ARTICLE pubs.acs.org/jpr
Proteomic Analysis of Cellular Response to Novel Proapoptotic Agents Related to Atypical Retinoids in Human IGROV-1 Ovarian Carcinoma Cells Alberto Milli,†,‡ Paola Perego,*,§ Giovanni L. Beretta,§ Alice Corvo,‡ Pier Giorgio Righetti,|| Nives Carenini,§ Elisabetta Corna,§ Valentina Zuco,§ Franco Zunino,§ and Daniela Cecconi*,‡ ‡
)
Dipartimento di Biotecnologie, Laboratorio di Proteomica e Spettrometria di Massa, University of Verona, Strada le Grazie 15, 37134, Verona, Italy Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”, Politecnico di Milano, Via Mancinelli 7, 20131, Milano, Italy § Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, 20133, Milano, Italy
bS Supporting Information ABSTRACT: Novel agents characterized by the scaffold of the atypical retinoid ST1926, but containing different chemical functions (carboxylic or hydroxamic acid), exhibit potent proapoptotic activity. In the present paper, we show that the treatment of the IGROV-1 ovarian cancer cell line with compounds sharing structural features with ST1926 (ST1898, ST3595, ST3056) determines a strong inhibition of proliferation mainly due to apoptotic cell death. In an effort to understand the mechanism of action of these compounds, we performed a proteomics analysis of IGROV-1 total lysates and nuclear extracts. Using this approach, we found that deregulation of calcium homeostasis, oxidative stress, cytoskeleton reorganization, and deregulation of proteasome function may represent important pathways involved in response of IGROV-1 cells to the studied compounds. The most prominent effect was down-regulation of factors involved in protein degradation, an event more marked in cells treated with ST3595. In addition, we identified proteins specifically modulated by each treatment, including prohibitin and cochaperone P23 (ST1898), pre-mRNA splicing factor SF2p32 and clathrin light chain (ST3595), as well as Far upstream element (FUSE) binding protein 1 and DNA-binding protein B (ST3056). By identifying proteins modulated by novel proapoptotic agents, this study provides insights into critical aspects of their mechanism of action. KEYWORDS: atypical retinoids, ovarian cancer, proteomics
1. INTRODUCTION Ovarian cancer is a disease characterized by the rapid growth and division of cells within one or both ovaries, mainly affecting cells on the surface of the ovary (ovarian epithelial carcinomas) and egg cells (malignant germ cell tumors).1 It is responsible for 5% of all cancer deaths among women, and it represents the leading cause of death from gynecological malignancies in Western countries. Ovarian cancer has the highest mortality rate of all female cancers, as a consequence of detection at an advanced stage.2 Despite treatments combining surgery and chemotherapy, relapse is observed in the majority of patients;3 therefore, intense investigation is focused on finding new therapeutic options. In recent years, atypical retinoids (ARs, or retinoid-related molecules4) have been developed as a promising class of potentially useful agents for cancer treatment.5-14 In contrast to classical retinoids, ARs exhibit a unique mechanism of action, resulting in growthinhibitory and pro-apoptotic activity that appears not to be mediated by nuclear retinoid receptors.6 Recently, we have r 2010 American Chemical Society
reported that RC307, a novel histone deacetylase inhibitor, was able to sensitize ovarian carcinoma cells to the AR ST1926,15 an effect associated with enhanced DNA damage response. Moreover, ST1926 was shown to induce phase-specific double-strand breaks preceding apoptotic cell death, thereby sugges-ting that growth inhibition and pro-apoptotic effects result from genotoxic damage.9,14 However, the cellular basis of the activity of such compounds remains largely unknown. Proteomics represents a useful approach to shed light on the molecular mechanism of action of drugs.16,17 In particular, protein profiling offers important clues to the drug effects on critical molecular processes and warning signals on the possible side-effects and potential resistance mechanisms of particular tumors.18 2-DE coupled to mass spectrometry is still the tool of choice for differential Received: September 20, 2010 Published: December 13, 2010 1191
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rent concentrations of ST1898, ST3595, ST3056 or to solvent for 72 h. At the end of treatment, culture medium was removed and adherent cells were harvested using trypsin and counted with a cell counter. IC50 is defined as the concentration causing a 50% inhibition of cell growth as compared with control. 2.3. Apoptosis and Cell Cycle Analysis
Exponentially growing cells were seeded in 75 cm2 flasks and 24 h later, they were exposed to drug for 24, 48 or 72 h. Under these conditions, the concentration of the drug solvent (0.005%) had no effects. At the end of treatment, floating and adherent cells were harvested for detection of apoptotic cells or cell cycle analysis. Apoptosis was evaluated by TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assay (Roche, Mannheim, Germany). After harvesting, the cells were fixed in para-formaldehyde, permeabilized in a solution of 0.1% Triton X-100 in 0.1% sodium citrate, and then incubated in the TUNEL reaction for 1 h. After washing, samples were analyzed by flow cytometry (Becton Dickinson, Mountain View, CA) using Cell Quest software. For cell cycle analysis, cells were fixed and stained with a propidium iodide (PI)-containing solution (30 μg/mL PI, 66 U/mL RNase A in PBS). The cell cycle perturbations were measured by using a flow cytometer. Samples were analyzed for DNA content and cell cycle distributions were calculated using Modfit software. 2.4. Total Lysates and Nuclear Protein Extraction
Figure 1. Chemical structures of ST1898, ST3595, and ST3056. The structure of the atypical retinoid ST1926 and of related compounds is shown.
