The Cytotoxic Pathway Triggered by Palytoxin Involves a Change in

Chem. Res. Toxicol. 2009, 22, 2009–2016

2009

The Cytotoxic Pathway Triggered by Palytoxin Involves a Change in the Cellular Pool of Stress Response Proteins Gian Luca Sala,† Mirella Bellocci,† and Gian Paolo Rossini* Dipartimento di Scienze Biomediche, UniVersita` di Modena e Reggio Emilia, Via G. Campi 287, I-41125 Modena, Italy ReceiVed August 24, 2009

We have analyzed the proteome of MCF-7 cells exposed to palytoxin (PlTX), to characterize protein components involved in the death response induced by the toxin. The protein profiles of cell lysates were obtained by two-dimensional (2D) electrophoresis, and we found that four components were increased by PlTX treatment. By tryptic digestion of protein spots in the gels and LC-ESI-MS/MS analysis of resulting peptides, those four components were found to include three isoforms of the heat shock protein (hsp) 27 differing with regard to their phosphrylation state, as well as DJ-1/PARK7. The effects exerted by PlTX on hsp 27 and DJ-1 proteins were further quantified by immunoblotting analyses of proteins separated by monodimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and using antibodies recognizing total hsp 27, the hsp 27 forms phosphorylated in Ser82, and DJ-1 protein. Dose-response and time-course experiments yielded results that only partially confirmed those found by protein staining after 2D electrophoresis. These findings were further checked by immunoblotting of proteins after fractionation by 2D electrophoresis, and we found that only some forms of those comigrating in a single band upon monodimensional SDS-PAGE were actually increased in extracts from PlTXtreated cells. We obtained evidence that the three hsp 27 isoforms whose relative abundance was increased in MCF-7 cells exposed to PlTX comprised two proteins phosphorylated in Ser82, whereas the third form most likely contains a phosphorylated amino acid residue other than Ser82. Moreover, we could show that PlTX treatment determined the accumulation of an oxidized isoform of DJ-1 in MCF-7 cells. We conclude that the toxicity pathway of PlTX in MCF-7 cells involves post-translational modifications of hsp 27 and DJ-1 stress response proteins, comprising a shift in the equilibria among hsp 27 isoforms toward those phosphorylated in Ser82, as well as the oxidation of DJ-1. Introduction Palytoxins (PlTXs) are large polyhydroxylated compounds that have been found in coelenterate zoanthids of the genus Palythoa (1) and are produced by benthic algae of the genus Ostreopsis (2, 3). Blooms of PlTX-producing algae have been associated with the death of benthic animals (4). In humans, PlTXs have been recognized to cause health problems following the ingestion of contaminated food (5), small skin injuries (6), and, most likely, the inhalation of aerosols contaminated with PlTXs in the coastal areas where Ostreopsis blooms were taking place (7). Animal studies have confirmed that PlTX can cause tissue injuries when administered by different routes (8-10). In the last 10 years, blooms of Ostreopsis algae have been recorded in the Mediterranean Sea, where their occurrence has been expanding and their frequency has been increasing (3, 4, 7, 11, 12), lending support to a growing concern about the chance that people might become exposed to the toxin more frequently during recreational activities as well as through the ingestion of contaminated seafood. Because of the variety of routes of possible human exposure to PlTX and the different tissues that may be affected by the toxin, a detailed understanding of the molecular pathways used by PlTX to bring about its effects is sought. * To whom correspondence should be addressed. [email protected]. † These authors have equally contributed to this study.

