Dehydroxymethylepoxyquinomicin, a novel nuclear factor-kappaB inhibitor, prevents inflammatory injury induced by interferon-gamma and histamine in NCTC 2544 keratinocytes.

1. The novel nuclear factor (NF)-kappaB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) is a derivative of the antibiotic epoxyquinomicin C from Amycolatopsis sp. that has been found to inhibit tumour necrosis factor (TNF)-alpha-induced activation of NF-kappaB by suppressing nuclear translocation of NF-kappaB. The aim of the present study was to determine the effects of DHMEQ on interferon (IFN)-gamma- and histamine-activated NCTC 2544 keratinocytes. 2. Keratinocytes were stimulated or not with 200 U/mL IFN-gamma and 10(-4) mol/L histamine in the absence or presence of different concentrations of DHMEQ (1, 5 and 10 microg/mL) or hydrocortisone (10(-5) mol/L), which was used as a reference anti-inflammatory drug. After 48 h, each sample was tested for the presence of intercellular adhesion molecule (ICAM)-1 by western blot analysis, as well as for the release of monocyte chemoattractant protein (MCP)-1, RANTES and interleukin (IL)-8 using specific sandwich ELISAs. To verify the effect of DHMEQ on cell viability of non-stimulated NCTC 2544 keratinocytes, the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used. 3. The results showed that 10 microg/mL DHMEQ potently inhibited ICAM-1 production (by 50%), as well as the release of MCP-1 (to 25% of control), RANTES (to 5% of control) and IL-8 (to 2% of control). The results of the MTT assay indicated that DHMEQ has no effect on cell viability. 4. In conclusion, DHMEQ inhibits the IFN-gamma- and histamine-induced activation of the keratinocyte cell line NCTC 2544. The anti-inflammatory effects of DHMEQ could be exploited by applying the drug topically alone or in combination with sub-toxic concentrations of anti-inflammatory drugs to producer a synergistic effect.

Melanoma: molecular pathogenesis and emerging target therapies (Review).

Malignant melanoma is an aggressive tumor of the skin with a poor prognosis for patients with advanced disease. It is resistant to current therapeutic approaches. In melanoma, both the Ras/Raf/MEK/ERK (MAPK) and the PI3K/AKT (AKT) signalling pathways are constitutively activated through multiple mechanisms. Mutations of BRAF have been proposed to contribute to melanoma development. Increased activity of the MAPK pathway prevents apoptosis and induces cell cycle progression. PTEN deletion results in Akt activation. Akt activation can result in the phosphorylation and inactivation of Raf. This decrease in downstream MEK and ERK activation may lead to loss of differentiation or senescence. This review summarizes the most relevant studies focused on the signalling pathways involved in melanomagenesis. New therapeutic strategies are also reported.

Novel nitric oxide-donating compound (S,R)-3-phenyl-4,5-dihydro-5-isoxazole acetic acid-nitric oxide (GIT-27NO) induces p53 mediated apoptosis in human A375 melanoma cells.

In this study we evaluated the effects of the new NO donating compound (S,R)-3-phenyl-4,5-dihydro-5-isoxazole acetic acid-nitric oxide (GIT-27NO) on the A375 human melanoma cell line. Treatment with the drug led to concentration-dependent reduction of mitochondrial respiration and number of viable cells in cultures. Decreased cell viability correlated with release and internalization of NO and was neutralized by the extracellular scavenger hemoglobin. GIT-27NO neither influenced cell division nor induced accidental or autophagic cell death. Early signs of apoptosis were observed upon coculture with the drug, and resulting in marked accumulation of hypodiploid cells, suggesting that the induction of apoptosis is one primary mode of action of the compound in A375 cells. GIT-27NO significantly inhibited the expression of the transcription repressor and apoptotic resistant factor YY1 and, in parallel, augmented the presence of total p53. The capacity of GIT-27NO to induce p53-mediated apoptosis along with inhibition of YY1 repressor in A375 melanoma cells indicates that GIT-27NO possesses an important anti-cancer pharmacological profile. The findings suggest the potential therapeutic use of GIT-27NO in the clinical setting.

