Sugars measured enzymatically in a fasting overnight urine sample are not sensitive biomarkers of dietary added sugar intake in postmenopausal women.

BACKGROUND: Restricting dietary sugar is a leading recommendation, but limited biomarkers assessing intake exist. Although 24-h urinary sucrose (U-Suc) and urinary fructose (U-Fruc) excretion has been used with mixed success, collection is burdensome. AIM: This study aimed to test the sensitivity of an enzymatic assay of U-Suc and U-Fruc to detect changing added sugar intake using low-burden overnight urine samples in 30 postmenopausal women. METHODS: Women consumed usual dietary intake during day 1 and usual intake plus a sugar sweetened beverage during day 2. Weighed, photographed food records assessed intake. Enzymatic assay measured U-Suc and U-Fruc from fasting overnight samples; liquid chromatography mass spectrometry (LC-MS) validated U-Suc findings. RESULTS: Dietary added sugars increased significantly during day 2 (p < 0.001), but urinary sugars were not significantly increased. Enzymatic assay detected urinary sugars in 75% (U-Suc) and 35% (U-Fruc) of samples. Dietary sucrose was not associated with U-Suc, however dietary fructose was significantly associated with U-Fruc [ß = 0.031; p < 0.05] among women with detectable urinary sugars. Participants with detectable U-Fruc consumed more energy from added sugars [12.6% kcal day 1; 21.5% kcal day 2] than participants with undetectable U-Fruc [9.3% kcal day 1; 17.4% kcal day 2], p < 0.05. Using LC-MS, U-Suc predicted sucrose and added sugar intake [ß = 0.017, ß = 0.013 respectively; both p < 0.05]. CONCLUSIONS: Urinary sugars measured enzymatically from overnight urine samples were not sensitive biomarkers of changing added sugar intake in postmenopausal women. However, urinary fructose measured by enzymatic assay or LC-MS may differentiate low versus high added sugar consumers.

The NCI-N87 Cell Line as a Gastric Epithelial Model to Study Cellular Uptake, Trans-Epithelial Transport, and Gastric Anti-Inflammatory Properties of Anthocyanins.

Anthocyanins are ubiquitous plant pigments with reported antioxidant, anti-inflammatory, and anti-cancer activities. To better understand these benefits, metabolism of anthocyanins requires further evaluation, especially in the stomach. Mammalian cell cultures provide useful models for investigating compound metabolism and absorption, but they are generally maintained at physiological pH. The NCI-N87 cell line is an acid-stable model of the gastric epithelium used to study gastric drug metabolism. The objective of this work was to investigate the uptake, trans-epithelial transport, and anti-inflammatory activity of anthocyanins by the NCI-N87 cell line. The cells formed a coherent monolayer, stable ≤32 days post confluency. Minimal effects on monolayer integrity were observed when the pH of the apical chamber was adjusted to pH 3.0, 5.0, or 7.4. Anthocyanins were transported across the NCI-N87 cell monolayer at 37 °C, but not at 0 °C, suggesting a facilitated process. Chokeberry anthocyanins (0-1500 µM) were not cytotoxic. At apical pH 3.0, they had anti-inflammatory properties by significantly attenuating IL-8 secretion when added to medium before, during, and after incubation with IL-1ß. These results suggest that the NCI-N87 cell line is a physiologically relevant model for in vitro studies of the transport, anti-inflammatory and potential anti-carcinogenic activities of anthocyanins in gastric tissue.

AFAP-110 is overexpressed in prostate cancer and contributes to tumorigenic growth by regulating focal contacts.

