David J. Boocock

Phase I Dose Escalation Pharmacokinetic Study in Healthy Volunteers of Resveratrol, a Potential Cancer Chemopreventive Agent

The red grape constituent resveratrol possesses cancer chemopreventive properties in rodents. The hypothesis was tested that, in healthy humans, p.o. administration of resveratrol is safe and results in measurable plasma levels of resveratrol. A phase I study of oral resveratrol (single doses of 0.5, 1, 2.5, or 5 g) was conducted in 10 healthy volunteers per dose level. Resveratrol and its metabolites were identified in plasma and urine by high-performance liquid chromatography-tandem mass spectrometry and quantitated by high-performance liquid chromatography-UV. Consumption of resveratrol did not cause serious adverse events. Resveratrol and six metabolites were recovered from plasma and urine. Peak plasma levels of resveratrol at the highest dose were 539 ± 384 ng/mL (2.4 μmol/L, mean ± SD; n = 10), which occurred 1.5 h post-dose. Peak levels of two monoglucuronides and resveratrol-3-sulfate were 3- to 8-fold higher. The area under the plasma concentration curve (AUC) values for resveratrol-3-sulfate and resveratrol monoglucuronides were up to 23 times greater than those of resveratrol. Urinary excretion of resveratrol and its metabolites was rapid, with 77% of all urinary agent-derived species excreted within 4 h after the lowest dose. Cancer chemopreventive effects of resveratrol in cells in vitro require levels of at least 5 μmol/L. The results presented here intimate that consumption of high-dose resveratrol might be insufficient to elicit systemic levels commensurate with cancer chemopreventive efficacy. However, the high systemic levels of resveratrol conjugate metabolites suggest that their cancer chemopreventive properties warrant investigation. (Cancer Epidemiol Biomarkers Prev 2007;16(6):1246–52)

Pharmacokinetics in mice and growth-inhibitory properties of the putative cancer chemopreventive agent resveratrol and the synthetic analogue trans 3,4,5,4′-tetramethoxystilbene

Resveratrol (trans-3,5,4′-trihydroxystilbene) is a naturally occurring polyphenol with cancer chemopreventive properties in preclinical models of carcinogenesis, including those of colorectal cancer. Recently, a variety of analogues of resveratrol have been synthesised and investigated in in vitro assays. One analogue, 3,4,5,4′-tetramethoxystilbene (DMU 212), showed preferential growth-inhibitory and proapoptotic properties in transformed cells, when compared with their untransformed counterparts. As part of a chemoprevention drug development programme, the pharmacokinetic properties of DMU 212 were compared with those of resveratrol in the plasma, liver, kidney, lung, heart, brain and small intestinal and colonic mucosa of mice. DMU 212 or resveratrol (240 mg kg−1) were administered intragastrically, and drug concentrations were measured by HPLC. Metabolites were characterised by cochromatography with authentic reference compounds and were identified by mass spectrometry. The ratios of area of plasma or tissue concentration vs time curves of resveratrol over DMU 212 (AUCres/AUCDMU212) for the plasma, liver, small intestinal and colonic mucosa were 3.5, 5, 0.1 and 0.15, respectively. Thus, resveratrol afforded significantly higher levels than DMU 212 in the plasma and liver, while DMU 212 exhibited superior availability compared to resveratrol in the small intestine and colon. Resveratrol was metabolised to its sulphate or glucuronate conjugates, while DMU 212 underwent metabolic hydroxylation or single and double O-demethylation. DMU 212 and resveratrol inhibited the growth of human-derived colon cancer cells HCA-7 and HT-29 in vitro with IC50 values of between 6 and 26 μM. In the light of the superior levels achieved in the gastrointestinal tract after the administration of DMU 212, when compared to resveratrol, the results provide a good rationale to evaluate DMU 212 as a colorectal cancer chemopreventive agent.

