WO1999040912A1 - Procede de stimulation de l'apoptose au moyen de mycotoxines du type trichothecenes - Google Patents

Procede de stimulation de l'apoptose au moyen de mycotoxines du type trichothecenes Download PDF

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WO1999040912A1
WO1999040912A1 PCT/US1999/002665 US9902665W WO9940912A1 WO 1999040912 A1 WO1999040912 A1 WO 1999040912A1 US 9902665 W US9902665 W US 9902665W WO 9940912 A1 WO9940912 A1 WO 9940912A1
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trichothecene
activation
protein synthesis
anisomycin
trichothecenes
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Victor I. Shifrin
Paul J. Anderson
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The Brigham And Women's Hospital, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients

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  • the present invention is directed to methods useful in treating pathological conditions, particularly cancer, associated with abnormalities in cellular apoptosis.
  • the agents used in these methods are trichothecene mycotoxins.
  • the trichothecenes are a structurally related family of low molecular weight mycotoxins synthesized by various species of Fusarium.
  • the ability of the trichothecenes to inhibit the growth of rapidly proliferating cells in vitro and to selectively target tissues with a high mitotic index led to the clinical testing of diacetoxyscirpenol as a treatment of human cancer (Bukowski et al., Cancer Treat. Rep. (5(5:381-383 (1982); Adler, et al, Cancer Treat. Rep. (5S423-425 (1984); DeSimone, et al., Am. J. Clin. Oncol. 9:187-188 (1986); Goodwin, et al, Cancer Treat. Rep.
  • Trichothecenes inhibit the peptidyl transferase reaction by binding to the 60S ribsomal subunit in eukaryotic cells and their antiproliferative activity has been presumed to be a consequence of inhibition of protein synthesis. Because the same mechanism appeared responsible for both therapeutic action and toxicity, interest in trichothecenes as a cancer treatment largely subsided. The identification of trichothecenes that inhibit cancer cell proliferation but which have substantially reduced toxicity would represent a significant advance in clinical medicine.
  • the present invention is based, in part, on the discovery that, contrary to accepted belief, the effect of trichothecenes on cell survival does not depend upon their inhibiting protein synthesis. Instead, it appears that the antiproliferative effect is due to the activation of MAP kinases inducing cellular apoptosis. By carefully screening compounds for high apoptotic activity and low inhibition of protein translation, trichothecenes suitable for the clinical treatment of cancer may be identified.
  • the present invention is directed to a method of treating a patient for a condition associated with an abnormally low level of cellular apoptosis by administering a 2 trichothecene mycotoxin in an amount and for a duration sufficient to significantly increase the rate of apoptosis.
  • a 2 trichothecene mycotoxin in an amount and for a duration sufficient to significantly increase the rate of apoptosis.
  • they may be stained, microscopically examined, and the results compared with comparable cells known to be normal. Other assays, e.g., those described in the Examples section, may also be used.
  • a "significant increase" is one that is statistically significant according to scientifically accepted standards.
  • the condition treated is cancer and the trichothecene induces the death of cancerous cells.
  • Preferred trichothecenes are those that do not substantially inhibit protein synthesis.
  • a trichothecene does not substantially inhibit protein synthesis if, when tested at a concentration of 10 ⁇ M in the in vitro assay described in the Examples section herein, protein synthesis is maintained at a level of at least
  • the preferred trichothecenes are T-2 tetraol and 3- acetyldiacetoxyscirpentriol.
  • the invention is directed to a method for assaying a trichothecene mycotoxin for its potential as a clinically useful anticancer therapeutic by determining both its apoptotic activity and its effect on protein synthesis. Any method for evaluating these properties may be used but the caspase-3 and protein synthesis assays described in the Examples section herein are generally preferred. The higher the ratio of apoptotic to protein synthesis inhibitory activity, the greater the potential that the compound has as a clinically useful drug.
  • the invention is also directed to a method for inducing apoptosis in cultured cells by contacting the cells with a solution that contains one or more trichothecene mycotoxins.
  • Preferred compounds for use in the assay are deoxynivalenol, scirpentriol and T-2 triol. Other compounds that may be used include nivalenol, diacetoxyscirpentriol, HT-2 and diacetylverrucarol.
