Detection of baicalin metabolites baicalein and oroxylin-a in mouse pancreas and pancreatic xenografts.

OBJECTIVES: Scutellaria baicalensis has been a subject of research interest due to its potential multiple therapeutic benefits. This study was to examine the distribution of baicalein, wogonin, oroxylin A and their glucuronide/sulfate-conjugated metabolites in plasma, colon, small intestine, lung, liver, pancreas, kidney, and prostate tissues and in pancreatic tumor in a xenograft animal model. In addition, we examined metabolic stability of baicalin in these tissues. METHODS: A mouse xenograft model was prepared by injection of 3 × 10 human pancreatic cancer MiaPaCa-2 cells subcutaneously into nude mice. Mice were randomly allocated to control diet (AIN-76A) and 1% S. baicalensis diet (n = 8 per group) for 13 weeks. Levels of baicalein, wogonin, oroxylin A, and their conjugates in mouse tissues were measured by high-pressure liquid chromatography after enzymatic hydrolysis and then extraction. RESULTS: A substantial amount of baicalin (34%-63%) was methylated to oroxylin A and its conjugates in various organs during absorption. Whereas plasma contained predominantly conjugates of baicalein, wogonin, and oroxylin A, both aglycones and conjugates were found in all other tissues investigated and in tumor. CONCLUSIONS: Substantial accumulation of bioactive metabolites are found in target tissues, suggesting strong potential for S. baicalensis use as a preventive or adjuvant supplement for pancreatic cancer.

Baicalein, a component of Scutellaria baicalensis, induces apoptosis by Mcl-1 down-regulation in human pancreatic cancer cells.

Scutellaria baicalensis (SB) and SB-derived polyphenols possess anti-proliferative activities in several cancers, including pancreatic cancer (PaCa). However, the precise molecular mechanisms have not been fully defined. SB extract and SB-derived polyphenols (wogonin, baicalin, and baicalein) were used to determine their anti-proliferative mechanisms. Baicalein significantly inhibited the proliferation of PaCa cell lines in a dose-dependent manner, whereas wogonin and baicalin exhibited a much less robust effect. Treatment with baicalein induced apoptosis with release of cytochrome c from mitochondria, and activation of caspase-3 and -7 and PARP. The general caspase inhibitor zVAD-fmk reversed baicalein-induced apoptosis, indicating a caspase-dependent mechanism. Baicalein decreased expression of Mcl-1, an anti-apoptotic member of the Bcl-2 protein family, presumably through a transcriptional mechanism. Genetic knockdown of Mcl-1 resulted in marked induction of apoptosis. The effect of baicalein on apoptosis was significantly attenuated by Mcl-1 over-expression, suggesting a critical role of Mcl-1 in this process. Our results provide evidence that baicalein induces apoptosis in pancreatic cancer cells through down-regulation of the anti-apoptotic Mcl-1 protein.

Ellagitannin-rich pomegranate extract inhibits angiogenesis in prostate cancer in vitro and in vivo.

Angiogenesis is critical to tumor growth and is stimulated by tissue hypoxia due to poor oxygen delivery. In turn, cellular hypoxia leads to angiogenesis via the induction of hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) at a cellular level. Pomegranate juice and extracts, which are rich sources of ellagitannins, have been shown to have chemopreventive potential against prostate cancer, but there have been no studies on the effects of an ellagitannin-rich pomegranate extract on angiogenesis. Human prostate cancer cells (LNCaP) and human umbilical vein endothelial cells (HUVEC) were incubated with a pomegranate extract standardized to ellagitannin content (POMx), under normoxic and hypoxic conditions in vitro. Human prostate cancer cells (LAPC4) were injected subcutaneously into severe combined immunodeficient (SCID) mice and the effects of oral administration of POMx on tumor growth, microvessel density, and HIF-1alpha and VEGF expression were determined after 4 weeks of treatment. POMx inhibited the proliferation of LNCaP and HUVEC cells significantly under both normoxic and hypoxic conditions. HIF-1alpha and VEGF protein levels were also reduced by POMx under hypoxic conditions. POMx decreased prostate cancer xenograft size, tumor vessel density, VEGF peptide levels and HIF-1alpha expression after 4 weeks of treatment in SCID mice. These results demonstrate that an ellagitannin-rich pomegranate extract can inhibit tumor-associated angiogenesis as one of several potential mechanisms for slowing the growth of prostate cancer in chemopreventive applications. Further studies in humans are needed to confirm that angiogenesis can be inhibited by an ellagitannin-rich pomegranate extract administered orally as a dietary supplement.

