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Sample records for activation inhibits melanoma

  1. Compound 13, an α1-selective small molecule activator of AMPK, potently inhibits melanoma cell proliferation.

    PubMed

    Hu, Xueqing; Jiang, Fangzhen; Bao, Qi; Qian, Huan; Fang, Quan; Shao, Zheren

    2016-01-01

    It is vital to develop new therapeutic agents for the treatment of melanoma. In the current study, we studied the potential effect of Compound 13 (C13), a novel α1-selective AMP-activated protein kinase (AMPK) activator, in melanoma cells. We showed that C13 exerted mainly cytostatic, but not cytotoxic activities in melanoma cells. C13 potently inhibited proliferation in melanoma cell lines (A375, OCM-1 and B16), but not in B10BR melanocytes. Meanwhile, the AMPK activator inhibited melanoma cell cycle progression by inducing G1-S arrest. Significantly, we failed to detect significant melanoma cell death or apoptosis after the C13 treatment. For the mechanism study, we showed that C13 activated AMPK and inhibited mammalian target of rapamycin complex 1 (mTORC1) signaling in melanoma cells through interaction with the α1 subunit. Short hairpin RNA (shRNA)-mediated knockdown of AMPKα1 not only blocked C13-mediated AMPK activation but also abolished its antiproliferative activity against melanoma cells. Together, these results show that C13 inhibits melanoma cell proliferation through activating AMPK signaling. Our data suggest that C13 along with other small molecular AMPK activators may be beneficial for patients with melanoma. PMID:26271666

  2. Lumican Inhibits SNAIL-Induced Melanoma Cell Migration Specifically by Blocking MMP-14 Activity.

    PubMed

    Stasiak, Marta; Boncela, Joanna; Perreau, Corinne; Karamanou, Konstantina; Chatron-Colliet, Aurore; Proult, Isabelle; Przygodzka, Patrycja; Chakravarti, Shukti; Maquart, François-Xavier; Kowalska, M Anna; Wegrowski, Yanusz; Brézillon, Stéphane

    2016-01-01

    Lumican, a small leucine rich proteoglycan, inhibits MMP-14 activity and melanoma cell migration in vitro and in vivo. Snail triggers epithelial-mesenchymal transitions endowing epithelial cells with migratory and invasive properties during tumor progression. The aim of this work was to investigate lumican effects on MMP-14 activity and migration of Snail overexpressing B16F1 (Snail-B16F1) melanoma cells and HT-29 colon adenocarcinoma cells. Lumican inhibits the Snail induced MMP-14 activity in B16F1 but not in HT-29 cells. In Snail-B16F1 cells, lumican inhibits migration, growth, and melanoma primary tumor development. A lumican-based strategy targeting Snail-induced MMP-14 activity might be useful for melanoma treatment. PMID:26930497

  3. Lumican Inhibits SNAIL-Induced Melanoma Cell Migration Specifically by Blocking MMP-14 Activity

    PubMed Central

    Stasiak, Marta; Boncela, Joanna; Perreau, Corinne; Karamanou, Konstantina; Chatron-Colliet, Aurore; Proult, Isabelle; Przygodzka, Patrycja; Chakravarti, Shukti; Maquart, François-Xavier; Kowalska, M. Anna; Wegrowski, Yanusz; Brézillon, Stéphane

    2016-01-01

    Lumican, a small leucine rich proteoglycan, inhibits MMP-14 activity and melanoma cell migration in vitro and in vivo. Snail triggers epithelial-mesenchymal transitions endowing epithelial cells with migratory and invasive properties during tumor progression. The aim of this work was to investigate lumican effects on MMP-14 activity and migration of Snail overexpressing B16F1 (Snail-B16F1) melanoma cells and HT-29 colon adenocarcinoma cells. Lumican inhibits the Snail induced MMP-14 activity in B16F1 but not in HT-29 cells. In Snail-B16F1 cells, lumican inhibits migration, growth, and melanoma primary tumor development. A lumican-based strategy targeting Snail-induced MMP-14 activity might be useful for melanoma treatment. PMID:26930497

  4. Activation of Notch1 signaling in stromal fibroblasts inhibits melanoma growth by upregulating WISP-1.

    PubMed

    Shao, H; Cai, L; Grichnik, J M; Livingstone, A S; Velazquez, O C; Liu, Z-J

    2011-10-20

    The tumor microenvironment is emerging as an important target for cancer therapy. Fibroblasts (Fbs) within the tumor stroma are critically involved in promoting tumor growth and angiogenesis through secretion of soluble factors, synthesis of extracellular matrix and direct cell-cell interaction. In this work, we aim to alter the biological activity of stromal Fbs by modulating the Notch1 signaling pathway. We show that Fbs engineered to constitutively activate the Notch1 pathway significantly inhibit melanoma growth and tumor angiogenesis. We determine that the inhibitory effect of 'Notch-engineered' Fbs is mediated by increased secretion of Wnt-induced secreted protein-1 (WISP-1) as the effects of Notch1 activation in Fbs are reversed by shRNA-mediated blockade of WISP-1. When 'Notch-engineered' Fbs are co-grafted with melanoma cells in SCID mice, shRNA-mediated blockade of WISP-1 reverses the tumor-suppressive phenotype of the 'Notch-engineered' Fbs, significantly increases melanoma growth and tumor angiogenesis. Consistent with these findings, supplement of recombinant WISP-1 protein inhibits melanoma cell growth in vitro. In addition, WISP-1 is modestly expressed in melanoma-activated Fbs but highly expressed in inactivated Fbs. Evaluation of human melanoma skin biopsies indicates that expression of WISP-1 is significantly lower in melanoma nests and surrounding areas filled with infiltrated immune cells than in the adjacent dermis unaffected by the melanoma. Overall, our study shows that constitutive activation of the Notch1 pathway confers Fbs with a suppressive phenotype to melanoma growth, partially through WISP-1. Thus, targeting tumor stromal Fbs by activating Notch signaling and/or increasing WISP-1 may represent a novel therapeutic approach to combat melanoma. PMID:21516124

  5. Tissue inhibitor of metalloproteinase 2 inhibits activation of the β-catenin signaling in melanoma cells.

    PubMed

    Xia, Yuxuan; Wu, Shaoping

    2015-01-01

    The tissue inhibitor of metalloproteinase (TIMP) family, including TIMP-2, regulates the activity of multifunctional metalloproteinases in pathogenesis of melanoma. The Wnt/β-catenin pathway is constitutively activated and plays a critical role in melanoma progression. However, the relationship between TIMP-2 expression and β-catenin activity is still unclear. We hypothesize that TIMP-2 over expression inhibits the activation of the Wnt/β-catenin pathway in melanoma cells. Protein expression, distribution, and transcriptional activity of β-catenin were assayed in established stable melanoma cell lines: parental A2058 expressing, A2058 T2-1 over-expressing (T2-1), and A2058 T2R-7 under-expressing (T2R-7) TIMP-2. Compared to T2-1 cells at the basal level, T2R-7 showed significantly lower amount protein and weaker immunofluorescence staining of β-catenin. This regulation is through posttranslational level via ubiquitination. Functionally, proliferation and cell growth were lower in T2R-7 compared to A2058 and T2-1. Lithium treatment was used to mimics activation of the Wnt/β-catenin pathway. In T2R-7 cells under-expressing TIMP2, lithium significantly increased total β-catenin, nuclear β-catenin, and its downstream protein phosphor-c-Myc (S62). Nuclear β-catenin staining was enhanced in T2R-7. Beta-catenin transcriptional activity and cell proliferation were also increased significantly. Axins inhibit β-catenin pathway via GSK-3 β. We further found the ratio of p-GSK-3 β (S9) to β-catenin and protein levels of Axins were significantly lower, whereas downstream Wnt 11 was high in T2R-7 treated with lithium. Collectively, the high level of TIMP-2 protein inhibits the activation of the Wnt/β-catenin pathway, thus suppressing proliferation. Insights in the molecular mechanisms of TIMP-2 may provide promising opportunities for anti-proliferative therapeutic intervention. PMID:25839957

  6. Inhibition of the mitogen-activated protein kinase pathway triggers B16 melanoma cell differentiation.

    PubMed

    Englaro, W; Bertolotto, C; Buscà, R; Brunet, A; Pagès, G; Ortonne, J P; Ballotti, R

    1998-04-17

    In B16 melanoma cells, mitogen-activated protein (MAP) kinases are activated during cAMP-induced melanogenesis (Englaro, W., Rezzonico, R., Durand-Clément, M., Lallemand, D., Ortonne, J. P., and Ballotti, R. (1995) J. Biol. Chem. 270, 24315-24320). To establish the role of the MAP kinases in melanogenesis, we studied the effects of a specific MAP kinase kinase (MEK) inhibitor PD 98059 on different melanogenic parameters. We showed that PD 98059 inhibits the activation of MAP kinase extracellular signal-regulated kinase 1 by cAMP, but does not impair the effects of cAMP either on the morphological differentiation, characterized by an increase in dendrite outgrowth, or on the up-regulation of tyrosinase that is the key enzyme in melanogenesis. On the contrary, PD 98059 promotes by itself cell dendricity and increases the tyrosinase amount and activity. Moreover, down-regulation of the MAP kinase pathway by PD 98059, or with dominant negative mutants of p21(ras) and MEK, triggers a stimulation of the tyrosinase promoter activity and enhances the effect of cAMP on this parameter. Conversely, activation of the MAP kinase pathway, using constitutive active mutants of p21(ras) and MEK, leads to an inhibition of basal and cAMP-induced tyrosinase gene transcription. These results demonstrate that the MAP kinase pathway activation is not required for cAMP-induced melanogenesis. Furthermore, the inhibition of this pathway induces B16 melanoma cell differentiation, while a sustained activation impairs the melanogenic effect of cAMP-elevating agents. PMID:9545341

  7. Shikonin Derivative DMAKO-05 Inhibits Akt Signal Activation and Melanoma Proliferation.

    PubMed

    Yang, Yao-Yao; He, Hui-Qiong; Cui, Jia-Hua; Nie, Yun-Juan; Wu, Ya-Xian; Wang, Rui; Wang, Gang; Zheng, Jun-Nian; Ye, Richard D; Wu, Qiong; Li, Shao-Shun; Qian, Feng

    2016-06-01

    DMAKO-05((S)-1-((5E,8E)-5,8-bis(hydroxyimino)-1,4-dimethoxy-5,8-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 3-methylbutanoate) is a novel oxime derivative of shikonin, the major component extracted from Chinese herb Lithospermun erythrorhizon. Here, we report that DMAKO-05 had an antitumor activity against mouse melanoma cell line B16F0. Our studies indicated that DMAKO-05 not only inhibited B16F0 proliferation and migration but also led to cell cycle arrest at G1 phase and cell apoptosis, in which DMAKO-05 triggered mitochondrial-mediated apoptosis signal including caspase-9/3 and PARP. In response to DMAKO-05 treatment, the Akt-mediated survival signals were remarkably attenuated in B16F0 cells. Collectively, DMAKO-05 has a strong cytotoxicity in B16F0 cells via inhibiting Akt activation, inducing G1 arrest, and promoting B16F0 cell apoptosis. DMAKO-05 might serve as a potential candidate lead compound for melanoma. PMID:26804061

  8. Comparative analysis of MAPK and PI3K/AKT pathway activation and inhibition in human and canine melanoma.

    PubMed

    Fowles, J S; Denton, C L; Gustafson, D L

    2015-09-01

    The lack of advanced animal models of human cancers is considered a barrier to developing effective therapeutics. Canine and human melanomas are histologically disparate but show similar disease progression and response to therapies. The purpose of these studies was to compare human and canine melanoma tumours and cell lines regarding MAPK and PI3K/AKT signalling dysregulation, and response to select molecularly targeted agents. Pathway activation was investigated via microarray and mutational analysis. Growth inhibition and cell cycle effects were assessed for pathway inhibitors AZD6244 (MAPK) and rapamycin (PI3K/AKT) in human and canine melanoma cells. Human and canine melanoma share similar differential gene expression patterns within the MAPK and PI3K/AKT pathways. Constitutive pathway activation and similar sensitivity to AZD6244 and rapamycin was observed in human and canine cells. These results show that human and canine melanoma share activation and sensitivity to inhibition of cancer-related signalling pathways despite differences in activating mutations. PMID:23745794

  9. Multifunctional bioscaffolds for 3D culture of melanoma cells reveal increased MMP activity and migration with BRAF kinase inhibition.

    PubMed

    Leight, Jennifer L; Tokuda, Emi Y; Jones, Caitlin E; Lin, Austin J; Anseth, Kristi S

    2015-04-28

    Matrix metalloproteinases (MMPs) are important for many different types of cancer-related processes, including metastasis. Understanding the functional impact of changes in MMP activity during cancer treatment is an important facet not typically evaluated as part of preclinical research. With MMP activity being a critical component of the metastatic cascade, we designed a 3D hydrogel system to probe whether pharmacological inhibition affected human melanoma cell proteolytic activity; metastatic melanoma is a highly aggressive and drug-resistant form of skin cancer. The relationship between MMP activity and drug treatment is unknown, and therefore we used an in situ fluorogenic MMP sensor peptide to determine how drug treatment affects melanoma cell MMP activity in three dimensions. We encapsulated melanoma cells from varying stages of progression within PEG-based hydrogels to examine the relationship between drug treatment and MMP activity. From these results, a metastatic melanoma cell line (A375) and two inhibitors that inhibit RAF (PLX4032 and sorafenib) were studied further to determine whether changes in MMP activity led to a functional change in cell behavior. A375 cells exhibited increased MMP activity despite an overall decrease in metabolic activity with PLX4032 treatment. The changes in proteolytic activity correlated with increased cell elongation and increased single-cell migration. In contrast, sorafenib did not alter MMP activity or cell motility, showing that the changes induced by PLX4032 were not a universal response to small-molecule inhibition. Therefore, we argue the importance of studying MMP activity with drug treatment and its possible implications for unwanted side effects. PMID:25870264

  10. Angiostatin inhibits endothelial and melanoma cellular invasion by blocking matrix-enhanced plasminogen activation.

    PubMed Central

    Stack, M S; Gately, S; Bafetti, L M; Enghild, J J; Soff, G A

    1999-01-01

    Angiostatin, a kringle-containing fragment of plasminogen, is a potent inhibitor of angiogenesis. The mechanism(s) responsible for the anti-angiogenic properties of angiostatin are unknown. We now report that human angiostatin blocks plasmin(ogen)-enhanced in vitro invasion of tissue plasminogen activator (t-PA)-producing endothelial and melanoma cells. Kinetic analyses demonstrated that angiostatin functions as a non-competitive inhibitor of extracellular-matrix (ECM)-enhanced, t-PA-catalysed plasminogen activation, with a Ki of 0.9+/-0.03 microM. This mechanism suggests that t-PA has a binding site for the inhibitor angiostatin, as well as for its substrate plasminogen that, when occupied, prevents ternary complex formation between t-PA, plasminogen and matrix protein. Direct binding experiments confirmed that angiostatin bound to t-PA with an apparent Kd [Kd(app)] of 6.7+/-0.7 nM, but did not bind with high affinity to ECM proteins. Together, these data suggest that angiostatin in the cellular micro-environment can inhibit matrix-enhanced plasminogen activation, resulting in reduced invasive activity, and suggest a biochemical mechanism whereby angiostatin-mediated regulation of plasmin formation could influence cellular migration and invasion. PMID:10229661

  11. CB2 Receptor Activation Inhibits Melanoma Cell Transmigration through the Blood-Brain Barrier

    PubMed Central

    Haskó, János; Fazakas, Csilla; Molnár, Judit; Nyúl-Tóth, Ádám; Herman, Hildegard; Hermenean, Anca; Wilhelm, Imola; Persidsky, Yuri; Krizbai, István A.

    2014-01-01

    During parenchymal brain metastasis formation tumor cells need to migrate through cerebral endothelial cells, which form the morphological basis of the blood-brain barrier (BBB). The mechanisms of extravasation of tumor cells are highly uncharacterized, but in some aspects recapitulate the diapedesis of leukocytes. Extravasation of leukocytes through the BBB is decreased by the activation of type 2 cannabinoid receptors (CB2); therefore, in the present study we sought to investigate the role of CB2 receptors in the interaction of melanoma cells with the brain endothelium. First, we identified the presence of CB1, CB2(A), GPR18 (transcriptional variant 1) and GPR55 receptors in brain endothelial cells, while melanoma cells expressed CB1, CB2(A), GPR18 (transcriptional variants 1 and 2), GPR55 and GPR119. We observed that activation of CB2 receptors with JWH-133 reduced the adhesion of melanoma cells to the layer of brain endothelial cells. JWH-133 decreased the transendothelial migration rate of melanoma cells as well. Our results suggest that changes induced in endothelial cells are critical in the mediation of the effect of CB2 agonists. Our data identify CB2 as a potential target in reducing the number of brain metastastes originating from melanoma. PMID:24815068

  12. Combined inhibition of MEK and Plk1 has synergistic anti-tumor activity in NRAS mutant melanoma

    PubMed Central

    Vujic, I; Sanlorenzo, M; Ma, J; Kim, ST; Kleffel, S; Schatton, T; Rappersberger, K; Gutteridge, R; Ahmad, N; Ortiz/Urda, S

    2015-01-01

    About one third of cancers harbor activating mutations in rat sarcoma viral oncogene homolog (RAS) oncogenes. In melanoma, aberrant neuroblastoma-RAS (NRAS) signaling fuels tumor progression in about 20% of patients. Current therapeutics for NRAS driven malignancies barely impact overall survival. To date, pathway interference downstream of mutant NRAS seems to be the most promising approach. In this study, data revealed that mutant NRAS induced Plk1 expression, and pharmacologic inhibition of Plk1 stabilized the size of NRAS mutant melanoma xenografts. The combination of MEK and Plk1 inhibitors resulted in a significant growth reduction of NRAS mutant melanoma cells in vitro, and regression of xenografted NRAS mutant melanoma in vivo. Independent cell cycle arrest and increased induction of apoptosis underlies the synergistic effect of this combination. Data further suggest that the p53 signaling pathway is of key importance to the observed therapeutic efficacy. This study provides in vitro, in vivo and first mechanistic data, that a MEK/Plk1 inhibitor combination might be a promising treatment approach for patients with NRAS driven melanoma. Since mutant NRAS signaling is similar across different malignancies, this inhibitor combination could also offer a previously unreported treatment modality for NRAS mutant tumors of other cell origins. PMID:26016894

  13. Upstream mitogen-activated protein kinase (MAPK) pathway inhibition: MEK inhibitor followed by a BRAF inhibitor in advanced melanoma patients.

    PubMed

    Goldinger, Simone M; Zimmer, Lisa; Schulz, Carsten; Ugurel, Selma; Hoeller, Christoph; Kaehler, Katharina C; Schadendorf, Dirk; Hassel, Jessica C; Becker, Juergen; Hauschild, Axel; Dummer, Reinhard

    2014-01-01

    BRAF-mutant melanoma can be successfully treated by BRAF kinase inhibitors (BRAFi) and MEK kinase inhibitors (MEKi). However, the administration of BRAFi followed by MEKi did not generate promising response rate (RR). The purpose of this investigation was to evaluate the time to progression (TTP) with a mitogen-activated protein kinase (MAPK) pathway upstream inhibition strategy in BRAF mutated melanoma patients. BRAF mutation positive metastatic melanoma patients were identified within the Dermatology Cooperative Oncology Group (DeCOG) network and were treated first with a MEKi and upon progression with a selective BRAFi. A total of 23 melanoma patients (six females, 17 males, aged 47-80 years) were retrospectively analysed for TTP. The total median TTP was 8.9 months. The median TTP for MEKi was 4.8 (1.2-23.2) and subsequent for BRAFi 4.5 (1.2-15.7) months, respectively. A higher RR for MEKi (39%, nine partial responses and 0 complete responses) than previously reported was observed. Our analysis suggests that the reversed inhibition of the MAPK pathway is feasible in BRAF mutated melanoma. The median TTP (8.9 months) is close to the promising BRAF- and MEKi combination therapy (median progression-free survival (PFS) 9.4 months). The total treatment duration of the MAPK inhibition when a MEKi is administered first is similar compared to the reversed sequence, but TTP shifts in favour to the MEKi. This approach is feasible with reasonable tolerability. This clinical investigation encourages further studies in prospective clinical trials to define the optimal treatment schedule for the MAPK pathway inhibition and should be accompanied by molecular monitoring using repeated biopsies. PMID:24183461

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

    PubMed Central

    Spinella, Francesca; Rosanò, Laura; Del Duca, Martina; Di Castro, Valeriana; Nicotra, Maria Rita; Natali, Pier Giorgio; Bagnato, Anna

    2010-01-01

    Background The endothelin B receptor (ETBR) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1α 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. Principal Findings Here we found that in melanoma cells ET-1, ET-2, and ET-3 through ETBR, enhance the expression and activity of HIF-1α and HIF-2α 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-α stability by inhibiting its degradation, as determined by impaired degradation of a reporter gene containing the HIF-1α oxygen-dependent degradation domain encompassing the PHD-targeted prolines. In particular, ETs through ETBR 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 ETBR-mediated PHD2 inhibition, HIF-1α, HIF-2α, 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-1α, ETBR expression is associated with low PHD2 levels. In melanoma xenografts, ETBR blockade by ETBR antagonist results in a concomitant reduction of tumor growth, angiogenesis, HIF-1α, and HIF-2α expression, and an increase in PHD2 levels. Conclusions In this study we identified the underlying mechanism by which ET-1, through the regulation of PHD2, controls HIF-1α stability and

  15. Inhibition of NADPH oxidase 1 activity and blocking the binding of cytosolic and membrane-bound proteins by honokiol inhibit migratory potential of melanoma cells.

    PubMed

    Prasad, Ram; Kappes, John C; Katiyar, Santosh K

    2016-02-16

    Overexpression of NADPH oxidase 1 (Nox1) in melanoma cells is often associated with increased migration/metastasis rate. To develop effective treatment options, we have examined the effect of honokiol, a phytochemical from Magnolia plant, on the migratory potential of human melanoma cell lines (A375, Hs294t, SK-Mel119 and SK-Mel28) and assessed whether Nox1 is the target. Using an in vitro cell migration assay, we observed that treatment of different melanoma cell lines with honokiol for 24 h resulted in a dose-dependent inhibition of cell migration that was associated with reduction in Nox1 expression and reduced levels of oxidative stress. Treatment of cells with N-acetyl-L-cysteine, an anti-oxidant, also inhibited the migration of melanoma cells. Treatment of cells with diphenyleneiodonium chloride, an inhibitor of Nox1, significantly decreased the migration ability of Hs294t and SK-Mel28 cells. Further, we examined the effect of honokiol on the levels of core proteins (p22phox and p47phox) of the NADPH oxidase complex. Treatment of Hs294t and SK-Mel28 cells with honokiol resulted in accumulation of the cytosolic p47phox protein and decreased levels of the membrane-bound p22phox protein, thus blocking their interaction and inhibiting Nox1 activation. Our in vivo bioluminescence imaging data indicate that oral administration of honokiol inhibited the migration/extravasation and growth of intravenously injected melanoma cells in internal body organs, such as liver, lung and kidney in nude mice, and that this was associated with an inhibitory effect on Nox1 activity in these internal organs/tissues. PMID:26760964

  16. Inhibition of NADPH oxidase 1 activity and blocking the binding of cytosolic and membrane-bound proteins by honokiol inhibit migratory potential of melanoma cells

    PubMed Central

    Prasad, Ram; Kappes, John C.; Katiyar, Santosh K.

    2016-01-01

    Overexpression of NADPH oxidase 1 (Nox1) in melanoma cells is often associated with increased migration/metastasis rate. To develop effective treatment options, we have examined the effect of honokiol, a phytochemical from Magnolia plant, on the migratory potential of human melanoma cell lines (A375, Hs294t, SK-Mel119 and SK-Mel28) and assessed whether Nox1 is the target. Using an in vitro cell migration assay, we observed that treatment of different melanoma cell lines with honokiol for 24 h resulted in a dose-dependent inhibition of cell migration that was associated with reduction in Nox1 expression and reduced levels of oxidative stress. Treatment of cells with N-acetyl-L-cysteine, an anti-oxidant, also inhibited the migration of melanoma cells. Treatment of cells with diphenyleneiodonium chloride, an inhibitor of Nox1, significantly decreased the migration ability of Hs294t and SK-Mel28 cells. Further, we examined the effect of honokiol on the levels of core proteins (p22phox and p47phox) of the NADPH oxidase complex. Treatment of Hs294t and SK-Mel28 cells with honokiol resulted in accumulation of the cytosolic p47phox protein and decreased levels of the membrane-bound p22phox protein, thus blocking their interaction and inhibiting Nox1 activation. Our in vivo bioluminescence imaging data indicate that oral administration of honokiol inhibited the migration/extravasation and growth of intravenously injected melanoma cells in internal body organs, such as liver, lung and kidney in nude mice, and that this was associated with an inhibitory effect on Nox1 activity in these internal organs/tissues. PMID:26760964

  17. Protease activated receptor-1 inhibits the Maspin tumor-suppressor gene to determine the melanoma metastatic phenotype

    PubMed Central

    Villares, Gabriel J.; Zigler, Maya; Dobroff, Andrey S.; Wang, Hua; Song, Renduo; Melnikova, Vladislava O.; Huang, Li; Braeuer, Russell R.; Bar-Eli, Menashe

    2011-01-01

    The thrombin receptor protease activated receptor-1 (PAR-1) is overexpressed in metastatic melanoma cell lines and tumor specimens. Previously, we demonstrated a significant reduction in tumor growth and experimental lung metastasis after PAR-1 silencing via systemic delivery of siRNA encapsulated into nanoliposomes. Gene expression profiling identified a 40-fold increase in expression of Maspin in PAR-1–silenced metastatic melanoma cell lines. Maspin promoter activity was significantly increased after PAR-1 silencing, suggesting that PAR1 negatively regulates Maspin at the transcriptional level. ChIP analyses revealed that PAR-1 decreases binding of Ets-1 and c-Jun transcription factors to the Maspin promoter, both known to activate Maspin transcription. PAR-1 silencing did not affect Ets-1 or c-Jun expression; rather it resulted in increased expression of the chromatin remodeling complex CBP/p300, as well as decreased activity of the CBP/p300 inhibitor p38, resulting in increased binding of Ets-1 and c-Jun to the Maspin promoter and higher Maspin expression. Functionally, Maspin expression reduced the invasive capability of melanoma cells after PAR-1 silencing, which was abrogated after rescuing with PAR-1. Furthermore, tumor growth and experimental lung metastasis was significantly decreased after expressing Maspin in a metastatic melanoma cell line. Moreover, silencing Maspin in PAR-1–silenced cells reverted the inhibition of tumor growth and experimental lung metastasis. Herein, we demonstrate a mechanism by which PAR-1 negatively regulates the expression of the Maspin tumor-suppressor gene in the acquisition of the metastatic melanoma phenotype, thus attributing an alternative function to PAR-1 other than coagulation. PMID:21187389

  18. Protease activated receptor-1 inhibits the Maspin tumor-suppressor gene to determine the melanoma metastatic phenotype.

    PubMed

    Villares, Gabriel J; Zigler, Maya; Dobroff, Andrey S; Wang, Hua; Song, Renduo; Melnikova, Vladislava O; Huang, Li; Braeuer, Russell R; Bar-Eli, Menashe

    2011-01-11

    The thrombin receptor protease activated receptor-1 (PAR-1) is overexpressed in metastatic melanoma cell lines and tumor specimens. Previously, we demonstrated a significant reduction in tumor growth and experimental lung metastasis after PAR-1 silencing via systemic delivery of siRNA encapsulated into nanoliposomes. Gene expression profiling identified a 40-fold increase in expression of Maspin in PAR-1-silenced metastatic melanoma cell lines. Maspin promoter activity was significantly increased after PAR-1 silencing, suggesting that PAR1 negatively regulates Maspin at the transcriptional level. ChIP analyses revealed that PAR-1 decreases binding of Ets-1 and c-Jun transcription factors to the Maspin promoter, both known to activate Maspin transcription. PAR-1 silencing did not affect Ets-1 or c-Jun expression; rather it resulted in increased expression of the chromatin remodeling complex CBP/p300, as well as decreased activity of the CBP/p300 inhibitor p38, resulting in increased binding of Ets-1 and c-Jun to the Maspin promoter and higher Maspin expression. Functionally, Maspin expression reduced the invasive capability of melanoma cells after PAR-1 silencing, which was abrogated after rescuing with PAR-1. Furthermore, tumor growth and experimental lung metastasis was significantly decreased after expressing Maspin in a metastatic melanoma cell line. Moreover, silencing Maspin in PAR-1-silenced cells reverted the inhibition of tumor growth and experimental lung metastasis. Herein, we demonstrate a mechanism by which PAR-1 negatively regulates the expression of the Maspin tumor-suppressor gene in the acquisition of the metastatic melanoma phenotype, thus attributing an alternative function to PAR-1 other than coagulation. PMID:21187389

  19. Lauroside B, a megastigmane glycoside from Laurus nobilis (bay laurel) leaves, induces apoptosis in human melanoma cell lines by inhibiting NF-κB activation.

    PubMed

    Panza, Elisabetta; Tersigni, Mariaroberta; Iorizzi, Maria; Zollo, Franco; De Marino, Simona; Festa, Carmen; Napolitano, Maria; Castello, Giuseppe; Ialenti, Armando; Ianaro, Angela

    2011-02-25

    Malignant melanoma is a highly aggressive tumor that frequently resists chemotherapy, so the search for new agents for its treatment is of great importance. In the present study, the antiproliferative propensity against human melanoma cell lines of lauroside B (1), a megastigmane glycoside isolated from Laurus nobilis (bay laurel) leaves, was investigated. This compound suppressed the proliferation of three human melanoma cell lines, namely, A375, WM115, and SK-Mel-28. The 1-induced inhibition of human melanoma cell proliferation was due to the induction of apoptosis, as demonstrated by FACS analysis with annexin V/PI staining and confirmed by activation of caspase-3 and by the cleavage of poly(ADP-ribose) polymerase (PARP). Growing evidence implicates NF-κB as an important contributor to metastasis and increased chemoresistance of melanoma. Thus, it was hypothesized that 1-induced apoptosis could be associated with suppression of NF-κB activation. The results showed that exposure of human melanoma cells to 1 inhibited IκB-α degradation and constitutive NF-κB DNA-binding activity as well as the expression, regulated by NF-κB, of two antiapoptotic genes, XIAP and c-FLIP. Induction of apoptosis by 1 in human aggressive melanoma cell lines has a potential high biological value. PMID:21188975

  20. Uveal Melanoma Cell Growth Is Inhibited by Aminoimidazole Carboxamide Ribonucleotide (AICAR) Partially Through Activation of AMP-Dependent Kinase

    PubMed Central

    Al-Moujahed, Ahmad; Nicolaou, Fotini; Brodowska, Katarzyna; Papakostas, Thanos D.; Marmalidou, Anna; Ksander, Bruce R.; Miller, Joan W.; Gragoudas, Evangelos; Vavvas, Demetrios G.

    2014-01-01

    Purpose. To evaluate the effects and mechanism of aminoimidazole carboxamide ribonucleotide (AICAR), an AMP-dependent kinase (AMPK) activator, on the growth of uveal melanoma cell lines. Methods. Four different cell lines were treated with AICAR (1–4 mM). Cell growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Cell cycle analysis was conducted by flow cytometry; additionally, expression of cell-cycle control proteins, cell growth transcription factors, and downstream effectors of AMPK were determined by RT-PCR and Western blot. Results. Aminoimidazole carboxamide ribonucleotide inhibited cell growth, induced S-phase arrest, and led to AMPK activation. Aminoimidazole carboxamide ribonucleotide treatment was associated with inhibition of eukaryotic translation initiation factor 4E-BP1 phosphorylation, a marker of mammalian target of rapamycin (mTOR) pathway activity. Aminoimidazole carboxamide ribonucleotide treatment was also associated with downregulation of cyclins A and D, but had minimal effects on the phosphorylation of ribosomal protein S6 or levels of the macroautophagy marker LC3B. The effects of AICAR were abolished by treatment with dipyridamole, an adenosine transporter inhibitor that blocks the entry of AICAR into cells. Treatment with adenosine kinase inhibitor 5-iodotubericidin, which inhibits the conversion of AICAR to its 5′-phosphorylated ribotide 5-aminoimidazole-4-carboxamide-1-D-ribofuranosyl-5′-monophosphate (ZMP; the direct activator of AMPK), reversed most of the growth-inhibitory effects, indicating that some of AICAR's antiproliferative effects are mediated at least partially through AMPK activation. Conclusions. Aminoimidazole carboxamide ribonucleotide inhibited uveal melanoma cell proliferation partially through activation of the AMPK pathway and downregulation of cyclins A1 and D1. PMID:24781943

  1. Fad104, a Positive Regulator of Adipocyte Differentiation, Suppresses Invasion and Metastasis of Melanoma Cells by Inhibition of STAT3 Activity

    PubMed Central

    Katoh, Daiki; Nishizuka, Makoto; Osada, Shigehiro; Imagawa, Masayoshi

    2015-01-01

    Metastasis is the main cause of death in patients with cancer, and understanding the mechanisms of metastatic processes is essential for the development of cancer therapy. Although the role of several cell adhesion, migration or proliferation molecules in metastasis is established, a novel target for cancer therapy remains to be discovered. Previously, we reported that fad104 (factor for adipocyte differentiation 104), a regulatory factor of adipogenesis, regulates cell adhesion and migration. In this report, we clarify the role of fad104 in the invasion and metastasis of cancer cells. The expression level of fad104 in highly metastatic melanoma A375SM cells was lower than that in poorly metastatic melanoma A375C6 cells. Reduction of fad104 expression enhanced the migration and invasion of melanoma cells, while over-expression of FAD104 inhibited migration and invasion. In addition, melanoma cells stably expressing FAD104 showed a reduction in formation of lung colonization compared with control cells. FAD104 interacted with STAT3 and down-regulated the phosphorylation level of STAT3 in melanoma cells. These findings together demonstrate that fad104 suppressed the invasion and metastasis of melanoma cells by inhibiting activation of the STAT3 signaling pathway. These findings will aid a comprehensive description of the mechanism that controls the invasion and metastasis of cancer cells. PMID:25671570

  2. Retinoic acid-induced AP-1 transcriptional activity regulates B16 mouse melanoma growth inhibition and differentiation.

    PubMed

    Huang, Ying; Boskovic, Goran; Niles, Richard M

    2003-02-01

    Retinoic acid (RA) inhibits growth and induces differentiation of B16 mouse melanoma cells. These effects are accompanied by a large increase in PKCalpha mRNA and protein levels and surprisingly an increase in activating protein-1 (AP-1) transcriptional activity. To further investigate the RA-induced AP-1 activity we established clones of B16 cells stably expressing an AP-1-luciferase reporter gene. Treatment of these clones with phorbol dibutyrate increased AP-1 activity which peaked at 2-4 h and returned to baseline level by 24 h. In contrast, RA treatment resulted in a slow increase in AP-1 activity that reached a maximum level at 48 h and was maintained for the duration of the treatment. We tested the importance of the RA-induced AP-1 activity by establishing clones which stably express a dominant negative fos gene (A-fos) and have greatly diminished AP-1 activity. Growth rates of untreated A-fos expressing cells were similar to wt B16 and clones not expressing A-fos. However, clones expressing the dominant-negative fos had a markedly decreased sensitivity to RA-induced inhibition of anchorage-dependent and -independent growth. Treatment of wt B16 cells for 48 h with RA increased melanin production by two to fourfold, but this effect was completely lost in the A-fos clones. The ability of RA to induce RARbeta and PKCalpha expression was retained in A-fos clones, suggesting that A-fos was not interfering with RAR transcription activation functions. We tested whether the RA-induced AP-1 activity might be mediated by the ERK1/2 MAPK pathway. Inhibition of ERK1/2 phosphorylation stimulated AP-1 activity, which was not additive to that induced by RA. This finding raises the possibility that this MAPK pathway may be a target of retinoid action. Our observations suggest that AP-1 transcriptional activity induced by RA likely plays an important role in the biological changes mediated by this retinoid in B16 melanoma cells. PMID:12494454

  3. A novel bioactive chalcone of Morus australis inhibits tyrosinase activity and melanin biosynthesis in B16 melanoma cells.

    PubMed

    Takahashi, Makoto; Takara, Kensaku; Toyozato, Tomonao; Wada, Koji

    2012-01-01

    The methanol extract of Morus australis (shimaguwa) acts as a whitening agent due to the inhibition of tyrosinase activity. In order to explore the mechanism(s) of the whitening action, constituents of the 95% methanol extract from the dried stems of shimaguwa were isolated and their skin-whitening capacity was examined. Bioassay-guided fractionation of the methanol soluble extract of shimaguwa led to the isolation of 2, 4, 2', 4'-hydroxycalcone (chalcone 1) and three analogues of chalcone 1 with 3'-substituted resorcinol moieties (chalcones 2-4). Chalcone derivative 4 proved to be a novel compound and was fully characterized. Chalcones 1-4 were evaluated for inhibition activity on mushroom tyrosinase using L-tyrosine as the substrate. The parent chalcone 1 was a highly effective inhibitor of tyrosinase activity (IC₅₀ = 0.21 μM) compared to arbutin (IC₅₀ = 164 μM). Compared to chalcone 1, chalcones 2 and 3, which possess 3'-substituted isoprenyl or bulky 2-benzoylbiphenyl, showed significantly decreased tyrosinase activity, while chalcone 4, possessing 3'-substituted 2-hydroxy-1-pentene group, showed slightly increased activity.The effects of chalcones 1-4 on melanin synthesis, without affecting cell growth, were assayed in melanin-producing B16 murine melanoma cells. Chalcone 3 significantly reduced cell viability before reaching the IC₅₀ value for melanin synthesis. In contrast, the inhibitory effects of chalcones 1, 2 and 4 were more than 100-fold greater than that of arbutin, with little or no cytotoxicity. More significantly, chalcone 2, which exhibited less tyrosinase inhibitory activity compared to the parent chalcone 1, showed the highest inhibition of melanin synthesis in B16 cells among the chalcones tested. Accordingly, chalcones 1 and 2, and the novel chalcone 4 might be the active components responsible for the whitening ability of shimaguwa. Moreover, whitening ability was not exclusively due to tyrosinase inhibition. PMID:23018855

  4. Inhibition of autotaxin production or activity blocks lysophosphatidylcholine-induced migration of human breast cancer and melanoma cells

    PubMed Central

    Gaetano, Cristoforo G.; Samadi, Nasser; Tomsig, Jose L.; Macdonald, Timothy L.; Lynch, Kevin R.; Brindley, David N.

    2009-01-01

    Increased expression of autotaxin is linked to several malignancies including glioblastoma, breast, renal, ovarian, lung and thyroid cancers. Autotaxin promotes metastasis as well as cell growth, survival, and migration of cancer cells. These actions could depend on the non-catalytic effects of autotaxin on cell adhesion, or the catalytic activity of autotaxin, which converts lysophosphatidylcholine into lysophosphatidate in the extracellular environment. Both lysophosphatidylcholine and lysophosphatidate have been reported to stimulate migration through their respective G-protein coupled receptors. The present study determines the roles of autotaxin, lysophosphatidylcholine and lysophosphatidate in controlling the migration two cancer cell lines MDA-MB-231 breast cancer cells, which produce little autotaxin and MDA-MB-435 melanoma cells that secrete significant levels of autotaxin. Lysophosphatidylcholine alone was unable to stimulate the migration of either cell type unless autotaxin was present. Knocking down autotaxin secretion, or inhibiting its catalytic activity, blocked cell migration by preventing lysophosphatidate production and the subsequent activation of LPA1/3 receptors. We conclude that inhibiting autotaxin production or activity of could provide a beneficial adjuvant to chemotherapy for preventing metastasis in patients with high autotaxin expression in their tumors. PMID:19204929

  5. Chaetocin inhibits IBMX-induced melanogenesis in B16F10 mouse melanoma cells through activation of ERK.

    PubMed

    Bae, Jung-Soo; Han, Mira; Yao, Cheng; Chung, Jin Ho

    2016-02-01

    Chaetocin is a natural product isolated from Chaetomium species that has anti-bacterial and anti-myeloma activities. In this study, we investigated the inhibitory effect of chaetocin on melanogenesis and the underlying mechanisms in B16F10 mouse melanoma cells. In the present study, chaetocin significantly inhibited IBMX-induced melanin production and tyrosinase activity without any cytotoxicity. Furthermore, chaetocin down-regulated both the protein and mRNA levels of tyrosinase, which is a specific enzyme that catalyzes the conversion of tyrosine to melanin. We also observed that the protein level of MITF was significantly reduced by chaetocin treatment. In addition, we found that the anti-melanogenic effect of chaetocin was suppressed by treatment with the specific ERK inhibitor (PD98059). Accordingly, chaetocin inhibited melanogenesis via suppressing the protein level of MITF followed by activation of the ERK signaling pathway. These data suggest that chaetocin may be a potential anti-melanogenic agent for use in skin-whitening cosmetics and a topical agent for treatment of hyperpigmentation disorders. PMID:26748310

  6. ERBB activation modulates sensitivity to MEK1/2 inhibition in a subset of driver-negative melanoma

    PubMed Central

    Hutchinson, Katherine E.; Johnson, Douglas B.; Johnson, Adam S.; Sanchez, Violeta; Kuba, Maria; Lu, Pengcheng; Chen, Xi; Kelley, Mark C.; Wang, Qingguo; Zhao, Zhongming; Kris, Mark; Berger, Michael F.; Sosman, Jeffrey A.; Pao, William

    2015-01-01

    Melanomas are characterized by activating “driver” mutations in BRAF, NRAS, KIT, GNAQ, and GNA11. Resultant mitogen-activated protein kinase (MAPK) pathway signaling makes some melanomas susceptible to BRAF (BRAF V600 mutations), MEK1/2 (BRAF V600, L597, fusions; NRAS mutations), or other kinase inhibitors (KIT), respectively. Among driver-negative (“pan-negative”) patients, an unexplained heterogeneity of response to MEK1/2 inhibitors has been observed. Analysis of 16 pan-negative melanoma cell lines revealed that 8 (50%; termed Class I) are sensitive to the MEK1/2 inhibitor, trametinib, similar to BRAF V600E melanomas. A second set (termed Class II) display reduced trametinib sensitivity, paradoxical activation of MEK1/2 and basal activation of ERBBs 1, 2, and 3 (4 lines, 25%). In 3 of these lines, PI3K/AKT and MAPK pathway signaling is abrogated using the ERBB inhibitor, afatinib, and proliferation is even further reduced upon the addition of trametinib. A potential mechanism of ERBB activation in Class II melanomas is minimal expression of the ERK1/2 phosphatase, DUSP4, as ectopic restoration of DUSP4 attenuated ERBB signaling through potential modulation of the ERBB ligand, amphiregulin (AREG). Consistent with these data, immunohistochemical analysis of patient melanomas revealed a trend towards lower overall DUSP4 expression in pan-negative versus BRAF- and NRAS-mutant tumors. This study is the first to demonstrate that differential ERBB activity in pan-negative melanoma may modulate sensitivity to clinically-available MEK1/2 inhibitors and provides rationale for the use of ERBB inhibitors, potentially in combination with MEK1/2 inhibitors, in subsets of this disease. PMID:26084293

  7. Targeting activating mutations of EZH2 leads to potent cell growth inhibition in human melanoma by derepression of tumor suppressor genes

    PubMed Central

    Tiffen, Jessamy C.; Gunatilake, Dilini; Gallagher, Stuart J.; Gowrishankar, Kavitha; Heinemann, Anja; Cullinane, Carleen; Dutton-Regester, Ken; Pupo, Gulietta M.; Strbenac, Dario; Yang, Jean Y.; Madore, Jason; Mann, Graham J.; Hayward, Nicholas K.; McArthur, Grant A.; Filipp, Fabian V.; Hersey, Peter

    2015-01-01

    The epigenetic modifier EZH2 is part of the polycomb repressive complex that suppresses gene expression via histone methylation. Activating mutations in EZH2 are found in a subset of melanoma that contributes to disease progression by inactivating tumor suppressor genes. In this study we have targeted EZH2 with a specific inhibitor (GSK126) or depleted EZH2 protein by stable shRNA knockdown. We show that inhibition of EZH2 has potent effects on the growth of both wild-type and EZH2 mutant human melanoma in vitro particularly in cell lines harboring the EZH2Y646 activating mutation. This was associated with cell cycle arrest, reduced proliferative capacity in both 2D and 3D culture systems, and induction of apoptosis. The latter was caspase independent and mediated by the release of apoptosis inducing factor (AIFM1) from mitochondria. Gene expression arrays showed that several well characterized tumor suppressor genes were reactivated by EZH2 inhibition. This included activating transcription factor 3 (ATF3) that was validated as an EZH2 target gene by ChIP-qPCR. These results emphasize a critical role for EZH2 in the proliferation and viability of melanoma and highlight the potential for targeted therapy against EZH2 in treatment of patients with melanoma. PMID:26304929

  8. Natural paniceins from mediterranean sponge inhibit the multidrug resistance activity of Patched and increase chemotherapy efficiency on melanoma cells.

    PubMed

    Fiorini, Laura; Tribalat, Marie-Aude; Sauvard, Lucy; Cazareth, Julie; Lalli, Enzo; Broutin, Isabelle; Thomas, Olivier P; Mus-Veteau, Isabelle

    2015-09-01

    Multidrug resistance has appeared to mitigate the efficiency of anticancer drugs and the possibility of successful cancer chemotherapy. The Hedgehog receptor Patched is a multidrug transporter expressed in several cancers and as such it represents a new target to circumvent chemotherapy resistance. We report herein that paniceins and especially panicein A hydroquinone, natural meroterpenoids produced by the Mediterranean sponge Haliclona (Soestella) mucosa, inhibit the doxorubicin efflux activity of Patched and enhance the cytotoxicity of this chemotherapeutic agent on melanoma cells in vitro. These results are supported by the molecular docking performed on the structure of the bacterial drug efflux pump AcrB and on the Patched model built from AcrB structure. Docking calculations show that panicein A hydroquinone interacts with AcrB and Patched model close to the doxorubicin binding site. This compound thus appears as the first antagonist of the doxorubicin efflux activity of Patched. The use of inhibitors of Patched drug efflux activity in combination with classical chemotherapy could represent a novel approach to reduce tumor drug resistance, recurrence and metastasis. PMID:26068979

  9. Natural paniceins from mediterranean sponge inhibit the multidrug resistance activity of Patched and increase chemotherapy efficiency on melanoma cells

    PubMed Central

    Fiorini, Laura; Tribalat, Marie-Aude; Sauvard, Lucy; Cazareth, Julie; Lalli, Enzo; Broutin, Isabelle; Thomas, Olivier P.; Mus-Veteau, Isabelle

    2015-01-01

    Multidrug resistance has appeared to mitigate the efficiency of anticancer drugs and the possibility of successful cancer chemotherapy. The Hedgehog receptor Patched is a multidrug transporter expressed in several cancers and as such it represents a new target to circumvent chemotherapy resistance. We report herein that paniceins and especially panicein A hydroquinone, natural meroterpenoids produced by the Mediterranean sponge Haliclona (Soestella) mucosa, inhibit the doxorubicin efflux activity of Patched and enhance the cytotoxicity of this chemotherapeutic agent on melanoma cells in vitro. These results are supported by the molecular docking performed on the structure of the bacterial drug efflux pump AcrB and on the Patched model built from AcrB structure. Docking calculations show that panicein A hydroquinone interacts with AcrB and Patched model close to the doxorubicin binding site. This compound thus appears as the first antagonist of the doxorubicin efflux activity of Patched. The use of inhibitors of Patched drug efflux activity in combination with classical chemotherapy could represent a novel approach to reduce tumor drug resistance, recurrence and metastasis. PMID:26068979

  10. Specificity of growth inhibition of melanoma by 4-hydroxyanisole

    SciTech Connect

    Kulkarni, G.A.; Nathanson, L.

    1989-01-01

    An experimental study using human melanoma (NEL-MI), rat hepatoma (Fu5-5), and human kidney (293-31) cell lines was undertaken in order to evaluate the antitumor activity of 4-hydroxyanisole (4-OHA) in vitro. Prior reports have indicated highly specific antitumor activity of 4-OHA against melanoma cells in vitro. This specific antitumor activity has been proposed to be due to the oxidation of 4-OHA by tyrosinase to cytotoxic oxidation products. Dose-dependent cytotoxicity was observed when cells were cultured for 72 h in the presence of 4-OHA. At 100 microM, 4-OHA produced growth inhibition of 62%, 32%, and 55% in melanoma, hepatoma, and kidney cell lines, respectively. No effect was seen at 10 microM 4-OHA. 1,000 microM 4-OHA produced 100% kill. Tyrosinase activity was detected only in melanoma cells. The effect of 100 microM 4-OHA on the incorporation of 3H DNA precursors in melanoma, hepatoma, and kidney cells was also studied. Thymidine incorporation was inhibited in all three cell lines at the lowest cell density tested, with the greatest inhibition seen on melanoma cells. As cell density increased, the effect of 4-OHA on thymidine incorporation decreased. With respect to RNA synthesis, 4-OHA significantly reduced the incorporation of uridine in all three cell lines, with the greatest effect in melanoma cells. Cell density also affected the inhibition of uridine incorporation, but to a lesser extent than that observed on thymidine incorporation. The effect of 4-OHA on leucine incorporation was modest and uninfluenced by cell density. Thus, cytotoxicity of 4-OHA may involve two different mechanisms.

  11. Resveratrol-loaded nanocapsules inhibit murine melanoma tumor growth.

    PubMed

    Carletto, Bruna; Berton, Juliana; Ferreira, Tamara Nascimento; Dalmolin, Luciana Facco; Paludo, Katia Sabrina; Mainardes, Rubiana Mara; Farago, Paulo Vitor; Favero, Giovani Marino

    2016-08-01

    In this study, resveratrol-loaded nanocapsules were developed and its antitumor activity tested on a melanoma mice model. These nanocapsules were spherically-shaped and presented suitable size, negative charge and high encapsulation efficiency for their use as a modified-release system of resveratrol. Nanoencapsulation leads to the drug amorphization. Resveratrol-loaded nanoparticles reduced cell viability of murine melanoma cells. There was a decrease in tumor volume, an increase in the necrotic area and inflammatory infiltrate of melanoma when resveratrol-loaded nanocapsules were compared to free resveratrol in treated mice. Nanoencapsulation of resveratrol also prevented metastasis and pulmonary hemorrhage. This modified-release technology containing resveratrol can be used as a feasible approach in order to inhibit murine melanoma tumor growth. PMID:27070053

  12. Regulating the response to targeted MEK inhibition in melanoma

    PubMed Central

    Conrad, William H.; Swift, Reyna D.; Biechele, Travis L.; Kulikauskas, Rima M.; Moon, Randall T.; Chien, Andy J.

    2012-01-01

    The limitations of revolutionary new mutation-specific inhibitors of BRAFV600E include the universal recurrence seen in melanoma patients treated with this novel class of drugs. Recently, our lab showed that simultaneous activation of the Wnt/β-catenin signaling pathway and targeted inhibition of BRAFV600E by PLX4720 synergistically induces apoptosis across a spectrum of BRAFV600E melanoma cell lines. As a follow-up to that study, treatment of BRAF-mutant and NRAS-mutant melanoma lines with WNT3A and the MEK inhibitor AZD6244 also induces apoptosis. The susceptibility of BRAF-mutant lines and NRAS-mutant lines to apoptosis correlates with negative regulation of Wnt/β-catenin signaling by ERK/MAPK signaling and dynamic decreases in abundance of the downstream scaffolding protein, AXIN1. Apoptosis-resistant NRAS-mutant lines can sensitize to AZD6244 by pretreatment with AXIN1 siRNA, similar to what we previously reported in BRAF-mutant cell lines. Taken together, these findings indicate that NRAS-mutant melanoma share with BRAF-mutant melanoma the potential to regulate apoptosis upon MEK inhibition through WNT3A and dynamic regulation of cellular AXIN1. Understanding the cellular context that makes melanoma cells susceptible to this combination treatment will contribute to the study and development of novel therapeutic combinations that may lead to more durable responses. PMID:22895053

  13. Activation of β2-adrenergic receptor by (R,R’)-4’-methoxy-1-naphthylfenoterol inhibits proliferation and motility of melanoma cells

    PubMed Central

    Wnorowski, Artur; Sadowska, Mariola; Paul, Rajib K.; Singh, Nagendra S.; Boguszewska-Czubara, Anna; Jimenez, Lucita; Abdelmohsen, Kotb; Toll, Lawrence; Jozwiak, Krzysztof; Bernier, Michel; Wainer, Irving W.

    2015-01-01

    (R,R’)-4’-methoxy-1-naphthylfenoterol [(R,R’)-MNF] is a highly-selective β2 adrenergic receptor (β2-AR) agonist. Incubation of a panel of human-derived melanoma cell lines with (R,R’)-MNF resulted in a dose- and time-dependent inhibition of motility as assessed by in vitro wound healing and xCELLigence migration and invasion assays. Activity of (R,R’)-MNF positively correlated with the β2-AR expression levels across tested cell lines. The anti-motility activity of (R,R’)-MNF was inhibited by the β2-AR antagonist ICI-118,551 and the protein kinase A inhibitor H-89. The adenylyl cyclase activator forskolin and the phosphodiesterase 4 inhibitor Ro 20–1724 mimicked the ability of (R,R’)-MNF to inhibit migration of melanoma cell lines in culture, highlighting the importance of cAMP for this phenomenon. (R,R’)-MNF caused significant inhibition of cell growth in β2-AR-expressing cells as monitored by radiolabeled thymidine incorporation and xCELLigence system. The MEK/ERK cascade functions in cellular proliferation, and constitutive phosphorylation of MEK and ERK at their active sites was significantly reduced upon β2-AR activation with (R,R’)-MNF. Protein synthesis was inhibited concomitantly both with increased eEF2 phosphorylation and lower expression of tumor cell regulators, EGF receptors, cyclin A and MMP-9. Taken together, these results identified β2-AR as a novel potential target for melanoma management, and (R,R’)-MNF as an efficient trigger of anti-tumorigenic cAMP/PKA-dependent signaling in β2-AR-expressing lesions. PMID:25703025

  14. Filamin A Expression Negatively Regulates Sphingosine-1-Phosphate-Induced NF-κB Activation in Melanoma Cells by Inhibition of Akt Signaling

    PubMed Central

    Campos, Ludmila S.; Rodriguez, Yamila I.; Leopoldino, Andreia M.; Hait, Nitai C.; Lopez Bergami, Pablo; Castro, Melina G.; Sanchez, Emilse S.; Maceyka, Michael

    2015-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that regulates many processes in inflammation and cancer. S1P is a ligand for five G-protein-coupled receptors, S1PR1 to -5, and also has important intracellular actions. Previously, we showed that intracellular S1P is involved in tumor necrosis factor alpha (TNF)-induced NF-κB activation in melanoma cell lines that express filamin A (FLNA). Here, we show that extracellular S1P activates NF-κB only in melanoma cells that lack FLNA. In these cells, S1P, but not TNF, promotes IκB kinase (IKK) and p65 phosphorylation, IκBα degradation, p65 nuclear translocation, and NF-κB reporter activity. NF-κB activation induced by S1P was mediated via S1PR1 and S1PR2. Exogenous S1P enhanced the phosphorylation of protein kinase Cδ (PKCδ), and its downregulation reduced S1P-induced the phosphorylation of IKK and p65. In addition, silencing of Bcl10 also inhibited S1P-induced IKK phosphorylation. Surprisingly, S1P reduced Akt activation in melanoma cells that express FLNA, whereas in the absence of FLNA, high phosphorylation levels of Akt were maintained, enabling S1P-mediated NF-κB signaling. In accord, inhibition of Akt suppressed S1P-mediated IKK and p65 phosphorylation and degradation of IκBα. Hence, these results support a negative role of FLNA in S1P-mediated NF-κB activation in melanoma cells through modulation of Akt. PMID:26552704

  15. Filamin A Expression Negatively Regulates Sphingosine-1-Phosphate-Induced NF-κB Activation in Melanoma Cells by Inhibition of Akt Signaling.

    PubMed

    Campos, Ludmila S; Rodriguez, Yamila I; Leopoldino, Andreia M; Hait, Nitai C; Lopez Bergami, Pablo; Castro, Melina G; Sanchez, Emilse S; Maceyka, Michael; Spiegel, Sarah; Alvarez, Sergio E

    2015-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that regulates many processes in inflammation and cancer. S1P is a ligand for five G-protein-coupled receptors, S1PR1 to -5, and also has important intracellular actions. Previously, we showed that intracellular S1P is involved in tumor necrosis factor alpha (TNF)-induced NF-κB activation in melanoma cell lines that express filamin A (FLNA). Here, we show that extracellular S1P activates NF-κB only in melanoma cells that lack FLNA. In these cells, S1P, but not TNF, promotes IκB kinase (IKK) and p65 phosphorylation, IκBα degradation, p65 nuclear translocation, and NF-κB reporter activity. NF-κB activation induced by S1P was mediated via S1PR1 and S1PR2. Exogenous S1P enhanced the phosphorylation of protein kinase Cδ (PKCδ), and its downregulation reduced S1P-induced the phosphorylation of IKK and p65. In addition, silencing of Bcl10 also inhibited S1P-induced IKK phosphorylation. Surprisingly, S1P reduced Akt activation in melanoma cells that express FLNA, whereas in the absence of FLNA, high phosphorylation levels of Akt were maintained, enabling S1P-mediated NF-κB signaling. In accord, inhibition of Akt suppressed S1P-mediated IKK and p65 phosphorylation and degradation of IκBα. Hence, these results support a negative role of FLNA in S1P-mediated NF-κB activation in melanoma cells through modulation of Akt. PMID:26552704

  16. Thymoquinone suppresses metastasis of melanoma cells by inhibition of NLRP3 inflammasome

    SciTech Connect

    Ahmad, Israr; Muneer, Kashiff M.; Tamimi, Iman A.; Chang, Michelle E.; Ata, Muhammad O.; Yusuf, Nabiha

    2013-07-01

    The inflammasome is a multi-protein complex which when activated regulates caspase-1 activation and IL-1β and IL-18 secretion. The NLRP3 (NACHT, LRR, and pyrin domain-containing protein 3) inflammasome is constitutively assembled and activated in human melanoma cells. We have examined the inhibitory effect of thymoquinone (2-isopropyl-5-methylbenzo-1,4-quinone), a major ingredient of black seed obtained from the plant Nigella sativa on metastatic human (A375) and mouse (B16F10) melanoma cell lines. We have assessed whether thymoquinone inhibits metastasis of melanoma cells by targeting NLRP3 subunit of inflammasomes. Using an in vitro cell migration assay, we found that thymoquinone inhibited the migration of both human and mouse melanoma cells. The inhibitory effect of thymoquinone on metastasis was also observed in vivo in B16F10 mouse melanoma model. The inhibition of migration of melanoma cells by thymoquinone was accompanied by a decrease in expression of NLRP3 inflammasome resulting in decrease in proteolytic cleavage of caspase-1. Inactivation of caspase-1 by thymoquinone resulted in inhibition of IL-1β and IL-18. Treatment of mouse melanoma cells with thymoquinone also inhibited NF-κB activity. Furthermore, inhibition of reactive oxygen species (ROS) by thymoquinone resulted in partial inactivation of NLRP3 inflammasome. Thus, thymoquinone exerts its inhibitory effect on migration of human and mouse melanoma cells by inhibition of NLRP3 inflammasome. Thus, our results indicate that thymoquinone can be a potential immunotherapeutic agent not only as an adjuvant therapy for melanoma, but also, in the control and prevention of metastatic melanoma. - Highlights: • Thymoquinone causes inhibition of migration of melanoma cells. • Thymoquinone causes inhibition of metastasis in vivo. • Thymoquinone causes inhibition of migration by activation of NLRP3 inflammasome.

  17. Counteracting oxidative phosphorylation-mediated resistance of melanomas to MAPK pathway inhibition

    PubMed Central

    McQuade, Jennifer L; Vashisht Gopal, Yn

    2015-01-01

    Mitochondrial oxidative phosphorylation (OxPhos) induces resistance to MAPK pathway inhibitors in melanoma. However, therapeutic targeting of mitochondria is challenging. In a recent study, we showed that inhibition of mTOR kinase activity resensitized resistant melanomas by indirectly inhibiting OxPhos via a novel mechanism. Here, we discuss the implications of these findings. PMID:27308473

  18. Counteracting oxidative phosphorylation-mediated resistance of melanomas to MAPK pathway inhibition.

    PubMed

    McQuade, Jennifer L; Vashisht Gopal, Yn

    2015-01-01

    Mitochondrial oxidative phosphorylation (OxPhos) induces resistance to MAPK pathway inhibitors in melanoma. However, therapeutic targeting of mitochondria is challenging. In a recent study, we showed that inhibition of mTOR kinase activity resensitized resistant melanomas by indirectly inhibiting OxPhos via a novel mechanism. Here, we discuss the implications of these findings. PMID:27308473

  19. RSK1 Activation Promotes Invasion in Nodular Melanoma

    PubMed Central

    Salhi, Amel; Farhadian, Joshua A.; Giles, Keith M.; Vega-Saenz de Miera, Eleazar; Silva, Ines P.; Bourque, Caitlin; Yeh, Karen; Chhangawala, Sagar; Wang, Jinhua; Ye, Fei; Zhang, David Y.; Hernando-Monge, Eva; Houvras, Yariv; Osman, Iman

    2016-01-01

    The two major melanoma histologic subtypes, superficial spreading and nodular melanomas, differ in their speed of dermal invasion but converge biologically once they invade and metastasize. Herein, we tested the hypothesis that distinct molecular alterations arising in primary melanoma cells might persist as these tumors progress to invasion and metastasis. Ribosomal protein S6 kinase, 90 kDa, polypeptide 1 (RSK1; official name RPS6KA1) was significantly hyperactivated in human melanoma lines and metastatic tissues derived from nodular compared with superficial spreading melanoma. RSK1 was constitutively phosphorylated at Ser-380 in nodular but not superficial spreading melanoma and did not directly correlate with BRAF or MEK activation. Nodular melanoma cells were more sensitive to RSK1 inhibition using siRNA and the pharmacological inhibitor BI-D1870 compared with superficial spreading cells. Gene expression microarray analyses revealed that RSK1 orchestrated a program of gene expression that promoted cell motility and invasion. Differential overexpression of the prometastatic matrix metalloproteinase 8 and tissue inhibitor of metalloproteinases 1 in metastatic nodular compared with metastatic superficial spreading melanoma was observed. Finally, using an in vivo zebrafish model, constitutive RSK1 activation increased melanoma invasion. Together, these data reveal a novel role for activated RSK1 in the progression of nodular melanoma and suggest that melanoma originating from different histologic subtypes may be biologically distinct and that these differences are maintained as the tumors invade and metastasize. PMID:25579842

  20. RSK1 activation promotes invasion in nodular melanoma.

    PubMed

    Salhi, Amel; Farhadian, Joshua A; Giles, Keith M; Vega-Saenz de Miera, Eleazar; Silva, Ines P; Bourque, Caitlin; Yeh, Karen; Chhangawala, Sagar; Wang, Jinhua; Ye, Fei; Zhang, David Y; Hernando-Monge, Eva; Houvras, Yariv; Osman, Iman

    2015-03-01

    The two major melanoma histologic subtypes, superficial spreading and nodular melanomas, differ in their speed of dermal invasion but converge biologically once they invade and metastasize. Herein, we tested the hypothesis that distinct molecular alterations arising in primary melanoma cells might persist as these tumors progress to invasion and metastasis. Ribosomal protein S6 kinase, 90 kDa, polypeptide 1 (RSK1; official name RPS6KA1) was significantly hyperactivated in human melanoma lines and metastatic tissues derived from nodular compared with superficial spreading melanoma. RSK1 was constitutively phosphorylated at Ser-380 in nodular but not superficial spreading melanoma and did not directly correlate with BRAF or MEK activation. Nodular melanoma cells were more sensitive to RSK1 inhibition using siRNA and the pharmacological inhibitor BI-D1870 compared with superficial spreading cells. Gene expression microarray analyses revealed that RSK1 orchestrated a program of gene expression that promoted cell motility and invasion. Differential overexpression of the prometastatic matrix metalloproteinase 8 and tissue inhibitor of metalloproteinases 1 in metastatic nodular compared with metastatic superficial spreading melanoma was observed. Finally, using an in vivo zebrafish model, constitutive RSK1 activation increased melanoma invasion. Together, these data reveal a novel role for activated RSK1 in the progression of nodular melanoma and suggest that melanoma originating from different histologic subtypes may be biologically distinct and that these differences are maintained as the tumors invade and metastasize. PMID:25579842

  1. Honokiol inhibits melanoma stem cells by targeting notch signaling.

    PubMed

    Kaushik, Gaurav; Venugopal, Anand; Ramamoorthy, Prabhu; Standing, David; Subramaniam, Dharmalingam; Umar, Shahid; Jensen, Roy A; Anant, Shrikant; Mammen, Joshua M V

    2015-12-01

    Melanoma is an aggressive disease with limited therapeutic options. Here, we determined the effects of honokiol (HNK), a biphenolic natural compound on melanoma cells and stemness. HNK significantly inhibited melanoma cell proliferation, viability, clonogenicity and induced autophagy. In addition, HNK significantly inhibited melanosphere formation in a dose dependent manner. Western blot analyses also demonstrated reduction in stem cell markers CD271, CD166, Jarid1b, and ABCB5. We next examined the effect of HNK on Notch signaling, a pathway involved in stem cell self-renewal. Four different Notch receptors exist in cells, which when cleaved by a series of enzymatic reactions catalyzed by Tumor Necrosis Factor-α-Converting Enzyme (TACE) and γ-secretase protein complex, results in the release of the Notch intracellular domain (NICD), which then translocates to the nucleus and induces target gene expression. Western blot analyses demonstrated that in HNK treated cells there is a significant reduction in the expression of cleaved Notch-2. In addition, there was a reduction in the expression of downstream target proteins, Hes-1 and cyclin D1. Moreover, HNK treatment suppressed the expression of TACE and γ-secretase complex proteins in melanoma cells. To confirm that suppression of Notch-2 activation is critical for HNK activity, we overexpressed NICD1, NICD2, and performed HNK treatment. NICD2, but not NICD1, partially restored the expression of Hes-1 and cyclin D1, and increased melanosphere formation. Taken together, these data suggest that HNK is a potent inhibitor of melanoma cells, in part, through the targeting of melanoma stem cells by suppressing Notch-2 signaling. PMID:25491779

  2. Inhibition of the RhoA GTPase Activity Increases Sensitivity of Melanoma Cells to UV Radiation Effects

    PubMed Central

    Espinha, Gisele; Osaki, Juliana Harumi; Costa, Erico Tosoni; Forti, Fabio Luis

    2016-01-01

    Ultraviolet radiation is the main cause of DNA damage to melanocytes and development of melanoma, one of the most lethal human cancers, which leads to metastasis due to uncontrolled cell proliferation and migration. These phenotypes are mediated by RhoA, a GTPase overexpressed or overactivated in highly aggressive metastatic tumors that plays regulatory roles in cell cycle progression and cytoskeleton remodeling. This work explores whether the effects of UV on DNA damage, motility, proliferation, and survival of human metastatic melanoma cells are mediated by the RhoA pathway. Mutant cells expressing dominant-negative (MeWo-RhoA-N19) or constitutively active RhoA (MeWo-RhoA-V14) were generated and subjected to UV radiation. A slight reduction in migration and invasion was observed in MeWo and MeWo-RhoA-V14 cells but not in MeWo-RhoA-N19 cells, which presented inefficient motility and invasiveness associated with stress fibers fragmentation. Proliferation and survival of RhoA-deficient cells were drastically reduced by UV compared to cells displaying normal or high RhoA activity, suggesting increased sensitivity to UV. Loss of RhoA activity also caused less efficient DNA repair, with elevated levels of DNA lesions such as strand breaks and cyclobutane pyrimidine dimers (CPDs). Thus, RhoA mediates genomic stability and represents a potential target for sensitizing metastatic tumors to genotoxic agents. PMID:26823948

  3. Blue light inhibits proliferation of melanoma cells

    NASA Astrophysics Data System (ADS)

    Becker, Anja; Distler, Elisabeth; Klapczynski, Anna; Arpino, Fabiola; Kuch, Natalia; Simon-Keller, Katja; Sticht, Carsten; van Abeelen, Frank A.; Gretz, Norbert; Oversluizen, Gerrit

    2016-03-01

    Photobiomodulation with blue light is used for several treatment paradigms such as neonatal jaundice, psoriasis and back pain. However, little is known about possible side effects concerning melanoma cells in the skin. The aim of this study was to assess the safety of blue LED irradiation with respect to proliferation of melanoma cells. For that purpose we used the human malignant melanoma cell line SK-MEL28. Cell proliferation was decreased in blue light irradiated cells where the effect size depended on light irradiation dosage. Furthermore, with a repeated irradiation of the melanoma cells on two consecutive days the effect could be intensified. Fluorescence-activated cell sorting with Annexin V and Propidium iodide labeling did not show a higher number of dead cells after blue light irradiation compared to non-irradiated cells. Gene expression analysis revealed down-regulated genes in pathways connected to anti-inflammatory response, like B cell signaling and phagosome. Most prominent pathways with up-regulation of genes were cytochrome P450, steroid hormone biosynthesis. Furthermore, even though cells showed a decrease in proliferation, genes connected to the cell cycle were up-regulated after 24h. This result is concordant with XTT test 48h after irradiation, where irradiated cells showed the same proliferation as the no light negative control. In summary, proliferation of melanoma cells can be decreased using blue light irradiation. Nevertheless, the gene expression analysis has to be further evaluated and more studies, such as in-vivo experiments, are warranted to further assess the safety of blue light treatment.

  4. In Vitro and In Vivo Anti-Melanoma Effects of Pituranthos tortuosus Essential Oil Via Inhibition of FAK and Src Activities.

    PubMed

    Krifa, Mounira; Meshri, Salah Edin El; Bentouati, Nawel; Pizzi, Antonio; Sick, Emilie; Chekir-Ghedira, Leila; Rondé, Philippe

    2016-05-01

    A large number of plants used in traditional medicines have been shown to possess antitumor activities. The aims of this study were to evaluate any anticancer effect of the essential oil (EO) extracted from P. tortuosus against B16F10 melanoma cancer cells in vitro as well as in vivo. In vitro, EO was shown to induce apoptosis and to inhibit migration and invasion processes. Further investigation revealed that EO decreased focal adhesion and invadopodia formation which was accompanied by a drastic downregulation of FAK, Src, ERK, p130Cas and paxillin. Moreover, EO treatment decreased the expression level of p190RhoGAP, and Grb2, which impair cell migration and actin assembly. Mice bearing the melanoma cells were used to confirm any in vivo effectiveness of the EO as an anti-tumor promoting agent. In mice dosed with 100 mg EO/kg/d (for 27 days), tumor weight was inhibited by 98% compared to that in mice that did not receive the product. In conclusion, these data suggested to us that an EO of P. tortuosus could evolve to be a potential medicinal resource for use in the treatment of cancers. J. Cell. Biochem. 117: 1167-1175, 2016. © 2015 Wiley Periodicals, Inc. PMID:26477879

  5. Convection-enhanced delivery of sorafenib and suppression of tumor progression in a murine model of brain melanoma through the inhibition of signal transducer and activator of transcription 3.

