B-RAF and N-RAS Mutations Are Preserved during Short Time In Vitro Propagation and Differentially Impact Prognosis

In melanoma, the RAS/RAF/MEK/ERK signalling pathway is an area of great interest, because it regulates tumor cell proliferation and survival. A varying mutation rate has been reported for B-RAF and N-RAS, which has been largely attributed to the differential source of tumor DNA analyzed, e.g., fixed tumor tissues or in vitro propagated melanoma cells. Notably, this variation also interfered with interpreting the impact of these mutations on the clinical course of the disease. Consequently, we investigated the mutational profile of B-RAF and N-RAS in biopsies and corresponding cell lines from metastatic tumor lesions of 109 melanoma patients (AJCC stage III/IV), and its respective impact on survival. 97 tissue biopsies and 105 biopsy-derived cell lines were screened for B-RAF and N-RAS mutations by PCR single strand conformation polymorphism and DNA sequencing. Mutations were correlated with patient survival data obtained within a median follow-up time of 31 months. B-RAF mutations were detected in 55% tissues and 51% cell lines, N-RAS mutations in 23% tissues and 25% cell lines, respectively. There was strong concordance between the mutational status of tissues and corresponding cell lines, showing a differing status for B-RAF in only 5% and N-RAS in only 6%, respectively. Patients with tumors carrying mutated B-RAF showed an impaired median survival (8.0 versus 11.8 months, p = 0.055, tissues; 7.1 versus 9.3 months, p = 0.068, cell lines), whereas patients with N-RAS-mutated tumors presented with a favorable prognosis (median survival 12.5 versus 7.9 months, p = 0.084, tissues; 15.4 versus 6.8 months, p = 0.0008, cell lines), each in comparison with wildtype gene status. Multivariate analysis qualified N-RAS (p = 0.006) but not B-RAF mutation status as an independent prognostic factor of overall survival. Our findings demonstrate that B-RAF and N-RAS mutations are well preserved during short term in vitro propagation and, most importantly, differentially impact the outcome of melanoma patients

Silymarin Targets β-Catenin Signaling in Blocking Migration/Invasion of Human Melanoma Cells

Metastatic melanoma is a leading cause of death from skin diseases, and is often associated with activation of Wnt/β-catenin signaling pathway. We have examined the inhibitory effect of silymarin, a plant flavanoid from Silybum marianum, on cell migration of metastasis-specific human melanoma cell lines (A375 and Hs294t) and assessed whether Wnt/β-catenin signaling is the target of silymarin. Using an in vitro invasion assay, we found that treatment of human melanoma cell lines with silymarin resulted in concentration-dependent inhibition of cell migration, which was associated with accumulation of cytosolic β-catenin, while reducing the nuclear accumulation of β-catenin (i.e., β-catenin inactivation) and reducing the levels of matrix metalloproteinase (MMP) -2 and MMP-9 which are the down-stream targets of β-catenin. Silymarin enhanced: (i) the levels of casein kinase 1α, glycogen synthase kinase-3β and phosphorylated-β-catenin on critical residues Ser45, Ser33/37 and Thr41, and (ii) the binding of β-transducin repeat-containing proteins (β-TrCP) with phospho forms of β-catenin in melanoma cells. These events play important roles in degradation or inactivation of β-catenin. To verify whether β-catenin is a potent molecular target of silymarin, the effect of silymarin was determined on β-catenin-activated (Mel 1241) and β-catenin-inactivated (Mel 1011) melanoma cells. Treatment of Mel 1241 cells with silymarin or FH535, an inhibitor of Wnt/β-catenin pathway, significantly inhibited cell migration of Mel 1241 cells, which was associated with the elevated levels of casein kinase 1α and glycogen synthase kinase-3β, and decreased accumulation of nuclear β-catenin and inhibition of MMP-2 and MMP-9 levels. However, this effect of silymarin and FH535 was not found in Mel 1011 melanoma cells. These results indicate for the first time that silymarin inhibits melanoma cell migration by targeting β-catenin signaling pathway

Endothelin-1 Inhibits Prolyl Hydroxylase Domain 2 to Activate Hypoxia-Inducible Factor-1α in Melanoma Cells

The endothelin B receptor (ET(B)R) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1alpha is essential for melanomagenesis and progression, and is controlled by site-specific hydroxylation carried out by HIF-prolyl hydroxylase domain (PHD) and subsequent proteosomal degradation.Here we found that in melanoma cells ET-1, ET-2, and ET-3 through ET(B)R, enhance the expression and activity of HIF-1alpha and HIF-2alpha that in turn regulate the expression of vascular endothelial growth factor (VEGF) in response to ETs or hypoxia. Under normoxic conditions, ET-1 controls HIF-alpha stability by inhibiting its degradation, as determined by impaired degradation of a reporter gene containing the HIF-1alpha oxygen-dependent degradation domain encompassing the PHD-targeted prolines. In particular, ETs through ET(B)R markedly decrease PHD2 mRNA and protein levels and promoter activity. In addition, activation of phosphatidylinositol 3-kinase (PI3K)-dependent integrin linked kinase (ILK)-AKT-mammalian target of rapamycin (mTOR) pathway is required for ET(B)R-mediated PHD2 inhibition, HIF-1alpha, HIF-2alpha, and VEGF expression. At functional level, PHD2 knockdown does not further increase ETs-induced in vitro tube formation of endothelial cells and melanoma cell invasiveness, demonstrating that these processes are regulated in a PHD2-dependent manner. In human primary and metastatic melanoma tissues as well as in cell lines, that express high levels of HIF-1alpha, ET(B)R expression is associated with low PHD2 levels. In melanoma xenografts, ET(B)R blockade by ET(B)R antagonist results in a concomitant reduction of tumor growth, angiogenesis, HIF-1alpha, and HIF-2alpha expression, and an increase in PHD2 levels.In this study we identified the underlying mechanism by which ET-1, through the regulation of PHD2, controls HIF-1alpha stability and thereby regulates angiogenesis and melanoma cell invasion. These results further indicate that targeting ET(B)R may represent a potential therapeutic treatment of melanoma by impairing HIF-1alpha stability

