SILAC-Based Mass Spectrometry Analysis Reveals That Epibrassinolide Induces Apoptosis via Activating Endoplasmic Reticulum Stress in Prostate Cancer Cells

Epibrassinolide (EBR) is a polyhydroxylated sterol derivative and biologically active compound of the brassinosteroids. In addition to well-described roles in plant growth, EBR induces apoptosis in the LNCaP prostate cancer cells expressing functional androgen receptor (AR). Therefore, it is suggested that EBR might have an inhibitory potential on androgen receptor signaling pathway. However, the mechanism by which EBR exerts its effects on LNCaP is poorly understood. To address this gap in knowledge, we used an unbiased global proteomics approach, i.e., stable-isotope labeling by amino acids in cell culture (SILAC). In total, 964 unique proteins were identified, 160 of which were differentially expressed after 12 h of EBR treatment. The quantification of the differentially expressed proteins revealed that the expression of the unfolded protein response (UPR) chaperone protein, calreticulin (CALR), was dramatically downregulated. The decrease in CALR expression was also validated by immunoblotting. Because our data revealed the involvement of the UPR in response to EBR exposure, we evaluated the expression of the other UPR proteins. We demonstrated that EBR treatment downregulated calnexin and upregulated BiP and IRE1α expression levels and induced CHOP translocation from the cytoplasm to nucleus. The translocation of CHOP was associated with caspase-9 and caspase-3 activation after a 12 h EBR treatment. Co-treatment of EBR with rapamycin, an upstream mTOR pathway inhibitor, prevented EBR-induced cell viability loss and PARP cleavage in LNCaP prostate cancer cells, suggesting that EBR could induce ER stress in these cells. In addition, we observed similar results in DU145 cells with nonfunctional androgen receptor. When proteasomal degradation of proteins was blocked by MG132 co-treatment, EBR treatment further induced PARP cleavage relative to drug treatment alone. EBR also induced Ca2+ sequestration, which confirmed the alteration of the ER pathway due to drug treatment. Therefore, we suggest that EBR promotes ER stress and induces apoptosis.


Introduction
Brassinosteroids (BRs) are steroid-derived molecules with numerous physiological effects, including the regulation of hormonal balance, the activation of protein and nucleic acid synthesis, enzymatic activity, the cell cycle and cell growth [1,2]. Beside the well-described effects in plants, their roles in mammalian cells are poorly understood and currently being investigated as anti-cancer agents [3][4][5]. The recent understanding is that EBR, a member of the BRs, induces apoptosis more effectively in nuclear hormone receptor (NHR)-expressing cancer cell lines, such as LNCaP prostate [with androgen receptor (AR)] [4] or MCF-7 breast cancer cell lines [with estrogen receptor (ER)] [3]. The structural similarity of EBR with mammalian steroids [6] has been suggested as possible reason for the hormonal specificity. However, the molecular basis of the EBR specificity has not been elucidated. Our previous experience indicated that although EBR (25 μM) was a strong apoptotic inducer in LNCaP (AR+) prostate cancer cells, it was also surprisingly effective in inducing apoptosis in DU 145 (AR-) cells. Importantly, EBR treatment was not cytotoxic for PNT1a normal prostate epithelial cells [4]. To better clarify the therapeutic potential of EBR, we investigated the whole proteome of LNCaP cells with or without EBR treatment.
The use of quantitative proteomic approaches is likely to provide information on the key molecular signatures and the detailed understanding of the involved targets [7]. SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture) analysis is a mass spectroscopy (MS)-combined proteomic approach without radioactive labeling. SILAC relies on the incorporation of a given 'light' ( 12 C labeled L-lysine or L-arginine) or 'heavy' ( 13 C labeled L-lysine or L-arginine) form of the amino acid into the proteins. After a number of cell divisions, each particular amino acid is replaced by its isotope analog and incorporated into newly synthesized proteins [8]. In this study, we used the SILAC approach to explore the novel apoptotic potential of EBR in androgen responsive LNCaP prostate cancer cells.
We observed that EBR significantly affected the expression profile of 160 proteins involving in different cellular functions (cell cytoskeleton, nucleic acid and energy metabolism, cell death and protein ubiquitination) compared with untreated control samples. Endoplasmic reticulum (ER) resident calreticulin (CALR), a chaperone protein, was significantly downregulated among those 160 proteins. We determined that the levels of ER stress proteins were altered after EBR treatment in LNCaP AR (+), and the same profile was also observed in the non-functional AR-expressing DU145 prostate cancer cell line. Alterations in the ER stress biomarkers triggered apoptosis in each cell line; in LNCaP cells, apoptosis was induced by CHOP transactivation and translocation to the nucleus. The addition of rapamycin, as a translational repressor of mTOR (mammalian target of rapamycin), or MG132, a proteasome inhibitor that reduces the degradation of ubiquitin-conjugated proteins, altered EBR-induced apoptosis, suggesting that ER stress was activated following EBR treatment in LNCaP cells. To prove the relationship between ER mediated cell death mechanism after EBR treatment, CALR plasmid transfection was performed. EBR-induced cell viability loss was prevented in CALR+ LNCaP cells. In addition, when CALR+ LNCaP cells were treated with EBR, we did not observe ER stress mediated apoptotic induction, suggesting that CALR is an important target of EBR.

