Progress 08/15/08 to 08/14/10
Outputs
OUTPUTS: The goals of this two year study were three-fold: 1) To further investigate the chemopreventive effects of freeze-dried black raspberries (BRBs) on the development of colon cancer in humans; 2) To identify the bioactive components in BRBs; and 3) To determine if other berry types might have chemopreventive potential. The potential inhibitory effects of BRBs on colon tumors/polyps were determined in 36 patients diagnosed with colon cancer at the Ohio State University Hospitals, and the University of Texas, San Antonio. BRBs were evaluated for their ability to regress rectal polyps in patients with familial adenomatous polyposis (FAP) at the Cleveland Clinic. At Ohio State University and the University of Texas, a total of 36 colon cancer patients were administered berry powder (20g/3x/day) in a slurry of water for an average of 3 weeks. Biopsies of normal colon and colon tumor specimens, serum and urine, were obtained before and after berry treatment. The berries were found to be well tolerated with an occasional patient developing either constipation or diarrhea for no more than 1-2 days. The effects of the berries on cell growth, apoptosis, angiogenesis, expression levels of genes in the Wnt signaling pathway, and on demethylation of selected tumor suppressor genes were measured in both normal colon and colon tumors. In the polyp regression study, a total of 18 FAP patients were administered either an oral placebo (60g/day) and two rectal suppositories (each containing 700mg BRB powder) daily or oral BRB powder (60g/day) and two rectal berry suppositories daily for nine months. The effects of these treatments on rectal polyp regression as well as on multiple biomarkers were determined. In general, the berries were found to be well tolerated by FAP patients when administered daily for nine months, with the exception of 4 patients who developed rectal fissure from the suppositories. These patients were removed from the trial. Towards the second goal, BRB powder was fractionated into different fractions containing predominately either berry anthocyanins or fiber plus ellagitannins. These fractions were evaluated for their relative ability to inhibit carcinogen-induced cancer in the rat esophagus when compared to whole berry powder. Towards the third goal, multiple berry types (black raspberries, strawberries, red raspberries, blueberries, acai, noni and goji) were compared for their ability to inhibit carcinogen-induced cancer in the rat esophagus when fed at 5% of the diet. Data from these studies has been presented in abstract form at multiple national and international meetings and at several academic and private institutions. In addition, the results have appeared in several publications from The Ohio State University, in numerous magazines and newspaper articles, and have received extensive television coverage. PARTICIPANTS: The Principal Investigator of this project was Dr. William Ravlin of the Ohio Agricultural Research and Development Center (OARDC). Dr. Ravlin was assisted by William Koshar in grant and report preparation. Dr. Gary Stoner oversaw all laboratory and clinical components of the colon cancer and FAP trials. He was assisted by Elizabeth Schockley of the OSU Comprehensive Cancer Center in budget preparation for the clinical trials and for monitoring expenses. The surgeons who worked on the colon cancer project were Drs. Mark Arnold and Edward Martin of the Department of Surgery, Ohio State University and Dr. John Winston of the University of Texas, San Antonio. The gastroenterologist at Texas Tech University in El Paso who worked on the colon cancer trial was Dr. Ann Morales. Dr. Carol Burke of the Department of Gastroenterology at the Cleveland Clinic conducted the FAP trial. The clinical managers who accrued patients to the projects and monitored compliance and toxicity were Christine Sardo at Ohio State, Hennie Hasson at the Cleveland Clinic and Sarah Sanchez of the University of Texas, San Antonio. Laboratory investigations for the clinical trials were conducted by Drs. Li-Shu Wang and John Lechner of Ohio State with technical assistance from Jennifer Melee, Claudio Rocha, Colleen McIntyre, Bethany Larue and Cassandra Henry. Pharmacokinetic studies of anthocyanins and ellagitannins in patient specimens were conducted by Drs. Steven Schwarts, Qingguo Tian and Kenneth Riedl. Anthocyanin studies were conducted by Drs. Joseph Scheerens and Raymond Miller of the OARDC with assistance from Drs. Artemio Tulio and Faith Wyzgoski. Statistical analysis of all trial data was performed by Dr. Dennis Pearl of Ohio State, and histopahological analysis of all tissue specimens was