    PubMed

    Zou, Zhaoxia; Yin, Yufang; Lin, Jenny; Hsu, Li-Chen J; Brandon, Vanessa L; Yang, Fan; Jove, Richard; Jandial, Rahul; Li, Gang; Chen, Mike Y

    2016-05-01

    OBJECT Despite recent advances, metastatic melanoma remains a terminal disease, in which life-threatening brain metastasis occurs in approximately half of patients. Sorafenib is a multikinase inhibitor that induces apoptosis of melanoma cells in vitro. However, systemic administration has been ineffective because adequate tissue concentrations cannot be achieved. This study investigated if convection-enhanced delivery (CED) of sorafenib would enhance tumor control and survival via inhibition of the signal transducer and activator of transcription 3 (Stat3) pathway in a murine model of metastatic brain melanoma. METHODS Melanoma cells treated with sorafenib in vitro were examined for signaling and survival changes. The effect of sorafenib given by CED was assessed by bioluminescent imaging and animal survival. RESULTS The results showed that sorafenib induced cell death in the 4 established melanoma cell lines and in 1 primary cultured melanoma cell line. Sorafenib inhibited Stat3 phosphorylation in HTB65, WYC1, and B16 cells. Accordingly, sorafenib treatment also decreased expression of Mcl-1 mRNA in melanoma cell lines. Because sorafenib targets multiple pathways, the present study demonstrated the contribution of the Stat3 pathway by showing that mouse embryonic fibroblast (MEF) Stat3 +/+ cells were significantly more sensitive to sorafenib than MEF Stat3 -/- cells. In the murine model of melanoma brain metastasis used in this study, CED of sorafenib increased survival by 150% in the treatment group compared with animals receiving the vehicle control (p < 0.01). CED of sorafenib also significantly abrogated tumor growth. CONCLUSIONS The data from this study indicate that local delivery of sorafenib effectively controls brain melanoma. These findings validate further investigation of the use of CED to distribute molecularly targeted agents. PMID:26544779

  6. RELIEF OF PROFOUND FEEDBACK INHIBITION OF MITOGENIC SIGNALING BY RAF INHIBITORS ATTENUATES THEIR ACTIVITY IN BRAFV600E MELANOMAS

    PubMed Central

    Lito, Piro; Pratilas, Christine A.; Joseph, Eric W.; Tadi, Madhavi; Halilovic, Ensar; Zubrowski, Matthew; Huang, Alan; Wong, Wai Lin; Callahan, Margaret K.; Merghoub, Taha; Wolchok, Jedd D.; de Stanchina, Elisa; Chandarlapaty, Sarat; Poulikakos, Poulikos I.; Fagin, James A.; Rosen, Neal

    2012-01-01

    SUMMARY BRAFV600E drives tumors by dysregulating ERK signaling. In these tumors, we show that high levels of ERK-dependent negative feedback potently suppress ligand-dependent mitogenic signaling and Ras function. BRAFV600E activation is Ras-independent and it signals as a RAF-inhibitor sensitive monomer. RAF inhibitors potently inhibit RAF monomers and ERK signaling, causing relief of ERK-dependent feedback, reactivation of ligand-dependent signal transduction, increased Ras-GTP and generation of RAF inhibitor-resistant RAF dimers. This results in a rebound in ERK activity and culminates in a new steady state, wherein ERK signaling is elevated compared to its initial nadir after RAF inhibition. In this state, ERK signaling is RAF inhibitor resistant, and MEK inhibitor sensitive, and combined inhibition results in enhancement of ERK-pathway inhibition and antitumor activity. PMID:23153539

  7. Combined PKC and MEK inhibition for treating metastatic uveal melanoma.

    PubMed

    Sagoo, M S; Harbour, J W; Stebbing, J; Bowcock, A M

    2014-09-25

    Uveal melanoma (UM) is the most common primary intraocular malignancy and the second most common form of melanoma. UM has a strong tendency for metastatic disease, and no effective treatments have yet been identified. Activating oncogenic mutations are commonly found in GNAQ and GNA11 in UM, and inhibiting key downstream effectors of the GNAQ/11 signaling pathway represents a rational therapeutic approach for treating metastatic UM. Chen et al., doi:10.1038/onc.2013.418, now confirm activation of the MAPK and PKC pathways as a result of GNAQ and GNA11 activating mutations in melanocytes, and they demonstrate that MAPK activation occurs downstream of PKC activation. PKC inhibitors disrupt MAPK signaling and block proliferation of GNAQ/11 mutant UM cell lines and slow the in vivo growth of xenografted UM tumors without inducing their shrinkage. However, a combination of PKC and MEK inhibition led to sustained MAPK pathway inhibition and tumor regression in vivo. Hence, the authors concluded that MEK and PKC inhibition is synergistic, with superior efficacy to treatment of GNAQ/GNA11 mutant UMs with either drug alone. PMID:24413085

  8. Combined PKC and MEK inhibition for treating metastatic uveal melanoma

    PubMed Central

    Sagoo, MS; Harbour, JW; Stebbing, J; Bowcock, AM

    2015-01-01

    Uveal melanoma (UM) is the most common primary intraocular malignancy and the second most common form of melanoma. UM has a strong tendency for metastatic disease, and no effective treatments have yet been identified. Activating oncogenic mutations are commonly found in GNAQ and GNA11 in UM, and inhibiting key downstream effectors of the GNAQ/11 signaling pathway represents a rational therapeutic approach for treating metastatic UM. Chen et al., doi:10.1038/onc.2013.418, now confirm activation of the MAPK and PKC pathways as a result of GNAQ and GNA11 activating mutations in melanocytes, and they demonstrate that MAPK activation occurs downstream of PKC activation. PKC inhibitors disrupt MAPK signaling and block proliferation of GNAQ/11 mutant UM cell lines and slow the in vivo growth of xenografted UM tumors without inducing their shrinkage. However, a combination of PKC and MEK inhibition led to sustained MAPK pathway inhibition and tumor regression in vivo. Hence, the authors concluded that MEK and PKC inhibition is synergistic, with superior efficacy to treatment of GNAQ/GNA11 mutant UMs with either drug alone. PMID:24413085

  9. Effects of BRAF mutations and BRAF inhibition on immune responses to melanoma

    PubMed Central

    Ilieva, Kristina M.; Correa, Isabel; Josephs, Debra H.; Karagiannis, Panagiotis; Egbuniwe, Isioma U.; Cafferkey, Michiala J.; Spicer, James F.; Harries, Mark; Nestle, Frank O.; Lacy, Katie E.; Karagiannis, Sophia N.

    2014-01-01

    Malignant melanoma is associated with poor clinical prognosis; however, novel molecular and immune therapies are now improving patient outcomes. Almost 50% of melanomas harbor targetable activating mutations of BRAF which promote RAS-RAF-MEK-ERK pathway activation and melanoma proliferation. Recent evidence also indicates that melanomas bearing mutant BRAF may also have altered immune responses, suggesting additional avenues for treatment of this patient group. The small molecule inhibitors selective for mutant BRAF induce significant but short-lived clinical responses in a proportion of patients, but also lead to immune stimulatory bystander events, which then subside with the emergence of resistance to inhibition. Simultaneous BRAF and MEK inhibition, and especially combination of BRAF inhibitors with new immunotherapies such as checkpoint blockade antibodies, may further enhance immune activation, or counteract immunosuppressive signals. Pre-clinical evaluation and ongoing clinical trials should provide novel insights into the role of immunity in the therapy of BRAF-mutant melanoma. PMID:25385327

  10. Pentoxifylline Inhibits WNT Signalling in β-Cateninhigh Patient-Derived Melanoma Cell Populations

    PubMed Central

    Talar, Beata; Gajos-Michniewicz, Anna; Talar, Marcin; Chouaib, Salem; Czyz, Malgorzata

    2016-01-01

    Background The heterogeneity of melanoma needs to be addressed and combination therapies seem to be necessary to overcome intrinsic and acquired resistance to newly developed immunotherapies and targeted therapies. Although the role of WNT/β-catenin pathway in melanoma was early demonstrated, its contribution to the lack of the melanoma patient response to treatment was only recently recognized. Using patient-derived melanoma cell populations, we investigated the influence of pentoxifylline on melanoma cells with either high or low expression of β-catenin. Findings Our results indicate that pentoxifylline inhibits the activity of the canonical WNT pathway in melanoma cell populations with high basal activity of this signalling. This is supported by lowered overall activity of transcription factors TCF/LEF and reduced nuclear localisation of active β-catenin. Moreover, treatment of β-cateninhigh melanoma cell populations with pentoxifylline induces downregulation of genes that are targets of the WNT/β-catenin pathway including connective tissue growth factor (CTGF) and microphthalmia-associated transcription factor (MITF-M), a melanocyte- and melanoma cell-specific regulator. Conclusions These results suggest that pentoxifylline, a drug approved by the FDA in the treatment of peripheral arterial disease, might be tested in a subset of melanoma patients with elevated activity of β-catenin. This pharmaceutical might be tested as an adjuvant drug in combination therapies when the response to immunotherapy is prevented by high activity of the WNT/β-catenin pathway. PMID:27351373

  11. MiR-26b inhibits melanoma cell proliferation and enhances apoptosis by suppressing TRAF5-mediated MAPK activation.

    PubMed

    Li, Meng; Long, Chaoqin; Yang, Guilan; Luo, Yang; Du, Hua

    2016-03-11

    Alterations in microRNA-26b (miR-26b) expression have been shown to participate in various malignant tumor developments. However, the possible function of miR-26b in human melanoma cells remains unclarified. In this study, quantitative polymerase chain reaction was used to explore the expression profiles of miR-26b in melanoma cells. The effect of miR-26b on cell viability was determined by using MTT assays and colony formation assay. The apoptosis levels were evaluated by using Annexin V/fluorescein isothiocyanate (FITC) apoptosis detection kit and the apoptosis cells were confirmed by Transmission Electron Microscopy (TEM). Luciferase reporter plasmids were constructed to confirm direct targeting. Our study found that the expression of miR-26b was downregulated in human melanoma specimens. Overexpression of miR-26b significantly increased the anti-proliferative effects and apoptosis in A375 and B16F10 melanoma cells. In addition, luciferase gene reporter assays confirmed that TRAF5 was a direct target gene of miR-26b and the anti-tumor effect of miR-26b in melanoma cells was significantly counteracted by treatment with TRAF5 overexpression. Furthermore, the molecular mechanisms underlying the tumor suppressor of miR-26b in malignant melanomas may be due to the dephosphorylation of MAPK pathway caused by the decrease in TRAF5 expression when miR-26b is up-regulated in melanoma cells. These findings indicate that miR-26b might influence TRAF5-MAPK signaling pathways to facilitate the malignant progression of melanoma cells. PMID:26872428

  12. Retinoids increase transglutaminase activity and inhibit ornithine decarboxylase activity in Chinese hamster ovary cells and in melanoma cells stimulated to differentiate.

    PubMed Central

    Scott, K F; Meyskens, F L; Russell, D H

    1982-01-01

    Transglutaminase (TGase; R-glutaminyl-peptide:amine gamma-glutamyltransferase, EC 2.3.2.13) and ornithine decarboxylase (ODCase; L-ornithine carboxy-lyase, EC 4.1.1.17) activities were measured after the addition of retinoid analogs to Chinese hamster ovary (CHO) cells released from quiescence and Cloudman S91 (CCL 53.1) mouse melanoma cells stimulated to differentiate with alpha-melanocyte-stimulating hormone (MSH, melanotropin). In both cell culture lines, we detected a biphasic increase in TGase activity and a single peak of ODCase activity within 7 hr after release or stimulation. Retinoid analogs altered the expression of the initial TGase peak in both CHO and melanoma cells. Retinol increased the activity of TGase 1 hr after release in CHO cells, and the activity remained elevated until hr 4. A broad peak of TGase activity also occurred after the addition of alpha-difluoromethylornithine, an irreversible inhibitor of ODCase, and after addition of alpha-difluoromethylornithine plus retinol. In mouse melanoma cells, retinoic acid plus MSH markedly enhanced the activity of the initial TGase peak compared to MSH alone. Retinoic acid alone also increased TGase activity biphasically in these cells without the addition of MSH. These studies suggest that retinoid effects that increase TGase activity may alter the ODCase expression in proliferation and differentiation. PMID:6125941

  13. Targeting Mitogen-Activated Protein Kinase/Extracellular Signal–Regulated Kinase Kinase in the Mutant (V600E) B-Raf Signaling Cascade Effectively Inhibits Melanoma Lung Metastases

    PubMed Central

    Sharma, Arati; Tran, Melissa A.; Liang, Shile; Sharma, Arun K.; Amin, Shantu; Smith, Charles D.; Dong, Cheng; Robertson, Gavin P.

    2009-01-01

    Malignant melanoma has a high propensity for metastatic spread, making it the most deadly form of skin cancer. B-RAF has been identified as the most mutated gene in these invasive cells and therefore an attractive therapeutic target. However, for uncertain reasons, chemotherapy inhibiting B-Raf has not been clinically effective. This has raised questions whether this pathway is important in melanoma metastasis or whether targeting a protein other than B-Raf in the signaling cascade could more effectively inhibit this pathway to reduce lung metastases. Here, we investigated the role played by V600EB-Raf in melanoma metastasis and showed that targeting this signaling cascade significantly reduces lung metastases. Small interfering RNA (siRNA)–mediated inhibition was used in mice to reduce expression (activity) of each member of the signaling cascade and effects on metastasis development were measured. Targeting any member of the signaling cascade reduced metastasis but inhibition of mitogen-activated protein kinase/extracellular signal–regulated kinase kinase (Mek) 1 and Mek 2 almost completely prevented lung tumor development. Mechanistically, metastatic inhibition was mediated through reduction of melanoma cell extravasation through the endothelium and decreased proliferative capacity. Targeting B-Raf with the pharmacologic inhibitor BAY 43-9006, which was found ineffective in clinical trials and seems to act primarily as an angiogenesis inhibitor, did not decrease metastasis, whereas inhibition of Mek using U0126 decreased cellular proliferative capacity, thereby effectively reducing number and size of lung metastases. In summary, this study provides a mechanistic basis for targeting Mek and not B-Raf in the mutant V600EB-Raf signaling cascade to inhibit melanoma metastases. PMID:16912199

  14. Oncogenic BRAFV600E inhibits BIM expression to promote melanoma cell survival

    PubMed Central

    Cartlidge, Robert A.; Thomas, G. R.; Cagnol, Sebastien; Jong, Kimberly A.; Molton, Sarah A.; Finch, Andrew J.; McMahon, Martin

    2016-01-01

    Summary Somatic activating mutations of BRAF are the earliest and most common genetic abnormality detected in the genesis of human melanoma. However, the mechanism(s) by which activated BRAF promotes melanoma cell cycle progression and/or survival remain unclear. Here we demonstrate that expression of BIM, a pro-apoptotic member of the BCL-2 family, is inhibited by BRAF → MEK → ERK signaling in mouse and human melanocytes and in human melanoma cells. Trophic factor deprivation of melanocytes leads to elevated BIM expression. However, re-addition of trophic factors or activation of a conditional form of BRAFV600E leads to rapid inhibition of BIM expression. In both cases, inhibition of BIM expression was dependent on the activity of MEK1/2 and the proteasome. Consistent with these observations, pharmacological inhibition of BRAFV600E or MEK1/2 in human melanoma cells (using PLX4720 and CI-1040 respectively) led to a striking elevation of BIM expression. Re-activation of BRAF → MEK → ERK signaling led to phosphorylation of BIM-EL on serine 69 and its subsequent degradation. Interestingly, endogenous expression of BIM in melanoma cells was insufficient to induce apoptosis unless combined with serum deprivation. Under these circumstances, inhibition of BIM expression by RNA interference provided partial protection from apoptosis. These data suggest that regulation of BIM expression by BRAF → MEK → ERK signaling is one mechanism by which oncogenic BRAFV600E can influence the aberrant physiology of melanoma cells. PMID:18715233

  15. NF-κB activation in melanoma

    PubMed Central

    Ueda, Yukiko; Richmond, Ann

    2009-01-01

    Summary Metastatic melanoma is an aggressive skin cancer that is notoriously resistant to current cancer therapies. In human melanoma, nuclear factor-kappa B (NF-κB) is upregulated, leading to the deregulation of gene transcription. In this review, we discuss (i) the relationship between gene alteration in melanoma and upregulation of NF-κB, (ii) mechanisms by which activated NF-κB switch from pro-apoptotic to anti-apoptotic functions in melanoma and (iii) autocrine mechanisms that promote constitutive activation of NF-κB in metastatic melanoma. PMID:16524427

  16. Inhibiting Drivers of Non-mutational Drug Tolerance Is a Salvage Strategy for Targeted Melanoma Therapy

    PubMed Central

    Smith, Michael P.; Brunton, Holly; Rowling, Emily J.; Ferguson, Jennifer; Arozarena, Imanol; Miskolczi, Zsofia; Lee, Jessica L.; Girotti, Maria R.; Marais, Richard; Levesque, Mitchell P.; Dummer, Reinhard; Frederick, Dennie T.; Flaherty, Keith T.; Cooper, Zachary A.; Wargo, Jennifer A.; Wellbrock, Claudia

    2016-01-01

    Summary Once melanomas have progressed with acquired resistance to mitogen-activated protein kinase (MAPK)-targeted therapy, mutational heterogeneity presents a major challenge. We therefore examined the therapy phase before acquired resistance had developed and discovered the melanoma survival oncogene MITF as a driver of an early non-mutational and reversible drug-tolerance state, which is induced by PAX3-mediated upregulation of MITF. A drug-repositioning screen identified the HIV1-protease inhibitor nelfinavir as potent suppressor of PAX3 and MITF expression. Nelfinavir profoundly sensitizes BRAF and NRAS mutant melanoma cells to MAPK-pathway inhibitors. Moreover, nelfinavir is effective in BRAF and NRAS mutant melanoma cells isolated from patients progressed on MAPK inhibitor (MAPKi) therapy and in BRAF/NRAS/PTEN mutant tumors. We demonstrate that inhibiting a driver of MAPKi-induced drug tolerance could improve current approaches of targeted melanoma therapy. PMID:26977879

  17. Inhibiting Drivers of Non-mutational Drug Tolerance Is a Salvage Strategy for Targeted Melanoma Therapy.

    PubMed

    Smith, Michael P; Brunton, Holly; Rowling, Emily J; Ferguson, Jennifer; Arozarena, Imanol; Miskolczi, Zsofia; Lee, Jessica L; Girotti, Maria R; Marais, Richard; Levesque, Mitchell P; Dummer, Reinhard; Frederick, Dennie T; Flaherty, Keith T; Cooper, Zachary A; Wargo, Jennifer A; Wellbrock, Claudia

    2016-03-14

    Once melanomas have progressed with acquired resistance to mitogen-activated protein kinase (MAPK)-targeted therapy, mutational heterogeneity presents a major challenge. We therefore examined the therapy phase before acquired resistance had developed and discovered the melanoma survival oncogene MITF as a driver of an early non-mutational and reversible drug-tolerance state, which is induced by PAX3-mediated upregulation of MITF. A drug-repositioning screen identified the HIV1-protease inhibitor nelfinavir as potent suppressor of PAX3 and MITF expression. Nelfinavir profoundly sensitizes BRAF and NRAS mutant melanoma cells to MAPK-pathway inhibitors. Moreover, nelfinavir is effective in BRAF and NRAS mutant melanoma cells isolated from patients progressed on MAPK inhibitor (MAPKi) therapy and in BRAF/NRAS/PTEN mutant tumors. We demonstrate that inhibiting a driver of MAPKi-induced drug tolerance could improve current approaches of targeted melanoma therapy. PMID:26977879

  18. Microtubule-Associated Protein 2, a Marker of Neuronal Differentiation, Induces Mitotic Defects, Inhibits Growth of Melanoma Cells, and Predicts Metastatic Potential of Cutaneous Melanoma

    PubMed Central

    Soltani, Mohammad H.; Pichardo, Rita; Song, Ziqui; Sangha, Namrata; Camacho, Fabian; Satyamoorthy, Kapaettu; Sangueza, Omar P.; Setaluri, Vijayasaradhi

    2005-01-01

    Dynamic instability of microtubules is critical for mitotic spindle assembly and disassembly during cell division, especially in rapidly dividing tumor cells. Microtubule-associated proteins (MAPs) are a family of proteins that influence this property. We showed previously that MAP2, a neuron-specific protein that stabilizes microtubules in the dendrites of postmitotic neurons, is induced in primary cutaneous melanoma but is absent in metastatic melanomas. We proposed that induction of a microtubule-stabilizing protein in primary melanoma could disrupt the dynamic instability of microtubules, inhibit cell division and prevent or delay tumor progression. Here we show, by Kaplan-Meier survival and multivariate Cox regression analysis, that patients diagnosed with MAP2+ primary melanomas have significantly better metastatic disease-free survival than those with MAP2− disease. Investigation of the mechanisms that underlie the effect of MAP2 on melanoma progression showed that MAP2 expression in metastatic melanoma cell lines leads to microtubule stabilization, cell cycle arrest in G2-M phase and growth inhibition. Disruption of microtubule dynamics by MAP2 resulted in multipolar mitotic spindles, defects in cytokinesis and accumulation of cells with large nuclei, similar to those seen in vivo in MAP2+ primary melanomas cells. These data suggest that ectopic activation of a neuronal differentiation gene in melanoma during early tumor progression inhibits cell division and correlates with inhibition or delay of metastasis. PMID:15920168

  19. Phosphorylation of eIF2α triggered by mTORC1 inhibition and PP6C activation is required for autophagy and is aberrant in PP6C-mutated melanoma.

    PubMed

    Wengrod, Jordan; Wang, Ding; Weiss, Sarah; Zhong, Hua; Osman, Iman; Gardner, Lawrence B

    2015-03-10

    Amino acid deprivation promotes the inhibition of the kinase complex mTORC1 (mammalian target of rapamycin complex 1) and activation of the kinase GCN2 (general control nonrepressed 2). Signaling pathways downstream of both kinases have been thought to independently induce autophagy. We showed that these two amino acid-sensing systems are linked. We showed that pharmacological inhibition of mTORC1 led to activation of GCN2 and phosphorylation of the eukaryotic initiation factor 2α (eIF2α) in a mechanism dependent on the catalytic subunit of protein phosphatase 6 (PP6C). Autophagy induced by pharmacological inhibition of mTORC1 required PP6C, GCN2, and eIF2α phosphorylation. Although some of the PP6C mutants found in melanoma did not form a strong complex with PP6 regulatory subunits and were rapidly degraded, these mutants paradoxically stabilized PP6C encoded by the wild-type allele and increased eIF2α phosphorylation. Furthermore, these PP6C mutations were associated with increased autophagy in vitro and in human melanoma samples. Thus, these data showed that GCN2 activation and phosphorylation of eIF2α in response to mTORC1 inhibition are necessary for autophagy. Additionally, we described a role for PP6C in this process and provided a mechanism for PP6C mutations associated with melanoma. PMID:25759478

  20. Metformin inhibits melanoma development through autophagy and apoptosis mechanisms

    PubMed Central

    Tomic, T; Botton, T; Cerezo, M; Robert, G; Luciano, F; Puissant, A; Gounon, P; Allegra, M; Bertolotto, C; Bereder, J-M; Tartare-Deckert, S; Bahadoran, P; Auberger, P; Ballotti, R; Rocchi, S

    2011-01-01

    Metformin is the most widely used antidiabetic drug because of its proven efficacy and limited secondary effects. Interestingly, recent studies have reported that metformin can block the growth of different tumor types. Here, we show that metformin exerts antiproliferative effects on melanoma cells, whereas normal human melanocytes are resistant to these metformin-induced effects. To better understand the basis of this antiproliferative effect of metformin in melanoma, we characterized the sequence of events underlying metformin action. We showed that 24 h metformin treatment induced a cell cycle arrest in G0/G1 phases, while after 72 h, melanoma cells underwent autophagy as demonstrated by electron microscopy, immunochemistry, and by quantification of the autolysosome-associated LC3 and Beclin1 proteins. In addition, 96 h post metformin treatment we observed robust apoptosis of melanoma cells. Interestingly, inhibition of autophagy by knocking down LC3 or ATG5 decreased the extent of apoptosis, and suppressed the antiproliferative effect of metformin on melanoma cells, suggesting that apoptosis is a consequence of autophagy. The relevance of these observations were confirmed in vivo, as we showed that metformin treatment impaired the melanoma tumor growth in mice, and induced autophagy and apoptosis markers. Taken together, our data suggest that metformin has an important impact on melanoma growth, and may therefore be beneficial in patients with melanoma. PMID:21881601

  1. Anti-tumour immunity in malignant melanoma assay by tube leucocyte adherence inhibition.

    PubMed Central

    Marti, J. H.; Thomson, D. M.

    1976-01-01

    Tumour antigen-induced inhibition of leucocyte adherence was modified for use in glass test tubes (Tube LAI assay) for the study of cell-mediated anti-tumour immunity to human malignant melanoma. Peripheral blood leucocytes (PBL) of 20 out of 25 patients (80%) with active malignant melanoma responded to an extract of malignant melanoma with LAI, whereas only 4-5% of 475 control subjects showed a response. The malignant melanoma patients reacted to both allogeneic and autologous extracts of malignant melanoma which indicates a common cross-reacting antigen. Malignant melanoma patients did not respond to unrelated tumour extracts. The LAI was mediated by PBL (monocytes) "armed" with cytophilic anti-tumour antibody specific for the sensitizing tumour antigen. The anti-tumour response of the malignant melanoma patients was dependent on the stage of the cancer, and 11 out of 13 Stage I patients had a positive NAI, whereas patients with disseminated cancer had decreased response. The diminished LAI in patients with large tumour burdens appeared to be the result of release of tumour antigen systemically. Also, surgery and chemotherapy depressed LAI. Although LAI was depressed after surgical excision of the cutaneous melanoma, most patients showed LAI 1-3 months later. Tumour-free melanoma patients monitored for one year by the Tube LAI assay showed a decline in their anti-tumour immunity 5-6 months after surgery. The NAI was low or negative after the 8th post-surgical month in tumour-free patients. Patients with residual malignant melanoma showed persistent or recurrent LAI after the 8th post-surgical month. LAI reactivity monitored after "curative" surgery for malignant melanoma may assist in determining whether the patient is tumour-free or has a recurrence. PMID:962991

  2. Acid Ceramidase in Melanoma: EXPRESSION, LOCALIZATION, AND EFFECTS OF PHARMACOLOGICAL INHIBITION.

    PubMed

    Realini, Natalia; Palese, Francesca; Pizzirani, Daniela; Pontis, Silvia; Basit, Abdul; Bach, Anders; Ganesan, Anand; Piomelli, Daniele

    2016-01-29

    Acid ceramidase (AC) is a lysosomal cysteine amidase that controls sphingolipid signaling by lowering the levels of ceramides and concomitantly increasing those of sphingosine and its bioactive metabolite, sphingosine 1-phosphate. In the present study, we evaluated the role of AC-regulated sphingolipid signaling in melanoma. We found that AC expression is markedly elevated in normal human melanocytes and proliferative melanoma cell lines, compared with other skin cells (keratinocytes and fibroblasts) and non-melanoma cancer cells. High AC expression was also observed in biopsies from human subjects with Stage II melanoma. Immunofluorescence studies revealed that the subcellular localization of AC differs between melanocytes (where it is found in both cytosol and nucleus) and melanoma cells (where it is primarily localized to cytosol). In addition to having high AC levels, melanoma cells generate lower amounts of ceramides than normal melanocytes do. This down-regulation in ceramide production appears to result from suppression of the de novo biosynthesis pathway. To test whether AC might contribute to melanoma cell proliferation, we blocked AC activity using a new potent (IC50 = 12 nM) and stable inhibitor. AC inhibition increased cellular ceramide levels, decreased sphingosine 1-phosphate levels, and acted synergistically with several, albeit not all, antitumoral agents. The results suggest that AC-controlled sphingolipid metabolism may play an important role in the control of melanoma proliferation. PMID:26553872

  3. Hair dyes resorcinol and lawsone reduce production of melanin in melanoma cells by tyrosinase activity inhibition and decreasing tyrosinase and microphthalmia-associated transcription factor (MITF) expression.

    PubMed

    Lee, Shu-Mei; Chen, Yi-Shyan; Lin, Chih-Chien; Chen, Kuan-Hung

    2015-01-01

    Hair coloring products are one of the most important cosmetics for modern people; there are three major types of hair dyes, including the temporary, semi-permanent and permanent hair dyes. The selected hair dyes (such as ammonium persulfate, sodium persulfate, resorcinol and lawsone) are the important components for hair coloring products. Therefore, we analyzed the effects of these compounds on melanogenesis in B16-F10 melanoma cells. The results proved that hair dyes resorcinol and lawsone can reduce the production of melanin. The results also confirmed that resorcinol and lawsone inhibit mushroom and cellular tyrosinase activities in vitro. Resorcinol and lawsone can also downregulate the protein levels of tyrosinase and microphthalmia-associated transcription factor (MITF) in B16-F10 cells. Thus, we suggest that frequent use of hair dyes may have the risk of reducing natural melanin production in hair follicles. Moreover, resorcinol and lawsone may also be used as hypopigmenting agents to food, agricultural and cosmetic industry in the future. PMID:25584612

  4. Activation of MITF by Argan Oil Leads to the Inhibition of the Tyrosinase and Dopachrome Tautomerase Expressions in B16 Murine Melanoma Cells.

    PubMed

    Villareal, Myra O; Kume, Sayuri; Bourhim, Thouria; Bakhtaoui, Fatima Zahra; Kashiwagi, Kenichi; Han, Junkyu; Gadhi, Chemseddoha; Isoda, Hiroko

    2013-01-01

    Argan (Argania spinosa L.) oil has been used for centuries in Morocco as cosmetic oil to maintain a fair complexion and to cure skin pimples and chicken pox pustules scars. Although it is popular, the scientific basis for its effect on the skin has not yet been established. Here, the melanogenesis regulatory effect of argan oil was evaluated using B16 murine melanoma cells. Results of melanin assay using B16 cells treated with different concentrations of argan oil showed a dose-dependent decrease in melanin content. Western blot results showed that the expression levels of tyrosinase (TYR), tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (DCT) proteins were decreased. In addition, there was an increase in the activation of MITF and ERK1/2. Real-time PCR results revealed a downregulation of Tyr, Trp1, Dct, and Mitf mRNA expressions. Argan oil treatment causes MITF phosphorylation which subsequently inhibited the transcription of melanogenic enzymes, TYR and DCT. The inhibitory effect of argan oil on melanin biosynthesis may be attributed to tocopherols as well as the synergistic effect of its components. The results of this study provide the scientific basis for the traditionally established benefits of argan oil and present its therapeutic potential against hyperpigmentation disorders. PMID:23935660

  5. Hair Dyes Resorcinol and Lawsone Reduce Production of Melanin in Melanoma Cells by Tyrosinase Activity Inhibition and Decreasing Tyrosinase and Microphthalmia-Associated Transcription Factor (MITF) Expression

    PubMed Central

    Lee, Shu-Mei; Chen, Yi-Shyan; Lin, Chih-Chien; Chen, Kuan-Hung

    2015-01-01

    Hair coloring products are one of the most important cosmetics for modern people; there are three major types of hair dyes, including the temporary, semi-permanent and permanent hair dyes. The selected hair dyes (such as ammonium persulfate, sodium persulfate, resorcinol and lawsone) are the important components for hair coloring products. Therefore, we analyzed the effects of these compounds on melanogenesis in B16-F10 melanoma cells. The results proved that hair dyes resorcinol and lawsone can reduce the production of melanin. The results also confirmed that resorcinol and lawsone inhibit mushroom and cellular tyrosinase activities in vitro. Resorcinol and lawsone can also downregulate the protein levels of tyrosinase and microphthalmia-associated transcription factor (MITF) in B16-F10 cells. Thus, we suggest that frequent use of hair dyes may have the risk of reducing natural melanin production in hair follicles. Moreover, resorcinol and lawsone may also be used as hypopigmenting agents to food, agricultural and cosmetic industry in the future. PMID:25584612

  6. Activation of MITF by Argan Oil Leads to the Inhibition of the Tyrosinase and Dopachrome Tautomerase Expressions in B16 Murine Melanoma Cells

    PubMed Central

    Villareal, Myra O.; Kume, Sayuri; Bourhim, Thouria; Bakhtaoui, Fatima Zahra; Han, Junkyu; Gadhi, Chemseddoha; Isoda, Hiroko

    2013-01-01

    Argan (Argania spinosa L.) oil has been used for centuries in Morocco as cosmetic oil to maintain a fair complexion and to cure skin pimples and chicken pox pustules scars. Although it is popular, the scientific basis for its effect on the skin has not yet been established. Here, the melanogenesis regulatory effect of argan oil was evaluated using B16 murine melanoma cells. Results of melanin assay using B16 cells treated with different concentrations of argan oil showed a dose-dependent decrease in melanin content. Western blot results showed that the expression levels of tyrosinase (TYR), tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (DCT) proteins were decreased. In addition, there was an increase in the activation of MITF and ERK1/2. Real-time PCR results revealed a downregulation of Tyr, Trp1, Dct, and Mitf mRNA expressions. Argan oil treatment causes MITF phosphorylation which subsequently inhibited the transcription of melanogenic enzymes, TYR and DCT. The inhibitory effect of argan oil on melanin biosynthesis may be attributed to tocopherols as well as the synergistic effect of its components. The results of this study provide the scientific basis for the traditionally established benefits of argan oil and present its therapeutic potential against hyperpigmentation disorders. PMID:23935660

  7. HIV protease inhibitor nelfinavir inhibits growth of human melanoma cells by induction of cell cycle arrest.

    PubMed

    Jiang, Wei; Mikochik, Peter J; Ra, Jin H; Lei, Hanqin; Flaherty, Keith T; Winkler, Jeffrey D; Spitz, Francis R

    2007-02-01

    HIV protease inhibitors (HIV PI) are a class of antiretroviral drugs that are designed to target the viral protease. Unexpectedly, this class of drugs is also reported to have antitumor activity. In this study, we have evaluated the in vitro activity of nelfinavir, a HIV PI, against human melanoma cells. Nelfinavir inhibits the growth of melanoma cell lines at low micromolar concentrations that are clinically attainable. Nelfinavir promotes apoptosis and arrests cell cycle at G(1) phase. Cell cycle arrest is attributed to inhibition of cyclin-dependent kinase 2 (CDK2) and concomitant dephosphorylation of retinoblastoma tumor suppressor. We further show that nelfinavir inhibits CDK2 through proteasome-dependent degradation of Cdc25A phosphatase. Our results suggest that nelfinavir is a promising candidate chemotherapeutic agent for advanced melanoma, for which novel and effective therapies are urgently needed. PMID:17283158

  8. The small molecule curcumin analog FLLL32 induces apoptosis in melanoma cells via STAT3 inhibition and retains the cellular response to cytokines with anti-tumor activity

    PubMed Central

    2010-01-01

    Background We characterized the biologic effects of a novel small molecule STAT3 pathway inhibitor that is derived from the natural product curcumin. We hypothesized this lead compound would specifically inhibit the STAT3 signaling pathway to induce apoptosis in melanoma cells. Results FLLL32 specifically reduced STAT3 phosphorylation at Tyr705 (pSTAT3) and induced apoptosis at micromolar amounts in human melanoma cell lines and primary melanoma cultures as determined by annexin V/propidium iodide staining and immunoblot analysis. FLLL32 treatment reduced expression of STAT3-target genes, induced caspase-dependent apoptosis, and reduced mitochondrial membrane potential. FLLL32 displayed specificity for STAT3 over other homologous STAT proteins. In contrast to other STAT3 pathway inhibitors (WP1066, JSI-124, Stattic), FLLL32 did not abrogate IFN-γ-induced pSTAT1 or downstream STAT1-mediated gene expression as determined by Real Time PCR. In addition, FLLL32 did not adversely affect the function or viability of immune cells from normal donors. In peripheral blood mononuclear cells (PBMCs), FLLL32 inhibited IL-6-induced pSTAT3 but did not reduce signaling in response to immunostimulatory cytokines (IFN-γ, IL 2). Treatment of PBMCs or natural killer (NK) cells with FLLL32 also did not decrease viability or granzyme b and IFN-γ production when cultured with K562 targets as compared to vehicle (DMSO). Conclusions These data suggest that FLLL32 represents a lead compound that could serve as a platform for further optimization to develop improved STAT3 specific inhibitors for melanoma therapy. PMID:20576164

  9. Essential role for acid sphingomyelinase-inhibited autophagy in melanoma response to cisplatin.

    PubMed

    Cervia, Davide; Assi, Emma; De Palma, Clara; Giovarelli, Matteo; Bizzozero, Laura; Pambianco, Sarah; Di Renzo, Ilaria; Zecchini, Silvia; Moscheni, Claudia; Vantaggiato, Chiara; Procacci, Patrizia; Clementi, Emilio; Perrotta, Cristiana

    2016-05-01

    The sphingolipid metabolising enzyme Acid Sphingomyelinase (A-SMase) has been recently shown to inhibit melanoma progression and correlate inversely to tumour grade. In this study we have investigated the role of A-SMase in the chemo-resistance to anticancer treatmentusing mice with melanoma allografts and melanoma cells differing in terms of expression/activity of A-SMase. Since autophagy is emerging as a key mechanism in tumour growth and chemo-resistance, we have also investigated whether an action of A-SMase in autophagy can explain its role. Melanoma sensitivity to chemotherapeutic agent cisplatin in terms of cell viability/apoptosis, tumour growth, and animal survival depended directly on the A-SMase levels in tumoural cells. A-SMase action was due to inhibition of autophagy through activation of Akt/mammalian target of rapamycin (mTOR) pathway. Treatment of melanoma-bearing mice with the autophagy inhibitor chloroquine restored sensitivity to cisplatin of tumours expressing low levels of A-SMase while no additive effects were observed in tumours characterised by sustained A-SMase levels. The fact that A-SMase in melanomas affects mTOR-regulated autophagy and plays a central role in cisplatin efficacy encourages pre-clinical testing on the modulation of A-SMase levels/activity as possible novel anti-neoplastic strategy. PMID:27107419

  10. Synchronized Targeting of Notch and ERBB Signaling Suppresses Melanoma Tumor Growth through Inhibition of Notch1 and ERBB3.

    PubMed

    Zhang, Keman; Wong, Poki; Salvaggio, Christine; Salhi, Amel; Osman, Iman; Bedogni, Barbara

    2016-02-01

    Despite significant advances in melanoma therapy, melanoma remains the deadliest form of skin cancer, with a 5-year survival rate of only 15%. Thus, novel treatments are required to address this disease. Notch and ERBB are evolutionarily conserved signaling cascades required for the maintenance of melanocyte precursors. We show that active Notch1 (Notch1(NIC)) and active (phosphorylated) ERBB3 and ERBB2 correlate significantly and are similarly expressed in both mutated and wild-type BRAF melanomas, suggesting these receptors are co-reactivated in melanoma to promote survival. Whereas blocking either pathway triggers modest effects, combining a ?-secretase inhibitor to block Notch activation and a tyrosine kinase inhibitor to inhibit ERBB3/2 elicits synergistic effects, reducing cell viability by 90% and hampering melanoma tumor growth. Specific inhibition of Notch1 and ERBB3 mimics these results, suggesting these are the critical factors triggering melanoma tumor expansion. Notch and ERBB inhibition blunts AKT and NF?B signaling. Constitutive expression of NF?B partially rescues cell death. Blockade of both Notch and ERBB signaling inhibits the slow cycling JARID1B-positive cell population, which is critical for long-term maintenance of melanoma growth. We propose that blocking these pathways is an effective approach to treatment of melanoma patients regardless of whether they carry mutated or wild-type BRAF. PMID:26967479

  11. Synchronized targeting of Notch and ERBB signaling suppresses melanoma tumor growth through inhibition of Notch1 and ERBB3*

    PubMed Central

    Zhang, Keman; Wong, Poki; Salvaggio, Christine; Salhi, Amel; Osman, Iman; Bedogni, Barbara

    2015-01-01

    Despite significant advances in melanoma therapy, melanoma remains the deadliest form of skin cancer, with a five-year survival of only 15%. Novel treatments are therefore required to address this disease. Notch and ERBB are evolutionarily conserved signaling cascades required for the maintenance of melanocyte precursors. We show that active Notch1 (Notch1NIC) and active (phosphorylated) ERBB3 and ERBB2 correlate significantly and are similarly expressed in both mutated and wild type BRAF melanomas, suggesting these receptors are co-reactivated in melanoma to promote survival. Indeed, while blocking either pathway triggers modest effects, combining a γ-secretase inhibitor to block Notch activation, and a tyrosine kinase inhibitor to inhibit ERBB3/2 elicits synergistic effects, reducing cell viability by 90% and by hampering melanoma tumor growth. Specific inhibition of Notch1 and ERBB3 mimics these results, suggesting these are the critical factors triggering melanoma tumor expansion. Notch and ERBB inhibition blunts AKT and NFκB signaling; Constitutive expression of NFκB partially rescues cell death. Finally, blockade of both Notch and ERBB signaling inhibits the slow cycling JARID1B positive cell population, which is critical for long-term maintenance of melanoma growth. We propose that blocking these pathways is an effective approach to treat melanoma patients regardless of whether they carry mutated or wild type BRAF. PMID:26967479

  12. Bioactive proanthocyanidins inhibit growth and induce apoptosis in human melanoma cells by decreasing the accumulation of β-catenin

    PubMed Central

    VAID, MUDIT; SINGH, TRIPTI; PRASAD, RAM; KATIYAR, SANTOSH K.

    2016-01-01

    Melanoma is a highly aggressive form of skin cancer with poor survival rate. Aberrant activation of Wnt/β-catenin has been observed in nearly one-third of human melanoma cases thereby indicating that targeting Wnt/β-catenin signaling could be a promising strategy against melanoma development. In the present study, we determined chemotherapeutic effect of grape seed proanthocyanidins (GSPs) on the growth of melanoma cells and validated their protective effects in vivo using a xenograft mouse model, and assessed if β-catenin is the target of GSP chemotherapeutic effect. Our in vitro data show that treatment of A375 and Hs294t human melanoma cells with GSPs inhibit the growth of melanoma cells, which was associated with the reduction in the levels of β-catenin. Administration of dietary GSPs (0.2 and 0.5%, w/w) in supplementation with AIN76A control diet significantly inhibited the growth of melanoma tumor xenografts in nude mice. Furthermore, dietary GSPs inhibited the xenograft growth of Mel928 (β-catenin-activated), while did not inhibit the xenograft growth of Mel1011 (β-catenin-inactivated) cells. These observations were further verified by siRNA knockdown of β-catenin and forced overexpression of β-catenin in melanoma cells using a cell culture model. PMID:26676402

  13. CREB Inhibits AP-2α Expression to Regulate the Malignant Phenotype of Melanoma

    PubMed Central

    Zigler, Maya; Villares, Gabriel J.; Braeuer, Russell R.; Wang, Hua; Huang, Li; Bar-Eli, Menashe

    2010-01-01

    Background The loss of AP-2α and increased activity of cAMP-responsive element binding (CREB) protein are two hallmarks of malignant progression of cutaneous melanoma. However, the molecular mechanism responsible for the loss of AP-2α during melanoma progression remains unknown. Methodology/Principal Findings Herein, we demonstrate that both inhibition of PKA-dependent CREB phosphorylation, as well as silencing of CREB expression by shRNA, restored AP-2α protein expression in two metastatic melanoma cell lines. Moreover, rescue of CREB expression in CREB-silenced cell lines downregulates expression of AP-2α. Loss of AP-2α expression in metastatic melanoma occurs via a dual mechanism involving binding of CREB to the AP-2α promoter and CREB-induced overexpression of another oncogenic transcription factor, E2F-1. Upregulation of AP-2α expression following CREB silencing increases endogenous p21Waf1 and decreases MCAM/MUC18, both known to be downstream target genes of AP-2α involved in melanoma progression. Conclusions/Significance Since AP-2α regulates several genes associated with the metastatic potential of melanoma including c-KIT, VEGF, PAR-1, MCAM/MUC18, and p21Waf1, our data identified CREB as a major regulator of the malignant melanoma phenotype. PMID:20805990

  14. ACTIBIND, a T2 RNase, competes with angiogenin and inhibits human melanoma growth, angiogenesis, and metastasis.

    PubMed

    Schwartz, Betty; Shoseyov, Oded; Melnikova, Vladislava O; McCarty, Marya; Leslie, Michael; Roiz, Levava; Smirnoff, Patricia; Hu, Guo-Fu; Lev, Dina; Bar-Eli, Menashe

    2007-06-01

    Melanoma is a very aggressive and highly angiogenic tumor in which standard treatments have had only limited success. Patients with advanced disease have a 5-year survival rate of 5%. In search for alternatives, we identified a natural product extracted from the fungus Aspergillus niger, termed ACTIBIND, that inhibits tumor growth and metastasis of melanoma in vivo. ACTIBIND, a T2 RNase, exerts antitumorigenic and antiangiogenic activities by competing with the angiogenic factor angiogenin (itself an RNase homologue). Thus, there was decreased expression and activity of the matrix metalloproteinase 2 in melanoma and vascular endothelial cells, decreased vascularization, and increased tumor cell apoptosis in vivo. ACTIBIND significantly inhibited angiogenesis in an in vivo angiogenesis assay with sponges containing angiogenin. In vitro, ACTIBIND was internalized by both melanoma and human umbilical vein endothelial cells, reached the cell nuclei, and inhibited the activity of angiogenin response elements in a dose-dependent manner. Collectively, our data indicate that ACTIBIND should be tested for its potential as a new antiangiogenic modality for the treatment of melanoma. PMID:17545605

  15. Targeting TBK1 Inhibits Migration and Resistance to MEK Inhibitors in Mutant NRAS Melanoma

    PubMed Central

    Vu, Ha Linh; Aplin, Andrew E.

    2015-01-01

    Melanoma is a devastating form of skin cancer with limited therapeutic options. Fifteen to twenty percent of melanoma patients have an activating mutation in the GTPase, NRAS. The major downstream effectors of RAS are RAFs (ARAF, BRAF, CRAF), phosphatidylinositol 3-kinase (PI3K), and the Ral guanine exchange factors (RalGEFs). TANK-binding kinase 1 (TBK1) is an atypical IκB kinase family member that acts downstream of RalGEFs. While many studies have analyzed RAF and PI3K signaling in mutant NRAS melanoma, the role of RalGEF/Ral is understudied and TBK1 has not been examined. To address this, TBK1 was modulated with knockdown approaches and targeted therapies to determine TBK1's role in motility, apoptosis and signaling. In melanoma, NRAS overexpression increased TBK1 phosphorylation. TBK1 depletion inhibited migration and invasion, while its constitutive overexpression led to an increase in invasion. In three dimensional (3D) systems that mimic the dermal microenvironment, TBK1 depletion or inhibition cooperated with MEK inhibitors to promote apoptosis, particularly in the context of MEK-insensitive mutant NRAS. This effect was absent in melanoma cells that are wild-type for NRAS. These results suggest the utility of TBK1 inhibitors as part of a treatment regimen for mutant NRAS melanoma patients, for whom there are no current effective therapies. PMID:24962318

  16. Oxidative stress inhibits distant metastasis by human melanoma cells

    PubMed Central

    Piskounova, Elena; Agathocleous, Michalis; Murphy, Malea M.; Hu, Zeping; Huddlestun, Sara E.; Zhao, Zhiyu; Leitch, A. Marilyn; Johnson, Timothy M.; DeBerardinis, Ralph J.; Morrison, Sean J.

    2015-01-01

    Solid cancer cells commonly enter the blood and disseminate systemically but are highly inefficient at forming distant metastases for poorly understood reasons. We studied human melanomas that differed in their metastasis histories in patients and in their capacity to metastasize in NSG mice. All melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficient metastasizers. Melanoma cells in the blood and visceral organs experienced oxidative stress not observed in established subcutaneous tumours. Successfully metastasizing melanomas underwent reversible metabolic changes during metastasis that increased their capacity to withstand oxidative stress, including increased dependence upon NADPH-generating enzymes in the folate pathway. Anti-oxidants promoted distant metastasis in NSG mice. Folate pathway inhibition using low-dose methotrexate, ALDH1L2 knockdown, or MTHFD1 knockdown inhibited distant metastasis without significantly affecting the growth of subcutaneous tumors in the same mice. Oxidative stress thus limits distant metastasis by melanoma cells in vivo. PMID:26466563

  17. Fucose-containing sulfated polysaccharides from brown seaweeds inhibit proliferation of melanoma cells and induce apoptosis by activation of caspase-3 in vitro.

    PubMed

    Ale, Marcel Tutor; Maruyama, Hiroko; Tamauchi, Hidekazu; Mikkelsen, Jørn D; Meyer, Anne S

    2011-12-01

    Fucose-containing sulfated polysaccharides (FCSPs) extracted from seaweeds, especially brown macro-algae, are known to possess essential bioactive properties, notably growth inhibitory effects on tumor cells. In this work, we conducted a series of in vitro studies to examine the influence of FCSPs products from Sargassumhenslowianum C. Agardh (FSAR) and Fucus vesiculosus (FVES), respectively, on proliferation of melanoma B16 cells and to investigate the underlying apoptosis promoting mechanisms. Cell viability analysis showed that both FCSPs products, i.e., FSAR and FVES, decreased the proliferation of the melanoma cells in a dose-response fashion, with FSAR being more potent at lower dosages, and FVES being relatively more anti-proliferative than FSAR at higher dosages. Flow cytometric analysis by Annexin V staining of the melanoma cells exposed to the FCSPs products confirmed that both FSAR and FVES induced apoptosis. The FCSPs-induced apoptosis was evidenced by loss of plasma membrane asymmetry and translocation of the cell membrane phospholipids and was accompanied by the activation of caspase-3. The FCSPs bioactivity is proposed to be attributable to distinct structural features of the FCSPs, particularly the presence of sulfated galactofucans (notably in S.henslowianum) and sulfated fucans (notably in F. vesiculosus). This study thus indicates that unfractionated FCSPs may exert bioactive effects on skin cancer cells via induction of apoptosis through cascades of reactions that involve activation of caspase-3. PMID:22363242

  18. Halofuginone inhibits the establishment and progression of melanoma bone metastases

    PubMed Central

    Juárez, Patricia; Mohammad, Khalid S.; Yin, Juan Juan; Fournier, Pierrick GJ.; McKenna, Ryan C.; Davis, Holly W.; Peng, Xiang H.; Niewolna, Maria; Javelaud, Delphine; Chirgwin, John M.; Mauviel, Alain; Guise, Theresa A.

    2015-01-01

    Transforming growth factor (TGF-β) derived from bone fuels melanoma bone metastases by inducing tumor secretion of pro-metastatic factors that act on bone cells to change the skeletal microenvironment. Halofuginone is a plant alkaloid derivative that blocks TGF-β signaling with antiangiogenic and antiproliferative properties. Here, we demonstrate for the first time that halofuginone therapy decreases development and progression of bone metastasis caused by melanoma cells through inhibition of TGF-β signaling. Halofuginone treatment of human melanoma cells inhibited cell proliferation, phosphorylation of SMAD proteins in response to TGF-β, and TGF-β-induced SMAD-driven transcription. In addition, halofuginone reduced expression of TGF-β target genes that enhance bone metastases, including PTHrP, CTGF, CXCR4, and IL11. Also, cell apoptosis was increased in response to halofuginone. In nude mice inoculated with 1205Lu melanoma cells, a preventive protocol with halofuginone inhibited bone metastasis. The beneficial effects of halofuginone treatment were comparable to those observed with other anti-TGF-β strategies, including systemic administration of SD208, a small molecule inhibitor of TGF-β receptor I kinase, or forced overexpression of Smad7, a negative regulator of TGF-β signaling. Furthermore, mice with established bone metastases treated with halofuginone had significantly less osteolysis than mice receiving placebo assessed by radiographys. Thus, halofuginone is also effective in reducing the progression of melanoma bone metastases. Moreover, halofuginone treatment reduced melanoma metastasis to the brain, showing the potential of this novel treatment against cancer metastasis. PMID:23002206

  19. IL-1-induced ERK1/2 activation up-regulates p21{sup Waf1/Cip1} protein by inhibition of degradation via ubiquitin-independent pathway in human melanoma cells A375

    SciTech Connect

    Arakawa, Tomohiro; Hayashi, Hidetoshi; Itoh, Saotomo; Takii, Takemasa; Onozaki, Kikuo

    2010-02-12

    IL-1 inhibits the proliferation of human melanoma cells A375 by arresting the cell cycle at G0/G1 phase, which accompanies the increase of p21{sup Waf1/Cip1} (p21) protein. Here, we demonstrate that IL-1 induces the stabilization of p21 protein via ERK1/2 pathway. The degradation of p21 was inhibited by IL-1, however the ubiquitination level of p21 was not affected. In addition, the degradation of non-ubiquitinated form of lysine less mutant p21-K6R was also inhibited by IL-1, suggesting that IL-1 stabilized p21 protein via ubiquitin-independent pathway. Furthermore, the inhibition of p21 protein degradation was prevented by a selective inhibitor of ERK1/2 pathway, PD98059. These results suggest that IL-1-induced ERK1/2 activation leads to the up-regulation of p21 by inhibiting degradation via ubiquitin-independent pathway in human melanoma cells A375.

  20. Solanum nigrum Linn. water extract inhibits metastasis in mouse melanoma cells in vitro and in vivo.

    PubMed

    Wang, Hsueh-Chun; Wu, Dun-Hao; Chang, Yun-Ching; Li, Yi-Ju; Wang, Chau-Jong

    2010-11-24

    Metastatic melanoma is an aggressive skin cancer notoriously resistant to current cancer therapies. Thus, new treatment strategies are urgently needed. Solanum nigrum Linn., commonly used in Oriental medicine, has showed antineoplastic activity in human cancer cell lines. The aim of this study was to evaluate the inhibitive effect of S. nigrum Linn. water extract (SNWE) on melanoma metastasis and dissect the underlying mechanisms of SNWE actions. B16-F1 cells were analyzed for migrating and invasive abilities with SNWE treatment, and several putative targets involved in metastatic melanoma were examined. In parallel, primary mouse xenograft and lung metastasis of melanoma models were established to examine the therapeutic potential of SNWE. The results indicated SNWE significantly inhibited B16-F1 cell migration and invasion. Meanwhile, decreased Akt activity and PKCα, Ras, and NF-κB protein expressions were detected in dose-dependent manners. In line with this notion, >50% reduced tumor weight and lung metastatic nodules were observed in 1% SNWE fed mice. This was associated with reduced serum MMP-9 as well as Akt activity and PKCα, Ras, and NF-κB protein expressions. Thus, this work indicates SNWE has potential application for treating metastatic melanoma. PMID:21028816

  1. Green tea polyphenol epigallocatechin-3-gallate suppresses melanoma growth by inhibiting inflammasome and IL-1{beta} secretion

    SciTech Connect

    Ellis, Lixia Z.; Liu, Weimin; Luo, Yuchun; Okamoto, Miyako; Qu, Dovina; Dunn, Jeffrey H.; Fujita, Mayumi

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer EGCG inhibits melanoma cell growth at physiological doses (0.1-1 {mu}M). Black-Right-Pointing-Pointer EGCG inhibits melanoma cell growth via inflammasomes and IL-1{beta} suppression. Black-Right-Pointing-Pointer Inflammasomes and IL-1{beta} could be potential targets for future melanoma therapeutics. -- Abstract: Epigallocatechin-3-gallate (EGCG), the major polyphenolic component of green tea, has been demonstrated to possess anti-inflammatory, antioxidant, anti-mutagenic and anti-carcinogenic properties. The anti-melanoma effect of EGCG has been previously suggested, but no clear mechanism of action has been established. In this study, we demonstrated that EGCG inhibits melanoma cell growth at physiological doses (0.1-1 {mu}M). In the search for mechanisms of EGCG-mediated melanoma cell suppression, we found that NF-{kappa}B was inhibited, and that reduced NF-{kappa}B activity was associated with decreased IL-1{beta} secretion from melanoma cells. Since inflammasomes are involved in IL-1{beta} secretion, we investigated whether IL-1{beta} suppression was mediated by inflammasomes, and found that EGCG treatment led to downregulation of the inflammasome component, NLRP1, and reduced caspase-1 activation. Furthermore, silencing the expression of NLRP1 abolished EGCG-induced inhibition of tumor cell proliferation both in vitro and in vivo, suggesting a key role of inflammasomes in EGCG efficacy. This paper provides a novel mechanism for EGCG-induced melanoma inhibition: inflammasome downregulation {yields} decreased IL-1{beta} secretion {yields} decreased NF-{kappa}B activities {yields} decreased cell growth. In addition, it suggests inflammasomes and IL-1{beta} could be potential targets for future melanoma therapeutics.

  2. Inhibition of peroxisome proliferator-activated receptor gamma prevents the melanogenesis in murine B16/F10 melanoma cells.

    PubMed

    Chen, Jiun-Han; Chang, Junn-Liang; Chen, Pei-Ru; Chuang, Yun-Ju; Tang, Shih-Tsang; Pan, Shwu-Fen; Lin, Tzer-Bin; Chen, Kang-Hua; Chen, Mei-Jung

    2014-01-01

    The purpose of this study was to investigate if PPARγ plays a role in the melanogenesis. B16/F10 cells were divided into five groups: control, melanin stimulating hormone (α-MSH), α-MSH+retinol, α-MSH+GW9662 (PPARγ antagonist), and GW9662. Cells in the control group were cultured in the Dulbecco's modified Eagle's medium (DMEM) for 48 hrs. To initiate the melanogenesis, cells in all α-MSH groups were cultured in medium containing α-MSH (10 nM) for 48 hrs. Cells were treated simultaneously with retinol (5 μM) in the α-MSH+retinol group. Instead of retinol, GW9662 (10 μM) was cocultured in the α-MSH+GW9662 group. Cells in the final group were cultured in the DMEM with GW9662. All the analyses were carried out 48 hours after treatments. The α-MSH was able to increase cell number, melanin production, and the activity of tyrosinase, the limiting enzyme in melanogenesis. These α-MSH-induced changes were prevented either by retinol or by GW9662. Further analyses of the activities of antioxidant enzymes including glutathione, catalase, and the superoxide dismutase (SOD) showed that α-MSH treatment raised the activity of SOD which was dependent on PPARγ level. According to our results, the α-MSH-induced melanogenesis was PPARγ dependent, which also modulated the expression of SOD. PMID:25250328

  3. RAF inhibition overcomes resistance to TRAIL-induced apoptosis in melanoma cells.

    PubMed

    Berger, Anja; Quast, Sandra-Annika; Plötz, Michael; Kuhn, Nicholas-Frederik; Trefzer, Uwe; Eberle, Jürgen

    2014-02-01

    Mutated BRAF represents a critical oncogene in melanoma, and selective inhibitors have been approved for melanoma therapy. However, the molecular consequences of RAF inhibition in melanoma cells remained largely elusive. Here, we investigated the effects of the pan-RAF inhibitor L-779,450, which inhibited cell proliferation both in BRAF-mutated and wild-type melanoma cell lines. It furthermore enhanced apoptosis in combination with the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and overcame TRAIL resistance in melanoma cells. Enhanced apoptosis coincided with activation of mitochondrial pathways, seen by loss of mitochondrial membrane potential and release of cytochrome c, Smac (second mitochondria-derived activator of caspases), and apoptosis-inducing factor (AIF). Subsequently, caspase-9 and -3 were activated. Apoptosis induction by L-779,450/TRAIL was prevented by Bcl-2 overexpression and was dependent on Bax. Thus, activation of Bax by L-779,450 alone was demonstrated by Bax conformational changes, whereas Bak was not activated. Furthermore, the BH3-only protein Bim was upregulated in response to L-779,450. The significant roles of Smac, Bax, and Bim in this setting were proven by small interfering RNA (siRNA)-mediated knockdown experiments. L-779,450 also resulted in morphological changes indicating autophagy confirmed by the autophagy marker light chain 3-II (LC3-II). The pro-apoptotic effects of L-779,450 may explain the antitumor effects of RAF inhibition and may be considered when evaluating RAF inhibitors for melanoma therapy. PMID:23955071

  4. Combined PKC and MEK inhibition in uveal melanoma with GNAQ and GNA11 mutations.

    PubMed

    Chen, X; Wu, Q; Tan, L; Porter, D; Jager, M J; Emery, C; Bastian, B C

    2014-09-25

    Uveal melanoma (UM) is a genetically and biologically distinct type of melanoma, and once metastatic there is no effective treatment currently available. Eighty percent of UMs harbor mutations in the Gαq family members GNAQ and GNA11. Understanding the effector pathways downstream of these oncoproteins is important to identify opportunities for targeted therapy. We report consistent activation of the protein kinase C (PKC) and MAPK pathways as a consequence of GNAQ or GNA11 mutation. PKC inhibition with AEB071 or AHT956 suppressed PKC and MAPK signalling and induced G1 arrest selectively in melanoma cell lines carrying GNAQ or GNA11 mutations. In contrast, treatment with two different MEK inhibitors, PD0325901 and MEK162, inhibited the proliferation of melanoma cell lines irrespective of their mutation status, indicating that in the context of GNAQ or GNA11 mutation MAPK activation can be attributed to activated PKC. AEB071 significantly slowed the growth of tumors in an allograft model of GNAQ(Q209L)-transduced melanocytes, but did not induce tumor shrinkage. In vivo and in vitro studies showed that PKC inhibitors alone were unable to induce sustained suppression of MAP-kinase signaling. However, combinations of PKC and MEK inhibition, using either PD0325901or MEK162, led to sustained MAP-kinase pathway inhibition and showed a strong synergistic effect in halting proliferation and in inducing apoptosis in vitro. Furthermore, combining PKC and MEK inhibition was efficacious in vivo, causing marked tumor regression in a UM xenograft model. Our data identify PKC as a rational therapeutic target for melanoma patients with GNAQ or GNA11 mutations and demonstrate that combined MEK and PKC inhibition is synergistic, with superior efficacy compared to treatment with either approach alone. PMID:24141786

  5. MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

    SciTech Connect

    Yu, Teng; Ji, Jiang; Guo, Yong-li

    2013-11-08

    Highlights: •Curcumin activates MST1 in melanoma cells. •MST1 mediates curcumin-induced apoptosis of melanoma cells. •ROS production is involved in curcumin-induced MST1 activation. •MST1 mediates curcumin-induced JNK activation in melanoma cells. •MST1 mediates curcumin-induced Foxo3a nuclear translocation and Bim expression. -- Abstract: Different groups including ours have shown that curcumin induces melanoma cell apoptosis, here we focused the role of mammalian Sterile 20-like kinase 1 (MST1) in it. We observed that curcumin activated MST1-dependent apoptosis in cultured melanoma cells. MST1 silencing by RNA interference (RNAi) suppressed curcumin-induced cell apoptosis, while MST1 over-expressing increased curcumin sensitivity. Meanwhile, curcumin induced reactive oxygen species (ROS) production in melanoma cells, and the ROS scavenger, N-acetyl-cysteine (NAC), almost blocked MST1 activation to suggest that ROS might be required for MST1 activation by curcumin. c-Jun N-terminal protein kinase (JNK) activation by curcumin was dependent on MST1, since MST1 inhibition by RNAi or NAC largely inhibited curcumin-induced JNK activation. Further, curcumin induced Foxo3 nuclear translocation and Bim-1 (Foxo3 target gene) expression in melanoma cells, such an effect by curcumin was inhibited by MST1 RNAi. In conclusion, we suggested that MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells.

  6. TORC1 Suppression Predicts Responsiveness to RAF and MEK Inhibition in BRAF-Mutant Melanoma

    PubMed Central

    Corcoran, Ryan B.; Rothenberg, Stephen Michael; Hata, Aaron N.; Faber, Anthony C.; Piris, Adriano; Nazarian, Rosalynn M.; Brown, Ronald D.; Godfrey, Jason T.; Winokur, Daniel; Walsh, John; Mino-Kenudson, Mari; Maheswaran, Shyamala; Settleman, Jeffrey; Wargo, Jennifer A.; Flaherty, Keith T.; Haber, Daniel A.; Engelman, Jeffrey A.

    2013-01-01

    RAF and MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase) inhibitors are effective in treating patients with BRAF-mutant melanoma. However, most responses are partial and short-lived, and many patients fail to respond at all. We found that suppression of TORC1 activity in response to RAF or MEK inhibitors, as measured by decreased phosphorylation of ribosomal protein S6 (P-S6), effectively predicted induction of cell death by the inhibitor in BRAF-mutant melanoma cell lines. In resistant melanomas, TORC1 activity was maintained after treatment with RAF or MEK inhibitors, in some cases despite robust suppression of mitogen-activated protein kinase (MAPK) signaling. In in vivo mouse models, suppression of TORC1 after MAPK inhibition was necessary for induction of apoptosis and tumor response. Finally, in paired biopsies obtained from patients with BRAF-mutant melanoma before treatment and after initiation of RAF inhibitor therapy, P-S6 suppression predicted significantly improved progression-free survival. Such a change in P-S6 could be readily monitored in real time by serial fine-needle aspiration biopsies, making quantitation of P-S6 a valuable biomarker to guide treatment in BRAF-mutant melanoma. PMID:23903755

  7. Melanoma

    MedlinePlus

    Melanoma is the most serious type of skin cancer. Often the first sign of melanoma is a change in the size, shape, color, or feel of a mole. Most melanomas have a black or black-blue area. Melanoma ...

  8. Crosstalk between Protease-activated Receptor 1 and Platelet-activating Factor Receptor Regulates Melanoma Cell Adhesion Molecule (MCAM/MUC18) Expression and Melanoma Metastasis*

    PubMed Central

    Melnikova, Vladislava O.; Balasubramanian, Krishnakumar; Villares, Gabriel J.; Dobroff, Andrey S.; Zigler, Maya; Wang, Hua; Petersson, Frederik; Price, Janet E.; Schroit, Alan; Prieto, Victor G.; Hung, Mien-Chie; Bar-Eli, Menashe

    2009-01-01

    The cellular and molecular pathways that regulate platelet activation, blood coagulation, and inflammation are emerging as critical players in cancer progression and metastasis. Here, we demonstrate a novel signaling mechanism whereby protease-activated receptor 1 (PAR1) mediates expression of melanoma cell adhesion molecule MCAM/MUC18 (MUC18), a critical marker of melanoma metastasis, via activation of platelet-activating factor receptor (PAFR) and cAMP-responsive element-binding protein (CREB). We found that PAR1 silencing with small hairpin RNA inhibits MUC18 expression in metastatic melanoma cells by inhibiting CREB phosphorylation, activity, and binding to the MUC18 promoter. We further demonstrate that the PAF/PAFR pathway mediates MUC18 expression downstream of PAR1. Indeed, PAR1 silencing down-regulates PAFR expression and PAF production, PAFR silencing blocks MUC18 expression, and re-expression of PAFR in PAR1-silenced cells rescues MUC18 expression. We further demonstrate that the PAR1-PAFR-MUC18 pathway mediates melanoma cell adhesion to microvascular endothelial cells, transendothelial migration, and metastatic retention in the lungs. Rescuing PAFR expression in PAR1-silenced cells fully restores metastatic phenotype of melanoma, indicating that PAFR plays critical role in the molecular mechanism of PAR1 action. Our results link the two pro-inflammatory G-protein-coupled receptors, PAR1 and PAFR, with the metastatic dissemination of melanoma and suggest that PAR1, PAFR, and MUC18 are attractive therapeutic targets for preventing melanoma metastasis. PMID:19703903

  9. Crosstalk between protease-activated receptor 1 and platelet-activating factor receptor regulates melanoma cell adhesion molecule (MCAM/MUC18) expression and melanoma metastasis.