Cell culture in SILAC media
SILAC DMEM (Pierce Biotechnology) was supplemented with 10% dialyzed fetal bovine serum (Thermo Scientific, Waltham, MA, USA), 1% streptomycin/penicillin. The heavy medium was supplemented with 13 C 6 L-arginine and 13 C 6 , 15 N 2 -L-lysine. The light medium was supplemented with normal L-arginine and L-lysine. For SILAC experiments, LNCaP cells were grown in parallel in either light or heavy media for 5 days, with media replacement every 24 h.

1-D SDS-PAGE separation and in-gel trypsin digestion
Total cell protein was isolated from LNCaP cells using RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). Protein quantification was performed according to the Bradford method (Bio-Rad Protein Assay) [9]. Samples containing a combined 40 μg of total protein (20 μg ''heavy" and 20 μg ''light") were diluted with Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) containing 5% β-mercaptoethanol. The mixture were then heated for 5 min at 90°C and loaded onto 10% polyacrylamide gels.1-D SDS-PAGE separation was performed with mini Protean II system (Bio-Rad) at 200 V for 45 min. Bands were visualized with Simply Blue Safe Stain (Life Technologies, CA, USA), and lanes were sliced into 12 sections, which were diced into~1x1 mm squares. After distaining with 50% v/v acetonitrile (ACN) in 25 mM ammonium bicarbonate buffer (bicarbonate buffer), proteins in gel pieces were reduced with 10 mM dithiothreitol (DTT) in bicarbonate buffer and alkylated by incubation with 50 mM iodoacetamide in bicarbonate buffer. After gel dehydration with 100% ACN, gel pieces were covered with approximately 50 μl of 12.5 μg/ml trypsin in bicarbonate buffer for in-gel digestion. Incubation for digestion was performed at 37°C for 12 h. Trypsin was inactivated with formic acid at 2% final volume, and peptides were extracted and cleaned using a C18 Tip column (ZipTips, Millipore, Medford, MA, USA), as previously described [10]. Michrom Bio-resources) and separated using a C18 capillary column (15 cm 75 mm, Agilent) with an Agilent 1100 LC pump delivering mobile phase at 300 nl/min. Gradient elution using mobile phases A (1% ACN/0.1% formic acid, balance H 2 O) and B (80% ACN/0.1% formic acid, balance H 2 O) was as follows (percentages for B, balance A): linear from 0 to 15% at 10 min, linear to 60% at 60 min, linear to 100% at 65 min. The nano ESI MS/MS was performed using an HCT Ultra ion trap mass spectrometer (Bruker). ESI was delivered using a distal-coating spray Silica tip (id 20 μm, tip inner id 10 μm, New Objective, Ringoes, NJ). Mass spectra were acquired in positive ion mode, capillary voltage at -1100 V and active ion charge control trap scanning from 300 to 1500 m/z; using an automatic switching between MS and MS/MS modes, MS/MS fragmentation was performed on the two most abundant ions on each spectrum using collision-induced dissociation with active exclusion (excluded after two spectra and released after 2 min). The complete system was fully controlled by HyStar 3.2 software.