The actin filament-associated protein AFAP-110 is an actin cross-linking protein first identified as a substrate of the viral oncogene v-Src. AFAP-110 regulates actin cytoskeleton integrity but also functions as an adaptor protein that affects crosstalk between Src and PKC. Here we investigated the roles of AFAP-110 in the tumorigenic process of prostate carcinoma. Using immunohistochemistry of human tissue arrays, we found that AFAP-110 was absent or expressed at very low levels in normal prostatic epithelium and benign prostatic hyperplasia but significantly increased in prostate carcinomas. The level of AFAP-110 in carcinomas correlated with the Gleason scores. Downregulation of AFAP-110 in PC3 prostate cancer cells inhibited cell proliferation in vitro and tumorigenicity and growth in orthotopic nude mouse models. Furthermore, downmodulation of AFAP-110 resulted in decreased cell-matrix adhesion and cell migration, defective focal adhesions, and reduced integrin beta1 expression. Reintroduction of avian AFAP-110 or a mutant disabling its interaction with Src restored these properties. However, expression of an AFAP-110 lacking the PKC-interacting domain failed to restore properties of parental cells. Thus, increased expression of AFAP-110 is associated with progressive stages of prostate cancer and is critical for tumorigenic growth, in part by regulating focal contacts in a PKC-dependent mechanism.

Green and black tea inhibit cytokine-induced IL-8 production and secretion in AGS gastric cancer cells via inhibition of NF-κB activity.

Consumption of tea is associated with a reduced risk for several gastrointestinal cancers. Inflammatory processes, such as secretion of IL-8 from the gastric epithelium in response to chronic chemokine or antigen exposure, serve both as a chemoattractant for white blood cells and a prerequisite for gastric carcinogenesis. In this study, the gastric adenocarcinoma cell line AGS was used to investigate the effect of green tea extract, black tea extract, and epigallocatechin gallate (EGCG), the most abundant catechin in tea, on cytokine-induced inflammation. AGS cells were stimulated with interleukin-1ß (IL-1ß) to initiate inflammation, followed by exposure to either tea extracts or EGCG. We found that both green and black tea extracts at concentrations of 20 and 2 µM total catechins, respectively, significantly (p < 0.05) inhibited IL-1ß-induced IL-8 production and secretion to a similar extent. Treatment of AGS cells with EGCG (8 µM) produced similar reductions in IL-1ß-induced IL-8 production and secretion. Inhibition of NF-κB activity was found to be responsible, in part, for these observed effects. Our findings demonstrate that both green and black tea extracts with distinctly different catechin profiles, are capable of disrupting the molecular link between inflammation and carcinogenesis via inhibition of NF-κB activity in AGS cells.

Knowledge, Attitudes, and Practices Regarding Dietary Sodium in College Students.

OBJECTIVE: Test a dietary sodium survey in a US adult population of college students using a survey previously validated in a non-US adult population. METHODS: Cross-sectional study of a convenience sample of college students from a Midwest (n = 168) and Pacific Island (n = 152) university. Main outcome measures were knowledge, attitudes, and practices regarding dietary sodium (38 items). Sum scores and percentages for constructs were calculated. A score <75% was considered unfavorable; t test or ANOVA were used to examine group differences. RESULTS: Midwest students were primarily non-Hispanic White individuals (81%) and 65% female. Pacific Island students were predominantly Asian (51%) and 66% female. Mean ± SD construct scores (percentage) for knowledge, attitudes, and practices were 58.69 ± 10.62, 63.96 ± 16.18, 66.00 ± 12.34 (Midwest) and 57.54 ± 10.93, 64.84 ± 14.96, 64.94 ± 13.18 (Pacific Island), respectively; there were no significant differences between schools or race. CONCLUSIONS AND IMPLICATIONS: College students scored low in knowledge, attitudes, and practices regarding sodium. Results from this formative study may inform assessment strategies in future dietary sodium interventions.

Basal cell induced differentiation of noncancerous prostate epithelial cells (RWPE-1) by glycitein.

Increased consumption of soy and soy isoflavones is associated with a reduced risk for prostate cancer (PCa). PCa progression is characterized, in part, by a loss of luminal/basal epithelial differentiation; however, the effects of soy isoflavones on cellular differentiation in the prostate are unknown. The present study examined the effects of the soy isoflavone glycitein on cellular differentiation in prostate epithelial cells (RWPE-1, WPE1-NB14, and RWPE-2). Glycitein significantly inhibited RWPE-1 cellular proliferation at concentrations ranging from 0.4 to 50 microM. Expression of the luminal epithelial cell marker cytokeratin 18 was not affected by glycitein treatment in the WPE1-NB14 and RWPE-2 cell lines. However, expression of cytokeratin 18 and prostate specific antigen (PSA) was decreased in the RWPE-1 cell line in response to glycitein treatment, whereas the expression of the basal epithelial cell markers p63 and cytokeratin 5 remained unchanged. These data suggest that glycitein may induce basal cell differentiation in the RWPE-1 cell line.