Inflammatory status in patients with chronic renal failure: the role of PTX3 and pro-inflammatory cytokines.

Increased plasma levels of several acute phase proteins, such as C-reactive protein (CRP), have been documented among different patients with chronic renal failure (CRF). The aim of the present study was to determine whether pentraxin-3 (PTX3) is a reliable marker of inflammation in CRF. Plasma samples and monocytes were taken from 43 patients before and after undergoing haemodialysis (HD), from 45 uraemic patients (UR) without HD treatment and from 25 healthy controls. Plasma and monocyte samples were analyzed by ELISA for levels of PTX3, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6); all of these protein levels were higher in CRF patients with respect to the controls. After HD, plasma PTX3 and cytokine levels increased. Inter- and intra-individual variations in CRP were observed in HD patients, while PTX3 plasma levels were stable. Release of PTX3, TNF-alpha, IL-1beta and IL-6 by unstimulated monocytes from patients, before and after HD, was higher with respect to UR patients and controls. After lipopolysaccharide stimulation, all values were higher in patients before HD than those in UR patients, but lower when compared to those in the controls. In contrast, no changes were observed after HD. A significant correlation among plasma PTX3 versus fibrinogen, TNF-alpha and IL-1beta was observed in HD and UR patients. Collectively, these data suggest that PTX3 protein may represent an additional and stable marker of inflammation in CRF.

Nectin like-5 overexpression correlates with the malignant phenotype in cutaneous melanoma.

NECL-5 is involved in regulating cell-cell junctions, in cooperation with cadherins, integrins and platelet-derived growth factor receptor, that are essential for intercellular communication. Its role in malignant transformation was previously described. It has been reported that transformation of melanocytes is associated with altered expression of adhesion molecules suggesting the potential involment of NECL-5 in melanoma development and prognosis. To shed light on this issue, the expression and the role of NECL-5 in melanoma tissues was investigated by bioinformatic and molecular approaches. NECL-5 was up-regulated both at the mRNA and the protein levels in WM35, M14 and A375 cell lines compared with normal melanocytes. A subsequent analysis in primary and metastatic melanoma specimens confirmed "in vitro" findings. NECL-5 overexpression was observed in 53 of 59 (89.8%) and 12 of 12 (100%), primary melanoma and melanoma metastasis, respectively; while, low expression of NECL-5 was detected in 12 of 20 (60%) benign nevi. A significant correlation of NECL-5 overexpression was observed with most of known negative melanoma prognostic factors, including lymph-node involvement (P = 0.009) and thickness (P = 0.004). Intriguingly, by analyzing the large series of melanoma samples in the Xu dataset, we identified the transcription factor YY1 among genes positively correlated with NECL-5 (r = 0.5). The concordant computational and experimental data of the present study indicate that the extent of NECL-5 expression correlates with melanoma progression.

The Role of B-RAF Mutations in Melanoma and the Induction of EMT via Dysregulation of the NF-κB/Snail/RKIP/PTEN Circuit.

Melanoma is a highly metastatic cancer, and there are no current therapeutic modalities to treat this deadly malignant disease once it has metastasized. Melanoma cancers exhibit B-RAF mutations in up to 70% of cases. B-RAF mutations are responsible, in large part, for the constitutive hyperactivation of survival/antiapoptotic pathways such as the MAPK, NF-κB, and PI3K/AKT. These hyperactivated pathways regulate the expression of genes targeting the initiation of the metastatic cascade, namely, the epithelial to mesenchymal transition (EMT). EMT is the result of the expression of mesenchymal gene products such as fibronectin, vimentin, and metalloproteinases and the invasion and inhibition of E-cadherin. The above pathways cross-talk and regulate each other's activities and functions. For instance, the NF-κB pathway directly regulates EMT through the transcription of gene products involved in EMT and indirectly through the transcriptional up-regulation of the metastasis inducer Snail. Snail, in turn, suppresses the expression of the metastasis suppressor gene product Raf kinase inhibitor protein RKIP (inhibits the MAPK and the NF-κB pathways) as well as PTEN (inhibits the PI3K/AKT pathway). The role of B-RAF mutations in melanoma and their direct role in the induction of EMT are not clear. This review discusses the hypothesis that B-RAF mutations are involved in the dysregulation of the NF-κB/Snail/RKIP/PTEN circuit and in both the induction of EMT and metastasis. The therapeutic implications of the dysregulation of the above circuit by B-RAF mutations are such that they offer novel targets for therapeutic interventions in the treatment of EMT and metastasis.

