WO2007116404A2 - Glucosides de cucurbitacine et leur utilisaton dans le traitment du cancer - Google Patents

Glucosides de cucurbitacine et leur utilisaton dans le traitment du cancer Download PDF

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WO2007116404A2
WO2007116404A2 PCT/IL2007/000454 IL2007000454W WO2007116404A2 WO 2007116404 A2 WO2007116404 A2 WO 2007116404A2 IL 2007000454 W IL2007000454 W IL 2007000454W WO 2007116404 A2 WO2007116404 A2 WO 2007116404A2
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cucurbitacin
glucoside
cells
glucosides
isolated
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PCT/IL2007/000454
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WO2007116404A3 (fr
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Tehila Tannin
Shlomo Grossman
Margalit Bergman
Sara Dovrat
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Bar-Ilan University
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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/42—Cucurbitaceae (Cucumber family)

Definitions

  • the present invention relates to cucurbitacin glucosides isolated from Citrullus colocynthis and their synergistic effects on inhibiting growth of human cancer cells including effects on cellular growth, cell cycle distribution, apoptosis and the expression of proteins involved in cell cycle regulation.
  • the plant contains cucurbitacins A, B, C and D and ⁇ -elaterin and probably other biologically active constituents (Adam et al., 2001; Small Rumin Res 40:239-244).
  • the cucurbitacins (highly oxygenated tetracyclic triterpens) are of great interest because of the wide range of biological activity they exhibit in plants and animals. They are predominantly found in the family Cucurbitaceae but are also present in several other families of the plant kingdom. Despite their toxicity, species of the plants in which they are found have been used for centuries in various pharmacopeia. A number of compounds of this group have been investigated for their cytotoxic, hepatoprotective, anti-inflammatory and cardiovascular effects (Jayaprakasam et al., 2003; Cancer Lett 189:11-16).
  • cucurbitacin E The general phenomenon that particular types of cucurbitacins are capable of inhibiting growth of certain types of cancer cells is known.
  • Duncan et al. have identified cucurbitacin E as having potent in vitro growth inhibitory activity against prostate carcinoma explants. It has been shown that cucurbitacin E causes marked disruption of the actin cytoskeleton, and the antiproliferative activity was directly correlated with the disruption of the F-actin cytoskeleton.
  • the distribution of vimentin was also altered in cells exposed to cucurbitacin E, as was shown by the association of vimentin with drug-induced membrane blebs (Duncan et al. 1996; Biochem Pharmacol. 52(10): 1553-1560).
  • US Application No. 2004/0138189 discloses use of cucurbitacin I for treating tumors and cancerous tissues through the modulation of JAK/STAT3 intracellular signaling, particularly by inhibiting the STAT3 activation pathways.
  • the present invention relates to cucurbitacin B and E glucoside, isolated from Citrullus colocynthis and their use for treating cancer, particularly breast cancer.
  • the present invention discloses for the first time the synergistic effects of cucurbitacin glucosides on inhibiting growth of human cancer cells including effects on cellular growth, cell cycle distribution, apoptosis and the expression of proteins involved in cell cycle regulation.
  • the present invention provides for the first time isolated cucurbitacin glucosides from C. colocynthis and has characterized by means of NMR analysis the molecular structures of both cucurbitacin B and E glucosides. These compounds are now shown to have anti proliferative activity on tumor cells.
  • the present invention is based in part on the finding that Citrullus colocynthis and specific constituents extracted therefrom, including cucurbitacins inhibit growth of estrogen dependent (MCF-7) and estrogen-independent (MDA-MB-231) human breast cancer cell lines.
  • inhibition of breast cancer cells may be the result of accumulation of cells in the G 2 ZM phase of the cell cycle accompanied with induction of apoptosis by the cucurbitacin B/E glucosides combination.
  • the inventors of the present invention also identified one of the possible signaling mechanisms through which the cucurbitacin glucosides exert their beneficial effects in the chemoprevention of breast cancer cells.
  • cucurbitacin glucosides treatment caused elevation in phosphorylated STAT3, an active form of STAT3, and in p21 WAF , which was proven to be a STAT3 positive target in the absence of survival signals.
  • the present invention provides an isolated cucurbitacin glucoside from Citrullus colocynthis.
  • the cucurbitacins are characterized by means of NMR.