Pomegranate ellagitannin-derived metabolites inhibit prostate cancer growth and localize to the mouse prostate gland.

Our group has shown in a phase II clinical trial that pomegranate juice (PJ) increases prostate specific antigen (PSA) doubling time in prostate cancer (CaP) patients with a rising PSA. Ellagitannins (ETs) are the most abundant polyphenols present in PJ and contribute greatly towards its reported biological properties. On consumption, ETs hydrolyze to release ellagic acid (EA), which is then converted by gut microflora to 3,8-dihydroxy-6H-dibenzo[b, d]pyran-6-one (urolithin A, UA) derivatives. Despite the accumulating knowledge of ET metabolism in animals and humans, there is no available data on the pharmacokinetics and tissue disposition of urolithins. Using a standardized ET-enriched pomegranate extract (PE), we sought to further define the metabolism and tissue distribution of ET metabolites. PE and UA (synthesized in our laboratory) were administered to C57BL/6 wild-type male mice, and metabolite levels in plasma and tissues were determined over 24 h. ET metabolites were concentrated at higher levels in mouse prostate, colon, and intestinal tissues as compared to other tissues after administration of PE or UA. We also evaluated the effects of PE on CaP growth in severe combined immunodeficient (SCID) mice injected subcutaneously with human CaP cells (LAPC-4). PE significantly inhibited LAPC-4 xenograft growth in SCID mice as compared to vehicle control. Finally, EA and several synthesized urolithins were shown to inhibit the growth of human CaP cells in vitro. The chemopreventive potential of pomegranate ETs and localization of their bioactive metabolites in mouse prostate tissue suggest that pomegranate may play a role in CaP treatment and chemoprevention. This warrants future human tissue bioavailability studies and further clinical studies in men with CaP.

Tea polyphenols and theaflavins are present in prostate tissue of humans and mice after green and black tea consumption.

Green and black tea have shown promise in the chemoprevention of prostate cancer. The objective of this study was to determine the bioavailability and bioactivity of tea polyphenols (PP) and theaflavins in human serum and human and mouse tissues. A decaffeinated black tea diet was administered to C57BL/6 mice. PPs and theaflavins were found in the small and large intestine, liver, and prostate in conjugated and free forms. The relative prostate bioavailability of theaflavin was 70% higher than that of epigallocatechin gallate (EGCG). In the second mouse study, a green tea (GT) diet was administered followed by the control diet for 1-5 d. Epicatechin (EC), EGCG, and epicatechin gallate (ECG) concentrations in prostate tissue were significantly decreased after 1 d of consuming the control diet. Epigallocatechin gallate (EGC), however, did not decrease significantly. For the human study, 20 men scheduled for surgical prostatectomy were randomly assigned to consume 1.42 L daily of GT, BT, or a caffeine-matched soda control (SC) for 5 d before radical prostatectomy. Tea PPs were greater in prostate samples from men consuming BT and GT than in men consuming SC (P = 0.0025). Although tea PP were not detectable in serum, ex vivo LNCaP prostate cancer cell proliferation was less when cells were grown in media containing patient serum collected after BT (P < 0.001) and GT (P = 0.025) consumption relative to baseline serum This is the first human study to show that tea polyphenols and theaflavins are bioavailable in the prostate where they may be active in the prevention of prostate cancer.

Nutrient-gene interaction: metabolic genotype-phenotype relationship.

The U.S. Department of Health and Human Services (DHHS)/USDA Dietary Guidelines for Americans is a science and population evidence-based guide on diet and physical activity, providing advice and recommendations to promote a healthier lifestyle and reduce the risk of chronic diseases, including cancer. These recommendations are supported by the comprehensive evidence-based review on diet and cancer prevention conducted by the American Institute for Cancer Research, National Cancer Institute, World Health Organization/International Agency for Research on Cancer, and others. However, influencing dietary effects are the individual genetic predispositions that are the basis for considerable interindividual variations in cancer risk within the population and in nutrient homeostasis, which is maintained by genomic-nutrient and metabolic-phenotype interactions. Although genetics is an important component, it accounts for only a portion of this variation. An individual's overall phenotype, including health status, is achieved and maintained by the sum of metabolic activities functioning under differing circumstances within the life cycle and the complex interactions among genotype, metabolic phenotype, and the environment. In this postgenomic era, high-throughput groups of technologies in genomics, proteomics, and metabolomics measure and analyze DNA sequences, RNA transcripts, proteins, and nutrient-metabolic fluxes in a single experiment. These advances have transformed biomarker studies on nutrient-gene interactions from a reductionist concept into a holistic practice in which many regulated genes involved in metabolism, along with its metabolic phenotypes, can be measured through functional genomics and metabolic profiling. The overall integration of data and information from the building blocks of metabolism-based nutrient-gene interaction can lead to future individualized dietary recommendations to diminish cancer risk.

