prevention with with ¢  ¢â‚¬¢Esophageal. Squamous cell carcinoma, adenocarcinoma ¢â‚¬¢ In U.S.,

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  • PREVENTION OF GASTROINTESTINAL TRACT CANCERS WITH LYOPHILIZED BERRIES

    Gary D. Stoner, Ph.D. Department of Internal Medicine College of Medicine & Public Health The Ohio State University

  • • Esophageal

    . Squamous cell carcinoma, adenocarcinoma

    • In U.S., 3% of cancers in males (less in females)

    • 5-year survival = 6-7%

    • Colon

    • Adenocarcinoma

    • Third most common cancer and third leading cause of cancer mortality in U.S.

    • 5-year survival = 55%

    Gastrointestinal Tract Cancers

  • 1548 142Black Raspberries

    630 90Strawberries

    < 100Peaches

    < 100Apples 120 4Cranberries

    < 100Tangerines < 100Pears

    < 100Oranges (navel) < 100Grapefruit (white, pink) < 100Grapes (white, red) < 100Elderberry < 100Cherries (sour, bing) < 100Blue Plums < 100Blueberries

    1473 108Red Raspberries

    g EA/gm dry weightFruit

    Concentration of Ellagic Acid in Fruits

  • Content of Ellagic Acid in Different Fruit Parts

    n.d.*Juice 52.3 0.08Seed

    7.3 0.03PulpRaspberries

    n.d.*Juice

    38.4 1.5Seed

    52.2 0.4PulpStrawberries

    g EA/gm Wet WeightFruit PartFruit

    E.M. Daniel, et al., J. Food Comp. Anal. 2: 338, 1989. *n.d. is non-detectable

  • Preparation of Freeze-Dried Berries

    Frozen Berries

    Line Trays for Freeze-Drying

    Freeze-Dried Berries

    2,000 lb batches

    1-2” thick

    Freeze-Dry in Virtis Sublimator

    Under a vacuum starting at -40C

    Berry Powder

    Pulverize

  • Freeze-dried black raspberries

    Freeze-dried strawberries

  • 17152705478Anthocyanins:

    34

    9

    8

    165

    -

    -

    Blackberries (mg/100 g)

    41

    < 3

    < 3

    67

    9

    32

    Strawberries (mg/100 g)

    89

    4

    < 3

    -sitosterol

    campesterol

    stigmasterol

    Phytosterols:

    175

    17-32

    8

    ellagic acid

    ferulic acid

    p-coumaric acid

    Phenols:

    Black Raspberries (mg/100 g)Substance

    Nutrient Content of Freeze-Dried Berries

  • Animal Model Tumor Systems

    50%

    50 – 80%

    30 – 60%

    Percent Tumor Inhibition

    spontaneouscolonmouse (Min)

    AOMbcolonrat

    NMBAaesophagusrat

    CarcinogenOrganSpecies

    aNMBA = N-nitrosomethylbenzylamine; bAOM = azoxymethane

  • 0 2 7 8 25

    NMBA (3x/wk/5 wks)

    Berries

    Protocol to Identify Suppressing Activity of Berries in the Esophagus

  • 500.7 0.3aNMBA + 10% BRB

    650.5 0.2aNMBA + 5% BRB

    -1.4 0.3NMBA (0.25 mg/kg)

    % Inhibition Tumors per EsophagusGroup

    aSignificantly different from NMBA control; p

  • Photomicrographs of Proliferating Cell Nuclear Antigen Staining

    Normal High-Grade Dysplasia

    Papilloma Positive Colon

  • Effects of Black Raspberries on PCNA Labeling Index

    22.63a

    22.40a

    31.54

    18.33

    18.46

    PCNA LI (%)

    28.2NMBA + 10% BRB

    29.0NMBA + 5% BRB

    0NMBA

    010% BRB

    0Control

    % ReductionTreatment

    aSignificantly different from NMBA controls, p < 0.05

  • COOH

    Arachidonic Acid

    PGG2

    PGH2

    ProstaglandinsProstacyclin Thromboxane

    Cyclooxygenase Activity

    Peroxidase Activity

    COX-1 COX-2

    PGE2, PGF2, PGD2 TxA2PCI2

    Arachidonic Acid Metabolism

  • Effect of Black Raspberries on COX-2 Expression and Activity in NMBA-Treated Rat Esophagus

    Group

    *

    *

  • Nitric Oxide Synthesis

    L-Arginine L-Citrulline + NO

    Peroxynitrite (ONOO-)

    DNA damage

    (O2-)

    COX-2 Activity

    iNOS

  • iNOS Staining

    Normal Hyperplasia

    Dysplasia Papilloma

  • Effect of Black Raspberries on iNOS Expression and Activity in NMBA-Treated Rat Esophagus

    Group

    *

    *

  • Effect of Black Raspberries on c-Jun Expression in NMBA-Treated Rat Esophagus

    * P < 0.05

    *

    *

  • Western Blot Analysis of iNOS, COX-2 and c-Jun

  • Effect of Black Raspberries on VEGF Expression in NMBA-Treated Rat Esophagus

    * * P < 0.005

    **

  • Effect of Black Raspberries on Microvessel Density (MVD) in NMBA-Treated Rat Esophagus

    * * * P < 0.0005

    ***

  • Y-axis: Stoner Rat esophogus NMBA, BRB, combined GEN... Colored by: CONTROL

    Error Bars: between-sample std. error Gene List: 578 significant in NMBA vs Cntl AND (NMBA + BRB) v...

