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The serine/threonine kinase Nek2 (NIMA‐related kinase 2) regulates centrosome separation and mitotic progression, with overexpression causing inductionofaneuploidyinvitro.Overexpression mayalsoenabletumourprogressionthrougheffects uponAktsignalling, celladhesionmarkersand the Wnt pathway. The objective of this study was to examine Nek2 protein expression in colorectal cancer (CRC). Nek2 protein expression was examined in a panel of CRC cell lines using Western blotting and immunofluorescence microscopy. Nek2 and beta‐catenin expression were examined by immunohistochemistry in a series of resected CRC, as well as their matched lymph node and liver metastases, and correlated with clinicopathological characteristics. Nek2 protein expression in all CRC lines examined was higher than in the immortalised colonocyte line HCEC. Nek2 overexpression was present in 86.4% of resected CRC and was significantly associated with advancing AJCC tumour stage and shortened cancer‐specific survival. Elevated Nek2 expression was maintained within all matched metastases from overexpressing primary tumours. Nek2 overexpression was significantly associated with lower tumour membranous beta‐catenin expression and higher cytoplasmic and nuclear beta‐ catenin accumulation. These data support a role for Nek2 in CRC progression and confirm potential for Nek2 inhibition as a therapeutic avenue in CRC. J. Surg. Oncol. . 2014 Wiley Periodicals, Inc.

Dietary phytochemicals exhibit chemopreventive potential in vivo through persistent low-dose exposures, whereas mechanistic in vitro studies with these agents generally use a high-dose single treatment. Because the latter approach is not representative of an in vivo steady state, we investigated antitumor activity of curcumin, 3,3′-diindolylmethane (DIM), epigallocatechin gallate (EGCG), genistein, or indole-3-carbinol (I3C) in breast cancer MDA-MB-231 cells, exposed in long-term culture to low concentrations, achievable in vivo. Curcumin and EGCG increased cell doubling time. Curcumin, EGCG, and I3C inhibited clonogenic growth by 55% to 60% and induced 1.5- to 2-fold higher levels of the basal caspase-3/7 activity. No changes in expression of cell cycle–related proteins or survivin were found; however, I3C reduced epidermal growth factor receptor expression, contributing to apoptosis. Because some phytochemicals are shown to inhibit DNA and histone modification, modulation of expression by the agents in a set of genes (cadherin-11, p21Cip1, urokinase-type plasminogen activator, and interleukin-6) was compared with changes induced by inhibitors of DNA methylation or histone deacetylation. The phytochemicals modified protein and/or RNA expression of these genes, with EGCG eliciting the least and DIM the most changes in gene expression. DIM and curcumin decreased cadherin-11 and increased urokinase-type plasminogen activator levels correlated with increased cell motility. Curcumin, DIM, EGCG, and genistein reduced cell sensitivity to radiation-induced DNA damage without affecting DNA repair. This model has revealed that apoptosis and not arrest is likely to be responsible for growth inhibition. It also implicated new molecular targets and activities of the agents under conditions relevant to human exposure. [Mol Cancer Ther 2007;6(11):3071–9]

Accumulating data suggest that dietary phytochemicals have the potential to moderate deregulated signalling or reinstate checkpoint pathways and apoptosis in damaged cells, while having minimal impact on healthy cells. These are ideal characteristics for chemopreventive and combination anticancer strategies, warranting substantial research effort into harnessing the biological activities of these agents in disease prevention and treatment. However, this requires further investigation into their mode of action and novel approaches to the development of reliable biomarkers.

Background and Objectives Reactivation of the Notch signalling pathway occurs in a range of human malignancies. Previous research suggests that Notch3 is expressed in pancreatic adenocarcinomas, but neither cellular location nor association with clinical parameters has been described. The relationship between Notch3, clinical endpoints, and other proteins with potential to interact with Notch was therefore examined. Methods An immunohistochemical study was performed on human pancreatic adenocarcinoma (n = 23) and normal pancreas (n = 12), to assess expression of Notch3, cyclin D1, pAkt, STAT3 and pSTAT3. Immunohistochemical data were then correlated with clinicopathological characteristics. Results Notch3 was significantly overexpressed in the cytoplasm of 73.9% of tumours. Nuclear expression was not observed in normal pancreatic ductal tissue, but was noted in 43.5% of tumours. No tumour expressing nuclear Notch3 was resectable. There were significant correlations between expression and intracellular location of Notch3 and each of STAT3, pSTAT3 and pAkt, but not cyclin D1. Conclusion The presence of Notch3 in tumour nuclei is likely to represent functional activation of the protein, and is clearly linked to a more aggressive tumour phenotype. The correlation with STAT3, pSTAT3 and pAkt expression has not previously been described and the concurrent intracellular localisation of these proteins suggests a functional relationship between them. J. Surg. Oncol. 2008;97:63–68. © 2007 Wiley-Liss, Inc.

Purpose and Experimental Design: Indole-3-carbinol has been proposed to induce apoptosis via a mechanism involving inhibition of protein kinase B (PKB) signaling in breast and prostate tumor cell lines. However, no functional data exist, and the effect of indole-3-carbinol on viability is known to be highly cell type specific. Here, we examine any requirement for PKB inhibition in induction of apoptosis by indole-3-carbinol in the MDA MB468 cell line using in vitro kinase assays, transfection, Western blotting, and flow cytometry. Comparison is also made with MCF10CA1 breast and PC3 prostate tumor cells. Results: Indole-3-carbinol directly inhibited activity of phosphatidylinositol 3-kinase (PI3K) immunoprecipitated from HBL100 or MDA MB468 cells in vitro. Nonetheless, we present three lines of evidence that inhibition of PI3K/PKB signaling is not required for induction of apoptosis by indole-3-carbinol. First, 50% inhibition of PKB phosphorylation by LY294002 resulted in only 15% apoptosis after 72 hours, whereas similar PKB inhibition by indole-3-carbinol coincided with 30% apoptosis after only 24 hours. Second, induction of phospho-PKB (p-PKB) levels following stimulation with epidermal growth factor did not prevent indole-3-carbinol–induced apoptosis. Third, overexpression of active PKBα did not prevent induction of apoptosis by indole-3-carbinol. Inhibition of PKB phosphorylation by LY294002 in the PC3 and MCF10CA1 tumor cell lines similarly failed to result in a significant increase in apoptosis. Conclusions: Our results show that inhibition of PI3K/PKB signaling by indole-3-carbinol or LY294002 is not directly correlated with induction of apoptosis in several breast or prostate cell lines.

Epidemiological studies have long hinted at the possibility that what we eat greatly influences our state of health, in particular our relative risk of developing cancer. In recent years there has been an exponential increase in the number of studies investigating how individual components of the diet interact at the molecular level to determine the fate of a cell. It is now apparent that many such molecules can preferentially inhibit the growth of tumour cells, by inducing cell cycle arrest or apoptosis. The number of signalling pathways and molecular targets involved is continually expanding. Consequently, the picture is becoming ever more complicated, not least because results often appear to be cell-type specific, dose-response relationships are critical, and any one agent appears to have multiple mechanisms of action. In addition most studies have been conducted in cell culture, often with physiologically unachievable concentrations of single agents, making extrapolation to the clinical situation difficult. In this review the mechanisms of action of a few well-studied dietary polyphenols (curcumin, epigallocatechin gallate and resveratrol) and indole-3 carbinol are considered in the light of these issues.

