Christine Sardo

Pharmacokinetics of anthocyanins and ellagic acid in healthy volunteers fed freeze-dried black raspberries daily for 7 days.

Eleven subjects completed a clinical trial to determine the safety/tolerability of freeze‐dried black raspberries (BRB) and to measure, in plasma and urine, specific anthocyanins‐cyanidin‐3‐glucoside, cyanidin‐3‐sambubioside, cyanidin‐3‐rutinoside, and cyanidin‐3‐xylosylrutinoside, as well as ellagic acid. Subjects were fed 45 g of freeze‐dried BRB daily for 7 days. Blood samples were collected predose on days 1 and 7 and at 10 time points postdose. Urine was collected for 12 hours predose on days 1 and 7 and at three 4‐hour intervals postdose. Maximum concentrations of anthocyanins and ellagic acid in plasma occurred at 1 to 2 hours, and maximum quantities in urine appeared from 0 to 4 hours. Overall, less than 1% of these compounds were absorbed and excreted in urine. None of the pharmacokinetic parameters changed significantly between days 1 and 7. In conclusion, 45 g of freeze‐dried BRB daily are well tolerated and result in quantifiable anthocyanins and ellagic acid in plasma and urine.

Modulation of Genetic and Epigenetic Biomarkers of Colorectal Cancer in Humans by Black Raspberries: A Phase I Pilot Study

Purpose: This study evaluated the effects of black raspberries (BRBs) on biomarkers of tumor development in the human colon and rectum including methylation of relevant tumor suppressor genes, cell proliferation, apoptosis, angiogenesis, and expression of Wnt pathway genes. Experimental Design: Biopsies of adjacent normal tissues and colorectal adenocarcinomas were taken from 20 patients before and after oral consumption of BRB powder (60 g/d) for 1–9 weeks. Methylation status of promoter regions of five tumor suppressor genes was quantified. Protein expression of DNA methyltransferase 1 (DNMT1) and genes associated with cell proliferation, apoptosis, angiogenesis, and Wnt signaling were measured. Results: The methylation of three Wnt inhibitors, SFRP2, SFRP5, and WIF1, upstream genes in Wnt pathway, and PAX6a, a developmental regulator, was modulated in a protective direction by BRBs in normal tissues and in colorectal tumors only in patients who received BRB treatment for an average of 4 weeks, but not in all 20 patients with 1–9 weeks of BRB treatment. This was associated with decreased expression of DNMT1. BRBs modulated expression of genes associated with Wnt pathway, proliferation, apoptosis, and angiogenesis in a protective direction. Conclusions: These data provide evidence of the ability of BRBs to demethylate tumor suppressor genes and to modulate other biomarkers of tumor development in the human colon and rectum. While demethylation of genes did not occur in colorectal tissues from all treated patients, the positive results with the secondary endpoints suggest that additional studies of BRBs for the prevention of colorectal cancer in humans now appear warranted. Clin Cancer Res; 17(3); 598–610. ©2010 AACR.

Plasma Cytokines as Potential Response Indicators to Dietary Freeze-Dried Black Raspberries in Colorectal Cancer Patients

Oral consumption of freeze-dried black raspberries attenuated neoplastic changes in colorectal tissue markers of apoptosis, cell proliferation, and angiogenesis in colorectal cancer (CRC) patients. To determine whether plasma concentrations of interleukin (IL)-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, granulocyte macrophage colony stimulating factor (GM-CSF), interferon-γ, and tumor necrosis factor-α (TNF-α) were associated with berry treatment and changes in colorectal tissue markers of apoptosis, cell proliferation, and angiogenesis, plasma and biopsy samples of adenocarcinoma and adjacent normal-appearing colorectal tissue were collected before and during berry treatment from 24 CRC patients who had not received prior therapy and drank a slurry of black raspberry powder (20 g in 100 ml drinking water) 3 times a day for 1 to 9 wk. Plasma concentrations of GM-CSF (+0.12 ± 0.04 pg/mL; P = 0.01) and IL-8 (-1.61 ± 0.71 pg/mL; P = 0.04) changed in patients receiving berries for more than 10 days. These changes were correlated with beneficial changes in markers of proliferation (rΔGM-CSF, ΔKi67 carcinoma - normal = −0.51) and apoptosis (rΔIL-8, ΔTUNEL carcinoma - normal = −0.52) observed in colorectal tissue taken within the same week. Plasma concentrations of GM-CSF and IL-8 may serve as noninvasive indicators to monitor tissue response to berry-based interventions for CRC.

