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FEBS Letters 584 (2010) 3185–3192
journal homepage: www.FEBSLetters.org
The role of enoyl-CoA hydratase short chain 1 and peroxiredoxin 3 in
PP2-induced apoptosis in human breast cancer MCF-7 cells
Xiang Liu, Renqing Feng *, Liying Du
College of Life Sciences, Peking University, Beijing 100871, PR China
a r t i c l e
i n f o
Article history:
Received 12 April 2010
Revised 25 May 2010
Accepted 2 June 2010
Available online 10 June 2010
Edited by Quan Chen
Keywords:
Breast cancer
Apoptosis
4-Amino-5-(4-chlorophenyl)-7-(tbutyl)pyrazolo[3,4-d]pyrimidine
Enoyl-CoA hydratase short chain 1
Peroxiredoxin 3
a b s t r a c t
We show that 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) induces
apoptosis and down-regulates the expression of enoyl-CoA hydratase short chain 1 (ECHS1) and peroxiredoxin 3 (PRDX3) in human breast cancer MCF-7 cells. The decrease of ECHS1 and PRDX3 was
validated by Western blot and quantitative real-time reverse transcription-PCR in MCF-7 and other
carcinoma cells. Knockdown and over-expression of ECHS1 and/or PRDX3 further supported the key
role of ECHS1 and PRDX3 in regulation of PP2-induced apoptosis. These results suggest a possible
apoptotic pathway whereby down-regulation of ECHS1 and PRDX3 potentiates PP2-induced apoptosis in MCF-7 cells.
Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction
Breast cancer is now the most prevalent cancer in women
worldwide [1]. Although endocrine therapy brings improvement
to breast cancer, it inevitably results in recurrence with uncontrolled growth and metastasis [2,3]. c-Src, a non-receptor tyrosine
kinase, plays key roles in mediating many functions during the
progression of cancer. c-Src itself is weakly oncogenic [4], but
phosphorylation of c-Src activates downstream targets, and therefore it can regulate many cellular processes. Because of its pivotal
roles in many signal pathways that regulate cell growth, adhesion,
migration and survival, c-Src may be characterized as a therapeutic
target for cancers [5–7].
Recent interest in c-Src as a therapeutic target for cancer has
led to the development of small molecule inhibitors [8,9]. Among
these inhibitors, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)
pyrazolo[3,4-d]pyrimidine) was ﬁrst identiﬁed as a pyrazolo-
Abbreviations:
PP2,
4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4d]pyrimidine; ECHS1, enoyl-CoA hydratase short chain 1; PRDX3, peroxiredoxin
3; MMP, mitochondrial membrane potential
* Corresponding author. Address: Department of Biochemistry and Molecular
Biology, College of Life Sciences, Peking University, Beijing 100871, PR China. Fax:
+86 10 62751526.
E-mail address: [email protected] (R. Feng).
pyrimidine compound that ﬁts into the ATP binding site of cSrc and therefore is a potent and selective inhibitor of the cSrc family kinases [10]. PP2 has been reported to induce apoptosis in murine B cell leukemia [11]. PP2 has also been reported to
potentiate the apoptosis in human neuroblastoma cells [12].
Although many characters of PP2-induced apoptosis have been
described, the molecular mechanism remains unknown. In addition, PP2 has not been studied for its effect on mitochondrial
protein expression in breast cancer cells.
In the present study, we observed that PP2 induced apoptosis in
MCF-7 cells with a concomitant decrease of mitochondrial membrane potential (MMP) and an alteration of Bcl-2 family proteins,
suggesting the possible mitochondria-mediated apoptotic pathway. Using two-dimensional electrophoresis and mass spectrometry, we identiﬁed two signiﬁcantly down-regulated mitochondrial
proteins, enoyl-CoA hydratase short chain 1 (ECHS1) and peroxiredoxin 3 (PRDX3) after c-Src suppression by PP2. RT-PCR and Western blot analysis demonstrated the down-regulation of ECHS1 and
PRDX3 at the transcription and protein levels in MCF-7 and other
carcinoma cells. In addition, we provide evidence that ECHS1 and
PRDX3 may play a key role in PP2-induced apoptosis by using
knockdown and over-expression strategy. These ﬁndings provide
important insights into c-Src suppression by PP2. Thus, for the ﬁrst
time ECHS1 and PRDX3 are shown to be down-regulated after c-Src
suppression.