expression proteomics,19 especially when associated with sample prefractionation (e.g., isolation of organelles such as nuclei). The aim of this study was to investigate at protein level the response of human ovarian carcinoma IGROV-1 cell line to three novel proapoptotic agents (ST1898, ST3595, ST3056) related to the AR ST1926 (Figure 1), to gain insights into their mechanisms of action. Herein, we report that treatments with the three compounds inhibit the growth of IGROV-1 cell line by inducing apoptosis and cell cycle arrest. Proteomic analyses of total lysates and nuclei support that the cellular response to drug treatment involves modulation of critical pathways which is consistent with the biological effects of these agents.
2. MATERIAL AND METHODS 2.1. Cell Culture and Drug Treatments
The human ovarian cancer IGROV-1 cell line was grown and maintained as monolayer in RPMI-1640 medium supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, CA). ST1898, ST3595, and ST3056, provided by Sigma-Tau (Pomezia, Italy), were dissolved and diluted in dimethylsufoxide. 2.2. Growth-Inhibition Assays
Cell sensitivity to the compounds was assessed by growthinhibition assay. Exponentially growing cells were harvested, seeded into 6-well plates and 24 h later cells were exposed to diffe-
Exponentially growing cells were seeded in 150 cm2 flasks and, 24 h later, they were exposed to 2 μM ST1898, ST3595, or ST3056 for 24 h. Total lysates protein extraction from 108 untreated and AR-treated cells was performed in a 2-D solubilizing/ lysing solution: 7 M urea (Sigma, Sigma-Aldrich Corporation, St. Louis, MO), 2 M thiourea (Sigma), 3% CHAPS (Sigma), 20 mM Tris (Sigma), 1% pH 3-10 Ampholyte (Fluka, Buchs SG Switzerland) and 1 protease inhibitor cocktail tablet (Comp lete, Mini; Roche, Basel, Switzerland). The samples were then sonicated 5 30 s on ice with 1 min rest in between times and centrifuged for 10 min at 10 000 g at 4 °C to remove the nucleic acids complexed with ampholytes. Concerning the nuclear proteins, the extraction from 109 untreated and AR-treated cells was obtained with the CelLytic NuCLEAR Extraction Kit (Sigma) following manufacturer’s instructions. Then 1% pH 3-10 Ampholyte was added and samples were centrifuged as above to remove the nucleic acids. Both total lysates and nuclear extracts were incubated with 5 mM tributyl phosphine and 20 mM acrylamide for 60 min at room temperature to reduce protein disulfide bonds and alkylate the cysteine thiolic groups. The reaction was blocked by the addition of 10 mM DTT (Sigma) and the samples were collected and stored at -80 °C. Protein concentration was evaluated with DC Protein assay (Bio-Rad, Laboratories., Hercules, CA) based on the Lowry method.
2.5. 2D-PAGE Analysis of Nuclear and Total Protein Extract
Protein fractionation by 2-DE was performed as previously described.20 Briefly, 400 μL of each sample (containing 2 mg/mL of protein) was separated in pH 3-10 IPG strips, and the total product time voltage applied was 70 000 Vh for each strip. The second dimensional separation was done using 8-18%T gradient SDS-PAGE, applying 40 mA for each gel for 3 min, then 2 mA/gel for 1 h, and 20 mA/gel until the track dye, Bromophenol-Blue, reached the anodic end of the gels. After 2-DE, the proteins were detected by Sypro Ruby. Five gels per group were generated for total proteins analysis, while for nuclear extracts six 1192
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Journal of Proteome Research replicated gels were obtained for each group. The image analysis of all the 2-DE gels replica was performed by PDQuest software (Bio-Rad), version 7.3. Each gel was analyzed for spot detection, background subtraction and protein spot OD intensity quantification. The gel image showing the highest number of spots and the best protein pattern was chosen as a reference template, and spots in a standard gel were then matched across all gels. Spot quantity values were normalized in each gel dividing the raw quantity of each spot by the total quantity of all the spots included in the standard gel. Gels were divided into four separated groups (control, ST1898, ST3595, ST3056), and for each protein spot, the average spot quantity value and its variance coefficient in each group were determined. A Student’s t test was performed to compare the groups and identify sets of proteins that showed a statistically significant difference with a confidence level of 0.05. Moreover, to better evaluate the results, and find the really most important modulated proteins, the false discovery rate (FDR) adjusted p-values ( 101) are presented. FL1 = relative fluorescence intensity, and FSC = forward light scatter.
