WO2005099721A2 - Compositions comportant des composes polyphenoliques derives de plantes et des inhibiteurs d'especes reactive d'oxygene et leurs procedes d'utilisation - Google Patents

Compositions comportant des composes polyphenoliques derives de plantes et des inhibiteurs d'especes reactive d'oxygene et leurs procedes d'utilisation Download PDF

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WO2005099721A2
WO2005099721A2 PCT/US2005/011741 US2005011741W WO2005099721A2 WO 2005099721 A2 WO2005099721 A2 WO 2005099721A2 US 2005011741 W US2005011741 W US 2005011741W WO 2005099721 A2 WO2005099721 A2 WO 2005099721A2
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pkc
inhibitor
cell
activation
cells
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PCT/US2005/011741
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WO2005099721A3 (fr
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Stephen Pandol
Anna Gukovskaya
Moussa Yazbeck
Guido Eibl
Laszlo Boros
Akihiko Sato
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The Regents Of The University Of California
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Priority claimed from US10/824,597 external-priority patent/US20040259816A1/en
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Publication of WO2005099721A2 publication Critical patent/WO2005099721A2/fr
Publication of WO2005099721A3 publication Critical patent/WO2005099721A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the present invention generally relates to plant-derived polyphenolic compounds, inhibitors of reactive oxygen species (ROS) and compositions thereof and methods for treating, preventing, or inhibiting diseases and disorders associated with NF- ⁇ B activation, such as pancreatic cancer and pancreatitis.
  • ROS reactive oxygen species
  • Pancreatic cancer is the fifth leading cause of cancer death in the United States Cures for this type of cancer are unusual with the cancer recurring as metastatic disease in most cases after the removal of the primary tumor at surgery. See DiMagno et al. (1999) Gastroenterology 117:1464-1484; and Todd et al. (1999) Pancreatic Adenocarcinoma. TEXTBOOK OF GASTROENTEROLOGY. Philadelphia: Lippincott Williams & Wilkins, p. 2178-2192.
  • the present invention relates to a method of treating, preventing, or inhibiting cancer in a subject comprising administering at least one polyphenolic compound and at least one inhibitor of reactive oxygen species to the subject.
  • the polyphenolic compound may be derived or isolated from plants.
  • the polyphenolic compound is a flavonoid. In other embodiments, the polyphenolic compound is a non-flavonoid.
  • the polyphenolic compound is quercetin, rutin, genistein, curcumin, rottlerin or trans-resveratrol.
  • the inhibitor is diphenylene iodonium, N-acetylcysteine, or Tiron.
  • the method further comprises administering at least one antioxidant to the subject.
  • the subject is mammalian, more preferably, the subject is human.
  • the present invention provides a method of inducing apoptosis in a tumor comprising contacting the tumor with at least one polyphenolic compound. In some embodiments, the method further includes contacting the tumor with at least one inhibitor of reactive oxygen species.
  • the polyphenolic compound may be derived or isolated from plants. In some embodiments, the polyphenolic compound is a flavonoid. In other embodiments, the polyphenolic compound is a non-flavonoid.
  • the polyphenolic compound is selected from the group consisting of flavenoids, anthrocyanins, anthrocyanidins, isoflavones, catechins, epigallocatechin gallate, gallic acid, chlorgenic acid, curcumin, kaempferol, quercetin, isoquercitrin, myricetin, rutin, pelargonidin, cyanidin, delphinidin, peonidin, malvidin, malvin, oenin, cyanidin, kuromanin, diadzein, daidzin, genitein, genistin, tannic acid, caffeic acid, ferulic acid, ellagic acid, rottlerin and traxol.
  • the polyphenolic compound is quercetin, rutin, genistein, curcumin, rottlerin or trans-resveratrol.
  • the inhibitor is diphenylene iodonium, N-acetylcysteine, or Tiron.
  • the method further comprises contacting the tumor with at least one antioxidant.
  • the tumor is a primary tumor. In other embodiments, the tumor is metastatic.
  • the polyphenolic compound is quercetin, rutin, genistein, curcumin, rottlerin or trans-resveratrol.
  • the inhibitor is diphenylene iodonium, N-acetylcysteine, or Tiron.
  • the method further comprises contacting the protein target with at least one antioxidant.
  • the present invention provides a method of activating caspase-3 with at least one polyphenolic compound and at least one inhibitor of reactive oxygen species. In some embodiments, the method further includes contacting caspase-3 with at least one inhibitor of reactive oxygen species.
  • the polyphenolic compound may be derived or isolated from food. In some embodiments, the polyphenolic compound is a flavonoid. In other embodiments, the polyphenolic compound is a non-flavonoid.
  • the polyphenolic compound is selected from the group consisting of flavenoids, anthrocyanins, anthrocyanidins, isoflavones, catechins, epigallocatechin gallate, gallic acid, chlorgenic acid, curcumin, kaempferol, quercetin, isoquercitrin, myricetin, rutin, pelargonidin, cyanidin, delphinidin, peonidin, malvidin, malvin, oenin, cyanidin, kuromanin, diadzein, daidzin, genitein, genistin, tannic acid, caffeic acid, ferulic acid, ellagic acid, rottlerin and traxol.
  • the polyphenolic compound is quercetin, rutin, genistein, curcumin, rottlerin or trans-resveratrol.
  • the inhibitor is diphenylene iodonium, N-acetylcysteine, or Tiron.
  • the method further comprises contacting caspase-3 with at least one antioxidant.
  • the present invention provides a method of preventing, inhibiting, or modulating NF- ⁇ B activation in a cell comprising administering to the cell at least one polyphenolic compound and MG-132, diphenylene iodonium, or both.
  • the polyphenolic compound may be derived or isolated from plants.
  • the polyphenolic compound is a flavonoid. In other embodiments, the polyphenolic compound is a non-flavonoid.
  • the polyphenolic compound is selected from the group consisting of flavenoids, anthrocyanins, anthrocyanidins, isoflavones, catechins, epigallocatechin gallate, gallic acid, chlorgenic acid, curcumin, kaempferol, quercetin, isoquercitrin, myricetin, rutin, pelargonidin, cyanidin, delphinidin, peonidin, malvidin, malvin, oenin, cyanidin, kuromanin, diadzein, daidzin, genitein, genistin, tannic acid, caffeic acid, ferulic acid, ellagic acid, rottlerin and traxol.
  • the present invention provides a method of making a cancer cell susceptible to apoptosis induced by a polyphenolic compound comprising inhibiting NF- ⁇ B activity in the cell.
  • the polyphenolic compound may be derived or isolated from plants.
  • the polyphenolic compound is a flavonoid. In other embodiments, the polyphenolic compound is a non-flavonoid.
  • the polyphenolic compound is selected from the group consisting of flavenoids, anthrocyanins, anthrocyanidins, isoflavones, catechins, epigallocatechin gallate, gallic acid, chlorgenic acid, curcumin, kaempferol, quercetin, isoquercitrin, myricetin, rutin, pelargonidin, cyanidin, delphinidin, peonidin, malvidin, malvin, oenin, cyanidin, kuromanin, diadzein, daidzin, genitein, genistin, tannic acid, caffeic acid, ferulic acid, ellagic acid, rottlerin and traxol.
  • the polyphenolic compound is quercetin, rutin, genistein, curcumin, rottlerin or trans-resveratrol.
  • the present invention provides a method of preventing, inhibiting, or attenuating the activation of Akt/PKB in a cell comprising administering to the cell at least one polyphenolic compound, at least one inhibitor of reactive oxygen species and at least one PI 3 -kinase inhibitor, or at least one polyphenolic compound and at least one inhibitor of NADPH oxidase or at least one inhibitor of reactive oxygen species.
  • the polyphenolic compound may be derived or isolated from plants.
  • the polyphenolic compound is a flavonoid. In other embodiments, the polyphenolic compound is a non-flavonoid.
  • the polyphenolic compound is selected from the group consisting of flavenoids, anthrocyanins, anthrocyanidins, isoflavones, catechins, epigallocatechin gallate, gallic acid, chlorgenic acid, curcumin, kaempferol, quercetin, isoquercitrin, myricetin, rutin, pelargonidin, cyanidin, delphinidin, peonidin, malvidin, malvin, oenin, cyanidin, kuromanin, diadzein, daidzin, genitein, genistin, tannic acid, caffeic acid, ferulic acid, ellagic acid, rottlerin and traxol.
  • the present invention provides a pharmaceutical composition comprising at least one polyphenolic compound, at least one inhibitor of reactive oxygen species, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may further comprise at least one antioxidant.
  • the pharmaceutical composition may further comprise at least one anti-neoplastic agent.
  • the polyphenolic compound may be derived or isolated from plants. In some embodiments, the polyphenolic compound is a flavonoid. In other embodiments, the polyphenolic compound is a non-flavonoid.
  • the polyphenolic compound is quercetin, rutin, genistein, curcumin, rottlerin or trans-resveratrol.
  • the inhibitor is diphenylene iodonium, N-acetylcysteine, or Tiron.
  • the polyphenolic compound is selected from the group consisting of flavenoids, anthrocyanins, anthrocyanidins, isoflavones, catechins, epigallocatechin gallate, gallic acid, chlorgenic acid, curcumin, kaempferol, quercetin, isoquercitrin, myricetin, rutin, pelargonidin, cyanidin, delphinidin, peonidin, malvidin, malvin, oenin, cyanidin, kuromanin, diadzein, daidzin, genitein, genistin, tannic acid, caffeic acid, ferulic acid, ellagic acid, rottlerin and traxol.
  • the polyphenolic compound is quercetin, rutin, genistein, curcumin, rottlerin or trans-resveratrol.
  • the inhibitor is diphenylene iodonium, N-acetylcysteine, or Tiron.
  • the present invention relates to a method of depolarizing a mitochondrial membrane comprising contacting the mitochondrial membrane with at least one polyphenolic compound and at least one inhibitor of reactive oxygen species.
  • the polyphenolic compound may be derived or isolated from plants.
  • the polyphenolic compound is a flavonoid. In other embodiments, the polyphenolic compound is a non-flavonoid.
  • the polyphenolic compound is quercetin, rutin, genistein, curcumin, rottlerin or trans-resveratrol.
  • the inhibitor is diphenylene iodonium, N-acetylcysteine, or Tiron.
  • the method further comprises contacting the mitochondrial membrane with at least one antioxidant.
  • the polyphenolic compound is selected from the group consisting of flavenoids, anthrocyanins, anthrocyanidins, isoflavones, catechins, epigallocatechin gallate, gallic acid, chlorgenic acid, curcumin, kaempferol, quercetin, isoquercitrin, myricetin, rutin, pelargonidin, cyanidin, delphinidin, peonidin, malvidin, malvin, oenin, cyanidin, kuromanin, diadzein, daidzin, genitein, genistin, tannic acid, caffeic acid, ferulic acid, ellagic acid, rottlerin and traxol.
  • the polyphenolic compound is quercetin, rutin, genistein, curcumin, rottlerin or trans-resveratrol.
  • the inhibitor is diphenylene iodonium, N-acetylcysteine, or Tiron.
  • the method further comprises contacting the mitochondiral PTP with at least one antioxidant.
  • the present invention provides a method of treating, preventing, inhibiting, or modulating NF- B activation in a cell or in a subject which comprises administering at least one polyphenolic compound, an inhibitor of PKC ⁇ translocation, an inhibitor of PKC ⁇ translocation, or a combination thereof to the cell or the subject.
  • the polyphenolic compound is rottlerin or a derivative thereof.
  • the method further comprises administering a second polyphenolic compound to the cell or the subject.
  • the second polyphenolic compound is selected from the group consisting of flavenoids, anthrocyanins, anthrocyanidins, isoflavones, catechins, epigallocatechin gallate, gallic acid, chlorgenic acid, curcumin, kaempferol, quercetin, isoquercitrin, myricetin, rutin, pelargonidin, cyanidin, delphinidin, peonidin, malvidin, malvin, oenin, cyanidin, kuromanin, diadzein, daidzin, genitein, genistin, tannic acid, caffeic acid, ferulic acid, ellagic acid, rottlerin and traxol.
  • the second polyphenolic compound is quercetin, rutin, genistein, curcumin or trans-resveratrol.
  • the method further comprises administering at least one inhibitor of a reactive oxygen species to the cell or the subject.
  • the inhibitor is diphenylene iodonium, N-acetylcysteine, or Tiron.
  • the method further comprises administering at least one antioxidant to the cell or the subject.
  • the inhibitor of PKC ⁇ translocation or the inhibitor of PKC ⁇ translocation is a peptide.
  • the peptide is ⁇ Vl-1 or ⁇ Vl- 2.
  • the present invention also provides a method of treating, preventing, or inhibiting a disease or disorder associated with NF- ⁇ B activation in a subject which comprises treating, preventing, inhibiting, or modulating NF- ⁇ B activation in a cell or in a subject which comprises administering at least one polyphenolic compound, an inhibitor of PKC ⁇ translocation, an inhibitor of PKC ⁇ translocation, or a combination thereof to the cell or the subject.
  • the polyphenolic compound is rottlerin or a derivative thereof.
  • the method further comprises administering a second polyphenolic compound to the cell or the subject.
  • the second polyphenolic compound is selected from the group consisting of flavenoids, anthrocyanins, anthrocyanidins, isoflavones, catechins, epigallocatechin gallate, gallic acid, chlorgenic acid, curcumin, kaempferol, quercetin, isoquercitrin, myricetin, rutin, pelargonidin, cyanidin, delphinidin, peonidin, malvidin, malvin, oenin, cyanidin, kuromanin, diadzein, daidzin, genitein, genistin, tannic acid, caffeic acid, ferulic acid, ellagic acid, rottlerin and traxol.
  • the disease or disorder is a cancer, preferably pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, kidney cancer, pancreatic cancer, colon cancer, thyroid cancer, melanoma, Hodgkin's lymphoma, acute lymphoblastic leukemia, acute myelogenous leukemia, diffuse large B- cell lymphoma, astrocytoma, glioblastoma, a head or neck cancer, or vulva cancer.
  • the cancer is related to in vitro transformation of BCR-ABL, DBL/DBS, RAF, RAS, TEL-JAK2, or TEL-PDGFR.
  • the cancer is related to viral oncogenesis caused by Epstein-Barr virus, hepatitis B virus, human herpesvirus-8, or human T-cell leukemia virus-1.
  • the disease or disorder is an inflammatory disease, preferably pancreatitis, inflammatory bowel disease, asthma, arthritis, rheumatoid arthritis, asthma, psoriasis, cystitis, or nephritis.
  • the disease or disorder is viral hepatitis, alcoholic liver disease, lung inflammation, Alzheimer's Disease, or atherosclerosis.
  • the method further comprises administering at least one antiproliferative agent, at least one anti-inflammatory agent, or both.
  • administration of the polyphenolic compound, the inhibitor of PKC ⁇ translocation, the inhibitor of PKC ⁇ translocation, or the combination thereof causes, induces, increases, or modulates cell cycle arrest, apoptosis, mitochondrial cytochrome c release, dissipation of mitochondrial polarity, caspase activation, mitochondrial permeability transition pore activation, or a combination thereof, in the cancer.
