Cancer Prevention Research Pilot Study of Oral Anthocyanins for Colorectal Cancer Chemoprevention

Naturally occurring anthocyanins possess colorectal cancer chemopreventive properties in rodent models. We investigated whether mirtocyan, an anthocyanin-rich standardized bilberry extract, causes pharmacodynamic changes consistent with chemopreventive efficacy and generates measurable levels of anthocyanins in blood, urine, and target tissue. Twenty-five colorectal cancer patients scheduled to undergo resection of primary tumor or liver metastases received mirtocyan 1.4, 2.8, or 5.6 grams (containing 0.5-2.0 grams anthocyanins) daily for 7 days before surgery. Bilberry anthocyanins were analyzed by high performance liquid chromatography (HPLC) with visible or mass spectrometric detection. Proliferation was determined by immunohistochemistry of Ki-67 in colorectal tumor. Concentrations of insulin-like growth factor (IGF)-I were measured in plasma. Mirtocyan anthocyanins and methyl and glucuronide metabolites were identified in plasma, colorectal tissue, and urine, but not in liver. Anthocyanin concentrations in plasma and urine were roughly dose-dependent, reaching ∼179 ng/gram in tumor tissue at the highest dose. In tumor tissue from all patients on mirtocyan, proliferation was decreased by 7% compared with preintervention values. The low dose caused a small but nonsignificant reduction in circulating IGF-I concentrations. In conclusion, repeated administration of bilberry anthocyanins exerts pharmacodynamic effects and generates concentrations of anthocyanins in humans resembling those seen in Apcmice, a model of FAP adenomas sensitive to the chemopreventive properties of anthocyanins. Studies of doses containing <0.5 gram bilberry anthocyanins are necessary to adjudge whether they may be appropriate for development as colorectal cancer chemopreventive agents. Lack of toxicity is a pivotal requirement for agents to be potentially useful in cancer chemoprevention particularly considering the necessity for prolonged durations of administration. Intervention trials using drugs such as aspirin or celecoxib have been complicated by unwanted side effects (1, 2). There is an absolute requirement to discover and develop novel efficacious and safe alternatives to such drugs. Dietary phytochemicals exemplified by anthocyanins, bright blueor red-colored polyphenols, which occur ubiquitously in vegetables and fruits, are attractive candidates for clinical evaluation, as they possess a good safety record. Studies in preclinical carcinogenesis models suggest that anthocyanins may prevent malignancies, notably in the gastrointestinal tract (3–8). However, many aspects of their clinical pharmacology, such as concentrations achievable in organs targeted for prevention of malignancy, are only poorly understood. Mechanistic properties of anthocyanins potentially related to chemoprevention encompass antiproliferation, apoptogenicity, and antioxidation (9). The insulin-like growth factor (IGF) signaling system, especially IGF-I and IGF binding protein-3 (IGFBP-3), which possess putative procarcinogenic and anticarcinogenic properties, respectively, has been implicated as a potential target of the polyphenols silibinin (10) and curcumin (11), two cancer chemopreventive phytochemicals with structural similarities to anthocyanins. There is the growing realization in cancer chemoprevention agent development that mixtures—exemplified by dietary anthocyanins—may have distinct advantages over single agents, because of potential mechanistic synergy of mixture constituents and their decreased propensity to exert untoward effects when administered together at very low doses (12, 13). In the light of all these considerations, we conducted a clinical pilot study with mirtocyan, a standardized anthocyanin mixture extracted from bilberries, currently marketed as a nutraceutical with potential benefit in the treatment of vascular permeability, capillary vessel fragility, and ophthalmologic Authors' Affiliations: Cancer Biomarkers and Prevention Group, Department of Cancer Studies and Molecular Medicine, University of Leicester; Department of Hepatobiliary and Pancreatic Surgery, Leicester General Hospital, University Hospitals Leicester; Departments of Pathology and Colorectal Surgery, Leicester Royal Infirmary, Leicester, United Kingdom Received 10/28/08; revised 12/2/08; accepted 1/28/09. Grant support: C325/A6691 from Cancer Research UK, an Experimental Cancer Medicine Centre grant from Cancer Research UK and the UK Department of Health