Peptides generated ex vivo from serum proteins by tumor-specific exopeptidases are not useful biomarkers in ovarian cancer

BACKGROUND: The serum peptidome may be a valuable source of diagnostic cancer biomarkers. Previous mass spectrometry (MS) studies have suggested that groups of related peptides discriminatory for different cancer types are generated ex vivo from abundant serum proteins by tumor-specific exopeptidases. We tested 2 complementary serum profiling strategies to see if similar peptides could be found that discriminate ovarian cancer from benign cases and healthy controls. METHODS: We subjected identically collected and processed serum samples from healthy volunteers and patients to automated polypeptide extraction on octadecylsilane-coated magnetic beads and separately on ZipTips before MALDI-TOF MS profiling at 2 centers. The 2 platforms were compared and case control profiling data analyzed to find altered MS peak intensities. We tested models built from training datasets for both methods for their ability to classify a blinded test set. RESULTS: Both profiling platforms had CVs of approximately 15% and could be applied for high-throughput analysis of clinical samples. The 2 methods generated overlapping peptide profiles, with some differences in peak intensity in different mass regions. In cross-validation, models from training data gave diagnostic accuracies up to 87% for discriminating malignant ovarian cancer from healthy controls and up to 81% for discriminating malignant from benign samples. Diagnostic accuracies up to 71% (malignant vs healthy) and up to 65% (malignant vs benign) were obtained when the models were validated on the blinded test set. CONCLUSIONS: For ovarian cancer, altered MALDI-TOF MS peptide profiles alone cannot be used for accurate diagnoses.

Variation in the ovarian reserve Is linked to alterations in intrafollicular estradiol production and ovarian biomarkers of follicular differentiation and oocyte quality in cattle

The mechanisms whereby the high variation in numbers of morphologically healthy oocytes and follicles in ovaries (ovarian reserve) may have an impact onovarian function, oocyte quality, and fertility are poorly understood. The objective was to determine whether previously validated biomarkers for follicular differentiation and function, as well as oocyte quality differed between cattle with low versus a high antral follicle count (AFC). Ovaries were removed (n = 5 per group) near the beginning of the nonovulatory follicular wave, before follicles could be identified via ultrasonography as being dominant, from heifers with high versus a low AFC. The F1, F2, and F3 follicles were dissected and diameters determined. Follicular fluid and thecal, granulosal, and cumulus cells and the oocyte were isolated and subjected to biomarker analyses. Although the size and numerous biomarkers of differentiation, such as mRNAs for the gonadotropin receptors, were similar, intrafollicular concentrations of estradiol and the abundance of mRNAs for CYP19A1 in granulosal cells and ESR1, ESR2, and CTSB in cumulus cells were greater, whereas mRNAs for AMH in granulosal cells and TBC1D1 in thecal cells were lower for animals with low versus a high AFC during follicle waves. Hence, variation in the ovarian reserve may have an impact on follicular function and oocyte quality via alterations in intrafollicular estradiol production and expression of key genes involved in follicle-stimulating hormone action (AMH) and estradiol (CYP19A1) production by granulosal cells, function and survival of thecal cells (TBC1D1), responsiveness of cumulus cells to estradiol (ESR1, ESR2), and cumulus cell determinants of oocyte quality (CTSB).

Clonal variation in acetylcholinesterase biomarkers and life history traits following OP exposure in Daphnia magna

Two clones of Daphnia magna (Standard and Ruth) were exposed for 7 days to sub-lethal concentrations of acephate (5.0 and 10.0 mg/L). Survivorship, individual growth, reproduction and the population growth rate (lambda) were evaluated over three weeks. Acetylcholinesterase (AChE) activity was measured on days 2, 7 and 21. Acephate exposure inhibited AChE activity but had no direct effect on life history (LH) traits. There was also no effect of clone on AChE activity, LH and lambda. However, a significant interaction between clone and acephate concentration was found on both fecundity and AChE inhibition at 48 h was associated with a decrease in lambda the Standard clone and an increase in lambda in clone Ruth. Therefore, our findings show that genotypic variation will influence the link between AChE activity and toxic effects at higher levels of biological organisation in D. magna. (c) 2007 Elsevier Inc. All rights reserved.