done by Dr. Wendy Frenkel of the Department of Pathology, Ohio State University Medical Center. Collaborations on this project include Mr. Dale Stokes, whose farm suppliled all of the BRB for the clinical trials; Dr. Neil Reese of South Dakota State University, and Dr. Peter Rinaldi and Ms. Ruiling Fu of the University of Akron who assisted with anthocyanin measurements using NMR. Additional collaborations on the analysis of colon tissue specimens have been initiated with Drs. Daniel Rosenberg and Charles Guardina of the University of Connecticut and Drs. Michael Goodman and Robert Bostick of Emory University. Postdoctoral training on these projects was provided to Drs. Li-Shu Wang, Kenneth Riedl and Artemio Tulio, Jr., and Roycelynn Mentor-Marcel. Lab oratory training in molecular biology was provided to Cassandra Henry who matriculated in graduate school at the University of Pittsburg and Colleen McIntyre who entered medical school at Emory University in the Autumn of 2008. TARGET AUDIENCES: The target audience for these studies include individuals at high risk for the development of colon cancer and possibly, other cancer types. The general population is also a target audience since it is known that fruit and vegetable consumption provides protection against the development of cancer in many organ sites, and berries would appear to be one of the more active foodstuffs in cancer prevention. PROJECT MODIFICATIONS: For the most part, both clinical trials were conducted according to protocol. Deviations from protocol were explained in an earlier progress report. Since the colon tumor specimens were heterogeneous with respect to the relative amounts of tumor, non-tumor and inflammatory tissues, our laboratory evaluations of the measured biomarkers were performed using immunohistochemistry only. We did not have access to laser capture microdissection techniques to select out tumor cells only for molecular analysis. The FAP trial was slowed by the fact that we were requested to obtain an IND from the FDA to conduct this trial even though the test substance is a natural product.
Impacts
The results from the study in colon cancer patients at The Ohio State University and the University of Texas in San Antonio have been described, in part, in the final reports for Project Number OHO00979-SS, CRIS Number 0204452 and Project Number OHO00004-S, CRIS Number 0207157. Treatment of colon cancer patients with BRBs led to a significant reduction in the growth rate of malignant cells in colon tumors with a lesser effect on the growth of normal colonic epithelium. The treatment also led to an increased rate of apoptosis (programmed cell death) and a non-significant reduction in angiogenesis in colon tumor tissues. The berries also down-regulated the mRNA expression levels of c-Myc and cyclin-D1 genes which are downstream of the Wnt signaling pathway, a pathway known to be altered in about 70% of colon cancers. Finally, berry treatment led to demethylation of the promoter sequences of suppressor genes associated with Wnt signaling as well as the p16 gene. This observation was published recently in Clinical Cancer Research and is the first report of the ability of berries to cause gene demethylation and subsequent re-expression. Results from the trial in FAP patients showed that BRB treatment for nine months led to a 20% rectal polyp regression rate in patients treated with the berries both orally and rectally (by suppository) and a 36% regression rate in patients treated with the suppository alone. This result suggests that treatment with the rectal suppository is superior to the dual treatment however, there were only 7 patients in each arm and three of the patients in the dual treatment arm were non-responsive to berry treatment. The bio-fractionation studies provided evidence that the anthocyanins in BRBs have chemopreventive potential and appear to be responsible for a significant portion of their chemopreventive effects. The ellagitannins appear to be of lesser importance however, the fiber fraction is active and studies are underway to identify the active components in the fiber. Interestingly, when fed in the diet at a concentration of 5%, all seven berry types (black raspberries, red raspberries, strawberries, blueberries, acai, noni, and goji) caused a significant reduction in carcinogen-induced cancer in the rat esophagus when compared to rats treated with carcinogen only. These data suggest that, irrespective of their content of anthocyanins and ellagitannins, all berry types have chemopreventive potential in the rodent esophagus. Further studies are underway to determine if they also exhibit similar preventative effects in other organ systems.