    PubMed

    Melnikova, Vladislava O; Balasubramanian, Krishnakumar; Villares, Gabriel J; Dobroff, Andrey S; Zigler, Maya; Wang, Hua; Petersson, Frederik; Price, Janet E; Schroit, Alan; Prieto, Victor G; Hung, Mien-Chie; Bar-Eli, Menashe

    2009-10-16

    The cellular and molecular pathways that regulate platelet activation, blood coagulation, and inflammation are emerging as critical players in cancer progression and metastasis. Here, we demonstrate a novel signaling mechanism whereby protease-activated receptor 1 (PAR1) mediates expression of melanoma cell adhesion molecule MCAM/MUC18 (MUC18), a critical marker of melanoma metastasis, via activation of platelet-activating factor receptor (PAFR) and cAMP-responsive element-binding protein (CREB). We found that PAR1 silencing with small hairpin RNA inhibits MUC18 expression in metastatic melanoma cells by inhibiting CREB phosphorylation, activity, and binding to the MUC18 promoter. We further demonstrate that the PAF/PAFR pathway mediates MUC18 expression downstream of PAR1. Indeed, PAR1 silencing down-regulates PAFR expression and PAF production, PAFR silencing blocks MUC18 expression, and re-expression of PAFR in PAR1-silenced cells rescues MUC18 expression. We further demonstrate that the PAR1-PAFR-MUC18 pathway mediates melanoma cell adhesion to microvascular endothelial cells, transendothelial migration, and metastatic retention in the lungs. Rescuing PAFR expression in PAR1-silenced cells fully restores metastatic phenotype of melanoma, indicating that PAFR plays critical role in the molecular mechanism of PAR1 action. Our results link the two pro-inflammatory G-protein-coupled receptors, PAR1 and PAFR, with the metastatic dissemination of melanoma and suggest that PAR1, PAFR, and MUC18 are attractive therapeutic targets for preventing melanoma metastasis. PMID:19703903

  10. Platelet-Derived Growth Factor-Receptor α Strongly Inhibits Melanoma Growth In Vitro and In Vivo1

    PubMed Central

    Faraone, Debora; Aguzzi, Maria Simona; Toietta, Gabriele; Facchiano, Angelo M; Facchiano, Francesco; Magenta, Alessandra; Martelli, Fabio; Truffa, Silvia; Cesareo, Eleonora; Ribatti, Domenico; Capogrossi, Maurizio C; Facchiano, Antonio

    2009-01-01

    Cutaneous melanoma is the most aggressive skin cancer; it is highly metastatic and responds poorly to current therapies. The expression of platelet-derived growth factor receptors (PDGF-Rs) is reported to be reduced in metastatic melanoma compared with benign nevi or normal skin; we then hypothesized that PDGF-Rα may control growth of melanoma cells. We show here that melanoma cells overexpressing PDGF-Rα respond to serum with a significantly lower proliferation compared with that of controls. Apoptosis, cell cycle arrest, pRb dephosphorylation, and DNA synthesis inhibition were also observed in cells overexpressing PDGF-Rα. Proliferation was rescued by PDGF-Rα inhibitors, allowing to exclude nonspecific toxic effects and indicating that PDGF-Rα mediates autocrine antiproliferation signals in melanoma cells. Accordingly, PDGF-Rα was found to mediate staurosporine cytotoxicity. A protein array-based analysis of the mitogen-activated protein kinase pathway revealed that melanoma cells overexpressing PDGF-Rα show a strong reduction of c-Jun phosphorylated in serine 63 and of protein phosphatase 2A/Bα and a marked increase of p38γ, mitogen-activated protein kinase kinase 3, and signal regulatory protein α1 protein expression. In a mouse model of primary melanoma growth, infection with the Ad-vector overexpressing PDGF-Rα reached a significant 70% inhibition of primary melanoma growth (P < .001) and a similar inhibition of tumor angiogenesis. All together, these data demonstrate that PDGF-Rα strongly impairs melanoma growth likely through autocrine mechanisms and indicate a novel endogenous mechanism involved in melanoma control. PMID:19649203

  11. Platelet-derived growth factor-receptor alpha strongly inhibits melanoma growth in vitro and in vivo.

    PubMed

    Faraone, Debora; Aguzzi, Maria Simona; Toietta, Gabriele; Facchiano, Angelo M; Facchiano, Francesco; Magenta, Alessandra; Martelli, Fabio; Truffa, Silvia; Cesareo, Eleonora; Ribatti, Domenico; Capogrossi, Maurizio C; Facchiano, Antonio

    2009-08-01

    Cutaneous melanoma is the most aggressive skin cancer; it is highly metastatic and responds poorly to current therapies. The expression of platelet-derived growth factor receptors (PDGF-Rs) is reported to be reduced in metastatic melanoma compared with benign nevi or normal skin; we then hypothesized that PDGF-Ralpha may control growth of melanoma cells. We show here that melanoma cells overexpressing PDGF-Ralpha respond to serum with a significantly lower proliferation compared with that of controls. Apoptosis, cell cycle arrest, pRb dephosphorylation, and DNA synthesis inhibition were also observed in cells overexpressing PDGF-Ralpha. Proliferation was rescued by PDGF-Ralpha inhibitors, allowing to exclude nonspecific toxic effects and indicating that PDGF-Ralpha mediates autocrine antiproliferation signals in melanoma cells. Accordingly, PDGF-Ralpha was found to mediate staurosporine cytotoxicity. A protein array-based analysis of the mitogen-activated protein kinase pathway revealed that melanoma cells overexpressing PDGF-Ralpha show a strong reduction of c-Jun phosphorylated in serine 63 and of protein phosphatase 2A/Balpha and a marked increase of p38gamma, mitogen-activated protein kinase kinase 3, and signal regulatory protein alpha1 protein expression. In a mouse model of primary melanoma growth, infection with the Ad-vector overexpressing PDGF-Ralpha reached a significant 70% inhibition of primary melanoma growth (P < .001) and a similar inhibition of tumor angiogenesis. All together, these data demonstrate that PDGF-Ralpha strongly impairs melanoma growth likely through autocrine mechanisms and indicate a novel endogenous mechanism involved in melanoma control. PMID:19649203

  12. Resistance to combination BRAF and MEK inhibition in metastatic melanoma: Where to next?

    PubMed

    Welsh, Sarah J; Rizos, Helen; Scolyer, Richard A; Long, Georgina V

    2016-07-01

    Treatment of BRAF-mutant metastatic melanoma with mitogen-activated protein kinase (MAPK) pathway targeted therapies (BRAF/MEK inhibitors) and immune checkpoint inhibitors has revolutionised management and improved outcomes for patients with advanced stage disease. However, acquired resistance to MAPK inhibitor therapy develops in the majority of patients at approximately 12 months and multiple mechanisms lead to resistance. Understanding the mechanisms of resistance is therefore critical for the development of more effective therapeutic strategies in BRAF-mutant melanoma. Recently, several distinct mechanisms of resistance to BRAF-inhibition have been proposed based on data obtained in experimental melanoma cell models and small series of human tumour samples. These include reactivation of the MAPK pathway resulting in continued extracellular signal-regulated kinase activation and activation of parallel signalling pathways including the PI3K-mTOR (phosphoinositide 3-kinase-mammalian target of rapamycin) pathway. Alterations in how the cells of the immune system respond to melanoma cells treated with targeted therapy may also influence response and progression. In this review, we discuss these mechanisms and identify potential therapeutic strategies to overcome resistance which, in turn, will lead to improved outcomes for patients with metastatic melanoma. PMID:27232329

  13. Branched-chain amino acids complex inhibits melanogenesis in B16F0 melanoma cells.

    PubMed

    Cha, Jae-Young; Yang, Hyun-Ju; Moon, Hyung-In; Cho, Young-Su

    2012-04-01

    Present study was investigated the effect of each or complex of three branched-chain amino acids (BCAAs; isoleucine, leucine, and valine) on melanin production in B16F0 melanoma cells treated with various concentrations (1-16 mM) for 72 h. Among the 20 amino acids, lysine and glycine showed the highest activities of DPPH radical scavenging and mushroom tyrosinase inhibition, respectively. Each and combination of BCAAs reduced melanogenesis in a concentration-dependent manner without any morphological changes and cell viability in melanoma cells. Present study was also investigated the inhibitory effects of each or complex of BCAAs at each 10 mM concentration on the 100 μM IBMX-mediated stimulation of melanogenesis in melanoma cells for 72 h and found that IBMX treatment was stimulated to enhance melanin synthesis and that the complex of BCAAs was the most effectively inhibited in the melanin amounts of cellular and extracellular and the whitening the cell pellet. When the inhibitory effect of BCAAs on tyrosinase was examined by intracellular tyrosinase assay, both isoleucine and valine exhibit slightly inhibition, but leucine and combination of BCAAs did not inhibit the cell-derived tyrosinase activity. Present study demonstrated that complex of BCAAs inhibited melanin production without changes intercellular tyrosinase activity. Thus, the complex of BCAAs may be used in development of safe potentially depigmenting agents. PMID:21854182

  14. MEK inhibition affects STAT3 signaling and invasion in human melanoma cell lines

    PubMed Central

    Vultur, Adina; Villanueva, Jessie; Krepler, Clemens; Rajan, Geena; Chen, Quan; Xiao, Min; Li, Ling; Gimotty, Phyllis A.; Wilson, Melissa; Hayden, James; Keeney, Frederick; Nathanson, Katherine L.; Herlyn, Meenhard

    2013-01-01

    Elevated activity of the MAPK signaling cascade is found in the majority of human melanomas and is known to regulate proliferation, survival, and invasion. Current targeted therapies focus on decreasing the activity of this pathway; however, we do not fully understand how these therapies impact tumor biology, especially given that melanoma is a heterogeneous disease. Using a three-dimensional (3D), collagen-embedded spheroid melanoma model, we observed that MEK and BRAF inhibitors can increase the invasive potential of approximately 20% of human melanoma cell lines. The invasive cell lines displayed increased receptor tyrosine kinase (RTK) activity and activation of the Src/FAK/STAT3 signaling axis, also associated with increased cell-to-cell adhesion and cadherin engagement following MEK inhibition. Targeting various RTKs, Src, FAK, and STAT3 with small molecule inhibitors in combination with a MEK inhibitor prevented the invasive phenotype, but only STAT3 inhibition caused cell death in the 3D context. We further show that STAT3 signaling is induced in BRAF-inhibitor resistant cells. Our findings suggest that MEK and BRAF inhibitors can induce STAT3 signaling, causing potential adverse effects such as increased invasion. We also provide the rationale for the combined targeting of the MAPK pathway along with inhibitors of RTKs, SRC, or STAT3 to counteract STAT3-mediated resistance phenotypes. PMID:23624919

  15. SKI knockdown inhibits human melanoma tumor growth in vivo.

    PubMed

    Chen, Dahu; Lin, Qiushi; Box, Neil; Roop, Dennis; Ishii, Shunsuke; Matsuzaki, Koichi; Fan, Tao; Hornyak, Thomas J; Reed, Jon A; Stavnezer, Ed; Timchenko, Nikolai A; Medrano, Estela E

    2009-12-01

    The SKI protein represses the TGF-beta tumor suppressor pathway by associating with the Smad transcription factors. SKI is upregulated in human malignant melanoma tumors in a disease-progression manner and its overexpression promotes proliferation and migration of melanoma cells in vitro. The mechanisms by which SKI antagonizes TGF-beta signaling in vivo have not been fully elucidated. Here we show that human melanoma cells in which endogenous SKI expression was knocked down by RNAi produced minimal orthotopic tumor xenograft nodules that displayed low mitotic rate and prominent apoptosis. These minute tumors exhibited critical signatures of active TGF-beta signaling including high levels of nuclear Smad3 and p21(Waf-1), which are not found in the parental melanomas. To understand how SKI promotes tumor growth we used gain- and loss-of-function approaches and found that simultaneously to blocking the TGF-beta-growth inhibitory pathway, SKI promotes the switch of Smad3 from tumor suppression to oncogenesis by favoring phosphorylations of the Smad3 linker region in melanoma cells but not in normal human melanocytes. In this context, SKI is required for preventing TGF-beta-mediated downregulation of the oncogenic protein c-MYC, and for inducing the plasminogen activator inhibitor-1, a mediator of tumor growth and angiogenesis. Together, the results indicate that SKI exploits multiple regulatory levels of the TGF-beta pathway and its deficiency restores TGF-beta tumor suppressor and apoptotic activities in spite of the likely presence of oncogenic mutations in melanoma tumors. PMID:19845874

  16. Nifuroxazide exerts potent anti-tumor and anti-metastasis activity in melanoma

    PubMed Central

    Zhu, Yongxia; Ye, Tinghong; Yu, Xi; Lei, Qian; Yang, Fangfang; Xia, Yong; Song, Xuejiao; Liu, Li; Deng, Hongxia; Gao, Tiantao; Peng, Cuiting; Zuo, Weiqiong; Xiong, Ying; Zhang, Lidan; Wang, Ningyu; Zhao, Lifeng; Xie, Yongmei; Yu, Luoting; Wei, Yuquan

    2016-01-01

    Melanoma is a highly malignant neoplasm of melanocytes with considerable metastatic potential and drug resistance, explaining the need for new candidates that inhibit tumor growth and metastasis. The signal transducer and activator of the transcription 3 (Stat3) signaling pathway plays an important role in melanoma and has been validated as promising anticancer target for melanoma therapy. In this study, nifuroxazide, an antidiarrheal agent identified as an inhibitor of Stat3, was evaluated for its anti-melanoma activity in vitro and in vivo. It had potent anti-proliferative activity against various melanoma cell lines and could induce G2/M phase arrest and cell apoptosis. Moreover, nifuroxazide markedly impaired melanoma cell migration and invasion by down-regulating phosphorylated-Src, phosphorylated-FAK, and expression of matrix metalloproteinase (MMP) -2, MMP-9 and vimentin. It also significantly inhibited tumor growth without obvious side effects in the A375-bearing mice model by inducing apoptosis and reducing cell proliferation and metastasis. Notably, nifuroxazide significantly inhibited pulmonary metastases, which might be associated with the decrease of myeloid-derived suppressor cells (MDSCs). These findings suggested that nifuroxazide might be a potential agent for inhibiting the growth and metastasis of melanoma. PMID:26830149

  17. Nifuroxazide exerts potent anti-tumor and anti-metastasis activity in melanoma.

    PubMed

    Zhu, Yongxia; Ye, Tinghong; Yu, Xi; Lei, Qian; Yang, Fangfang; Xia, Yong; Song, Xuejiao; Liu, Li; Deng, Hongxia; Gao, Tiantao; Peng, Cuiting; Zuo, Weiqiong; Xiong, Ying; Zhang, Lidan; Wang, Ningyu; Zhao, Lifeng; Xie, Yongmei; Yu, Luoting; Wei, Yuquan

    2016-01-01

    Melanoma is a highly malignant neoplasm of melanocytes with considerable metastatic potential and drug resistance, explaining the need for new candidates that inhibit tumor growth and metastasis. The signal transducer and activator of the transcription 3 (Stat3) signaling pathway plays an important role in melanoma and has been validated as promising anticancer target for melanoma therapy. In this study, nifuroxazide, an antidiarrheal agent identified as an inhibitor of Stat3, was evaluated for its anti-melanoma activity in vitro and in vivo. It had potent anti-proliferative activity against various melanoma cell lines and could induce G2/M phase arrest and cell apoptosis. Moreover, nifuroxazide markedly impaired melanoma cell migration and invasion by down-regulating phosphorylated-Src, phosphorylated-FAK, and expression of matrix metalloproteinase (MMP) -2, MMP-9 and vimentin. It also significantly inhibited tumor growth without obvious side effects in the A375-bearing mice model by inducing apoptosis and reducing cell proliferation and metastasis. Notably, nifuroxazide significantly inhibited pulmonary metastases, which might be associated with the decrease of myeloid-derived suppressor cells (MDSCs). These findings suggested that nifuroxazide might be a potential agent for inhibiting the growth and metastasis of melanoma. PMID:26830149

  18. Disruption of the protein interaction between FAK and IGF-1R inhibits melanoma tumor growth.

    PubMed

    Ucar, Deniz A; Kurenova, Elena; Garrett, Timothy J; Cance, William G; Nyberg, Carl; Cox, Audrey; Massoll, Nicole; Ostrov, David A; Lawrence, Nicholas; Sebti, Said M; Zajac-Kaye, Maria; Hochwald, Steven N

    2012-09-01

    FAK (focal adhesion kinase) and IGF-1R (insulin-like growth factor receptor-1) directly interact with each other and thereby activate crucial signaling pathways that benefit cancer cells. Inhibition of FAK and IGF-1R function has been shown to significantly decrease cancer cell proliferation and increase sensitivity to chemotherapy and radiation treatment. As a novel approach in human melanoma, we evaluated the effect of a small-molecule compound that disrupts the protein interaction of FAK and IGF-1R. Previously, using virtual screening and functional testing, we identified a lead compound (INT2-31) that targets the known FAK-IGF-1R protein interaction site. We studied the ability of this compound to disrupt FAK-IGF-1R protein interactions, inhibit downstream signaling, decrease human melanoma cell proliferation, alter cell cycle progression, induce apoptosis and decrease tumor growth in vivo. INT2-31 blocked the interaction of FAK and IGF-1R in vitro and in vivo in melanoma cells and tumor xenografts through precluding the activation of IRS-1, leading to reduced phosphorylation of AKT upon IGF-1 stimulation. As a result, INT2-31 significantly inhibited cell proliferation and viability (range 0.05-10 μM). More importantly, 15 mg/kg of INT2-31 given for 21 d via intraperitoneal injection disrupted the interaction of FAK and IGF-1R and effectively decreased phosphorylation of tumor AKT, resulting in significant melanoma tumor regression in vivo. Our data suggest that the FAK-IGF-1R protein interaction is an important target, and disruption of this interaction with a novel small molecule (INT2-31) has potential anti-neoplastic therapeutic effects in human melanoma. PMID:22894899

  19. Characterization of xanthatin: anticancer properties and mechanisms of inhibited murine melanoma in vitro and in vivo.

    PubMed

    Li, Wei D; Wu, Yu; Zhang, Lei; Yan, Ling G; Yin, Fang Z; Ruan, Jun S; Chen, Zhi P; Yang, Guang M; Yan, Cui P; Zhao, Ding; Lu, Yin; Cai, Bao C

    2013-07-15

    Anti-cancer investigations on Xanthatin mainly focus on in vitro experiments. We herein reported the anti-tumor effects of Xanthatin both in vitro and in vivo. MTS assay results showed that Xanthatin had a remarkable anti-proliferative effect on B16-F10 cells. Moreover, the expression of β-catenin was up-regulated both in vitro and in vivo. Animal studies further revealed that Xanthatin killed the tumor cells around the blood vessels which contributes to reduce microvascular density extremely. All these results indicate that Xanthatin inhibited murine melanoma B16-F10 cell proliferation possibly associated with activation of Wnt/β-catenin pathway and its activity against melanoma tumor might also be relevant to inhibition of angiogenesis. PMID:23664560

  20. Timosaponin AIII inhibits melanoma cell migration by suppressing COX-2 and in vivo tumor metastasis.

    PubMed

    Kim, Ki Mo; Im, A-Rang; Kim, Seung Hyung; Hyun, Jin Won; Chae, Sungwook

    2016-02-01

    Melanoma is the leading cause of death from skin disease, due in large part to its propensity to metastasize. We examined the effects of timosaponin AIII, a compound isolated from Anemarrhena asphodeloides Bunge, on melanoma cancer cell migration and the molecular mechanisms underlying these effects using B16-F10 and WM-115 melanoma cells lines. Overexpression of COX-2, its metabolite prostaglandin E2 (PGE2 ), and PGE2 receptors (EP2 and EP4) promoted cell migration in vitro. Exposure to timosaponin AIII resulted in concentration-dependent inhibition of cell migration, which was associated with reduced levels of COX-2, PGE2 , and PGE2 receptors. Transient transfection of COX-2 siRNA also inhibited cell migration. Exposure to 12-O-tetradecanoylphorbal-13-acetate enhanced cell migration, whereas timosaponin AIII inhibited 12-O-tetradecanoylphorbal-13-acetate-induced cell migration and reduced basal levels of EP2 and EP4. Moreover, timosaponin AIII inhibited activation of nuclear factor-kappa B (NF-κB), an upstream regulator of COX-2 in B16-F10 cells. Consistent with our in vitro findings, in vivo studies showed that timosaponin AIII treatment significantly reduced the total number of metastatic nodules in the mouse lung and improved histological alterations in B16-F10-injected C57BL/6 mice. In addition, C57BL/6 mice treated with timosaponin AIII showed reduced expression of COX-2 and NF-κB in the lung. Together, these results indicate that timosaponin AIII has the capacity to inhibit melanoma cell migration, an essential step in the process of metastasis, by inhibiting expression of COX-2, NF-κB, PGE2, and PGE2 receptors. PMID:26595378

  1. In vitro and in vivo anti-tumor activity of CoQ0 against melanoma cells: inhibition of metastasis and induction of cell-cycle arrest and apoptosis through modulation of Wnt/β-catenin signaling pathways

    PubMed Central

    Hseu, You-Cheng; Thiyagarajan, Varadharajan; Tsou, Hsiao-Tung; Lin, Kai-Yuan; Chen, Hui-Jye; Lin, Chung-Ming; Liao, Jiuun-Wang; Yang, Hsin-Ling

    2016-01-01

    Coenzyme Q0 (CoQ0, 2,3-dimethoxy-5-methyl-1,4-benzoquinone), a novel quinone derivative, has been shown to modulate cellular redox balance. However, effect of this compound on melanoma remains unclear. This study examined the in vitro or in vivo anti-tumor, apoptosis, and anti-metastasis activities of CoQ0 (0-20 μM) through inhibition of Wnt/β-catenin signaling pathway. CoQ0 exhibits a significant cytotoxic effect on melanoma cell lines (B16F10, B16F1, and A2058), while causing little toxicity toward normal (HaCaT) cells. The suppression of β-catenin was seen with CoQ0 administration accompanied by a decrease in the expression of Wnt/β-catenin transcriptional target c-myc, cyclin D1, and survivin through GSK3β-independent pathway. We found that CoQ0 treatment caused G1 cell-cycle arrest by reducing the levels of cyclin E and CDK4. Furthermore, CoQ0 treatment induced apoptosis through caspase-9/-3 activation, PARP degradation, Bcl-2/Bax dysregulation, and p53 expression. Notably, non- or sub-cytotoxic concentrations of CoQ0 markedly inhibited migration and invasion, accompanied by the down-regulation of MMP-2 and -9, and up-regulation of TIMP-1 and -2 expressions in highly metastatic B16F10 cells. Furthermore, the in vivo study results revealed that CoQ0 treatment inhibited the tumor growth in B16F10 xenografted nude mice. Histological analysis and western blotting confirmed that CoQ0 significantly decreased the xenografted tumor progression as demonstrated by induction of apoptosis, suppression of β-catenin, and inhibition of cell cycle-, apoptotic-, and metastatic-regulatory proteins. The data suggest that CoQ0 unveils a novel mechanism by down-regulating Wnt/β-catenin pathways and could be used as a potential lead compound for melanoma chemotherapy. PMID:26968952

  2. Inhibition of the STAT3 signaling pathway contributes to apigenin-mediated anti-metastatic effect in melanoma

    PubMed Central

    Cao, Hui-Hui; Chu, Jian-Hong; Kwan, Hiu-Yee; Su, Tao; Yu, Hua; Cheng, Chi-Yan; Fu, Xiu-Qiong; Guo, Hui; Li, Ting; Tse, Anfernee Kai-Wing; Chou, Gui-Xin; Mo, Huan-Biao; Yu, Zhi-Ling

    2016-01-01

    Signal transducer and activator of transcription 3 (STAT3) signaling is constantly activated in human melanoma, and promotes melanoma metastasis. The dietary flavonoid apigenin is a bioactive compound that possesses low toxicity and exerts anti-metastatic activity in melanoma. However, the anti-metastasis mechanism of apigenin has not been fully elucidated. In the present study, we showed that apigenin suppressed murine melanoma B16F10 cell lung metastasis in mice, and inhibited cell migration and invasion in human and murine melanoma cells. Further study indicated that apigenin effectively suppressed STAT3 phosphorylation, decreased STAT3 nuclear localization and inhibited STAT3 transcriptional activity. Apigenin also down-regulated STAT3 target genes MMP-2, MMP-9, VEGF and Twist1, which are involved in cell migration and invasion. More importantly, overexpression of STAT3 or Twist1 partially reversed apigenin-impaired cell migration and invasion. Our data not only reveal a novel anti-metastasis mechanism of apigenin but also support the notion that STAT3 is an attractive and promising target for melanoma treatment. PMID:26911838

  3. 67-kDa laminin receptor-dependent protein phosphatase 2A (PP2A) activation elicits melanoma-specific antitumor activity overcoming drug resistance.

    PubMed

    Tsukamoto, Shuntaro; Huang, Yuhui; Umeda, Daisuke; Yamada, Shuhei; Yamashita, Shuya; Kumazoe, Motofumi; Kim, Yoonhee; Murata, Motoki; Yamada, Koji; Tachibana, Hirofumi

    2014-11-21

    The Ras/Raf/MEK/ERK pathway has been identified as a major, druggable regulator of melanoma. Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, resulting in constitutive melanoma hyperproliferation. A selective BRAF inhibitor showed remarkable clinical activity in patients with mutated BRAF. Unfortunately, most patients acquire resistance to the BRAF inhibitor, highlighting the urgent need for new melanoma treatment strategies. Green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) inhibits cell proliferation independently of BRAF inhibitor sensitivity, suggesting that increased understanding of the anti-melanoma activity of EGCG may provide a novel therapeutic target. Here, by performing functional genetic screening, we identified protein phosphatase 2A (PP2A) as a critical factor in the suppression of melanoma cell proliferation. We demonstrated that tumor-overexpressed 67-kDa laminin receptor (67LR) activates PP2A through adenylate cyclase/cAMP pathway eliciting inhibitions of oncoproteins and activation of tumor suppressor Merlin. Activating 67LR/PP2A pathway leading to melanoma-specific mTOR inhibition shows strong synergy with the BRAF inhibitor PLX4720 in the drug-resistant melanoma. Moreover, SET, a potent inhibitor of PP2A, is overexpressed on malignant melanoma. Silencing of SET enhances 67LR/PP2A signaling. Collectively, activation of 67LR/PP2A signaling may thus be a novel rational strategy for melanoma-specific treatment. PMID:25294877

  4. Cuprous oxide nanoparticles inhibit the growth and metastasis of melanoma by targeting mitochondria

    PubMed Central

    Wang, Y; Yang, F; Zhang, H-X; Zi, X-Y; Pan, X-H; Chen, F; Luo, W-D; Li, J-X; Zhu, H-Y; Hu, Y-P

    2013-01-01

    Metal and its oxide nanoparticles show ideal pharmacological activity, especially in anti-tumor therapy. Our previous study demonstrated that cuprous oxide nanoparticles (CONPs) selectively induce apoptosis of tumor cells in vitro. To explore the anti-tumor properties of CONPs in vivo, we used the particles to treat mouse subcutaneous melanoma and metastatic lung tumors, based on B16-F10 mouse melanoma cells, by intratumoral and systemic injections, respectively. The results showed that CONPs significantly reduced the growth of melanoma, inhibited the metastasis of B16-F10 cells and increased the survival rate of tumor-bearing mice. Importantly, the results also indicated that CONPs were rapidly cleared from the organs and that these particles exhibited little systemic toxicity. Furthermore, we observed that CONPs targeted the mitochondria, which resulted in the release of cytochrome C from the mitochondria and the activation of caspase-3 and caspase-9 after the CONPs entered the cells. In conclusion, CONPs can induce the apoptosis of cancer cells through a mitochondrion-mediated apoptosis pathway, which raises the possibility that CONPs could be used to cure melanoma and other cancers. PMID:23990023

  5. Activation of CCR9/CCL25 in Cutaneous Melanoma Mediates Preferential Metastasis to the Small Intestine

    PubMed Central

    Amersi, Farin F.; Terando, Alicia M.; Goto, Yasufumi; Scolyer, Richard A.; Thompson, John F.; Tran, Andy N.; Faries, Mark B.; Morton, Donald L.; Hoon, Dave S.B.

    2009-01-01

    Purpose Specific chemokines and their respective receptors have been implicated in distant tumor cell metastasis. Cutaneous melanoma has a distinct pattern of metastasis, preferentially targeting the submucosa of the small intestine. However, the underlying pathogenic mechanism remains unknown. Migration of CCR9(+) lymphocytes to the small intestine is known to occur in response to the chemoattractant effects of CCL25 (thymus-expressed chemokine). The integrin heterodimers αβ are also known to be important mediators of cellular adhesion. We hypothesize that the mechanism of small intestinal metastasis by melanoma is via the CCR9-CCL25 axis and specific integrins. Experimental Design Quantitative reverse transcription-PCR, flow cytometry, and immunohistochemistry were used to assess melanoma tumors for CCR9 and CCL25. Integrin expression was assessed using flow cytometry. CCR9 expression by quantitative reverse transcription-PCR was assessed in primary (n = 23) and metastatic (n = 198) melanomas, and melanoma lines derived from small intestinal metastases (n = 23). Results We showed CCR9 expression in 88 of 102 paraffin-embedded metastatic melanomas from the small intestine, 8 of 8 melanoma lines derived from metastases in the small intestine, and 0 of 96 metastatic melanomas from other sites. In vitro migration and invasion studies done on CCR9(+) melanoma lines showed migration in response to CCL25 that was inhibited by anti-CCR9 antibody or by short interfering RNA CCR9. Flow cytometric analysis confirmed CCR9 expression by melanomas to the small intestine and showed concomitant α4β1 integrin expression. Conclusions Our findings show that functionally active CCR9 on melanoma cells facilitates metastasis to the small intestine. The CCR9-CCL25 axis may explain the high incidence of melanoma metastasis to this specific location. PMID:18245522

  6. Molecular pathway activation features linked with transition from normal skin to primary and metastatic melanomas in human.

    PubMed

    Shepelin, Denis; Korzinkin, Mikhail; Vanyushina, Anna; Aliper, Alexander; Borisov, Nicolas; Vasilov, Raif; Zhukov, Nikolay; Sokov, Dmitry; Prassolov, Vladimir; Gaifullin, Nurshat; Zhavoronkov, Alex; Bhullar, Bhupinder; Buzdin, Anton

    2016-01-01

    Melanoma is the most aggressive and dangerous type of skin cancer, but its molecular mechanisms remain largely unclear. For transcriptomic data of 478 primary and metastatic melanoma, nevi and normal skin samples, we performed high-throughput analysis of intracellular molecular networks including 592 signaling and metabolic pathways. We showed that at the molecular pathway level, the formation of nevi largely resembles transition from normal skin to primary melanoma. Using a combination of bioinformatic machine learning algorithms, we identified 44 characteristic signaling and metabolic pathways connected with the formation of nevi, development of primary melanoma, and its metastases. We created a model describing formation and progression of melanoma at the level of molecular pathway activation. We discovered six novel associations between activation of metabolic molecular pathways and progression of melanoma: for allopregnanolone biosynthesis, L-carnitine biosynthesis, zymosterol biosynthesis (inhibited in melanoma), fructose 2, 6-bisphosphate synthesis and dephosphorylation, resolvin D biosynthesis (activated in melanoma), D-myo-inositol hexakisphosphate biosynthesis (activated in primary, inhibited in metastatic melanoma). Finally, we discovered fourteen tightly coordinated functional clusters of molecular pathways. This study helps to decode molecular mechanisms underlying the development of melanoma. PMID:26624979

  7. Molecular pathway activation features linked with transition from normal skin to primary and metastatic melanomas in human

    PubMed Central

    Shepelin, Denis; Korzinkin, Mikhail; Vanyushina, Anna; Aliper, Alexander; Borisov, Nicolas; Vasilov, Raif; Zhukov, Nikolay; Sokov, Dmitry; Prassolov, Vladimir; Gaifullin, Nurshat; Zhavoronkov, Alex; Bhullar, Bhupinder; Buzdin, Anton

    2016-01-01

    Melanoma is the most aggressive and dangerous type of skin cancer, but its molecular mechanisms remain largely unclear. For transcriptomic data of 478 primary and metastatic melanoma, nevi and normal skin samples, we performed high-throughput analysis of intracellular molecular networks including 592 signaling and metabolic pathways. We showed that at the molecular pathway level, the formation of nevi largely resembles transition from normal skin to primary melanoma. Using a combination of bioinformatic machine learning algorithms, we identified 44 characteristic signaling and metabolic pathways connected with the formation of nevi, development of primary melanoma, and its metastases. We created a model describing formation and progression of melanoma at the level of molecular pathway activation. We discovered six novel associations between activation of metabolic molecular pathways and progression of melanoma: for allopregnanolone biosynthesis, L-carnitine biosynthesis, zymosterol biosynthesis (inhibited in melanoma), fructose 2, 6-bisphosphate synthesis and dephosphorylation, resolvin D biosynthesis (activated in melanoma), D-myo-inositol hexakisphosphate biosynthesis (activated in primary, inhibited in metastatic melanoma). Finally, we discovered fourteen tightly coordinated functional clusters of molecular pathways. This study helps to decode molecular mechanisms underlying the development of melanoma. PMID:26624979

  8. Involvement of ER stress and activation of apoptotic pathways in fisetin induced cytotoxicity in human melanoma.

    PubMed

    Syed, Deeba N; Lall, Rahul K; Chamcheu, Jean Christopher; Haidar, Omar; Mukhtar, Hasan

    2014-12-01

    The prognosis of malignant melanoma remains poor in spite of recent advances in therapeutic strategies for the deadly disease. Fisetin, a dietary flavonoid is currently being investigated for its growth inhibitory properties in various cancer models. We previously showed that fisetin inhibited melanoma growth in vitro and in vivo. Here, we evaluated the molecular basis of fisetin induced cytotoxicity in metastatic human melanoma cells. Fisetin treatment induced endoplasmic reticulum (ER) stress in highly aggressive A375 and 451Lu human melanoma cells, as revealed by up-regulation of ER stress markers including IRE1α, XBP1s, ATF4 and GRP78. Time course analysis indicated that the ER stress was associated with activation of the extrinsic and intrinsic apoptotic pathways. Fisetin treated 2-D melanoma cultures displayed autophagic response concomitant with induction of apoptosis. Prolonged treatment (16days) with fisetin in a 3-D reconstituted melanoma model resulted in inhibition of melanoma progression with significant apoptosis, as evidenced by increased staining of cleaved Caspase-3 in the treated constructs. However, no difference in the expression of autophagic marker LC-3 was noted between treated and control groups. Fisetin treatment to 2-D melanoma cultures resulted in phosphorylation and activation of the multifunctional AMP-activated protein kinase (AMPK) involved in the regulation of diverse cellular processes, including autophagy and apoptosis. Silencing of AMPK failed to prevent cell death indicating that fisetin induced cytotoxicity is mediated through both AMPK-dependent and -independent mechanisms. Taken together, our studies confirm apoptosis as the primary mechanism through which fisetin inhibits melanoma cell growth and that activation of both extrinsic and intrinsic pathways contributes to fisetin induced cytotoxicity. PMID:25016296

  9. Involvement of ER stress and activation of apoptotic pathways in fisetin induced cytotoxicity in human melanoma

    PubMed Central

    Chamcheu, Jean Christopher; Haidar, Omar; Mukhtar, Hasan

    2014-01-01

    The prognosis of malignant melanoma remains poor in spite of recent advances in therapeutic strategies for the deadly disease. Fisetin, a dietary flavonoid is currently being investigated for its growth inhibitory properties in various cancer models. We previously showed that fisetin inhibited melanoma growth in vitro and in vivo. Here, we evaluated the molecular basis of fisetin induced cytoxicity in metastatic human melanoma cells. Fisetin treatment induced endoplasmic reticulum (ER) stress in highly aggressive A375 and 451Lu human melanoma cells, as revealed by up- regulation of ER stress markers including IRE1α, XBP1s, ATF4 and GRP78. Time course analysis indicated that the ER stress was associated with activation of the extrinsic and intrinsic apoptotic pathways. Fisetin treated 2-D melanoma cultures displayed autophagic response concomitant with induction of apoptosis. Prolonged treatment (16 days) with fisetin in a 3-D reconstituted melanoma model resulted in inhibition of melanoma progression with significant apoptosis, as evidenced by increased staining of cleaved Caspase-3 in the treated constructs. However, no difference in the expression of autophagic marker LC-3 was noted between treated and control groups. Fisetin treatment to 2-D melanoma cultures resulted in phosphorylation and activation of the multifunctional AMPK-activated protein kinase (AMPK) involved in the regulation of diverse cellular processes, including autophagy and apoptosis. Silencing of AMPK failed to prevent cell death indicating that fisetin induced cytotoxicity is mediated through both AMPK-dependent and -independent mechanisms. Taken together, our studies confirm apoptosis as the primary mechanism through which fisetin inhibits melanoma cell growth and that activation of both extrinsic and intrinsic pathways contributes to fisetin induced cytotoxicity. PMID:25016296

  10. Enhanced Histone Deacetylase Activity in Malignant Melanoma Provokes RAD51 and FANCD2-Triggered Drug Resistance.

    PubMed

    Krumm, Andrea; Barckhausen, Christina; Kücük, Pelin; Tomaszowski, Karl-Heinz; Loquai, Carmen; Fahrer, Jörg; Krämer, Oliver Holger; Kaina, Bernd; Roos, Wynand Paul

    2016-05-15

    DNA-damaging anticancer drugs remain a part of metastatic melanoma therapy. Epigenetic reprogramming caused by increased histone deacetylase (HDAC) activity arising during tumor formation may contribute to resistance of melanomas to the alkylating drugs temozolomide, dacarbazine, and fotemustine. Here, we report on the impact of class I HDACs on the response of malignant melanoma cells treated with alkylating agents. The data show that malignant melanomas in situ contain a high level of HDAC1/2 and malignant melanoma cells overexpress HDAC1/2/3 compared with noncancer cells. Furthermore, pharmacologic inhibition of class I HDACs sensitizes malignant melanoma cells to apoptosis following exposure to alkylating agents, while not affecting primary melanocytes. Inhibition of HDAC1/2/3 caused sensitization of melanoma cells to temozolomide in vitro and in melanoma xenografts in vivo HDAC1/2/3 inhibition resulted in suppression of DNA double-strand break (DSB) repair by homologous recombination because of downregulation of RAD51 and FANCD2. This sensitized cells to the cytotoxic DNA lesion O(6)-methylguanine and caused a synthetic lethal interaction with the PARP-1 inhibitor olaparib. Furthermore, knockdown experiments identified HDAC2 as being responsible for the regulation of RAD51. The influence of class I HDACs on DSB repair by homologous recombination and the possible clinical implication on malignant melanoma therapy with temozolomide and other alkylating drugs suggests a combination approach where class I HDAC inhibitors such as valproic acid or MS-275 (entinostat) appear to counteract HDAC- and RAD51/FANCD2-mediated melanoma cell resistance. Cancer Res; 76(10); 3067-77. ©2016 AACR. PMID:26980768

  11. Modulation of WNT/β-catenin pathway in melanoma by biologically active components derived from plants.

    PubMed

    Gajos-Michniewicz, Anna; Czyz, Malgorzata

    2016-03-01

    Metastatic melanoma is an aggressive cancer, often resistant to treatment. Therefore, it is essential to determine the molecular mechanisms leading to melanoma or underlying resistance to therapy, and the response to targeted inhibition of the RAS/BRAF/MEK/ERK pathway was a good lesson in this respect. Aberrant WNT/β-catenin pathway is observed in melanoma, and the modulators of this signaling cascade have been under investigation in the context of therapy as well as chemoprevention. Several natural compounds were recognized as being capable of targeting elements of the WNT/β-catenin pathway in various cancers, however, only a few of them can modulate this pathway in melanoma. This review examines recent research on the role of the WNT/β-catenin pathway in tumor development and maintenance, as well as summarizes the current knowledge concerning the modulation of this pathway in melanoma by active compounds of natural origin. PMID:26851176

  12. Norcantharidin induces melanoma cell apoptosis through activation of TR3 dependent pathway

    PubMed Central

    Liu, Shujing; Yu, Hong; Kumar, Suresh M.; Martin, James S.; Bing, Zhanyong; Sheng, Weiqi; Bosenberg, Marcus

    2011-01-01

    Norcantharidin (NCTD) has been reported to induce tumor cell apoptosis. However, the underlying mechanism behinds its antitumor effect remains elusive. We have previously shown that TR3 expression is significantly decreased in metastatic melanomas and involved in melanoma cell apoptosis. In this study, we showed that NCTD inhibited melanoma cell proliferation and induced apoptosis in a dose related manner. NCTD induced translocation of TR3 from nucleus to mitochondria where it co-localized with Bcl-2 in melanoma cells. NCTD also increased cytochome c release from mitochondria to the cytoplasm. These changes were accompanied by increased expression of Bax and cleaved caspase-3 along with decreased expression of Bcl2 and NF-κB2. The effects of NCTD were inhibited by knockdown of TR3 expression using TR3 specific shRNA in melanoma cells. Furthermore, NCTD significantly decreased tumor volume and improved survival of Tyr::CreER; BRAFCa/+; Ptenlox/lox transgenic mice. Our data indicates that NCTD inhibits melanoma growth by inducing tumor cell apoptosis via activation of a TR3 dependent pathway. These results suggest that NCTD is a potential therapeutic agent for melanoma. PMID:22123174

  13. Bisphosphonamidate Clodronate Prodrug Exhibits Selective Cytotoxic Activity Against Melanoma Cell Lines

    PubMed Central

    Webster, Marie R.; Kamat, Chandrashekhar; Connis, Nick; Zhao, Ming; Weeraratna, Ashani T.; Rudek, Michelle A.; Hann, Christine L.; Freel Meyers, Caren L.

    2014-01-01

    Bisphosphonates are used clinically to treat disorders of calcium metabolism and malignant bone disease and are known to inhibit cancer cell growth, adhesion, and invasion. However, clinical use of these agents for the treatment of extraskeletal disease is limited due to low cell permeability. We recently described a bisphosphonamidate prodrug strategy for efficient intracellular release of bisphosphonates, including clodronate (CLO), in NSCLC cells. To evaluate anticancer activity of this prodrug class across many cancer cell types, the bisphosphonamidate clodronate prodrug (CLO prodrug) was screened against the NCI-60 cell line panel, and was found to exhibit selectivity toward melanoma cell lines. Here, we confirm efficient cellular uptake and intracellular activation of this prodrug class in melanoma cells. We further demonstrate inhibition of melanoma cell proliferation, induction of apoptosis, and an anti-tumor effect of CLO prodrug in a xenograft model. These data suggest a novel therapeutic application for the CLO prodrug and potential to selectively target melanoma cells. PMID:24310621

  14. Rac1 activity regulates proliferation of aggressive metastatic melanoma

    SciTech Connect

    Bauer, Natalie N. Chen Yihwen; Samant, Rajeev S.; Shevde, Lalita A.; Fodstad, Oystein

    2007-11-01

    Molecular mechanisms underlying the different capacity of two in vivo selected human melanoma cell variants to form experimental metastases were studied. The doubling times of the FEMX-I and FEMX-V cell sublines in vitro were 15 and 25 h, respectively. The invasive capacity of FEMX-I cells was 8-fold higher than FEMX-V cells, and the time to form approximately 10 mm s.c. tumors in nude mice was 21 versus 35 days. FEMX-I displayed a spindle-like formation in vitro, whereas FEMX-V cells had a rounded shape. Hence, we examined known determinants of cell shape and proliferation, the small GTPases. The four studied showed equal expression in both cell types, but Rac1 activity was significantly decreased in FEMX-V cells. Rac1 stimulates NF{kappa}B, and we found that endogenous NF{kappa}B activity of FEMX-V cells was 2% of that of FEMX-I cells. Inhibition of Rac1 resulted in blocked NF{kappa}B activity. Specific inhibition of either Rac1 or NF{kappa}B significantly reduced proliferation and invasion of FEMX-I cells, the more pronounced effects observed with Rac1 inhibition. These data indicate that Rac1 activity in FEMX cells regulates cell proliferation and invasion, in part via its effect on NF{kappa}B, signifying Rac1 as a key molecule in melanoma progression and metastasis.

  15. Broad-spectrum therapeutic suppression of metastatic melanoma through nuclear hormone receptor activation.

    PubMed

    Pencheva, Nora; Buss, Colin G; Posada, Jessica; Merghoub, Taha; Tavazoie, Sohail F

    2014-02-27

    Melanoma metastasis is a devastating outcome lacking an effective preventative therapeutic. We provide pharmacologic, molecular, and genetic evidence establishing the liver-X nuclear hormone receptor (LXR) as a therapeutic target in melanoma. Oral administration of multiple LXR agonists suppressed melanoma invasion, angiogenesis, tumor progression, and metastasis. Molecular and genetic experiments revealed these effects to be mediated by LXRβ, which elicits these outcomes through transcriptional induction of tumoral and stromal apolipoprotein-E (ApoE). LXRβ agonism robustly suppressed tumor growth and metastasis across a diverse mutational spectrum of melanoma lines. LXRβ targeting significantly prolonged animal survival, suppressed the progression of established metastases, and inhibited brain metastatic colonization. Importantly, LXRβ activation displayed melanoma-suppressive cooperativity with the frontline regimens dacarbazine, B-Raf inhibition, and the anti-CTLA-4 antibody and robustly inhibited melanomas that had acquired resistance to B-Raf inhibition or dacarbazine. We present a promising therapeutic approach that uniquely acts by transcriptionally activating a metastasis suppressor gene. PMID:24581497

  16. Inhibition of cell proliferation, migration and invasion of B16-F10 melanoma cells by α-mangostin.

    PubMed

    Beninati, Simone; Oliverio, Serafina; Cordella, Martina; Rossi, Stefania; Senatore, Cinzia; Liguori, Immacolata; Lentini, Alessandro; Piredda, Lucia; Tabolacci, Claudio

    2014-08-01

    In this study, we have evaluated the potential antineoplastic effects of α-mangostin (α-M), the most representative xanthone in Garcinia mangostana pericarp, on melanoma cell lines. This xanthone markedly inhibits the proliferation of high-metastatic B16-F10 melanoma cells. Furthermore, by deeply analyzing which steps in the metastatic process are influenced by xanthone it was observed that α-M strongly interferes with homotypic aggregation, adhesion, plasticity and invasion ability of B16-F10 cells, probably by the observed reduction of metalloproteinase-9 activity. The antiproliferative and antimetastatic properties of α-M have been established in human SK-MEL-28 and A375 melanoma cells. In order to identify pathways potentially involved in the antineoplastic properties of α-M, a comparative mass spectrometry proteomic approach was employed. These findings may improve our understanding of the molecular mechanisms underlying the anti-cancer effects of α-M on melanoma. PMID:25019992

  17. The Pan-Aurora Kinase Inhibitor, PHA-739358, Induces Apoptosis and Inhibits Migration in Melanoma Cell Lines

    PubMed Central

    Xie, Lifang; Meyskens, Frank L

    2014-01-01

    Treatment of metastatic melanoma has long been a challenge due to its resistance to traditional chemotherapeutics leading to the search for alternative strategies. Aurora kinases are key mitotic regulators that are frequently overexpressed in various cancers including melanoma, making them ideal targets for anticancer therapeutics. Several Aurora kinase inhibitors have been developed and tested pre-clinically and clinically. PHA-739358 is currently the most advanced clinical compound; however its antitumor effect has not been tested in melanoma. In this study, the anti-proliferative and anti-invasive effects of PHA-739358 were investigated in melanoma cell lines. The results demonstrated that PHA-739358 produces a time and dose dependent inhibition of cell proliferation, induction of apoptosis, and inhibition of cell migration. Downregulation of MMP-2 via inhibition of NFκB signaling pathway may contribute to PHA-739358-induced migration inhibition. Furthermore, PHA-739358 enhanced temozolomide-induced caspase activation. This study provides a promising new strategy for the treatment of advanced melanoma. PMID:23344158

  18. Methylthioadenosine (MTA) inhibits melanoma cell proliferation and in vivo tumor growth

    PubMed Central

    2010-01-01

    Background Melanoma is the most deadly form of skin cancer without effective treatment. Methylthioadenosine (MTA) is a naturally occurring nucleoside with differential effects on normal and transformed cells. MTA has been widely demonstrated to promote anti-proliferative and pro-apoptotic responses in different cell types. In this study we have assessed the therapeutic potential of MTA in melanoma treatment. Methods To investigate the therapeutic potential of MTA we performed in vitro proliferation and viability assays using six different mouse and human melanoma cell lines wild type for RAS and BRAF or harboring different mutations in RAS pathway. We also have tested its therapeutic capabilities in vivo in a xenograft mouse melanoma model and using variety of molecular techniques and tissue culture we investigated its anti-proliferative and pro-apoptotic properties. Results In vitro experiments showed that MTA treatment inhibited melanoma cell proliferation and viability in a dose dependent manner, where BRAF mutant melanoma cell lines appear to be more sensitive. Importantly, MTA was effective inhibiting in vivo tumor growth. The molecular analysis of tumor samples and in vitro experiments indicated that MTA induces cytostatic rather than pro-apoptotic effects inhibiting the phosphorylation of Akt and S6 ribosomal protein and inducing the down-regulation of cyclin D1. Conclusions MTA inhibits melanoma cell proliferation and in vivo tumor growth particularly in BRAF mutant melanoma cells. These data reveal a naturally occurring drug potentially useful for melanoma treatment. PMID:20529342

  19. Conditional ablation of Ikkb inhibits melanoma tumor development in mice

    PubMed Central

    Yang, Jinming; Splittgerber, Ryan; Yull, Fiona E.; Kantrow, Sara; Ayers, Gregory D.; Karin, Michael; Richmond, Ann

    2010-01-01

    Several lines of evidence suggest that tumor cells show elevated activity of the NF-κB transcription factor, a phenomenon often resulting from constitutive activity of IκB kinase β (IKKβ). However, others have found that loss of NF-κB activity or IKKβ is tumor promoting. The role of NF-κB in tumor progression is therefore controversial and varies with tumor type. We sought to more extensively investigate the role IKKβ in melanoma tumor development by specifically disrupting Ikkb in melanocytes in an established mouse model of spontaneous melanoma, whereby HRasV12 is expressed in a melanocyte-specific, doxycycline-inducible manner in mice null for the gene encoding the tumor suppressor inhibitor cyclin-dependent kinase 4/alternative reading frame (Ink4a/Arf). Our results show that Ink4a/Arf–/– mice with melanocyte-specific deletion of Ikkb were protected from HRasV12-initiated melanoma only when p53 was expressed. This protection was accompanied by cell cycle arrest, with reduced cyclin-dependent kinase 2 (Cdk2), Cdk4, Aurora kinase A, and Aurora kinase B expression. Increased p53-mediated apoptosis was also observed, with decreased expression of the antiapoptotic proteins Bcl2 and survivin. Enhanced stabilization of p53 involved increased phosphorylation at Ser15 and reduced phosphorylation of double minute 2 (Mdm2) at Ser166. Together, our findings provide genetic and mechanistic evidence that mutant HRas initiation of tumorigenesis requires Ikkβ-mediated NF-κB activity. PMID:20530876

  20. Notch1 Pathway Activity Determines the Regulatory Role of Cancer-Associated Fibroblasts in Melanoma Growth and Invasion

    PubMed Central

    Shao, Hongwei; Kong, Ranran; Ferrari, Massimiliano L.; Radtke, Freddy; Capobianco, Anthony J.; Liu, Zhao-Jun

    2015-01-01

    Cancer-associated fibroblasts (CAF) play a crucial role in regulating cancer progression, yet the molecular determinant that governs the tumor regulatory role of CAF remains unknown. Using a mouse melanoma model in which exogenous melanoma cells were grafted on the skin of two lines of mice where the genetic activation or inactivation of Notch1 signaling specifically occurs in natural host stromal fibroblasts, we demonstrated that Notch1 pathway activity could determine the tumor-promoting or tumor-suppressing phenotype in CAF. CAF carrying elevated Notch1 activity significantly inhibited melanoma growth and invasion, while those with a null Notch1 promoted melanoma invasion. These findings identify the Notch1 pathway as a molecular determinant that controls the regulatory role of CAF in melanoma skin growth and invasion, unveiling Notch1 signaling as a potential therapeutic target for melanoma and potentially other solid tumors. PMID:26562315

  1. Epigenetic Silencing of SPINT2 promotes Cancer Cell Motility via HGF-MET Pathway Activation in Melanoma

    PubMed Central

    Hwang, Soonyean; Kim, Hye-Eun; Min, Michelle; Raghunathan, Rekha; Panova, Izabela P.; Munshi, Ruchi; Ryu, Byungwoo

    2015-01-01

    Aberrant HGF-MET signaling activation via interactions with surrounding stromal cells in tumor microenvironment plays significant roles in malignant tumor progression. However, extracellular proteolytic regulation of HGF activation which is influenced by the tumor microenvironment and its consequential effects on melanoma malignancy remain uncharacterized. In this study we identified SPINT2: a proteolytic inhibitor of hepatocyte growth factor activator (HGFA), which plays a significant role in the suppression of the HGF-MET pathway and malignant melanoma progression. SPINT2 expression is significantly lower in metastatic melanoma tissues compared to those in early stage primary melanomas which also corresponded with DNA methylation levels isolated from tissue samples. Treatment with the DNA hypomethylating agent decitabine in cultured melanoma cells induced transcriptional reactivation of SPINT2, suggesting that this gene is epigenetically silenced in malignant melanomas. Furthermore, we show that ectopically expressed SPINT2 in melanoma cells inhibits HGF induced MET-AKT signaling pathway and decreases malignant phenotype potential such as cell motility, and invasive growth of melanoma cells. These results suggest that SPINT2 is associated with tumor suppressive functions in melanoma by inhibiting an extracellular signal regulator of HGF which is typically activated by tumor-stromal interactions. These findings indicate that epigenetic impairment of the tightly regulated cytokine-receptor communications in tumor microenvironment may contribute to malignant tumor progression. PMID:25910030

  2. Melanoma

    MedlinePlus

    ... to other parts of the body very quickly. Melanoma treatment can cause side effects, including pain, nausea, and ... Livingstone; 2013:chap 69. National Cancer Institute: PDQ Melanoma Treatment. Bethesda, MD: National Cancer Institute. Last modified March ...

  3. Inhibition of BRAF and BRAF+MEK drives a metastatic switch in melanoma

    PubMed Central

    Smalley, Keiran SM; Fedorenko, Inna V

    2015-01-01

    Recent analyses by our group and others showed that the majority of melanoma patients who fail BRAF inhibitor therapy do so at new disease sites. Using phosphoproteomics we showed that BRAF inhibition mediates a switch to an aggressive/metastatic melanoma phenotype that is driven by ligand-independent erythropoietin-producing hepatocellular receptor A2 (EphA2) signaling. PMID:27308505

  4. Inhibition of BRAF and BRAF+MEK drives a metastatic switch in melanoma.

    PubMed

    Smalley, Keiran Sm; Fedorenko, Inna V

    2015-01-01

    Recent analyses by our group and others showed that the majority of melanoma patients who fail BRAF inhibitor therapy do so at new disease sites. Using phosphoproteomics we showed that BRAF inhibition mediates a switch to an aggressive/metastatic melanoma phenotype that is driven by ligand-independent erythropoietin-producing hepatocellular receptor A2 (EphA2) signaling. PMID:27308505

  5. Genistein enhances the cisplatin-induced inhibition of cell growth and apoptosis in human malignant melanoma cells.

    PubMed

    Tamura, Shingo; Bito, Toshinori; Ichihashi, Masamitsu; Ueda, Masato

    2003-10-01

    Genistein, a naturally occurring isoflavone found chiefly in soybeans, has been reported to be a potent antitumor agent. Genistein is presumed to exert multiple effects related to the inhibition of cancer growth. Metastatic melanoma is a chemotherapy-refractory neoplasm. The present study was designed to explore the possible activity of genistein to inhibit the aberrant proliferation and to induce apoptosis of human malignant melanoma cells in cooperation with cisplatin treatment. Five human melanoma cell lines were utilized for these experiments. Genistein at physiologic concentrations (20 microM) did not induce apoptosis by itself but did enhance cisplatin-induced apoptosis in all five human melanoma cell lines tested. The enhanced susceptibility among the cell lines was diverse. Changes in the expression of two anti-apoptotic proteins, bcl-2 and bcl-xL, and one pro-apoptotic protein, apoptotic protease activating factor-1 (Apaf-1), were examined. Genistein alone or cisplatin alone generally did not alter bcl-2 expression or bcl-xL expression, but slightly increased Apaf-1 in some cell lines. The combined treatment with genistein and cisplatin significantly reduced bcl-2 and bcl-xL protein and increased Apaf-1 protein expression. These data suggest that genistein therapy may enhance the chemosensitivity of melanoma patients. PMID:12950722

  6. [Pulsed electric fields inhibit tumor growth but induce myocardial injury of melanoma-bearing mice].

    PubMed

    Pan, Fengying; Wu, Sha; Wang, Xiaoxu; Zhang, Xiaogang

    2016-07-01

    Objective To investigate the tumor inhibiting effect of pulsed electric fields (PEFs) on melanoma-bearing mice, and understand its influence on myocardial cells and cardial electrical activity. Methods The melanoma models of the BALB/c mice were established by subcutaneously injecting B16 melanoma cells. These mice were randomly divided into four groups. The treated groups received pulsed electric stimulation at pulse width of 1, 3, 5 ms, with field strength of 100 V/cm and frequency of 10 Hz for 10 minutes daily in 15 consecutive days. ECG of mice was recorded. Tumor volume was measured with vernier caliper. Morphological changes of tumors were observed by HE staining. The expression of proliferating cell nuclear antigen (PCNA) mRNA was tested by real-time quantitative PCR, and the expression of PCNA protein was detected by immunofluorescence histochemistry. The ultrastructural changes of the cardiac tissues were observed by transmission electron microscopy (TEM). The serum levels of cardial troponin T (cTnT) and creatine kinase isoenzyme MB (CK-MB) were detected by ELISA. Results Compared with the control group, tumor volumes in all treated groups were reduced 7 days after PEFs treatment; more melanin granules in tumor cells were found in the treated groups; the expressions of PCNA mRNA and protein were down-regulated in all treated groups, and there were greater changes in the groups receiving the bigger pulse width. Myocardial injury was found in 3 ms group and 5 ms group, and the expressions of cTnT and CK-MB were significantly higher than those in the control group. Conclusion PEFs can inhibit tumor growth in melanoma-bearing mice. Increase of pulse width will aggravate myocardial injury. PMID:27363271

  7. Ribozyme-Mediated Targeting of IκBγ Inhibits Melanoma Invasion and Metastasis

    PubMed Central

    Torabian, Sima Z.; de Semir, David; Nosrati, Mehdi; Bagheri, Sepideh; Dar, Altaf A.; Fong, Sylvia; Liu, Yong; Federman, Scot; Simko, Jeff; Haqq, Chris; Debs, Robert J.; Kashani-Sabet, Mohammed

    2009-01-01

    IκBγ is one member of a family of proteins that can inhibit the nuclear localization of nuclear factor-κB. However, the other specific functions of IκBγ are still poorly understood, and its effects on tumor metastasis have not yet been characterized. We examined the consequences of targeting IκBγ in melanoma cells using a hammerhead ribozyme. We developed stable transformant B16-F10 melanoma cell lines that express a ribozyme that targets mouse IκBγ (IκBγ-144-Rz). Tail-vein injection of B16-F10 cells that stably express IκBγ-144-Rz into mice resulted in a significant reduction of the metastatic potential of these cells. IκBγ-144-Rz-expressing B16 cells were shown to have increased transcriptional activity of nuclear factor-κB. We then showed that IκBγ-144-Rz-expressing cells demonstrated both reduced invasion and increased apoptosis, suggesting the existence of pathways through which IκBγ promotes melanoma metastasis. Using gene expression profiling, we identified a differentially expressed gene set that is regulated by the stable suppression of IκBγ that may participate in mediating its anti-metastatic effects; we also confirmed the altered expression levels of several of these genes by quantitative real time polymerase chain reaction. Plasmid-mediated expression of IκBγ-144-Rz produced a significant inhibition of the metastatic progression of B16-F10 cells to the lung and resulted in significant anti-invasive and pro-apoptotic effects on murine Lewis lung carcinoma cells. Our results suggest a novel role for IκBγ in promoting the metastatic progression of melanoma. PMID:19179607

  8. Fully humanized neutralizing antibodies to interleukin-8 (ABX-IL8) inhibit angiogenesis, tumor growth, and metastasis of human melanoma.

    PubMed

    Huang, Suyun; Mills, Lisa; Mian, Badar; Tellez, Carmen; McCarty, Marya; Yang, X-D; Gudas, Jean M; Bar-Eli, Menashe

    2002-07-01

    Interleukin-8 (IL-8) has recently been shown to contribute to human melanoma progression by functioning as a mitogenic and angiogenic factor. In the present study, we investigated whether targeting IL-8 by a fully human anti-IL-8 antibody (ABX-IL8) could be a potential therapeutic strategy to control angiogenesis, growth, and metastasis of melanoma. The human melanoma cells A375SM (high IL-8 producer) and TXM-13 (intermediate IL-8 producer) were injected subcutaneously into nude mice, which were then treated with ABX-IL8 (1 mg/3 times weekly, i.p., for 3 weeks). Tumor growth of both melanomas in ABX-IL8-treated mice was significantly inhibited when compared with control IgG-treated animals. ABX-IL8 treatment also suppressed experimental metastasis when the melanoma cells were injected intravenously. IL-8 blockade by ABX-IL8 significantly inhibited the promoter activity and the collagenase activity of matrix metalloproteinase-2 in human melanoma cells, resulting in decreased invasion through reconstituted basement membrane in vitro. In vivo, ABX-IL8 treatment resulted in decreased expression of matrix metalloproteinase-2, and decreased vascularization (angiogenesis) of tumors concomitant with increased apoptosis of tumor cells. Moreover, in an in vitro vessel formation assay, ABX-IL8 directly interfered with the tubule formation by human umbilical vein endothelial cells. Taken together, these results point to the potential utility of ABX-IL8 as a modality to treat melanoma and other solid tumors either alone or in combination with conventional chemotherapy or other anti-tumor agents. PMID:12107097

  9. Vertical inhibition of the MAPK pathway enhances therapeutic responses in NRAS-mutant melanoma

    PubMed Central

    Rebecca, Vito W.; Alicea, Gretchen M.; Paraiso, Kim H.T.; Lawrence, Harshani; Gibney, Geoffrey T.; Smalley, Keiran S.M.

    2014-01-01

    Summary The MEK inhibitor MEK162 is the first targeted therapy agent with clinical activity in patients whose melanomas harbor NRAS mutations; however, median PFS is 3.7 months, suggesting the rapid onset of resistance in the majority of patients. Here we show that treatment of NRAS-mutant melanoma cell lines with the MEK inhibitors AZD6244 or trametinib resulted in a rebound activation of phospho-ERK (pERK). Functionally, the recovery of signaling was associated with the maintenance of cyclin-D1 expression and therapeutic escape. The combination of a MEK inhibitor with an ERK inhibitor suppressed the recovery of cyclin-D1 expression and was associated with a significant enhancement of apoptosis and the abrogation of clonal outgrowth. The MEK/ERK combination strategy induced greater levels of apoptosis compared to dual MEK/CDK4 or MEK/PI3K inhibition across a panel of cell lines. These data provide the rationale for further investigation of vertically co-targeting the MAPK pathway as a potential treatment option for NRAS-mutant melanoma patients. PMID:25130256

  10. Aloin enhances cisplatin antineoplastic activity in B16-F10 melanoma cells by transglutaminase-induced differentiation.

    PubMed

    Tabolacci, Claudio; Rossi, Stefania; Lentini, Alessandro; Provenzano, Bruno; Turcano, Lorenzo; Facchiano, Francesco; Beninati, Simone

    2013-01-01

    Aloin, a natural anthracycline from aloe plant, is a hydroxyanthraquinone derivative shown to have antitumor properties. This study demonstrated that aloin exerted inhibition of cell proliferation, adhesion and invasion abilities of B16-F10 melanoma cells under non-cytotoxic concentrations. Furthermore, aloin induced melanoma cell differentiation through the enhancement of melanogenesis and transglutaminase activity. To improve the growth-inhibiting effect of anticancer agents, we found that the combined treatment of cells with aloin and low doses of cisplatin increases the antiproliferative activity of aloin. The results suggest that aloin possesses antineoplastic and antimetastatic properties, exerted likely through the induction of melanoma cell differentiation. PMID:22139409

  11. Inhibition by Tyroserleutide (YSL) on the Invasion and Adhesion of the Mouse Melanoma Cell

    PubMed Central

    Yao, Zhi; Che, Xu-chun; Lu, Rong; Zheng, Min-na; Zhu, Zhi-feng; Li, Jin-ping; Jian, Xu; Shi, Lin-xi; Liu, Jun-yan; Gao, Wen-yuan

    2007-01-01

    Tyroserleutide (YSL) is an active, low-molecular-weight polypeptide, comprised of three amino acids, that has shown antitumor effects on human hepatocarcinoma BEL-7402 in vitro and in vivo. In this study, we evaluated the inhibition of YSL on invasion and adhesion of the mouse B16-F10 melanoma cell line by injecting B16-F10 cells into the tail veins of C57BL/6 mice to establish an experimental lung metastasis model. YSL inhibited B16-F10 cell metastasis to lung, reducing the number and area of metastasis lesions. When we treated B16-F10 cells with YSL (0.01, 0.1, 1, 10, or 100 μg/mL) in vitro, we found that YSL inhibited the proliferation of B16-F10 cells with a 28.11% rate of inhibition. YSL significantly decreased the adhesiveness of B16-F10 cells to Matrigel with a 29.15% inhibition rate; YSL also significantly inhibited the invasion of B16-F10 cells, producing an inhibition of 35.31%. By analyses with Western blot and real-time RT-PCR, we found that YSL markedly inhibited the expression of ICAM-1 in B16-F10 cells. These data suggest that YSL inhibits the growth, invasion, and adhesion of B16-F10 cells. PMID:17515953

  12. The pharmacological NF-κB inhibitor BAY11-7082 induces cell apoptosis and inhibits the migration of human uveal melanoma cells.

    PubMed

    Hu, Shuiqing; Luo, Qingqiong; Cun, Biyun; Hu, Dan; Ge, Shengfang; Fan, Xianqun; Chen, Fuxiang

    2012-01-01

    Uveal melanomas are highly metastatic and have high rate of recurrence due to the lack of effective systemic therapy. The identification of important survival pathways in uveal melanomas provides novel therapeutic targets for effective treatment. In the present study, we found that the NF-κB signaling pathway was constitutively and highly activated in uveal melanoma cells. Treatment with the pharmacological NF-κB specific inhibitor BAY11-7082 markedly decreased the nuclear translocation of NF-κB. In a dose-dependent setting, BAY11-7082 inhibited the proliferation and growth of uveal melanoma cells by inducing apoptosis without effect on cell cycle. The migration capacity of uveal melanoma cells was also significantly suppressed by BAY11-7082 treatment. Mechanistically, BAY11-7082 increased the activity of caspase 3 and reduced the expression of anti-apoptotic protein Bcl-2, but did not influence the expression of pro-apoptotic protein Bax. Furthermore, BAY11-7082 induced uveal melanoma cell apoptosis and inhibited xenograft tumor growth in vivo. Collectively, the present study identified NF-κB as an important survival signal for uveal melanoma cells and suggested that administration of specific NF-κB inhibitor BAY11-7082 could serve as an effective treatment for patients with uveal melanoma. PMID:23443086

  13. The Pharmacological NF-κB Inhibitor BAY11-7082 Induces Cell Apoptosis and Inhibits the Migration of Human Uveal Melanoma Cells

    PubMed Central

    Hu, Shuiqing; Luo, Qingqiong; Cun, Biyun; Hu, Dan; Ge, Shengfang; Fan, Xianqun; Chen, Fuxiang

    2012-01-01

    Uveal melanomas are highly metastatic and have high rate of recurrence due to the lack of effective systemic therapy. The identification of important survival pathways in uveal melanomas provides novel therapeutic targets for effective treatment. In the present study, we found that the NF-κB signaling pathway was constitutively and highly activated in uveal melanoma cells. Treatment with the pharmacological NF-κB specific inhibitor BAY11-7082 markedly decreased the nuclear translocation of NF-κB. In a dose-dependent setting, BAY11-7082 inhibited the proliferation and growth of uveal melanoma cells by inducing apoptosis without effect on cell cycle. The migration capacity of uveal melanoma cells was also significantly suppressed by BAY11-7082 treatment. Mechanistically, BAY11-7082 increased the activity of caspase 3 and reduced the expression of anti-apoptotic protein Bcl-2, but did not influence the expression of pro-apoptotic protein Bax. Furthermore, BAY11-7082 induced uveal melanoma cell apoptosis and inhibited xenograft tumor growth in vivo. Collectively, the present study identified NF-κB as an important survival signal for uveal melanoma cells and suggested that administration of specific NF-κB inhibitor BAY11-7082 could serve as an effective treatment for patients with uveal melanoma. PMID:23443086

  14. The FGF-2-Derived Peptide FREG Inhibits Melanoma Growth In Vitro and In Vivo

    PubMed Central

    Aguzzi, Maria S; Faraone, Debora; D'Arcangelo, Daniela; De Marchis, Francesco; Toietta, Gabriele; Ribatti, Domenico; Parazzoli, Alberto; Colombo, Paolo; Capogrossi, Maurizio C; Facchiano, Antonio

    2011-01-01

    Previous data report that fibroblast growth factor-2 (FGF-2)-derived peptide FREG potently inhibits FGF-2-dependent angiogenesis in vitro and in vivo. Here, we show that FREG inhibits up to 70% in vitro growth and invasion/migration of smooth muscle and melanoma cells. Such inhibition is mediated by platelet-derived growth factor-receptor-α (PDGF-Rα); in fact, proliferation and migration were restored upon PDGF-Rα neutralization. Further experiments demonstrated that FREG interacts with PDGF-Rα both in vitro and in vivo and stimulates its phosphorylation. We have previously shown that overexpressing PDGF-Rα strongly inhibits melanoma growth in vivo; we, therefore, hypothesized that PDGF-Rα agonists may represent a novel tool to inhibit melanoma growth in vivo. To support this hypothesis, FREG was inoculated intravenously (i.v.) in a mouse melanoma model and markedly inhibited pulmonary metastases formation. Immunohistochemical analyses showed less proliferation, less angiogenesis, and more apoptosis in metastasized lungs upon FREG treatment, as compared to untreated controls. Finally, in preliminary acute toxicity studies, FREG showed no toxicity signs in healthy animals, and neither microscopic nor macroscopic toxicity at the liver, kidney, and lungs level. Altogether, these data indicate that FREG systemic treatment strongly inhibits melanoma metastases development and indicate for the first time that agonists of PDGF-Rα may control melanoma both in vitro and in vivo. PMID:20924364

  15. PI3′-Kinase Inhibition Forestalls the Onset of MEK1/2 Inhibitor Resistance in BRAF-Mutated Melanoma

    PubMed Central

    Deuker, Marian M.; Durban, Victoria Marsh; Phillips, Wayne A.; McMahon, Martin

    2014-01-01

    Phosphatidylinositide 3′ (PI3′)-lipid signaling cooperates with oncogenic BRAFV600E to promote melanomagenesis. Sustained PI3′-lipid production commonly occurs via silencing of the PI3′-lipid phosphatase PTEN or, less commonly, through mutational activation of PIK3CA, encoding the 110kDa catalytic subunit of PI3′-kinase-α (PI3Kα). To define the PI3K catalytic isoform dependency of BRAF-mutated melanoma, we utilized pharmacologic, isoform-selective PI3K inhibitors in conjunction with melanoma-derived cell lines and genetically engineered mouse (GEM) models. While BRAFV600E/PIK3CAH1047R melanomas were sensitive to the anti-proliferative effects of selective PI3Kα blockade, inhibition of BRAFV600E/PTENNull melanoma proliferation required combined blockade of PI3Kα, δ and γ, and was insensitive to PI3Kβ blockade. In GEM models, isoform-selective PI3K inhibition elicited cytostatic effects, but significantly potentiated melanoma regression in response to BRAFV600E pathway-targeted inhibition. Interestingly, PI3K inhibition forestalled the onset of MEK inhibitor resistance in two independent GEM models of BRAFV600E-driven melanoma. These results suggest that combination therapy with PI3K inhibitors may be a useful strategy to extend the duration of clinical response of BRAF-mutated melanoma patients to BRAFV600E pathway-targeted therapies. (Words: 165) PMID:25472943

  16. PAX3 and ETS1 synergistically activate MET expression in melanoma cells

    PubMed Central

    Kubic, Jennifer D.; Little, Elizabeth C.; Lui, Jason W.; Iizuka, Takumi; Lang, Deborah

    2014-01-01

    Melanoma is a highly aggressive disease that is difficult to treat due to rapid tumor growth, apoptotic resistance, and high metastatic potential. The MET tyrosine kinase receptor promotes many of these cellular processes, and while MET is often overexpressed in melanoma, the mechanism driving this overexpression is unknown. Since the MET gene is rarely mutated or amplified in melanoma, MET overexpression may be driven by to increased activation through promoter elements. In this report, we find that transcription factors PAX3 and ETS1 directly interact to synergistically activate MET expression. Inhibition of PAX3 and ETS1 expression in melanoma cells leads to a significant reduction of MET receptor levels. The 300 bp 5′ proximal MET promoter contains a PAX3 response element and two ETS1 consensus motifs. While ETS1 can moderately activate both of these sites without cofactors, robust MET promoter activation of the first site is PAX-dependent and requires the presence of PAX3, while the second site is PAX-independent. The induction of MET by ETS1 via this second site is enhanced by HGF-dependent ETS1 activation, thereby MET indirectly promotes its own expression. We further find that expression of a dominant negative ETS1 reduces the ability of melanoma cells to grow both in culture and in vivo. Thus, we discover a pathway where ETS1 advances melanoma through the expression of MET via PAX-dependent and independent mechanisms. PMID:25531327

  17. Honokiol bis-dichloroacetate (Honokiol DCA) demonstrates activity in vemurafenib-resistant melanoma in vivo

    PubMed Central

    Bonner, Michael Y.; Karlsson, Isabella; Rodolfo, Monica; Arnold, Rebecca S.; Vergani, Elisabetta; Arbiser, Jack L.

    2016-01-01

    The majority of human melanomas bears BRAF mutations and thus is treated with inhibitors of BRAF, such as vemurafenib. While patients with BRAF mutations often demonstrate an initial dramatic response to vemurafenib, relapse is extremely common. Thus, novel agents are needed for the treatment of these aggressive melanomas. Honokiol is a small molecule compound derived from Magnolia grandiflora that has activity against solid tumors and hematopoietic neoplasms. In order to increase the lipophilicity of honokiol, we have synthesized honokiol DCA, the dichloroacetate ester of honokiol. In addition, we synthesized a novel fluorinated honokiol analog, bis-trifluoromethyl-bis-(4-hydroxy-3-allylphenyl) methane (hexafluoro). Both compounds exhibited activity against A375 melanoma in vivo, but honokiol DCA was more active. Gene arrays comparing treated with vehicle control tumors demonstrated induction of the respiratory enzyme succinate dehydrogenase B (SDHB) by treatment, suggesting that our honokiol analogs induce respiration in vivo. We then examined its effect against a pair of melanomas, LM36 and LM36R, in which LM36R differs from LM36 in that LM36R has acquired vemurafenib resistance. Honokiol DCA demonstrated in vivo activity against LM36R (vemurafenib resistant) but not against parental LM36. Honokiol DCA and hexafluoro inhibited the phosphorylation of DRP1, thus stimulating a phenotype suggestive of respiration through mitochondrial normalization. Honokiol DCA may act in vemurafenib resistant melanomas to increase both respiration and reactive oxygen generation, leading to activity against aggressive melanoma in vivo. PMID:26871475

  18. Antiproliferative and pro-apoptotic activity of eugenol-related biphenyls on malignant melanoma cells

    PubMed Central

    Pisano, Marina; Pagnan, Gabriella; Loi, Monica; Mura, Maria Elena; Tilocca, Maria Giovanna; Palmieri, Giuseppe; Fabbri, Davide; Dettori, Maria Antonietta; Delogu, Giovanna; Ponzoni, Mirco; Rozzo, Carla

    2007-01-01

    Background Malignant melanoma is one of the most aggressive skin cancer and chemotherapeutic agents currently in use are still unsatisfactory. Prevention and early diagnosis are the only effective tools against this tumour whose incidence and mortality rates are highly increased during the last decades in fair skin populations. Therefore the search for novel therapeutic approaches is warranted. Aim of this work was to identify and test new compounds with antiproliferative and cytotoxic activity on melanoma cells. We tested eugenol together with six natural and synthetic eugenol-related compounds for their capability to inhibit cell growth on primary melanoma cell lines established from patients' tissue samples. Results Eugenol and isoeugenol monomers and their respective O-methylated forms did not show to inhibit melanoma cells proliferation. Conversely, the dimeric forms (biphenyls) showed some antiproliferative activity which was mild for dehydrodieugenol, higher for its O,O'-methylated form (O,O'-dimethyl-dehydrodieugenol), and markedly pronounced for the racemic mixture of the brominated biphenyl (6,6'-dibromo-dehydrodieugenol) (S7), being its enantiomeric form (S) the most effective compared to the other compounds. Such activity resulted to be selective against tumour cells, without affecting cultured normal human skin fibroblasts. Dose and time dependence curves have been obtained for the enantiomeric form S7-(S). Then IC50 and minimal effective doses and times have been established for the melanoma cell lines tested. TUNEL and phosphatidylserine exposure assays demonstrated the occurrence of apoptotic events associated with the antiproliferative activity of S7-(S). Cytotoxic activity and apoptosis induced by treating melanoma cells with eugenol-related biphenyls was partially dependent by caspase activation. Conclusion Our findings demonstrate that the eugenol related biphenyl (S)-6,6'-dibromo-dehydrodieugenol elicits specific antiproliferative activity on

  19. Sarcophine-diol, a skin cancer chemopreventive agent, inhibits proliferation and stimulates apoptosis in mouse melanoma B₁₆F₁₀ cell line.

    PubMed

    Szymanski, Pawel T; Kuppast, Bhimanna; Ahmed, Safwat A; Khalifa, Sherief; Fahmy, Hesham

    2012-01-01

    Sarcodiol (SD) is a semi-synthetic derivative of sarcophine, a marine natural product. In our previous work, we reported the significant chemopreventive effects of SD against non-melanoma skin cancer both in vitro and in vivo mouse models. In this investigation, we extended this work to study the effect of sarcodiol on melanoma development, the more deadly form of skin cancer, using the mouse melanoma B₁₆F₁₀ cell line. In this study we report that SD inhibits the de novo DNA synthesis and enhances fragmentation of DNA. We also evaluated the antitumor effect of SD on melanoma cell viability using several biomarkers for cell proliferation and apoptosis. SD inhibits the expression levels of signal transducers and activators of transcription protein (STAT-3) and cyclin D1, an activator of cyclin-dependent kinase 4 (Cdk4). SD treatment also enhances cellular level of tumor suppressor protein 53 (p53) and stimulates cleavage of the nuclear poly (ADP-ribose) polymerase (cleaved-PARP). SD also enhances cellular levels of cleaved Caspase-3, -8, -9 and stimulates enzymatic activities of Caspase-3, -8 and -9. These results, in addition to inhibition of cell viability, suggest that SD inhibits melanoma cell proliferation by arresting the cell-division cycle in a Go quiescent phase and activates programmed cell death (apoptosis) via extrinsic and intrinsic pathways. Finally, these studies demonstrate that SD shows a very promising chemopreventive effect in melanoma B₁₆F₁₀ tumor cells. PMID:22363217

  20. Ardisia crenata extract stimulates melanogenesis in B16F10 melanoma cells through inhibiting ERK1/2 and Akt activation.

    PubMed

    Yao, Cheng; Jin, Cheng Long; Oh, Jang-Hee; Oh, Inn Gyung; Park, Chi-Hyun; Chung, Jin Ho

    2015-01-01

    Melanin protects the skin against ultraviolet radiation by scattering incoming light and absorbing diverse free radicals. Agents that increase melanin synthesis in melanocytes may reduce the risk of photodamage and skin cancer. The present study investigated the effect of a methanol extract of Ardisia crenata (AC) on melanogenesis in B16F10 cells. Treatment of cultured B16F10 cells with AC extract (10, 20 and 40 µg/ml) stimulated an increase in melanin levels in a concentration-dependent manner, without cytotoxicity. Tyrosinase is key in the regulation of melanin production, thus the effect of AC extract on tyrosinase activity and protein expression was analyzed. AC extract was observed to significantly increase tyrosinase activity and protein expression in B16F10 cells. Furthermore, AC extract was found to markedly increase the protein expression of microphthalmia-associated transcription factor, which is an important transcription factor involved in tyrosinase gene expression. In addition, AC extract (40 µg/ml) was observed to suppress the activation of extracellular signal-regulated kinase (ERK) and Akt, which negatively regulate melanin synthesis in B16F10 cells. In conclusion, to the best of our knowledge, the present study is the first to show that a methanol extract of AC stimulates melanogenesis by increasing tyrosinase expression via the inhibition of ERK and Akt. Thus, methanol extract of AC may be a potential treatment for hypopigmentation diseases and may be a candidate for skin-tanning cosmetic products. PMID:25333888

  1. Structurally Modified Curcumin Analogs Inhibit STAT3 Phosphorylation and Promote Apoptosis of Human Renal Cell Carcinoma and Melanoma Cell Lines

    PubMed Central

    Bill, Matthew A.; Nicholas, Courtney; Mace, Thomas A.; Etter, Jonathan P.; Li, Chenglong; Schwartz, Eric B.; Fuchs, James R.; Young, Gregory S.; Lin, Li; Lin, Jiayuh; He, Lei; Phelps, Mitch; Li, Pui-Kai; Lesinski, Gregory B.