Effects of dredging in Goteborg Harbor, Sweden, assessed by biomarkers in eelpout (Zoarces viviparus)

We used a battery of biomarkers in fish to study the effects of the extensive dredging in Goteborg harbor situated at the river Gota alv estuary, Sweden. Eelpout (Zoarces viviparus) were sampled along a gradient into Goteborg harbor, both before and during the dredging. Biomarker responses in the eelpout before the dredging already indicated that fish in Goteborg harbor are chronically affected by pollutants under normal conditions compared to those in a reference area. However, the results during the dredging activities clearly show that fish were even more affected by remobilized pollutants. Elevated ethoxyresorufin-O-deethylase activities and cytochrome P4501A levels indicated exposure to polycyclic aromatic hydrocarbons. Elevated metallothionein gene expression indicated an increase in metal exposure. An increase in general cell toxicity, measured as a decrease in lysosomal membrane stability, as well as effects on the immune system also could be observed in eelpout sampled during the dredging. The results also suggest that dredging activities in the Gota alv estuary can affect larger parts of the Swedish western coast than originally anticipated. The present study demonstrates that the application of a set of biomarkers is a useful approach in monitoring the impact of anthropogenic activities on aquatic environments.

Longitudinal selenium status in healthy British adults: assessment using biochemical and molecular biomarkers

Human selenium (Se) requirements are currently based on biochemical markers of Se status. In rats, tissue glutathione peroxidase-1 (Gpx1) mRNA levels can be used effectively to determine Se requirements; blood Gpx1 mRNA levels decrease in Se-deficient rats, so molecular biology-based markers have potential for human nutrition assessment. To study the efficacy of molecular biology markers for assessing Se status in humans, we conducted a longitudinal study on 39 subjects (age 45 +/- 11) in Reading, UK. Diet diaries (5 day) and blood were obtained from each subject at 2, 8, 17 and 23 weeks, and plasma Se, glutathione peroxidase (Gpx3) enzyme activity, and selenoprotein mRNA levels were determined. There were no significant longitudinal effects on Se biomarkers. Se intake averaged 48 +/- 14 mu g/d. Plasma Se concentrations averaged 1.13 +/- 0.16 mu mol/l. Plasma Se v. energy-corrected Se intake (ng Se/kJ/d) was significantly correlated, but neither Gpx3 activity v. Se intake (ng Se/kJ/d) nor Gpx3 activity v. plasma Se was significantly correlated. Collectively, this indicates that subjects were on the plateaus of the response curves. Selenoprotein mRNAs were quantitated in total RNA isolated from whole blood, but mRNA levels for Gpx1, selenoprotein H, and selenoprotein W (all highly regulated by Se in rodents), as well selenoprotein P, Gpx3, and phospholipid hydroperoxide glutathione peroxidase were also not significantly correlated with plasma Se. Thus selenoprotein molecular biomarkers, as well as traditional biochemical markers, are unable to further distinguish differences in Se status in these Se replete subjects. The efficacy of molecular biomarkers to detect Se deficiency needs to be tested in Se-deficient populations.

Biomarkers of the intake of dietary polyphenols: strengths, limitations and application in nutrition research

In order to establish firm evidence for the health effects of dietary polyphenol consumption, it is essential to have quantitative information regarding their dietary intake. The usefulness of the current methods, which rely mainly on the assessment of polyphenol intake using food records and food composition tables, is limited as they fail to assess total intake accurately. This review highlights the problems associated with such methods with regard to polyphenol-intake predictions. We suggest that the development of biological biomarkers, measured in both blood and urine, are essential for making accurate estimates of polyphenol intake. However, the relationship between dietary intakes and nutritional biomarkers are often highly complex. This review identifies the criteria that must be considered in the development of such biomarkers. In addition, we provide an assessment of the limited number of potential biomarkers of polyphenol intake currently available.

