WO2004093891A1 - Vegetable extract compositions comprising inodole acetic, methods for the preparation of said compsitions, and their use as cell growth inhibiting agents - Google Patents

Vegetable extract compositions comprising inodole acetic, methods for the preparation of said compsitions, and their use as cell growth inhibiting agents Download PDF

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Publication number
WO2004093891A1
WO2004093891A1 PCT/EP2004/004382 EP2004004382W WO2004093891A1 WO 2004093891 A1 WO2004093891 A1 WO 2004093891A1 EP 2004004382 W EP2004004382 W EP 2004004382W WO 2004093891 A1 WO2004093891 A1 WO 2004093891A1
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vegetable extract
plant material
acid
cells
biologically active
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PCT/EP2004/004382
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French (fr)
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Hubert Jean Marie François GILLESSEN
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Veijlen N.V.
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Publication of WO2004093891A1 publication Critical patent/WO2004093891A1/en

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    • 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)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • 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)
    • 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)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • 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)
    • A61K36/31—Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi
    • 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)
    • 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)
    • A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed

Definitions

  • the present invention relates to biologically active compositions inhibiting cellular growth and especially inhibiting cellular growth by induction of apoptosis.
  • the present invention further relates to methods for the preparation of said biologically active compositions and the
  • compositions as growth inhibiting agents and especially the use of said biologically active compositions for the induction of apoptosis in for example cancer cells.
  • apoptosis is an active process that is subject to intricate control
  • the aspartate-specific proteases are critically involved in the apoptotic process in mammalian cells and serve to incapacitate specific substrates, thereby leading to the disassembly of nuclear and cytoskeletal structures, disabling of cell repair and tagging 0 of the apoptotic cell for engulfment by phagocytes.
  • indole acetic acid like growth inhibiting properties, and especially the apoptosis inducing properties, can synergistically be enhanced by combining indole acetic acid (IAA) with a vegetable extract, for example, obtained from the potato tuber, or one or more components thereof.
  • the present invention relates to a biologically active composition comprising indole acetic acid (IAA), and a vegetable extract (i) or one or more components thereof, which extract is obtainable by a method comprising: a) grounding a plant material; b) extracting the grounded plant material; c) charging a Q hyper DTM20 gel with the extracted and grounded plant material to ionically bind the extracted and grounded plant material to the gel; d) subjecting the charged Q hyper DTM20 gel to an increasing NaCl gradient to elute the bound extracted and grounded plant material form the gel; e) collecting the fraction which elutes at 1.02 M NaCl to obtain vegetable extract (i) .
  • the biologically active composition according to the present invention does not only relate to a composition comprising indole acetic acid as such, but also to biologically active compositions comprising analogues or derivatives of indole acetic acid (IAA) .
  • Indole acetic acid can be substituted.
  • in nature halogenated indole alkaloids can be found, particularly in marine organisms (i.e. 6-bromoindigotin) .
  • indole acetic acid can be conjugated with other molecules, such as conjugates via an ester bond, in particular with various sugars, for example IAA-glucose, IAA-alfa-aspartic acid lN-glucoside, IAA- inositol, IAA-myoinositols, IAA-various carbohydrates or conjugates as amides for example with amino acids and peptides.
  • Examples thereof are acetamide, alfa-leucine, alfa- alanine, alfa-aspartate (most important conjugate of IAA) , alfa-glutamate, alfa-lysine, alfa-glycine, alfa-valine and alfa-phenylalanine . Conjugation with peptides is common, whereas also conjugates with other amino acids occur in different plants. In addition, this group comprises 3-acetonitrile derivatives, which easily are converted into the corresponding acid, like indole-3-acetonitrile that decomposes in indole acetic acid (IAA) both chemically (under basic conditions) and catalytically (by. nitrilases) .
  • IAA indole acetic acid
  • amide linked indole acetic acid (IAA) constitutes approximately 90%, whereas approximately 10% is ester-linked and approximately 1% is free indole acetic acid (IAA) .
  • the levels of free + bound indole acetic acid (IAA) are about 1.2 ⁇ g/g Dry Weight (for Arabldopsis 9 days old, later lower) . Of this only approximately 1% is free indole acetic acid (IAA) .
  • indole acetic acid (IAA) or analogues or derivatives thereof are combined with a vegetable extract (i) , or one or more components thereof, which vegetable extract (i) is obtainable by a) grounding a plant material; b) extracting the grounded plant material; c) charging a Q hyper DTM20 gel with the extracted and grounded plant material to ionically bind the extracted and grounded plant material to the gel; d) subjecting the charged Q hyper DTM20 gel to an increasing NaCl gradient to elute the bound extracted and grounded plant material form the gel; e) collecting the fraction which elutes at
  • vegetable extract (i) 1.02 M NaCl to obtain vegetable extract (i) .
  • the term "one or more components thereof" is used to indicate those components of the vegetable extract (i) that provide the observed synergistic effect in combination with indole acetic acid (IAA) .
  • These components can be a single compound present vegetable extract (i) , like 5-alpha- pregnane-3-alpha 17.20 beta thriylthoxy, or a combination of compounds present in vegetable extract (i) , like carboiimide, octonoic acid, sucrose, 5-alpha-pregnane-3-alpha 17.20 beta thriylthoxy, L-norvaline, ribonic acid, propylene glycol, 1- amino ethanol, butanoic acid, L-valine, L-alanine, L- isoleucine, L-proline, L-serine, L-threonine, 3,4- dihydroxybutanoic acid, tetronic acid,
  • the plant material to be grounded is of vegetable origin containing glucide, protein, glycoprotein and terpene .
  • plant material are potato tubers, radish, turnip, beet, carrot, Jerusalem artichoke, sweet potato tubers, rape, cucumber, pumpkin, courgette, tomato, and the like.
  • Any method known in the art can be used for grounding the plant material. Examples of such methods are methods involving pressing of the plant material to pulp, milling of the plant material, cutting of the plant material using for example in a blender, freezing, for example in liquid nitrogen, and subsequent grounding of the plant material, a combination of a hot/cold treatment and grounding, and the like.
  • the grounded plant material is subjected to an extraction step. This extraction step can also simultaneously be performed with the grounding step of the plant material like grounding the plant material in the presence of an extraction liquid such as water or an organic liquid.
  • Extraction methods are generally known in the art and the present invention is not restricted to a specific extraction method or to a specific extraction liquid as long as the extraction method and/or liquid do not significantly reduce the synergistic properties of the vegetable extract obtained.
  • Suitable extraction methods according to the present invention are for example described by Rebiere C.J.P.; "Plant extract used as cell growth factor medicament with antitumoural and anticancer activity”; patent application no. FR2732347 and by Parinaud J. , Milhet P., Vietez G., Richoilley G.; "Human sperm capacitation and in-vitro fertilization in a chemically defined and protein-free medium SMART1®”; Human Reproduction (1998), 13: 2579-2582.
  • a Q hyper DTM20 gel is charged with the material to ionically bind the material to the gel.
  • a specific example of this gel charging step is for example described in FR2732347 and by Parinaud et al . (supra) .
  • the charged Q hyper DTM20 gel is subjected to an increasing NaCl gradient, for example from 0 to 1.5 M NaCl, see also FR2732347 and Parinaud et al. (supra) , resulting in, depending on the strength of the ionic interaction between the gel and compounds in the extracted and grounded plant material, in different fractions comprising compounds eluted at a specific NaCl concentration or interval.
  • the vegetable extract (i) providing the synergistic properties in combination with indole acetic acid (IAA) is comprised in the fraction eluting at an interval or concentration of 1.02 M NaCl.
  • the amount vegetable extract (i) in the biologically active composition according to the present invention ranges from 1 to 10 mg/ml, like 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/ml.
  • the vegetable extract (i) is in the form of a vegetable extract marketed under the name LoxatTM available from Optipharma B.V., Maastricht, The Netherlands.
  • optimal growth inhibiting properties of the biologically active composition are obtained at concentration indole acetic acid (IAA) ranging from 2*10 "4 M to 3*10 "3 M, like 2*10 "4 , 3*10 "4 , 4*10 "4 , 5*10 ⁇ 4 , 6*10 ⁇ 4 , 7*10 "4 , 8*10 "4 , 9*10 "4 , 1*10 "3 , 2*10 "3 , or 3*10 "3 M.
  • IAA concentration indole acetic acid
  • the molar concentrations are calculated using the molecular weight of free indole acetic acid (IAA, 175 g/mol) . It should be understood that the specific values of these molar concentrations will vary depending if the above- mentioned derivatives and/or analogues of indole acetic acid (IAA) comprised in the biologically active composition. In the above molar concentrations range, a preferable balance is provided between the desired growth inhibiting properties of the biologically active composition according to the present invention and the cytotoxic effect of indole acetic acid (IAA) at high concentrations.
  • indole acetic acid (IAA) is comprised in a second vegetable extract (ii) obtainable by a method comprising: a) grounding a plant material; b) extracting the grounded plant material; c) charging a Q hyper DTM20 gel with the extracted and grounded plant material to ionically bind the extracted and grounded plant material to the gel; d) subjecting the charged Q hyper DTM20 gel to an increasing NaCl gradient to elute the bound extracted and grounded plant material form the gel; e) collecting the fraction which elutes at 0.78 M NaCl to obtain vegetable extract (ii) comprising indole acetic acid (IAA) .
  • a second vegetable extract ii) obtainable by a method comprising: a) grounding a plant material; b) extracting the grounded plant material; c) charging a Q hyper DTM20 gel with the extracted and grounded plant material to ionically bind the extracted and grounded plant material to the gel; d) subjecting the charged Q hyper DTM
  • any plant material can be used.
  • the plant material to be grounded is of vegetable origin containing glucide, protein, glycoprotein and terpene.
  • examples of such plant material are potato tubers, radish, turnip, beet, carrot, Jerusalem artichoke, sweet potato tubers, rape, cucumber, pumpkin, courgette, tomato, and the like.