0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2010.06.002
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X. Liu et al. / FEBS Letters 584 (2010) 3185–3192
2. Materials and methods
2.1. Cell culture
All cell lines used in this work were obtained from American
Type Culture Collection (Manassas, VA). Cells were seeded at an
initial concentration of 105 cells/ml in DMEM (Invitrogen, Carlsbad,
CA) supplemented with 10% fetal bovine serum (FBS, Hyclone,
Pittsburgh, PA), 3.75 g/L sodium bicarbonate, 100 U/mL penicillin
and 100 lg/mL streptomycin. Cells were cultured at 37 °C in a
humidiﬁed incubator containing 5% CO2.
2.2. Cell growth assay
MCF-7 cells were seeded in 96-well plates (Costar, Corning, NY)
with complete culture medium (DMEM containing 10% FBS) and
grown for 24 h. Cells were then cultured with different concentrations of PP2 (Merck, Whitehouse Station, NJ) or without PP2 for
48 h. At the end of the treatment period, 20 lL 3-(4,5)-dimethylthiahiazo (zy1)-3,5-di-phenytetrazoliumromide (MTT,
Amresco, Solon, OH) was added to each well and cells were incubated at 37 °C for 3 h. The reacting product formazan crystals were
dissolved in 200 lL DMSO (Sigma, St. Louis, MO) after removing
the supernatant. After shaking plates for 15 min, the optical density of the wells was measured at the wavelength of 570 nm with
Microplate Reader (Bio-Rad, Hercules, CA). Each concentration of
PP2 was performed in three wells and cell growth assay was repeated for three times.
at an accelerating voltage of 20 kV using CHCA as the matrix. The
spectra were internally calibrated using trypsin autolysis products.
All PMFs obtained were used to search the NCBI database using the
MASCOT search engine (http://www.matrixscience.com/) with a
tolerance of ±100 ppm and one missed cleavage site.
2.5. Western blot analysis
Twenty micrograms of cell lysates of each sample was separated by 12.5% SDS–PAGE. Proteins were transferred onto PVDF
membrane (Millipore) and incubated with primary antibodies at
optimal dilution at 4 °C overnight, followed by incubating with
secondary antibody (HRP-conjugated goat anti-mouse IgG was
used, Jackson ImmunoResearch, West Grove, PA) at 37 °C for 1 h.
The immunoblot was visualized with Enhanced Western Luminescent Detection Kit (Vigorous, Beijing, China). All Western blots
were performed at least three times for each experiment. The relative amount of immunoblot band was measured by densitometry
using Imagemaster 2D Software.
Monoclonal antibodies to peroxiredoxin 3, Bcl-2 and Bax were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody to glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was obtained from Proteintech Group (Chicago, IL).
Monoclonal antibody to enoyl-CoA hydratase short chain 1 was a
generous gift from Dr. Jian’en Gao (Beijing Proteome Research Center, China).
2.6. Quantitative real-time reverse transcription-PCR (qRT-PCR)
analysis
2.3. Protein extraction for two-dimensional gel electrophoresis (2-DE)
MCF-7 cells were cultured for 30 h in DMEM containing 0.2%
FBS for the synchronization of cell cycle, which was also evaluated
by FACS analysis. Cells were collected before PP2 treatment or after
10 lM PP2 treatment for 12 h, 24 h and 30 h. Cells were washed
three times with washing buffer (0.25 M sorbitol, 0.01 M Tris, pH
7.5) and centrifuged at 500g for 10 min at 4 °C. The cell pellet
was resuspended in lysis buffer (8 M urea, 2 M thiourea, 4% CHAPS,
40 mM DTT, containing protease inhibitor cocktail, Roche, Mannheim, Germany). Total cell lysates were centrifuged at 16 000g
for 1 h at 4 °C. The supernatant was collected and puriﬁed with
2-D Clean-Up Kit (Amersham Pharmacia Biotech, Piscataway, NJ).
Protein concentration was determined by 2-D Quant Kit (Amersham Pharmacia Biotech).
