Chad W. Skaer

Beneficial regulation of metabolic profiles by black raspberries in human colorectal cancer patients

Dietary intervention of freeze-dried black raspberries (BRBs) in a group of human colorectal cancer patients has demonstrated beneficial effects, including proapoptosis, antiproliferation, and antiangiogenesis. The aim of this study was to investigate BRB-mediated metabolite changes from this same cohort of patients. Twenty-eight colorectal cancer patients were given 60 g BRB powder daily for 1 to 9 weeks. Urine and plasma specimens were collected before and after BRB intervention. A mass spectrometry–based nontargeted metabolomic analysis was conducted on each specimen. A total of more than 400 metabolites were annotated in each specimen. Of these 34 and 6 metabolites were significantly changed by BRBs in urine and plasma, respectively. Increased levels of 4-methylcatechol sulfate in both post-BRB urine and post-BRB plasma were significantly correlated with a higher level of apoptotic marker (TUNEL) in post-BRB tumors. One tricarboxylic acid (TCA) cycle metabolites, cis-aconitate, was increased in post-BRB urine. Furthermore, BRB-derived polyphenols were absorbed and metabolized to various benzoate species, which were significantly increased in post-BRB specimens. Increased benzoate levels were positively correlated with enhanced levels of amino acid metabolite. These results suggest that BRBs induce significant metabolic changes and affect energy generating pathways.This study supports the hypothesis that BRBs might be beneficial to colorectal cancer patients through the regulation of multiple metabolites. Cancer Prev Res; 8(8); 743–50. ©2015 AACR.

Loss of Free Fatty Acid Receptor 2 enhances colonic adenoma development and reduces the chemopreventive effects of black raspberries in ApcMin/+ mice

We previously showed that black raspberries (BRBs) have beneficial effects in human colorectal cancer and a mouse model of colorectal cancer (ApcMin/+). The current study investigated the role of free fatty acid receptor 2 (FFAR2) in colon carcinogenesis and whether the FFAR2 signaling pathway contributes to BRB-mediated chemoprevention in mice. FFAR2 (also named GPR43) is a member of the G-protein-coupled receptor family that is expressed in leukocytes and colon. ApcMin/+ and ApcMin/+-FFAR2−/− mice were given a control diet or the control diet supplemented with 5% BRBs for 8 weeks. FFAR2 deficiency promoted colonic polyp development, with 100% incidence and increased polyp number and size. The ApcMin/+ mice developed colonic tubular adenoma, whereas the ApcMin/+-FFAR2−/− mice developed colonic tubular adenoma with high-grade dysplasia. FFAR2 deficiency also enhanced the cAMP-PKA-CREB-HDAC pathway, downstream of FFAR2 signaling, and increased activation of the Wnt pathway, and raised the percentage of GR-1+ neutrophils in colonic lamina propria (LP) and increased infiltration of GR-1+ neutrophils into colonic polyps. BRBs suppressed colonic polyp development and inhibited the cAMP-PKA-CREB-HDAC and Wnt pathways in the ApcMin/+ mice but not the ApcMin/+-FFAR2−/− mice. They also increased the percentage of GR-1+ neutrophils and cytokine secretion in colonic LP and decreased the infiltration of GR-1+ neutrophils and IL-1&bgr; expression in colon polyps of ApcMin/+ mice but not ApcMin/+-FFAR2−/− mice. These results suggest that loss of FFAR2 drives colon tumorigenesis and that BRBs require functional FFAR2 to be chemopreventive. BRBs have the potential to modulate the host immune system, thereby enhancing the antitumor immune microenvironment.

Black raspberries suppress colonic adenoma development in ApcMin/+ mice: relation to metabolite profiles