GeLC-MS/MS and data analysis
Mass spectra data processing was performed using Mascot Distiller (Version 2.4.3.3) with search and quantitation toolbox options. The generated de-isotoped peak list was submitted to an in-house Mascot server 2.4.0 for searching against the SwissProt database version 2013_01 (538849 sequences; 191337357 residues). Mascot search parameters were set as follows: species, Homo sapiens (20,233 sequences); enzyme, trypsin with maximal 2 missed cleavage; fixed modification: cysteine carbamidomethylation; variable modifications: methionine oxidation, Gln->pyro-Glu (N-term Q), Glu->pyro-Glu (N-term E), Label: 13C(6)15N(2) (K), and Label:13C(6) (R); 0.90-Da mass tolerance for precursor peptide ions; and 0.6 Da for MS/MS fragment ions. SILAC quantitation was performed in Mascot Distiller using SILAC K+8 R+6 quantitation method; SILAC ratios for heavy and light peptide pairs were calculated using the Simpsons integration method, minimum 1 peptide with unique sequence and 0.05 of significant threshold. The following criteria were used to evaluate protein identification: one or more unique peptides with ion score 45 and two or more unique peptides with ion score 30 (p<0.05 threshold); proteins identified were extracted using MS Data Miner (MDM) [11]. Quantified proteins with 2 and 0.5-fold change were selected and clustered by biological functions, pathway and network analysis using Ingenuity Pathway Analysis (IPA) software (www.ingenuity.com) for bioinformatics analysis.

Construction of p-EGFP CALR plasmid
Total RNA was isolated from HEK-293 human embryonic kidney cells using TRIPure (Roche) according to the manufacturer's indications. First strand cDNA was transcribed using iScript cDNA Synthesis Kit (Bio-Rad). CALR cDNA was amplified using designed CALR gene-specific cloning primers: 5'-CGGAGTCAACGGATTTGGTCGTAT-3'; reverse, 5'-GCCTTCTC CATGGTGGTGAAGAC-3'. The protocol included an initial denaturation step at 94°C for 3 min, followed by 30 cycles with 30 sec. of denaturation at 94°C, 30 sec of annealing at 55°C and 45 sec. of elongation at 72°C, followed by a final elongation step 72°C for 10 min. Ten microliters of PCR product and 1 μg pEGF-LC3.1 plasmids were digested with 10 U/μl EcoRI and BglII (Fermentas, St. Leon-Rot, Germany) for 30 minutes at 37°C. The digestion products were subjected to 1.5% agarose gel electrophoresis and then extracted from the agarose gel. Ligation of the PCR product:plasmid was produced using 1:1, 1:3 and 1:5 ratios with T4 DNA ligase (Fermentas, St. Leon-Rot, Germany). Recombinant DNA was transformed into HB101 cells that were prepared using the CaCl 2 method. Positive clones were selected by using ampicillin (Amp) LB agar plates. All of the colonies were picked and grown in 1 ml of Amp+LB and incubated overnight at 37°C. Plasmids from every colony were isolated by using QIAPrep Spin Miniprep columns (Qiagen, Valencia, CA, USA). Plasmids from selected ten colonies were used as templates to amplify the CALR gene using cloning primers in PCR. One selected amplified positive plasmid was sequenced to confirm the Open Reading Frame (ORF) of the EGF-CALR fusion gene (İontek, Turkey). The sequenced recombinant plasmid was used for the overexpression experiments.

Transient transfection of the pEGFP-CALR plasmid
LNCaP cells were seeded overnight on 6-well plates at a density of 3x10 5 cells/well. Approximately 1 μg/μl pCMV-CALR in the presence of transfection reagent (Fugene HD, Roche, Mannheim, Germany) was prepared in serum-free media. The mixture was incubated for 15 minutes at room temperature and gently added dropwise onto cells. After 48-h plasmid transfection, cells were treated with 25 μM EBR, and total RNA was isolated for CALR expression analysis or protein extraction for western blotting.

