Download Differential toxicological endpoints of di(2-ethylhexyl

Indian Journal of Experimental Biology
Vol. 52, November 2014, pp. 1052-1061
Differential toxicological endpoints of di(2-ethylhexyl) phthalate (DEHP)
exposure in MCF-7 and MDA-MB-231 cell lines: Possible estrogen receptor
α (ERα) independent modulations
Mihir Tanay Das, Manoj Kumar Singh & Indu Shekhar Thakur*
School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110 067, India
Received 25 December 2013; revised 19 May 2014
Wide spread use of Di-(2-ethylhexyl) phthalate (DEHP) has made it a ubiquitous contaminant in today’s environment,
responsible for possible carcinogenic and endocrine disrupting effects. In the present investigation an integrative toxicoproteomic approach was made to study the estrogenic potential of DEHP. In vitro experiments carried out with DEHP
(0.1-100 µM) induced proliferations (E-screen assay) in human estrogen receptors-α (ERα) positive MCF-7 and ERα
negative MDA-MB-231 breast cancer cells irrespective of their ERα status. Further, DEHP suppressed tamoxifen (a potent
anti-breast cancer drug) induced apoptosis in both cell types as shown by flowcytometric cell cycle analysis. Label-free
quantitative proteomics analysis of the cell secretome of both the cell lines indicated a wide array of stress related, structural
and receptor binding proteins that were affected due to DEHP exposure. The secretome of DEHP treated MCF-7 cells
revealed the down regulation of lactotransferrin, an ERα responsive iron transport protein. The results indicated that
toxicological effects of DEHP did not follow an ERα signaling pathway. However, the differential effects in MCF-7 and
MDA-MB-231 cell lines indicate that ERα might have an indirect modulating effect on DEHP induced toxicity.
Keywords: Cell cycle, DEHP, Estrogen receptors-α, Label-free quantification, Secretome
Phthalates are ubiquitous in the environment due to
their wide spectrum use in industries, consumer
products and biomedical devices. Among several
congener of phthalates, di(2-ethylhexyl) phthalate
(DEHP) is the most important one with respect to
its large volume production, wide range of use
and prevalence in the environment1,2. The abundant
anthropogenic sources of DEHP result in human
exposure which can reach as high as 124 µg/kg/d3.
DEHP demonstrates a wide range of toxic effects,
in both animal and human population. Most of the
toxicological effects of DEHP indicate the possible
endocrine disruption function, however, the mechanisms
by which DEHP exert its endocrine disrupting effects
are not yet fully elucidated. The xenoestrogenic
behaviour of DEHP still remains as a matter of
debate due to differences in in vitro bioassay results.
Stimulation of cell proliferation by DEHP has been
reported in ERα positive MCF-7 cells which was
completely suppressed by the addition of pure antiestrogen ICI 182,7804. DEHP inhibited tamoxifeninduced apoptosis and also induced significant cell
——————
*Correspondent author
Telephone: 0091 11 2670 4321 (O)
E-mail: [email protected]; [email protected]
proliferation only in MCF-7 cells but not in ERα
negative MDA-MB-231 cells5. These studies indicate
the estrogenic nature of DEHP. However, in ERα
mediated recombinant yeast screen assay6 and kidney
fibroblast (CV-1) cell line reporter gene assay7, DEHP
showed no estrogenic activity. Moreover, recently
some other phthalate members (DBP, BBP) were also
shown to induce proliferation and invasiveness of
MDA-MB-231 cells8. Hence, some ERα independent
mechanisms may have a role in the proliferating
action which needs to be studied further.
In vitro e-screen assay method using the MCF-7
breast cancer cell line has been frequently employed
to assess the estrogenic activity of several
environmental chemicals9-11. In addition, tamoxifen
(TAM) has also been used for further confirmation of
estrogenic activity5,9. TAM is a potent ER antagonist
which has been extensively used in the chemotherapy
of breast cancer12-14. TAM is cytotoxic to both
in MCF-7 and MDA-MB-231 cells. ER-mediated
genomic effects of TAM cause cytotoxicity in MCF-7
cells15 whereas; ER-independent non-genomic effects
are responsible for the apoptotic effects in MDA-MB231 cells16. Natural estrogen and xenoestrogens
suppress the effect of TAM in ER positive breast
DAS et al.: DEHP EXPOSURE IN MCF-7 & MDA-MB-231 CELL LINES
cancer cells5,9,17. Therefore, to evaluate the chemical’s
proliferating potential, effects of DEHP and TAM
have been studied individually and in combination in
both MCF-7 and MDA-MB-231 cell lines. Eestrogen
responsive (MCF-7) and estrogen nonresponsive
(MDA-MB-231) cell lines were selected as in vitro
models in order to study whether ERα status of cells
plays a role in DEHP induced toxicological endpoints.