2.4. 2-DE and mass spectrometry analysis
2-DE was carried out according to the protocol described before
[13,14]. Brieﬂy, protein samples (70 lg for analytical gel, 1 mg for
preparative gel) were loaded on 24 cm Immobiline 4–7 linear DryStrips (Amersham Pharmacia Biotech) and IEF was run at 30 V for
8 h, 50 V for 4 h, 100 V for 1 h, 300 V for 1 h, 500 V for 1 h, 1000 V
for 1 h, and 8000 V for 12 h. Then second dimensional SDS–PAGE
was carried out in the Ettan Dalt Six Elect Unit 230 (Amersham
Pharmacia Biotech) at 2 W/gel for 45 min and then 15 W/gel until
the dye front reached the bottom of the gel. The analytical gels
for quantiﬁcation were silver stained and the preparative gels were
stained with PhastGel Blue R (CBB R350, Amersham Pharmacia Biotech). Spot detection, quantiﬁcation and analysis were performed
using Imagemaster 2D Software (version 4.01, Amersham Pharmacia Biotech). The ‘‘Total Spot Volume Normalization” method was
used in order to correct the differences in sample loading or stain
intensity among gels. Spots of interest were excised from 2-D gels
that were stained with CBB R350 and followed by in gel digestion.
All mass spectra from MALDI-TOF MS were obtained on an Ultraﬂex
TOF/TOF (Bruker Daltonics, Bremen, Germany) in positive ion mode
Total RNA was extracted by TRIzol reagent (Invitrogen) according to the suggested protocol. RNA (5 lg) was used to synthesize
the ﬁrst-strand cDNA with the Superscript ﬁrst-strand synthesis
system (Invitrogen) for RT-PCR according to the manufacturer’s
recommendation. Gene-speciﬁc primers for RT- and qRT-PCR analyses were synthesized commercially at AuGCT Biotechnology (Beijing, China) as follows, ECHS1: 50 -CGCTGCTGTCAATGGCTATG-30
(forward) and 50 -CTTGGCGTCCTGGGCTGAGA-30 (reverse); PRDX3:
50 -GCCGTTGTCAATGGAGAGTTC-30 (forward) and 50 - GCAAGATGGCTAAAGTGGGAA-30 (reverse). Human GAPDH gene was used as
internal control (forward primer, 50 -GGGAGCCAAAAGGGTCATCATC-30 ; reverse primer, 50 -CCATGCCAGTGAGCTTCCCGTTC-30 ).
The qRT-PCR was performed using the SYBR Green qPCR Kits
(NEB, Ipswich, MA) and a DNA Engine Opticon continuous ﬂuorescence detection system (Bio-Rad) according to the manufacturer’s
instruction. All results were obtained from at least three independent experiments using different RNA samples.
2.7. Analysis of apoptosis by ﬂow cytometry
After different treatment, apoptotic cells were detected by
staining with FITC-conjugated Annexin V and propidium iodide
(PI) using a commercially available apoptosis detection kit (Biosea,
Beijing, China). Brieﬂy, 2 105 cells were collected, washed three
times with PBS and resuspended in binding buffer. After incubation
for 15 min with FITC-conjugated Annexin V and PI according to the
manufacturer’s instructions, apoptotic cells were analyzed by FACS
Calibur ﬂow cytometer. The Summit software (version 4.0, DakoCytomation, CO) was used to determine the number of apoptotic
cells.
2.8. Measurement of mitochondrial membrane potential (MMP)
Mitochondrial membrane potential was measured using ﬂuorescent probe tetramethylrhodamine, methyl ester, perchlorate
(TMRM, Invitrogen). After different treatment, 20 nM TMRM was
X. Liu et al. / FEBS Letters 584 (2010) 3185–3192
added to the external solution and cells were incubated for 20 min
in darkness at 37 °C. Fluorescent signals were detected by a confocal microscopy at the wavelength of Ex540 nm/Em595 nm. The
ﬂuorescent intensity was determined using a FACSCalibur ﬂow
cytometer. Totally, 10 000 cells per sample were analyzed in the
gate region used for calculation.