  • the method further comprises administering a second polyphenolic compound, at least one inhibitor of reactive oxygen species, at least one inhibitor of PI 3 -kinase, at least one inhibitor of ⁇ ADPH oxidase, or a combination thereof.
  • the present invention provides a method of inducing apoptosis in a cell or making the cell susceptible to apoptosis which comprises treating, preventing, inhibiting, or modulating NF- ⁇ B activation in a cell or in a subject which comprises administering at least one polyphenolic compound, an inhibitor of PKC ⁇ translocation, an inhibitor of PKC ⁇ translocation, or a combination thereof to the cell or the subject.
  • the polyphenolic compound is rottlerin or a derivative thereof.
  • the method further comprises administering a second polyphenolic compound to the cell or the subject.
  • the second polyphenolic compound is quercetin, rutin, genistein, curcumin or trans-resveratrol.
  • the method further comprises administering at least one inhibitor of a reactive oxygen species to the cell or the subject.
  • the inhibitor is diphenylene iodonium, N-acetylcysteine, or Tiron.
  • the method further comprises administering at least one antioxidant to the cell or the subject.
  • the inhibitor of PKC ⁇ translocation or the inhibitor of PKC ⁇ translocation is a peptide.
  • the peptide is ⁇ Nl-1 or ⁇ Nl- 2.
  • the cell is a tumor cell or a cancer cell.
  • the tumor is a primary tumor.
  • the tumor is metastatic.
  • the second polyphenolic compound is selected from the group consisting of flavenoids, anthrocyanins, anthrocyanidins, isoflavones, catechins, epigallocatechin gallate, gallic acid, chlorgenic acid, curcumin, kaempferol, quercetin, isoquercitrin, myricetin, rutin, pelargonidin, cyanidin, delphinidin, peonidin, malvidin, malvin, oenin, cyanidin, kuromanin, diadzein, daidzin, genitein, genistin, tannic acid, caffeic acid, ferulic acid, ellagic acid, rottlerin and traxol.
  • the second polyphenolic compound is quercetin, rutin, genistein, curcumin or trans-resveratrol.
  • the pharmaceutical composition further comprises an inhibitor of PKC ⁇ translocation, an inhibitor of PKC ⁇ translocation, or both.
  • the inhibitor of PKC ⁇ translocation or the inhibitor of PKC ⁇ translocation is SFNSYELGSLRQIKIWFQNRRMKWKK (SEQ ID NO: 10) or EANSLKPTRQIKIWFQNRRMKWKK (SEQ ID NO:l 1).
  • the pharmaceutical composition further comprises an inhibitor of a reactive oxygen species.
  • the pharmaceutical composition further comprises an antioxidant.
  • FIGS. 3C are Western blots that illustrate that poly(ADP-ribose) polymerase (PARP) was cleaved in cell lines treated with quercetin and trans-resveratrol, but not rutin.
  • PARP poly(ADP-ribose) polymerase
  • Figure 5 shows the effects of inhibition of ROS and trans-resveratrol on Annexin V staining in Mia PACA-2 pancreatic cancer cells.
  • PI propidium iodide
  • AnV Annexin V
  • RS trans-resveratrol
  • Figure 6A is a Western blot showing that trans-resveratrol converts caspase-3 into the active form in BSp73AS cells.
  • Figure 6B shows the results of a fluorogenic assay that confirms that trans- resveratrol and quercetin activate caspase-3 in a time dependent manner in BSp73 AS cells.
  • Figure 6C shows the results of a fluorogenic assay that confirms that quercetin activates caspase-3 in a dose dependent manner in BSp73 AS cells.
  • Figure 9A is a Western blot showing that polyphenolic compounds stimulate mitochondrial release of cytochrome c in BSp73AS cells.
  • R ⁇ rutin
  • Q quercetin
  • RS trans-resveratrol
  • G ⁇ genistein.
  • FIG. 10A shows that polyphenolic compounds induce depolarization of mitochondrial membrane potential in BSp73AS cells.
  • Figure 12A shows that inhibition of PTP by cyclosporin A alone or in combination with aristolochic acid prevents release of mitochondrial cytochrome c release in MiaPACA-2 cancer cells treated with trans-resveratrol, quercetin and genistein.
  • Z-VAD prevents mitochondrial cytochrome c release in untreated (control) cells.
  • Q quercetin
  • RS trans-resveratrol
  • GN genistein.
  • Figure 12B shows that inhibition of PTP by cyclosporine A and aristolochic acid attenuates caspase-3 activity in Mia PACA-2 cells treated with polyphenolic compounds.
  • the caspase inhibitor Z-VAD blocked caspase activity in the MiaPACA-2 cells.
  • Q quercetin
  • RS trans-resveratrol
  • GN genistein.
  • Figure 12C shows that inhibition of PTP by cyclosporin A and aristolochic acid and inhibition of caspases by Z-VAD decreases apoptosis in MiaPACA-2 cells treated with polyphenolic compounds.
  • Q quercetin
  • RS trans-resveratrol
  • GN genistein.
  • Figure 13 A is an immunoblot that shows the effect of polyphenolic compounds alone and in combination on cytochrome c release in Mia PACA-2 cells.
  • Q quercetin
  • RS trans-resveratrol.
  • Figure 14B shows the relative NF- ⁇ B activities in cells treated with polyphenolic compounds or MG-132.
  • Figure 18 shows the effects of L Y294002 and DPI on NF- ⁇ B activation in Mia PACA-2 pancreatic cancer cells.
  • DPI diphenylene iodonium.
  • FIG. 19A shows subcellular distribution of PKC isoforms in response to CCK-8 in rat pancreatic acini. Shown are representative Western blots from 3 independent experiments.
  • Figure 20 A shows NF- ⁇ B binding activity by electromobility shift assay (EMS A) in rat pancreatic acini treated with or without CCK-8 in the presence or absence of inhibitors of specific isoforms of PKC.
  • EMS A electromobility shift assay
  • Figure 22A shows subcellular distribution of PKC isoforms in response to TNF- ⁇ in rat pancreatic acini. Shown are representative blots from 3 independent experiments.
  • Figure 23 A shows NF- ⁇ B binding activity in nuclear extracts from rat pancreatic acini treated with or without TNF- ⁇ in the presence or absence of inhibitors of specific isoforms of PKC.
  • Figure 23B shows NF- ⁇ B band intensities in rat pancreatic acini treated with or without TNF- ⁇ in the presence or absence of inhibitors of isoforms of PKC.
  • Figure 23 C shows I ⁇ B ⁇ degradation in cytosolic extracts by Western blot analysis of rat pancreatic acini treated with or without TNF- ⁇ in the presence of absence of inhibitors of specific isoforms of PKC. Representative of 4 independent experiments.
  • Figure 24A shows NF- ⁇ B binding activity in nuclear extracts from rat pancreatic acini treated with or without CCK-8 or TNF- ⁇ in the presence or absence of a Src kinase inhibitor, PP2.
  • Figure 24B shows I ⁇ B ⁇ degradation in cytosolic extracts by Western blot analysis of rat pancreatic acini treated with or without CCK-8 or TNF- ⁇ in the presence or absence of a Src kinase inhibitor, PP2. Representative of 4 independent experiments.
  • Figure 25 shows the effects of Src kinase inhibitor on tyrosine phosphorylation of PKC ⁇ in rat pancreatic acini treated with or without CCK-8 or TNF- ⁇ . Representative of 4 independent experiments.
  • Figure 26A shows NF- ⁇ B binding activity in nuclear extracts from rat pancreatic acini treated with or without CCK-8 or TNF- ⁇ in the presence or absence of a phosphatidylinositol (P ⁇ )-specific phospholipase C (PLC) inhibitor, U-73122, or a phosphatidylcholine (PC)-specific PLC inhibitor, D-609.
  • P ⁇ phosphatidylinositol
  • PC phosphatidylcholine
  • Figure 28B shows NF- ⁇ B binding activity in nuclear extracts from rat pancreatic acini treated with or without ethanol and CCK-8 in the presence or absence of rottlerin. Shown are representative blots from 3 independent experiments.
  • Figure 29 shows rottlerin but not protein kinase C inhibitors cause apoptosis as measured by oligonucleosomal DNA fragmentation in MIA PaCa-2 pancreatic cancer cells.
  • Figure 30 shows rottlerin but not protein kinase C inhibitors cause apoptosis as measured by oligonucleosomal DNA fragmentation in PANC-1 pancreatic cancer cells.
  • Figure 33B shows the effect of rottlerin on the percentage of MIA PaCa-2 pancreatic cancer cells with high ⁇ m.
  • Figure 34 shows the effects of rottlerin on mitochondrial cytochrome c release in MIA PaCa-2 pancreatic cancer cells.
  • Figure 35 shows the effects of rottlerin and GF109203X on NF- ⁇ B activation in MIA PaCa-2 pancreatic cancer cells.
  • Figure 36A1 -4 are histograms of intracellular H O as measured by flow cytometry using an H 2 O 2 -sensitive intracellular probe (DCF) showing the effects of rottlerin (Rt) and GF109203X (GF) on production of ROS in MIA PaCa-2 pancreatic cancer cells.
  • Figure 36B shows the percentage of cells with high DCF fluorescence.
  • Figure 37 shows the effect of rottlerin on the growth of MIA PaCa-2 tumors in nude mice.
  • Figure 38 shows the effect of rottlerin on deoxyribose (1) and ribose (2) 13 C tracer accumulation from glucose in MIA PaCa-2 cells.
  • Figure 39 shows the effect of rottlerin on oxidative deoxyribose (1) and non- oxidative deoxyribose (2) synthesis, as well as oxidative ribose (3) and non-oxidative ribose (4) synthesis based on positional 13 C tracer accumulation from glucose into nucleic acid of MIA PaCa-2 cells.
  • Figure 40 shows the effect of rottlerin on direct glucose oxidation and recycling in the pentose cycle in MIA PaCa-2 cells.
  • Figure 41 shows the effect of rottlerin on glucose oxidation relative to glucose anaplerosis in the TCA cycle of MIA PaCa-2 cells.
  • Figure 42 shows the effect of rottlerin on de novo myrystate (1), palmitate (2), stearate (3) and oleate (4) fatty acid synthesis of MIA PaCa-2 cells.
  • the present invention is directed to compounds, compositions, and methods for treating, preventing, and inhibiting cancer.
  • the present invention provides compositions comprising at least one plant-derived polyphenolic compound and at least one inhibitor of reactive oxygen species (ROS) to cause cancer cell death and prevent or treat cancer.
  • the present invention also provides methods for treating or preventing cancer in a subject which comprises administering at least one plant-derived polyphenolic compound and at least one inhibitor of reactive oxygen species (ROS) to the subject.
  • ROS reactive oxygen species
  • Flavonoids are characterized as molecules possessing two phenols joined by a pyran (oxygen-containing) carbon ring structure. Common flavonoids include quercetin, rutin and genistein. Flavonoids represent the most common and widely distributed group of plant polyphenolic compounds. Examples of nonflavonoid polyphenolic compounds include the resveratrol family of compounds.
  • Polyphenolic compounds of the present invention include compounds that have more than one phenol ring structure.
  • the present invention provides a method of treating, preventing, or inhibiting cancer, preferably pancreatic cancer, in a subject, preferably human, comprising administering to the subject an effective amount of a polyphenolic compound.
  • the present invention also provides a method of treating, inhibiting, preventing, or decreasing, metastatic cancer lesions in a subject, preferably human, comprising administering to the subject an effective amount of a polyphenolic compound.
  • the present invention further provides a method of treating, inhibiting, or decreasing the growth or growth rate of a primary tumor in a subject, preferably human, comprising administering to the subject an effective amount of a polyphenolic compound.
  • the polyphenolic compound is quercetin, trans-resveratrol, or genistein.
  • incubation media free of serum was used in order to determine the effects of the agents in the absence of growth factors.
  • serum was subsequently added to the incubation conditions, the effects of the polyphenolic compounds described above were attenuated, thereby indicating that agents in serum have an effect on regulating the apoptosis pathways.
  • ROS reactive oxygen species
  • IGF-1 insulin growth factor- 1
  • the methods of the present invention further comprise administration of at least one antioxidant.
  • quercetin, trans-resveratrol and genistein enhance apoptotic cancer cell death in pancreatic cancer cells by causing mitochondrial depolarization and cytochrome c release followed by caspase-3 activation. Inhibition of the mitochondrial PTP resulted in the prevention of mitochondrial depolarization, cytochrome c release, caspase-3 activation and apoptosis. Furthermore, both quercetin and genistein caused inhibition of growth of pancreatic cancer in a nude mouse model. The inhibition was most pronounced on metastatic spread of the tumor and included increased apoptosis in the tumor.
  • the present invention provides a method of inducing apoptosis in a primary tumor comprising contacting the primary tumor with an effective amount of at least one polyphenolic compound.
  • the present invention also provides a method of cleaving a protein target of caspases-3 activation, P ARP, comprising contacting PARP with an effective amount of at least one polyphenolic compound.
  • ROS ROS Inhibitors
  • ROS are produced in large quantities by phagocytes mediating host defense against a variety of microorganisms. See Thannickal & Fanburg, (2000) Am. J. Physiol. Lung Cell. Mol. Physiol. 279:L1005-L1028; Freeman & Crapo (1982) Lab. Invest. 47: 412-426; Rhee (1999) Exp. Mol. Med. 31:53-59; and Babior (1999) Blood 93:1464- 1476, which are herein incorporated by reference. There is accumulating evidence that ROS are produced in smaller quantities by non-phagocytes including cancer cells. See Nakamura et al. (1997) Ann. Rev. Immunol.
  • ROS reactive oxygen species
  • ROS ROS oxidase
  • the best-characterized source is the NADPH oxidase system in phagocytes. Recent studies suggest that a group of functional proteins analogous to the NADPH oxidase system are present and mediate ROS in non- phagocytic cells. The proteins are called NOX proteins, which are homologous to the NADPH oxidase catalytic subunit, gp91phox.
  • NOX proteins homologous to the NADPH oxidase catalytic subunit
  • Another source of ROS generation is the mitochondria where ROS are produced as "by-products" of the electron transfer reactions.
  • Other sources of ROS production include oxidation of the phospholipase A 2 product, arachidonic acid, by 5-lipoxygenases; and cytosolic xanthine oxidase.
  • trans-resveratrol and genistein were found to cause a small increase in ROS production in addition to the effect of serum. Both the effects of serum and the polyphenolic compounds on ROS were prevented by DPI. Thus, serum, trans-resveratrol, and genistein increase ROS in pancreatic cancer cells and that agents known to inhibit ROS production prevent the increases in ROS.
  • polyphenolic compounds alone and in combination with inhibitors of ROS formation on apoptosis of pancreatic cancer cells was studied. As provided herein, when cancer cells were treated with serum combinations of inhibitors of ROS and polyphenolic compounds resulted in a synergistic increases in cancer cell DNA fragmentation.
  • caspases the transcription factor
  • nuclear factor KB nuclear factor KB
  • PI 3-kinase phosphatidylinositol 3-kinase
  • Caspases are necessary for apoptosis to occur. More than a dozen caspases have been identified. The caspases are synthesized as inactive proenzymes requiring cleavage at Asp residues to be activated. At least some of these caspases can activate each other in the form of a proteolytic cascade. Caspases are generally divided into “initiator” caspases and “executioner” caspases. Caspases-8 and -9 are “initiator” caspases while caspases-3, -6 and -7 are “executioner” caspases.
