Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0025202 (melanoma)
 69,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gene therapy is a novel therapy for melanoma. To date, however, there is still no powerful tumor specific promoter (TSP) to restrict the transgene expression in melanoma cells. In order to define a useful TSP for targeting in the context of melanoma gene therapy, four promoters, the cyclooxygenase-2 (Cox-2), alpha-chemokine SDF-1 receptor (CXCR4), epithelial glycoprotein 2 (EGP-2), and survivin, were tested in both established melanoma cell lines and primary melanoma cells. We employed recombinant adenoviral vectors (reAds) each with a candidate TSP (the Cox-2, CXCR4, EGP-2, or survivin), a reporter luciferase gene, and a poly-A signal, all of which were inserted into the E1-deleted region. A reAdGL3Bcytomegalovirus (CMV), containing the CMV promoter and luciferase gene, was used as a positive control to normalize the luciferase activity. Luciferase activity was measured in multiple tumor cell lines and two primary melanoma cell cultures after infection with reAds. Human epithelial melanocytes, HEM, were used as normal control. In contrast to three other promoters, the survivin promoter exhibited the highest activities within both melanoma cell lines and primary melanoma cells, but not in HEMs. Additionally, the survivin promoter exhibited very low activities in major mouse organs including the liver, in vivo. EGP-2 is not active in melanoma; messenger RNA expressions were correlated to promoter activities both in melanoma cell lines and primary cell cultures. Thus, these data suggest that the survivin promoter achieved a 'tumor-on/liver-off' profile, and thus represents a potentially useful tumor-specific promoter with applications for transcriptional targeting of Ad vector-based cancer gene therapy or oncolysis to melanoma.
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PMID:Evaluation of tumor-specific promoter activities in melanoma. 1569 77

Silencing of oncogenes or other genes contributing to tumor malignancy or progression by RNA interference (RNAi) offers a promising approach to treating tumor patients. To achieve RNAi-based tumor therapy, a small interfering RNA (siRNA) or siRNA-expressing vector needs to be delivered to tumor cells, but little information about its in vivo delivery has been reported. In this study, we examined whether the expression of the target gene in tumor cells can be suppressed by the delivery of RNAi effectors to primary and metastatic tumor cells. To quantitatively evaluate the RNAi effects in tumor cells, mouse melanoma B16-BL6 cells were stably transfected with both firefly (a model target gene) and sea pansy (an internal standard gene) luciferase genes to obtain B16-BL6/dual Luc cells. The target gene expression in subcutaneous primary tumors of B16-BL6/dual Luc cells was significantly suppressed by direct injection of the RNAi effectors followed by electroporation. The expression in metastatic hepatic tumors was also significantly reduced by an intravenous injection of either RNAi effector by the hydrodynamics-based procedure. These results indicate that the both RNAi effectors have a potential to silence target gene in tumor cells in vivo when successfully delivered to tumor cells.
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PMID:Gene silencing in primary and metastatic tumors by small interfering RNA delivery in mice: quantitative analysis using melanoma cells expressing firefly and sea pansy luciferases. 1593 41

Interleukin (IL)-8 and transforming growth factor (TGF)-beta1 are proangiogenic factors overexpressed in advanced human melanoma. We investigated the effects of TGF-beta1 on IL-8 expression in the well-characterized A375 human melanoma system. We demonstrated by enzyme-linked immunoassay and Northern blot analysis that TGF-beta1 selectively induced IL-8 expression, at both protein and mRNA levels, in highly metastatic A375SM cells but not cells of their poorly metastatic parental line A375P. Transient transfection with luciferase reporter gene constructs revealed that TGF-beta1 activated IL-8 promoter activity in A375SM cells but not A375P cells. Studies with progressive 5' deletion constructs and site-specific mutations demonstrated that a construct containing -133 to +44 of the 5'-flanking sequence was necessary and sufficient for maximal TGF-beta1-induced transcription response and that TGF-beta1-induced activation of IL-8 promoter depended on AP-1 (-126 to -120 bp), NF-kappaB (-94 to -71 bp), and C/EBP-like factor NF-IL6 (-94 to -81 bp) in this region. Interestingly, both A375P and A375SM cells expressed type I and type II TGF-beta receptors and TGF-beta1 induced the nuclear translocation of Smad3 protein in both A375P and A375SM cells. Moreover, both A375P and A375SM cells were susceptible to TGF-beta1-induced growth inhibition. Our data thus demonstrated that TGF-beta1 selectively induced IL-8 expression in highly metastatic A375SM melanoma cells. This TGF-beta1-induced IL-8 expression could be an amplification cascade responsible for overexpression of IL-8 in human melanoma and one of potential mechanisms by which TGF-beta1 promotes angiogenesis, growth, and metastasis of human melanoma.
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PMID:Selective induction of interleukin-8 expression in metastatic melanoma cells by transforming growth factor-beta 1. 1597 19