Evaluation of apoptotic cell death by Annexin V-FITC staining
LNCaP cells were seeded in 6 well-plates (3x10 5 cells/well) and treated with 25 μM EBR for 24 h. Both floating and adherent cells were collected, resuspended in Annexin V binding buffer and incubated with Annexin V-FITC and PI following manufacturer instructions (BD Biosciences, Bedford, MA). One thousand events per sample were acquired on the Accuri C6 (BD Biosciences). Fluorescence emissions were collected through 530-nm and 570-nm band-pass filters for FITC and PI, respectively. Data are presented as dot plots (Annexin fluorescence on the x-axis; PI fluorescence on the y-axis). The numbers present in the four quadrants represent the percentage of viable (lower left), necrotic (upper left), early apoptotic (lower right), and late apoptotic (upper right) cells evaluated using BD Accuri C6 software (BD Biosciences).

Measurement of intracellular Ca 2+ levels
LNCaP prostate cancer cells were treated with EBR for 12 h, and intracellular Ca 2+ levels were determined by calcium green-1 (1 μmol/L, Molecular Probes, Eugene, OR, USA) staining for 30 minutes. Flow cytometric analysis of stained cells was performed with a flow cytometer Accuri C6 (BD Biosciences, San Jose, CA, USA). Calcium green-1-stained cells were observed by fluorescence microscopy (excitation 506 nm, emission: 531 nm).

Statistical analysis
Statistical significance was assessed using the one-tailed unpaired t-test. P<0.05 was taken as a level of significance. Western blot results were repeated at least three times. Band intensities were quantified using ImageJ software and normalized to β-actin.

Proteome of EBR-treated LNCaP cells
To obtain a global perspective of changes in the entire proteome, LNCaP cells were treated with EBR (25 μM) and subjected to SILAC treatment. In total, 964 unique proteins were identified by this technique. Changes greater than 2-fold ( 2 and 0.5) between light and heavy proteins were considered significant according to the cutoffs of previous studies [10]. Quantitative analysis between paired samples revealed that among the 964 proteins, 160 were significantly changed after 12 h EBR treatment (S1 Table).

Functional characterization of identified proteins and bioinformatics analysis
We next analyzed 160 differentially expressed proteins with respect to biological function, pathway and network using IPA software. As shown in Fig 1, according to the biological function analysis, EBR-altered proteins have functions in cellular growth and proliferation (16.6%), cell death and survival (14%), cellular assembly and organization (13.3%), cellular function and maintenance (13.3%), nucleic acid metabolism (5%), DNA replication (4%), protein synthesis and protein folding (3.7 and 1%, respectively) (Fig 1). The analysis of canonical pathways (p 0.05) identified aldosterone signaling in epithelial cells as well as cellular metabolic pathways (including UDP-N-acetyl-d-galactosamine biosynthesis, gluconeogenesis, fatty acid biosynthesis, protein ubiquitination pathway, cytoskeleton signaling and others) (Fig 2). CALR exhibited a significant alteration after 12 h EBR treatment with score 150, 11 matches, a heavy/ light ratio of 0.4372 and 4 peptides (S1 Table). In addition according to the molecules associated with the SILAC analysis results CALR was downregulated by 2.287 compared to control samples (Table 1).

EBR-induced modulation of CALR expression
Differentially expressed proteins following EBR treatment were mapped to 7 specific functional networks with each network containing 11 or more "focus" members. The networks of interest corresponded to cell death and survival, cellular assembly and organization, cellular compromise, cellular function and maintenance, drug metabolism, lipid metabolism, cell morphology and cellular function. CALR was found a major protein in the cellular response, cellular assembly and organization networks (Fig 3A). Because CALR is a chaperone protein and because alterations in the CALR expression lead to the unfolded protein response and ER stress, we detected the interactions of CALR with other molecules to detect evidence of the UPR following EBR treatment in LNCaP prostate cancer cells. While we run IPA for pathway analysis after EBR treatment, the system predicted that heat shock chaperone protein family might also been involved in EBR-induced stress conditions as shown in Fig 3B. In addition, the IPA comparison of the EBR effect with other known anticancer agents causing similar effects indicated that EBR may act as an ER stress inducer like tunicamycin and is able to alter heat shock proteins and CALR (Fig 3C).