  • the mechanism of mitochondrial permeabilization and release of cytochrome c is incompletely understood.
  • the permeabilization is usually associated with a loss of mitochondrial transmembrane potential and "opening" of the mitochondrial permeability transition pore (PTP).
  • the PTP inhibitor, cyclosporine A is frequently used to demonstrate the role of PTP in the involved in apoptosis, i.e. cytochrome c release and caspase activation.
  • both quercetin and trans-resveratrol convert caspase-3 from its inactive form (32 kDa doublet) to its active form (17 kDa).
  • caspase-3 activity is synergistically activated with a combination of an inhibitor of ROS production and a polyphenolic compound. Therefore, the present invention provides a method of activating caspase-3 comprising contacting the inactive caspase-3 with at least one ROS inhibitor or at least one polyphenolic compound.
  • the inactive caspase-3 is activated with at least one ROS inhibitor and at least one polyphenolic compound.
  • the present invention provides a method of increasing cytosolic cytochrome c, decreasing mitochondrial cytochrome c, dissipating mitochondrial membrane potential, or a combination thereof, comprising administering to a cell or a subject an effective amount of at least one polyphenolic compound.
  • NF- ⁇ B activation occurs as a result of phosphorylation and degradation of NF- ⁇ B-associated proteins-I ⁇ B ⁇ and I ⁇ B ⁇ (inhibitory KBS).
  • IKBS phosphatidylinositol 3- kinase
  • ROS phosphatidylinositol 3- kinase
  • the mechanisms of the anti-apoptotic action of NF- ⁇ B are not fully understood.
  • the known anti-apoptotic targets of activated NF- ⁇ B include the inhibitors of apoptosis (IAP) family of proteins, such as cIAP-1 and -2, and XIAP, as well as the anti-apoptotic Bcl-2 proteins.
  • the proteosome inhibitor, MG-132 blocks NF- ⁇ B activation in both cell lines and causes a small increase in caspase-3 activity. Additionally, trans-resveratrol in combination with MG-132 alone or MG-132 plus DPI increased apoptosis to a greater degree than that observed with MG- 132 alone or MG-132 plus DPI, thereby indicating that inhibition of NF- ⁇ B sensitizes the cancer cells to apoptosis caused by trans-resveratrol.
  • the present invention provides a method of preventing or inhibiting NF- ⁇ B activation in a cell comprising administering to the cell DPI and at least one polyphenolic compound or MG-132 and at least one polyphenolic compound or MG-132 and DPI. Since DPI is an antioxidant and MG-132 is a proteosomal inhibitor, the present invention provides a method of preventing or inhibiting NF- ⁇ B activation in a cell comprising administering to the cell an antioxidant, a proteosomal inhibitor, or both and at least one polyphenolic compound. The present invention also provides a method of making a cancer cell susceptible to apoptosis induced by a polyphenolic compound comprising inhibiting NF- ⁇ B activity in the cell.
  • NF- ⁇ B A key regulator of the expression of these inflammatory molecules is NF- ⁇ B.
  • NF- ⁇ B activation in acinar cells is one of the earliest events and the inhibition of NF- ⁇ B activation attenuates inflammatory response and the severity of pancreatitis. See Gukovsky et al. (2003) Am. J. Physiol. Gastrointest. Liver Physiol.
  • CCK-8 stimulation of isolated rat pancreatic acini can be also used to investigate the mechanism of NF- ⁇ B activation. See Han & Logsdon (1999) Am. J. Physiol. Cell. Physiol. 277:C74-C82, Han & Logsdon (2000) Am. J. Physiol. Cell. Physiol. 278:C344- C351, and Tando et al. (1999) Am. J. Physiol. Gastrointest. Liver Physiol. 277:G678- G686, which are herein incorporated by reference.
  • CCK is a physiologic regulator of pancreatic digestive enzyme secretion; however, supramaximally stimulating doses of CCK-8 cause the inflammatory response that underlies many of the features of human pancreatitis. See Williams (2001) Ann. Rev. Physiol. 63:77-97, which is herein incorporated by reference. Similar to TNF- ⁇ , the post receptor events mediating NF- ⁇ B activation by CCK-8 are poorly understood.
  • PKCs protein kinase Cs
  • PKCs In addition to the regulation by Ca and lipid messengers, the activity of PKCs is regulated by phosphorylation and one important mediator of this pathway is the family of Src kinases. See Gschwendt (1999) Eur. J. Biochem. 259:555-564, and Parekh et al. (2000) EMBO J. 19:496-503, which are herein incorporated by reference. Each PKC isoform has a different pattern of cell distribution, can be activated independently by specific stimuli, and mediates distinct biological functions. In general, the activation of PKCs is associated with their translocation to distinct intracellular compartments, and specific anchoring proteins target individual PKCs to different intracellular components and confer specificity for different substrates. See Mochly-Rosen & Gordon (1998) FASEB J. 12:35-42, and Gschwendt et al. (1996) FEBS Lett. 392:77-80, which are herein incorporated by reference.
  • PKC ⁇ and ⁇ are responsible for both CCK-8- induced and TNF- ⁇ -induced NF- ⁇ B activation in pancreatic acinar cells. Translocation but not phosphorylation of PKC ⁇ is necessary for mediating NF- ⁇ B activation. Pharmacologic analysis showed that both phosphatidylinositol (P ⁇ )-specific PLC and phosphatidylcholine (PC)-specific PLC are necessary for the activation of PKC ⁇ , PKC ⁇ , and NF- ⁇ B by CCK-8. In contrast, these responses occur only through PC-specific PLC in acini stimulated with TNF- ⁇ . Although CCK-8 and TNF- ⁇ initiate NF- ⁇ B activation by different PLC pathways, these pathways converge on the activation of PKC ⁇ and PKC ⁇ , leading to NF- ⁇ B activation in pancreatic acinar cells.
  • P ⁇ phosphatidylinositol
  • PC phosphatidylcholine
  • peptides were found to block the translocation of either PKC ⁇ or PKC ⁇ , thereby inhibiting activation of NF- ⁇ B in pancreatic acinar cells.
  • these peptide inhibitors and other agents that block the translocation of one or both of these PKC isoforms will result in inhibition of activation of NF- ⁇ B may be used to treat, prevent, or inhibit pancreatitis and other diseases in which NF- ⁇ B is involved in the pathogensesis such as inflammation, cancer, and the like.
  • Cancers associated with NF- ⁇ B activation also include cancers caused by in vitro transformation including BCR- ABL, DBL/DBS, RAF, RAS, TEL-JAK2, TEL-PDGFR, and the like, and viral oncogenesis caused by Epstein-Barr virus, hepatitis B virus, human herpesvirus-8, and human T-cell leukemia virus- 1, and the like.
  • Inflammatory diseases associated with NF- ⁇ B activation include inflammatory bowel disease, asthma, arthritis including rheumatoid arthritis, asthma, psoriasis, cystitis, nephritis, and the like. See Barnes & Karin (1997) N. Engl. J. Med. 336(15):1066-1071, Abdel-Mageed (2003) Urol. Res. 31(5):300-305, and Lopez-Franco et al. (2002) Am. J. Pathol. 161(4):1497-1505, which are herein incorporated by reference.
  • Alzheimer's disease is associated with inflammation, increased brain cytokines and activation of micorglia.
  • Lim GP et al. (2001) J. Neuroscience 21 -.8370-8377, which is herein incorporated by reference.
  • Epidemiologic studies demonstrate a role for the inflammatory response in the mechanism of the disease because of findings showing the use of nonsteriodal anti-inflammatory drugs (NSAIDs) may prevent of delay the onset of Alzheimer's disease.
  • NSAIDs nonsteriodal anti-inflammatory drugs
  • the methods and inhibitors of NF-kB activation provided herein may be used to prevent, inhibit, delay or modulate the onset of Alzheimer's disease.
  • the present invention provides methods for preventing, inhibiting, delaying, or modulating the onset of Alzheimer's disease in a subject which comprises inhibiting, preventing, or modulating NF-kB activation by administering to the subject (1) at least one plant derived polyphenolic compound, preferably rottlerin or a derivative thereof, alone or in combination with at least one antioxidant or at least one ROS inhibitor, or both, or (2) at least one inhibitor of activation of protein kinase C isoforms, epsilon and delta, such as a PKC ⁇ translocation inhibitor or a PKC ⁇ translocation inhibitor.
  • a PKC ⁇ translocation inhibitor ⁇ Vl-1 SFNSYELGSL (SEQ ID NO : l )
  • a PKC ⁇ translocation inhibitor ⁇ Vl-2 EAVSLKPT ( SEQ ID NO : 2 ) and control peptide LSETKPAV ( SEQ ID NO : 3 ) according to previous studies. See Chen et al. (2001) PNAS USA 98:11114-11119, and Dorn et al.
  • CCK-8 causes a rapid and prolonged NF- ⁇ B activation in pancreatic acinar cells in a dose and time dependent manner, and that the response of NF- ⁇ B to 100 nM CCK-8 reaches a maximum at 30 minutes after the stimulation. See Gukovsky (1998) Am. J. Physiol. 275:G1402-G1414, and Pandol et al. (1999) Gastroenterology 117:706-716, which are herein incorporated by reference.
  • pancreatic acini prepared from normal rats were preincubated with each PKC translocation inhibitor (10 ⁇ M) or same volume of DMSO for 3 hours, and then stimulated with CCK-8 (100 nM) or TNF- ⁇ (100 ng/ml) for 30 minutes in the series of experiments provided in the Examples.
  • Control peptide (10 ⁇ M) instead of DMSO was used as the control for the translocation inhibitors.
  • I- ⁇ B inl ⁇ ibitory- ⁇ B
  • the cell permeant peptide translocation inhibitors specifically blocked translocation of the isoform of PKC they were intended to inhibit. Furthermore, both inhibitors prevented NF- ⁇ B activation. This inhibition in activation of NF- ⁇ B may be used as provided in Section D above as well as the attenuation of inflammatory responses and pancreatitis as disclosed in U.S. Patent Application Publication No. 20040037902 published 26 February 2004, which is herein incorporated by reference.
  • CCK-8 activates PKC ⁇ , ⁇ , and ⁇ , but not PKC ⁇ , in rat pancreatic acini
  • the presence and translocation of each PKC isoform was assayed by Western blot methods known in the art. As previously reported in the art and as shown in Figure 19 A, immunoreactivities to four isoforms of PKC ( ⁇ , ⁇ , ⁇ , and ⁇ ) were detected in untreated rat pancreatic acini, with a large percentage of each isoform residing in the cytosolic fraction. Treatment with 100 nM CCK-8 decreased the presence of PKC ⁇ and PKC ⁇ in the cytosolic fraction and increased them in the membrane fraction, thereby indicating translocation from cytosol to cell membranes.
  • the CCK-8 induced NF- ⁇ B activation was inhibited by the broad spectrum PKC inhibitor, GF109203X, the PKC ⁇ translocation inhibitor, ⁇ Vl- 1, and the PKC ⁇ translocation inhibitor, ⁇ Vl-2, by about 98%, about 76%, and about 80%, respectively, as shown in Figure 20 A and Figure 20B.
  • the conventional PKC isoform inhibitor, Go6976 did not inhibit, but rather enhanced the NF- ⁇ B response. See Figure 20A and Figure 20B.
  • PKC ⁇ pseudosubstrate did not affect NF- ⁇ B activation as shown in Figure 20A and Figure 20B while abolishing the increase in kinase activity of PKC ⁇ (data not shown).
  • the present invention provides methods of inhibiting NF- ⁇ B activation comprising preventing PKC ⁇ translocation, PKC ⁇ translocation, or both.
  • preventing PKC ⁇ translocation comprises contacting ⁇ Nl-1 with PKC ⁇ .
  • preventing PKC ⁇ translocation comprises contacting ⁇ Vl-2 with PKC ⁇ .
  • the present invention also provides methods of treating, preventing, or inhibiting a disease or disorder associated with NF- ⁇ B activation, such as abnormal cell proliferation, e.g. cancer, and inflammation, e.g. pancreatitis, and the like, which comprises inhibiting NF- ⁇ B activation.
  • NF- ⁇ B activation is inhibited by preventing PKC ⁇ translocation, PKC ⁇ translocation, or both.
  • TNF- ⁇ caused NF- ⁇ B activation in pancreatic acini.
  • CCK-8 the responses of both PKC and NF- ⁇ B to TNF- ⁇ were relatively smaller, but see Figure 20B and Figure 23B.
  • acini were pretreated with GF109203X, the increase inNF- ⁇ B binding activity by TNF- ⁇ was abolished, indicated participation of PKC isoforms in the NF- ⁇ B activation by TNF- ⁇ .
  • the TNF- ⁇ induced NF- ⁇ B activation was inhibited by GF109203X, ⁇ Vl-1, and ⁇ Vl-2, by 81%, 57%, and 58%, respectively. See Figure 23A and Figure 23B.
  • the conventional PKC isoform inhibitor, Go6976 did not inhibit, but rather enhanced the NF- ⁇ B response.
  • PKC ⁇ pseudosubstrate did not affect NF- ⁇ B activation. See Figure 23A and Figure 23B. Consistent with the results of NF- ⁇ B binding activity, TNF- ⁇ - induced degradation of I ⁇ B ⁇ was blocked by ⁇ Nl-1 and ⁇ Vl-2, enhanced by Go6976, and unaffected by PKC ⁇ pseudosubstrate as shown in Figure 23 C. These results indicate that PKC ⁇ and PKC ⁇ are responsible for TNF- ⁇ -induced NF- ⁇ B activation and that PKC ⁇ may exert an inhibitory effect on the NF- ⁇ B activation.
  • the present invention provides methods of inhibiting, preventing, or modulating TNF- ⁇ induced NF- ⁇ B activation in a cell or a subject which comprises administering to the cell or the subject GF109203X, ⁇ Nl-1, ⁇ Nl-2, or a combination thereof.
  • Src kinase inhibitor does not prevent CCK-8- or TNF- ⁇ - induced NF- ⁇ B activation
  • Src kinases have been implicated as upstream modulators of PKC in response to CCK-8. See Ferris et al. (1999) Biochemistry 38:1497-1508, Tsunoda et al. (1996) Biochem. Biophys. Res. Commun. 227:876-884, Tapia et al. (2002) Biochim. Biophys. Acta. 1593:99-113, and Tapia et al. (2003) J. Biol. Chem. 278:35220-35230, which are herein incorporated by reference.
  • Src kinases have also been linked to NF- ⁇ B activation in a number of cell types. See Abu-Amer et al. (1998) J. Biol. Chem. 273:29417-29423, Devary et al. (1993) Science 261 :1442-1445, and Li et al. (1998) PNAS USA 95:5718-5723, which are herein incorporated by reference. [154] To investigate whether Src tyrosine kinases are involved in the activation of NF- KB in pancreatic acinar cells, PP2, a specific inhibitor of Src kinases was applied.
  • PKC ⁇ is the most efficiently tyrosine phosphorylated isoform.
  • PP2 almost completely inhibited tyrosine phosphorylation of PKC ⁇ induced by CCK-8 and TNF- ⁇ as shown in Figure 25.
