Berry Polyphenols in the Prevention of Primary and Recurrent Breast Cancer

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1 Berry Polyphenols in the Prevention of Primary and Recurrent Breast Cancer Harini S Aiyer, Ph.D. Lombardi Comprehensive Cancer Center Georgetown University Berry Health Symposium June 27, 211

2 Overview Breast cancer incidence and risk factors Selection of berries for breast cancer prevention Berries in mammary tumor prevention Mechanisms by which berries prevent breast cancer Future directions

3 Breast Cancer- Causes Causes Sporadic breast cancer 9% Familial breast cancer 1%

4 Breast cancer- risk factors Non-Modifiable Risk Factors Modifiable Risk Factors Hormonal Risk Factors Age Diet Cumulative exposure to estrogen Gender Alcohol Age at menarche and menopause Genetics Smoking Parity Family history Body weight Lactation Previous breast disease Exercise Radiation Hormone replacement therapy

5 Berry polyphenols Excellent antioxidant properties Interaction with Estrogen receptor Cause cell-cycle arrest Interfere with cell-signaling pathways Interact with multiple pathways involved in carcinogenesis Cyanidin Pterostilbene Stibenoids Quercetin Delphinidinn Anthocyanidins Berry Phenolics Flavonols Kaempferol Pelargonidinn Tannins Proanthocyanidins Condensed Tannins Hydrolysable Tannins Ellagitannins Metabolism Metabolism

6 Adducts/1 6 N Effect of ellagic acid on 4-hydroxy etsradiolinduced oxidative DNA damage P-1 8-oxodG EA dose in µm 4 Aiyer et al., Int J Mol Sci. 28 Mar;9(3):327-41

7 Next step-in vivo study ( Short-Term) Received Mice Day 5 Day 25 Day 1 Euthanized Control Diet Supplemented Diet 5% (w/w) berries based on EA content 1 Blueberry (low <1 ppm) Strawberry (moderate ppm) Red raspberry (high ppm) DNA damage ( 32 P-postlabeling) Liver Genomics (microarray) Ellagic acid (4 ppm) 1- Daniel et al., 1993

8 Short-term in vivo study 1 Raspberry supplemented diet 1 Ellagic acid supplemented diet 5 5 Fold Change Fold Change These diets also significantly reduced baseline hepatic-dna damage in a similar fashion -1 Aiyer et al., Int J Mol Sci. 28 Mar;9(3):327-41

9 Next step- tumorigenesis study -3-2 Weeks 24 Control diet Receive Animals Sham or 17ß-Estradiol Implants Experimental diet DIET DOSE Termination Control (AIN 93M) NA Blueberry 2.5% (w/w) Black raspberry 2.5% (w/w) Ellagic acid 4 ppm

10 Rationale for berry types used DIET DOSE Ellagic acid content Anthocyanin content Blueberry 2.5% (w/w) Low (<1 ppm) Moderate ( 4 ppm) Black raspberry 2.5% (w/w) High ( ppm) High ( 7 ppm) Ellagic acid 4 ppm Pure compound Blueberry Black Raspberry mg/ g dry wt Anthocyanins Total Phenolics % 2% 4% 6% 8% 1% Cyanidin Delphinidin Malvidin Petunidin Pelargonidin Peonidin Aiyer et al., Berries and Cancer Prevention, Eds Stoner GD and Seeram NP, Springer, 211

11 Berry diets increase mammary tumor latency 1 % Tumor Incidence Days after E 2 implant 18 Sham treatment E2+ Control diet E2+ Blueberry diet E2+ Black raspberry diet E2+ Ellagic acid diet

12 Estrogen treatment associated morbidity in ACI rats

13 Berry diets reduce treatment-associated morbidity in ACI rats

14 Berry diets diminish treatment-associated mortality Morbidity score 1 to 5 (1- best; 5-worst. ) Weight loss > 1 g per day Survival % Control diet Blueberry diet Hair loss Crouch Eye deposits Black raspberry diet Ellagic acid diet Loss of balance Tumor size in mortality index) Weeks after estrogen implant