  • the concentration of mycotoxins can be adjusted to optimize apoptosis using methods that are standard in the art. Studies performed in vitro can be used to help identify cells, e.g., cells obtained in biopsy samples, that are responsive to the mycotoxins. 3
  • the present invention is also directed to a method for assaying a test compound for an ability to induce cellular apoptosis by determining the extent to which the compound interferes with the interaction between trichothecene mycotoxins, or similar compounds, and ribosomes.
  • One way to carry out this method is to perform binding assays in which ribosomes or a ribosomal fragment containing the trichothecene mycotoxin binding site are incubated with a detectably labeled ligand known to modulate apoptosis by ribosomal interaction. The results obtained in the absence of test compound are compared with the results obtained from incubating ribosomes with a detectably labeled ligand in the presence of test compound.
  • Trichothecene mycotoxins that may be labeled and used in the assay include deoxynivalenol, scirpentriol, nevalenol, diacetoxyscirpentriol, HT-2 and diacetylverrucarol.
  • a compound identified as displacing ligand will also be directly assayed for apoptotic activity and for its effect on protein synthesis.
  • Figure 1 shows formulas for nivalenol, scirpenol and T-2 toxin subfamilies of trichothecenes. The arrows point out inter-family differences at the C7 and C8 side chains. For structural features of R1-R4 side chains see Figure 2 A.
  • Figure 2A shows trichothecenes used in experiments discussed herein.
  • the symbol + indicates more than a five-fold activation; the symbol +/- indicates a 2 to 5 fold activation; and the symbol - indicates less than a 2-fold activation.
  • the symbol + indicates strong inhibition, with protein synthesis occurring at a level below 15 percent of control; the symbol +/- indicates weak inhibition, with protein synthesis occurring at between 15 and 70 percent of the control level; and the symbol - indicates very weak inhibition with protein synthesis occurring at 70 percent or more of the control level.
  • Figure 2B shows the effect on protein synthesis of compounds other than trichothecenes. 4
  • Programmed cell death, apoptosis is a normal aspect of animal development and tissue homeostasis.
  • the process serves to regulate cell number, facilitate morphogenesis, remove harmful or abnormal cells, and eliminate cells that have already performed their function.
  • abnormal changes in the rate of cellular apoptosis sometimes occur and have been associated with a number of pathological conditions, including cancer.
  • the present invention is concerned with agents, the trichothecene mycotoxins, that interact with ribosomes and induce apoptotic activity.
  • agents may be used in vitro to determine whether cells are responsive and in experiments designed to elucidate the pathways by which apoptosis is controlled.
  • samples of cultured cells may be harvested, pelleted, and mixed with acridine orange diluted in phosphate-buffered saline.
  • the percentage of cells with apoptotic morphology may then be analyzed microscopically.
  • cells may be stained with propidium iodine as described by Nicoletti et al. (J. Immunol. Methods 139:271-279
  • the percentage of apoptotic cells may then be quantitated using Flow Cytometry with apoptotic cells being distinguished from non-apoptotic intact cells by a decreased DNA content as reflected by lower propidium iodine staining intensity.
  • Other methods for measuring cellular apoptosis are described in the Examples section and may also be used.
  • Agents inducing apoptosis such as the trichothecene mycotoxins may also be delivered in vivo to a patient suffering from a condition characterized by abnormal cellular apoptosis.
  • Biopsy samples obtained from the patient can be used to determine the percentage of cells that are apoptotic and, by comparing the results with similar tissue samples from normal individuals, a determination can be made as to whether abnormal apoptosis is occurring.
  • a physician should begin by administering a low dose of therapeutic agent and then determine whether any improvement has been observed in a patient's condition. For example, an improvement in a cancer patient would be evidenced by a reduction in tumor growth, a reduction in tumor size, a reduction in the number of metastases associated with a tumor, etc. If no response is seen at the initial dosage, it may then be raised until a therapeutic effect is achieved or side effects become unacceptable. 5
  • Preferred trichothecene mycotoxins induce apoptosis but have relatively little effect on protein synthesis.
  • a physician may begin by initially administering an agent at a dosage of, for example, 1 nmol/kg/day and increase the dosage over a period of weeks up to, for example, 1 ⁇ mol/kg/day. During this time, the symptoms of the patient would be periodically evaluated. If no improvement was observed over, for example, a period of three months, drug administration may be discontinued. These are simply guidelines, since the actual dosage will be carefully selected and titrated by the attending physician based upon clinical factors unique to each patient. The optimal daily dose will be determined by methods known in the art and will be influenced by factors such as the age of the patient, disease state, side effects associated with the particular agent being administered, and other clinically relevant factors.