  • any method known in the art can be used for grounding the plant material. Examples of such methods are methods involving pressing of the plant material to pulp, milling of the plant material, cutting of the plant material for example in a blender, freezing, for example in liquid nitrogen, and subsequent grounding of the plant material, a combination of a hot/cold treatment and grounding, and the like.
  • the grounded plant material is subjected to an extraction step.
  • this extraction step can also be simultaneously performed with the grounding step of the plant material like grounding the plant material in the presence of an extraction liquid such as water or an organic liquid.
  • Extraction methods are generally known in the art and the present invention is not restricted to a specific extraction method or to a specific extraction liquid as long as the extraction method and/or liquid do not disadvantageously reduce the synergistic properties of the vegetable extract obtained.
  • Suitable extraction methods according to the present invention are for example described in FR2732347 and by Parinaud et al . (supra).
  • a Q hyper DTM20 gel is charged with the material to ionically bind the material to the gel.
  • a specific example of this gel charging step is for example described in FR2732347 and by Parinaud et al . (supra) .
  • the charged Q hyper DTM20 gel is subjected to an increasing NaCl gradient, for example from 0 to 1.5 M NaCl, see also FR2732347 and Parinaud et al.
  • the vegetable extract (ii) comprising indole acetic acid (IAA) is comprised in the fraction eluting at an interval or concentration of 0.78 M NaCl.
  • the vegetable extract (ii) is in the form of a vegetable extract marketed under the name NoclosanTM available from Optipharma B.V., Maastricht, The Netherlands.
  • optimal growth inhibiting properties of the biologically active composition are obtained at concentration of the vegetable extract (ii), comprising indole acetic acid (IAA) , ranging from 1 to 10 mg/ml, like 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/ml.
  • IAA indole acetic acid
  • the above defined biologically active composition according to the present invention provides advantageously synergistic properties with respect to the, growth inhibiting characteristics of indole acetic acid (IAA) . Therefore, the present invention also relates to the use of this biologically active composition as a growth inhibiting agent, especially as an apoptosis inducing agent of eukaryotic cells.
  • IAA indole acetic acid
  • the growth inhibiting properties of the biologically active compositions according to the present invention are especially suitable to be used on cancer cells, either of human or animal origin, present in, for example, non-small and small cells lung carcinoma's, lung adenocarcinoma' s, colon carcinoma's and adenocarcinoma' s, breast carcinoma's and adenocarcinoma' s, ovary carcinoma's and adenocarcinoma' s, leukemic carcinoma's and adenocarcinoma' s, monocystic leukemia, T-cell lymphoma's, epidermal carcinoma's and adenocarcinoma' s, like skin carcinoma's and adenocarcinoma' s, umbilical cord carcinoma's and adenocarcinoma' s, microvascular endothelial carcinoma's and adenocarcinoma' s, rhabdomyosarcoma' s, and the like.
  • the present invention also relates to a method for the preparation of a biologically active composition comprising indole acetic acid (IAA) and a vegetable extract (i) comprising: a) grounding a plant material; b) extracting the grounded plant material; c) charging a Q hyper DTM20 gel with the extracted and grounded plant material to ionically bind the extracted and grounded plant material to the gel; d) subjecting the charged Q hyper DTM20 gel to an increasing NaCl gradient to elute the bound extracted and grounded plant material form the gel; e) collecting the fraction which elutes at
  • any plant material can be used as starting material and also grounding, extraction, and isolation of vegetable extract (i) can be performed using any method generally known in the art. Examples of suitable methods are described in FR2732347 and by Parinaud et al. (supra) .
  • the above method provides a biologically active composition
  • a biologically active composition comprising 2*10 ⁇ 4 M to 3*10 "3 M indole acetic acid (IAA), like 2*10 '4 , 3*10 4*10” , 5*10 "4 , 6*10 “4 , 7*10 “4 , 8*l ⁇ 9*10 ⁇ 4 , 1*10 ""3 , 2*10 "3 , or 3*10 "3 M, and 1 mg/ml to 10 mg/ml vegetable extract (i) , like 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/ml.
  • Optimal growth inhibiting properties of the biologically active composition obtained are provided using these concentration ranges.
  • the molar concentrations of indole acetic acid (IAA) are calculated using the molecular weight of free indole acetic acid (IAA, 175 g/mol) . It should be understood that the specific values of these molar concentrations will vary depending if the above derivatives and/or analogues of indole acetic acid (IAA) comprised in the biologically active composition. In the above concentration ranges, a preferable balance is provided between the desired growth inhibiting properties of the biologically active composition according to the present invention and the cytotoxic effect of indole acetic acid (IAA) and vegetable extract (i) at high concentrations.
  • the indole acetic acid, or analogues or derivatives thereof, in the above method according to the present invention is comprised in a vegetable extract (ii) , which extract is obtainable by: a) grounding a plant material; b) extracting the grounded plant material; c) charging a Q hyper DTM20 gel with the extracted and grounded plant material to ionically bind the extracted and grounded plant material to the gel; d) subjecting the charged Q hyper DTM20 gel to an increasing NaCl gradient to elute the bound extracted and grounded plant material form the gel; e) collecting the fraction which elutes at
  • any plant material can be used as starting material and also grounding, extraction, and isolation of vegetable extract (ii) can be performed using any method generally known in the art. Examples of suitable methods are described in FR2732347 and by Parinaud et al. (supra) .
  • the above method provides a biologically active composition comprising 1 mg/ml to 10 mg/ml vegetable extract (ii) , like 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/ml, and 1 mg/ml to 10 mg/ml vegetable extract (i), like 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/ml..
  • Optimal growth inhibiting properties of the biologically active composition obtained are provided using these concentration ranges.
  • concentration ranges a preferable balance is provided between the desired growth inhibiting properties of the biologically active composition according to the present invention and the cytotoxic effect of vegetable extract (ii) and vegetable extract (i) at high concentrations .
  • the invention will further be illustrated in the examples that follow and these examples are not intended to limit the invention in any way. In the Examples reference is made to the following figures:
  • Figure 1 Growth curves of CaCo2 cells (A), MR65 cells (B) and MCF7 cells (C, D) grown in control culture medium (squares) or in the presence of NoclosanTM (triangles) or LoxatTM (diamonds) .
  • A, B D 5 mg/ml of both compounds was used, while in Figure 1C 2.5 mg/ml of both reagents was applied.
  • the number of cells (ordinate) is plotted versus the time in culture (abscissa) . Note the significant delay in growth of all cell lines when cultured in the presence of 5 mg/ml NoclosanTM or LoxatTM.
  • Figure 2 The S-phase progression (bars) and percentage (lines) of apoptosis in MCF7 (A), CaCo2 (B) , MR65 (C) and U937 (D) cells, cultured in the presence of various doses of NoclosanTM (solid bars, solid diamonds) or LoxatTM (open bars, open diamonds) .
  • NoclosanTM solid bars, solid diamonds
  • LoxatTM open bars, open diamonds
  • Figure 3 MR65 (a-d) and CaCo2 (e-h) cells stained with the anti-tubulin antibody E7.
  • Cells were cultured for 5 hours (a,c,e,g) or 24 hours (b,d,f,h) in the presence of 5 mg/ml NoclosanTM (b,c,g,h) or LoxatTM (a,b,e,f). Note the metaphase arrest after 5 hours of culture and the appearance of apoptotic figures after 24 hours.
  • Figure 4 The G2/M-phase exit and percentage of apoptosis in CaCo2 (A), MR65 (B) and U937 (C) cells, cultured in the presence of various doses of NoclosanTM (solid bars, solid diamonds) or LoxatTM (open bars, open diamonds) .
  • NoclosanTM solid bars, solid diamonds
  • LoxatTM open bars, open diamonds
  • FIG. 5 The induction of apoptosis in MR65 cells by NoclosanTM or LoxatTM.
  • MR65 cells were cultured for 7 hours with 10 mg/ml NoclosanTM or LoxatTM.
  • Cells were either stained with FITC-labeled Annexin-V (solid bars) or harvested and assayed for caspase 3/7 activity using Ac-DEVD-AMC (open bars) or M30-CytoDeath reactivity (striped bars) .
  • the general caspase inhibitor z-VAD was used to block apoptosis.
  • the CDK- inhibitor Roscovitine was used as a positive control. Note that both NoclosanTM and LoxatTM induced apoptosis in MR65 cells. The caspase inhibitor was not able to inhibit the exposure of the M30-CytoDeath epitope in apoptotic cells.
  • FIG. 6 Western blotting analysis of M30 CytoDeath in MR65 cells treated with NoclosanTM or LoxatTM. The asterisks indicate the characteristic cleavage fragments of cytokeratin 18.
  • Figure 7 The effect of NoclosanTM or LoxatTM on the population doubling time (Td) in CaCo2 (solid bars), MR65 (diagonally downwards striped bars), OVCAR (diagonally upwards striped bars), MCF7 (open bars and vertically striped bars) , Jurkat (horizontally striped bars) and U937 (speckled bars) cells.
  • CaCo2, MR65, OVCAR and MCF7 (open bars) cells were cultured in the presence of 5 mg/ml NoclosanTM or LoxatTM, MCF7 (vertically striped bars), Jurkat and U937 cells were cultured in the presence of 2.5 mg/ml NoclosanTM or LoxatTM. Doubling times were expressed as percentage of the unperturbed exponential growth.
  • Figure 8 A,B) The effect of repeated administration of LoxatTM (A) or NoclosanTM (B) to CaCo2 cells. LoxatTM or
  • NoclosanTM was administered either at a single dose of 5 mg/ml and the cultures were left unchanged (diamonds) or cultures were changed every 24 hours with a fresh dose (triangles) . Exponentially growing cells (squares) were used as a control. The number of cells (solid figures) is plotted versus the time in culture (abscissa) . The number of apoptotic cells (open figures) remains low. Note that there is no difference in either growth behaviour of the cells or the frequency of apoptotic cells when the two culture conditions are compared. C,D,E) The effect of repeated administration of NoclosanTM or LoxatTM on the induction of apoptosis in MCF7 (C) , MR65 (D) .
  • LoxatTM was administered either at a single dose of 5 mg/ml and the cultures were left unchanged (solid bars) or cultures were changed every 24 hours with a fresh dose (open bars) .
  • NoclosanTM was administered as a single dose (diagonally downwards striped bars) or culture media were repeatedly renewed (diagonally upwards striped bars) .
  • Exponentially growing cells were used as a control. The number of M30 CYTODEATH-positive cells is shown.
  • Figure 9 The effect of combined administration of NoclosanTM and LoxatTM on MR65 (A) and CaCo2 (B) cells.
  • Cells were cultured with various combinations of doses of both compounds as indicated on the abscissa. After 7 hours cells were assayed for M30 CYTODEATH reactivity (ordinate) and the measured (open bars) and the expected (solid bars) number of apoptotic cells are plotted. The expected number of apoptotic cells is calculated as the sum of the numbers of apoptotic cells measured when the compounds were administered individually in the indicated dose. Note that the measured effect is higher than the expected, indicating a synergistic effect of both compounds.