Association between circulating concentrations of 25(OH)D and colorectal adenoma: A pooled analysis

The relationship between the biomarker of vitamin D status, 25(OH)D, and the risk for colorectal neoplasia is suggestive but equivocal. Questions remain regarding whether there are differential associations between 25(OH)D and colorectal adenoma by gender, colorectal subsite or features of baseline and recurrent adenomas. We sought to investigate the relationship between 25(OH)D and both baseline and recurrent adenoma characteristics. Our study was conducted among 2,074 participants in a pooled population of two clinical intervention trials of colorectal adenoma recurrence. A cross‐sectional analysis of 25(OH)D and baseline adenoma characteristics and a prospective study of recurrent adenomas and their characteristics were conducted. There was a statistically significant inverse association between the concentrations of 25(OH)D and the presence of three or more adenomas at baseline. Compared to participants with 25(OH)D levels of <20 ng/mL, the adjusted odds ratios (ORs) (95% condifdence intervals [CIs]) were 0.99 (0.70–1.41) for those with concentrations of ≥20 and <30 ng/mL, and 0.73 (0.50–1.06) among participants with levels of ≥30 ng/mL (p‐trend = 0.05). Baseline villous histology was also significantly inversely related to 25(OH)D levels (p‐trend = 0.04). Conversely, 25(OH)D concentrations were not associated with overall colorectal adenoma recurrence, with ORs (95% CIs) of 0.91 (0.71–1.17) and 0.95 (0.73–1.24; p‐trend = 0.85). These findings support the concept that the relationship between vitamin D and colorectal neoplasia may vary by stage of adenoma development.

Associations between vitamin D-binding protein isotypes, circulating 25(OH)D levels, and vitamin D metabolite uptake in colon cancer cells.

Vitamin D metabolites have been extensively studied as cancer chemopreventive agents. Gc-globulin (GC) isotypes, based on rs7041 and rs4588 diplotypes, have varying affinities for 1α,25-dihydroxyvitamin D (1,25(OH)2D) and 25-hydroxyvitamin D (25(OH)D), which may affect circulating metabolite concentration as well as delivery at the cellular level. We evaluated associations between GC isotype and circulating vitamin D metabolite concentrations in 403 ursodeoxycholic acid (UDCA) clinical trial participants. Metabolite uptake was evaluated in human colon cancer (HCT-116) cells treated with ethanol vehicle, 1,25(OH)2D, or 25(OH)D, and with plasma from individuals with known GC isotype. Mammalian-2-hybrid and vitamin D–responsive element-based luciferase assays were used to measure the vitamin D receptor pathway activation as a marker for metabolite uptake. Regression analysis demonstrated significantly lower serum 25(OH)D concentration for clinical trial participants with 1F_2, 1S_2, or 2_2 isotypes (P < 0.01) compared with 1S_1S. Consistent with these in vivo observations, cellular data revealed that 25(OH)D uptake varied less by GC isotype only at the higher concentration tested (P = 0.05), while 1,25(OH)2D uptake differed markedly by GC isotype across concentration and assay (P < 0.01). The 1F_1S and 1F_2 isotypes produced the greatest reporter gene induction with 1,25(OH)2D treatment and, while activation varied less with 25(OH)D, the 2_2 isotype demonstrated increased induction at the lower concentration. These results suggest that vitamin D metabolite concentration and delivery to colon cells may vary not only by GC isotype, but also that certain isotypes may more effectively deliver 1,25(OH)2D versus 25(OH)D. Overall, these results may help identify populations at risk for cancer and potential recipients of targeted chemoprevention. Cancer Prev Res; 7(4); 426–34. ©2014 AACR.

An Open-Label Randomized Crossover Trial of Lyophilized Black Raspberries on Postprandial Inflammation in Older Overweight Males: A Pilot Study.