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The molecular mechanism of action of PlTX involves toxin binding to the Na+, K+-ATPase. The binding of PlTX to the N-terminal, extracellular segment of the R subunit of the Na+, K+-ATPase converts the ion pump into a nonspecific cation channel (13-15). This change in the functional properties of the Na+, K+-ATPase determines Na+ entrance into the cells and membrane depolarization (13-15). The perturbed ion homeostasis caused by PlTX then affects the transmembrane equilibria of other ions, such as Ca2+ and H+ (15-18), resulting in a collapse of the mechanisms controlling ion and water balances at the cellular level and triggering a variety of secondary effects in cells exposed to PlTX (reviewed in ref 19). The cytotoxic effects of PlTX have long been recorded in many experimental models (13, 20-22) and have been shown to depend on the impairment of the cellular Na+, K+-ATPase by PlTX (13, 20-22). The molecular pathway that brings about the cytotoxic response to PlTX, however, has not been fully characterized. In this study, we have used the analysis of the proteome of a biological system exposed to PlTX and have investigated the cellular components that are affected by the toxin. The MCF-7 cell line in culture represents the model system of our study because it is sensitive to PlTX and responds by cell death (22). This model system then allowed us to characterize protein components that are modified during the death response induced by the toxin, and we found that changes in the cellular pool of

10.1021/tx900297g  2009 American Chemical Society Published on Web 11/23/2009

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stress proteins are part of the cytotoxic pathway triggered by PlTX in MCF-7 cells.

Experimental Procedures Materials. PlTX was obtained from Wako Chemicals (Germany). Stock PlTX solutions (50 µM) were prepared in absolute ethanol, and their working dilutions were prepared in 60% ethanol. Toxin solutions were stored in glass vials protected from light at -20 °C. The monoclonal antibody recognizing total heat shock protein (hsp) 27 (both nonphosphorylated and phosphorylated isoforms of the protein) and the polyclonal antibody recognizing the hsp 27 isoforms phosphorylated in Ser82 were purchased from Cell Signaling Technology. The monoclonal antibody recognizing the DJ-1 protein was obtained from Abcam. The monoclonal antiactin antibody was obtained from Chemicon International. Trypsin was a product from Roche. Peroxidase-linked antirabbit and antimouse Ig antibodies and the enhanced chemioluminescence (ECL) detection reagents were from GE Healthcare. The nitrocellulose membrane Protran BA 83 was obtained from Whatman. The chemicals used to carry out electrophoretic procedures were from Bio-Rad. All other chemicals were from Sigma. Cell Culture Conditions and Toxin Treatments. MCF-7 cells were obtained from the European Collection of Animal Cell Cultures (ECACC No. 86012803 CB No. 2705) and were grown in 5% carbon dioxide in air at 37 °C, in 90 mm diameter Petri dishes, with a culture medium composed of Dulbecco’s modified Eagle’s medium, containing 1% nonessential amino acids and 10% fetal calf serum, as previously described (23). Cells in logarithmic growth received the indicated concentrations of PlTX, and control cells received an equal volume of 60% ethanol. The final concentration of ethanol in culture medium in our experiments was 0.06%. If not stated otherwise, cells were incubated in the presence or in the absence of 0.03 nM PlTX for 8 h at 37 °C. Preparation of Cell Extracts. At the end of incubations, cells were harvested and processed to obtain extracts. All of the steps were performed at 4 °C. Cells from every dish were washed with 5 mL of 20 mM phosphate buffer, pH 7.4, and 0.15 M NaCl (PBS buffer). Cells were then mechanically detached from culture dishes with a scraper and were washed three times by resuspension in 5 mL of PBS and centrifugation for 10 min at 800g. Cells were then lysed by resuspension in 8 M urea, 2% CHAPS, 0.2% Biolyte Ampholyte, 50 mM DTT, 0.1 mg/mL PMSF, and 1 mM Na3VO4 (0.16 mL/dish), followed by vortexing of the cell suspension, and the lysate was next centrifuged for 60 min at 110000g. The supernatant of this centrifugation was saved, its protein content was determined according to Bradford (24), and the extract was then used for protein separation by either onedimensional (1D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional (2D) electrophoresis. Fractionation of Proteins by Electrophoresis. In 1D SDSPAGE, samples containing the same amount of protein were fractionated according to Laemmli (25), using a 10% separating gel and a 4% stacking gel. At the end of the electrophoresis, the fractionated proteins in the gels were analyzed by immunoblotting. Protein separation by 2D electrophoresis was carried out as already reported (26). The 3-10 nonlinear pH gradient was used for protein separation by isoelectrofocusing in the first dimension. The SDS-PAGE conditions of our gels allowed a separation of proteins in the 5-80 kDa range. At the end of the electrophoresis, the proteins in the gels were either detected by silver stain (27) or subjected to immunoblotting. Detection of Protein Components by Immunoblotting. After completion of electrophoresis, proteins were electrophoretically transferred onto a nitrocellulose membrane (Protran BA 83), and binding sites remaining on the membrane were blocked by incubation of blots for 1 h at room temperature with a solution composed of 20 mM Tris-HCl, pH 7.5, at 25 °C, 0.15 M NaCl, and 0.05% (v/v) Tween20 (immunoblotting buffer), containing 3-5% nonfat dry milk. After unspecific sites were blocked, the

