u-0126 and Skin-Neoplasms

Ultraviolet B (UVB) radiation from the sun may lead to photocarcinogenesis of the skin. Sunscreens were used to protect the skin by reducing UVB irradiance, but sunscreen use did not reduce sunburn episodes. It was shown that UVB-induced erythema depends on surface exposure but not irradiance of UVB. We previously showed that irradiance plays a critical role in UVB-induced cell differentiation. This study investigated the impact of irradiance on UVB-induced photocarcinogenesis. For hairless mice receiving equivalent exposure of UVB radiation, the low irradiance (LI) UVB treated mice showed more rapid tumor development, larger tumor burden, and more keratinocytes harboring mutant p53 in the epidermis as compared to their high irradiance (HI) UVB treated counterpart. Mechanistically, using cell models, we demonstrated that LI UVB radiation allowed more keratinocytes harboring DNA damages to enter cell cycle via ERK-related signaling as compared to its HI UVB counterpart. These results indicated that at equivalent exposure, UVB radiation at LI has higher photocarcinogenic potential as compared to its HI counterpart. Since erythema is the observed sunburn at moderate doses and use of sunscreen was not found to associate with reduced sunburn episodes, the biological significance of sunburn with or without sunscreen use warrants further investigation.

Topics: Adult; Animals; Bromodeoxyuridine; Butadienes; Carcinogenesis; Cell Count; Cell Survival; Cells, Cultured; Dermatitis, Contact; DNA Damage; Extracellular Signal-Regulated MAP Kinases; G2 Phase; Humans; Immunosuppression Therapy; Keratinocytes; Mice, Hairless; Mitosis; Mutation; Nitriles; Protein Kinase Inhibitors; Pyrimidine Dimers; Skin Neoplasms; Tumor Suppressor Protein p53; Ultraviolet Rays

Oncogenesis reflects an orchestrated interaction between misguided growth signals. Although much effort has been launched to pharmacologically disable activated oncogenes, one sidelined approach is the restoration of tumor suppressive signals. As TP53 is often structurally preserved, but functionally crippled, by CDKN2A/ARF loss in melanoma, rescue of p53 function represents an attractive point of vulnerability in melanoma. In this study, we showed that both p53 protein and activity levels in melanoma cells were strongly induced by nutlin-3, a canonical HDM2 antagonist. Among a test panel of 51 cell lines, there was a marked reduction in melanoma viability that was directly linked to TP53 status. Moreover, we also found that the melanoma growth suppression mediated by mitogen-activated protein kinase/extracellular signal-regulated kinase inhibition was potentiated by HDM2 antagonism. These results provide fundamental insights into the intact p53 circuitry, which can be restored through small molecule inhibitors and potentially deployed for therapeutic gain.

Topics: Apoptosis; Butadienes; Cell Cycle Checkpoints; Cell Line, Tumor; Humans; Imidazoles; Melanoma; Mitogen-Activated Protein Kinase Kinases; Nitriles; Piperazines; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-mdm2; Skin Neoplasms; Tumor Suppressor Protein p53

This study investigated the growth-regulating effects of progesterone (Prog) on nPR-negative malignant melanoma cells and the possible mechanisms. A375 and A875 cells were cultured and treated with Prog of different concentrations. For signal transduction pathway studies, the cells were pretreated with Prog receptor antagonist (RU486, 1 x 10(-7) mol/L) or MAPK inhibitor (U0126, 5 x 10(-6) mol/L) for 1 h and then co-incubated with prog (10(-9) mol/L) for another 24 h. Indirect immunofluorescence assay, MTT, flow cytometry and Western blotting were used for assessing the nPR expression, cell growth, cell apoptosis and ERK1/2 Phosphorylation, respectively. Our results showed that lower progesterone concentration promoted the proliferation of both A375 and A875 cells, but this growth-stimulatory effect decreased at progesterone concentration of 1 x 10(-7) mol/L or higher. The response could be abolished by MAPK inhibitor U0126, but could not be blocked by progesterone antagonist RU486. Flow cytometry exhibited that high concentration ([Symbol: see text]1 x 10(-7) mol/L) progesterone increased the apoptosis of the two cells in a dose-dependent manner. The level of ERK1/2 phosphorylation was increased by a lower progesterone concentration, but reduced by a higher concentration (1 x 10(-6) mol/L). These results suggest progesterone exerts growth-regulating effects on nPR-negative tumor cells through a non-genomic mechanism.

