US National Cancer Institute-China Collaborative Studies on Chinese Medicine and Cancer.

Since 2007, the US National Cancer Institute (NCI) Office of Cancer Complementary and Alternative Medicine (OCCAM), together with the Cancer Institute of the China Academy of Chinese Medical Sciences (CICACMS), institutes at China Academy of Sciences and Chinese Academy of Medical Sciences, have engaged in collaborations on Chinese medicine (CM) and cancer research. Through these collaborations, CM drugs and compounds have been studied at NCI labs. This paper summarizes the discoveries and progress on these research projects, exploring the aspects of cancer prevention, botanical drug mechanisms of action and component analysis/quality control (QC), and anticancer activity screening. These and other related projects have been presented in various jointly convened workshops and have provided the backdrop for establishing a new organization, the International Consortium for CM and Cancer, to promote international collaborations in this field.

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

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.

Diallyl Disulfide (DADS), a Constituent of Garlic, Inactivates NF-κB and Prevents Colitis-Induced Colorectal Cancer by Inhibiting GSK-3ß.

There is a strong belief that garlic has medicinal properties and may even reduce the risk of developing certain cancers including those of the gastrointestinal tract. The chemopreventive effects of garlic may be attributed to the anti-inflammatory properties of the sulfur-containing constituents of garlic, which includes diallyl disulfide (DADS). Here, we demonstrate that DADS prevented colorectal tumorigenesis in a mouse model of colitis-induced colorectal cancer. Supplementation with 85 ppm of DADS (60 mg daily human equivalent dose) in the diet of FVB/N mice treated with chemical carcinogen azoxymethane (AOM) and colonic irritant dextran sodium sulfate (DSS) resulted in the reduction in tumor incidence, tumor number, and tumor burden by 21.54%, 47.3%, and 66.4%, respectively. Further analysis revealed that mice fed the DADS-supplemented diet resolved the initial DSS-induced inflammation faster than those on the control diet, preventing prolonged inflammation and cellular transformation. Subsequent mechanistic studies in vitro suggest that DADS chemopreventive effects are mediated through NF-κB signaling. When SW480 colorectal cancer cells were treated with DADS, NF-κB nuclear localization and activity were diminished. Interestingly, NF-κB suppression was found to be dependent on DADS inhibition of GSK-3ß, a positive regulator of NF-κB. Inhibition of GSK-3ß and loss of nuclear NF-κB activity were also observed in vivo in AOM/DSS-treated mice fed a diet supplemented with 85 ppm DADS. Our results indicate that DADS can prevent tumorigenesis by suppressing inflammation, a process largely involving GSK-3ß inhibition and consequential reduction in NF-κB nuclear localization. Cancer Prev Res; 9(7); 607-15. ©2016 AACR.

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.

Cryptotanshinone, a Stat3 inhibitor, suppresses colorectal cancer proliferation and growth in vitro.

Cryptotanshinone (CPT) is a natural compound extracted from herbal medicine that has been previously shown to possess antitumor properties in various types of human cancer cells. In the present study, we examined the potential role of CPT in the treatment of colorectal cancer. Using SW480, HCT116, and LOVO colorectal cancer cell lines, the effects of CPT on cell viability, apoptosis, and tumorigenicity were evaluated. The results showed that CPT significantly inhibited the growth and viability of SW480, HCT116, and LOVO cell lines by inducing apoptosis and prevented anchorage dependent growth on agar. In addition, CPT inhibited the activation of Signal transducer and activator of transcription 3 (Stat3) pathways in colorectal cancer cells. Stat3 is a transcription factor that mediates the expression of various genes associated with many cellular processes, such as inflammation and cell growth, and has been shown to promote several cancer types, including colorectal cancer. These findings indicate that CPT may be a potential candidate for the treatment and prevention of colorectal cancer in part by inhibiting the activation of Stat3.

Identification and monitoring of metabolite markers of dry bean consumption in parallel human and mouse studies.

SCOPE: Aim of the study was to identify and monitor metabolite markers of dry bean consumption in parallel human and mouse studies that each had shown chemopreventive effects of dry bean consumption on colorectal neoplasia risk. METHODS AND RESULTS: Using LC/mass spectroscopy ± ESI and GC/mass spectroscopy, serum metabolites of dry beans were measured in 46 men before and after a 4-week dry bean enriched diet (250 g/day) and 12 mice that received a standardized diet containing either 0 or 10% navy bean ethanol extract for 6 weeks; we also investigated fecal metabolites in the mice. The serum metabolites identified in these controlled feeding studies were then investigated in 212 polyp-free participants from the Polyp Prevention Trial who self-reported either increased (≥+31 g/day from baseline), high dry bean intake of ≥42 g/day in year 3 or low, unchanged dry bean consumption of <8 g/day; serum was analyzed from baseline and year 3. Serum pipecolic acid and S-methyl cysteine were elevated after dry bean consumption in human and mouse studies and reflected dry bean consumption in the Polyp Prevention Trial. CONCLUSION: Serum levels of pipecolic acid and S-methyl cysteine are useful biomarkers of dry bean consumption.

