Induction of Tissue Factor Expression in Endothelial Cells by Basic Fibroblast Growth Factor and its Modulation by Fenofibric acid

BACKGROUND: Tissue factor (TF), expressed in endothelial cells (ECs) and enriched in human atherosclerotic lesions, acts as a critical initiator of blood coagulation in acute coronary syndrome. Basic fibroblast growth factor (bFGF) induces the proliferation and migration of ECs and plays a role in angiogenesis and restoration of endothelial integrity. As TF is implicated in angiogenesis, we studied the effect of bFGF on TF gene and protein expression. Methods: Human umbilical vein ECs (HUVECs) were exposed to bFGF. TF mRNA was assessed by Northern blot and TF protein was assessed by Western blot. TF promoter activity was assessed by transient transfection assay and transcription factor was identified by electro mobility shift assay. RESULTS: bFGF increased TF mRNA and protein expression in HUVECs. Increased TF mRNA was attenuated by inhibition of extracellular signal-regulated kinase kinase in human ECV304 cells. Transient transfection assays of the human TF promoter-luciferase construct (-786/+121 bp) demonstrated that bFGF induced transcription was dependent on the elements within the -197 to -176 bp relative to the transcription start site of the human TF gene. This region contains NF-κB like binding site. Electro mobility shift assay showed that bFGF increased nuclear translocation or DNA binding of NF-κB transcription factor to TF promoter. Nucleotide substitution to disrupt NF-κB like site reduced bFGF stimulated promoter activity. Fenofibric acid, an agonist ligand for the peroxisome proliferator activated receptor-α, reduced basal and bFGF stimulated TF expression. CONCLUSIONS: These results indicate that bFGF may increase TF production in ECs through activation of transcription at NF-κB binding site, and control coagulation in vessel walls. Fibrate can inhibit TF expression and therefore reduce the thrombogenecity of human atherosclerotic lesions

HOXD3-overexpression increases integrin alphavbeta3 expression and deprives E-cadherin while it enhances cell motility in A549 cells.

We have previously shown that transduction of HOXD3, one of homeobox genes, into human lung cancer A549 cells enhances cell motility, invasion and metastasis. In the present study, we examined the roles of integrin β3 which was up-regulated by HOXD3-overexpression in the HOXD3-induced motility of A549 cells. We first established integrin β3-transfectants and compared their motile activity to those of the HOXD3-transfected, control-transfected and parental cells by three different assays. The integrin β3-transfectants as well as the HOXD3-transfectants formed heterodimer with integrin αv subunit, and showed highly motile activities assessed by haptotaxis or phagokinetic track assay compared to the control transfectants or parental cells. In vitro wound-healing assay revealed that migratory activities were graded as the HOXD3-transfectants > the integrin β3-transfectants > the control transfectants or parental cells. E-cadherin was expressed in the integrin β3-transfectants but not expressed in the HOXD3-transfectants. An addition of function-blocking antibody to E-cadherin into the wound-healing assay promoted the migratory activity of the integrin β3-transfectants, suggesting that E-cadherin prevented the cells from dissociating from the wound edges. These results indicate that increased expression of integrin αv β3 and loss of E-cadherin by HOXD3-overexpression are responsible for the enhanced motility and dissociation

p53 dominant-negative mutant R273H promotes invasion and migration of human endometrial cancer HHUA cells

Dominant negative (DN) mutations of tumor suppressor p53 (TP53) are clinically associated with cancer progression and metastasis of endometrial malignancy. To investigate the DN effect on tumor migration and invasion, we generated cells that stably co-expressed wild-type (wt) and R273H DN mutant TP53 (273H cells), and wt and R213Q recessive mutant TP53 (213Q cells), by transfection in endometrial cancer cells HHUA that expressed wt p53. R273H, but not R213Q, repressed wt p53-stimulated transcription of p21, Bax, and MDM2. 273H cells also showed markedly increased in vitro invasion and migration potentials, and displayed reduced Maspin, PAI-1, and KAI1 mRNA expressions as compared with 213Q and wt cells. The induction of wt p53 function by use of Adriamycin resulted in the inhibition of the invasion/migration capacity in association with the up-regulation of p53 target genes to a far greater degree in 213Q and wt cells than in 273H cells. R273H expression in p53-null cancer cell SK-OV-3 and Saos-2 did not significantly affect cell invasion and migration activities. Taken together, these results suggest that transdominance of R273H mutant over wt p53 rather than a gain-of-function promotes tumor metastasis by increasing invasion and migration in HHUA cells