  • Src kinases mediate tyrosine phosphorylation of PKC ⁇ but are not involved in NF- ⁇ B activation induced by CCK-8 and TNF- ⁇ in pancreatic acini.
  • CCK-8 activates the novel PKC isoforms and NF- ⁇ B through both Pi-specific PLC and PC-specific PLC, whereas TNF- ⁇ activates them through only PC-specific PLC
  • Both phosphatidylinositol and phosphatidylcholine are the main precursors of DAG generation in pancreatic acinar cells after CCK stimulation. See Hermans et al. (1996) Eur. J. Biochem. 235:73-81, Pandol & Schoeffield (1986) J. Biol. Chem. 261:4438-4444, Pandol. Et al. (1985) Am. J. Physiol. Gastrointest. Liver Physiol. 248:G551-G560, which are herein incorporated by reference.
  • TNF- ⁇ activated the novel PKCs and NF- ⁇ B in pancreatic acini through only PC-specific PLC.
  • TNF receptor 1 has been shown to mediate the inflammatory response in pancreatitis and is functional on pancreatic acinar cells.
  • TNF- ⁇ may accelerate the inflammatory response by producing DAG through PC-specific PLC.
  • DAG may mediate NF- ⁇ B activation by promoting translocation of PKC ⁇ and PKC ⁇ .
  • the translocation inhibitor peptides ⁇ N 1 - 1 and ⁇ N 1 -2, designed to competitively inhibit the binding of PKC ⁇ and PKC ⁇ to specific anchoring proteins, prevented the increases in kinase activity and translocation of their target PKC isoforms.
  • These results indicate that translocation of PKC ⁇ or PKC ⁇ is involved in activation by CCK-8 and TNF- ⁇ in pancreatic acini.
  • ⁇ Nl-1 and ⁇ Nl-2 did not cross-inhibit the PKC isoforms, indicating the high specificity of these peptide inhibitors.
  • these peptide inhibitors Prior to the present invention, these peptide inhibitors have not been previously applied to study the role of PKC in pancreatic acinar cells.
  • PKC ⁇ and ⁇ translocation inhibitors prevented both the CCK-8-induced and TNF- ⁇ -induced NF- ⁇ B activation, determined by NF- ⁇ B binding activity and I ⁇ B ⁇ degradation. These results indicate that PKC ⁇ and PKC ⁇ are key mediators of the NF- KB activation in pancreatic acinar cells. Because each isoform-specific inhibitor prevented NF- ⁇ B activation to about the same degree without affecting the kinase activity and localization of the other PKC isoform, PKC ⁇ and ⁇ regulate NF- ⁇ B activation independently at the level of I ⁇ B ⁇ degradation or upstream.
  • PI 3-Kinase [164] Next, the possibility that phosphatidylinositol 3-kinase (PI 3-kinase) and Akt/PKB mediate the effects of serum onNF- ⁇ B; and that the effects of the polyphenols on NF- ⁇ B activation are due, at least in part, to an ability to inhibit PI 3-kinase were studied.
  • the PI 3-kinase signaling system was used because it is an important mediator of responses to growth factors and because there is evidence that polyphenolic compounds such as quercetin and genistein inhibit PI 3-kinase and/or Akt/PKB.
  • PI 3-kinase one commonly used inhibitor of PI 3-kinase is LY294002 (Calbiochem, San Diego, CA), a derivative of quercetin. Also, there are some suggestions for a role of ROS in activation of PI 3-kinase. Finally, and most importantly, there are several publications indicating that one of the effects of PI 3-kinase signaling is the activation of NF- ⁇ B. See Kane et al. (1999) Nature 401:86-99; Ozes et al. (1999) Nature 401:82-85; Madrid et al. (2000) Mol. Cell. Biol. 20:1626-1638; Xie et al. (2000) J. Biol. Chem. 275: 24907-24914; and Madrid et al. (2001) J. Biol. Chem. 276:18934-18940, which are herein incorporated by reference.
  • PI 3-kinase is an important signaling system that is activated by growth factors and G protein-coupled receptors that have been determined to regulate various cellular processes including proliferation, survival, inflammation and metabolism. See Katada et al. (1999) Chem. Phys. Lipids 98:79-86; and Leevers et al. (1999) Curr. Opin. Cell. Biol. 11 -.219-225, which are herein incorporated by reference.
  • PI 3-kinase results in an increase in D-3 phosphorylated phosphoinositides such as phosphatidylinositol-3 phosphate, phosphatidylinositol-3,4 bisphosphate and phosphatidylinositol-3,4,5 trisphosphate.
  • D-3 phosphorylated phosphoinositides such as phosphatidylinositol-3 phosphate, phosphatidylinositol-3,4 bisphosphate and phosphatidylinositol-3,4,5 trisphosphate.
  • the PI 3-kinase that is stimulated by tyrosine kinase activating receptors is relevant to the present application.
  • This PI 3-kinase is structurally characterized as a heterodimer consisting of a 110-kD catalytic subunit (pl 10- ⁇ , ⁇ or - ⁇ ) and an 85-kD regulatory subunit (p85). Stimulation of tyrosine kinase activating receptors by extracellular signals, i.e. insulin and insulin-related growth factors results in phosphorylation of the receptor or receptor associated adapter proteins. The phosphorylated receptor or adapter proteins then bind to regulatory p85, which, in turn, activates catalytic pl 10.
  • the combination of LY294002 and DPI inhibits NF-KB activation in a manner similar to the combination of a polyphenolic compound and DPI.
  • the combination a ROS inhibitor and the PI 3- kinase inhibitor, LY294002 inhibit NF-icB activation. As indicated above, inhibition of NF- ⁇ B activation can, in turn, sensitize the cancer cell to apoptosis.
  • the present invention provides a preventing, inhibiting, or attenuating the activation of Akt/PKB in a cell comprising administering to the cell at least one polyphenolic compound or a ROS inhibitor and a PI 3-kinase inhibitor, or at least one polyphenolic compound and an inhibitor of NADPH oxidase or inhibitor of ROS formation.
  • the present invention provides methods of treating, preventing, or inhibiting cancer in a subject comprising administering to the subject at least one polyphenolic compound.
  • the methods of the present invention may further comprise one or more of the following: 1. Administering a ROS inhibitor; 2. Administering a PI 3-kinase inhibitor; 3. Administering a NADPH oxidase inhibitor; 4. Preventing or inhibiting NF- ⁇ B activation; 5. Inducing apoptosis; 6. Inducing caspase-3 activation; 7. Inducing mitochondrial cytochrome c release; 8. Inducing dissipation of mitochondrial polarity; and 9. Activating mitochondrial PTP; and 10. Activating PARP.
  • rottlerin is a plant derived polyphenolic compound that is more potent than other polyphenolic compounds and may be used alone in order to prevent, treat, or inhibit proliferative diseases such as cancers including pancreatic cancer; and inflammatory diseases in which NF- ⁇ B is involved in the pathogensesis such as pancreatitis, and other diseases or disorders associated with NF- ⁇ B activation.
  • Rottlerin causes apoptotic cell death of pancreatic cancer cells cultures in vitro by "opening" the cancer cell's mitochondrial permeability transition by (1) releasing cytochrome c from the mitochondria, (2) activating cellular caspases, (3) inhibiting the formation of ROS, and (4) inhibiting the activation of NF- ⁇ B. Therefore, the present invention provides methods of using rottlerin or a derivative thereof to prevent, treat, or inhibit cancers and cancer recurrence.
  • the present invention also provides methods of using rottlerin to sensitize cancers to chemotherapy, radiotherapy, thermal therapy, and the like known in the art because the major reason for the lack of efficacy of these therapies is that cancers can become resistant to these therapies because the cancer cell upregulates survival factors, i.e. NF- ⁇ B, to prevent it from undergoing apoptosis. See Soltoff (2001) J. Biol. Chem. 276:37986-37992, which is herein incorporated by reference.
  • Rottlerin a polyphenolic phytochemical derived from the plant Mallotus philippinensis, has the following structural formula:
  • rottlerin derivatives have the following general structural formula:
  • each R are independently selected from the group consisting of hydrogen, hydroxyl, a halo, alkyl, or alkoxyl.
  • the alkyl may be methyl or ethyl.
  • the alkoxyl groups may be methoxyl or ethoxyl.
  • the halo is fluoro.
  • the ring structures of either the rottlerin compound or the rottlerin derivatives according to the present invention may be optionally substituted.
  • halo means a halogen radical such as fluoro, chloro, bromo or iodo.
  • an "alkyl” is intended to mean a straight or branched chain monovalent radical of saturated and/or unsaturated carbon atoms and hydrogen atoms, such as methyl (Me), ethyl (Et), propyl (Pr), isopropyl (i-Pr), butyl (n-Bu), isobutyl (i- Bu), t-butyl (t-Bu), (sec-Bu), ethenyl, pentenyl, butenyl, propenyl, ethynyl, butynyl, propynyl, pentynyl, hexynyl, and the like, which may be unsubstituted (i.e., contain only carbon and hydrogen) or substituted by one or more suitable sustituents as defined below (e.g., one or more halogen, such as F, Cl, Br, or I, with F and Cl being preferred).
  • suitable sustituents e.g., one
  • a "hydroxyl” is intended to mean the radical -OH.
  • alkoxyl is intended to mean the radical -OR a , where R a is an alkyl group.
  • alkoxyl groups include methoxyl, ethoxyl, propoxyl, and the like.
  • Such moieties may also be optionally substituted by a fused-ring structure or bridge, for example OCH 2 -O. All of these substituents may optionally be further substituted with a substituent selected from groups such as hydroxyl groups, halogens, oxo groups, alkyl groups, acyl groups, sulfonyl groups, mercapto groups, alkylthio groups, alkyloxyl groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, carboxyl groups, amino groups, alkylamino groups, dialkylamino groups, carbamoyl groups, aryloxyl groups, heteroaryloxyl groups, arylthio groups, heteroarylthio groups, and the like.
  • groups such as hydroxyl groups, halogens, oxo groups, alkyl groups, acyl groups, sulfonyl groups, mercapto groups, alkylthio groups, alkyloxyl
  • Rottlerin has been used as an inhibitor of protein kinase C ⁇ (PKC ⁇ ) and has recently been reported to have mitochondrial effects when used in experiments to define the role of PKC ⁇ in certain cellular functions. See Arlt et al. (2003) Oncogene 22:32- 43-3251, which is herein incorporated by reference.
  • DENDase activity, as well as the effect of the broad-spectrum caspase inhibitor, Z-NAD, on apoptosis by measuring the effect of caspase inhibition on oligonucleosomal D ⁇ A fragmentation caused by rottlerin was examined as provided in Example 9.
  • caspase-3 activity is markedly activated by rottlerin and that Z-NAD by inhibiting the activation of the caspase reverses the apoptosis-inducing effect of rottlerin as measured by oligonucleosomal D ⁇ A fragmentation.
  • Example 9 The experiments according to Example 9 were designed to determine the effects of rottlerin on ⁇ F- ⁇ B activation and production of ROS, two factors that promote survival in pancreatic cancer cells by preventing apoptosis. As illustrated in Figure 35 and Figure 36, rottlerin but not the PKC inhibitor prevented the activation of NF- ⁇ B in the cancer cells and rottlerin decreased the formation of ROS in the cancer cells.
  • Ribose and deoxyribose molecules labeled with a single 13 C atom on the first carbon position (ml) recovered from RNA were used to gauge the ribose fraction produced by direct oxidation of glucose through the G6PD pathway according to Example 9. Ribose molecules labeled with 13 C on the first two carbon positions (m2) were used to measure the fraction produced by the non-oxidative steps of the pentose cycle via transketolase.
  • Figure 39 shows oxidative and non-oxidative nucleic acid precursor synthesis for DNA and RNA production in response to 2.5 and 5.0 mM rottlerin.
  • rottlerin primarily affects DNA precursor synthesis through the non-oxidative transketolase pathway and there is a dose dependent increase in the oxidative synthesis of deoxyribose.
  • RNA ribose synthesis was not affected by rottlerin treatment indicating that this phytochemical affects metabolic pathways and non-oxidative precursor synthesis during the S cycle phase when DNA is synthesized in rapidly proliferating MIA PaCa-2 cells.
  • inhibiting non-oxidative deoxyribose synthesis is a very effective and selective mechanism of controlling cell proliferation in pancreatic cancer.
  • rottlerin dose- about 2.5 ⁇ M to about 10 ⁇ M
  • time- about 24 hours to about 72 hours
  • apoptosis in MIA PaCa-2 and PANC-1 cells.
  • Rottlerin did not increase necrosis, i.e. cells positive for PI, and did not decrease cellular ATP.
  • the pro-apoptotic effect of rottlerin was much greater than of other polyphenols studied.
  • Rottlerin inhibited both ROS generation and NF- ⁇ B binding activity, resulting in pro-apoptotic cytochrome c release, mitochondrial depolarization, and activation of caspase-3. Rottlerin inhibited both ROS and NF- ⁇ B to a much greater extent than genistein or resveratrol.
  • rottlerin causes apoptosis in pancreatic cancer cells by causing mitochondrial depolarization and release of its cytochrome c.
  • the cytochrome c causes activation of cellular caspases, which mediate apoptosis.
  • Rottlerin also acts by inhibiting the formation of ROS and by inliibiting the activation of NF- ⁇ B.
  • ROS and activated NF- ⁇ B act as survival factors for cancer cells. Thus, their inhibition promotes cell death through apoptosis.
  • rottlerin significantly reduces the growth of pancreatic tumors.
  • Rottlerin treated tumor cells also exhibit a significant decrease in macromolecule DNA/RNA precursor synthesis and that of fatty acids which are molecules are necessary for proliferation and growth of tumor cells.
  • rottlerin has multiple effects on the cancer cells that promote their death while having no significant toxic effects on normal tissues in vivo.
  • the present invention provides methods of inducing apoptosis in cancer cells, such as pancreatic cancer cell, which comprises contacting the cells with rottlerin.
  • the present invention also provides methods of treating, preventing, or inhibiting tumor growth in a subject which comprises administering to the subject a therapeutically effective amount of rottlerin.
  • Rottlerin may be used in combination with other therapies for treating, preventing, or inhibiting diseases and disorders associated with NF- ⁇ B activation.
  • rottlerin may be used in combination with other chemotherapeutic agents such as gemcitabine, the most effective chemotherapeutic for pancreatic cancer. See Li et al. (2004) Lancet 363 (9414): 1049, which is herein incorporated by reference.
  • Other chemotherapeutics may be used in combination with rottlerin.
  • One skilled in the art may readily ascertain the effectiveness and suitable dosages of the combinations according to the methods disclosed herein as well as methods known in the art.
  • rottlerin since rotterlin inhibits NF- ⁇ B activation which is involved in abnormal cell proliferation such as cancer and inflammatory diseases such as pancreatitis, rottlerin may be used alone or in combination with antiproliferative agents and anti-inflammatory agents known in the art to treat both cancer and inflammatory disorders.