Publications
- Stoner, G.D., Wang, L-S, Sardo, C., Zikri, N., Hecht, S.S., and Mallery, S.R. (2010) Cancer prevention with berries: Role of anthocyanins. In: Bioactive Compounds and Cancer, J.A. Milner and D.F. Romagnolo (Eds). Humana Press/Springer, Towata, New Jersey, Chapter 29, pp 703-724.
- Tokusoglu, O. and Stoner, G.D. (2010) Phytochemical bioactives in berries. In: Fruit and Cereal Bioactives: Sources, Chemistry and Applications, O. Tokusoglu and C. Hall III (Eds), CRC Press, Boca Raton, Florida, Chapter 7, part II.
- Wang, L-S., Dombkowski, A.A., Rocha, C.., Seguin, C., Cukovic, D., Mukundan, A., Henry, C., and Stoner, G.D. (2010) Effects of black raspberries on late events in N-nitrosomethylbenzylamine-induced rat esophageal carcinogenesis as determined by DNA microarray (in press, Molecular Carcinogenesis).
- Wang, L.S., Hecht, S.S., Carmella, S.G., Seguin, C., Rocha, C., Yu, N., Stoner, K. and Chiiu, S., and Stoner, G.D. (2010) Berry ellagitannins may not be sufficient for prevention of tumors in the rodent esophagus. J. Agric. Food Chem. 58:3992-3995.
- Desai, K.G.H., Olsen, K.F., Mallery, S.R., Stoner. G.D., and Schwendeman, S.P. (2010) Formulation and In Vitro-In Vivo evaluation of black raspberry extract-loaded PLGA/PLA injectable millicylindrical implants for sustained delivery of chemopreventive anthocyanins. Pharm Res. 27:628-643.
- Stoner, G.D., Wang, L-S., Seguin, C., Rocha, C., Stoner, K., Chiu, S., and Kinghorn, A.D. (2010) Multiple berry types prevent N-nitrosomethylbenzylamine-induced esophageal cancer in rats. Pharm Res. 27:1138-45.
- Ozgen, M., Wyzgoski, F.J., Tulio Jr., A.Z., Gazula, A., Miller, A.R., Scheerens, J.C., Reese, R.N., and Wright, S.R. (2008) Antioxidant capacity and phenolic antioxidants of Midwestern black raspberries grown for direct markets are influenced by production site. Hort. Science 43:2039-2047.
- Tulio Jr., A.Z., Reese, R.N., Wyzgoski, F.J., Rinaldi, P.L., Fu, R., Scheerens, J.C., Miller, A.R. (2008) Cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside as primary phenolic antioxidants in black raspberry. J. Agric. Food Chem. 56:1880-1888.
- Wang, L-S, Hecht, S., Carmella, S., Yu, N., Larue, B., Henry C., McIntyre C., Rocha, C., Lechner, J.F., and Stoner, G.D. (2009) Anthocyanins in black raspberries prevent esophageal tumors in rats. Cancer Prev. Res. 2:84-93.
- Stoner, G.D. (2009) Foodstuffs for cancer prevention: The preclinical and clinical development of berries. Cancer Prev. Res. 2:187-92.
- Ugalde, C.M., Liu, Z., Ren, C., Chan, K., Rodrigo, K.A., Ling, Y., Larsen, P.E., Chacon, Stoner, G.D., Mumper, R.J., Fields, H.W., and Mallery, S.R. (2009) Distribution of anthocyanins delivered from a bioadhesive black raspberry gel following typical intraoral application in normal healthy volunteers. Pharm. Res. 26:977-86.
- Zikri, N.N., Riedl, K.M., Wang, L-S., Lechner, J.F., Schwartz, S.J., and Stoner, G.D. (2009) Black raspberry components inhibit proliferation, induce apoptosis and modulate gene expression in rat esophageal epithelial cells. Nutrition and Cancer 61:816-826.