  • the cucurbitacin glucoside is selected from the group consisting of cucurbitacin B glucoside and cucurbitacin E glucoside.
  • the cucurbitacin B and E glucosides have characteristic NMR parameters as presented in Table 1 hereinbelow.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of at least one isolated cucurbitacin glucoside from Citrullus colocynthis or a salt thereof and a pharmaceutically acceptable excipient, diluent or carrier.
  • the cucurbitacin glucoside is selected from the group consisting of cucurbitacin B glucoside and cucurbitacin E glucoside.
  • the cucurbitacin B and E glucosides are characterized by certain NMR parameters presented in Table 1 hereinbelow.
  • the pharmaceutical composition comprises a plurality of cucurbitacins isolated from C. colocynthis.
  • the pharmaceutical composition comprises a therapeutically effective amount of a combination of cucurbitacin B glucoside and cucurbitacin E glucoside.
  • the present invention provides a method for treating a tumor in a subject in need thereof comprising administering a pharmaceutical composition comprising an isolated cucurbitacin glucoside of the invention.
  • the method comprises administering a pharmaceutical composition comprising a plurality of isolated cucurbitacin glucosides that act synergistically to reduce the proliferation of the tumor cells.
  • the cucurbitacin glucosides are cucurbitacin B glucoside and cucurbitacin E glucoside isolated from C. colocynthis.
  • the cucurbitacin B glucoside and cucurbitacin E glucoside have characteristic NMR parameters as presented in Table 1 hereinbelow.
  • the pharmaceutical composition of the present invention is administered in combination with at least one additional anti-tumor treatment.
  • the additional anti tumor treatment is selected from the group consisting of, but not limited to, radiation therapy, chemotherapy, immunotherapy, hormonal therapy and genetic therapy.
  • the tumor is human breast tumor.
  • FIG. 2 illustrates a dose dependent effect of cucurbitacin glucosides combination (B and E in 1 :1 ratio) on proliferation of estrogen-independent MDA-MB-231 cells.
  • the data represent the mean ⁇ SE of 3 experiments where each treatment was performed in 3 wells.
  • FIG. 3 shows the effect of cucurbitacin B/E glucosides on cell cycle distribution in MDA-MB-231 and MCF7 cells.
  • the data shown are representative of three independent experiments with similar findings.
  • FIG. 4 shows the effect of cucurbitacin glucosides combination (E+B, 1:1) on cell cycle distribution.
  • A MCF7 cells treated with cucurbitacin glucosides combination (5.8, 11.6 and 14.5 ⁇ g/ml). The values indicate the percentage of cells in the indicated phases of the cell cycle. The data shown are representative of three independent experiments with similar findings; B: Histograms showing the number of cells per channel (vertical axis) versus DNA content (horizontal axis).
  • FIG. 5 is a Western blot analysis of extracts obtained from MCF7 and MDA-MB-231 cells treated with the cucurbitacin glucosides combination.
  • FIG. 6 shows the effect of cucurbitacin glucosides combination treatment on MDA- MB-231 cell morphology.
  • FIG. 7 is a Western blot analysis of extracts obtained from MCF7 and MDA-MB-231 cells treated with the cucurbitacin glucosides combination.
  • Cells were treated with 0 (-) and 14.5 ⁇ g/ml (+) cucurbitacin glucosides combination for 24 h.
  • Extracts were prepared and analyzed by Western blotting with an antibody to STAT3, phospho- STAT3 (Tyr-705).
  • A antibody to p21
  • B antibody to survivin
  • C An antibody to ⁇ - actin was used as a loading control.
  • Western blots are representative of three independent experiments.
  • FIG. 8 shows annexin V binding and propidium iodide uptake induced by the cucurbitacin glucosides combination treatment.
  • the horizontal (FLl- H) and vertical (FL2-H) axes represent labeling with annexin V and propidium iodide (PI), respectively;
  • B Graphic presentation of data obtained by Annexin/PI staining after 24h treatment using 5.8 ⁇ g/ml and 14.5 ⁇ g/ml cucurbitacin glucosides combination.
  • UR represents late apoptotic cells (positive for both Annexin and PI);
  • LL represents live cells. The data shown are representative of three independent experiments with similar findings.
  • FIG. 9 shows the effect of cucurbitacin glucoside combination on membrane potential.
  • Cells were analyzed on a FACScan cytometer.