  • Figure 10 The kinetics of various caspase activities in MR65 cells after induction of apoptosis by either LoxatTM (A) or NoclosanTM (B) .
  • Caspase 2 open circle
  • 3/7 square
  • 5/4/1 solid diamond
  • 6 open diamond
  • 8 triangles
  • 9 solid circle
  • Figure 11 MR65 (a) and HaCat (b) cells stained with the anti-active caspase 8 antibody 11G10. Cells were cultured for 6 h. in the presence of 5 mg/ml NoclosanTM and 10 mg/ml LoxatTM
  • (c) MR65 control cells without the addition of the apoptosis inducing agent.
  • Figure 12 Western blotting analysis of active
  • the compounds carboiimide, octonoic acid, sucrose, 5-alpha-pregnane-3-alpha 17.20 beta thriylthoxy, L-norvaline, and ribonic acid form the major components of loxatTM.
  • MR65 cells non-small cell lung cancer
  • NCI-H125 lung adenocarcinoma
  • MCF7 cells breast cancer
  • CaCo2 cells colon carcinoma
  • OVCAR3 ovary carcinoma
  • U937 cells monocytic leukemia
  • Jurkat T cell lymphoma
  • the cell lines were cultured in RPMI 1640 medium without L-Glutamine (Life Technologies, GIBCO-BRL, Scotland) , supplemented with 0.1 % gentamycin (Eurovet, Bladel, The Netherlands), 1 % L-glutamine (22942, Serva, Heidelberg,
  • HaCat cells As a source of human keratinocytes immortalized HaCat cells (Boukamp P., Petrussevska R.T., Breitkreutz D. , Hornung J., Markham A., Fusenig N.E.; "Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line"; J. Cell . Biol . (1988), 106: 761-771.) were used. These cells were grown as adherent cell cultures in EMEM, supplemented with 10% fetal calf serum and non essential amino acids.
  • Human umbilical vein endothelial cells were obtained as primary cultures after trypsinisation of umbilical cord tissue.
  • Trypsin (2.5 % trypsin (Flow #16-893-49), 2 % di- natrium EDTA (Titriplex) and 2 % glucose in PBS) was used to harvest the cells.
  • NoclosanTM and LoxatTM were isolated from the potato tuber ( Solanum tuberosum) .
  • the raw plant material is grounded, water extracted and further purified by ion exchange chromatography on Q hyper DTM20 gel, using increasing concentrations of NaCl.
  • the extended purification protocols FR2732347 and Parinaud et al.
  • NoclosanTM and LoxatTM on the induction of apoptosis HaCat cells were treated for 7 h with either NoclosanTM and LoxatTM alone or a combination of both compounds.
  • the following combinations were tested: NoclosanTM (10, 5 and 1 mg/ml), LoxatTM (10, 5 and 1 mg/ml) and NoclosanTM + LoxatTM (10 mg/ml + 10 mg/ml; 5 mg/ml + 10 mg/ml; 1 mg/ml + 10 mg/ml; 10 mg/ml + 2 mg/ml; 5 mg/ml + 2 mg/ml; 1 mg/ml + 2 mg/ml) .
  • BrdUrd pulse chase experiment Briefly, BrdUrd (Serva) was added to the culture medium in a final concentration of 10 ⁇ M for 30 minutes. Then, the cells were rinsed twice with prewarmed PBS and chased in prewarmed culture medium supplemented with 5 ⁇ M deoxythymidine (Serva) and various doses of NoclosanTM and LoxatTM. After approximately 6 hours ' cells were harvested. 1.5 Kinase inhibitors
  • erk-2 inhibitor (PD 98059) was used (Cell Signalling Technology Inc., Beverly MA, USA). Staurosporine (C 2S H 26 N 4 0 3 ) (Sigma-Aldrich, Zwijndrecht, The Netherlands) was used as an inhibitor of the protein kinase C-pathway.
  • phosphatidylserine (PS) exposure at the outer leaflet of the membrane cells were labelled with annexin V OregonGreen (Nexins Research BV, Hoeven, The Netherlands). Propidium iodide (PI) was added in a final concentration of 5 mg/ml to probe for loss of membrane integrity.
  • BrdUrd was detected as described previously (14). Briefly, approximately 10 6 ethanol fixed cells were rinsed once in PBS and resuspended in 2 ml of 0.4 mg/ml pepsin in 0.1 N HC1 (Serva, Heidelberg, Germany). After 30 minutes at room temperature cells were pelleted, resuspended in 2 N HC1 and incubated for another 30 minutes at 37 °C. Cells were rinsed in 0.1 M borate buffer, pH 8.5 and PBS/BSA (lmg/ml BSA in PBS).
  • anti-BrdUrd antibody (clone IIB5, available form Euro-Diagnostica B.V., Arnhem, The Netherlands) was added to the cell pellet, resuspended in 100 ml PBS/BSA. After incubation for 1 h at room temperature, the cells were rinsed twice in PBS/BSA. For visualization FITC conjugated Fab2 fragments of rabbit anti-mouse Ig (DAKO A/S, ' Glostrup, Denmark) antibody was added in a 1:10 dilution.
  • Apoptosis was detected and quantified as described previously (15) . Briefly, methanol fixed cells were rinsed once in PBS. Appropriately diluted M30 CytoDeath antibody (Boeringer, Mannheim) was added to approximately 10 5 cells, resuspended in 100 ml PBS/BSA. After incubation for 1 h at room temperature, the cells were rinsed twice in PBS/BSA. For visualization FITC conjugated Fab 2 fragments of rabbit anti- mouse Ig (DAKO A/S, Glostrup, Denmark) antibody was added in a 1:10 dilution.
  • Cleaved (activated) caspase 8 was detected with mouse monoclonal antibody 11G10 obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA)
  • Tubulin staining was performed. using the monoclonal antibody E7 (Developmental Studies Hybridoma Bank at the University of Iowa, Iowa, USA) using the protocol as described above for M30 CytoDeath.
  • Scraped cells were combined with cells from the supernatants and allowed to lysate on ice for 20 minutes. After addition of an equal volume of electrophoresis sample buffer (62.5 mM Tris-HCl, 10 % glycerol, 2.3 % SDS, 5 % ?-mercaptoethanol (Bio-Rad) , 0.05 % bromophenol blue (Sigma) the cell lysates were boiled for 5 minutes and subsequently centrifuged for 5 minutes in a microfuge (Sigma) .
  • electrophoresis sample buffer 62.5 mM Tris-HCl, 10 % glycerol, 2.3 % SDS, 5 % ?-mercaptoethanol (Bio-Rad)
  • 0.05 % bromophenol blue (Sigma) the cell lysates were boiled for 5 minutes and subsequently centrifuged for 5 minutes in a microfuge (Sigma) .
  • Blots were incubated with the respective antibodies (diluted in blocking buffer) for 1 hour at RT or overnight at 4°C. After washing three times with PBS containing 0.5 % Triton X-100 (Scintran, Brunschwig Chemie, Amsterdam, The Netherlands) blots were incubated with horseradish peroxidase conjugated rabbit anti mouse immunoglobulins (DAKO) , diluted in blocking buffer, for 1 hour at RT. Signals were visualized using ECL detection reagent (Amersham Pharmacia, Roosendaal, The Netherlands), according to the manufacturer's instructions and subsequent exposure to X-ray film (Fuji Photo Film Europe, Dusseldorf, Germany) .
  • ECL detection reagent Amersham Pharmacia, Roosendaal, The Netherlands
  • NCI-H125 and MR65 cells were washed 3 times with PBS and harvested in cold 50 mM Tris/HCl, pH 7.5 with a disposable cell scraper. Subsequently 1 X 10 7 cells/ml were sonicated for 5 seconds using a Soniprep 150 sonicator (MSE Scientific Instruments, Beun-De Ronde) , (amplitude 12 micron). Cell lysates were pelleted at 10,000 g at 4°C in a microfuge (Sigma, Ostenrode Am Harz, Germany) and supernatants were either immediately assayed or frozen at -20°C.
  • Caspase activity was measured in 50 ⁇ l aliquots of cell lysate, which is the.
  • the following 7-amino-4-methyl coumarin (AMC) -labeled caspase substrates were used: WEHD (caspase 1, 4 and 5), DEVD (caspase 3,7), IETD (caspase 3- processing and 8), VEID (caspase 6), VDVAD (caspase 2), LEHD (caspase 9), YVAD (caspase 1, 4). All the substrates were purchased from Alexis (Kordia, Leiden, NL) .
  • BrdU positive divided cells N ld
  • BrdU negative G2 cells ' G 2 _
  • BrdU negative Gl cells G ⁇
  • BrdU negative Gl and S cells G X S-
  • the means of the four parameters were plotted as a function of time after BrdUrd pulse labeling for each dose of olomoucine and roscovitine tested.
  • linear portions of the kinetic curves were fitted by linear regression analysis.
  • the linear regression analysis was performed on the logarithmically transformed data. The slopes of the regression lines were used to elucidate the changes in the rate of cell cycle progression.
  • NoclosanTM and LoxatTM showed a dose dependent effect on cell cycle progression in all cell lines tested. For instance, at low to intermediate doses (i.e. 1.25-5 mg/ml) the progression of different cancer cell lines through the S-phase of the cell cycle was unaffected (Fig. 2). At high doses (i.e. 10-20 mg/ml) a complete cell cycle arrest was observed. At intermediate doses cells tend to accumulate in the G2/M phase of the cell cycle. Both G2/M-exit and Gl- entry rates were decreased in a dose dependent manner. Cells were in general more sensitive to the NoclosanTM as compared to LoxatTM.
  • NoclosanTM and LoxatTM were effective in the induction of apoptosis at high doses. In some cell lines a toxic effect prevailed.
  • Accumulation of cells in the G2/M phase of the cell cycle at intermediate doses did not lead to an increase in apoptosis (Fig. 4), but it was obvious that after 24 hours both compounds induced apoptosis in particular in those cells that were arrested in metaphase (Fig. 3).
  • Apoptosis was confirmed by presence of ⁇ DEVD-like' activity, the immunocytochemical detection of cells positive for active caspase-3 and the absence of apoptotic cells when cells were treated in the presence of the general caspase inhibitor zVAD-fmk (Fig. 5) .
  • CaCo2 cells were insensitive for the induction of apoptosis by NoclosanTM or LoxatTM at a dose of 10 mg/ml. In contrast a sudden rise in the number of necrotic cells (i.e. Annexin V+/PI+) cells was observed when these cells were cultured in the presence of NoclosanTM, indicating a toxic effect of the compound (data not shown) .
  • MR65 cells responded almost immediately to LoxatTM treatment by an exponential rise in caspase 2, 3, 6 and 3/8 processing activities. Caspase 1 and 9 activities remained at almost baseline values. In contrast NoclosanTM treatment resulted in an almost linear increase of caspase 2, 3, 6, 9 and 3/8 processing activities.
  • MR65 and HaCat cells were immunocytochemically examined for the presence of active caspase 8 after treatment with a combination of LoxatTM and NoclosanTM, both cell lines showed positively in a fraction of the cells (figure 11a and lib) 2.6 Signalling pathways involved
  • the potato tuber extract NoclosanTM and LoxatTM were studied for their effects on the cell cycle and their ability to induce apoptosis in an in vitro cell culture system using (human) cancer cell lines of different origin. Despite the fact that both compounds are used as additives in serum free culture media (12,13), a growth stimulatory effect could not be confirmed when added to standard culture media supplemented with serum. However, both compounds showed a dose dependent delay in cell cycle progression of exponentially growing cells. The transition through the G 2 /M- to G x -phase of the cell cycle seemed to be the most sensitive. Prolonged exposure of cells to lower doses NoclosanTM or LoxatTM resulted in growth retardation in some cell lines, whilst in others a decline in cell number was observed. This decline in cell number arrest was accompanied by the appearance of apoptotic cells. These observations were consistently found in all cell lines tested. Therefore, we conclude that the observed cell cycle effects are not a cell type specific phenomenon.
  • NoclosanTM and LoxatTM showed a dose dependent delay of cell cycle progression with complete cell cycle arrest at the highest doses tested. This cell cycle arrest was accompanied by induction of apoptosis.
  • NCI-H125 cells lung adenocarcinoma, example 3
  • MCF7 cells breast cancer, example 4
  • OVCAR3 ovary carcinoma, example 5
  • U937 cells monocytic leukemia, example 6
  • Jurkat cells T cell lymphoma, example 7
  • HaCat cells human epidermis, example 8
  • HUVEC cells human umbilical cord endothelium, example 9)
  • HAMEC cells human microvascular endothelium, example 10
  • NCI-24H human small-cell lung carcinoma, example 11
  • NCI-H69 human small-cell lung carcinoma, example 12
  • Rl cells rat rhabdo yosarcoma, example 13

