prevention with berries with berries.pdf · •esophageal. squamous cell carcinoma, adenocarcinoma...
TRANSCRIPT
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PREVENTION OF GASTROINTESTINAL TRACTCANCERS WITH LYOPHILIZED BERRIES
Gary D. Stoner, Ph.D.Department of Internal MedicineCollege of Medicine & Public HealthThe Ohio State University
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• 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 causeof cancer mortality in U.S.
• 5-year survival = 55%
Gastrointestinal Tract Cancers
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1548 142Black Raspberries
630 90Strawberries
< 100Peaches
< 100Apples120 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
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Content of Ellagic Acid in Different Fruit Parts
n.d.*Juice52.3 0.08Seed
7.3 0.03PulpRaspberries
n.d.*Juice
38.4 1.5Seed
52.2 0.4PulpStrawberries
g EA/gm WetWeightFruit PartFruit
E.M. Daniel, et al., J. Food Comp. Anal. 2: 338, 1989.*n.d. is non-detectable
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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 -40C
Berry Powder
Pulverize
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Freeze-dried blackraspberries
Freeze-driedstrawberries
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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-coumaricacid
Phenols:
Black Raspberries(mg/100 g)Substance
Nutrient Content of Freeze-Dried Berries
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Animal Model Tumor Systems
50%
50 – 80%
30 – 60%
Percent TumorInhibition
spontaneouscolonmouse (Min)
AOMbcolonrat
NMBAaesophagusrat
CarcinogenOrganSpecies
aNMBA = N-nitrosomethylbenzylamine; bAOM = azoxymethane
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0 2 7 8 25
NMBA (3x/wk/5 wks)
Berries
Protocol to Identify Suppressing Activityof Berries in the Esophagus
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500.7 0.3aNMBA + 10% BRB
650.5 0.2aNMBA + 5% BRB
-1.4 0.3NMBA (0.25 mg/kg)
% InhibitionTumors perEsophagusGroup
aSignificantly different from NMBA control; p<0.05;L.A. Kresty, et al., Cancer Res. 61:6112, 2001.
Suppressing Effects of Freeze-Dried BlackRaspberries on NMBA Tumorigenesis
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Photomicrographs of Proliferating Cell NuclearAntigen Staining
Normal High-Grade Dysplasia
Papilloma Positive Colon
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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
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COOH
Arachidonic Acid
PGG2
PGH2
ProstaglandinsProstacyclin Thromboxane
Cyclooxygenase Activity
Peroxidase Activity
COX-1COX-2
PGE2, PGF2, PGD2 TxA2PCI2
Arachidonic Acid Metabolism
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Effect of Black Raspberries on COX-2 Expression andActivity in NMBA-Treated Rat Esophagus
Group
*
*
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Nitric Oxide Synthesis
L-Arginine L-Citrulline + NO
Peroxynitrite (ONOO-)
DNA damage
(O2-)
COX-2 Activity
iNOS
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iNOS Staining
Normal Hyperplasia
Dysplasia Papilloma
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Effect of Black Raspberries on iNOS Expressionand Activity in NMBA-Treated Rat Esophagus
Group
*
*
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Effect of Black Raspberries on c-Jun Expression inNMBA-Treated Rat Esophagus
* P < 0.05
*
*
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Western Blot Analysis of iNOS, COX-2 and c-Jun
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Effect of Black Raspberries on VEGF Expression inNMBA-Treated Rat Esophagus
* * P < 0.005
**
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Effect of Black Raspberries on Microvessel Density(MVD) in NMBA-Treated Rat Esophagus
* * * P < 0.0005
***
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Y-axis: Stoner Rat esophogus NMBA, BRB, combined GEN...Colored by: CONTROL
Error Bars: between-sample std. errorGene 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
0.70.8
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
0.70.8
2
3
4
5 Normalized Intensity(log scale)
Two distinct clusters in the 578 genes. The overall trend is that 5% BRB treatment modulatesthe 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.
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0 2 4 36
(AOM 15 mg/kg x 2)
2.5, 5, 10% black raspberries
Protocol for AOM-Induced Tumors in RatColon
3
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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
Tum
ors
/col
onSuppressing Activity of Black Raspberries on Rat
Colon Adenocarcinomas
****
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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.
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Scheme for Extraction of Freeze-Dried Berries
ETEtOH eluate
Chromatographed on silica gelEluated by CH2Cl2: EtOH (1:1) andEtOH, respectively
Chromatography:
DMCH2Cl2: EtOH eluate
F001
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Summary of Studies Conducted Using AlcoholExtracts 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 cavityand colon cancer cells
• Exhibits potent radical scavenging activity• Downregulates expression of AP-1, NFB, VEGF and
their associated kinases in mouse epidermal JB-6 clone41 cells
• Inhibits UVB-induced skin carcinogenesis in mice
Ethanol Extract of Black Raspberries
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Rel
ativ
eV
EG
FIn
duct
ion
B[a]PDE (2 M) Extract RO- (25 g/ml) F003 F004 DM ET
* P < 0.05
Inhibition of VEGF by BRB Extracts
*
*
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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
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Inhibition of Signaling Pathways by Alcohol Extract
•Akt
AP-1
PI(3,4,5)P3
PI-3K
ERKs/p38K
RO ET
IkBa Degradation
NFkBNFAT
COX-2 and VEGF
JNks
B[a]PDE
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Anthocyanins in Black Raspberry EtOH Fraction
Cyanidin 3-O-glucoside
Cyanidin 3-O-rutinoside Cyanidin 3-O-(2G-xylosylrutinoside)
Cyanidin 3-O-sambubioside
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Phase I Clinical Trial of Freeze-DriedBlack Raspberries
• 10 normal volunteers, > 18 years ofage
• “Phenol-free” diet• 45 g BRB / day, 14 days• Blood and urine• Clinical signs of toxicity
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Results of Phase I Clinical Trial ofFreeze-Dried Black Raspberries
• Well tolerated
• blood levels of ellagic acid andseveral anthocyanins
• Reduced levels of 8-OH-dG in urine
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Ongoing Human Clinical Trials With BerryPreparations
• 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.
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Eat 1 hour before or 1 hour after regularmeals
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Collaborators
OSU Current Collaborators
Tong Chen
Alan Dahl
Laura Kresty
Susan Mallery
Jennifer Mele
Raymond Miller
Ron Nines
Rashmeet Reen
Christine Sardo
Joseph Scheerens
Steven Schwartz
Qingguo Tian
Anne Vanbuskirk
Christopher Weghorst
Nancy Zikri
University of Minnesota
Stephen Hecht
New York University
Chuanshu Huang
Wayne State University
Alan Dombkowski
Columbus Children’s Research Institute
Herbert Auer
OSU Former Collaborators
Robeena Aziz
Peter Carlton
Beth Liston
Keith Harris
Supported byODAUSDANCICSGA