Overexpression of cAMP-response element (CRE)-binding protein (CREB) and activating transcription factor (ATF) 1 contributes to melanoma progression and metastasis at least in part by promoting tumor cell survival and stimulating matrix metalloproteinase (MMP) 2 expression. However, little is known about the regulation of CREB and ATF-1 activities and their phosphorylation within the tumor microenvironment. We analyzed the effect of platelet-activating factor (PAF), a potent phospholipid mediator of inflammation, for its ability to activate CREB and ATF-1 in eight cultured human melanoma cell lines, and we found that PAF receptor (PAFR) was expressed in all eight lines. In metastatic melanoma cell lines, PAF induced CREB and ATF-1 phosphorylation via a PAFR-mediated signal transduction mechanism that required pertussis toxin-insensitive Galphaq protein and adenylate cyclase activity and was antagonized by a cAMP-dependent protein kinase A and p38 MAPK inhibitors. Addition of PAF to metastatic A375SM cells stimulated CRE-dependent transcription, as observed in a luciferase reporter assay, without increasing the CRE DNA binding capacity of CREB. Furthermore, PAF stimulated the gelatinase activity of MMP-2 by activating transcription and MMP-2 expression. MMP-2 activation correlated with the PAF-induced increase in the expression of an MMP-2 activator, membrane type 1 MMP. PAF-induced expression of pro-MMP-2 was causally related to PAF-induced CREB and ATF-1 phosphorylation; it was prevented by PAFR antagonist and inhibitors of p38 MAPK and protein kinase A and was abrogated upon quenching of CREB and ATF-1 activities by forced overexpression of a dominant-negative form of CREB. PAF-induced MMP-2 activation was also down-regulated by p38 MAPK and protein kinase A inhibitors. Finally, PAFR antagonist PCA4248 inhibited the development of A375SM lung metastasis in nude mice. This result indicated that PAF acts as a promoter of melanoma metastasis in vivo. We proposed that metastatic melanoma cells overexpressing CREB/ATF-1 are better equipped than nonmetastatic cells to respond to PAF within the tumor microenvironment.
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PMID:Platelet-activating factor mediates MMP-2 expression and activation via phosphorylation of cAMP-response element-binding protein and contributes to melanoma metastasis. 1630 50

We have previously shown that tumour necrosis factor-alpha (TNF-alpha) upregulates human melanoma cell integrin expression, migration and invasion in vitro. The aim of this study was to investigate the effect of the non-steroidal anti-inflammatory agent sodium salicylate on TNF-alpha-induced activation of the transcription factor nuclear factor-kappaB (NF-kappaB) and upregulation of intercellular adhesion molecule-1 (ICAM-1), and TNF-alpha-stimulated cell migration and invasion through fibronectin. HBL human melanoma cells were pre-incubated with sodium salicylate prior to stimulation with TNF-alpha for 24 h. NF-kappaB activation was measured using an assay that detects changes in the expression of a luciferase reporter gene under the direct control of NF-kappaB transcriptional activity. The effect of sodium salicylate and TNF-alpha on HBL cell invasion over 20 h and migration over 24 h was studied using fibronectin invasion and 'scratch wound' migration models in vitro, as described previously. Sodium salicylate inhibited TNF-alpha-stimulated NF-kappaB activation in melanoma cells in a concentration-dependent manner, and this was achieved with pre-incubation times as short as 15 min. TNF-alpha-stimulated ICAM-1 expression in HBL cells was also downregulated by sodium salicylate, although in a manner inversely related to the concentration of this agent. In functional assays, TNF-alpha stimulated migration and invasion, and sodium salicylate significantly reduced the extent of melanoma invasion and migration in both the presence and absence of TNF-alpha. In conclusion, sodium salicylate effectively inhibited TNF-alpha-induced upregulation of NF-kappaB, ICAM-1 expression, in-vitro migration and invasion in human melanoma cells, indicating that non-steroidal anti-inflammatory drugs may be a useful therapeutic approach to oppose inflammation-induced melanoma invasion and metastasis in vivo.
Melanoma Res 2006 Feb
PMID:Sodium salicylate inhibits TNF-alpha-induced NF-kappaB activation, cell migration, invasion and ICAM-1 expression in human melanoma cells. 1643 51

