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DOC file, 112KB, Enzymes involved in DNA and chromatin folding, which are targeted by dietary agents.

PDF file, 67KB, MDA-MB- 231 cells were treated for 48 h with phytochemicals in indicated concentrations (μM).

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]

PDF file, 93KB, MDA-MB-231 cells were grown in the presence of curcumin, DIM or DMSO.

DOC file, 111KB, Effect of dietary phytochemicals on metastasis and apoptosis in primary tumors in animal models.

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.

This is the pre-peer reviewed version of the following article: Rapid Communications in Mass Spectrometry, 2010, 24 (16), pp. 2329-2340, which has been published in final form at www3.interscience.wiley.com, Doi: 10.1002/rcm.4645

The worldwide incidence of cholangiocarcinoma (CC) is steadily rising, with the incidence in United Kingdom (UK) now exceeding 1000 cases per year. It is an aggressive malignancy typified by unresponsiveness to the existing chemotherapy and radiotherapy regimes in the vast majority of cases. Surgery offers the only hope of a cure, though post-operative disease recurrence is common, with 5-year survival rates of less than 25% following resection. Developments in molecular techniques and improved understanding of the basis of carcinogenesis in CC has led to examination of the role of biomarkers in predicting poor outcome. This systematic review examines published evidence relating to the prognostic significance of these molecular markers in CC. Of the molecular markers which have been investigated to date, p53 mutation, cyclins, proliferation indices, mucins, CA19-9, CRP and aneuploidy appear to hold significant potential as predictors of outcome in CC. These and other biomarkers may themselves represent novel therapeutic targets for CC.

Consumption of fruits and vegetables has generally been associated with a decrease in cancer incidence and cardiovascular disease. Over the years, numerous bioactive compounds have been identified that contribute to these beneficial health effects. More recently, evidence is emerging that specific combinations of phytochemicals may be far more effective in protecting against cancer than isolated compounds. Combinatorial effects have been observed where any one of the single agents is inactive. Apart from interactions among dietary micronutrients, drug-phytochemical interactions have also been observed, indicating possibilities for improved cancer therapeutic strategies. Our understanding of the molecular mechanisms underlying such synergistic effects is still limited, but it appears that different combinations of complementary modes of actions are involved. In this review, we discuss the molecular mechanisms that are likely to be involved in cancer chemoprevention and summarize the most important findings of those studies that report synergistic chemopreventive effects of dietary compounds.

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.

Colorectal cancer remains a leading cause of cancer death worldwide, despite markedly improved response rates to current systemic therapies. Oxaliplatin either alone or incorporated into 5-fluorouracil/leucovorin regimes has resulted in increased survival rates, particularly with regards to metastatic colorectal carcinoma. The chemopreventive polyphenol curcumin, which is currently in clinical trial, has been advocated for use in colorectal cancer either singly or in combination with chemotherapeutic drugs. In this study, the antiproliferative capacity of both compounds was compared in HCEC (normal-derived), HT29 (p53 mutant adenocarcinoma) and HCT116 (p53wt adenocarcinoma) colorectal cell lines to determine whether effects were cell-type specific at pharmacologically achievable doses, and whether the combination resulted in enhanced efficacy. Both oxaliplatin and curcumin displayed marked antiproliferative capacity at therapeutic concentrations in the two tumor cell lines. Order of sensitivity to oxaliplatin was HCT116>HT29>HCEC, whereas order of sensitivity to curcumin was HT29>HCT116>HCEC. HCT116 cells underwent induction of G2/M arrest in response to both oxaliplatin (irreversible) and curcumin (reversible). Apoptosis was induced by both agents, and up to 16-fold induction of p53 protein was observed in response to the combination. Antiproliferative effects in HT29 cells were largely cell cycle independent, and were mediated by induction of apoptosis. Effects were greatly enhanced in both cell lines when agents were combined. This study provides further evidence that curcumin may be of use in therapeutic regimes directed against colorectal cancer, and suggests that in combination with oxaliplatin it may enhance efficacy of the latter in both p53wt and p53 mutant colorectal tumors.