Serum markers of cutaneous melanoma.

Malignant melanoma currently accounts for approximately 1%, of all cancer deaths. The incidence of cutaneous melanoma is rising worldwide. The treatment of early-stage melanoma consists primarily of surgical removal of the tumour. The overall 5-years survival rate for malignant melanoma is 81%. Recently, many efforts have been made to analyse the potential significance and the possible relationship of disease progression and circulating markers in malignant melanoma. Several serum biomarkers appear to hold significant potential both as prognostic indicators and as targets for future therapeutic agents. The application of these markers in clinical practice possibly holds the key to significant advances in melanoma. This review summarizes the principal characteristics of serum markers of melanoma. Serum lactate dehydrogenase (ldh), protein S-100 beta, melanoma-inhibiting activity (MIA) may correlate with melanoma progression. Tenascin-c, Hyaluronan, Laminin-1 and type VI Collagen are involved in melanoma development and extracellular matrix remodelling during melanoma progression.

Co-regulated expression of matrix metalloproteinase-2 and transforming growth factor-beta in melanoma development and progression.

Previous studies have found that matrix metalloproteinase-2 (MMP-2) and transforming growth factor-beta (TGF-beta) can be considered as biomarkers and indices of disease progression in several human cancers. In this study, we investigated the plasma levels of MMP-2 and TGF-beta and their correlation in 49 primary cutaneous melanoma and 10 metastatic melanoma. Plasma MMP-2 and TGF-beta levels in patients with primary melanoma were significantly higher than those of healthy controls. These protein levels were significantly higher in patients with metastatic melanoma. A positive correlation between plasma levels of MMP-2 and TGF-beta in melanoma patients supports the hypothesis that TGF-beta triggers the release of MMP-2. The immunohistochemistry analysis shows that MMP-2 and TGF-beta were highly expressed in tumor tissues as well as in matched plasma samples. This finding suggests that these proteins are released from tumor cells. Overall, our data indicate that MMP-2 and TGF-beta may represent novel diagnostic markers and therapeutic targets in melanoma and the determination of their concentration could be a useful diagnostic and prognostic indicator. TGF-beta, leading the tissue invasion mediated by MMP-2, is a strong promoter of tumor progression. Therefore, reducing or blocking the activity of TGF-beta may represent a promising target in therapeutic strategies for limiting the growth of melanoma.

Genista sessilifolia DC. and Genista tinctoria L. inhibit UV light and nitric oxide-induced DNA damage and human melanoma cell growth.

Many Genista species (Leguminosae), containing isoflavones as biologically active substances, show interesting biological properties such as hypoglycemic, antiinflammatory, antiulcer, spasmolytic, antioxidant, estrogenic and cytotoxic activity against different human cancer cell lines. In this work, we describe the chemical composition of the methanolic extracts from aerial parts of Genista sessilifolia DC. and Genista tinctoria L., and their biological activity testing the effect on pBR322 DNA cleavage induced by hydroxyl radicals (*OH), generated from UV-photolysis of hydrogen peroxide (H(2)O(2)) and by nitric oxide (NO). In addition, we investigated the growth inhibitory activity of these natural products against human melanoma cell line (M14). The extracts of G. sessilifolia and G. tinctoria, for their isoflavone components, showed a protective effect on UV light and nitric oxide-mediated plasmid DNA damage, and inhibited the growth of melanoma cells. The data of the present study also suggest that these natural products could trigger apoptotic death in M14 cells. In fact, a high DNA fragmentation (COMET assay) and a significant increase of caspase-3 activity, not correlated to LDH release, a marker of membrane breakdown, occurred in melanoma cells exposed to these extracts. The significant production of reactive oxygen species (ROS) evidenced in these experimental conditions could contribute to trigger the apoptosis cascades.