Validation of protein identification and quantification
Because the protein interaction network and pathway analysis revealed the alteration of the UPR response in prostate cancer cells exposed to EBR, we next determined the expression levels of other UPR proteins by western blotting. We confirmed that the changes in the ratios of CALR in LNCaP cells were consistent with the ones derived from SILAC studies (Fig 4A, left  panel). As shown in Fig 4, although EBR treatment downregulated the expression level of CALNX, the molecular chaperone BiP was upregulated. An excessive accumulation of misfolded proteins in ER triggers the dissociation of BiP from ER membrane-located receptors such as IRE1α, PERK and ATF6 as a significant ER stress response. In agreement with the altered BiP expression profile, IRE1α, PERK and ATF6 were upregulated following EBR treatment in LNCaP cells (Fig 4A, left panel). We also determined the expression levels of other ER stress-related proteins such as CHOP, ATF4 and PDI in the total protein lysates. We observed that the exposure of LNCaP cells to EBR increased the expression of CHOP in both cytoplasmic and nuclear fractions ( Fig 4C). Interestingly, PDI, a stress protein abundant in ER, did not elevated in response to EBR treatment. Excessive and prolonged ER stress triggers apoptosis. Consistent with this finding, EBR time-dependently activated caspase-12, caspase-9 and caspase-3 and induced the cleavage of PARP (Fig 4A, left panel). Similar results were observed in DU145 prostate cancer cells exposed to EBR (Fig 4A, right panel). Annexin-V/PI staining results confirmed that EBR induced apoptosis in LNCaP cells (Fig 4B). These results indicate that EBR induced apoptosis via the UPR axis in both prostate cancer cells regardless of the functional AR expression.
To verify that the result of EBR-induced apoptosis was related to the UPR, LNCaP cells were transfected with the reporter construct of CHOP promoter (-649/+136) pmCherry-1 plasmid. As shown in Fig 4D, EBR clearly induced CHOP activity and the resulting puncta pattern in LNCaP cells. To further validate the effect of EBR on ER stress-related apoptosis, we inhibited mTOR by rapamycin treatment to block de novo mRNA and protein synthesis. Inhibition of mRNA synthesis leads to the accumulation of unfolded/misfolded proteins in the ER and therefore can potentially alleviate ER stress-induced cell death [12]. We observed that co-treatment with rapamycin significantly prevented EBR-induced cell viability loss ( Fig 5A) and apoptosis (Fig 5B, left panel) in LNCaP cells. In contrary, 26S proteasome inhibitor MG132 co-treatment further increased cell viability loss ( Fig 5A) and apoptosis (Fig 5B, right panel). According to the obtained data, we suggest that the presence of MG132 prevented the EBR-induced degradation of misfolded proteins and exacerbated the apoptotic response in LNCaP prostate cancer cells. Collectively, these results support the hypothesis that the EBR-induced cell death mechanism is mediated by the UPR in prostate cancer cells.

CALR is an important target of EBR
To begin to understand the molecular mechanism behind EBR-induced UPR, we transiently transfected LNCaP cells with a GFP-tagged pCMV-CALR plasmid (Fig 6A). Concomitantly we utilized tunicamycin as a positive control to activate ER stress in AR-sensitive LNCaP cells. Although CALR overexpression prevented the EBR-dependent loss of cell viability, it did not exert same effect following tunicamycin treatment (Fig 6B). Although tunicamycin activated the ER stress pathway by upregulating the expression of CALR, CALNX, BiP and CHOP, EBR treatment only induced the upregulation of CHOP but downregulated CALR and CALNX. CALR overexpression diminished the potential effect of EBR on ER signaling players, which indicated that CALR is a critical target of EBR in LNCaP cells. In addition, we suggest that EBR differs from tunicamycin through activating different targets of the ER stress machinery (Fig 6C).
Next, we determined the apoptotic efficiency of EBR in both wt and CALR+ LNCaP cells. As shown in Fig 6C, EBR activated caspase-12, which culminated in PARP cleavage in LNCaP wt cells but not in CALR+ LNCaP cells (Fig 6C). As CALR is an important regulator of intracellular Ca 2+ buffering capacity, which also triggers apoptosis, we examined Ca 2+ levels  following EBR treatment (Fig 7). We observed that EBR treatment increase the release of Ca 2+ by 6-fold in LNCaP cells. Finally, we proposed that EBR induced apoptosis in prostate cancer cells by causing ER stress related to CALR downregulation and Ca 2+ release into the cytosol (Fig 8).