Figure 5a and b shows standard map of total lysate and nuclear proteome of IGROV-1 cell line, respectively, together with the differentially expressed spots (p < 0.05) between control and treated samples. Spots selected as regulated from the differential analysis were subjected to RP-HPLC-ESI-MS/MS analysis for protein identification. The unique differentially expressed proteins identified were 84. In Table 2, the identity of the successfully identified proteins corresponding to up- or down-regulated spots (confidence level of 0.05) are shown, together with the standard spot number (SSP), the MS identification parameters (a supplemental Table 2 with peptide sequences is provided in Supporting Information), and the indication of their gene ontology (GO) annotation (cellular component, biological process and molecular function). The overlap between the identified differentially expressed proteins modulated by three compounds is shown in Figure 6. The diagram obtained demonstrates that a total of 20 proteins are regulated by all the three atypical retinoids, and that ST1898, ST3595, ST3056 also modulated 12, 10, and 8 specific proteins, respectively (Figure 6). 3.3. Validation of Selected Proteins by Western Blot
To validate the findings obtained by 2-DE, the regulation in level of expression of three candidate proteins were further investigated by immunoblot analysis. Western blot results are shown in Figure 7. Trends of changes in the same direction as those detected in the 2D gel analyses were detected for all the three proteins. The quantitative difference between the results obtained by 2D electrophoresis and by Western blot suggested
that most changes detected by the former technique specifically involve post-translationally modified forms, which can be separated only in 2D maps.
4. DISCUSSION We performed a proteomic analysis of total lysates and nuclear extracts of cells treated with agents characterized by the scaffold of the AR ST1926. Here, we describe their putative mechanism of action and discuss some of the differentially expressed proteins related to cell death and apoptosis. We focus first on proteins that are regulated in a similar manner by the three ARs and then on proteins specifically modulated by each single treatment. 4.1. Mechanism of Action Shared by the Three Atypical Retinoids
4.1.1. Deregulation of Calcium Homeostasis. The deregulation of calcium homeostasis (mainly maintained by endoplasmic reticulum, ER, and mitochondria) represents an early and critical event requested for apoptosis.24 Among the modulated proteins involved in regulation of calcium homeostasis, we identified calreticulin (CALR), a multifunctional protein that acts as a major Ca2þ-binding and storage protein in the lumen of the ER, which is also found in the nucleus where it has a role in transcription regulation. CALR overexpression increases Ca2þ fluxes across the ER and decreases mitochondrial Ca2þ; the increased Ca2þ turnover between the two organelles contributes to the damage of mitochondria increasing the susceptibility of cells to apoptotic stimuli.25 In addition, it has been reported that CALR 1194
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Figure 3. Cell cycle distribution in IGROV-1 cells exposed to ST1898, ST3595, and ST3056, analyzed 24 h after exposure to 2 μM drug in propidium iodide stained cells.
Figure 4. Western blot analysis of selected acetylated proteins and DNA damage response proteins in IGROV-1 cells exposed to ST1898, ST3595, or ST3056. IGROV-1 cells were exposed for 4 h to the indicated compounds (drug concentrations producing a growth inhibitory effect around 80-90%) and then processed for Western blot analysis of total cell lysates. Control loading is shown by β-tubulin.
