EFS
(Etablissement Français du Sang) Aquitaine-Limousin,
after written informed consent was obtained from
participants. Monocytes were depleted by a 2h adherence
step, and the naive PBLs were stimulated as described
previously [54]. HeLa and Jurkat T-leukemic cell lines
were maintained in RPMI supplemented with 8% (v/v)
heat-inactivated FCS and 2mM L-glutamine at 37°C in a
5% CO2 incubator.
Mitochondrial calcium imaging
Jurkat, PBLs and HeLa cells were loaded with
Rhod-2 AM fluorescent probe (1µM, 2h, and 37°C).
Cells were grown on glass coverslips and placed in an
Attofluor observation chamber for confocal microscopy
(Zeiss LSM 510 Meta, with Planapochromat x63 oil
immersion objective). Helium/Neon laser (543 nm)
was used for excitation, and emitted fluorescence
was recorded through a low-pass filter (560nm).
Analysis was made with Zeiss software (Axiovision).
Mitochondria were stained using Mitotracker green
(200nM, 2h, and 37°C).
Real time membrane PIP2 and cytosolic IP3
imaging
Pharmacological tools
Anisomycin, fluoxetine and ML9 were purchased
from Sigma-Aldrich (L’Isle d’Abeau Chesnes, St-QuentinFallavier, France). RU360 were purchased from Santa
Cruz Biotechnology (Heidelberg, Germany). Oligomycin
and rhTRAIL were obtained from Merck Millipore
(Fontenay sous Bois, France) and AdipoGen (Liestal,
Switzerland), respectively. 2-APB, BAPTA-AM, BTP2,
TG and xestospongin C were obtained from Calbiochem
(Merck Chemicals Ltd., Nottingham, UK).
A fusion protein between PH domain (Pleckstrin
Homology domain) of PLCγ1 and GFP (GFP-PH) was
used. This PH domain has a high selectivity for PIP2
and a higher affinity for IP3 than for PIP2. It translocates
thus from membrane to cytosol with IP3. This system
enables thus real time IP3 production in living cells, with
migration of green fluorescence (GFP) from the membrane
to cytoplasm [57]. Construct was obtained from Pr. T.
Meyer (Stanford University, CA, USA). HeLa cells
were transfected with the Exgen500 kit (Euromedex) for
plasmid expression.
Ca2+ monitoring
In cell populations, [Ca2+]i was measured
ratiometrically in Indo-1–loaded cells using a Hitachi
F2500 spectrophotometer, as described previously [55].
Cells bathed in Hank’s Balanced Salt Solution (HBSS)
were placed in a quartz cuvette under continuous stirring.
The Fura 2 fluorescence response to [Ca2+]i was recorded
as the F340nm/F380nm fluorescence ratio. The values of the
emitted fluorescence for each cell (F) were normalized to
the starting fluorescence (F0) and reported as F/F0 (relative
[Ca2+]cyt). Each experimental condition was repeated
independently at least three times; values are reported as
mean ± SD.
Single-cell [Ca2+]i imaging was performed
ratiometrically as described previously [54]. Cells were
loaded with 5μM Fura2-PE3-AM for 30min at room
temperature in (HBSS). Fura2-PE3-AM exhibits limited
compartmentalization in intracellular stores and is leakageresistant [56]. Imaging was controlled by Universal
Imaging software, including MetaFluor and MetaMorph.
All images were background-subtracted. Data processing
was performed using OriginPro 7.5 software (OriginLab).
In some experiments, cells were placed in a Ca2+free medium, consisting of the HBSS described above in
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Western blot
Cells were lysed into lysis buffer containing 1% Na
deoxycholate (Sigma), 150mM NaCl, 10mM PO4Na2/K,
pH 7.2 and supplemented with inhibitor cocktail (Sigma),
2mM EDTA, 1mM sodium orthovanadate (New England
Biolabs), phosphatase inhibitor (Thermo Fisher) and
5mM PMSF (phenylmethanesulfonyl fluoride, Sigma).
Benzonase nuclease (Santa Cruz) was added to reduce
viscosity in protein extracts. Protein content was
measured using BCA method (Biorad). Each sample was
dissolved in Laemmli 5X buffer, and then denatured at
100°C for 10min. After SDS-PAGE and transfer onto
nitrocellulose membrane (Hybond, GE Healthcare),
membranes were incubated with goat polyclonal primary
antibodies against p-AMPk (1/1000, Millipore) or AMPk
(1/1000, Sigma) overnight at 4°C. Membranes were
incubated with anti-goat peroxidase-conjugated anti-IgG
secondary antibody (1/5000, Santa Cruz), developed
using ECL substrate solution, exposed to the Fusion
Fx7 (Thermo Fisher) and analyzed using Quantity One
software (Biorad).
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Respiration experiments
Abbreviations
Respiration assays were performed using an
Oroboros Oxygraph-2k. The oxygen consumption was
measured polarographically at 37°C using a Clark
oxygen electrode in a thermostatically controlled
chamber. Respiratory rates (JO2) were determined from
the slope of a plot of O2 concentration versus time, and
normalized by million cells. Respiration assays were
performed in the growth medium supplemented with
HEPES 10mM, pH 7.2.
AICAR, 5-aminoimidazole-4-carboxamide-1-β-Dribofuranoside.
CRAC channels, Calcium Release-Activated
Calcium channels.
ER, Endoplasmic Reticulum.
IP3, Inositol 1,4,5-tris-Phosphate.
IP3R, IP3 Receptors.
MCU, Mitochondrial Calcium Uniporter.
PLC, PhosphoLipase C.
PTP, Permeability Transition Pore.
SERCA, Sarcoplasmic/Endoplasmic Reticulum
Ca2+-ATPase.
SOC, Store-Operated Channel.
SOCE, Store-Operated Calcium Entry.
SSRI, Selective Serotonin Re-uptake Inhibitor.
ATP measurements
Mitochondrial ATP levels were measured by
transfecting HeLa cells with the mitochondrially targeted
ATP-sensitive FRET-based probe (Mito-ATeam), as
previously described [38]. Briefly, cells seeded on
coverslips were washed in a modified Ringer’s medium
(in mM: 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES,
and 10 glucose, pH 7.3) and images were collected using
440nm excitation and alternate 485/535 nm emission on
an Axiovert S100 TV microscope through a ×40, 1.3 NA
oil-immersion objective (Carl Zeiss AG, Switzerland)
equipped with a 16-bit CCD camera, Xenon lamp and
filter-based wavelength switcher (Visitron Systems
GmbH, Germany). Minimum FRET emission ratios
were obtained by washing cells in Ringer’s were glucose
was replaced by 2-deoxyglucose and contained 10µg/
ml oligomycin. Relative changes in mitochondrial ATP
are reported as background-subtracted FRET ratios
normalized to baseline and minimum FRET emission
ratios where Relative Mito-ATeam FRET = ((F335/485 Fmin) / Fbaseline). The loss of ATP is reported as Loss ATP
= (1 - Relative Mito-ATeam FRET) at t=10min after
stimulation. Bars show mean ± SEM of 3 independent
experiments.
ACKNOWLEDGMENTS
The authors wish to thank Dr P. Legembre (ER440OSS CLCC Eugène Marquis, Université de Rennes 1) for
the gift of Jurkat cells and Jurkat variants.
CONFLICTS OF INTEREST
There is no conflict of interest.
FUNDING
This work was supported by the National Cancer
Institute (#2012-119).
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