Structure−Activity Relationships of Yessotoxins in Cultured Cells

Aug 5, 2004 - Citation data is made available by participants in Crossref's Cited-by Linking service. For a more comprehensive list of ... Patrizia Ci...
0 downloads 11 Views 109KB Size
Download PDF
Chem. Res. Toxicol. 2004, 17, 1251-1257

1251

Structure-Activity Relationships of Yessotoxins in Cultured Cells Sara Ferrari,† Patrizia Ciminiello,‡ Carmen Dell’Aversano,‡ Martino Forino,‡ Claudia Malaguti,† Aurelia Tubaro,§ Roberto Poletti,|| Takeshi Yasumoto,⊥ Ernesto Fattorusso,‡ and Gian Paolo Rossini*,† Dipartimento di Scienze Biomediche, Universita` di Modena e Reggio Emilia, Via G. Campi 287, I-41100 Modena, Italy, Dipartimento di Chimica delle Sostanze Naturali, Universita` di Napoli “Federico II”, Via D. Montesano 49, I-80131 Napoli, Italy, Dipartimento di Economia e Merceologia delle Risorse Naturali e della Produzione, Universita` di Trieste, Via A. Valerio 6, I-34127 Trieste, Italy, Centro Ricerche Marine, Via A. Vespucci 2, I-47042 Cesenatico, Italy, and Japan Food Research Laboratories, Tama Laboratories, Tokyo, Japan Received November 7, 2003

The structure-activity relationship of yessotoxins (YTX) has been probed by measuring the potency of several YTX analogues to cause the accumulation of a 100 kDa MW fragment of E-cadherin in MCF-7 breast cancer cells. Under our experimental conditions, the EC50 of YTX, the reference compound, was 0.55 nM. The introduction of a methylene unit adjacent to one of the sulfate groups, as is the case with the homoyessotoxin molecule, did not appear to greatly affect the potency of the analogue, as the measured EC50 for this compound was 0.62 nM. The EC50 values we measured for 45-hydroxyhomoyessotoxin and carboxyyessotoxin were about 9.4 and 26 nM, respectively, whereas the EC50 of noroxoyessotoxin, lacking most of the C9 chain, was about 50 nM. Thus, significant differences in the potencies of YTX analogues were found when structural changes involved the C9 terminal chain of these compounds, leading to the conclusion that this portion of the molecule is essential for the activity of YTX in MCF-7 cells. A comparison of our findings with available information regarding the potency of YTX and its analogues in other experimental systems shows that the EC50’s we measured for the different compounds are up to 200-fold lower and vary in a wider concentration range. We speculate that YTX effects could involve two separate receptorial systems.

Introduction Yessotoxins (YTX) are sulfated polyether compounds (Figure 1) that are produced by dinophyceae algae, such as Protoceratium reticulatum and Lingulodinium polyedrum (1-3). By filter feeding, bivalve mollusks, such as mussels and scallops, accumulate YTX and its analogues (3-15). The chemical modifications found in YTX analogues include the addition of a methylene unit adjacent to one of the sulfate group, as in the homoyessotoxin (HYTX) molecule (7), and more or less extensive changes of the C9 terminal chain. These latter modifications include a further functionalization (-OH or -COOH) of the side chain (7, 10, 12, 13, 16), its partial cleavage (14, 15), or its total removal accompanied by loss of four carbon atoms of the adjacent ether ring (9). Finally, loss (17) or addition (9) of sulfate groups are further structural modifications observed in YTX analogues. The observation that intraperitoneal (ip) injection of YTX in mice leads to death even at very low doses of toxin has raised some concern with regard to the risk posed to consumers by the ingestion of mollusks contaminated by * To whom correspondence should be addressed. Tel: +39.059.205.5388. Fax: +39.059.205.5410. E-mail: rossini.gianpaolo@ unimore.it. † Universita ` di Modena e Reggio Emilia. ‡ Universita ` di Napoli “Federico II”. § Universita ` di Trieste. || Centro Ricerche Marine. ⊥ Tama Laboratories.

this class of compounds. Toxicological studies have shown that every YTX analogue described so far can cause mouse death when it is injected ip, at doses comprised between 80 and 1000 µg/kg of body weight (6, 7, 12, 1421). Interestingly, major differences have been found about the lethal doses of single components, such as YTX, rather than among different analogues, and this variability is believed to depend on experimental conditions, particularly with regard to the strain, sex, and age of mice used in those investigations. This type of finding would indicate that the side chain of YTX might not be a major structural determinant of its activity, in keeping with the contention that the ladder-shaped structure of YTX resembles brevetoxins (4, 22). The analysis of responses in the intact animals, however, would have to take into account the absorption, metabolism, and excretion of the individual compounds. More information about the structure-activity relationship of YTXs, in turn, could be obtained from analyses of binding characteristics of YTX receptor, but essentially nothing is known with regard to the primary molecular target, i.e. the receptor of YTX. The use of cultured cells, which allows a more tight control over experimental conditions, and the availability of a molecular response to YTX amenable of quantitative analysis, in turn, could provide appropriate means for probing the relative potencies of YTX analogues. The

10.1021/tx030054x CCC: $27.50 © 2004 American Chemical Society Published on Web 08/05/2004

1252

Chem. Res. Toxicol., Vol. 17, No. 9, 2004

Ferrari et al.