Sala et al. membranes were incubated for either 1 h at room temperature or overnight at 4 °C with the primary antibody solutions, following the indications of the information sheet of individual products. After incubation, membranes were washed five times with immunoblotting buffer and incubated for 1 h at room temperature with a peroxydase-linked secondary antibody at either 1:3000 or 1:5000 dilution in immunoblotting buffer containing 1-5% nonfat dry milk. After they were washed, the membranes were developed by the ECL detection system, and results were visualized by autoradiography. For quantitative estimates of antigens detected by immunoblotting, autoradiographs were subjected to densitometric scanning, and the values (arbitrary units) thus recorded were used to estimate the cellular levels of individual components that were expressed with reference to the values obtained in control samples. The results shown in figures have been obtained in the 2-5 replicate experiments that we performed. The data that we obtained in immmunoblotting analyses after protein fractionation by 1D electrophoresis were subjected to analysis of variance (ANOVA) testing. Digital Elaboration and Statistical Analysis of Data Obtained by 2D Electrophoresis and Identification of Proteins by Mass Spectrometry. To reduce the effect of sample variability, results were derived from at least three distinct experiments, in which each cell extract was analyzed by at least five gels by 2D electrophoresis, run in parallel, and used for the digital analysis. Thirty gels were then used for our analyses, when comparing proteomes in the extracts from control and PlTX-treated cells. The stained gels were converted into digital images by GS800 (Bio-Rad) scanner densitometer acquisition. The files so acquired were processed with the software PDQuest (Bio-Rad), to obtain a virtual gel from the superimposition of all of the single gels subjected to analysis. The protein profiles from treated samples were then matched with those of controls to identify qualitative and quantitative differences. The significance of experimental data was set by the software, with a cutoff value of 1 ( 0.2 for the detection of n-fold differences. The data sets yielded by the software were analyzed by the F test, and only one outlier was identified by the Grubbs test (R ) 0.05). After removal of that outlier, the detected changes were retained when they reached a significance at the 95th percentile by the Student’s t test. The relevant protein spots were excised from the gels and were subjected to tryptic digestion (28) before being analyzed by LC-electrospray ionization (ESI)-quantitative time-of-flight (QTOF) on a 6520 Accurate Mass Q-Tof LC/MS system equipped with a nanoLC-ChipESI interface (Agilent Technologies).

Results Effect of PlTX on the Protein Profile of MCF-7 Cells. The treatment of MCF-7 cells with 0.1-1 nM PlTX for 1 h can be sufficient to induce cell lysis (22). Such an exposure, however, might be too short to allow the recording of changes in protein profiles, because turnover of cellular proteins occurs in longer time frames (29). Preliminary experiments were then carried out to establish the sensitivity of our cellular system to a PlTX treatment of 8 h, which would be sufficient to detect changes in the proteome of MCF-7 cells (26). To this end, we treated MCF-7 cells with increasing concentrations of PlTX for 8 h and measured the total content of proteins that could be recovered from culture dishes after toxin treatment, as a general indication of the cell death response triggered by the toxin under our experimental conditions. The results that we obtained (Figure 1) showed that MCF-7 cell lysis was induced by PlTX concentrations higher than 0.01 nM, and an EC50 of about 0.03 nM was measured under our experimental conditions. On the basis of those data, we chose to treat cells with 0.03 nM PlTX for 8 h, and our experiments were aimed at the characterization of the effects of PlTX on the MCF-7 cell proteome under conditions corresponding to a system that is