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Abstract

The present invention relates to biologically active compositions inhibiting cellular growth and especially inhibiting cellular growth by induction of apoptosis, which biologically active composition comprises indole acetic acid (IAA), and a vegetable extract (i) or components thereof, which vegetable extract (i) is obtainable by a method comprising: grounding a plant material; extracting the grounded plant material; c) charging a Q hyper DTM20 gel with the extracted and grounded plant material; d) subjecting the charged Q hyper DTM20 gel to an increasing NaC1 gradient; e) collecting the fraction which elutes at 1.02 M NaC1 to obtain vegetable extract (i).

Description

VEGETABLE EXTRACT COMPOSITIONS COMPRISING INDOLE ACETIC ACID, METHODS FOR THE PREPARATION OF SAID COMPOSITIONS, AND THEIR USE AS CELL GROWTH INHIBITING
AGENTS
5 The present invention relates to biologically active compositions inhibiting cellular growth and especially inhibiting cellular growth by induction of apoptosis. The present invention further relates to methods for the preparation of said biologically active compositions and the
10 use of said compositions as growth inhibiting agents and especially the use of said biologically active compositions for the induction of apoptosis in for example cancer cells.
Cell proliferation and programmed cell death (apoptosis) are two fundamental processes which are required
15 for the maintenance of tissue homeostasis in the human and animal body. Both processes involve highly orchestrated series of molecular events that control, for example, entry of cells into the cell cycle as well as the progression of cells through the various phases of this cycle, see for
20 example Schutte B., Ramaekers F.C.; "Molecular switches that govern the balance between proliferation and apoptosis"; Prog. Cell . Cycle Res . (2000), 4: 207-17.
Like proliferation, programmed cell death (apoptosis) is an active process that is subject to intricate control
25 (Schutte and Ramaekaers, supra) . The aspartate-specific proteases (caspases) are critically involved in the apoptotic process in mammalian cells and serve to incapacitate specific substrates, thereby leading to the disassembly of nuclear and cytoskeletal structures, disabling of cell repair and tagging 0 of the apoptotic cell for engulfment by phagocytes.
Recent studies have emphasized the importance of mitochondria as sensors and/or executioners of apoptosis, involving several mitochondrial proteins such as cytochrome c, and members of the Bcl-2 family during the apoptotic process, see for example Green D.R. and Reed J.C.; "Mitochondria and apoptosis"; Science (1998), 281: 1309-1312. On the one hand, disturbances in the molecular mechanisms controlling either proliferation or apoptosis can lead to malignancy, but on the other hand specific interference in these signalling pathways might be helpful in designing therapeutic strategies to control or cure cancer. Such therapeutic strategies could, for example, aim at suppressing proliferation and survival and/or activate death pathways .
Many of the traditional chemical treatment protocols aim at eradication of cancer cells by inhibition of the cell division mechanism or by introducing a lethal dose of DNA damage to the cancer cells. Despite the improved effectiveness of these approaches, a quest for a more subtle therapeutic approach is ongoing. In this quest several powerful compounds are tested that interfere in specific signalling pathways. Amongst these compounds are several compounds and extracts of phytogenic origin. One such compound is the major plant growth hormone indole acetic acid (IAA) .
According to the present invention it has now been found that the biological properties of indole acetic acid (IAA), like growth inhibiting properties, and especially the apoptosis inducing properties, can synergistically be enhanced by combining indole acetic acid (IAA) with a vegetable extract, for example, obtained from the potato tuber, or one or more components thereof. Therefore the present invention relates to a biologically active composition comprising indole acetic acid (IAA), and a vegetable extract (i) or one or more components thereof, which extract is obtainable by a method comprising: a) grounding a plant material; b) extracting the grounded plant material; c) charging a Q hyper DTM20 gel with the extracted and grounded plant material to ionically bind the extracted and grounded plant material to the gel; d) subjecting the charged Q hyper DTM20 gel to an increasing NaCl gradient to elute the bound extracted and grounded plant material form the gel; e) collecting the fraction which elutes at 1.02 M NaCl to obtain vegetable extract (i) .
The biologically active composition according to the present invention does not only relate to a composition comprising indole acetic acid as such, but also to biologically active compositions comprising analogues or derivatives of indole acetic acid (IAA) .
Indole acetic acid (IAA) can be substituted. In nature halogenated indole alkaloids can be found, particularly in marine organisms (i.e. 6-bromoindigotin) .
Synthetically all types of substituents can be introduced on the aromatic ring, e.g. methyl, amino, nitro, fluoride, chloride, bromide, and iodide on the positions 4, 5, 6 and 7. Furthermore, indole acetic acid (IAA) can be conjugated with other molecules, such as conjugates via an ester bond, in particular with various sugars, for example IAA-glucose, IAA-alfa-aspartic acid lN-glucoside, IAA- inositol, IAA-myoinositols, IAA-various carbohydrates or conjugates as amides for example with amino acids and peptides. Examples thereof are acetamide, alfa-leucine, alfa- alanine, alfa-aspartate (most important conjugate of IAA) , alfa-glutamate, alfa-lysine, alfa-glycine, alfa-valine and alfa-phenylalanine . Conjugation with peptides is common, whereas also conjugates with other amino acids occur in different plants. In addition, this group comprises 3-acetonitrile derivatives, which easily are converted into the corresponding acid, like indole-3-acetonitrile that decomposes in indole acetic acid (IAA) both chemically (under basic conditions) and catalytically (by. nitrilases) .
Of the total indole acetic acid (IAA) pool in plants, amide linked indole acetic acid (IAA) constitutes approximately 90%, whereas approximately 10% is ester-linked and approximately 1% is free indole acetic acid (IAA) . In plants, the levels of free + bound indole acetic acid (IAA) are about 1.2 μg/g Dry Weight (for Arabldopsis 9 days old, later lower) . Of this only approximately 1% is free indole acetic acid (IAA) .
According to the present invention indole acetic acid (IAA) or analogues or derivatives thereof are combined with a vegetable extract (i) , or one or more components thereof, which vegetable extract (i) is obtainable by a) grounding a plant material; b) extracting the grounded plant material; c) charging a Q hyper DTM20 gel with the extracted and grounded plant material to ionically bind the extracted and grounded plant material to the gel; d) subjecting the charged Q hyper DTM20 gel to an increasing NaCl gradient to elute the bound extracted and grounded plant material form the gel; e) collecting the fraction which elutes at
1.02 M NaCl to obtain vegetable extract (i) . The term "one or more components thereof" is used to indicate those components of the vegetable extract (i) that provide the observed synergistic effect in combination with indole acetic acid (IAA) . These components can be a single compound present vegetable extract (i) , like 5-alpha- pregnane-3-alpha 17.20 beta thriylthoxy, or a combination of compounds present in vegetable extract (i) , like carboiimide, octonoic acid, sucrose, 5-alpha-pregnane-3-alpha 17.20 beta thriylthoxy, L-norvaline, ribonic acid, propylene glycol, 1- amino ethanol, butanoic acid, L-valine, L-alanine, L- isoleucine, L-proline, L-serine, L-threonine, 3,4- dihydroxybutanoic acid, tetronic acid, gamma-lactone, arabino-hexanoic acid, D-fructose, cyclopentane carboxylic acid, linear alkane, fructose oxi , talose, D-gluconic acid, hexadecanoic acid, cyclododecylamine, octadecanoic acid, 9- octadocenamide, N,N-diethyl forma ide, ethylamine, trans-3 methyl-2-nonene, ethylene glycol, urea, acetetic acid, 3- methylene 1,4 butanediol, D, L-isoborneol, glycerol, Myo- inositol, hexadecanamide, galactunoic acid, butanoic acid, and/or 6-hydroxyhexanoic acid.
It should be understood that the source of the above- described compounds is not limited to vegetable extract (i) . These compounds can also be commercially obtained, chemically synthesized, and/or biologically provided.
According to the present invention any plant material can be used. Preferably, the plant material to be grounded is of vegetable origin containing glucide, protein, glycoprotein and terpene . Examples of such plant material are potato tubers, radish, turnip, beet, carrot, Jerusalem artichoke, sweet potato tubers, rape, cucumber, pumpkin, courgette, tomato, and the like. Any method known in the art can be used for grounding the plant material. Examples of such methods are methods involving pressing of the plant material to pulp, milling of the plant material, cutting of the plant material using for example in a blender, freezing, for example in liquid nitrogen, and subsequent grounding of the plant material, a combination of a hot/cold treatment and grounding, and the like. After grounding of the plant material, the grounded plant material is subjected to an extraction step. This extraction step can also simultaneously be performed with the grounding step of the plant material like grounding the plant material in the presence of an extraction liquid such as water or an organic liquid.
Extraction methods are generally known in the art and the present invention is not restricted to a specific extraction method or to a specific extraction liquid as long as the extraction method and/or liquid do not significantly reduce the synergistic properties of the vegetable extract obtained.
Suitable extraction methods according to the present invention are for example described by Rebiere C.J.P.; "Plant extract used as cell growth factor medicament with antitumoural and anticancer activity"; patent application no. FR2732347 and by Parinaud J. , Milhet P., Vietez G., Richoilley G.; "Human sperm capacitation and in-vitro fertilization in a chemically defined and protein-free medium SMART1®"; Human Reproduction (1998), 13: 2579-2582. After obtaining the extracted and grounded plant material according to the present invention, a Q hyper DTM20 gel is charged with the material to ionically bind the material to the gel. A specific example of this gel charging step is for example described in FR2732347 and by Parinaud et al . (supra) .
Subsequently, the charged Q hyper DTM20 gel is subjected to an increasing NaCl gradient, for example from 0 to 1.5 M NaCl, see also FR2732347 and Parinaud et al. (supra) , resulting in, depending on the strength of the ionic interaction between the gel and compounds in the extracted and grounded plant material, in different fractions comprising compounds eluted at a specific NaCl concentration or interval.
According to the present invention, the vegetable extract (i) providing the synergistic properties in combination with indole acetic acid (IAA) is comprised in the fraction eluting at an interval or concentration of 1.02 M NaCl.
Taken into account the cytotoxic properties of vegetable extract (i) at high concentrations, preferably the amount vegetable extract (i) in the biologically active composition according to the present invention ranges from 1 to 10 mg/ml, like 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/ml.
Preferably the vegetable extract (i) is in the form of a vegetable extract marketed under the name Loxat™ available from Optipharma B.V., Maastricht, The Netherlands. According to the present invention, optimal growth inhibiting properties of the biologically active composition are obtained at concentration indole acetic acid (IAA) ranging from 2*10"4 M to 3*10"3 M, like 2*10"4, 3*10"4, 4*10"4, 5*10~4, 6*10~4, 7*10"4, 8*10"4, 9*10"4, 1*10"3, 2*10"3, or 3*10"3 M. The molar concentrations are calculated using the molecular weight of free indole acetic acid (IAA, 175 g/mol) . It should be understood that the specific values of these molar concentrations will vary depending if the above- mentioned derivatives and/or analogues of indole acetic acid (IAA) comprised in the biologically active composition. In the above molar concentrations range, a preferable balance is provided between the desired growth inhibiting properties of the biologically active composition according to the present invention and the cytotoxic effect of indole acetic acid (IAA) at high concentrations.
According to one embodiment of the present invention, indole acetic acid (IAA) is comprised in a second vegetable extract (ii) obtainable by a method comprising: a) grounding a plant material; b) extracting the grounded plant material; c) charging a Q hyper DTM20 gel with the extracted and grounded plant material to ionically bind the extracted and grounded plant material to the gel; d) subjecting the charged Q hyper DTM20 gel to an increasing NaCl gradient to elute the bound extracted and grounded plant material form the gel; e) collecting the fraction which elutes at 0.78 M NaCl to obtain vegetable extract (ii) comprising indole acetic acid (IAA) .