Figure 1. Structures of yessotoxin and some of its analogues.

molecular response to YTX in epithelial cells in culture, for instance, involves the accumulation of a 100 kDa fragment of E-cadherin (23). This type of response, induced by sub-nanomolar concentrations of the compound, can be subjected to quantitative analysis and is very reproducible (23). We have then exploited this response and have analyzed the potencies of several YTX analogues in MCF-7 breast cancer cells. In this paper we show that YTX analogues can display striking differences in their relative potencies in cultured epithelial cells and, based on our experimental data, discuss a model of the binding site of YTX on its primary molecular target. Furthermore, by comparing our findings with the effective concentrations of the agent in other experimental systems, we speculate that YTX effects described so far could involve two separate receptorial systems.

Experimental Procedures Materials. The monoclonal anti-E-cadherin antibody was purchased from Alexis Corp. (clone HECD-1). Peroxidase-linked anti-mouse Ig antibody and the enhanced chemioluminescence (ECL) detection reagents were from Amersham Biosciences. Prestained molecular mass markers were obtained from Sigma. The nitrocellulose membrane Protran BA83 was obtained from Schleicher & Schuell. All other reagents were of analytical grade. Preparation of Yessotoxin Analogues. Yessotoxin and its analogues were obtained from several collections of Italian contaminated mussels Mytilus galloprovincialis carried out during 1998-2000. Specimens of the toxic mussels were collected along the coastal area of the northern Adriatic Sea (Cesenatico, Emilia Romagna, Italy) when routine control tests had shown mussels to be positive for DSP toxicity. The following extraction and purification procedure was used for all the investigated samples. Digestive glands were extracted with acetone twice. After evaporation of the acetone, the aqueous concentrate was extracted three times with EtOAc. Isolation of YTXs was carried

out from the combined EtOAc extracts. After removal of EtOAc, the extract was partitioned between 80% MeOH and n-hexane. The hydromethanolic layer was further partitioned between 40% aqueous methanol and methylene chloride. The dichloromethanesoluble material was then chromatographed on a Develosil ODS column, washing stepwise with MeOH-H2O (8:2 and 9:1) and MeOH in this order. Toxins eluted in the last fraction were passed through a Toyopearl HW-40 SF column with MeOH. The toxins were dissolved in MeOH-H2O 6:4 and further purified on a RP-8 column equilibrated with the same solvent. The column was then washed stepwise with MeOH-H2O (6:4 and 8:2, in this order). The presence of YTXs in the dichloromethane extract and in the eluates was checked by NMR on a Bru¨ker AMX-500 spectrometer and by LC-MS. The final HPLC purification was carried out on a RP 18 column with CH3CNMeOH-H2O 1:2:2 as eluent. It is to be noted that composition and relative abundance of YTXs in contaminated mussels vary seasonally and annually. Therefore, the YTX analogues used in the present investigation were obtained from different mussel collections and here we only report the retention time of each compound on the final HPLC column. In particular, yessotoxin (tR 5.15) (6), homoyessotoxin (tR 5.20), and 45-hydroxyhomoyessotoxin (tR 3.91) were obtained in pure forms, while mixtures of carboxyhomoyessotoxin (tR 3.12) (13) and carboxyyessotoxin (tR 3.09) (12) as well as noroxohomoYTX (tR 2.92) (14) and noroxoYTX (tR 2.89) (15) were not further resolved. Working solutions of yessotoxin standards and their analogues were prepared in absolute ethanol, obtained by serial dilutions of a 500 µM stock solution and were stored at -20 °C, in glass vials, protected from light. Acetylation of 45-Hydroxyyessotoxin. 45-Hydroxyyessotoxin (10 µg) was treated with Ac2O in anhydrous pyridine for 12 h, thus obtaining 5.8 µg of the acetylated derivative. The acetylated compound was analyzed by LC-MS using a highpressure pump SP model P 4000 (ThermoFinnigan Separation Products, San Jose, CA) coupled to an Applied Biosystem API2000 triple quadrupole mass spectrometer equipped with a turbo-ion-spray source (Thornhill, ON, Canada). LC separation was performed by using a Hypersil C8 BDS, 50 × 2.00 mm, 3 µm column (Phenomenex, Torrance, CA). Eluent A was water and B was a 95% acetonitrile/water solution, both eluents