Tricin, a flavone found in rice bran, inhibits the growth of human-derived malignant MDA-MB-468 breast tumour cells at submicromolar concentrations. As part of the exploration of tricin as a potential cancer chemopreventive agent, we investigated the duration and cell cycle specificity of growth inhibition elicited by tricin in vitro and the effect of tricin on the development of MDA-MB-468 tumours grown in immune-compromised MF-1 mice in vivo. Preincubation of MDA-MB-468 cells with tricin (1-40 microM) for 72 h compromised cell growth after tricin removal, and such irreversibility was not observed in human breast-derived nonmalignant HBL-100 cells. Tricin (>/=5 microM) arrested MDA-MB-468 cells in the G2/M phase of the cell cycle without inducing apoptosis as adjudged by annexin V staining. In nude mice consumption of tricin with the diet (0.2%, w w(-1)) from 1 week prior to MDA-MB-468 cell implantation failed to impede tumour development. Steady-state levels of tricin in plasma, breast tumour tissue and intestinal mucosa, as measured by HPLC, were 0.13 microM and 0.11 and 63 nmol g(-1), respectively. Cells were exposed to tricin (0.11, 1.1 or 11 microM) in vitro for 72 h and then implanted into mice. The volume of tumours in animals bearing cells pre-exposed to 11 microM tricin was less than a third of that in mice with control cells, while tumours from cells incubated with 0.1 or 1.1 microM tricin were indistinguishable from controls. These results suggest that the potent breast tumour cell growth-inhibitory activity of tricin in vitro does not directly translate into activity in the nude mouse bearing the MDA MB-468 tumour. While the results do not support the notion that tricin is a promising candidate for breast cancer chemoprevention, its high levels in the gastrointestinal tract after dietary intake render exploration of its ability to prevent colorectal carcinogenesis propitious.

The regulation of the aldo-keto reductase AKR7A1 was examined in the livers of male and female rats during development by using Western blots, and its contribution to carbonyl metabolism was assessed by using enzyme assays. Hepatic levels of AKR7A1 are low in fetal rats and rise to a peak at around 6 weeks of age in animals of both sexes. Higher levels of the enzyme are found in adult male rat liver than in adult female rat liver. The reductase, therefore, appears to be subject to sex-specific regulation. The effect of growth hormone in mediating this difference in expression was examined by using hypophysectomized animals whose serum growth hormone levels had been feminized by continuous administration. Results demonstrate that such treatment leads to a reduction in AKR7A1 expression. AKR7A1 was found to be constitutively expressed in rat tissues such as liver, kidney, small intestine, and testis, but it was not detected in nasal mucosa, skeletal muscle, heart, adrenal gland, brain, or spleen. However, AKR7A1 was inducible by the synthetic antioxidant ethoxyquin in liver, kidney, and small intestine, but not in the other tissues examined. These results show that levels of this important detoxication enzyme vary considerably according to age and sex and that dietary antioxidants can also influence its level in several tissues.

Genistein is thought to contribute to the putative breast cancer preventive activity of soya. The mechanisms by which it arrests the growth of breast cells are incompletely understood. In order to explore generic features of the modulation of human breast cell growth by genistein, its effects on cell lines MCF-7, ZR-75.1, T47-D, MDA-MB 468, MDA-MB 231 and HBL 100 were compared. Genistein at 1 μM stimulated growth only in MCF-7 cells. At 10 μM it arrested the growth of all 6 cell types, however that of T47-D and HBL 100 cells only in medium with reduced (2%) fetal calf serum. Genistein induced apoptosis in only MDA-MB 468 cells. It arrested cells in the G2 stage of the cell cycle in all cell lines except ZR-75.1. Cells differed in their susceptibility towards inhibition by genistein of phorbol ester-induced proto-oncogene c-fos levels, transcription factor activator protein-1 (AP-1) activity and extracellular signal-regulated kinase (ERK) activity. Genistein augmented anisomycin-induced levels of proto-oncogene c-jun in ZR 75.1 and MCF-7 cells. The results suggest that induction of apoptosis, G2 cell cycle arrest and inhibition of c-fos expression, AP-1 transactivation and ERK phosphorylation may contribute to the growth-inhibitory effect of genistein in some breast cell types, but none of these effects of genistein constitutes a generic mode of growth-arresting action. © 2001 Cancer Research Campaign http://www.bjcancer.com

Many dietary constituents are chemopreventive in animal models, and experiments with cultured cells are revealing various potential mechanisms of action. Compounds classified as blocking agents can prevent, or greatly reduce, initiation of carcinogenesis, while suppressing agents affect later stages of the process by reducing cell proliferation. Many compounds have both types of activity. Blocking mechanisms include alteration of drug metabolising activities and scavenging of reactive oxygen species. Mechanisms which suppress tumorigenesis often involve modulation of signal transduction pathways, leading to altered gene expression, cell cycle arrest or apoptosis. As our knowledge of how these dietary components affect cell biochemistry improves, so the likelihood of success in chemoprevention trials and in provision of dietary advice to the general population to optimise the chances of preventing disease is increased.

For a disease such as cancer, where a number of alterations to normal cell function accumulate over time, there are several opportunities to inhibit, slow down or even reverse the process. Many of the changes which drive the disease process occur in cell-signalling pathways that regulate proliferation and apoptosis. As our knowledge of these complicated signalling networks improves, it is becoming clear that many molecules, both drugs and naturally occurring dietary constituents, can interact beneficially with deregulated pathways. Aspirin and other non-steroidal anti-inflammatory drugs, as well as natural compounds present in plants such as green vegetables and tea, can modulate signalling by affecting kinase activity and therefore phosphorylation of key molecules. Examples of pathways which can be modulated by these agents include activation of the transcription factor nuclear factor κB by tumour promoters or cytokines, signalling by growth factors through the growth-factor receptor/extracellular-regulated protein kinase pathways and by a number of other molecules through the stress-activated c-Jun N-terminal kinase and p38 pathways. These mitogen-activated protein kinase pathways regulate a number of transcription factors including c-Fos and c-Jun. Evidence exists, at least from in vitro experiments, that by targeting such pathways, certain dietary compounds may be able to restore abnormal rates of apoptosis and proliferation to more normal levels.

gamma-Glutamyl transpeptidase is normally not present in adult rat hepatocytes, but its expression is induced by a range of xenobiotics, including carcinogens and chemopreventive agents. Synthesis of the enzyme is mediated by at least six mRNAs transcribed from tandemly arranged promoters on a single gene. We previously identified and partially characterized promoter III as being responsible for upregulation of gamma-glutamyl transpeptidase in rat liver in response to inducing agents. In this study, we examined response elements involved in the regulation of this promoter by using reporter gene assays and in vitro DNase I footprinting and electrophoretic mobility shift assays. Among the response elements was a region with negative regulatory activity upstream of a 240-bp basal regulatory region covering the transcriptional start site. This negative regulatory region, lying between nt -465 and -185, contained sequences with considerable homology to silencer elements in the glutathione s-transferase P gene. The region of basal regulation (nt -185 to +55) contained a CCAAT box, a TFIID binding site, and a GAGA box. A hepatocyte nuclear factor 3-like sequence was identified at nt -234 to -254 and had a DNase I hypersensitive site characteristic of binding of members of the hepatocyte nuclear factor 3/fork head family of proteins and may be involved in the regulation of this promoter.