Probiotics and cancer: from in vitro to human studies

Studies in cell cultures and animal models provide evidence that probiotics can beneficially influence various stages in development of colon cancer including tumor initiation, promotion and metastasis. For example, oral administration of Lactobacillus and Bifidobacterium strains can prevent genotoxic damage to the colonic epithelium (considered to be an early stage of the carcinogenic process). Administration to rats of probiotics reduced the incidence of carcinogen-induced pre-cancerous lesions (aberrant crypt foci) in the colon. Furthermore a combination of Bifidobacterium longum and inulin (a prebiotic) was more effective than either treatment alone. In this latter study, the dietary treatments were given after exposure to the carcinogen, which suggests that the protective effects were being exerted at the promotional phase of carcinogenesis. L. acidophilus feeding has been shown to decrease the incidence of colon tumors in rats challenged with a carcinogen and B. longum reduced the incidence of carcinogeninduced colon, liver and mammary tumors. There is limited evidence from epidemiological studies for protective effects of products containing probiotics in humans, but a number of recent dietary intervention studies in healthy subjects and in polyp and cancer patients have yielded promising results on the basis of biomarkers of cancer risk and grade of colorectal tumors.

Dietary synbiotics reduce cancer risk factors in polypectomized and colon cancer patients

Background: Animal studies suggest that prebiotics and probiotics exert protective effects against tumor development in the colon, but human data supporting this suggestion are weak. Objective: The objective was to verify whether the prebiotic concept (selective interaction with colonic flora of nondigested carbohydrates) as induced by a synbiotic preparation-oligofructose-enriched inulin (SYN1) + Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (BB12)-is able to reduce the risk of colon cancer in humans. Design: The 12-wk randomized, double-blind, placebo-controlled trial of a synbiotic food composed of the prebiotic SYN1 and probiotics LGG and BB12 was conducted in 37 colon cancer patients and 43 polypectomized patients. Fecal and blood samples were obtained before, during, and after the intervention, and colorectal biopsy samples were obtained before and after the intervention. The effect of synbiotic consumption on a battery of intermediate biomarkers for colon cancer was examined. Results: Synbiotic intervention resulted in significant changes in fecal flora: Bifidobacterium and Lactobacillus increased and Clostridium perfringens decreased. The intervention significantly reduced colorectal proliferation and the capacity of fecal water to induce necrosis in colonic cells and improve epithelial barrier function in polypectomized patients. Genotoxicity assays of colonic biopsy samples indicated a decreased exposure to genotoxins in polypectomized patients at the end of the intervention period. Synbiotic consumption prevented an increased secretion of interleukin 2 by peripheral blood mononuclear cells in the polypectomized patients and increased the production of interferon gamma in the cancer patients. Conclusions: Several colorectal cancer biomarkers can be altered favorably by synbiotic intervention.

Effect of apoE genotype and vitamin E on biomarkers of oxidative stress in cultured neuronal cells and the brain of targeted replacement mice

The aetiology of apoE4 genotype-Alzheimer's disease (AD) association are complex. The current study emphasizes the impact of apoE genotype and potential beneficial effects of vitamin E (VE) in relation to oxidative stress. Agonist induced neuronal cell death was examined 1) in the presence of conditioned media containing equal amounts of apoE3 or apoE4 obtained from stably transfected macrophages, and 2) after pretreatment with alpha- and gamma-tocopherol, and -tocotrienol. ApoE3 and apoE4 transgenic mice were fed a diet poor or rich in VE to study the interplay of both apoE genotype and VE status, on membrane lipid peroxidation, antioxidative enzyme activity and glutathione levels in the brain. Cytotoxicity of hydrogen peroxide and glutamate was higher in neuronal cells cultured with apoE4 than apoE3 conditioned media. VE pre-treatment of neurons counteracted the cytotoxicity of a peroxide challenge but not of nitric oxide. No significant effects of apoE genotype or VE supplementation were observed on lipid peroxidation or antioxidative status in the brain of apoE3 and apoE4 mice. VE protects against oxidative insults in vitro, however, no differences in brain oxidative status were observed in mice. Unlike in cultured cells, apoE4 may not contribute to higher neuronal oxidative stress in the brain of young targeted replacement mice.

