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Introduction
The anticancer drug
1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine
(ET-18-OCH3, edelfosine), is the prototypic compound of a promising family of antitumor
drugs, collectively known as alkyl-lysophospholipid
analogs or antitumor ether lipids, which induce apoptosis in a
wide variety of cancer cells[1_4]. Edelfosine has been also
found to be cytotoxic to Saccharomyces
cerevisiae yeasts[5,6], but the type of cell death induced by edelfosine in yeasts
remains to be established. Recent evidence suggests
that edelfosine-induced cytotoxicity in both tumor cells and
yeasts is mediated through a modification of lipid raft
composition[6_8]. In addition,
mitochondria[9,10] and endoplasmic
reticulum[11] have been suggested to play a major role in
edelfosine-mediated apoptosis in tumor cells.
Mitochondria have a central role in apoptosis, and many
important aspects of the apoptotic process converge in this
organelle[12]. Mitochondria are the richest source of reactive
oxygen species (ROS) in the cell, converting 1%_2% of
reduced oxygen into superoxide
anion[13]. The inhibition of the mitochondrial electron transport chain, resulting in
subsequent release of ROS, is an early event in apoptotic cell
death induced by different agents (eg, ceramide,
dexametha-sone, and bleomycin)[14_16].
Although it was initially assumed that apoptosis arose
with multicellularity and would have been counterselected
in unicellular organisms, several findings indicate that a
similar process operates in single-celled
eukaryotes[17,18]. In particular, the yeast
S cerevisiae has been reported to undergo apoptosis-like cell death in response to various harsh
treatments[19].
In the present report, we have found that edelfosine
induces apoptosis in S cerevisiae cells via
mitochondrial-derived ROS generation. In addition, edelfosine-induced
apoptosis in human tumor cells was also mediated by
mitochondria and correlated with ROS generation, suggesting
that a similar process mediates cytotoxicity of this drug in
both yeasts and human tumor cells.
Materials and methods
Cell culture and reagents The S
cerevisiae strain used in this study was BY4742
(MATα his3D leu2D
lys2D ura3D). The cells were incubated at 32 ºC in a 25-mL flask with 10
mL YPAD (1% yeast extract, 2% peptone, 2% D-glucose,
and 0.024 mg/ml adenine hemisulfate) medium with
constant shaking at 200×g. Cell growth was monitored by
measuring the optical density of the cultures at 600 nm
(OD600) by using an spectrophotometer. After 2 d incubation
at 32 ºC, the yeasts were at the stationary phase
(OD600 = ~ 6_10) and then cells were diluted for the experiments.
Human acute T-cell leukemia Jurkat and CEM-C7H2 cells
were cultured at 37 °C in a humidified atmosphere of 5%
CO2 in RPMI-1640 medium supplemented with 10%
(v/v) heat-inactivated fetal calf serum (FCS), 2 mmol/L
L-gluta-mine, 100 U/mL penicillin, and 100 µg/mL strepto-mycin.
The CEM-C7H2-9F3 cells, stably transfected with human
transgenic Bcl-2[20], were kindly provided by Dr S Geley
(Institute for General and Experimental Pathology,
University of Innsbruck, Innsbruck, Austria) and cultured in the
presence of 500 μg/mL hygromycin as previously
described[9].
Edelfosine was obtained from Huabei Biochem (Beijing,
China) and INKEYSA (Barcelona, Spain). Stock solutions of
edelfosine in sterile water (for yeast studies) and RPMI-1640
culture medium containing 10% heat-inactivated FCS (for
Jurkat cell studies) were prepared as previously
described[9]. All other chemicals were from Sigma (St Louis, MO, USA).
Edelfosine treatment of S
cerevisiae The yeasts were inoculated into a 25-mL flask containing 10 mL YPAD medium.