Pilot Study of Oral Silibinin, a Putative Chemopreventive Agent, in Colorectal Cancer Patients: Silibinin Levels in Plasma, Colorectum, and Liver and Their Pharmacodynamic Consequences

Silibinin, a flavonolignan from milk thistle, has intestinal cancer chemopreventive efficacy in rodents. It is a strong antioxidant and modulates the insulin-like growth factor (IGF) system by increasing circulating levels of IGF-binding protein 3 (IGFBP-3) and decreasing levels of IGF-I. Here, the hypothesis was tested that administration of oral silibinin generates agent levels in human blood and colorectal and hepatic tissues consistent with pharmacologic activity. Patients with confirmed colorectal adenocarcinoma received silibinin formulated with phosphatidylcholine (silipide) at dosages of 360, 720, or 1,440 mg silibinin daily for 7 days. Blood and biopsy samples of normal and malignant colorectum or liver were obtained before dosing, and blood and colorectal or hepatic tissues were collected at resection surgery after the final silipide dose. Levels of silibinin were quantified by high-pressure liquid chromatography-UV, and plasma metabolites were identified by liquid chromatography-mass spectrometry. Blood levels of IGFBP-3, IGF-I, and the oxidative DNA damage pyrimidopurinone adduct of deoxyguanosine (M1dG) were determined. Repeated administration of silipide was safe and achieved levels of silibinin of 0.3 to 4 μmol/L in the plasma, 0.3 to 2.5 nmol/g tissue in the liver, and 20 to 141 nmol/g tissue in colorectal tissue. Silibinin monoglucuronide, silibinin diglucuronide, silibinin monosulfate, and silibinin glucuronide sulfate were identified in the plasma. Intervention with silipide did not affect circulating levels of IGFBP-3, IGF-I, or M1dG. The high silibinin levels achieved in the human colorectal mucosa after consumption of safe silibinin doses support its further exploration as a potential human colorectal cancer chemopreventive agent.

Effect of cyanidin-3-glucoside and an anthocyanin mixture from bilberry on adenoma development in the ApcMin mouse model of intestinal carcinogenesis : Relationship with tissue anthocyanin levels

Anthocyanins are dietary flavonoids, which can prevent carcinogen‐induced colorectal cancer in rats. Here, the hypotheses were tested that Mirtoselect, an anthocyanin mixture from bilberry, or isolated cyanidin‐3‐glucoside (C3G), the most abundant anthocyanin in diet, interfere with intestinal adenoma formation in the ApcMin mouse, a genetic model of human familial adenomatous polyposis, and that consumption of C3G or Mirtoselect generates measurable levels of anthocyanins in the murine biophase. ApcMin mice ingested C3G or Mirtoselect at 0.03, 0.1 or 0.3% in the diet for 12 weeks, and intestinal adenomas were counted. Plasma, urine and intestinal mucosa were analyzed for presence of anthocyanins by high‐pressure liquid chromatography with detection by UV spectrophotometry (520 nm) or tandem mass spectrometry (multiple reaction monitoring). Ingestion of either C3G or Mirtoselect reduced adenoma load dose‐dependently. At the highest doses of C3G and Mirtoselect adenoma numbers were decreased by 45% (p < 0.001) or 30% (p < 0.05), respectively, compared to controls. Anthocyanins were found at the analytical detection limit in the plasma and at quantifiable levels in the intestinal mucosa and urine. Anthocyanin glucuronide and methyl metabolites were identified in intestine and urine. Total anthocyanin levels in mice on C3G or Mirtoselect were 43 ng and 8.1 μg/g tissue, respectively, in the intestinal mucosa, and 7.2 and 12.3 μg/ml in the urine. The efficacy of C3G and Mirtoselect in the ApcMin mouse renders the further development of anthocyanins as potential human colorectal cancer chemopreventive agents worthwhile.

Effect of cyanidin-3-glucoside and an anthocyanin mixture from bilberry on adenoma development in the ApcMin mouse model of intestinal carcinogenesis--relationship with tissue anthocyanin levels.