  • FL2 red fluorescence
  • FLl green fluorescence
  • Citrullus colocynthis leaves contain constituents that inhibit the growth of human breast cancer cells, and therefore may be useful in the prevention or treatment of breast cancer, or other forms of cancer.
  • the present invention discloses that these novel ingredients act synergistically to retard the growth of exemplary human breast tumor cell lines.
  • the present invention provides for the first time a detailed NMR analysis of cucurbitacin glucosides isolated from C. colocynthis leaves and their inhibiting activity on breast cancer cells through G 2 /M arrest and apoptosis.
  • treating a tumor or “inhibiting a tumor” as used herein is intended to encompass tumor formation, primary tumors, tumor progression or tumor metastasis.
  • reduction of growth in relation to cancer cells, in the context of the present invention refers to a decrease in at least one of the following: number of cells
  • Reduction of growth of cancer cells may be utilized for the treatment of cancer by the administration, to an individual in need of such treatment, of a therapeutically effective amount of the compound of the present invention, as described herein.
  • the terms "subject” or “patient” refers to a human or non-human mammal. Subjects in need of treatment involving inhibition of tumor growth can be identified using standard techniques known to those in the relevant art. Preferred embodiments of the invention
  • the cucurbitacin glucosides of the present invention were extracted from the leaves of Citriillus colocynthis with water, followed by few more extractions to yield cucurbitacin B glucoside and cucurbitacin E glucoside purified fractions as described hereinbelow. Cucurbitacin I glucoside was also extracted using the same methods. It is to be understood that cucurbitacin B/E glucosides extracted by other methods as are known in the art are also encompassed within the scope of the present invention. Analogues of the cucurbitacin glucosides of the present invention as well as salt thereof, as along as they exert growth inhibitory activity on tumor cells may be also used according to the teaching of the present invention.
  • Cucurbitacin B/E glucoside combination (1 :1) exhibited growth inhibitory activity of human breast cancer cell lines in a dose- and time-dependent manner as can be seen by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay ( Figure 2).
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay ( Figure 2).
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay ( Figure 2).
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
  • Figure 2 Cucurbitacin B and E combination treatment inhibited cell growth with IC 5O value of 5.8 ⁇ g/ml after 48h for the estrogen
  • the present invention now shows a significant synergistic effect on cell cycle arrest when a combination of both cucurbitacin was applied to estrogen dependent as well as to estrogen independent cancerous breast cells (Figure 3).
  • p34 and cyclin Bl are known as the key proteins regulating G 2 /M transition.
  • the p34 DC protein kinase is generally acknowledged to be an important mediator of G 2 /M phase transition in all eukaryotic cells (Liang et al. 2003; Biochem Pharmacol 65:1053-1060).
  • the active mitotic kinase (MPF, or mitosis-promoting factor) is a dimer comprised of catalytic subunit, a B-type cyclin and a cyclin dependent kinase termed p34 CDC2 or CDKl .
  • the cyclins are a class of proteins that are synthesized during the interphase of each cell cycle and are rapidly degraded at the end of mitosis (Xiao et al. 2003; Carcinogenesis 24:891-897).
  • the activity of the p34 CDC2 kinase not only depends on its association with cyclin Bl, but also on its phosphorylation state. Phosphorylation of either Thrl4 or Tyrl5 inhibits p34 CDC2 kinase activity, while phosphorylation on Thrl ⁇ l by CDK7 kinase is required for kinase activity.
  • the dephosphorylation ofThrH or Tyrl5 by CD C25C phosphatase is a final step for p34 CDC2 kinase activity (Molinari M. 2000; Cell Prolif 33:261-274) and also serves as a main component in regulating G 2 /M arrest in respond to DNA damaging agents.
  • Treating cells with the cucurbitacin B/E glucosides combination of the invention caused a reduction at the protein level of both p34 CDC2 and Cyclin Bl. This reduction was very rapid and could be detected as early as 1 h post treatment. By 24 h only remnant of p34 and cyclin Bl protein were observed ( Figure 5A). This phenomenon indicates a highly effective activity, as by 24 h of treatment most of the cells were arrested in G 2 /M stage in which p34 and cyclin Bl are mostly expressed. Moreover, when phosphorylated p34 was followed, a sharp decrease in phosphorylation status was observed, with a similar kinetics compared to the inhibition at the protein level (Figure 5B).
