15-Lipoxygenase-1 re-expression in colorectal cancer alters endothelial cell features through enhanced expression of TSP-1 and ICAM-1

15-lipoxygenase-1 (15-LOX-1) oxygenates linoleic acid to 13(S)-hydroxyoctadecadienoic acid (RODE). The enzyme is widely suppressed in different cancers and its re-expression has tumor suppressive effects. 15-LOX-1 has been shown to inhibit neoangiogenesis in colorectal cancer (CRC); in the present study we confirm this phenomenon and describe the mechanistic basis. We show that re-expression of 15-LOX-1 in CRC cell lines resulted in decreased transcriptional activity of HIF1 alpha and reduced the expression and secretion of VEGF in both normoxic and hypoxic conditions. Conditioned medium (CM) was obtained from CRC or prostate cancer cell lines re-expressing 15-LOX-1 (15-LOX-1CM). 15-LOX-1CM treated aortic rings from 6-week old C57BL/6 mice showed significantly less vessel sprouting and more organized structure of vascular network. Human umbilical vein endothelial cells (HUVECs) incubated with 15-LOX-1CM showed reduced motility, enhanced expression of intercellular cell adhesion molecule (ICAM-1) and reduced tube formation but no change in proliferation or cell cycle distribution. HUVECs incubated with 13(S)-HODE partially phenocopied the effects of 15-LOX-1CM, showed reduced motility and enhanced expression of ICAM-1, but did not reduce tube formation, implying the importance of additional factors. Therefore, a Proteome Profiler Angiogenesis Array was carried out, which showed that Thrombospondin-1 (TSP-1), a matrix glycoprotein known to strongly inhibit neovascularization, was expressed significantly more in HUVECs incubated with 15-LOX-1CM. TSP-1 blockage in HUVECs reduced the expression of ICAM-1 and enhanced cell motility, thereby providing a mechanism for reduced angiogenesis. The anti-angiogenic effects of 15-LOX-1 through enhanced expressions of ICAM-1 and TSP-1 are novel findings and should be explored further to develop therapeutic options

Quantification of SLIT-ROBO transcripts in hepatocellular carcinoma reveals two groups of genes with coordinate expression

Background: SLIT-ROBO families of proteins mediate axon pathfinding and their expression is not solely confined to nervous system. Aberrant expression of SLIT-ROBO genes was repeatedly shown in a wide variety of cancers, yet data about their collective behavior in hepatocellular carcinoma (HCC) is missing. Hence, we quantified SLIT-ROBO transcripts in HCC cell lines, and in normal and tumor tissues from liver

Dose- and time-dependent expression patterns of zebrafish orthologs of selected E2F target genes in response to serum starvation/replenishment

Targets of E2F transcription factors effectively regulate the cell cycle from worms to humans. Furthermore, the dysregulation of E2F transcription modules plays a highly conserved role in cancers of human and zebrafish. Studying E2F target expression under a given cellular state, such as quiescence, might lead to a better understanding of the conserved patterns of expression in different taxa. In the present study, we used literature searches and phylogeny to identify several targets of E2F transcription factors that are known to be serum-responsive; namely, PCNA, MYBL2, MCM7, TYMS, and CTGF. The transcriptional serum response of zebrafish orthologs of these genes were quantified under different doses (i.e., 0, 0.1, 1, 3, and 10% FBS) and time points (i.e., 6, 24 and 48 hours, h) using quantitative RT-PCR (qRT-PCR) in the zebrafish fibroblast cells (ZF4). Our results indicated that mRNA expression of zebrafish pcna, mybl2, mcm7 and tyms drastically decreased while that of ctgf increased with decreasing serum levels as observed in mammals. These genes responded to serum starvation at 24 and 48 h and to the mitogenic stimuli as early as 6 h except for ctgf whose expression was significantly altered at 24 h. The zebrafish Mcm7 protein levels also were modulated by serum starvation/replenishment. The present study provides a foundation for the comparative analysis of quantitative expression patterns for genes involved in regulation of cell cycle using a zebrafish serum response model

Investigation and comparison of the preprocessing algorithms for microarrayanalysis for robust gene expression calculation and performance analysis of technical replicates

Preprocessing of microarray data involves the necessary steps of background correction, normalization and summarization of the raw intensity data obtained from cDNA or oligo-arrays before statistical analysis. Several algorithms, namely RMA, dChip, and MAS5 exist for the preprocessing of Affymetrix microarray data. Previous studies have identified RMA as one of most accurate algorithms while MAS5 was characterized with lower accuracy and sensitivity levels. In this study, performance of different preprocessing algorithms have been compared in terms of ROC characteristics of pairwise intensity differences of microarray replicates. Our findings indicated that all three algorithms predicted in similar order the quality of the technical replicates obtained from a selected set of latin square experiments [1]. On the other hand, RMA exhibited higher performance in terns of accuracy by maximizing the area under the receiver operating curve. The proposed method also is useful for detection of global and/or local artifacts associated within the technical replicas of a microarray experiment. Therefore this study is unique in the sense that it provides an extensive investigation and comparison of preprocessing algorithms and proposes a novel method for the detection and identification of fine technical replicate pair