This study was a 14-day, outpatient, open-label randomized crossover trial of lyophilized black raspberries (BRBs) in older overweight or obese males to determine whether BRB consumption affects postprandial inflammation associated with consumption of a high-fat high-calorie (HFHC) meal. Ten study participants consumed 45 g/d of lyophilized BRBs for 4 days, followed by a HFHC breakfast plus BRBs on day 6 or consumed the HFHC breakfast on day 6 without previous consumption of BRBs and then crossed over to the other treatment after a 2-day washout period. Blood samples were obtained before and 1, 2, 4, 8, and 12 hours after consumption of the HFHC breakfast. The primary study outcomes were changes in area under the concentration–time curve (AUC) for interleukin-6 (IL-6), C-reactive protein (CRP), and tumor necrosis factor-alpha (TNF-&agr;). The secondary outcomes were safety and tolerability of lyophilized BRB powder. The chronology and values of measured serum concentrations for IL-6, TNF-&agr;, and CRP were consistent with those described previously by other investigators. The AUC of serum IL-6 was lowered significantly (P = 0.03, n = 10) with BRB consumption (34.3 ± 7.6 pg·mL−1·h−1 compared with 42.4 ± 17.9 pg·mL−1·h−1 for consumption of the HFHC meal alone). However, no significant differences (change in AUC) were calculated for serum CRP and TNF-&agr;. The findings of this pilot study suggest that consumption of lyophilized BRBs may attenuate postprandial inflammation in overweight or obese males consuming a HFHC meal. Further investigation of BRBs is warranted to better elucidate their inflammomodulatory potential.

Cancer Prevention with Berries: Role of Anthocyanins

1. Berries have been part of the human diet for many centuries. They are a rich source of known chemopreventive agents including provitamin A carotenoids, C, E, and folate, calcium and selenium, simple and complex phenols, and phytosterols.

Effects of Black Raspberries on Cellular and Epigenetic Biomarkers of Colon Cancer Development in Humans

Colorectal cancer (CRC) is the leading digestive tract cancer in the Western world, particularly in the United States where it accounts for approximately 10% of all cancer deaths. The multistage development of CRC is associated with chromosomal instability, DNA-repair defects, aberrant DNA methylation, and mutational events in oncogenes and tumor suppressor genes. Preclinical studies using cultured human colon tumor cells and animal models of colorectal cancer indicate that black raspberries (BRBs) and their constituent anthocyanins and ellagitannins elicit chemopreventive effects against CRC. A 7-day Phase I trial indicated that BRBs are well tolerated by humans and their component anthocyanins and ellagic acid are absorbed into blood. The results of a Phase Ib trial in which BRBs were administered daily to colorectal cancer patients from the time of diagnosis of the disease until surgical removal of the tumor (average = 3 weeks) are presented in this chapter. BRB treatment resulted in reduced proliferation and increased apoptosis of CRC cells. In addition, tumor angiogenesis was inhibited by berries. BRB treatment reduced s-catenin expression and enhanced E-cadherin expression in CRC indicating a protective effect on the Wnt signaling pathway. DNA methylation assays showed that BRBs are capable of demethylating tumor suppressor genes associated with the Wnt pathway, in part, through inhibiting the expression of DNMT1, a DNA methyltransferase that is commonly overexpressed in CRC. These data suggest that BRBs should be further examined for chemopreventive effects against CRC.

Abstract 4680: Biomarkers of adherence and efficacy: modulation of biomarkers of DNA damage in a Phase 1b trial of post-surgical oral cancer patients on a long-term food-based prevention study with black raspberries.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Consumption of phytochemical-rich fruits and vegetables is associated with a lower risk of oral cancer and protection against oxidative stress-mediated damage from reactive oxygen species (ROS). We proposed that antioxidant components in black raspberries (BRBs) could “scavenge” ROS and diminish oxidative stress burden. We tested the hypothesis that a long-term, low-dose administration of BRBs to a population of disease-free oral cancer survivors is both (i) achievable and (ii) will result in the attenuation of oxidative DNA damage. Methods: Participants were assigned to consume 0, 4, or 8 gms of BRBs daily for 6 months and provided self-report logbooks of adherence. Mass spectrometry (MS) of urine for dimethyl ellagic acid (DMEA) at baseline, 10, and 20 wks was utilized as a biomarker of adherence. ELISA of urine was used to measure 8-hydroxy-2’-deoxyguanosine (8-OHdG). A logistic regression model was used to determine factors associated with willingness to continue on the regimen once enrolled. Results: 112 participants were enrolled and assigned to a BRB regimen. 81 participants (72%) provided self-reported adherence logbooks. Adherence at wks 2, 10 and 20 was 92% (n=77), 85% (n=55), and 86% (n=44) respectively. The regimen was well tolerated with no significant adverse events. MS/MS measurements of DMEA was 10 fold higher (p<0.0001; CI: 4-25) at 10 and 20 wks in participants treated with BRBs vs control. ELISA measurements of 8-OHdG in urine at 10 wks was 1.28 fold lower (p<0.0183; CI: 1.04-1.58) in patients treated with BRBs vs control. Logistic regression analysis revealed that those having the least travel distance (OR: 0.98 [10 and 20 wks] p<0.03) and those having government insurance (OR: 3.96 [10 wks]; 7.04 [20 wks]; p<0.02) or private insurance (OR: 5.1 [20 wks]; p<0.01) vs those with no insurance were more likely to complete the study. Also, the analysis showed that past (OR: 3.85; p<0.02) and current (OR: 3.82; p<0.03) tobacco users were more likely reach 20 wks than never smokers. Furthermore, those having had no adjuvant therapy (OR: 4.5; p<0.004) vs those having had chemotherapy and/or radiation were more likely to complete 20 wks of intervention. Conclusions: A food-based prevention strategy utilizing BRBs in oral cancer survivors is safe and feasible. Compliance for those staying on regimen was high and factors identified that influenced continued participation included: having insurance, travel distance, having had adjuvant therapy, and tobacco use. While the number of active participants declined, those completing the intervention maintained outstanding levels of adherence (>85%). Biomarkers of adherence were identified as an objective means of validating self-reported adherence. Moreover, 8-OHdG levels were reduced, providing a potentially useful biomarker of BRB efficacy. Citation Format: Lana K. Uhrig, Thomas J. Knobloch, Bruce C. Casto, Amit Agrawal, David E. Schuller, Theodoros N. Teknos, Enver Ozer, Matthew O. Old, Christine L. Sardo, Jeff Xueliang, Steven K. Clinton, Dennis K. Pearl, Christopher M. Weghorst. Biomarkers of adherence and efficacy: modulation of biomarkers of DNA damage in a Phase 1b trial of post-surgical oral cancer patients on a long-term food-based prevention study with black raspberries. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4680. doi:10.1158/1538-7445.AM2013-4680