    2012-01-01

    The Janus kinase-2 (Jak2)-signal transducer and activator of transcription-3 (STAT3) pathway is critical for promoting an oncogenic and metastatic phenotype in several types of cancer including renal cell carcinoma (RCC) and melanoma. This study describes two small molecule inhibitors of the Jak2-STAT3 pathway, FLLL32 and its more soluble analog, FLLL62. These compounds are structurally distinct curcumin analogs that bind selectively to the SH2 domain of STAT3 to inhibit its phosphorylation and dimerization. We hypothesized that FLLL32 and FLLL62 would induce apoptosis in RCC and melanoma cells and display specificity for the Jak2-STAT3 pathway. FLLL32 and FLLL62 could inhibit STAT3 dimerization in vitro. These compounds reduced basal STAT3 phosphorylation (pSTAT3), and induced apoptosis in four separate human RCC cell lines and in human melanoma cell lines as determined by Annexin V/PI staining. Apoptosis was also confirmed by immunoblot analysis of caspase-3 processing and PARP cleavage. Pre-treatment of RCC and melanoma cell lines with FLLL32/62 did not inhibit IFN-γ-induced pSTAT1. In contrast to FLLL32, curcumin and FLLL62 reduced downstream STAT1-mediated gene expression of IRF1 as determined by Real Time PCR. FLLL32 and FLLL62 significantly reduced secretion of VEGF from RCC cell lines in a dose-dependent manner as determined by ELISA. Finally, each of these compounds inhibited in vitro generation of myeloid-derived suppressor cells. These data support further investigation of FLLL32 and FLLL62 as lead compounds for STAT3 inhibition in RCC and melanoma. PMID:22899991

  2. Structurally modified curcumin analogs inhibit STAT3 phosphorylation and promote apoptosis of human renal cell carcinoma and melanoma cell lines.

    PubMed

    Bill, Matthew A; Nicholas, Courtney; Mace, Thomas A; Etter, Jonathan P; Li, Chenglong; Schwartz, Eric B; Fuchs, James R; Young, Gregory S; Lin, Li; Lin, Jiayuh; He, Lei; Phelps, Mitch; Li, Pui-Kai; Lesinski, Gregory B

    2012-01-01

    The Janus kinase-2 (Jak2)-signal transducer and activator of transcription-3 (STAT3) pathway is critical for promoting an oncogenic and metastatic phenotype in several types of cancer including renal cell carcinoma (RCC) and melanoma. This study describes two small molecule inhibitors of the Jak2-STAT3 pathway, FLLL32 and its more soluble analog, FLLL62. These compounds are structurally distinct curcumin analogs that bind selectively to the SH2 domain of STAT3 to inhibit its phosphorylation and dimerization. We hypothesized that FLLL32 and FLLL62 would induce apoptosis in RCC and melanoma cells and display specificity for the Jak2-STAT3 pathway. FLLL32 and FLLL62 could inhibit STAT3 dimerization in vitro. These compounds reduced basal STAT3 phosphorylation (pSTAT3), and induced apoptosis in four separate human RCC cell lines and in human melanoma cell lines as determined by Annexin V/PI staining. Apoptosis was also confirmed by immunoblot analysis of caspase-3 processing and PARP cleavage. Pre-treatment of RCC and melanoma cell lines with FLLL32/62 did not inhibit IFN-γ-induced pSTAT1. In contrast to FLLL32, curcumin and FLLL62 reduced downstream STAT1-mediated gene expression of IRF1 as determined by Real Time PCR. FLLL32 and FLLL62 significantly reduced secretion of VEGF from RCC cell lines in a dose-dependent manner as determined by ELISA. Finally, each of these compounds inhibited in vitro generation of myeloid-derived suppressor cells. These data support further investigation of FLLL32 and FLLL62 as lead compounds for STAT3 inhibition in RCC and melanoma. PMID:22899991

  3. Anticancer activity of 7,8-dihydroxyflavone in melanoma cells via downregulation of α-MSH/cAMP/MITF pathway.

    PubMed

    Sim, Deok Yong; Sohng, Jae Kyung; Jung, Hye Jin

    2016-07-01

    Malignant melanoma is one of the most aggressive skin cancer and highly resistant to most conventional treatment. In the present study, we aimed to investigate the anticancer effects and mechanisms of action of 7,8-dihydroxyflavone (7,8-DHF), a monophenolic flavone, in melanoma cells. At concentrations not exhibiting cytotoxicity, 7,8-DHF potently inhibited growth and clonogenic survival of alpha-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 melanoma cells. Furthermore, it significantly blocked migration and invasion of the metastatic melanoma cells. We also observed that 7,8-DHF exhibits anti-melanogenic activity through inhibition of tyrosinase activity in α-MSH-stimulating condition. Notably, the suppressive activities of 7,8-DHF on melanoma progression were associated with the downregulation of microphthalmia-associated transcription factor (MITF) and its main downstream transcription targets, including hypoxia-inducible factor 1α (HIF1α) and c-MET, by a decrease in cyclic adenosine monophosphate (cAMP) level. In addition, combination treatment with 7,8-DHF and resveratrol, a known therapeutic agent against melanoma, had greater anticancer activities and MITF inhibition than treatment with each single agent in α-MSH-treated B16F10 cells. Collectively, these findings may contribute to the potential application of 7,8-DHF in the prevention and treatment of malignant melanoma. PMID:27220989

  4. Cucurbitacins: potential candidates targeting mitogen-activated protein kinase pathway for treatment of melanoma.

    PubMed

    Ahmed, Mahmoud S; Halaweish, Fathi T

    2014-04-01

    Cucurbitacins (Cucs) have been classified as signal transducer and activator of transcription 3 inhibitors. Kinase inhibition has been a validated drug target in multiple types of malignancies. B-RAF mutations are highly expressed in the melanoma. Our hypothesis is the Cucs can be a potential candidate to inhibit the signaling kinase pathway. The research presented is the evaluation of Cucs, as B-RAF and MEK1 kinase inhibitors. Virtual screening methods were employed to identify lead compounds. The hypothesis was tested on mutant B-RAF cell lines, A-375 and Sk-Mel-28 cell lines to determine the activity toward melanoma. A series of natural Cucs show an improved activity toward Sk-Mel-28 and A-375 cell lines. Cucs show potential inhibition for the total and phosphorylated ERK using ELISA kits. Cucs could be potential candidate for inhibiting cell growth. PMID:23368732

  5. MAGE-A1 promotes melanoma proliferation and migration through C-JUN activation.

    PubMed

    Wang, Dong; Wang, Junyun; Ding, Nan; Li, Yongjun; Yang, Yaran; Fang, Xiangdong; Zhao, Hua

    2016-05-13

    MAGE-A1 belongs to the chromosome X-clustered genes of cancer-testis antigen family and is normally expressed in the human germ line but is also overexpressed in various tumors. Previous studies of MAGE-A1 in melanoma mainly focused on methylation changes or its role in immunotherapy, however, its biological functions in melanoma have remained unknown. In order to determine the role of MAGE-A1 in melanoma growth and metastasis, we manipulated melanoma cell lines with overexpression and knockdown of MAGE-A1. Integration of cell proliferation assays, transwell migration and invasion assays, and RNA-Seq analysis revealed that up-regulation of MAGE-A1 dramatically promoted proliferation, migration, and invasion of human melanoma cell lines in vitro, while down-regulation of MAGE-A1 inhibited those characteristics associated with tumor cells. Furthermore, transcriptome sequencing revealed that MAGE-A1 exerts its tumor promoting activity by activating p-C-JUN directly or through ERK-MAPK signaling pathways. Based on our findings, we propose that MAGE-A1 may be a potential therapeutic target for melanoma patients. PMID:27045082

  6. Biofunctional Activities of Equisetum ramosissimum Extract: Protective Effects against Oxidation, Melanoma, and Melanogenesis.

    PubMed

    Li, Pin-Hui; Chiu, Yu-Pin; Shih, Chieh-Chih; Wen, Zhi-Hong; Ibeto, Laura Kaodichi; Huang, Shu-Hung; Chiu, Chien Chih; Ma, Dik-Lung; Leung, Chung-Hang; Chang, Yaw-Nan; Wang, Hui-Min David

    2016-01-01

    Equisetum ramosissimum, a genus of Equisetaceae, is a medicinal plant that can be separated into ethyl acetate (EA), dichloromethane (DM), n-hexane (Hex), methanol (MeOH), and water extracts. EA extract was known to have potent antioxidative properties, reducing power, DPPH scavenging activity, and metal ion chelating activity. This study compared these five extracts in terms of their inhibiting effects on three human malignant melanomas: A375, A375.S2, and A2058. MTT assay presented the notion that both EA and DM extracts inhibited melanoma growth but did not affect the viabilities of normal dermal keratinocytes (HaCaT) or fibroblasts. Western blot analyses showed that both EA and DM extracts induced overexpression of caspase proteins in all three melanomas. To determine their roles in melanogenesis, this study analyzed their in vitro suppressive effects on mushroom tyrosinase. All extracts except for water revealed moderate suppressive effects. None of the extracts affected B16-F10 cells proliferation. EA extract inhibited cellular melanin production whereas DM extract unexpectedly enhanced cellular pigmentation in B16-F10 cells. Data for modulations of microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related protein 1, and tyrosinase-related protein 2 showed that EA extract inhibited protein expression mentioned above whereas DM extract had the opposite effect. Overall, the experiments indicated that the biofunctional activities of EA extract contained in food and cosmetics protect against oxidation, melanoma, and melanin production. PMID:27403230

  7. Biofunctional Activities of Equisetum ramosissimum Extract: Protective Effects against Oxidation, Melanoma, and Melanogenesis

    PubMed Central

    Li, Pin-Hui; Chiu, Yu-Pin; Shih, Chieh-Chih; Wen, Zhi-Hong; Ibeto, Laura Kaodichi; Huang, Shu-Hung; Chiu, Chien Chih; Ma, Dik-Lung; Leung, Chung-Hang; Chang, Yaw-Nan; Wang, Hui-Min David

    2016-01-01

    Equisetum ramosissimum, a genus of Equisetaceae, is a medicinal plant that can be separated into ethyl acetate (EA), dichloromethane (DM), n-hexane (Hex), methanol (MeOH), and water extracts. EA extract was known to have potent antioxidative properties, reducing power, DPPH scavenging activity, and metal ion chelating activity. This study compared these five extracts in terms of their inhibiting effects on three human malignant melanomas: A375, A375.S2, and A2058. MTT assay presented the notion that both EA and DM extracts inhibited melanoma growth but did not affect the viabilities of normal dermal keratinocytes (HaCaT) or fibroblasts. Western blot analyses showed that both EA and DM extracts induced overexpression of caspase proteins in all three melanomas. To determine their roles in melanogenesis, this study analyzed their in vitro suppressive effects on mushroom tyrosinase. All extracts except for water revealed moderate suppressive effects. None of the extracts affected B16-F10 cells proliferation. EA extract inhibited cellular melanin production whereas DM extract unexpectedly enhanced cellular pigmentation in B16-F10 cells. Data for modulations of microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related protein 1, and tyrosinase-related protein 2 showed that EA extract inhibited protein expression mentioned above whereas DM extract had the opposite effect. Overall, the experiments indicated that the biofunctional activities of EA extract contained in food and cosmetics protect against oxidation, melanoma, and melanin production. PMID:27403230

  8. Xeroderma Pigmentosum Group A Promotes Autophagy to Facilitate Cisplatin Resistance in Melanoma Cells through the Activation of PARP1.

    PubMed

    Ge, Rui; Liu, Lin; Dai, Wei; Zhang, Weigang; Yang, Yuqi; Wang, Huina; Shi, Qiong; Guo, Sen; Yi, Xiuli; Wang, Gang; Gao, Tianwen; Luan, Qi; Li, Chunying

    2016-06-01

    Xeroderma pigmentosum group A (XPA), a key protein in the nucleotide excision repair pathway, has been shown to promote the resistance of tumor cells to chemotherapeutic drugs by facilitating the DNA repair process. However, the role of XPA in the resistance of melanoma to platinum-based drugs like cisplatin is largely unknown. In this study, we initially found that XPA was expressed at higher levels in cisplatin-resistant melanoma cells than in cisplatin-sensitive ones. Furthermore, the knockdown of XPA not only increased cellular apoptosis but also inhibited cisplatin-induced autophagy, which rendered the melanoma cells more sensitive to cisplatin. Moreover, we discovered that the increased XPA in resistant melanoma cells promoted poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) activation and that the inhibition of PARP1 could attenuate the cisplatin-induced autophagy. Finally, we proved that the inhibition of PARP1 and the autophagy process made resistant melanoma cells more susceptible to cisplatin treatment. Our study shows that XPA can promote cell-protective autophagy in a DNA repair-independent manner by enhancing the activation of PARP1 in melanoma cells resistant to cisplatin and that the XPA-PARP1-mediated autophagy process can be targeted to overcome cisplatin resistance in melanoma chemotherapy. PMID:26880244

  9. Targeted inhibition of metastatic melanoma through interference with Pin1-FOXM1 signaling.

    PubMed

    Kruiswijk, F; Hasenfuss, S C; Sivapatham, R; Baar, M P; Putavet, D; Naipal, K A T; van den Broek, N J F; Kruit, W; van der Spek, P J; van Gent, D C; Brenkman, A B; Campisi, J; Burgering, B M T; Hoeijmakers, J H J; de Keizer, P L J

    2016-04-28

    Melanoma is the most lethal form of skin cancer and successful treatment of metastatic melanoma remains challenging. BRAF/MEK inhibitors only show a temporary benefit due to rapid occurrence of resistance, whereas immunotherapy is mainly effective in selected subsets of patients. Thus, there is a need to identify new targets to improve treatment of metastatic melanoma. To this extent, we searched for markers that are elevated in melanoma and are under regulation of potentially druggable enzymes. Here, we show that the pro-proliferative transcription factor FOXM1 is elevated and activated in malignant melanoma. FOXM1 activity correlated with expression of the enzyme Pin1, which we found to be indicative of a poor prognosis. In functional experiments, Pin1 proved to be a main regulator of FOXM1 activity through MEK-dependent physical regulation during the cell cycle. The Pin1-FOXM1 interaction was enhanced by BRAF(V600E), the driver oncogene in the majority of melanomas, and in extrapolation of the correlation data, interference with\\ Pin1 in BRAF(V600E)-driven metastatic melanoma cells impaired both FOXM1 activity and cell survival. Importantly, cell-permeable Pin1-FOXM1-blocking peptides repressed the proliferation of melanoma cells in freshly isolated human metastatic melanoma ex vivo and in three-dimensional-cultured patient-derived melanoids. When combined with the BRAF(V600E)-inhibitor PLX4032 a robust repression in melanoid viability was obtained, establishing preclinical value of patient-derived melanoids for prognostic use of drug sensitivity and further underscoring the beneficial effect of Pin1-FOXM1 inhibitory peptides as anti-melanoma drugs. These proof-of-concept results provide a starting point for development of therapeutic Pin1-FOXM1 inhibitors to target metastatic melanoma. PMID:26279295

  10. Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

    PubMed Central

    Pal, Harish Chandra; Sharma, Samriti; Strickland, Leah Ray; Katiyar, Santosh K.; Ballestas, Mary E.; Athar, Mohammad; Elmets, Craig A.; Afaq, Farrukh

    2014-01-01

    Malignant melanoma is responsible for approximately 75% of skin cancer-related deaths. BRAF plays an important role in regulating the mitogen-activated protein kinase (MAPK) signaling cascade in melanoma with activating mutations in the serine/threonine kinase BRAF occurring in 60–70% of malignant melanomas. The BRAF-MEK-ERK (MAPK) pathway is a key regulator of melanoma cell invasion. In addition, activation of NFκB via the MAPK pathway is regulated through MEK-induced activation of IKK. These pathways are potential targets for prevention and treatment of melanoma. In this study, we investigated the effect of fisetin, a phytochemical present in fruits and vegetables, on melanoma cell invasion and epithelial-mesenchymal transition, and delineated the underlying molecular mechanism. Treatment of multiple human malignant melanoma cell lines with fisetin (5–20 µM) resulted in inhibition of cell invasion. BRAF mutated melanoma cells were more sensitive to fisetin treatment, and this was associated with a decrease in the phosphorylation of MEK1/2 and ERK1/2. In addition, fisetin inhibited the activation of IKK leading to a reduction in the activation of the NFκB signaling pathway. Treatment of cells with an inhibitor of MEK1/2 (PD98059) or of NFκB (caffeic acid phenethyl ester) also reduced melanoma cell invasion. Furthermore, treatment of fisetin promoted mesenchymal to epithelial transition in melanoma cells, which was associated with a decrease in mesenchymal markers (N-cadherin, vimentin, snail and fibronectin) and an increase in epithelial markers (E-cadherin and desmoglein). Employing three dimensional skin equivalents consisting of A375 cells admixed with normal human keratinocytes embedded onto a collagen-constricted fibroblast matrix, we found that treatment of fisetin reduced the invasive potential of melanoma cells into the dermis and increased the expression of E-cadherin with a concomitant decrease in vimentin. These results indicate that fisetin

  11. Cyclohexanediol bis-ethylhexanoate inhibits melanogenesis of murine B16 melanoma and UV-induced pigmentation in human skin.

    PubMed

    Lim, Joo Hyuck; Park, So-Hyun; Kim, Myoung Rae; Yoo, Byoung-Sam; Yang, Jae Chan; Cheong, In Woo; Kim, Jung Hyun; Cho, Jin Hun

    2013-01-01

    The role of cyclohexane diester analogues in the formation of melanin has been recently reported. In the present study, we investigated the inhibitory effect of cyclohexanediol bis-ethylhexanoate (CHEH) on melanogenesis in B16 melanoma cells and on UV-B-induced pigmentation in human skin. CHEH significantly reduced the melanin content in a dose-dependent manner, without cytotoxic effects at the effective concentrations. Moreover, CHEH dose-dependently inhibited tyrosinase activity in B16 melanoma cells, as confirmed by Western blot analysis of the tyrosinase protein levels. However, tyrosinase transcript levels remained unchanged under the same experimental conditions. These results indicate that CHEH inhibited melanogenesis in B16 melanoma cells by regulating tyrosinase activity at the post-transcriptional level. On the other hand, in a cell-free system, CHEH did not inhibit tyrosinase activity. This indicated that CHEH suppressed the pigmentation of melanocytes by indirectly regulating tyrosinase activity. Finally, in a clinical trial, a cream containing 1.0% CHEH showed good whitening effect on UV-induced pigmented human skin without adverse effects. In conclusion, we suggest that CHEH may be an effective inhibitor of melanogenesis and useful effects in the treatment of hyperpigmented disorders. PMID:23258078

  12. Targeting metabolic flexibility by simultaneously inhibiting respiratory complex I and lactate generation retards melanoma progression

    PubMed Central

    Chaube, Balkrishna; Malvi, Parmanand; Singh, Shivendra Vikram; Mohammad, Naoshad; Meena, Avtar Singh; Bhat, Manoj Kumar

    2015-01-01

    Melanoma is a largely incurable skin malignancy owing to the underlying molecular and metabolic heterogeneity confounded by the development of resistance. Cancer cells have metabolic flexibility in choosing either oxidative phosphorylation (OXPHOS) or glycolysis for ATP generation depending upon the nutrient availability in tumor microenvironment. In this study, we investigated the involvement of respiratory complex I and lactate dehydrogenase (LDH) in melanoma progression. We show that inhibition of complex I by metformin promotes melanoma growth in mice via elevating lactate and VEGF levels. In contrast, it leads to the growth arrest in vitro because of enhanced extracellular acidification as a result of increased glycolysis. Inhibition of LDH or lactate generation causes decrease in glycolysis with concomitant growth arrest both in vitro and in vivo. Blocking lactate generation in metformin-treated melanoma cells results in diminished cell proliferation and tumor progression in mice. Interestingly, inhibition of either LDH or complex I alone does not induce apoptosis, whereas inhibiting both together causes depletion in cellular ATP pool resulting in metabolic catastrophe induced apoptosis. Overall, our study suggests that LDH and complex I play distinct roles in regulating glycolysis and cell proliferation. Inhibition of these two augments synthetic lethality in melanoma. PMID:26484566

  13. Pluripotency markers are differentially induced by MEK inhibition in thyroid and melanoma BRAFV600E cell lines

    PubMed Central

    Dorris, Emma R.; Blackshields, Gordon; Sommerville, Gary; Alhashemi, Mohsen; Dias, Andrew; McEneaney, Victoria; Smyth, Paul; O'Leary, John J.; Sheils, Orla

    2016-01-01

    ABSTRACT Oncogenic mutations in BRAF are common in melanoma and thyroid carcinoma and drive constitutive activation of the MAPK pathway. Molecularly targeted therapies of this pathway improves survival compared to chemotherapy; however, responses tend to be short-lived as resistance invariably occursCell line models of melanoma and thyroid carcinoma, +/− BRAFV600E activating mutation, were treated with the MEK inhibitor PD0325901. Treated and naive samples were assayed for expression of key members of the MAPK pathway. Global microRNA expression profiling of naive and resistant cells was performed via next generation sequencingand indicated pluripotency pathways in resistance. Parental cell lines were progressed to holoclones to confirm the miRNA stemness profileMembers of the MIR302/373/374/520 family of embryonic stem cell specific cell cycle regulating (ESCC) microRNAs were identified as differentially expressed between resistant BRAFV600E melanoma and thyroid cell lines. Upregulated expression of gene and protein stemness markers, upregulated expression of MAPK pathway genes and downregulation of the ESCC MIR302 cluster in BRAFV600E melanoma indicated an increased stem-like phenotype in resistant BRAFV600E melanoma. Conversely, downregulated expression of gene and protein stemness markers, downregulated expression of MAPK pathway genes, upregulation of the ESCC MIR520 cluster, reeexpression of cell surface receptors, and induced differentiation-associated morphology in resistant BRAFV600E indicate a differentiated phenotype associated with MEK inhibitor resistance in BRAFV600E thyroid cellsThe differential patterns of resistance observed between BRAFV600E melanoma and thyroid cell lines may reflect tissue type or de novo differentiation, but could have significant impact on the response of primary and metastatic cells to MEK inhibitor treatment. This study provides a basis for the investigation of the cellular differentiation/self-renewal access and its role

  14. Decursin from Angelica gigas Nakai Inhibits B16F10 Melanoma Growth Through Induction of Apoptosis.

    PubMed

    Kim, Byung Soo; Seo, Hyobin; Kim, Ha-Jeong; Bae, Sang Mun; Son, Hye-Nam; Lee, Yoon Jeong; Ryu, Sungpil; Park, Rang-Woon; Nam, Ju-Ock

    2015-10-01

    Decursin, a bioactive phytochemical isolated from Angelica gigas Nakai (danggwi), has shown preclinical anticancer efficacy in various cancer models. However, the antitumor effect of decursin in melanoma models remains undefined. The antitumor activities of decursin were investigated in B16F10 cells in vitro and in vivo. In this study, we show that treatment with decursin inhibited cell proliferation in a dose-dependent manner in B16F10 cells, but not in normal cells. Decursin also induced apoptosis in B16F10 cells, as determined by annexin V-staining assay and transferase-mediated nick-end labeling (TUNEL) staining assay. Decursin increased the phosphorylation of p38 as well as the expression of Bax while decreasing the phosphorylation of extracellular signaling-regulated kinase (ERK) and the expression of Bcl-2 in B16F10 cells. Moreover, decursin activated caspase-3 in B16F10 cells and xenograft tumor tissue. Together, these findings support further investigations into the potential use of decursin in the treatment of melanoma cells. PMID:26336081

  15. Transcription factor LSF (TFCP2) inhibits melanoma growth

    PubMed Central

    Goto, Yuji; Yajima, Ichiro; Kumasaka, Mayuko; Ohgami, Nobutaka; Tanaka, Asami; Tsuzuki, Toyonori; Inoue, Yuji; Fukushima, Satoshi; Ihn, Hironobu; Kyoya, Mikiko; Ohashi, Hiroyuki; Kawakami, Tamihiro; Bennett, Dorothy C.; Kato, Masashi

    2016-01-01

    Late SV40 factor 3 (LSF), a transcription factor, contributes to human hepatocellular carcinoma (HCC). However, decreased expression level of LSF in skin melanoma compared to that in benign melanocytic tumors and nevi in mice and humans was found in this study. Anchorage-dependent and -independent growth of melanoma cells was suppressed by LSF overexpression through an increased percentage of G1 phase cells and an increased p21CIP1 expression level in vitro and in vivo. Anchorage-dependent growth in LSF-overexpressed melanoma cells was promoted by depletion of LSF in the LSF-overexpressed cells. Integrated results of our EMSA and chromatin immunoprecipitation assays showed binding of LSF within a 150-bp upstream region of the transcription start site of p21CIP1 in melanoma cells. Taken together, our results suggest potential roles of LSF as a growth regulator through control of the transcription of p21CIP1 in melanocytes and melanoma cells as well as a biomarker for nevus. PMID:26506241

  16. Transcription factor LSF (TFCP2) inhibits melanoma growth.

    PubMed

    Goto, Yuji; Yajima, Ichiro; Kumasaka, Mayuko; Ohgami, Nobutaka; Tanaka, Asami; Tsuzuki, Toyonori; Inoue, Yuji; Fukushima, Satoshi; Ihn, Hironobu; Kyoya, Mikiko; Ohashi, Hiroyuki; Kawakami, Tamihiro; Bennett, Dorothy C; Kato, Masashi

    2016-01-19

    Late SV40 factor 3 (LSF), a transcription factor, contributes to human hepatocellular carcinoma (HCC). However, decreased expression level of LSF in skin melanoma compared to that in benign melanocytic tumors and nevi in mice and humans was found in this study. Anchorage-dependent and -independent growth of melanoma cells was suppressed by LSF overexpression through an increased percentage of G1 phase cells and an increased p21CIP1 expression level in vitro and in vivo. Anchorage-dependent growth in LSF-overexpressed melanoma cells was promoted by depletion of LSF in the LSF-overexpressed cells. Integrated results of our EMSA and chromatin immunoprecipitation assays showed binding of LSF within a 150-bp upstream region of the transcription start site of p21CIP1 in melanoma cells. Taken together, our results suggest potential roles of LSF as a growth regulator through control of the transcription of p21CIP1 in melanocytes and melanoma cells as well as a biomarker for nevus. PMID:26506241

  17. Inhibition of colony formation in agarose of metastatic human breast carcinoma and melanoma cells by synthetic glycoamine analogs.

    PubMed

    Glinsky, G V; Mossine, V V; Price, J E; Bielenberg, D; Glinsky, V V; Ananthaswamy, H N; Feather, M S

    1996-05-01

    We studied the influence of 10 synthetic glycoamine analogs on colony formation in 0.3 and 0.9% agarose by metastatic human breast carcinoma (MDA-MB-435) and melanoma (TXM-13) cells. Nine synthetic analogs significantly inhibited the colony formation in 0.9% agarose of MDA-MB-435 human breast carcinoma cells; five compounds caused a 73-83% reduction of colony formation. Seven synthetic glycoamines caused a significant inhibition of colony formation in 0.9% agarose by TXM-13 melanoma cells with the inhibitory effect ranging from 71 to 87%. The 50% inhibition (I50) doses and relative activity rank of the compounds were similar for both breast carcinoma and melanoma cell lines. The murine B16 melanoma cell aggregation assay was employed to elucidate the potential mechanism(s) of the inhibitory activity of synthetic glycoamines. The relative activity ranks of the compounds based on the independently determined I50 doses for both cell aggregation and clonogenic growth assays were very similar for the four most active synthetic analogs and clearly indicated the importance of hydrophobic amino acid in mediating the bioactivity of synthetic glycoamines. In both experimental systems (clonogenic growth in agarose and cell aggregation assay) the leading compound was N-(1-deoxy-D-fructos-1-yl)-D-leucine (Fru-D-Leu) and the least active analog was N-(l-deoxy-D-fructos-1-yl)-glycine (Fru-Gly). These results show that synthetic glycoamines may act by competing for specific carbohydrate-lectin interactions, particularly those involving beta-galactoside-specific lectins expressed on metastatic cells. PMID:8674280

  18. Gold (I) N-heterocyclic carbene complex inhibits mouse melanoma growth by p53 upregulation

    PubMed Central

    2014-01-01

    Background Cancer treatment using gold (I) complexes is becoming popular. In this study, a gold (I) N-heterocyclic complex designated as complex 3 was synthesized, its cytotoxicity was examined, and its anti-melanoma activity was evaluated in vitro and in vivo. Methods Viability of cancer cells was determined by MTT assay upon treatment with various concentrations of a gold (I) N-heterocyclic carbene complex (complex 3) in a dose and time dependent manner. Mouse melanoma cells B16F10 were selected for further apoptotic studies, including flowcytometric analysis of annexin binding, cell cycle arrest, intracellular ROS generation and loss in the mitochondrial membrane potential. ELISA based assays were done for caspase activities and western blots for determining the expression of various survival and apoptotic proteins. Immunocytology was performed to visualize the translocation of p53 to the nucleus. B16F10 cells were inoculated into mice and post tumor formation, complex 3 was administered. Immunohistology was performed to determine the expressions of p53, p21, NF-κB (p65 and p50), MMP-9 and VEGF. Student’s t test was used for determining statistical significance. The survival rate data were analyzed by Kaplan-Meier plots. Results Complex 3 markedly inhibited the growth of HCT 116, HepG2, and A549, and induced apoptosis in B16F10 cells with nuclear condensation, DNA fragmentation, externalization of phosphatidylserine, activation of caspase 3 and caspase 9, PARP cleavage, downregulation of Bcl-2, upregulation of Bax, cytosolic cytochrome c elevation, ROS generation, and mitochondrial membrane potential loss indicating the involvement of an intrinsic mitochondrial death pathway. Further, upregulation of p53, p-p53 (ser 15) and p21 indicated the role of p53 in complex 3 mediated apoptosis. The complex reduced tumor size, and caused upregulation of p53 and p21 along with downregulation of NF-κB (p65 and p50), VEGF and MMP-9. These results suggest that it induced

  19. The Complement C3a Receptor Contributes to Melanoma Tumorigenesis by Inhibiting Neutrophil and CD4+ T Cell Responses.

    PubMed

    Nabizadeh, Jamileh A; Manthey, Helga D; Steyn, Frederik J; Chen, Weiyu; Widiapradja, Alexander; Md Akhir, Fazrena N; Boyle, Glen M; Taylor, Stephen M; Woodruff, Trent M; Rolfe, Barbara E

    2016-06-01

    The complement peptide C3a is a key component of the innate immune system and a major fragment produced following complement activation. We used a murine model of melanoma (B16-F0) to identify a hitherto unknown role for C3a-C3aR signaling in promoting tumor growth. The results show that the development and growth of B16-F0 melanomas is retarded in mice lacking C3aR, whereas growth of established melanomas can be arrested by C3aR antagonism. Flow cytometric analysis showed alterations in tumor-infiltrating leukocytes in the absence of C3aR. Specifically, neutrophils and CD4(+) T lymphocyte subpopulations were increased, whereas macrophages were reduced. The central role of neutrophils was confirmed by depletion experiments that reversed the tumor inhibitory effects observed in C3aR-deficient mice and returned tumor-infiltrating CD4(+) T cells to control levels. Analysis of the tumor microenvironment showed upregulation of inflammatory genes that may contribute to the enhanced antitumor response observed in C3aR-deficient mice. C3aR deficiency/inhibition was also protective in murine models of BRAF(V600E) mutant melanoma and colon and breast cancer, suggesting a tumor-promoting role for C3aR signaling in a range of tumor types. We propose that C3aR activation alters the tumor inflammatory milieu, thereby promoting tumor growth. Therapeutic inhibition of C3aR may therefore be an effective means to trigger an antitumor response in melanoma and other cancers. PMID:27183625

  20. Pharmacodynamic Characterization of the Efficacy Signals Due to Selective BRAF Inhibition with PLX4032 in Malignant Melanoma12

    PubMed Central

    Tap, William D; Gong, Ke-Wei; Dering, Judy; Tseng, Yiou; Ginther, Charles; Pauletti, Giovanni; Glaspy, John A; Essner, Richard; Bollag, Gideon; Hirth, Peter; Zhang, Chao; Slamon, Dennis J

    2010-01-01

    Purpose About 65% to 70% of melanomas harbor a mutation in v-raf murine sarcoma viral oncogene homolog B1 (BRAF) that causes the steady-state activation of extracellular signal-regulated kinase (ERK). We sought to investigate the efficacy of PLX4032 (BRAF inhibitor) to identify patterns/predictors of response/resistance and to study the effects of BRAF in melanoma. Experimental Design Well-characterized melanoma cell lines, including several with acquired drug resistance, were exposed to PLX4032. Growth inhibition, phosphosignaling, cell cycle, apoptosis, and gene expression analyses were performed before and after exposure to drug. Results Using a growth-adjusted inhibitory concentration of 50% cutoff of 1 µM, 13 of 35 cell lines were sensitive to PLX4032, 16 resistant, and 6 intermediate (37%, 46%, and 17% respectively). PLX4032 caused growth inhibition, G0/G1 arrest, and restored apoptosis in the sensitive cell lines. A BRAF mutation predicted for but did not guarantee a response, whereas a neuroblastoma RAS viral oncogene homolog mutation or wild-type BRAF conferred resistance. Cells with concurrent BRAF mutations and melanocortin 1 receptor germ line variants and/or a more differentiated melanocyte genotype had a preferential response. Acquired PLX4032 resistance reestablishes ERK signaling, promotes a nonmelanocytic genotype, and is associated with an increase in the gene expression of certain metallothioneins and mediators of angiogenesis. Conclusions PLX4032 has robust activity in BRAF mutated melanoma. The preclinical use of this molecule identifies criteria for its proper clinical application, describes patterns of and reasons for response/resistance, and affords insight into the role of a BRAF mutation in melanoma. PMID:20689758

  1. A novel binuclear palladacycle complex inhibits melanoma growth in vitro and in vivo through apoptosis and autophagy.

    PubMed

    Aliwaini, Saeb; Swarts, Andrew J; Blanckenberg, Angelique; Mapolie, Selwyn; Prince, Sharon

    2013-12-15

    Malignant melanoma is an aggressive skin cancer and it is reported to be the most treatment-resistant human cancer. Here we describe the anti-tumour activity of a novel binuclear palladacycle complex (AJ-5) in vertical growth phase (ME1402) and metastatic (WM1158) melanoma cell lines. We show that compared to normal control cell lines, AJ-5 is more effective in inhibiting the proliferation of ME1402 and WM1158 melanoma cells with IC50 values of 0.19 and 0.20μM, respectively. Flow cytometry analyses showed that AJ-5 induced apoptosis (sub-G1 peak) which was confirmed by Annexin V-FITC/propidium iodide double-staining, nuclear fragmentation and an increase in the levels of PARP cleavage. Furthermore, AJ-5 was shown to induce both intrinsic and extrinsic apoptotic pathways as measured by PUMA, Bax and active caspases. Interestingly, AJ-5 treatment also simultaneously induced the formation of autophagosomes and led to an increase in the autophagy markers LC3II and Beclin1. Inhibition of autophagy reduced AJ-5 cytotoxicity suggesting that AJ-5 induced autophagy was a cell death and not cell survival mechanism. Moreover we show that AJ-5 induces the ATM-CHK2 DNA damage pathway and that its anti-tumour function is mediated by the p38 and ERK1/2 signalling pathways. Importantly, AJ-5 treatment efficiently reduced tumour growth in melanoma bearing mice and induced high levels of autophagy and apoptosis markers. Together these findings suggest that AJ-5 may be an effective chemotherapeutic drug in the treatment of melanoma, a highly aggressive and intractable cancer. PMID:24099796

  2. In vivo Effects in Melanoma of ROCK Inhibition-Induced FasL Overexpression

    PubMed Central

    Teiti, Iotefa; Florie, Bertrand; Pich, Christine; Gence, Rémi; Lajoie-Mazenc, Isabelle; Rochaix, Philippe; Favre, Gilles; Tilkin-Mariamé, Anne-Françoise

    2015-01-01

    Ectopic Fas-ligand (FasL) expression in tumor cells is responsible for both tumor escape through tumor counterattack of Fas-positive infiltrating lymphocytes and tumor rejection though inflammatory and immune responses. We have previously shown that RhoA GTPase and its effector ROCK negatively control FasL membrane expression in murine melanoma B16F10 cells. In this study, we found that B16F10 treatment with the ROCK inhibitor H1152 reduced melanoma development in vivo through FasL membrane overexpression. Although H1152 treatment did not reduce tumor growth in vitro, pretreatment of tumor cells with this inhibitor delayed tumor appearance, and slowed tumor growth in C57BL/6 immunocompetent mice. Thanks to the use of mice-bearing mutated Fas receptors (B6/lpr), we found that reduced tumor growth, observed in immunocompetent mice, was linked to FasL overexpression induced by H1152 treatment. Tumor growth analysis in immunosuppressed NUDE and IFN-γ-KO mice highlighted major roles for T lymphocytes and IFN-γ in the H1152-induced tumor growth reduction. Histological analyses of subcutaneous tumors, obtained from untreated versus H1152-treated B16F10 cells, showed that H1152 pretreatment induced a strong intratumoral infiltration of leukocytes. Cytofluorometric analysis showed that among these leukocytes, the number of activated CD8 lymphocytes was increased. Moreover, their antibody-induced depletion highlighted their main responsibility in tumor growth reduction. Subcutaneous tumor growth was also reduced by repeated intravenous injections of a clinical ROCK inhibitor, Fasudil. Finally, H1152-induced ROCK inhibition also reduced pulmonary metastasis implantation independently of T cell-mediated immune response. Altogether, our data suggest that ROCK inhibitors could become interesting pharmacological molecules for melanoma immunotherapy. PMID:26236689

  3. In vivo Effects in Melanoma of ROCK Inhibition-Induced FasL Overexpression.

    PubMed

    Teiti, Iotefa; Florie, Bertrand; Pich, Christine; Gence, Rémi; Lajoie-Mazenc, Isabelle; Rochaix, Philippe; Favre, Gilles; Tilkin-Mariamé, Anne-Françoise

    2015-01-01

    Ectopic Fas-ligand (FasL) expression in tumor cells is responsible for both tumor escape through tumor counterattack of Fas-positive infiltrating lymphocytes and tumor rejection though inflammatory and immune responses. We have previously shown that RhoA GTPase and its effector ROCK negatively control FasL membrane expression in murine melanoma B16F10 cells. In this study, we found that B16F10 treatment with the ROCK inhibitor H1152 reduced melanoma development in vivo through FasL membrane overexpression. Although H1152 treatment did not reduce tumor growth in vitro, pretreatment of tumor cells with this inhibitor delayed tumor appearance, and slowed tumor growth in C57BL/6 immunocompetent mice. Thanks to the use of mice-bearing mutated Fas receptors (B6/lpr), we found that reduced tumor growth, observed in immunocompetent mice, was linked to FasL overexpression induced by H1152 treatment. Tumor growth analysis in immunosuppressed NUDE and IFN-γ-KO mice highlighted major roles for T lymphocytes and IFN-γ in the H1152-induced tumor growth reduction. Histological analyses of subcutaneous tumors, obtained from untreated versus H1152-treated B16F10 cells, showed that H1152 pretreatment induced a strong intratumoral infiltration of leukocytes. Cytofluorometric analysis showed that among these leukocytes, the number of activated CD8 lymphocytes was increased. Moreover, their antibody-induced depletion highlighted their main responsibility in tumor growth reduction. Subcutaneous tumor growth was also reduced by repeated intravenous injections of a clinical ROCK inhibitor, Fasudil. Finally, H1152-induced ROCK inhibition also reduced pulmonary metastasis implantation independently of T cell-mediated immune response. Altogether, our data suggest that ROCK inhibitors could become interesting pharmacological molecules for melanoma immunotherapy. PMID:26236689

  4. Extract of Cordyceps militaris inhibits angiogenesis and suppresses tumor growth of human malignant melanoma cells.

    PubMed

    Ruma, I Made Winarsa; Putranto, Endy Widya; Kondo, Eisaku; Watanabe, Risayo; Saito, Ken; Inoue, Yusuke; Yamamoto, Ken-Ichi; Nakata, Susumu; Kaihata, Masaji; Murata, Hitoshi; Sakaguchi, Masakiyo

    2014-07-01

    Angiogenesis is essential for tumor development and metastasis. Among several angiogenic factors, vascular endothelial growth factor receptor (VEGF) is important for tumor-derived angiogenesis and commonly overexpressed in solid tumors. Thus, many antitumor strategies targeting VEGF have been developed to inhibit cancer angiogenesis, offering insights into the successful treatment of solid cancers. However, there are a number of issues such as harmful effects on normal vascularity in clinical trials. Taking this into consideration, we employed Cordyceps militaris as an antitumor approach due to its biological safety in vivo. The herbal medicinal mushroom Cordyceps militaris has been reported to show potential anticancer properties including anti-angiogenic capacity; however, its concrete properties have yet to be fully demonstrated. In this study, we aimed to elucidate the biological role of Cordyceps militaris extract in tumor cells, especially in regulating angiogenesis and tumor growth of a human malignant melanoma cell line. We demonstrated that Cordyceps militaris extract remarkably suppressed tumor growth via induction of apoptotic cell death in culture that links to the abrogation of VEGF production in melanoma cells. This was followed by mitigation of Akt1 and GSK-3β activation, while p38α phosphorylation levels were increased. Extract treatment in mouse model xenografted with human melanoma cells resulted in a dramatic antitumor effect with down-regulation of VEGF expression. The results suggest that suppression of tumor growth by Cordyceps militaris extract is, at least, mediated by its anti-angiogenicity and apoptosis induction capacities. Cordyceps militaris extract may be a potent antitumor herbal drug for solid tumors. PMID:24789042

  5. A new melanoma diagnosis active support system.

    PubMed

    Fiorini, R A; Dacquino, G; Laguteta, G

    2004-01-01

    The aim of this paper is to present the operational performance of a new MDASS (Melanoma Diagnosis Active Support System) prototype able to distil optimal knowledge from acquired data to automatically capture and reliably discriminate and quantify the stage of disease evolution. Automated classification dermatoscopical parameters can be divided into two main classes: Size Descriptor (point size, local, and global) and Intrinsic Descriptor (morphological, geometrical, chromatic, others). Usually elementary geometric shape robust and effective characterization, invariant to environment and optical geometry transformations, on a rigorous mathematical level is a key and computational intensive problem. MDASS uses GEOGINE (GEOmetrical enGINE), a state-of-the-art OMG (Ontological Model Generator) based on n-D Tensor Moment Invariants for shape/texture effective description. MDASS main results show robust disease classification procedure with distillation of minimal reference grids for pathological cases and they ultimately achieve effective early diagnosis of melanocytic lesion. System results are validated by carefully designed experiments with certified clinical reference database. Overall system operational performance is presented. Finally, MDASS error analysis and computational complexity are addressed and discussed. PMID:17270962

  6. Curcumin induces autophagy, inhibits proliferation and invasion by downregulating AKT/mTOR signaling pathway in human melanoma cells.

    PubMed

    Zhao, Guangming; Han, Xiaodong; Zheng, Siwen; Li, Zhen; Sha, Yang; Ni, Jing; Sun, Zhe; Qiao, Song; Song, Zhiqi

    2016-02-01

    Melanoma is the foremost malignant cutaneous cancer and it is extremely resistant to chemotherapy and radiotherapy. Curcumin is an active component of turmeric, the yellow spice derived from the rhizome of Curcuma longa, and is widely known for its anti-inflammatory and anti-cancerogenic properties. Several recent studies suggest that curcumin induces apoptosis by modulating multiple signaling pathways to exert its anticancer effect. In the present study, we investigated the effect of curcumin on the viability, invasion potential, cell cycle, autophagy and the AKT, mTOR, P70S6K proteins of AKT/mTOR signaling pathway in human melanoma A375 and C8161 cell lines in vitro and in an in vivo tumorigenesis model. Curcumin effectively inhibited the proliferation of melanoma cells in vitro and in vivo. It suppressed cell invasion, arrested the cancer cells at G2/M phase of the cell cycle, and induced autophagy. Furthermore, curcumin suppressed the activation of AKT, mTOR and P70S6K proteins. Curcumin, therefore, is a potent suppressor of cell viability and invasion, and simultaneously an inducer of autophagy in A375 and C8161 cells. Accordingly, curcumin could be a novel therapeutic candidate for the management of melanoma. PMID:26573768

  7. Inhibition of cell proliferation, migration and invasion of B16-F10 melanoma cells by α-mangostin

    SciTech Connect

    Beninati, Simone; Oliverio, Serafina; Cordella, Martina; Rossi, Stefania; Senatore, Cinzia; Liguori, Immacolata; Lentini, Alessandro; Piredda, Lucia; Tabolacci, Claudio

    2014-08-08

    Highlights: • We studied the anticancer potential of a new emerging molecule, α-mangostin (α-M). • We provide first evidences on the effects of α-M on transglutaminase activity. • We deeply examined the antimetastatic effects of α-M through many in vitro assays. • Proteomic analysis revealed that α-M promotes a reorganization at cellular level. - Abstract: In this study, we have evaluated the potential antineoplastic effects of α-mangostin (α-M), the most representative xanthone in Garcinia mangostana pericarp, on melanoma cell lines. This xanthone markedly inhibits the proliferation of high-metastatic B16-F10 melanoma cells. Furthermore, by deeply analyzing which steps in the metastatic process are influenced by xanthone it was observed that α-M strongly interferes with homotypic aggregation, adhesion, plasticity and invasion ability of B16-F10 cells, probably by the observed reduction of metalloproteinase-9 activity. The antiproliferative and antimetastatic properties of α-M have been established in human SK-MEL-28 and A375 melanoma cells. In order to identify pathways potentially involved in the antineoplastic properties of α-M, a comparative mass spectrometry proteomic approach was employed. These findings may improve our understanding of the molecular mechanisms underlying the anti-cancer effects of α-M on melanoma.

  8. Caspase dependent apoptotic inhibition of melanoma and lung cancer cells by tropical Rubus extracts.

    PubMed

    George, Blassan Plackal Adimuriyil; Abrahamse, Heidi; Hemmaragala, Nanjundaswamy M

    2016-05-01

    Rubus fairholmianus Gard. inhibits human melanoma (A375) and lung cancer (A549) cell growth by the caspase dependent apoptotic pathway. Herbal products have a long history of clinical use and acceptance. They are freely available natural compounds that can be safely used to prevent various ailments. The plants and plant derived products became the basis of traditional medicine system throughout the world for thousands of years. The effects of R. fairholmianus root acetone extract (RFRA) on the proliferation of A375 and A549 cells was examined in this study. RFRA led to a decrease in cell viability, proliferation and an increase in cytotoxicity in a dose dependent manner when compared with control and normal skin fibroblast cells (WS1). The morphology of treated cells supported apoptotic cell death. Annexin V/propidium iodide staining indicated that RFRA induced apoptosis in A375 and A549 cells and the percentages of early and late apoptotic populations significantly increased. Moreover, the apoptotic inducing ability of RFRA when analysing effector caspase 3/7 activity, indicated a marked increase in treated cells. In summary, we have shown the anticancer effects of RFRA in A375 and A549 cancer cells via induction of caspase dependent apoptosis in vitro. The extract is more effective against melanoma; which may suggest the usefulness of RFRA-based anticancer therapies. PMID:27133056

  9. Identification of a second active site in laminin for promotion of cell adhesion and migration and inhibition of in vivo melanoma lung colonization.

    PubMed

    Kleinman, H K; Graf, J; Iwamoto, Y; Sasaki, M; Schasteen, C S; Yamada, Y; Martin, G R; Robey, F A

    1989-07-01

    Previously we reported that a pentapeptide (Tyr-Ile-Gly-Ser-Arg or YIGSR) from domain III of the B1 chain of laminin is a cell attachment site with the ability to stimulate cell adhesion and migration and to block experimental metastases. Here we report studies on the activities of synthetic peptides that cover domain III and report a second biologically active peptide PDSGR from this domain with activities similar to YIGSR. We also show that cyclic YIGSR is more potent in these assays than the linear peptide as expected since this sequence on laminin is bracketed by cysteines. Due to their proximity and similar spectrum of activities, it is possible that these sequences act in concert in the native laminin molecule. PMID:2735766

  10. Ultrasound-mediated interferon {beta} gene transfection inhibits growth of malignant melanoma

    SciTech Connect

    Yamaguchi, Kazuki; Feril, Loreto B.; Tachibana, Katsuro; Takahashi, Akira; Matsuo, Miki; Endo, Hitomi; Harada, Yoshimi; Nakayama, Juichiro

    2011-07-22

    Highlights: {yields} Successful ultrasound-mediated transfection of melanoma (C32) cells with IFN-{beta} genes both in vitro and in vivo. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited proliferation of melanoma cells in vitro. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited melanoma tumor growth in vivo. -- Abstract: We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon {beta} (IFN-{beta}) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-{beta} in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-{beta} genes mixed with microbubbles. Successful sonotransfection with IFN-{beta} gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-{beta} gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.

  11. Melanoma

    MedlinePlus

    ... have melanoma that has spread. Help the patient’s immune system fight the cancer Ipilimumab (Yervoy®), which was FDA ... How ipilimumab works : This drug helps the patient’s immune system to recognize, target, and attack cancer cells. Healthy ...

  12. VS-5584, a Novel PI3K-mTOR Dual Inhibitor, Inhibits Melanoma Cell Growth In Vitro and In Vivo

    PubMed Central

    Shao, Zheren; Bao, Qi; Jiang, Fangzhen; Qian, Huan; Fang, Quan; Hu, Xueqing

    2015-01-01

    Melanomas cause over 76% of skin cancer deaths annually. Phosphatidylinositol 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) signaling pathway is important for melanoma initiation and progression. In the current study, we evaluated the potential anti-melanoma effect of VS-5584, a novel and highly potent PI3K-mTOR dual inhibitor. We demonstrated that VS-5584 potently inhibited survival and proliferation of established (A375, A-2058 and SK-MEL-3 lines) and primary human melanoma cells, but was non-cytotoxic to non-cancerous human skin keratinocytes and B10BR murine melanocytes. At the meantime, VS-5584 induced caspase-dependent apoptotic death in melanoma cells, and its cytotoxicity was alleviated by the caspase inhibitors. At the molecular level, VS-5584 blocked AKT-mTOR activation and downregulated cyclin D1 expression in melanoma cells, while the expressions of Bcl-xL and Bcl-2 were not affected by VS-5584 treatment. On the other hand, a BH-3 mimetic Bcl-xL/Bcl-2 inhibitor ABT-737, as well as siRNA-mediated knockdown of Bcl-xL or Bcl-2, enhanced the activity of VS-5584 in melanoma cells. In vivo, oral administration of VS-5584 suppressed A375 melanoma xenograft growth in nude mice, and its activity was further enhanced by co-administration of ABT-737. These results provide the rationale for the clinical assessment of VS-5584 in melanoma patients and development of ABT-737 and other Bcl-xL/Bcl-2 inhibitors as the possible adjuvants. PMID:26204252

  13. The effects of Salacia reticulata on anti-cellular oxidants and melanogenesis inhibition in alpha-MSH-stimulated and UV irradiated B16 melanoma cells.

    PubMed

    Suwannalert, Prasit; Kariya, Ryusho; Suzu, Ikuko; Okada, Seiji

    2014-04-01

    The purposes of this study were to investigate the inhibitory effects of Salacia reticulata Tul. root extract on cellular oxidants and melanogenesis in B16 melanoma cells. Cells treated with non-toxic doses of S. reticulata root extract were investigated for their effects on melanogenesis, cellular tyrosinase activity and cellular oxidant scavenging activity. The results indicated that S. reticulata extract inhibited melanin synthesis and tyrosinase activity in alpha-MSH-induced or UV-irradiated B16 melanoma cells in a dose dependent manner. Additionally, the extract also exhibited anti-cellular oxidants in UV-induced radical melanoma cells. Altogether, these results suggested that S. reticulata root extract has roles in suppression of melanogenesis and oxidant inhibition. S. reticulata root extract may be a potential source for the development of pharmaceutical products for treatment of skin hyperpigmentation disorders. PMID:24868882

  14. Simultaneous blocking of IL-6 and IL-8 is sufficient to fully inhibit CAF-induced human melanoma cell invasiveness.

    PubMed

    Jobe, Njainday Pulo; Rösel, Daniel; Dvořánková, Barbora; Kodet, Ondřej; Lacina, Lukáš; Mateu, Rosana; Smetana, Karel; Brábek, Jan

    2016-08-01

    Tumour microenvironment plays a critical role in cell invasion and metastasis. To investigate the role of cancer-associated fibroblasts (CAFs) in melanoma cell invasiveness, we used 3D spheroid invasion assay. The effect of conditioned media from normal fibroblasts and CAFs cultivated alone or co-cultivated with melanoma cells on BLM or A2058 melanoma spheroid invasion was analysed. We found that conditioned media from CAFs and CAFs co-cultured with melanoma cells, especially, promote invasion and migration, without significant effect on melanoma cell proliferation. We further analysed the expression of pro-invasive cytokines IL-8 and IL-6 in media and found that melanoma cells are dominant producers of IL-8 and fibroblasts are dominant producers of IL-6 in 2D monocultures, while co-cultivation of CAFs with melanoma cells induces production/secretion of IL-6 and IL-8 into the media. The analyses of IL-6 levels in 3D cultures and human melanoma samples, however, revealed that at least in some cases IL-6 is also produced directly by melanoma cells. Analysis of the role of IL-6 and IL-8 in CAF-induced melanoma invasion, using neutralising antibodies, revealed that simultaneous blocking of IL-6 and IL-8 is sufficient to fully inhibit CAF-induced human melanoma cell invasiveness. In summary, these experiments indicate the important role of CAFs and IL-8 and IL-6 cytokines in melanoma cell invasiveness. PMID:27102177

  15. Alternol inhibits the proliferation and induces the differentiation of the mouse melanoma B16F0 cell line.

    PubMed

    Wang, Caixia; Xu, Wenjuan; Hao, Wenjin; Wang, Bingsheng; Zheng, Qiusheng

    2016-08-01

    High malignant potential and low susceptibility to treatment are characteristics of malignant melanoma. Alternol, a novel compound purified from microbial fermentation products obtained from the bark of the yew tree, exhibits a variety of antitumor activities. Based on these findings, the aim of the present study was to extend the knowledge on the antineoplastic effect of alternol in the mouse melanoma B16F0 cell line. Alternol significantly inhibited the proliferation and colony formation of B16F0 cells in a dose-dependent manner as detected by MTT and soft agar colony formation assays. NaOH alkaline lysis and oxidation of Dopa indicated that alternol enhanced the melanin content and tyrosinase activity of the B16F0 cells and results also showed a dose‑response relationship. Morphologic changes accompanied by extended dendrites were discovered in the B16F0 cells after treatment with alternol. Furthermore, the mRNA levels of tyrosinase, Trp1 and Trp2 were increased by alternol. Our results confirmed that alternol possesses marked antineoplastic properties against melanoma cells, indicating that this microbial fermentation product is a promising agent for the differentiation therapy of cancer. The inhibition of cell proliferation and colony formation by alternol was associated with both cytotoxicity and induction of differentiation. PMID:27278753

  16. Fisetin inhibits human melanoma cell growth through direct binding to p70S6K and mTOR: findings from 3-D melanoma skin equivalents and computational modeling

    PubMed Central

    Syed, Deeba N.; Chamcheu, Jean-Christopher; Khan, Mohammad Imran; Sechi, Mario; Lall, Rahul K.; Adhami, Vaqar M.; Mukhtar, Hasan

    2014-01-01

    The incidence of melanoma continues to rise. Inspite of treatment advances, the prognosis remains grim once the disease has metastasized, emphasizing the need to explore additional therapeutic strategies. One such approach is through the use of mechanism-based dietary intervention. We previously showed that the flavonoid fisetin inhibits melanoma cell proliferation, in vitro and in vivo. Here, we studied fisetin-mediated regulation of kinases involved in melanoma growth and progression. Time-course analysis in 3-D melanoma constructs that transitioned from radial to vertical growth showed that fisetin treatment resulted in significant decrease in melanocytic lesions in contrast to untreated controls that showed large tumor nests and invading disseminated cells. Further studies in melanoma cultures and mouse xenografts showed that fisetin-mediated growth inhibition was associated with dephosphorylation of AKT, mTOR and p70S6K proteins. In silico modeling indicated direct interaction of fisetin with mTOR and p70S6K with favorable free energy values. These findings were validated by cell-free competition assays that established binding of fisetin to p70S6K and mTOR while little affinity was detected with AKT. Kinase activity studies reflected similar trend with % inhibition observed for p70S6K and mTOR at lower doses than AKT. Our studies characterized, for the first time, the differential interactions of any botanical agent with kinases involved in melanoma growth and demonstrate that fisetin inhibits mTOR and p70S6K through direct binding while the observed inhibitory effect of fisetin on AKT is mediated indirectly, through targeting interrelated pathways. PMID:24675012

  17. Overexpression of protease-activated receptor-1 contributes to melanoma metastasis via regulation of connexin 43.

    PubMed

    Villares, Gabriel J; Dobroff, Andrey S; Wang, Hua; Zigler, Maya; Melnikova, Vladislava O; Huang, Li; Bar-Eli, Menashe

    2009-08-15

    Protease-activated receptor-1 (PAR-1) is a key player in melanoma metastasis with higher expression seen in metastatic melanoma cell lines and tissue specimens. cDNA microarray and Western blot analyses reveal that the gap junctional intracellular communication molecule connexin 43 (Cx-43), known to be involved in tumor cell diapedesis and attachment to endothelial cells, is significantly decreased after PAR-1 silencing in metastatic melanoma cell lines. Furthermore, Cx-43 promoter activity was significantly inhibited in PAR-1-silenced cells, suggesting that PAR-1 regulates Cx-43 at the transcriptional level. Chromatin immunoprecipitation studies showed a reduction in the binding of SP-1 and AP-1 transcription factors to the promoter of Cx-43. Both transcription factors have been shown previously to be required for maximal Cx-43 promoter activity. These results were corroborated by mutating the AP-1 and SP-1 binding sites resulting in decreased Cx-43 promoter activity in PAR-1-positive cells. Moreover, as Cx-43 has been shown to facilitate arrest of circulating tumor cells at the vascular endothelium, melanoma cell attachment to endothelial cells was significantly decreased in PAR-1-silenced cells, with this effect being abrogated after PAR-1 rescue. Herein, we report that up-regulation of PAR-1 expression, seen in melanoma progression, mediates high levels of Cx-43 expression. As both SP-1 and AP-1 transcription factors act as positive regulators of Cx-43, our data provide a novel mechanism for the regulation of Cx-43 expression by PAR-1. Indeed, Cx-43 expression was restored following PAR-1 rescue in PAR-1-silenced cells. Taken together, our data support the tumor promoting function of Cx-43 in melanoma. PMID:19679555

  18. Overexpression of Protease Activated Receptor-1 Contributes to Melanoma Metastasis via Regulation of Connexin 43

    PubMed Central

    Villares, Gabriel J.; Dobroff, Andrey S.; Wang, Hua; Zigler, Maya; Melnikova, Vladislava O.; Huang, Li; Bar-Eli, Menashe

    2009-01-01

    Protease Activated Receptor-1 (PAR-1) is a key player in melanoma metastasis with higher expression seen in metastatic melanoma cell lines and tissue specimens. cDNA microarray and Western blot analyses reveal that the gap junctional intracellular communication molecule, Connexin 43 (Cx-43), known to be involved in tumor cell diapedesis and attachment to endothelial cells, is significantly decreased after PAR-1 silencing in metastatic melanoma cell lines. Furthermore, Cx-43 promoter activity was significantly inhibited in PAR-1 silenced cells suggesting that PAR-1 regulates Cx-43 at the transcriptional level. Chromatin Immunoprecipitation studies found a reduction in the binding of SP-1 and AP-1 transcription factors to the promoter of Cx-43. Both transcription factors have previously been shown to be required for maximal Cx-43 promoter activity. These results were corroborated by mutating the AP-1 and SP-1 binding sites resulting in decreased Cx-43 promoter activity in PAR-1 positive cells. Moreover, as Cx-43 has been shown to facilitate arrest of circulating tumor cells at the vascular endothelium, melanoma cell attachment to endothelial cells was significantly decreased in PAR-1 silenced cells with this effect being abrogated after PAR-1 rescue. Herein, we report that upregulation of PAR-1 expression seen in melanoma progression, mediates high levels of Cx-43 expression. As both SP-1 and AP-1 transcription factors act as positive regulators of Cx-43, our data provide a novel mechanism for the regulation of Cx-43 expression by PAR-1. Indeed, Cx-43 expression was restored following PAR-1 rescue in PAR-1 silenced cells. Taken together, our data support the tumor promoting function of Connexin 43 in melanoma. PMID:19679555

  19. Biotransformed Soybean Extract (BSE) Inhibits Melanoma Cell Growth and Viability In Vitro: Involvement of Nuclear Factor-Kappa B Signaling

    PubMed Central

    Vilela, Fernanda Maria Pinto; Syed, Deeba N.; Chamcheu, Jean Christopher; Calvo-Castro, Laura A.; Fortes, Vanessa Silveira; Fonseca, Maria José Vieira; Mukhtar, Hasan

    2014-01-01

    Melanoma is recognized as one of the most aggressive cancers with a relatively high propensity for metastasis. The prognosis of melanoma remains poor in spite of treatment advances, emphasizing the importance of additional preventive measures. Isoflavonoids have become not only potential chemopreventive, but also important therapeutic natural agents. We evaluated the antiproliferative and proapoptotic properties of biotransformed soybean extract (BSE) in A375 melanoma cells. Previous analyses demonstrated that the concentration of daidzein, genistein and aminoacids/peptides present in BSE, fermented by Aspergillus awamori is much higher than in the non biotransformed extract (NBSE). Experiments comparing the efficacy of the extracts in preventing cancer cell growth showed that treatment (24 h) of aggressive melanoma cells (A375 and 451Lu) with BSE resulted in a dose-dependent inhibition of growth and viability. In contrast, treatment with similar doses of NBSE failed to inhibit melanoma cell viability. Further studies in A375 cells showed that decrease in cell viability with BSE treatment (1.5–1.9 mg/ml; 24 h) was associated with induction of apoptosis. Immunoblot analysis revealed that BSE treatment resulted in induction of PARP cleavage, activation of caspase-3, -7, and -8 and increased expression of TRAIL and its receptor DR4. BSE did not activate the intrinsic apoptotic pathway in A375 cells, as no change was observed in caspase-9 expression. The expression of Bcl-2 apoptotic proteins such as Bid and Bax remained unaffected with BSE treated cells. Interestingly, we also showed that BSE treatment increased the phosphorylation and activation of IKK, IκBα degradation and p65/NF-κB translocation to the nucleus, and that stimulation of the NF-???B pathway was required for BSE-induced apoptosis of A375 cells. Our findings indicate that the biotransformation of soybean plays a crucial role in the extract anti-cancer effect observed in melanoma cells. However

  20. Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

    SciTech Connect

    Matsuoka, Hiroshi; Tsubaki, Masanobu; Yamazoe, Yuzuru; Ogaki, Mitsuhiko; Satou, Takao; Itoh, Tatsuki; Kusunoki, Takashi; Nishida, Shozo

    2009-07-15

    In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKC{alpha} and PKC{delta} phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.

  1. Cell context-dependent activities of parthenolide in primary and metastatic melanoma cells

    PubMed Central

    Czyz, M; Lesiak-Mieczkowska, K; Koprowska, K; Szulawska-Mroczek, A; Wozniak, M

    2010-01-01

    Background and purpose: Growing evidence implicates NF-κB as an important contributor to metastasis and increased chemoresistance of melanoma. Here, we report the effects of parthenolide on either untreated, cisplatin- or TNFα-treated melanoma cell lines A375, 1205Lu and WM793, exhibiting different levels of constitutive NF-κB activity. Experimental approach: Electrophoretic mobility shift assay was used to assess changes in NF-κB activity, and real-time PCR to evaluate expression of NF-κB-regulated genes. Cell cycle arrest and apoptosis were assessed by flow cytometry. Cell death was also visualized by fluorescence microscopy. Migration was determined by scratch assay and invasiveness by Matrigel assay. Key results: Parthenolide suppressed both constitutive and induced NF-κB activity in melanoma cells. This was accompanied by down-regulation of cancer-related genes, with NF-κB-binding sites in their promoters, including: Bcl-XL, survivin, cyclin D1, interleukin 8 and matrix metalloproteinase 9. When the various effects of 6 µM parthenolide were compared, apoptosis associated with loss of mitochondrial membrane potential was most efficiently induced in 1205Lu cells, cell cycle arrest in G0/G1 phase was observed in WM793 cells, and high metastatic potential was markedly reduced in A375 cells. These findings not only reflected differences between melanoma cell lines in basal expression of NF-κB-regulated genes, but also suggested other parthenolide targets involved in cell cycle progression, migration, invasiveness and survival. Conclusions: Inhibition of constitutive and therapeutically induced NF-κB pathway by parthenolide might be useful in the treatment of melanoma, although the diversity of changes induced in melanoma cells with different genetic backgrounds indicate context-dependent poly-pharmacological properties of this compound. PMID:20590608

  2. Dexamethasone and zinc in combination inhibit the anchorage-independent growth of S-91 Cloudman murine melanoma

    SciTech Connect

    Kreutzfeld, K.L.; Lei, K.Y.; Bregman, M.D.; Meyskens, F.L. Jr.

    1985-03-04

    Zinc inhibited the colony formation of Cloudman S-91 murine melanoma cells in a dose dependent manner with an ID/sub 50/ of 3.4 ..mu..g/ml. Total inhibition of the melanoma colony-forming units occurred at a zinc concentration of 4.42 ..mu..g/ml. In the presence of dexamethasone the ID/sub 50/ for zinc inhibition was reduced by 49% and total inhibition of anchorage-independent growth occurred at the achievable in vivo zinc concentration of 3.0 ..mu..g/ml. Dexamethasone and zinc in combination effected a greater than additive inhibition of the murine melanoma colony-forming units. Statistical evaluation of these results showed that zinc and dexamethasone interacted synergistically to inhibit the formation of murine melanoma colonies. 29 references, 1 figure, 1 table.

  3. Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma

    NASA Astrophysics Data System (ADS)

    Sun, Chong; Wang, Liqin; Huang, Sidong; Heynen, Guus J. J. E.; Prahallad, Anirudh; Robert, Caroline; Haanen, John; Blank, Christian; Wesseling, Jelle; Willems, Stefan M.; Zecchin, Davide; Hobor, Sebastijan; Bajpe, Prashanth K.; Lieftink, Cor; Mateus, Christina; Vagner, Stephan; Grernrum, Wipawadee; Hofland, Ingrid; Schlicker, Andreas; Wessels, Lodewyk F. A.; Beijersbergen, Roderick L.; Bardelli, Alberto; di Nicolantonio, Federica; Eggermont, Alexander M. M.; Bernards, Rene

    2014-04-01

    Treatment of BRAF(V600E) mutant melanoma by small molecule drugs that target the BRAF or MEK kinases can be effective, but resistance develops invariably. In contrast, colon cancers that harbour the same BRAF(V600E) mutation are intrinsically resistant to BRAF inhibitors, due to feedback activation of the epidermal growth factor receptor (EGFR). Here we show that 6 out of 16 melanoma tumours analysed acquired EGFR expression after the development of resistance to BRAF or MEK inhibitors. Using a chromatin-regulator-focused short hairpin RNA (shRNA) library, we find that suppression of sex determining region Y-box 10 (SOX10) in melanoma causes activation of TGF-β signalling, thus leading to upregulation of EGFR and platelet-derived growth factor receptor-β (PDGFRB), which confer resistance to BRAF and MEK inhibitors. Expression of EGFR in melanoma or treatment with TGF-β results in a slow-growth phenotype with cells displaying hallmarks of oncogene-induced senescence. However, EGFR expression or exposure to TGF-β becomes beneficial for proliferation in the presence of BRAF or MEK inhibitors. In a heterogeneous population of melanoma cells having varying levels of SOX10 suppression, cells with low SOX10 and consequently high EGFR expression are rapidly enriched in the presence of drug, but this is reversed when the drug treatment is discontinued. We find evidence for SOX10 loss and/or activation of TGF-β signalling in 4 of the 6 EGFR-positive drug-resistant melanoma patient samples. Our findings provide a rationale for why some BRAF or MEK inhibitor-resistant melanoma patients may regain sensitivity to these drugs after a `drug holiday' and identify patients with EGFR-positive melanoma as a group that may benefit from re-treatment after a drug holiday.

  4. RasGRP3, a Ras activator, contributes to signaling and the tumorigenic phenotype in human melanoma

    PubMed Central

    Li, Luowei; Kedei, Noemi; Tóth, Zsuzsanna E.; Czap, Alexandra; Velasquez, Julia F.; Mihova, Daniela; Michalowski, Aleksandra M.; Yuspa, Stuart H.; Blumberg, Peter M.

    2014-01-01

    RasGRP3, an activator for H-Ras, R-Ras and Rap1/2, has emerged as an important mediator of signaling downstream from receptor coupled phosphoinositide turnover in B and T cells. Here, we report that RasGRP3 showed a high level of expression in multiple human melanoma cell lines as well as in a subset of human melanoma tissue samples. Suppression of endogenous RasGRP3 expression in these melanoma cell lines reduced Ras-GTP formation as well as c-Met expression and Akt phosphorylation downstream from HGF or EGF stimulation. RasGRP3 suppression also inhibited cell proliferation and reduced both colony formation in soft agar and xenograft tumor growth in immunodeficient mice, demonstrating the importance of RasGRP3 for the transformed phenotype of the melanoma cells. Reciprocally, overexpression of RasGRP3 in human primary melanocytes altered cellular morphology, markedly enhanced cell proliferation, and rendered the cells tumorigenic in a mouse xenograft model. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses and suppressed cell growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. The identification of the role of RasGRP3 in melanoma highlights its importance, as a Ras activator, in the phosphoinositide signaling pathway in human melanoma and provides a new potential therapeutic target. PMID:21602881

  5. Anti-proliferative and cytotoxic activity of rosuvastatin against melanoma cells

    PubMed Central

    Czajkowski, Rafal; Zegarska, Barbara; Kowaliszyn, Bogna; Pokrywczynska, Marta; Drewa, Tomasz

    2016-01-01

    Introduction Statins are considered potential candidate agents for melanoma chemoprevention. Statin-induced mevalonate pathway inhibition leads to reduction of cholesterol synthesis and also to decreased cellular levels of non-steroidal isoprenoids, geranylgeranyl pyrophosphate and farnesyl pyrophosphate. This results in the impairment of protein prenylation which affects carcinogenesis. Aim To analyze anti-proliferative and cytotoxic activity of rosuvastatin against melanoma cells. Material and methods Melanoma cell lines (A375 and WM1552C) and normal fibroblasts (BJ) were used as the primary research material. Cells were treated with rosuvastatin at concentrations ranging from 0.01 µM to 10 µM. Cell viability was analyzed with the use of an MTT assay. Expression of proliferation marker Ki67 was assessed on the basis of immunofluorescence staining. Results Rosuvastatin reduced A375 and BJ cell viability in a time- and dose-dependent manner. After 72 h incubation, the IC50, half maximal inhibitory concentration, was 2.3 µM for melanoma cells and 7.4 µM for normal fibroblasts. In turn, rosuvastatin exhibited relatively lower activity against WM1552C cells. A significant reduction of Ki67 expression was also noted for BJ fibroblasts after prolonged incubation with the tested drug. Conclusions The results indicate that the anti-melanoma properties of rosuvastatin are highly dependent on the tumor cell line assessed. However, the concentrations required to decrease melanoma cell viability in vitro exceed the plasma concentrations reached in patients treated with rosuvastatin at well-tolerated doses. What is more disturbing, reduction of proliferation and viability observed in BJ fibroblasts indicated that rosuvastatin at high doses may be toxic for normal cells. PMID:27605895

  6. Antiproliferative Activity of Cyanophora paradoxa Pigments in Melanoma, Breast and Lung Cancer Cells

    PubMed Central

    Baudelet, Paul-Hubert; Gagez, Anne-Laure; Bérard, Jean-Baptiste; Juin, Camille; Bridiau, Nicolas; Kaas, Raymond; Thiéry, Valérie; Cadoret, Jean-Paul; Picot, Laurent

    2013-01-01

    The glaucophyte Cyanophora paradoxa (Cp) was chemically investigated to identify pigments efficiently inhibiting malignant melanoma, mammary carcinoma and lung adenocarcinoma cells growth. Cp water and ethanol extracts significantly inhibited the growth of the three cancer cell lines in vitro, at 100 µg·mL−1. Flash chromatography of the Cp ethanol extract, devoid of c-phycocyanin and allophycocyanin, enabled the collection of eight fractions, four of which strongly inhibited cancer cells growth at 100 µg·mL−1. Particularly, two fractions inhibited more than 90% of the melanoma cells growth, one inducing apoptosis in the three cancer cells lines. The detailed analysis of Cp pigment composition resulted in the discrimination of 17 molecules, ten of which were unequivocally identified by high resolution mass spectrometry. Pheophorbide a, β-cryptoxanthin and zeaxanthin were the three main pigments or derivatives responsible for the strong cytotoxicity of Cp fractions in cancer cells. These data point to Cyanophora paradoxa as a new microalgal source to purify potent anticancer pigments, and demonstrate for the first time the strong antiproliferative activity of zeaxanthin and β-cryptoxanthin in melanoma cells. PMID:24189278

  7. miR-17 regulates melanoma cell motility by inhibiting the translation of ETV1.