  • the present methods are not limited to any particular dosage form or route of administration. Although oral administration will generally be most convenient, the invention is compatible with parenteral, transdermal, sublingual, buccal, or implantable routes of administration as well. Agents may be given in a substantially purified form or, preferably, as part of a pharmaceutical composition containing one or more excipients or flavoring agents.
  • compositions may also include other active ingredients for the treatment of patients.
  • the preparations may be solid or liquid and take any of the pharmaceutical forms presently used in medicine, e.g., tablets, gel capsules, granules, suppositories, transdermal compositions, or injectable preparations.
  • the active ingredient or ingredients may be incorporated into dosage forms in conjunction with the vehicles that are commonly employed in pharmaceutical preparations, e.g. , talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives, glycols, etc.
  • vehicles that are commonly employed in pharmaceutical preparations, e.g. , talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives, glycols, etc.
  • Methods for preparing appropriate formulations are well known in the art (see, e.g.. Remington's Pharmaceutical Sciences. 16th ed., A. Oslo ed., Easton, PA (1980)).
  • a treatment on disease In order to determine the effect of a treatment on disease, patients should be evaluated on a regular basis over an extended period of time. It may take several weeks for the full therapeutic effect of a treatment to become apparent.
  • the effect of treatment on apoptotic activity can be determined by obtaining biological samples from the patient and then staining 6 them in one of the manners discussed above.
  • the effect of treatment on parameters such as tumor size, tumor growth and tumor metastasis may be determined using standard radiological procedures.
  • the present invention is directed to a method for assaying a test compound to determine if it will induce cellular apoptosis.
  • binding assays isolated ribosomes, or ribosomal fragments containing the site at which trichothecene mycotoxins interact, are incubated together with a ligand known to bind to this site and with the compound being tested.
  • the ligand will be a detectably labeled trichothecene mycotoxin.
  • ribosomes are separated from the solution containing the ligand and test compound, e.g., by centrifugation, and the amount of binding that has occurred is determined.
  • Non-specific binding may be determined by carrying out the binding reaction in the presence of a large excess of unlabeled ligand.
  • labeled ligand may be incubated with ribosome and test compound in the presence of a thousandfold excess of unlabeled ligand.
  • Nonspecific binding should be subtracted from the total binding, i.e., binding in the absence of unlabeled ligand, to arrive at the specific binding for each sample tested.
  • Other steps such as washing, stirring, shaking, filtering and the like may be included in the assays as necessary.
  • compounds identified as displacing the binding of ligand to ribosome be re-examined in a concentration range sufficient to perform a Scatchard analysis on the results. This type of analysis is well known in the art and can be used for determining the affinity of a test compound for receptor (see, e.g., Ausubel et al., Current Protocols in
  • Example 1 Trichothecene Mycotoxins Regulate a Ribosomal Signaling Pathway that Activates JNK/p38 MAP Kinases
  • Trichothecenes and other protein synthesis and protease inhibitors were obtained from Sigma (St. Louis, MO) unless indicated otherwise. Stock solutions were prepared in DMSO at 3.3 mM, or in water at 10 mg/ml (puromycin, emetin and cycloheximide).
  • cells were incubated with the first reagent (or solvents for control samples) for 30 min at 37 °C, before addition of the second reagent (or solvents for control samples) and continued in culture at 37°C for an additional 2 hrs.
  • Cells were collected by centrifugation at 2000x g for 1 min at 4°C, and washed twice with ice-cold phosphate-buffered saline (PBS) before freezing cell pellets in dry-ice ethanol for storage at -80 °C until further analysis.