In vivo MRI virtual colonography in a mouse model of colon cancer.

We have developed a reliable noninvasive method for monitoring colonic tumors and mucosal inflammation in a mouse model of colon cancer using magnetic resonance colonography (MRC). After a mild cleansing enema, the colon is filled with Fluorinert, a perfluorinated liquid that does not produce a proton MR signal. The mouse is placed in a dedicated volume MR receiver coil, and high-resolution images are acquired in three planes. The Fluorinert enema distends the mouse colon, creating an artifact-free black homogeneous background, allowing clear delineation of the inflamed colonic wall and visualization of luminal tumors in various stages of development. A gadolinium-based contrast agent can be administered i.v. to the animal to detect mural inflammation or tumor vascularity. This technique is useful for serial monitoring of the effects of preventive or therapeutic strategies on tumor development without killing the animal or requiring invasive endoscopies. The animal preparation and imaging can be completed in ∼1.5 h.

Chemopreventive activity of plant flavonoid isorhamnetin in colorectal cancer is mediated by oncogenic Src and ß-catenin.

Analysis of the Polyp Prevention Trial showed an association between an isorhamnetin-rich diet and a reduced risk of advanced adenoma recurrence; however, the mechanism behind the chemoprotective effects of isorhamnetin remains unclear. Here, we show that isorhamnetin prevents colorectal tumorigenesis of FVB/N mice treated with the chemical carcinogen azoxymethane and subsequently exposed to colonic irritant dextran sodium sulfate (DSS). Dietary isorhamnetin decreased mortality, tumor number, and tumor burden by 62%, 35%, and 59%, respectively. MRI, histopathology, and immunohistochemical analysis revealed that dietary isorhamnetin resolved the DSS-induced inflammatory response faster than the control diet. Isorhamnetin inhibited AOM/DSS-induced oncogenic c-Src activation and ß-catenin nuclear translocation, while promoting the expression of C-terminal Src kinase (CSK), a negative regulator of Src family of tyrosine kinases. Similarly, in HT-29 colon cancer cells, isorhamnetin inhibited oncogenic Src activity and ß-catenin nuclear translocation by inducing expression of csk, as verified by RNA interference knockdown of csk. Our observations suggest the chemoprotective effects of isorhamnetin in colon cancer are linked to its anti-inflammatory activities and its inhibition of oncogenic Src activity and consequential loss of nuclear ß-catenin, activities that are dependent on CSK expression.

Elevated phosphate activates N-ras and promotes cell transformation and skin tumorigenesis.

Recent results suggest a paradigm shift from viewing inorganic phosphate as a passive requirement for basic cell functions to an active regulator of cell behavior. We have previously shown that elevated concentrations of phosphate increased cell proliferation and expression of protumorigenic genes such as Fra-1 and osteopontin in a preosteoblast cell line. Therefore, we hypothesized that elevated phosphate concentrations would promote cell transformation in vitro and tumorigenesis in vivo. Supplementation of medium with phosphate increased anchorage-independent transformation and proliferation of BALB/c mouse JB6 epidermal cells, activation of N-ras, ERK1/2, and activator protein-1, and increased gene expression of Fra-1, COX-2, and osteopontin in a dose-dependent manner. These in vitro results led to the hypothesis that varying the levels of dietary inorganic phosphate would alter tumorigenesis in the mouse model of skin carcinogenesis. Female FVB/N mice were treated with 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate and fed high- or low-phosphate diets (1.2% versus 0.2% of the diet) for 19 weeks. The high-phosphate diet increased skin papilloma number by approximately 50% without changing feed intake and body weights. High dietary phosphate increased serum concentrations of phosphate, parathyroid hormone, and osteopontin and decreased serum concentrations of calcium. Thus, we conclude that elevated phosphate promotes cell transformation and skin tumorigenesis partly by increasing the availability of phosphate for activation of N-ras and its downstream targets, which defines reducing dietary phosphate as a novel target for chemoprevention.

Monitoring of tumor promotion and progression in a mouse model of inflammation-induced colon cancer with magnetic resonance colonography.

Early detection of precancerous tissue has significantly improved survival of most cancers including colorectal cancer (CRC). Animal models designed to study the early stages of cancer are valuable for identifying molecular events and response indicators that correlate with the onset of disease. The goal of this work was to investigate magnetic resonance (MR) colonography in a mouse model of CRC on a clinical MR imager. Mice treated with azoxymethane and dextran sulfate sodium were imaged by serial MR colonography (MRC) from initiation to euthanasia. Magnetic resonance colonography was obtained with both T1- and T2-weighted images after administration of a Fluorinert enema to remove residual luminal signal and intravenous contrast to enhance the colon wall. Individual tumor volumes were calculated and validated ex vivo. The Fluorinert enema provided a clear differentiation of the lumen of the colon from the mucosal lining. Inflammation was detected 3 days after dextran sulfate sodium exposure and subsided during the next week. Tumors as small as 1.2 mm(3) were detected and as early as 29 days after initiation. Individual tumor growths were followed over time, and tumor volumes were measured by MR imaging correlated with volumes measured ex vivo. The use of a Fluorinert enema during MRC in mice is critical for differentiating mural processes from intraluminal debris. Magnetic resonance colonography with Fluorinert enema and intravenous contrast enhancement will be useful in the study of the initial stages of colon cancer and will reduce the number of animals needed for preclinical trials of prevention or intervention.