Palytoxin Alters Stress Response Proteins

Figure 1. Effect of increasing PlTX concentrations on MCF-7 cells. Cells have been treated with the indicated concentrations of PlTX for 8 h at 37 °C, before being processed for the measurement of total protein in cell lysates, as described in the Experimental Procedures. Data represent means ( SDs obtained in three separate experiments.

responding to the toxin but whose functioning is not completely collapsed by the toxin treatment. We then prepared total protein extracts from control and PlTX-treated cells and carried out the separation of proteins in the extracts by 2D electrophoresis, obtaining profiles containing several hundreds of different components. The protein profile obtained with extracts from MCF-7 cells exposed to PlTX is reported in Figure 2. The

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statistical analysis of the results obtained in three separate experiments showed four components whose levels were significantly (p < 0.05) increased by PlTX treatment (Figures 2 and 3). In particular, SSP 2114 and SSP 3105 were barely detectable in samples from control cells, but our densitometric analyses showed that their content was increased many-fold in PlTX-treated cells (Figure 3). The cellular levels of component SSP 6002, which will be further considered in other experiments (see below), did not show any significant change under our experimental conditions (Figure 3). Identification of Protein Components Affected by PlTX Treatment in MCF-7 Cells. The protein components whose relative abundance was changed in extracts from cells exposed to PlTX were then identified by tryptic digestion of protein spots in the gels and LC-ESI-MS/MS analysis of resulting peptides (Table 1). In the case of SSP 2114, 3105, and 4107, the peptides identified by Mascot search are part of a member of the superfamily of heat shock proteins involved in cell stress responses (30-33), the stress protein hsp 27. Because hsp 27 is known to be phosphorylated in many amino acid residues

Figure 2. Protein profile of MCF-7 cell lysates from cells treated with PlTX. Cells have been treated with 0.03 nM PlTX for 8 h at 37 °C, before being used for the preparation of cell lysates, whose proteins were fractionated by 2D electrophoresis, using 17 cm strips for the separation in the first dimension, as described in the Experimental Procedures. The four components whose levels are affected in MCF-7 cells exposed to PlTX are circled by a solid line, whereas component SSP 6002, which is not significantly affected by PlTX but will be further considered in other experiments, has been circled by a dashed line.

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Figure 3. Effect of PlTX treatment of MCF-7 cells on the protein profile of total cell lysates. MCF-7 cells were treated with 0.03 nM PlTX for 8 h at 37 °C, before being used for the preparation of cell lysates. A 150 µg amount of protein from each cell extract was subjected to 2D electrophoresis, using 17 cm strips for the separation in the first dimension. Top: protein profiles in MCF-7 cell lysates from control and PlTX-treated cells in the area including the spots altered by the toxin treatment. Center: list of components (SSP) whose levels have been affected by PlTX treatment, as determined by the absorbances measured for indicated spots, which have been expressed as means ( SDs of values obtained from three separate experiments in pentaplicate. The data on component SSP 6002, which was not affected by PlTX but will be further considered in other experiments, have also been included. Bottom: graphical representation of the changes detected in the cellular levels of components affected by PlTX.

Table 1. Identification of Protein Components Whose Levels Are Affected in MCF-7 Cells Exposed to PlTX SSP

accession number

name

mass

score

sequence coverage (%)