Discussion
EBR, a plant growth regulator, has been recently suggested as a candidate chemotherapeutic drug because of its ability to induce cell cycle arrest and apoptosis in different cancer cell lines without affecting normal epithelial cells [4]. Given that EBR-induced apoptosis is more EBR induced ER stress and apoptosis. A) Total protein was isolated after EBR treatment, and the expression profiles of ER stress biomarkers, caspases and PARP cleavage were determined by immunoblotting using appropriate antibodies. β-actin was used as a loading control. B) Approximately 2 x 10 5 cells were seeded into a six-well plate and treated with EBR for 12 and 24 h. Annexin V-PI staining was performed to determine apoptotic cell populations. Fluorescence signals from Annexin V-FITC and from PI are reported on the x-axis and y-axis, respectively. Numbers presented in the four quadrants represent the percentage of viable (lower left), necrotic (upper left), early apoptotic (lower right) and late apoptotic (upper right) cells. C) Following 12-h EBR treatment, cytosolic and nuclear proteins were isolated and separated in a 12% SDS gel, transferred onto a PVDF membrane and blotted with an anti-CHOP antibody. D) LNCaP cells were transfected with the reporter construct CHOP promoter (-649/+136) pmCherry-1. The CHOP activation was visualized with fluorescence microscopy. Excitation: 575 nm Emission: 601.
doi:10.1371/journal.pone.0135788.g004 effective in NHR-expressing cells than in non-NHR-expressing cells, it has been suggested that its steroid-like structure acts on NHRs and triggers apoptosis [3]. However, the clear apoptotic effect on prostate or breast cancer cell lines with non-functional NHR, such as DU 145 or MDA-MB-231 cells [4,5], indicated an unknown common target for apoptosis. Although there are reports suggesting that different cellular mechanisms are involved in EBR-induced apoptosis, little is known about its mechanistic action. Therefore, in our study, we were focused on the idea of proposing a mutual target using a global approach.
Proteomic studies are powerful approaches to examine the molecular mechanisms of chemotherapeutic drugs and their interacting signaling mechanisms. Therefore, to clarify the apoptotic effect of EBR through evaluating changes in the proteome, we performed SILAC-based mass spectrometry analysis in LNCaP cells. Our study indicated that 160 different proteins exhibited significant alterations after 12 h EBR treatment (S1 Table). According to our biological function data, those proteins played roles in cellular growth and proliferation (16.6%), cell death and survival (14%), cellular assembly and organization (13.3%), cellular function and maintenance (13.3%), nucleic acid metabolism (5%), DNA replication (4%), protein synthesis (3.7%) and protein folding (1%) (Fig 1). In addition, the pathway analysis revealed that aldosterone signaling, gluconeogenesis, fatty acid biosynthesis, the protein ubiquitination pathway and cytoskeleton signaling were affected (Fig 2). The proteins in the cell death and survival, cellular function and maintenance pathways were also mapped (Fig 3A and 3B). Among the significantly altered proteins following EBR treatment, CALR was noteworthy based on the following parameters: score: 150, matches: 11, heavy/light ratio: 0.4372 and number of peptides: 4 according to the MDM analysis ( Fig 4A). The significant downregulation of CALR prompted us to investigate the potential role of EBR-induced ER stress. CALR, as an ER chaperone, plays role in the folding process of newly synthesized proteins as well as in the decoding of both normal and pathological Ca 2+ signals due to its buffering capacity [13]. Downregulation of CALR has been shown to lead to rapid and severe alterations in ER Ca 2+ homeostasis [14]. CALR, as an androgen-responsive gene, has been shown to be downregulated in castrated rat prostate or in vitro models [15]. CALR binding to misfolded proteins prevents their export from the ER lumen to the cytosol. In addition, CALR antisense oligonucleotides significantly increase the sensitivity of LNCaP cells to Ca 2+ ionophore A23187-induced cell death [16]. Together, these findings validated the approach from SILAC analysis