Electrically-assisted gene delivery is a non-viral gene delivery technique, using application of square wave electric pulses to facilitate uptake of plasmid DNA into the cells. Feasibility and effectiveness of this method in vivo was already demonstrated, elaborating on pulse parameters and plasmid construction. However, there were no studies performed on sequencing and timing of plasmid DNA injection into the tumors and application of electric pulses. For this purpose we measured luciferase expression in two tumor models (LPB fibrosarcoma, B16F1 melanoma) after electrically-assisted gene delivery at varying time intervals between the pCMV-Luc plasmid injection and electroporation. Expression of luciferase was determined by measurement of its activity using luminometer. The results demonstrated that pCMV-Luc plasmid has to be injected before the application of electric pulses, since no measurable expression was detected in the tumors when pCMV-Luc plasmid was injected after electroporation of tumors. In both tumor models the highest transfection efficiency was obtained when pCMV-Luc plasmid was injected not less than 5 minutes but also not more than 30 minutes before the application of electric pulses. The results also demonstrated variability in the transfection efficiency depending on the tumor model. High expression was obtained in B16F1 tumor model (approximately 5500 pg luc/mg tumor) and lower in LPB fibrosarcoma (approximately 200 pg luc/mg tumor). In conclusion, our results demonstrate that regardless of the susceptibility of the tumors to electrically-assisted gene delivery, the best timing for pCMV-Luc plasmid is between 30 to 5 minutes prior to the application of electric pulses to the tumors.
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PMID:Sequence and time dependence of transfection efficiency of electrically-assisted gene delivery to tumors in mice. 1647 96

Transforming growth factor-beta3 (TGF-beta3) has a specific role in vivo in the patterning of embryonic and tissue-specific gene expression. We have cloned and sequenced the mouse TGF-beta3 5'-flanking region to study the transcriptional regulation of this gene. Promoter fragments were cloned into a promoterless luciferase reporter plasmid to study functional activity in a human skin melanoma cell line A375 (A375). Sequential 5'-deletion encompassing DNA sequences from -2297 to -1003 bp exhibited high promoter activity in A375 cells, whereas the promoter activity decreased to minimal in the -742 to 104 bp regions, suggesting both positive and negative transcriptional regulation in the TGF-beta3 promoter. The fragment containing 1.8 kb had the highest luciferase activity. Characterization of this 1.8 kb 5'-flanking region upstream of the translation start site showed a putative hunchback-binding site consensus sequence. The electrophoretic mobility shift assay (EMSA) and transient transfection experiments showed that the putative hunchback-binding site is functional and regulated TGF-beta3 promoter transcriptional activity. The DNA-complex including the hunchback sequence binding protein (HbSBP) was important for suppression of the promoter activity in A375 cells. Mutation of the hunchback consensus sequence resulted in up to 2-fold higher promoter activity than the wild type construct. There was an absence of HbSBP in other cell lines tested including 3T3 fibroblast and B-16 mouse skin melanoma as determined by EMSA and Western blot analysis. HbSBP may function as a TGF-beta3 gene transcriptional regulator and may be expressed in a cell type-specific manner.
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PMID:Hunchback sequence binding protein suppresses mouse TGF-beta3 promoter in vitro. 1678 76