In the present study, the toxicological biomarkers
of DEHP have been evaluated in secreted proteins of
MCF-7 and MDA-MB-231 breast cancer cells lines.
The study has been carried out with the help of a
nano-scale multi dimensional LC/MS instrument and
using a recently developed label-free quantitative
method. Classical 2DE based methods suffer from
poor reproducibility between gels, and low sensitivity
to detect low abundant and hydrophobic proteins.
LC-MS based methods overcome these drawbacks,
and hence, it is helpful in exploring potential
biomarkers from the proteins of the cell secretome18.
Quantifying changes in protein expression in different
samples is an issue of major concern for differential
secretome analysis. Isotope-labelling methods, such
as isotope coded affinity tag (ICAT) have been used
for quantification purpose19. However, these label
based approaches are costly and time-consuming. In
this context label-free quantitation method is an
attractive alternative which is based on the
measurements of mass spectral peak signal intensities
or spectral counts that are linearly proportional to
analyte concentrations20,21.
Materials and Methods
Chemicals and reagents—Foetal bovine serum
(FBS) and charcoal-dextran-stripped foetal bovine
serum (CD-FBS) were purchased from HyClone
(USA). Mass spectrometry grade acetonitrile (ACN),
ammonium bicarbonate, trifluroacetic acid (TFA),
dithiothritol (DTT), iodacetamide and sequence grade
porcine trypsin were purchased from Sigma-Aldrich
(St. Louis, MO, USA). RapiGest SF surfactant and
Mass spectrometry grade yeast enolase digestion
standard were obtained from Waters Corporation
(Milford, MA, USA). Other chemicals were
purchased from Sigma-Aldrich (St. Louis, MO, USA)
or Merck (Darmstadt, Germany) unless stated
otherwise.
Cell culture conditions—Breast cancer cell lines
MCF-7 and MDA-MB-231 were obtained from the
National Centre for Cell Science (NCCS, Pune, India).
Both cell lines were routinely grown as monolayer in
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DMEM:Ham's F12 medium (1:1 mixture) supplemented
with 10% foetal bovine serum, 1% antibiotic antimycotic
solution (final concentrations: penicillin, 100 units/mL;
streptomycin, 0.1 µg/mL; amphotericin B, 2.5 ng/mL)
at 37 °C in a humidified atmosphere of 5% CO2. Cells
were passaged at 70–80% confluence, about twice a
week by trypsinization.
Analysis of estrogenic nature of DEHP by E-screen
assay—E-screen assay was carried out in both cell
lines according to Soto et al.10 with some
modifications. Both the cell lines were seeded with
normal growth medium in 96 well plates at a density
of approximately 5×104 cells/mL for MCF-7 cells
and 2.5×104 cells/mL for MDA-MB-231 cells.
After attachment of cells (24 h), growth medium
was removed and cells were washed with PBS
(with Ca2+ and Mg2+) to remove all estrogenic
compounds. Growth medium was replaced by phenol
red-free DMEM with similar supplements as growth
medium, but containing 5% CD-FBS and cells were
allowed to grow for 48 h. CD-FBS was used to
minimize the estrogenic and steroid activity of serum
and to synchronize the cells in the G0/G1- phase of the
cell cycle11,22. After 48 h growth, exposure medium
was refreshed and test chemicals were added. DEHP
was added to wells in triplicate at different
concentrations. A positive (with E2) and a solvent
control (only 0.5% DMSO, without E2 or DEHP)
were included on each plate. After 4 days of
incubation exposure medium was removed and cell
proliferation was measured using the MTT assay9.
The results of E-screen assay were expressed as
changes in proliferation relative to the hormone-free
control. The Estrogenic activity of DEHP in terms of
the relative proliferative effect (RPE) and the relative
proliferative potency (RPP) were calculated using
following parameters10,11:
Cmax: The concentration of test chemicals (DEHP
or E2) at which the maximum proliferation of cells
occurred.