SAR of Yessotoxins in Cultured Cells containing 3.5 mM ammonium formate and 50 mM formic acid. A gradient elution (from 10 to 100% B in 10 min and then 100% B for 15 min) was used. The flow rate was 200 µL/min and a sample injection volume of 5 µL was used. The ions at m/z 1199.1 [M - 2Na + H]- and 1119.1 [M - 2Na + H - SO3]- were monitored in negative selected ion monitoring (SIM) experiments. Direct comparison to a standard solution of 45-hydroxyyessotoxin at similar concentration injected under the same experimental conditions allowed us to determine the acetylated 45-hydroxyyessotoxin content in the sample to be 5.8 µg. Methylation of Carboxyyessotoxin. A solution of 10 µg of carboxyyessotoxin in dichloromethane was treated with an excess of CH2N2 in ether at room temperature for 1 h. The solution was taken to dryness under a stream of N2 and analyzed by LC-MS by using the same experimental procedure as described above. The ions at m/z 1187.1 [M - 2Na + H]and 1107.1 [M - 2Na + H - SO3]- were monitored in negative selected ion monitoring (SIM) experiments. Direct comparison to a standard solution of carboxyyessotoxin at similar concentration injected under the same experimental conditions allowed us to determine the methylated carboxyyessotoxin content in the sample to be 6.1 µg. Cell Culture Conditions and Treatments. MCF-7 were obtained from the European Collection of Animal Cell Cultures (ECACC No. 86012803, CB No. CB2705), were routinely seeded at a density of (2-4) × 104 cells/cm2, 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 medium, containing 1% nonessential amino acids and 10% fetal calf serum, as previously described (24). Cells in logarithmic growth were seeded in 24-well (φ 15 mm) plates, as described above, and samples (0.6 mL/well), in triplicates, received increasing concentrations of either YTX or YTX analogues. Cells were incubated for 20 h at 37 °C, before being processed to prepare and analyze extracts, as specified below. Preparation of Cell Extracts. Cells from culture wells were washed three times with 20 mM phosphate buffer, pH 7.4, 0.15 M NaCl (PBS), and used to prepare cytosoluble extracts. These have been prepared by cell dispersion in 50 µL of PBS/well, containing 1% (v/v) Triton x-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/ v) SDS, 0.1 mg/mL phenylmethylsulfonyl fluoride. After 10 min at 4 °C, the material was recovered from each well, the triplicate samples were combined, and cytosoluble extracts were then obtained by centrifugation for 30 min at 16 000g. The supernatants of this centrifugation were used for colorimetric determinations of protein content, with bicinchoninic acid (25), and then brought to 2% SDS and 5% β-mercaptoethanol, to be used for fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fractionation of Proteins by Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoresis and Immunoblotting (SDS-PAGE). Samples were fractionated by SDS-PAGE, according to the method of Laemmli (26), using a 10% separating gel and a 3% stacking gel. If not stated otherwise, identical amounts of protein were loaded onto each lane of the gel. After completion of electrophoresis, proteins were electrophoretically transferred onto a nitrocellulose membrane (Protran BA83), and binding sites remaining on the membrane were blocked by incubation of blots for 1 h at room temperature with 20 mM Tris-HCl, pH 7.5 at 25 °C, 0.15 M NaCl, and 1 mM CaCl2 containing 3% nonfat dry milk. Membranes were then incubated for 1 h at room temperature with 20 mM Tris-HCl, pH 7.5 at 25 °C, 0.15 M NaCl, and 1 mM CaCl2 containing 1% nonfat dry milk and anti-E-cadherin antibody at a final 2 µg/mL concentration. After incubation, membranes were washed five times with 20 mM Tris-HCl, pH 7.5 at 25 °C, 0.15 M NaCl, 0.05% (v/v) Tween 20 (immunoblotting buffer) and incubated for 1 h at room temperature with a peroxidase-linked secondary antibody at a 1:3000 dilution in immunoblotting buffer containing 1% nonfat dry milk. After washing, the membrane was developed by the

Chem. Res. Toxicol., Vol. 17, No. 9, 2004 1253 Table 1. Yessotoxin Samples Used in This Study sample YTX (H)YTX 45OH-HYTX CB-(H)YTX N-(H)YTX

toxins

molar ratios

yessotoxin homoyessotoxin yessotoxin 45-hydroxyhomoyess otoxin 44-carboxyyessotoxin 44-carboxyhomoyess otoxin noroxohomoyessotoxi n noroxoyessotoxin