    PubMed

    Cohen, Ronit; Greenberg, Eyal; Nemlich, Yael; Schachter, Jacob; Markel, Gal

    2015-08-01

    Melanoma is an aggressive malignancy with a high metastatic potential. microRNA-17 (miR-17) is a member of the oncogenic miR-17/92 cluster. Here we study the effect of miR-17 on melanoma cell motility. Over expression of the mature or pri-microRNA form of miR-17 in WM-266-4 and 624mel melanoma lines enhances cell motility, evident in both wound healing and transwell migration assays. TargetScan algorithm predicts the PEA3-subfamily member ETV1 as a direct target of miR-17. Indeed, a 3-4-fold decrease of ETV1 protein levels are observed following miR-17 transfection into the various melanoma lines, with no significant change in ETV1 mRNA expression. Dual luciferase experiments demonstrate direct binding of miR-17 to the 3'-untranslated region of ETV1, confirmed by abolishing point mutations in the putative binding site. These combined results suggest regulation of ETV1 by miR-17 by a direct translational repression. Further, in both melanoma cell lines ETV1 knockdown by selective siRNA successfully pheno-copies the facilitated cell migration, while overexpression of ETV1 inhibits cell motility and migration. Altered ETV1 expression does not affect melanoma net-proliferation. In conclusion, we show a new role for miR-17 in melanoma, facilitating cell motility, by targeting the translation of ETV1 protein, which may support the development of metastasis. PMID:26158900

  8. Epigenetic deregulation of TCF21 inhibits metastasis suppressor KISS1 in metastatic melanoma.

    PubMed

    Arab, Khelifa; Smith, Laura T; Gast, Andreas; Weichenhan, Dieter; Huang, Joseph Po-Hsien; Claus, Rainer; Hielscher, Thomas; Espinosa, Allan V; Ringel, Matthew D; Morrison, Carl D; Schadendorf, Dirk; Kumar, Rajiv; Plass, Christoph

    2011-10-01

    Metastatic melanoma is a fatal disease due to the lack of successful therapies and biomarkers for early detection and its incidence has been increasing. Genetic studies have defined recurrent chromosomal aberrations, suggesting the location of either tumor suppressor genes or oncogenes. Transcription factor 21 (TCF21) belongs to the class A of the basic helix-loop-helix family with reported functions in early lung and kidney development as well as tumor suppressor function in the malignancies of the lung and head and neck. In this study, we combined quantitative DNA methylation analysis in patient biopsies and in their derived cell lines to demonstrate that TCF21 expression is downregulated in metastatic melanoma by promoter hypermethylation and TCF21 promoter DNA methylation is correlated with decreased survival in metastatic skin melanoma patients. In addition, the chromosomal location of TCF21 on 6q23-q24 coincides with the location of a postulated metastasis suppressor in melanoma. Functionally, TCF21 binds the promoter of the melanoma metastasis-suppressing gene, KiSS1, and enhances its gene expression through interaction with E12, a TCF3 isoform and with TCF12. Loss of TCF21 expression results in loss of KISS1 expression through loss of direct interaction of TCF21 at the KISS1 promoter. Finally, overexpression of TCF21 inhibits motility of C8161 melanoma cells. These data suggest that epigenetic downregulation of TCF21 is functionally involved in melanoma progression and that it may serve as a biomarker for aggressive tumor behavior. PMID:21771727

  9. Epigenetic deregulation of TCF21 inhibits metastasis suppressor KISS1 in metastatic melanoma

    PubMed Central

    Arab, Khelifa; Smith, Laura T.; Gast, Andreas; Weichenhan, Dieter; Huang, Joseph Po-Hsien; Claus, Rainer; Hielscher, Thomas; Espinosa, Allan V.; Ringel, Matthew D.; Morrison, Carl D.; Schadendorf, Dirk; Kumar, Rajiv; Plass, Christoph

    2011-01-01

    Metastatic melanoma is a fatal disease due to the lack of successful therapies and biomarkers for early detection and its incidence has been increasing. Genetic studies have defined recurrent chromosomal aberrations, suggesting the location of either tumor suppressor genes or oncogenes. Transcription factor 21 (TCF21) belongs to the class A of the basic helix-loop-helix family with reported functions in early lung and kidney development as well as tumor suppressor function in the malignancies of the lung and head and neck. In this study, we combined quantitative DNA methylation analysis in patient biopsies and in their derived cell lines to demonstrate that TCF21 expression is downregulated in metastatic melanoma by promoter hypermethylation and TCF21 promoter DNA methylation is correlated with decreased survival in metastatic skin melanoma patients. In addition, the chromosomal location of TCF21 on 6q23–q24 coincides with the location of a postulated metastasis suppressor in melanoma. Functionally, TCF21 binds the promoter of the melanoma metastasis-suppressing gene, KiSS1, and enhances its gene expression through interaction with E12, a TCF3 isoform and with TCF12. Loss of TCF21 expression results in loss of KISS1 expression through loss of direct interaction of TCF21 at the KISS1 promoter. Finally, overexpression of TCF21 inhibits motility of C8161 melanoma cells. These data suggest that epigenetic downregulation of TCF21 is functionally involved in melanoma progression and that it may serve as a biomarker for aggressive tumor behavior. PMID:21771727

  10. [Melanoma].

    PubMed

    Uhara, Hisashi

    2016-04-01

    Since 2011, several effective drugs for patients with metastatic melanoma, including BRAF inhibitors, MEK inhibitors, and immune checkpoint inhibitors, have been approved. The combination of BRAF and MEK inhibitors achieve response rates of 70% and a median progression-free survival of >11 months in patients. The combination of ipilimumab and nivolumab has shown response rates of up to 60-70% and a median progression-free survival of 11-14 months, despite increased toxicities. Moreover, many clinical trials for new combination therapies are still ongoing. PMID:27220785

  11. MiR-21 Enhances Melanoma Invasiveness via Inhibition of Tissue Inhibitor of Metalloproteinases 3 Expression: In Vivo Effects of MiR-21 Inhibitor

    PubMed Central

    Martin del Campo, Sara E.; Latchana, Nicholas; Levine, Kala M.; Grignol, Valerie P.; Fairchild, Ene T.; Jaime-Ramirez, Alena Cristina; Dao, Thao-Vi; Karpa, Volodymyr I.; Carson, Mary; Ganju, Akaansha; Chan, Anthony N.; Carson III, William E.

    2015-01-01

    Metastatic melanoma is the most aggressive form of this cancer. It is important to understand factors that increase or decrease metastatic activity in order to more effectively research and implement treatments for melanoma. Increased cell invasion through the extracellular matrix is required for metastasis and is enhanced by matrix metalloproteinases (MMPs). Tissue inhibitor of metalloproteinases 3 (TIMP3) inhibits MMP activity. It was previously shown by our group that miR-21, a potential regulator of TIMP3, is over-expressed in cutaneous melanoma. It was therefore hypothesized that increased levels of miR-21 expression would lead to decreased expression of TIMP3 and thereby enhance the invasiveness of melanoma cells. miR-21 over-expression in the melanoma cell lines WM1552c, WM793b, A375 and MEL 39 was accomplished via transfection with pre-miR-21. Immunoblot analysis of miR-21-overexpressing cell lines revealed reduced expression of TIMP3 as compared to controls. This in turn led to a significant increase in the invasiveness of the radial growth phase cell line WM1552c and the vertical growth phase cell line WM793b (p < 0.05), but not in the metastatic cell lines A375 or MEL 39. The proliferation and migration of miR-21 over-expressing cell lines was not affected. Reduced expression of TIMP3 was achieved by siRNA knockdown and significantly enhanced invasion of melanoma cell lines, mimicking the effects of miR-21 over-expression. Treatment of tumor cells with a linked nucleic acid antagomir to miR-21 inhibited tumor growth and increased tumor expression of TIMP3 in vivo in 01B74 Athymic NCr-nu/nu mice. Intra-tumoral injections of anti-miR-21 produced similar effects. This data shows that increased expression of miR-21 enhanced the invasive potential of melanoma cell lines through TIMP3 inhibition. Therefore, inhibition of miR-21 in melanoma may reduce melanoma invasiveness. PMID:25587717

  12. Silymarin inhibits melanoma cell growth both in vitro and in vivo by targeting cell cycle regulators, angiogenic biomarkers and induction of apoptosis.

    PubMed

    Vaid, Mudit; Singh, Tripti; Prasad, Ram; Katiyar, Santosh K

    2015-11-01

    Cutaneous malignant melanoma is the leading cause of death from skin diseases and is often associated with activating mutations of the proto-oncogene BRAF. To develop more effective strategies for the prevention or treatment of melanoma, we have examined the inhibitory effects of silymarin, a flavanoid from Silybum marianum, on melanoma cells. Using A375 (BRAF-mutated) and Hs294t (non BRAF-mutated but highly metastatic) human melanoma cell lines, we found that in vitro treatment with silymarin resulted in a dose-dependent: (i) reduction in cell viability; (ii) enhancement of either Go/G1 (A375) or G2-M (Hs294t) phase cell cycle arrest with corresponding alterations in cyclins and cyclin-dependent kinases; and (iii) induction of apoptosis. The silymarin-induced apoptosis of human melanoma cells was associated with a reduction in the levels of anti-apoptotic proteins (Bcl-2 and Bcl-xl), an increase in the levels of pro-apoptotic protein (Bax), and activation of caspases. Further, oral administration of silymarin (500 mg/kg body weight/2× a week) significantly inhibited (60%, P < 0.01) the growth of BRAF-mutated A375 melanoma tumor xenografts, and this was associated with: (i) inhibition of cell proliferation; (ii) induction of apoptosis of tumor cells; (iii) alterations in cell cycle regulatory proteins; and (iv) reduced expression of tumor angiogenic biomarkers in tumor xenograft tissues. These results indicate that silymarin may have a chemotherapeutic effect on human melanoma cell growth and warrant its further evaluation. PMID:25174976

  13. IGF-1R inhibition induces schedule-dependent sensitization of human melanoma to temozolomide.

    PubMed

    Ramcharan, Roger; Aleksic, Tamara; Kamdoum, Wilfride Petnga; Gao, Shan; Pfister, Sophia X; Tanner, Jordan; Bridges, Esther; Asher, Ruth; Watson, Amanda J; Margison, Geoffrey P; Woodcock, Mick; Repapi, Emmanouela; Li, Ji-Liang; Middleton, Mark R; Macaulay, Valentine M

    2015-11-24

    Prior studies implicate type 1 IGF receptor (IGF-1R) in mediating chemo-resistance. Here, we investigated whether IGF-1R influences response to temozolomide (TMZ), which generates DNA adducts that are removed by O6-methylguanine-DNA methyltransferase (MGMT), or persist causing replication-associated double-strand breaks (DSBs). Initial assessment in 10 melanoma cell lines revealed that TMZ resistance correlated with MGMT expression (r = 0.79, p = 0.009), and in MGMT-proficient cell lines, with phospho-IGF-1R (r = 0.81, p = 0.038), suggesting that TMZ resistance associates with IGF-1R activation. Next, effects of IGF-1R inhibitors (IGF-1Ri) AZ3801 and linsitinib (OSI-906) were tested on TMZ-sensitivity, cell cycle progression and DSB induction. IGF-1Ri sensitized BRAF wild-type and mutant melanoma cells to TMZ in vitro, an effect that was independent of MGMT. Cells harboring wild-type p53 were more sensitive to IGF-1Ri, and showed schedule-dependent chemo-sensitization that was most effective when IGF-1Ri followed TMZ. This sequence sensitized to clinically-achievable TMZ concentrations and enhanced TMZ-induced apoptosis. Simultaneous or prior IGF-1Ri caused less effective chemo-sensitization, associated with increased G1 population and reduced accumulation of TMZ-induced DSBs. Clinically relevant sequential (TMZ → IGF-1Ri) treatment was tested in mice bearing A375M (V600E BRAF, wild-type p53) melanoma xenografts, achieving peak plasma/tumor IGF-1Ri levels comparable to clinical Cmax, and inducing extensive intratumoral apoptosis. TMZ or IGF-1Ri caused minor inhibition of tumor growth (gradient reduction 13%, 25% respectively), while combination treatment caused supra-additive growth delay (72%) that was significantly different from control (p < 0.01), TMZ (p < 0.01) and IGF-1Ri (p < 0.05) groups. These data highlight the importance of scheduling when combining IGF-1Ri and other targeted agents with drugs that induce replication-associated DNA damage. PMID

  14. IGF-1R inhibition induces schedule-dependent sensitization of human melanoma to temozolomide

    PubMed Central

    Ramcharan, Roger; Aleksic, Tamara; Kamdoum, Wilfride Petnga; Gao, Shan; Pfister, Sophia X.; Tanner, Jordan; Bridges, Esther; Asher, Ruth; Watson, Amanda J.; Margison, Geoffrey P.; Woodcock, Mick; Repapi, Emmanouela; Li, Ji-Liang; Middleton, Mark R.; Macaulay, Valentine M.

    2015-01-01

    Prior studies implicate type 1 IGF receptor (IGF-1R) in mediating chemo-resistance. Here, we investigated whether IGF-1R influences response to temozolomide (TMZ), which generates DNA adducts that are removed by O6-methylguanine-DNA methyltransferase (MGMT), or persist causing replication-associated double-strand breaks (DSBs). Initial assessment in 10 melanoma cell lines revealed that TMZ resistance correlated with MGMT expression (r = 0.79, p = 0.009), and in MGMT-proficient cell lines, with phospho-IGF-1R (r = 0.81, p = 0.038), suggesting that TMZ resistance associates with IGF-1R activation. Next, effects of IGF-1R inhibitors (IGF-1Ri) AZ3801 and linsitinib (OSI-906) were tested on TMZ-sensitivity, cell cycle progression and DSB induction. IGF-1Ri sensitized BRAF wild-type and mutant melanoma cells to TMZ in vitro, an effect that was independent of MGMT. Cells harboring wild-type p53 were more sensitive to IGF-1Ri, and showed schedule-dependent chemo-sensitization that was most effective when IGF-1Ri followed TMZ. This sequence sensitized to clinically-achievable TMZ concentrations and enhanced TMZ-induced apoptosis. Simultaneous or prior IGF-1Ri caused less effective chemo-sensitization, associated with increased G1 population and reduced accumulation of TMZ-induced DSBs. Clinically relevant sequential (TMZ → IGF-1Ri) treatment was tested in mice bearing A375M (V600E BRAF, wild-type p53) melanoma xenografts, achieving peak plasma/tumor IGF-1Ri levels comparable to clinical Cmax, and inducing extensive intratumoral apoptosis. TMZ or IGF-1Ri caused minor inhibition of tumor growth (gradient reduction 13%, 25% respectively), while combination treatment caused supra-additive growth delay (72%) that was significantly different from control (p < 0.01), TMZ (p < 0.01) and IGF-1Ri (p < 0.05) groups. These data highlight the importance of scheduling when combining IGF-1Ri and other targeted agents with drugs that induce replication-associated DNA damage. PMID

  15. Inhibition of melanocortin 1 receptor slows melanoma growth, reduces tumor heterogeneity and increases survival.

    PubMed

    Kansal, Rita G; McCravy, Matthew S; Basham, Jacob H; Earl, Joshua A; McMurray, Stacy L; Starner, Chelsey J; Whitt, Michael A; Albritton, Lorraine M

    2016-05-01

    Melanoma risk is increased in patients with mutations of melanocortin 1 receptor (MC1R) yet the basis for the increased risk remains unknown. Here we report in vivo evidence supporting a critical role for MC1R in regulating melanoma tumor growth and determining overall survival time. Inhibition of MC1R by its physiologically relevant competitive inhibitor, agouti signaling protein (ASIP), reduced melanin synthesis and morphological heterogeneity in murine B16-F10 melanoma cells. In the lungs of syngeneic C57BL/6 mice, mCherry-marked, ASIP-secreting lung tumors inhibited MC1R on neighboring tumors lacking ASIP in a dose dependent manner as evidenced by a proportional loss of pigment in tumors from mice injected with 1:1, 3:1 and 4:1 mixtures of parental B16-F10 to ASIP-expressing tumor cells. ASIP-expressing B16-F10 cells formed poorly pigmented tumors in vivo that correlated with a 20% longer median survival than those bearing parental B16-F10 tumors (p=0.0005). Mice injected with 1:1 mixtures also showed survival benefit (p=0.0054), whereas injection of a 4:1 mixture showed no significant difference in survival. The longer survival time of mice bearing ASIP-expressing tumors correlated with a significantly slower growth rate than parental B16-F10 tumors as judged by quantification of numbers of tumors and total tumor load (p=0.0325), as well as a more homogeneous size and morphology of ASIP-expressing lung tumors. We conclude that MC1R plays an important role in regulating melanoma growth and morphology. Persistent inhibition of MC1R provided a significant survival advantage resulting in part from slower tumor growth, establishing MC1R as a compelling new molecular target for metastatic melanoma. PMID:27028866

  16. Raspberry pulp polysaccharides inhibit tumor growth via immunopotentiation and enhance docetaxel chemotherapy against malignant melanoma in vivo.

    PubMed

    Yang, Yong-Jing; Xu, Han-Mei; Suo, You-Rui

    2015-09-01

    It has been reported previously that the systemic efficacy of chemotherapeutic agents is substantially restricted for some cancer types, including malignant melanoma. Therefore, the development of more effective treatment modalities remains a critical, albeit elusive, goal in anticancer therapy. The study presented here evaluates the antitumor activity of raspberry pulp polysaccharides (RPPs) against malignant melanoma using a murine tumor-bearing model. Furthermore, the underlying mechanism of this antitumor activity has also been investigated. The results show that while RPP exhibits no direct cytotoxic effect on HT-29, MGC-803, HeLa, Bel-7402, L02 and B16F10 cells in vitro, it does demonstrate a dose-dependent growth inhibition of melanoma in vivo with an inhibition ratio of 59.95% at a dose of 400 mg kg(-1). Besides this, the body weight and spleen index in tumor-bearing mice have also been improved in RPP-treated groups. RPP is also found to induce splenocyte proliferation and is able to upregulate the activity of immune-related enzymes, including acid phosphatase (ACP), alkaline phosphatase (AKP), lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in the spleen of tumor-bearing mice. The levels of tumor necrosis factor α (TNF-α), interferon γ (IFN-γ) and interleukin 2 (IL-2) in the serum of tumor-bearing mice show to be effectively increased upon RPP treatment. Histopathological analyses show that RPP induces tumor tissue necrosis by increasing inflammatory cell infiltration and causes no lesions to liver and kidney tissues. Remarkably, RPP further enhances the antitumor effect of the chemotherapeutic drug docetaxel and alleviates docetaxel-induced liver and kidney lesions in tumor-bearing mice. These findings indicate that RPP exhibits antitumor activity in vivo against malignant melanoma, partly by enhancing the cellular immune response of the host organism. In summary, RPP features critical properties to potentially find use as an

  17. Inhibition of Autophagy Enhances Curcumin United light irradiation-induced Oxidative Stress and Tumor Growth Suppression in Human Melanoma Cells.

    PubMed

    Niu, Tianhui; Tian, Yan; Mei, Zhusong; Guo, Guangjin

    2016-01-01

    Malignant melanoma is the most aggressive form of skin carcinoma, which possesses fast propagating and highly invasive characteristics. Curcumin is a natural phenol compound that has various biological activities, such as anti-proliferative and apoptosis-accelerating impacts on tumor cells. Unfortunately, the therapeutical activities of Cur are severely hindered due to its extremely low bioavailability. In this study, a cooperative therapy of low concentration Cur combined with red united blue light irradiation was performed to inspect the synergistic effects on the apoptosis, proliferation and autophagy in human melanoma A375 cell. The results showed that red united blue light irradiation efficaciously synergized with Cur to trigger oxidative stress-mediated cell death, induce apoptosis and inhibit cell proliferation. Meanwhile, Western blotting revealed that combined disposure induced the formation of autophagosomes. Conversely, inhibition of the autophagy enhanced apoptosis, obstructed cell cycle arrest and induced reversible proliferation arrest to senescence. These findings suggest that Cur combined with red united blue light irradiation could generate photochemo-preventive effects via enhancing apoptosis and triggering autophagy, and pharmacological inhibition of autophagy convert reversible arrested cells to senescence, therefore reducing the possibility that damaged cells might escape programmed death. PMID:27502897

  18. Inhibition of Autophagy Enhances Curcumin United light irradiation-induced Oxidative Stress and Tumor Growth Suppression in Human Melanoma Cells

    PubMed Central

    Niu, Tianhui; Tian, Yan; Mei, Zhusong; Guo, Guangjin

    2016-01-01

    Malignant melanoma is the most aggressive form of skin carcinoma, which possesses fast propagating and highly invasive characteristics. Curcumin is a natural phenol compound that has various biological activities, such as anti-proliferative and apoptosis-accelerating impacts on tumor cells. Unfortunately, the therapeutical activities of Cur are severely hindered due to its extremely low bioavailability. In this study, a cooperative therapy of low concentration Cur combined with red united blue light irradiation was performed to inspect the synergistic effects on the apoptosis, proliferation and autophagy in human melanoma A375 cell. The results showed that red united blue light irradiation efficaciously synergized with Cur to trigger oxidative stress-mediated cell death, induce apoptosis and inhibit cell proliferation. Meanwhile, Western blotting revealed that combined disposure induced the formation of autophagosomes. Conversely, inhibition of the autophagy enhanced apoptosis, obstructed cell cycle arrest and induced reversible proliferation arrest to senescence. These findings suggest that Cur combined with red united blue light irradiation could generate photochemo-preventive effects via enhancing apoptosis and triggering autophagy, and pharmacological inhibition of autophagy convert reversible arrested cells to senescence, therefore reducing the possibility that damaged cells might escape programmed death. PMID:27502897

  19. Compounds isolated from the aerial part of Crataegus azarolus inhibit growth of B16F10 melanoma cells and exert a potent inhibition of the melanin synthesis.

    PubMed

    Mustapha, Nadia; Bzéouich, Imèn Mokdad; Ghedira, Kamel; Hennebelle, Thierry; Chekir-Ghedira, Leila

    2015-02-01

    Poor therapeutic results have been reported for treatment of malignant melanoma; therefore in this study, we have investigated inhibitory capacity of vitexin-2''-O-rhamnoside as well as the extract from which it was isolated, i.e. the ethyl acetate extract obtained from the leaves of Crataegus azarolus, on mouse melanoma (B16F10) proliferation. Cell viability was determined using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475nm. Ethyl acetate extract and vitexin-2''-O-rhamnoside exhibited significant anti-proliferative activity against B16F10 melanoma cells after incubation for 48hours with IC50s of 50μg/mL and 20μM, respectively. Furthermore, these two compounds have the ability to reduce the melanin content by inhibiting the tyrosinase activity of B16F10 cells. Thus, further investigations are merited to ascertain their potential application in treating hyperpigmentation disorders. PMID:25661350

  20. Activity of the MEK Inhibitor Trametinib (GSK1120212) in Advanced Melanoma in a Phase I, Dose-escalation Trial

    PubMed Central

    Falchook, Gerald S; Lewis, Karl D; Infante, Jeffrey R; Gordon, Michael S; Vogelzang, Nicholas J; DeMarini, Douglas J; Sun, Peng; Moy, Christopher; Szabo, Stephen A.; Roadcap, Lori T; Peddareddigari, Vijay G R; Lebowitz, Peter F; Le, Ngocdiep T; Burris, Howard A; Messersmith, Wells A; O'Dwyer, Peter J; Kim, Kevin B.; Flaherty, Keith; Bendell, Johanna C.; Gonzalez, Rene; Kurzrock, Razelle; Fecher, Leslie A

    2014-01-01

    Summary Purpose The mitogen-activated extracellular signal-related kinase kinase (MEK) is a member of the RAS/RAF/MEK/ERK signalling cascade, which is commonly activated in melanoma. Direct inhibition of MEK inhibits ERK signalling. Methods We conducted a multicentre, first-in-human, three-part study (dose escalation, cohort expansion, and pharmacodynamic evaluation) to evaluate the oral small-molecule MEK inhibitor trametininb (GSK1120212) in advanced cancer. Intermittent and continuous dosing regimens were evaluated. Safety and efficacy data in patients with melanoma are presented here, with exploratory analyses of available tumour tissues performed on an Illumina genotyping platform. This completed study is registered with ClinicalTrials.gov, number NCT00687622. Findings Ninety-seven melanoma patients, including 81 with cutaneous or unknown primary melanoma (36 BRAF-mutant, 39 BRAF wild-type, six BRAF status unknown) and 16 uveal melanoma patients were enrolled. The most common treatment-related adverse events were rash/dermatitis acneiform (80 out of 97; 82%) and diarrhoea (n=44; 45%), most of which were grade 2 or lower. No cutaneous squamous cell carcinomas were observed. Among the 36 BRAF-mutant patients, 30 were BRAF-inhibitor naïve. Among these 30 patients, 2 complete responses (CRs) and 10 partial responses (PRs) were observed (unconfirmed response rate=40%) including 2 confirmed CRs and 8 confirmed PRs (confirmed response rate=33%); the median progression-free survival was 5·7 months (95% CI, 4·0–7·4). Among the 6 BRAF-mutant patients who received prior BRAF inhibitor therapy, 1 unconfirmed PR was observed. Among 39 patients with BRAF wild-type melanoma, 4 PRs (all confirmed) were observed (confirmed response rate=10%). Conclusions To our knowledge, this is the first demonstration of substantial clinical activity by a MEK inhibitor in melanoma. These data suggest that MEK is a valid therapeutic target. PMID:22805292

  1. VEGF Secretion is Inhibited by Interferon-Alpha in Several Melanoma Cell Lines

    PubMed Central

    Raig, Ene T.; Jones, Natalie B.; Varker, Kimberly A.; Benniger, Kristen; Go, Michael R.; Biber, Jennifer L.; Lesinski, Gregory B.

    2008-01-01

    Interferon-alpha (IFN-α) is employed in the treatment of malignant melanoma; however, it mediates regression of disease in only 10–15% of patients. Currently, its mechanism of action is uncharacterized. Low-dose IFN-α exerts anti-angiogenic effects when used in the treatment of life-threatening hemangiomas of infancy, suggesting anti-angiogenesis as a mechanism of action. IFN-α may exert its anti-tumor effect in the setting of advanced malignancy by inhibiting the secretion of vascular endothelial growth factor (VEGF), a pro-angiogenic substance. We hypothesized that IFN-α would decrease the release of VEGF by melanoma tumors. We studied the effect of IFN-α on VEGF production in nine human melanoma cell lines. We also examined VEGF levels in 49 patients with advanced malignancies who received low-dose IFN-α and interleukin-12 (IL-12) on an NCI-sponsored phase I trial. Human melanoma cell lines produced varying amounts of VEGF in vitro (60–1500 pg/mL at 48 h). Certain melanoma cell lines such as 18105 MEL secreted low levels of VEGF (152 pg/mL) after 48 h of culture, whereas other lines secreted very high levels (FO-1 3,802 pg/mL). Treatment of melanoma cells with IFN-α (2000 U/mL) decreased VEGF secretion by 40–60% in VEGF-high cell lines; however, this effect was not demonstrated in VEGF-low cell lines. In cancer patients, pretreatment VEGF plasma levels varied from 471 to 4200 pg/mL. A decrease in VEGF plasma levels after treatment directly correlated with the number of treatment cycles administered (Pearson correlation, p = 0.04). In summary, IFN-α inhibits VEGF secretion by melanoma cell lines in vitro and may have similar actions in malignancies that respond to IFN-α treatment. PMID:18771339

  2. Involvement of autophagy in recombinant human arginase-induced cell apoptosis and growth inhibition of malignant melanoma cells.

    PubMed

    Wang, Ziyu; Shi, Xunlong; Li, Yubin; Zeng, Xian; Fan, Jiajun; Sun, Yun; Xian, Zongshu; Zhang, Guoping; Wang, Shaofei; Hu, Haifeng; Ju, Dianwen

    2014-03-01

    Recombinant human arginase (rhArg) has been developed for arginine derivation therapy of cancer and is currently in clinical trials for a variety of malignant solid tumors. In this study, we reported for the first time that rhArg could induce obvious autophagy in human melanoma cells; inhibition of autophagy by chloroquine (CQ) significantly increased rhArg-induced cell apoptosis and growth inhibition of A375 cells. A significant increase in mitochondrial membrane potential loss and elevated intracellular reactive oxygen species (ROS) levels were detected in A375 cells after rhArg treatment when compared with control. Membrane transition inhibitor cyclosporine A blocked autophagy and accelerated cell death induced by rhArg, indicating that rhArg induced autophagy via mitochondria pathway. Furthermore, antioxidant N-acetyl-L-cysteine suppressed rhArg-induced autophagy and rescued cells from cell growth inhibition, suggesting that ROS played an important role in rhArg-induced A375 cell growth inhibition and autophagy. Akt/mTOR signaling pathway was involved in autophagy induced by rhArg in a time-dependent manner. Moreover, rhArg could induce ERK1/2 activation in a dose- and time-dependent manner and rhArg-induced autophagy was attenuated when p-ERK1/2 was inhibited by MEK 1/2 inhibitor, U0126. Taken together, this study provides new insight into the molecular mechanism of autophagy involved in rhArg-induced cell apoptosis and growth inhibition, which facilitates the development of rhArg in combination with CQ as a potential therapy for malignant melanoma. PMID:23917632

  3. Berberine, an isoquinoline alkaloid, inhibits melanoma cancer cell migration by reducing the expressions of cyclooxygenase-2, prostaglandin E2 and prostaglandin E2 receptors

    PubMed Central

    Singh, Tripti; Vaid, Mudit; Katiyar, Nandan; Sharma, Samriti; Katiyar, Santosh K.

    2011-01-01

    Melanoma is the leading cause of death from skin disease due, in large part, to its propensity to metastasize. We have examined the effect of berberine, an isoquinoline alkaloid, on human melanoma cancer cell migration and the molecular mechanisms underlying these effects using melanoma cell lines, A375 and Hs294. Using an in vitro cell migration assay, we show that over expression of cyclooxygenase (COX)-2, its metabolite prostaglandin E2 (PGE2) and PGE2 receptors promote the migration of cells. We found that treatment of A375 and Hs294 cells with berberine resulted in concentration-dependent inhibition of migration of these cells, which was associated with a reduction in the levels of COX-2, PGE2 and PGE2 receptors (EP2 and EP4). Treatment of cells with celecoxib, a COX-2 inhibitor, or transient transfection of cells with COX-2 small interfering RNA, also inhibited cell migration. Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of COX-2 or PGE2, enhanced cell migration, whereas berberine inhibited TPA- or PGE2-promoted cell migration. Berberine reduced the basal levels as well as PGE2-stimulated expression levels of EP2 and EP4. Treatment of the cells with the EP4 agonist stimulated cell migration and berberine blocked EP4 agonist-induced cell migration activity. Moreover, berberine inhibited the activation of nuclear factor-kappa B (NF-κB), an upstream regulator of COX-2, in A375 cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, inhibited cell migration. Together, these results indicate for the first time that berberine inhibits melanoma cell migration, an essential step in invasion and metastasis, by inhibition of COX-2, PGE2 and PGE2 receptors. PMID:20974686

  4. Wnt/β-catenin signaling and AXIN1 regulate apoptosis triggered by inhibition of the mutant kinase BRAFV600E in human melanoma.

    PubMed

    Biechele, Travis L; Kulikauskas, Rima M; Toroni, Rachel A; Lucero, Olivia M; Swift, Reyna D; James, Richard G; Robin, Nick C; Dawson, David W; Moon, Randall T; Chien, Andy J

    2012-01-10

    Because the Wnt/β-catenin signaling pathway is linked to melanoma pathogenesis and to patient survival, we conducted a kinome small interfering RNA (siRNA) screen in melanoma cells to expand our understanding of the kinases that regulate this pathway. We found that BRAF signaling, which is constitutively activated in many melanomas by the BRAF(V600E) mutation, inhibits Wnt/β-catenin signaling in human melanoma cells. Because inhibitors of BRAF(V600E) show promise in ongoing clinical trials, we investigated whether altering Wnt/β-catenin signaling might enhance the efficacy of the BRAF(V600E) inhibitor PLX4720. We found that endogenous β-catenin was required for PLX4720-induced apoptosis of melanoma cells and that activation of Wnt/β-catenin signaling synergized with PLX4720 to decrease tumor growth in vivo and to increase apoptosis in vitro. This synergistic enhancement of apoptosis correlated with reduced abundance of an endogenous negative regulator of β-catenin, AXIN1. In support of the hypothesis that AXIN1 is a mediator rather than a marker of apoptosis, siRNA directed against AXIN1 rendered resistant melanoma cell lines susceptible to apoptosis in response to treatment with a BRAF(V600E) inhibitor. Thus, Wnt/β-catenin signaling and AXIN1 may regulate the efficacy of inhibitors of BRAF(V600E), suggesting that manipulation of the Wnt/β-catenin pathway could be combined with BRAF inhibitors to treat melanoma. PMID:22234612

  5. Concurrent MEK and autophagy inhibition is required to restore cell death associated danger-signalling in Vemurafenib-resistant melanoma cells.

    PubMed

    Martin, S; Dudek-Perić, A M; Maes, H; Garg, A D; Gabrysiak, M; Demirsoy, S; Swinnen, J V; Agostinis, P

    2015-02-01

    Vemurafenib (PLX4032), an inhibitor of BRAF(V600E), has demonstrated significant clinical anti-melanoma effects. However, the majority of treated patients develop resistance, due to a variety of molecular mechanisms including MAPK reactivation through MEK. The induction of a cancer cell death modality associated with danger-signalling resulting in surface mobilization of crucial damage-associated-molecular-patterns (DAMPs), e.g. calreticulin (CRT) and heat shock protein-90 (HSP90), from dying cells, is emerging to be crucial for therapeutic success. Both cell death and danger-signalling are modulated by autophagy, a key adaptation mechanism stimulated during melanoma progression. However, whether melanoma cell death induced by MAPK inhibition is associated with danger-signalling, and the reliance of these mechanisms on autophagy, has not yet been scrutinized. Using a panel of isogenic PLX4032-sensitive and resistant melanoma cell lines we show that PLX4032-induced caspase-dependent cell death and DAMPs exposure in the drug-sensitive cells, but failed to do so in the drug-resistant cells, displaying heightened MEK activation. MEK inhibitor, U0126, treatment sensitized PLX4032-resistant cells to death and re-established their danger-signalling capacity. Only melanoma cells exposing death-induced danger-signals were phagocytosed and induced DC maturation. Although the PLX4032-resistant melanoma cells displayed higher basal and drug-induced autophagy, compromising autophagy, pharmacologically or by ATG5 knockdown, was insufficient to re-establish their PLX4032 sensitivity. Interestingly, autophagy abrogation was particularly efficacious in boosting cell death and ecto-CRT/ecto-HSP90 in PLX4032-resistant cells upon blockage of MEK hyper-activation by U0126. Thus combination of MEK inhibitors with autophagy blockers may represent a novel treatment regime to increase both cell death and danger-signalling in Vemurafenib-resistant metastatic melanoma. PMID:25529535

  6. Activin receptor-like kinase5 inhibition suppresses mouse melanoma by ubiquitin degradation of Smad4, thereby derepressing eomesodermin in cytotoxic T lymphocytes

    PubMed Central

    Yoon, Jeong-Hwan; Jung, Su Myung; Park, Seok Hee; Kato, Mitsuyasu; Yamashita, Tadashi; Lee, In-Kyu; Sudo, Katsuko; Nakae, Susumu; Han, Jin Soo; Kim, Ok-Hee; Oh, Byung-Chul; Sumida, Takayuki; Kuroda, Masahiko; Ju, Ji-Hyeon; Jung, Kyeong Cheon; Park, Seong Hoe; Kim, Dae-Kee; Mamura, Mizuko

    2013-01-01

    Varieties of transforming growth factor-β (TGF-β) antagonists have been developed to intervene with excessive TGF-β signalling activity in cancer. Activin receptor-like kinase5 (ALK5) inhibitors antagonize TGF-β signalling by blocking TGF-β receptor-activated Smad (R-Smad) phosphorylation. Here we report the novel mechanisms how ALK5 inhibitors exert a therapeutic effect on a mouse B16 melanoma model. Oral treatment with a novel ALK5 inhibitor, EW-7197 (2.5 mg/kg daily) or a representative ALK5 inhibitor, LY-2157299 (75 mg/kg bid) suppressed the progression of melanoma with enhanced cytotoxic T-lymphocyte (CTL) responses. Notably, ALK5 inhibitors not only blocked R-Smad phosphorylation, but also induced ubiquitin-mediated degradation of the common Smad, Smad4 mainly in CD8+ T cells in melanoma-bearing mice. Accordingly, T-cell-specific deletion of Smad4 was sufficient to suppress the progression of melanoma. We further identified eomesodermin (Eomes), the T-box transcription factor regulating CTL functions, as a specific target repressed by TGF-β via Smad4 and Smad3 in CD8+ T cells. Thus, ALK5 inhibition enhances anti-melanoma CTL responses through ubiquitin-mediated degradation of Smad4 in addition to the direct inhibitory effect on R-Smad phosphorylation. PMID:24127404

  7. Activin receptor-like kinase5 inhibition suppresses mouse melanoma by ubiquitin degradation of Smad4, thereby derepressing eomesodermin in cytotoxic T lymphocytes.

    PubMed

    Yoon, Jeong-Hwan; Jung, Su Myung; Park, Seok Hee; Kato, Mitsuyasu; Yamashita, Tadashi; Lee, In-Kyu; Sudo, Katsuko; Nakae, Susumu; Han, Jin Soo; Kim, Ok-Hee; Oh, Byung-Chul; Sumida, Takayuki; Kuroda, Masahiko; Ju, Ji-Hyeon; Jung, Kyeong Cheon; Park, Seong Hoe; Kim, Dae-Kee; Mamura, Mizuko

    2013-11-01

    Varieties of transforming growth factor-β (TGF-β) antagonists have been developed to intervene with excessive TGF-β signalling activity in cancer. Activin receptor-like kinase5 (ALK5) inhibitors antagonize TGF-β signalling by blocking TGF-β receptor-activated Smad (R-Smad) phosphorylation. Here we report the novel mechanisms how ALK5 inhibitors exert a therapeutic effect on a mouse B16 melanoma model. Oral treatment with a novel ALK5 inhibitor, EW-7197 (2.5 mg/kg daily) or a representative ALK5 inhibitor, LY-2157299 (75 mg/kg bid) suppressed the progression of melanoma with enhanced cytotoxic T-lymphocyte (CTL) responses. Notably, ALK5 inhibitors not only blocked R-Smad phosphorylation, but also induced ubiquitin-mediated degradation of the common Smad, Smad4 mainly in CD8(+) T cells in melanoma-bearing mice. Accordingly, T-cell-specific deletion of Smad4 was sufficient to suppress the progression of melanoma. We further identified eomesodermin (Eomes), the T-box transcription factor regulating CTL functions, as a specific target repressed by TGF-β via Smad4 and Smad3 in CD8(+) T cells. Thus, ALK5 inhibition enhances anti-melanoma CTL responses through ubiquitin-mediated degradation of Smad4 in addition to the direct inhibitory effect on R-Smad phosphorylation. PMID:24127404

  8. The combination of glutamate receptor antagonist MK-801 with tamoxifen and its active metabolites potentiates their antiproliferative activity in mouse melanoma K1735-M2 cells

    SciTech Connect

    Ribeiro, Mariana P.C.; Nunes-Correia, Isabel; Santos, Armanda E.; Custódio, José B.A.

    2014-02-15

    Recent reports suggest that N-methyl-D-aspartate receptor (NMDAR) blockade by MK-801 decreases tumor growth. Thus, we investigated whether other ionotropic glutamate receptor (iGluR) antagonists were also able to modulate the proliferation of melanoma cells. On the other hand, the antiestrogen tamoxifen (TAM) decreases the proliferation of melanoma cells, and is included in combined therapies for melanoma. As the efficacy of TAM is limited by its metabolism, we investigated the effects of the NMDAR antagonist MK-801 in combination with TAM and its active metabolites, 4-hydroxytamoxifen (OHTAM) and endoxifen (EDX). The NMDAR blockers MK-801 and memantine decreased mouse melanoma K1735-M2 cell proliferation. In contrast, the NMDAR competitive antagonist APV and the AMPA and kainate receptor antagonist NBQX did not affect cell proliferation, suggesting that among the iGluR antagonists only the NMDAR channel blockers inhibit melanoma cell proliferation. The combination of antiestrogens with MK-801 potentiated their individual effects on cell biomass due to diminished cell proliferation, since it decreased the cell number and DNA synthesis without increasing cell death. Importantly, TAM metabolites combined with MK-801 promoted cell cycle arrest in G1. Therefore, the data obtained suggest that the activity of MK-801 and antiestrogens in K1735-M2 cells is greatly enhanced when used in combination. - Highlights: • MK-801 and memantine decrease melanoma cell proliferation. • The combination of MK-801 with antiestrogens inhibits melanoma cell proliferation. • These combinations greatly enhance the effects of the compounds individually. • MK-801 combined with tamoxifen active metabolites induces cell cycle arrest in G1. • The combination of MK-801 and antiestrogens is an innovative strategy for melanoma.

  9. Nicotinamide Inhibits Vasculogenic Mimicry, an Alternative Vascularization Pathway Observed in Highly Aggressive Melanoma

    PubMed Central

    Shalmon, Bruria; Kubi, Adva; Treves, Avraham J.; Shapira-Frommer, Ronnie; Avivi, Camilla; Ortenberg, Rona; Ben-Ami, Eytan; Schachter, Jacob; Besser, Michal J.; Markel, Gal

    2013-01-01

    Vasculogenic mimicry (VM) describes functional vascular channels composed only of tumor cells and its presence predicts poor prognosis in melanoma patients. Inhibition of this alternative vascularization pathway might be of clinical importance, especially as several anti-angiogenic therapies targeting endothelial cells are largely ineffective in melanoma. We show the presence of VM structures histologically in a series of human melanoma lesions and demonstrate that cell cultures derived from these lesions form tubes in 3D cultures ex vivo. We tested the ability of nicotinamide, the amide form of vitamin B3 (niacin), which acts as an epigenetic gene regulator through unique cellular pathways, to modify VM. Nicotinamide effectively inhibited the formation of VM structures and destroyed already formed ones, in a dose-dependent manner. Remarkably, VM formation capacity remained suppressed even one month after the complete withdrawal of Nicotimamid. The inhibitory effect of nicotinamide on VM formation could be at least partially explained by a nicotinamide-driven downregulation of vascular endothelial cadherin (VE-Cadherin), which is known to have a central role in VM. Further major changes in the expression profile of hundreds of genes, most of them clustered in biologically-relevant clusters, were observed. In addition, nicotinamide significantly inhibited melanoma cell proliferation, but had an opposite effect on their invasion capacity. Cell cycle analysis indicated moderate changes in apoptotic indices. Therefore, nicotinamide could be further used to unravel new biological mechanisms that drive VM and tumor progression. Targeting VM, especially in combination with anti-angiogenic strategies, is expected to be synergistic and might yield substantial anti neoplastic effects in a variety of malignancies. PMID:23451174

  10. New imidazoquinoxaline derivatives: Synthesis, biological evaluation on melanoma, effect on tubulin polymerization and structure-activity relationships.

    PubMed

    Zghaib, Zahraa; Guichou, Jean-François; Vappiani, Johanna; Bec, Nicole; Hadj-Kaddour, Kamel; Vincent, Laure-Anaïs; Paniagua-Gayraud, Stéphanie; Larroque, Christian; Moarbess, Georges; Cuq, Pierre; Kassab, Issam; Deleuze-Masquéfa, Carine; Diab-Assaf, Mona; Bonnet, Pierre-Antoine

    2016-06-01

    Microtubules are considered as important targets of anticancer therapy. EAPB0503 and its structural imidazo[1,2-a]quinoxaline derivatives are major microtubule-interfering agents with potent anticancer activity. In this study, the synthesis of several new derivatives of EAPB0503 is described, and the anticancer efficacy of 13 novel derivatives on A375 human melanoma cell line is reported. All new compounds show significant antiproliferative activity with IC50 in the range of 0.077-122μM against human melanoma cell line (A375). Direct inhibition of tubulin polymerization assay in vitro is also assessed. Results show that compounds 6b, 6e, 6g, and EAPB0503 highly inhibit tubulin polymerization with percentages of inhibition of 99%, 98%, 90%, and 84% respectively. Structure-activity relationship studies within the series are also discussed in line with molecular docking studies into the colchicine-binding site of tubulin. PMID:27094151

  11. Hinokitiol, a tropolone derivative, inhibits mouse melanoma (B16-F10) cell migration and in vivo tumor formation.

    PubMed

    Huang, Chien-Hsun; Lu, Shing-Hwa; Chang, Chao-Chien; Thomas, Philip Aloysius; Jayakumar, Thanasekaran; Sheu, Joen-Rong

    2015-01-01

    Invasion and metastasis are the major causes of treatment failure in patients with cancer. Hinokitiol, a natural bioactive compound found in Chamacyparis taiwanensis, has been used in hair tonics, cosmetics, and food as an antimicrobial agent. In this study, we investigated the effects and possible mechanisms of action of hinokitiol on migration by the metastatic melanoma cell line, B16-F10, in which matrix metalloproteinase-1 (MMP-1) is found to be highly- expressed. Treatment with hinokitiol revealed a concentration-dependent inhibition of migration of B16-F10 melanoma cells. Hinokitiol appeared to achieve this effect by reducing the expression of MMP-1 and by suppressing the phosphorylation of mitogen- activated protein kinase (MAPK) signaling molecules such as extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK and c-Jun N-terminal kinases (JNK). On the other hand, hinokitiol treatment reversed IκB-α degradation and inhibited the phosphorylation of p65 nuclear factor kappa B (NF-κB) and cJun in B16-F10 cells. In addition, hinokitiol suppressed the translocation of p65 NF-κB from the cytosol to the nucleus, suggesting reduced NF-κB activation. Consistent with these in vitro findings, our in vivo study demonstrated that hinokitiol treatment significantly reduced the total number of mouse lung metastatic nodules and improved histological alterations in B16-F10 injected C57BL/6 mice. These findings suggest that treatment of B16-F10 cells with hinokitiol significantly inhibits metastasis, possibly by blocking MMP-1 activation, MAPK signaling pathways and inhibition of the transcription factors, NF-κB and c-Jun, involved in cancer cell migration. These results may accelerate the development of novel therapeutic agents for the treatment of malignant cancers. PMID:25449038

  12. Apoptosis and melanogenesis in human melanoma cells induced by anthrax lethal factor inactivation of mitogen-activated protein kinase kinase

    NASA Astrophysics Data System (ADS)

    Koo, Han-Mo; Vanbrocklin, Matt; McWilliams, Mary Jane; Leppla, Stephan H.; Duesbery, Nicholas S.; Vande Woude, George F.

    2002-03-01

    Lethal factor, the principal virulence factor of Bacillus anthracis, inhibits mitogen-activated protein kinase (MAPK) signaling by proteolytically cleaving MAPK kinases. Edema factor, another component of anthrax toxin, is an adenylate cyclase, which increases intracellular cAMP. Inhibition of MAPK signaling with either anthrax lethal toxin (LeTx) or small molecule MAPK kinase inhibitors triggers apoptosis in human melanoma cells. Normal melanocytes do not undergo apoptosis in response to MAPK inhibition but arrest in the G1 phase of the cell cycle. Importantly, in vivo treatment of human melanoma xenograft tumors in athymic nude mice with LeTx results in significant or complete tumor regression without apparent side effects, suggesting that inhibiting the MAPK signaling pathway may be a useful strategy for treating melanoma. Additionally, interrupting MAPK signaling with LeTx and elevating cAMP with anthrax edema toxin in both melanoma cells and melanocytes lead to dramatic melanin production, perhaps explaining the formation of blackened eschars in cutaneous anthrax.

  13. Overcoming resistance to BRAF inhibition in BRAF-mutated metastatic melanoma

    PubMed Central

    Spagnolo, Francesco; Ghiorzo, Paola; Queirolo, Paola

    2014-01-01

    Almost 50% of metastatic melanoma patients harbor a BRAFV600 mutation andthe introduction of BRAF inhibitors has improved their treatment options. BRAF inhibitors vemurafenib and dabrafenib achieved improved overall survival over chemotherapy and have been approved for the treatment of BRAF-mutated metastatic melanoma. However, most patients develop mechanisms of acquired resistance and about 15% of them do not achieve tumor regression at all, due to intrinsic resistance to therapy. Moreover, early adaptive responses limit the initial efficacy of BRAF inhibition, leading mostly to incomplete responses that may favor the selection of a sub-population of resistant clones and the acquisition of alterations that cause tumor regrowth and progressive disease. The purpose of this paper is to review the mechanisms of resistance to therapy with BRAF inhibitors and to discuss the strategies to overcome them based on pre-clinical and clinical evidences. PMID:25344914

  14. Novel Pyrrolidine Diketopiperazines Selectively Inhibit Melanoma Cells via Induction of Late-Onset Apoptosis

    PubMed Central

    2015-01-01

    A common liability of cancer drugs is toxicity to noncancerous cells. Thus, molecules are needed that are potent toward cancer cells while sparing healthy cells. The cost of traditional cell-based HTS is dictated by the library size, which is typically in the hundreds of thousands of individual compounds. Mixture-based combinatorial libraries offer a cost-effective alternative to single-compound libraries while eliminating the need for molecular target validation. Presently, lung cancer and melanoma cells were screened in parallel with healthy cells using a mixture-based library. A novel class of compounds was discovered that selectively inhibited melanoma cell growth via apoptosis with submicromolar potency while sparing healthy cells. Additionally, the cost of screening and biological follow-up experiments was significantly lower than in typical HTS. Our findings suggest that mixture-based phenotypic HTS can significantly reduce cost and hit-to-lead time while yielding novel compounds with promising pharmacology. PMID:24471466

  15. Proteome analysis identified the PPARγ ligand 15d-PGJ2 as a novel drug inhibiting melanoma progression and interfering with tumor-stroma interaction.

    PubMed

    Paulitschke, Verena; Gruber, Silke; Hofstätter, Elisabeth; Haudek-Prinz, Verena; Klepeisz, Philipp; Schicher, Nikolaus; Jonak, Constanze; Petzelbauer, Peter; Pehamberger, Hubert; Gerner, Christopher; Kunstfeld, Rainer

    2012-01-01

    Peroxisome proliferator-activated receptors (PPARs) have been originally thought to be restricted to lipid metabolism or glucose homeostasis. Recently, evidence is growing that PPARγ ligands have inhibitory effects on tumor growth. To shed light on the potential therapeutic effects on melanoma we tested a panel of PPAR agonists on their ability to block tumor proliferation in vitro. Whereas ciglitazone, troglitazone and WY14643 showed moderate effects on proliferation, 15d-PGJ2 displayed profound anti-tumor activity on four different melanoma cell lines tested. Additionally, 15d-PGJ2 inhibited proliferation of tumor-associated fibroblasts and tube formation of endothelial cells. 15d-PGJ2 induced the tumor suppressor gene p21, a G(2)/M arrest and inhibited tumor cell migration. Shot gun proteome analysis in addition to 2D-gel electrophoresis and immunoprecipitation of A375 melanoma cells suggested that 15d-PGJ2 might exert its effects via modification and/or downregulation of Hsp-90 (heat shock protein 90) and several chaperones. Applying the recently established CPL/MUW database with a panel of defined classification signatures, we demonstrated a regulation of proteins involved in metastasis, transport or protein synthesis including paxillin, angio-associated migratory cell protein or matrix metalloproteinase-2 as confirmed by zymography. Our data revealed for the first time a profound effect of the single compound 15d-PGJ2 on melanoma cells in addition to the tumor-associated microenvironment suggesting synergistic therapeutic efficiency. PMID:23049949

  16. Proteome Analysis Identified the PPARγ Ligand 15d-PGJ2 as a Novel Drug Inhibiting Melanoma Progression and Interfering with Tumor-Stroma Interaction

    PubMed Central

    Paulitschke, Verena; Gruber, Silke; Hofstätter, Elisabeth; Haudek-Prinz, Verena; Klepeisz, Philipp; Schicher, Nikolaus; Jonak, Constanze; Petzelbauer, Peter; Pehamberger, Hubert; Gerner, Christopher; Kunstfeld, Rainer

    2012-01-01

    Peroxisome proliferator-activated receptors (PPARs) have been originally thought to be restricted to lipid metabolism or glucose homeostasis. Recently, evidence is growing that PPARγ ligands have inhibitory effects on tumor growth. To shed light on the potential therapeutic effects on melanoma we tested a panel of PPAR agonists on their ability to block tumor proliferation in vitro. Whereas ciglitazone, troglitazone and WY14643 showed moderate effects on proliferation, 15d-PGJ2 displayed profound anti-tumor activity on four different melanoma cell lines tested. Additionally, 15d-PGJ2 inhibited proliferation of tumor-associated fibroblasts and tube formation of endothelial cells. 15d-PGJ2 induced the tumor suppressor gene p21, a G2/M arrest and inhibited tumor cell migration. Shot gun proteome analysis in addition to 2D-gel electrophoresis and immunoprecipitation of A375 melanoma cells suggested that 15d-PGJ2 might exert its effects via modification and/or downregulation of Hsp-90 (heat shock protein 90) and several chaperones. Applying the recently established CPL/MUW database with a panel of defined classification signatures, we demonstrated a regulation of proteins involved in metastasis, transport or protein synthesis including paxillin, angio-associated migratory cell protein or matrix metalloproteinase-2 as confirmed by zymography. Our data revealed for the first time a profound effect of the single compound 15d-PGJ2 on melanoma cells in addition to the tumor-associated microenvironment suggesting synergistic therapeutic efficiency. PMID:23049949

  17. Protease-Activated Receptors and other G-Protein-Coupled Receptors: the Melanoma Connection

    PubMed Central

    Rosero, Rebecca A.; Villares, Gabriel J.; Bar-Eli, Menashe

    2016-01-01

    The vast array of G-protein-coupled receptors (GPCRs) play crucial roles in both physiological and pathological processes, including vision, coagulation, inflammation, autophagy, and cell proliferation. GPCRs also affect processes that augment cell proliferation and metastases in many cancers including melanoma. Melanoma is the deadliest form of skin cancer, yet limited therapeutic modalities are available to patients with metastatic melanoma. Studies have found that both chemokine receptors and protease-activated receptors, both of which are GPCRs, are central to the metastatic melanoma phenotype and may serve as potential targets in novel therapies against melanoma and other cancers. PMID:27379162

  18. Curcumin combined with FAPαc vaccine elicits effective antitumor response by targeting indolamine-2,3-dioxygenase and inhibiting EMT induced by TNF-α in melanoma.

    PubMed

    Jiang, Guan-Min; Xie, Wan-Ying; Wang, Hong-Sheng; Du, Jun; Wu, Bai-Ping; Xu, Wei; Liu, Hui-Fang; Xiao, Ping; Liu, Zhi-Gang; Li, Hong-Yan; Liu, Shuang-Quan; Yin, Wen-Jun; Zhang, Qiu-Gui; Liang, Jian-Ping; Huang, Hong-Jun

    2015-09-22

    Fibroblast activation protein α (FAPα) is a potential target for cancer therapy. However, elimination of FAPα+ fibroblasts activates secretion of IFN-γ and TNF-α. IFN-γ can in turn induce expression indolamine-2,3-dioxygenase (IDO), thereby contributing to immunosuppression, while TNF-α can induce EMT. These two reactive effects would limit the efficacy of a tumor vaccine. We found that curcumin can inhibit IDO expression and TNF-α-induced EMT. Moreover, FAPαc vaccine and CpG combined with curcumin lavage inhibited tumor growth and prolonged the survival of mice implanted with melanoma cells. The combination of FAPαc vaccine, CpG and curcumin stimulated FAPα antibody production and CD8+ T cell-mediated killing of FAPα-expressing stromal cells without adverse reactive effects. We suggest a combination of curcumin and FAPαc vaccine for melanoma therapy. PMID:26305550

  19. Antitumor Activity of Kielmeyera Coriacea Leaf Constituents in Experimental Melanoma, Tested in Vitro and in Vivo in Syngeneic Mice

    PubMed Central

    Figueiredo, Carlos Rogério; Matsuo, Alisson Leonardo; Massaoka, Mariana Hiromi; Girola, Natalia; Azevedo, Ricardo Alexandre; Rabaça, Aline Nogueira; Farias, Camyla Fernandes; Pereira, Felipe Valença; Matias, Natalia Silva; Silva, Luciana Pereira; Rodrigues, Elaine Guadelupe; Lago, João Henrique Guilardi; Travassos, Luiz Rodolpho; Silva, Regildo Márcio Gonçalves

    2014-01-01

    Purpose: The antitumor activity of Kielmeyera coriacea (Clusiaceae), a medicinal plant used in the treatment of parasitic, as well as fungal and bacterial infections by the Brazilian Cerrado population, was investigated. Methods: A chloroform extract (CE) of K. coriacea was tested in the murine melanoma cell line (B16F10-Nex2) and a panel of human tumor cell lines. Tumor cell migration was determined by the wound-healing assay and the in vivo antitumor activity of CE was investigated in a melanoma cell metastatic model. 1H NMR and GC/MS were used to determine CE chemical composition. Results: We found that CE exhibited strong cytotoxic activity against murine melanoma cells and a panel of human tumor cell lines in vitro. CE also inhibited growth of B16F10-Nex2 cells at sub lethal concentrations, inducing cell cycle arrest at S phase, and inhibition of tumor cell migration. Most importantly, administration of CE significantly reduced the number of melanoma metastatic nodules in vivo. Chemical analysis of CE indicated the presence of the long chain fatty compounds, 1-eicosanol, 1-docosanol, and 2-nonadecanone as main constituents. Conclusion: These results indicate that K. coriacea is a promising medicinal plant in cancer therapy exhibiting antitumor activity both in vitro and in vivo against different tumor cell lines. PMID:25364658

  20. Activation of hERG3 channel stimulates autophagy and promotes cellular senescence in melanoma

    PubMed Central

    Perez-Neut, Mathew; Haar, Lauren; Rao, Vidhya; Santha, Sreevidya; Lansu, Katherine; Rana, Basabi; Jones, Walter K.; Gentile, Saverio

    2016-01-01

    Ion channels play a major factor in maintaining cellular homeostasis but very little is known about the role of these proteins in cancer biology. In this work we have discovered that, the Kv11.3 (hERG3) a plasma-membrane potassium channel plays a critical role in the regulation of autophagy in a cancer cell model. We have found that pharmacologic stimulation of the Kv11.3 channel with a small molecule activator, NS1643 induced autophagy via activation of an AMPK-dependent signaling pathway in melanoma cell line. In addition, we have found that NS1643 produced a strong inhibition of cell proliferation by activating a cellular senescence program. Furthermore, inhibition of autophagy via siRNA targeting AMPK or treatment with hydroxychloroquine an autophagy inhibitor activates apoptosis in NS1643-treated cells. Thus, we propose that, Kv11.3 is a novel mediator of autophagy, autophagy can be a survival mechanism contributing to cellular senescence, and that use of a combinatorial pharmacologic approach of Kv11.3 activator with inhibitors of autophagy represents a novel therapeutic approach against melanoma. PMID:26942884

  1. Combined MTOR and autophagy inhibition: phase I trial of hydroxychloroquine and temsirolimus in patients with advanced solid tumors and melanoma.

    PubMed

    Rangwala, Reshma; Chang, Yunyoung C; Hu, Janice; Algazy, Kenneth M; Evans, Tracey L; Fecher, Leslie A; Schuchter, Lynn M; Torigian, Drew A; Panosian, Jeffrey T; Troxel, Andrea B; Tan, Kay-See; Heitjan, Daniel F; DeMichele, Angela M; Vaughn, David J; Redlinger, Maryann; Alavi, Abass; Kaiser, Jonathon; Pontiggia, Laura; Davis, Lisa E; O'Dwyer, Peter J; Amaravadi, Ravi K

    2014-08-01

    The combination of temsirolimus (TEM), an MTOR inhibitor, and hydroxychloroquine (HCQ), an autophagy inhibitor, augments cell death in preclinical models. This phase 1 dose-escalation study evaluated the maximum tolerated dose (MTD), safety, preliminary activity, pharmacokinetics, and pharmacodynamics of HCQ in combination with TEM in cancer patients. In the dose escalation portion, 27 patients with advanced solid malignancies were enrolled, followed by a cohort expansion at the top dose level in 12 patients with metastatic melanoma. The combination of HCQ and TEM was well tolerated, and grade 3 or 4 toxicity was limited to anorexia (7%), fatigue (7%), and nausea (7%). An MTD was not reached for HCQ, and the recommended phase II dose was HCQ 600 mg twice daily in combination with TEM 25 mg weekly. Other common grade 1 or 2 toxicities included fatigue, anorexia, nausea, stomatitis, rash, and weight loss. No responses were observed; however, 14/21 (67%) patients in the dose escalation and 14/19 (74%) patients with melanoma achieved stable disease. The median progression-free survival in 13 melanoma patients treated with HCQ 1200mg/d in combination with TEM was 3.5 mo. Novel 18-fluorodeoxyglucose positron emission tomography (FDG-PET) measurements predicted clinical outcome and provided further evidence that the addition of HCQ to TEM produced metabolic stress on tumors in patients that experienced clinical benefit. Pharmacodynamic evidence of autophagy inhibition was evident in serial PBMC and tumor biopsies only in patients treated with 1200 mg daily HCQ. This study indicates that TEM and HCQ is safe and tolerable, modulates autophagy in patients, and has significant antitumor activity. Further studies combining MTOR and autophagy inhibitors in cancer patients are warranted. PMID:24991838

  2. BRAF inhibition decreases cellular glucose uptake in melanoma in association with reduction in cell volume

    PubMed Central

    Theodosakis, Nicholas; Held, Matthew A.; Marzuka-Alcala, Alexander; Meeth, Katrina M.; Micevic, Goran; Long, Georgina V.; Scolyer, Richard A.; Stern, David F.; Bosenberg, Marcus W.

    2015-01-01

    BRAF kinase inhibitors have dramatically impacted treatment of BRAFV600E/K-driven metastatic melanoma. Early responses assessed using [18F]fluorodeoxyglucose uptake-positron emission tomography (FDG-PET) have shown dramatic reduction of radiotracer signal within two weeks of treatment. Despite high response rates, relapse occurs in nearly all cases, frequently at sites of treated metastatic disease. It remains unclear whether initial loss of 18FDG uptake is due to tumor cell death or other reasons. Here we provide evidence of melanoma cell volume reduction in a patient cohort treated with BRAF inhibitors. We present data demonstrating that BRAF inhibition reduces melanoma glucose uptake per cell, but that this change is no longer significant following normalization for cell volume changes. We also demonstrate that volume normalization greatly reduces differences in transmembrane glucose transport and hexokinase-mediated phosphorylation. Mechanistic studies suggest that this loss of cell volume is due in large part to decreases in new protein translation as a consequence of vemurafenib treatment. Ultimately, our findings suggest that cell volume regulation constitutes an important physiologic parameter that may significantly contribute to radiographic changes observed in clinic. PMID:25948295

  3. Nuclear heparanase-1 activity suppresses melanoma progression via its DNA-binding affinity.

    PubMed

    Yang, Y; Gorzelanny, C; Bauer, A T; Halter, N; Komljenovic, D; Bäuerle, T; Borsig, L; Roblek, M; Schneider, S W

    2015-11-19

    Heparanase-1 (HPSE) plays a pivotal role in structural remodeling of the ECM and the glycocalyx, thus conferring protumorigenic, proangiogenic and prometastatic properties to many cancer entities. In addition to its extracellular function, recent studies suggest an intracellular activity of HPSE with a largely unknown significance during tumor progression. Therefore, we investigated the relevance of the dual functions of HPSE to malignant melanoma in vitro, as well as in different mouse melanoma models based on the intradermal or intravenous injection of melanoma cells. Consistent with its extracellular action, an HPSE deficiency led to a reduced shedding of the glycocalyx accompanied by a reduced availability of vascular endothelial growth factor, affecting tumor growth and vascularization. In contrast, we measured an elevated expression of the protumorigenic factors pentraxin-3, tissue factor, TNF-α and most prominently, MMP-9, upon HPSE knockdown. In vivo, an HPSE deficiency was related to increased lymph node metastasis. Since the inhibition of its extracellular function with heparin was unable to block the gene regulatory impact of HPSE, we proposed an intracellular mechanism. Immunostaining revealed a counter-staining of HPSE and NF-κB in the nucleus, suggesting a close relationship between both proteins. This finding was further supported by the discovery of a direct charge-driven molecular interaction between HPSE and DNA by using atomic force microscopy and a co-precipitation approach. Our findings are novel and point towards a dual function for HPSE in malignant melanoma with a protumorigenic extracellular activity and a tumor-suppressive nuclear action. The identification of molecular strategies to shuttle extracellular HPSE into the nuclei of cancer cells could provide new therapeutic options. PMID:25745999

  4. A Natural Bacterial-Derived Product, the Metalloprotease Arazyme, Inhibits Metastatic Murine Melanoma by Inducing MMP-8 Cross-Reactive Antibodies

    PubMed Central

    Pereira, Felipe V.; Ferreira-Guimarães, Carla A.; Paschoalin, Thaysa; Scutti, Jorge A. B.; Melo, Filipe M.; Silva, Luis S.; Melo, Amanda C. L.; Silva, Priscila; Tiago, Manoela; Matsuo, Alisson L.; Juliano, Luiz; Juliano, Maria A.; Carmona, Adriana K.; Travassos, Luiz R.; Rodrigues, Elaine G.

    2014-01-01

    The increased incidence, high rates of mortality and few effective means of treatment of malignant melanoma, stimulate the search for new anti-tumor agents and therapeutic targets to control this deadly metastatic disease. In the present work the antitumor effect of arazyme, a natural bacterial-derived metalloprotease secreted by Serratia proteomaculans, was investigated. Arazyme significantly reduced the number of pulmonary metastatic nodules after intravenous inoculation of B16F10 melanoma cells in syngeneic mice. In vitro, the enzyme showed a dose-dependent cytostatic effect in human and murine tumor cells, and this effect was associated to the proteolytic activity of arazyme, reducing the CD44 expression at the cell surface, and also reducing in vitro adhesion and in vitro/in vivo invasion of these cells. Arazyme treatment or immunization induced the production of protease-specific IgG that cross-reacted with melanoma MMP-8. In vitro, this antibody was cytotoxic to tumor cells, an effect increased by complement. In vivo, arazyme-specific IgG inhibited melanoma lung metastasis. We suggest that the antitumor activity of arazyme in a preclinical model may be due to a direct cytostatic activity of the protease in combination with the elicited anti-protease antibody, which cross-reacts with MMP-8 produced by tumor cells. Our results show that the bacterial metalloprotease arazyme is a promising novel antitumor chemotherapeutic agent. PMID:24788523

  5. Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo.

    PubMed

    Girola, Natalia; Figueiredo, Carlos R; Farias, Camyla F; Azevedo, Ricardo A; Ferreira, Adilson K; Teixeira, Sarah F; Capello, Tabata M; Martins, Euder G A; Matsuo, Alisson L; Travassos, Luiz R; Lago, João H G

    2015-11-27

    Natural monoterpenes were isolated from the essential oil of Piper cernuum Vell. (Piperaceae) leaves. The crude oil and the individual monoterpenes were tested for cytotoxicity in human tumor cell lineages and B16F10-Nex2 murine melanoma cells. In the present work we demonstrate the activity of camphene against different cancer cells, with its mechanism of action being investigated in vitro and in vivo in murine melanoma. Camphene induced apoptosis by the intrinsic pathway in melanoma cells mainly by causing endoplasmic reticulum (ER) stress, with release of Ca(2+) together with HmgB1 and calreticulin, loss of mitochondrial membrane potential and up regulation of caspase-3 activity. Importantly, camphene exerted antitumor activity in vivo by inhibiting subcutaneous tumor growth of highly aggressive melanoma cells in a syngeneic model, suggesting a promising role of this compound in cancer therapy. PMID:26471302

  6. Overcoming acquired BRAF inhibitor resistance in melanoma via targeted inhibition of Hsp90 with ganetespib.

    PubMed

    Acquaviva, Jaime; Smith, Donald L; Jimenez, John-Paul; Zhang, Chaohua; Sequeira, Manuel; He, Suqin; Sang, Jim; Bates, Richard C; Proia, David A

    2014-02-01

    Activating BRAF kinase mutations serve as oncogenic drivers in over half of all melanomas, a feature that has been exploited in the development of new molecularly targeted approaches to treat this disease. Selective BRAF(V600E) inhibitors, such as vemurafenib, typically induce initial, profound tumor regressions within this group of patients; however, durable responses have been hampered by the emergence of drug resistance. Here, we examined the activity of ganetespib, a small-molecule inhibitor of Hsp90, in melanoma lines harboring the BRAF(V600E) mutation. Ganetespib exposure resulted in the loss of mutant BRAF expression and depletion of mitogen-activated protein kinase and AKT signaling, resulting in greater in vitro potency and antitumor efficacy compared with targeted BRAF and MAP-ERK kinase (MEK) inhibitors. Dual targeting of Hsp90 and BRAF(V600E) provided combinatorial benefit in vemurafenib-sensitive melanoma cells in vitro and in vivo. Importantly, ganetespib overcame mechanisms of intrinsic and acquired resistance to vemurafenib, the latter of which was characterized by reactivation of extracellular signal-regulated kinase (ERK) signaling. Continued suppression of BRAF(V600E) by vemurafenib potentiated sensitivity to MEK inhibitors after acquired resistance had been established. Ganetespib treatment reduced, but not abolished, elevations in steady-state ERK activity. Profiling studies revealed that the addition of a MEK inhibitor could completely abrogate ERK reactivation in the resistant phenotype, with ganetespib displaying superior combinatorial activity over vemurafenib. Moreover, ganetespib plus the MEK inhibitor TAK-733 induced tumor regressions in vemurafenib-resistant xenografts. Overall these data highlight the potential of ganetespib as a single-agent or combination treatment in BRAF(V600E)-driven melanoma, particularly as a strategy to overcome acquired resistance to selective BRAF inhibitors. PMID:24398428

  7. Elastin-derived peptides enhance melanoma growth in vivo by upregulating the activation of Mcol-A (MMP-1) collagenase

    PubMed Central

    Devy, J; Duca, L; Cantarelli, B; Joseph-Pietras, D; Scandolera, A; Rusciani, A; Parent, L; Thevenard, J; Pasco, S Brassart; Tarpin, M; Martiny, L; Debelle, L

    2010-01-01

    Background: Elastin peptides possess several biological activities and in vitro data suggest they could be involved in the early phase of melanoma growth. Methods: Using diverse in vitro and in vivo techniques (cell proliferation, invasion and migration assays, zymography, western blots, collagen degradation assay, reverse transcription PCR, melanoma allographs and immunohistochemistry), we analysed the effect of elastin-derived peptides (EDPs) on B16F1 melanoma growth and invasion, as well as on the proteolytic systems involved. Results: We found that EDPs dramatically promote in vivo tumour development of B16F1 melanoma, as well as their in vitro migration and invasion. The inhibition of serine proteases and matrix metalloproteinases (MMPs) activities, by aprotinin and galardin, respectively, demonstrated that these enzymes were involved in these processes. However, we found that EDPs did not increase urokinase-type plasminogen activator, tissue-type plasminogen activator or MMP-2 expression and/or activation, neither in vitro nor in vivo. Nevertheless, we observed a strong increase of pro-MMP-9 secretion in EDPs-treated tumours and, more importantly, an increase in the expression and activation of the murine counterpart of MMP-1, named murine collagenase-A (Mcol-A). Moreover, we show that plasminogen system inhibition decreases collagen degradation by this enzyme. Finally, the use of a specific blocking antibody against Mcol-A abolished EDP-induced B16F1 invasion in vitro, showing that this MMP was directly involved in this process. Conclusion: Our data show that in vivo, EDPs are involved in melanoma growth and invasion and reinforced the concept of elastin fragmentation as a predictive factor. PMID:20959825

  8. Inhibition of melanoma cell proliferation by resveratrol is correlated with upregulation of quinone reductase 2 and p53

    SciTech Connect

    Hsieh Tzechen; Wang Zhirong; Hamby, Carl V.; Wu, Joseph M. . E-mail: Joseph_Wu@nymc.edu

    2005-08-19

    Resveratrol (trans-3,4',5-trihydroxystilbene) is a grape-derived polyphenol under intensive study for its potential in cancer prevention. In the case of cultured human melanoma cells, no one to our knowledge has investigated whether resveratrol exerts similar anti-proliferative activities in cells with different metastatic potential. Therefore, we examined the effects of this polyphenol on the growth of weakly metastatic Line IV clone 3 and on autologous, highly metastatic Line IV clone 1 cultured melanoma cells. Comparable inhibition of growth and colony formation resulted from treatment by resveratrol in both cell lines. Flow cytometric analysis revealed that resveratrol-treated clone 1 cells had a dose-dependent increase in S phase and a concomitant reduction in the G{sub 1} phase. No detectable change in cell cycle phase distribution was found in similarly treated clone 3 cells. Western blots demonstrated a significant increase in the expression of the tumor suppressor gene p53, without a commensurate change in p21 and several other cell cycle regulatory proteins in both cell types. Chromatography of Line IV clone 3 and clone 1 cell extracts on resveratrol affinity columns revealed that the basal expression of dihydronicotinamide riboside quinone reductase 2 (NQO2) was higher in Line IV clone 1 than clone 3 cells. Levels of NQO2 but not its structural analog NQO1 were dose-dependently increased by resveratrol in both cell lines. We propose that induction of NQO2 may relate to the observed increased expression of p53 that, in turn, contributes to the observed suppression of cell growth in both melanoma cell lines.