EC50: It is the concentration of the test chemicals at
which 50% maximum proliferative effect was
observed. It is estimated from the dose-response curve
constructed using four parameter logistic curve
equation.
Proliferative effect (PE): It is calculated as the ratio
between the highest cell yield in terms of maximum
optical density value obtained with the chemical
(DEHP or E2), versus the cell yield of the solvent
control (0.5% DMSO).
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INDIAN J EXP BIOL, NOVEMBER 2014
PE = OD max (DEHP or E2)/OD (solvent control)
Thus, PE of hormone free solvent control is 1.
Relative proliferative effect (RPE): It is the ratio
of the maximum proliferation with DEHP and that of
E2. The RPE value is expressed as a percentage.
RPE (%) = [PE (DEHP) -1/PE (E2) - 1] ×100
Here, 1 represents the PE of hormone free solvent
control.
Relative proliferative potency (RPP): It is
calculated as ratio of EC50 concentration of E2 and
that of the DEHP11.
RPP = EC50 (E2)/EC50 (DEHP)
Thus, the RPP value for E2 is 1.
Cell cycle analysis–Effect of DEHP on tamoxifen
induced apoptosis—Potential of DEHP to revert
tamoxifen-induced apoptosis in human breast cancer
cells was studied by flow-cytometric cell cycle
analysis23. Analogous to the E-screen assay after
attachment of cells, growth medium was replaced
by phenol red-free DMEM supplemented with 5%
CD-FBS. After 48 h, the medium was again removed
and the cells were incubated for 60 min in serum-free
medium containing 100 µM DEHP, and/or 10 nM E2,
and/or 5 µM TAM in different combinations as
indicated in the results. Serum-free conditions were
used, as previous studies have shown that rapid
effects can be most clearly demonstrated under such
conditions24. After exposure, cells were harvested by
trypsinization, washed with PBS (with Ca2+ and Mg2+)
and fixed in 70% ice cold ethanol. Cells were stained
with 1mL of freshly prepared propidium iodide (PI)
staining solution (0.1% Triton X-100, 0.1% sodium
citrate, 50 µg/mL PI) and RNase A (10 µg/mL)
according to Krishan25. Flow-cytometric analysis
was performed using a FACS Verse flow cytometer
(Becton Dickerson, San Jose, CA, USA). PI
fluorescence was collected with a 575/25 nm band
pass filter (orange–red fluorescence (FL2)) and for
each sample, 10,000 events were acquired. Cell cycle
data with low robust coefficient of variation (RCV) of
the G0/G1 peak (RCV < 8), were used for further
statistical analysis.
Collection of conditioned medium for secretome
analysis—For secretome analysis experiment, both
the cell lines were seeded in normal growth medium
in 100 mm culture dishes. Approximately at 60%
confluence growth medium was removed and cells
were washed with serum free media to remove all
traces of serum proteins. Growth medium was
replaced by serum free DMEM containing 100 µM
DEHP (dissolved in DMSO) and cells were allowed
to grow for another 48 h. DMSO (0.5%) treated cells
served as the control.
Preparation of the secreted proteins—After the
exposure duration, the conditioned media were
collected and centrifuged at 4 °C at 2000 rpm for
5 min to remove cell debris. The clear supernatants
were collected and were concentrated using a
molecular cut off column (3 kDa; Amicon, Millipore,
Bradford, MA, USA). The samples were further
concentrated to dryness by means of speed vac
concentrator and stored at −80 °C till further analysis.
Preparation of protein digest—The protein samples
were prepared for LC-MS according to Brioschi
et al.20 with minor modifications. The dry protein
pellets were dissolved in 50 mM NH4HCO3
containing 0.1% RapiGest SF (Waters Corporation,
Milford, MA, USA), and vortexed. Then the protein
concentrations were determined using Bradford's
method employing bovine serum albumin as a
standard. The concentration of proteins in both
control and treated samples of both the cell lines were
within the range 1–1.2 µg mL-1. Further each sample
was heated at 80 °C for 15 min and reduced with
5 mM DTT at 60 °C for 15 min. The samples were
cooled down and carbamidomethylated with 10 mM
iodacetamide in dark for 30 min at room temperature.