- Ling, Y., Ren, C., Mallery, S.R., Ugalde, C.M., Pei, P., Saradhi, V., Stoner, G.D., Chan, K.K., and Liu, Z. (2009) A rapid and sensitive LC-MS/MS method for quantification of four anthocyanins and its application in a clinical pharmacological study of a bioadhesive black raspberry gel. J. of Chromatography B 877:4027-34.
- Wang, L-S., and Stoner, G.D. (2009) Anthocyanidins. In: Chemoprevention of Cancer and DNA Damage by Dietary Factors, S. Knasmuller, D.M. DeMarini, I.T. Johnson, C. Gerhauser (Eds),Wiley-VCH, Weinheim, Germany, Chapter 30, pp. 516-524.
- Duncan, F.J., Martin, J.R., Wulff, B.C., Stoner, G.D., Tober, K.L., Oberyszyn, T.M., Kusewitt, D.F., and VanBuskirk, A.M. (2009) Topical treatment with black raspberry extract reduces cutaneous UVB-induced carcinogenesis and inflammation. Cancer Prevention Res. 2:665-672.
- Garzon G.A., Narvaez C.A., Riedl K., and Schwartz S.J. (2010) Chemical composition, anthocyanins, nonanthocanin phenolics and antioxidant activity of wild bilberry (Vaccinium merdionale Swartz) from Colombia. Food Chem. 122:980-986.
- Wyzgoski, F.J., Paudel, L., Rinaldi, P.L., Reese, R.N., Ozgen, M., Tulio, Jr., A.Z., Miller, A.R., Scheerens J.C., and Hardy, J.K. (2010). Modeling relationships among active components in black raspberry (Rubus occidentalis L.) fruit extracts using high-resolution 1H NMR spectroscopy and multivariate statistical analysis. J. Agric. Food Chem. 58:3407-3414.
- Estupinan D.C., Schwartz S.J., and Garzon, G.A. (2011) Antioxidant Activity, Total Phenolics Content, Anthocyanin, and Color Stability of Isotonic Model Beverages Colored with Andes Berry (Rubus glaucus Benth) Anthocyanin Powder. J. Food Sci.
- Johnson, J.L., Bomser, J.A., Scheerens, J.C., and Giusti, M.M. (2011). Effect of black raspberry (Rubus occidentalis L.) extract variation conditioned by cultivar, production site, and fruit maturity stage on colon cancer cell proliferation. J. Agric. Food Chem.
|
Progress 08/15/08 to 08/14/09
Outputs
OUTPUTS: While quantifying anthocyanins in some anthocyanin-rich berry extracts we found significant non-anthocyanin peaks including several ellagitannins, ellagic acid, quercetin glycosides and a series of unknown peaks with absorbance at 360nm. Using the accurate mass capabilities of our quadrupole time-of-flight mass spectrometer we were able to identify the latter peaks as methylated ellagic acid derivatives. Urolithins, metabolites of ellagic acid formed in the human colon from the action of resident bacteria, have been detected in various biological specimens including urine, although no comprehensive method exists to quantify the entire suite of metabolites. Determining urolithin levels in urine involves digesting urolithin conjugates (glucuronides and sulfates) with enzyme preparations (glucuronidase/aryl sulfatase) followed by extraction into ether. Spike recovery experiments were conducted to test whether the liberated aglycones could be fully extracted from the enzymatic digestions. We found using our current approach that 70-90% of spiked urolithins were recovered from blank urines but that when enzyme was added the recovery was more typically only 50%. A UPLC-MS/MS method has been developed to determine urolithins A, B, C, D, methyl A, dimethyl A and ellagic acid in digested urine samples. Within eight minutes we are able to resolve and detect these species by tandem MS. Other metabolites may including methyl ellagic acid and dimethyl ellagic acid in subjects consuming ellagic acid and ellagitannins. We noticed peaks in our full MS scan chromatograms that correspond to m/z for these species and are synthesizing standards quantify their levels in biological samples. Our plant-based studies continue to develop statistical models identifying the chemoprotective components of black raspberries using high-field NMR spectroscopy and bioassay. In 2009, we completed NMR analysis of 75 berry extracts and used Pirouette and Intellibucket software to align all spectra and determined the antiproliferation effects of the extracts using HT-29 colon cancer cells. Statistical