Epigallocatechin-3-gallate (EGCG) inhibits PC-3 prostate cancer cell proliferation via MEK-independent ERK1/2 activation.

Epigallocatechin-3-gallate (EGCG), a tea polyphenol, inhibits the proliferation of many cancer cell lines; however, the antiproliferative mechanism(s) are not well-characterized. The objective of this study is to identify the cellular signaling mechanism(s) responsible for the antiproliferative effects of EGCG in the PC-3 prostate cancer cell line. EGCG inhibited PC-3 cell proliferation in a concentration-dependent manner with an IC(50) value of 39.0 microM, but had no effect on the proliferation of a nontumorigenic prostate epithelial cell line (RWPE-1). Treatment of PC-3 cells with EGCG (0-50 microM) resulted in time and concentration-dependent activation of the extracellular signal-regulated kinase (ERK1/2) pathway. EGCG treatment did not induce ERK1/2 activity in RWPE-1 cells. The activation of ERK1/2 by EGCG was not inhibited using PD98059, a potent inhibitor of mitogen-activated protein kinase kinase (MEK), the immediate upstream kinase responsible for ERK1/2 activation; suggesting a MEK-independent signaling mechanism. Pretreatment of PC-3 cells with a phosphoinositide-3 kinase (PI3K) inhibitor partially reduced both EGCG-induced ERK1/2 activation and the antiproliferative effects of this polyphenol. These results suggest that ERK1/2 activation via a MEK-independent, PI3-K-dependent signaling pathway is partially responsible for the antiproliferative effects of EGCG in PC-3 cells.

Apoptotic effects of dietary and synthetic sphingolipids in androgen-independent (PC-3) prostate cancer cells.

Stress-induced activation and metabolism of plasma membrane sphingolipids results in intracellular ceramide accumulation and has been shown to induce apoptosis in human prostate cancer cells. This effect has been observed using synthetic ceramide analogs, such as C6-ceramide; however, the effects of naturally-occurring sphingolipids, such as C18-ceramide and sphingomyelin (CerPCho), on apoptosis and prostate cancer cell proliferation have not been examined. The results of the present study demonstrate that natural (CerPCho, C18-ceramide) and synthetic (C6-ceramide) sphingolipids reduced PC-3 cell proliferation by 15 +/- 1.8, 17 +/- 2.5, and 46 +/- 2.1%, respectively (P < 0.05). These reductions in proliferation were due, in part, to increased cellular apoptosis. Treatment of PC-3 cells with CerPCho and C18-ceramide significantly increased apoptosis by 3.0 +/- 0.8 and 3.6 +/- 0.6%, respectively, compared to the untreated control, while the synthetic C6-ceramide significantly increased apoptosis by 55.7 +/- 0.4%. C6-ceramide-induced apoptosis was associated with cell cycle arrest in the G(2)/M phase, decreased extracellular signal-regulated kinase (ERK1/2) signaling and activation of the cell cycle regulatory protein, retinoblastoma (pRb). Treatment of PC-3 cells with C18-ceramide and CerPCho did not alter cell cycle distribution, pRb or ERK1/2 activation. Taken together, these results suggest that natural and synthetic sphingolipids induce apoptosis in PC-3 cells via distinct signaling mechanisms and potencies.

Effect of grape polyphenols on oxidative stress in canine lens epithelial cells.