that EBR might be effective in triggering ER stress to induce apoptosis. In addition, when we investigated the CALRrelated pathways using IPA, we detected that the downregulation of CALR could be related to ER stress. Therefore, we confirmed our results by western blotting. As shown in Fig 4A, EBR decreased the CALR and CALNX expression profiles time-dependently by causing caspasedependent apoptotic cell death in both AR-expressing and AR-non-expressing prostate cancer cells (Fig 4A and 4B). In addition, the levels of BiP and CHOP in total lysates and CHOP translocation to the nucleus were upregulated (Fig 4C), suggesting that EBR is a candidate to induce ER stress. The effect of EBR was also confirmed by the transfection of the CHOP promoter (-649/+136) tagged with pmCherry-1, suggesting that EBR is a candidate to induce ER stress (Fig 4D). The upregulation in BiP is crucial to initiate the ER stress response via ER membrane-located proteins such as IRE1α, PERK and ATF6 which in turn activates XBP1, ATF4 and ATF6 itself as transcription factors to induce ER stress. CHOP expression [17]. Particulary, CHOP expression is induced in response to XBP1 transactivation. Consistent with this fact, EBR treatment was also found to upregulate IRE1α and CHOP expression in LNCaP prostate cancer cells. Once CHOP is expressed, it can trigger the expression of pro-apoptotic proteins, acts on the mitochondrial membrane to release cytochrome c, and triggers the caspase cascade via caspase-9 and caspase-3 [18,19]. Many agents inducing apoptosis via ER-stress have been shown to upregulate IRE1 family members and the downstream targets XBP1 and CHOP [20]. Caspase-12, caspase-9, caspase-3 and PARP cleavage profiles supported our hypothesis ( Fig  4A).
To clarify the mechanism of EBR-activated ER stress, we suppressed de novo protein synthesis through mTOR complex inhibition by rapamycin [21]. Rapamycin is a well-known translational inhibitor that has been shown to prevent tunicamycin-and bortezomib-induced ERstress in MEF and Elt3 cells, respectively [22,23]. Similar to this finding, we found that rapamycin co-treatment prevented EBR-induced apoptotic cell death (Fig 5A and 5B). In contrary, inhibition of proteasomal degradation by MG132 further induced EBR-induced apoptosis. Supporting this finding, recent data suggested that inhibition of proteasomal degradation resulted in a quick apoptotic induction response in proliferating cells [24].
Recently, a number of reports have suggested that CALR is a fine-tuning target to modulate cell survival and death-related signaling pathways. According to the expression level of CALR, protein tyrosine kinases or phosphatases might be altered to decide cell fate under apoptotic stimuli. CALR overexpression was suggested as a promoting factor in differentiation-induced apoptosis in H9c2 embryonic rat heart cells under trans-retinoic acid stimuli via modulating the Akt signaling cascade [25]. However, the overexpression of CALR might prevent druginduced apoptosis via the enhanced buffering potential of Ca 2+ [26]. Increased cellular Ca 2+ influx is a mediator of apoptosis and should be sensed and corrected by CALR. In this study, CALR overexpression abolished the cytotoxic effect of EBR by preventing EBR-induced BiP and CHOP upregulation, leading to the ER-dependent apoptosis cascade. This result was also confirmed by caspase-12 and PARP cleavage profiles. Therefore, we suggest that EBR might act on a Ca 2+ buffering mechanism via altering CALR expression regardless of NHR status in prostate cancer cells. Increased Ca 2+ levels, which was shown by calcium green staining, was observed following EBR treatment.
In conclusion, all data in this study suggest that EBR is an effective apoptotic agent through modulating the CALR expression profile and thus causing deficient Ca 2+ buffering potential regardless of NHR expression in prostate cancer cells (Fig 8). In addition, this study is the first to present the proteomic alterations due to EBR treatment investigated by SILAC assay, which also presents other novel targets for EBR.
Supporting Information S1 Table. One hundred sixty significantly altered proteins after 12 h EBR treatment identified with SILAC LC-MS/MS. (DOCX)