  • Antiproliferative agents include asparaginase, alemtuzumab, bleomycin, busulfan, beracizumab, carboplatin, cisplatin, cetuximab, cyclophosphamide, daunorubicin, docetaxel, epirubicin, floxuridine, fluoruracil, foscarnet, gentuzamab izogamicin, hydroxyurea, idarubicin, ifosfamide, iriotecan, lomustine, leamisole, melphalan, mercaptopurine, methotrexate, methyl CCNU, oxoliplatin, paclitaxel, rituximab, streptozocin, tamoxifen, temozolomide, tenipozide, thioguanine, thiotepa, tumor necrosis factor, tositumomab, trastuzmab, vinblastine
  • Antiinflammatory agents include acetylsalicylic acid, aspirin, Ecotrin, choline magnesium salicylate, Trilisate, Cox-2 inhibitors, diclofenac, Noltaren, Cataflam, Noltaren-XR, diflunisal, Dolobid, etodolac, Lodine, fenoprofen, ⁇ alfon, flurbiprofen, Ansaid, ibuprofen, Advil, Motrin, Medipren, ⁇ uprin, indomethacin, Indocin, Indocin- SR, ketoprofen, Orudis, Oruvail, meclofenamate, Meclomen, nabumetone, Relafen, naproxen, ⁇ aprosyn, ⁇ aprelan, Anaprox, Aleve, oxaprozin, Daypro, phenylbutazone, Butazolidine, salsalate, Disalcid, Salflex, tolmetin
  • rottlerin and an inhibitor of PKC ⁇ translocation, PKC ⁇ translocation, or both may be combined to treat, prevent, or inhibit diseases and disorders mediated by ⁇ F- ⁇ B activation such as proliferative diseases including cancer and inflammatory diseases including pancreatitis.
  • the present invention provides methods of inhibiting NF- ⁇ B activation in a cell or a subject comprising administering to the cell or the subject rottlerin alone or in combination with at least one an inhibitor of PKC ⁇ translocation, PKC ⁇ translocation, or both.
  • the inhibitors include rottlerin, GF109203X (Sigma, St. Louis, MO), and the peptide inhibitors provided herein.
  • At least one polyphenolic compound may be administered in a therapeutically effective amount to a mammal such as a human.
  • a therapeutically effective amount may be readily determined by standard methods known in the art.
  • An effective amount of a polyphenolic compound is an amount that treats, prevents, or inhibits cancer or tumor growth as compared to a control using methods known in the art.
  • An effective amount of a polyphenolic compound may also mean an amount that induces apoptosis in a cancer cell as compared to a control using methods known in the art.
  • an effective amount of an agent that inhibits, prevents or modulates NF- ⁇ B activation is an amount that reduces the amount of NF- ⁇ B activity as compared to a control.
  • the dosages to be administered can be determined by one of ordinary skill in the art depending on the clinical severity of the disease, the age and weight of the subject, or the exposure of the subject to carcinogens and neoplastic conditions.
  • Preferred effective amounts of the compounds of the invention ranges from about 1 to about 2400 mg/kg body weight, preferably about 10 to about 1000 mg/kg body weight, and more preferably about 10 to about 500 mg/kg body weight.
  • Preferred topical concentrations include about 0.1% to about 10% in a formulated salve.
  • treatment of a subject with a compound or composition of the present invention can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with a compound of the invention in the range of between about 1 to about 2400 mg/kg body weight, at least one time per week for between about 1 to about 24 weeks, and preferably between about 1 to about 10 weeks.
  • the effective dosage of the compound used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some conditions chronic administration may be required.
  • the pharmaceutical compositions of the invention may be prepared in a unit- dosage form appropriate for the desired mode of administration.
  • compositions of the present invention may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal). It will be appreciated that the preferred route will vary with the condition and age of the recipient, the nature of the condition to be treated, and the chosen active compound.
  • compositions of this invention will vary according to the particular complex being used, the particular composition formulated, the mode of administration, and the particular site, host, and disease being treated. Optimal dosages for a given set of conditions may be ascertained by those skilled in the art using conventional dosage-determination tests in view of the experimental data for a given compound. Administration of prodrugs may be dosed at weight levels that are chemically equivalent to the weight levels of the fully active forms.
  • compositions of this invention comprise a therapeutically effective amount of a polyphenolic compound, and an inert, pharmaceutically acceptable carrier or diluent.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the pharmaceutical carrier employed may be either a solid or liquid. Exemplary of solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
  • liquid carriers are syrup, peanut oil, olive oil, water and the like.
  • the carrier or diluent may include time-delay or time- release material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate and the like.
  • time-delay or time- release material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate and the like.
  • Supplementary active compounds can also be incorporated into the compositions.
  • Supplementary active compounds include ROS inhibitors such as N-acetylcysteine, vitamins C, A, and E, beta- carotene, allopurinol, carvediol, coenzyme Q, Tiron, DPI, and any other antioxidant or inhibitor of ROS.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • a variety of pharmaceutical forms can be employed.
  • the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge.
  • the amount of solid carrier may vary, but generally will be from about 25 mg to about 1 g.
  • a liquid carrier is used, the preparation will be in the form of syrup, emulsion, soft gelatin capsule, sterile injectable solution or suspension in an ampoule or vial or non-aqueous liquid suspension.
  • a pharmaceutically acceptable salt of a polyphenolic compound is dissolved in an aqueous solution of an organic or inorganic acid, such as 0.3 M solution of succinic acid or citric acid.
  • an organic or inorganic acid such as 0.3 M solution of succinic acid or citric acid.
  • the compound may be dissolved in a suitable cosolvent or combinations of cosolvents.
  • suitable cosolvents include, but are not limited to, alcohol, propylene glycol, polyethylene glycol 300, polysorbate 80, glycerin and the like in concentrations ranging from 0-60% of the total volume.
  • the polyphenolic compound of the present invention is dissolved in DMSO and diluted with water.
  • the composition may also be in the form of a solution of a salt form of the active ingredient in an appropriate aqueous vehicle such as water or isotonic saline or dextrose solution.
  • compositions of the invention may be manufactured in manners generally known for preparing pharmaceutical compositions, e.g., using conventional techniques such as mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing.
  • Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers, which may be selected from excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the agents of the invention may be formulated into aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use can be obtained using a solid excipient in admixture with the active ingredient (agent), optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients include: fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; and cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP).
  • PVP polyvinylpyrrolidone
  • disintegrating agents may be added, such as crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings may be used, which may optionally comprise gum arabic, polyvinyl pyrrolidone, Carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active agents.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can comprise the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active agents may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can comprise any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of gelatin for use in an inhaler or insufflator and the like may be formulated comprising a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit-dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may comprise formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • Aqueous injection suspensions may comprise substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also comprise suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • suspensions of the active agents may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or lipo somes.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating a therapeutically effective amount of a compound of the invention in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating at least one polyphenolic compound into a sterile vehicle which comprises a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active compound plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, foams, powders, sprays, aerosols or creams as generally known in the art.
  • pharmaceutically acceptable excipients may comprise solvents, emollients, humectants, preservatives, emulsif ⁇ ers, and pH agents.
  • suitable solvents include ethanol, acetone, glycols, polyurethanes, and others known in the art.
  • Suitable emollients include petrolatum, mineral oil, propylene glycol dicaprylate, lower fatty acid esters, lower alkyl ethers of propylene glycol, cetyl alcohol, cetostearyl alcohol, stearyl alcohol, stearic acid, wax, and others known in the art.
  • Suitable humectants include glycerin, sorbitol, and others known in the art.
  • Suitable emulsif ⁇ ers include glyceryl monostearate, glyceryl monoleate, stearic acid, polyoxyethylene cetyl ether, polyoxyethylene cetostearyl ether, polyoxyethylene stearyl ether, polyethylene glycol stearate, propylene glycol stearate, and others known in the art.
  • Suitable pH agents include hydrochloric acid, phosphoric acid, diethanolamine, triethanolamine, sodium hydroxide, monobasic sodium phosphate, dibasic sodium phosphate, and others known in the art.
  • Suitable preservatives include benzyl alcohol, sodium benzoate, parabens, and others known in the art.
  • the compound of the invention is delivered in a pharmaceutically acceptable ophthalmic vehicle such that the compound is maintained in contact with the ocular surface for a sufficient time period to allow the compound to penetrate the corneal and internal regions of the eye, including, for example, the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris/cilary, lens, choroid/retina and selera.
  • the pharmaceutically acceptable ophthalmic vehicle may be an ointment, vegetable oil, or an encapsulating material.
  • a compound of the invention may also be injected directly into the vitreous and aqueous humor.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., comprising conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion- exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a pharmaceutical carrier for hydrophobic compounds is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • the cosolvent system may be a VPD co-solvent system.
  • VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
  • the VPD co-solvent system (VPD:5W) comprises VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.
  • co-solvent component may be varied considerably without destroying its solubility and toxicity characteristics.
  • identity of the co-solvent components may be varied, for example: other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may be substituted for dextrose.
  • hydrophobic pharmaceutical compounds may be employed.
  • Liposomes and emulsions are known examples of delivery vehicles or carriers for hydrophobic drugs.
  • Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers comprising the therapeutic agent.
  • sustained-release materials have been established and are known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
  • compositions also may comprise suitable solid- or gel-phase carriers or excipients.
  • suitable solid- or gel-phase carriers or excipients include calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • Some of the compounds of the invention may be provided as salts with pharmaceutically compatible counter ions.
  • Pharmaceutically compatible salts may be formed with many acids, including hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free-base forms.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit comprising a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 5 o.
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inliibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound which achieves a half-maximal inliibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • polyphenolic compounds of the present invention may be prepared using reaction routes, synthesis schemes and techniques available in the art using starting materials that are readily available.
  • Example 1 Pancreatic Cancer Growth Assay To determine the effect of a polyphenolic compound on pancreatic cancer cell growth, the following assay using a nude mouse model was conducted. Specifically, the effect of quercetin was tested in a nude mouse model of pancreatic cancer using the highly malignant pancreatic cancer cell line, Mia PACA-2. [242] Tumor induction in nude mice was performed as described by Hotz et al. (2001) Pancreas 22:113-121, which is herein incorporated by reference. For subcutaneous n tumor formation, 1 x 10 Mia PACA-2 tumor cells were subcutaneously injected in the medio-dorsal region of a nude mouse.
  • a small tumor fragment about 1 mm in diameter, was removed from the subcutaneous tumor and transplanted into the pancreatic tail of mice of two study groups.
  • the treated animals received daily intraperitoneal injections of 1.3 mg quercetin dissolved in DMSO; the control group received DMSO only by intraperitoneal injection.
  • the animals were sacrificed once clinical tumor signs including severe cachexia ascites with abdominal distension or heavy tumor burden, larger than 1.5 cm, became apparent.
  • Tumor volume was calculated as described by Hotz et al. (2001) as 0.5 x length x width x depth.
  • Metastatic tumor spread was determined macroscopically at autopsy in all thoracic, abdominal, retroperitoneal and pelvic organs. All macroscopic suspicious lesions were further confirmed as tumor dissemination by microscopic analysis. Each value in the metastatic score represented a different organ of metastatic tumor spread.
  • the mean number of organs with metastatic lesions was 4.4 in control animals as compared to the 0.6 in quercetin-treated animals. Therefore, quercetin treatment prevents metastatic cancer lesions. Furthermore, quercetin treatment significantly decreased the growth of the primary tumor.
  • Example 2 Effects of Serum and Growth Factors [245] To determine the effect of serum in cancer cells, the following assay was conducted. Mia PACA-2 pancreatic cancer cells were cultured for 72 hours in the absence and presence of serum (15% FBS) or 100 ng/ml insulin growth factor-1 (IGF-1). Dichloroflyorescein diocetate (DCF-DA) was used to label the cells. Intracellular H 2 O 2 was measured by flow cytometry of DCF-labeled cells.
  • DCF-DA Dichloroflyorescein diocetate
  • Mia PACA-2 cells were cultured for 72 hours in the presence of serum or IGF-1 with or without antioxidants, intracellular superoxide scavenger tiron (10 mM) or NADPH oxidase inhibitor, diphenylene iodonium (DPI, 15 ⁇ M) and trans-resveratrol (100 ⁇ M), genistein (100 ⁇ M), or a combination of polyphenolic compounds and antioxidants.
  • Intracellular H O 2 was measured by flow cytometry of DCF-labeled cells.
  • Mia PACA-2 cells and BSp73AS cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% heat inactivated FBS, penicillin G (100 U/ml) and streptomycin (100 mg/ml) in a humidified atmosphere comprising 5% (v/v) CO 2 .
  • FBS Dulbecco's Modified Eagle's Medium
  • penicillin G 100 U/ml
  • streptomycin 100 mg/ml
  • Example 3 Apoptosis Assays I. POLYPHENOLIC COMPOUND ALONE [250] In order to determine the mechanism of the suppressive effects of quercetin on the growth of the pancreatic cancer, apoptosis in the primary tumors using the TUNEL assay was conducted. See Gukovskaya et al. (1997) Clin Invest 100:1853-1862; Gukovskaya et al. (1996) Gastroenterology 110:875-884; and Sandoval et al. (1996) Gastroenterology 111:1081-1091, which are herein incorporated by reference. Specifically, 3 ⁇ m tissue section were deparaffinized and rehydrated through a graded series of ethanol and redistilled water.
  • Tissue sections were refixed in 4% paraformaldehyde for 15 minutes at room temperature and then incubated with proteinase K (20 ⁇ g/ml in 10 mM Tirs/HCL, pH 7.4-8.0) for 15 minutes at 37 °C. DNA breaks were then labeled with terminal deoxytransferase (TdT) and biotinylated deoxyUTP. Staining without TdT enzyme or the biotinylated substrate were used as negative controls. For positive controls, slides were treated with DNase I. Measurements were made by light microscpy observations and values calculated as the percentage of cells positively stained as a percentage of the total number of cells.
  • proteinase K 20 ⁇ g/ml in 10 mM Tirs/HCL, pH 7.4-8.0
  • BSp73AS cells are derived from a rat pancreatic carcinoma and both Mia PACA-2 and BSp73AS cells have mutated p53 and express K-ras.
  • Human pancreatic carcinoma cell line Mia PACA-2 and rat pancreatic carcinoma BSp73 AS were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% heat inactivated FBS, penicillin G (100 U/ml) and streptomycin (100 mg/ml) in a humidified atmosphere comprising 5% (v/v) CO 2 .
  • FBS Dulbecco's Modified Eagle's Medium
  • penicillin G 100 U/ml
  • streptomycin 100 mg/ml
  • a humidified atmosphere comprising 5% (v/v) CO 2 .
  • Oligonucleotide DNA fragmentation, annexin staining, and PARP proteolysis assays were conducted as follows:
  • BSp73 AS pancreatic cancer cells were cultured for 6 hours in the presence or absence of 100 ⁇ M of rutin, quercetin, or trans-resveratrol. DNA was isolated as described by Gukovskaya AS, et al. (1997) Clin Invest 100:1853-1862, which is herein incorporated by reference.
  • pancreatic cancer cells growing on plates were removed by treatment with trypsin, collected by centrifugation and lysed by resuspension in a buffer comprising 10 mM Tris/HCl (pH 8.0) 10 mM NaCl, 10 mM EDTA, 300 ⁇ g/ml proteinase K and 1% SDS.
  • Cell lysates were incubated overnight at 45 °C; and DNA was purified by phenol/chloroform extraction (1 :1 v/v), precipitated overnight at 20 °C with 0.3 M sodium acetate and collected by centrifugation at 15,000 g for 15 minutes at 4 °C.