2.9. Small interfering RNA (siRNA)
Small interfering RNAs were synthesized by Genepharma Company (Shanghai, China). The target sequences for ECHS1 siRNAs
were: 50 -AACCATGATGTGTGATATCAT-30 (508–528 bp, clone #2)
and 50 -AAGAAGTAAGTTGGAGAAGAA-30 (835–855 bp, clone #3)
and for PRDX3 siRNAs were: 50 -AAGGTTCTGGTCTTGCACTAA-30
(521–541 bp, clone #2) and 50 -AAGGCGTTCCAGTATGTAGAA-30
(649–669 bp, clone #3). Scramble siRNA duplexes were used as a
negative control (NC). Cells were transfected with 100 nM target
siRNAs or control siRNA using Lipofectamine 2000 Reagent (Invitrogen) according to the instructions of the manufacturers, and
transfection was carried out for 5 h. After that, culture medium
was replaced with complete DMEM supplemented with 10% FBS.
Cells were collected after transfection for 48 h and silencing effects
were evaluated by Western blot analysis.
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2.10. Plasmid constructs and transfection
cDNAs of human PRDX3 and ECHS1 were obtained using RT-PCR
with RNA extraction from MCF-7 cells (for PRDX3, Forward primer:
50 -CGGGATCCCGATGGCGGCTGCTGTAGGA-30 , Reverse primer: 50 CCGAATTCCTACTGATTTACCTTCTGAAAGTAC-30 ; for ECHS1, Forward primer: 50 -CGGGATCCCGATGGCCGCCCTGCGTGTC-30 , Reverse
primer: 50 -CCGAATTCTCACTGGTCTTTGAAGTTGGC-30 ). Full length
of ECHS1 or PRDX3 was cloned into pcDNA3.1/myc-His A mammalian expression vector (Invitrogen) using BamHI and EcoRI sites.
Correct integrity of both genes was conﬁrmed by restriction digests
and sequencing. MCF-7 cells were transfected by 2 lg pcDNA3.1PRDX3 vector, pcDNA3.1-ECHS1 vector or with empty vector using
Lipofectamine 2000 Reagent (Invitrogen), and transfection was
carried out for 5 h. After that, culture medium was replaced with
complete DMEM supplemented with 10% FBS. Cells were collected
after transfection for 48 h and over-expression of both proteins
was evaluated by Western blot analysis.
2.11. Statistical analysis
Statistic signiﬁcance of differences in each condition was estimated using One Way ANOVA with SigmaStat software (Version
Fig. 1. PP2 inhibited cell proliferation, induced apoptosis with a decrease of mitochondrial membrane potential and an alteration of Bcl-2 family proteins in MCF-7 cells. (A)
PP2 caused a dose-dependent inhibition on the proliferation of MCF-7 cells. Cells were treated with different concentrations of PP2 for 48 h. Relative cell number (y-axis) was
shown as percentage of control (with the same concentration of DMSO) after treatment with indicated concentrations of PP2. (B) PP2 caused a time-dependent inhibition on
proliferation of MCF-7 cells. Cells were treated with 10 lM PP2 for indicated times. (C) The apoptosis was observed after 10 lM PP2 treatment for 12 h, 24 h and 30 h. Cells
were stained with Annexin V-FITC/PI and the apoptosis was analyzed by ﬂow cytometry. (D) The percentage of apoptotic cells was determined by counting the number of
cells only with Annexin V-FITC-positive and normalizing to the number of total cells. (E) Mitochondrial membrane potential of MCF-7 cells after 10 lM PP2 treatment was
measured by TMRM staining. Fluorescent signal was detected by confocal microscopy (left lane). The ﬂuorescent intensity was measured by ﬂow cytometric analysis and the
numbers gated in each phase areas are the mean of three independent experiments (right lane). Bar scale, 30 lm. (F) PP2 induced the decrease of Bcl-2 and increase of Bax in
MCF-7 cells. Cells were collected before PP2 treatment or after 10 lM PP2 treatment for 12 h, 24 h and 30 h. Total protein lysates (20 lg) were separated by SDS–PAGE
followed by Western blot analysis using antibodies speciﬁc to Bcl-2, Bax and GAPDH (as internal control).
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3.5, Germany). Differences were considered to be signiﬁcant when
P < 0.05. In all experiments, * indicates P < 0.05.