  • PBS ice-cold phosphate-buffered saline
  • JNK kinase activity was assayed as described previously (Shifrin, et al, J. Biol Chem. 272:2957-2962 (1997)) with slight modifications. Aliquots of 1.0 x IO 7 frozen cells were lysed in 200 ⁇ l of lysis buffer A (20 mM HEPES, pH 7.1, 1% Triton X-100, 50 raM KC1, 5 mM EDTA, 5 mM EGTA, 50 mM ⁇ -glycerophosphate, 2 mM DTT, 1 mM Na 3 PO 4 , 50 mM NaF,
  • calyculin A 10 ⁇ g/ml leupeptin, 1 ⁇ g/ml aprotinin, 1 ⁇ g/ml antipain, 250 mg/ml benzamidine and 20 ⁇ g/ml PMSF
  • incubated on ice for 10 min vortexed for 10 sec and clarified by centrifugation at 15,000x g for 5 min at 4°C.
  • Protein synthesis inhibition assays Protein synthesis was assayed by measuring the incorporation of labeled amino acids into cellular proteins, essentially as described in Ausubel et al, (Current Protocols in Molecular Biology. John Wiley and Sons (1995))with the following modifications: Jurkat cells were grown and collected as described above, washed once with Hank's balanced salt solution and resuspended at 2xl0 6 /ml in cysteine- and methionine-free RPMI-1640 medium supplemented with 10% dialyzed heat-inactivated fetal bovine serum, incubated for 15 min at 37 °C and treated in triplicate with protein synthesis inhibitors (or with corresponding solvents for control samples) for 20 min at 37 °C before the addition of 50 ⁇ Ci/ml of 35 S-labeled methionine/cysteine mixture (NEN, Boston, MA) and incubation for an additional 20 min at 37°.
  • INK andp38 MAP kinases Activation of INK andp38 MAP kinases by selected trichothecenes
  • Jurkat T cells were used to compare the relative ability of anisomycin and trichothecene mycotoxins to inhibit protein synthesis (Table 1) and activate IN and p38 SAMKs.
  • the trichothecenes are divided into three structural subfamilies which differ in the side groups at the C7 and C8 positions of the trichothecene molecule (Fig. 1, arrows; see also Fig. 2A for individual structures). Within all three subfamilies (derivatives of nivalenol, scirpenol and T-2 toxin), members were identified that strongly activate SAMKs (e.g., nivalenol, scirpentriol and
  • T-2 triol as well as members that more weakly activate SAMKs (e.g., 3-acetyldeoxynivalenol, HT-2).
  • SAMKs 3-acetyldeoxynivalenol
  • T-2 triol members that more weakly activate SAMKs
  • Trichothecene-induced translational arrest is not sufficient to activate SAMKs: although T-2 toxin, acetyl T-2 toxin and T-2 triol are strong protein synthesis inhibitors, only T-2 triol activates SAMKs. Moreover, trichothecene-induced translational arrest is not necessary for
  • T-2 tetraol activates SAMKs without significantly inhibiting protein synthesis.
  • the inability of T-2 tetraol to inhibit protein synthesis was reported previously (Middlebrook, et al, J. Pharmacol. Exp. Ther. 250:860-866 (1989)), where it was estimated that T-2 tetraol is about 300-fold less potent, on a molar basis, than T-2 toxin. This reduced potency is not due to decreased ribosome binding, since T-2 tetraol binds to isolated ribosomes as efficiently as T-2 toxin (Middlebrook et al, J. Pharmacol. Exp. Ther. 250:860-866 (1989)). Taken together, these results suggest that the ability of trichothecenes to inhibit protein synthesis is independent of their ability to activate SAMKs.
  • Deacetylanisomycin does not activate JNKs and inhibits anisomycin- and trichothecene-induced .JNK activation
  • SAMKs should prevent the binding of structurally related SAMK-activating compounds to ribosomes.
  • the ability of these compounds to inhibit SAMK activation would be consistent with a requirement ribosome binding for activation of SAMKs.
  • DA As an indirect measure of the ability of DA to penetrate the plasma membrane and interact with ribosomes, the effects of DA on anisomycin-induced translational arrest were measured. At a concentration of 1350 ⁇ M, DA inhibits protein synthesis by approximately 70%). Pre-treatment with DA significantly impairs the ability of both anisomycin and trichothecenes to inhibit protein synthesis in intact cells, as well as in a reticulocyte lysate-based in vitro translation system. Thus, whereas deacetylation of anisomycin greatly reduces its ability to inhibit protein synthesis and eliminates its ability to activate SAMKs, it does not eliminate its ability to enter cells and interact with ribosomes.