Although transcription regulation of human cytochrome P450 enzymes (CYP) is known to play an important role in drug metabolism and homeostasis, factors influencing the expression of various CYP genes in humans remain largely undefined. We used three cell lines and CD-1 mice to assess the activity of genomic promoter sequences of human CYP2D6, 1A2, 3A4, 2C9, 2C18, and 2E1 genes. CYP promoter sequences were amplified by PCR using human liver genomic DNA as the template and cloned into pGL3-Basic vectors that contain a luciferase reporter gene but lack promoter or enhancer sequences. Each plasmid construct was transfected into cells in vitro using polyethylenimine (PEI) as the transfection reagent and into mice using the recently developed hydrodynamics-based procedure. Relative promoter strength was determined by the level of luciferase expression in transfected cells. All six human CYP promoters are active in driving reporter gene expression in cultured hepatic HepG2 cells and non-hepatic cells such as human embryonic kidney fibroblasts (293 cells) and murine melanoma cells (BL-16) as well as cells in intact mouse liver, lung, heart, kidney and spleen. The order of strength among CYP promoters examined was found to be 2D6 > 1A2 > 3A4 > 2C9 > 2C18 > 2E1.
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PMID:Direct assessment of promoter activity of human cytochrome p450 genes using optimized transfection in vitro and in vivo. 1685 Feb 52

Hypoxia-inducible factor-alpha (Hif-alpha) plays an important role in tumor growth by increasing resistance to apoptosis and the production of angiogenic factors, such as vascular endothelial growth factor (VEGF). Therefore, Hif-alpha is an attractive target for development of novel cancer therapeutics. We have generated Chinese hamster ovary cells, which stably express luciferase reporter construct under the control of a hypoxia response element to screen 15,000 compounds. We identified 40 compounds that inhibited hypoxic up-regulation of luciferase, and the top 30 compounds were further screened in a secondary assay using MDA-468 breast cancer cell line. Eight compounds were shown to inhibit VEGF expression in hypoxic cells at subtoxic concentrations. Three top putative Hif inhibitors, DJ12, DJ15, and DJ30, were chosen for further analysis. Transient transfection of cells with hypoxia-regulated luciferase reporter plasmids further validated that these compounds inhibit hypoxia up-regulated genes. All three compounds failed to inhibit Hif-1alpha protein levels but they did inhibit induction of downstream targets of Hif-alpha under hypoxia. Two of the three compounds were cell type specific, whereas compound DJ12 inhibited VEGF at subtoxic levels in breast cancer cell lines MDA-468 and ZR-75, melanoma cell line MDA-435, and pVHL mutant renal cancer cell lines RCC4 and 786-0. Compound DJ12 down-regulated mRNA of downstream targets of Hif-alpha, and significantly inhibited Hif-1alpha transactivation activity by blocking Hif-1alpha hypoxia response element-DNA binding. Our cell-based approach and deconvolution of the inhibitory effect of DJ12 has identified a novel compound that targets the hypoxia pathway by inhibiting Hif-alpha-inducible transcription.
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PMID:Identification of novel small-molecule inhibitors of hypoxia-inducible factor-1 transactivation and DNA binding. 1698 52

Although obesity is known as a risk factor for several human cancers, the association of obesity with cancer recurrence and metastasis remains to be characterized. Here, B16-BL6 melanoma and Lewis lung carcinoma cells were intravenously injected into diabetic (db/db) and obese (ob/ob) mice. The number of experimental lung colonies was markedly promoted in these mice when compared with C57BL/6 mice. In contrast, tumor growth at the implanted site was comparable when cells were inoculated orthotopically. The use of B16-BL6 cells stably transfected with the luciferase gene revealed that the increased metastasis reflected a difference mainly within 6 hr after the intravenous inoculation of tumor cells. Administration of recombinant leptin in ob/ob mice abolished the increase in metastasis early on as well as the decrease in the splenic NK cell number. In addition, depletion of NK cells by an anti-asialo-GM1 antibody abrogated the enhanced metastasis in db/db mice. These results demonstrate that metastasis is markedly promoted in diabetic and obese mice mainly because of decreased NK cell function during the early phase of metastasis.
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PMID:Severe pulmonary metastasis in obese and diabetic mice. 1699 95


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