2114 3105 4107 5003

P04792 P04792 P04792 Q99497

HSPB1 HSPB1 HSPB1 DJ-1/PARK7

22826 22826 22826 20050

171 427 653 195

29 60 72 43

(32, 33), the detection of three hsp 27 isoforms that had been separated by isoelectrofocusing was an indication that they could differ according to their phosphorylation state. Accordingly, the Mascot search identified these three proteins as isoforms of hsp 27 differing according to their phosphorylation state (see the Supporting Information). Although phosphorylated amino acid residues in individual forms could not be defined with certainty, the Mascot search indicated the most likely structure of the proteins that we have analyzed. In particular, the best matching

of results indicated SSP 3105 as an isoform having a phosphorylated Ser82 and SSP 2114 as another isoform containing a phosphorylation of Ser82 and/or another amino acid residue, which might consist of Ser83. In any case, the lower pI of SSP 2114, as compared to SSP 3105, implies that this latter isoform contains a lower number of phosphate groups than SSP 2114. Other less abundant phosphorylated isoforms of hsp 27 could cofractionate with SSP 2114 and SSP 3105. In turn, SSP 4107 could represent both nonphopsphorylated hsp 27 and an hsp 27 isoform containing a single phosphate group (see the Supporting Information). The fourth component (SSP 5003), whose relative abundance was changed in extracts from cells exposed to PlTX under our experimental conditions, was identified as DJ1/PARK7. This protein is involved in cell stress responses (34-38), and its alterations have been found to be responsible for the onset of different forms of Parkinson’s disease (38-40). Validation of Results by Immunoblotting Analyses. The effects exerted by PlTX on hsp 27 and DJ-1 proteins were further analyzed by immunoblotting, after proteins had been separated by monodimensional SDS-PAGE and using antibodies recognizing total hsp 27 (being able to bind both nonphosphorylated and phosphorylated isoforms of the protein), the hsp 27 isoforms phosphorylated in Ser82, as well as DJ-1 protein. Our choice of antibody for the detection of phosphorylated hsp 27 was made on the basis of the Mascot search, indicating that isoforms phosphorylated in Ser82 gave the best scores for SSP 2114 and SSP 3105 (see the Supporting Information). When extracts were prepared from MCF-7 cells that had been exposed to PlTX concentrations in the 10-11 M range (Figure 4), we found a dose-dependent decrease in the cellular abundance of total hsp 27, DJ-1, and actin, used as an internal reference of cellular proteins, in keeping with the cytotoxic effect the toxin exerted in our experimental system (Figure 1). The cellular abundance of hsp 27 phosphorylated in Ser82, in turn, was increased by PlTX in the concentration range used in these experiments (Figure 4). We then analyzed the effect of 0.03 nM PlTX on the cellular abundance of those components, by measuring their levels in MCF-7 cells that had been exposed to the toxin for different times. The results that we obtained in our time-course experiments (Figure 5) showed an about 5-fold increase in the cellular abundance of hsp 27 phosphorylated in Ser82. The slight increase in total hsp 27 was also significant in these time-course experiments, whereas the analysis of DJ-1 did not lead to detection of significant changes over time under those experimental conditions. Overall, the results obtained by immunoblotting analysis of proteins separated by monodimensional electrophoresis only partially confirmed the effects exerted by PlTX, as detected by silver staining of proteins separated by 2D electrophoresis. No increase in the content of DJ-1, in particular, could be detected in extracts from cells exposed to PlTX by these analyses. Taking into consideration that the separation of proteins by 1D SDS-PAGE may not resolve components having similar molecular masses but different isoelectric points (pI), the results that we obtained by immunoblotting could be explained if the relevant component affected by PlTX treatment was only one of several protein isoforms comigrating in the gel and being recognized by the same antibody. Indeed, a plurality of hsp 27 isoforms with masses that may not be resolved by SDS-PAGE is expected for both phosphorylated and total hsp 27 proteins in the extracts that we prepared from MCF-7 cells (Figure 3


Figure 4. Dose-dependent effect of PlTX on the levels of total hsp 27, hsp 27 phosphorylated in Ser82, DJ-1, and actin in MCF-7 cells. Cells have been treated with the indicated PlTX concentrations for 8 h at 37 °C, before being processed for the preparation of cell extracts. Ten micrograms of protein from each cell extract was loaded onto each lane, separated by 1D SDS-PAGE, and then subjected to immunoblotting. The significance of results based on ANOVA testing is indicated.