  • DA ability of DA to inhibit translational arrest by structurally unrelated compounds that bind to a common site on the large ribosomal subunit (i.e., the trichothecenes) is consistent with a role for DA in the competitive displacement of anisomycin and trichothecenes from this binding site.
  • T-2 triol, T-2 tetraol, and anisomycin potently activate JNK in Jurkat cells
  • pre-treatment of Jurkat cells with DA eliminates JNK activation by these compounds in a dose-dependent manner.
  • Pre-treatment with DA also inhibits nivalenol-, fusarenon- and trichothecene-induced JNK activation.
  • T-2 toxin inhibits anisomycin- and trichothecene-induced JNK activation independently of translational arrest
  • T-2 toxin a trichothecene that inhibits protein synthesis without activating SAMKs, should also 12 inhibit the activation of SAMKs by anisomycin and trichothecenes.
  • T-2 toxin has previously been shown to block anisomycin-induced JNK activation (Iordanov, et al. , Mol Cell. Biol 77:3373-3381 (1997)). It was found that T-2 toxin also blocks trichothecene- induced JNK activation.
  • T-2 toxin or verrucarin A a non-activating trichothecene from the scirpenol/verrucarol subfamily, see Fig. 2 and Table 1
  • verrucarin A a non-activating trichothecene from the scirpenol/verrucarol subfamily, see Fig. 2 and Table 1
  • non-activating trichothecenes block JNK activation by both JNK- activating trichothecenes and anisomycin.
  • T-2 toxin (10 ⁇ M) was used to inhibit protein synthesis in Jurkat cells by >98%> prior to the addition of graded concentrations of anisomycin.
  • T-2 toxin inhibits JNK activation at low concentrations of anisomycin, the inhibitory effect is overcome at higher concentrations of anisomycin, indicating that anisomycin can activate JNK in the absence of protein synthesis.
  • T-2 toxin blocks anisomycin-induced JNK activation not by imposing a translational arrest, but by competing with anisomycin for biding to a common ribosomal site, an effect that can be overcome by increasing the concentration of anisomycin.
  • Protein synthesis inhibitors that bind ribosomes also block trichothecene- and anisomycin-induced JNK activation Competition between anisomycin and trichothecenes, as well as between trichothecenes and harringtonines for binding to a common site on the 60S ribosomal subunit is well documented (Jimenez, et al, Eur. J. Biochem. 54:483-492 (1975); Cannon, et al, Biochem. J. 160:137-45 (1976); Middlebrook, et al, Biochem. Pharmacol. 755:3103-3110 (1989); Fresno, et al, Eur. J. Biochem. 72:323-330 (1977)).
  • Harrington a plant alkaloid structurally unrelated to either anisomycin or the trichothecenes (Fig. 2B) was tested for its ability to activate JNK 13 and/or interfere with JNK activation induced by anisomycin or trichothecenes. It was found that harringtonine does not activate JNKs, but efficiently blocks both anisomycin- and trichothecene- induced JNK activation. This effect is not a consequence of translational arrest per se, since a similar level of protein synthesis inhibition produced by puromycin (which does not bind to ribosomes, but causes premature termination), does not block anisomycin- or trichothecene- induced JNK activation.
  • emetine Another ribosome-binding protein synthesis inhibitor, emetine, was previously reported to block JNK activation by anisomycin (Iordanov, et al., Mol. Cell. Biol. 17:3373-3381 (1997)). Although emetine's binding site is located on the small ribosomal subunit, its close proximity to the trichothecene binding site on the large ribosomal subunit allows it to compete with T-2 toxin for ribosome binding (Leatherman, et al. J. Pharmacol. Exp. Ther. 266:732-740 (1993)).
  • T-2 triol differs from T-2 tetraol only in the addition of a 3-methylbutyryloxy group (R4 in Fig. 2) at the C8 position. It is possible that this side group somehow stabilizes the binding of T-2 triol to the ribosome, preventing its competitive displacement by emetine.
  • pactomycin like emetine, binds to the small ribosomal subunit, it is not known to interfere with the binding of anisomycin or trichothecenes.