    CONTROL NMBA 5% BRB + NMBA 5% BRB

    Treatment

    1

    0.2

    0.3

    0.4

    0.5

    0.6

    0.7 0.8

    2

    3

    4

    5 Normalized Intensity (log scale)

    CONTROL NMBA 5% BRB + NMBA 5% BRB

    Treatment

    1

    0.2

    0.3

    0.4

    0.5

    0.6

    0.7 0.8

    2

    3

    4

    5 Normalized Intensity (log scale)

    Two distinct clusters in the 578 genes. The overall trend is that 5% BRB treatment modulates the NMBA effect. Note that in nearly all cases the combined treatment gene expression level is

    closer to the control level, as compared to NMBA alone.

  • 0 2 4 36

    (AOM 15 mg/kg x 2)

    2.5, 5, 10% black raspberries

    Protocol for AOM-Induced Tumors in Rat Colon

    3

  • AOM only 2.5% BRB +

    AOM

    5.0% BRB +

    AOM

    10.0% BRB +

    AOM

    **

    0.00

    0.25

    0.50

    0.75

    1.00

    1.25

    1.50

    1.75

    2.00

    2.25

    Tu m

    o rs

    /c ol

    on Suppressing Activity of Black Raspberries on Rat

    Colon Adenocarcinomas

    ** **

  • Summary of Preclinical Studies

    • Black raspberries inhibit: • a. NMBA-induced tumors in rat esophagus.

    Mechanisms include reduction in:  NMBA-induced DNA adducts  Cell proliferation  COX-2, iNOS, VEGF, and c-Jun expression and

    activities • b. Tumor progression rat colon and oxidative

    stress in AOM-treated animals. • c. Tumor development in the Min mouse model.

  • Scheme for Extraction of Freeze-Dried Berries

    ET EtOH eluate

    Chromatographed on silica gel Eluated by CH2Cl2: EtOH (1:1) and EtOH, respectively

    Chromatography:

    DM CH2Cl2: EtOH eluate

    F001

  • Summary of Studies Conducted Using Alcohol Extracts of Black Raspberries

    • Inhibits transformation of SHE cells with B(a)P • Selectively inhibits growth and stimulates apoptosis in

    tumorigenic rat esophageal cells, and human oral cavity and colon cancer cells

    • Exhibits potent radical scavenging activity • Downregulates expression of AP-1, NFB, VEGF and

    their associated kinases in mouse epidermal JB-6 clone 41 cells

    • Inhibits UVB-induced skin carcinogenesis in mice

    Ethanol Extract of Black Raspberries

  • R el

    at iv

    e V

    E G

    F In

    du ct

    io n

    B[a]PDE (2 M)       Extract RO- (25 g/ml)   F003 F004 DM ET

    * P < 0.05

    Inhibition of VEGF by BRB Extracts

    *

    *

  • Inhibition of pAKT and p70S6k by BRB Extracts

    P-Akt at Thr308

    Akt

    p70S6K at

    P-Akt at Ser473

    P-p70S6K at Thr389

              B[a]PDE (2 M)   50 12 3   50 12 3 RO-ET (g/ml)

    60 min 120 min

    P-p70S6K at Thr421/Ser424

  • Inhibition of Signaling Pathways by Alcohol Extract

    •Akt

    AP-1

    PI(3,4,5)P3

    PI-3K

    ERKs/p38K

    RO ET

    IkBa Degradation

    NFkB NFAT

    COX-2 and VEGF

    JNks

    B[a]PDE

  • Anthocyanins in Black Raspberry EtOH Fraction

    Cyanidin 3-O-glucoside

    Cyanidin 3-O-rutinoside Cyanidin 3-O-(2G-xylosylrutinoside)

    Cyanidin 3-O-sambubioside

  • Phase I Clinical Trial of Freeze-Dried Black Raspberries

    • 10 normal volunteers, > 18 years of age

    • “Phenol-free” diet • 45 g BRB / day, 14 days • Blood and urine • Clinical signs of toxicity

  • Results of Phase I Clinical Trial of Freeze-Dried Black Raspberries

    • Well tolerated

    • blood levels of ellagic acid and several anthocyanins

    • Reduced levels of 8-OH-dG in urine

  • Ongoing Human Clinical Trials With Berry Preparations

    • Barrett’s Esophagus – Laura Kresty, Ph.D. • Esophageal Squamous Cell Carcinoma – Tong Chen,

    M.D., Ph.D. • Colon Cancer – Yiqing Xu, M.D. • Colon Polyps – Carol Burke, M.D. • Oral Leukoplakia – Susan Mallery – D.D.S., Ph.D.

  • Eat 1 hour before or 1 hour after regular meals

  • Collaborators

    OSU Current Collaborators

    Tong Chen

    Alan Dahl

    Laura K