The chemopreventive agent curcumin has anti-proliferative effects in many tumour types, but characterization of cell cycle arrest, particularly with physiologically relevant concentrations, is still incomplete. Following oral ingestion, the highest concentrations of curcumin are achievable in the gut. Although it has been established that curcumin induces arrest at the G(2)/M stage of the cell cycle in colorectal cancer lines, it is not clear whether arrest occurs at the G(2)/M transition or in mitosis. To elucidate the precise stage of arrest, we performed a direct comparison of the levels of curcumin-induced G(2)/M boundary and mitotic arrest in eight colorectal cancer lines (Caco-2, DLD-1, HCA-7, HCT116p53+/+, HCT116p53(-)/(-), HCT116p21(-)/(-), HT-29 and SW480). Flow cytometry confirmed that these lines underwent G(2)/M arrest following treatment for 12h with clinically relevant concentrations of curcumin (5-10 μM). In all eight lines, the majority of this arrest occurred at the G(2)/M transition, with a proportion of cells arresting in mitosis. Examination of the mitotic index using fluorescence microscopy showed that the HCT116 and Caco-2 lines exhibited the highest levels of curcumin-induced mitotic arrest. Image analysis revealed impaired mitotic progression in all lines, exemplified by mitotic spindle abnormalities and defects in chromosomal congression. Pre-treatment with inhibitors of the DNA damage signalling pathway abrogated curcumin-induced mitotic arrest, but had little effect at the G(2)/M boundary. Moreover, pH2A.X staining seen in mitotic, but not interphase, cells suggests that this aberrant mitosis results in DNA damage.

gamma-Glutamyl transpeptidase (GGT) is normally absent from adult rat hepatocytes but is induced by a range of xenobiotics, including carcinogens and chemoprotective agents. As many as six mRNA species for this enzyme have been described in both rat and mouse, with various degrees of tissue specificity. These originate from one gene and have separate promoters within alternative 5' untranslated sequences. By using a cDNA-derived sequence specific for GGT mRNA III to screen a rat genomic library, a clone that contains the promoter region for this mRNA was isolated and characterized. The transcriptional start site lay some 3.5 kb upstream from that already characterized for mRNA II in rat kidney. Luciferase activity was obtained after transfection of rat hepatoma-derived cell lines with constructs containing the putative promoter III fused to a luc reporter. Although this promoter lacks a TATA box, a sequence close to the start site that binds the transcription factor TFIID in vitro was identified. By using PCR techniques, mRNA III (homologous to both mouse III and IV) and an mRNA (IV) with homology to VI in mouse were found in ethoxyquin- and aflatoxin B1-treated rat liver and kidney as well as in a hepatoma-derived cell line. No evidence was found for a product homologous to mRNA from promoter V described in the mouse.

Tamoxifen was administered in the diet (420 p.p.m.) to female F344 (Fischer), Wistar (LAC-P) and LEW (Lewis) rats to determine for each strain the early morphological and biochemical changes associated with the subsequent development of liver cancer. Hepatic DNA damage, as determined by 32P-postlabelling, showed a cumulative increase with time from 500 adducts/10(8) nucleotides at 30 days to almost 3000 adducts/10(8) nucleotides after 180 days, with little difference between strains at this time point. A significant strain difference was found in the number of adducts present in the Fischer rats at 90 days, compared to the Wistar and Lewis strains. There was a marked strain differences in the time to development of liver tumours. After 6 months treatment, both Wistar and Lewis rats had tumours while none were seen in the Fischer animals. After 11 months, all of the Wistar and Lewis rats had developed liver carcinoma, while the Fischer rats developed liver carcinoma by 20 months. Depression in cell proliferation, relative to age-matched controls, was seen in the livers of Fischer rats after six months of exposure to tamoxifen, in contrast to an increase in the Wistar and Lewis rats. This observation is consistent with the promotion of foci to tumours and the subsequent progression of tumours to carcinomas in the latter two strains. These data may assist in establishing the possible risk factors, such as extent of DNA damage and increased liver cell proliferation, to women with long-term prophylactic exposure to tamoxifen.

Female F344 rats received an i.p. injection of iron-dextran (600 mg Fe/kg) and then after 1 week were fed a diet containing 0.02% hexachlorobenzene (HCB) for up to 65 weeks. All rats (8/8) which received HCB after iron overload developed multiple hepatic nodules whereas only 3/8 rats administered HCB alone had nodules (average of one per positive liver). These hyperplastic regions were depleted of iron and were often positive for gamma-glutamyl transpeptidase (GGT) and glutathione S-transferase P (GST-P). Telangiectasis and peliosis were prominent features in the lesions. Short-term experiments (5-15 weeks of iron/HCB treatments) showed that GGT and GST-P were induced early in the neoplastic process but not in discrete focal areas. Iron alone also caused some induction of these enzymes. Some cells with induced GST-P in either short or long term experiments also stained positively for this enzyme in the nucleus. Studies of cytochrome P450 mediated activities showed that at 5 and 15 weeks HCB had induced EROD (an estimate of CYP1A1), PROD (CYP2B1 activity) and BROD activities (CYP2B1 but also other isoenzymes). Under the influence of iron overload EROD was significantly depressed from HCB alone, but not the others or cytochrome P450 reductase. Cytosolic glutathione S-transferase activities were also induced by HCB, but, unlike microsomal EROD, preloading with iron enhanced the effects. In contrast, although cytosolic diaphorase activity was induced by HCB, this response was depressed in combination with iron. Glutathione peroxidase (with H2O2 as substrate) was depressed by both iron and HCB. Clearly, iron overload potentiates the neoplastic process induced by HCB in rats, with both enhancing and depressing effects on various enzyme activities induced by this chemical.

gamma-Glutamyltranspeptidase (gamma-GT) was localized in slices of rat lung, at the ultrastructural level, by pre-embedding immunogold labelling. Antiserum was raised against the protein purified from rat kidney. The enzyme was found to be concentrated on the lumenal surface of the non-ciliated ("Clara") cells of the bronchiolar epithelium and, to a lesser degree, on the surface of type II alveolar pneumocytes. This immunological localization was consistent with the distribution of reaction product, in both slices and resin sections incubated to demonstrate gamma-GT activity. gamma-GT is probably involved in the utilization of reduced glutathione (GSH) present in the fluid lining the airway epithelium.

There is a large body of evidence that the consumption of fruit and vegetables can decrease the risk of cancer. However, the link between diet and health is extremely complex. Some dietary phytochemicals seem to offer protection in an exposure-related manner and many molecular targets and signaling pathways affected by phytochemicals have been discovered. Although in vitro studies have contributed significantly to our understanding, quite a number use concentrations orders of magnitude greater than those achievable in humans or toxic to normal tissues (exemplified by toxic concentrations of indole-3-carbinol, epigallocatechin-3-gallate, curcumin, and genistein for breast cells). Such studies may produce results that are physiologically irrelevant, thus hindering predictions of efficacy. Here, we argue for careful consideration to be given to the in vitro experimental conditions under which dietary phytochemicals are investigated. Design features, such as the use of appropriate nontoxic concentrations, extended treatment times, three-dimensional cultures, primary tumor cultures, and comparison of susceptibility of various cancer subtypes, should improve our understanding of their molecular targets. This in turn would facilitate predictions as to their potential usefulness in the clinic.