and ref 26). Furthermore, multiple DJ-1 isoforms, having similar mass but differing in their pI, have been reported (34, 36, 38, 41). On the basis of these considerations, we repeated our immunoblotting analyses after proteins were fractionated by 2D electrophoresis. The results that we obtained are reported in Figure 6 and show that multiple isoforms can be detected by the antibodies used in our immunoblotting analyses after they have been resolved by isoelectrofocusing. Ten major spots were detected with the antibody recognizing total hsp 27, including nonphosphorylated and phosphorylated isoforms, whereas two major spots were detected by the antibody binding the hsp 27 isoforms phosphorylated in Ser82. Likewise, two spots were detected by the antibody specific for the DJ-1 protein. By sequential incubation of the same membrane with antibodies recognizing total hsp 27, the hsp 27 isoforms phosphorylated in Ser82, as well as DJ-1 protein, we could align the protein spots in our immunoblots, and the correspondence with SSP 4107, 2114, 3105, as well as 5003 and 6002 could be established (Figure 6). Although the error inherent into the matching of results from films developed using different membranes prevents precise quantitative estimates, a direct matching of spots within the same


Figure 5. Time-dependent effect of PlTX on the levels of total hsp 27, hsp 27 phosphorylated in Ser82, DJ-1, and actin in MCF-7 cells. Cells have been treated with 0.03 nM PlTX for the indicated times at 37 °C, before being processed for the preparation of cell extracts. Ten micrograms of protein from each cell extract was loaded onto each lane, separated by 1D SDS-PAGE, and then subjected to immunoblotting. The significance of results based on ANOVA testing is indicated.

membrane can provide information regarding the cellular pools of protein forms and the effects exerted by PlTX on their relative abundance. The results that we obtained with the antibody recognizing both nonphosphorylated and phosphorylated hsp 27, for instance, showed a clear increase in the relative abundance of the SSP 2114 and a more limited enrichment of isoforms SSP 4107 and SSP 3105 (Figure 6, top). The results that we obtained with the antibody recognizing the hsp 27 isoforms phosphorylated in Ser82 confirmed these results and those obtained by gel staining (Figure 3), as the extracts obtained from PlTX-treated cells were enriched in the phosphorylated hsp 27 isoform SSP 2114, taking the other hsp 27 phosphorylated in Ser82 (SSP 3105) as an internal reference (Figure 6, middle). Taking into consideration that these three hsp 27 isoforms are separated during isoelectrofocusing, the results that we obtained by immunoblotting analysis using an antibody recognizing both nonphosphorylated and phosphorylated hsp 27 (Figure 6, top) show that SSP 4107 possesses an intermediate acidity. Thus, our results indicated that SSP 2114 and SSP 3105 contain multiple phosphorylated residues, including Ser82, whereas SSP 4107 most likely represents an hsp 27 isoform

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Figure 6. Immunoblotting analysis of total hsp 27, hsp 27 phosphorylated in Ser82, and DJ-1 in MCF-7 cells exposed to PlTX. Cells were treated with 0.03 nM PlTX for 8 h at 37 °C, before being processed for the preparation of cell extracts that were subjected to 2D electrophoresis, using 7 cm strips for the separation in the first dimension. Proteinfractionationby2Delectrophoresiswasfollowedbyimmunoblotting.

phosphorylated in some amino acid residue(s) other than Ser82 (Figure 6, middle). The immunoblotting of DJ-1 also confirmed the results obtained by gel staining (Figure 3), showing a net increase of an isoform, corresponding to SSP 5003, with a pI more acidic than that of the major immunoreactive spot, corresponding to SSP 6002 (Figure 6, bottom). The theoretical estimates of the pI of the two isoforms present in MCF-7 cell lysates were 5.94 and 6.38, which are in excellent agreement with those estimated in other studies (36, 38, 41), for the oxidized (pI ) 5.5-6.0) and the reduced (pI ) 6.2-6.4) DJ-1 isoforms, respectively. The results that we obtained by immunoblotting after protein fractionation by 2D electrophoresis (Figure 6) then explained those obtained when the analysis was carried out using proteins separated by monodimensional SDS-PAGE (Figures 4 and 5), showing that only some isoforms of those comigrating in a single band upon SDS-PAGE were actually increased in the extracts prepared from MCF-7 cells that had been treated with PlTX. Thus, the visualization of the entire set of nonresolved isoforms sharing the epitopes recognized by the antibodies used in immunoblotting analyses, comprising PlTX-insensitive components, masked the detection of changes in the relative abundance of those that had been selectively affected by PlTX and were present in the same bands after protein separation by monodimensional SDS-PAGE (Figures 4 and 5).