Freeze-dried black raspberries (BRBs) have demonstrated chemopreventive effects in a dietary intervention trial with human colorectal cancer patients. The aim of this study was to investigate BRB-caused metabolite changes using the Apc(Min/+) mouse as a model of human colorectal cancer. Wild-type (WT) mice were fed control diet, and Apc(Min/+) mice were fed either control diet or control diet supplemented with 5% BRBs for 8 weeks. Colonic and intestinal polyp size and number were measured. A non-targeted metabolomic analysis was conducted on colonic mucosa, liver and fecal specimens. Eight weeks of BRB treatment significantly decreased intestinal and colonic polyp number and size in Apc(Min/+) mice. The apc gene mutation significantly changed 52 metabolites in colonic mucosa associated with increased amino acid and decreased lipid metabolites, as well as 39 liver and 8 fecal metabolites. BRBs significantly reversed 23 apc-regulated metabolites, including 13 colonic mucosa, 8 liver and 2 fecal metabolites that were involved in amino acid, glutathione, lipid and nucleotide metabolism. Of these, changes in eight metabolites were linearly correlated with decreased colonic polyp number and size in BRB-treated Apc(Min/+) mice. Elevated levels of putrescine and linolenate in Apc(Min/+) mice were significantly decreased by BRBs. Ornithine decarboxylase expression, the key enzyme in putrescine generation, was fully suppressed by BRBs. These results suggest that BRBs produced beneficial effects against colonic adenoma development in Apc(Min/+) mice and modulated multiple metabolic pathways. The metabolite changes produced by BRBs might potentially reflect the BRB-mediated chemopreventive effects in colorectal cancer patients.

Berries and other natural products in pancreatic cancer chemoprevention in human clinical trials

Pancreatic ductal adenocarcinoma (PDAC) was the 12th and 11th most common cancer in men and women worldwide in 2012, with the highest incidence in North America and Europe and the lowest in Africa and Asia. Due to the lack of efficient early diagnosis and rapid disease progression, PDAC patients have a 5-year survival rate of just 5%. Epidemiological studies suggest that smoking, obesity, type II diabetes, and pancreatitis are common risk factors for PDAC development. By contrast, high intake of fresh fruit, vegetables, and nuts rich in phytochemicals could reduce PDAC risk. This review summarizes the human clinical studies that have used berries or other natural products for chemoprevention of PDAC. Developing chemopreventive agents against PDAC would be tremendously valuable for the high-risk population and patients with premalignant lesions. Although some clinical trials of these agents have been completed, most are in early phases, and the results are not promising, which may be due to administration of the natural products at advanced stages of PDAC. Thus, further mechanistic studies using genetic animal models that recapitulate the tumor microenvironment and immunology of human PDAC would be informative for selecting an effective window for intervention with berries or other natural compounds.

Systemic Metabolite Changes in Wild-type C57BL/6 Mice Fed Black Raspberries

ABSTRACT Introduction: Freeze-dried black raspberries (BRBs) elicit chemopreventive effects against colorectal cancer in humans and in rodents. The objective of this study was to investigate potential BRB-caused metabolite changes using wild-type (WT) C57BL/6 mice. Methods and results: WT mice were fed either control diet or control diet supplemented with 5% BRBs for 8 wk. A nontargeted metabolomic analysis was conducted on colonic mucosa, liver, and fecal specimens collected from both diet groups. BRBs significantly changed the levels of 41 colonic mucosa metabolites, 40 liver metabolites, and 34 fecal metabolites compared to control diet-fed mice. BRBs reduced 34 lipid metabolites in colonic mucosa and increased levels of amino acids in liver. One metabolite, 3-[3-(sulfooxy) phenyl] propanoic acid, might be a useful biomarker of BRB consumption. In addition, BRB powder was found to contain 30-fold higher levels of linolenate compared to control diets. Consistently, multiple omega-3 polyunsaturated fatty acids (ω-3 PUFAs), including stearidonate, docosapentaenoate (ω-3 DPA), eicosapentaenoate (EPA), and docosahexaenoate (DHA), were significantly elevated in livers of BRB-fed mice. Conclusion: The data from the current study suggest that BRBs produce systemic metabolite changes in multiple tissue matrices, supporting our hypothesis that BRBs may serve as both a chemopreventive agent and a beneficial dietary supplement.

Abstract B59: Black raspberries inhibit pancreatic carcinogenesis by suppressing Raf/MEK/ERK/STAT3 signaling pathways and promoting apoptosis