Survival rate of patients diagnosed with the invasive form of bladder cancer is low suggesting an urgent need to implement novel treatments. GTC (gemcitabine, paclitaxel and cisplatin) is a new chemotherapeutic regimen, which has shown promise in clinical trials. Given that receptor tyrosine kinases of the ErbB family are overexpressed in a high proportion of metastatic bladder tumours, approaches involving small-molecule inhibitors of ErbB receptors in combination with conventional cytostatic drugs are of potential interest. Here, we show that the dual inhibitor of ErbB receptors, lapatinib, enhances cytostatic and induces cytotoxic effects of GTC in two bladder cancer cell lines which differ with regard to expression levels of proteins taking part in the ErbB pathway. Lapatinib inhibited phosphorylation of ErbB receptors and also reduced the level of phosphorylated AKT. Flow cytometry analysis demonstrated that GTC treatment affects cell cycle distribution differently in the presence or absence of lapatinib. In RT112 cells, which express high levels of ErbB receptors and harbour wild-type p53, combined GTC/lapatinib treatment resulted in the phosphorylation of p53 at Ser46 and accumulation of sub-G1 cell populations. Our data indicate that a combinatorial approach involving GTC and lapatinib may have therapeutic potential in a subset of bladder tumours depending on the genetic context.

The methylation status of the rat gamma-glutamyl transpeptidase (GGT) gene was investigated during liver development and hepatocarcinogenesis. The analysis with the restriction enzymes MspI/HpaII revealed that, during ontogeny, there is a progressive methylation of the GGT gene that coincides with a progressive decrease in GGT activity. Thus, there is an inverse correlation between methylation and expression of the GGT gene, suggesting a role for DNA methylation in the regulation of the gene during normal differentiation. The methylation patterns of the GGT gene in liver tumours induced by aflatoxin B1 exhibit heterogeneity. Nevertheless, a band of 5.7 kb was observed in all the DNA samples from aflatoxin B1-induced tumours which was not present in control liver DNA. The specificity of the DNA methylation changes was assessed using nafenopin, which induces hepatic tumours without elevation of GGT activity. We conclude that, during hepatocarcinogenesis, there is a modification of the DNA methylation pattern of the GGT gene, but there is no simple correlation with GGT activity. In no case was the GGT gene methylation in hepatocarcinogenesis found to be equivalent to the pattern observed in fetal liver. Thus if methylation is involved in the regulation of GGT gene transcription, the mechanisms must be different in fetal liver and hepatocarcinoma.

A marked sex difference in the development of uroporphyria occurred after administration of polychlorinated and polybrominated biphenyls (PCBs and PBBs), as well as hexachlorobenzene (HCB), to F344 rats for 15 weeks. Thus the propensity of female rats to develop uroporphyria appears to be a general response to this class of halogenated chemicals. A heat-stable inhibitor(s) of liver uroporphyrinogen decarboxylase was extractable from uroporphyric livers. Although oxidation of uroporphyrinogen I to uroporphyrin I by hepatic microsomes from rats pretreated with porphyrogenic regimes of HCB and PCBs was induced, there was no correlation with the in vivo sex difference in porphyria development. Levels of total cytochrome P450 and pentoxyresorufin and benzyloxyresorufln dealkylase activities (associated with cytochrome P450IIB1) were greater in microsomes from control, HCB, PCB and PBB treated male rats than females. In contrast, ethoxyresorufin deethylase activity (associated with cytochrome P450IA1) was always significantly greater in females. These findings were confirmed by immunoblotting with polyclonal antibodies to cytochromes P450IA1, IA2 and IIB1. Immunocytochemical studies showed that, even after 30 weeks of HCB exposure, cytochromes P450IA1 and P450IA2 were still more highly induced in female liver, especially in the centrilobular region. The results are consistent with the association of cytochrome P450IA isoenzymes with uroporphyria development, although the sex difference in P450IA levels alone may not be marked enough to provide the complete explanation for the pronounced susceptibility of females to HCB.

There is growing interest in the ability of phytochemicals to prevent chronic diseases, such as cancer and heart disease. However, some of these agents have poor bioavailability and many of the in-depth studies into their mechanisms of action have been carried out in vitro using doses which are unachievable in humans. In order to optimize the design of chemopreventive treatment, it is important to determine which of the many reported mechanisms of action are clinically relevant. In this review we consider the physiologically achievable doses for a few of the best studied agents (indole-3-carbinol, diindolylmethane, curcumin, epigallocatechin-3-gallate and resveratrol) and summarize the data derived from studies using these low concentrations in cell culture. We then cite examples of in vitro effects which have been observed in vivo. Finally, the ability of agent combinations to act synergistically or antagonistically is considered. We conclude that each of the compounds shows an encouraging range of activities in vitro at concentrations which are likely to be physiologically relevant. There are also many examples of in vivo studies which validate in vitro observations. An important consideration is that combinations of agents can result in significant activity at concentrations where any single agent is inactive. Thus, for each of the compounds reviewed here, in vitro studies have provided useful insights into their mechanisms of action in humans. However, data are lacking on the full range of activities at low doses in vitro and the benefits or otherwise of combinations in vivo. The corresponding author's laboratory is supported by the UK Medical Research Council and the EU Network of Excellence, ECNIS.

Glycogen synthase kinase (GSK) was initially described as a key enzyme involved in glycogen metabolism. However, since that time it has been found to regulate a diverse range of cell functions. In addition to having a major role in the regulation of the important onco-protein beta-catenin, GSK is also a critical regulator of NF-kappaB. NF-kappaB comprises a family of transcription factors which activate the expression of a wide array of genes involved in inflammation, tumourigenesis, metastasis, differentiation, embryonic development, apoptosis. Inflammation mediated by the NF-kappaB family has been implicated in the initiation of pancreatic cancer, resistance to chemotherapy and the development of the debilitating cancer cachexia seen with advanced disease. Hence, GSK has potential as an important new target both in the treatment of resectable pancreatic cancer as an adjuvant to surgery, and in the palliation of inoperable tumours.

Natural products represent a rich resource for drug delivery and are currently being exploited to target tumour angiogenesis. A vast array of products of natural origin have been shown to have anti-angiogenic potential in preclinical models, including purified endogenous inhibitors, and exogenous compounds derived from varied species of plant, animal and micro-organism. Over a dozen of these agents have now entered clinical trial. This review discusses evidence for the efficacy of this drug class and key issues in the translation of pre-clinical results into the development of efficacious drugs for clinical use.

Indole-3-carbinol (I3C) is a promising anticancer dietary compound, which inhibits breast cancer in animal models. The objective of the current study was to characterize I3C-induced cell death in a panel of human breast tumorigenic cells (MCF7, MDA-MB-468, MDA-MB-231 and HBL100) in comparison with normal fibroblasts. Since epithelial cells are protected from cell death by a three-dimensional environment, 3D cell culture (collagen I gel and spheroids) was employed to investigate susceptibility to I3C. Cell viability in the presence of 256 microM I3C, a concentration close to the physiologically achievable range, was in the order fibroblasts = HBL100MDA-MB-231MCF7MDA-MB-468 in monolayer culture. However, 3D culture conditions increased the susceptibility of MCF7 and MDA-MB-468 cancer cells towards I3C. I3C induced cell death in breast cancer MCF7, MDA-MB-468 and MDA-MB-231 cells via the mitochondrial apoptotic pathway. I3C significantly reduced levels of epidermal growth factor receptor (EGFR) in MDA-MB-468 after 6 h and in MDA-MB-231 and HBL100 cells after 30 h. Downregulation of EGFR in MDA-MB468 and MDA-MB-231 cells using an EGFR inhibitor resulted in apoptosis. EGFR modulation using EGF or an EGFR inhibitor markedly influenced viability and response to I3C in MDA-MB-468 cells in 3D conditions. EGFR expression was modulated by 3D conditions. Therefore, I3C-induced EGFR reduction in these cells is likely to be responsible for I3C-induced apoptosis.