The sample proteolysis was performed overnight at
37 °C with sequencing grade trypsin at 1:100 (w/w)
enzyme/protein ratio. After digestion, 2% TFA was
added to hydrolyse RapiGest and inactivate trypsin
and the solution was incubated at 37 °C for 20 min.
The samples were then centrifuged at 15,000 rpm for
10 min and the clear supernatants were collected for
LC-MS analysis.
Label-free quantitative LC-MSE analysis—The LCMS analysis involved two steps including Nano-scale
LC separations of trypsin digested proteins followed
by mass spectrometry analysis. Nano-scale LC
separation was carried out using a nanoACQUITY
system (Waters Corporation, Milford, MA, USA).
Before LC separation the digested protein sample
were mixed with 50 fmol yeast enolase digestion mix
as an internal standard for quantitative molar amount
estimation. The samples were then injected into a
180 µm×2 cm Symmetry C18 (5 µm) trap column
(Waters Corporation, Milford, MA, USA) for
pre-concentration and desalting. Subsequently the
DAS et al.: DEHP EXPOSURE IN MCF-7 & MDA-MB-231 CELL LINES
1055
concentrated peptide mix was directed to a 1.7 µm
BEH 75 µm×250 mm analytical column (Waters
Corporation, Milford, MA, USA) for chromatographic
separation. Sample elution was carried out at a
flow rate 300 nL min-1. The reverse phase solvent
A (0.1% formic acid in H2O) and B (0.1% formic acid
in acetonitrile) were used to increase the organic
solvent concentration from 1% to 40% B over 90 min.
A hybrid quadruple orthogonal acceleration
time-of-flight (Q-Tof) mass spectrometer, SYNAPT
G5-MS, (Waters Corporation, Milford, MA, USA)
directly coupled to the chromatographic system, was
used to measure the fragment ion spectra. Accurate
mass data were collected in data-independent analysis
(DIA) mode according to specifications given by
Brioschi et al20.
Data processing and protein identification—All
MS data were processed and searched using Protein
Lynx Global SERVER (PLGS) version 2.5 (Waters
Corporation, Milford, MA, USA). Ion detection, data
clustering, and the normalisation of LC–MSE data in
DIA mode were carried out as described previously26,27.
The proteins were identified by searching a human
specific UniProt database (release 57.0; 20,333
entries) following the algorithm developed by Li
et al27. The search parameters included specifications
as given by Brioschi et al20.
Results and Discussion
DEHP stimulates proliferation of MCF-7 and
MDA-MB-231 cells grown in estrogen free medium—
To investigate the estrogenic activity of DEHP, MCF7 and MDA-MB-231 cells were grown in estrogen
free medium supplemented with E2 or DEHP. As an
ERα negative cell line, the growth of MDA-MB-231
cells was not enhanced by E2. But, DEHP was found
to stimulate dose dependent proliferation of both
MCF-7 and MDA-MB-231 cells within 0.1 to 1000 µM
concentration range (Fig. 1a and b). However, the
proliferation in terms of PE value for DEHP, obtained
on MDA-MB-231 cells was lower compared to that of
the MCF-7 cells (Fig. 2). Further, even though DEHP
causes proliferation, the relative proliferative potential
(RPP) of DEHP was nearly 3000 times less than that
of E2 in MCF-7 cells (Table 1). The RPP value for
MDA-MB-231 cells was not calculated as E2 did not
show significant proliferative effect in these cells.
The results of E-screen assay indicate that DEHP
induces proliferation of ER-negative MDA-MB-231
cells through some ERα independent signaling
pathway. Present data contradict Kim et al.5 who
Fig. 1—Effects of DEHP on proliferation of MCF-7 (a) and
MDA-MB-231 (b) cells. Fold proliferation was calculated over
solvent control (DMSO, 0.5%). E2 was used as positive control.
Values are mean ± SD from 4 observations each.
Fig. 2—Proliferative effect (PE) values in MCF-7 and MDA-MB231 cells after exposure to DEHP and E2. Values are mean ± SD
from 4 observations each.