Background: Despite improved understanding of the pathophysiology of pancreatic ductal adenocarcinoma (PDAC) in the past two decades, PDAC has an extremely low 5-year survival rate (7%). However, diets rich in fruits and vegetables have been reported to reduce the risk of cancer development. We previously demonstrated that black raspberries (BRBs) have chemopreventive effects in human colorectal cancer patients and mouse models of colorectal cancer. Our current study aimed to investigate the potential effects of BRBs against PDAC in mice. Methods: Mice that carry a constitutively activated KrasLSL.G12D/+ oncogene and loss-of-function Trp53LSL.R172H/+ tumor-suppressor gene in the pancreas (KrasLSL.G12D/+-Trp53LSL.R172H/+-Pdx-1-Cre) spontaneously develop PDAC that recapitulates the human PDAC. Four-week-old KrasLSL.G12D/+-Trp53LSL.R172H/+-Pdx-1-Cre mice were given a control diet or a control diet supplemented with 5% BRBs. In addition, luciferase-transfected human Panc-1 (Panc-1-Luc) cells were orthotopically injected into the pancreas of nonobese, diabetic, severe combined immunodeficient (NOD.SCID) mice. Four weeks after the inoculation of Panc-1-Luc cells, the NOD.SCID mice were given a control diet or a control diet supplemented with 5% BRBs for 6 weeks. Gross tumor volume of orthotopic pancreatic tumors was measured. Results: The 4-week-old KrasLSL.G12D/+-Trp53LSL.R172H/+-Pdx-1-Cre mice developed pancreatic intraepithelial neoplasia (PanIN) lesions, which are the precancerous lesions of PDAC, and they were fed control or 5% BRB diet. Kaplan-Meier survival analysis showed that 5% BRBs significantly prolonged the survival of those PDAC mice with a median survival of 189 days versus 154 days for the mice fed the control diet. Molecular studies suggested that 5% BRBs suppress the Raf/MEK/ERK/STAT3 pathway, which is the downstream of Kras activation, and inhibit cancer cell proliferation in pancreatic tumor tissues. In addition, 5% BRBs significantly decreased the gross tumor volume of orthotopic tumors produced by injecting Panc-1-Luc cells into the pancreas. Orthotopic pancreatic tumors in BRB-treated NOD.SCID mice had a higher rate of apoptosis than tumors from mice fed the control diet. Conclusions: These results support the hypothesis that BRBs have the clinical potential to delay pancreatic cancer progression by suppressing cancer cell proliferation and/or promoting apoptosis. Citation Format: Pan Pan, Chad W. Skaer, Hsin-Tzu Wang, Susan Tsai, Kiyoko Oshima, Yi-Wen Huang, Li-Shu Wang.{Authors}. Black raspberries inhibit pancreatic carcinogenesis by suppressing Raf/MEK/ERK/STAT3 signaling pathways and promoting apoptosis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B59.

Abstract B80: Beneficial regulation of metabolic profiles by black raspberries in human colorectal cancer patients

Objectives: Dietary intervention of freeze-dried black raspberries (BRBs) in a group of human colorectal cancer patients has demonstrated beneficial effects, including pro-apoptosis, anti-proliferation and anti-angiogenesis. The aim of this study was to investigate BRB-mediated metabolite changes from this same cohort of patients. Methods: 28 colorectal cancer patients were given 60 g BRB powder daily for 1-9 weeks. Urine and plasma specimens were collected before and after BRB intervention. A comprehensive mass spectrometry-based nontargeted metabolomic analysis was conducted on each specimen. Results: A total of more than 400 metabolites were annotated in each specimen. Of these 69 and 58 metabolites were significantly changed by BRBs in urine and plasma, respectively. An increased level of 4-methylcatechol sulfate was correlated with a higher apoptotic marker (TUNEL) in post-BRB tumors, while a decreased level of N-acetylglycine was correlated with a lower cell proliferation marker (Ki67) in post-BRB tumors. Cis-aconitate and isocitrate, two tricarboxylic acid (TCA) cycle metabolites, were increased in post-BRB urine. Fatty acid β-oxidation and synthesis were decreased in post-BRB plasma. In addition, BRB-derived polyphenols were absorbed and metabolized to various benzoate species, which were significantly increased in post-BRB specimens. Increased benzoate levels were positively correlated with enhanced amino acid and carbohydrate metabolism but were negatively correlated with decreased fatty acid metabolism. Conclusion: These results suggest that BRBs may induce systemic changes that affect energy generating pathways. This study supports the hypothesis that BRBs might be beneficial to colorectal cancer patients through altering multiple intrinsic metabolic pathways. Citation Format: Pan Pan, Chad W. Skaer, Steven M. Stirdivant, Matthew R. Young, Gary D. Stoner, John F. Lechner, Yi-Wen Huang, Li-Shu Wang. Beneficial regulation of metabolic profiles by black raspberries in human colorectal cancer patients. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B80.