Background Determination of prognosis in patients with resectable colorectal liver metastases (CLM) is desirable in order to improve case selection for surgery and tailor adjuvant treatment according to individual recurrence risk. Conventional clinicopathological factors lack the sensitivity to accurately achieve this goal. Consideration of tumour biology and the identification of molecular prognostic markers may allow more accurate risk stratification. Method This systematic review examines evidence from published manuscripts looking at molecular markers in resectable colorectal liver metastases and their correlation with disease recurrence and survival following hepatectomy. Results Studies have yielded promising results in the search for prognostic molecular markers of CLM. Molecular biomarkers from varied aspects of tumour biology have been examined and a number of these, including proliferation indices, telomerase, thymidylate synthase, microvessel density and thrombospondin-1 appear to have prognostic utility in this context. Validation of other markers, notably p53, has been limited by a failure of methodologies to account for their biological complexity. Conclusions A biomarker-based approach may yield significant benefits through informed treatment of resectable metastatic colorectal malignancy. Standardised retrospective analyses are necessary to confirm preliminary findings and identify existing and novel markers for inclusion into prospective studies. Assessment and verification of multiple molecular markers in this manner may allow molecular profiling of metastases and tailoring of therapy according to the biological aggressiveness of individual tumours. The advent of genomic- and proteomic-based technologies will allow the simultaneous analysis of multiple molecular markers and the derivation of disease profiles associated with disease recurrence and poor survival.

There are many facets to cancer prevention: a good diet, weight control and physical activity, a healthy environment, avoidance of carcinogens such as those in tobacco smoke, and screening of populations at risk to allow early detection. But there is also the possibility of using drugs or naturally occurring compounds to prevent initiation of, or to suppress, tumour growth. Only a few such agents have been used to date in the clinic with any success, and these include non-steroidal anti-inflammatory drugs for colon, finasteride for prostate and tamoxifen or raloxifene for breast tumours. An ideal chemopreventive agent would restore normal growth control to a preneoplastic or cancerous cell population by modifying aberrant signalling pathways or inducing apoptosis (or both) in cells beyond repair. Characteristics for such an agent include selectivity for damaged or transformed cells, good bioavailability and more than one mechanism of action to foil redundancy or crosstalk in signalling pathways. As more research effort is being targeted towards this area, the distinction between chemotherapeutic and chemopreventive agents is blurring. Chemotherapeutic drugs are now being designed to target over- or under-active signalling molecules within cancer cells, a philosophy which is just as relevant in chemoprevention. Development of dietary agents is particularly attractive because of our long-standing exposure to them, their relative lack of toxicity, and encouraging indications from epidemiology. The carcinogenic process relies on the cell's ability to proliferate abnormally, evade apoptosis, induce angiogenesis and metastasise to distant sites. In vitro studies with a number of different diet-derived compounds suggest that there are molecules capable of modulating each of these aspects of tumour growth. However, on the negative side many of them have rather poor bioavailability. The challenge is to uncover their multiple mechanisms of action in order to predict their efficacy, to learn how to use them effectively in combination, and in some cases to redesign them to improve potency or bioavailability. These ideas are illustrated by dietary agents such as indole-3-carbinol (I3C), epigallocatechin gallate (EGCG), curcumin and resveratrol, all of which appear to have a number of different molecular targets, impinging on several signalling pathways. Ultimately it may be possible not only to suppress tumours and to extend quality of life by administering appropriate diet-derived molecules, but also to refine the definition of a cancer chemopreventive diet.

trans- Resveratrol (t-RVT) has been shown to have a wide range of anti-inflammatory properties, some of which have been suggested to contribute to the molecular explanation of the French Paradox, a possible reason for the low incidence of heart disease in France. The ability of t-RVT to inhibit the production of reactive oxygen species (ROS) from monocytes (differentiated U937) was investigated using isoluminol, luminol, lucigenin, and 2′,7′-dichlorofluorescein (DCF). t-RVT (0.1–50 μM) was found to significantly inhibit cellular ROS production stimulated by f-Met-Leu-Phe (fMLP), 12-phorbol 13-myristate, and arachidonic acid after a 1-h preincubation. The efficacy of t - RVT could be increased if it was added directly into the assay. NADPH-dependent superoxide production was measured in cell homogenates and t-RVT (10–50 μM) was found to have no effect on this activity. The majority of these redox probes require a peroxidase to be oxidized; therefore, the inhibitory effect of t-RVT on ROS measured by these probes is complicated by its ability to be oxidized by peroxidase enzymes and thus compete with the probe. t - RVT, known to be oxidized by the horseradish peroxidase (HRP)/H 2 O 2 system, was found to inhibit the HRP-dependent oxidation of the fluorescent probe DCF and the chemiluminescent probe isoluminol. However, using a redox probe that did not require oxidation by a peroxidase (lucigenin), significant inhibition was still observed. Moreover, the inhibitory effects of t-RVT on fMLP-induced ROS production correlated with significant inhibitory effects on fMLP-induced phosphatidylinositol 3-kinase (PI3K) activity at 50 μM and Akt phosphorylation (10–50 μM). Other known inhibitors of both PI3K and Akt were also found to inhibit this response. Therefore, inhibition of signaling through the PI3K to NADPH oxidase by t-RVT might represent an important anti-inflammatory mechanism.

Following observations that curcumin inhibited proliferation (IC(50)=1-5 microM), invasiveness and progression through S/G2/M phases of the cell cycle in the non-tumourigenic HBL100 and tumourigenic MDA-MB-468 human breast cell lines, it was noted that apoptosis was much more pronounced in the tumour line. Therefore, the ability of curcumin to modulate signalling pathways which might contribute to cell survival was investigated. After pre-treatment of cells for 20 min, curcumin (40 microM) inhibited EGF-stimulated phosphorylation of the EGFR in MDA-MB-468 cells and phosphorylation of extracellular signal regulated kinases (ERKs) 1 and 2, as well as ERK activity and levels of nuclear c-fos in both cell lines. At a lower dose (10 microM), it also inhibited the ability of anisomycin to activate JNK, resulting in decreased c-jun phosphorylation, although it did not inhibit JNK activity directly. In contrast, the activation of p38 mitogen activated protein kinase (MAPK) by anisomycin was not inhibited. Curcumin inhibited basal phosphorylation of Akt/protein kinase B (PKB) in both cell lines, but more consistently and to a greater extent in the MDA-MB-468 cells. The MAPK kinase (MKK) inhibitor U0126 (10 microM), while preventing ERK phosphorylation in MDA-MB-468 cells, did not induce apoptosis. The PI3K inhibitor LY294002 (50 microM) inhibited PKB phosphorylation in both cells lines, but only induced apoptosis in the MDA-MB-468 line. These results suggest that while curcumin has several different molecular targets within the MAPK and PI3K/PKB signalling pathways that could contribute to inhibition of proliferation and induction of apoptosis, inhibition of basal activity of Akt/PKB, but not ERK, may facilitate apoptosis in the tumour cell line.