INDIAN J EXP BIOL, NOVEMBER 2014
1056
reported that DEHP (10 µM) was only able to induce
proliferation of MCF-7 cells but not that of MDAMB-231 cells. However, the above study focused on a
single concentration and does not take dose response
relation into account. Moreover, phthalates (DBP
and BBP) were shown to induce proliferation of
ER-negative MDA-MB-231 cells by an ERα
independent signalling pathway8.
DEHP suppresses tamoxifen induced cytotoxicity
in MCF-7 and MDA-MB-231 cells—To study effects
of DEHP on TAM-induced apoptosis, growth arrested
MCF-7 and MDA-MB-231 cells were treated
with TAM (5 µM) in presence or absence of DEHP
(100 µM) or E2 (10 nM) in serum free medium for
60 min. After exposure morphological and flowcytometric cell cycle analysis were carried out. TAM
(5 µM) induced rapid death in both MCF-7 and
MDA-MB-231 cells. After treatment with both TAM
and E2, the proportions of dead cells decreased in
MCF-7 but not in MDA-MB-231 cells. Whereas,
when the cells were treated with both TAM and
DEHP the numbers of dead cells did not increase in
both MCF-7 and MDA-MB-231 cells lines. The
results of morphological analysis were also reflected
in cell cycle analysis.
Table 1—Estrogenic parameters of DEHP measured with the
E-screen assay
[Values are mean ± SD]
Test
PE
RPE
EC50
RPP
compound
(%)
(M)
MCF-7
E2
DEHP
3.93 e-8
1.34 e-5
4.46 ± 0.16
100
2.24 ± 0.25 35.92 ± 8.34
1
2.94 e-3
MDA-MB-231
E2
DEHP
1.12 ± 0.06
100
E2 was not effective
1.65 ± 0.08 677.80 ± 337.59
1.96 e-5
-----
The results of cell cycle analysis are shown in
Table 2 and Figs 3 and 4. In cell cycle analysis, the
ratio between percentage of post G0-G1 and sub G0-G1
populations was used to define growth effect of the
test chemicals. In a propidium iodide (PI) stained cell
cycle analysis report, the post G0-G1 population
indicates the actively proliferating cells which
includes the S phase and G2-M phase cells. On the
other hand, the sub G0-G1 population corresponds to
the late apoptotic cells with damaged nuclei. Thus,
both these populations are helpful in defining the
growth status. Some earlier studies have used either
only sub G0-G1 population5 or only the S phase
population11 to define growth status. But the ratio of
populations can be a good indicator in experimental
combinations where both apoptosis and proliferation
are of interest like the present case.
Differential secretome analysis by label-free
quantitative LC/MSE proteomics—MCF-7 and MDAMB-231 cells were used as the in vitro models to
study the DEHP induced change in secretome profile.
After cell treatment with DEHP or vehicle, the
conditioned media were collected and processed for
LC/MSE application. The yields of the recovered
soluble-secreted proteins from 3×106 cells was within
the range 50-60 µg for both control and treated
samples of both the cell lines. The differential
expression of proteins was quantified on the basis
of the molar estimate of internal standard (IS).
Pre-digested protein (yeast enolase) was used as
the IS which was spiked into the samples after
trypsin digestion. The average MS signal response
from the IS protein was used to determine a universal
signal response factor (counts/moles of protein),
which was further applied to the other proteins in
order to determine their corresponding absolute
concentration20.
Table 2—Effects of DEHP on the redistribution of growth arrested MCF-7 and MDA-MB-231 cells in the different phases of the
cell cycle
Treatments
MCF-7
MDA-MB-231
Sub
G0-G1
G0-G1
S
G2-M
RCV
(G0-G1)
Post
G0-G1/
Sub G0-G1
Sub
G0-G1
G0-G1
S
G2-M
RCV
(G0-G1)
Post
G0-G1/
Sub G0-G1
Control
13.64
50.86
20.69
12.98
6.27
2.47
2.47
55.35
21.48
18.94
4.09
16.36
TAM (5 µM)
25.65
44.78
17.94
10.09
6.44
1.09
2.6
67.38
15.03
13.68
3.93
11.04
E2 (10 nM)
11.89
49.97
22.45
14.34
5.64
3.09
3.07
53.89
17.56
23.67
4.27
13.43
DEHP (100 µM)
14.15
44.60
26.96
12.70
7.70
2.80
2.31
52.68
19.19
23.97
4.13
18.68
TAM + DEHP
14.92
49.34
22.32
11.77
6.44
2.28
2.47
59.83
19.48
16.45
4.01
14.55
TAM + E2
11.95
51.35
22.05
13.24
5.80
2.95
2.32
66.76
15.94
13.8
4.02
12.82
DAS et al.: DEHP EXPOSURE IN MCF-7 & MDA-MB-231 CELL LINES
1057
Fig. 3—Flow-cytometry analysis: Cell cycle histogram of MCF-7 (a) and MDA-MB-231 (b) cells treated with TAM in presence or
absence of DEHP or E2. DNA content was measured by a flow-cytometer after propidium iodide (PI) staining. PI fluorescence of 10,000
events were analyzed, cell doublets, aggregates and debris were gated out using a two parameter dot plot of PI-Area versus PI-Width and
the gated in population were plotted in histogram. Horizontal lines demarcate different cell populations from each other.