OBJECTIVE: To evaluate whether the effects of oxidative stress could be attenuated in cultures of canine lens epithelial cells (LECs) by incubation with grape seed proanthocyanidin extract (GSE), resveratrol (RES), or a combination of both (GSE+RES). SAMPLE POPULATION: Primary cultures of canine LECs. PROCEDURES: LECs were exposed to 100MM tertiary butyl-hydroperoxide (TBHP) with or without GSE, RES, or GSE+RES. The dichlorofluorescein assay was used to detect production of reactive oxygen species (ROS), and immunoblot analysis was used to evaluate the expression of stress-induced cell-signaling markers (ie, the mitogen-activated protein kinase [MAPK] and phosphoinositide-3 kinase [PI3K] pathways). RESULTS: GSE and GSE+RES significantly reduced ROS production after a 30-minute exposure to TBHP. Only GSE significantly reduced ROS production after a 120-minute exposure to TBHP. Incubation with GSE reduced TBHP-induced activity of the MAPK and PI3K pathways. CONCLUSIONS AND CLINICAL RELEVANCE: GSE inhibited key components associated with cataractogenesis, ROS production, and stress-induced cell signaling. On the basis of the data reported here, there is strong evidence that GSE could potentially protect LECs from the damaging effects of oxidative stress.

Glycitein activates extracellular signal-regulated kinase via vascular endothelial growth factor receptor signaling in nontumorigenic (RWPE-1) prostate epithelial cells.

Increased consumption of soy is associated with a decreased risk for prostate cancer; however, the specific cellular mechanisms responsible for this anticancer activity are unknown. Dietary modulation of signaling cascades controlling cellular growth, proliferation and differentiation has emerged as a potential chemopreventive mechanism. The present study examined the effects of four soy isoflavones (genistein, daidzein, glycitein and equol) on extracellularsignal-regulated kinase (ERK1/2) activity in a nontumorigenic prostate epithelial cell line (RWPE-1). All four isoflavones (10 micromol/L) significantly increased ERK1/2 activity in RWPE-1 cells, as determined by immunoblotting. Isoflavone-induced ERK1/2 activation was rapid and sustained for approximately 2 h posttreatment. Glycitein, the most potent activator of ERK1/2, decreased RWPE-1 cell proliferation by 40% (P<.01). Glycitein-induced ERK1/2 activation was dependent, in part, on tyrosine kinase activity associated with vascular endothelial growth factor receptor (VEGFR). The presence of both VEGFR1 and VEGFR2 in the RWPE-1 cell line was confirmed by immunocytochemistry. Treatment of RWPE-1 cells with VEGF(165) resulted in transient ERK1/2 activation and increased cellular proliferation. The ability of isoflavones to modulate ERK1/2 signaling cascade via VEGFR signaling in the prostate may be responsible, in part, for the anticancer activity of soy.

Catechin degradation with concurrent formation of homo- and heterocatechin dimers during in vitro digestion.

Catechins were subjected to in vitro gastric and small intestinal digestion. EGCG, EGC, and ECG were significantly degraded at all concentrations tested, with losses of 71-91, 72-100, and 60-61%, respectively. EC and C were comparatively stable, with losses of 8-11 and 7-8%, respectively. HLPC-ESI-MS/MS indicated that EGCG degradation under simulated digestion resulted in production of theasinensins (THSNs) A and D (m/z 913) and P-2 (m/z 883), its autoxidation homodimers. EGC dimerization produced the homodimers THSN C and E (m/z 609) and homodimers analogous to P-2 (m/z 579). ECG homodimers were not observed. EGCG and EGC formed heterodimers analogous to the THSNs (m/z 761) and P-2 (m/z 731). EGCG and ECG formed homodimers analogous to the THSNs (m/z 897). This study provides an expanded profile of catechin dimers of digestive origin that may potentially form following consumption of catechins. These data provide a logical basis for initial screening to detect catechin digestive products in vivo.

Regulation of gap junction intercellular communication in primary canine lens epithelial cells: role of protein kinase C.

