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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 1053 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). 1054 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. References 1 2 3 4 5 6 7 8 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 DEHP affected multitude of targets including stress relate proteins, structural proteins and several receptor binding proteins of the breast cancer cells. The evidence of down regulation of lactotransferrin, an ERα responsive gene, is the most significant finding from proteomics analysis. This finding further established that DEHP does not act as a ligand activator for ERα. Future research needs to focus on the possible role of ERα, lactotransferrin, and TNF-α 9 10 11 12 13 USFDA, Safety assessment of di (2-ethylhexyl) phthalate (DEHP) release from medical devices. US Food and drug administration. Washington, DC, 2002. http://www.fda.gov/ cdrh/ost/dehp-pvc.pdf. ATSDR, Toxicological Profile for Di (2-ethylhexyl) Phthalate (Agency for Toxic Substances and Disease. Atlanta, GA) 2002. 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