Fig. 4—Proliferation fold change [fold change in post G0-G1 and
sub G0-G1 population ratio (Table 2)] of MCF-7 and MDA-MB-231
cells treated with TAM (5 µM) in presence or absence of DEHP
(100 µM) or E2 (10 nM).
In MCF-7 cells a total of 233 proteins
were detected from both control and treated
samples out of which 20 proteins were up or
down regulated in treated cell secretome or
uniquely found in either control or treated cell
secretome. These are the proteins of interest
(Table 3). Similarly in the secretome of MDAMB-231 cells a total of 110 proteins were detected
out of which 12 were found to be significant
for present purpose (Table 4). PLGS score, molecular
weight, pI, sequence coverage, fold expression
ratio, and gene ontology (GO) of each of
these significant proteins have been shown in
the tables. Gene ontology of the proteins was
investigated using UniProt Consortium database
(www.uniprot.org).
INDIAN J EXP BIOL, NOVEMBER 2014
1058
Table 3—Differentially expressed proteins in DEHP treated MCF-7 cells vs. control MCF-7 cells
UniPort
Description
Accession
PLGS score
pI/
mW(Da)
Coverage
(%)
treated/
control
Gene Ontology
(Molecular function)
8.01/ 78131
16.0563
0.4956
Alpha fetoprotein
387.8546 5.35/ 68633
Unconventional myosin Ia 299.3816 9.57/118325
Alpha 2 HS glycoprotein 2894.628 5.33/ 39299
9.3596
8.9166
18.2561
0.3973
0.7316
0.1275
DNA binding, Ferric ion binding,
Heparin binding, serine-type endo-peptidase
activity
Metal ion binding
ATP binding, Motor activity
Cysteine-type endo-peptidase inhibitor
activity, kinase inhibitor activity
17.9181
2.5946
MCF-7 treated down regulated proteins
P02788
Lactotransferrin
731.8861
P02771
Q9UBC5
P02765
MCF-7 treated up regulated proteins
P08107-2 Isoform 2 of Heat shock
70 kDa protein
191.4132
5.36/63897
protein binding involved in protein folding,
unfolded protein binding, ATP binding
P68104
Elongation factor 1 alpha
248.7394
9.34/50109
19.2641
P06733
Alpha enolase
248.5028
7.17/47139
23.2719
Q9BQ04
RNA binding protein 4B
202.517
6.28/40124
29.805
L lactate dehydrogenase
B chain
P22392-2 Isoform 3 of Nucleoside
diphosphate kinase B
197.3917
5.63/36629
16.4671
271.4644
9.26/30117
23.221
P29622
P68363
Kallistatin
Tubulin alpha 1B chain
289.9682
377.6599
7.17/48527
4.76/50119
16.6276
37.2506
P08238
Heat shock protein HSP
90 beta
352.9948
4.77/ 83212
11.7403
P50225
Sulfotransferase 1A1
338.0493
6.16/ 34143
6.7797
Q12931
314.6082
8.28/80059
16.1932
469.5851
1252.646
6.75/77013
5.31/70009
17.0487
29.6412
P60709
Heat shock protein 75
kDa mitochondrial
Serotransferrin
Heat shock 70 kDa
protein 1A 1B
Actin cytoplasmic 1
1772.405
5.14/ 41709
25.0667
P04217
Alpha 1B glycoprotein
2175.994
5.13/53520
47.1173
Amount
(fmol)
50.9459 GTP binding, translation elongation factor
activity, GTPase activity
38.888 Sequence-specific DNA binding, magnesium
ion binding, phosphopyruvate hydratase
activity, transcription factor activity,
transcription corepressor activity
19.0444 RNA binding, Nucleotide binding, zinc ion
binding
38.2657 L-lactate dehydrogenase activity, NAD
binding
40.4653 ATP, DNA and metal ion binding, nucleoside
diphosphate kinase activity, protein histidine
kinase activity, sequence-specific DNA
binding transcription factor activity
1576.481 serine-type endopeptidase inhibitor activity
52.6441 structural constituent of cytoskeleton, structural
molecule activity, GTPase activity, GTP binding
86.5625 ATP, CTP, GTP, TPR domain, UTP and
dATP binding, nitric-oxide synthase regulator
activity
126.8159 aryl sulfotransferase activity, flavonol 3sulfotransferase activity, steroid