Anthocyanins are dietary flavonoids, which can prevent carcinogen‐induced colorectal cancer in rats. Here, the hypotheses were tested that Mirtoselect, an anthocyanin mixture from bilberry, or isolated cyanidin‐3‐glucoside (C3G), the most abundant anthocyanin in diet, interfere with intestinal adenoma formation in the ApcMin mouse, a genetic model of human familial adenomatous polyposis, and that consumption of C3G or Mirtoselect generates measurable levels of anthocyanins in the murine biophase. ApcMin mice ingested C3G or Mirtoselect at 0.03, 0.1 or 0.3% in the diet for 12 weeks, and intestinal adenomas were counted. Plasma, urine and intestinal mucosa were analyzed for presence of anthocyanins by high‐pressure liquid chromatography with detection by UV spectrophotometry (520 nm) or tandem mass spectrometry (multiple reaction monitoring). Ingestion of either C3G or Mirtoselect reduced adenoma load dose‐dependently. At the highest doses of C3G and Mirtoselect adenoma numbers were decreased by 45% (p < 0.001) or 30% (p < 0.05), respectively, compared to controls. Anthocyanins were found at the analytical detection limit in the plasma and at quantifiable levels in the intestinal mucosa and urine. Anthocyanin glucuronide and methyl metabolites were identified in intestine and urine. Total anthocyanin levels in mice on C3G or Mirtoselect were 43 ng and 8.1 μg/g tissue, respectively, in the intestinal mucosa, and 7.2 and 12.3 μg/ml in the urine. The efficacy of C3G and Mirtoselect in the ApcMin mouse renders the further development of anthocyanins as potential human colorectal cancer chemopreventive agents worthwhile.

Identification of SPARC-like 1 protein as part of a biomarker panel for Alzheimer's disease in cerebrospinal fluid.

We have used proteomic fingerprinting to investigate diagnosis of Alzheimers disease (AD). Samples of lumbar cerebrospinal fluid (CSF) from clinically-diagnosed AD cases (n = 33), age-matched controls (n = 20), and mild cognitive impairment (MCI) patients (n = 10) were used to obtain proteomic profiles, followed by bioinformatic analysis that generated a set of potential biomarkers in CSF samples that could discriminate AD cases from controls. The identity of the biomarker ions was determined using mass spectroscopy. The panel of seven peptide biomarker ions was able to discriminate AD patients from controls with a median accuracy of 95% (sensitivity 85%, specificity 97%). When this model was applied to an independent blind dataset from MCI patients, the intensity of signals was intermediate between the control and AD patients implying that these markers could potentially predict patients with early neurodegenerative disease. The panel were identified, in order of predictive ability, as SPARC-like 1 protein, fibrinogen alpha chain precursor, amyloid-β, apolipoprotein E precursor, serum albumin precursor, keratin type I cytoskeletal 9, and tetranectin. The 7 ion ANN model was further validated using an independent cohort of samples, where the model was able to classify AD cases from controls with median accuracy of 84.5% (sensitivity 93.3%, specificity 75.7%). Validation by immunoassay was performed on the top three identified markers using the discovery samples and an independent sample cohort which was from postmortem confirmed AD patients (n = 17).

Tamoxifen forms DNA adducts in human colon after administration of a single [14C]-labeled therapeutic dose.

Tamoxifen is widely prescribed for the treatment of breast cancer and is also licensed in the United States for the prevention of this disease. However, tamoxifen therapy is associated with an increased occurrence of endometrial cancer in women, and there is also evidence that it may elevate the risk of colorectal cancer. The underlying mechanisms responsible for tamoxifen-induced carcinogenesis in women have not yet been elucidated, but much interest has focused on the role of DNA adduct formation. We investigated the propensity of tamoxifen to bind irreversibly to colorectal DNA when given to 10 women as a single [(14)C]-labeled therapeutic (20 mg) dose, approximately 18 h before undergoing colon resections. Using the sensitive technique of accelerator mass spectrometry, coupled with high-performance liquid chromatography separation of enzymatically digested DNA, a peak corresponding to authentic dG-N(2)-tamoxifen adduct was detected in samples from three patients, at levels ranging from 1 to 7 adducts/10(9) nucleotides. No [(14)C]-radiolabel associated with tamoxifen or its major metabolites was detected. The presence of detectable CYP3A4 protein in all colon samples suggests that this tissue has the potential to activate tamoxifen to alpha-hydroxytamoxifen, in addition to that occurring in the systemic circulation, and direct interaction of this metabolite with DNA could account for the binding observed. Although the level of tamoxifen-induced damage displayed a degree of interindividual variability, when present, it was approximately 10 to 100 times higher than that reported for other suspect human colon carcinogens such as 2-amino-1-methyl-6-phenyimidazo[4,5-b]pyridine. These findings provide a mechanistic basis through which tamoxifen could increase the incidence of colon cancers in women.