  9. miR-200c Inhibits Melanoma Progression and Drug Resistance through Down-Regulation of Bmi-1

    PubMed Central

    Liu, Shujing; Tetzlaff, Michael T.; Cui, Rutao; Xu, Xiaowei

    2013-01-01

    MicroRNAs (miRNAs) are short noncoding RNAs that play crucial roles in tumorigenesis and tumor progression. Melanoma is the most aggressive skin cancer that is resistant or rapidly develops resistance to a variety of chemotherapeutic agents. The role of miRNAs in melanoma progression and drug resistance has not been well studied. Herein, we demonstrate that miR-200c is down-regulated in melanomas (primary and metastatic) compared with melanocytic nevi. Overexpression of miR-200c in melanoma cells resulted in significantly decreased cell proliferation and migratory capacity as well as drug resistance. miR-200c overexpression resulted in significant down-regulation of BMI-1, ABCG2, ABCG5, and MDR1 expression and in a concomitant increase in E-cadherin levels. Knockdown of BMI-1 showed similar effects as miR-200c overexpression in melanoma cells. In addition, miR-200c overexpression significantly inhibited melanoma xenograft growth and metastasis in vivo, and this correlated with diminished expression of BMI-1 and reduced levels of E-cadherin in these tumors. The effects of miR-200c on melanoma cell proliferation and migratory capacity and on self-renewal were rescued by overexpression of Bmi-1, and the reversal of these phenotypes correlated with a reduction in E-cadherin expression and increased levels of ABCG2, ABCG5, and MDR1. Taken together, these findings demonstrate a key role for miR-200c in melanoma progression and drug resistance. These results suggest that miR-200c may represent a critical target for increasing melanoma sensitivity to clinical therapies. PMID:22982443

  10. Growth-Inhibitory and Antiangiogenic Activity of the MEK Inhibitor PD0325901 in Malignant Melanoma with or without BRAF Mutations12

    PubMed Central

    Ciuffreda, Ludovica; Del Bufalo, Donatella; Desideri, Marianna; Di Sanza, Cristina; Stoppacciaro, Antonella; Ricciardi, Maria Rosaria; Chiaretti, Sabina; Tavolaro, Simona; Benassi, Barbara; Bellacosa, Alfonso; Foà, Robin; Tafuri, Agostino; Cognetti, Francesco; Anichini, Andrea; Zupi, Gabriella; Milella, Michele

    2009-01-01

    The Raf/MEK/ERK pathway is an important mediator of tumor cell proliferation and angiogenesis. Here, we investigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC50 in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G1-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27KIP1) and apoptosis (Bcl-2 and survivin) regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma. PMID:19649202

  11. Inhibition of Cell Proliferation in an NRAS Mutant Melanoma Cell Line by Combining Sorafenib and α-Mangostin.

    PubMed

    Xia, Yun; Li, Ying; Westover, Kenneth D; Sun, Jiaming; Chen, Hongxiang; Zhang, Jianming; Fisher, David E

    2016-01-01

    α-Mangostin is a natural product commonly used in Asia for cosmetic and medicinal applications including topical treatment of acne and skin cancer. Towards finding new pharmacological strategies that overcome NRAS mutant melanoma, we performed a cell proliferation-based combination screen using a collection of well-characterized small molecule kinase inhibitors and α-Mangostin. We found that α-Mangostin significantly enhances Sorafenib pharmacological efficacy against an NRAS mutant melanoma cell line. The synergistic effects of α-Mangostin and Sorafenib were associated with enhanced inhibition of activated AKT and ERK, induced ER stress, and reduced autophagy, eventually leading to apoptosis. The structure of α-Mangostin resembles several inhibitors of the Retinoid X receptor (RXR). MITF expression, which is regulated by RXR, was modulated by α-Mangostin. Molecular docking revealed that α-Mangostin can be accommodated by the ligand binding pocket of RXR and may thereby compete with RXR-mediated control of MITF expression. In summary, these data demonstrate an unanticipated synergy between α-Mangostin and sorafenib, with mechanistic actions that convert a known safe natural product to a candidate combinatorial therapeutic agent. PMID:27152946

  12. Inhibition of Cell Proliferation in an NRAS Mutant Melanoma Cell Line by Combining Sorafenib and α-Mangostin

    PubMed Central

    Xia, Yun; Li, Ying; Westover, Kenneth D.; Sun, Jiaming; Chen, Hongxiang; Zhang, Jianming; Fisher, David E.

    2016-01-01

    α-Mangostin is a natural product commonly used in Asia for cosmetic and medicinal applications including topical treatment of acne and skin cancer. Towards finding new pharmacological strategies that overcome NRAS mutant melanoma, we performed a cell proliferation-based combination screen using a collection of well-characterized small molecule kinase inhibitors and α-Mangostin. We found that α-Mangostin significantly enhances Sorafenib pharmacological efficacy against an NRAS mutant melanoma cell line. The synergistic effects of α-Mangostin and Sorafenib were associated with enhanced inhibition of activated AKT and ERK, induced ER stress, and reduced autophagy, eventually leading to apoptosis. The structure of α-Mangostin resembles several inhibitors of the Retinoid X receptor (RXR). MITF expression, which is regulated by RXR, was modulated by α-Mangostin. Molecular docking revealed that α-Mangostin can be accommodated by the ligand binding pocket of RXR and may thereby compete with RXR-mediated control of MITF expression. In summary, these data demonstrate an unanticipated synergy between α-Mangostin and sorafenib, with mechanistic actions that convert a known safe natural product to a candidate combinatorial therapeutic agent. PMID:27152946

  13. Alteration of tyrosinase activity in human melanocytes and melanoma cells by histamine H2 and H3 ligands.

    PubMed

    Le Gros, G; Zhang, X M; Parsons, P G

    1994-12-01

    Nontoxic doses of the histamine H2 antagonists ranitidine, cimetidine, lamtidine and mifentidine rapidly and reversibly increased tyrosinase activity in an amelanotic human melanoma cell line (MM96L) with low constitutive activity. The H2 antagonists, famotidine and MGTI, and the imidazol(in)e receptor ligand clonidine had no effect either alone or in competition with ranitidine, whilst metiamide decreased tyrosinase activity. Lysosomotropic amines had a similar effect to ranitidine, except that induction reached a plateau at 6 h and was insensitive to amiloride. Human melanocytes and pigmented human melanoma cell lines exhibited minimal levels of tyrosinase induction, which was dependent on protein synthesis but not on RNA or DNA synthesis. Constitutive tyrosinase activity in MM96L cells was much less stable than in melanocytes and pigmented melanoma cells. No change was observed in expression of gp75, neural specific octamer binding proteins, or in mRNA levels of tyrosinase, Pmel-17 and gp75 (TRP-1). Tyrosinase was inhibited by the H3 agonist imetit but not by alpha-methylhistamine or the H3 antagonist thioperamide. Overall, this work showed that certain H2 antagonists activate an unstable form of tyrosinase in amelanotic melanoma cells by a post-transcriptional mechanism dependent on protein synthesis. An imidazoline/guanidinium receptor site rather than the H2 receptor appeared to be involved. PMID:7535606

  14. Inhibition of melanoma cells tumorigenicity by the snake venom toxin jararhagin.

    PubMed

    Corrêa, Mário César; Maria, Durvanei A; Moura-da-Silva, Ana M; Pizzocaro, Kazumi F; Ruiz, Itamar R G

    2002-06-01

    Skmel-28 human melanoma cells were treated with jararhagin (Jara), a metalloproteinase disintegrin isolated from Bothrops jararaca snake venom, and Jari (Jara with the catalytic domain inactivated). Following treatments, monolayer cells lost cytoplasmic expansions acquiring round shapes, detached and formed cell clusters in suspension. Cytotoxicity effect of Jari was dramatically increased at concentrations higher than 0.4 microM, whereas cell adhesion responses did not differ significantly between similar concentrations of Jara and Jari. Treated cells were significantly inhibited to adhere to non-coated wells, as to ECM proteins-coated plates. Migration and invasion were also significantly inhibited in vitro. A decreased proliferation rate was observed in toxin-treated cells. Immunofluorescence staining showed a wide distribution of Jari across the cells. Jara treated cells (67.5%) steady bound anti-jara antibodies after 90 min, while Jari treated cells steady bound only after 6h (57.3%), as determined by FACS. Skmel-28 melanoma cells tumorigenicity was evaluated 180 days after s.c. injections in AIRmin mice. A statistically significant decrease in the ability of Jara and Jari treated cells to promote lung metastasis was observed. These results point to the potential use of this toxin as a tool for applied researches in the clinical field. PMID:12175610

  15. Hinokitiol Inhibits Melanogenesis via AKT/mTOR Signaling in B16F10 Mouse Melanoma Cells.

    PubMed

    Tsao, Yu-Tzu; Huang, Yu-Fen; Kuo, Chun-Yu; Lin, Yu-Chiang; Chiang, Wei-Cheng; Wang, Wei-Kuang; Hsu, Chia-Wei; Lee, Che-Hsin

    2016-01-01

    H inokitiol purified from the heartwood of cupressaceous plants has had various biological functions of cell differentiation and growth. Hinokitiol has been demonstrated as having an important role in anti-inflammation and anti-bacteria effect, suggesting that it is potentially useful in therapies for hyperpigmentation. Previously, hinokitiol inhibited the production of melanin by inhibiting tyrosinase activity. The autophagic signaling pathway can induce hypopigmentation. This study is warranted to investigate the mechanism of hinokitiol-induced hypopigmentation through autophagy in B16F10 melanoma cells. The melanin contents and expression of microthphalmia associated transcription factor (MITF) and tyrosinase were inhibited by treatment with hinokitiol. Moreover, the phosphorylation of the protein express levels of phospho-protein kinase B (P-AKT) and phospho-mammalian targets of rapamycin (P-mTOR) were reduced after hinokitiol treatment. In addition, the microtubule associated protein 1 light chain 3 (LC3) -II and beclin 1 (autophagic markers) were increased after the B16F10 cell was treated with hinokitiol. Meanwhile, hinokitiol decreased cellular melanin contents in a dose-dependent manner. These findings establish that hinokitiol inhibited melanogenesis through the AKT/mTOR signaling pathway. PMID:26901194

  16. Imiquimod-induced apoptosis of melanoma cells is mediated by ER stress-dependent Noxa induction and enhanced by NF-κB inhibition.

    PubMed

    El-Khattouti, Abdelouahid; Selimovic, Denis; Hannig, Matthias; Taylor, Erin B; Abd Elmageed, Zakaria Y; Hassan, Sofie Y; Haikel, Youssef; Kandil, Emad; Leverkus, Martin; Brodell, Robert T; Megahed, Mosaad; Hassan, Mohamed

    2016-02-01

    Melanoma is characterized by dysregulated intracellular signalling pathways including an impairment of the cell death machinery, ultimately resulting in melanoma resistance, survival and progression. This explains the tumour's extraordinary resistance to the standard treatment. Imiquimod is a topical immune response modifier (imidazoquinoline) with both antiviral and antitumour activities. The mechanism by which imiquimod triggers the apoptosis of melanoma cells has now been carefully elucidated. Imiquimod-induced apoptosis is associated with the activation of apoptosis signalling regulating kinase1/c-Jun-N-terminal kinase/p38 pathways and the induction of endoplasmic stress characterized by the activation of the protein kinase RNA-like endoplasmic reticulum kinase signalling pathway, increase in intracellular Ca(2+) release, degradation of calpain and subsequent cleavage of caspase-4. Moreover, imiquimod triggers the activation of NF-κB and the expression of the inhibitor of apoptosis proteins (IAPs) such as, X-linked IAP (XIAP) together with the accumulation of reactive oxygen species (ROS). Also, imiquimod triggers mitochondrial dysregulation characterized by the loss of mitochondrial membrane potential (Δψm), the increase in cytochrome c release, and cleavage of caspase-9, caspase-3 and poly(ADP-ribose) polymerase (PARP). Inhibitors of specific pathways, permit the elucidation of possible mechanisms of imiquimod-induced apoptosis. They demonstrate that inhibition of NF-kB by the inhibitor of nuclear factor kappa-B kinase (IKK) inhibitor Bay 11-782 or knockdown of XIAP induces melanoma apoptosis in cells exposed to imiquimod. These findings support the use of either IKK inhibitors or IAP antagonists as adjuvant therapies to improve the effectiveness topical imiquimod in the treatment of melanoma. PMID:26578344

  17. Subcutaneous Adipocytes Promote Melanoma Cell Growth by Activating the Akt Signaling Pathway

    PubMed Central

    Kwan, Hiu Yee; Fu, Xiuqiong; Liu, Bin; Chao, Xiaojuan; Chan, Chi Leung; Cao, Huihui; Su, Tao; Tse, Anfernee Kai Wing; Fong, Wang Fun; Yu, Zhi-Ling

    2014-01-01

    Tumorigenesis involves constant communication between tumor cells and neighboring normal cells such as adipocytes. The canonical function of adipocytes is to store triglyceride and release fatty acids for other tissues. This study was aimed to find out if adipocytes promoted melanoma cell growth and to investigate the underlying mechanism. Here we isolated adipocytes from inguinal adipose tissue in mice and co-cultured with melanoma cells. We found that the co-cultured melanoma had higher lipid accumulation compared with mono-cultured melanoma. In addition, fluorescently labeled fatty acid BODIPY® FLC16 signal was detected in melanoma co-cultured with the adipocytes that had been loaded with the fluorescent dye, suggesting that the adipocytes provide fatty acids to melanoma cells. Compared with mono-cultured melanoma, co-cultured melanoma cells had a higher proliferation and phospho-Akt (Ser-473 and Thr-450) expression. Overexpression of Akt mutants in melanoma cells reduced the co-culture-enhanced proliferation. A lipidomic study showed that the co-cultured melanoma had an elevated palmitic acid level. Interestingly, we found that palmitic acid stimulated melanoma cell proliferation, changed the cell cycle distribution, and increased phospho-Akt (Ser-473 and Thr-450) and PI3K but not phospho-PTEN (phosphophosphatase and tensin homolog) expressions. More importantly, the palmitic acid-stimulated proliferation was further enhanced in the Akt-overexpressed melanoma cells and was reduced by LY294002 or knockdown of endogenous Akt or overexpression of Akt mutants. We also found that palmitic acid-pretreated B16F10 cells were grown to a significantly larger tumor in mice compared with control cells. Taken together, we suggest that adipocytes may serve as an exogenous source of palmitic acid that promotes melanoma cell growth by activating Akt. PMID:25228694

  18. Inhibition of autophagy enhances the effects of the AKT inhibitor MK-2206 when combined with paclitaxel and carboplatin in BRAF wild-type melanoma.

    PubMed

    Rebecca, Vito W; Massaro, Renato R; Fedorenko, Inna V; Sondak, Vernon K; Anderson, Alexander R A; Kim, Eunjung; Amaravadi, Ravi K; Maria-Engler, Silvya S; Messina, Jane L; Gibney, Geoffrey T; Kudchadkar, Ragini R; Smalley, Keiran S M

    2014-05-01

    This study investigates the mechanism of action behind the long-term responses (12-16 months) of two BRAF WT melanoma patients to the AKT inhibitor MK-2206 in combination with paclitaxel and carboplatin. Although single agent MK-2206 inhibited phospho-AKT signaling, it did not impact in vitro melanoma growth or survival. The combination of MK-2206 with paclitaxel and carboplatin was cytotoxic in long-term colony formation and 3D spheroid assays, and induced autophagy. Autophagy was initially protective with autophagy inhibitors and deletion of ATG5 found to enhance cytotoxicity. Although prolonged autophagy induction (>6 days) led to caspase-dependent apoptosis, drug resistant clones still emerged. Autophagy inhibition enhanced the cell death response through reactive oxygen species and could be reversed by anti-oxidants. We demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma and show that an autophagy inhibitor may prevent resistance to these drugs. PMID:24490764

  19. Expression of the platelet-activating factor receptor enhances benzyl isothiocyanate-induced apoptosis in murine and human melanoma cells.

    PubMed

    Sahu, Ravi Prakash

    2015-07-01

    Melanoma cells often express platelet-activating factor receptor (PAF-R), which has been demonstrated to increase metastatic behavior. However, the effect of PAF-R on the responsiveness of melanoma to naturally occurring cytotoxic agents remains to be elucidated. The present study aimed to determine the relative cytotoxicity and mechanism of benzyl isothiocyanate (BITC), a component of cruciferous vegetables, in melanoma cells expressing PAF-R. To evaluate the importance of PAF-R signaling in melanoma cell growth, PAF-R-negative murine B16F10 cells were transduced with a retrovirus containing the cDNA for PAF-R to generate cells stably expressing PAF-R (B16-PAF-R) or an empty vector (MSCV) to generate PAF-R-deficient B16-MSCV control cells. Activation of PAF-R, using the PAF-R agonist, 1-hexadecyl-2-N-methylcarbamoyl-3-glycerophosphocholine, induced an increase in the proliferation of B16-PAF-R cells compared with the B16-MSCV cells. Reverse transcription quantitative polymerase chain reaction revealed the presence of functional PAF-R in human melanoma SK23MEL cells, but not in SK5MEL cells. The present study investigated the effect of BITC treatments on the survival of murine and human melanoma cells, in the presence or absence of functional PAF-R. The results revealed that treatment with BITC decreased the survival rate of the PAF-R-positive and negative murine and human melanoma cells. However, the expression of PAF-R substantially augmented BITC-mediated cytotoxicity in the PAF-R-positive cells at lower concentrations compared with the PAF-R-negative cells. In order to determine the underlying mechanism, flow cytometric analysis was used, which demonstrated a significant increase in the generation of reactive oxygen species (ROS) in the B16-PAF-R cells compared with the B16-MSCV cells, which enhanced apoptosis by BITC, as measured by increased caspase-3/7 luminescence. Notably, the BITC-mediated decreased cell survival rate, increased ROS and increased

  20. Inhibition of gallium-67 uptake in melanoma by an anti-human transferrin receptor monoclonal antibody

    SciTech Connect

    Chan, S.M.; Hoffer, P.B.; Maric, N.; Duray, P.

    1987-08-01

    The effect of an anti-human transferrin receptor (anti-TFR) monoclonal antibody (MoAb), designated B3/25, and an anti-melanoma antibody, designated 96.5, on the uptake of gallium-67 (/sup 67/Ga) by tumor was studied. Three groups of six athymic mice bearing a human melanoma were injected via tail vein with (a) 0.55 mg human serum albumin (HSA) (control group), (b) 0.5 mg MoAb B3/25 + 0.55 mg HSA, and (c) 0.5 mg MoAb 96.5 + 0.55 mg HSA, respectively. Twenty-four hours later, each mouse was given an intravenous dose of 5 microCi (/sup 67/Ga) citrate. Biodistribution of activity (percent injected dose per gram) determined 48 hr after injection of /sup 67/Ga showed a 75% decrease in tumor uptake in the group of mice that received B3/25 (anti-TFR MoAb) compared with the control group. In contrast, MoAb 96.5 did not show any effect on melanoma uptake of /sup 67/Ga. Histologic findings suggest that the decreased uptake was not due to cellular damage resulting from binding of B3/25 to TFR. The results of this study strongly suggest the involvement of TFR in the in vivo tumor uptake of /sup 67/Ga.

  1. Non-canonical Smads phosphorylation induced by the glutamate release inhibitor, riluzole, through GSK3 activation in melanoma.

    PubMed

    Abushahba, Walid; Olabisi, Oyenike O; Jeong, Byeong-Seon; Boregowda, Rajeev K; Wen, Yu; Liu, Fang; Goydos, James S; Lasfar, Ahmed; Cohen-Solal, Karine A

    2012-01-01

    Riluzole, an inhibitor of glutamate release, has shown the ability to inhibit melanoma cell xenograft growth. A phase 0 clinical trial of riluzole as a single agent in patients with melanoma resulted in involution of tumors associated with inhibition of both the mitogen-activated protein kinase (MAPK) and phophoinositide-3-kinase/AKT (PI3K/AKT) pathways in 34% of patients. In the present study, we demonstrate that riluzole inhibits AKT-mediated glycogen synthase kinase 3 (GSK3) phosphorylation in melanoma cell lines. Because we have demonstrated that GSK3 is involved in the phosphorylation of two downstream effectors of transforming growth factor beta (TGFβ), Smad2 and Smad3, at their linker domain, our aim was to determine whether riluzole could induce GSK3β-mediated linker phosphorylation of Smad2 and Smad3. We present evidence that riluzole increases Smad2 and Smad3 linker phosphorylation at the cluster of serines 245/250/255 and serine 204 respectively. Using GSK3 inhibitors and siRNA knock-down, we demonstrate that the mechanism of riluzole-induced Smad phosphorylation involved GSK3β. In addition, GSK3β could phosphorylate the same linker sites in vitro. The riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFβ. We also demonstrate that riluzole-induced Smad linker phosphorylation is independent of the expression of the metabotropic glutamate receptor 1 (GRM1), which is one of the glutamate receptors whose involvement in human melanoma has been documented. We further show that riluzole upregulates the expression of INHBB and PLAU, two genes associated with the TGFβ signaling pathway. The non-canonical increase in Smad linker phosphorylation induced by riluzole could contribute to the modulation of the pro-oncogenic functions of Smads in late stage melanomas. PMID:23077590

  2. Phase II Study of Nilotinib in Melanoma Harboring KIT Alterations Following Progression to Prior KIT Inhibition

    PubMed Central

    Carvajal, Richard D.; Lawrence, Donald P.; Weber, Jeffrey S.; Gajewski, Thomas F.; Gonzalez, Rene; Lutzky, Jose; O’Day, Steven J.; Hamid, Omid; Wolchok, Jedd D.; Chapman, Paul B.; Sullivan, Ryan J.; Teitcher, Jerrold B.; Ramaiya, Nikhil; Giobbie-Hurder, Anita; Antonescu, Cristina R.; Heinrich, Michael C.; Bastian, Boris C.; Corless, Christopher L.; Fletcher, Jonathan A.; Hodi, F. Stephen

    2016-01-01

    Purpose Although durable responses can be achieved with tyrosine kinase inhibitors such as imatinib in melanomas harboring KIT mutations, the efficacy of alternative inhibitors after progression to imatinib and the activity of these agents on brain metastases is unknown. Experimental Design We conducted a phase II study of nilotinib 400 mg BID in two cohorts of patients with melanomas harboring KIT mutations or amplification: A) those refractory or intolerant to a prior KIT inhibitor; and B) those with brain metastases. The primary endpoint was 4-month disease control rate. Secondary endpoints included response rate, time-to-progression and overall survival. A Simon two-stage and a single-stage design was planned to assess for the primary endpoint in Cohorts A and B, respectively. Results Twenty patients were enrolled and 19 treated (11-Cohort A; 8-Cohort B). Three patients on Cohort A (27%; 95% CI, 8% – 56%) and 1 on Cohort B (12.5%; 90% CI, 0.6% – 47%) achieved the primary endpoint. Two partial responses were observed in Cohort A (18.2%, 90% CI, 3% – 47%); none were observed in Cohort B. The median time-to-progression and overall survival was 3·3 (90% CI, 2.1 – 3.9 months) and 9.1 months (90% CI, 4.3 – 14.2 months), respectively, in all treated patients. Conclusion Nilotinib may achieve disease control in patients with melanoma harboring KIT alterations and whose disease progressed after imatinib therapy. The efficacy of this agent in KIT altered melanoma with brain metastasis is limited. PMID:25695690

  3. Simultaneous knockdown of BRAF and expression of INK4A in melanoma cells leads to potent growth inhibition and apoptosis.

    PubMed

    Zhao, Yanhua; Zhang, Yan; Yang, Zhen; Li, Albert; Dong, Jianli

    2008-06-01

    Abnormal BRAF and p16INK4A co-exist in 60% of melanomas. BRAF mutation also occurs in 80% of benign nevi where it turns-on p16INK4A resulting in proliferative senescence; loss of p16INK4A removes the inhibitory block leading to melanoma development. Since only melanomas with wild-type BRAF have amplified CDK4 and cyclin D1 genes, p16INK4A-CDK4/6-cyclin D pathway is viewed as linearly downstream of BRAF. Thus, co-occurrence of aberrant BRAF and INK4A may be remnant of changes during melanoma formation without functional significance. To explore this notion, we simultaneously knocked down BRAF (via siRNA) and expressed INK4A cDNA in melanoma cells and observed enhanced growth inhibition. Notably, although each alone had no statistically significant effect on apoptosis, co-expression of BRAF siRNA and INK4A cDNA caused potent apoptosis, which was associated with up-regulation of BIM and down-regulation of BCL2. Our results suggest that aberrant BRAF and INK4A cooperate to promote proliferation and survival of melanoma cells. PMID:18402768

  4. Constitutive Smad linker phosphorylation in melanoma: a mechanism of resistance to transforming growth factor-β-mediated growth inhibition.

    PubMed

    Cohen-Solal, Karine A; Merrigan, Kim T; Chan, Joseph L-K; Goydos, James S; Chen, Wenjin; Foran, David J; Liu, Fang; Lasfar, Ahmed; Reiss, Michael

    2011-06-01

    Melanoma cells are resistant to transforming growth factor-β (TGFβ)-induced cell-cycle arrest. In this study, we investigated a mechanism of resistance involving a regulatory domain, called linker region, in Smad2 and Smad3, main downstream effectors of TGFβ. Melanoma cells in culture and tumor samples exhibited constitutive Smad2 and Smad3 linker phosphorylation. Treatment of melanoma cells with the MEK1/2 inhibitor, U0126, or the two pan-CDK and GSK3 inhibitors, Flavopiridol and R547, resulted in decreased linker phosphorylation of Smad2 and Smad3. Overexpression of the linker phosphorylation-resistant Smad3 EPSM mutant in melanoma cells resulted in an increase in expression of p15(INK4B) and p21(WAF1) , as compared with cells transfected with wild-type (WT) Smad3. In addition, the cell numbers of EPSM Smad3-expressing melanoma cells were significantly reduced compared with WT Smad3-expressing cells. These results suggest that the linker phosphorylation of Smad3 contributes to the resistance of melanoma cells to TGFβ-mediated growth inhibition. PMID:21477078

  5. MicroRNA-206 induces G1 arrest in melanoma by inhibition of CDK4 and Cyclin D.

    PubMed

    Georgantas, Robert W; Streicher, Katie; Luo, Xiaobing; Greenlees, Lydia; Zhu, Wei; Liu, Zheng; Brohawn, Philip; Morehouse, Christopher; Higgs, Brandon W; Richman, Laura; Jallal, Bahija; Yao, Yihong; Ranade, Koustubh

    2014-03-01

    Expression profiling of microRNAs in melanoma lesional skin biopsies compared with normal donor skin biopsies, as well as melanoma cell lines compared with normal melanocytes, revealed that hsa-miR-206 was down-regulated in melanoma (-75.4-fold, P = 1.7 × 10(-4)). MiR-206 has been implicated in a large number of cancers, including breast, lung, colorectal, ovarian, and prostate cancers; however, its role in tumor development remains largely unknown, its biologic function is poorly characterized, and its targets affecting cancer cells are largely unknown. MiR-206 reduced growth and migration/invasion of multiple melanoma cell lines. Bioinformatics identified cell cycle genes CDK2, CDK4, Cyclin C, and Cyclin D1 as strong candidate targets. Western blots and 3'UTR reporter gene assays revealed that miR-206 inhibited translation of CDK4, Cyclin D1, and Cyclin C. Additionally, hsa-miR-206 transfection induced G1 arrest in multiple melanoma cell lines. These observations support hsa-miR-206 as a tumor suppressor in melanoma and identify Cyclin C, Cyclin D1, and CDK4 as miR-206 targets. PMID:24289491

  6. A melanoma subtype with intrinsic resistance to BRAF inhibition identified by receptor tyrosine kinases gene-driven classification

    PubMed Central

    Dugo, Matteo; Nicolini, Gabriella; Tragni, Gabrina; Bersani, Ilaria; Tomassetti, Antonella; Colonna, Valentina; Del Vecchio, Michele; De Braud, Filippo; Canevari, Silvana

    2015-01-01

    Dysregulation of receptor tyrosine kinases (RTKs) contributes to several aspects of oncogenesis including drug resistance. In melanoma, distinct RTKs have been involved in BRAF inhibitors (BRAFi) resistance, yet the utility of RTKs expression pattern to identify intrinsically resistant tumors has not been assessed. Transcriptional profiling of RTKs and integration with a previous classification, reveals three robust subtypes in two independent datasets of melanoma cell lines and one cohort of melanoma samples. This classification was validated by Western blot in a panel of patient-derived melanoma cell lines. One of the subtypes identified here for the first time displayed the highest and lowest expression of EGFR and ERBB3, respectively, and included BRAF-mutant tumors all intrinsically resistant to BRAFi PLX4720, as assessed by analysis of the Cancer Cell Line Encyclopedia pharmacogenomic study and by in vitro growth inhibition assays. High levels of EGFR were detected, even before therapy, in tumor cells of one of three melanoma patients unresponsive to BRAFi. Use of different pharmacological inhibitors highlighted the relevance of PI3K/mTOR signaling for growth of this PLX4720-resistant subtype. Our results identify a specific molecular profile of melanomas intrinsically resistant to BRAFi and suggest the PI3K/mTOR pathway as a potential therapeutic target for these tumors. PMID:25742786

  7. Simultaneous knockdown of BRAF and expression of INK4A in melanoma cells leads to potent growth inhibition and apoptosis

    SciTech Connect

    Zhao Yanhua; Zhang Yan; Yang Zhen; Li, Albert; Dong Jianli

    2008-06-06

    Abnormal BRAF and p16INK4A co-exist in 60% of melanomas. BRAF mutation also occurs in 80% of benign nevi where it turns-on p16INK4A resulting in proliferative senescence; loss of p16INK4A removes the inhibitory block leading to melanoma development. Since only melanomas with wild-type BRAF have amplified CDK4 and cyclin D1 genes, p16INK4A-CDK4/6-cyclin D pathway is viewed as linearly downstream of BRAF. Thus, co-occurrence of aberrant BRAF and INK4A may be remnant of changes during melanoma formation without functional significance. To explore this notion, we simultaneously knocked down BRAF (via siRNA) and expressed INK4A cDNA in melanoma cells and observed enhanced growth inhibition. Notably, although each alone had no statistically significant effect on apoptosis, co-expression of BRAF siRNA and INK4A cDNA caused potent apoptosis, which was associated with up-regulation of BIM and down-regulation of BCL2. Our results suggest that aberrant BRAF and INK4A cooperate to promote proliferation and survival of melanoma cells.

  8. NUAK2 Amplification Coupled with PTEN Deficiency Promotes Melanoma Development via CDK Activation.

    PubMed

    Namiki, Takeshi; Yaguchi, Tomonori; Nakamura, Kenta; Valencia, Julio C; Coelho, Sergio G; Yin, Lanlan; Kawaguchi, Masakazu; Vieira, Wilfred D; Kaneko, Yasuhiko; Tanemura, Atsushi; Katayama, Ichiro; Yokozeki, Hiroo; Kawakami, Yutaka; Hearing, Vincent J

    2015-07-01

    The AMPK-related kinase NUAK2 has been implicated in melanoma growth and survival outcomes, but its therapeutic utility has yet to be confirmed. In this study, we show how its genetic amplification in PTEN-deficient melanomas may rationalize the use of CDK2 inhibitors as a therapeutic strategy. Analysis of array-CGH data revealed that PTEN deficiency is coupled tightly with genomic amplification encompassing the NUAK2 locus, a finding strengthened by immunohistochemical evidence that phospho-Akt overexpression was correlated with NUAK2 expression in clinical specimens of acral melanoma. Functional studies in melanoma cells showed that inactivation of the PI3K pathway upregulated p21 expression and reduced the number of cells in S phase. NUAK2 silencing and inactivation of the PI3K pathway efficiently controlled CDK2 expression, whereas CDK2 inactivation specifically abrogated the growth of NUAK2-amplified and PTEN-deficient melanoma cells. Immunohistochemical analyses confirmed an association of CDK2 expression with NUAK2 amplification and p-Akt expression in melanomas. Finally, pharmacologic inhibition of CDK2 was sufficient to suppress the growth of NUAK2-amplified and PTEN-deficient melanoma cells in vitro and in vivo. Overall, our results show how CDK2 blockade may offer a promising therapy for genetically defined melanomas, where NUAK2 is amplified and PTEN is deleted. PMID:25832654

  9. The mitogen-activated protein kinase pathway plays a critical role in regulating immunological properties of BRAF mutant cutaneous melanoma cells.

    PubMed

    Whipple, Chery A; Boni, Andrea; Fisher, Jan L; Hampton, Thomas H; Tsongalis, Gregory J; Mellinger, Diane L; Yan, Shaofeng; Tafe, Laura J; Brinckerhoff, Constance E; Turk, Mary J; Mullins, David W; Fadul, Camilo E; Ernstoff, Marc S

    2016-06-01

    The advent of drugs targeting the mitogen-activated protein kinase (MAPK) pathway has markedly changed the treatment of advanced-stage melanoma harboring BRAF mutations. However, drug resistance, through mechanisms not well elucidated, often occurs. A better understanding of how melanoma-derived immunologically active molecules change in response to MAPK inhibition of BRAF mutated (BRAF) and BRAF wild type (BRAF) melanomas could help identify promising treatment combinations of small molecule inhibitors and immunotherapy. To this aim, we treated 13 BRAF and 13 BRAF mutated human melanoma cell lines with either a specific BRAF inhibitor or an MEK1/2 inhibitor and analyzed changes in the secretion of 42 selected cytokines, chemokines, and growth factors. We also measured changes in the expression levels of immunologically relevant melanoma cell surface markers. The BRAF melanomas showed minimal changes in response to the inhibitors, whereas the BRAF cell lines showed, on average, a significant decrease in IFNα2, interleukin-7, Fractalkine, GCSF, GRO, TGFα2, interleukin-8, and VEGF, as well as a reduction in pERK and pMEK protein levels, upon MAPK pathway blockade. BRAF inhibition in BRAF cell lines also resulted in significant changes in the expression of several surface markers including upregulation of β2-microglobulin as well as a decrease in MIC A/B and TRAIL-R2. These results indicate that MAPK pathway inhibition leads to changes in the immunological properties of mutant BRAF melanoma cells and lends support for future studies aimed at designing effective treatment strategies that combine BRAF and MEK inhibition with immunotherapy. PMID:26974965

  10. Carnosic Acid Inhibits the Epithelial-Mesenchymal Transition in B16F10 Melanoma Cells: A Possible Mechanism for the Inhibition of Cell Migration

    PubMed Central

    Park, So Young; Song, Hyerim; Sung, Mi-Kyung; Kang, Young-Hee; Lee, Ki Won; Park, Jung Han Yoon

    2014-01-01

    Carnosic acid is a natural benzenediol abietane diterpene found in rosemary and exhibits anti-inflammatory, antioxidant, and anti-carcinogenic activities. In this study, we evaluated the effects of carnosic acid on the metastatic characteristics of B16F10 melanoma cells. When B16F10 cells were cultured in an in vitro Transwell system, carnosic acid inhibited cell migration in a dose-dependent manner. Carnosic acid suppressed the adhesion of B16F10 cells, as well as the secretion of matrix metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1, urokinase plasminogen activator (uPA), and vascular cell adhesion molecule (VCAM)-1. Interestingly, secretion of TIMP-2 increased significantly in B16F10 cells treated with 10 μmol/L carnosic acid. Additionally, carnosic acid suppressed the mesenchymal markers snail, slug, vimentin, and N-cadherin and induced epithelial marker E-cadherin. Furthermore, carnosic acid suppressed phosphorylation of Src, FAK, and AKT. These results indicate that inhibition of the epithelial-mesenchymal transition may be important for the carnosic acid-induced inhibition of B16F10 cell migration. PMID:25036034

  11. Carnosic acid inhibits the epithelial-mesenchymal transition in B16F10 melanoma cells: a possible mechanism for the inhibition of cell migration.

    PubMed

    Park, So Young; Song, Hyerim; Sung, Mi-Kyung; Kang, Young-Hee; Lee, Ki Won; Park, Jung Han Yoon

    2014-01-01

    Carnosic acid is a natural benzenediol abietane diterpene found in rosemary and exhibits anti-inflammatory, antioxidant, and anti-carcinogenic activities. In this study, we evaluated the effects of carnosic acid on the metastatic characteristics of B16F10 melanoma cells. When B16F10 cells were cultured in an in vitro Transwell system, carnosic acid inhibited cell migration in a dose-dependent manner. Carnosic acid suppressed the adhesion of B16F10 cells, as well as the secretion of matrix metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1, urokinase plasminogen activator (uPA), and vascular cell adhesion molecule (VCAM)-1. Interestingly, secretion of TIMP-2 increased significantly in B16F10 cells treated with 10 μmol/L carnosic acid. Additionally, carnosic acid suppressed the mesenchymal markers snail, slug, vimentin, and N-cadherin and induced epithelial marker E-cadherin. Furthermore, carnosic acid suppressed phosphorylation of Src, FAK, and AKT. These results indicate that inhibition of the epithelial-mesenchymal transition may be important for the carnosic acid-induced inhibition of B16F10 cell migration. PMID:25036034

  12. The combination of glutamate receptor antagonist MK-801 with tamoxifen and its active metabolites potentiates their antiproliferative activity in mouse melanoma K1735-M2 cells.

    PubMed

    Ribeiro, Mariana P C; Nunes-Correia, Isabel; Santos, Armanda E; Custódio, José B A

    2014-02-15

    Recent reports suggest that N-methyl-d-aspartate receptor (NMDAR) blockade by MK-801 decreases tumor growth. Thus, we investigated whether other ionotropic glutamate receptor (iGluR) antagonists were also able to modulate the proliferation of melanoma cells. On the other hand, the antiestrogen tamoxifen (TAM) decreases the proliferation of melanoma cells, and is included in combined therapies for melanoma. As the efficacy of TAM is limited by its metabolism, we investigated the effects of the NMDAR antagonist MK-801 in combination with TAM and its active metabolites, 4-hydroxytamoxifen (OHTAM) and endoxifen (EDX). The NMDAR blockers MK-801 and memantine decreased mouse melanoma K1735-M2 cell proliferation. In contrast, the NMDAR competitive antagonist APV and the AMPA and kainate receptor antagonist NBQX did not affect cell proliferation, suggesting that among the iGluR antagonists only the NMDAR channel blockers inhibit melanoma cell proliferation. The combination of antiestrogens with MK-801 potentiated their individual effects on cell biomass due to diminished cell proliferation, since it decreased the cell number and DNA synthesis without increasing cell death. Importantly, TAM metabolites combined with MK-801 promoted cell cycle arrest in G1. Therefore, the data obtained suggest that the activity of MK-801 and antiestrogens in K1735-M2 cells is greatly enhanced when used in combination. PMID:24240127

  13. Casticin Induced Apoptosis in A375.S2 Human Melanoma Cells through the Inhibition of NF-[Formula: see text]B and Mitochondria-Dependent Pathways In Vitro and Inhibited Human Melanoma Xenografts in a Mouse Model In Vivo.

    PubMed

    Shiue, Yin-Wen; Lu, Chi-Cheng; Hsiao, Yu-Ping; Liao, Ching-Lung; Lin, Jing-Pin; Lai, Kuang-Chi; Yu, Chien-Chih; Huang, Yi-Ping; Ho, Heng-Chien; Chung, Jing-Gung

    2016-01-01

    Casticin, a polymethoxyflavone occurring in natural plants, has been shown to have anticancer activities. In the present study, we aims to investigate the anti-skin cancer activity of casticin on melanoma cells in vitro and the antitumor effect of casticin on human melanoma xenografts in nu/nu mice in vivo. A flow cytometric assay was performed to detect expression of viable cells, cell cycles, reactive oxygen species production, levels of [Formula: see text] and caspase activity. A Western blotting assay and confocal laser microscope examination were performed to detect expression of protein levels. In the in vitro studies, we found that casticin induced morphological cell changes and DNA condensation and damage, decreased the total viable cells, and induced G2/M phase arrest. Casticin promoted reactive oxygen species (ROS) production, decreased the level of [Formula: see text], and promoted caspase-3 activities in A375.S2 cells. The induced G2/M phase arrest indicated by the Western blotting assay showed that casticin promoted the expression of p53, p21 and CHK-1 proteins and inhibited the protein levels of Cdc25c, CDK-1, Cyclin A and B. The casticin-induced apoptosis indicated that casticin promoted pro-apoptotic proteins but inhibited anti-apoptotic proteins. These findings also were confirmed by the fact that casticin promoted the release of AIF and Endo G from mitochondria to cytosol. An electrophoretic mobility shift assay (EMSA) assay showed that casticin inhibited the NF-[Formula: see text]B binding DNA and that these effects were time-dependent. In the in vivo studies, results from immuno-deficient nu/nu mice bearing the A375.S2 tumor xenograft indicated that casticin significantly suppressed tumor growth based on tumor size and weight decreases. Early G2/M arrest and mitochondria-dependent signaling contributed to the apoptotic A375.S2 cell demise induced by casticin. In in vivo experiments, A375.S2 also efficaciously suppressed tumor volume in a

  14. miR-203 inhibits melanoma invasive and proliferative abilities by targeting the polycomb group gene BMI1

    SciTech Connect

    Chang, Xiao; Sun, Yong; Han, Siqi; Zhu, Wei; Zhang, Haiping; Lian, Shi

    2015-01-02

    Highlights: • First reported deregulation of miR-203 and up-regulation of BMI1 in metastatic melanoma. • miR-203 decreased BMI1 expression by directly binding to 3′UTR. • Further found miR-203 overexpression suppressed cell invasion and stemness. • Re-expression of BMI1 rescued miR-203-mediated suppression. • miR-203-BMI1 axis may be potential therapeutic targets of melanoma metastasis. - Abstract: Metastasis is the major problem in malignant melanoma, posing a therapeutic challenge to clinicians. The investigation of the underlying mechanism driving this progress remains a large unmet need. In this study, we revealed a miR-203-BMI1 axis that regulated melanoma metastasis. We found significantly deregulation of miR-203 and up-regulation of BMI1 in melanoma, particularly in metastatic melanoma. An inverse correlation between the levels of miR-203 and BMI1 was further observed in melanoma tissues and cell lines. We also identified BMI1 as a downstream target gene of miR-203, which bound to the 3′UTR of BMI1. Overexpression of miR-203 was associated with decreased BMI1 expression and impaired cell invasion and tumor sphere formation activities. Re-expression of BMI1 markedly rescued miR-203-mediated suppression of these events. Taken together, our results demonstrated that miR-203 regulated melanoma invasive and proliferative abilities in part by targeting BMI1, providing new insights into potential mechanisms of melanoma metastasis.

  15. Four new ginsenosides from red ginseng with inhibitory activity on melanogenesis in melanoma cells.

    PubMed

    Zhou, Qi-Le; Yang, Xiu-Wei

    2015-08-15

    During a search for novel melanogenesis inhibitors originating from nature sources, four new ginsenosides, including three dammarane-type triterpenoid saponins, 20(S)-ginsenoside-Rf-1a (1), 20Z-ginsenoside-Rs4 (2), 23-O-methylginsenoside-Rg11 (3), and one oleanane-type saponin, ginsenoside-Ro-6'-O-butyl ester (4) were isolated from red ginseng (the steamed ginseng) to evaluate their protective effects against melanogenesis. Compounds 2 and 3 exhibited potent inhibitory effects against both melanin synthesis and tyrosinase activity in a dose-dependent manner in the α-MSH-stimulated B16 melanoma cells, and were more potent than the positive control arbutin, a well-known tyrosinase inhibitor. The results indicated that just the two carbon-20(22) double-bond-type ginsenosides showed strong inhibiting activity on melanogenesis through reducing tyrosinase activity. Thus, ginsenosides with such similar chemical structure in red ginseng may be potential natural products as tyrosinase inhibitors against malignant melanoma. PMID:26087936

  16. Melanin biosynthesis inhibitory activity of a compound isolated from young green barley (Hordeum vulgare L.) in B16 melanoma cells.

    PubMed

    Meng, Tian Xiao; Irino, Nobuto; Kondo, Ryuichiro

    2015-07-01

    In the course to find compounds that inhibit melanin biosynthesis (i.e., whitening agents), we evaluated the effects of the methanol-soluble fraction (i.e., the water-soluble portion of methanol extracts-CHP20P-MeOH eluted fraction) from young green barley leaves on melanin production in B16 melanoma cells. Activity-guided fractionation led to an isolate called tricin (compound 1) as an inhibitory compound of melanin production in B16 melanoma cells. Furthermore, tricin analogs such as tricetin, tricetin trimethyl ether, luteolin, and apigenin were used for analyzing the structure-activity relationships (SAR) of 5,7-dihydroxyflavones studies. Tricin demonstrated stronger inhibitory activity compared to three other compounds. The results suggest that a hydroxyl group at the C-4' position and methoxy groups at the C-3',5' positions of the tricin skeleton may have important roles in this inhibitory activity in B16 melanoma cells. Our results suggest that tricin inhibits melanin biosynthesis with higher efficacy than arbutin, and it could be used as a whitening agent. PMID:25827948

  17. Novel synthetic derivatives of the natural product berbamine inhibit Jak2/Stat3 signaling and induce apoptosis of human melanoma cells

    PubMed Central

    Nam, Sangkil; Xie, Jun; Perkins, Angela; Ma, Yuelong; Yang, Fan; Wu, Jun; Wang, Yan; Xu, Rong-zhen; Huang, Wendong; Horne, David A.; Jove, Richard

    2012-01-01

    Persistent Jak/Stat3 signal transduction plays a crucial role in tumorigenesis and immune development. Activated Jak/Stat3 signaling has been validated as a promising molecular target for cancer therapeutics discovery and development. Berbamine (BBM), a natural bis-benzylisoquinoline alkaloid, was identified from the traditional Chinese herbal medicine Berberis amurensis used for treatment of cancer patients. While BBM has been shown to have potent antitumor activities with low toxicity in various cancer types, the molecular mechanism of action of BBM remains largely unknown. Here, we determine the antitumor activities of thirteen synthetic berbamine derivatives (BBMDs) against human solid tumor cells. BBMD3, which is the most potent in this series of novel BBMDs, exhibits over 6-fold increase in biological activity compared to natural BBM. Moreover, BBMD3, directly inhibits Jak2 autophosphorylation kinase activity in vitro with IC50 = 0.69 μM. Autophosphorylation of Jak2 kinase at Tyr1007/1008 sites also was strongly inhibited in the range of 1 μM to 5 μM of BBMD3 in human melanoma cells at 4 h after treatment. Following inhibition of autophosphorylation of Jak2, BBMD3 blocked constitutive activation of downstream Stat3 signaling in melanoma cells. BBMD3 also down-regulated expression of the Stat3 target proteins Mcl-1 and Bcl-xL, associated with induction of apoptosis. In sum, our findings demonstrate that the novel berbamine derivative BBMD3 is an inhibitor of the Jak2/Stat3 signaling pathway, providing evidence for a molecular mechanism whereby BBMD3 exerts at least in part the apoptosis of human melanoma cells. In addition, BBMD3 represents a promising lead compound for development of new therapeutics for cancer treatment. PMID:22717603

  18. Detention of copper by sulfur nanoparticles inhibits the proliferation of A375 malignant melanoma and MCF-7 breast cancer cells.

    PubMed

    Liu, Hao; Zhang, Yikai; Zheng, Shanyuan; Weng, Zeping; Ma, Jun; Li, Yangqiu; Xie, Xinyuan; Zheng, Wenjie

    2016-09-01

    Selective induction of cell death or growth inhibition of cancer cells is the future of chemotherapy. Clinical trials have found that cancer tissues are enriched with copper. Based on this finding, many copper-containing compounds and complexes have been designed to "copper" cancer cells using copper as bait. However, recent studies have demonstrated that copper boosts tumor development, and copper deprivation from serum was shown to effectively inhibit the promotion of cancer. Mechanistically, copper is an essential cofactor for mitogen-activated protein kinase (MAPK)/extracellular activating kinase (ERK) kinase (MEK), a central molecule in the BRAF/MEK/ERK pathway. Therefore, depleting copper from cancer cells by directly sequestering copper has a wider field for research and potential for combination therapy. Based on the affinity between sulfur and copper, we therefore designed sulfur nanoparticles (Nano-S) that detain copper, achieving tumor growth restriction. We found that spherical Nano-S could effectively bind copper and form a tighter surficial structure. Moreover, this Nano-S detention of copper effectively inhibited the proliferation of A375 melanoma and MCF-7 breast cancer cells with minimum toxicity to normal cells. Mechanistic studies revealed that Nano-S triggered inactivation of the MEK-ERK pathway followed by inhibition of the proliferation of the A375 and MCF-7 cells. In addition, lower Nano-S concentrations and shorter exposure stimulated the expression of a copper transporter as compensation, which further increased the cellular uptake and anticancer activities of cisplatin. Collectively, our results highlight the potential of Nano-S as an anticancer agent or adjuvant through its detention of copper. PMID:27392714

  19. Callicarpa longissima extract, carnosol-rich, potently inhibits melanogenesis in B16F10 melanoma cells.

    PubMed

    Yamahara, Minori; Sugimura, Koji; Kumagai, Ayako; Fuchino, Hiroyuki; Kuroi, Azusa; Kagawa, Mai; Itoh, Yumi; Kawahara, Hidehisa; Nagaoka, Yasuo; Iida, Osamu; Kawahara, Nobuo; Takemori, Hiroshi; Watanabe, Hideto

    2016-01-01

    Cosmetic industries focus on developing materials and resources that regulate skin pigmentation. Melanin, the major pigment in human skin, protects the skin against damage from ultraviolet light. An ethanolic extract of the leaves of Callicarpa longissima inhibits melanin production in B16F10 mouse melanoma cells by suppressing microphthalmia-associated transcription factor (MITF) gene expression. Following purification and analysis using liquid chromatography-mass spectrometry (LC-MS), NMR, and biochemical assays, carnosol was determined to be responsible for the major inhibitory effect of the C. longissima extract on melanin production. Carnosol is an oxidative product of carnosic acid, whose presence in the extract was also confirmed by an authentic reference. The carnosol and carnosic acid content in the extract was approximately 16% (w/w). These results suggest that C. longissima is a novel, useful, and attractive source of skin-whitening agents. PMID:26267810

  20. Melanoma cell galectin-1 ligands functionally correlate with malignant potential*

    PubMed Central

    Yazawa, Erika M.; Geddes-Sweeney, Jenna E.; Cedeno-Laurent, Filiberto; Walley, Kempland C.; Barthel, Steven R.; Opperman, Matthew J.; Liang, Jennifer; Lin, Jennifer Y.; Schatton, Tobias; Laga, Alvaro C.; Mihm, Martin C.; Qureshi, Abrar A.; Widlund, Hans R.; Murphy, George F.; Dimitroff, Charles J.

    2015-01-01

    Galectin-1 (Gal-1)-binding to Gal-1 ligands on immune and endothelial cells can influence melanoma development through dampening anti-tumor immune responses and promoting angiogenesis. However, whether Gal-1 ligands are functionally expressed on melanoma cells to help control intrinsic malignant features remains poorly understood. Here, we analyzed expression, identity and function of Gal-1 ligands in melanoma progression. Immunofluorescent analysis of benign and malignant human melanocytic neoplasms revealed that Gal-1 ligands were abundant in severely-dysplastic nevi as well as in primary and metastatic melanomas. Biochemical assessments indicated that melanoma cell adhesion molecule (MCAM) was a major Gal-1 ligand on melanoma cells that was largely dependent on its N-glycans. Other melanoma cell Gal-1 ligand activity conferred by O-glycans was negatively regulated by α2,6 sialyltransferase ST6GalNAc2. In Gal-1-deficient mice, MCAM-silenced (MCAMKD) or ST6GalNAc2-overexpressing (ST6O/E) melanoma cells exhibited slower growth rates, underscoring a key role for melanoma cell Gal-1 ligands and host Gal-1 in melanoma growth. Further analysis of MCAMKD or ST6O/E melanoma cells in cell migration assays indicated that Gal-1 ligand-dependent melanoma cell migration was severely inhibited. These findings provide a refined perspective on Gal-1 – melanoma cell Gal-1 ligand interactions as contributors to melanoma malignancy. PMID:25756799

  1. Combined inhibition of Hsp90 and heme oxygenase-1 induces apoptosis and endoplasmic reticulum stress in melanoma.

    PubMed

    Barbagallo, Ignazio; Parenti, Rosalba; Zappalà, Agata; Vanella, Luca; Tibullo, Daniele; Pepe, Francesco; Onni, Toniangelo; Li Volti, Giovanni

    2015-10-01

    Heat shock proteins are ubiquitous molecular chaperones involved in post-translational folding, stability, activation and maturation of many proteins that are essential mediators of signal transduction and cell cycle progression. Heat shock protein 90 (Hsp90) has recently emerged as an attractive therapeutic target in cancer treatment since it may act as a key regulator of various oncogene products and cell-signaling molecules. Heme oxygenase-1 (HO-1; also known as Hsp32) is an inducible enzyme participating in heme degradation and involved in oxidative stress resistance. Recent studies indicate that HO-1 activation may play a role in tumor development and progression. In the present study we investigated the chemotherapic effects of combining an Hsp90 inhibitor (NMS E973) and an HO-1 inhibitor (SnMP) on A375 melanoma cells. NMS E973 treatment was able to reduce cell viability and induce endoplasmic reticulum (ER) stress (i.e. Ire1α, ERO1, PDI, BIP and CHOP). Interestingly, no significant effect was observed in reactive oxygen species (ROS) formation. Finally, NMS E973 treatment resulted in a significant HO-1 overexpression, which in turn serves as a possible chemoresistance molecular mechanism. Interestingly, the combination of NMS E973 and SnMP produced an increase of ROS and reduced cell viability compared to NMS E973 treatment alone. The inhibitors combination exhibited higher ER stress, apoptosis as evidenced by bifunctional apoptosis regulator (BFAR) mRNA expression and lower phosphorylation of Akt when compared to NMS E973 alone. In conclusion, these data suggest that HO-1 inhibition potentiates NMS E973 toxicity and may be exploited as a strategy for melanoma treatment. PMID:26493719

  2. FKBP51 employs both scaffold and isomerase functions to promote NF-κB activation in melanoma

    PubMed Central

    Romano, Simona; Xiao, Yichuan; Nakaya, Mako; D'Angelillo, Anna; Chang, Mikyoung; Jin, Jin; Hausch, Felix; Masullo, Mariorosario; Feng, Xixi; Romano, Maria Fiammetta; Sun, Shao-Cong

    2015-01-01

    Melanoma is the most aggressive skin cancer; its prognosis, particularly in advanced stages, is disappointing largely due to the resistance to conventional anticancer treatments and high metastatic potential. NF-κB constitutive activation is a major factor for the apoptosis resistance of melanoma. Several studies suggest a role for the immunophilin FKBP51 in NF-κB activation, but the underlying mechanism is still unknown. In the present study, we demonstrate that FKBP51 physically interacts with IKK subunits, and facilitates IKK complex assembly. FKBP51-knockdown inhibits the binding of IKKγ to the IKK catalytic subunits, IKK-α and -β, and attenuates the IKK catalytic activity. Using FK506, an inhibitor of the FKBP51 isomerase activity, we found that the IKK-regulatory role of FKBP51 involves both its scaffold function and its isomerase activity. Moreover, FKBP51 also interacts with TRAF2, an upstream mediator of IKK activation. Interestingly, both FKBP51 TPR and PPIase domains are required for its interaction with TRAF2 and IKKγ, whereas only the TPR domain is involved in interactions with IKKα and β. Collectively, these results suggest that FKBP51 promotes NF-κB activation by serving as an IKK scaffold as well as an isomerase. Our findings have profound implications for designing novel melanoma therapies based on modulation of FKBP51. PMID:26101251

  3. CARI III inhibits tumor growth in a melanoma-bearing mouse model through induction of G0/G1 cell cycle arrest.

    PubMed

    Park, Hye-Jin

    2014-01-01

    Mushroom-derived natural products have been used to prevent or treat cancer for millennia. In this study, we evaluated the anticancer effects of CARI (Cell Activation Research Institute) III, which consists of a blend of mushroom mycelia from Phellinus linteus grown on germinated brown rice, Inonotus obliquus grown on germinated brown rice, Antrodia camphorata grown on germinated brown rice and Ganoderma lucidum. Here, we showed that CARI III exerted anti-cancer activity, which is comparable to Dox against melanoma in vivo. B16F10 cells were intraperitoneally injected into C57BL6 mice to develop solid intra-abdominal tumors. Three hundred milligrams of the CARI III/kg/day p.o. regimen reduced tumor weight, comparable to the doxorubicin (Dox)-treated group. An increase in life span (ILS% = 50.88%) was observed in the CARI III-administered group, compared to the tumor control group. CARI III demonstrates anti-proliferative activity against B16F10 melanoma cells through inducing G0/G1 cell cycle arrest. CARI III inhibits the expression of cyclin D1, CDK4 and CDK2 and induces p21. Therefore, CARI III could be a potential chemopreventive supplement to melanoma patients. PMID:25221864

  4. The disintegrin tzabcanin inhibits adhesion and migration in melanoma and lung cancer cells.

    PubMed

    Saviola, Anthony J; Burns, Patrick D; Mukherjee, Ashis K; Mackessy, Stephen P

    2016-07-01

    Integrins play an essential role in cancer survival and invasion, and they have been major targets in drug development and design. Disintegrins are small (4-16kDa) viperid snake venom proteins that exhibit a canonical integrin-binding site (often RGD). These non-enzymatic proteins inhibit integrin-mediated cell-cell and cell-extracellular matrix interactions, making them potential candidates as therapeutics in cancer and numerous other human disorders. The present study examined the cytotoxic, anti-adhesion, and anti-migration effects of a recently characterized disintegrin, tzabcanin, towards melanoma (A-375) and lung (A-549) cancer cell lines. Tzabcanin inhibits adhesion of both cells lines to vitronectin and exhibited very weak cytotoxicity towards A-375 cells; however, it had no effect on cell viability of A-549 cells. Further, tzabcanin significantly inhibited migration of both cell lines in cell scratch/wound healing assays. Flow cytometric analysis indicates that both A-375 and A-549 cell lines express integrin αvβ3, a critical integrin in tumor motility and invasion, and a major receptor of the extracellular matrix protein vitronectin. Flow cytometric analysis also identified αvβ3 as a binding site of tzabcanin. These results suggest that tzabcanin may have utility in the development of anticancer therapies, or may be used as a biomarker to detect neoplasms that over-express integrin αvβ3. PMID:27060015

  5. BRAF inhibition for advanced locoregional BRAF V600E mutant melanoma: a potential neoadjuvant strategy.

    PubMed

    Sloot, Sarah; Zager, Jonathan S; Kudchadkar, Ragini R; Messina, Jane L; Benedict, Jacob J; Gonzalez, Ricardo J; DeConti, Ronald; Turner, Leslie M; McCardle, Timothy; Smalley, Keiran S M; Weber, Jeffrey S; Sondak, Vernon K; Gibney, Geoffrey T

    2016-02-01

    Selective BRAF inhibitors (BRAFi) yield objective responses in 50% of patients with metastatic BRAF V600E mutant melanoma. Adding an MEK inhibitor increases this response rate to 70%. Limited data are available on the outcomes of unresectable stage III patients, and it remains unclear whether BRAF-targeted therapy can be utilized as a neoadjuvant strategy. Data on patients with advanced locoregional BRAF V600E mutant melanoma treated with BRAF-targeted therapy at Moffitt Cancer Center were analyzed to determine response rates, subsequent resection rates after tumor downsizing, pathologic responses, and patient survival. Fifteen patients with locoregional disease treated with BRAF-targeted therapy, either BRAFi alone (vemurafenib; 11 patients) or a combination of BRAFi and an MEK inhibitor (dabrafenib plus trametinib or placebo; four patients), were identified. The median age was 50 years; the median follow-up was 25.4 months. The median BRAF-targeted therapy treatment duration was 6.0 months (range 1.2-29.4 months). Response Evaluation Criteria In Solid Tumors-based evaluation demonstrated objective response in 11 patients (73.3%). Six patients underwent resection of the remaining disease after therapy. Pathological analysis showed complete pathologic response (n=2), partial pathologic response (n=2), or no pathologic response (n=2). Four of six patients undergoing surgery have been alive for more than 2 years, including three patients currently free from active disease. No complications attributable to BRAF-targeted therapy were observed in the perioperative period. Dose reduction or discontinuation because of toxicities occurred in 10/15 patients. Neoadjuvant BRAF-targeted therapy may be effective in advanced locoregional BRAF V600E mutant melanoma patients in increasing resectability, yielding pathological responses, and achieving prolonged survival. PMID:26731560

  6. Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma.

    PubMed

    Tan, Justin L; Fogley, Rachel D; Flynn, Ryan A; Ablain, Julien; Yang, Song; Saint-André, Violaine; Fan, Zi Peng; Do, Brian T; Laga, Alvaro C; Fujinaga, Koh; Santoriello, Cristina; Greer, Celeste B; Kim, Yoon Jung; Clohessy, John G; Bothmer, Anne; Pandell, Nicole; Avagyan, Serine; Brogie, John E; van Rooijen, Ellen; Hagedorn, Elliott J; Shyh-Chang, Ng; White, Richard M; Price, David H; Pandolfi, Pier Paolo; Peterlin, B Matija; Zhou, Yi; Kim, Tae Hoon; Asara, John M; Chang, Howard Y; Young, Richard A; Zon, Leonard I

    2016-04-01

    Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation, while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma. PMID:27058786

  7. Redox control of retinoic acid receptor activity: a novel mechanism for retinoic acid resistance in melanoma cells.

    PubMed

    Demary, K; Wong, L; Liou, J S; Faller, D V; Spanjaard, R A

    2001-06-01

    Retinoic acid (RA) slows growth and induces differentiation of tumor cells through activation of RA receptors (RARs). However, melanoma cell lines display highly variable responsiveness to RA, which is a poorly understood phenomenon. By using Northern and Western blot analyses, we show that RA-resistant A375 and RA-responsive S91 melanoma cells express comparable levels of major components of RAR-signaling pathways. However, A375 cells have substantially higher intracellular reactive oxygen species (ROS) levels than S91 cells. Lowering ROS levels in A375 cells through hypoxic culture conditions restores RAR-dependent trans-activity, which could be further enhanced by addition of the antioxidant N-acetyl-cysteine. Hypoxia also enhances RAR activity in the moderately RA-responsive C32 cells, which have intermediate ROS levels. Conversely, increasing oxidative stress in highly RA-responsive S91 and B16 cells, which have low ROS levels, by treatment with H(2)O(2) impairs RAR activity. Consistent with these observations, RA more potently inhibited the proliferation of hypoxic A375 cells than that of normoxic cells. Oxidative states diminish, whereas reducing conditions enhance, DNA binding of retinoid X receptor/RAR heterodimers in vitro, providing a molecular basis for the observed inverse correlation between RAR activity and ROS levels. The redox state of melanoma cells provides a novel, epigenetic control mechanism of RAR activity and RA resistance. PMID:11356710

  8. Clinical value of protein S100 and melanoma-inhibitory activity (MIA) in malignant melanoma.

    PubMed

    Tas, Faruk; Yasasever, Vildan; Duranyildiz, Derya; Camlica, Hakan; Ustuner, Zeki; Aydiner, Adnan; Topuz, Erkan

    2004-06-01

    Serum protein S100 and melanoma-inhibitory protein (MIA) have been described as useful tumor markers for malignant melanoma. In this study, these two serum proteins were compared in 48 patients with melanoma at different stages of disease. Serum concentrations of S100 and MIA were measured by immunoradiometric and enzyme-linked immunosorbent assays, respectively. We found that the cut-off values were 17.4 ng/ml for MIA and 0.09 microg/l for S100. Five patients had stage I-II, 22 had stage III, and 21 had stage IV disease. Serum levels of two markers were elevated with metastatic disease (p < 0.05). Sensitivities of the MIA were found higher compared with S100 in patients with extensive (M1c) metastatic disease and with chemotherapy nonresponders (p > 0.05). We showed a trend for worsened outcome in patients with elevated MIA level in univariate analysis. MIA was found to be more sensitive and is a potential prognostic marker for patients with metastatic malignant melanoma in comparison with S100. PMID:15170138

  9. Studies on the mechanisms responsible for inhibition of experimental metastasis of B16-F10 murine melanoma by pentoxifylline.

    PubMed

    Gude, R P; Binda, M M; Presas, H L; Klein-Szanto, A J; Bonfil, R D

    1999-01-01

    Pentoxifylline (PTX), a methylxanthine derivative widely used as a hemorheological agent in the treatment of peripheral vascular disease, was studied to unveil the mechanisms responsible for its inhibitory action on B16-F10 experimental metastasis. In vitro pretreatment of B16-F10 cells with noncytotoxic concentrations of PTX significantly inhibited their adhesion to reconstituted basement membrane Matrigel(R) and type IV collagen as well as the relative activity of secreted 92 kD metalloproteinase. However, PTX pretreatment of B16-F10 cells did not affect their in vitro invasiveness. Heterotypic organ adhesion assays carried out with B16-F10 cells and suspended organ tissues demonstrated that pretreatment with noncytotoxic concentrations of PTX of both, tumor cells or lung tissue, brought about a dose-dependent inhibition of melanoma cell adhesion to lung. Immunohistochemical studies using antibodies against CD31 adhesion molecule (PECAM-1) revealed that B16-F10 cells adhere to lung endothelial cells. Our results suggest that PTX may exert its inhibitory effect on tumor lodgment, and as a consequence of that on experimental metastases, through an inhibitory action on cell adhesion molecules. PMID:10087444

  10. Digitized quantitative electroencephalographic patterns applied as magnetic fields inhibit melanoma cell proliferation in culture.

    PubMed

    Karbowski, Lukasz M; Harribance, Sean L; Buckner, Carly A; Mulligan, Bryce P; Koren, Stanley A; Lafrenie, Robert M; Persinger, Michael A

    2012-08-15

    Weak (1 μT) physiologically patterned magnetic fields produce changes in behavioral, physiological, and cellular activity. In the present experiments 12 temporal samples of the electroencephalographic anomaly and normal activity of a person (SLH) whose proximity reliably affected the brain activity of others were extracted from QEEG data, digitized, and presented as equivalent magnetic field patterns to B16 mouse melanoma cells. Only two of the patterns, both originating from the primary source (right temporal lobe) of the EEG anomaly reduced the cell growth by one-third compared to the other patterns extracted from his QEEG or sham field exposures. In previous experiments these EEG transients were also associated with marked increases in photon emissions from the right side of SLH's head. The results suggest that the intrinsic complexity of electroencephalographic patterns of some people, when amplified appropriately and applied as computer-generated magnetic fields in the three spatial planes, could diminish cancer cell growth. PMID:22750152

  11. Enhanced anti-tumor activity of a new curcumin-related compound against melanoma and neuroblastoma cells

    PubMed Central

    2010-01-01

    Background Sharing the common neuroectodermal origin, melanoma and neuroblastoma are tumors widely diffused among adult and children, respectively. Clinical prognosis of aggressive neuroectodermal cancers remains dismal, therefore the search for novel therapies against such tumors is warranted. Curcumin is a phytochemical compound widely studied for its antioxidant, anti-inflammatory and anti-cancer properties. Recently, we have synthesized and tested in vitro various curcumin-related compounds in order to select new anti-tumor agents displaying stronger and selective growth inhibition activity on neuroectodermal tumors. Results In this work, we have demonstrated that the new α,β-unsaturated ketone D6 was more effective in inhibiting tumor cells growth when compared to curcumin. Normal fibroblasts proliferation was not affected by this treatment. Clonogenic assay showed a significant dose-dependent reduction in both melanoma and neuroblastoma colony formation only after D6 treatment. TUNEL assay, Annexin-V staining, caspases activation and PARP cleavage unveiled the ability of D6 to cause tumor cell death by triggering apoptosis, similarly to curcumin, but with a stronger and quicker extent. These apoptotic features appear to be associated with loss of mitochondrial membrane potential and cytochrome c release. In vivo anti-tumor activity of curcumin and D6 was surveyed using sub-cutaneous melanoma and orthotopic neuroblastoma xenograft models. D6 treated mice exhibited significantly reduced tumor growth compared to both control and curcumin treated ones (Melanoma: D6 vs control: P < 0.001 and D6 vs curcumin P < 0.01; Neuroblastoma: D6 vs both control and curcumin: P < 0.001). Conclusions Our data indicate D6 as a good candidate to develop new therapies against neural crest-derived tumors. PMID:20525240

  12. Inhibition effect of phenyl compounds from the Oryza sativa roots on melanin production in murine B16-F10 melanoma cells.

    PubMed

    Cho, Jin-Gyeong; Huh, Jeongran; Jeong, Rak-Hun; Cha, Byeong-Ju; Shrestha, Sabina; Lee, Dong-Geol; Kang, Hee-Cheol; Kim, Ji-Young; Baek, Nam-In

    2015-01-01

    Five phenyl compounds, vanillin (1), methyl trans-ferulate (2), trans-p-coumaric acid methyl ester (3), N-benzoyltryptamine (4), and N-(trans-cinnamoyl)tryptamine (5), were isolated from the roots of Oryza sativa L. and identified on the basis of spectroscopic data. Compounds 3 and 5 showed strong inhibition effect on melanin production in murine B16-F10 melanoma cells and tyrosinase activity. Also, the quantitative analysis of the compounds was carried out using LC/MS/MS experiment. Compounds 3 and 5 could be used as skin-whitening agents. PMID:25299734

  13. MicroRNA-18b inhibits the growth of malignant melanoma via inhibition of HIF-1α-mediated glycolysis.

    PubMed

    Chen, Yao; Zhang, Ziqing; Luo, Chengqun; Chen, Zizi; Zhou, Jianda

    2016-07-01

    MicroRNAs (miRs) have been demonstrated to play critical roles in the development and progression of malignant melanoma (MM). However, the exact role and underlying mechanism of miR-18b in MM growth remains unclear. In the present study, real-time PCR data indicated that miR-18b was significantly downregulated in MM tissues compared to their matched adjacent non-tumor tissues. Low miR-18b expression was significantly associated with the tumor thickness and stage, although no significant association was observed between the miR-18b expression and the age, gender, or lymph node metastasis. Besides, miR-18b was also significantly downregulated in MM B16 and A375 cells compared to normal skin HACAT cells. Ectopic expression of miR-18b decreased the proliferation of A375 and B16 cells, while induced a remarkable cell cycle arrest at G1 stage. Besides, miR-18b overexpression also inhibited the glycolysis in A375 and B16 cells. HIF-1α, a key regulator in glycolysis, was then identified as a target gene of miR-18b, and its expression was negatively mediated by miR-18b in A375 and B16 cells. Overexpression of HIF-1α rescued the suppressive effect of miR-18b on MM cell proliferation and glycolysis. In vivo study further showed that overexpression of miR-18b inhibited the MM growth as well as the tumor-related death, accompanied with HIF-1α downregulation. Taken together, the present study suggests that miR-18b inhibits the growth of MM cells in vitro and in vivo through directly targeting HIF-1α. PMID:27220837

  14. Nonlethal Levels of Zeaxanthin Inhibit Cell Migration, Invasion, and Secretion of MMP-2 via NF-κB Pathway in Cultured Human Uveal Melanoma Cells.

    PubMed

    Bi, Ming-Chao; Hose, Nicole; Xu, Cai-Lian; Zhang, Chen; Sassoon, Jodi; Song, E

    2016-01-01

    Zeaxanthin at nonlethal dosages (3-10 μM) significantly inhibited the cell migration of cultured uveal melanoma cells (C918 cell line) as determined by wound healing assay and Boyden chamber assay. Matrigel invasion assay showed that cell invasion of uveal melanoma cells could be significantly inhibited by zeaxanthin. Secretion of MMP-2 by melanoma cells was significantly inhibited by zeaxanthin in a dose-dependent manner as measured by ELISA kit. Zeaxanthin also significantly inhibited the NF-κB levels in nuclear extracts of the UM cells, which is the upstream of the MMP-2 secretion. These results suggest that zeaxanthin might be a potentially therapeutic approach in the prevention of metastasis in uveal melanoma. PMID:26942004

  15. Nonlethal Levels of Zeaxanthin Inhibit Cell Migration, Invasion, and Secretion of MMP-2 via NF-κB Pathway in Cultured Human Uveal Melanoma Cells

    PubMed Central

    Bi, Ming-Chao; Hose, Nicole; Xu, Cai-Lian; Zhang, Chen; Sassoon, Jodi; Song, E.

    2016-01-01

    Zeaxanthin at nonlethal dosages (3–10 μM) significantly inhibited the cell migration of cultured uveal melanoma cells (C918 cell line) as determined by wound healing assay and Boyden chamber assay. Matrigel invasion assay showed that cell invasion of uveal melanoma cells could be significantly inhibited by zeaxanthin. Secretion of MMP-2 by melanoma cells was significantly inhibited by zeaxanthin in a dose-dependent manner as measured by ELISA kit. Zeaxanthin also significantly inhibited the NF-κB levels in nuclear extracts of the UM cells, which is the upstream of the MMP-2 secretion. These results suggest that zeaxanthin might be a potentially therapeutic approach in the prevention of metastasis in uveal melanoma. PMID:26942004

  16. Antiproliferative Activity of Double Point Modified Analogs of 1,25-Dihydroxyvitamin D2 Against Human Malignant Melanoma Cell Lines

    PubMed Central

    Piotrowska, Anna; Wierzbicka, Justyna; Nadkarni, Sharmin; Brown, Geoffrey; Kutner, Andrzej; Żmijewski, Michał A.