Epigenetic silencing of E- and P-cadherin gene expression in human melanoma cell lines

The degree of E- and P-cadherin expressions inversely correlate with the progression stage of human melanoma. In the present study, we analyzed mechanisms of down-regulation of E- and P-cadherin gene expressions in 8 human melanoma cell lines. In 5 of the 8 melanoma cell lines, E-cadherin expression was lost or markedly decreased compared to that in normal melanocytes, and 4 of the 5 melanoma cell lines lost P-cadherin expression. All of the melanoma cell lines expressed snail, which is known to encode a transcription repressor for E-cadherin, at a higher level than melanocytes whereas expression levels of the snail varied among cell lines. Transduction of snail gene into MMAc cells which expressed a high level of E-cadherin and an extremely low level of snail decreased expression of E-cadherin but not P-cadherin. In contrast, transduction of antisense-snail gene into A375M cells which expressed no E-cadherin and a high level of snail restored expression of E-cadherin but not P-cadherin. Methylation-specific PCR analysis revealed CpG methylation in the promoter region of E-cadherin of MeWo and AKI cells. Further, the treatment with a demethylating agent, 5-azacytidine led AKI and A375M cells to re-express both E- and P-cadherin. The results show E-cadherin gene is silenced by at least two distinct mechanisms (methylation and transrepression by Snail) in human melanoma cell lines whereas P-cadherin gene seems to be silenced by methylation but not by snail

Disordered Expression of HOX Genes in Human Non-Small Cell Lung Cancer

We hypothesized that the disordered tissue architecture in cancer results from the steps that the cells execute the program designed during ontogeny in a spatiotemporally inappropriate manner. HOX genes are known as master regulators of embryonic morphogenesis, and encode transcription factors which regulate the transcription of the downstream genes to realize the program of body plan. In this study, we quantified the expression levels of 39 HOX genes in 41 human non-small cell lung cancer (non-SCLC) and non-cancerous lung tissues by a comprehensive analysis system based on the realtime RT-PCR method. We found that the expression levels of HOXA1, A5, A10 and C6 in squamous cell carcinoma tissues (and HOXA5 and A10 in adenocarcinoma tissues) were significantly higher than those in the non-cancerous tissues. Comparison of HOX gene expressions between adenocarcinoma and squamous cell carcinoma tissues showed higher expressions of HOXA1, D9, D10 and D11 in squamous cell carcinoma tissues than in adenocarcinoma tissues. Immunohistochemical analysis revealed that HOXA5 and A10 proteins were localized in the cytoplasm of tumor cells in both adenocarcinoma and squamous cell carcinoma tissues. These results suggest that the disordered patterns of HOX gene expressions were involved in not only the development of non-SCLC but also histological diversity such as adenocarcinoma and squamous cell carcinoma

PAX4 has the potential to function as a tumor suppressor in human melanoma

We hypothesize that dysregulated expression levels of the developmental regulatory genes in the adult body result in tumor development and malignant progression. PAX genes discovered as human orthologous genes of Drosophila 'paired' encode transcription factors, which control the expression of target genes to go on along the program of development. In this study, we first quantified expression of 9 PAX genes in human nevus pigmentosus tissues, melanoma tissues and melanoma cell lines by the real-time reverse transcription-PCR method. As a result, we found that the expression levels of PAX4 and PAX9 were extremely low in melanoma tissues and cell lines compared to nevus pigmentosus tissues. We then established melanoma cells overexpressing PAX4 and examined roles of PAX4 in cell growth. PAX4-overexpression reduced in vitro cell growth of human melanoma C8161 and MeWo cells. BrdU-uptake assay and cell cycle analysis by flow cytometry indicated that the retardation of cell proliferation by PAX4-overexpression was due to decreased DNA synthesis and cell cycle arrest at the G0/G1 phase. Furthermore, treatment of C8161 and MeWo cells with 5-azacytidine, a DNA demethylating agent, induced the expression of PAX4, suggesting that DNA methylation repressed the PAX4 gene expression in human melanoma. These results suggest that PAX4 functions as a potent tumor suppressor