Cytoplasmic PML promotes TGF-β-associated epithelial-mesenchymal transition and invasion in prostate cancer.

Epithelial–mesenchymal transition (EMT) is a key event that is involved in the invasion and dissemination of cancer cells. Although typically considered as having tumour-suppressive properties, transforming growth factor (TGF)-β signalling is altered during cancer and has been associated with the invasion of cancer cells and metastasis. In this study, we report a previously unknown role for the cytoplasmic promyelocytic leukaemia (cPML) tumour suppressor in TGF-β signalling-induced regulation of prostate cancer-associated EMT and invasion. We demonstrate that cPML promotes a mesenchymal phenotype and increases the invasiveness of prostate cancer cells. This event is associated with activation of TGF-β canonical signalling pathway through the induction of Sma and Mad related family 2 and 3 (SMAD2 and SMAD3) phosphorylation. Furthermore, the cytoplasmic localization of promyelocytic leukaemia (PML) is mediated by its nuclear export in a chromosomal maintenance 1 (CRM1)-dependent manner. This was clinically tested in prostate cancer tissue and shown that cytoplasmic PML and CRM1 co-expression correlates with reduced disease-specific survival. In summary, we provide evidence of dysfunctional TGF-β signalling occurring at an early stage in prostate cancer. We show that this disease pathway is mediated by cPML and CRM1 and results in a more aggressive cancer cell phenotype. We propose that the targeting of this pathway could be therapeutically exploited for clinical benefit.

Characterization of outer membrane vesicles from a neonatal meningitic strain of Cronobacter sakazakii

Cronobacter sakazakii is associated with severe and often fatal cases of infant meningitis and necrotizing enterocolitis. The form of meningitis differs from that due to Neisseria meningitidis and Streptococcus spp., in that it is highly invasive and destructive towards human brain cells. However, there is relatively little understanding of the cytopathogenic interaction of C. sakazakii with host cells which results in stimulation of an inflammatory immune response. The production of Cronobacter outer membrane vesicles (OMV) and their potential pathogenic functions have not yet been elucidated. This study is the first to show that C. sakazakii produce OMV, which may play a role in the activation of cytopathogenic and host cell responses on human intestinal epithelial cells. Cronobacter sakazakii strain 767 was used which had been isolated from a fatal outbreak of neonatal meningitis and necrotizing enterocolitis. Cronobacter sakazakii OMV were internalized by Caco-2 cells, increased cell proliferation and stimulated the hosts innate proinflammatory response without inducing overt toxicity. A total of 18 OMV-associated proteins were identified by mass spectrometry and their potential pathogenicity roles were evaluated. Collectively, these data indicate that C. sakazakii OMV could play a role in pathogenesis by delivering bacterial toxins into host epithelial cells, driving proliferative and proinflammatory responses.

A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival

The regulation of tissue transglutaminase (TG2) activity by the GPCR family is poorly understood. In this study, we investigated the modulation of TG2 activity by the A1 adenosine receptor in cardiomyocyte-like H9c2 cells. H9c2 cells were lysed following stimulation with the A1 adenosine receptor agonist N(6)-cyclopentyladenosine (CPA). Transglutaminase activity was determined using an amine incorporating and a protein cross linking assay. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1 adenosine receptor-induced cytoprotection was investigated by monitoring hypoxia-induced cell death. CPA induced time and concentration-dependent increases in amine incorporating and protein crosslinking activity of TG2. CPA-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA were blocked by PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) inhibitors and by removal of extracellular Ca(2+). CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Fluorescence microscopy revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (Histone H4) and novel (Hexokinase 1) protein substrates for TG2. CPA pre-treatment reversed hypoxia-induced LDH release and decreases in MTT reduction. TG2 inhibitors R283 and Z-DON attenuated A1 adenosine receptor-induced cytoprotection. TG2 activity was stimulated by the A1 adenosine receptor in H9c2 cells via a multi protein kinase dependent pathway. These results suggest a role for TG2 in A1 adenosine receptor-induced cytoprotection.