  • treating cancerous cells with cucurbitacin B/E glucosides may cause cell cycle arrest at G 2 /M by reducing the amount and hence the activity of p34 CDC2 /cyclin Bl complex, which is necessary for G 2 to M transition, and not by inhibition of p34 CDC2 activity by its phosphorylation.
  • Cucurbitacin B/E treatment also caused a marked change in cell morphology with a disruption of the elongated shape of the cells toward a round shape appearance, as was demonstrated by both tubulin staining and by light microscopy visualization (Figure 6). These results suggest that cucurbitacin treatment cause impairment in actin filament organization. This profound morphological change might as well influence intracellular signaling by molecules such as PKB, resulting in inhibition in transmission of survival signals. Indeed, as is shown in Figure 7, cucurbitacin glucosides treatment caused a sharp reduction in survivin expression and, on the other hand, induced p21 w expression as expected in a situation where STAT3 is enhanced in the presence of inhibited PKB signaling.
  • the inhibitory effect of cucurbitacin B/E glucoside on breast cell proliferation can be attributed to both STAT3 activation and at the same time inhibition of PKB signaling through disruption of cytoskeleton filaments causing inhibition in survivin expression.
  • apoptosis The preferred outcome of a chemotherapeutic drug is apoptosis.
  • cell surface annexin V binding which measure the appearance of phosphatidylserine on the external plasma membrane and by binding of a fluorescent dye, known as JC-I that indicates changes in mitochondrial membrane potential ( ⁇ ) (Bijl et al. 2003; Arthritis Rheum 48:248-254; Nihal et al. 2005; Int J Cancer 114:513-521).
  • JC-I a fluorescent dye that indicates changes in mitochondrial membrane potential ( ⁇ )
  • cucurbitacin glucosides effectively inhibit proliferation of both estrogen-dependent and estrogen-independent human breast cancer cells by causing G 2 /M phase arrest and apoptosis.
  • the use of the cucurbitacin glucosides combination of the present invention is advantageous to the use of non- glucosilated cucurbitacin for the treatment of cancer, as the glucosilated forms were found to have minor toxic effects as compared to cucurbitacins that do not contain a glucoside moiety (Bartalis and Halaweish 2005; J Chromatogr B Analyt Technol
  • cucurbitacin E glucoside, cucurbitacin B glucoside, or preferably, a combination thereof are formulated as a pharmaceutical composition to be used for treating a tumor according to the teaching of the present invention.
  • the pharmaceutical compositions of the invention will be administered to the patient by standard procedures.
  • the amount of compound to be administered and the route of administration will be determined according to the kind of rumor, stage of the disease, age and health conditions of the patient.
  • the pharmaceutical composition of the present invention may be administered by any suitable means, such as topically, orally or parenterally including intranasal, subcutaneous, intramuscular, intravenous, intraarterial, intraarticular, or intralesional administration. Ordinarily, intravenous (i.v.), intraarticular or oral administration will be preferred.
  • the compound used according to the invention can be formulated by any required method to provide pharmaceutical compositions suitable for administration to a patient.
  • the cucurbitacin compounds of the present invention include all hydrates and salts that can be prepared by those of skill in the art. Under conditions where the compounds of the present invention are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate.
  • pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, alpha-ketoglutarate, and alpha-glycerophosphate.
  • Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
  • the novel compositions contain, in addition to the active ingredient, conventional pharmaceutically acceptable carriers, diluents and the like as are known in the art (see, for example, Ansel, Pharmaceutical Dosage Forms and Drug Delivery Systems. Malvern, PA: Williams and Wilkins, 1995; Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, flavoring agents, binders, surface active agents, thickeners, lubricants, preservatives, (including antioxidants) and the like.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, flavoring agents, binders, surface active agents, thickeners, lubricants, preservatives, (including antioxidants) and the like.
  • compositions for use in accordance with the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Solid compositions for oral administration such as tablets, pills, capsules or the like, may be prepared by mixing the active ingredient with conventional, pharmaceutically acceptable ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate and gums, with pharmaceutically acceptable diluents.
  • the tablets or pills can be coated or otherwise compounded with pharmaceutically acceptable materials known in the art to provide a dosage form affording prolonged action or sustained release.
  • Other solid compositions can be prepared as microscapsules for parenteral administration.
  • Liquid forms may be prepared for oral administration or for injection, the term including subcutaneous, intramuscular, intravenous, and other parenteral routes of administration.