Abstract 163: Metabolomic profiling reveals a protective modulation on fatty acid metabolism in colorectal cancer patients following consumption of freeze-dried black raspberries.

Metabolic reprogramming which refers to altered nutrient uptake and use is thought to be essential for rapid cancer cell proliferation. Accelerated phospholipid biosynthesis is another metabolic signature of cancer because proliferating cells have a significant need for membrane production. Fatty acids that compose the hydrophobic tails of membrane phospholipids can regulate gene transcription through enzyme-mediated pathways, e.g., cyclooxygenase, lipoxygenase, and changing lipid raft composition that affect receptor-mediated signalings. We previously showed that dietary intervention with freeze-dried black raspberries (BRBs) decreased cell proliferation in colorectal tumors and IL-8, a pro-inflammatory cytokine, in plasma in colorectal cancer patients. The goal of the current study was to determine if BRBs affect fatty acid metabolism which may contribute to their anti-proliferative and anti-inflammatory activities. Plasma samples were collected from 28 colorectal cancer patients before and after oral consumption of BRB powder (60g/day) for 1-to-9 wks for metabolomic profiling analysis. 421 biochemicals were analyzed using UHPLC/Gas chromatography and mass spectrometry. When data from all 28 patients were combined, the top 30 ranking biochemicals suggest that berry intervention led to alterations mostly in lipids, following by carbohydrates, amino acids, and cofactors and vitamins. Both monounsaturated fatty acids (MUFAs), e.g., eicosenoate (20:1n9 or 11), and polyunsaturated fatty acids (PUFAs), e.g., linoleate (18:2n6), arachidonate (20:4n6), were lower in the post plasma samples. PUFAs can be synthesized from linoleate and they also can be released by phospholipase A from phospholipid membrane. Therefore, berry intervention might alter activities of phospholipase A, elongases, and desaturases which in turn reduce levels of PUFAs. Alternatively, berry intervention increased secondary bile acids, e.g., glycodeoxycholate, produced by the action of enzymes existing in the microbial flora of the colonic environment, suggesting dietary BRBs could alter colonic microflora. Berry intervention associated alternations in bile acid metabolism could affect fat absorption and subsequently impact fatty acid metabolism. In conclusion, our results suggest that dietary berry consumption protectively modulates enzymes associated with fatty acid metabolism in the host as well as in gut microflora leading to decreased proliferation and inflammation in colorectal cancer patients. Supported by R01 CA148818 to L-S Wang Citation Format: Li-Shu Wang, Matthew Young, Chieh-Ti Kuo, Mark Arnold, Edward Martin, Christine Sardo, Gary Stoner. Metabolomic profiling reveals a protective modulation on fatty acid metabolism in colorectal cancer patients following consumption of freeze-dried black raspberries. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 163. doi:10.1158/1538-7445.AM2013-163