  • RNA and DNA were resuspended in TE buffer (10 mM Tris/HCl (pH 8.0), 1.0 mM EDTA) and treated subsequently with RNase (200 ⁇ g/ml) for 2 hours at room temperature, followed by an incubation overnight with proteinase K (200 ⁇ g/ml) at 45 °C. Finally, the mixture was re-extracted with phenol/chloroform and chloroform, precipitated with ethanol and resuspended in TE buffer.
  • TE buffer 10 mM Tris/HCl (pH 8.0), 1.0 mM EDTA
  • RNase 200 ⁇ g/ml
  • proteinase K 200 ⁇ g/ml
  • DNA fragments were separated electrophoretically on 1.8% agarose gel comprising 0.5 ⁇ g/ml ethidium bromide in 0.5 x TBE buffer (TBE: 89 mM Tris base, 89 mM boric acid and 2 mM EDTA). The experiment was repeated twice with similar results.
  • Mia PACA-2 cells were cultured for 72 hours in the presence of 0, 12, 24, 50, and 100 ⁇ M rutin, quercetin, or trans-resveratrol. About 1 x 10 cells as determined with a hemocytometer were analyzed for annexin-V binding using an Annexin V-FLUOS Staining Kit (Boehringer Mannheim, Germany). Briefly, cells were washed twice with PBS and incubated for 10 minutes at room temperature with fluorescein isothiocyanate (FITC)-conjugated, annexin-V reagent (20 ⁇ g/ml) and propidium iodide (50 ⁇ g/ml).
  • FITC fluorescein isothiocyanate
  • BSp73AS cells were cultured for 6 hours and Mia PACA-2 cells were cultured for 24 hours in the presence or absence of 100 ⁇ M of each rutin, quercetin or trans- resveratrol and with or without 50 ⁇ M of each K-VAD FMK(K-VAD).
  • the cells were ashed twice with PBS and lysed by incubating for 20 minutes at 4 °C in lysis buffer comprising 0.15 M NaCl, 50 mM Tris (pH 7.2), 1% deoxycholic acid (wt/vol), 1% Triton X-100 (wt/vol), 0.1% SHS (wt/vol) and 1 mM PMSF, as well as 5 ⁇ g/ml each of protease inhibitors, pepstatin, leupeptin, chymostatin, antipain, and aprotinin. Then the cell lysates were centrifuged for 20 minutes at 15,000 g at 4 °C.
  • the supematants were separated by 4-20% SDS-PAGE for 2 hours at 120 V using precast Tris-glycine gels and a Mini-Cell gel apparatus (No vex, San Diego, CA). Separated proteins were electrophoretically transferred to a nitrocellulose membrane for 2 hours at 30 V using a Novex Blot Module (Novex, San Diego, CA). Nonspecific binding was blocked by 1 hour incubation of nitrocellulose membranes in 5% (wt/vol) nonfat dry milk in Tris- buffered saline (TBS; pH 7.5).
  • TBS Tris- buffered saline
  • Blots were then incubated overnight at 4 °C with rabbit polyclonal antibody against poly (ADP-ribose) polymerase (PARP) (Santa Cruz Biotechnology, Santa Cruz, CA) (1;3,000) in an antibody buffer comprising (1% (wt/vol) non-fat dry milk in TTBS (0.05% vol/vol) Tween-20 in TBS), washed 3 times with TTBS and finally incubated for 1 hour with a peroxidase-labeled secondary antibody in the antibody buffer. Blots were developed for visualization using ECL detection kit. To test for equal protein loading, the blots were stripped and re-probed with an antibody against tubulin.
  • PARP poly (ADP-ribose) polymerase
  • FIG. 3B The dose-response evaluation in Figure 3B indicates that quercetin is more potent in causing apoptosis than trans-resveratrol.
  • Figure 3C illustrates that a protein target of caspases-3 activation, PARP, was cleaved to its activated form in cell lines treated with quercetin and trans-resveratrol, but not rutin. The cleavage did not occur in the presence of the specific caspase inhibitor, Z- VAD.
  • Z- VAD the specific caspase inhibitor
  • Mia PACA-2 cells were cultured for 72 hours in the presence of serum with or without 15 ⁇ M DPI, 10 mM Tiron, 100 ⁇ M genistein, 100 ⁇ M or 50 ⁇ M trans- resveratrol, or a combination thereof. Oligonucleosomal DNA fragmentation was measured in cell lysates by cell death ELISA.
  • Mia PACA-2 cells were cultured for 72 hours in the presence of serum with or without 10 mM Tiron, 100 ⁇ M trans-resveratrol, or a combination thereof. Phosphatidylserine externalization was measured by flow cytometry in cells stained with Annexin V and propidium iodide (PI). Cells positive for Annexin V (AnV) and negative for PI were considered apoptotic.
  • PI propidium iodide
  • BSp73 AS cells were cultured for 0, 1, 3, and 6 hours and Mia PACA-2 cells were cultured in the absence of serum or growth factors for 0, 1, 4, 6, and 24 hours in the presence of 100 ⁇ M or quercetin, trans-resveratrol, rutin, or control.
  • Caspase-3 activity was measured in cell lysates with a fluorogenic assay using DEVD-AMC as a substrate. The results were normalized to the DEVDase activity in untreated cells.
  • both quercetin and trans-resveratrol convert caspase-3 from its inactive form (32 kDa doublet) to its active form (17 kDa) as illustrated by a decrease in the inactive form and an increase in the active form using Western blot analysis and an antibody that recognizes both forms.
  • the results show a dose dependency with effects occurring with as little as 20 ⁇ M for both compounds.
  • Figures 6B, 6C, and 7B show that both quercetin and trans-resveratrol, but not rutin, caused caspase-3 activation as provided in the specific fluorogenic assay for caspase-3.
  • the ELISA assay for DNA fragmentation was conducted as described above.
  • a fluorimetric assay for caspase-3 activity was conducted. Specifically, cells were collected, washed with ice-cold PBS and resuspended in lysis buffer comprising 0.5% Nonidet P-40 or manufactured by the name IGEPAL CA-630, 0.5 mM EDTA, 150 mM NaCl and 50 mM Tris at pH 7.5. Cell lysates were placed for 30 minutes on a rotator at 4 °C and then centrifuged for 15 minutes at 15,000 g. Cytosolic protein extracts (supematants) were collected, protein concentrations were determined and the extracts were aliquoted and stored at - 80 °C.
  • Enzyme assays were carried out at 37 °C in a buffer comprising 25 mM HEPES (pH 7.5), 10% sucrose, 0.1% CHAPS and 10 mM DTT with 800 g cytosolic protein and 20 ⁇ M of specific fluorogenic substrate.
  • the substrate was z-DEVD.
  • Cleavage of the caspase substrate releases 7- amino-4-methylcoumarin (AMC), which emits a fluorescent signal with excitation at 38 nm and emission at 440 nm.
  • AMC 7- amino-4-methylcoumarin
  • the reaction was started by addition of caspase-3 substrate, the readings were taken at 0, 60, 90, and 120 minutes. Fluorescence was calibrated using a standard curve for AMC. The data were expressed as mol AMC/mg protein/min.
  • the caspase-3 activity and apoptosis are synergistically activated with a combination of an inhibitor of ROS production and a polyphenolic compound and Z-VAD inhibits apoptosis caused by the combinations.
  • the supernatant was removed and the pellet was washed twice with PBS by alternate centrifugation and resuspension. The pellet was then lysed by addition of 1 ml of H 2 O and homogenized.
  • the concentration of DiOC 6 (3) was read on a Perkin-Elmer LS-5 fluorescence spectrometer at 488 nm excitation and 500 nm emission. An aliquot of the cells was used for determining the DiOC 6 (3) fluorescence that was retained by the cells.
  • Mia PACA-2 cells were cultured for 72 hours in the presence of serum and trans- resveratrol or genistein in the presence or absence of DPI or Tiron. Mitochondrial membrane potential was measured as described above.
  • trans-resveratrol alone and genistein alone have no effects on mitochondrial membrane potential. Additionally, DPI and Tiron markedly depolarized the mitochondrial membrane and the depolarization was not changed by the addition of trans-resveratrol or genistein. Thus, in the presence of serum, the production of ROS is essential for the maintenance of mitochondrial membrane polarity and the ability of polyphenolic compounds to potentiate the effects of ROS inhibitors on apoptosis is not due to changes in membrane polarity.
  • Example 6 Effects of Inhibitors of PTP A. POLYPHENOLIC COMPOUND ALONE [277]
  • Mia PACA-2 cells were cultured in the absence of serum or growth factors for 24 hours in the presence or absence of quercetin (100 ⁇ M), trans-resveratrol (100 ⁇ M), Z-VAD FMK (50 ⁇ M), cyclosporin A (5 ⁇ M) and/or aristolochic acid (50 ⁇ M).
  • Mia PACA-2 cells were cultured in the absence of serum or growth factors for 24 hours in the presence or absence of quercetin (100 ⁇ M), trans-resveratrol (100 ⁇ M), Z- VAD FMK (50 ⁇ M), cyclosporin A (5 ⁇ M) and/or aristolochic acid (50 ⁇ M).
  • Enzyme assays were carried out at 37 °C in a buffer comprising 25 mM HEPES (pH 7.5), 10% sucrose, 0.1% CHAPS and 10 mM DTT with 800 g cytosolic protein and 20 ⁇ M of specific fluorogenic substrate.
  • the substrate was K-AspGluValAsp-AMC (z-DEVD).
  • AMC (7-amino-4- methylcoumarin), which emits a fluorescent signal with excitation at 380 nm and emission at 440 nm.
  • the reaction was started by addition of caspase-3 substrate, the readings were taken at 0, 60, 90, and 120 minutes. Fluorescence was calibrated using a standard curve for AMC. The results were normalized to the DEVDase activity in cells not treated with polyphenolic compounds. The data were expressed as mol AMC/mg protein/min.
  • Mia PACA-2 cells were cultured in the absence of serum or growth factors for 24 hours in the presence or absence of quercetin (100 ⁇ M), trans-resveratrol (100 ⁇ M), Z- VAD FMK (50 ⁇ M), cyclosporin A (5 ⁇ M) and aristolochic acid (50 ⁇ M). The samples were analyzed by annexin staining as provided above.
  • Cyclosporin A inhibits PTP channels by interacting with one of the key subunits of the PTP, cyclophilin, and cyclosporin A by itself or in combination with aristolochic acid blocks cytochrome c release on several cell types.
  • Z- VAD inhibited the release of cytochrome c into the cytoplasm in control (untreated) cells.
  • Z-VAD had no effect on cytosolic cytochrome c release caused by quercetin or trans-resveratrol, thereby indicating that their action is directly on the mitochondria and not through the pathway involving caspase-8 and Bid.
  • cytochrome c release caused by quercetin and genistein were inhibited by cyclosporin A alone, whereas the cytochrome c release caused by trans-resveratrol required both cyclosporin A and aristolochic acid for inhibition.
  • cyclosporin A alone inhibited caspase-3 activity in quercetin and genistein treated cells, whereas both cyclosporin A and aristolochic acid were required to inhibit caspase-3 activity in trans-res veratrol-treated cells.
  • Mia PACA-2 cells were cultured in the absence of serum or growth factors for 24 hours in the presence or absence of trans-resveratrol (25 ⁇ M), quercetin (25 ⁇ M), or the combination of trans-resveratrol (25 ⁇ M) and quercetin (25 ⁇ M).
  • Cytosolic extracts were prepared and subject to SDS-PAGE followed by protein transfer. Immunoblot was performed with an antibody against tubulin to confirm equal protein loading.
  • Mia PACA-2 cells were cultured in the absence of serum or growth factors for 24 hours in the presence or absence of trans-resveratrol (25 ⁇ M), quercetin (25 ⁇ M), or the combination of trans-resveratrol (25 ⁇ M) and quercetin (25 ⁇ M).
  • Caspase-3 activity was measured in cell lysates with a fluorgenic assay using DEVD-AMC as a substrate. The results were normalized to the DEVDase activity in untreated cells. As illustrated in Figure 13, the combinations resulted in responses of cytochrome c release and caspase-3 activity that were significantly greater than the additive responses.
  • Example 7 NF- ⁇ B Assays I. POLYPHENOLIC COMPOUND ALONE [284] In order to determine the role of activated NF- ⁇ B in the regulation of apoptosis caused by the polyphenolic compound, the following assay was conducted. BSp73AS cells were cultured for 6 hours and Mia PACA-2 cells were cultured in the absence of serum or growth factors for 24 hours in the absence (controls) or presence of rutin, quercetin, trans-resveratrol, or genistein, each at 100 ⁇ M and 20 ⁇ M proteosome inhibitor MG-132. Nuclear proteins were isolated and analyzed for NF- ⁇ B DNA binding activity with electrophoretic mobility shift assay (EMSA).
  • ESA electrophoretic mobility shift assay
  • the oligonucleotide probe 5 ' -GCAGAGGG6ACTTTCCGAGA ( SEQ ID NO : 5 ) containing the KB binding motif (underlined) was annealed to the complementary oligonucleotide with a 5'-G overhang and end-labeled using Klenow DNA polymerase I.
  • the samples were electrophoresed at room temperature in 0.5 x TBE buffer (1 x TBE 89 mM Tris base, 89 mM boric acid and 2 mM EDTA) on nondenaturing 4.5% polyacrylamide gel at 200 V. Gels were dried and directly analyzed in the Phosphorlmager (Molecular Dynamics, Sunnyvale, CA). The experiment was repeated twice.
  • NF- ⁇ B is constitutively active in both cancer cell lines.
  • Figures 14A and 14B show that quercetin and trans-resveratrol inhibit NF- ⁇ B activation in both pancreatic cell lines, rutin activates NF- ⁇ B in BSp73 AS cells but not Mia PACA- 2 cells, and genistein has no effect on NF- ⁇ B in the Mia PACA-2 cells.
  • the proteosome inhibitor, MG-132 blocks NF- ⁇ B activation in both cell lines.
  • MG-132 causes a small increase in caspase-3 activity that adds to the caspase-3 activity caused by quercetin.
  • MG-132 Complete inhibition of NF- ⁇ B by MG-132 does not increase apoptosis rates to the same extent as trans-resveratrol which only partially inhibits NF- KB activation. Additionally, genistein causes significant apoptosis in the absence of an effect on NF-icB activation.
  • Mia PACA-2 cells were cultured for 72 hours in the presence of serum with or without 100 ⁇ M trans-resveratrol, 15 ⁇ M DPI, a combination of trans- resveratrol, 100 ⁇ M genistein, or a combination of genistein and DPI.
  • NF- ⁇ B binding activity was measured in nuclear extracts by gel shift assay as described above.
  • NF- ⁇ B activation was inhibited and the effects on apoptosis in the presence and absence of polyphenolic compounds and ROS inhibitors were studied.
  • Mia PACA-2 cells were cultured for 72 hours in the presence of serum with or without 50 ⁇ M trans-resveratrol (RS), 15 ⁇ M DPI, a combination of DPI and trans-resveratrol (RS+DPI), 10 ⁇ M MG-132 alone and in combination with RS, DPI, and RS+DPI. Intemucleosomal DNA fragmentation was measured in cell lysates by cell death ELISA.
