Profiling tumour heterogeneity through circulating tumour DNA in patients with pancreatic cancer.

The majority of pancreatic ductal adenocarcinomas (PDAC) are diagnosed late so that surgery is rarely curative. Earlier detection could significantly increase the likelihood of successful treatment and improve survival. The aim of the study was to provide proof of principle that point mutations in key cancer genes can be identified by sequencing circulating free DNA (cfDNA) and that this could be used to detect early PDACs and potentially, premalignant lesions, to help target early effective treatment. Targeted next generation sequencing (tNGS) analysis of mutation hotspots in 50 cancer genes was conducted in 26 patients with PDAC, 14 patients with chronic pancreatitis (CP) and 12 healthy controls with KRAS status validated by digital droplet PCR. A higher median level of total cfDNA was observed in patients with PDAC (585 ng/ml) compared to either patients with CP (300 ng/ml) or healthy controls (175 ng/ml). PDAC tissue showed wide mutational heterogeneity, whereas KRAS was the most commonly mutated gene in cfDNA of patients with PDAC and was significantly associated with a poor disease specific survival (p=0.018). This study demonstrates that tNGS of cfDNA is feasible to characterise the circulating genomic profile in PDAC and that driver mutations in KRAS have prognostic value but cannot currently be used to detect early emergence of disease. Importantly, monitoring total cfDNA levels may have utility in individuals "at risk" and warrants further investigation.

Overexpression of the Nek2 kinase in colorectal cancer correlates with beta-catenin relocalization and shortened cancer-specific survival.

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.

Induction of apoptosis and cell cycle arrest in human colon carcinoma cells by Corema album leaves.

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.

Inhibition of STAT signalling in bladder cancer by diindolylmethane: relevance to cell adhesion, migration and proliferation.

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.

Curcumin-induced mitotic arrest is characterized by spindle abnormalities, defects in chromosomal congression and DNA damage.

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.

Notch3 and HEY-1 as prognostic biomarkers in pancreatic adenocarcinoma.

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.

Curcumin ameliorates oxaliplatin-induced chemoresistance in HCT116 colorectal cancer cells in vitro and in vivo.

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.

Development of a targeted adductomic method for the determination of polycyclic aromatic hydrocarbon DNA adducts using online column-switching liquid chromatography/tandem mass spectrometry.

Human exposure to polycyclic aromatic hydrocarbons (PAHs) from sources such as industrial or urban air pollution, tobacco smoke and cooked food is not confined to a single compound, but instead to mixtures of different PAHs. The interaction of different PAHs may lead to additive, synergistic or antagonistic effects in terms of DNA adduct formation and carcinogenic activity resulting from changes in metabolic activation to reactive intermediates and DNA repair. The development of a targeted DNA adductomic approach using liquid chromatography/tandem mass spectrometry (LC/MS/MS) incorporating software-based peak picking and integration for the assessment of exposure to mixtures of PAHs is described. For method development PAH-modified DNA samples were obtained by reaction of the anti-dihydrodiol epoxide metabolites of benzo[a]pyrene, benzo[b]fluoranthene, dibenzo[a,l]pyrene (DB[a,l]P) and dibenz[a,h]anthracene with calf thymus DNA in vitro and enzymatically hydrolysed to 2'-deoxynucleosides. Positive LC/electrospray ionisation (ESI)-MS/MS collision-induced dissociation product ion spectra data showed that the majority of adducts displayed a common fragmentation for the neutral loss of 116 u (2'-deoxyribose) resulting in a major product ion derived from the adducted base. The exception was the DB[a,l]P dihydrodiol epoxide adduct of 2'-deoxyadenosine which resulted in major product ions derived from the PAH moiety being detected. Specific detection of mixtures of PAH-adducted 2'-deoxynucleosides was achieved using online column-switching LC/MS/MS in conjunction with selected reaction monitoring (SRM) of the [M+H](+) to [M+H-116](+) transition plus product ions derived from the PAH moiety for improved sensitivity of detection and a comparison was made to detection by constant neutral loss scanning. In conclusion, different PAH DNA adducts were detected by employing SRM [M+H-116](+) transitions or constant neutral loss scanning. However, for improved sensitivity of detection optimised SRM transitions relating to the PAH moiety product ions are required for certain PAH DNA adducts for the development of targeted DNA adductomic methods.

Dietary chemopreventive phytochemicals: too little or too much?

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.

Lapatinib, a dual inhibitor of ErbB-1/-2 receptors, enhances effects of combination chemotherapy in bladder cancer cells.

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.

Phytochemicals as potential chemopreventive and chemotherapeutic agents in hepatocarcinogenesis.

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.

Prognostic molecular markers in cholangiocarcinoma: a systematic review.

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.

Mechanisms of combined action of different chemopreventive dietary compounds: a review.

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.

Indole-3-carbinol enhances anti-proliferative, but not anti-invasive effects of oxaliplatin in colorectal cancer cell lines.

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 beta-catenin levels in both cell lines (assessed by in-cell westerns). In SW480 cells confocal microscopy revealed an increase in membrane-associated beta-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.

Expression of nuclear Notch3 in pancreatic adenocarcinomas is associated with adverse clinical features, and correlates with the expression of STAT3 and phosphorylated Akt.

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.

Extended treatment with physiologic concentrations of dietary phytochemicals results in altered gene expression, reduced growth, and apoptosis of cancer cells.

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.

Predicting the physiological relevance of in vitro cancer preventive activities of phytochemicals.

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.

Comparison of oxaliplatin- and curcumin-mediated antiproliferative effects in colorectal cell lines.

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.

Prognostic molecular markers in hepatocellular carcinoma: a systematic review.

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.

EGFR and Src are involved in indole-3-carbinol-induced death and cell cycle arrest of human breast cancer cells.

Indole-3-carbinol (I3C), a dietary chemopreventive compound, induced marked reduction in epidermal growth factor receptor (EGFR) prior to cell death in cells representing three breast cancer subtypes. Signalling pathways, linking these events were investigated in detail. I3C modulated tyrosine phosphorylation from 30 min in four cell lines. In MDA-MB-468 and HBL100 cells, it induced Src activation after 5 h. In MDA-MB-468 cells, I3C induced signalling between 4.5 and 7 h, which involved sequential activation of Src, EGFR, STAT-1 and STAT-3, followed by EGFR degradation. It also induced physical association between activated Src and EGFR. In MCF7 and MDA-MB-231 cells, I3C modulated expression of cell cycle-related proteins, p21Cip1, p27Kip1, cyclin E, cyclin D1 and CDK6, with upregulation of p21Cip1 and cyclin E being dependent on Src. Inhibition of EGFR by specific inhibitors PD153035 or ZD1839 increased susceptibility to I3C-induced apoptosis of MCF7, MDA-MB-468 and MDA-MB-231 cells. Inhibition of Src sensitized MDA-MB-468 and MDA-MB-231 cells to I3C, whereas overexpression of c-Src increased resistance to I3C in MDA-MB-468 and HBL100 cells. Modulation of Src in MDA-MB-468 cells influenced the basal level of EGFR expression and cell viability; the latter being positively correlated with EGFR activation levels. Therefore, EGFR and Src activities are essential for I3C-induced cell cycle arrest and death; however, I3C-induced pathways depend on specific features of breast cancer cells. The cancer types, which rely on 'EGFR addiction' or Src deregulation, are likely to be susceptible to I3C.