Vitamin D and colon carcinogenesis.

Colorectal cancer is the third most commonly occurring cancer in the United States and accounts for approximately 11% of cancer deaths. Many epidemiological studies have shown an association between dietary factors, including calcium and vitamin D, and the incidence of colon cancer. Recently the Calcium Polyp Prevention Study demonstrated that calcium supplementation can reduce the recurrence of colon polyps, but the effect depends on serum vitamin D levels. We used the Apc(min) mouse model of intestinal cancer to investigate the effects of vitamin D treatment and calcium intake independently on polyp formation. We found that 1,25-dihydroxycholecaliferol was potent in inhibiting tumor load; however, the dose used to achieve this antiproliferative effect led to deleterious effects on serum calcium homeostasis. These effects were minimized by use of a synthetic analogue with reduced toxicity. Additionally, we tested the effect of a modified-calcium diet in Apc(min) mice but did not find a protective effect, perhaps because of a reduction in circulating levels of 25-hydroxycholecaliferol with increasing levels of dietary calcium. A number of other studies that use rodent models with vitamin D supplementation or deficiency illustrate the efficacy of vitamin D in colon cancer prevention. The mechanisms of direct action of vitamin D on colonic epithelium include regulation of growth factor and cytokine synthesis and signaling, as well as modulation of the cell cycle, apoptosis, and differentiation. Because of the apparent synergistic effect of vitamin D and calcium, cosupplementation of both nutrients in cancer prevention programs may be advised.

Intestinal polyp formation in the Apcmin mouse: effects of levels of dietary calcium and altered vitamin D homeostasis.

This study evaluated the effects of various levels of dietary calcium on polyp formation, vitamin D homeostasis, and fecal bile acids in the Apcmin mouse. Female Apcmin mice were randomized to three groups and fed a purified diet with either half or double the level of calcium in control AIN-93G. Serum 25-OH-D and fecal bile acids were measured at weeks 0 and 12 of treatment. Mice were killed for polyp scoring by two observers blinded to treatment after 12 weeks. Results show there was no difference in polyp number or tumor load with dietary calcium in any treatment group. Serum 25-OH-D was reduced and total fecal bile acids were increased in animals that received the high calcium diet. We have previously shown that vitamin D supplementation diminishes polyp load; the lack of effect of an altered calcium diet seen here may be due to a disturbance in vitamin D homeostasis.

Antidiabetic actions of arachidonic acid and zinc in genetically diabetic Goto-Kakizaki rats.

In previous studies, we showed that feeding arachidonic acid (AA) supplemented with a fixed amount of zinc lowered blood glucose concentrations in the fed state and water intake in rats with streptozotocin-induced diabetes. The present study was designed to determine dose-dependent effects of AA supplemented with a fixed amount of zinc on fed blood glucose levels, water intake, and glucose tolerance in genetically type 2 diabetic Goto-Kakizaki (G-K) Wistar rats. In an acute study, 20 mg/kg AA plus 10 mg/kg zinc administered via gastric gavage significantly improved oral glucose tolerance in G-K rats when compared to rats given distilled water (DW) only. When rats were treated chronically (2 weeks) with increasing doses of AA in drinking water, fed blood glucose concentrations and water intake were maximally decreased with diets containing 20 or 30 mg/L AA plus 10 mg/L zinc. Three-hour average area-above-fasting glucose concentrations (TAFGC; index of oral glucose tolerance) in diabetic G-K rats treated with 10, 20, or 30 mg/L AA plus 10 mg/L zinc for 2 weeks were significantly decreased relative to DW-treated rats. The effect on TAFGC values was maintained for an additional 2 weeks after cessation of treatment. Plasma insulin levels significantly increased in rats treated with 20 mg/L AA only or 10 mg/L AA plus 10 mg/L zinc, but not in rats treated with 20 or 30 mg/L AA plus 10 mg/L zinc, which are the most effective doses for the improvement of clinical signs of diabetes in G-K rats. In in vitro assays, 0.2 mg/mL AA in the incubation media was optimal for glucose uptake in isolated soleus muscle slices. These results suggest that treatment of genetically diabetic G-K rats with AA plus zinc lowers blood glucose levels via improvement of insulin sensitivity.