  • CCK-8 was from American Peptide Company (Sunnyvale, CA); recombinant TNF- ⁇ , from BD Biosciences (San Diego, CA); medium 199, from GIBCO BRL (Grand Island, NY); [ ⁇ - 32 P]ATP, from ICN Biomedicals (Costa Mesa, CA); GF-109203X, G ⁇ - 6976, PKC ⁇ peptide substrate, PKC ⁇ peptide substrate, from Calbiochem (La Jolla, CA); PKC ⁇ substrate and PKC ⁇ pseudosubstrate, from Biosource International (Camarillo, CA); antibodies against I ⁇ B ⁇ , PKC ⁇ , PKC ⁇ , PKC ⁇ , and PKC ⁇ , from Santa Cruz Biotechnology (Santa Cruz, CA); PP2, D-609, U-73122, from Biomol (Plymouth Meeting, PA); conventional PKC substrate and anti-phosphotyros
  • Pancreatic acini were prepared from Sprague-Dawley rats (about 75 to about 100 g) using a collagenase digestion method known in the art and then incubated in 199 medium supplemented with penicillin (100 U/ml) and streptomycin (0.1 mg/ml) for 3 hours at 37 °C in a 5% CO 2 humidified atmosphere. These incubation conditions are referred to herein as "standard incubation conditions”.
  • the supernatant (cytosolic proteins) was saved for Western blot analysis of I ⁇ B ⁇ , and the nuclear pellet resuspended in a high-salt buffer C supplemented with 1 mM PMSF, 1 mM DTT, and the protease inhibitor cocktail described above. After incubating at 4 °C, membrane debris were pelleted by microcentrifugation for 10 minutes, and the clear supernatant (nuclear extract) was aliquoted and stored at -80 °C. Protein concentration in the extracts was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA).
  • EMSA For EMSA, aliquots of nuclear extracts with equal amounts of protein (about 5 to about 10 ⁇ g) were mixed in 20 ⁇ l reactions with a buffer containing 10 mM HEPES (pH 7.8), 50 mM KC1, 0.1 mM EDTA, 1 mM DTT, 10% glycerol, and 3 ⁇ g poly(dl-dC). Binding reactions were started by addition of P-labeled DNA probe and incubated at room temperature for 20 minutes.
  • the oligo probe 5 ' -GCAGAGGGG ⁇ CTTTCCGAGA ( SEQ ID NO : 5 ) containing KB binding motif (underlined) was annealed to the complementary oligonucleotide and end-labeled using T4 polynucleotide kinase.
  • Samples were electrophoresed on a native 4.5% polyacrylamide gel at 200 V in 0.5 x TBE buffer (1 x TBE: 89 mM Tris base, 89 mM boric acid, 2 mM EDTA). Gels were dried and densitometrically quantified in the Phosphorlmager (Molecular Dynamics, Sunnyvale, CA).
  • the NF- ⁇ B band has 2 components: the upper component corresponds to the p50/p65 heterodimer and the lower component to the p50/p50 homodimer. See Pandol et al. (1999) Gastroenterology 117:706-716, which is herein incorporated by reference. In the present study, the total (combined) intensity of the NF- KB band was quantified.
  • the dispersed acini were homogenized with 50 strokes in a Dounce homoginizer in an ice-cold homogenization buffer containing 130 mM NaCl, 50 mM Tris/HCl (pH 7.5), 5 mM EGTA, 5 mM EDTA, 1.5 mM MgC12, 10 mM NaF, 1 mM Na3VO4, 10 mM Na4P2O7, 10% (vol/vol) glycerol, 1 mM PMSF, and 5 ⁇ g/ml each of pepstatin, leupeptin, chymostatin, antipain, and aprotinin.
  • Pancreatic acini were suspended in 1 ml of ice-cold homogenization buffer, sonicated 5 times for 10 seconds on ice, and incubated for 45 minutes at 4 °C. After the centrifugation for 15 minutes at 15,000 g, specific antibody against individual PKC (Santa Cruz, Santa Cruz, CA) isoform (1:100 dilution) was added to the lysate, which was rotated overnight at 4 °C. Protein A-Sepharose beads (50% slurry) were added and rotated for 2 hours at 4 °C.
  • the beads were washed twice in the lysis buffer followed by additional 3 washes with the kinase buffer (20 mM MOPS (pH 7.2), 25 mM ⁇ - glycerophosphate, 5 mM EGTA, 1 mM Na 3 VO 4 , and 1 mM DTT). The beads were resuspended in a final 50 ⁇ l of kinase buffer.
  • Isoform specific PKC kinase assay [296] The kinase assay was performed using the PKC assay kit (Upstate Biotechnology, Charlottesville, NA) according to the manufacturer's instruction with minor modifications. Substrates optimized for individual PKC isoforms were used.
  • the substrates used are as follows: For PKC ⁇ : QKRPSQRSKYL ( SEQ ID ⁇ O : ⁇ ) For PKC ⁇ : RFAVRDMRQTVAVGVIKAVDKK (SEQ ID NO : 7 ) For PKC ⁇ : ERMRPRKRQGSVRRRV ( SEQ ID NO : 8 ) For PKC ⁇ : SIYRRGSRRWRKL ( SEQ ID NO : 9 ) . [297] The kinase buffer as provided in Section E above was used for the measurement of PKC ⁇ , ⁇ , and ⁇ , and supplemented it with 1 mM CaCl 2 for PKC ⁇ .
  • the assay was started with the addition of a magnesium/ATP mixture (75 mM MgCl 2 and 0.5 mM ATP) containing 10 ⁇ Ci of [ ⁇ - 32 P]ATP to the sample containing 10 ⁇ l of the PKC isoform specific immunoprecipitate, 30 ⁇ l of kinase buffer and 40 ⁇ M of substrate, and the reaction incubated for 10 minutes at 30 °C. Reactions were stopped by the addition of 50 ⁇ l of 0.75% phosphoric acid, and the samples were applied onto p81 phosphocellulose paper (Upstate Biotechnology, Charlottesville, NA).
  • the membranes were blocked by overnight incubation in Tris-buffered saline (TBS) supplemented with 5% nonfat dry milk and probed with an antibody against I ⁇ B ⁇ (1 :100 dilution), PKC ⁇ , PKC ⁇ , PKC ⁇ , PKC ⁇ (1:200 dilution each), or phosphotyrosine (1 :500 dilution) for 2 hours at room temperature.
  • TBS Tris-buffered saline
  • the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase for 1 hour at room temperature. Blots were developed using the enhanced chemiluminescence detection kit (Pierce, Rockford, IL). When reprobing was necessary, the membrane was stripped of bound antibody by incubating in stripping buffer at room temperature for 20 minutes.
  • these peptides correspond to specific sequences in the VI regions, which is responsible for anchoring the individual isoform to its translocation site.
  • the peptides competitively inhibit the binding of a specific isoform of PKC to its anchoring protein.
  • Each of these peptides was conjugated to a Drosophila antennapedia peptide RQIKIWFQNRRMKWKK ( SEQ ID NO : 4 ) to make them cell-permeable.
  • Figure 19A shows subcellular distribution of PKC isoforms in response to CCK-8 in cytosolic and membrane fractions us ing isoform specific PKC antibodies and Western blot analysis.
  • Figure 19B shows the changes in PKC kinase activities stimulated by CCK-8. Individual PKC isoforms were immunoprecipitated from whole cell lysates and PKC activities were measured by kinase assay using isoform-optimized substrates.
  • Pancreatic acini were preincubated for 3 hours with a PKC broad spectrum inhibitor, GF109203X (GF); a conventional PKC isoform inhibitor, Go6976 (Go); a PKC ⁇ translocation inhibitor ( ⁇ Vl-1); a PKC ⁇ translocation inhibitor ( ⁇ Vl-2); a PKC ⁇ inhibitor, a PKC ⁇ pseudosubstrate ( ⁇ pseudo), 10 ⁇ M each, or with DMSO, and then stimulated with 100 nM CCK-8 for 30 minutes.
  • GF109203X GF109203X
  • Go PKC isoform inhibitor
  • Go6976 Go
  • a PKC ⁇ translocation inhibitor ⁇ Vl-1
  • ⁇ Vl-2 PKC ⁇ translocation inhibitor
  • ⁇ pseudo PKC ⁇ pseudosubstrate
  • Figure 20A shows NF- ⁇ B binding activity measured in nuclear extracts measured by EMSA.
  • Figure 20B shows NF- ⁇ B band intensities quantified in the Phosphorlmager and normalized on the band intensity in unstimulated control acini.
  • Pancreatic acini were preincubated with PKC translocation inhibitors, ⁇ Vl -1 or ⁇ Vl-2 (10 ⁇ M each), scrambled peptide (10 ⁇ M), delivered in DMSO for 3 hours, and then stimulated with 100 nM CCK-8 for 30 minutes. At the end of this incubation, subcellular fractions were obtained as described in Section D above and used for Western blot analysis as described in Section G above. Kinase activity measurements were performed on samples that were immunoprecipitated as described in Section E above with a specific antibody for the isoform of PKC to be measured. The kinase activity measurement was as described in Section F above.
  • Figure 21 A shows cytosolic and membrane fractions subjected to Western blot analysis. SDS-PAGE and blotted using antibodies specific for PKC ⁇ or ⁇ were used.
  • Figure 21B shows the effects of PKC translocation inhibitors on kinase activity. For each PKC isoform, activity values were normalized on its basal activity in unstimulated control acini.
  • Pancreatic acini were preincubated for 3 hours with in standard incubation conditions with 1% DMSO (vol/vol) for 3 hours, and then stimulated with 100 ng/ml TNF- ⁇ for 30 minutes.
  • the scrambled peptide (10 ⁇ M) was used as a control and inhibitors and their controls were delivered in DMSO so that final DMSO concentration was 1% (vol/vol) in this a subsequent experiments.
  • subcellular fractions were obtained as described in Section D above and used for Western blot analysis as described in Section G above.
  • Kinase activity measurements were performed on samples that were immunoprecipitated as described in Section E above with a specific antibody for the isoform of PKC to be measured. The kinase activity measurement was as described in Section F above.
  • Figure 22A shows subcellular distribution of PKC isoforms in response to TNF- ⁇ in cytosolic and membrane fractions using isoform specific PKC antibodies and Western blot analysis.
  • Figure 22B shows changes in PKC kinase activities stimulated by TNF- ⁇ .
  • Individual PKC isoforms were immunoprecipitated from whole cell lysates and PKC activities were measured by kinase assay using isoform-optimized substrates. For each PKC isoform, activity values were normalized on its basal activity in unstimulated control acini.
  • Pancreatic acini were preincubated for 3 hours with PKC broad spectrum inhibitor, GF109203X (GF); conventional PKC isoform inhibitor, G ⁇ 6976 (Go); PKC ⁇ translocation inhibitor ( ⁇ Vl-1); PKC ⁇ translocation inhibitor ( ⁇ Vl-2); PKC ⁇ inhibitor, PKC ⁇ pseudosubstrate ( ⁇ pseudo), 10 ⁇ M each, or with DMSO, and then stimulated with 100 ng/ml TNF- ⁇ for 30 minutes.
  • PKC broad spectrum inhibitor GF109203X (GF)
  • G ⁇ 6976 Go
  • PKC ⁇ translocation inhibitor ⁇ Vl-1
  • PKC ⁇ translocation inhibitor ⁇ Vl-2
  • PKC ⁇ inhibitor, PKC ⁇ pseudosubstrate ( ⁇ pseudo) 10 ⁇ M each, or with DMSO and then stimulated with 100 ng/ml TNF- ⁇ for 30 minutes.
  • Figure 23A shows NF- ⁇ B binding activity in nuclear extracts measured by EMSA.
  • Figure 23 B shows NF- ⁇ B band intensities quantified in the Phosphorlmager and normalized on the band intensity in unstimulated control acini.
  • Figure 23 C shows I ⁇ B ⁇ degradation in cytosolic extracts by Western blot analysis.
  • Figure 24A shows NF- ⁇ B binding activity in nuclear extracts measured by EMSA.
  • Figure 24B shows I ⁇ B ⁇ degradation in cytosolic extracts by Western blot analysis.
  • Pancreatic acini were preincubated with with Src kinase inhibitor, PP2 (20 ⁇ M), or DMSO (vehicle) for 3 hours, and then stimulated with 100 nM CCK-8 or 100 ng/ml of TNF- ⁇ for 30 minutes.
  • Src kinase inhibitor PP2 (20 ⁇ M)
  • DMSO DMSO
  • whole cell lysates were immunoprecipitated with an antibody specific to PKC ⁇ as described in Section E above.
  • the resulting immunoprecipitate was subjected to Western blot analysis with an antibody to phosphotyrosine as described in Section G above.
  • the upper panel of Figure 25 shows whole cell lysates immunoprecipitated with anti-PKC ⁇ antibody, and then subjected to SDS-PAGE and blotted using anti- phosphotyrosine antibody.
  • the lower panel of Figure 25 shows equal protein loading was verified using PKC ⁇ antibody after stripping the membranes.
  • Pancreatic acini were preincubated for 3 hours with Pi-specific PLC inhibitor U- 73122 (10 ⁇ M), or for 30 minutes with PC-specific PLC inhibitor D-609 (50 ⁇ M), and then stimulated for 30 minutes with 100 nM CCK-8 or 100 ng/ml TNF- ⁇ . D-609 was added to the culture medium 30 minutes before the stimulation because longer incubation with this inhibitor was toxic for pancreatic acini.
  • N-609 electomobility shift assay
  • Figure 26A shows NF- ⁇ B binding activity in nuclear extracts measured by EMSA.
  • Figure 26B shows I ⁇ B ⁇ degradation in cytosolic extracts measured by Western blot analysis.
  • Pancreatic acini were preincubated for 3 hours with Pi-specific PLC inhibitor U- 73122 (10 ⁇ M), or for 30 minutes with PC-specific PLC inhibitor D-609 (50 ⁇ M), and then stimulated for 30 minutes with 100 nM CCK-8 ( Figure 27 A) or 100 ng/ml TNF- ⁇ ( Figure 27B). At the end of this incubation, subcellular fractions were obtained as described in Section D above and used for Western blot analysis as described in Section G above.
  • FIG. 28 A is a schematic of the signaling pathways involved in NF- ⁇ B activation induced by CCK-8 and TNF- ⁇ in pancreatic acinar cells. Binding of CCK-8 to its receptor activates both Pi-specific and PC-specific PLC, whereas TNF- ⁇ only activates PC-specific PLC. Activation of PLC leads to DAG generation, promotes translocation of PKC ⁇ and PKC ⁇ , which, in turn, mediates I ⁇ B ⁇ degradation and NF- KB activation. CCK-8 and TNF- ⁇ also induce PKC ⁇ activation, but it is not involved in NF- ⁇ B activation. Constitutive activity of PKC ⁇ exerts an inhibitory effect onNF- ⁇ B activation. Although tyrosine phosphorylation of PKC ⁇ is induced by Src, this event is not involved in NF- ⁇ B activation induced by CCK-8 and TNF- ⁇ .