Discussion The analysis of the MCF-7 cell proteome has shown that the cytotoxic response triggered by PlTX involves changes in the cellular pool of hsp 27 and DJ-1, which represent proteins participating to stress responses (30-38). In particular, PlTX

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treatment changed the relative concentrations of three isoforms of phosphorylated hsp 27, including two isoforms phosphorylated in Ser82, and caused the accumulation of an acidic isoform of DJ-1. The modulation of post-translational modifications of two stress response proteins, hsp 27 and DJ-1, then represents the major effect of PlTX detectable under our experimental conditions. In molecular terms, the changes that we have detected in MCF-7 cells exposed to PlTX correspond to those that have been found in other systems exposed to noxious stimuli and contribute to the cell’s adaptive response to survive stress conditions (31-38, 41, 42). As far as hsp 27 is concerned, our findings show that PlTX treatment of MCF-7 cells causes an increased phosphorylation of hsp 27 in Ser82 and in other as yet unidentified amino acid residue(s). The phosphorylation of hsp 27 has a role in the control of its functioning. In cells under normal conditions, the hsp 27 pool mostly includes nonphosphorylated proteins, but a noxious stimulus induces the phosphorylation of hsp 27 in several sites, including Ser82, causing down-regulation of the protective function of hsp 27 (33). The results obtained in this study, therefore, are in line with the model of hsp 27 functioning. DJ-1 is another protein playing a protective role in the cells, where it favors cell survival to noxious stimuli, particularly oxidative stress (34-38, 41). Furthermore, mutations of the gene coding DJ-1, or decreased cellular levels of the protein, have been linked to the onset of neurodegenerative diseases (38-40). The mechanism by which DJ-1 protects the cells has been attributed to its capacity to function as an antioxidant protein, due to its property to become oxidized in Cys106, thereby protecting cells from oxidative stress and cell death (34, 36, 38, 41). Because the pI of the acidic DJ-1 isoform that we detected coincides with that of oxidized DJ-1 (36, 38, 41), our observations imply that PlTX induces the oxidation of DJ-1 in MCF-7. We have no data on the status of Cys106 in our samples, because the corresponding peptide was not covered by our MS/MS analyses, but its oxidation in PlTX-treated MCF-7 cells would be suggested by our observations (Figure 6). The cell death induced by PlTX in MCF-7 cells is ouabainsensitive (22), confirming that an impairment of Na+, K+ATPase is triggering the response under our experimental conditions. The molecular mechanisms that cause the change in the cellular pools of hsp 27 and DJ-1 as a consequence of PlTX impairment of Na+, K+-ATPase in MCF-7 cells, however, remain undetermined. The phosphorylation of hsp 27 in Ser82 is catalyzed by the mitogen-activated protein kinase-activated protein kinase 2 and 3 isoforms, in signaling pathways involving the p38 mitogenactivated protein kinase in vivo (33, 42-45). The impairment of Na+, K+-ATPase by PlTX, in turn, has been shown to activate p38 protein kinases in intact cells (46, 47). Thus, it seems likely that the stress response induced by PlTX in MCF-7 cells, through a shift in the equilibria among hsp 27 isoforms toward those phosphorylated in Ser82, is brought about by activation of p38 protein kinases. The p38 mitogen-activated protein kinases are key effectors of stress responses (42-45) and have also been involved in the molecular mechanisms of cell protection by noxious stimuli exerted by DJ-1 (48, 49). Thus, a mechanistic link between the PlTX-induced activation of p38 kinase and the oxidation of DJ-1 in MCF-7 cells might exist. In more general terms, p38 protein kinases could represent common effectors of the molecular mechanisms of cytotoxicity triggered by different marine biotoxins. The activation of p38