3. Results
3.1. PP2 inhibited cell proliferation in MCF-7 cells
We ﬁrst examined the c-Src inhibitor PP2 for its effect on cell
growth to determine the suitable concentration of PP2. As shown
in Fig. 1A, PP2 caused a dose-dependent inhibition on the proliferation of MCF-7 cells. As the PP2 concentration increased from 1 lM
to 50 lM, the number of living cells decreased dramatically
according to cell growth assay (Fig. 1A). In general, 25.8 lM PP2
induced about 50% growth inhibition of MCF-7 cells, which was
in accordance with a previous study [15]. Thus, MCF-7 cells were
cultured in DMEM with 10 lM PP2 in the subsequent experiments.
Fig. 1B shows 10 lM PP2 inhibited cell proliferation in a timedependent manner. The phosphorylation of c-Src was greatly
inhibited by 10 lM PP2 while the total level of c-Src was not changed (our unpublished data).
3.2. PP2 induced apoptosis with a decrease of MMP and alteration of
Bcl-2 family proteins in MCF-7 cells
As shown in Fig. 1C and D, PP2 treatment for 30 h induced about
30% apoptosis in MCF-7 cells. We then conducted experiments to
Fig. 1 (continued)
X. Liu et al. / FEBS Letters 584 (2010) 3185–3192
measure MMP in MCF-7 cells treated with PP2. Using TMRM as the
marker of mitochondrial membrane integrity, confocal microscopy
images revealed evident depolarization of mitochondrial membrane due to the decrease in ﬂuorescent intensity and the blurring
of mitochondrial morphology (Fig. 1E). FACS quantiﬁcation showed
a 21% decrease in ﬂuorescent intensity after PP2 treatment for
30 h. Because the mitochondria-mediated apoptotic pathway is
regulated by Bcl-2 family proteins [16,17], we next examine the
expression of Bcl-2 (anti-apoptotic protein) and Bax (pro-apoptotic
protein) after PP2 treatment. Western blot analysis showed that
Bcl-2 was suppressed rapidly after PP2 treatment for 12 h, whereas
Bax was up-regulated (Fig. 1F).
3.3. PP2 down-regulated ECHS1 and PRDX3 at the protein and
transcription levels in MCF-7 and other carcinoma cells
To investigate changes of protein expression in the entire cell
cycle, MCF-7 cells were cultured in the presence of 10 lM PP2
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for 12 h, 24 h and 30 h. Total proteins were extracted at each time
point and separated by IEF followed by SDS–PAGE. Of the identiﬁed
proteins that expressed differentially after PP2 treatment, ECHS1
and PRDX3 are considered interesting because they play important
roles in fatty acid metabolism and redox balance of mitochondria
(Fig. 2A).
Because both 2-DE (Fig. 2A) and Western blot (Fig. 2B) validated
the decrease of ECHS1 and PRDX3, we next examined the effect of
PP2 on the transcription of these two genes. The qRT-PCR results
indicated that ECHS1 mRNA decreased to 58.2% while PRDX3 mRNA
decreased to 38.6% (Fig. 2C) after PP2 treatment for 30 h compared
to the control sample (100%). The decreased protein and transcription levels of ECHS1 and PRDX3 were also validated in other carcinoma cells (Supplementary Fig. 1).
3.4. Knockdown of ECHS1 and PRDX3 by RNAi increased apoptosis
induced by PP2 in MCF-7 cells
To conﬁrm the role of ECHS1 and/or PRDX3 in PP2-induced
apoptosis, we used the siRNA strategy to deplete ECHS1 and/or
PRDX3. Western blot analysis demonstrated that ECHS1 and
PRDX3 were signiﬁcantly suppressed in two siRNA clones and in
a double knockdown clone (Fig. 3A). But there was no difference
between parental cells and mock cells transfected with scramble
siRNA.
Depletion of ECHS1 and/or PRDX3 with concomitant PP2 treatment led to signiﬁcant increase in the number of apoptotic cells.
Treatment of MCF-7 cells with 10 lM PP2 for 12 h increased the
number of apoptotic cells from 15.57 ± 1.31% in negative control
Fig. 2. PP2 induced Down-regulation of ECHS1 and PRDX3 in MCF-7 cells. (A)
Decreased protein expression of ECHS1 and PRDX3 on 2D gels. The gel images of
protein spot were displayed on top of the graph as representative of three
independent experiments. Relative protein abundance (y-axis) was determined by
ImageMaster 2D software using the ‘‘Total Spot Volume Normalization” method to
correct the differences in sample loading or stain intensity among gels. Column, the
mean of three independent experiments; bars, S.D. *P < 0.05. (B) Western blot
analysis of ECHS1 and PRDX3 after 10 lM PP2 treatment for the indicated times.