Certain dietary constituents can protect against chemically induced carcinogenesis in rodents. A principal mechanism by which these chemopreventive compounds exert their protective effects is likely to be via induction of carcinogen detoxification. This can be mediated by conjugation with glutathione, which is synthesized by the sequential actions of glutamate-cysteine ligase (GLCL) and glutathione synthetase. We have demonstrated that dietary administration of the naturally occurring chemopreventive agents, ellagic acid, coumarin or alpha-angelicalactone caused an increase in GLCL activity of between approximately 3- and 5-fold in rat liver. Treatment with the synthetic antioxidant ethoxyquin or the classic inducer phenobarbital caused < 2-fold induction of GLCL activity in rat liver, which was not found to be significant. The increases in GLCL activity were accompanied by increases (between 2- and 4-fold) in levels of both the catalytic heavy subunit (GLCLC) and regulatory light subunit (GLCLR). No substantial induction of GLCL was observed in rat kidney. The glutathione S-transferase (GST) subunits A1, A3, A4, A5, P1 and M1 were all found to be inducible in rat liver by most of the agents. The greatest levels of induction were observed for GST P1, following treatment with coumarin (20-fold), alpha-angelicalactone (10-fold) or ellagic acid (6-fold), and GST A5, following treatment with coumarin (7-fold), alpha-angelicalactone (6-fold) and ethoxyquin (6-fold). Glutathione synthetase was induced approximately 1.5-fold by coumarin, alpha-angelicalactone, ellagic acid and ethoxyquin. The expression of glutathione-related enzymes was also examined in preneoplastic lesions induced in rat liver by aflatoxin B(1). The majority of gamma-glutamyltranspeptidase (GGT)-positive preneoplastic foci contained increased levels of GLCLC relative to the surrounding tissue. This was usually found to be accompanied by an increase in GLCLR. Cells in the inner cortex of rat kidney were found to contain the highest levels of both GLCLC and GLCLR. The same cells showed the strongest staining for GGT activity.

Colorectal cancer is a major cause of cancer deaths in Western countries, but epidemiological data suggest that dietary modification might reduce these by as much as 90%. Cyclo-oxygenase 2 (COX2), an inducible isoform of prostaglandin H synthase, which mediates prostaglandin synthesis during inflammation, and which is selectively overexpressed in colon tumours, is thought to play an important role in colon carcinogenesis. Curcumin, a constituent of turmeric, possesses potent anti-inflammatory activity and prevents colon cancer in animal models. However, its mechanism of action is not fully understood. We found that in human colon epithelial cells, curcumin inhibits COX2 induction by the colon tumour promoters, tumour necrosis factor alpha or fecapentaene-12. Induction of COX2 by inflammatory cytokines or hypoxia-induced oxidative stress can be mediated by nuclear factor kappa B (NF-kappaB). Since curcumin inhibits NF-kappaB activation, we examined whether its chemopreventive activity is related to modulation of the signalling pathway which regulates the stability of the NF-kappaB-sequestering protein, IkappaB. Recently components of this pathway, NF-kappaB-inducing kinase and IkappaB kinases, IKKalpha and beta, which phosphorylate IkappaB to release NF-kappaB, have been characterised. Curcumin prevents phosphorylation of IkappaB by inhibiting the activity of the IKKs. This property, together with a long history of consumption without adverse health effects, makes curcumin an important candidate for consideration in colon cancer prevention.

The relationship between food and cancer is extremely complex. It is generally accepted that diet is a contributory factor in the aetiology of a large proportion of cancers, but with very few exceptions, we are unable to identify specific causal agents. Many food components have genotoxic potential and more are produced endogenously during digestion. Conversely, there is increasing evidence that consumption of some foods may decrease the risk of cancer, and a number of plant constituents have been shown to have the potential to inhibit various stages of the carcinogenic process. Yet we have little understanding of the interactions between the different food-related genotoxic and protective factors. A further complication is the variation in individual susceptibility and vulnerability. As a result we are still not able to determine the optimal diet for minimising cancer risk. In recognition of these issues, the UK Ministry of Agriculture, Fisheries and Food (MAFF) is funding a number of projects aimed at providing greater mechanistic understanding of the links between food and cancer, in order to offer detailed advice to the public. This report summarises the proceedings of a workshop entitled 'Factors influencing the carcinogenicity of food chemicals', held in London on 1 June 1998, providing overviews of some of the key issues, and demonstrating how the MAFF-funded research is contributing to advances in these areas. It includes discussion of genetic polymorphisms and how they may contribute to individual susceptibility and help to identify causal links between food components and colorectal cancer. Biomarkers of DNA damage in human studies and of inhibition of carcinogen activation and endogenous formation of genotoxic reactive nitrogen species are examined. Also considered are the potential uses of physiologically based pharmacokinetic modelling techniques for providing more accurate estimates of risk and reducing the uncertainty in extrapolation between species and doses. Research now in progress will help to establish the critical risk and protective factors involved in diet-related colorectal cancers, in order to provide a sound scientific basis for formulation of dietary advice to the public.

Indole-3-carbinol (I3C) was examined for its ability to inhibit aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in male Fischer rats when administered either before or after the carcinogen. After 13 weeks, animals pretreated with I3C (0.5% in the diet) for 2 weeks prior to administration of AFB1 and with continuing treatment during exposure to the carcinogen were protected from development of preneoplastic lesions, as determined by the classical markers gamma-glutamyltranspeptidase (GGT) and glutathione S-transferase (GST) P. In animals receiving AFB1 for 6 weeks before treatment with I3C, there was no obvious protective effect at 13 weeks compared with animals receiving only AFB1. Using cytokeratin 18 expression as a marker, animals fed AFB1 alone had a small number of positive foci at 13 weeks. However, no cytokeratin-positive foci were visible in the majority of livers from either group receiving I3C in combination with AFB1 and after 43 weeks all animals in these groups were protected from liver tumour formation. These results suggest that expression of cytokeratin 18, a later phenotypic change in foci than induction of GST-P and GGT, correlates more closely with tumour outcome in this model. I3C appeared to retard progression of AFB1-induced carcinogenesis at both the initiation and promotion stages. Continuous treatment with I3C for 13 weeks caused significant induction of CYP1A1, 1A2, 3A and 2B1/2, GST Yc2, aflatoxin B1 aldehyde reductase and quinone reductase. Such alteration of the drug metabolizing capacity of the liver by I3C contributes to blocking of initiation, while the observed inhibition of ornithine decarboxylase, a rate limiting enzyme in polyamine biosynthesis, and of tyrosine kinase activity may contribute to the suppressive effect of I3C.

A characteristic feature of the class Theta glutathione S-transferase (GST) T1-1 is its ability to activate dichloromethane and dibromoethane by catalysing the formation of mutagenic conjugates. The level of the GSTT1 subunit within tissues is an important determinant of susceptibility to the carcinogenic effects of these dihaloalkanes. In the present study it is demonstrated that hepatic GST activity towards these compounds can be elevated significantly in female and male Fischer-344 rats by feeding these animals on diets supplemented with cancer chemopreventive agents. Immunoblotting experiments showed that increased activity towards the dihaloalkanes is associated with elevated levels of the GSTT1 subunit in rat liver. Sex-specific effects were observed in the induction of GSTT1 protein. Amongst the chemopreventive agents tested, indole-3-carbinol proved to be the most potent inducer of hepatic GSTT1 in male rats (6.2-fold), whereas coumarin was the most potent inducer of this subunit in the livers of female rats (3.5-fold). Phenobarbital showed significant induction of GSTT1 only in male rat liver and had little effect in female rat liver. Western blotting showed that class Alpha, Mu and Pi GST subunits are not co-ordinately induced with GSTT1, indicating that the expression of GSTT1 is determined, at least in part, by mechanisms distinct from those that regulate levels of other transferases. The increase in amount of hepatic GSTT1 protein was also reflected by an increase in the steady-state level of mRNA in response to treatment with chemopreventive agents and model inducers. Immunohistochemical detection of GSTT1 in rat liver supported the Western blotting data, but showed, in addition to cytoplasmic staining, significant nuclear localization of the enzyme in hepatocytes from some treated animals, including those fed on an oltipraz-containing diet. Significantly, the hepatic level of cytochrome P-450 2E1, an enzyme which offers a detoxification pathway for dihaloalkanes, was unchanged by the various inducing agents studied. It is concluded that the induction of GSTT1 by dietary components and its localization within cells are important factors that should be considered when assessing the risk dihaloalkanes pose to human health.