1 1 0.32 1 1 0.97 1 0.31

ECL detection system, and results were visualized by autoradiography. Calculations and Data Presentation. The electropherograms obtained by autoradiographies were subjected to densitometric scanning, and the absorbances were recorded as the peak area of bands of two protein components, corresponding to intact E-cadherin and an E-cadherin-related antigen (ECRA100), as detailed below. The parameters used in our calculations included the total immunoreactivity (TI) of components and the relative immunoreactivity of ECRA100 (RIECRA100):

TI ) AUE-cadherin + AUECRA100 RIECRA100 ) AUECRA100/TI × 100 The fractional activity (FA) of samples was then calculated with reference to the maximal molecular response measurable in the assay (23), corresponding to the RIECRA100 in samples prepared from MCF-7 cells that had been treated with 2 nM YTX (RIECRA100MAX):

FA ) RIECRA100/RIECRA100MAX The plot of FA vs the concentration of the tested compounds yielded the value of its EC50, defined as the molar concentration of the compound that could elicit the half-maximal molecular response in our system.

Results Characterization of Samples Used in Our Study. Different YTX analogues have been employed in this study, whose structures are reported in Figure 1, including HYTX, 45-hydroxyhomoyessotoxin (45OH-HYTX), 44carboxyyessotoxin (CB-YTX), and 44-carboxyhomoyessotoxin (CB-HYTX), as well as noroxoyessotoxin (N-YTX) and noroxohomoyessotoxin (N-HYTX). As is shown in Table 1, only the YTX and 45OH-HYTX standards were pure, whereas the other samples consisted of mixtures of analogues, due to the copurification of YTX and HYTX [abbreviated as (H)YTX] or their analogues. Determination of the Potency of Homoyessotoxin. The presence of HYTX analogues in our samples led us to carry out preliminary experiments to assess its potency with reference to YTX. To this end, we treated MCF-7 cells with increasing concentrations of pure YTX and of a mixture of YTX and HYTX (sample (H)YTX in Table 1). The data we obtained are reported in Figure 2, and it can be observed that cell treatment with a mixture of YTX and HYTX led to a response which, on a molar basis, was indistinguishable from that elicited by YTX alone (Figure 2, panels A and B). Repeated experiments confirmed that dose-response analyses for YTX and the mixture of YTX and HYTX did not significantly differ (Figure 2, panel C), and the EC50 calculated from those curves was 0.55 nM for YTX and 0.62 nM for HYTX, respectively.

1254

Chem. Res. Toxicol., Vol. 17, No. 9, 2004

Ferrari et al.

Figure 3. Effect of MCF-7 cell treatment with increasing concentrations of yessotoxin (YTX) and 45-hydroxyhomoyessotoxin (45OH-HYTX) on E-cadherin. MCF-7 cells have been treated with the indicated concentrations of YTX (solid circles) and 45OH-HYTX (open circles) for 20 h at 37 °C. The experimental conditions were as described in the legend to Figure 2, and the data represent means ( SD obtained in at least three separate experiments.

Figure 2. Effect of MCF-7 cell treatment with increasing concentrations of yessotoxin (YTX) and homoyessotoxin (HYTX) on E-cadherin. MCF-7 cells have been treated with the indicated concentrations of YTX (panels A and C) and a mixture of YTX and HYTX (panels B and C) for 20 h at 37 °C. At the end of the incubation, cells were processed to obtain cytosoluble extracts, which have been fractionated by SDS-PAGE and subjected to immunoblotting using the anti-E-cadherin antibody. Panel A: electropherogram obtained by ECL detection and autoradiography in a typical experiment after cell treatment with YTX. Panel B: as in A, but cells were treated with a mixture of YTX and HYTX. The electrophoretic mobility of the β-galactosidase (116 kDa) subunit used as marker protein running in a parallel lane is shown on the left. Panel C: fractional activity of YTX (solid circles) and the mixture of YTX and HYTX (open circles), measured as described in the methods section. Data represent means ( SD obtained in at least three separate experiments.

Measurement of the Relative Potency of YTX Analogues. On the basis of the substantial equipotency of YTX and HYTX, we could simplify the evaluation of potencies of analogues using the samples described in Table 1. We then turned to YTX analogues and determined the relative potency of 45-hydroxylated YTX, by the use of the 45OH-HYTX sample. The data we obtained are reported in Figure 3 and show that the presence of an hydroxy group in the C45 position of the C9 terminal chain leads to a measurable (about 20-fold) decrease in the potency of the tested compound with reference to YTX, and the measured EC50 for 45OH-HYTX was 9.35 nM. The relative potency of carboxy-YTX was next assessed. On the basis of the substantial equipotency of YTX and HYTX, we could simplify the evaluation of potency of the CB-(H)YTX sample by expressing the concentrations of carboxyl-containing YTXs as the sum of CB-YTX and CB-HYTX. The results we obtained are reported in Figure 4, which shows that the oxidation of C-55 in the terminal C9 chain of YTX causes a great loss of activity, as compared to (H)YTX, yielding an EC50 for CB(H)YTX of 26.0 nM. Another series of experiments was then carried out to evaluate the potency of N-(H)YTX, and the EC50 we measured for this sample was 49.9 nM (Figure 5). Thus, the data we obtained showed that N-(H)YTX displayed