  • compositions include aqueous solutions, with or without organic cosolvents, aqueous or oil suspensions, emulsions with edible oils, as well as similar pharmaceutical vehicles.
  • compositions of the present invention may be formed as encapsulated pellets or other depots, for sustained delivery.
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective to prevent, delay, alleviate or ameliorate one or more symptoms associated with a disorder being treated over a period of time. Particularly, a disorder according to the teaching of the present invention is tumor growth.
  • cucurbitacin B or E glucoside, or a pharmaceutically acceptable salt or analogue thereof can be administered to a patient as sole active ingredient, or co-administered with another compound.
  • a combination of cucurbitacin B/E or a pharmaceutically acceptable salt or analogue thereof is administered.
  • the cucurbitacin B/E combination of the present invention can be administered to a subject as adjunctive therapy with an additional antitumor agent. Co-administration can be carried out simultaneously (in the same or separate formulations) or consecutively.
  • the adjunctive therapy is selected from the group consisting of radiation therapy, chemotherapy, immunotherapy, hormonal therapy and genetic therapy.
  • the cucurbitacin formulations of the present invention can include various other components as additives.
  • acceptable components or adjuncts which can be employed in relevant circumstances include antioxidants, free radical scavenging agents, peptides, growth factors, antibiotics, bacteriostatic agents, immunosuppressive, anticoagulants, buffering agents, anti-inflammatory agents, antipyretics, time-release binders, anesthetics, steroids, and corticosteroids.
  • Such components can provide additional therapeutic benefit, act to affect the therapeutic action of the cucurbitacin compound, or act towards preventing any potential side effects which may be posed as a result of administration of the cucurbitacin compound.
  • the cucurbitacin compounds of the subject invention can be conjugated to a therapeutic agent, as well.
  • Additional agents that can be co -administered to a subject in the same or as a separate formulation include those that modify a given biological response, such as immunomodulators.
  • proteins such as tumor necrosis factor (TNF), interferon (such as alpha-interferon and beta-interferon), nerve growth factor (NGF), platelet derived growth factor (PDGF), and tissue plasminogen activator can be administered.
  • Biological response modifiers such as lymphokines, interleukins (such as interleukin-1 (IL-I), interleukin-2 (IL-2), and interleukin-6 (IL-6)), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), or other growth factors can be administered.
  • lymphokines such as interleukin-1 (IL-I), interleukin-2 (IL-2), and interleukin-6 (IL-6)
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • Toxicity and therapeutic efficacy of the cucurbitacin compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC50 (the concentration which provides
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (e.g. Fingl, et al, 1975, in "The
  • the dosage may be an escalating dosage so that low dosage may be administered first, and subsequently higher dosages may be administered until an appropriate response is achieved.
  • the dosage of the composition can be administered to the subject in multiple administrations in the course of the treatment period in which a portion of the dosage is administered at each administration.
  • dosing can also be a single administration of a slow release composition, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • the therapeutically effective amount of the molecule according to the present invention will depend, inter alia upon the administration schedule, the unit dose of molecule administered, whether the molecule is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the molecule administered and the judgment of the treating physician.
  • Fresh leaves of Citrullus colocynthis (L.) Shrad were mixed 1 :4 (w/v) with distilled water and homogenized in a blender for about 5 min. The homogenate was filtered and centrifuged at 20,000xg for 10 min. The supernatant was frozen in liquid nitrogen and was dried in lyophilizer (0.07 mbar, -48 0 C). The powder was further extracted in 70% chloroform/methanol overnight, filtered and evaporated under reduced pressure. The resulting extract was examined by thin layer chromatography (silica gel 60 F 254 plates, Merck Eurolab SA, France) using the solvent system: chloroform/methanol (9:1).
  • the two cucurbitacin glucosides were separated by another TLC (0.25 mm layer thickness) using the solvent system benzene-ethanol (8:2). The compounds were visualized under UV.
  • MCF7 estrogen receptor positive
  • MDA-MB-231 estrogen receptor negative
  • FCS fetal calf serum
  • the effect of the cucurbitacin glucoside treatment on survival/proliferation of MCF7 and MDA-MB-231 cells was measured by a modified 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma, MO) assay based on the ability of live cells to cleave in active mitochondria the tetrazolium ring to a molecule that absorbs at 570 nm (Mosmann 1983; J Immunol Methods 65:55-63).