MCF-7 cells were collected before PP2 treatment or after 10 lM PP2 treatment for
indicated times. Total protein lysates (20 lg) were separated by SDS–PAGE
followed by Western blot analysis using antibodies speciﬁc to ECHS1, PRDX3 and
GAPDH (as internal control). The relative amounts of ECHS1 and PRDX3 were
measured by densitometry. Data were calculated as the mean optical density of
treated samples as a percent of untreated cells normalized against GAPDH. (C) qRTPCR analysis of ECHS1 and PRDX3 after 10 lM PP2 treatment for the indicated times.
Relative transcription level (y-axis) was determined according to the manufacturer’s instruction with control as calibrator (1-fold). Column, the mean of three
independent experiments; bars, S.D. *P < 0.05.
Fig. 3. Knockdown of ECHS1 and PRDX3 potentiated PP2-induced apoptosis. (A)
Depletion of ECHS1 and PRDX3 in MCF-7 cells. Western blot analysis indicated that
ECHS1 and PRDX3 were suppressed in two separated siRNA clones and a double
knockdown clones compared to that of parental cells (no treatment) or mock cells
(negative control siRNA, NC). (B) Knockdown of ECHS1 and PRDX3 increased PP2induced apoptosis. After 36 h transfection with 100 nM siRNAs or scramble siRNAs
(NC, negative control), MCF-7 cells were treated with 10 lM PP2 for additional 12 h.
Apoptosis was measured by ﬂow cytometry after staining with Annexin V-FITC/PI.
Data represent the percentage of apoptotic cells relative to the total cells. (C)
Knockdown of ECHS1 and PRDX3 induced the decrease of mitochondrial membrane
potential compared to the NC in MCF-7 cells when exposure to 10 lM PP2 for 12 h.
(D) Knockdown of ECHS1 and PRDX3 induced the alteration of Bcl-2 and Bax in
MCF-7 cells in the presence of PP2.
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cells to about 23.75 ± 2.21% in cells with depletion of ECHS1 and/or
PRDX3 (Fig. 3B and Supplementary Fig. 2). The addition of PP2 further increased the dissipation of MMP in siRNA-ECHS1 and/or siRNA-PRDX3-transfected cells compared to cells transfected with
scramble siRNAs (Fig. 3C). Knockdown of ECHS1 and/or PRDX3 induced the decrease of Bcl-2 after PP2 treatment. But knockdown of
ECHS1 and/or PRDX3 had less effect on Bax expression in MCF-7
cells when exposure to PP2 (Fig. 3D).
3.5. Over-expression of PRDX3 inhibited PP2-induced apoptosis in
MCF-7 cells
Next, we transiently transfected MCF-7 cells with expression
vectors pcDNA3.1-ECHS1 or pcDNA3.1-PRDX3. Western blot analysis demonstrated the over-expression of ECHS1 and/or PRDX3
(Fig. 4A). Over-expression of PRDX3 induced less reduction in the
number of apoptotic cells compared to cells transfected with
empty vector in the presence of PP2. The reduction in apoptotic
levels was more evident in the cells over-expressing both PRDX3
and ECHS1, whereas no signiﬁcant difference in cells over-expressing ECHS1 (Fig. 4B and Supplementary Fig. 3). Furthermore, less
reduction of MMP was observed after over-expression of PRDX3
compared to cells transfected with empty vector when exposure
to PP2 (Fig. 4C). Over-expression of ECHS1 and/or PRDX3 induced
the decrease of Bax in the presence of PP2. Over-expression of
ECHS1 had less effect on Bcl-2 expression, whereas PRDX3 overexpression induced the increase of Bcl-2 after PP2 treatment
Fig. 4. Over-expression of PRDX3 attenuated PP2-induced apoptosis in MCF-7 cells.