Abstract B81: Effects of black raspberry diet on the metabolomic profile of C57BL/6J mouse tissues

In our previous work, a diet containing 5% freeze-dried black raspberries (BRB) was shown to significantly inhibit colorectal cancer in ApcMin mice. Studies in recent years have shown that metabolomics may help to elucidate the anti-cancer effects of dietary factors. Initially, therefore, we evaluated the effects of black raspberries on the metabolomic profiles of C57BL/6J mice, the mouse strain from which ApcMin mice were derived. In this study, we report the effects of the BRB diet on the metabolomic profiles from C57BL/6J mice. After 8 weeks on a 5% BRB or control AIN-76A diet, feces, liver, and colon mucosa tissues from 9 BRB-fed mice and 7 control-diet fed mice were collected and profiled using mass spectrometry. A total of 424 named metabolites were observed in the liver, feces, and colon mucosa, with 115 metabolites whose levels were significantly different between BRB and control-diet mice (P Citation Format: Chad W. Skaer, Pan Pan, Matthew R. Young, Hsin Tzu Wang, Kiyoko Oshima, Yi-Wen Huang, Gary D. Stoner, John Lechner, Li-Shu Wang. Effects of black raspberry diet on the metabolomic profile of C57BL/6J mouse tissues. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B81.

Abstract LB-275: Beneficial regulation of metabolic profiles by black raspberries in human colorectal cancer patients

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Objectives: Dietary intervention of freeze-dried black raspberries (BRBs) in a group of human colorectal cancer patients has demonstrated beneficial effects, including pro-apoptosis, anti-proliferation and anti-angiogenesis. The aim of this study was to investigate BRB-mediated metabolite changes from this same cohort of patients. Methods: 28 colorectal cancer patients were given 60 g BRB powder daily for 1-9 weeks. Urine and plasma specimens were collected before and after BRB intervention. A comprehensive mass spectrometry-based nontargeted metabolomic analysis was conducted on each specimen. Results: A total of more than 400 metabolites were annotated in each specimen. Of these 69 and 58 metabolites were significantly changed by BRBs in urine and plasma, respectively. An increased level of 4-methylcatechol sulfate was correlated with a higher apoptotic marker (TUNEL) in post-BRB tumors, while a decreased level of N-acetylglycine was correlated with a lower cell proliferation marker (Ki67) in post-BRB tumors. Cis-aconitate and isocitrate, two tricarboxylic acid (TCA) cycle metabolites, were increased in post-BRB urine. Fatty acid β-oxidation and synthesis were decreased in post-BRB plasma. In addition, BRB-derived polyphenols were absorbed and metabolized to various benzoate species, which were significantly increased in post-BRB specimens. Increased benzoate levels were positively correlated with enhanced amino acid and carbohydrate metabolism but were negatively correlated with decreased fatty acid metabolism. Conclusion: These results suggest that BRBs may induce systemic changes that affect energy generating pathways. This study supports the hypothesis that BRBs might be beneficial to colorectal cancer patients through altering multiple intrinsic metabolic pathways. Citation Format: Pan Pan, Chad Skaer, Steven Stirdivant, Matthew R. Young, Gary Stoner, John Lechner, Yi-Wen Huang, Li-Shu Wang. Beneficial regulation of metabolic profiles by black raspberries in human colorectal cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-275. doi:10.1158/1538-7445.AM2015-LB-275

Abstract LB-263: Effects of black raspberry diet on the metabolomic profile of C57BL/6J mouse tissues

In our previous work, a diet containing 5% freeze-dried black raspberries (BRB) was shown to significantly inhibit colorectal cancer in ApcMin mice. Studies in recent years have shown that metabolomics may help to elucidate the anti-cancer effects of dietary factors. Initially, therefore, we evaluated the effects of black raspberries on the metabolomic profiles of C57BL/6J mice, the mouse strain from which ApcMin mice were derived. In this study, we report the effects of the BRB diet on the metabolomic profiles from C57BL/6J mice. After 8 weeks on a 5% BRB or control AIN-76A diet, feces, liver, and colon mucosa tissues from 9 BRB-fed mice and 7 control-diet fed mice were collected and profiled using mass spectrometry. A total of 424 named metabolites were observed in the liver, feces, and colon mucosa, with 115 metabolites whose levels were significantly different between BRB and control-diet mice (P Citation Format: Chad W. Skaer, Pan Pan, Matthew Young, Hsin Tzu Wang, Kiyoko Oshima, Yi-Wen Huang, Gary D. Stoner, John Lechner, Li-Shu Wang. Effects of black raspberry diet on the metabolomic profile of C57BL/6J mouse tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-263. doi:10.1158/1538-7445.AM2015-LB-263