Pancreatic cancer has a poor prognosis. The use of drugs or natural agents which inhibit or slow down tumour growth therefore has important potential in the development of future therapies.A literature search of the PubMed and ISI Web of Science databases was undertaken to review the current data available on the alterations in signalling pathways found in pancreatic carcinogenesis, in order to identify sites that could be targeted by chemopreventive agents. Several agents of particular relevance to pancreatic cancer were identified, and their possible mechanisms of action reviewed.Chemopreventive agents such as non-steroidal anti-inflammatory drugs, green tea constituents, and antioxidants have been shown to target various steps in intracellular signalling pathways, particularly those controlling cell proliferation and survival. Work on cell lines and animal models has shown that some of these agents may be able to modulate the growth of pancreatic tumours. Initial clinical trials of some chemopreventives in pancreatic cancer have been undertaken, and have yielded mixed results, prompting the need for further studies.As the molecular pathology of pancreatic cancer becomes better understood, sites of action of chemopreventive substances have been uncovered. Several agents have shown promising results by their ability to inhibit pancreatic carcinogenesis in laboratory studies. If these effects can be successfully translated into human studies then these agents may prove to be valuable adjuvant therapies in the future.

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.

Indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) are promising cancer chemopreventive agents in rodent models, but there is a paucity of data on their pharmacokinetics and tissue disposition. The disposition of I3C and its acid condensation products, DIM, [2-(indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane (LTr1), indolo[3,2b]carbazole (ICZ) and 1-(3-hydroxymethyl)-indolyl-3-indolylmethane (HI-IM) was studied, after oral administration of I3C (250 mg/kg) to female CD-1 mice. Blood, liver, kidney, lung, heart, and brain were collected between 0.25 and 24 h after administration and the plasma and tissue concentrations of I3C and its derivatives determined by high-performance liquid chromotography. I3C was rapidly absorbed, distributed, and eliminated from plasma and tissues, falling below the limit of detection by 1 h. Highest concentrations of I3C were detected in the liver where levels were approximately 6-fold higher than those in the plasma. Levels of DIM, LTr1, and HI-IM were much lower, although they persisted in plasma and tissues for considerably longer. DIM and HI-IM were still present in the liver 24 h after I3C administration. Tissue levels of DIM and LTr1 were found to be in equilibrium with plasma at almost every time point measured. In addition to acid condensation products of I3C, a major oxidative metabolite (indole-3-carboxylic acid) and a minor oxidative metabolite (indole-3-carboxaldehyde) were detected in plasma of mice after oral administration of I3C. ICZ was also tentatively identified in the liver of these mice. This study shows for the first time that, after oral administration to mice, I3C, in addition to its acid condensation products, is absorbed from the gut and distributed systemically into a number of well-perfused tissues, thus allowing the possibility for some pharmacological activity of the parent compound in vivo.

3,3'-Diindolylmethane (DIM) is a naturally occurring indole, which is currently under investigation as a potential chemopreventive agent. The concentrations of DIM in plasma, liver, kidney, lung, heart, and brain tissues were determined following oral administration of two different formulations to mice (250 mg/kg). Mice were sacrificed periodically from 0 to 24 h after administration of either a crystalline or an absorption-enhanced formulation (Bio-Response-DIM; Indolplex) of DIM, and plasma and tissue concentrations were determined by high-performance liquid chromatography (UV detection, 280 nm). A physiologically based pharmacokinetic (PBPK) model was developed to characterize the pharmacokinetic properties of the two different formulations. The final model included parameters reflecting linear first-order absorption, systemic clearance, and distributional clearance in the remainder compartment, which were considered independent of formulation. All pharmacokinetic profiles from the two formulations were fitted simultaneously to estimate unknown model parameters. Plasma and tissue concentration-time profiles exhibited a rapid rise to peak values at 0.5 to 1 h, followed by a polyexponential decline with an extended terminal phase. These profiles were well described by the final model and unknown parameters were estimated with relatively low coefficients of variation. Relative drug exposure and absorption parameters suggest that BioResponse-DIM exhibited approximately 50% higher bioavailability than the crystalline formulation. Clearance of DIM was estimated as 7.18 ml/h. This is the first study to characterize the pharmacokinetics of DIM in mice, and the established PBPK model should prove useful in the design and analysis of future preclinical studies aimed at evaluating the in vivo pharmacological effects of DIM.