    2016-01-01

    Vitamin D is a lipid soluble steroid hormone with pleiotropic biological properties, including regulation of cell proliferation, differentiation and apoptosis. As to these desirable anticancer actions, 1,25-dihydroxyvitamins D and analogs have been reported to inhibit the proliferation and to induce differentiation of a wide variety of cancer cell types, including human malignant melanoma. However, there is a need for novel and more efficacious vitamin D analogs, and how best to design such is still an open issue. A series of double point modified (DPM) analogs of 1,25-dihydroxyvitamin D2 (1,25(OH)2D2) induced differentiation of the vitamin D receptor (VDR) positive A375 and VDR negative SK-MEL 188b human malignant melanoma cell lines. Surprisingly, the dose of 1,25(OH)2D2 required to inhibit the proliferation of the A375 melanoma cell line by was several fold lower than that required in the case of 1,25(OH)2D3. To evaluate the impact of the modification in the side chain (additional 22-hydroxyl) and in the A-ring (5,6-trans modification), the regular side-chain of vitamin D2 or D3 was retained in the structure of our analogs. As expected, 5,6-trans modification was advantageous to enhancing the anti-proliferative activity of analogs, but not as a single point modification (SPM). Very unexpectedly, the additional 22-hydroxyl in the side-chain reduced significantly the anti-proliferative activity of both the natural and 5,6-trans series analogs. Finally, an induction of pigmentation in melanoma SK-MEL 188b cells was observed to sensitized cells to the effect of vitamin D analogs. PMID:26760999

  17. MicroRNA-199a-5p inhibits tumor proliferation in melanoma by mediating HIF-1α.

    PubMed

    Yang, Xinghua; Lei, Shaorong; Long, Jianhong; Liu, Xiaojin; Wu, Qizhen

    2016-06-01

    The expression of hypoxia-inducible factor 1α (HIF-1α) is often abundant in human cancer and it is associated with poor prognosis. The present study aimed to investigate its regulation by microRNA (miRNA). The expression of miRNA-199a-5p (miR-199a-5p) in melanoma was detected by quantitative polymerase chain reaction on samples from 25 melanoma patients. The target of miR-199a-5p was predicted and demonstrated by a dual‑luciferase reporter system. The effects of miR-199a-5p on melanoma cells were assayed in B16 and HME1 melanoma cell lines. Furthermore, the potential of miR‑199a‑5p as a therapeutic target was illustrated in xenograft nude mice models. Low expression of miR‑199a‑5p in tumor melanoma tissue samples from patients was associated with high histological grade and advanced tumor stage. The 3'-untranslated region of HIF‑1α was identified as a target of miR‑199a‑5p by Targetscan software. The dual-luciferase reporter assay demonstrated that miR‑199a‑5p transfection of mimics decreased the luciferase activity significantly (P<0.05). In the B16 and HME1 cell lines, overexpression of miR‑199a‑5p suppressed cell proliferation and arrested the cell cycle in the G1 phase. In vivo overexpression of miR‑199a‑5p significantly suppressed xenograft growth and downregulated the expression of HIF‑1α (P<0.05). The results from the present study suggest that miR‑199a‑5p suppressed melanoma proliferation via HIF‑1α, suggesting it may be a potential therapeutic target for melanoma treatment. PMID:27122154

  18. Calpain-dependent clearance of the autophagy protein p62/SQSTM1 is a contributor to ΔPK oncolytic activity in melanoma.

    PubMed

    Colunga, A; Bollino, D; Schech, A; Aurelian, L

    2014-04-01

    Oncolytic virotherapy is a promising strategy for reducing tumor burden through selective virus replication in rapidly proliferating cells. However, the lysis of slowly replicating cancer stem cells (CSCs), which maintain neoplastic clonality, is relatively modest and the potential contribution of programmed cell death pathways to oncolytic activity is still poorly understood. We show that the oncolytic virus ΔPK lyses CSC-enriched breast cancer and melanoma 3D spheroid cultures at low titers (0.1 pfu/cell) without resistance development and it inhibits the 3D growth potential (spheroids and agarose colonies) of melanoma and breast cancer cells. ΔPK induces calpain activation in both melanoma and breast cancer 3D cultures as determined by the loss of the p28 regulatory subunit, and 3D growth is restored by treatment with the calpain inhibitor PD150606. In melanoma, ΔPK infection also induces light chain 3 (LC3)-II accumulation and p62/SQSTM1 clearance, both markers of autophagy, and 3D growth is restored by treatment with the autophagy inhibitor chloroquine (CQ). However, expression of the autophagy-required protein Atg5 is not altered and CQ does not restore p62/SQSTM1 expression, suggesting that the CQ effect may be autophagy-independent. PD150606 restores expression of p62/SQSTM1 in ΔPK-infected melanoma cultures, suggesting that calpain activation induces anti-tumor activity through p62/SQSTM1 clearance. PMID:24553345

  19. Phorbol ester phorbol-12-myristate-13-acetate promotes anchorage-independent growth and survival of melanomas through MEK-independent activation of ERK1/2

    SciTech Connect

    Jorgensen, Kjersti; Skrede, Martina; Cruciani, Veronique; Mikalsen, Svein-Ole; Slipicevic, Ana; Florenes, Vivi Ann . E-mail: v.a.florenes@labmed.uio.no

    2005-04-01

    The phorbol ester, phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, is known to stimulate the in vitro growth of monolayer cultures of normal human melanocytes whereas it inhibits the growth of most malignant melanoma cell lines. We examined the effect of PMA on proliferation and survival of melanoma cells grown as multicellular aggregates in suspension (spheroids), and aimed to elucidate downstream targets of PKC signaling. In contrast to monolayer cultures, PMA increased cell proliferation as well as protected melanoma cells from suspension-mediated apoptosis (anoikis). Supporting the importance of PKC in anchorage-independent growth, treatment of anoikis-resistant melanoma cell lines with antisense oligonucleotides against PKC-{alpha}, or the PKC inhibitor Goe6976, strongly induced anoikis. PMA induced activation of ERK1/2, but this effect was not prevented by the MEK inhibitors PD98059 or by U0126. Whereas PD98059 treatment alone led to marked activation of the pro-apoptotic Bim and Bad proteins and significantly increased anoikis, these effects were clearly reversed by PMA. In conclusion, our results indicate that the protective effect of PMA on anchorage-independent survival of melanoma cells at least partly is mediated by MEK-independent activation of ERK1/2 and inactivation of downstream pro-apoptotic effector proteins.

  20. A novel microtubule de-stabilizing complementarity-determining region C36L1 peptide displays antitumor activity against melanoma in vitro and in vivo

    PubMed Central

    Figueiredo, Carlos R.; Matsuo, Alisson L.; Azevedo, Ricardo A.; Massaoka, Mariana H.; Girola, Natalia; Polonelli, Luciano; Travassos, Luiz R.

    2015-01-01

    Short peptide sequences from complementarity-determining regions (CDRs) of different immunoglobulins may exert anti-infective, immunomodulatory and antitumor activities regardless of the specificity of the original monoclonal antibody (mAb). In this sense, they resemble early molecules of innate immunity. C36L1 was identified as a bioactive light-chain CDR1 peptide by screening 19 conserved CDR sequences targeting murine B16F10-Nex2 melanoma. The 17-amino acid peptide is readily taken up by melanoma cells and acts on microtubules causing depolymerization, stress of the endoplasmic reticulum and intrinsic apoptosis. At low concentrations, C36L1 inhibited migration, invasion and proliferation of B16F10-Nex2 cells with cell cycle arrest at G2/M phase, by regulating the PI3K/Akt signaling axis involving Rho-GTPase and PTEN mediation. Peritumor injection of the peptide delayed growth of subcutaneously grafted melanoma cells. Intraperitoneal administration of C36L1 induced a significant immune-response dependent anti-tumor protection in a syngeneic metastatic melanoma model. Dendritic cells stimulated ex-vivo by the peptide and transferred to animals challenged with tumor cells were equally effective. The C36 VL CDR1 peptide is a promising microtubule-interacting drug that induces tumor cell death by apoptosis and inhibits metastases of highly aggressive melanoma cells. PMID:26391685

  1. [Mesenchymal stem cells expressing cytosine deaminase inhibit growth of murine melanoma B16F10 in vivo].

    PubMed

    Krasikova, L S; Karshieva, S S; Cheglakov, I B; Belyavsky, A V

    2015-01-01

    The aim of this study was to estimate the efficacy of mesenchymal stem cell-based suicide gene therapy in mice bearing murine melanoma B16F10. Adipose mesenchymal stem cells (MSCs) were transfected with plasmid constructs expressing cytosine deaminase fused with uracil phosphoribosyltransferase (CDA/UPRT) or CDA/UPRT fused with HSV-1 tegument protein VP22 (CDA/UPRT/VP22). In this study, we demonstrate that direct intratumoral transplantation of MSCs expressing CDA/UPRT or CDA/UPRT/VP22 followed by systemic administration of 5-fluorocytosine (5-FC) results in a significant inhibition of tumor growth. There was a 53% reduction in tumor volume in mice treated with CDA/UPRT-MSCs and 58% reduction in tumor volume in mice treated with CDA/UPRT/VP22-MSCs as compared with control animals transplanted with B16F10 melanoma alone. Injection of CDA/UPRT-MSC and CDA/UPRT/VP22-MSC prolonged the life span of mice bearing B16F10 melanoma by 15 and 26%, respectively. The data indicate that in murine B16F10 melanoma model, MSCs encoding CDA/UPRT suicide gene have a significant antitumor effect. PMID:26710783

  2. Anticancer Activity of Saponins from Allium chinense against the B16 Melanoma and 4T1 Breast Carcinoma Cell

    PubMed Central

    Yu, Zhihui; Zhang, Tong; Zhou, Fengjuan; Xiao, Xiuqing; Ding, Xuezhi; He, Hao; Rang, Jie; Quan, Meifang; Wang, Ting; Zuo, Mingxing; Xia, Liqiu

    2015-01-01

    The cytotoxic substance of A. chinense saponins (ACSs) was isolated using ethanol extraction and purified with the D101 macroporous adsorption resin approach. We investigated the anticancer activity of ACSs in the B16 melanoma and 4T1 breast carcinoma cell lines. Methylthioninium chloride and hematoxylin-eosin staining with Giemsa dyestuff were used when the cells were treated with ACSs. The results showed that the cells morphologies changed significantly; ACSs induced cell death in B16 and 4T1 cells based on acridine orange/ethidium bromide double fluorescence staining, with the number and degree of apoptotic tumor cells increasing as ACS concentration increased. ACSs inhibited the proliferation of B16 and 4T1 cells in a dose-dependent manner. They also inhibited cell migration and colony formation and exhibited a concentration-dependent effect. In addition, ACSs apparently inhibited the growth of melanoma in vivo. The preliminary antitumor in vivo assay revealed that early medication positively affected tumor inhibition action and effectively protected the liver and spleen of C57 BL/6 mice from injury. This study provides evidence for the cytotoxicity of ACSs and a strong foundation for further research to establish the theoretical basis for cell death and help in the design and development of new anticancer drugs. PMID:26146506

  3. Simultaneous Inhibition of Key Growth Pathways in Melanoma Cells and Tumor Regression by a Designed Bidentate Constrained Helical Peptide

    PubMed Central

    Dhar, Amlanjyoti; Mallick, Shampa; Ghosh, Piya; Maiti, Atanu; Ahmed, Israr; Bhattacharyya, Seemana; Mandal, Tapashi; Manna, Asit; Roy, Koushik; Singh, Sandeep; Nayak, Dipak Kumar; Wilder, Paul T.; Markowitz, Joseph; Weber, David J.; Ghosh, Mrinal K.; Chattopadhyay, Samit; Guha, Rajdeep; Konar, Aditya; Bandyopadhyay, Santu; Roy, Siddhartha

    2014-01-01

    Protein-protein interactions are part of a large number of signaling networks and potential targets for drug development. However, discovering molecules that can specifically inhibit such interactions is a major challenge. S100B, a calcium-regulated protein, plays a crucial role in the proliferation of melanoma cells through protein-protein interactions. In this article, we report the design and development of a bidentate conformationally constrained peptide against dimeric S100B based on a natural tight binding peptide, TRTK-12. The helical conformation of the peptide was constrained by substitution of α-amino isobutyric acid----an amino acid having high helical propensity----in positions which do not interact with S100B. A branched bidentate version of the peptide, bound to S100B tightly with a dissociation constant of 8 nM. When conjugated to a cell penetrating peptide, it caused growth inhibition and rapid apoptosis in melanoma cells. The molecule exerts anti-proliferative action through simultaneous inhibition of key growth pathways including reactivation of wild-type p53 and inhibition of Akt and STAT-3 phosphorylation. The apoptosis induced by the bidentate constrained helix is caused by direct migration of p53 to mitochondria. At moderate intravenous dose, the peptide completely inhibits melanoma growth in a mouse model without any significant observable toxicity. The specificity was shown by lack of ability of a double mutant peptide to cause tumor regression at the same dose level. The methodology described here for direct protein-protein interaction inhibition may be effective for rapid development of inhibitors against relatively weak protein-protein interactions for de novo drug development. PMID:24839139

  4. Improved antitumor activity of immunotherapy with BRAF and MEK inhibitors in BRAFV600E melanoma

    PubMed Central

    Hu-Lieskovan, Siwen; Mok, Stephen; Moreno, Blanca Homet; Tsoi, Jennifer; Faja, Lidia Robert; Goedert, Lucas; Pinheiro, Elaine M.; Koya, Richard C.; Graeber, Thomas; Comin-Anduix, Begoña; Ribas, Antoni

    2016-01-01

    Combining immunotherapy and BRAF targeted therapy may result in improved antitumor activity with the high response rates of targeted therapy and the durability of responses with immunotherapy. However, the first clinical trial testing the combination of the BRAF inhibitor vemurafenib and the CTLA-4 antibody ipilimumab was terminated early due to substantial liver toxicities. MEK inhibitors can potentiate the MAPK inhibition in BRAF mutant cells, while potentially alleviating the unwanted paradoxical MAPK activation in BRAF wild type cells that lead to side effects when using BRAF inhibitors alone. However, there is the concern of MEK inhibitors being detrimental to T cell functionality. Using a mouse model of syngeneic BRAFV600E driven melanoma, we tested whether addition of the MEK inhibitor trametinib would enhance the antitumor activity of combined immunotherapy with the BRAF inhibitor dabrafenib. Combination of dabrafenib and trametinib with pmel-1 adoptive cell transfer (ACT) showed complete tumor regression, increased T cell infiltration into tumors and improved in vivo cytotoxicity. Single agent dabrafenib increased tumor-associated macrophages and T regulatory cells (Tregs) in tumors, which decreased with the addition of trametinib. The triple combination therapy resulted in increased melanosomal antigen and MHC expression, and global immune-related gene up-regulation. Given the up-regulation of PD-L1 seen with dabrafenib and/or trametinib combined with antigen-specific ACT, we tested combination of dabrafenib, trametinib with anti-PD1 therapy in SM1 tumors, and observed superior anti-tumor effect. Our findings support the testing of triple combination therapy of BRAF and MEK inhibitors with immunotherapy in patients with BRAFV600E mutant metastatic melanoma. PMID:25787767

  5. Stimulation of lymphocyte anti-melanoma activity by co-cultured macrophages activated by complex homeopathic medication

    PubMed Central

    2009-01-01

    Background Melanoma is the most aggressive form of skin cancer, and the most rapidly expanding cancer in terms of worldwide incidence. Chemotherapeutic approaches to treat melanoma have been uniformly disappointing. A Brazilian complex homeopathic medication (CHM), used as an immune modulator, has been recommended for patients with depressed immune systems. Previous studies in mice have demonstrated that the CHM activates macrophages, induces an increase in the number of leukocytes and improves the murine response against Sarcoma-180. Methods Here we studied the interaction of mouse lymph node lymphocytes, co-cultured in vitro with macrophages in the presence or absence of the CHM, with B16F10 melanoma cells. Results Lymphocytes co-cultured with macrophages in the presence of the CHM had greater anti-melanoma activity, reducing melanoma cell density and increasing the number of lysed tumor cells. There was also a higher proportion of activated (CD25+) lymphocytes with increased viability. Overall, lymphocytes activated by treatment destroyed growing cancer cells more effectively than control lymphocytes. Conclusion Co-culture of macrophages with lymphocytes in the presence of the CHM enhanced the anti-cancer performance of lymphocytes against a very aggressive lineage of melanoma cells. These results suggest that non-toxic therapies using CHMs are a promising alternative approach to the treatment of melanomas. In addition, they are attractive combination-therapy candidates, which may enhance the efficacy of conventional medicines by improving the immune response against tumor cells. PMID:19698142

  6. Kaurenic acid: Evaluation of the in vivo and in vitro antitumor activity on murine melanoma

    PubMed Central

    Sosa-Sequera, Miriam C.; Chiurillo, Miguel; Moscoso, Jaime; Dolinar, Josefina; Suarez, Omar; Neira, Natalia; Mendoza, Hernan; Rivero-Paris, María

    2011-01-01

    Objective: The in vivo and in vitro antitumor activity of kaurenic acid [kaur 16-en-19 oic acid] (KA) in melanoma was evaluated in a murine model in comparison with taxol (Tx). Materials and Methods: B16F1 melanoma was developed in C57BL/6 mice and cell cultures. Survival test, tumor growth, dissected-tumor measurements, histology, cytoxicity assay on cultured cells, and changes of apoptotic gene expression at mRNA level were analyzed. Results: KA showed antitumor effect in vivo and in vitro and compared with Tx, its antimelanoma activity was greater (P < 0.001). These results were confirmed by morphological analysis (P < 0.001). In melanoma cell cultures, KA IC50 was 0.79 μM vs. 18.94 μM for Tx (P < 0.001). RT-PCR analysis demonstrated that Bcl-xL mRNA expression was altered in B16F1 mouse melanoma cells obtained from mice treated with either KA or Tx. Conclusion: The data suggest that KA is active in animal melanoma models, both in vitro and in vivo, being its cytotoxic effects stronger than those exhibited by Tx. Further trials should be conducted to elucidate its mechanism of action in melanoma with respect to necrosis or apoptotic processes. Our results support other evidences indicating that KA is a potential chemotherapeutic agent against cancer that has to be widely explored. PMID:22144774

  7. Isoangustone A, a novel licorice compound, inhibits cell proliferation by targeting PI3K, MKK4, and MKK7 in human melanoma.

    PubMed

    Song, Nu Ry; Lee, Eunjung; Byun, Sanguine; Kim, Jong-Eun; Mottamal, Madhusoodanan; Park, Jung Han Yoon; Lim, Soon Sung; Bode, Ann M; Lee, Hyong Joo; Lee, Ki Won; Dong, Zigang

    2013-12-01

    Licorice root is known to possess various bioactivities, including anti-inflammatory and anticancer effects. Glycyrrhizin, a triterpene compound, is the most abundant constituent of dried licorice root. However, high intake or long-term consumption of glycyrrhizin causes several side effects, such as hypertension, hypertensive encephalopathy, and hypokalemia. Therefore, finding additional active compounds other than glycyrrhizin in licorice that exhibit anticancer effects is worthwhile. We found that isoangustone A (IAA), a novel flavonoid from licorice root, suppressed proliferation of human melanoma cells. IAA significantly blocked cell-cycle progression at the G1-phase and inhibited the expression of G1-phase regulatory proteins, including cyclins D1 and E in the SK-MEL-28 human melanoma cell line. IAA suppressed the phosphorylation of Akt, GSK-3β, and JNK1/2. IAA also bound to phosphoinositide 3-kinase (PI3K), MKK4, and MKK7, strongly inhibiting their kinase activities in an ATP-competitive manner. Moreover, in a xenograft mouse model, IAA significantly decreased tumor growth, volume, and weight of SK-MEL-28 xenografts. Collectively, these results suggest that PI3K, MKK4, and MKK7 are the primary molecular targets of IAA in the suppression of cell proliferation. This insight into the biologic actions of IAA provides a molecular basis for the potential development of a new chemotherapeutic agent. PMID:24104352

  8. Dehydroleucodine inhibits tumor growth in a preclinical melanoma model by inducing cell cycle arrest, senescence and apoptosis.

    PubMed

    Costantino, Valeria V; Lobos-Gonzalez, Lorena; Ibañez, Jorge; Fernandez, Dario; Cuello-Carrión, F Darío; Valenzuela, Manuel A; Barbieri, Manuel A; Semino, Silvana N; Jahn, Graciela A; Quest, Andrew F G; Lopez, Luis A

    2016-03-01

    Malignant melanoma represents the fastest growing public health risk of all cancer types worldwide. Several strategies and anti-cancer drugs have been used in an effort to improve treatments, but the development of resistance to anti-neoplastic drugs remains the major cause of chemotherapy failure in melanomas. Previously, we showed that the sesquiterpene lactone, dehydroleucodine (DhL), promotes the accumulation of DNA damage markers, such as H2AX and 53BP1, in human tumor cells. Also DhL was shown to trigger either cell senescence or apoptosis in a concentration-dependent manner in HeLa and MCF7 cells. Here, we evaluated the effects of DhL on B16F0 mouse melanoma cells in vitro and in a pre-clinical melanoma model. DhL inhibited the proliferation of B16F0 cells by inducing senescence or apoptosis in a concentration-dependent manner. Also, DhL reduced the expression of the cell cycle proteins cyclin D1 and B1 and the inhibitor of apoptosis protein, survivin. In melanomas generated by subcutaneous injection of B16F0 cells into C57/BL6 mice, the treatment with 20 mg DhL /Kg/day in preventive, simultaneous and therapeutic protocols reduced tumor volumes by 70%, 60% and 50%, respectively. DhL treatments reduced the number of proliferating, while increasing the number of senescent and apoptotic tumor cells. To estimate the long-term effects of DhL, a mathematical model was applied to fit experimental data. Extrapolation beyond experimental time points revealed that DhL administration following preventive and therapeutic protocols is predicted to be more effective than simultaneous treatments with DhL in restricting tumor growth. PMID:26718258

  9. FOXP3 over-expression inhibits melanoma tumorigenesis via effects on proliferation and apoptosis.

    PubMed Central

    Tan, BeeShin; Anaka, Matthew; Deb, Siddhartha; Freyer, Claudia; Ebert, Lisa M.; Chueh, Anderly C.; Al-Obaidi, Sheren; Behren, Andreas; Jayachandran, Aparna; Cebon, Jonathan; Chen, Weisan; Mariadason, John M.

    2014-01-01

    The Forkhead box P3 (FOXP3) transcription factor is the key driver of regulatory T cell (Treg cells) differentiation and immunosuppressive function. In addition, FOXP3 has been reported to be expressed in many tumors, including melanoma. However, its role in tumorigenesis is conficting, with both tumor suppressive and tumor promoting functions described. The aim of the current study was to characterize the expression and function of FOXP3 in melanoma. FOXP3 expression was detected by immunohistochemistry (IHC) in 12% (18/146) of stage III and IV melanomas. However expression was confined to fewer than 1% of cells in these tumors. Stable over-expression of FOXP3 in the SK-MEL-28 melanoma cell line reduced cell proliferation and clonogenicity in vitro, and reduced xenograft growth in vivo. FOXP3 over-expression also increased pigmentation and the rate of apoptosis of SK-MEL-28 cells. Based on its infrequent expression in human melanoma, and its growth inhibitory and pro-apoptotic effect in over-expressing melanoma cells, we conclude that FOXP3 is not likely to be a key tumor suppressor or promoter in melanoma. PMID:24406338

  10. Melanoma Expressed-CD70 Is Regulated by RhoA and MAPK Pathways without Affecting Vemurafenib Treatment Activity

    PubMed Central

    Sarrabayrouse, Guillaume; Gallardo, Franck; Gence, Rémi; Tilkin-Mariamé, Anne-Françoise

    2016-01-01

    CD70 is a costimulatory molecule member of the Tumor Necrosis Factor family that is expressed on activated immune cells. Its ectopic expression has been described in several types of cancer cells including lymphomas, renal cell carcinomas and glioblastomas. We have recently described its expression in a part of tumor cells from the vast majority of melanoma biopsies and human melanoma cell lines, and found that CD70 expression decreased over time as the disease progressed. Here, we show that RhoA, BRAF and Mitogen Activating Protein Kinase pathways are involved in the positive transcriptional regulation of CD70 expression in melanomas. Interestingly, the clinical inhibitor of the common BRAF V600E/D variants, Vemurafenib (PLX-4032), which is currently used to treat melanoma patients with BRAF V600E/D-mutated metastatic melanomas, decreased CD70 expression in human CD70+ melanoma cell lines. This decrease was seen in melanoma cells both with and without the BRAFV600E/D mutation, although was less efficient in those lacking the mutation. But interestingly, by silencing CD70 in CD70+ melanoma cell lines we show that PLX-4032-induced melanoma cell killing and its inhibitory effect on MAPK pathway activation are unaffected by CD70 expression. Consequently, our work demonstrates that CD70 ectopic expression in melanomas is not a valuable biomarker to predict tumor cells sensitivity to BRAF V600 inhibitors. PMID:26828592

  11. Melanoma Expressed-CD70 Is Regulated by RhoA and MAPK Pathways without Affecting Vemurafenib Treatment Activity.

    PubMed

    Pich, Christine; Teiti, Iotefa; Sarrabayrouse, Guillaume; Gallardo, Franck; Gence, Rémi; Tilkin-Mariamé, Anne-Françoise

    2016-01-01

    CD70 is a costimulatory molecule member of the Tumor Necrosis Factor family that is expressed on activated immune cells. Its ectopic expression has been described in several types of cancer cells including lymphomas, renal cell carcinomas and glioblastomas. We have recently described its expression in a part of tumor cells from the vast majority of melanoma biopsies and human melanoma cell lines, and found that CD70 expression decreased over time as the disease progressed. Here, we show that RhoA, BRAF and Mitogen Activating Protein Kinase pathways are involved in the positive transcriptional regulation of CD70 expression in melanomas. Interestingly, the clinical inhibitor of the common BRAF V600E/D variants, Vemurafenib (PLX-4032), which is currently used to treat melanoma patients with BRAF V600E/D-mutated metastatic melanomas, decreased CD70 expression in human CD70+ melanoma cell lines. This decrease was seen in melanoma cells both with and without the BRAFV600E/D mutation, although was less efficient in those lacking the mutation. But interestingly, by silencing CD70 in CD70+ melanoma cell lines we show that PLX-4032-induced melanoma cell killing and its inhibitory effect on MAPK pathway activation are unaffected by CD70 expression. Consequently, our work demonstrates that CD70 ectopic expression in melanomas is not a valuable biomarker to predict tumor cells sensitivity to BRAF V600 inhibitors. PMID:26828592

  12. RASSF8 regulates progression of cutaneous melanoma through nuclear factor-κb

    PubMed Central

    Wang, Jinhua; Hua, Wei; Huang, Sharon K.; Fan, Kun; Takeshima, Ling; Mao, Ying; Hoon, Dave S.B.

    2015-01-01

    Our group previously demonstrated that the RASSF1 gene has a significant tumor suppressor role in cutaneous melanoma. The RASSF8 gene is a member of the N-terminal RASSF gene family. Previously, we identified RASSF8 (HOJ1, NCBI Gene ID:11228) expression in cutaneous melanoma; however the functional role of RASSF8 in melanoma is not known. RASSF8 expression was assessed in melanoma cell lines and tumors of different AJCC stages. Results indicated that RASSF8 expression was low in metastatic melanoma lines and decreased with melanoma progression. We then explored the mechanism of RASSF8 downregulation in melanoma by assessing methylation of RASSF8 and demonstrated that methylation of RASSF8 gene promoter was higher in advanced than in early stages melanomas. Functional activity of RASSF8 in melanoma lines by knockdown and overexpression of RASSF8 demonstrated that RASSF8 expression significantly inhibited cell growth, cell migration and invasion, whereas knockdown of RASSF8 expression significantly increased cell growth, cell migration and invasion of melanoma cells by increasing expression of P65 and its downstream target IL-6. Moreover RASSF8 was found to induce apoptosis in melanoma cells by activating the P53-P21 pathway, and also in vivo studies demonstrated that inhibiting RASSF8 increases the tumorigenic properties of human melanoma xenografts. These results suggest that RASSF8 plays a significant role in suppressing the progression of cutaneous melanoma. PMID:26334503

  13. Antitumor activity of placenta-derived mesenchymal stem cells producing pigment epithelium-derived factor in a mouse melanoma model.

    PubMed

    Chen, Qiaoling; Cheng, Ping; Song, Na; Yin, Tao; He, Hong; Yang, Li; Chen, Xiancheng; Wei, Yuquan

    2012-09-01

    Mesenchymal stem cells (MSCs) are a new tool that can be used for the delivery of therapeutic agents to tumor cells. Among the various types of MSCs, placenta-derived MSCs (PDMSCs) have emerged as one of the most attractive vehicles for gene therapy due to their high throughput, lack of ethical concerns, non-invasive procedure for their harvesting and ease of isolation. In this study, we evaluated the antitumor activity of human PDMSCs loaded with recombinant adenoviruses expressing pigment epithelium-derived factor (PEDF). PDMSCs were transduced with adenovirus PEDF and the expression of PEDF was confirmed by western blotting and ELISA. The inhibition of angiogenesis mediated by PEDF-expressing PDMSCs (PDMSC-PEDF) was determined using human umbilical vein endothelial cell (HUVEC) proliferation inhibition assay and migration inhibition assay in vitro. In in vivo experiments, C57BL/6 mice bearing B16-F10 melanoma were treated with intratumoral injection of PDMSC-PEDF twice at a 4-day interval. The tumor volume and weight were recorded. The results demonstrated that the administration of PDMSC-PEDF resulted in marked suppression of tumor growth in an established melanoma model, which was associated with a decreased number of microvessels and increased apoptosis of tumor cells compared with the controls. The results suggest that human PDMSCs have potential use as effective delivery vehicles for cancer gene therapy. PMID:23741242

  14. Mechanisms of resistance to RAF inhibition in melanomas harboring a BRAF mutation.

    PubMed

    Chapman, Paul B

    2013-01-01

    Treatment of V600E/K BRAF-mutated melanomas with RAF inhibitors (either vemurafenib or dabrafenib) results in rapid and dramatic responses in most patients-results that are associated with improved progression-free survival (PFS) and in the case of vemurafenib, overall survival (OS). However, resistance develops at a median time of approximately 6 months. Understanding the mechanisms of resistance is critical to develop strategies to prolong PFS and OS. Negative feedback mechanisms inherent in the MAPK pathway serve to modulate responses to these drugs. However, genetic changes develop within the tumor, which lead to reactivation of the MAPK and resistance to these drugs. The mechanisms that have been demonstrated in many patients by multiple investigators are (1) development of an activating mutation in NRAS, and (2) appearance of a BRAFV600E splice variant that encourages RAF dimerization. Several other mechanisms of resistance have also been described in individual patients or in preclinical models of resistance. In addition, there is evidence that activation of parallel pathways, such as the PI3K/AKT pathway, may represent another mechanism of resistance. Understanding the various mechanisms of resistance will inform our attempts to prevent resistance to RAF inhibitors. PMID:23714462

  15. Natural Compounds' Activity against Cancer Stem-Like or Fast-Cycling Melanoma Cells

    PubMed Central

    Majchrzak, Kinga; Hartman, Mariusz; Czyz, Malgorzata

    2014-01-01

    Background Accumulating evidence supports the concept that melanoma is highly heterogeneous and sustained by a small subpopulation of melanoma stem-like cells. Those cells are considered as responsible for tumor resistance to therapies. Moreover, melanoma cells are characterized by their high phenotypic plasticity. Consequently, both melanoma stem-like cells and their more differentiated progeny must be eradicated to achieve durable cure. By reevaluating compounds in heterogeneous melanoma populations, it might be possible to select compounds with activity not only against fast-cycling cells but also against cancer stem-like cells. Natural compounds were the focus of the present study. Methods We analyzed 120 compounds from The Natural Products Set II to identify compounds active against melanoma populations grown in an anchorage-independent manner and enriched with cells exerting self-renewing capacity. Cell viability, cell cycle arrest, apoptosis, gene expression, clonogenic survival and label-retention were analyzed. Findings Several compounds efficiently eradicated cells with clonogenic capacity and nanaomycin A, streptonigrin and toyocamycin were effective at 0.1 µM. Other anti-clonogenic but not highly cytotoxic compounds such as bryostatin 1, siomycin A, illudin M, michellamine B and pentoxifylline markedly reduced the frequency of ABCB5 (ATP-binding cassette, sub-family B, member 5)-positive cells. On the contrary, treatment with maytansine and colchicine selected for cells expressing this transporter. Maytansine, streptonigrin, toyocamycin and colchicine, even if highly cytotoxic, left a small subpopulation of slow-dividing cells unaffected. Compounds selected in the present study differentially altered the expression of melanocyte/melanoma specific microphthalmia-associated transcription factor (MITF) and proto-oncogene c-MYC. Conclusion Selected anti-clonogenic compounds might be further investigated as potential adjuvants targeting melanoma stem

  16. MicroRNA-9 up-regulates E-cadherin through inhibition of NF-κB1–Snail1 pathway in melanoma

    PubMed Central

    Liu, Shujing; Kumar, Suresh M; Lu, Hezhe; Liu, Aihua; Yang, Ruifeng; Pushparajan, Anitha; Guo, Wei; Xu, Xiaowei

    2013-01-01

    MicroRNAs (miRNAs) are short non-coding RNAs that post-transcriptionally regulate gene expression. Hsa-miR-9 has been shown to have opposite functions in different tumour types; however, the underlying mechanism is unclear. Here we show that hsa-miR-9 is down-regulated in metastatic melanomas compared to primary melanomas. Overexpression of miR-9 in melanoma cells resulted in significantly decreased cell proliferation and migratory capacity with decreased F-actin polymerization and down-regulation of multiple GTPases involved in cytoskeleton remodelling. miR-9 overexpression induced significant down-regulation of Snail1 with a concomitant increase in E-cadherin expression. In contrast, knockdown of miR-9 increased Snail1 expression as well as melanoma cell proliferation and migration capacity. Mechanistically, miR-9 expression down-regulated NF-κB1 in melanoma and the effect was abolished by mutations in the putative miR-9 binding sites within the 3′-untranslated region (UTR) of NF-κB1. Anti-miR-9 miRNA inhibitor also increased the expression of NF-κB1. The effects of miR-9 on Snail1 expression and melanoma cell proliferation and migration were rescued by overexpression of NF-κB1 in these cells. Furthermore, miR-9 overexpression resulted in significantly decreased melanoma growth and metastasis in vivo. In summary, miR-9 inhibits melanoma proliferation and metastasis through down-regulation of the NF-κB1-Snail1 pathway. This study finds a new mechanism that miR-9 utilizes to decrease E-cadherin expression and inhibit melanoma progression. The results suggest that function of microRNAs is context and tumour type-specific. PMID:22131135

  17. Activated Notch1 expression is associated with angiogenesis in cutaneous melanoma.

    PubMed

    Murtas, Daniela; Piras, Franca; Minerba, Luigi; Maxia, Cristina; Ferreli, Caterina; Demurtas, Paolo; Lai, Simone; Mura, Ester; Corrias, Michela; Sirigu, Paola; Perra, Maria Teresa

    2015-08-01

    An early event in melanocytic tumor growth is the upregulation of Notch signaling. When an active form of Notch1 is overexpressed in primary human melanocytes, it increases cell growth, survival and invasive properties, promoting melanoma progression. Recent evidence suggested that tumor initiation and growth are driven by a subset of tumor-initiating cells termed cancer stem cells. Notch1 plays a predominant role in the maintenance of melanoblasts, including melanocyte stem cells, by preventing initiation of apoptosis. Moreover, the importance of Notch1 in the regulation of tumor angiogenesis is supported by growing evidence in various cancers. Nestin has been widely used as a marker for melanocyte stem cells as well as an angiogenic marker to evaluate neovascularity of endothelial cells in tumors. To gain an insight into the impact of Notch1 activation on the maintenance of melanocyte stem cells and angiogenesis in melanoma, the expression levels of activated Notch1 and nestin were analyzed by immunohistochemistry in 114 primary cutaneous melanomas and 35 lymph node metastases. Activated Notch1 and nestin expression was also evaluated in four dysplastic melanocytic nevi. This study provides evidence that activated Notch1 is overexpressed in cutaneous melanoma, in tumor cells as well as in microvessel endothelium, and that it can promote tumor angiogenesis. Indeed, the overexpression of activated Notch1 in both tumor and vascular endothelial cells was significantly associated with microvascular density in melanoma samples. Thus, activated Notch1 inhibitors may provide a therapeutic strategy in the treatment of melanoma by blocking tumor-associated vascularization. PMID:25034654

  18. The DNA intercalating alkaloid cryptolepine interferes with topoisomerase II and inhibits primarily DNA synthesis in B16 melanoma cells.

    PubMed

    Bonjean, K; De Pauw-Gillet, M C; Defresne, M P; Colson, P; Houssier, C; Dassonneville, L; Bailly, C; Greimers, R; Wright, C; Quetin-Leclercq, J; Tits, M; Angenot, L

    1998-04-14

    Cryptolepine hydrochloride is an indoloquinoline alkaloid isolated from the roots of Cryptolepis sanguinolenta. It is characterized by a multiplicity of host-mediated biological activities, including antibacterial, antiviral, and antimalarial properties. To date, the molecular basis for its diverse biological effects remains largely uncertain. Several lines of evidence strongly suggest that DNA might correspond to its principal cellular target. Consequently, we studied the strength and mode of binding to DNA of cryptolepine by means of absorption, fluorescence, circular, and linear dichroism, as well as by a relaxation assay using DNA topoisomerases. The results of various optical and gel electrophoresis techniques converge to reveal that the alkaloid binds tightly to DNA and behaves as a typical intercalating agent. In DNAase I footprinting experiments it was found that the drug interacts preferentially with GC-rich sequences and discriminates against homo-oligomeric runs of A and T. This study has also led to the discovery that cryptolepine is a potent topoisomerase II inhibitor and a promising antitumor agent. It stabilizes topoisomerase II-DNA covalent complexes and stimulates the cutting of DNA at a subset of preexisting topoisomerase II cleavage sites. Taking advantage of the fluorescence of the indoloquinoline chromophore, fluorescence microscopy was used to map cellular uptake of the drug. Cryptolepine easily crosses the cell membranes and accumulates selectively into the nuclei rather than in the cytoplasm of B16 melanoma cells. Quantitative analyses of DNA in cells after Feulgen reaction and image cytometry reveal that the drug blocks the cell cycle in G2/M phases. It is also shown that the alkaloid is more potent at inhibiting DNA synthesis rather than RNA and protein synthesis. Altogether, the results provide direct evidence that DNA is the primary target of cryptolepine and suggest that this alkaloid is a valid candidate for the development of tumor

  19. HIF1α and HIF2α independently activate SRC to promote melanoma metastases

    PubMed Central

    Hanna, Sara C.; Krishnan, Bhavani; Bailey, Sean T.; Moschos, Stergios J.; Kuan, Pei-Fen; Shimamura, Takeshi; Osborne, Lukas D.; Siegel, Marni B.; Duncan, Lyn M.; O’Brien, E. Tim; Superfine, Richard; Miller, C. Ryan; Simon, M. Celeste; Wong, Kwok-Kin; Kim, William Y.

    2013-01-01

    Malignant melanoma is characterized by a propensity for early lymphatic and hematogenous spread. The hypoxia-inducible factor (HIF) family of transcription factors is upregulated in melanoma by key oncogenic drivers. HIFs promote the activation of genes involved in cancer initiation, progression, and metastases. Hypoxia has been shown to enhance the invasiveness and metastatic potential of tumor cells by regulating the genes involved in the breakdown of the ECM as well as genes that control motility and adhesion of tumor cells. Using a Pten-deficient, Braf-mutant genetically engineered mouse model of melanoma, we demonstrated that inactivation of HIF1α or HIF2α abrogates metastasis without affecting primary tumor formation. HIF1α and HIF2α drive melanoma invasion and invadopodia formation through PDGFRα and focal adhesion kinase–mediated (FAK-mediated) activation of SRC and by coordinating ECM degradation via MT1-MMP and MMP2 expression. These results establish the importance of HIFs in melanoma progression and demonstrate that HIF1α and HIF2α activate independent transcriptional programs that promote metastasis by coordinately regulating cell invasion and ECM remodeling. PMID:23563312

  20. Initial experience with combined BRAF and MEK inhibition with stereotactic radiosurgery for BRAF mutant melanoma brain metastases.

    PubMed

    Patel, Bindiya G; Ahmed, Kamran A; Johnstone, Peter A S; Yu, Hsiang-Hsuan Michael; Etame, Arnold B

    2016-08-01

    The combined use of the BRAF inhibitor dabrafenib and MEK inhibitor trametinib has been found to improve survival over dabrafenib alone. The management of melanoma brain metastases continues to present challenges. In this study, we report our initial experience in the management of melanoma brain metastases with stereotactic radiosurgery (SRS) with the use of BRAF and MEK inhibitors. We identified six patients treated with SRS for 17 brain metastases within 3 months of BRAF and MEK inhibitor administration. The median planning target volume was 0.42 cm (range: 0.078-2.08 cm). The median treatment dose was 21 Gy (range 18-24 Gy). The median follow-up of all lesions from SRS was 10.6 months (range 5.8-28.5 months). One lesion was found to undergo local failure 21.7 months following SRS treatment. The median overall survival was 20.0 months (range 6.1-31.8 months) from the time of SRS treatment and 23.1 months (range: 12.1-30.9 months) from the date of BRAFi and MEKi administration. There was no evidence of increased nor unexpected toxicity with the two modalities combined. In this initial experience of melanoma brain metastases treated with BRAF and MEK inhibition with SRS, we find the two modalities can be combined safely. These outcomes should be assessed further in prospective evaluations. PMID:26926151

  1. Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells

    PubMed Central

    Martin-Orozco, Natalia; Li, Yufeng; Wang, Yijun; Liu, Shijuan; Hwu, Patrick; Liu, Yong-Jun; Dong, Chen; Radvanyi, Laszlo

    2010-01-01

    CD4+CD25+Foxp3+ T-regulatory cells (Tregs) accumulate in tumors, however little is known about how the tumor environment influences this process. Here we show that human melanomas express ICOS-ligand (ICOS-L/B7H) that can provide costimulation through ICOS for the expansion of activated Tregs maintaining high Foxp3 and CD25 expression as well as suppressive function. Thus, ICOS-L expression by melanoma tumor cells may directly drive Treg activation and expansion in the tumor microenvironment as another mechanism of immune evasion. PMID:21098714

  2. miR-146a promotes the initiation and progression of melanoma by activating Notch signaling

    PubMed Central

    Forloni, Matteo; Dogra, Shaillay Kumar; Dong, Yuying; Conte, Darryl; Ou, Jianhong; Zhu, Lihua Julie; Deng, April; Mahalingam, Meera; Green, Michael R; Wajapeyee, Narendra

    2014-01-01

    Oncogenic mutations in BRAF and NRAS occur in 70% of melanomas. In this study, we identify a microRNA, miR-146a, that is highly upregulated by oncogenic BRAF and NRAS. Expression of miR-146a increases the ability of human melanoma cells to proliferate in culture and form tumors in mice, whereas knockdown of miR-146a has the opposite effects. We show these oncogenic activities are due to miR-146a targeting the NUMB mRNA, a repressor of Notch signaling. Previous studies have shown that pre-miR-146a contains a single nucleotide polymorphism (C>G rs2910164). We find that the ability of pre-miR-146a/G to activate Notch signaling and promote oncogenesis is substantially higher than that of pre-miR-146a/C. Analysis of melanoma cell lines and matched patient samples indicates that during melanoma progression pre-miR-146a/G is enriched relative to pre-miR-146a/C, resulting from a C-to-G somatic mutation in pre-miR-146a/C. Collectively, our results reveal a central role for miR-146a in the initiation and progression of melanoma. DOI: http://dx.doi.org/10.7554/eLife.01460.001 PMID:24550252

  3. Enhancement of melphalan activity by buthionine sulfoximine and electroporation in melanoma cells.

    PubMed

    Ongaro, Alessia; Pellati, Agnese; De Mattei, Monica; De Terlizzi, Francesca; Rossi, Carlo R; Campana, Luca G

    2015-03-01

    Melphalan represents the reference drug for locoregional chemotherapy of melanoma; nevertheless, treatment failure may occur because of resistance to chemotherapy. Refractory melanoma cells show either an increased capability of drug inactivation, which is known to be associated with elevated intracellular levels of glutathione (GSH), or a decreased melphalan uptake. The aim of this study was to explore a biochemical and a biophysical strategy, and their combination, to overcome melphalan resistance in melanoma cells. The biochemical strategy was based on the treatment of melanoma cells with DL-buthionine (S,R)-sulfoximine (BSO) to deplete the GSH levels, thus reducing melphalan inactivation. In the biophysical strategy, cell membrane electroporation was used to increase melphalan uptake. The SK-MEL 28-resistant human melanoma cell line was pretreated with 50 μmol/l BSO for 24 h and then treated with increasing melphalan doses, with or without electroporation. Spectrophotometric quantification of cell viability was used to determine melphalan cytotoxicity. Intracellular total GSH was measured using a kinetic enzymatic assay. BSO induced 3.50-fold GSH depletion in untreated cells and a similar reduction was also maintained in melphalan-treated cells. BSO pretreatment produced a 2.46-fold increase in melphalan cytotoxicity. Electroporation increased melphalan cytotoxicity 1.42-fold. The combination of both BSO pretreatment with melphalan plus electroporation led to a 4.40-fold increase in melphalan cytotoxicity compared with melphalan alone. Pretreatment with BSO and cell membrane permeabilization by electroporation enhanced the cytotoxic activity of melphalan in melanoma cells. Their rational combination deserves further investigation and may improve the efficacy of locoregional chemotherapy of melanoma. PMID:25514113

  4. Rational targeting of BRAF and PI3-Kinase signaling for melanoma therapy

    PubMed Central

    Deuker, Marian M.; McMahon, Martin

    2016-01-01

    ABSTRACT Although mitogen-activated protein kinase (MAPK) inhibitors elicit initial regression of BRAF-mutated melanoma, drug resistance is an inevitable and fatal event. We recently reported that in genetically engineered mouse models of BRAF-mutated melanoma, isoform-selective phosphatidylinositol 3-kinase inhibition cooperates with MAPK pathway inhibition to forestall the onset of MAPK pathway inhibitor resistance. PMID:27314067

  5. Rational targeting of BRAF and PI3-Kinase signaling for melanoma therapy.

    PubMed

    Deuker, Marian M; McMahon, Martin

    2016-05-01

    Although mitogen-activated protein kinase (MAPK) inhibitors elicit initial regression of BRAF-mutated melanoma, drug resistance is an inevitable and fatal event. We recently reported that in genetically engineered mouse models of BRAF-mutated melanoma, isoform-selective phosphatidylinositol 3-kinase inhibition cooperates with MAPK pathway inhibition to forestall the onset of MAPK pathway inhibitor resistance. PMID:27314067

  6. Huntingtin inhibits caspase-3 activation

    PubMed Central

    Zhang, Yu; Leavitt, Blair R; van Raamsdonk, Jeremy M; Dragatsis, Ioannis; Goldowitz, Dan; MacDonald, Marcy E; Hayden, Michael R; Friedlander, Robert M

    2006-01-01

    Huntington's disease results from a mutation in the HD gene encoding for the protein huntingtin. The function of huntingtin, although beginning to be elucidated, remains largely unclear. To probe the prosurvival function of huntingtin, we modulate levels of wild-type huntingtin in a number of cellular and in vivo models. Huntingtin depletion resulted in caspase-3 activation, and overexpression of huntingtin resulted in caspase-3 inhibition. Additionally, we demonstrate that huntingtin physically interacts with active caspase-3. Interestingly, mutant huntingtin binds active caspase-3 with a lower affinity and lower inhibitory effect on active caspase-3 than does wild-type huntingtin. Although reduction of huntingtin levels resulted in caspase-3 activation in all conditions examined, the cellular response was cell-type specific. Depletion of huntingtin resulted in either overt cell death, or in increased vulnerability to cell death. These data demonstrate that huntingtin inhibits caspase-3 activity, suggesting a mechanism whereby caspase-mediated huntingtin depletion results in a detrimental amplification cascade leading to further caspase-3 activation, resulting in cell dysfunction and cell death. PMID:17124493

  7. The genetic landscape of clinical resistance to RAF inhibition in metastatic melanoma

    PubMed Central

    Van Allen, Eliezer M.; Wagle, Nikhil; Sucker, Antje; Treacy, Daniel; Johannessen, Cory; Goetz, Eva M.; Place, Chelsea S.; Taylor-Weiner, Amaro; Whittaker, Steven; Kryukov, Gregory; Hodis, Eran; Rosenberg, Mara; McKenna, Aaron; Cibulskis, Kristian; Farlow, Deborah; Zimmer, Lisa; Hillen, Uwe; Gutzmer, Ralf; Goldinger, Simone M.; Ugurel, Selma; Gogas, Helen J.; Egberts, Friederike; Berking, Carola; Trefzer, Uwe; Loquai, Carmen; Weide, Benjamin; Hassel, Jessica C.; Gabriel, Stacey B.; Carter, Scott L.; Getz, Gad; Garraway, Levi A.; Schadendorf, Dirk

    2013-01-01

    Most patients with BRAFV600 metastatic melanoma develop resistance to selective RAF kinase inhibitors. The spectrum of clinical genetic resistance mechanisms to RAF inhibitors and options for salvage therapy are incompletely understood. We performed whole exome sequencing on formalin-fixed, paraffin embedded (FFPE) tumors from 45 patients with BRAFV600 metastatic melanoma who received vemurafenib or dabrafenib monotherapy. Genetic alterations in known or putative RAF inhibitor resistance genes were observed in 23 of 45 patients (51%). Besides previously characterized alterations, we discovered a “long tail” of new MAPK pathway alterations (MAP2K2, MITF) that confer RAF inhibitor resistance. In three cases, multiple resistance gene alterations were observed within the same tumor biopsy. Overall, RAF inhibitor therapy leads to diverse clinical genetic resistance mechanisms, mostly involving MAPK pathway reactivation. Novel therapeutic combinations may be needed to achieve durable clinical control of BRAFV600 melanoma. Integrating clinical genomics with preclinical screens may model subsequent resistance studies. PMID:24265153

  8. MERTK receptor tyrosine kinase is a therapeutic target in melanoma

    PubMed Central

    Schlegel, Jennifer; Sambade, Maria J.; Sather, Susan; Moschos, Stergios J.; Tan, Aik-Choon; Winges, Amanda; DeRyckere, Deborah; Carson, Craig C.; Trembath, Dimitri G.; Tentler, John J.; Eckhardt, S. Gail; Kuan, Pei-Fen; Hamilton, Ronald L.; Duncan, Lyn M.; Miller, C. Ryan; Nikolaishvili-Feinberg, Nana; Midkiff, Bentley R.; Liu, Jing; Zhang, Weihe; Yang, Chao; Wang, Xiaodong; Frye, Stephen V.; Earp, H. Shelton; Shields, Janiel M.; Graham, Douglas K.

    2013-01-01

    Metastatic melanoma is one of the most aggressive forms of cutaneous cancers. Although recent therapeutic advances have prolonged patient survival, the prognosis remains dismal. C-MER proto-oncogene tyrosine kinase (MERTK) is a receptor tyrosine kinase with oncogenic properties that is often overexpressed or activated in various malignancies. Using both protein immunohistochemistry and microarray analyses, we demonstrate that MERTK expression correlates with disease progression. MERTK expression was highest in metastatic melanomas, followed by primary melanomas, while the lowest expression was observed in nevi. Additionally, over half of melanoma cell lines overexpressed MERTK compared with normal human melanocytes; however, overexpression did not correlate with mutations in BRAF or RAS. Stimulation of melanoma cells with the MERTK ligand GAS6 resulted in the activation of several downstream signaling pathways including MAPK/ERK, PI3K/AKT, and JAK/STAT. MERTK inhibition via shRNA reduced MERTK-mediated downstream signaling, reduced colony formation by up to 59%, and diminished tumor volume by 60% in a human melanoma murine xenograft model. Treatment of melanoma cells with UNC1062, a novel MERTK-selective small-molecule tyrosine kinase inhibitor, reduced activation of MERTK-mediated downstream signaling, induced apoptosis in culture, reduced colony formation in soft agar, and inhibited invasion of melanoma cells. This work establishes MERTK as a therapeutic target in melanoma and provides a rationale for the continued development of MERTK-targeted therapies. PMID:23585477

  9. Constitutive activation of the ETS-1-miR-222 circuitry in metastatic melanoma

    PubMed Central

    Mattia, Gianfranco; Errico, M Cristina; Felicetti, Federica; Petrini, Marina; Bottero, Lisabianca; Tomasello, Luisa; Romania, Paolo; Boe, Alessandra; Segnalini, Patrizia; Di Virgilio, Antonio; Colombo, Mario P; Carè, Alessandra

    2011-01-01

    MicroRNAs-221 and -222 are highly upregulated in several solid tumors, including melanomas. We demonstrate that the proto-oncogene ETS-1, involved in the pathogenesis of cancers of different origin, is a transcriptional regulator of miR-222 by direct binding to its promoter region. Differently from 293FT cells or early stage melanomas, where unphosphorylated ETS-1 represses miR-222 transcription, in metastatic melanoma the constitutively Thr-38 phosphorylated fraction of ETS-1 induces miR-222. Despite its stepwise decreased expression along with melanoma progression, the oncogenic activity of ETS-1 relies on its RAS/RAF/ERK-dependent phosphorylation status more than on its total amount. To close the loop, we demonstrate ETS-1 as a direct target of miR-222, but not miR-221, showing the novel option of their uncoupled functions. In addition, a spatial redistribution of ETS-1 protein from the nucleus to the cytoplasm is also evidenced in advanced melanoma cells. Finally, in vivo studies confirmed the contribution of miR-222 to the increased invasive potential obtained by ETS- silencing. PMID:21711453

  10. Fluorescent peptide biosensor for monitoring CDK4/cyclin D kinase activity in melanoma cell extracts, mouse xenografts and skin biopsies.

    PubMed

    Prével, Camille; Pellerano, Morgan; González-Vera, Juan A; Henri, Pauline; Meunier, Laurent; Vollaire, Julien; Josserand, Véronique; Morris, May C

    2016-11-15

    Melanoma constitutes the most aggressive form of skin cancer, which further metastasizes into a deadly form of cancer. The p16(INK4a)-Cyclin D-CDK4/6-pRb pathway is dysregulated in 90% of melanomas. CDK4/Cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of Cyclin D or loss of p16(INK4a) leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker in melanoma and an emerging pharmacological target, however there are no tools enabling direct detection or quantification of its activity. Here we report on the design and application of a fluorescent peptide biosensor to quantify CDK4 activity in melanoma cell extracts, skin biopsies and melanoma xenografts. This biosensor provides sensitive means of comparing CDK4 activity between different melanoma cell lines and further responds to CDK4 downregulation by siRNA or small-molecule inhibitors. By affording means of monitoring CDK4 hyperactivity consequent to cancer-associated molecular alterations in upstream signaling pathways that converge upon this kinase, this biosensor offers an alternative to immunological identification of melanoma-specific biomarkers, thereby constituting an attractive tool for diagnostic purposes, providing complementary functional information to histological analysis, of particular utility for detection of melanoma onset in precancerous lesions. This is indeed the first fluorescent peptide biosensor which has been successfully implemented to monitor kinase activity in skin samples and melanoma tumour xenografts. Moreover by enabling to monitor response to CDK4 inhibitors, this biosensor constitutes an attractive companion assay to identify compounds of therapeutic relevance for melanoma. PMID:27203461

  11. Intravenous Administration of Manuka Honey Inhibits Tumor Growth and Improves Host Survival When Used in Combination with Chemotherapy in a Melanoma Mouse Model

    PubMed Central

    Fernandez-Cabezudo, Maria J.; El-Kharrag, Rkia; Torab, Fawaz; Bashir, Ghada; George, Junu A.; El-Taji, Hakam; al-Ramadi, Basel K.

    2013-01-01

    Manuka honey has been recognized for its anti-bacterial and wound-healing activity but its potential antitumor effect is poorly studied despite the fact that it contains many antioxidant compounds. In this study, we investigated the antiproliferative activity of manuka honey on three different cancer cell lines, murine melanoma (B16.F1) and colorectal carcinoma (CT26) as well as human breast cancer (MCF-7) cells in vitro. The data demonstrate that manuka honey has potent anti-proliferative effect on all three cancer cell lines in a time- and dose-dependent manner, being effective at concentrations as low as 0.6% (w/v). This effect is mediated via the activation of a caspase 9-dependent apoptotic pathway, leading to the induction of caspase 3, reduced Bcl-2 expression, DNA fragmentation and cell death. Combination treatment of cancer cells with manuka and paclitaxel in vitro, however, revealed no evidence of a synergistic action on cancer cell proliferation. Furthermore, we utilized an in vivo syngeneic mouse melanoma model to assess the potential effect of intravenously-administered manuka honey, alone or in combination with paclitaxel, on the growth of established tumors. Our findings indicate that systemic administration of manuka honey was not associated with any alterations in haematological or clinical chemistry values in serum of treated mice, demonstrating its safety profile. Treatment with manuka honey alone resulted in about 33% inhibition of tumor growth, which correlated with histologically observable increase in tumor apoptosis. Although better control of tumor growth was observed in animals treated with paclitaxel alone or in combination with manuka honey (61% inhibition), a dramatic improvement in host survival was seen in the co-treatment group. This highlights a potentially novel role for manuka honey in alleviating chemotherapy-induced toxicity. PMID:23409104

  12. Simvastatin rises reactive oxygen species levels and induces senescence in human melanoma cells by activation of p53/p21 pathway

    SciTech Connect

    Guterres, Fernanda Augusta de Lima Barbosa; Martinez, Glaucia Regina; Rocha, Maria Eliane Merlin; Winnischofer, Sheila Maria Brochado

    2013-11-15

    Recent studies demonstrated that simvastatin has antitumor properties in several types of cancer cells, mainly by inducing apoptosis and inhibiting growth. The arrest of proliferation is a feature of cellular senescence; however, the occurrence of senescence in melanoma cells upon simvastatin treatment has not been investigated until now. Our results demonstrated that exposure of human metastatic melanoma cells (WM9) to simvastatin induces a senescent phenotype, characterized by G1 arrest, positive staining for senescence-associated β-galactosidase assay, and morphological changes. Also, the main pathways leading to cell senescence were examined in simvastatin-treated human melanoma cells, and the expression levels of phospho-p53 and p21 were upregulated by simvastatin, suggesting that cell cycle regulators and DNA damage pathways are involved in the onset of senescence. Since simvastatin can act as a pro-oxidant agent, and oxidative stress may be related to senescence, we measured the intracellular ROS levels in WM9 cells upon simvastatin treatment. Interestingly, we found an increased amount of intracellular ROS in these cells, which was accompanied by elevated expression of catalase and peroxiredoxin-1. Collectively, our results demonstrated that simvastatin can induce senescence in human melanoma cells by activation of p53/p21 pathway, and that oxidative stress may be related to this process. - Highlights: • Lower concentrations of simvastatin can induce senescent phenotype in melanoma cells. • Simvastatin induces senescence in human melanoma cells via p53/p21 pathway. • Senescent phenotype is related with increased intracellular ROS. • Partial detoxification of ROS by catalase/peroxiredoxin-1 could lead cells to senescence rather than apoptosis.

  13. Selumetinib for the treatment of metastatic uveal melanoma: past and future perspectives.

    PubMed

    Komatsubara, Kimberly M; Manson, Daniel K; Carvajal, Richard D

    2016-06-01

    Uveal melanoma is a rare but aggressive subtype of melanoma. Nearly 50% of patients will develop metastatic disease despite primary enucleation or radiation therapy. There is currently no standard of care therapy for metastatic uveal melanoma, and no therapy that has been shown to prolong overall survival. Uveal melanoma is characterized by activation of signaling pathways including the MAPK pathway and the PI3K/AKT pathway, among others, via mutations in the G-α-proteins GNAQ and GNA11. MEK inhibition with selumetinib has been evaluated as a therapeutic strategy in metastatic uveal melanoma. This review will discuss preclinical and clinical studies evaluating selumetinib in metastatic uveal melanoma, as well as potential future perspectives on MEK inhibition in the management of metastatic uveal melanoma. PMID:27044592

  14. Renin inhibition activity by chitooligosaccharides.

    PubMed

    Park, Pyo-Jam; Ahn, Chang-Bum; Jeon, You-Jin; Je, Jae-Young

    2008-04-01

    Six kinds of chitooligosaccharides (COSs) with different molecular weight (MW) and degree of deacetylation (DD) were prepared using ultrafiltration membrane reactor, and their renin inhibition modes were evaluated. All the COSs showed the renin-inhibitory activities with dose-dependent manner, and 90-COSs had the potent renin-inhibitory activity than that of 50-COSs. Among them, 90-MMWCOS (1000-5000Da) exhibits the highest activity with IC(50) value of 0.51mg/mL and acts as competitive inhibitor with K(i) value of 0.28mg/mL by Lineweaver-Burk and Dixon plots. These results indicated that DD value and MW of COSs are important factors affecting renin-inhibitory activity. PMID:18313296

  15. Melanoma cells show a heterogeneous range of sensitivity to ionizing radiation and are radiosensitized by inhibition of B-RAF with PLX-4032

    PubMed Central

    Sambade, Maria J.; Peters, Eldon C.; Thomas, Nancy E.; Kaufmann, William K.; Kimple, Randall J.; Shields, Janiel M.

    2011-01-01

    Purpose To assess the relative radiosensitivities of a large collection of melanoma cell lines and to determine whether pharmacologic inhibition of mutant B-RAF with PLX-4032 can radiosensitize B-Raf+ melanoma cells. Methods and Materials A large collection of melanoma cell lines (n=37) were treated with 0 – 8 Gy IR and clonogenic survival assays used to generate survival curves to rank relative radiosensitivities among the cell lines. The ability of a B-RAF inhibitor, PLX-4032, to radiosensitize highly radioresistant, B-Raf+ cells was also assessed by clonogenic cell survival and spheroid invasion assays and the effects of treatment on the cell cycle assessed by FACS. Results Melanoma cell lines displayed a very large, heterogeneous range of SF2 values (1.002 – 0.053) with a mean of 0.51. Cell lines with surviving fractions of 0.29 or less at SF2 and SF4 were observed at a high frequency of 18.9% and 70.2%, respectively. Treatment of B-Raf+ cells with the B-RAF inhibitor PLX-4032 in combination with radiation provided enhanced inhibition of both colony formation and invasion, and radiosensitized cells through an increase in G1 arrest. Conclusions Our data suggest that melanomas are not uniformly radioresistant with a significant subset displaying inherent radiosensitivity. Pharmacologic inhibition of B-RAF with PLX-4032 effectively radiosensitized B-Raf+ melanoma cells suggesting this combination approach could provide improved radiotherapeutic response in B-Raf+ melanoma patients. PMID:21295875

  16. Gab2-Mediated Signaling Promotes Melanoma Metastasis

    PubMed Central

    Horst, Basil; Gruvberger-Saal, Sofia K.; Hopkins, Benjamin D.; Bordone, Lindsey; Yang, Ying; Chernoff, Karen A.; Uzoma, Ijeoma; Schwipper, Volker; Liebau, Jutta; Nowak, Norma J.; Brunner, Georg; Owens, David; Rimm, David L.; Parsons, Ramon; Celebi, Julide Tok

    2009-01-01

    Metastatic melanoma is a disease with a poor prognosis that currently lacks effective treatments. Critical biological features of metastasis include acquisition of migratory competence, growth factor independence, and invasive potential. In an attempt to identify genes that contribute to melanoma pathogenesis, a genome-wide search using bacterial artificial chromosome array comparative genomic hybridization and single nucleotide polymorphism arrays in a series of 64 metastatic melanoma samples and 20 melanoma cell lines identified increased copy numbers of Gab2 located on 11q14.1. Gab2 is an adaptor protein that potentiates the activation of the Ras-Erk and PI3K-Akt pathways and has recently been implicated in human cancer; however, its role in melanoma has not been explored. In this study, we found that Gab2 was either amplified (∼11%) and/or overexpressed (∼50%) in melanoma. Gab2 protein expression correlated with clinical melanoma progression, and higher levels of expression were seen in metastatic melanomas compared with primary melanoma and melanocytic nevi. We found that overexpression of Gab2 potentiates, whereas silencing of Gab2 reduces, migration and invasion of melanoma cells. Gab2 mediated the hyperactivation of Akt signaling in the absence of growth factors, whereas inhibition of the PI3K-Akt pathway decreased Gab2-mediated tumor cell migration and invasive potential. Gab2 overexpression resulted in enhanced tumor growth and metastatic potential in vivo. These studies demonstrate a previously undefined role for Gab2 in melanoma tumor progression and metastasis. PMID:19342374

  17. Melanoma Cell-Intrinsic PD-1 Receptor Functions Promote Tumor Growth.

    PubMed

    Kleffel, Sonja; Posch, Christian; Barthel, Steven R; Mueller, Hansgeorg; Schlapbach, Christoph; Guenova, Emmanuella; Elco, Christopher P; Lee, Nayoung; Juneja, Vikram R; Zhan, Qian; Lian, Christine G; Thomi, Rahel; Hoetzenecker, Wolfram; Cozzio, Antonio; Dummer, Reinhard; Mihm, Martin C; Flaherty, Keith T; Frank, Markus H; Murphy, George F; Sharpe, Arlene H; Kupper, Thomas S; Schatton, Tobias

    2015-09-10

    Therapeutic antibodies targeting programmed cell death 1 (PD-1) activate tumor-specific immunity and have shown remarkable efficacy in the treatment of melanoma. Yet, little is known about tumor cell-intrinsic PD-1 pathway effects. Here, we show that murine and human melanomas contain PD-1-expressing cancer subpopulations and demonstrate that melanoma cell-intrinsic PD-1 promotes tumorigenesis, even in mice lacking adaptive immunity. PD-1 inhibition on melanoma cells by RNAi, blocking antibodies, or mutagenesis of melanoma-PD-1 signaling motifs suppresses tumor growth in immunocompetent, immunocompromised, and PD-1-deficient tumor graft recipient mice. Conversely, melanoma-specific PD-1 overexpression enhances tumorigenicity, as does engagement of melanoma-PD-1 by its ligand, PD-L1, whereas melanoma-PD-L1 inhibition or knockout of host-PD-L1 attenuate growth of PD-1-positive melanomas. Mechanistically, the melanoma-PD-1 receptor modulates downstream effectors of mTOR signaling. Our results identify melanoma cell-intrinsic functions of the PD-1:PD-L1 axis in tumor growth and suggest that blocking melanoma-PD-1 might contribute to the striking clinical efficacy of anti-PD-1 therapy. PMID:26359984

  18. Octaphlorethol A isolated from Ishige foliacea inhibits α-MSH-stimulated induced melanogenesis via ERK pathway in B16F10 melanoma cells.

    PubMed

    Kim, Kil-Nam; Yang, Hye-Mi; Kang, Sung-Myung; Kim, Daekyung; Ahn, Ginnae; Jeon, You-Jin

    2013-09-01

    In this study, the potent skin-whitening effects of Octaphlorethol A (OPA) isolated from Ishige foliacea was investigated through inhibitory effect of melanin synthesis and tyrosinase activity in alpha-melanocyte stimulating hormone (α-MSH) induced B16F10 melanoma cells. OPA markedly inhibited melanin synthesis and tyrosinase activity in a concentration-dependent manner. We also found that OPA decreased microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 and -2 (TRP-1 and TRP-2) protein expressions. Moreover, OPA reduces p38 MAPK protein levels and activates extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinases (JNKs) protein expressions in B16F10 cells. A specific ERK inhibitor PD98059 significantly blocks OPA-inhibited melanin synthesis and tyrosinase activity, whereas a p38MAP and JNK inhibitor had no effect. These findings provide evidence demonstrating that the anti-melanogenic effect of OPA is mediated through the activation of ERK signal pathway in B16F10 cells. These results indicate that OPA has the potential to be used as a melanogenesis inhibitor in the food and cosmetics industry. PMID:23810793

  19. Basal and treatment-induced activation of AKT mediates resistance to cell death by AZD6244 (ARRY-142886) in Braf-mutant human cutaneous melanoma cells

    PubMed Central

    Gopal, Y.N. Vashisht; Deng, Wanleng; Woodman, Scott E.; Komurov, Kakajan; Ram, Prahlad; Smith, Paul D.; Davies, Michael A.

    2014-01-01

    The majority of melanomas demonstrate constitutive activation of the RAS-RAF-MEK-MAPK pathway. AZD6244 is a selective MEK1/2 inhibitor which markedly reduces tumor P-MAPK levels, but it produced few clinical responses in melanoma patients. An improved understanding of the determinants of resistance to AZD6244 may lead to improved patient selection and effective combinatorial approaches. The effects of AZD6244 on cell growth and survival were tested in a total of 14 Braf-mutant and 3 wild-type human cutaneous melanoma cell lines. Quantitative assessment of phospho-protein levels in the Braf-mutant cell lines by reverse phase protein array (RPPA) analysis showed no significant association between P-MEK or P-MAPK levels and AZD6244 sensitivity, but activation-specific markers in the PI3K-AKT pathway correlated with resistance. We also identified resistant cell lines without basal activation of the PI3K-AKT pathway. RPPA characterization of the time-dependent changes in signaling pathways revealed that AZD6244 produced durable and potent inhibition of P-MAPK in sensitive and resistant Braf-mutant cell lines, but several resistant lines demonstrated AZD6244-induced activation of AKT. In contrast, sensitive cell lines demonstrated AZD6244 treatment-induced upregulation of PTEN protein and mRNA expression. Inhibition of AKT, TORC1/2, or IGF1R blocked AZD6244-induced activation of AKT and resulted in synergistic cell killing with AZD6244. These findings identify basal and treatment-induced regulation of the PI3K-AKT pathway as a critical regulator of AZD6244 sensitivity in Braf-mutant cutaneous melanoma cells, the novel regulation of PTEN expression by AZD6244 in sensitive cells, and suggest new combinatorial approaches for patients. PMID:20959481

  20. Concomitant Inhibition of PI3Kβ and BRAF or MEK in PTEN-Deficient/BRAF-Mutant Melanoma Treatment: Preclinical Assessment of SAR260301 Oral PI3Kβ-Selective Inhibitor.

    PubMed

    Bonnevaux, Hélène; Lemaitre, Olivier; Vincent, Loic; Levit, Mikhail N; Windenberger, Fanny; Halley, Frank; Delorme, Cécile; Lengauer, Christoph; Garcia-Echeverria, Carlos; Virone-Oddos, Angela

    2016-07-01

    Class IA PI3K pathway activation resulting from PTEN deficiency has been associated with lack of sensitivity of melanoma to BRAF kinase inhibitors. Although previous studies have shown synergistic activity when pan-PI3K inhibitors were combined with MAPK inhibitors in the treatment of melanoma exhibiting concurrent genetic abnormalities, overlapping adverse events in patients limit optimal dosing and clinical application. With the aim of specifically targeting PTEN-deficient cancers and minimizing the potential for on-target toxicity when inhibiting multiple PI3K isoforms, we developed a program to discover PI3Kβ-selective kinase inhibitors and identified SAR260301 as a potent PI3Kβ-selective, orally available compound, which is now in clinical development. Herein, we provide a detailed biological characterization of SAR260301, and show that this compound has outstanding biochemical and cellular selectivity for the PI3Kβ isoform versus the α, δ, and γ isoforms and a large panel of protein and lipid kinases. We demonstrate that SAR260301 blocks PI3K pathway signaling preferentially in PTEN-deficient human tumor models, and has synergistic antitumor activity when combined with vemurafenib (BRAF inhibitor) or selumetinib (MEK inhibitor) in PTEN-deficient/BRAF-mutated human melanoma tumor models. Combination treatments were very well tolerated, suggesting the potential for a superior safety profile at optimal dosing using selective compounds to inhibit multiple signaling pathways. Together, these experiments provide a preclinical proof-of-concept for safely combining inhibitors of PI3Kβ and BRAF or MEK kinase modulators to improve antitumor activity in PTEN-deficient/BRAF-mutant melanoma, and support the evaluation of SAR260301-based combinations in clinical studies. Mol Cancer Ther; 15(7); 1460-71. ©2016 AACR. PMID:27196754

  1. Quantitative expression profiling of G-protein-coupled receptors (GPCRs) in metastatic melanoma: the constitutively active orphan GPCR GPR18 as novel drug target.

    PubMed

    Qin, Y; Verdegaal, E M E; Siderius, M; Bebelman, J P; Smit, M J; Leurs, R; Willemze, R; Tensen, C P; Osanto, S

    2011-02-01

    G-protein-coupled receptors (GPCRs) have been implicated in the tumorigenesis and metastasis of human cancers and are considered amongst the most desirable targets for drug development. Utilizing a robust quantitative PCR array, we quantified expression of 94 human GPCRs, including 75 orphan GPCRs and 19 chemokine receptors, and 36 chemokine ligands, in 40 melanoma metastases from different individuals and benign nevi. Inter-metastatic site comparison revealed that orphan GPR174 and CCL28 are statistically significantly overexpressed in subcutaneous metastases, while P2RY5 is overexpressed in brain metastases. Comparison between metastases (all three metastatic sites) and benign nevi revealed that 16 genes, including six orphan receptors (GPR18, GPR34, GPR119, GPR160, GPR183 and P2RY10) and chemokine receptors CCR5, CXCR4, and CXCR6, were statistically significantly differentially expressed. Subsequent functional experiments in yeast and melanoma cells indicate that GPR18, the most abundantly overexpressed orphan GPCR in all melanoma metastases, is constitutively active and inhibits apoptosis, indicating an important role for GPR18 in tumor cell survival. GPR18 and five other orphan GPCRs with yet unknown biological function may be considered potential novel anticancer targets in metastatic melanoma. PMID:20880198

  2. MiR-211 is epigenetically regulated by DNMT1 mediated methylation and inhibits EMT of melanoma cells by targeting RAB22A.