Similar to the above method for obtaining vegetable extract (i) any plant material can be used. Preferably, the plant material to be grounded is of vegetable origin containing glucide, protein, glycoprotein and terpene. Examples of such plant material are potato tubers, radish, turnip, beet, carrot, Jerusalem artichoke, sweet potato tubers, rape, cucumber, pumpkin, courgette, tomato, and the like.
Also, any method known in the art can be used for grounding the plant material. Examples of such methods are methods involving pressing of the plant material to pulp, milling of the plant material, cutting of the plant material for example in a blender, freezing, for example in liquid nitrogen, and subsequent grounding of the plant material, a combination of a hot/cold treatment and grounding, and the like.
After grounding of the plant material, the grounded plant material is subjected to an extraction step. The person skilled in the art will recognize that this extraction step can also be simultaneously performed with the grounding step of the plant material like grounding the plant material in the presence of an extraction liquid such as water or an organic liquid. Extraction methods are generally known in the art and the present invention is not restricted to a specific extraction method or to a specific extraction liquid as long as the extraction method and/or liquid do not disadvantageously reduce the synergistic properties of the vegetable extract obtained.
Suitable extraction methods according to the present invention are for example described in FR2732347 and by Parinaud et al . (supra).
After obtaining the extracted and grounded plant material according to the present invention, a Q hyper DTM20 gel is charged with the material to ionically bind the material to the gel. A specific example of this gel charging step is for example described in FR2732347 and by Parinaud et al . (supra) . Subsequently, the charged Q hyper DTM20 gel is subjected to an increasing NaCl gradient, for example from 0 to 1.5 M NaCl, see also FR2732347 and Parinaud et al. (supra) , resulting in, depending on the strength of the ionic interaction between the gel and compounds in the extracted and grounded plant' material, in different fractions comprising compounds eluted at a specific NaCl concentration or interval . According to the present invention, the vegetable extract (ii) comprising indole acetic acid (IAA) is comprised in the fraction eluting at an interval or concentration of 0.78 M NaCl. Preferably the vegetable extract (ii) is in the form of a vegetable extract marketed under the name Noclosan™ available from Optipharma B.V., Maastricht, The Netherlands.
According to the present invention optimal growth inhibiting properties of the biologically active composition are obtained at concentration of the vegetable extract (ii), comprising indole acetic acid (IAA) , ranging from 1 to 10 mg/ml, like 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/ml.
At the above concentrations a preferable balance is provided between the desired growth inhibiting properties of the biologically active composition according to the present invention and the cytotoxic effect of vegetable extract (ii) at high concentrations.
The above defined biologically active composition according to the present invention provides advantageously synergistic properties with respect to the, growth inhibiting characteristics of indole acetic acid (IAA) . Therefore, the present invention also relates to the use of this biologically active composition as a growth inhibiting agent, especially as an apoptosis inducing agent of eukaryotic cells.
The growth inhibiting properties of the biologically active compositions according to the present invention are especially suitable to be used on cancer cells, either of human or animal origin, present in, for example, non-small and small cells lung carcinoma's, lung adenocarcinoma' s, colon carcinoma's and adenocarcinoma' s, breast carcinoma's and adenocarcinoma' s, ovary carcinoma's and adenocarcinoma' s, leukemic carcinoma's and adenocarcinoma' s, monocystic leukemia, T-cell lymphoma's, epidermal carcinoma's and adenocarcinoma' s, like skin carcinoma's and adenocarcinoma' s, umbilical cord carcinoma's and adenocarcinoma' s, microvascular endothelial carcinoma's and adenocarcinoma' s, rhabdomyosarcoma' s, and the like.
The present invention also relates to a method for the preparation of a biologically active composition comprising indole acetic acid (IAA) and a vegetable extract (i) comprising: a) grounding a plant material; b) extracting the grounded plant material; c) charging a Q hyper DTM20 gel with the extracted and grounded plant material to ionically bind the extracted and grounded plant material to the gel; d) subjecting the charged Q hyper DTM20 gel to an increasing NaCl gradient to elute the bound extracted and grounded plant material form the gel; e) collecting the fraction which elutes at
1.02 M NaCl to obtain vegetable extract (i) ; g) combining indole acetic acid (IAA) , or analogues or derivatives thereof, with the vegetable extract (i) to provide a biologically active composition.
As already outlined above any plant material can be used as starting material and also grounding, extraction, and isolation of vegetable extract (i) can be performed using any method generally known in the art. Examples of suitable methods are described in FR2732347 and by Parinaud et al. (supra) .
According to the present invention, the above method provides a biologically active composition comprising 2*10~4 M to 3*10"3 M indole acetic acid (IAA), like 2*10'4, 3*10 4*10" , 5*10"4, 6*10"4, 7*10"4, 8*lθ 9*10~4, 1*10""3, 2*10"3, or 3*10"3 M, and 1 mg/ml to 10 mg/ml vegetable extract (i) , like 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/ml. Optimal growth inhibiting properties of the biologically active composition obtained are provided using these concentration ranges. As already set out above, the molar concentrations of indole acetic acid (IAA) are calculated using the molecular weight of free indole acetic acid (IAA, 175 g/mol) . It should be understood that the specific values of these molar concentrations will vary depending if the above derivatives and/or analogues of indole acetic acid (IAA) comprised in the biologically active composition. In the above concentration ranges, a preferable balance is provided between the desired growth inhibiting properties of the biologically active composition according to the present invention and the cytotoxic effect of indole acetic acid (IAA) and vegetable extract (i) at high concentrations.
Preferably the indole acetic acid, or analogues or derivatives thereof, in the above method according to the present invention is comprised in a vegetable extract (ii) , which extract is obtainable by: a) grounding a plant material; b) extracting the grounded plant material; c) charging a Q hyper DTM20 gel with the extracted and grounded plant material to ionically bind the extracted and grounded plant material to the gel; d) subjecting the charged Q hyper DTM20 gel to an increasing NaCl gradient to elute the bound extracted and grounded plant material form the gel; e) collecting the fraction which elutes at
0.78 M NaCl to provide a vegetable extract (ii) comprising indole acetic acid (IAA) .
Similar, as outlined above, any plant material can be used as starting material and also grounding, extraction, and isolation of vegetable extract (ii) can be performed using any method generally known in the art. Examples of suitable methods are described in FR2732347 and by Parinaud et al. (supra) .
According to the present invention, the above method provides a biologically active composition comprising 1 mg/ml to 10 mg/ml vegetable extract (ii) , like 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/ml, and 1 mg/ml to 10 mg/ml vegetable extract (i), like 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/ml..
Optimal growth inhibiting properties of the biologically active composition obtained are provided using these concentration ranges. In the above concentration ranges a preferable balance is provided between the desired growth inhibiting properties of the biologically active composition according to the present invention and the cytotoxic effect of vegetable extract (ii) and vegetable extract (i) at high concentrations . The invention will further be illustrated in the examples that follow and these examples are not intended to limit the invention in any way. In the Examples reference is made to the following figures:
Figure 1: Growth curves of CaCo2 cells (A), MR65 cells (B) and MCF7 cells (C, D) grown in control culture medium (squares) or in the presence of Noclosan™ (triangles) or Loxat™ (diamonds) . In Figures 1 A, B, D 5 mg/ml of both compounds was used, while in Figure 1C 2.5 mg/ml of both reagents was applied. The number of cells (ordinate) is plotted versus the time in culture (abscissa) . Note the significant delay in growth of all cell lines when cultured in the presence of 5 mg/ml Noclosan™ or Loxat™.
Figure 2: The S-phase progression (bars) and percentage (lines) of apoptosis in MCF7 (A), CaCo2 (B) , MR65 (C) and U937 (D) cells, cultured in the presence of various doses of Noclosan™ (solid bars, solid diamonds) or Loxat™ (open bars, open diamonds) . The relative movement of cells (left ordinate) at a fixed interval (see text) after pulse labeling with BrdU is plotted versus the dose of Noclosan™ or Loxat™ (abscissa) . Note that for all cell lines the S-phase progression declines starting from a dose of 5 mg/ml Noclosan™. Simultaneously the number of apoptotic cells increases (right ordinate) . Similar effects were observed for Loxat™ at a slightly higher dose.
Figure 3: MR65 (a-d) and CaCo2 (e-h) cells stained with the anti-tubulin antibody E7. Cells were cultured for 5 hours (a,c,e,g) or 24 hours (b,d,f,h) in the presence of 5 mg/ml Noclosan™ (b,c,g,h) or Loxat™ (a,b,e,f). Note the metaphase arrest after 5 hours of culture and the appearance of apoptotic figures after 24 hours.
Figure 4: The G2/M-phase exit and percentage of apoptosis in CaCo2 (A), MR65 (B) and U937 (C) cells, cultured in the presence of various doses of Noclosan™ (solid bars, solid diamonds) or Loxat™ (open bars, open diamonds) . The number of BrdU-negative G2/M-phase cells (left ordinate) at a fixed interval after BrdU-pulse labeling is plotted versus the dose of Noclosan™ or Loxat™ (abscissa) . Note that for all cell lines the number of BrdU-negative G2/M-phase cells remains high starting from a dose of 2.5 mg/ml Noclosan™ and higher. At this dose the number of apoptotic cells (right ordinate) remains however low. Similar effects were observed for Loxat™ at a slightly higher dose.
Figure 5: The induction of apoptosis in MR65 cells by Noclosan™ or Loxat™. MR65 cells were cultured for 7 hours with 10 mg/ml Noclosan™ or Loxat™. Cells were either stained with FITC-labeled Annexin-V (solid bars) or harvested and assayed for caspase 3/7 activity using Ac-DEVD-AMC (open bars) or M30-CytoDeath reactivity (striped bars) . The general caspase inhibitor z-VAD was used to block apoptosis. The CDK- inhibitor Roscovitine was used as a positive control. Note that both Noclosan™ and Loxat™ induced apoptosis in MR65 cells. The caspase inhibitor was not able to inhibit the exposure of the M30-CytoDeath epitope in apoptotic cells.
Figure 6: Western blotting analysis of M30 CytoDeath in MR65 cells treated with Noclosan™ or Loxat™. The asterisks indicate the characteristic cleavage fragments of cytokeratin 18.
Figure 7: The effect of Noclosan™ or Loxat™ on the population doubling time (Td) in CaCo2 (solid bars), MR65 (diagonally downwards striped bars), OVCAR (diagonally upwards striped bars), MCF7 (open bars and vertically striped bars) , Jurkat (horizontally striped bars) and U937 (speckled bars) cells. CaCo2, MR65, OVCAR and MCF7 (open bars) cells were cultured in the presence of 5 mg/ml Noclosan™ or Loxat™, MCF7 (vertically striped bars), Jurkat and U937 cells were cultured in the presence of 2.5 mg/ml Noclosan™ or Loxat™. Doubling times were expressed as percentage of the unperturbed exponential growth.
Figure 8: A,B) The effect of repeated administration of Loxat™ (A) or Noclosan™ (B) to CaCo2 cells. Loxat™ or
Noclosan™ was administered either at a single dose of 5 mg/ml and the cultures were left unchanged (diamonds) or cultures were changed every 24 hours with a fresh dose (triangles) . Exponentially growing cells (squares) were used as a control. The number of cells (solid figures) is plotted versus the time in culture (abscissa) . The number of apoptotic cells (open figures) remains low. Note that there is no difference in either growth behaviour of the cells or the frequency of apoptotic cells when the two culture conditions are compared. C,D,E) The effect of repeated administration of Noclosan™ or Loxat™ on the induction of apoptosis in MCF7 (C) , MR65 (D) . Loxat™ was administered either at a single dose of 5 mg/ml and the cultures were left unchanged (solid bars) or cultures were changed every 24 hours with a fresh dose (open bars) . Similarly, Noclosan™ was administered as a single dose (diagonally downwards striped bars) or culture media were repeatedly renewed (diagonally upwards striped bars) . Exponentially growing cells were used as a control. The number of M30 CYTODEATH-positive cells is shown.