kinases, in fact, has been found in cytotoxic responses induced by a variety of marine biotoxins other than PlTX, including okadaic acid (50), maitotoxin (51), azaspiracid-1 (52), and domoic acid (53). In a separate study, we have found that the death response of MCF-7 cells to okdaic acid is accompanied by a prominent accumulation of hsp 27 isoforms phosphorylated in Ser82 (26), corresponding to the components SSP 2114 and 3105 detected in this report. Thus, the results of our investigations onto the changes induced by the cytotoxic marine biotoxins okadaic acid (26) and PlTX (this study) in the MCF-7 cell proteome show that the cell death pathways of both toxins involve a shift of the equilibrium in the cellular pool of hsp 27 toward isoforms phosphorylated in Ser82, independently of the noxious stimulus that has triggered the response. The accumulation of hsp 27 isoforms phosphorylated in Ser82 can then be induced through different mechanisms of action in MCF-7 cells, because okadaic acid is known to act by binding and inhibiting Ser/Thr phosphoprotein phosphatases (19, 54), whereas the Na+, K+-ATPase represents the molecular target of PlTX (13-15). Those different molecular mechanisms of action would then converge to some common effector(s) that could control the cellular pool of hsp 27 and the equilibria between phosphorylated hsp 27 isoforms. The p38 protein kinase could then represent such a common effector. Although the accumulation of hsp 27 isoforms phosphorylated in Ser82 appears an important event in the stress response of MCF-7 cells to marine biotoxins, additional factors are also involved in the pathways, affecting the hsp 27 and other protein components. For instance, the treatment of MCF-7 with gambierol, an algal toxin that blocks voltage-dependent potassium channels (55, 56), triggers a stress response characterized by the accumulation of nonphosphorylated hsp 27, which results in cell survival to this noxious stimulus (26), in keeping with the contention that increased levels of hsp 27 protect cells from stressors (30-33). Thus, the toxicity pathways of marine biotoxins that affect MCF-7 cell survival could control the cellular equilibria among different hsp 27 isoforms, and the accumulation of those possessing defined patterns of phosphorylated residues, including Ser82, would characterize cell death responses. As far as other protein components are concerned, different patterns can be found in responses to distinct toxins. On the one hand, we have not detected any significant change in the cellular pool of DJ-1 when MCF-7 cells have been exposed to okadaic acid (26), as opposed to PlTX (this study), implying that the same biological end point of exposure (cell death) to biotoxins acting through different mechanisms of action can be attained by toxicity pathways that only partly overlap. On the other hand, one protein component can be altered by biotoxins acting through different mechanisms of action, as is the case of the ATP synthase subunit δ (26), but the responses to different toxins may result in distinct effects, such as cell death or survival, as it occurs in MCF-7 cells exposed to okadaic acid and gambierol, respectively (26). In conclusion, our investigations into the changes that marine biotoxins with distinct mechanisms of action exert onto the proteome of the same biological system, the MCF-7 cell line, show that the toxicity pathways triggered by different compounds include both common and toxin-related molecular responses, which most likely stem from both converging and diverging series of steps at different levels of the pathways. Work is in progress to further characterize the mechanistic details of those pathways.


Acknowledgment. We thank an anonymous reviewer, whose constructive criticism led us to improve our paper. We thank the Centro Interdipartimentale Grandi Strumenti at Universita` di Modena e Reggio Emilia for their skillful technical assistance in MS analyses, as well as Fondazione Cassa di Risparmio di Modena. This investigation was supported by the Italian MUR (Grant 2007FXSCL2). Supporting Information Available: Information on the Mascot search results of the tryptic peptides of hsp 27 isoforms affected by PlTX treatment of MCF-7 cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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