The concept that cancer can be prevented, or its onset postponed, by certain diet-derived substances is currently eliciting considerable interest. Agents which interfere with tumour development at the stage of promotion and progression in particular are of potential clinical value. As chemopreventive agents have to be administered over a long period of time in order to establish whether they possess efficacy in humans, it is of paramount importance to establish their lack of toxicity. The desire to select the best chemopreventive drug candidates for clinical trial, and the necessity to monitor efficacy in the short and intermediate term, render the identification of specific mechanism-based in vivo markers of biological activity a high priority. Antioxidation, inhibition of arachidonic acid metabolism, modulation of cellular signal transduction pathways, inhibition of hormone and growth factor activity and inhibition of oncogene activity are discussed as mechanisms by which the soya constituent genistein, the curry ingredient curcumin and the vitamin A analogue 13-cis retinoic acid exert tumour suppression. A better understanding of these mechanisms will help the establishment of screens for the discovery of new and better chemopreventive agents and the identification of surrogate markers to assess the outcome of clinical chemoprevention trials.

The synthesis of gamma-glutamyl-7-amino-4-(trifluoromethyl)coumarin and its use as a substrate for gamma-glutamyltranspeptidase is described. The reaction product 7-amino-4-(trifluoromethyl)coumarin was fluorescent at neutral pH values and with excitation and emission wavelengths of 400 and 490 nm, respectively, concentration was linearly related to fluorescence over the range of 10 to 300 pmol/3 ml reaction mixture. At pH 8.4, the optimum for purified gamma-glutamyltranspeptidase, continuous fluorometric determination of enzyme activity with time could be carried out. This permitted accurate estimations to be made of initial reaction rates. Low levels of gamma-glutamyltranspeptidase activity could be shown in isolated rat hepatocytes (1 x 10(5) cells). Such activity could be inhibited by the addition acivicine (alpha-amino-3-chloro-5-isoxazoleacetic acid), suggesting the absence of peptidase-like activity. Following 2-week pretreatment of rats with the antiestrogen, tamoxifen, a fourfold increase in gamma-glutamyltranspeptidase activity was observed. The present method offers a convenient, sensitive method for the determination of gamma-glutamyltranspeptidase activity without the need for elaborate workup procedures.

Exposure of iron-loaded C57BL/10ScSn mice to the polychlorinated biphenyls (PCBs) mixture Aroclor 1254 in the diet (0.01%) for 5 weeks caused massive hepatic porphyria far greater than occurred with PCBs alone. This regime eventually causes hepatocellular carcinoma. Hepatic microsomal ethoxy-, pentoxy-, and benzyloxyresorufin dealkylase activities (respectively EROD, PROD, and BROD) catalyzed primarily by cytochrome P4501A1 and 2B isoenzymes were markedly induced after 2 weeks of diet (when no porphyria had developed) but showed little effect of iron. EROD activity in the nuclear membrane was also induced by the PCBs as was CYP1A1 protein when shown by immunoblotting. Nuclear dealkylase activities of PCBs-treated mice were considerably less than microsomal activities but were stimulated by iron pretreatment. The mechanism of the iron-enhanced toxicity may be due to oxidative damage associated with chronic induction of CYP1A1 isoforms. Lucigenin-enhanced chemiluminescence (CL) by microsomes and nuclear membranes was used as a method to estimate their potential to form reactive oxygen species. Despite CL being induced by PCBs it was less with microsomes from iron-treated mice. In a comparison of a variety of inducers of microsomal cytochrome P450 there was no correlation between inducer, uroporphyrogenic agent, and intensity of CL. On the other hand, cytosolic glutathione S-transferase (GST) activities with 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene (DCNB) as substrates, were also induced by the PCBs mixture, the induction with DCNB being synergistically potentiated by iron pretreatment. Complementary results were observed by immunocytochemistry using anti alpha-GST antibody. In contrast, total glutathione peroxidase activity and selenium-dependent glutathione peroxidase activity were depressed by PCBs but particularly in mice also administered iron. The results illustrate that PCBs not only induce CYP1A1 in microsomes but also in the nuclear membrane, which may be of significance in the mechanism of the iron-enhanced carcinogenicity of these chemicals. The iron-enhanced induction of GST with accompanying depletion of glutathione peroxidase provides evidence for oxidative processes induced in vivo by the PCBs.

In order to achieve a better outcome for pancreatic cancer patients, reliable biomarkers are required which allow for improved diagnosis. These may emanate from a more detailed molecular understanding of the aggressive nature of this disease. Having previously reported that Notch3 activation appeared to be associated with more aggressive disease, we have now examined components of this pathway (Notch1, Notch3, Notch4, HES-1, HEY-1) in more detail in resectable (n = 42) and non-resectable (n = 50) tumours compared to uninvolved pancreas. All three Notch family members were significantly elevated in tumour tissue, compared to uninvolved pancreas, with expression maintained within matched lymph node metastases. Furthermore, significantly higher nuclear expression of Notch1, -3 and -4, HES-1, and HEY-1 (all p ≤ 0.001) was noted in locally advanced and metastatic tumours compared to resectable cancers. In survival analyses, nuclear Notch3 and HEY-1 expression were significantly associated with reduced overall and disease-free survival following tumour resection with curative intent, with nuclear HEY-1 maintaining independent prognostic significance for both outcomes on multivariate analysis. These data further support a central role for Notch signalling in pancreatic cancer and suggest that nuclear expression of Notch3 and its target gene, HEY-1, merit validation in biomarker panels for diagnosis, prognosis and treatment efficacy. A peptide fragment of Notch3 was detected in plasma from patients with inoperable pancreatic cancer, but due to wide inter-individual variation, mean levels were not significantly different compared to age-matched controls.

The toxicity of ethoxyquin (EQ) to rat kidney was examined in males which were either weanling or adult at the beginning of the experiment, and also in adult females. Female rats were much less susceptible to the toxic effects of EQ than males of the same age. In males damage to the cortex, mainly as an acceleration of the normal ageing process, was similar in both age groups, but rats exposed to EQ as weanlings also suffered from extensive papillary necrosis. Male rats were more prone than females to proteinuria, which was greatly exacerbated by EQ in both age groups. Thus there is very little evidence of nephrotoxicity in adult female rats on exposure to EQ at 0.5% in the diet for 26 weeks. In males, the initial age of the animal, as well as the length of treatment, influences the extent of damage.