  • photoaffinity labeling experiments indicate that it also interacts with the large ribosomal subunit (Synetos, et al, Biochim. Biophys. Ada 5(55:249-253
  • trichothecene derivatives e.g., T-2 tetraol and, to a smaller degree
  • R1-R4 side groups within trichothecene subfamilies and the ability to activate JNKs
  • one structural feature that might favor SAMK activation is the presence of a hydroxyl group at the C3 position of the pentane ring (i.e., Rl in Fig. 2 A; witness nivalenol, deoxynivalenol, fusarenon, scirpenetriol, T-2 triol, and T-2 tetraol).
  • Verrucarol 10 ⁇ M 92.4 (6.5) Diacetyl verrucarol, 10 ⁇ M 7.1 (1.5) Verrucarin A, 10 ⁇ M 1.6 (0.2) Scirpenetriol, 10 ⁇ M 3.6 (0.34) Diacetoxyscirpentriol, 10 ⁇ M 2.4 (0.19) 3-Acetyldiacetoxyscirpentriol, 10 ⁇ M 70.5 (1.8)
  • Example 2 Trichothecene Mycotoxins Trigger a Ribotoxic Stress Response that Activates JNK and p38 MAP Kinases and Induces Apoptosis
  • Trichothecenes and other protein synthesis and protease inhibitors were obtained from Sigma (St. Louis, MO) unless indicated otherwise. Stock solutions were prepared in DMSO at 3.3 mM, or in water at 10 mg/ml (puromycin, emetin and cycloheximide).
  • cells were incubated with the first reagent (or solvents for control samples) for 30 min at 37°C, before addition of the second reagent (or solvents for control samples) and continued in culture at 37 °C for indicated periods of time.
  • Cells were collected by centrifugation at 2000x g for 1 min at 4°C, and washed twice with ice-cold phosphate-buffered saline (PBS) and then frozen in liquid nitrogen for storage at -80 °C until further analysis.
  • PBS ice-cold phosphate-buffered saline
  • DNA fragmentation assay (first apoptosis induction assay)
  • Jurkat human T-lymphoid cells per treatment were lysed in 0.5 ml of 10 mM Tris (pH 7.5), 1% Triton X-100, 5 mM EDTA, incubated on ice for 10 min, vortexed for 5 sec, and lysates were clarified for 5 min at 4°C in an Eppendorf microcentrifuge at top speed. 0.45 ml of the supematants were extracted once with an equal volume of phenol/chloroform (1 :1) and aqueous phases were adjusted to 0.5 M NaCl and precipitated with equal volumes of isopropanol, followed by overnight incubation at -20°C.
  • Precipitates were collected by centrifugation (10 min) at 4°C in an Eppendorf 18 microcentrifiige at top speed, pellets were washed with 70% ethanol, air dried and resuspended in 40 ⁇ l of 10 mM Tris (pH 7.5), 1 mM EDTA, 50 ⁇ g/ml RNase A. Following a 30 min incubation at 37°C, 10 ⁇ l aliquots were separated on 1.2% agarose gels in TAE buffer as described (Tian, et al, Cell 67:629-39 (1991)).
  • Fluorescent assay of caspase-3 activity (second apoptosis induction assay)
  • DEVD-specific caspase activity was determined as described (Nicholson, et al., Nature 376:37-43 (1995)) with modifications: IO 7 cells were resuspended in 0.1 ml of lysis buffer (20 mM HEPES, pH 7.1, 1% Triton X-100, 10 mM KCI, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 5 ⁇ g/ml pepstatin, 10 ⁇ g/ml leupeptin, 2 ⁇ g/ml aprotinin, 25 ⁇ g/ml ALLN (Broehringer Mannheim, Indianapolis, IN)), incubated on ice for
  • DEVD-specific caspase activity was determined in triplicate by mixing 10 ⁇ l of supematants (50 ⁇ g of protein) with 0.2 ml of reaction buffer (100 mM HEPES (pH 7.1), 10% sucrose, 0.1% CHAPS, 10 mM DTT, 0.1 mg/ml BSA with 2 ⁇ M DEVD-AMC) and incubating at 30° C for 20 min.
  • the DEVD-specific caspase activity was calculated by measuring fluorescence of released AMC using a CytoFluor 4000 MultiWell Plate Reader (PerSeptive Biosystems, Framingham, MA) with excitation at 360 nm and emission at 460 nm.
  • the cell lysates used for enzymatic assay of caspase-3 were also subjected to Western blotting analysis with caspase-3 (CPP32)-specific antibodies (PharMingen, San).