ET-743, an experimental antitumor drug with promising activity in sarcoma, breast and ovarian carcinoma, is currently under phase 2 clinical evaluation. It is hepatotoxic in animals and patients. We tested the hypothesis that indole-3-carbinol (I3C), the hydrolysis product of glucosinolates occurring in cruciferous vegetables, may protect against ET-743-induced hepatotoxicity in the female Wistar rat, the animal species with the highest sensitivity toward the adverse hepatic effect of this drug. Hepatotoxicity was adjudged by measurement of plasma levels of bilirubin, alkaline phosphatase (ALP) and aspartate aminotransferase (AST) and by liver histopathology. The effect of I3C on the kinetics of ET-743 in rat plasma and liver was investigated by high-pressure liquid chromatography. The effect of I3C on the antitumor efficacy of ET-743 was explored in rats bearing the 13762 mammary carcinoma. ET-743 (40 microg/kg i.v.) alone caused an elevation of plasma bilirubin, ALP and AST levels and degeneration and patchy focal necrosis of bile duct epithelial cells. Addition of I3C to the diet (0.5%) for 6 days prior to ET-743 administration almost completely abolished manifestations of hepatotoxicity. In contrast, a dietary concentration of 0.1% I3C did not protect, nor did dietary diindolylmethane (0.2%), an acid-catalyzed condensation product of I3C. Ingestion by rats of I3C for 6 days prior to ET-743 (40 microg/kg i.v.) decreased plasma but not hepatic concentrations of ET-743 compared to animals that received ET-743 alone. I3C did not interfere with the antitumor efficacy of ET-743. The results suggest that ingestion of I3C may counteract the unwanted effect of ET-743 in the liver. I3C should be investigated as a hepatoprotectant in patients who receive ET-743 therapy.

A high-performance liquid chromatographic method was developed for the simultaneous determination of indole-3-carbinol (I3C), 3,3'-diindolylmethane (DIM), [2-(indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane (LTr(1)), and indolo[3,2b]carbazole (ICZ). Compounds were extracted from mouse plasma using tert.-butyl methyl ether, incorporating 4-methoxy-indole as internal standard. Chromatographic separation utilized a Waters Symmetry RP18 in tandem with a Thermoquest BDS C(18) column, an acetonitrile-water gradient and UV (280 nm) in series with fluorescence (ex. 335 nm; em. 415 nm) detection. Calibration curves were linear (r(2)>0.99) between 50 and 15,000 ng/ml for I3C; 150 and 15,000 ng/ml for LTr(1); and 0.15 and 37.5 ng/ml for ICZ and the method was reproducible and precise (within-day and between-day coefficients of variation below 9.7 and 13%, respectively). The method described is suitable for comprehensive pharmacokinetic studies with indole-3-carbinol.

The serine/threonine kinase Nek2 (NIMA-related kinase 2) regulates centrosome separation and mitotic progression, with overexpression causing induction of aneuploidy in vitro. Overexpression may also enable tumour progression through effects upon Akt signalling, cell adhesion markers and 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.

The leaves of Corema album (Ericaceae), an endemic shrub which grows in Atlantic coastal areas of the Iberian Peninsula, are rich in flavonoids and other secondary metabolites. Silica gel column chromatography of the ethyl acetate extract from dried leaves was performed and a flavonic active fraction was obtained. The cytotoxic activity of this fraction was assessed using the colon cancer cell lines HCT116 and HT29. After 48 hours of treatment, cell viability was determined with luminescence-based ATPLite assay, showing IC50 values of 7.2 +/- 0.7 and 6.8 +/- 1.2 microg/mL, respectively. The study by flow cytometry revealed that the cytotoxicity of this fraction was mediated, at least in part, by induction of apoptosis and G2/M cell cycle arrest. The active fraction was then subjected to Sephadex LH-20 chromatography and two flavonoids were separated and identified as the flavanone pinocembrin and 2',4'-dihydroxychalcone after UV, MS and NMR analysis.

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

Colorectal cancer (CRC) remains a cause of significant mortality in developed countries despite extensive knowledge of its epidemiology and molecular basis. Since multiple molecular steps that collectively bring about this disease are known, its chemoprevention is a realistic proposition. Biochemical targets of CRC chemopreventive agents include carcinogen metabolising enzymes, arachidonic acid metabolism, the transcription factor nuclear factor-kappa beta (NF-κB), enzymes responsible for polyamine metabolism, and events associated with proliferation and apoptosis of preneoplastic cells. Aspirin, celecoxib, calcium and α-difluoromethylornithine are examples of drugs that have undergone clinical testing. Critical evaluation of these trials allows optimisation of methodologies for clinical advancement of novel chemopreventive agents. Cancer patients can be a suitable cohort of subjects for pilot studies of certain new agents. Such studies and larger trials in high-risk healthy individuals require the stringent use of carefully validated ‘preneoplastic’ biomarkers which are intrinsically related to defined stages of colorectal carcinogenesis and/or to mechanisms of action of the agent under investigation.

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.

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.

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.