and an award from the Leicester Rutland Hope Foundation for Cancer Research. Requests for reprints: Andreas J. Gescher, Department of Cancer Studies and Molecular Medicine, RKCSB, University of Leicester, Leicester LE2 7LX, United Kingdom. Phone: 44-1162231856; Fax: 44-1162231855; E-mail: [email protected]. ©2009 American Association for Cancer Research. doi:10.1158/1940-6207.CAPR-08-0201 625 Cancer Prev Res 2009;2(7) July 2009 www.aacrjournals.org Cancer Research. on April 13, 2017. © 2009 American Association for cancerpreventionresearch.aacrjournals.org Downloaded from disorders. Mirtocyan contains glycosides of the anthocyanidins cyanidin, delphinidin, malvidin, peonidin, and petunidin bound to glucose, galactose, or arabinose. The aim of this study was to generate pharmacodynamic and pharmacokinetic information to help plan the future clinical evaluation of anthocyanins in colorectal cancer chemoprevention studies. Colorectal cancer patients due to undergo surgical resection of the primary tumor or liver metastases received mirtocyan before surgery and provided tissue for assessment. This pilot study design, in which experimental cancer chemopreventive agents are administered to patients with established cancer, has been used, for example, with perillyl alcohol (14), curcumin (15), silibinin (16), and lycopene (17). Although issues germane to long-term safety and efficacy of such agents need evaluation in healthy humans and/or patients with preneoplasia, pilot studies in patients with cancer can be very useful as they allow early valuable insights into pharmacodynamic and pharmacokinetic properties. In general, information on tissue levels and pharmacodynamics would be difficult to accrue in healthy individuals or patients with preneoplasia. Using this approach, we wished to establish whether oral administration of mirtocyan generates measurable levels of anthocyanins in patients' blood, urine, colorectal, and liver tissue, and whether it causes inhibition of proliferation in colorectal tissue and affects circulating levels of IGF-I and IGFBP-3. Materials and Methods Patients and procurement of blood and tissues The study was approved by the Leicestershire Northamptonshire & Rutland Research Ethics Committee. Fifteen patients with histologically confirmed colorectal adenocarcinomas and 10 patients with confirmed colorectal liver metastases, all amenable to surgical resection, were recruited into the trial between October 2006 and May 2008 and gave written informed consent. The study was pilot in nature, it was not blinded, and it was designed around the patients' routine care. Subjects were selected based on a consecutive colorectal cancer patient population and randomized with respect to dose level. Patient characteristics are shown in Table 1. Peripheral blood was collected in heparinized tubes before intervention and 1 h after the penultimate mirtocyan dose, portal blood at surgery. Blood was centrifuged to generate plasma, which was kept on ice until storage. Patients were recruited into the study after their diagnosis of cancer. This means that they had their endoscopy, in which cancer tissue but not normal tissue is taken routinely to enable diagnosis, before recruitment. Retrospective consent was obtained from them to use biopsies. Therefore presurgery biopsy samples of normal colorectal tissue could not be obtained. Colectomy and liver surgery occurred 6.0 ± 2.7 h and 4.7 ± 1.2 h, respectively, after the last mirtocyan dose. Samples (∼1 gram) of tissue were taken from resection specimens, in the case of normal colorectal tissue from sites 5 and 10 cm proximal and distal, respectively, to the tumor. Utmost care was taken to perform tissue processing as rapidly as possible, within minutes after tissues has been resected. Surgical tissue samples did not show histologic evidence of necrosis when reviewed by a pathologist. Tissue samples were snap frozen (liquid nitrogen). Biomatrices were stored at −80° C for up to 2 mo. Preliminary HPLC analysis established that bilberry anthocyanins are stable under these conditions in tissues and plasma. Intervention and dose Mirtocyan (formerly mirtoselect), a standardized extract of bilberries (supplied by Indena S.p.A.), was prepared by an industrial proprietary process ensuring constant and reproducible anthocyanin composition (36%, w/w). Predominant anthocyanin constituents are delphinidin-3-galactoside, delphinidin-3-glucoside, and delphinidin3-arabinoside, and cyanidin-3-galactoside and cyanidin-3-glucoside (Indena datasheet). Other anthocyanins in