  • MTT 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl tetrazolium bromide
  • 7x10 3 cells were grown on a 96-well microliter plate and incubated with cucurbitacin B/E glucoside or their combination (2.9, 5.8 and 14.5 ⁇ g/ml) in RPM-10% FCS medium. After 24, 48, and 72h, the medium was changed and 130 ⁇ l/well of fresh RPMI- 1640 media were added. Next, 20 ⁇ l MTT reagent (5 mg/1 ml PBS) was added to each well, and the cells were further incubated at 37 0 C for 2h.
  • MCF-7 and MDA-MB-231 cells (5x10 5 ) were seeded in 100 mm culture dishes, and allowed to attach overnight. The medium was replaced with fresh complete medium containing desired concentration of cucurbitacin B/E glucoside (B: 15 and 23 ⁇ g/ml; E: 10 and 18 ⁇ g/ml) or their combination (5.8, 11.6, 14.5 ⁇ g/ml). Cells were incubated for 24 h at 37°C. The cells were washed with PBS and then spun at 300g. The pellet was resuspended in 250 ⁇ l PBS and 250 ⁇ l propidium iodide (50 ⁇ g/ml final concentration) for 15 min, and analyzed using a flow cytometer.
  • Apoptosis was determined based on morphological change. Apoptotic cells were quantified by annexin V-FITC and propidium iodide (PI) double staining by using a staining kit purchased from MBL Co., Ltd. (Watertown, MA).
  • PI propidium iodide
  • JC-I (5,5 ' ,6,6 ' -tetrachloro- 1 , 1 ' ,3 ,3 ' -tetraethyl-benzamidazolocarbocyanin iodide, sigma, MO) was used for in-situ detection of mitochondrial membrane transition events in live cells, which provides an early indication of the initiation of cellular apoptosis.
  • JC-I used to indicate the collapse in the electrochemical gradient across the mitochondrial membrane ( ⁇ ).
  • JC-I exists as a monomer in the cytosol (green) and also accumulates as aggregates in the mitochondria (red).
  • JC-I exists in monomeric form and stains the cytosol green.
  • cells were treated with the cucurbitacin glucoside combination (0- control, 5.8, 14.5 ⁇ g/ml) and maintained at 37 0 C in a humidified 5% CO 2 atmosphere for 24h. Cells were washed with PBS and spun at 30Og.
  • the pellet was resuspended in 500 ⁇ l PBS, and 2 ⁇ l of 1 mg/ml JC-I reagent was added for 20 minutes in 37 0 C in the dark. Cells were washed with PBS and analyzed using a flow cytometer. The analysis was performed using Cell Quest software (BD Biosciences, San Jose, CA) for apoptosis.
  • MCF7 and MDA-MB-231 cells (1x106) were seeded in 100 mm culture dishes.
  • the cells were treated with the cucurbitacin glucosides combination (14.5 ⁇ g/ml) in RPMI- 1640 media for 24h. The media was then aspirated, and the cells were washed with cold PBS. The cells were scraped and washed twice by centrifugation at 500xg for 5 min at 4 0 C. The pellet was resuspended in lysis buffer supplemented with proteases and phosphatase inhibitors and incubated for Ih at 4°C.
  • the lysate was collected by centrifugation at 14,000xg for 40 min at 4°C, and the supernatant (total cell lysate) was stored at -2O 0 C.
  • total cell lysate was stored at -2O 0 C.
  • Western blot analysis 30 ⁇ g protein were resolved over 12% polyacrylamide gels and transferred to a nitrocellulose membrane.
  • the blot was blocked in blocking buffer (1% nonfat dry milk/1% Tween 20 in PBS) for 1 h at room temperature, incubated with appropriate monoclonal primary antibodies (human reactive anti- p34 , anti-Survivin, anti- STAT3 from Santa Cruz Biotechnology, anti- Cyclin Bl from Biosource and anti-p21 WAF from BD Biosciences) or polyclonal primary antibodies (human reactive anti-phospho- p34 CDC2 (Thrl4 and Tyrl5) from Santa Cruz and anti-phospho- STAT3 from cell signaling) in blocking buffer overnight at 4°C.