has a role in the regulation of apoptosis by modulating p53 function and expression and affecting its rate of degradation and nuclear localization.26 Interestingly, CALR inhibits the DNAbinding activity of both heterodimeric RAR/RXR and homodimeric RXR complexes;27,28 in particular, CALR overexpression resulted in a decrease in retinoic acid-stimulated reporter gene expression.29 In agreement with these observations, we found that CALR was up-regulated by ST1898, ST3595, and ST3056
(2.74, 2.93, and 1.59-fold, respectively). Our findings suggest that the antiproliferative effect of the three may be related to the deregulation of calcium homeostasis and that the action of these compounds does not involve RAR/RXR receptor pathways. The maintenance of calcium homeostasis is also related to nuclear chloride channel (CLIC1). Recently, CLIC channels have been recognized as potential targets for cancer therapy due to their contributions in modifying cell cycle, apoptosis, adhesion, and motility.30 CLIC proteins are involved in protecting cells with ryanodine receptors calcium-channels, from apoptosis induced by Ca2þ mobilization from intracellular stores.31 Moreover, Cl-channel blockers have been demonstrated to inhibit cell proliferation and the cell cycle progression,32 as well as arrest, adhesion, and invasion of ovarian cancer cells, through a mechanism involving an increase in intracellular Ca2þ concentration.33 Consistent with the IGROV-1 cells growth inhibition induced by ST1898, ST3595, ST3056, we found that CLIC1 was down-regulated (1.50, 2.91, and 1.48-fold, respectively), supporting that the deregulation of calcium homeostasis may contribute to the mechanism of action of these novel ARs. 4.1.2. Oxidative Stress. Oxidative stress in cancer cells may have significant consequences, such as stimulation of cellular proliferation, promotion of mutations and genetic instability, as well as alterations in sensitivity to anticancer agents. In line with the above observations, a cross-talk between calcium and ROS signaling has been amply demonstrated.34,35 As an indication that oxidative stress may represent a mechanism of action of ST1898, ST3595, and ST3056, we identified peroxiredoxin 3 (PRDX3), a mitochondrion-specific H2O2-scavenging enzyme. PRDX3 is a 1195
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Figure 5. Differentially expressed proteins after the treatments. Standard maps of (A) total lysates and (B) nuclear proteins extracted from IGROV-1 cell line, the differentially expressed protein spots are marked by an open circle plus the spot number as reported in Table 2. On the right, PDQuest (version 7.3) output showing representative differentially expressed spots (p < 0.05) between control and treated samples. For each differentially expressed proteins an enlarged region of the respective 2-DE map is shown which contains the referred spot (highlighted by a circle) and the corresponding fold of change. Each spot was identified by MS/MS analysis.
c-Myc target gene required for mitochondrial homeostasis and neoplastic transformation.36 The correlation between PRDX3 depletion and the induction of oxidative stress has been demonstrated in vivo using knockout mice.37 It has also been shown that depletion of PRDX3 resulted in increased intracellular levels of ROS and sensitized cells to induction of apoptosis.38,39 In agreement with the inhibition of cell growth induced by ST1898, ST3595 and ST3056, we found that the tested compounds strongly downregulated (20 fold of variation), ** = differentially expressed proteins at p < 0.01 after FDR correction.
a
NUTF2 PSMA2
Nuclear transport factor 2 Proteasome alpha 2 subunit
PSMB4
PPIA
Cyclophilin A
Proteasome beta 4 subunit
PPIA
Cyclophilin A
PSMB2
CLTA
Clathrin light chain
Proteasome beta 2 subunit
TUBB5
Tubulin, beta, 5
PSMB1
TUBB2C
Tubulin, beta, 2
Proteasome beta 1 subunit
PFN1
gene name
Profilin 1
protein name
Table 2. Continued
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dx.doi.org/10.1021/pr100963n |J. Proteome Res. 2011, 10, 1191–1207
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Figure 6. Modulated protein overlapping between the three compounds. Summary of the spots corresponding to differentially expressed proteins after the indicated treatments. Numbers on the overlapping circles indicate the numbers of spots that are regulated by more than one treatment. The Venn diagram obtained demonstrates that a total of 20 proteins are regulated by all three atypical retinoids and that ST1898, ST3595, ST3056 also modulated 12, 10, and 8 specific proteins, respectively.
4.1.3. Cytoskeleton Reorganization. Cytoskeleton plays an important role in the processes of growth and differentiation and may be involved in signal transduction modulation. Multiple evidence suggests that cells from lower to more highly evolved systems have adopted the dynamic state of the cytoskeleton as an indicator of the cells’ overall health. It has been shown that reorganization of cytoskeletal proteins correlates with the induction of apoptosis,42,43 and it has been proved that ROS are involved in remodelling of the actin cytoskeleton.44,45 Among the more significant proteins involved in cytoskeletal reorganization
we identified actin, gamma 1 (ACTG1) a cytoplasmic actin found in nonmuscle cells which is thought to play a role in cell motility.46 It has been shown that ACTG1 is required for reinforcement and long-term stability of F-actin-based structures, that is, microfilaments of the cytoskeleton.47 In line with the above observations, we reported here that ACTG1 is strongly down-regulated (