The aims of this study were to determine potency of oxaliplatin in combination with curcumin in oxaliplatin-resistant cell lines in vitro and to evaluate the efficacy of a novel curcumin formulation (Meriva®) alone and in combination with oxaliplatin in colorectal tumor-bearing mice, exploring relevant pharmacodynamic markers in vivo. Oxaliplatin-resistant HCT116 p53wt and p53(-/-) cell lines were generated, and the effects of oxaliplatin in combination with curcumin on resistance- and proliferation-associated proteins investigated. Eighty nude mice were implanted with HCT116 p53wt colorectal cancer cells before randomization into the following treatment groups: control; Meriva only; oxaliplatin only; Meriva + oxaliplatin. Tumor volume was assessed, as was the expression of Ki-67, cleaved caspase-3 and Notch-1. Curcumin in combination with oxaliplatin was able to decrease proliferative capacity of oxaliplatin-resistant p53 wildtype and p53(-/-) cell lines more effectively than oxaliplatin alone. It also decreased markers associated with proliferation. After 21 days of treatment in the xenograft model, the order of efficacy was combination > Meriva > oxaliplatin > control. The decrease in tumor volume when compared to vehicle-treated animals was 53, 35 and 16%, respectively. Ki-67 and Notch-1 immunoreactivity was decreased by the combination when compared to vehicle-treated animals, with cleaved caspase-3 rising by 4.4-fold. Meriva did not adversely affect the DNA-platinating ability of oxaliplatin. Curcumin enhanced the cytotoxicity of oxaliplatin in models of oxaliplatin resistance in vitro. In vivo, Meriva greatly enhanced oxaliplatin efficacy, without affecting the mode of action of oxaliplatin. Addition of formulated curcumin to oxaliplatin-based chemotherapy regimens has the potential for clinical benefit.

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.

Hepatocellular carcinoma (HCC) is the fifth commonest malignancy worldwide and the incidence is rising. Surgery, including transplantation resection, is currently the most effective treatment for HCC; however, recurrence rates are high and long-term survival is poor. Identifying novel chemopreventive and chemotherapeutic agents and targeting them to patients at high risk of developing HCC or following curative treatment may go some way towards improving prognosis. This review examines current knowledge regarding the chemopreventive and chemotherapeutic potential of phytochemicals in heptocarcinogenesis. Both in-vitro and animal studies demonstrate that several phytochemicals, including curcumin, resveratrol, green tea catechins, oltipraz and silibinin, possess promising chemopreventive and chemotherapeutic properties. Despite this, very few clinical trials have been performed. Problems regarding validation of biomarkers, agent delivery, side effects and patient selection are barriers that need to be overcome to determine the potential of such agents in clinical practice.

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.

The primary aim of this study was to determine whether combination of the chemopreventive agent indole-3-carbinol (I3C) with oxaliplatin would decrease proliferative index and invasive potential of human colorectal tumour cells. Combination of the agents resulted in a 170-fold decrease in proliferative capacity in SW480 and SW620 cell lines, which was approximately 6-fold greater than for oxaliplatin alone. Decreased proliferation was attributed to enhanced S-phase cell cycle arrest for SW480, and increased apoptosis for SW620 cells. The combined agents resulted in significantly increased E-cadherin levels in SW480 cells, and β-catenin levels in both cell lines (assessed by in-cell westerns). In SW480 cells confocal microscopy revealed an increase in membrane-associated β-catenin levels, with oxaliplatin treatments enhancing nuclear export and cytoplasmic localisation. In SW620 cells, all treatments increased membrane localisation of E-cadherin. Whilst both oxaliplatin and I3C decreased invasive capacity of SW480 cells, this was not further enhanced by the combined treatment.

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.

Hepatocellular carcinoma (HCC) is the fifth commonest malignancy worldwide and its incidence is rising. Surgery, including transplantation, remains the only potentially curative modality for HCC, yet recurrence rates are high and long-term survival poor. The ability to predict individual recurrence risk and subsequently prognosis would help guide surgical and chemotherapeutic treatment. As understanding of hepatocarcinogenesis has increased, the myriad of genetic and molecular events that drive the hepatocarcinogenic disease process, including angiogenesis, invasion and metastasis, have been identified. This systematic review examines the evidence from published manuscripts reporting the prognostic potential of molecular biomarkers in hepatocellular carcinoma. In summary, a number of molecular biomarkers with prognostic significance have been identified in hepatocellular carcinoma. Not only might these molecules allow more accurate prediction of prognosis for patients with HCC, but they may also provide targets for potential therapeutic agents.

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.