15 Berry diets decrease tumor volume and multiplicity Aiyer et al., Nutr Cancer. 28;6(2):

16 % of animals with tumors BRB diet BB diet EA diet A B C # # # # Weeks after E 2 implantation Aiyer et al., Cancer Prev Res (Phila). 21 Jun;3(6):727-37

17 BERry diets modulate phase I enzymes Relative fold change HSD * CYP1A CYP1B1 Weeks after estrogen treatment Aiyer et al., Cancer Prev Res (Phila). 21 Jun;3(6): Sham-Control diet E2-Control diet E2-Blueberry diet E2-Black raspberry diet E2-Ellagic acid diet

18 BERRY diets also modulate phase II enzyme Relative fold change COMT * CYP1A1 Weeks after estrogen treatment Sham-Control diet E2-Control diet Aiyer et al., Cancer Prev Res (Phila). 21 Jun;3(6): E2-Blueberry diet E2-Ellagic acid diet E2-Black raspberry diet

19 Berry diets inhibit E2-induced cell proliferation 35 PCNA positive cells (%) * Estrogen Diet (2.5% w/w)

20 Pituitary wet weight (mg) Serum Prolactin levels (mg/ml) , Berry phytochemicals act as Sham + Control diet Sham + Control diet antiestrogens E2 + Control diet E % Blueberry diet ** E % Black raspberry diet E2 + Control diet E2 + 5% Blueberry diet * E2 + 4 ppm Ellagic acid diet E2 + 5% Black raspberry diet Adapted from Aiyer et al., Berries and Cancer Prevention, Eds Stoner GD and Seeram NP, Springer, 211

21 Berry phytochemicals act as antiestrogens 1, 9 # P <.5 Ovary and Uterus combined wet weight (mg) * * * Sham + Control diet E2 + Control diet E % Blueberry diet E % Black raspberry diet E2 + 4 ppm Ellagic acid diet Adapted from Aiyer et al., Berries and Cancer Prevention, Eds Stoner GD and Seeram NP, Springer, 211

22 Overall mechanism of primary cancer prevention by berries Effectors Mediators Effects Cancer Stage Tumor Promotion? Tumor Initiation Aiyer et al., Berries and Cancer Prevention, Eds Stoner GD and Seeram NP, Springer, 211

23 So how much berries do you need to eat? 1 cup a day Control Diet Blueberry diet Black raspberry diet Units 1% 2.5% 5% 1% 2.5% 5% Berries consumed by rats based on caloric intake (1g feed = 4 kcal) Allometric scaling to human consumption (2, kcal/day). mg/kcal /day g dried berry powder Common conversion for dried berries Tbsp/d Common conversion for fresh berries Cups/d Source: Aiyer et al., Berries and Cancer Prevention, Eds Stoner GD and Seeram NP, Springer, 211

24 Future- questions & directions Mechanistic What is the effect of berry diets on E2 metabolism and clearance in the liver? What is the effect of berry diets on pituitary-ovarian axis? Do berry diets affect other cell-signaling pathways (Erbb2, MAPK etc) involved in E2-induced tumorigenesis? Translational What is the effect of berry-intervention on women who have been diagnosed with breast cancer? What are the possible interactions between berry constituents and current chemotherapy regimens in breast cancer?

25 Proposed mechanisms of AntiEstrogen action Effect on Bone/Ovary Pituitary- Ovarian axis Liver Metabolism of and to estrogens StromalCells Paracrine effects AE Immune function Cell membrane Autocrine effects Membrane Receptors Cytoplasm E2 CoA ER CoR Free radicals NFkB mediated signaling ER-independent signaling Kinasesignaling Nucleus Transcriptional effects Adapted from Clarke et al., 21

26 Acknowledgements Mentors Ramesh Gupta, PhD Robert Clarke, PhD, DSc Leena Hilakivi-Clarke, PhD Labs Clarke Labs Gupta Lab Special Gary Stoner, PhD Jim Shull, PhD Funding AICR postdoctoral Fellowship to HSA NCI (CA-9892 and ) to RCG

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