(A) Over-expression of ECHS1 and/or PRDX3 in MCF-7 cells. Western blot analysis
demonstrated the over-expression of ECHS1 and/or PRDX3 compared to cells
transfected with empty vector (OX: over-expression). (B) Over-expression of PRDX3
attenuated PP2-induced apoptosis. After 36 h transfection, MCF-7 cells were treated
with 10 lM PP2 for additional 12 h. Apoptosis was measured by ﬂow cytometry
after staining with Annexin V-FITC/PI. Data represent the percentage of apoptotic
cells relative to the total cells. (C) Over-expression of PRDX3 in MCF-7 cells led to
less reduction of MMP compared to cells transfected with empty vector when
exposure to 10 lM PP2 for 12 h. (D) Over-expression of ECHS1 and/or PRDX3
induced alteration of Bcl-2 family proteins in MCF-7 cells in the presence of PP2.
(Fig. 4D). These results suggest that PRDX3 over-expression may
alleviate PP2-induced apoptosis, whereas over-expression of
ECHS1 itself has no such effect.
4. Discussion
In this study, we have investigated PP2-induced apoptosis in
human breast cancer MCF-7 cells. The decrease of MMP and alteration of Bcl-2 family proteins suggest the possible mitochondriamediated apoptotic pathway after PP2 treatment. Using a proteomic approach, we identiﬁed thirteen differentially expressed proteins (Supplementary Fig. 4 and Supplementary Table 1). The
small number of differences is likely due to the low sensitivity of
the method in which differential 2-D gel spots are identiﬁed by
mass spectrometry. Because of their low expression level or low
molecular weight, ﬁve protein spots were not identiﬁed. We then
focused on two mitochondrial proteins that are involved in fatty
acid metabolism and redox balance.
Protein spot 1 with a decrease of 2.07-fold after PP2 treatment
for 30 h was identiﬁed as ECHS1 (Fig. 2A). ECHS1 is a key enzyme
that catalyzes the second step in the physiologically important boxidation pathway of fatty acid metabolism. This enzyme is located in the matrix of mitochondria and catalyzes the hydration
of a,b-unsaturated enoyl-CoA thioesters to the corresponding bhydroxybutyryl-CoA thioesters [18,19]. It is reported that ECHS1
is expressed 1.89 times higher in breast cancer tissue than in normal tissue [20]. The decrease of ECHS1 may cause a reduction of boxidation of fatty acid. Protein spot 11 with a decrease of 2.73-fold
after PP2 treatment for 30 h was identiﬁed as PRDX3 (Fig. 2A).
There are six known isoforms of PRDX family in mammalian cells,
and all of them play important roles in the removal of harmful
reactive oxygen species (ROS) [21]. PRDX3 is located in the matrix
of mitochondria and is therefore a mitochondria-speciﬁc ROS-scavenging protein. PRDX3 is found to be over-expressed in some human cancers, which might protect growing tumor cells against
apoptosis [22,23]. It is reported that PRDX3 is expressed 15.86
times higher in breast cancer tissue than in normal tissue [20].
The decrease of PRDX3 may weaken the capability of mitochondria
to neutralize the production of harmful ROS derived from physiological reaction. Because the continuous accumulation of oxidative
stress and depolarization of mitochondrial membrane is a part of
the mitochondria-mediated apoptotic pathway [24], the downregulation of ECHS1 and PRDX3 may functionally impair the integrity of the mitochondria and subsequently activate and initiate
apoptosis.
Depletion of ECHS1 and/or PRDX3 by siRNAs in MCF-7 cells
potentiated the early apoptosis when exposure to PP2 for 12 h.
The over-expression of PRDX3 alleviated the responsiveness of
MCF-7 cells to PP2 treatment, which conﬁrmed the involvement
of PRDX3 during PP2-induced apoptosis. It was reported that
over-expression of PRDX3 in mouse thymoma cells protected cells
against H2O2-induced apoptosis [21]. On the other hand, PP2-induced apoptosis was not signiﬁcantly inhibited in cells overexpressing ECHS1 in our experiments.