mirtocyan are cyanidin-3arabinoside, petunidin-3-galactoside, petunidin-3-glucoside, petunidin-3-arabinoside, peonidin-3-galactoside, peonidin-3-glucoside, peonidin-3-arabinoside, malvidin-3-galactoside, malvidin-3-glucoside, and malvidin-3-arabinoside. Mirtocyan also contains other polyphenols (phenolic acids, flavonols, proanthocyanidins; ∼18%), carbohydrates and aliphatic organic alcohols (∼29%), fats (∼0.04%), nitrogen compounds (∼1%), ash (∼0.7%), with the remaining 15% undefined. Mirtocyan was formulated in gelatin capsules (0.47 gram per capsule; Nova Pharmaceuticals) and administered thrice a day for 7 d at total daily doses of 1.4, 2.8, or 5.6 grams. These doses span the dietary dose of mirtocyan in Apc mice (∼450 mg/kg mouse = ∼2.6 g per 80 kg human extrapolated by dose-surface area comparison; see ref. 18), which reduced adenoma number by 30% (8). The last third of the daily dose was administered in the early morning before surgery, when the patients were fasting. Analysis of anthocyanins by HPLC-VIS and -MS/MS Anthocyanins exist in a pH-dependent dynamic equilibrium of at least four tautomers (19). Only one, the flavylium cation (Fig. 1) predominating at pH of <2, is colored. Acidification of biomatrix extracts transformed colorless tautomers into flavylium ions detectable by HPLC with visible spectroscopy (VIS). Biomatrices were thawed, and plasma was centrifuged. Colorectal tumor and normal tissue samples (150 mg) were homogenized (PBS, 1:10 w/v) and centrifuged (5, 000 × g, 20 min); the residue was rehomogenized and recentrifuged. Aliquots of plasma supernatant (2 mL), urine (1 mL), or colorectal tissue supernatant were subjected to solid phase extraction as described before (7). In the case of liver, tissue was homogenized with PBS (1:2.5 w/v) containing formic acid (2%). The homogenate was mixed with acetone/formic acid (9:1), and the mixture was centrifuged. The supernatant was dried (nitrogen), and the residue was reconstituted in water/formic acid. Solid phase eluate (for plasma, urine, colorectal tissue) and liver extract were analyzed by HPLC with detection by VIS (510 nm) or tandem mass spectrometry (MS/MS), the latter with selected reaction monitoring (SRM), operated in the positive ion mode, as described and validated previously (20). Anthocyanin concentrations were estimated semiquantitatively using a standard curve for authentic cyanidin-3-glucoside, based on the simplifying assumption that all detected anthocyanins possess similar molar absorption coefficients. The molecular weight of cyanidin-3-glucoside was applied to all anthocyanins measured; therefore, results are approximate values prefaced by the ∼ sign. Pharmacodynamic analyses Colorectal tumor sections, obtained before and after the consumption of mirtocyan, were stained for Ki-67 (mouse anti-human monoclonal antibody; Dako) or cleaved caspase-3 (cleaved caspase-3 Asp 175 polyclonal antibody; New England Biolabs). Tissue-antibody reaction was visualized by a commercial kit (Dako). Representative fields were selected in the biopsies and from superficial regions of the resected tumor specimens. The total number of epithelial cells and the number of positively staining epithelial cells were counted in six, adjacent high power fields (magnification, ×400; Leitz Orthoplan microscope, Leica DC 300 camera) for each sample by two independent observers. Analyses were done blinded. Differences in counts between these observers were <10%, and both observed the same differences between cohorts. Acqusition software was Adobe Photoshop version 7. Numbers of epithelial cells counted in each preintervention and 5 http://www.mirtoselect.com Cancer Prevention Research 626 Cancer Prev Res 2009;2(7) July 2009 www.aacrjournals.org Cancer Research. on April 13, 2017. © 2009 American Association for cancerpreventionresearch.aacrjournals.org Downloaded from postintervention sample stained for Ki-67 were 1,161 ± 312 and 1,330 ± 465, respectively, and for caspase-3 analysis, they were 1,161 ± 403 and 1,516 ± 703, respectively. Preintervention samples obtained from two patients contained insufficient tissue and were excluded. IGF-I and IGFBP-3 concentrations in plasma were determined using ELISA kits DG 100 and BAF675, respectively (R&D Systems). Assays were validated and done according to the manufacturer's instructions. Statistical analyses Statistical comparisons were made by paired Student's t test or, in the case of the analysis of plasma IGF-I concentrations, Wilcoxon signed-rank test for nonparametric data, using SPSS version 13 (Windows XP).