Figure 4. Effect of MCF-7 cell treatment with increasing concentrations of yessotoxin (YTX) and a mixture of 44-carboxyyessotoxin (CB-YTX) and 44-carboxyhomoyessotoxin (CBHYTX) on E-cadherin. MCF-7 cells have been treated with the indicated concentrations of YTX (solid circles) and a mixture of CB-YTX and CB-HYTX (open circles) for 20 h at 37 °C. The experimental conditions were as described in the legend to Figure 2, and the data represent means ( SD obtained in at least three separate experiments.

the lowest activity among the YTX analogues we have tested, with effective concentrations 2 orders of magnitude higher than those of the reference compound (Figure 2). The results obtained with 45OH-HYTX and CB(H)YTX showed that structural changes in the C9 terminal chain of YTX profoundly affect its activity. To probe the effects of increasing the size and hydrophobicity of the C9 terminal chain on the activity of YTX, the acetate of 45hydroxyyessotoxin and the methyl ester of carboxyyessotoxin were obtained and their activity was assessed, compared to their parent compound. On the basis of the capacity to induce the fragmentation of E-cadherin, the results we obtained showed that acetylation of 45OH-YTX leads to an increased potency, as compared to the parent 45OH-YTX (Figure 6). Similarly, methylation of CB-YTX led to an increase in the activity of the parent compound (not shown). On the basis of these data, we can conclude that an increase in the hydrophobicity of the C9 terminal chain of YTX potentiates the activity of the toxin analogues, and the relative

SAR of Yessotoxins in Cultured Cells

Figure 5. Effect of MCF-7 cell treatment with increasing concentrations of yessotoxin (YTX) and a mixture of noroxoyessotoxin (N-YTX) and noroxohomoyessotoxin (N-HYTX) on E-cadherin. MCF-7 cells have been treated with the indicated concentrations of YTX (solid circles) and a mixture of N-YTX and N-HYTX (open circles) for 20 h at 37 °C. The experimental conditions were as described in the legend to Figure 2, and the data represent means ( SD obtained in at least three separate experiments.

Figure 6. Effect of acetylation of 45OH-YTX on the activity of the YTX analogue. MCF-7 cells have been treated with 5 nM 45OH-YTX (lane 2), 5 nM of its acetylated derivative (lane 3), or with vehicle (lane 1), for 20 h at 37 °C. At the end of the incubation, cells were processed to obtain cytosoluble extracts, which have been fractionated by SDS-PAGE and subjected to immunoblotting using the anti-E-cadherin antibody. The electrophoretic mobilities of β-galactosidase (116 kDa) and fructose6-phosphate kinase (90 kDa) subunits used as marker proteins running in a parallel lane are shown on the left.

increase in the size of the chain, at least in the case of the analogues we have tested, is not detrimental to the activity of the toxin.

Discussion The measurement of ECRA100 accumulation in MCF-7 cells has allowed a preliminary analysis of the structureactivity relationship of YTX analogues in cultured cells. On the basis of the results we obtained, the introduction of a methylene unit adjacent to one of the sulfate groups, as is the case in the HYTX molecule, does not significantly affect the potency of the toxin (Figure 2). Structural changes of the C9 terminal chain of YTX, instead, greatly influences the potency of the compounds, so that the activities of 45OH-HYTX and CB-(H)YTX are between 20- and 50-fold lower than that of the reference compound (Figures 3 and 4). Our findings on the effect of increasing the size and hydrophobicity of the C9 terminal chain of YTX further support the notion that this portion of the molecule controls the activity of the toxin. In particular, the hydrophobicity of the C9 terminal chain of the molecule, rather than its size, has a major role in determining the activity of yessotoxins. The presence of the C9 chain, however, is essential to display

Chem. Res. Toxicol., Vol. 17, No. 9, 2004 1255

Figure 7. Working model for the binding site of YTX on its primary molecular target and of its interaction with the ligand. The symbol “Ψ” denotes the portions of the binding site for the C9 terminal chain of YTX containing amino acids with hydrophobic side chains.

