Elevated beta1,4-galactosyltransferase I in highly metastatic human lung cancer cells. Identification of E1AF as important transcription activator.

The elevated levels of beta1,4-galactosyltransferase I (GalT I; EC 2.4.1.38) are detected in highly metastatic lung cancer PGBE1 cells compared with its less metastatic partner PGLH7 cells. Decreasing the GalT I surface expression by small interfering RNA or interfering with the surface of GalT I function by mutation inhibited cell adhesion on laminin, the invasive potential in vitro, and tyrosine phosphorylation of focal adhesion kinase. The mechanism by which GalT I activity is up-regulated in highly metastatic cells remains unclear. To investigate the regulation of GalT I expression, we cloned the 5'-region flanking the transcription start point of the GalT I gene (-1653 to +52). Cotransfection of the GalT I promoter/luciferase reporter and the Ets family protein E1AF expression plasmid increased the luciferase reporter activity in a dose-dependent manner. By deletion and mutation analyses, we identified an Ets-binding site between nucleotides -205 and -200 in the GalT I promoter that was critical for responsiveness to E1AF. It was identified that E1AF could bind to and activate the GalT I promoter by electrophoretic mobility shift assay in PGLH7 cells and COS1 cells. A stronger affinity of E1AF for DNA has contributed to the elevated expression of GalT I in PGBE1 cells. Stable transfection of the E1AF expression plasmid resulted in increased GalT I expression in PGLH7 cells, and stable transfectants migrated faster than control cells. Meanwhile, the content of the beta1,4-Gal branch on the cell surface was increased in stably transfected PGLH7 cells. GalT I expression can also be induced by epidermal growth factor and dominant active Ras, JNK1, and ERK1. These data suggest an essential role for E1AF in the activation of the human GalT I gene in highly metastatic lung cancer cells.

Increased anti-tumour efficacy of doxorubicin when combined with sulindac in a xenograft model of an MRP-1-positive human lung cancer

Background: A number of cellular proteins, including P-glycoprotein (P-gp) and Multiple drug Resistance Protein (MRP-1), act as drug efflux pumps and are important in the resistance of many cancers to chemotherapy. We previously reported that a small number of NSAIDs could inhibit the activity of MRP-1. Materials and Methods: We chose sulindac as a candidate agent for further investigation as it has the most favourable efficacy and toxicity profile of the agents available for a potential specific MRP-1 inhibitor. NCI H460 cells expressed MRP-1 protein (by Western blot) and also the toxicity, of doxorubicin (a substrate of MRP-1) could be potentiated in this line using non-toxic concentrations of the MRP-1 substrate/inhibitor sulindac. These cells were implanted in nude mice and the animals divided into various groups which were administered doxorubicin and/or sulindac. Results: Sulindac was shown to significantly potentiate the tumour growth inhibitor activity of doxorubicin in this MRP-1-overexpressing human tumour xenograft model. Conclusion: Sulindac may be clinically useful as an inhibitor of the MRP-1 cancer resistance mechanism.

Effects of the amphibian skin-derived bradykinin B2 receptor antagonist peptide, kinestatin, on the proliferation of human prostate cancer cell lines

Bradykinin and related peptides are found in the defensive skin secretions of many frogs and toads. While the physiological roles of bradykinin-related peptides in sub-mammalian vertebrates remains obscure, in mammals, including humans, canonical bradykinin mediates a multitude of biological effects including the proliferation of many types of cancer cell. Here we have examined the effect of the bradykinin B2 receptor antagonist peptide, kinestatin, originally isolated by our group from the skin secretion of the giant fire-bellied toad, Bombina maxima, on the proliferation of the human prostate cancer cell lines, PC3, DU175 and LnCAP. The bradykinin receptor status of all cell lines investigated was established through PCR amplification of transcripts encoding both B1 and B2 receptor subtypes. Following this demonstration, all cell lines were grown in the presence or absence of kinestatin and several additional bradykinin receptor antagonists of amphibian skin origin and the effects on proliferation of the cell lines was investigated using the MTT assay and by counting of the cells in individual wells of 96-well plates. All of the amphibian skin secretion-derived bradykinin receptor antagonists inhibited proliferation of all of the prostate cancer lines investigated in a dose-dependent manner. In addition, following incubation of peptides with each cell line and analysis of catabolites by mass spectrometry, it was found that bradykinin was highly labile and each antagonist was highly stable under the conditions employed. Bradykinin signalling pathways are thus worthy of further investigation in human prostate cancer cell lines and the evidence presented here would suggest the testing of efficacy in animal models of prostate cancer as a positive outcome could lead to a drug development programme for the treatment of this disease.

A study of HER2 gene amplification and protein expression in gastric cancer

Background Gastric cancer is a leading cause of cancer-related mortality, and current treatment outcomes for advanced disease remain poor. HER2 has been identified as a potential candidate for targeted therapy in gastric cancers displaying HER2 gene amplification and protein overexpression.

MicroRNA-130b regulates the tumour suppressor RUNX3 in gastric cancer

Aim: Accumulating evidence indicates that RUNX3 is an important tumour suppressor that is inactivated in many cancer types. This study aimed to assess the role of microRNA (miRNA) in the regulation of RUNX3.

Claudin-1 Has Tumor Suppressive Activity and Is a Direct Target of RUNX3 in Gastric Epithelial Cells

BACKGROUND & AIMS: The transcription Factor RUNX3 is a gastric tumor suppressor. Tumorigenic Runx3(-/-) gastric epithelial cells attach weakly to each other, compared with nontumorigenic Runx3(+/+) cells. We alined to identify RUNX3 target genes that promote cell-cell contact to Improve our understanding of RUNX3's role in Suppressing gastric carcinogenesis. METHODS: We compared gene expression profiles of Runx3(+/+) and Runx3(-/-) cells and observed down-regulation of genes associated with cell-cell adhesion in Runx3(-/-) cells. Reporter, mobility shift, and chromatin immunoprecipitation assays were used to examine the regulation of these genes by RUNX3. Tumorigenesis assays and immunohistologic, analyses of human gastric tumors were performed to confirm the role of the candidate genes ill gastric tumor development. RESULTS: Mobility shift and chromatin immunoprecipitation assays revealed that the promoter activity of the gene that encodes the tight Junction protein claudin-1 was up-regulated via the binding of RUNX3 to the RUNX consensus sites. The tumorigenicity of gastric epithelial cells From Runx3(-/-) mice was significantly reduced by restoration of claudin-1 expression, whereas knockdown of claudin-1. increased the tumorigenicity of human gastric cancer cells. Concomitant expression of RUNX3 and claudin-1 was observed in human normal gastric epithelium and cancers. CONCLUSIONS: The tight junction protein claudin-1 has gastric tumor suppressive activity and is a direct transcriptional target of RUNX3. Claudin-1 is down-regulated during the epithelial-mesenchymal transition; RUNX3 might therefore act as a tumor suppressor to antagonize the epithelial-mesenchymal transition.