  • MAP kinases in Jurkat T cells was compared.
  • structural subfamily i.e., derivatives of nivalenol, scirpenol and T-2 toxin
  • trichothecenes were identified that induce strong (e.g., nivalenol, scirpentriol and T-2 triol), intermediate (e.g., acetyldeoxynivalenol, acetoxyscirpenol, and HT-2), or weak (e.g., verrucarin, T-2 toxin) activation of JNK/p38 kinases.
  • the data indicate that structural differences between individual trichothecenes can influence their ability to trigger the ribotoxic stress response.
  • trichothecenes During analysis of JNK activation by various trichothecenes, it was noticed that many trichothecenes induce what appears to be a typical apoptotic cell death in Jurkat cells. The relative ability of individual trichothecenes to induce various manifestations of apoptosis was assessed by monitoring intemucleosomal DNA fragmentation, processing of pro-caspase-3, activation of DEVD-specific caspases, and cleavage of one of the major caspase-3 substrates - poly (ADP) ribose polymerase (PARP).
  • ADP ribose polymerase
  • nivalenol, diacetoxyscirpentriol, HT-2) and weak (e.g., 3- acetyldeoxynivalenol, varrucarin, T-2) inducers of apoptosis.
  • trichothecenes that similarly activate stress kinases e.g., T-2 triol and T-2 tetraol
  • T-2 triol and T-2 tetraol can differ significantly in their ability to induce apoptosis as measured by caspase-3 activation.
  • the most potent apoptotic trichothecenes strongly activate stress kinases, suggesting that kinase activation contributes to the efficient induction of rapid apoptosis.
  • Inhibition of protein synthesis by trichothecenes can signal cell survival or induce cell death in different cell types under different conditions. Inhibitors of protein synthesis can promote the induction of apoptosis in response to inflammatory cytokines that activate stress kinases (e.g., Fas-ligand, TNF- ), suggesting that the survival pathway, but not the death pathway, requires new protein synthesis (Leist, et al, J. Immunol. 755:1778-88 (1994); Nagata, Cell 55:355-365 (1997)). Table 2 compares the ability of individual trichothecenes to inhibit protein synthesis, activate caspase-3 and activate JNK.
  • inflammatory cytokines e.g., Fas-ligand, TNF-
  • T-2 toxin, and verrucarin block the activation of JNK and caspase-3 by both anisomycin and apoptotic trichothecenes
  • T-2 toxin, verrucarin might inhibit the function (i.e., JNK/p38 kinase and caspase-3 activation) of apoptotic trichothecenes and/or anisomycin.
  • Deacetylanisomycin is an anisomycin analog that enters cells, binds to ribosomes and inhibits protein synthesis (albeit with 10,000-fold lower potency than anisomycin). When used at a concentration that inhibits protein synthesis by 65%> (300 ⁇ g/ml), it fails to activate JNKs on its own, and inhibits activation of JNKs by T-2 triol, T-2 tetraol and anisomycin. At similar concentrations, DA also inhibits anisomycin-induced translational arrest in rabbit reticulocyte lysates, suggesting that its functional effects are a consequence of ribosome binding. T-2 toxin and verrucarin similarly inhibit the activation of JNKs by these compounds.
  • Pre-treatment with either DA, T-2 toxin or verrucarin also prevents caspase-3 activation in Jurkat cells cultured with apoptotic trichothecenes (T-2 triol, diacetylverrucarol and deoxynivalenol.
  • T-2 triol, diacetylverrucarol and deoxynivalenol The ability of DA and non-apoptotic trichothecenes to bind to the peptidyl transferase site and inhibit the function of anisomycin and apoptotic trichothecenes suggests that ribosome binding is required for the activation of stress kinases and, subsequently, caspase- 3. 22
  • T-2 triol differs from T-2 tetraol only in the addition of a 3-methylbutyryloxy group at the C8 position.
  • Harringtonine a plant alkaloid that is structurally unrelated to either anisomycin or trichothecenes, competes with these compounds for binding to the ribosomal peptidyl transferase site (Fresno, et al, Eur. J. Biochem. 72:323-330 (1977); Hobden, et al, Biochem.
  • harringtonine weakly activates JNKs on its own, but efficiently blocks both anisomycin- and trichothecene-induced JNK activation. Both emetine 23 and harringtonine also inhibit caspase activation by apoptotic trichothecenes.