a proper activity of YTX in cultured cells, as the effective concentrations of N-(H)YTX, lacking this moiety, are 2 orders of magnitude higher than those of the reference compound (Figure 5). On the basis of those findings, and assuming that YTX triggers responses by binding to primary molecular targets (receptors) in sensitive systems, some preliminary conclusions about the interaction between YTX and its receptor can be drawn. The C9 terminal chain of YTX, in particular, should represent a primary structural determinant of a productive interaction between YTX and its receptor under our experimental conditions. Furthermore, the chemical modification of the C9 chain would greatly affect the interaction of YTX with its putative receptor, and the finding that changes in different portions of the chain, as in the case of 45OH-HYTX and CB-(H)YTX (Figures 3 and 4), cause a loss of activity suggests that the terminal portion of YTX could be enveloped in a pocket of the YTX binding site on the receptor molecule in MCF-7 cells. Figure 7 shows our working model of the ligand binding site of the YTX receptor, based on the data discussed above. In this model, the YTX molecule is properly positioned in the receptor binding site by salt bridges between the sulfate groups and positive charges on the surface of the protein, and the C9 chain is buried in a pocket of the receptor. The hydrophobic nature of the ladder-shaped portion of YTX could contribute to the interaction, but this would not suffice to bring about the transduction of the YTX signal, as the lack of the C9 chain in N-(H)YTX is accompanied by severe loss of activity. Thus, the C9 chain of YTX would have a role in inducing or stabilizing a “productive” structure of the primary molecular target of the toxin (the receptor), competent to transduce the YTX signal, by establishing proper interactions with the receptor molecule. Our data show that the hydrophobic characteristics of the C9 chain of YTX is a key determinant of its activity and suggest that the proposed pocket in the receptor would involve hydrophobic residues of the polypeptide chain. Thus, the presence of a hydroxy group in 45OH(H)YTX and the carboxy moiety of CB-(H)YTX would cause a relative loss of activity, primarily due to an increase in the polarity of the C9 chain, rather than to an increase in its size, leading to a decrease in the affinity of the YTX receptor for these ligands.

1256

Chem. Res. Toxicol., Vol. 17, No. 9, 2004

Table 2. Potencies of Yessotoxin Analogues in MCF-7 Cells and in Mouse toxin

EC50a (nM)

EC50b (µg/L)

LD50c (µg/kg)

YTX HYTX 45OH-(H)YTX CB-(H)YTX N-(H)YTX

0.6 0.6 9.4 26.0 49.9

0.6 0.6 11.2 31.7 54.5

80-1000 100-400 500d 500 500

a The values indicated have been approximated to the first decimal digit. b On the basis of the substantial equipotency of YTX and HYTX, the EC50 of each toxin analogue has been calculated on the basis of the MW of the reference compound. c The values have been approximated using the data available in the literature and are based on the death times after ip injection in mice. d The lethal dose has been indicated by Satake et al. (7, 16) using male ICR mice, but in a recent study, 45OH-HYTX could not kill female CD-1 mice at a dose of 750 µg/kg in 24 h (21).

An experimental evaluation of this working model, and direct measurements of affinity constants of YTX receptor for its ligands, will be possible after the identification of the receptorial component. Some considerations, however, can be put forth on the basis of the analysis of the data obtained in vitro. The cellular systems used to study YTX have involved hepatocytes (27), glioma cells (19), lymphocytes (28, 29), neuroblastoma cells (30), HeLaS3 cells (31), and MCF-7 cells (23). The effective doses of YTX in those systems have been shown to span two distinct ranges, represented by 10-10-10-9 M (19, 23, 31) and 10-7-10-6 M (27-29), respectively. On the basis of those observations, and the 3 orders of magnitude existing between the ranges of effective doses of YTX in cultured cells, it seems likely that YTX might trigger its responses by interacting with two different receptorial components, whose expression could be cellspecific. The YTX receptor in MCF-7 and glioma cells, in particular, would have a higher affinity for YTX (KD ≈ 10-10 M) than its counterpart in human lymphocytes (KD ≈ 10-7 M). If data on the activity of YTX and its analogues are considered in the light of available information on the molecular mechanism of YTX action, our interpretation of the existence of two separate receptorial components might have some impact on the current views regarding the mode of action of this toxin. On the basis of the YTX concentrations capable to alter ion movements in several subcellular compartments (28, 29), it seems likely that the low affinity YTX receptor might be involved in this type of response. A high-affinity YTX receptor, in turn, would be involved in some cell-specific responses, such as loss of cell adhesion to culture dishes (19, 31) and E-cadherin fragmentation (23). The relevance of our working hypothesis on the tworeceptor systems with regard to the toxicology of YTX in vivo is presently undetermined. The LD50’s reported in the literature for YTX and its analogues, measured by ip injection in mice, vary according to different experimental conditions (6, 7, 12, 1421). If these LD50’s are compared with the EC50’s we have measured in our system (Table 2), then the inspection of data reveals that the values we measured for the EC50 of YTX analogues in our cellular system are up to 1000fold lower than lethal doses recorded in the mouse bioassay. Furthermore, the relative potency of YTX analogues in the mouse bioassay varies in a narrow range as compared to our cellular system (100-fold).