The Bcl-2 family proteins have been reported to play key roles
in determining whether cells will undergo apoptosis [25]. It has
been reported that the ratio of Bax and Bcl-2, rather than Bcl-2
alone, is important for the survival of drug-induced apoptosis in
cancer cells [26]. Moreover, Bax translocation into mitochondria
may target the mitochondrial intermembrane contact sites and release cytochrome c, resulting in the sequence of apoptotic processes [27,28]. It has been shown that Akt inhibits a
conformational change in Bax protein and prevents its translocation into mitochondria, thus inhibiting the disruption of mitochondrial inner membrane potential and apoptosis [29,30]. The
inactivation of Akt will facilitate the translocation of Bax from
X. Liu et al. / FEBS Letters 584 (2010) 3185–3192
cytosol to mitochondria [30]. c-Src has been shown to be involved
in the activation of Akt [5]. It is reported that Akt activity is inhibited by PP2 in preadipocytes and lung cancer cells [31,32]. Besides,
the intracellular ROS may also induce Bax conformational change
and its subsequent translocation to mitochondria [33,34]. In the
present study, depletion of PRDX3 with concomitant PP2 treatment led to signiﬁcant increase in the intracellular ROS levels.
Treatment of MCF-7 cells with 10 lM PP2 for 12 h increased the
ROS levels from 56.88 ± 4.61% in negative control cells to about
72.12 ± 4.57% in cells with depletion of PRDX3 (Supplementary
Fig. 5A and B). Furthermore, the decrease in ROS levels was observed after over-expression of PRDX3 compared to cells transfected with empty vector when exposure to PP2 (Supplementary
Fig. 5C and D). Cell fractionation results indicated that Bax was decreased in the cytosolic fractions and increased in the mitochondrial fractions after PRDX3 silencing by siRNA (Supplementary
Fig. 6). These results suggest that intracellular ROS levels may be
upstream factor for Bax activation. Further study may be required
to investigate the details of how ROS regulate Bax activation.
Down-regulation of ECHS1 and PRDX3 after PP2 treatment was
conﬁrmed in MCF-7 cells by Western blot and qRT-PCR. The discrepancy between 2-DE and Western blot analysis (especially between samples with PP2 treatment for 12 h) may be due to the
application of different experimental methods. Western blot may
be more sensitive and reliable because of the speciﬁcity of antibody. 2-DE may be a useful tool for preliminary separation and
identiﬁcation for differentially expressed proteins. But 2-DE is
inﬂuenced by many factors, such as loading efﬁciency of protein
sample, sensitivity of silver staining. Despite the existing discrepancy, both ECHS1 and PRDX3 were signiﬁcantly decreased after
PP2 treatment for 30 h in 2-DE and Western blot analysis. There
could be several reasons for the discrepancy between RNA and protein levels. One possibility is that translation and transcription of
ECHS1 or PRDX3 may be differently regulated after c-Src suppression by PP2. The discrepancy may also be associated with posttranscriptional regulation or half-life period. But RNA and protein
levels of ECHS1 or PRDX3 were consistently decreased after PP2
treatment for 30 h. Further study may be required to investigate
the details of regulational mechanism that lead to the down-regulation of ECHS1 and PRDX3 after PP2 treatment. However, we have
found that ECHS1 and PRDX3 were not only down-regulated in
MCF-7 cells but also in other carcinoma cells, such as MDA-MB231 (breast adenocarcinoma cell line), T-47D (breast ductal carcinoma cell line), HepG2 (hepatocellular carcinoma cell line), DU
145 (prostate carcinoma cell line), HCT-8 (colon adenocarcinoma
cell line) and SKOV-3 (ovarian adenocarcinoma cell line). Therefore, down-regulation of ECHS1 and PRDX3 may be a common response to PP2 treatment in different carcinoma cells.
In conclusion, our study demonstrated that PP2 induced apoptosis through the mitochondria-mediated pathway and down-regulated the expression of ECHS1 and PRDX3. Change of ECHS1 and
PRDX3 may be a direct response for carcinoma cells when face to
PP2 or other pro-apoptotic stimuli.
Acknowledgments
We thank Professor Jeang Kuan-Teh (NIH/NIAID) for revision of
our manuscript. We thank Professor Yuxian Zhu and Zhenquan
Guo (College of Life Sciences, Peking University) for guidance in
accomplishing this work. We thank Dr. Jian’en Gao (Beijing Proteome Research Center, China) for the generous gift of anti-enoylCoA hydratase short chain 1 monoclonal antibody. This work was
supported by the National Natural Science Foundation of China
(No. 30571711) and Beijing Natural Science Foundation (No.
5092012) to Renqing Feng.
3191
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.febslet.2010.06.002.
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