Figure 9: The effect of combined administration of Noclosan™ and Loxat™ on MR65 (A) and CaCo2 (B) cells. Cells were cultured with various combinations of doses of both compounds as indicated on the abscissa. After 7 hours cells were assayed for M30 CYTODEATH reactivity (ordinate) and the measured (open bars) and the expected (solid bars) number of apoptotic cells are plotted. The expected number of apoptotic cells is calculated as the sum of the numbers of apoptotic cells measured when the compounds were administered individually in the indicated dose. Note that the measured effect is higher than the expected, indicating a synergistic effect of both compounds.
Figure 10: The kinetics of various caspase activities in MR65 cells after induction of apoptosis by either Loxat™ (A) or Noclosan™ (B) . Caspase 2 (open circle), 3/7 (square)/ 5/4/1 (solid diamond), 6 (open diamond), 8 (triangles) and 9 (solid circle) activities were plotted as function of time after administration of either of both compounds, as is the number of M30 CYTODEATH-positive cells (open squares) . Note that the kinetics of induction is different for both compounds . Figure 11: MR65 (a) and HaCat (b) cells stained with the anti-active caspase 8 antibody 11G10. Cells were cultured for 6 h. in the presence of 5 mg/ml Noclosan™ and 10 mg/ml Loxat™, (c) : MR65 control cells without the addition of the apoptosis inducing agent. Figure 12: Western blotting analysis of active
Erkl/2, p38 MAPK, SAPK/JNK and Akt kinase in MR65 cells treated with Noclosan™ or Loxat™ in the presence or absence of the general caspase inhibitor z-VAD-fmk. Note the increase in p38 MAPK and SAPK/JNK kinase activity in the presence of Noclosan™ and Loxat™.
EXAMPLES
EXAMPLE 1
Using standard chemical analysis techniques, like NMR, HPLC, mass spectrometry, gas chromatography, etc., both vegetable extracts Noclosan™ and Loxat™ were analyzed with respect to their detectable components. It was found that the active component in Noclosan™ is the compound indole acetic acid (IAA).
With respect to Loxat™, a complex mixture of the following compounds was found: carboiimide, octonoic acid, sucrose, 5-alpha-pregnane-3-alpha 17.20 beta thriylthoxy, L- norvaline, ribonic acid, propylene glycol, 1-amino ethanol, butanoic acid, L-valine, L-alanine, L-isoleucine, L-proline,' L-serine, L-threonine, 3, -dihydroxybutanoic acid, tetronic acid, gamma-lactone, arabino-hexanoic acid, D-fructose, cyclopentane carboxylic acid, linear alkane, fructose oxim, talose, D-gluconic acid, hexadecanoic acid, cyclododecylamine, octadecanoic acid, 9-octadocenamide, N,N- diethyl formamide, ethylamine, trans-3 methyl-2-nonene, ethylene glycol, urea, acetetic acid, 3-methylene 1,4 butanediol, D, L-isoborneol, glycerol, Myo-inositol, hexadecanamide, galactunoic acid, butanoic acid, and 6- hydroxyhexanoic acid. Of these, the compounds carboiimide, octonoic acid, sucrose, 5-alpha-pregnane-3-alpha 17.20 beta thriylthoxy, L-norvaline, and ribonic acid form the major components of loxat™.
EXAMPLE 2
1.Materials and Methods
1.1 Cell lines and cell culture
To investigate the effects of Noclosan™ and Loxat™ on cell cycle kinetics and induction of apoptosis the following cell lines were used: MR65 cells (non-small cell lung cancer) and NCI-H125 (lung adenocarcinoma) , MCF7 cells (breast cancer) , CaCo2 cells (colon carcinoma) , OVCAR3 (ovary carcinoma) , U937 cells (monocytic leukemia) and Jurkat (T cell lymphoma) . Except for the U937 and Jurkat cell lines, which grow in suspension, all cell lines grow as adherent cell cultures.
The cell lines were cultured in RPMI 1640 medium without L-Glutamine (Life Technologies, GIBCO-BRL, Scotland) , supplemented with 0.1 % gentamycin (Eurovet, Bladel, The Netherlands), 1 % L-glutamine (22942, Serva, Heidelberg,
Germany) and 10 % heat inactivated newborn calf serum (GIBCO, 021-6010M) , except for the MR65 and CaCo2 cell lines, which were grown in EMEM, supplemented with 10% fetal calf serum and non essential amino acids.
As a source of human keratinocytes immortalized HaCat cells (Boukamp P., Petrussevska R.T., Breitkreutz D. , Hornung J., Markham A., Fusenig N.E.; "Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line"; J. Cell . Biol . (1988), 106: 761-771.) were used. These cells were grown as adherent cell cultures in EMEM, supplemented with 10% fetal calf serum and non essential amino acids.
Human umbilical vein endothelial cells (HUVEC) were obtained as primary cultures after trypsinisation of umbilical cord tissue.
Trypsin (2.5 % trypsin (Flow #16-893-49), 2 % di- natrium EDTA (Titriplex) and 2 % glucose in PBS) was used to harvest the cells.
For immunocytochemistry the cells were fixed overnight in methanol at -20°C.
1.2 Preparation of Noclosan™ and Loxat™
Both Noclosan™ and Loxat™ were isolated from the potato tuber ( Solanum tuberosum) . In brief the raw plant material is grounded, water extracted and further purified by ion exchange chromatography on Q hyper DTM20 gel, using increasing concentrations of NaCl. The fractions eluted at 0.78 M and 1.02 M NaCl, represent Noclosan™ and Loxat™, respectively. The extended purification protocols (FR2732347 and Parinaud et al. (supra)) as well as applications of both products in tissue culture have been described before (Parinaud J., Milhet P., Vietez G., Richoilley G.; "Human sperm capacitation and in-vitro fertilization in a chemically defined and protein-free medium SMARTl®"; Human Reproduction (1998), 13: 2579-2582 and Parinaud J., Milhet P., Vietez G., Richoilley G.; "Use of a medium devoid of any human or animal compound (SMART21) for embryo culture in intracytoplasmic sperm injection"; J. Ass. Reprod. Gen. (1999), 16: 13-16.). Stock solutions were made by carefully dissolving Noclosan™ and Loxat™ (available from Optipharma B.V., Maastricht, The Netherlands) to a final dose of 20 mg/ml in the proper culture medium. The stock solutions were centrifuged at 600 xg to remove any insoluble remnants.
1.3 Dose dependent effects of Noclosan™ and Loxat™ To test the general nature of the effects of Noclosan™ and Loxat™ on cell cycle progression and apoptosis induction, the cell lines were exposed to various doses of Noclosan™ and Loxat™ , i.e. 1, 2.5, 5, 10 and 20 mg/ml. To test the effect of combined administration of
Noclosan™ and Loxat™ on the induction of apoptosis, HaCat cells were treated for 7 h with either Noclosan™ and Loxat™ alone or a combination of both compounds. The following combinations were tested: Noclosan™ (10, 5 and 1 mg/ml), Loxat™ (10, 5 and 1 mg/ml) and Noclosan™ + Loxat™ (10 mg/ml + 10 mg/ml; 5 mg/ml + 10 mg/ml; 1 mg/ml + 10 mg/ml; 10 mg/ml + 2 mg/ml; 5 mg/ml + 2 mg/ml; 1 mg/ml + 2 mg/ml) .
1.4 BrdUrd pulse labeling The effect on cell cycle kinetics was analysed by a
BrdUrd pulse chase experiment. Briefly, BrdUrd (Serva) was added to the culture medium in a final concentration of 10 μM for 30 minutes. Then, the cells were rinsed twice with prewarmed PBS and chased in prewarmed culture medium supplemented with 5 μM deoxythymidine (Serva) and various doses of Noclosan™ and Loxat™. After approximately 6 hours ' cells were harvested. 1.5 Kinase inhibitors
To study the involvement of the MAP kinase-pathway the erk-2 inhibitor (PD 98059) was used (Cell Signalling Technology Inc., Beverly MA, USA). Staurosporine (C2SH26N403) (Sigma-Aldrich, Zwijndrecht, The Netherlands) was used as an inhibitor of the protein kinase C-pathway.
1.6 The annexin V assay
To analyse phosphatidylserine (PS) exposure at the outer leaflet of the membrane, cells were labelled with annexin V OregonGreen (Nexins Research BV, Hoeven, The Netherlands). Propidium iodide (PI) was added in a final concentration of 5 mg/ml to probe for loss of membrane integrity.
1.7 Immunocytochemistry 1.7.1 BrdUrd staining
Incorporated BrdUrd was detected as described previously (14). Briefly, approximately 106 ethanol fixed cells were rinsed once in PBS and resuspended in 2 ml of 0.4 mg/ml pepsin in 0.1 N HC1 (Serva, Heidelberg, Germany). After 30 minutes at room temperature cells were pelleted, resuspended in 2 N HC1 and incubated for another 30 minutes at 37 °C. Cells were rinsed in 0.1 M borate buffer, pH 8.5 and PBS/BSA (lmg/ml BSA in PBS).
Appropriately diluted anti-BrdUrd antibody (clone IIB5, available form Euro-Diagnostica B.V., Arnhem, The Netherlands) was added to the cell pellet, resuspended in 100 ml PBS/BSA. After incubation for 1 h at room temperature, the cells were rinsed twice in PBS/BSA. For visualization FITC conjugated Fab2 fragments of rabbit anti-mouse Ig (DAKO A/S,' Glostrup, Denmark) antibody was added in a 1:10 dilution. After incubation for 45 minutes at room temperature samples were rinsed twice in PBS/BSA and the cells were finally resuspended in 0.5 ml cold PBS supplemented with 100 mg/ml RNAse (Serva) and 20 mg/ml propidium iodide (PI; Calbiochem, La Jolla, CA) . The samples were allowed to stand for 15 minutes on ice in the dark before flow cytometric analysis. In the negative control the primary antibody was omitted.
1.7.2 M30 CytoDeath apoptosis assay
Apoptosis was detected and quantified as described previously (15) . Briefly, methanol fixed cells were rinsed once in PBS. Appropriately diluted M30 CytoDeath antibody (Boeringer, Mannheim) was added to approximately 105 cells, resuspended in 100 ml PBS/BSA. After incubation for 1 h at room temperature, the cells were rinsed twice in PBS/BSA. For visualization FITC conjugated Fab2 fragments of rabbit anti- mouse Ig (DAKO A/S, Glostrup, Denmark) antibody was added in a 1:10 dilution. After incubation for 45 minutes at room temperature samples were rinsed twice in PBS/BSA and the cells were finally resuspended in 0.5 ml cold PBS supplemented with 100 mg/ml RNAse (Serva) and 20 mg/ml propidium iodide (PI; Calbiochem, La Jolla, CA) . The samples were allowed to stand for 15 minutes on ice in the dark before flow cytometric analysis. In the negative control the primary antibody was omitted.
1.7.3 Caspase 8
Cleaved (activated) caspase 8 (Asp384) was detected with mouse monoclonal antibody 11G10 obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA)
1.7.4 Tubulin
Tubulin staining was performed. using the monoclonal antibody E7 (Developmental Studies Hybridoma Bank at the University of Iowa, Iowa, USA) using the protocol as described above for M30 CytoDeath.
1.8 Western blotting We used the semi-dry electrophoretic transfer equipment for Western blotting. After incubation of the cells with Ac-DEVD-aldehyde or zVAD-fmk (an irreversible, cell permeable, broad spectrum caspase inhibitor, Biomol) and apoptosis inducers (A and B) for the indicated time periods the supernatants and detached cells were harvested and pelleted by centrifugation. Adherent cells were washed twice with PBS and detached by scraping in lysis buffer (62.5 mM Tris-HCl, pH 6.8, 12.5 % glycerol (GibcoBRL) , 2 % Nonidet P- 40 (Fluka, Sigma-Aldrich Chemie) . Scraped cells were combined with cells from the supernatants and allowed to lysate on ice for 20 minutes. After addition of an equal volume of electrophoresis sample buffer (62.5 mM Tris-HCl, 10 % glycerol, 2.3 % SDS, 5 % ?-mercaptoethanol (Bio-Rad) , 0.05 % bromophenol blue (Sigma) the cell lysates were boiled for 5 minutes and subsequently centrifuged for 5 minutes in a microfuge (Sigma) .
Samples (the equivalent of 2.5.105 cells in 25 μl) were resolved on appropriate SDS-PAGE gels using a Mini- Protean II system (Bio Rad Laboratories) . Proteins were transferred to nitrocellulose membranes (Schleicher and Schuell, Dasel, Germany) at 100 volts for 1 hour (transfer buffer = 25 mM Tris (Merck), 190 mM glycine (Merck), 20 % v/v methanol (Rathburn, Walkerburn, Scotland) .
Blots were blocked for 1 hour at RT in 10 mM Tris- HC1, pH 7.4, 150 mM NaCl, 0.05 % Tween 20 (Sigma-Aldrich Chemie) , 5 % non-fat dry milk (Bio-Rad) . For immunoblot analysis the following antibodies were used: M30 CytoDeath, directed against the cytokeratin 18 neo-epitope formed during apoptosis (15), phospho-p44/42 MAPK, phospho-SAPK/JNK, phospho-p38 MAPK and AKT (60 kDa) (New England Biolabs) .
Blots were incubated with the respective antibodies (diluted in blocking buffer) for 1 hour at RT or overnight at 4°C. After washing three times with PBS containing 0.5 % Triton X-100 (Scintran, Brunschwig Chemie, Amsterdam, The Netherlands) blots were incubated with horseradish peroxidase conjugated rabbit anti mouse immunoglobulins (DAKO) , diluted in blocking buffer, for 1 hour at RT. Signals were visualized using ECL detection reagent (Amersham Pharmacia, Roosendaal, The Netherlands), according to the manufacturer's instructions and subsequent exposure to X-ray film (Fuji Photo Film Europe, Dusseldorf, Germany) .
1.9 Preparation of cell lysates
NCI-H125 and MR65 cells were washed 3 times with PBS and harvested in cold 50 mM Tris/HCl, pH 7.5 with a disposable cell scraper. Subsequently 1 X 107 cells/ml were sonicated for 5 seconds using a Soniprep 150 sonicator (MSE Scientific Instruments, Beun-De Ronde) , (amplitude 12 micron). Cell lysates were pelleted at 10,000 g at 4°C in a microfuge (Sigma, Ostenrode Am Harz, Germany) and supernatants were either immediately assayed or frozen at -20°C.
1.10 Analysis of caspase-activity
Caspase activity was measured in 50 μl aliquots of cell lysate, which is the. The following 7-amino-4-methyl coumarin (AMC) -labeled caspase substrates were used: WEHD (caspase 1, 4 and 5), DEVD (caspase 3,7), IETD (caspase 3- processing and 8), VEID (caspase 6), VDVAD (caspase 2), LEHD (caspase 9), YVAD (caspase 1, 4). All the substrates were purchased from Alexis (Kordia, Leiden, NL) . To 50 μl of cell lysate, an equivalent of 50,000 cells, 50 μl of substrate mix (50 mM Tris/HCl, pH 7.5, containing 5 mM DTT and 40 μM substrate solution was added. Following incubation for 60 minutes at 37 °C 1.8 ml PBS was added.