Ferrari et al.

These striking differences between the effective concentrations of YTX and its analogues in intact animals, as compared to cellular systems, could be explained by pharmacokinetic considerations. If YTX absorption, metabolism, and excretion in intact animals do not entirely justify effective doses of the agent that are 3 orders of magnitude higher than those observed with some cellular systems, the relatively high YTX concentrations inducing mouse death could indicate that a low affinity YTX receptor might be also involved in responses triggered in animal models. This interpretation could be supported by the observation that the relative potency of YTX analogues in triggering E-cadherin fragmentation differ by 2 orders of magnitude, whereas only a 5-fold change in potency is found among YTX analogues when mouse death is concerned (Table 2), indicating relevant differences in the structural determinants of YTX involved in eliciting those effects. Work is in progress to probe the involvement of a highaffinity receptor in responses induced by YTX in the intact animals, particularly in the case of long term/ delayed responses.

Acknowledgment. This work was supported by grants from the Italian MIUR (grants MM05171533, and 2002058477).

References (1) Satake, M., MacKenzie, L., and Yasumoto, T. (1997) Identification of Protoceratium reticulatum as the biogenetic origin of yessotoxin. Nat. Toxins 5, 164-167. (2) Tubaro, A., Sidari, L., Della Loggia, R., and Yasumoto, T. (1998) Occurrence of yessotoxin-like toxins in phytoplankton and mussels from northern Adriatic Sea. In Harmful Algae (Reguera, B., Blanco, J., Fernandez, M. L., and Wyatt, T., Eds.) pp 470-472, Xunta de Galicia and IOC/UNESCO, Vigo, Spain. (3) Ciminiello, P., Dell’Aversano, C., Fattorusso, E., Forino, M., Magno, S., Guerrini, F., Pistocchi, R., and Boni, L. (2003) Complex yessotoxins profile in Protoceratium reticulatum from northwestern Adriatic sea revealed by LC-MS analysis. Toxicon 42, 7-14. (4) Murata, M., Kumagai, M., Lee, S. J., and Yasumoto, T. (1987) Isolation and structure of yessotoxin, a novel polyether compound implicated in diarrhetic shellfish poisoning. Tetrahedron Lett. 28, 5869-5872. (5) Lee, J. S., Tangen, K., Darhl, E., Hovgaard, P., and Yasumoto, T. (1988) Diarrhetic shellfish toxins in Norwegian mussels. Nippon Suisan Gakkaishi 54, 1953-1957. (6) Ciminiello, P., Fattorusso, E., Forino, M., Magno, S., Poletti, R., Satake, M., Viviani, R., and Yasumoto, T. (1997) Yessotoxin in mussels of the northern Adriatic Sea. Toxicon 35, 177-183. (7) Satake, M., Tubaro, A., Lee, J. S., and Yasumoto, T. (1997) Two new analogues of yessotoxin, homoyessotoxin and 45-hydroxyhomoyessotoxin, isolated from mussels of the Adriatic Sea. Nat. Toxins 5, 107-110. (8) Yasumoto, T., and Takizawa, A. (1997) Fluorometric measurement of yessotoxins in shellfish by high-pressure liquid chromatography. Biosci. Biotechnol. Biochem. 61, 1775-1777. (9) Ciminiello, P., Fattorusso, E., Forino, M., Magno, S., Poletti, R., and Viviani, R. (1998) Isolation of adriatoxin, a new analogue of yessotoxin from mussels of the Adriatic Sea. Tetrahedron Lett. 39, 8897-8900. (10) Ciminiello, P., Fattorusso, E., Forino, M., Magno, S., Poletti, R., and Viviani, R. (1999) Isolation of 45-hydroxyyessotoxin from mussels of the Adriatic Sea. Toxicon 37, 689-693. (11) Draisci, R., Ferretti, E., Palleschi, L., Marchiafava, C., Poletti, R., Milandri, A., Ceredi, A., and Pompei, M. (1999) High levels of yessotoxin in mussels and presence of yessotoxin and homoyessotoxin in dinoflagellates of the Adriatic Sea. Toxicon 37, 11871193. (12) Ciminiello, P., Fattorusso, E., Forino, M., Poletti, R., and Viviani, R. (2000) A new analogue of yessotoxin, carboxyyessotoxin, isolated from Adriatic Sea mussels. Eur. J. Org. Chem. 291-295. (13) Ciminiello, P., Fattorusso, E., Forino, M., Poletti, R., and Viviani, R. (2000) Structure determination of carboxyhomoyessotoxin, a