  • monoclonal primary antibodies human reactive anti- p34 , anti-Survivin, anti- STAT3 from Santa Cruz Biotechnology, anti- Cyclin Bl from Biosource and anti-p21 WAF from BD Biosciences
  • polyclonal primary antibodies human reactive anti-phospho- p34 CDC2 (Thrl4 and Tyrl5) from Santa Cruz and anti-phospho- STAT3 from cell signaling
  • the blot was then incubated with anti-mouse secondary antibody horseradish peroxidase conjugate and detected by chemiluminescence and autoradiography using X- RAY film, ⁇ -actin detected on the same membrane and used as a loading control.
  • Cells were grown on glass coverslips and fixed after treatment (14.5 ⁇ g/ml of the cucurbitacin glucosides combination for 24h) in 4% paraformaldehyde solution for 30 min. Cells were washed twice with PBS, and blocked by 90% blocking solution (1% BSA, 0.5% Triton in PBS) and 10% Fetal calf serum, for Ih. Incubation with ⁇ -tubulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was performed in blocking solution over night at 4°C. Cells were washed four times with PBST (0.2% Tween) and incubated with secondary antibody (in blocking solution) for Ih. The glass slides were analyzed using a confocal microscope. Statistical analysis
  • MDA-MB-231 cells The proliferation of MDA-MB-231 cells was reduced by 50% upon a 48 h exposure to 5.8 ⁇ g/ml cucurbitacin glucoside combination, while using cucurbitacin B or E glucoside alone in this concentration was less effective. Antiproliferative activity of the cucurbitacin glucoside combination was further confirmed by counting live cells (using trypan blue exclusion dye). Proliferation of MDA-MB-231 cells was inhibited upon treatment in dose- and time-dependent manner.
  • Cucurbitacin E was previously shown to interfere with cell cytoskeleton (Duncan and Duncan 1996; 1997; supra), and to cause marked disruption of the actin cytoskeleton. Staining analysis using light microscopy was employed to measure the effect of the cucurbitacin glucosides combination of the invention on cell morphology, particularly on cytoskeleton elements. Viewing cells after treatment (14.5 ⁇ g/ml) compared to control, using light microscopy, showed clearly a profound change in the overall morphology of the cell from an elongated typical MDA-MB -231 cells to a round shape cells (Figure 6A). Histochemical staining with ⁇ -tubulin confirmed this result (Figure 6B).
  • cucurbitacin treatment may brings about the specific condition in which STAT3 activity is exploited in favor of preventing cell proliferation rather than enhancing it. Indeed, the present invention now demonstrates that cucurbitacin treatment elevates p21 waf expression and inhibits survivin expression, exactly as expected ( Figure. 7B-7C). Thus, without wishing to be bound to a specific mechanism or theory, it is postulated that cucurbitacin exhibit pleiotropic effects on cells causing both disruption of actin which reduces PKB signaling and enhancement of STAT3 activity culminating in both elevated expression of p21 waf and reduction in survivin expression level.
  • the apoptosis inducing effect of cucurbitacin glucoside combination was evaluated by annexin V/PI binding.
  • One of the earliest events of apoptosis is loss of plasma membrane polarity, which is accompanied by translocation of phosphatidylserine (PS) from the inner to outer membrane leaflets, thereby exposing PS to the external environment (Vermes et al. 1995; J Immunol Methods 184:39-51).
  • PS phosphatidylserine
  • the phospholipid-binding protein Annexin V has a high affinity for PS and can bind to cells with externally exposed PS.
  • annexin V positive, PI negative positive staining with fluorescently labeled annexin V correlates with loss of membrane polarity but precedes the complete loss of membrane integrity that accompanies later stages of cell death resulting from either apoptosis or necrosis.
  • PI can only enter cells after loss of membrane integrity.
  • dual staining with annexin V and PI allows clear discrimination between unaffected cells (annexinV negative, PI negative), early apoptotic cells (annexin V positive, PI negative), and late apoptotic cells (annexin V positive, PI positive).

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Abstract

La présente invention concerne des glucosides de cucurbitaciii isolés à partir de Citrullus colocynthis ainsi que leurs effets synergiques sur l'inhibition de la croissance des cellules cancéreuses humaines, notamment leurs effets sur la croissance cellulaire, la distribution du cycle cellulaire, l'apoptose et l'expression des protéines impliquées dans la régulation du cycle cellulaire.