Starter yeast cultures were grown to saturation (about 2 d) at
32 °C (approximately 1×108 cells/mL). After saturation, cells
were diluted with fresh YPAD medium to an
OD600=0.1. Then, 10 mL of this diluted cell suspension was added to pre-warmed
25 mL Erlenmeyer flasks containing 10 mL YPAD medium,
and incubated with increasing concentrations of edelfosine
at 32 °C, with shaking at 200×g. Then, 1 mL of cultured cells
were taken at 2-h intervals to determine cell proliferation by
absorbance readings at OD600 and to measure the generation
of ROS. Furthermore, to identify whether ROS generation is
the trigger of apoptosis, the antioxidant α-tocopherol
succinate (Sigma, USA) was used with edelfosine.
α-Tocopherol succinate was dissolved in sterile absolute ethanol at a
concentration of 10 mmol/L and stored at -20 °C. For the cell
treatment, α-tocopherol was added to the cell culture
medium to achieve a final concentration of 10 μmol/L.
ROS measurement in yeasts A modified version of a
previously described assay for the intracellular conversion
of nitro blue tetrazolium (NBT) to formazan by superoxide
anion was used to measure the generation of
ROS[21]. Briefly, the yeasts were incubated with 50
µmol/L NBT for 15 min at 32 °C, fixed in absolute ethanol, and air dried. The
formazan content of the cells was then solubilized with 960
µL 2 mol/L potassium hydroxide and 1120
µL DMSO. The absorbance at 630 nm was measured
spectrophoto-metrically. As a positive control, 100
µmol/L H2O2 was added to the cells and the amount of formazan formed was
measured.
Inhibition of mitochondrial function Rotenone, an
inhibitor of complex I of the mitochondria electron transport
chain[22], was dissolved in tissue culture grade DMSO to
achieve a final concentration of 1 mmol/L. For the cell
treat-ments, rotenone was added at a final concentration of 50
nmol/L to the cell culture, and incubated at 32 °C, with
constant shaking at 200×g for 6 h. The solvent concentration in
the media never exceeded 0.001%.
Assessment of apoptosis by terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling (TUNEL)
assay The cells were loaded on coverslides coated with
poly-L-lysine (Sigma, USA) and dried at 37 °C. After
incubation for 5 min, the cells were fixed in 4% paraformaldehyde
for 20 min at 4 °C. The cells were then gently washed twice
with phosphate buffered saline (PBS) and permeabilized with
0.2% Triton X-100 for 5 min. Following 2 PBS washes, TUNEL
assay was carried out with the Promega kit (DeadEnd
Fluorometric TUNEL System, Promega, Madison, WI, USA)
according to the manufacturer's instructions. Finally, the
cells were analyzed immediately using a Leica DMRXA
microscope (Leica, Wetzlar, Germany) equipped with
Nomarski optics and epifluorescence and photographed with
a Photometrics Sensys CCD camera (Tucson, AZ, USA). The
percentage of cells undergoing apoptosis was determined
by counting the number of cells with apoptotic staining in 10
randomly selected microscope fields (10×ocular, 40×
objective).
Assay of mitochondrial membrane potential
In order to explore whether edelfosine changes mitochondrial membrane
potential in yeast and human acute T-cell leukemia Jurkat
cells, the cells were incubated with 2 mmol/L rhodamine123
for 10 min at 37 °C. After the incorporation of the fluorescent
probe, the cells were incubated with or without 5
μmol/L edelfosine and treated for 6 h. At the end of the incubation,
the cells were washed twice with PBS, and then resuspended
in 1.5 mL PBS. For the yeast cells, the samples were
sonicated for 5 s at 8 microns just before passing through the
flow cytometer. The fluorescence of each individual cell
was measured with a flow cytometer at an excitation
wavelength of 480 nm and an emission wavelength of 530 nm, and
analyzed with CellQuest 3.1f Analysis Software (Becton
Dickinson, Franklin Lakes, NJ, USA).
Cytofluorimetric analysis of DNA fragmentation and
generation of ROS in Jurkat cells The quantitation of
apoptotic cells was calculated by flow cytometry as the
percentage of cells in the sub-G1 region (hypodiploidy) in cell
cycle analysis as previously
described[23]. To evaluate the generation of ROS, the cells were incubated in PBS with 2
μmol/L dihydroethidine (HE) for 20 min at 37 ºC, followed by
analysis on a Becton-Dickinson FACSCalibur (Becton
Dickinson, Franklin Lakes, NJ, USA) flow cytometer as
previously described[9].