PURPOSE: Gap junction intercellular communication (GJIC) is important in maintaining lens epithelial cell homeostasis and reductions in GJIC may be associated with the development of cataract. Protein kinase C (PKC) activation can disrupt gap junction communication via phosphorylation of connexin 43 (C x 43) proteins that compose gap junction channels. This study examined the role of PKC activation in modulating GJIC in a primary canine lens epithelial cell (LEC) line. METHODS: TPA (12-O-tetradecanoyl-phorbol-acetate), a potent PKC activator and inhibitor of GJIC, was utilized in the present study. Primary cultures of canine LEC were treated with TPA (0-1000 ng/ml) for 0.5 hr and GJIC was assessed by scrape loading/dye transfer (SL/DT), and immunoblotting to detect phosphorylation of C x 43 protein. Inhibition of general and calcium-dependent PKC activity was achieved by pretreatment of cells with GF109203X and Gö6976, respectively. RESULTS: Treatment with TPA (1-1000 ng/ml) significantly decreased GJIC in canine LEC as assessed by SL/DT. Pretreatment with 10 and 100 ng/ml TPA decreased GJIC by 80% as compared to controls and increased Cx43 phosphorylation as assessed by immunoblotting. Pretreatment of cells with GF109203X and Gö6976, partially restored TPA-inhibited GJIC by 40% and 60%, respectively, and reduced C x 43 phosphorylation. Expression of calcium dependent PKC isoforms was detected in canine whole lens and LEC. CONCLUSIONS: Treatment with TPA significantly reduces GJIC in canine LEC. These effects are mediated, in part, by activation of calcium-dependent PKC isoforms. Primary canine LEC are a useful model in the study of the molecular mechanisms involved in GJIC and cataractogenesis.

Genistein modulates prostate epithelial cell proliferation via estrogen- and extracellular signal-regulated kinase-dependent pathways.

Epidemiological evidence suggests that consumption of soy is associated with a decreased risk for prostate cancer. Genistein, the most abundant isoflavone present in soy, is thought to be responsible, in part, for these anticancer effects. The present study examined the effects of genistein on cellular proliferation, extracellular signal-regulated kinase (ERK1/2) activity and apoptosis in a nontumorigenic human prostate epithelial cell line (RWPE-1). Low concentrations of genistein (0-12.5 micromol/L) significantly increased cell proliferation and ERK1/2 activity (P<.01) in RWPE-1 cells, while higher concentrations (50 and 100 micromol/L) of genistein significantly inhibited cell proliferation and ERK1/2 activity (P<.001). A similar biphasic effect of genistein on MEK1 activity, an ERK1/2 kinase, was also observed. Pretreatment of cells with a MEK1 inhibitor (PD 098059) significantly blocked genistein-induced proliferation and ERK1/2 activity (P<.01). In addition, treatment of cells with ICI 182,780, a pure antiestrogen, inhibited genistein-induced RWPE-1 proliferation and ERK1/2 signaling. Taken together, these results suggest that genistein modulates RWPE-1 cell proliferation and signal transduction via an estrogen-dependent pathway involving ERK1/2 activation.

Comparison of effects of high-pressure processing and heat treatment on immunoactivity of bovine milk immunoglobulin G in enriched soymilk under equivalent microbial inactivation levels.

Immunoglobulin-rich foods may provide health benefits to consumers. To extend the refrigerated shelf life of functional foods enriched with bovine immunoglobulin G (IgG), nonthermal alternatives such as high-pressure processing (HPP) may offer advantages to thermal processing for microbial reduction. To evaluate the effects of HPP on the immunoactivity of bovine IgG, a soymilk product enriched with milk protein concentrates, derived from dairy cows that were hyperimmunized with 26 human pathogens, was subjected to HPP or heat treatment. To achieve a 5 log reduction in inoculated Escherichia coli 8739, the HPP or heat treatment requirements were 345 MPa for 4 min at 30 degrees C or for 20 s at 70 degrees C, respectively. To achieve a 5 log reduction in natural flora in the enriched soymilk, the HPP or heat treatments needed were 552 MPa for 4 min at 30 degrees C or for 120 s at 78.2 degrees C, respectively. At equivalent levels for a 5 log reduction in E. coli, HPP and heat treatment caused 25% and no detectable loss in bovine IgG activity, respectively. However, at equivalent levels for a 5 log reduction in natural flora, HPP and heat resulted in 65 and 85% loss of bovine IgG activity, respectively. Results of combined pressure-thermal kinetic studies of bovine milk IgG activity were provided to determine the optimal process conditions to preserve product function.