Fluorescence-activated flow cytometry has been used to separate rat liver parenchymal cells into five equal computer-generated regions on the basis of their mixed function activities towards diethoxyfluorescein. Enzymic activities in the sorted fractions showed 2.3–2.9-fold enrichment of 3-cyano-7-ethoxycoumarin O -deethylase, lactate dehydrogenase and pyruvate kinase activities in the cells of region 5. Cytochrome P-450 PBla and glutamine synthase activities were enriched 1.8- and 7-fold respectively in region 2 hepatocytes. Pretreatment of rats with phenobarbitone or 3-methylcholanthrene did not change the pattern of enrichment with respect to monooxygenase or lactate dehydrogenase activities. However, cytochrome P-450 PB3a and MC1a respectively were concentrated in the cells from region 5. In contrast, ethoxyquin caused enrichment of cytochrome P-450 in the hepatocytes in region 2. This fraction also contained the highest percentage of γ-glutamyltranspeptidase positive cells.

We have studied the induction of rat liver gamma-glutamyl transpeptidase (GGT) mRNA by the antioxidant ethoxyquin and during aflatoxin B1-induced carcinogenesis. Using a rat kidney GGT cDNA probe, Northern blot analysis revealed that GGT mRNA induced in liver by either compounds was slightly larger than that found in untreated kidney. GGT mRNA was not detected in untreated liver or freshly isolated hepatocytes, but induction of the message in treated tissues correlated with the increase in enzymic activity observed by histochemistry and quantitative assay. Slot-blot analysis of poly(A)+ mRNA indicated that constitutive GGT mRNA levels in kidney were at least 5-fold greater than those in the most GGT-positive liver-derived tissue examined.

Hexachlorobenzene (HCB) was fed to male and female F344 rats as 0.02% of the diet for 15 weeks. Females developed a massive porphyria, due to depression of uroporphyrinogen decarboxylase activity, whereas males did not. Although hepatic non-haem iron levels in control females were 3-5 times greater than males (iron is implicated in the pathogenesis of this condition) preloading the latter with iron did not increase their susceptibility. After 90 weeks of HCB treatment 100% of surviving females had multiple liver tumours which were strongly gamma-glutamyl transpeptidase (GGT) positive and histologically classified as neoplastic nodules or hepatocellular carcinomas. In contrast, only 16% of males developed tumours which were smaller and fewer in number per liver than those in females. Accumulation of porphyrins was still significantly less in males than females although no uroporphyrinogen decarboxylase activity was detected in treated livers of either sex. No differences in porphyrin levels or enzyme activity were found between tumours and surrounding tissue showing that tumours did not revert to a non-porphyric state. The sex difference in tumour response could not be explained by differences in hepatic HCB concentrations. Non-haem iron concentrations of livers fell after HCB treatment for 90 weeks in both sexes and were even lower in tumours. These studies demonstrate that not only are female rats far more sensitive than males to the porphyrinogenic effects of HCB but also to the hepatocarcinogenic actions, suggesting a link between these two manifestations of toxicity that may also apply to other polyhalogenated aromatics.

Summary We previously observed a biphasic response in rat hepatic gamma glutamyl transpeptidase (GGT) activity to aflatoxin B 1 (AFB 1 ) feeding [9]. We have extended this observation to examine the effect of partial hepatectomy (PH) on the activity and distribution of GGT at different stages of the feeding regime. In control-fed animals GGT levels were elevated 3–7 days after PH with increased activity in periportal hepatocytes. In animals fed a sub-carcinogenic dose of AFB 1 (up to 4 weeks) the effect of PH on GGT activity was similar to that in control animals, but increased activity was mainly due to biliary hyperplasia. There was no obvious difference between animals returned to control diet after PH and those returned to toxic diet. In animals fed 4–15 weeks the percentage increase in GGT activity 1 week after PH correlated with length of time on AFB 1 diet before operation, with an increase in number and size of altered foci. These results further support the idea that there is a preliminary toxic response in GGT activity followed by a secondary response more closely related to the carcinogenic process.

Effective treatments to prevent recurrence or progression of non-muscle-invasive bladder cancer, or to inhibit metastasis of muscle-invasive forms of the disease, would deliver significant patient benefit. Here the involvement of STAT signalling and the chemopreventive potential of diindolylmethane (DIM) in human bladder cancer were investigated. Muscle-invasive bladder cancer tissues were characterised by nuclear expression of phosphorylated STAT1, 3 and 5. In E-cadherin positive tumour cell lines (RT112, RT4, HT1376), STAT5 was constitutively phosphorylated, while E-cadherin negative lines (J82, T24, UMUC3) contained phosphoSTAT3. Knockdown of STAT3 induced G₀/G₁ arrest and inhibited adhesion in J82 cells. Knockdown of STAT1inhibited migration in J82 and RT112 lines. No significant increase in apoptosis was observed. In response to the Janus kinase inhibitor, AG490, RT112 and J82 cells initially underwent G₀/G₁ arrest, with RT112 cells subsequently exhibiting S phase arrest. Phosphorylation of STAT1(Tyr701), STAT3(Tyr705) and (Ser727) and STAT5(Tyr694) was inhibited by DIM, as was adhesion of J82 cells to collagen, an effect that was enhanced when STAT1 or 3 was reduced by siRNA. However, over-expression of STAT3C partially rescued the DIM inhibitory effect on collagen-mediated adhesion. Migration of both lines was inhibited by DIM, while transfection of constitutively active STAT3C enhanced migration of RT112 cells. DIM induced cell cycle arrest and apoptosis in three cell lines with different degrees of radioresistance. Taken together, these results suggest that inhibition of STAT signalling and/or treatment with DIM may decrease invasiveness of bladder cancer. DIM can induce apoptosis in cell lines which are radioresistant, so in combination with radiotherapy may be useful in overcoming such resistance.

A range of potential chemoprotective agents, most of them natural dietary constituents, has been examined for ability to modulate both phase I (cytochrome P450 1A1, 1A2, 2B1/2, 2C11, 2E1, 3A, 4A) and phase II drug metabolizing enzymes (glutathione S-transferases, in particular subunits Yc2 and P, aflatoxin B1-aldehyde reductase and quinone reductase) in rat liver. In addition to assays of total enzyme activity and Western blots for individual isozymes, the ability of microsomes to metabolize aflatoxin B1, and of cytosols to conjugate aflatoxin B1 (AFB1)-epoxide to GSH and to produce AFB1-dialcohol, were measured. Induction of gamma-glutamyl transpeptidase activity was examined by histochemistry. Differing patterns of induction were observed, reflecting differences in the control of expression of the individual enzymes studied. Of the compounds examined, butylated hydroxytoluene, ethoxyquin, indole-3-carbinol and phenethyl isothiocyanate were the most potent bifunctional agents (inducing both phase I and II activities). Oltipraz, while only weakly inducing CYP1A2 and 2B1/2, was a potent inducer of phase II enzymes. Caffeic acid, garlic oil, sinigrin and propyl gallate all showed some ability to induce phase II enzymes. 4-Methyl catechol, alpha-tocopherol and red wine decreased certain phase I enzyme activities, while inducing total GST activity. Butylated hydroxytoluene, ethoxyquin, garlic oil and indole-3-carbinol induced gamma glutamyltranspeptidase in periportal hepatocytes. Particularly because of their ability to induce the detoxifying activities of glutathione S-transferase Yc2 and aldehyde reductase, butylated hydroxytoluene, ethoxyquin, indole-3-carbinol, oltipraz, phenethyl isothiocyanate and sinigrin will be effective blocking agents in rodents, if administered prior to AFB1. While these studies indicate the relative contributions of phase I and II metabolism in the overall protective effect in rat, care should be taken that a similar balance is achieved in man, and that relevant enzymes or iso forms are induced.