Topics: Butadienes; Cell Line, Tumor; Cell Proliferation; Humans; Melanoma; Mifepristone; Mitogen-Activated Protein Kinase Kinases; Nitriles; Progesterone; Receptors, Progesterone; Signal Transduction; Skin Neoplasms

Tuberous sclerosis (TS), neurological disorder manifesting with the formation of tumors in numerous organ systems, is a disease associated with the upregulation of mammalian target of rapamycin (mTOR) pathway. It has been found that in healthy individuals two tumor suppressor genes, TSC1 and TSC2, encoding proteins called hamartin and tuberin, respectively, are responsible for the control over mTOR kinase. Loss of one of these genes constitutes the genetic background of TS. In the current study, we aimed at evaluating the fitness of the only TS-associated sarcoma cell line deposited in American Tissue Culture Collection, TSC2ang1, for the in vitro studies on TS. We found that the line shows a stable chromosome pattern with typical Robertsonian translocations. Similarly to primary tumors from TS patients, TSC2ang1 cells respond to rapamycin-induced mTOR inhibition. The cells demonstrate activation of both Akt and Erk pathways, but inhibition of neither of them is as effective as mTOR suppression when considering proliferation potential. Based on these results we propose TSC2ang1 as a good and stable model for pathophysiological and pharmacological studies on skin lesions in TS.

Topics: Androstadienes; Animals; Butadienes; Cell Line; Cell Proliferation; CHO Cells; Chromones; Chromosomes, Human; Cricetinae; Cricetulus; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Heterozygote; Humans; Karyotyping; Morpholines; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-akt; Skin Neoplasms; Substrate Specificity; TOR Serine-Threonine Kinases; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins; Wortmannin

The RAS-RAF-MEK-ERK and PI3K-AKT-mTOR signaling pathways are activated through multiple mechanisms and appear to play a major role in melanoma progression. Herein, we examined whether targeting the RAS-RAF-MEK-ERK pathway with the RAF inhibitor sorafenib and/or the PI3K-AKT-mTOR pathway with the mTOR inhibitor rapamycin has therapeutic effects against melanoma. A combination of sorafenib (4 microM) with rapamycin (10 nM) potentiated growth inhibition in all six metastatic melanoma cell lines tested. The absolute enhancement of growth inhibition rates ranged from 13.0-27.8% in different cell lines (P<0.05, combination treatment vs monotreatment). Similar results were obtained with combinations of the MEK inhibitors U0126 (30 microM) or PD98059 (50 microM) with rapamycin (10 nM). The combined treatment of melanoma cells with sorafenib and rapamycin led to an approximately twofold increase of cell death compared with sorafenib monotreatment (P<0.05) as assessed by propidium iodide staining and cell death detection ELISA. Moreover, sorafenib in combination with rapamycin completely suppressed invasive melanoma growth in organotypic culture mimicking the physiological context. These effects were associated with complete downregulation of the antiapoptotic proteins Bcl-2 and Mcl-1. Sorafenib combined with rapamycin appears to be a promising strategy for the effective treatment of melanoma and merits clinical investigation.

Topics: Androstadienes; Apoptosis; Benzenesulfonates; Butadienes; Cell Line, Tumor; Cell Proliferation; Chromones; Down-Regulation; Flavonoids; Humans; Mechanistic Target of Rapamycin Complex 1; Melanoma; Mitogen-Activated Protein Kinase Kinases; Morpholines; Multiprotein Complexes; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Invasiveness; Niacinamide; Nitriles; Phenylurea Compounds; Protein Kinase Inhibitors; Proteins; Proto-Oncogene Proteins c-bcl-2; Pyridines; Signal Transduction; Sirolimus; Skin Neoplasms; Sorafenib; TOR Serine-Threonine Kinases; Transcription Factors; Wortmannin

The matrix fibronectin protein is a multifunctional adhesive molecule that promotes migration and invasiveness of many tumors including melanomas. Increased fibronectin synthesis has been associated with the metastatic potential of melanoma cells; however, the molecular mechanisms underlying fibronectin overexpression during melanoma development are poorly understood. We report that hepatocyte growth factor/scatter factor (HGF) induces fibronectin expression and its extracellular assembly on the surface of melanoma cells through activation of mitogen-activated protein (MAP) kinase pathway, and induction and transcriptional activation of Early growth response-1 (Egr-1). Inhibition of B-RAF/MAP kinase pathway by dominant-negative mutants and by U0126-abrogated HGF-induced Egr-1, and chromatin immunoprecipitation showed that Egr-1 is bound to the fibronectin promoter in response to HGF. Exogenously expressed Egr-1 increased fibronectin levels, while blockage of Egr-1 activation by expression of the Egr-1 corepressor NAB2 interfered with the upregulation of fibronectin synthesis induced by HGF, indicating that Egr-1 exerts a significant role in fibronectin expression in response to HGF. Finally, analysis of the expression pattern of fibronectin in melanoma cells demonstrated that fibronectin levels are correlated with constitutive MAP kinase signaling. Our data define a novel mechanism that might have important implications in regulation of melanoma progression by autocrine HGF signaling or by constitutive activation of MAP kinase pathway.

Topics: Autocrine Communication; Butadienes; Cell Line, Tumor; DNA-Binding Proteins; Early Growth Response Protein 1; Extracellular Signal-Regulated MAP Kinases; Fibronectins; Genes, Reporter; Hepatocyte Growth Factor; Humans; Immediate-Early Proteins; Luciferases; MAP Kinase Signaling System; Melanoma; Nitriles; Phosphorylation; Promoter Regions, Genetic; ras Proteins; Signal Transduction; Skin Neoplasms; Transcription Factors; Up-Regulation