sulfotransferase activity
85.3997 ATP binding, tumour necrosis factor receptor
binding
36.9028 ferric iron binding
124.6725 protein binding involved in protein folding,
unfolded protein binding,
174.9266 structural constituent of cytoskeleton, ATP
binding
929.0521 ------
Q86XR8-2 Isoform 2 of Centrosomal 185.8288
protein of 57 kDa
Q86TP1 Protein prune homolog
96.843
9.63/ 54130
12.8692
1271.793 microtubule anchoring
5.15/ 50167
11.9205
52.3708
P19823
276.043
6.39/106396
5.814
285.58
1076.766
5.16/ 41765
28.5333
184.187
MCF-7 treated unique
P07195
P02787
P08107
MCF-7 control unique
P63261
Inter alpha trypsin
inhibitor heavy chain H2
Actin cytoplasmic 2
inorganic diphosphatase activity, manganese
ion binding
endopeptidase inhibitor activity, serine-type
endopeptidase inhibitor activity
structural constituent of cytoskeleton, ATP
binding
DAS et al.: DEHP EXPOSURE IN MCF-7 & MDA-MB-231 CELL LINES
1059
Table 4—Differentially expressed proteins in DEHP treated MDA-MB-231 cells vs. control MDA-MB-231 cells.
Accession Deccription
PLGS score
pI/
mW(Da)
Coverage
(%)
treated/
control
Gene Ontology
6.50/ 53972
23.4542
0.3750
calcium ion binding, metallo-endo-peptidase
activity, zinc ion binding
5.13/ 53520
5.12/ 38394
26.0437
52.3677
1.5347
1.0036
----Cysteine-type endo-peptidase inhibitor
activity, kinase inhibitor activity
5.77/121285
18.7907
Amount
(fmol)
11.2321
ATP binding
7.94/ 30037
7.9468
20.3342
L-lactate dehydrogenase activity
5.09/ 71595
16.0063
5.3423
axon guidance
612.9011
330.7459
9.24/ 58689
3.98/ 15585
12.2
36.1702
147.5101
6.2226
zinc ion binding, DNA binding
------
319.4367
4.84/ 58445
8.5389
10.2131
enzyme activator activity, lipid binding
293.0734 4.52/ 129299
16.9231
366.6636
TGF beta, proteoglycan, phosphatidylserine,
low-density lipoprotein particle, laminin,
integrin, heparin, fibronectin, fibroblast
growth factor, fibrinogen, collagen V,
calcium ion and identical protein binding
DNA-N1-methyladenine dioxygenase,
oxidative DNA demethylase, oxidative
RNA demethylase and DNA-N1methyladenine dioxygenase activity
structural constituent of cytoskeleton, ATP
binding
MDA-MB-231 treated down regulated proteins
P03956 Interstitial collagenase 483.6768
MDA-MB-231 treated up regulated proteins
P04217 Alpha 1B glycoprotein 711.6302
P02765 Alpha 2 HS
13866.7
glycoprotein
MDA-MB-231 treated unique
Q6S8J3
P00338-2
A0MZ66
POTE ankyrin domain 1074.194
family member E
Isoform 2 of L lactate
936.4359
dehydrogenase A chain
Shootin 1
232.9884
MDA-MB-231 control unique
P0CJ79
Zinc finger protein 888
Q5MJ09
Sperm protein
associated with the
nucleus on the X
chromosome
P07602-3 Isoform Sap mu 9 of
Proactivator
polypeptide
P07996
Thrombospondin
Q9C0B1
Alpha ketoglutarate
207.9361
dependent dioxygenase
4.82/ 57969
20.3187
15.4774
P60709
Actin cytoplasmic 1 OS 539.8703
Homo sapiens
5.14/ 41709
15.4667
26.0437
Some of the differentially detected significant
proteins—DEHP treated MCF-7 cells showed down
regulation of lactotransferrin secretion. In MDA-MB231 cells it was neither detected in control nor in
treated cell secretome. Lactotransferrin is an iron
transport protein that is an essential component for
iron metabolism and essential growth factor necessary
for cell proliferation, differentiation and cell
function28. Lactotransferrin expression is constitutive
in various tissues including hepatic cells under the
control of endogenous estrogen receptors28-30. Further,
it has also been shown that tamoxifen, a potent anti
estrogen, reduces its enhanced secretion31. Down
regulation of lactotransferrin in the present
experiment is in good agreement with the results of