  • the ability of emetine to prevent JNK activation by T-2 triol is reflected in its relative inability to block caspase activation by this trichothecene. Since extended treatment with many inhibitors of protein synthesis can induce apoptosis (Kochi, et al, Exp. Cell. Res.
  • JNK1 Activation of JNK1 by protein synthesis inhibitors that bind to, or alter the structure of, 28S ribosomal RNA (e.g., blastocidin S, gougerotin, anisomycin, ricin toxin, sarcin toxin) led Iordanov, et al. (Mol. Cell. Biol. 77:3373-3381 (1997)) to propose the existence of a ribotoxic stress response in eukaryotic cells.
  • the ability of ribosomes to sense cellular stress and activate signaling pathways that alter cellular function has been well characterized in prokaryotes.
  • prokaryotic ribosomes In response to amino acid starvation, prokaryotic ribosomes produce guanosine 3',5'- bispyrophosphate (ppGpp), a nucleoside analogue that arrests transcription of genes encoding translation factors. This response promotes survival under starvation conditions (Cashel, et al, in Escherichia coli and Salmonella Typhimurium: Cellular and Molecular Biology ,(Neidhardt, et al, eds.) vol. 1, pp. 1410-38, American Society for Microbiology, Washington, D.C. (1997)).
  • ppGpp guanosine 3',5'- bispyrophosphate
  • Trichothecenes that inhibit protein synthesis without activating JNKs e.g., acetyl T-2, T-2 toxin, and verrucarin
  • JNKs e.g., acetyl T-2, T-2 toxin, and verrucarin
  • induction of apoptosis is linearly correlated with the ability to inhibit protein synthesis.
  • this analysis reveals that the ability of individual trichothecenes to induce rapid apoptosis is a function of both translational arrest and stress kinase activation.
  • Pactomycin another compound that inhibits the ribotoxic stress response (Iordanov, et al, Mol. Cell. Biol. 17:3373-3381 (1997)), also binds to the small ribosomal subunit. Although it is not known to interfere with the binding of anisomycin or trichothecenes, photoaffmity labeling experiments indicate that it also interacts with the large ribosomal subunit (Synetos, et al, J, Biochim. Biophys. Acta 838:249-
  • trichothecene derivatives e.g., T-2 tetraol and, to a lesser degree

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Abstract

La présente invention concerne des procédés de modulation de l'apoptose à la fois in vitro et in vivo, au moyen de mycotoxines du type trichothécènes. Elle concerne également un procédé d'identification d'agents qui induisent l'apoptose sans inhiber la synthèse des protéines.
PCT/US1999/002665 1998-02-11 1999-02-08 Procede de stimulation de l'apoptose au moyen de mycotoxines du type trichothecenes WO1999040912A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6559178B1 (en) * 2002-01-29 2003-05-06 Mark Zamoyski Compositions and methods for apoptotic chemosurgery

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5733925A (en) * 1993-01-28 1998-03-31 Neorx Corporation Therapeutic inhibitor of vascular smooth muscle cells
US5855866A (en) * 1992-03-05 1999-01-05 Board Of Regenis, The University Of Texas System Methods for treating the vasculature of solid tumors
EP0896055A2 (fr) * 1996-04-19 1999-02-10 The Cancer Institute Of Japanese Foundation For Cancer Research Proteine inductrice de l'apoptose et gene codant pour elle

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5855866A (en) * 1992-03-05 1999-01-05 Board Of Regenis, The University Of Texas System Methods for treating the vasculature of solid tumors
US5733925A (en) * 1993-01-28 1998-03-31 Neorx Corporation Therapeutic inhibitor of vascular smooth muscle cells
EP0896055A2 (fr) * 1996-04-19 1999-02-10 The Cancer Institute Of Japanese Foundation For Cancer Research Proteine inductrice de l'apoptose et gene codant pour elle

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE MEDLINE 1 January 1900 (1900-01-01), XP002920703, Database accession no. 97101881 *
DATABASE STN CAPLUS 1 January 1900 (1900-01-01), XP002920704, Database accession no. 1997:726866 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6559178B1 (en) * 2002-01-29 2003-05-06 Mark Zamoyski Compositions and methods for apoptotic chemosurgery

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