  • FIG. 28B J. Rottlerin inhibits NF- ⁇ B activation in pancreatic acinar cells
  • Figure 28B was performed to test if a nonpeptide agent know to inhibit PKC ⁇ could inhibit NF-kB in pancreatic acinar cells.
  • Pancreatic acini were preincubated for 3 hours with or without ethanol (100 mM) and with or without rottlerin (2.5 ⁇ M) for 30 minutes and then stimulated for 30 minutes with 100 nM CCK-8.
  • nuclear extracts were prepared and subjected to electomobility shift assay (EMSA) for NF- ⁇ B binding activity as described in Section C above. The results show complete inhibition of NF-kB by rottlerin.
  • ESA electomobility shift assay
  • Example 8 PI 3 -Kinase Inhibition Assays [329] To determine whether phosphatidylinositol 3-kinase (PI 3-kinase) and Akt/PKB mediate the effects of serum on NF- ⁇ B activation and that the effects of polyphenolic compounds on NF- ⁇ B activation are due to an ability to inhibit PI 3-kinase, the following assays were conducted.
  • Mia PACA-2 cells were cultured for 72 hours in the absence or presence of serum with or without 100 ⁇ M genistein (GN) or 50 ⁇ M LY294002.
  • Western blots were performed on whole cell lysates as described above except that specific antibodies against phosphorylated and total Akt/PKB were used (Akt/PKB is Anti- ⁇ S473 AktpAB from Promega, Madison WI and total Akt is Akt 1/2 from Santa Cmz, Santa Cruz, CA). The membranes were then stripped and re-probed with an antibody against total Akt .
  • Mia P ACA- 2 cells were cultured for 72 hours in the presence of serum and 15 ⁇ M DPI with or without 50 ⁇ M LY294002.
  • NF- ⁇ B DNA binding activity was measured in nuclear extracts by gel shift assay as described above.
  • MIA PaCa-2 cells were cultured for up to about 72 hours in the presence of serum with or without rottlerin (Rt) and protein kinase C inhibitors, GF109203X (GF) and Ro-32-0432 (Ro). Oligonucleosomal DNA fragmentation was measured by an ELISA technique using Cell Death Detection ELISA Plus (Roche, Indianapolis, IN). Figure 29 represents three experiments with similar results.
  • Annexin V and propidium iodide staining were measured according to Example 3B above using methods known in the art.
  • Annexin V stains phosphatidylserine on the surface of the cell when the cell is impermeant. This is a specific measure of apoptosis because during apoptosis, phosphatidylserine externalizes to the outside surface of the cell.
  • the propidium iodide enters the cell and stains the nucleus when the cell is permeant as happens during necrosis. Cells that stain with Annexin V but not propidium iodide are apoptotic.
  • MIA PaCa-2 cells were cultured for about 72 hours in the presence of serum with or without the indicated concentrations of rottlerin (Rt). Phosphatidylserine externalization was measured by flow cytometry in cells stained with AnV and PI. AnVVPT cells were considered apoptotic. Cells positive for PI were considered necrotic. Figure 31 represents two experiments with similar results. The results indicate that rottlerin causes cell death through apoptosis and not necrosis.
  • MIA PaCa-2 pancreatic cancer cells were cultured for 48 hours in the presence of serum with or without the indicated concentrations of rottlerin (Rt), Ro-32-0432 (Ro) and 100 ⁇ M z-VAD.fmk (Z-VAD).
  • DEVDase activity was measured in whole cell lysates with a fluorimetric assay as Example 41 above using methods l ⁇ iown in the art.
  • FIG. 33 A is a histogram that shows changes in ⁇ m were measured by flow cytometry in cells labeled with membrane potential sensitive fluorescent dye DiOC 6 (3) as provided in Example 51 above using methods known in the art.
  • Figure 33B shows the percentage of cells with high ⁇ m.
  • MIA PaCa-2 pancreatic cancer cells were cultured for about 72 hours in the absence or presence of the indicated concentration of rottlerin. The cells were then lysed and cytosolic fractions were isolated. The cytoclirome c level in cytosolic fractions was measured by Western blot analysis. The membranes were stripped and re-probed with an antibody against actin to show equal protein loading.
  • Figure 34 represents two experiments with the similar results. The results demonstrate that rottlerin causes significant release of mitochondrial cytochrome c into the cytoplasm of the cancer cells. The combination of results indicate that rottlerin causes apoptosis by causing mitochondrial depolarization leading to cytochrome c release which, in turn, leads to caspase-3 activation and apoptosis.
  • MIA PaCa-2 pancreatic cancer cells were cultured for about 72 hours in the presence of serum with or without rottlerin (Rt, 2.5 ⁇ M) or GF109203X (GF, 10 ⁇ M).
  • NF- ⁇ B binding activity was measured in nuclear extracts by gel shift assay on the MIA PaCa-2 cells at the end of the incubation as described in Section C above.
  • Figures 35 and 36 represent three experiments with similar results.
  • MIA PaCa-2 cells were cultured for 72 hours in the presence of serum and with or without rottlerin or GF109203X. Intracellular ROS was measured using oxidation-sensitive cell-permeable fluorescent probe, dichlorofluorescein diacetate (DCF-DA) to measure H O 2 using methods known in the art. See Royall & Ischiropoulos (1993) Arch. Biochem. Biophys. 302:348-355, which is herein incorporated by reference. To measure ROS, cells were collected after incubation, washed with PBS, and incubated for 15 minutes with 8 mM DCF-DA. Samples were analyzed by flow cytometry. The amount of DCF-DA fluorescence correlated with the amount of ROS in the cells.
  • DCF-DA dichlorofluorescein diacetate
  • Figure 36B shows the percentage of cells with high DCF fluorescence. These figures represent two experiments with the similar results. The results demonstrate that rottlerin almost completely inhibits ROS production in the cancer cells. These results indicate that rottlerin promotes mitochondrial changes, caspase-3 activation and apoptosis in part because it inhibits ROS generation in the cancer cells.
  • MIA PACA-2 TUMOR GROWTH IN VIVO AND ROTTLERIN The effect of rottlerin on the growth of MIA PaCa-2 tumors in nude mice was studied. MIA PaCa-2 cells were injected subcutaneously into the flank of nude mice. Animals were thereafter treated with daily intraperitoneal injections of either rottlerin (0.5 mg/kg body weight) or control vehicle for 14 days. Tumor volume was assessed at the end of the treatment period using the formula for a hemi-ellipsoid (2/3* ⁇ *a*b*c with a, b, and c being the half diameters for height, width, and length of the tumor).
  • RNA and DNA synthesis and Rottlerin [347] RNA ribose and DNA deoxyribose were isolated by acid hydrolysis of cellular nucleic acid after Trizol purification of cell extracts. Total RNA amounts were assessed by spectrophotometric determination, in triplicate cultures. Ribose was derivatized to its aldonitrile acetate form using hydroxylamine in pyridine with acetic anhydride (Supelco, Bellefonte, PA) before mass spectral analyses.
  • the ion cluster was monitored around the m/z 256 (carbons 1-5 of ribose) (chemical ionization, Cl) and m/z 217 (carbons 3-5 of ribose) and m/z 242 (carbons 1-4 of ribose) (electron impact ionization, El) to determine molar enrichment and the positional distribution of C in ribose.
  • the base mass of C- compounds is given as m 0 as measured by mass spectrometry as described in the prior art. See Boros et al. (2002) Drug Discovery Today 7:364-372, which is herein incorporated by reference.
  • Figure 38 shows total tracer 13 C carbon incorporation into DNA deoxyribose and RNA ribose, respectively, from the tracer [l,2- I3 C 2 ]glucose.
  • Deoxyribose and ribose molecules labeled with a single C atom on the first carbon position (ml) recovered from DNA or RNA were used to guage the ribose fraction produced by direct oxidation of glucose through the G6PD pathway.
  • Deoxyribose and ribose molecules labeled with 13 C on the first two carbon positions (m2) were used to measure the fraction produced by the non-oxidative steps of the pentose cycle via transketolase.
  • Rottlerin induced a significant decrease in non-oxidative deoxyribose synthesis (2) with a compensatory increase in oxidative deoxyribose synthesis (1), but rottlerin affected neither oxidative (3) nor non-oxidative (4) RNA ribose synthesis.
  • Lactate from the cell culture media (0.2 ml) was extracted by ethylene chloride after acidification with HCL. Lactate was derivatized to its propylamine-HFB form and the m/z328 (carbons 1-3 of lactate) (chemical ionization, Cl) was monitored for the detection of ml (recycled lactate through the PC) and m2 (lactate produced by the Embden-Meyerhof-Parnas pathway) for the estimation of pentose cycle activity. See Lee et al. (1998) Am. J. Physiol. 274:E843-E851, which is herein incorporated by reference.
  • MIA PaCa-2 cells were treated with vehicle, 2.5 ⁇ M and 5.0 ⁇ M rottlerin for 72 hours in the presence of [l,2- 13 C 2 ]glucose in culture.
  • Rottlerin decreased direct glucose oxidation and recycling in the pentose cycle, although this effect was not dose dependent.
  • Figure 40 represents the mean of 3 cultures in each group. The ml/m2 ratios in lactate produced and released by MIA PaCa-2 pancreatic adenocarcinoma cells was recorded in order to determine pentose cycle activity versus glycolysis in response to rottlerin treatment.
  • Glutamate label distribution from glucose is suitable for determining glucose oxidation versus anabolic glucose use within the TCA cycle, also known as anaplerotic flux. See Lee et al. (1996) Dev. Neurosci. 18:469-477, which is herein incorporated by reference.
  • tissue culture medium was first treated with 6% perchloric acid. The supernatant was passed through a 3 cm 3 Dowex-50 (H+) column. Amino acids were eluted with 15 ml 2N ammonium hydroxide. To further separate glutamate from glutamine, the amino acid mixture was passed through a 3 cm 3 Dowex-1 (acetate) column, then collected with 15 ml 0.5 N acetic acid.
  • TAB-glutamate trifluoroacetyl butyl ester
  • ionization of TAB-glutamate gives rise to two fragments, m/zl98 and m/zl52, corresponding to C2-C5 and C2-C4 of glutamate.
  • Glutamate labeled on the 4-5 carbon positions indicates pyruvate dehydrogenase activity while glutamate labeled on the 2-3 carbon positions indicates pyruvate carboxylase activity for the entry of glucose carbons to the TCA cycle.
  • TCA cycle anabolic glucose utilization is calculated based on the m m 2 ratios of glutamate.
  • the TCA cycle metabolite alpha-ketoglutarate is in equilibrium with glutamate, which is released by the cells into the medium.
  • the m2/ml ratio in glutamate is proportional with the activity of glucose oxidation as CO 2 is released from alpha-ketoglutarate during each completed cycle.
  • TCA cycle anaplerotic flux is calculated based on the m2/ml ratios of glutamate.
  • Anaplerosis refers to the reactions that allow the entry of carbon into the TCA cycle intermediate pools other than via citrate synthase. Any carbon that enters the cycle as acetyl-CoA is oxidized to carbon dioxide and water; any carbon that enters the citric acid cycle via an anaplerotic pathway is not oxidized, but must be disposed of by some other route.
  • Glutamate dehydrogenase is one possible route providing equilibrium between alpha-ketoglutarate and glutamate, some other reactions include pyruvate carboxylation, transamination reactions and propionate carboxylation.
  • MIA PaCa-2 cells were treated with vehicle, 2.5 ⁇ M and 5.0 ⁇ M rottlerin for 72 hours in the presence of [l,2- 13 C ]glucose in culture.
  • Rottlerin increased glucose oxidation in the TCA cycle, which indicates that MIA cells utilize glucose for energy production more efficiently in the presence of rottlerin.
  • Glucose substrate flow in rottlerin treated MIA cells is from nucleotide synthesis toward glucose oxidation and energy production in the TCA cycle.
  • MIA PaCa-2 cells were treated with vehicle, 2.5 ⁇ M and 5.0 ⁇ M rottlerin for 72 hours in the presence of [l,2- 13 C 2 ]glucose in culture. Rottlerin induced a significant sharp decrease in the de novo synthesis of all fatty acid species.
  • the data in Figure 42 represent the mean of 3 cultures in each group.
  • Myrystate (C:14), palmitate (C:16), stearate (C:18) and oleate (C:18-l) were extracted after saponification of cell pellets in 30% KOH and 100%) ethanol using petroleum ether. Fatty acids were converted to their methylated derivative using 0.5N methanolic-HCL. Palmitate, stearate and oleate were monitored at m/z 270, m/z 298 and m/z 264, respectively, with the enrichment of C labeled acetyl units which reflect synthesis, elongation and desaturation of the new lipid fraction as determined by mass isotopomer distribution analysis (MID A) of different isotopomers. See Lee et al. (1995) Anal. Biochem. 226:100-112, and Lee et al. (1998) J. Biol. Chem. 273:20929-20934, which are herein incorporated by reference.
  • MID A mass isotopomer distribution analysis

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Abstract

La présente invention a trait à des procédés pour le traitement, la prévention, ou l'inhibition de maladies et de troubles associés à l'activation de NF-Kb comprenant des maladies proliférantes telles que le cancer et des maladies inflammatoires telles que la pancréatite chez un sujet comprenant l'administration d'au moins un composé polyphénolique et/ou au moins un inhibiteur d'espèce réactive d'oxygène, L'invention a également trait à des compositions pharmaceutiques comportant au moins un composé phénolique et/ou au moins un inhibiteur d'espèce réactive d'oxygène. Le composé polyphénolique peut être dérivé ou isolé à partir de plantes. Dans certains modes de réalisation, le composé polyphénolique est de type non flavonoïde. L'invention a trait en outre à d'autres procédés et trousses ainsi qu'à des compositions pharmaceutiques.
PCT/US2005/011741 2004-04-15 2005-04-07 Compositions comportant des composes polyphenoliques derives de plantes et des inhibiteurs d'especes reactive d'oxygene et leurs procedes d'utilisation WO2005099721A2 (fr)

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WO2008073294A2 (fr) * 2006-12-08 2008-06-19 The Board Of Trustees Of The Leland Stanford Junior University Methodes d'inhibition de l'angiogenese et de la croissance tumorale par inhibition de la proteine kinase c beta ou delta
WO2008026125A3 (fr) * 2006-09-01 2008-10-02 Nicholas Piramal India Ltd Utilisation d'acide caféique et de dérivés de ce composé comme anticancéreux
EP2119434A1 (fr) * 2008-05-13 2009-11-18 Institut National De La Sante Et De La Recherche Medicale (Inserm) Utilisation de dérivés flavonoïdes hétérosidiques pour la thérapie de cancers de cellules souches
FR2954352A1 (fr) 2009-12-21 2011-06-24 Roussy Inst Gustave Marqueur de predisposition a un cancer
WO2011104412A2 (fr) * 2010-02-25 2011-09-01 Universidad Del País Vasco Composés pour le traitement d'alzheimer
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EP2578210A1 (fr) * 2011-10-05 2013-04-10 ATB Innovation Ltd. Rottlerine pour le traitement de l'hypertension pulmonaire et de maladies et troubles apparentés
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US11497749B2 (en) * 2017-10-24 2022-11-15 Lunella Biotech, Inc. Mitoflavoscins: targeting flavin-containing enzymes eliminates cancer stem cells (CSCS) by inhibiting mitochondrial respiration

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