Statistical analysis Statistical evaluation of the effect of
edelfosine was performed by analysis of variance (ANOVA).
Statistical significance was recognized at
P<0.05.
Results
Edelfosine induces apoptosis in S
cerevisiae We found that edelfosine inhibited yeast proliferation in a
concentration-dependent manner (Figure 1A). This effect was
cytotoxic rather than cystostatic as cell viability declined with
increasing concentrations of edelfosine when cultured again
in drug-free liquid medium (data not shown). We next
determined whether edelfosine-induced yeast death by apoptosis.
One of the most important markers of apoptosis is
chromosomal DNA fragmentation as assessed by TUNEL staining,
a reliable marker of yeast
apoptosis[24,25]. Propidium iodide stains both apoptotic and non-apoptotic cells, but
fluorescein-12-dUTP incorporation results in localized green
fluorescence within the nuclei of only the
DNA-degraded apoptotic cells. Most of the cells treated with either 2.5 or 5
µmol/L edelfosine for 6 h were TUNEL positive, whereas all
of the untreated cells remained TUNEL negative (Figure 1B).
These results indicate that edelfosine-induced cell death in
yeasts by apoptosis as previously shown in tumor
cells[1,2,9].
Edelfosine induces apoptosis in S
cerevisiae via mitochondrial-derived ROS The external addition of
H2O2 has been reported as a major inducer of apoptosis in
yeasts[24]. Yeast strain YPH98gsh1, lacking glutathione due to a
deletion of the GSH1 gene coding γ-glutamylcysteine synthetase,
undergoes apoptosis after 3 d of being cultured with
glutathione-free medium[24,25]. Thus, ROS generation seems a
key event in yeast apoptosis. We next analyzed the putative
increase in ROS following yeast treatment with edelfosine.
We found that the alkyl-lysophospholipid analog induced a
concentration- and time-dependent increase in the
generation of ROS. The results showed that the generation of ROS
was significantly increased in S cerevisiae cells treated with
1.0, 2.5, and 5.0 µmol/L edelfosine for 6 and 8 h as compared
to the control (Figure 2A,B). The addition of rotenone, an
inhibitor of the complex I in the mitochondrial electron chain,
prior to edelfosine treatment prevented both ROS
generation (Figure 3A) and apoptosis (Figure 3B) in
S cerevisiae cells. Edelfosine-induced generation of intracellular ROS
and apoptosis were also abrogated by 10 µmol/L
α-tocopherol (Figure 4). These data suggest that ROS are
generated from mitochondria upon edelfosine treatment in yeasts
and that ROS generation was the trigger of apoptosis.
Edelfosine induces mitochondrial-derived ROS in
human tumor cells The above data indicated that ROS
generated in mitochondria played a role in edelfosine-induced
apoptosis in yeasts. We next studied whether the same
process took place in human tumor cells. We found that
edelfosine induced ROS generation in human leukemia Jurkat
and CEM-C7H2 cells that preceded apoptosis (Figure 5).
Since Bcl-2 acts as a safeguard in
mitochondria[12], we analyzed the effect of overexpressing Bcl-2 in edelfosine-
induced apoptosis and ROS generation in CEM-C7H2 cells.
The ectopic expression of Bcl-2 blocked both ROS
generation and apoptosis when the cells were incubated with
edelfosine (Figure 5), suggesting that mitochondria and
mitochondrial-derived ROS generation may play a role in
edelfosine-induced apoptosis in human tumor cells.
Edelfosine induces changes of mitochondrial membrane
potential In the present study, the results showed that
relative mitochondrial membrane potential in yeast and Jurkat
cell treated with 5 µmol/L edelfosine for 6 h decreased
significantly (Figure 6), suggesting functional alterations
in mitochondria.