PCT/IL2007/000454 2006-04-10 2007-04-10 Glucosides de cucurbitacine et leur utilisaton dans le traitment du cancer WO2007116404A2 (fr)

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WO2012115723A1 (fr) 2011-02-25 2012-08-30 Medtronic, Inc. Passage en mode d'urgence pour les modes sans stimulation
WO2012134520A2 (fr) * 2010-08-31 2012-10-04 Sami Labs Limited Régulation de la réponse immunitaire par la colocynthine et/ou ses dérivés
WO2013103384A1 (fr) 2012-01-06 2013-07-11 Elcelyx Therapeutics, Inc. Compositions à base de biguanide et procédés de traitement de troubles métaboliques
WO2013103919A2 (fr) 2012-01-06 2013-07-11 Elcelyx Therapeutics, Inc. Compositions et procédés de traitement de troubles métaboliques
CN103360452A (zh) * 2012-04-04 2013-10-23 浙江大学 甜瓜蒂四环三萜葫芦素类化合物的制备和应用
WO2014011926A1 (fr) 2012-07-11 2014-01-16 Elcelyx Therapeutics, Inc. Compositions comportant des statines, des biguanides et d'autres agents pour réduire un risque cardiométabolique
WO2014107617A1 (fr) 2013-01-05 2014-07-10 Elcelyx Therapeutics, Inc. Compositions et méthodes permettant de traiter des affections métaboliques
US9238069B2 (en) 2009-12-16 2016-01-19 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Method of sensitizing cancer cells to the cytotoxic effects of death receptor ligands in cancer treatment
CN107727787A (zh) * 2017-09-15 2018-02-23 四川辅正药业股份有限公司 一种鉴别雪胆品种的薄层色谱鉴别方法
EP3763419A1 (fr) 2011-01-07 2021-01-13 Anji Pharma (US) LLC Traitements à base de ligand de récepteur chimiosensoriel
ES2906476A1 (es) * 2020-10-13 2022-04-18 Univ Granada Extracto etanólico de semillas de Citrullus colocynthis, método para obtenerlo, composición farmacéutica que lo contiene y su uso como agente antitumoral

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9238069B2 (en) 2009-12-16 2016-01-19 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Method of sensitizing cancer cells to the cytotoxic effects of death receptor ligands in cancer treatment
WO2012134520A2 (fr) * 2010-08-31 2012-10-04 Sami Labs Limited Régulation de la réponse immunitaire par la colocynthine et/ou ses dérivés
WO2012134520A3 (fr) * 2010-08-31 2013-02-07 Sami Labs Limited Régulation de la réponse immunitaire par la colocynthine et/ou ses dérivés
EP3763419A1 (fr) 2011-01-07 2021-01-13 Anji Pharma (US) LLC Traitements à base de ligand de récepteur chimiosensoriel
WO2012115723A1 (fr) 2011-02-25 2012-08-30 Medtronic, Inc. Passage en mode d'urgence pour les modes sans stimulation
WO2013103384A1 (fr) 2012-01-06 2013-07-11 Elcelyx Therapeutics, Inc. Compositions à base de biguanide et procédés de traitement de troubles métaboliques
WO2013103919A2 (fr) 2012-01-06 2013-07-11 Elcelyx Therapeutics, Inc. Compositions et procédés de traitement de troubles métaboliques
CN103360452B (zh) * 2012-04-04 2016-02-24 浙江大学 甜瓜蒂四环三萜葫芦素类化合物的制备和应用
CN103360452A (zh) * 2012-04-04 2013-10-23 浙江大学 甜瓜蒂四环三萜葫芦素类化合物的制备和应用
WO2014011926A1 (fr) 2012-07-11 2014-01-16 Elcelyx Therapeutics, Inc. Compositions comportant des statines, des biguanides et d'autres agents pour réduire un risque cardiométabolique
WO2014107617A1 (fr) 2013-01-05 2014-07-10 Elcelyx Therapeutics, Inc. Compositions et méthodes permettant de traiter des affections métaboliques
CN107727787A (zh) * 2017-09-15 2018-02-23 四川辅正药业股份有限公司 一种鉴别雪胆品种的薄层色谱鉴别方法
ES2906476A1 (es) * 2020-10-13 2022-04-18 Univ Granada Extracto etanólico de semillas de Citrullus colocynthis, método para obtenerlo, composición farmacéutica que lo contiene y su uso como agente antitumoral

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