For the measurement of the caspase-activity, a spectrofluorometer (SLM-AMINCO, SPF-500CTM) was used. Excitation was done at a wavelength of 380 NM (excitation) , and emission was measured at 440 nm (HV = 650, gain at 10/1, filter = 3, the bandpass of the excitation was 5 nm and for the emission 10 nm) .
1.11 Flow cytometry
For flow cytometric analysis a FACSort (Becton Dickinson, Sunnyvale, CA) equipped with a single Argon ion laser was used. Excitation was done at 488 nm, and the emission filters used were 515-545 BP (green; FITC), 572-588 BP (orange; PE) and 600 LP (red; PI). A minimum of 10,000 cells per sample were analysed and data stored in list mode. FITC signals were recorded as logarithmic amplified data, while the PI signals were recorded as linear amplified data. For bivariate FITC/PI analysis no compensation was used. Data analysis was performed with the standard Cellquest software (Becton Dickinson) . As a standard procedure for all analyses, data were gated on pulse processed PI signals to exclude doublets and larger aggregates.
For detailed cell kinetic analysis, five cell cycle compartments were identified as described by Higashikubo et al. (16). Briefly, the following populations were quantified (see figure 1): 1. BrdU positive undivided cells (Nlu) , 2.
BrdU positive divided cells (Nld) , 3. BrdU negative G2 cells' (G2_) , 4. BrdU negative Gl cells (G^) , and 5. BrdU negative Gl and S cells (GXS-) . From populations 1 and 5 the mean DNA content was calculated and normalized to Gx- and G2/M-phase position (Xlu and XG1S, respectively) . From these, four parameters reflecting the modes of cell progression through the cell cycle were determined: i) relative movement (RM) of the cells through the S- phase, according tho Begg et al . (17), i.e. the mean DNA content of BrdUrd-positive undivided population normalized to Gx and G2/M peak position = (Xlu-XG1) / (XG2 IQ-XGI) ii) fraction of undivided cells among the BrdUrd- positive population (F+ undivided) = Nlu/ (Nlu+Nld/2) . iii) BrdUrd negative G2/M fraction (FG2M_) = NG2/M/NT iv) the position of negative cells excluding the initial G2/M cells normalized to Gl peak position (XG1) =
Figure imgf000027_0001
1.12 Data analysis
The means of the four parameters were plotted as a function of time after BrdUrd pulse labeling for each dose of olomoucine and roscovitine tested. To quantitatively assess cell cycle progression, linear portions of the kinetic curves were fitted by linear regression analysis. For the BrdUrd- negative G2/M fractions the linear regression analysis was performed on the logarithmically transformed data. The slopes of the regression lines were used to elucidate the changes in the rate of cell cycle progression.
2 Results
To test the general nature of the effects of Noclosan™ and Loxat™ the cell lines were exposed to various doses of either of both compounds, i.e. 1, 2.5, 5, 10 and 20 mg/ml. Microscopical examination and cell counts (Fig. 1) showed that both Noclosan™ and Loxat™ appeared to be cytotoxic at the highest doses in all cell lines. In adherent cultures, cells rounded up and massive debris was seen. At doses of 10 mg/ml cells showed retraction of their cytoplasm and tended to loose contact with the culture substratum. At doses of 1 mg/ml no effects on morphology were observed as compared to the control culture.
2.1 Short term effect on cell cycle kinetics
Both Noclosan™ and Loxat™ showed a dose dependent effect on cell cycle progression in all cell lines tested. For instance, at low to intermediate doses (i.e. 1.25-5 mg/ml) the progression of different cancer cell lines through the S-phase of the cell cycle was unaffected (Fig. 2). At high doses (i.e. 10-20 mg/ml) a complete cell cycle arrest was observed. At intermediate doses cells tend to accumulate in the G2/M phase of the cell cycle. Both G2/M-exit and Gl- entry rates were decreased in a dose dependent manner. Cells were in general more sensitive to the Noclosan™ as compared to Loxat™.
Accumulation in the G2/M-phase of the cell cycle was also confirmed by immunocytochemistry. Anti-tubulin staining showed an increased number of cells in metaphase of MR65 and CaCo2 cells after addition of Noclosan™ or Loxat™ for 5 hours (Fig. 3) .
The effects of both compounds on the cell cycle of HUVEC cells was less pronounced as compared to the results in the cancer cell lines (results not shown) .
2.2 Short term induction of apoptosis
Both Noclosan™ and Loxat™ were effective in the induction of apoptosis at high doses. In some cell lines a toxic effect prevailed. An increase in the apoptotic fraction, as measured by the M30 CytoDeath assay or the Annexin V binding assay, coincided with total cell cycle arrest (Fig. 2). Accumulation of cells in the G2/M phase of the cell cycle at intermediate doses did not lead to an increase in apoptosis (Fig. 4), but it was obvious that after 24 hours both compounds induced apoptosis in particular in those cells that were arrested in metaphase (Fig. 3).
Apoptosis was confirmed by presence of ΛDEVD-like' activity, the immunocytochemical detection of cells positive for active caspase-3 and the absence of apoptotic cells when cells were treated in the presence of the general caspase inhibitor zVAD-fmk (Fig. 5) .
Immunoblotting studies in MR65 cells treated with either Noclosan™ or Loxat™ at 10 or 20 mg/ml using the M30 CytoDeath antibody revealed the typical breakdown products of cytokeratin 18 in these cells (Fig. 6) . To study the kinetics of apoptosis, MR65 and CaCo2 cells were cultured for various periods of time in the presence of 10 mg/ml Noclosan™ or Loxat™. Cells were harvested and assayed using the M30 CytoDeath assay. Immediately after exposure to either of both compounds a gradual increase in the number of apoptotic MR65 cells was observed. CaCo2 cells were insensitive for the induction of apoptosis by Noclosan™ or Loxat™ at a dose of 10 mg/ml. In contrast a sudden rise in the number of necrotic cells (i.e. Annexin V+/PI+) cells was observed when these cells were cultured in the presence of Noclosan™, indicating a toxic effect of the compound (data not shown) .
2.3 Long term effects of Noclosan™ and Loxat™
In general, the short term effects of exposure to high doses of either Noclosan™ and Loxat™ were a block in cell cycle progression, which was parallelled by the rapid induction of apoptosis. Accumulation of cells in the G2/M phase of the cell cycle at intermediate doses of either of both compounds, did not result in short term induction of apoptosis. To study the effects of long term exposure of cells to these intermediate doses cells were grown cultured for a total period of 72 hours in the presence of 5 mg/ml Noclosan™ and Loxat™. Both in MR65 and CaCo2 cell cultures population doubling times (Td) increased (Fig. 1A,B). When studied in the different cancer cell lines the effect was most pronounced for Noclosan™ (Fig. 7) . In case of OVCAR3 cells a decrease in total cell number was observed. In general, no increase in the number of apoptotic cells was observed when cells were exposed for prolonged periods of time in the presence of Noclosan™ and Loxat™. However, in cultures of MCF7 cells exposed to Noclosan™ and of 0VCAR3, U937 and Jurkat cells exposed to either of both compounds, the numbers of apoptotic cells increased. In MCF7 cells a gradual increase in the number of apoptotic cells was observed. In U937 and Jurkat cells the number of apoptotic cells increased dramatically at 48 hours and at 72 hours many dead (secondary necrotic) cells were observed in the cultures . Since it can be argued that Noclosan™ and Loxat™ is rapidly metabolized in some cell lines, explaining the lack of effect, the experiment was repeated for CaCo2 cells. The effects of continuous growth in the presence of both compounds was compared to the effects after repeatedly refreshing culture conditions (Figures 8A-E) . Cell cycle effects and the frequency of apoptosis were similar for both experiments .
In the HUVEC cell cultures also a dose dependent effect of Loxat™ was detected with apoptotic fractions of up to 80% at concentrations of 10 mg Loxat™/ml (results not shown) .
2.4 Reversibility of the effects of Noclosan™ and Loxat™ The cell cycle effects of 24 h exposure to 5 mg/ml of Noclosan™ were reversible in MR65 cells. When excess of Noclosan™ was washed out of the culture medium after 24 hours of exposure, cells regained their ability to progress through the cell cycle after 8 hours.
2.5 Synergistic effect of Noclosan™ and Loxat™
Although both Noclosan™ and Loxat™ showed similar effects on the cell lines tested, both compounds seemed to act synergistically in the induction of apoptosis. When MR65 and CaCo2 cells were exposed to various combinations of both compounds the measured frequency of apoptosis was higher than the sum of the effects of either compound alone (Figures 9A,B) . To investigate whether Noclosan™ and Loxat™ use different apoptosis induction pathways the kinetics of caspase-activation was in detail. To this end, cultures were exposed to 20 mg/ml of Loxat™ and 10 mg/ml of Noclosan™ for various periods of time. Cell lysates were prepared and different caspase-activities were measured. For both compounds the rise in the number of apoptotic cells as measured with the M30-assay was parallelled by an increase in caspase activity (Figures 10A,B).
MR65 cells responded almost immediately to Loxat™ treatment by an exponential rise in caspase 2, 3, 6 and 3/8 processing activities. Caspase 1 and 9 activities remained at almost baseline values. In contrast Noclosan™ treatment resulted in an almost linear increase of caspase 2, 3, 6, 9 and 3/8 processing activities. When MR65 and HaCat cells were immunocytochemically examined for the presence of active caspase 8 after treatment with a combination of Loxat™ and Noclosan™, both cell lines showed positively in a fraction of the cells (figure 11a and lib) 2.6 Signalling pathways involved
To investigate the signalling pathways that lead to induction of apoptosis by Noclosan™ and Loxat™, Western blot analyses were performed on cells treated with either compound. To prevent caspase mediated proteolysis of kinases involved, the analyses were also performed in the presence of the general caspase inhibitor zVAD-fmk. Blots were incubated with antibodies directed against the phospho-p44/p42, phospho-SAPK/JNK, phospho-p38 MAPK and phospho-AKT kinase. As shown in Figure 11 the levels of phospho-p44/p42 erkl/2 remained constant, when comparing control cultures and cultures treated with Noclosan™ or Loxat™. In contrast, increased levels of phospho-p38 MAPK and phospho-SAPL/JNK activity were observed. Despite the relatively high background, a possible decrease in phospho-akt kinase activity was evident after induction of apoptosis by either Noclosan™ or Loxat™.
Discussion
The potato tuber extract Noclosan™ and Loxat™ were studied for their effects on the cell cycle and their ability to induce apoptosis in an in vitro cell culture system using (human) cancer cell lines of different origin. Despite the fact that both compounds are used as additives in serum free culture media (12,13), a growth stimulatory effect could not be confirmed when added to standard culture media supplemented with serum. However, both compounds showed a dose dependent delay in cell cycle progression of exponentially growing cells. The transition through the G2/M- to Gx-phase of the cell cycle seemed to be the most sensitive. Prolonged exposure of cells to lower doses Noclosan™ or Loxat™ resulted in growth retardation in some cell lines, whilst in others a decline in cell number was observed. This decline in cell number arrest was accompanied by the appearance of apoptotic cells. These observations were consistently found in all cell lines tested. Therefore, we conclude that the observed cell cycle effects are not a cell type specific phenomenon.
Next to an inhibitory effect on cell cycle progression, both Noclosan™ and Loxat™ were able to induce apoptosis in a dose dependent way.
In conclusion, Noclosan™ and Loxat™ showed a dose dependent delay of cell cycle progression with complete cell cycle arrest at the highest doses tested. This cell cycle arrest was accompanied by induction of apoptosis.
EXAMPLES 3-13
To demonstrate the general synergistic effect of the combination of Loxat™ and Noclosan™ on human cancer cells, the following cell lines were tested: NCI-H125 cells (lung adenocarcinoma, example 3), MCF7 cells (breast cancer, example 4), OVCAR3 (ovary carcinoma, example 5), U937 cells (monocytic leukemia, example 6) , Jurkat cells (T cell lymphoma, example 7), HaCat cells (human epidermis, example 8), HUVEC cells (human umbilical cord endothelium, example 9) , HAMEC cells (human microvascular endothelium, example 10), NCI-24H (human small-cell lung carcinoma, example 11), NCI-H69 (human small-cell lung carcinoma, example 12) , and Rl cells (rat rhabdo yosarcoma, example 13) . In brief, the above cells (examples 2-12) were exposed to various combinations of Noclosan™/indole acetic acid (IAA) and Loxat™ as described above (example 1, materials and methods, paragraph 1.5). As observed for MR65 cells and CaCo2 cells (see above) , the measured frequency of apoptosis was higher than the sum of the effects of either compound alone for all cell lines tested.