Discussion
Edelfosine has been reported to be preferentially
accumulated in the plasma membrane of transformed cells,
accounting for up to 17% of the purified membrane
phospho-lipids. Despite edelfosine exerts its major effects at the plasma
membrane level, it also affects gene expression by
modulating the expression and activity of transcription
factors[3,4]. Current evidence indicates that edelfosine induces apoptosis
through mitochondria. Edelfosine-induced apoptosis is
prevented by Bcl-2 or Bcl-xL overexpression by gene
transfer[2]. Edelfosine induces the disruption of the mitochondrial
transmembrane potential followed by the production of ROS and
the release of mitochondrial cytochrome
c[9]. In addition, edelfosine activates caspase-3 and its inactivation prevents
the apoptotic response elicited by the ether
lipid[9]. Moreover, recent evidence indicates that edelfosine is the first
antitumor drug acting through the intracellular activation of the
Fas/CD95 death receptor, involving a raft-mediated pro-
cess[7,8].
We have described here that the antitumor agent
edelfosine induces apoptosis in yeasts through a
mitochondrial-generated ROS process. It reacts similarly in human
tumor cells, which suggests that this mechanism is an
ancestral apoptotic pathway being preserved in both
single-celled and metazoan organisms. S
cerevisiae has become a powerful model organism with which to study the process of
apoptosis[26,27]. We propose that it may contribute to
exploring the molecular mechanism of edelfosine on apoptosis.
Moreover, some of its key attributes and advantages in this
regard are the ease with which genetic, molecular, and
cytological manipulations can be performed in this yeast.
Our results indicate that yeasts undergo apoptosis when
treated with edelfosine. DNA fragmentation in S
cerevisiae, for the determination of apoptosis, has so far only been
evidenced by TUNEL-positive
phenotypes[24,25]. This is apparently due to the induction of large DNA fragments (several
hundreds kilobases) in yeast
apoptosis[25], rather than the typical 180 bp multiple DNA fragments found in mammalian
cell apoptosis following internucleosomal DNA
fragmentation[28]. The TUNEL assay was developed for measuring
DNA fragmentation at the single-cell level by incorporating
biotylated or fluorescent dUTP at sites of free 3'
-hydroxy groups in DNA[29]. The TUNEL assay has been used in
numerous studies to classify apoptotic cell death in
yeasts[24,25]. Our results showed that a majority of yeasts treated with 2.5
or 5 µmol/L edelfosine were TUNEL-positive, suggesting
apoptosis in the yeast cell.
Apoptosis is characterized by biochemical processes that
are largely conserved throughout evolution, and
mitochondria seem to act as a central coordinator of cell
death[30,31]. A number of reports have revealed that perturbations in
mitochondria respiration occur early in the apoptotic process
and that the mitochondrion itself serves as a control switch
for apoptosis[32,33]. Complex I (NADH-quinone
oxido-reductase) of the mitochondrial electron transport chain
has been reported to be an important site for leakage of
electrons and the subsequent generation of ROS.
Complex I contains a single large inhibitor binding
pocket[34], and binding to this site of different types of inhibitors,
including rotenone[35], prevents mitochondrial ROS
production[14,16]. In this study, rotenone prevented both apoptosis
and ROS generation in yeasts following edelfosine
treat-ment. The results showed that edelfosine induces the
disruption of the mitochondrial transmembrane potential
followed by the production of ROS that later could lead to the
onset of apoptosis.
Our data also showed that edelfosine increased ROS in
human leukemic cells and the overexpression of Bcl-2 blocked
both apoptosis and ROS generation, in agreement with
previous findings[9]. Thus, a similar mitochondrial-mediated ROS
production is taking place in edelfosine-promoted cell death
in both yeasts and human tumor cells, suggesting that yeasts
might be used as a model to study the mechanism of action
of edelfosine. Our data also support a major role for
mitochondria and mitochondrial-derived ROS in
edelfosine-induced cell death.
Collectively, our findings have shown for the first time
that the antitumor agent edelfosine induces an apoptotic
response in yeasts that involves mitochondria, most likely
through the generation of mitochondrial-derived ROS. This
deadly process induced by edelfosine in yeasts is similar to
the cell death process triggered by this drug in human tumor
cells, suggesting that this mechanism is an ancestral
apoptotic pathway being preserved in both single-celled and
metazoan organisms. Interestingly, these results suggest
that S cerevisiae might be used as an interesting cell model
to study the mechanism of action of edelfosine.
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