models were developed to indicate NMR spectral components that actively inhibit cell growth. The chemoprotective superiority of under-ripe fruit extracts is an important finding of this phase, as it suggests phenolic compounds other than anthocyanin pigments are important to the antiproliferation effects of berries. We recently completed HPLC analyses of berry anthocyanins and non-anthocyanin phenolics as influenced by maturity. Anthocyanins increased during the ripening process, decreased during the first 12 hrs after harvest, then increased significantly throughout berry senescence. This decrease followed by apparent de-novo synthesis after harvest may be related to levels of oxidative stress. Ripening berry fruit also accumulates vitamin C, hydroxybenzoic acids and flavonol glycosides (e.g., quercetin 3-rutinoside). However, soluble ellagic acid derivatives decrease during the ripening and senescence processes. Hydroxybenzoic acids increased as berries ripened and then decreased as berries senesced. PARTICIPANTS: PROJECT STAFF: Dr. Ann M. Chanon, Project Coordinator, Ms. Deborah A. Miller, Research Associate. OSU COOPERATORS: Dr. Faith J. Wyzgoski (Chemistry), Dr. M. Monica Giusti (Food Science and Technology), Dr. Joshua A. Bomser (Human Nutrition), Dr. Jim Fuchs (Pharmacy) INTER-INSTITUTIONAL COLLABORATORS: Dr. R. Neil Reese (Dept. Biology/Micro-biology, South Dakota State Univ., Brookings, SD), Dr. Peter L. Rinaldi (Dept. Chemistry, Univ. Akron, Akron, OH) TARGET AUDIENCES: Medical practitioners, health researchers, food scientists, plant scientists, graduate and undergraduate students and fruit producers are target audiences for information generated by this research program. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Aero-digestive cancers account for a significant percentage of all diagnosed cancers; colon cancer alone is the second leading cause of death due to cancer in the US. The development of food-based approaches to reduce the incidence, extent and severity of cancer and other degenerative diseases of aging will have positive effects for US citizens. Berry-based clinical products are essential components of dietary intervention strategies. However, a relatively thorough understanding of the bioavailability, metabolism and mode of action of key bioactive berry compounds in humans will be required before these strategies can be included in common medical practice. The systematic examination of berry constituent metabolism in the blood and urine of clinical trial patients will provide insight for the design, application and efficacy of berry-based clinical products. The successful demonstration of a metabolomics-based approach to modeling the interactivity of BR bioactive compounds may aid oncology scientists as they delineate the human physiological mechanisms by which BR is chemoprotective and may offer interdisciplinary teams a mechanism to study other phytonutrient-rich fruits or vegetables. Genetic, cultural and environmental factors are known to substantially influence levels of pigments and other antioxidant constituents in ripening fruits. A thorough delineation of these factors and their effects on developing fruit at the physiological level coupled with extensive applied research examining differences in cultural strategies will lead to effective recommendations for producers enabling them to consistently deliver fruit with optimum phytonutrient levels for use in the manufacture of clinical products or for the fresh and processed fruit marketplace.
Publications
- Zikri, N.N., Riedl, K.M., Wang, L-S., Lechner, J.F., Schwartz, S.J., and Stoner, G.D. (2009) Black raspberry components inhibit proliferation, induce apoptosis and modulate gene expression in rat esophageal epithelial cells. Nutrition and Cancer 61:816-826.
- Johnson, J.L., J.A. Bomser, J.C. Scheerens and M.M. Giusti. 2009. Effects of different black raspberry extracts on colon cancer cell proliferation in an in vitro cell system. IFT Annual Meeting and Food Expo. 6-9 June 2009, Anaheim California Abstract 126-18.
|