Resveratrol reduces oxidation and proliferation of human retinal pigment epithelial cells via extracellular signal-regulated kinase inhibition.

Epidemiological evidence suggests that moderate wine consumption and antioxidant-rich diets may protect against age-related macular degeneration (AMD), the leading cause of vision loss among the elderly. Development of AMD and other retinal diseases, such as proliferative vitreoretinopathy (PVR), is associated with oxidative stress in the retinal pigment epithelium (RPE), a cell layer responsible for maintaining the health of the retina by providing structural and nutritional support. We hypothesize that resveratrol, a red wine polyphenol, may be responsible, in part, for the health benefits of moderate red wine consumption on retinal disease. To test this hypothesis, the antioxidant and antiproliferative effects of resveratrol were examined in a human RPE cell line (designated ARPE-19). Cell proliferation was determined using the bromodeoxyuridine (BrdU) assay, intracellular oxidation was assessed by dichlorofluorescein fluorescence, and activation of the mitogen-activated protein kinase (MAPK) cascade was measured by immunoblotting. Treatment with 50 and 100 micromol/L resveratrol significantly reduced proliferation of RPE cells by 10% and 25%, respectively (P<0.05). This reduction in proliferation was not associated with resveratrol-induced cytotoxicity. Resveratrol (100 micromol/L) inhibited basal and H2O2-induced intracellular oxidation and protected RPE cells from H2O2-induced cell death. The observed reduction in cell proliferation was associated with inhibition of mitogen activated protein kinase/ERK (MEK) and extracellular signal-regulated kinase (ERK 1/2) activities at concentrations of resveratrol as low as 5 micromol/L. These results suggest that resveratrol can reduce oxidative stress and hyperproliferation of the RPE.

Effects of pulsed electric fields and heat treatment on stability and secondary structure of bovine immunoglobulin G.

Bovine immunoglobulin G (IgG) solutions were subjected to pulsed electric fields (PEF) or heat treatment to investigate the effect of processing on secondary structure monitored using circular dichroism spectrometry. Under heat treatment, the critical temperature for bovine IgG to change secondary structure at neutral pH in borate buffer is 72 degrees C. A conversion of the secondary structure from beta-sheets into random coils along with the loss of immunoactivity of bovine IgG was observed when heated at 82 degrees C for 120 s. In contrast, PEF treatment at 41.1 kV/cm for 54 mus with bipolar pulses (outlet at 43.8 degrees C) caused no detectable changes in the secondary structure or the thermal stability of secondary structure. A shape factor, S (200nm) over (217nm), ratio of magnitude of the positive CD band at 200 nm to that of the negative CD band at 217 nm, was closely correlated to the immunoactivity of bovine IgG (r(2) = 0.99) and quantifies changes of secondary structure.

Apoptotic and necrotic mechanisms of stress-induced human lens epithelial cell death.

Exposure to ultraviolet radiation (UVR) and reactive oxygen species (ROS) can damage the human lens and contribute to cataract formation. Recent evidence suggests that apoptosis in lens epithelial cells (LEC) is an initiating event in noncongenital cataract formation in humans and animals. The present study examines the cellular and molecular mechanisms by which environmental (ultraviolet B [UVB]) and chemical (hydrogen peroxide [H(2)O(2)], t-butyl hydroperoxide [TBHP]) stress induces cell death in an SV-40 immortalized human lens epithelial (HLE) cell line. Treatment of HLE cells with UVB, H(2)O(2), and TBHP significantly decreased cell density with LD50 values of 350 J/m(2), 500 muM, and 200 muM, respectively. Cellular morphology, DNA fragmentation, and annexin/propidium iodide staining consistent with apoptosis was observed only in UVB-treated cells, whereas lactate dehydrogenase (LDH) release was significantly higher in H(2)0(2)- and TBHP-treated cells. In addition, activation of apoptotic stress-signaling proteins, including c-Jun NH2-terminal kinase (JNK), caspase-3, and DNA fragmentation factor 45 (DFF45) was observed only in UVB-treated cells. Inhibition of JNK activity increased UVB-induced cell death, suggesting that this pathway may serve a prosurvival role in HLE cells. These findings suggest UVB predominantly induces apoptosis in HLE cells, whereas H(2)O(2) and TBHP induce necrosis.

