PREVENTION OF GASTROINTESTINAL TRACTCANCERS WITH LYOPHILIZED BERRIES

Gary D. Stoner, Ph.D.Department of Internal MedicineCollege of Medicine & Public HealthThe 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 causeof cancer mortality in U.S.

• 5-year survival = 55%

Gastrointestinal Tract Cancers

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

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

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

Freeze-dried blackraspberries

Freeze-driedstrawberries

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

Animal Model Tumor Systems

50%

50 – 80%

30 – 60%

Percent TumorInhibition

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 Activityof Berries in the Esophagus

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

Photomicrographs of Proliferating Cell NuclearAntigen 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-1COX-2

PGE2, PGF2, PGD2 TxA2PCI2

Arachidonic Acid Metabolism

Effect of Black Raspberries on COX-2 Expression andActivity 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 Expressionand Activity in NMBA-Treated Rat Esophagus

Group

*

*

Effect of Black Raspberries on c-Jun Expression inNMBA-Treated Rat Esophagus

* P < 0.05

*

*

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

Effect of Black Raspberries on VEGF Expression inNMBA-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. 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.

0 2 4 36

(AOM 15 mg/kg x 2)

2.5, 5, 10% black raspberries

Protocol for AOM-Induced Tumors in RatColon

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

Tum

ors

/col

onSuppressing 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

ETEtOH eluate

Chromatographed on silica gelEluated by CH2Cl2: EtOH (1:1) andEtOH, respectively

Chromatography:

DMCH2Cl2: EtOH eluate

F001

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

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

*

*

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

NFkBNFAT

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-DriedBlack Raspberries

• 10 normal volunteers, > 18 years ofage

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

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

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.

Eat 1 hour before or 1 hour after regularmeals

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