Soy-isoflavone-enriched foods and inflammatory biomarkers of cardiovascular disease risk in postmenopausal women: interactions with genotype and equol production

Background: Dietary isoflavones are thought to be cardioprotective because of their structural similarity to estrogen. The reduction of concentrations of circulating inflammatory markers by estrogen may be one of the mechanisms by which premenopausal women are protected against cardiovascular disease. Objective: Our aim was to investigate the effects of isolated soy isoflavones on inflammatory biomarkers [von Willebrand factor, intracellular adhesion molecule 1, vascular cell adhesion molecule 1 (VCAM-1), E-selectin, monocyte chemoattractant protein 1, C-reactive protein (CRP), and endothelin 1 concentrations]. Differences with respect to single-nucleotide polymorphisms in selected genes [estrogen receptor alpha (XbaI and PvuII), estrogen receptor beta [ER beta (AluI) and ER beta[cx] (Tsp5091), endothelial nitric oxide synthase (Glu298Asp), apolipoprotein E (Apo E2, E3, and E4), and cholesteryl ester transfer protein (TaqIB)] and equol production were investigated. Design: One hundred seventeen healthy European postmenopausal women participated in this randomized, double-blind, placebo-controlled, crossover dietary intervention trial. Isoflavone-enriched (genistein-to-daidzein ratio of 2:1;50 mg/d) or placebo cereal bars were consumed for 8 wk, with a washout period of 8 wk between the crossover. Plasma inflammatory factors were measured at 0 and 8 wk of each study arm. Results: Isoflavones improved CRP concentrations [odds ratio (95% Cl) for CRP values >1 mg/L for isoflavone compared with placebo: 0.43 (0.27, 0.69)]; no significant effects of isoflavone treatment on other plasma inflammatory markers were observed. No significant differences in the response to isoflavones were observed according to subgroups of equol production. Differences in the VCAM-1 response to isoflavones and to placebo were found with ER beta AluI genotypes. Conclusion: Isoflavones have beneficial effects on CRP concentrations, but not on other inflammatory biomarkers of cardiovascular disease risk in postmenopausal women, and may improve VCAM-1 in an ER beta gene polymorphic subgroup.

Proteomic biomarkers of peripheral blood mononuclear cells obtained from postmenopausal women undergoing an intervention with soy isoflavones

Background: The incidence of cardiovascular diseases increases after menopause, and soy consumption is suggested to inhibit disease development. Objective: The objective was to identify biomarkers of response to a dietary supplementation with an isoflavone extract in postmenopausal women by proteome analysis of peripheral blood mononuclear cells. Design: The study with healthy postmenopausal woman was performed in a placebo-controlled sequential design. Peripheral mononuclear blood cells were collected from 10 volunteers after 8 wk of receiving daily 2 placebo cereal bars and after a subsequent 8 wk of intervention with 2 cereal bars each providing 25 mg of isoflavones. The proteome of the cells was visualized after 2-dimensional gel electrophoresis, and peptide mass fingerprinting served to identify proteins that by the intervention displayed altered protein concentrations. Results: Twenty-nine proteins were identified that showed significantly altered expression in the mononuclear blood cells under the soy-isoflavone intervention, including a variety of proteins involved in an antiinflammatory response. Heat shock protein 70 or a lymphocyte-specific protein phosphatase and proteins that promote increased fibrinolysis, such as a-enolase, were found at increased intensities, whereas those that mediate adhesion, migration, and proliferation of vascular smooth muscle cells, such as galectin-1, were found at reduced intensities after soy extract consumption. Conclusion: Protcome analysis identified in vivo markers that respond to a dietary intervention with isoflavone-enriched soy extract in postmenopausal women. The nature of the proteins identified suggests that soy isoflavones may increase the anti inflammatory response in blood mononuclear cells that might contribute to the atherosclerosis-preventive activities of a soy-rich diet.