Bovine pituitary extract provides remarkable protection against oxidative stress in human prostate epithelial cells.

Bovine pituitary extract (BPE) is routinely used as a mitogenic supplement in serum-free growth medium. In addition to its mitogenic activity, BPE contains a variety of growth factors and hormones with reported antioxidant activity. This study examines the antioxidant potential of BPE in nontumorigenic human prostate epithelial cells (RWPE-1). Treatment of RWPE-1 cells with BPE (50 microg/ml) provided significant protection against H(2)O(2)-induced cell death, deoxyribonucleic acid fragmentation, protein oxidation, and membrane damage. Treatment with heat (71 degrees C, 10 min) and proteolytic enzymes reduced the antioxidant activity of BPE, suggesting that proteins present in BPE may be responsible for the antioxidant activity. Residual catalase activity present in BPE was responsible for a portion (30%) of the antioxidant activity. Interestingly, RWPE-1 cells treated with BPE and H(2)O(2) rapidly accumulated intracellular reactive oxygen species (ROS) to a greater extent than cells receiving only H(2)O(2). Pretreatment of RWPE-1 cells with tyrosine kinase inhibitors (genistein, tyrphostin 47, and AG-1296) before the addition of H(2)O(2) diminished BPE protection against H(2)O(2)-induced cell death, whereas treatment with purified mitogens commonly found in BPE, growth hormone and basic fibroblast growth factor, did not protect against oxidative damage. Taken together, these data suggest that BPE contains proteins or protein complexes with remarkable antioxidant activity. These yet unidentified compounds appear to confer protection against H(2)O(2)-induced cell death by tyrosine kinase-dependent pathways that increase intracellular ROS generation. The antioxidant activity of BPE may represent a confounding variable when studying oxidative stress in cells maintained in BPE-supplemented serum-free medium.

NMR-based metabolomic investigation of bioactivity of chemical constituents in black raspberry (Rubus occidentalis L.) fruit extracts.

Black raspberry (Rubus occidentalis L.) (BR) fruit extracts with differing compound profiles have shown variable antiproliferative activities against HT-29 colon cancer cell lines. This study used partial least-squares (PLS) regression analysis to develop a high-resolution (1)H NMR-based multivariate statistical model for discerning the biological activity of BR constituents. This model identified specific bioactive compounds and ascertained their relative contribution against cancer cell proliferation. Cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside were the predominant contributors to the extract bioactivity, but salicylic acid derivatives (e.g., salicylic acid glucosyl ester), quercetin 3-glucoside, quercetin 3-rutinoside, p-coumaric acid, epicatechin, methyl ellagic acid derivatives (e.g., methyl ellagic acetyl pentose), and citric acid derivatives also contributed significantly to the antiproliferative activity of the berry extracts. This approach enabled the identification of new bioactive components in BR fruits and demonstrates the utility of the method for assessing chemopreventive compounds in foods and food products.

Effect of black raspberry ( Rubus occidentalis L.) extract variation conditioned by cultivar, production site, and fruit maturity stage on colon cancer cell proliferation.

Black raspberries have been shown to inhibit multiple stages of oral, esophageal, and colon cancer. The objective of this study was to evaluate how black raspberry extract variability conditioned by horticultural factors affected the antiproliferative activity of 75 black raspberry extracts using an in vitro colon cancer cell model. HT-29 cells grown in 96-well plates were treated with freeze-dried extracts at 0.6 and 1.2 mg of extract/mL of medium. Percent cell growth inhibition for each concentration of the extracts was determined using the sulforhodamine B assay. All extracts significantly inhibited the growth of HT-29 colon cancer cells in a dose-dependent manner. Cell proliferation was significantly influenced by cultivar, production site, and stage of maturity. The lack of correlation between growth inhibition and extract total phenolic and total monomeric anthocyanin assays suggested horticultural parameters influence bioactivity in a complex manner.