Prenylation Inhibition-Induced Cell Death in Melanoma: Reduced Sensitivity in BRAF Mutant/PTEN Wild-Type Melanoma Cells.

While targeted therapy brought a new era in the treatment of BRAF mutant melanoma, therapeutic options for non-BRAF mutant cases are still limited. In order to explore the antitumor activity of prenylation inhibition we investigated the response to zoledronic acid treatment in thirteen human melanoma cell lines with known BRAF, NRAS and PTEN mutational status. Effect of zoledronic acid on proliferation, clonogenic potential, apoptosis and migration of melanoma cells as well as the activation of downstream elements of the RAS/RAF pathway were investigated in vitro with SRB, TUNEL and PARP cleavage assays and videomicroscopy and immunoblot measurements, respectively. Subcutaneous and spleen-to-liver colonization xenograft mouse models were used to evaluate the influence of zoledronic acid treatment on primary and disseminated tumor growth of melanoma cells in vivo. Zoledronic acid more efficiently decreased short-term in vitro viability in NRAS mutant cells when compared to BRAF mutant and BRAF/NRAS wild-type cells. In line with this finding, following treatment decreased activation of ribosomal protein S6 was found in NRAS mutant cells. Zoledronic acid demonstrated no significant synergism in cell viability inhibition or apoptosis induction with cisplatin or DTIC treatment in vitro. Importantly, zoledronic acid could inhibit clonogenic growth in the majority of melanoma cell lines except in the three BRAF mutant but PTEN wild-type melanoma lines. A similar pattern was observed in apoptosis induction experiments. In vivo zoledronic acid did not inhibit the subcutaneous growth or spleen-to-liver colonization of melanoma cells. Altogether our data demonstrates that prenylation inhibition may be a novel therapeutic approach in NRAS mutant melanoma. Nevertheless, we also demonstrated that therapeutic sensitivity might be influenced by the PTEN status of BRAF mutant melanoma cells. However, further investigations are needed to identify drugs that have appropriate pharmacological properties to efficiently target prenylation in melanoma cells

Embryonic Remnants of the Umbilical-cord - Morphologic and Clinical Aspects

Embryonic vestiges of the umbilical cord are classic findings in routine morphologic examination of the placenta. In order to evaluate their clinicopathologic significance, we examined samples from the fetal and placental ends of 1,000 umbilical cords and collected the principal clinical findings of the corresponding newborns. Microscopic evidence of embryonic remnants were found in 231 cases (23.1%) divided into remnants of the allantoic duct (63%), the omphalomesenteric duct (6.6%), and the embryonic vessels (30.4%), including one case of hemangioma and an accessory small artery. There were no significant clinical differences between the three vestigial groups, and no particular association with congenital malformations or perinatal complications. In 70.9% of the cases, the embryonic remnants were found at the fetal end of the umbilical cord, where most tumors of the cord develop. © 1989.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Nonimmune Hydrops Fetalis Associated With Genetic Abnormalities

The purpose of this review of the literature on nonimmune hydrops fetalis was to evaluate whether recent clinicopathologic studies have modified the relative incidence of the different associated conditions and the management of these pregnancies. We found 600 cases of nonimmune hydrops fetalis published since 1982. These cases were reviewed with particular attention to genetic causes and were compared with a literature review of 298 cases published before 1982. The mean gestational age at diagnosis varied from 24-29 weeks in the recent series, compared with 31-33 weeks in the earlier series. Genetically transmitted conditions accounted for more than 35% of the fetal and maternal disorders associated with nonimmune hydrops fetalis in the recent series, compared with 21% before 1982. The most frequently identified genetic abnormalities in our review were chromosomal disorders (15.7%), α-thalassemia (10.3%), skeletal dysplasia (4%), arthrogryposis multiplex syndromes (1.8%), multiple pterygium syndrome (1.5%), and lysosomal storage disorders (1.0%). These results confirm the need for systematic chromosome analysis in fetuses with nonimmune hydrops. From this review, we conclude that prenatal noninvasive and invasive techniques combined with detailed pathologic studies have improved the accuracy of diagnosis of the underlying causes of nonimmune hydrops fetalis and have influenced the management of these pregnancies.SCOPUS: re.jinfo:eu-repo/semantics/publishe