previous section. If DEHP would have activated ERα
in the MCF-7 cells, then, secretion of lactotransferrin
would have been up-regulated. This evidence is in the
support of the hypothesis that DEHP does not mimic
estrogen at molecular level to activate ERα. DEHP
treated MCF-7 cell secretome showed down
regulation of another protein, alpha 2-HS glycoprotein
(AHSG); whereas, it is not affected in MDA-MB-231
cell secretome. AHSG is a secretory glycoprotein
found at high levels in serum, cerebrospinal fluid,
urine, saliva and bone. Yi et al32 have demonstrated
the role of AHSG in breast cancer development. The
study suggested exploring AHSG as a potential
1060
INDIAN J EXP BIOL, NOVEMBER 2014
biomarker for clinical serologic screening of breast
cancer32. Lactate dehydrogenase (LDH) was uniquely
detected in the DEHP treated cell secretome of both
the cell lines. LDH leakage has often been used as an
indicator to study plasma membrane integrity. Present
results indicate that DEHP potentially disrupts the
plasma membrane integrity. In an earlier study of
2-dimentional gel-electrophoresis (2DE) based
proteomic analysis, dose dependent up-regulation of
LDH was also reported in the secretome of DEHP
treated HepG-2 cells33. Heat shock proteins were also
found either up regulated or uniquely expressed in
MCF-7 cells after DEHP treatment. Production of
high level of heat shock proteins is an indicator of
stress responses of the DEHP treated cells.
Probable toxicity mechanism—Similar to the
results of present study, an in vivo study by Hasmall
et al.34 has shown DEHP induced down-regulation of
lactotransferrin in mouse liver cells. They
hypothesized that down regulation of lactotransferrin
increased the level of tumor necrosis factor-alpha
(TNF-α), which in turn enhanced the proliferation of
hepatocytes. Earlier reports have shown that TNF-α is
highly expressed in breast carcinomas and their
chronic expression supports tumor growth35,36.
Moreover, studies have supported the view that
lactotransferrin represses TNF-α34,37,38. Thus, in the
present context, down-regulation of lactotransferrin
may have a role in up-regulation of TNF-α, leading to
growth promotion of breast cancer cells. However, it
remains to be deciphered how DEHP down-regulates
lactotransferrin expression and whether the said
mechanism has any significance in ERα negative
MDA-MB-231 cells.
in modulating the growth regulations. Along with,
exploration of other signaling pathways is also needed
to decipher the exact toxicity mechanism of DEHP.
Acknowledgement
Thanks are due to Mr. Plabon Borah, of Advanced
Instrumentation Research Facility (AIRF) Jawaharlal Nehru
University, New Delhi for Label-Free Quantitative
LC/MSE analysis. One of the authors (MTD) also thanks
University Grants Commission (UGC), New Delhi,
Government of India, for research fellowship.
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Conclusion
As shown by E-screen assay and cell cycle
analysis, DEHP was found to be growth promoting
and was able to suppress the effects of TAM in both
the cell lines irrespective of their ERα status. This
indicates that some ERα independent mechanism may
be involved. The secretome analysis showed that
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relate proteins, structural proteins and several receptor
binding proteins of the breast cancer cells. The
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from proteomics analysis. This finding further
established that DEHP does not act as a ligand
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