SAR of Yessotoxins in Cultured Cells

(14)

(15)

(16) (17)

(18)

(19) (20) (21)

(22)

new yessotoxin analogue isolated from Adriatic mussels. Chem. Res. Toxicol. 13, 770-774. Ciminiello, P., Fattorusso, E., Forino, M., and Poletti, R. (2001) 42,43,44,45,46,47,55-Heptanor-41-oxohomoyessotoxin, a new biotoxin from mussels of the northern Adriatic Sea. Chem. Res. Toxicol. 14, 596-599. Ciminiello, P., Dell’Aversano, C., Fattorusso, E., Forino, M., Magno, S., and Poletti, R. (2002) The detection and identification of 42,43,44,45,46,47,55-Heptanor-41-oxoyessotoxin, a new marine toxin from Adriatic shellfish, by Liquid Chromatography-Mass Spectrometry. Chem. Res. Toxicol. 15, 979-984. Satake, M., Terasawa, K., Kadowaki, Y., and Yasumoto, T. (1996) Relative configuration of yessotoxin and isolation of two new analogues from toxic scallops. Tetrahedron Lett. 37, 5955-5958. Daiguji, M., Satake, M., Ramstad, H., Aune, T., Naoki, H., and Yasumoto, T. (1998) Structure and fluorometric HPLC determination of 1-desulfoyessotoxin, a new analogue isolated from mussels from Norway. Nat. Toxins 6, 235-239. Terao, K., Ito, E., Oarada, M., Murata, M., and Yasumoto, T. (1990) Histopathological studies on experimental marine toxin poisoning-5. The effects in mice of yessotoxin isolated from Patinopecten yessoensis and of a desulfated derivative. Toxicon 28, 1095-1104. Ogino, H., Kumagai, M., and Yasumoto, T. (1997) Toxicologic evaluation of yessotoxin. Nat. Toxins 5, 255-259. Aune, T., Sørby, R., Yasumoto, T., Ramstad, H., and Landsverk, T. (2002) Comparison of oral and intraperitoneal toxicity of yessotoxin towards mice. Toxicon 40, 77-82. Tubaro, A., Sosa, S., Carbonatto, M., Altinier, G., Vita, F., Melato, M., Satake, M., and Yasumoto, T. (2003) Oral and intraperitoneal toxicity studies of yessotoxin and homoyessotoxins in mice. Toxicon 41, 783-792. Yasumoto, T., and Murata, M. (1993) Marine toxins. Chem. Rev. 93, 1897-1909.

Chem. Res. Toxicol., Vol. 17, No. 9, 2004 1257 (23) Pierotti, S., Malaguti, C., Milandri, A., Poletti, R., and Rossini, G. P. (2003) Functional assay to measure yessotoxins in contaminated mussel samples. Anal. Biochem. 312, 208-216. (24) Rossini, G. P., Pinna, C., and Viviani, R. (1997) Inhibitors of phosphoprotein phosphatases 1 and 2A cause activation of a 53 kDa protein kinase accompanying the apoptotic response of breast cancer cells. FEBS Lett. 410, 347-350. (25) Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85. (26) Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. (27) Aune, T. (1989) Toxicity of marine and freshwater algal biotoxins towards freshly prepared hepatocytes. In Mycotoxins and Phycotoxins ‘88 (Natori, S., Hashimoto, K., Ueno, Y., Eds.) pp 461468, Elsevier Science Publishers BV, Amsterdam. (28) de la Rosa, L. A., Alfonso, A., Vilarino, N., Vieytes, M. R., and Botana, L. M. (2001) Modulation of cytosolic calcium levels of human lymphocytes by yessotoxin, a novel marine phycotoxin. Biochem. Pharmacol. 61, 827-833. (29) Alfonso, A., de la Rosa, L. A., Vieytes, M. R., Yasumoto, T., and Botana, L. M. (2003) Yessotoxin, a novel phycotoxin, activates phosphodiesterase activity. Effect of yessotoxin on cAMP levels in human lymphocytes. Biochem. Pharmacol. 65, 193-208. (30) Leira, F., Alvarez, C., Vieites, J. M., Vieytes, M. R., and Botana, L. M. (2002) Characterization of distinct apoptotic changes induced by okadaic acid and yessotoxin in the BE(2)-M17 neuroblastoma cell line. Toxicol. Vitro 16, 23-31. (31) Malaguti, C., Ciminiello, P., Fattorusso, E., and Rossini, G. P. (2002) Caspase activation and death induced by yessotoxin in HeLa cells. Toxicol. Vitro 16, 357-363.

TX030054X