Claims

1. Biologically active composition comprising indole acetic acid (IAA), and a vegetable extract (i) or one or more components thereof, which extract is obtainable by a method comprising: a) grounding a plant material; b) extracting the grounded plant material; c) charging a Q hyper DTM20 gel with the extracted and grounded plant material; d) subjecting the charged Q hyper DTM20 gel to an increasing NaCl gradient; e) collecting the fraction which elutes at 1.02 M NaCl to obtain vegetable extract (i) .
2. Biologically active composition according to claim 1, wherein the concentration indole acetic acid (IAA) ranges from 2*10-4 M to 3*10"3 M.
3. Biologically active composition according to claim
1 or claim 2, wherein the indole acetic acid (IAA) is comprised in a vegetable extract (ii) , which extract is obtainable by a method comprising: a) grounding a plant material; b) extracting the grounded plant material; c) charging a Q hyper DTM20 gel with the extracted and grounded plant material; d) subjecting the charged Q hyper DTM20 gel to an increasing NaCl gradient; e) collecting the fraction which elutes at
0.78 M NaCl to obtain vegetable extract (ii)' comprising indole acetic acid (IAA) .
4. Biologically active composition according to claim 3, wherein the vegetable extract (ii) comprising indole acetic acid (IAA) is Noclosan™.
5. Biologically active composition according to claim
3 or claim 4, wherein the concentration of the vegetable extract (ii) comprising indole acetic acid (IAA) ranges from 1 to 10 mg/ml.
6. Biologically active composition according to any of the claims 1 to 5, wherein one or more components of the vegetable extract (i) are chosen from the group consisting of carboiimide, octonoic acid, sucrose, 5-alpha-pregnane-3-alpha 17.20 beta thriylthoxy, L-norvaline, ribonic acid, propylene glycol, 1-amino ethanol, butanoic acid, L-valine, L-alanine, L-isoleucine, L-proline, L-serine, L-threonine, 3,4- dihydroxybutanoic acid, tetronic acid, gamma-lactone, arabino-hexanoic acid, D-fructose, cyclopentane carboxylic acid, linear alkane, fructose oxim, talose, D-gluconic acid, hexadecanoic acid, cyclododecylamine, octadecanoic acid, 9- octadocenamide, N,N-diethyl formamide, ethylamine, trans-3 methyl-2-nonene, ethylene glycol, urea, acetetic acid, 3- methylene 1,4 butanediol, D, L-isoborneol, glycerol, Myo- inositol, hexadecanamide, galactunoic acid, butanoic acid, and 6-hydroxyhexanoic acid.
7. Biologically active composition according to any of the claims 1 to 6, wherein the component of the vegetable extract (i) is 5-alpha-pregnane-3-alpha 17.20 beta thriylthoxy.
8. Biologically active composition according to any of the claims 1 to 7, wherein the vegetable extract (i) is Loxat™.
9. Biologically active composition according to any of the claims 1 to 8, wherein the concentration of the vegetable extract (i) ranges from 1 mg/ml to 10 mg/ml.
10. Biologically active composition according to any of the claims 1 to 9 for use as a growth inhibiting agent of eukaryotic cells.
11. Biologically active composition according to any of the claims 1 to 9 for use as an apoptosis inducing agent of eukaryotic cells.
12. Method for the preparation of a biologically active composition comprising indole acetic acid (IAA) and a vegetable extract (i) comprising: a) grounding a plant material; b) extracting the grounded plant material; c) charging a Q hyper DTM20 gel with the extracted and grounded plant material; d) subjecting the charged Q hyper DTM20 gel to an increasing NaCl gradient; e) collecting the fraction which elutes at
1.02 M NaCl to obtain vegetable extract (i); g) combining indole acetic acid (IAA) with the vegetable extract (i) to provide a biologically active composition.
13. Method according to claim 12, wherein the indole acetic acid is comprised in a vegetable extract (ii) obtainable by: a) grounding a plant material; b) extracting the grounded plant material; c) charging a Q hyper DTM20 gel with the extracted and grounded plant material; d) subjecting the charged Q hyper DTM20 gel to an increasing NaCl gradient; e) collecting the fraction which elutes at
0.78 M NaCl to provide a vegetable extract (ii) comprising indole acetic acid (IAA)..
14. Method according to claim 12, wherein the provided biologically active composition comprises 2*10-4 M to 3*10~3 M indole acetic acid (IAA) and 1 mg/ml to 10 mg/ml vegetable extract (i) .
15. Method according to claim 13, wherein the provided biologically active composition comprises 1 mg/ml to 10 mg/ml vegetable extract (ii) and 1 mg/ml to 10 mg/ml vegetable extract (i) .
PCT/EP2004/004382 2003-04-23 2004-04-23 Vegetable extract compositions comprising inodole acetic, methods for the preparation of said compsitions, and their use as cell growth inhibiting agents WO2004093891A1 (en)

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WO1998046244A1 (en) * 1997-04-16 1998-10-22 Feva N.V. Vegetable extract, method for preparation thereof and its applications in human and veterinary medicine

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WO1998046244A1 (en) * 1997-04-16 1998-10-22 Feva N.V. Vegetable extract, method for preparation thereof and its applications in human and veterinary medicine

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100130421A1 (en) * 2008-10-17 2010-05-27 Alfredo Gallegos Biomodulators for treatment or prevention of disease
US9063122B2 (en) * 2008-10-17 2015-06-23 Alfredo Gallegos Biomodulators for treatment or prevention of disease

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