Inflammation-associated serum and colon markers as indicators of dietary attenuation of colon carcinogenesis in ob/ob mice.

Although inflammatory cytokines and obesity-associated serum proteins have been reported as biomarkers of colorectal adenoma risk in humans, little is known of biomarkers of response to interventions that attenuate tumorigenesis. Dietary navy beans and their fractions attenuate colon carcinogenesis in carcinogen-induced genetically obese mice. We hypothesized that this attenuation would be associated with changes in inflammatory cytokines and obesity-related serum proteins that may serve as measures of efficacy. ob/ob mice (n = 160) were injected with the carcinogen azoxymethane (AOM) to induce colon cancer and randomly placed on one of four diets (control, whole navy bean, bean residue fraction, or bean extract fraction) for 26 to 28 wk. Serum was analyzed for 14 inflammation- or obesity-related proteins, and colon RNA was analyzed for expression of 84 inflammation-associated genes. Six of 14 serum proteins were increased [i.e., interleukin (IL)-4, IL-5, IL-6, IL-10, IFN gamma, granulocyte macrophage colony-stimulating factor] in hyperplastic/dysplastic stages of colon carcinogenesis. Bean-fed mice had significantly higher monocyte chemoattractant protein-1 and lower IL-6 levels in serum. In colon mucosa, 55 of 84 inflammation-associated genes differed between AOM-induced and noninduced mice. Of the 55 AOM-induced genes, 5 were counteracted by bean diets, including IL-6 whose increase in expression levels was attenuated by bean diets in AOM-induced mice. In summary, IL-6 emerged as a serum protein that was increased in hyperplastic/dysplastic stages of colon carcinogenesis, but attenuated with bean-based diet in serum and colon mucosa. Changes in a subset of inflammation-associated serum proteins and colon gene expression may serve as response indicators of dietary attenuation of colon carcinogenesis.

Dietary cooked navy beans and their fractions attenuate colon carcinogenesis in azoxymethane-induced ob/ob mice.

Based on the protective effects of cooked dry bean consumption in a human intervention study, we evaluated which fraction of cooked dry beans is responsible for its cancer-preventive effects. Cooked navy beans (whole beans), the insoluble fraction (bean residue) or soluble fraction of the 60% (vol:vol) ethanol extract of cooked navy beans (bean extract), or a modified AIN-93G diet (16.6% fat including 12.9% lard) as control diet were fed to 160 male obese ob/ob mice after 2 azoxymethane injections. In comparison to control-fed mice, dysplasia, adenomas, or adenocarcinomas were detected in fewer mice on either bean fraction diet (percent reduction from control: whole beans 54%, P=0.10; bean residue 81%, P=0.003; bean extract 91%, P=0.007), and any type of colon lesions, including focal hyperplasia, were found in fewer mice on each of the 3 bean diets percent reduction from control: whole bean 56%, P=0.04; bean residue 67%, P=0.01; bean extract 87%, P=0.0003. These results suggest that both the soluble and the insoluble fraction of the extract contribute to the cancer-protective effect of cooked navy beans.

A high-throughput cell-based assay to identify specific inhibitors of transcription factor AP-1.

The oncogenic transcription factor AP-1 (activator protein-1) is required for tumor promotion and progression. Identification of novel and specific AP-1 inhibitors would be beneficial for cancer prevention and therapy. The authors have developed a high-throughput assay to screen synthetic and natural product libraries for noncytotoxic inhibitors of mitogen-activated AP-1 activity. The cell-based high-throughput screen is conducted in a 384-well format using a fluorescent resonance energy transfer (FRET) substrate to quantify the activity of a beta-lactamase reporter under the control of an AP-1-dependent promoter. The ratiometric FRET readout makes this assay extremely robust and reproducible, particularly for use with natural product extracts. To eliminate false positives due to cell killing, a cytotoxicity assay was incorporated. The AP-1 beta-lactamase reporter was validated with inhibitors of kinases located upstream of AP-1 and with known natural product inhibitors of AP-1 (nordihydroguaiaretic acid and curcumin). The assay was able to identify other known AP-1 inhibitors and protein kinase C modulators, as well as a number of chemically diverse compounds with unknown mechanisms of action from natural products libraries. Application to natural product extracts identified hits from a range of taxonomic groups. Screening of synthetic compounds and natural products should identify novel AP-1 inhibitors that may be useful in the prevention and treatment of cancers.