Daily consumption of an aqueous green tea extract supplement does not impair liver function or alter cardiovascular disease risk biomarkers in healthy men

Regular consumption of green tea polyphenols (GTP) is thought to reduce the risk of cardiovascular disease (CVD) but has also been associated with liver toxicity. The present trial aimed to assess the safety and potential CVD health beneficial effects of daily GTP consumption. We conducted a placebo-controlled parallel study to evaluate the chronic effects of GTP on liver function and CVD risk biomarkers in healthy men. Volunteers (treatment: n = 17, BMI 26.7 +/- 3.3 kg/m(2), age 41 +/- 9 y; placebo, n = 16, BMI 25.4 +/- 3.3 kg/m(2), age 40 +/- 10 y) consumed for 3 wk 6 capsules per day (2 before each principal meal) containing green tea extracts (equivalent to 714 mg/d GTP) or placebo. At the beginning and end of the intervention period, we collected blood samples from fasting subjects and measured vascular tone using Laser Doppler lontophoresis. Biomarkers of liver function and CVD risk (including blood pressure, plasma lipids, and asymmetric dimethylarginine) were unaffected by GTP consumption. After treatment, the ratio of total:HDL cholesterol was significantly reduced in participants taking GTP capsules compared with baseline. Endothelial-dependent and -independent vascular reactivity did not significantly differ between treatments. In conclusion, the present data suggests that the daily consumption of high doses of GTP by healthy men for 3 wk is safe but without effects on CVD risk biomarkers other than the total:HDL cholesterol ratio. J. Nutr. 139: 58-62, 2009.

Antiproliferative activity of Titanocene Y against tumor colony-forming units

Bis-[(p-methoxybenzyl)cyclopentadienyl] titanium dichloride, better known as Titanocene Y, is a newly synthesized titanium-based anticancer drug. We studied the antitumor activity of Titanocene Y with concentrations of 2.1, 21 and 210 μmol/l against a range of freshly explanted human tumors, using an in-vitro soft agar cloning system. The sensitivity against Titanocene Y was highly remarkable in the case of renal cell, ovarian, nonsmall cell lung and colon cancer. In particular the surprisingly good response of nonsmall cell lung cancer and colon cancer against Titanocene Y at its lowest concentration of 2.1 μmol/l was well comparable or better with respect to cisplatin, given at a concentration of 1.0 μmol/l. Further clinical development of Titanocene Y appears to be warranted because of the broad cytotoxic activity shown and the specific activity of Titanocene Y against renal cell cancer.

Anti-tumor activity of Titanocene Y in xenografted Caki-1 tumors in mice

The benzyl-substituted unbridged titanocene bis-[(p-methoxybenzyl)cyclopentadienyl] titanium(IV) dichloride (Titanocene Y) was tested in vitro against human renal cancer cells (Caki-1), in which it showed an IC50 value of 36 x 10(-6) mol/l. Titanocene Y was then given in vivo in doses of 10, 20, 30, 40 and 50 mg/kg on 5 consecutive days to Caki-1-bearing mice, and it showed concentration-dependent and statistically significant tumor growth reduction with respect to a solvent-treated control cohort. The maximum tolerable dose of Titanocene Y was determined to be 40 mg/kg and it showed significantly better tumor volume growth reduction than cisplatin given at a dose of 2 mg/kg. This superior activity of Titanocene Y with respect to cisplatin will hopefully lead to clinical tests against metastatic renal cell cancer in the near future.

Antitumor activity of Titanocene Y against freshly explanted human breast tumor cells and in xenografted MCF-7 tumors in mice

Bis-[(p-methoxybenzyl)cyclopentadienyl] titanium dichloride, better known as Titanocene Y, is a newly synthesized transition metal-based anticancer drug. We studied the antitumor activity of Titanocene Y with concentrations of 2.1, 21 and 210 mu mol/l against a freshly explanted human breast cancer, using an in-vitro soft agar cloning system. The sensitivity against Titanocene Y was highly remarkable in the breast cancer tumor in the full concentration range. Titanocene Y showed cell death induction at 2.1 mu mol/l, well comparable to cisplatin, given at a concentration of 1.0 mu mol/l. A further preclinical development of Titanocene Y was warranted and therefore an MCF-7 human breast cancer xenograft nonobese diabetic/severe combined immunodeficient mouse model was used. Titanocene Y was given for 21 days at 30 mg/kg/ day (75% of the maximum tolerable dose of Titanocene Y), which resulted in the reduction of the tumor volume to around one-third, whereas no mouse was lost because of the surprisingly low toxicity of Titanocene Y.