    PubMed

    Yu, Haizhou; Yang, Weixi

    2016-08-01

    MiR-211 has strong inhibitive effects on melanoma cell growth, invasion and metastasis. However, how it is downregulated and whether other genes are involved its downstream regulation in melanoma are not clear. In this study, we firstly verified the expression of miR-211 in melanoma cell lines and observed that its downregulation is associated with increased DNMT1 expression. By performing qRT-PCR and MSP analysis, we confirmed that DNMT1 is negatively correlated with miR-211 expression and can modulate DNA methylation in the promoter region of miR-211. By performing bioinformatics analysis, we found that RAB22A is a possible target of miR-211, which has two broadly conversed binding sites with miR-211 in the 3'UTR. Following dual luciferase assay, qRT-PCR and western blot analysis confirmed the direct binding between miR-211 and RAB22A and the suppressive effect of miR-211 on RAB22A expression. Knockdown of RAB22A increased epithelial properties and impaired mesenchymal properties of the melanoma cells, suggesting that miR-211 modulates epithelial mesenchymal transition (EMT) of melanoma cells via downregulating RAB22A. In summary, the present study firstly demonstrated that DNMT1 mediated promoter methylation is a mechanism of miRNA suppression in melanoma and revealed a new tumor suppressor role of the miR-211 by targeting RAB22A in melanoma. The DNMT1/miR-211/RAB22A axis provides a novel insight into the pathogenesis of melanoma, particularly in the EMT process. PMID:27237979

  3. High intensity focused ultrasound enhances anti-tumor immunity by inhibiting the negative regulatory effect of miR-134 on CD86 in a murine melanoma model

    PubMed Central

    Yang, Min; Zha, He; Sun, Hui; Li, Xue-Ru; Li, Ai-Fang; Gu, Yue; Duan, Liang; Luo, Jin-Yong; Li, Chong-Yan; Wang, Yan; Wang, Zhi-Biao; He, Tong-Chuan; Zhou, Lan

    2015-01-01

    HIFU has been demonstrated to enhance anti-tumor immunity, however, the mechanism of which has not been well elucidated. Emerging evidence indicates that miRNAs play important roles in immune response. In this study, we used the B16F10 melanoma allograft mouse model to investigate the role of miRNAs in HIFU-enhanced anti-tumor immunity. We found that HIFU treatment decreased circulating B16F10 cells and pulmonary metastasis nodules while increased IFN-γ and TNF-α in the peripheral blood and cumulative mouse survival, which was associated with inhibition of miR-134 expression and activation of CD86 expression in tumor tissues. Further, we determined that miR-134 directly binds to the 3′UTR of CD86 mRNA to suppress its expression in B16F10 cells. When B16F10 cells transfected with miR-134 were co-cultured with normal splenic lymphocytes, the secretion of IFN-γ and TNF-α from lymphocytes was reduced and B16F10 cell survival was increased. HIFU exposure efficiently decreased miR-134 while increased CD86 expression in B16F10 cells in vitro. CD86 knockdown with siRNA markedly rescued the viability of HIFU-treated B16F10 cells that co-cultured with lymphocytes. Altogether, our results suggest that HIFU down-regulates miR-134 to release the inhibition of miR-134 on CD86 in melanoma cells, thereby enhancing anti-tumor immune response. PMID:26485753

  4. MK615, a Prunus mume Steb. Et Zucc ('Ume') extract, attenuates the growth of A375 melanoma cells by inhibiting the ERK1/2-Id-1 pathway.

    PubMed

    Tada, Ko-ichi; Kawahara, Ko-ichi; Matsushita, Shigeto; Hashiguchi, Teruto; Maruyama, Ikuro; Kanekura, Takuro

    2012-06-01

    The Japanese apricot, a commonly consumed food called 'Ume' in Japan, has been used for a traditional Japanese medicine for centuries. MK615, an extract of compounds from 'Ume', has strong antitumorigenic and antiinflammatory effects including the induction of apoptosis and autophagy, and inhibition of cytokine production mediated via the inhibition of MAPKs signaling including ERK-1/2, JNK and p38MAPK. The inhibitor of DNA binding 1 (Id-1), a basic helix-loop-helix (bHLH) transcription factor family, is essential for DNA binding and the transcriptional regulation of various proteins that play important roles in the development, progression and invasion of tumors. In melanoma, Id-1 is constitutively expressed in the late and early stages, suggesting it as a therapeutic target in patients with melanoma. This study reports that MK615 profoundly reduced both the mRNA- and protein expression levels of Id-1 and inhibited cell growth in A375 melanoma cells. MK615 markedly inhibited the phosphorylation of ERK1/2, which is associated with Id-1 protein expression in A375 cells. Id-1-specific RNAi induced the death of A375 cells. Moreover, the expression of Bcl-2 was decreased by both MK615 and Id-1-specific RNAi in A375 cells. The results suggest that MK615 is a potential therapeutic agent for treating malignant melanoma. PMID:22076920

  5. Control of melanoma cell invasion by type IV collagen.

    PubMed

    Pasco, Sylvie; Brassart, Bertrand; Ramont, Laurent; Maquart, François-Xavier; Monboisse, Jean-Claude

    2005-01-01

    Malignant melanoma is the leading cause of death from diseases of the skin. This review summarizes the data from the literature and our laboratory addressing the effects of type IV collagen on melanoma progression. Many different sequences from type IV collagen promote melanoma cell adhesion, migration and invasion. The triple helical conformation of the collagenous domain plays a critical role in some of these interactions. However, recent studies from our group demonstrated that a sequence from the alpha3(IV) NC1 domain inhibits melanoma cell proliferation, migration and invasion by decreasing MMP production and activation. Peptide sequences from the alpha1(IV), alpha2(IV) and alpha3(IV) chains named arresten, canstatin and tumstatin, respectively were shown to inhibit angiogenesis. Further investigations regarding the inhibitory effects of the alpha(IV) NC1 domains will have a paramount relevance for the design of efficient strategies to limit melanoma development. PMID:15936594

  6. Imatinib enhances human melanoma cell susceptibility to TRAIL-induced cell death: Relationship to Bcl-2 family and caspase activation.

    PubMed

    Hamaï, A; Richon, C; Meslin, F; Faure, F; Kauffmann, A; Lecluse, Y; Jalil, A; Larue, L; Avril, M F; Chouaib, S; Mehrpour, M

    2006-12-01

    In order to define genetic determinants of primary and metastatic melanoma cell susceptibility to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we have applied oligonucleotide microarrays to TRAIL-sensitive primary T1 cells and TRAIL-resistant metastatic G1 cells treated or not with TRAIL. T1 and G1 cells are isogenic melanoma cell subclones. We examined 22 000 spots, 4.2% of which displayed differential expression in G1 and T1 cells. Cell susceptibility to TRAIL-mediated apoptosis was found to be correlated with gene expression signatures in this model. Some of the differentially expressed genes were identified as involved in ATP-binding and signaling pathways, based on previously published data. Further analysis provided evidences that c-kit was overexpressed in G1 cells while it was absent in T1 cells. The c-kit inhibitor, imatinib, did not restore TRAIL sensitivity, excluding a role for c-kit in TRAIL resistance in G1 cells. Surprisingly, imatinib inhibited cell proliferation and TRAIL-mediated apoptosis in melanoma cells. We investigated the possible involvement of several molecules, including c-ABL, platelet-derived growth factor receptor (PDGFR), cellular FADD-like interleukin-1 alpha-converting enzyme-like inhibitory protein (c-FLIP)(L/S), Fas-associated DD kinase, p53, p21(WAF1), proteins of B-cell leukemia/lymphoma 2 (Bcl-2) family and cytochrome c. Imatinib did not modulate the expression or activation of its own targets, such as c-ABL, PDGFRalpha and PDGFRbeta, but it did affect the expression of c-FLIP(L), BCL2-associated X protein (Bax) and Bcl-2. Moreover, c-FLIP(L) knockdown sensitized T1 cells to TRAIL-mediated apoptosis, with a sensitivity similar to that of cells previously treated with imatinib. More notably, we found that the resistance to TRAIL in G1 cells was correlated with constitutive c-FLIP(L) recruitment to the DISC and the inhibition of caspase 8, 3 and 9 processing. Moreover, c-FLIP(L) knockdown partly restored TRAIL

  7. Holothurian glycosaminoglycan inhibits metastasis via inhibition of P-selectin in B16F10 melanoma cells.

    PubMed

    Yue, Zhiqiang; Wang, Aiyun; Zhu, Zhijie; Tao, Li; Li, Yao; Zhou, Liang; Chen, Wenxing; Lu, Yin

    2015-12-01

    P-selectin-mediated tumor cell adhesion to platelets is a well-established stage in the process of tumor metastasis. Through computerized structural analysis, we found a marine-derived polysaccharide, holothurian glycosaminoglycan (hGAG), behaved as a ligand-competitive inhibitor of P-selectin, indicating its potential to disrupt the binding of P-selectin to cell surface receptor and activation of downstream regulators of tumor cell migration. Our experimental data demonstrated that hGAG significantly inhibited P-selectin-mediated adhesion of tumor cells to platelets and tumor cell migration in vitro and reduced subsequent pulmonary metastasis in vivo. Furthermore, abrogation of the P-selectin-mediated adhesion of tumor cells led to down-regulation of protein levels of integrins, FAK and MMP-2/9 in B16F10 cells, which is a crucial molecular mechanism of hGAG to inhibit tumor metastasis. In conclusion, hGAG has emerged as a novel anti-cancer agent via blocking P-selectin-mediated malignant events of tumor metastasis. PMID:26318439

  8. Evaluation of surrogate tissues as indicators of drug activity in a melanoma skin model.

    PubMed

    Parekh, Palak R; Choudhuri, Rohini; Weyemi, Urbain; Martin, Olga A; Bonner, William M; Redon, Christophe E

    2016-08-01

    The development of novel cancer treatments is a challenging task, partly because results from model systems often fail to predict drug efficacy in humans, and also tumors are often inaccessible for biochemical analysis, preventing effective monitoring of drug activity in vivo. Utilizing a model system, we evaluated the use of drug-induced DNA damage in surrogate tissues as indicators of drug efficacy. Samples of a commercially available melanoma skin model (Mattek MLNM-FT-A375) containing keratinocyte and fibroblast layers with melanoma nodules were subjected to various chemotherapeutic regimens for one, four, or eight days. At these times they were analyzed for DNA double-stranded breaks (γH2AX foci) and apoptosis (TUNEL). A wide range of drug responses in both tumor and normal tissues were observed and cataloged. For the melanoma, the most common drug response was apoptosis. The basal keratinocyte layer, which was the most reliable indicator of drug response in the melanoma skin model, responded with γH2AX foci formation that was abrupt and transient. The relationships between tumor and surrogate tissue drug responses are complex, indicating that while surrogate tissue drug responses may be useful clinical tools, careful control of variables such as the timing of sampling may be important in interpreting the results. PMID:27339860

  9. Epigenetic activation of a cryptic TBC1D16 transcript enhances melanoma progression by targeting EGFR

    PubMed Central

    Vizoso, Miguel; Ferreira, Humberto J; Lopez-Serra, Paula; Javier Carmona, F; Martínez-Cardús, Anna; Girotti, Maria Romina; Villanueva, Alberto; Guil, Sonia; Moutinho, Catia; Liz, Julia; Portela, Anna; Heyn, Holger; Moran, Sebastian; Vidal, August; Martinez-Iniesta, Maria; Manzano, Jose L; Fernandez-Figueras, Maria Teresa; Elez, Elena; Muñoz-Couselo, Eva; Botella-Estrada, Rafael; Berrocal, Alfonso; Pontén, Fredrik; van den Oord, Joost; Gallagher, William M; Frederick, Dennie T; Flaherty, Keith T; McDermott, Ultan; Lorigan, Paul; Marais, Richard; Esteller, Manel

    2016-01-01

    Metastasis is responsible for most cancer-related deaths, and, among common tumor types, melanoma is one with great potential to metastasize. Here we study the contribution of epigenetic changes to the dissemination process by analyzing the changes that occur at the DNA methylation level between primary cancer cells and metastases. We found a hypomethylation event that reactivates a cryptic transcript of the Rab GTPase activating protein TBC1D16 (TBC1D16-47 kDa; referred to hereafter as TBC1D16-47KD) to be a characteristic feature of the metastatic cascade. This short isoform of TBC1D16 exacerbates melanoma growth and metastasis both in vitro and in vivo. By combining immunoprecipitation and mass spectrometry, we identified RAB5C as a new TBC1D16 target and showed that it regulates EGFR in melanoma cells. We also found that epigenetic reactivation of TBC1D16-47KD is associated with poor clinical outcome in melanoma, while conferring greater sensitivity to BRAF and MEK inhibitors. PMID:26030178

  10. Epigenetic activation of a cryptic TBC1D16 transcript enhances melanoma progression by targeting EGFR.

    PubMed

    Vizoso, Miguel; Ferreira, Humberto J; Lopez-Serra, Paula; Carmona, F Javier; Martínez-Cardús, Anna; Girotti, Maria Romina; Villanueva, Alberto; Guil, Sonia; Moutinho, Catia; Liz, Julia; Portela, Anna; Heyn, Holger; Moran, Sebastian; Vidal, August; Martinez-Iniesta, Maria; Manzano, Jose L; Fernandez-Figueras, Maria Teresa; Elez, Elena; Muñoz-Couselo, Eva; Botella-Estrada, Rafael; Berrocal, Alfonso; Pontén, Fredrik; Oord, Joost van den; Gallagher, William M; Frederick, Dennie T; Flaherty, Keith T; McDermott, Ultan; Lorigan, Paul; Marais, Richard; Esteller, Manel

    2015-07-01

    Metastasis is responsible for most cancer-related deaths, and, among common tumor types, melanoma is one with great potential to metastasize. Here we study the contribution of epigenetic changes to the dissemination process by analyzing the changes that occur at the DNA methylation level between primary cancer cells and metastases. We found a hypomethylation event that reactivates a cryptic transcript of the Rab GTPase activating protein TBC1D16 (TBC1D16-47 kDa; referred to hereafter as TBC1D16-47KD) to be a characteristic feature of the metastatic cascade. This short isoform of TBC1D16 exacerbates melanoma growth and metastasis both in vitro and in vivo. By combining immunoprecipitation and mass spectrometry, we identified RAB5C as a new TBC1D16 target and showed that it regulates EGFR in melanoma cells. We also found that epigenetic reactivation of TBC1D16-47KD is associated with poor clinical outcome in melanoma, while conferring greater sensitivity to BRAF and MEK inhibitors. PMID:26030178

  11. Activated mouse CD4(+)Foxp3(-) T cells facilitate melanoma metastasis via Qa-1-dependent suppression of NK-cell cytotoxicity.

    PubMed

    Wang, Xiaojuan; Cui, Yanyan; Luo, Gaoxing; Wang, Qinghong; Hu, Jie; He, Weifeng; Yuan, Jun; Zhou, Junyi; Wu, Yan; Sun, Xiaofeng; Robson, Simon C; Li, Xianchang; Tan, Jiangling; Peng, Yanmeng; Xue, Gang; Lu, Linrong; Gao, Wenda; Wu, Jun

    2012-12-01

    The regulatory activities of mouse CD4(+)Foxp3(+) T cells on various immune cells, including NK cells, have been well documented. Under some conditions, conventional CD4(+)Foxp3(-) T cells in the periphery are able to acquire inhibitory function on other T cells, but their roles in controlling innate immune cells are poorly defined. As a potential cellular therapy for cancer, ex vivo activated CD4(+)Foxp3(-) effector T cells are often infused back in vivo to suppress tumor growth and metastasis. Whether such activated T cells could affect NK-cell control of tumorigenesis is unclear. In the present study, we found that mitogen-activated CD4(+)Foxp3(-) T cells exhibited potent suppressor function on NK-cell proliferation and cytotoxicity in vitro, and notably facilitated B16 melanoma metastasis in vivo. Suppression of NK cells by activated CD4(+)Foxp3(-) T cells is cell-cell contact dependent and is mediated by Qa-1:NKG2A interaction, as administration of antibodies blocking either Qa-1 or NKG2A could completely reverse this suppression, and significantly inhibited otherwise facilitated melanoma metastasis. Moreover, activated CD4(+)Foxp3(-) cells from Qa-1 knockout mice completely lost the suppressor activity on NK cells, and failed to facilitate melanoma metastasis when transferred in vivo. Taken together, our findings indicate that innate anti-tumor response is counter regulated by the activation of adaptive immunity, a phenomenon we term as "activation-induced inhibition". Thus, the regulatory role of activated CD4(+)Foxp3(-) T cells in NK-cell activity must be taken into consideration in the future design of cancer therapies. PMID:22945357

  12. The X protein of hepatitis B virus activates hepatoma cell proliferation through repressing melanoma inhibitory activity 2 gene

    SciTech Connect

    Xu, Yilin; Yang, Yang; Cai, Yanyan; Liu, Fang; Liu, Yingle; Zhu, Ying; Wu, Jianguo

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer We demonstrated that HBV represses MIA2 gene expression both invitro and in vivo. Black-Right-Pointing-Pointer The X protein of HBV plays a major role in such regulation. Black-Right-Pointing-Pointer Knock-down of MIA2 in HepG2 cells activates cell growth and proliferation. Black-Right-Pointing-Pointer HBx activates cell proliferation, over-expression of MIA2 impaired such regulation. Black-Right-Pointing-Pointer HBx activates hepatoma cell proliferation through repressing MIA2 expression. -- Abstract: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer deaths globally. Chronic hepatitis B virus (HBV) infection accounts for over 75% of all HCC cases; however, the molecular pathogenesis of HCC is not well understood. In this study, we found that the expression of the newly identified gene melanoma inhibitory activity 2 (MIA2) was reduced by HBV infection invitro and invivo, and that HBV X protein (HBx) plays a major role in this regulation. Recent studies have revealed that MIA2 is a potential tumor suppressor, and that, in most HCCs, MIA2 expression is down-regulated or lost. We found that the knock-down of MIA2 in HepG2 cells activated cell growth and proliferation, suggesting that MIA2 inhibits HCC cell growth and proliferation. In addition, the over-expression of HBx alone induced cell proliferation, whereas MIA2 over-expression impaired the HBx-mediated induction of proliferation. Taken together, our results suggest that HBx activates hepatoma cell growth and proliferation through repression of the potential tumor suppressor MIA2.

  13. High LIFr expression stimulates melanoma cell migration and is associated with unfavorable prognosis in melanoma.

    PubMed

    Guo, Hongwei; Cheng, Yabin; Martinka, Magdalena; McElwee, Kevin

    2015-09-22

    Increased or decreased expression of LIF receptor (LIFr) has been reported in several human cancers, including skin cancer, but its role in melanoma is unknown. In this study, we investigated the expression pattern of LIFr in melanoma and assessed its prognostic value. Using tissue microarrays consisting of 441 melanomas and 96 nevi, we found that no normal nevi showed high LIFr expression. LIFr staining was significantly increased in primary melanoma compared to dysplastic nevi (P = 0.0003) and further increased in metastatic melanoma (P = 0.0000). Kaplan-Meier survival curve and univariate Cox regression analyses showed that increased expression of LIFr was correlated with poorer 5-year patient survival (overall survival, P = 0.0000; disease-specific survival, P = 0.0000). Multivariate Cox regression analyses indicated that increased LIFr expression was an independent prognostic marker for primary melanoma (P = 0.036). LIFr knockdown inhibited melanoma cell migration in wound healing assays and reduced stress fiber formation. LIFr knockdown correlated with STAT3 suppression, but not YAP, suggesting that LIFr activation might stimulate melanoma cell migration through the STAT3 pathway. Our data indicate that strong LIFr expression identifies potentially highly malignant melanocytic lesions at an early stage and LIFr may be a potential target for the development of early intervention therapeutics. PMID:26329521

  14. High LIFr expression stimulates melanoma cell migration and is associated with unfavorable prognosis in melanoma

    PubMed Central

    Guo, Hongwei; Cheng, Yabin; Martinka, Magdalena; McElwee, Kevin

    2015-01-01

    Increased or decreased expression of LIF receptor (LIFr) has been reported in several human cancers, including skin cancer, but its role in melanoma is unknown. In this study, we investigated the expression pattern of LIFr in melanoma and assessed its prognostic value. Using tissue microarrays consisting of 441 melanomas and 96 nevi, we found that no normal nevi showed high LIFr expression. LIFr staining was significantly increased in primary melanoma compared to dysplastic nevi (P = 0.0003) and further increased in metastatic melanoma (P = 0.0000). Kaplan–Meier survival curve and univariate Cox regression analyses showed that increased expression of LIFr was correlated with poorer 5-year patient survival (overall survival, P = 0.0000; disease-specific survival, P = 0.0000). Multivariate Cox regression analyses indicated that increased LIFr expression was an independent prognostic marker for primary melanoma (P = 0.036). LIFr knockdown inhibited melanoma cell migration in wound healing assays and reduced stress fiber formation. LIFr knockdown correlated with STAT3 suppression, but not YAP, suggesting that LIFr activation might stimulate melanoma cell migration through the STAT3 pathway. Our data indicate that strong LIFr expression identifies potentially highly malignant melanocytic lesions at an early stage and LIFr may be a potential target for the development of early intervention therapeutics. PMID:26329521

  15. Arthrophytum scoparium inhibits melanogenesis through the down-regulation of tyrosinase and melanogenic gene expressions in B16 melanoma cells.

    PubMed

    Chao, Hui-Chia; Najjaa, Hanen; Villareal, Myra O; Ksouri, Riadh; Han, Junkyu; Neffati, Mohamed; Isoda, Hiroko

    2013-02-01

    Melanin performs a crucial role in protecting the skin against harmful ultraviolet light. However, hyperpigmentation may lead to aesthetic problems and disorders such as solar lentigines (SL), melasma, postinflammatory hyperpigmentation and even melanoma. Arthrophytum scoparium grows in the desert in the North African region, and given this type of environment, A. scoparium exhibits adaptations for storing water and produces useful bioactive factors. In this study, the effect of A. scoparium ethanol extract (ASEE) on melanogenesis regulation in B16 murine melanoma cells was investigated. Cells treated with 0.017% (w/v) ASEE showed a significant inhibition of melanin biosynthesis in a time-dependent manner without cytotoxicity. To clarify the mechanism behind the ASEE-treated melanogenesis regulation, the expressions of tyrosinase enzyme and melanogenesis-related genes were determined. Results showed that the expression of tyrosinase enzyme was significantly decreased and Tyr, Trp-1, Mitf and Mc1R mRNA expressions were significantly down-regulated. LC-ESI-TOF-MS analysis of the extract identified the presence of six phenolic compounds: coumaric acid, cinnamic acid, chrysoeriol, cyanidin, catechol and caffeoylquinic acid. The melanogenesis inhibitory effect of ASEE may therefore be attributed to its catechol and tetrahydroisoquinoline derivative content. We report here that ASEE can inhibit melanogenesis in a time-dependent manner by decreasing the tyrosinase protein and Tyr, Trp-1, Mitf and Mc1R mRNA expressions. This is the first report on the antimelanogenesis effect of A. scoparium and on its potential as a whitening agent. PMID:23362872

  16. A new water soluble MAPK activator exerts antitumor activity in melanoma cells resistant to the BRAF inhibitor vemurafenib.

    PubMed

    Graziani, Grazia; Artuso, Simona; De Luca, Anastasia; Muzi, Alessia; Rotili, Dante; Scimeca, Manuel; Atzori, Maria Grazia; Ceci, Claudia; Mai, Antonello; Leonetti, Carlo; Levati, Lauretta; Bonanno, Elena; Tentori, Lucio; Caccuri, Anna Maria

    2015-05-01

    Recovery of mitogen activated protein kinase (MAPK) or activation of alternative pathways, such as the PI3K/AKT/mTOR, are involved in acquired resistance to BRAF inhibitors which represent the first-line treatment of BRAF-mutated metastatic melanoma. We recently demonstrated that 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and its water soluble analog 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)ethoxy)ethoxy)ethanol (MC3181) trigger apoptosis in BRAF V600E mutated melanoma cells through activation of the MAPK c-Jun N-terminal kinase (JNK). Herein, we investigated whether NBDHEX and MC3181 might exert antitumor activity against BRAF V600E mutated human melanoma cells rendered resistant to the BRAF inhibitor vemurafenib. To this aim we generated a subline of A375 melanoma resistant in vitro and in vivo to vemurafenib (A375-VR8) and characterized by NRAS G13R mutation, high basal levels of CRAF protein and phospho-activation of AKT. In these cells ERK phosphorylation was not significantly down-modulated by vemurafenib concentrations capable of abrogating ERK phosphorylation in sensitive A375 cells. Both NBDHEX and MC3181 induced marked antiproliferative and apoptotic effects in A375-VR8 cells and, at equitoxic concentrations, caused a strong phosphorylation of JNK, p38, and of the downstream mediators of apoptosis ATF2 and p53. Drug treatment further increased ERK phosphorylation, which was required for the cellular response to the NBD derivatives, as apoptosis was antagonized by the ERK inhibitor FR180204. Finally, in vivo administration of MC3181 provoked JNK activation at the tumor site and markedly reduced A375-VR8 growth. These evidences strongly suggest that the activation of multiple pro-apoptotic MAPK pathways by MC3181 might represent a new strategy for the treatment of melanoma resistant to BRAF inhibitors. PMID:25795251

  17. Cutaneous autoimmune effects in the setting of therapeutic immune checkpoint inhibition for metastatic melanoma.

    PubMed

    Mochel, Mark C; Ming, Michael E; Imadojemu, Sotonye; Gangadhar, Tara C; Schuchter, Lynn M; Elenitsas, Rosalie; Payne, Aimee S; Chu, Emily Y

    2016-09-01

    Therapeutic immune checkpoint blockade for metastatic melanoma has been associated with vitiligo, pruritus and morbilliform eruptions. Reports of other autoimmune skin disease in this setting are rare. We sought to expand the spectrum of cutaneous immune-mediated effects related to immune checkpoint inhibitor therapy. In this report, we describe two unusual cutaneous reactions related to checkpoint inhibitor therapy, namely bullous pemphigoid (BP) and dermatitis herpetiformis. The development of BP and dermatitis herpetiformis in the context of checkpoint inhibitor therapy is consistent with previous investigations supporting the importance of effector and regulatory T cells in the pathogenesis of these diseases. PMID:27161449

  18. Radiosensitization and downregulation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) upon inhibition of mitogen/extracellular signal-regulated kinase (MEK) in malignant melanoma cells

    PubMed Central

    Eder, Stefan; Lamkowski, Andreas; Priller, Markus; Port, Matthias; Steinestel, Konrad

    2015-01-01

    Background Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an important cofactor in the p53-mediated DNA damage response pathway upon ionizing radiation (IR) and exerts anti-apoptotic effects also independent of p53 pathway activation. Furthermore, hnRNP K is overexpressed in various neoplasms including malignant melanoma (MM). Here, we investigate the role of hnRNP K in the radioresistance of MM cells. Methods and results Our results show cytoplasmic expression of hnRNP K in human MM surgical specimens, but not in benign nevi, and a quick dose- and time-dependent upregulation in response to IR accompanied by cytoplasmic redistribution of the protein in the IPC-298 cellular tumor model carrying an activating NRAS mutation (p.Q61L). SiRNA-based knockdown of hnRNP K induced a delayed decline in γH2AX/53BP1-positive DNA repair foci upon IR. Pharmacological interference with MAPK signaling abrogated ERK phosphorylation, diminished cellular hnRNP K levels, impaired γH2AX/53BP1-foci repair and proliferative capability and increased apoptosis comparable to the observed hnRNP K knockdown phenotype in IPC-298 cells. Conclusion Our results indicate that pharmacological interference with MAPK signaling increases vulnerability of NRAS-mutant malignant melanoma cells to ionizing radiation along with downregulation of endogenous hnRNP K and point towards a possible use for combined MEK inhibition and localized radiation therapy of MM in the NRAS-mutant setting where BRAF inhibitors offer no clinical benefit. PMID:26136337

  19. High-dose intensity pulse interleukin-2 with famotidine has activity in metastatic melanoma.

    PubMed

    Quan, Walter D Y; Walker, Paul R; Picton, Maria; Quan, Francine M; King, Linda A; Tyre, Charley; Liles, Darla K

    2008-10-01

    Daily short intravenous (i.v.) infusions (pulses) of interleukin-2 (IL-2) have been developed to decrease toxicity while maintaining anticancer activity of this agent against melanoma. Such IL-2 schedules have previously been shown to promote lymphokine-activated killer (LAK) cell activity. Famotidine may increase LAK activity by increasing IL-2 internalization by the IL-2 receptor on lymphocytes. We treated 16 patients with metastatic melanoma using pulse IL-2 18 (15 patients) or 9 million IU/M2 (1 patient) i.v. over 15-30 minutes preceded by famotidine 20 mg i.v. daily for 5 days on an oncology inpatient unit. Cycles were repeated every 3 weeks until disease progression. Patient characteristics were as follows: 11 males, median age, 66, median ECOG performance status, 1; common metastatic sites: lymph nodes, lungs, subcutaneous, liver, and bone. Median number of cycles received was 3. Overall, 93% of planned doses were delivered. Most common toxicities were hypomagnesemia, fever, rigors, hypophosphatemia, and nausea/emesis. Three (3) patients had partial responses (19% response rate; 95% confidence interval: 6%-44%). A fourth patient, after resection of residual disease, remains a surgical complete responder at > 12 months. Responses occurred in lung, liver, lymph nodes, bone, and subcutaneous sites. Median response duration was 7 months. Pulse IL-2 with famotidine has activity in melanoma. PMID:18999936

  20. The activation of human endogenous retrovirus K (HERV-K) is implicated in melanoma cell malignant transformation

    SciTech Connect

    Serafino, A. Balestrieri, E.; Pierimarchi, P.; Matteucci, C.; Moroni, G.; Oricchio, E.; Rasi, G.; Mastino, A.; Spadafora, C.; Garaci, E.; Vallebona, P. Sinibaldi

    2009-03-10

    Melanoma development is a multi-step process arising from a series of genetic and epigenetic events. Although the sequential stages involved in progression from melanocytes to malignant melanoma are clearly defined, our current understanding of the mechanisms leading to melanoma onset is still incomplete. Growing evidence show that the activation of endogenous retroviral sequences might be involved in transformation of melanocytes as well as in the increased ability of melanoma cells to escape immune surveillance. Here we show that human melanoma cells in vitro undergo a transition from adherent to a more malignant, non-adherent phenotype when exposed to stress conditions. Melanoma-derived non-adherent cells are characterized by an increased proliferative potential and a decreased expression of both HLA class I molecules and Melan-A/MART-1 antigen, similarly to highly malignant cells. These phenotypic and functional modifications are accompanied by the activation of human endogenous retrovirus K expression (HERV-K) and massive production of viral-like particles. Down-regulation of HERV-K expression by RNA interference prevents the transition from the adherent to the non-adherent growth phenotype in low serum. These results implicate HERV-K in at least some critical steps of melanoma progression.

  1. The preferentially expressed antigen in melanoma (PRAME) inhibits myeloid differentiation in normal hematopoietic and leukemic progenitor cells

    PubMed Central

    Guthrie, Katherine A.; Cummings, Carrie L.; Sabo, Kathleen; Wood, Brent L.; Gooley, Ted; Yang, Taimei; Epping, Mirjam T.; Shou, Yaping; Pogosova-Agadjanyan, Era; Ladne, Paula; Stirewalt, Derek L.; Abkowitz, Janis L.; Radich, Jerald P.

    2009-01-01

    The preferentially expressed antigen in melanoma (PRAME) is expressed in several hematologic malignancies, but either is not expressed or is expressed at only low levels in normal hematopoietic cells, making it a target for cancer therapy. PRAME is a tumor-associated antigen and has been described as a corepressor of retinoic acid signaling in solid tumor cells, but its function in hematopoietic cells is unknown. PRAME mRNA expression increased with chronic myeloid leukemia (CML) disease progression and its detection in late chronic-phase CML patients before tyrosine kinase inhibitor therapy was associated with poorer therapeutic responses and ABL tyrosine kinase domain point mutations. In leukemia cell lines, PRAME protein expression inhibited granulocytic differentiation only in cell lines that differentiate along this lineage after all-trans retinoic acid (ATRA) exposure. Forced PRAME expression in normal hematopoietic progenitors, however, inhibited myeloid differentiation both in the presence and absence of ATRA, and this phenotype was reversed when PRAME was silenced in primary CML progenitors. These observations suggest that PRAME inhibits myeloid differentiation in certain myeloid leukemias, and that its function in these cells is lineage and phenotype dependent. Lastly, these observations suggest that PRAME is a target for both prognostic and therapeutic applications. PMID:19625708

  2. Cucurbitacins from Trichosanthes kirilowii as the inhibitory components on tyrosinase activity and melanin synthesis of B16/F10 melanoma cells.

    PubMed

    Oh, Hyuncheol; Mun, Yeun-Ja; Im, Sook-Jung; Lee, Seung Yon; Song, Ho Joon; Lee, Ho-Sub; Woo, Won-Hong

    2002-09-01

    Cucurbitacins 1 and 2 were isolated from the root of Trichosanthes kirilowii by tyrosinase inhibitory activity-guided fractionation. Spectroscopic analysis revealed that compounds 1 and 2 were cucurbitacin D and 23,24-dihydro-cucurbitacin D, respectively. Compounds 1 and 2 effectively inhibited the activity of tyrosinase (IC(50) = 0.18 microM and 6.7 microM, respectively), and the synthesis of melanin (IC(50) = 0.16 microM and 7.5 microM, respectively) in B16/F10 melanoma cells. PMID:12357397

  3. Factors significantly increasing or inhibiting early stages of malignant melanoma (M.M.) and non-invasive evaluation of new treatment by ingestion and external application of optimal doses of the most effective anti-M.M. substances: haritaki, cilantro, vitamin D3, nori, EPA with DHA, & application of special (+) solar energy stored paper, which reduced the M.M. active area & asbestos rapidly.

    PubMed

    Omura, Yoshiaki; Jones, Marilyn; Duvvi, Harsha; Paluch, Kamila; Shimotsuura, Yasuhiro; Ohki, Motomu

    2013-01-01

    Sterilizing the pre-cancer skin of malignant melanoma (M.M.) with 70% Isopropyl alcohol intensified malignancy & the malignant response extended to surrounding normal looking skin, while sterilizing with 80% (vodka) or 12% (plum wine) ethyl alcohol completely inhibited M.M. in the area (both effects lasted for about 90 minutes initially). Burnt food (bread, vegetables, meat, and fish), a variety of smoked & non-smoked fish-skin, many animal's skin, pepper, Vitamin C over 75 mg, mango, pineapple, coconut, almond, sugars, Saccharine & Aspartame, garlic, onion, etc & Electromagnetic field from cellular phones worsened M.M. & induced abnormal M.M. response of surrounding skin. We found the following factors inhibit early stage of M.M. significantly: 1) Increasing normal cell telomere, by taking 500 mg Haritaki, often reached between 400-1150 ng& gradually diminished, but the M.M. response was completely inhibited until normal cell telomeres are reduced to 150 ng, which takes 6-8 hours. More than 70 mg Vitamin C, Orange Juice, & other high Vitamin C containing substances shouldn't be taken because they completely inhibit the effects of Haritaki. 2) We found Chrysotile asbestos & Tremolite asbestos (% of the Chrysotile amount) coexist. A special Cilantro tablet was used to remove asbestos & some toxic metals. 3) Vitamin D3 400 I.U. has a maximum inhibiting effect on M.M. but 800 I.U. or higher promotes malignancy. 4) Noricontaining Iodine, etc., was used. 5) EPA 180 mm with DHA 120 mg was most effectively used after metastasis to the surrounding skin was eliminated. When we combined 1 Cilantro tablet & Vitamin D3 400 I.U. withsmall Nori pieces & EPA with DHA, the effect of complete inhibition of M.M. lasted 9-11 hours. When these anti-M.M.substances (Haritaki, Vitamin D3, Cilantro, Nori, EPA. with DHA) were taken together, the effect lasted 12-14 hoursand M.M. involvement in surrounding normal-looking skin disappeared rapidly & original dark brown or black are as

  4. Effective innate and adaptive anti-melanoma immunity through localized TLR-7/8 activation

    PubMed Central

    Singh, Manisha; Khong, Hiep; Dai, Zhimin; Huang, Xue-Fei; Wargo, Jennifer A.; Cooper, Zachary A.; Vasilakos, John P.; Hwu, Patrick; Overwijk, Willem W.

    2014-01-01

    Intratumoral immune activation can induce local and systemic anti-tumor immunity. Imiquimod is a cream-formulated, TLR-7 agonist that is FDA-approved for the treatment of non-melanoma skin cancers, but has limited activity against melanoma. We studied the anti-tumor activity and mechanism of action of a novel, injectable, tissue-retained TLR 7/8 agonist, 3M-052, which avoids systemic distribution. Intratumoral administration of 3M-052 generated systemic anti-tumor immunity, and suppressed both injected and distant, uninjected wild-type B16.F10 melanomas. Treated tumors showed increased level of CCL2 chemokines and infiltration of M1 phenotype-shifted macrophages, which could kill tumor cells directly through production of nitric oxide and CCL2, was essential for the anti-tumor activity of 3M-052. CD8+ T cells, B cells, Type I IFN, IFN-γ, and pDC were contributed to efficient tumor suppression whereas perforin, NK cells and CD4 T cells were not required. Finally, 3M-052 therapy potentiated checkpoint blockade therapy with anti-CTLA-4 and anti-PD-L1 antibodies, even when checkpoint blockade alone was ineffective. Our findings suggest that intratumoral treatment with 3M-052 is a promising approach for the treatment of cancer and establish a rational strategy and mechanistic understanding for combination therapy with intratumoral, tissue-retained TLR7/8 agonist and checkpoint blockade in metastatic cancer. PMID:25252955

  5. Stepwise multi-photon activation fluorescence reveals a new method of melanoma imaging for dermatologists

    NASA Astrophysics Data System (ADS)

    Lai, Zhenhua; Lian, Christine; Ma, Jie; Yu, Jingyi; Gu, Zetong; Rajadhyaksha, Milind; DiMarzio, Charles A.

    2014-02-01

    Previous research has shown that the stepwise multi-photon activated fluorescence (SMPAF) of melanin, activated by a continuous-wave (CW) mode near infrared (NIR) laser, is a low cost and reliable method of detecting melanin. SMPAF images of melanin in a mouse hair and a formalin fixed mouse melanoma were compared with conventional multiphoton fluorescence microscopy (MPFM) images and confocal reflectance microscopy (CRM) images, all of which were acquired at an excitation wavelength of 920 nm, to further prove the effectiveness of SMPAF in detecting melanin. SMPAF images add specificity for melanin detection to MPFM images and CRM images. Melanin SMPAF can be a promising technology to enable melanoma imaging for dermatologists.

  6. Maximizing the Efficacy of MAPK-Targeted Treatment in PTENLOF/BRAFMUT Melanoma through PI3K and IGF1R Inhibition.

    PubMed

    Herkert, Barbara; Kauffmann, Audrey; Mollé, Sandra; Schnell, Christian; Ferrat, Thomas; Voshol, Hans; Juengert, Janina; Erasimus, Hélène; Marszalek, Grégory; Kazic-Legueux, Malika; Billy, Eric; Ruddy, David; Stump, Mark; Guthy, Daniel; Ristov, Mitko; Calkins, Keith; Maira, Sauveur-Michel; Sellers, William R; Hofmann, Francesco; Hall, Michael N; Brachmann, Saskia M

    2016-01-15

    The introduction of MAPK pathway inhibitors paved the road for significant advancements in the treatment of BRAF-mutant (BRAF(MUT)) melanoma. However, even BRAF/MEK inhibitor combination therapy has failed to offer a curative treatment option, most likely because these pathways constitute a codependent signaling network. Concomitant PTEN loss of function (PTEN(LOF)) occurs in approximately 40% of BRAF(MUT) melanomas. In this study, we sought to identify the nodes of the PTEN/PI3K pathway that would be amenable to combined therapy with MAPK pathway inhibitors for the treatment of PTEN(LOF)/BRAF(MUT) melanoma. Large-scale compound sensitivity profiling revealed that PTEN(LOF) melanoma cell lines were sensitive to PI3Kβ inhibitors, albeit only partially. An unbiased shRNA screen (7,500 genes and 20 shRNAs/genes) across 11 cell lines in the presence of a PI3Kβ inhibitor identified an adaptive response involving the IGF1R-PI3Kα axis. Combined inhibition of the MAPK pathway, PI3Kβ, and PI3Kα or insulin-like growth factor receptor 1 (IGF1R) synergistically sustained pathway blockade, induced apoptosis, and inhibited tumor growth in PTEN(LOF)/BRAF(MUT) melanoma models. Notably, combined treatment with the IGF1R inhibitor, but not the PI3Kα inhibitor, failed to elevate glucose or insulin signaling. Taken together, our findings provide a strong rationale for testing combinations of panPI3K, PI3Kβ + IGF1R, and MAPK pathway inhibitors in PTEN(LOF)/BRAF(MUT) melanoma patients to achieve maximal response. PMID:26577700

  7. Malignant Melanoma

    PubMed Central

    Perera, Eshini; Gnaneswaran, Neiraja; Jennens, Ross; Sinclair, Rodney

    2013-01-01

    Melanomas are a major cause of premature death from cancer. The gradual decrease in rates of morbidity and mortality has occurred as a result of public health campaigns and improved rates of early diagnosis. Survival of melanoma has increased to over 90%. Management of melanoma involves a number of components: excision, tumor staging, re-excision with negative margins, adjuvant therapies (chemo, radiation or surgery), treatment of stage IV disease, follow-up examination for metastasis, lifestyle modification and counseling. Sentinel lymph node status is an important prognostic factor for survival in patients with a melanoma >1 mm. However, sentinel lymph node biopsies have received partial support due to the limited data regarding the survival advantage of complete lymph node dissection when a micrometastasis is detected in the lymph nodes. Functional mutations in the mitogen-activated pathways are commonly detected in melanomas and these influence the growth control. Therapies that target these pathways are rapidly emerging, and are being shown to increase survival rates in patients. Access to these newer agents can be gained by participation in clinical trials after referral to a multidisciplinary team for staging and re-excision of the scar. PMID:27429256

  8. Exceptional antineoplastic activity of a dendritic-cell-targeted vaccine loaded with a Listeria peptide proposed against metastatic melanoma

    PubMed Central

    Calderon-Gonzalez, Ricardo; Bronchalo-Vicente, Lucia; Freire, Javier; Frande-Cabanes, Elisabet; Alaez-Alvarez, Lidia; Gomez-Roman, Javier; Yañez-Diaz, Sonsóles; Alvarez-Dominguez, Carmen

    2016-01-01

    Vaccination with dendritic cells (DCs) is proposed to induce lasting responses against melanoma but its survival benefit in patients needs to be demonstrated. We propose a DC-targeted vaccine loaded with a Listeria peptide with exceptional anti-tumour activity to prevent metastasis of melanoma. Mice vaccinated with vaccines based on DCs loaded with listeriolysin O peptide (91–99) (LLO91–99) showed clear reduction of metastatic B16OVA melanoma size and adhesion, prevention of lung metastasis, enhanced survival, and reversion of immune tolerance. Robust innate and specific immune responses explained the efficiency of DC-LLO91–99 vaccines against B16OVA melanoma. The noTable features of this vaccine related to melanoma reduction were: expansion of immune-dominant LLO91–99-specific CD8 T cells that helped to expand melanoma-specific CD8+ T cells; high numbers of tumour-infiltrating lymphocytes with a cytotoxic phenotype; and a decrease in CD4+CD25high regulatory T cells. This vaccine might be a useful alternative treatment for advanced melanoma, alone or in combination with other therapies. PMID:26942874

  9. Casticin Inhibits A375.S2 Human Melanoma Cell Migration/Invasion through Downregulating NF-κB and Matrix Metalloproteinase-2 and -1.

    PubMed

    Wu, Zih-Yun; Lien, Jin-Cherng; Huang, Yi-Ping; Liao, Ching-Lung; Lin, Jen-Jyh; Fan, Ming-Jen; Ko, Yang-Ching; Hsiao, Yu-Ping; Lu, Hsu-Feng; Chung, Jing-Gung

    2016-01-01

    Casticin is one of the main components from Fructus Viticis, which is widely used as an anti-inflammatory agent. The mechanism of how casticin affects melanoma cell migration and invasion is still not well known. Here we studied the anti-metastasis effects of casticin on A375.S2 melanoma cells by using a non-lethal concentration. First; we used an adhesion assay to test the A375.S2 cells' adhesion ability after treatment with casticin. We next investigated the cell migration ability after casticin treatment by using a wound healing assay to prove that the migration of A375.S2 cells can be inhibited by casticin and double checked the results using the transwell-migration assay. The suppressive effects on matrix metalloproteinase-2; and -9 (MMP-2; and -9) activities were examined by gelatin zymography. Furthermore, western blotting was used to investigate the protein level changes in A375.S2 cells. We found that p-EGFR; Ras and p-ERK1/2 are decreased by casticin, indicating that casticin can down-regulate the migration and invasion ability of A375.S2 cells via the p-EGFR/Ras/p-ERK pathway. The NF-κB p65 and p-ERK levels in nuclear proteins are also decreased by treatment with casticin. An EMSA assay also discovered that the NF-κB p65 and DNA interaction is decreased. NF-κB p65 protein level was examined by immunofluorescence staining and also decreased. Our findings suggest that casticin has anti-metastatic potential by decreasing the invasiveness of A375.S2 cells. We also found that casticin suppressed A375.S2 cell proliferation and cell adhesion ability, but did not affect cell death, as examined using cytometry and a collagen adhesion assay. Based on these observations, casticin could be used as an inhibitor of migration and invasion of human melanoma cells in the future. PMID:27007357

  10. Basic and clinical aspects of malignant melanoma

    SciTech Connect

    Nathanson, L. )

    1987-01-01

    This book contains the following 10 chapters: The role of oncogenes in the pathogenesis of malignant melanoma; Laminin and fibronectin modulate the metastatic activity of melanoma cells; Structure, function and biosynthesis of ganglioside antigens associated with human tumors derived from the neuroectoderm; Epidemiology of ocular melanoma; Malignant melanoma: Prognostic factors; Endocrine influences on the natural history of human malignant melanoma; Psychosocial factors associated with prognostic indicators, progression, psychophysiology, and tumor-host response in cutaneous malignant melanoma; Central nervous system metastases in malignant melanoma; Interferon trials in the management of malignant melanoma and other neoplasms: an overview; and The treatment of malignant melanoma by fast neutrons.

  11. SIRT1 regulates lamellipodium extension and migration of melanoma cells.

    PubMed

    Kunimoto, Risa; Jimbow, Kowichi; Tanimura, Akihiko; Sato, Masahiro; Horimoto, Kouhei; Hayashi, Takashi; Hisahara, Shin; Sugino, Toshiya; Hirobe, Tomohisa; Yamashita, Toshiharu; Horio, Yoshiyuki

    2014-06-01

    Melanoma is highly metastatic, but the mechanism of melanoma cell migration is still unclear. We found that melanoma cells expressed the nicotinamide adenine dinucleotide-dependent protein deacetylase SIRT1 in the cytoplasm. Cell membrane extension and migration of melanoma cells were inhibited by SIRT1 inhibitors or SIRT1 knockdown, whereas SIRT1 activators enhanced elongation of protrusion and cellular motility. In B16F1 cells, growth factor stimulation induced lamellipodium extension, a characteristic feature at the leading edge of migrating cells, and SIRT1 was found in the lamellipodium. SIRT1 inhibitor nicotinamide (NAM) or SIRT1 small interfering RNAs suppressed the lamellipodium extension by serum or platelet-derived growth factor (PDGF). The lamellipodium formation by dominant-active Rac1 was also inhibited by NAM, a SIRT1 inhibitor. NAM inhibited the accumulation of phosphorylated Akt at the submembrane by serum or PDGF. Using fluorescence resonance energy transfer, we found that NAM impaired PDGF-dependent increase in the phosphatidylinositol-3,4,5-trisphosphate level at the leading edge. NAM inhibited the abdominal metastasis of transplanted B16F1 melanoma cells in C57BL6/J mice and improved survival. Finally, SIRT1-knockdown B16F1 cells showed significantly reduced metastasis in transplanted mice compared with that in control B16F1 cells. These results indicate that SIRT1 inhibition is a strategy to suppress metastasis of melanoma cells. PMID:24480879

  12. Inhibition of autophagy enhances the anticancer activity of silver nanoparticles

    PubMed Central

    Lin, Jun; Huang, Zhihai; Wu, Hao; Zhou, Wei; Jin, Peipei; Wei, Pengfei; Zhang, Yunjiao; Zheng, Fang; Zhang, Jiqian; Xu, Jing; Hu, Yi; Wang, Yanhong; Li, Yajuan; Gu, Ning; Wen, Longping

    2014-01-01

    Silver nanoparticles (Ag NPs) are cytotoxic to cancer cells and possess excellent potential as an antitumor agent. A variety of nanoparticles have been shown to induce autophagy, a critical cellular degradation process, and the elevated autophagy in most of these situations promotes cell death. Whether Ag NPs can induce autophagy and how it might affect the anticancer activity of Ag NPs has not been reported. Here we show that Ag NPs induced autophagy in cancer cells by activating the PtdIns3K signaling pathway. The autophagy induced by Ag NPs was characterized by enhanced autophagosome formation, normal cargo degradation, and no disruption of lysosomal function. Consistent with these properties, the autophagy induced by Ag NPs promoted cell survival, as inhibition of autophagy by either chemical inhibitors or ATG5 siRNA enhanced Ag NPs-elicited cancer cell killing. We further demonstrated that wortmannin, a widely used inhibitor of autophagy, significantly enhanced the antitumor effect of Ag NPs in the B16 mouse melanoma cell model. Our results revealed a novel biological activity of Ag NPs in inducing cytoprotective autophagy, and inhibition of autophagy may be a useful strategy for improving the efficacy of Ag NPs in anticancer therapy. PMID:25484080

  13. Inhibition of autophagy enhances the anticancer activity of silver nanoparticles.

    PubMed

    Lin, Jun; Huang, Zhihai; Wu, Hao; Zhou, Wei; Jin, Peipei; Wei, Pengfei; Zhang, Yunjiao; Zheng, Fang; Zhang, Jiqian; Xu, Jing; Hu, Yi; Wang, Yanhong; Li, Yajuan; Gu, Ning; Wen, Longping

    2014-01-01

    Silver nanoparticles (Ag NPs) are cytotoxic to cancer cells and possess excellent potential as an antitumor agent. A variety of nanoparticles have been shown to induce autophagy, a critical cellular degradation process, and the elevated autophagy in most of these situations promotes cell death. Whether Ag NPs can induce autophagy and how it might affect the anticancer activity of Ag NPs has not been reported. Here we show that Ag NPs induced autophagy in cancer cells by activating the PtdIns3K signaling pathway. The autophagy induced by Ag NPs was characterized by enhanced autophagosome formation, normal cargo degradation, and no disruption of lysosomal function. Consistent with these properties, the autophagy induced by Ag NPs promoted cell survival, as inhibition of autophagy by either chemical inhibitors or ATG5 siRNA enhanced Ag NPs-elicited cancer cell killing. We further demonstrated that wortmannin, a widely used inhibitor of autophagy, significantly enhanced the antitumor effect of Ag NPs in the B16 mouse melanoma cell model. Our results revealed a novel biological activity of Ag NPs in inducing cytoprotective autophagy, and inhibition of autophagy may be a useful strategy for improving the efficacy of Ag NPs in anticancer therapy. PMID:25484080

  14. Fully Phosphorothioate-Modified CpG ODN with PolyG Motif Inhibits the Adhesion of B16 Melanoma Cells In Vitro and Tumorigenesis In Vivo

    PubMed Central

    Wang, Xueju; Wang, Liying; Wan, Min; Wu, Xiuli; Yu, Yongli

    2013-01-01

    Adhesion to the extracellular matrix and endothelial lining of blood vessels is critical for tumor cells to grow at original or metastatic sites. Inhibition of tumor cell adhesion can be an antitumor strategy. Guanosine-rich (G-rich) oligodeoxynucleotides (ODNs) can inhibit the adhesion of certain tumor cells. However, no data exist on how inclusion of the CpG motif in the G-rich sequence influences tumor cell adhesion and subsequent tumorigenesis. In this study, in vitro and in vivo assays were used to evaluate how a panel of ODN-containing contiguous guanosines and the CpG motif influenced adhesion of B16 melanoma cells. The results showed that a self-designed ODN, named BW001, containing the polyG motif and a full phosphorothioate modification backbone could inhibit B16 melanoma cell adhesion on a culture plate or on a plate coated with various substances. In vivo data revealed that B16 melanoma cells co-administered with BW001 and intraperitoneally injected into mice formed fewer tumor colonies in peritoneal cavities. This effect was related to the polyG motif and the full phosphorothioate modification backbone and enhanced by the existence of the CpG motif. Additional in vivo data showed that survival of tumor-bearing mice in the BW001 group was significantly prolonged, subcutaneous melanoma developed much more slowly, and lung dissemination colonies formed much less often than in mice inoculated with B16 melanoma cells only. The effect was CpG motif-dependent. These results suggest that BW001 may exert an integrated antitumor effect. PMID:23848522

  15. AZD1480 delays tumor growth in a melanoma model while enhancing the suppressive activity of myeloid-derived suppressor cells.

    PubMed

    Maenhout, Sarah K; Du Four, Stephanie; Corthals, Jurgen; Neyns, Bart; Thielemans, Kris; Aerts, Joeri L

    2014-08-30

    AZD1480 is a potent, competitive small-molecule inhibitor of JAK1/2 kinase which inhibits STAT3 phosphorylation and tumor growth. Here we investigated the effects of AZD1480 on the function of different immune cell populations in a melanoma model. When MO4 tumor-bearing mice were treated with AZD1480 we observed a strong inhibition of tumor growth as well as a prolonged survival. Moreover, a significant decrease in the percentage of myeloid-derived suppressor cells (MDSCs) was observed after treatment with AZD1480. However, AZD1480 enhanced the suppressive capacity of murine MDSCs while at the same time impairing the proliferative as well as the IFN-γ secretion capacity of murine T cells. The addition of AZD1480 to co-cultures of human MDSCs and T cells does not affect the suppressive activity of MDSCs but it does reduce the IFN-γ secretion and the proliferative capacity of T cells. We showed that although AZD1480 has the ability to delay the tumor growth of MO4 tumor-bearing mice, this drug has detrimental effects on several aspects of the immune system. These data indicate that systemic targeting of the JAK/STAT pathway by JAK1/2 inhibition can have divergent effects on tumor growth and anti-tumor immune responses. PMID:25149535

  16. AZD1480 delays tumor growth in a melanoma model while enhancing the suppressive activity of myeloid-derived suppressor cells

    PubMed Central

    Maenhout, Sarah K.; Four, Stephanie Du; Corthals, Jurgen; Neyns, Bart; Thielemans, Kris; Aerts, Joeri L.

    2014-01-01

    AZD1480 is a potent, competitive small-molecule inhibitor of JAK1/2 kinase which inhibits STAT3 phosphorylation and tumor growth. Here we investigated the effects of AZD1480 on the function of different immune cell populations in a melanoma model. When MO4 tumor-bearing mice were treated with AZD1480 we observed a strong inhibition of tumor growth as well as a prolonged survival. Moreover, a significant decrease in the percentage of myeloid-derived suppressor cells (MDSCs) was observed after treatment with AZD1480. However, AZD1480 enhanced the suppressive capacity of murine MDSCs while at the same time impairing the proliferative as well as the IFN-γ secretion capacity of murine T cells. The addition of AZD1480 to co-cultures of human MDSCs and T cells does not affect the suppressive activity of MDSCs but it does reduce the IFN-γ secretion and the proliferative capacity of T cells. We showed that although AZD1480 has the ability to delay the tumor growth of MO4 tumor-bearing mice, this drug has detrimental effects on several aspects of the immune system. These data indicate that systemic targeting of the JAK/STAT pathway by JAK1/2 inhibition can have divergent effects on tumor growth and anti-tumor immune responses. PMID:25149535

  17. Piperine Causes G1 Phase Cell Cycle Arrest and Apoptosis in Melanoma Cells through Checkpoint Kinase-1 Activation

    PubMed Central

    Fofaria, Neel M.; Kim, Sung-Hoon; Srivastava, Sanjay K.

    2014-01-01

    In this study, we determined the cytotoxic effects of piperine, a major constituent of black and long pepper in melanoma cells. Piperine treatment inhibited the growth of SK MEL 28 and B16 F0 cells in a dose and time-dependent manner. The growth inhibitory effects of piperine were mediated by cell cycle arrest of both the cell lines in G1 phase. The G1 arrest by piperine correlated with the down-regulation of cyclin D1 and induction of p21. Furthermore, this growth arrest by piperine treatment was associated with DNA damage as indicated by phosphorylation of H2AX at Ser139, activation of ataxia telangiectasia and rad3-related protein (ATR) and checkpoint kinase 1 (Chk1). Pretreatment with AZD 7762, a Chk1 inhibitor not only abrogated the activation of Chk1 but also piperine mediated G1 arrest. Similarly, transfection of cells with Chk1 siRNA completely protected the cells from G1 arrest induced by piperine. Piperine treatment caused down-regulation of E2F1 and phosphorylation of retinoblastoma protein (Rb). Apoptosis induced by piperine was associated with down-regulation of XIAP, Bid (full length) and cleavage of Caspase-3 and PARP. Furthermore, our results showed that piperine treatment generated ROS in melanoma cells. Blocking ROS by tiron protected the cells from piperine mediated cell cycle arrest and apoptosis. These results suggest that piperine mediated ROS played a critical role in inducing DNA damage and activation of Chk1 leading to G1 cell cycle arrest and apoptosis. PMID:24804719

  18. A novel o-naphtoquinone inhibits N-cadherin expression and blocks melanoma cell invasion via AKT signaling.

    PubMed

    Montenegro, Raquel Carvalho; de Vasconcellos, Marne Carvalho; Barbosa, Gleyce Dos Santos; Burbano, Rommel M R; Souza, Luciana G S; Lemos, Telma L G; Costa-Lotufo, Letícia V; de Moraes, Manoel Odorico

    2013-10-01

    The down-regulation or loss of epithelial markers is often accompanied by the up-regulation of mesenchymal markers. E-cadherin generally suppresses invasiveness, whereas N-cadherin promotes invasion and metastasis in vitro. The aim of this work is to investigate the role of biflorin, a naphthoquinone with proven anticancer properties, on the expression of N-cadherin and AKT proteins in MDA-MB-435 invasive melanoma cancer cells after 12h of exposure to 1, 2.5 and 5 μM biflorin. Biflorin inhibited MDA-MB-435 invasion in a dose-dependent manner (p<0.01). Likewise, biflorin down-regulated N-cadherin and AKT-1 expression in a dose-dependent manner. Biflorin did not inhibit the adhesion of MDA-MB-435 cells to any tested substrates. Additionally, biflorin blocked the invasiveness of cells by down-regulating N-cadherin, most likely via AKT-1 signaling. As such, biflorin may be a novel anticancer agent and a new prototype for drug design. PMID:23912027

  19. Effects of Wnt-10b on proliferation and differentiation of murine melanoma cells

    SciTech Connect

    Misu, Masayasu; Ouji, Yukiteru; Kawai, Norikazu; Nishimura, Fumihiko; Nakamura-Uchiyama, Fukumi; Yoshikawa, Masahide

    2015-08-07

    In spite of the strong expression of Wnt-10b in melanomas, its role in melanoma cells has not been elucidated. In the present study, the biological effects of Wnt-10b on murine B16F10 (B16) melanoma cells were investigated using conditioned medium from Wnt-10b-producing COS cells (Wnt-CM). After 2 days of culture in the presence of Wnt-CM, proliferation of B16 melanoma cells was inhibited, whereas tyrosinase activity was increased. An in vitro wound healing assay demonstrated that migration of melanoma cells to the wound area was inhibited with the addition of Wnt-CM. Furthermore, evaluation of cellular senescence revealed prominent induction of SA-β-gal-positive senescent cells in cultures with Wnt-CM. Finally, the growth of B16 melanoma cell aggregates in collagen 3D-gel cultures was markedly suppressed in the presence of Wnt-CM. These results suggest that Wnt-10b represses tumor cell properties, such as proliferation and migration of B16 melanoma cells, driving them toward a more differentiated state along a melanocyte lineage. - Highlights: • Wnt-10b inhibited proliferation and migration of melanoma cells. • Wnt-10b induced tyrosinase activity and senescence of melanoma cells. • Wnt-10b suppressed growth of cell aggregates in collagen 3D-gel cultures. • Wnt-10b represses tumor cell properties, driving them toward a more differentiated state along a melanocyte lineage.

  20. ADAM15 expression is downregulated in melanoma metastasis compared to primary melanoma

    SciTech Connect

    Ungerer, Christopher; Doberstein, Kai; Boehm, Beate; Pfeilschifter, Josef; Mihic-Probst, Daniela; Gutwein, Paul

    2010-10-22

    Research highlights: {yields} Strong ADAM15 expression is found in normal melanocytes. {yields} ADAM15 expression is significantly downregulated in patients with melanoma metastasis. {yields} TGF-{beta} can downregulate ADAM15 expression in melanoma cells. {yields} Overexpression of ADAM15 in melanoma cells inhibits migration, proliferation and invasion of melanoma cells. {yields} Conclusion: ADAM15 represents an tumor suppressor protein in melanoma. -- Abstract: In a mouse melanoma metastasis model it has been recently shown that ADAM15 overexpression in melanoma cells significantly reduced the number of metastatic nodules on the lung. Unfortunately, the expression of ADAM15 in human melanoma tissue has not been determined so far. In our study, we characterized the expression of ADAM15 in tissue micro-arrays of patients with primary melanoma with melanoma metastasis. ADAM15 was expressed in melanocytes and endothelial cells of benign nevi and melanoma tissue. Importantly, ADAM15 was significantly downregulated in melanoma metastasis compared to primary melanoma. We further demonstrate that IFN-{gamma} and TGF-{beta} downregulate ADAM15 protein levels in melanoma cells. To investigate the role of ADAM15 in melanoma progression, we overexpressed ADAM15 in melanoma cells. Importantly, overexpression of ADAM15 in melanoma cells reduced the migration, invasion and the anchorage dependent and independent cell growth of melanoma cells. In summary, the downregulation of ADAM15 plays an important role in melanoma progression and ADAM15 act as a tumorsuppressor in melanoma.

  1. Dynamic interplay between autophagic flux and Akt during melanoma progression in vitro.

    PubMed

    Maes, Hannelore; Martin, Shaun; Verfaillie, Tom; Agostinis, Patrizia

    2014-02-01

    Despite advances in cancer diagnosis and therapy, metastatic melanoma remains untreatable, due to its notorious resistance to apoptosis, deeming traditional therapies obsolete. Deregulated PI3K/Akt signalling is a common oncogenic event enabling melanocyte transformation and represents a significant and 'druggable' pathway in melanoma. Emerging data show that the ability of cancer cells to survive is also facilitated by alteration of vital homoeostatic mechanisms, such as autophagy. Although the role of autophagy in melanoma is still controversial, recent studies suggest that basal autophagy is down-modulated in primary melanomas. However, the dynamic connection between pro-tumorigenic PI3K/Akt and autophagy during melanoma progression has not been systematically studied. By using human primary melanocytes, incipient melanoma and metastatic melanoma cell lines, we show that early in melanomagenesis, increased Akt activity is associated with a low baseline autophagic flux. However, during melanoma progression, metastatic melanoma cells regain the ability to stimulate autophagic flux, supporting survival. Heightened autophagy is associated with an attenuated Akt activation status and can be suppressed by overexpressing a constitutive active mutant of Akt. On the other hand, blocking the higher Akt activity of primary melanoma is sufficient to incite autophagy. Interestingly, we found that although Akt supports survival of melanocytes and all melanoma cell lines, autophagy inhibition specifically targeted the metastatic melanoma cells, thus indicating a stage-specific requirement for Akt and autophagic flux, throughout melanoma progression. Therefore, this study highlights a dynamic interplay between Akt signalling and autophagic rescue in melanoma, which should be considered in the design of therapeutic strategies targeting these pathways. PMID:24313465

  2. Prophylactic Dendritic Cell-Based Vaccines Efficiently Inhibit Metastases in Murine Metastatic Melanoma

    PubMed Central

    Sennikov, Sergey V.; Vlassov, Valentin V.; Zenkova, Marina A.

    2015-01-01

    Recent data on the application of dendritic cells (DCs) as anti-tumor vaccines has shown their great potential in therapy and prophylaxis of cancer. Here we report on a comparison of two treatment schemes with DCs that display the models of prophylactic and therapeutic vaccination using three different experimental tumor models: namely, Krebs-2 adenocarcinoma (primary tumor), melanoma (B16, metastatic tumor without a primary node) and Lewis lung carcinoma (LLC, metastatic tumor with a primary node). Dendritic cells generated from bone marrow-derived DC precursors and loaded with lysate of tumor cells or transfected with the complexes of total tumor RNA with cationic liposomes were used for vaccination. Lipofectamine 2000 and liposomes consisting of helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and cationic lipid 2D3 (1,26-Bis(1,2-de-O-tetradecyl-rac-glycerol)-7,11,16,20-tetraazahexacosan tetrahydrocloride) were used for RNA transfection. It was shown that DCs loaded with tumor lysate were ineffective in contrast to tumor-derived RNA. Therapeutic vaccination with DCs loaded by lipoplexes RNA/Lipofectamine 2000 was the most efficient for treatment of non-metastatic Krebs-2, where a 1.9-fold tumor growth retardation was observed. Single prophylactic vaccination with DCs loaded by lipoplexes RNA/2D3 was the most efficient to treat highly aggressive metastatic tumors LLC and B16, where 4.7- and 10-fold suppression of the number of lung metastases was observed, respectively. Antimetastatic effect of single prophylactic DC vaccination in metastatic melanoma model was accompanied by the reductions in the levels of Th2-specific cytokines however the change of the levels of Th1/Th2/Th17 master regulators was not found. Failure of double prophylactic vaccination is explained by Th17-response polarization associated with autoimmune and pro-inflammatory reactions. In the case of therapeutic DC vaccine the polarization of Th1-response was found nevertheless

  3. Prophylactic Dendritic Cell-Based Vaccines Efficiently Inhibit Metastases in Murine Metastatic Melanoma.

    PubMed

    Markov, Oleg V; Mironova, Nadezhda L; Sennikov, Sergey V; Vlassov, Valentin V; Zenkova, Marina A

    2015-01-01

    Recent data on the application of dendritic cells (DCs) as anti-tumor vaccines has shown their great potential in therapy and prophylaxis of cancer. Here we report on a comparison of two treatment schemes with DCs that display the models of prophylactic and therapeutic vaccination using three different experimental tumor models: namely, Krebs-2 adenocarcinoma (primary tumor), melanoma (B16, metastatic tumor without a primary node) and Lewis lung carcinoma (LLC, metastatic tumor with a primary node). Dendritic cells generated from bone marrow-derived DC precursors and loaded with lysate of tumor cells or transfected with the complexes of total tumor RNA with cationic liposomes were used for vaccination. Lipofectamine 2000 and liposomes consisting of helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and cationic lipid 2D3 (1,26-Bis(1,2-de-O-tetradecyl-rac-glycerol)-7,11,16,20-tetraazahexacosan tetrahydrocloride) were used for RNA transfection. It was shown that DCs loaded with tumor lysate were ineffective in contrast to tumor-derived RNA. Therapeutic vaccination with DCs loaded by lipoplexes RNA/Lipofectamine 2000 was the most efficient for treatment of non-metastatic Krebs-2, where a 1.9-fold tumor growth retardation was observed. Single prophylactic vaccination with DCs loaded by lipoplexes RNA/2D3 was the most efficient to treat highly aggressive metastatic tumors LLC and B16, where 4.7- and 10-fold suppression of the number of lung metastases was observed, respectively. Antimetastatic effect of single prophylactic DC vaccination in metastatic melanoma model was accompanied by the reductions in the levels of Th2-specific cytokines however the change of the levels of Th1/Th2/Th17 master regulators was not found. Failure of double prophylactic vaccination is explained by Th17-response polarization associated with autoimmune and pro-inflammatory reactions. In the case of therapeutic DC vaccine the polarization of Th1-response was found nevertheless

  4. β-Catenin Signaling Increases during Melanoma Progression and Promotes Tumor Cell Survival and Chemoresistance

    PubMed Central

    Sinnberg, Tobias; Menzel, Moritz; Ewerth, Daniel; Sauer, Birgit; Schwarz, Michael; Schaller, Martin; Garbe, Claus; Schittek, Birgit

    2011-01-01

    Beta-catenin plays an important role in embryogenesis and carcinogenesis by controlling either cadherin-mediated cell adhesion or transcriptional activation of target gene expression. In many types of cancers nuclear translocation of beta-catenin has been observed. Our data indicate that during melanoma progression an increased dependency on the transcriptional function of beta-catenin takes place. Blockade of beta-catenin in metastatic melanoma cell lines efficiently induces apoptosis, inhibits proliferation, migration and invasion in monolayer and 3-dimensional skin reconstructs and decreases chemoresistance. In addition, subcutaneous melanoma growth in SCID mice was almost completely inhibited by an inducible beta-catenin knockdown. In contrast, the survival of benign melanocytes and primary melanoma cell lines was less affected by beta-catenin depletion. However, enhanced expression of beta-catenin in primary melanoma cell lines increased invasive capacity in vitro and tumor growth in the SCID mouse model. These data suggest that beta-catenin is an essential survival factor for metastatic melanoma cells, whereas it is dispensable for the survival of benign melanocytes and primary, non-invasive melanoma cells. Furthermore, beta-catenin increases tumorigenicity of primary melanoma cell lines. The differential requirements for beta-catenin signaling in aggressive melanoma versus benign melanocytic cells make beta-catenin a possible new target in melanoma therapy. PMID:21858114

  5. Melanoma resistance to photodynamic therapy: new insights

    PubMed Central

    Huang, Ying-Ying; Vecchio, Daniela; Avci, Pinar; Yin, Rui; Garcia-Diaz, Maria; Hamblin, Michael R.

    2012-01-01

    Melanoma is the most dangerous form of skin cancer, with a steeply rising incidence and a poor prognosis in its advanced stages. Melanoma is highly resistant to traditional chemotherapy and radiotherapy, although modern targeted therapies such as BRAF inhibitors are showing some promise. Photodynamic therapy (PDT, the combination of photosensitizing dyes and visible light) has been tested for melanoma with some promising results, but melanoma is generally considered to also be resistant to PDT. Optical interference by the highly-pigmented melanin, the anti-oxidant effect of melanin, the sequestration of photosensitizers inside melanosomes, defects in apoptotic pathways, and the efflux of photosensitizers by ATP-binding cassette (ABC) transporters have all been implicated in melanoma resistance to PDT. Approaches to overcoming melanoma resistance to PDT include: the discovery of highly active photosensitizers absorbing in the 700–800-nm near infrared spectral region; interventions that can temporarily reduce the amount or the pigmentation of the melanin; compounds that can reverse apoptotic defects or inhibit drug-efflux of photosensitizers; and immunotherapy approaches that can take advantage of the ability of PDT to activate the host immune system to the treated tumor. PMID:23152406

  6. Control of NF-kB activity in human melanoma by bromodomain and extra-terminal protein inhibitor I-BET151.

    PubMed

    Gallagher, Stuart J; Mijatov, Branka; Gunatilake, Dilini; Gowrishankar, Kavitha; Tiffen, Jessamy; James, Wilmott; Jin, Lei; Pupo, Gulietta; Cullinane, Carleen; McArthur, Grant A; Tummino, Peter J; Rizos, Helen; Hersey, Peter

    2014-11-01

    The transcription factor NF-kappaB (NF-kB) is a key regulator of cytokine and chemokine production in melanoma and is responsible for symptoms such as anorexia, fatigue, and weight loss. In addition, NF-kB is believed to contribute to progression of the disease by upregulation of cell cycle and anti-apoptotic genes and to contribute to resistance against targeted therapies and immunotherapy. In this study, we have examined the ability of the bromodomain and extra-terminal (BET) protein inhibitor I-BET151 to inhibit NF-kB in melanoma cells. We show that I-BET151 is a potent, selective inhibitor of a number of NF-kB target genes involved in induction of inflammation and cell cycle regulation and downregulates production of cytokines such as IL-6 and IL-8. SiRNA studies indicate that BRD2 is the main BET protein involved in regulation of NF-kB and that I-BET151 caused transcriptional downregulation of the NF-kB subunit p105/p50. These results suggest that BET inhibitors may have an important role in treatment of melanoma where activation of NF-kB may have a key pathogenic role. PMID:24924589

  7. Combinatorial drug screening and molecular profiling reveal diverse mechanisms of intrinsic and adaptive resistance to BRAF inhibition in V600E BRAF mutant melanomas

    PubMed Central

    Roller, Devin G.; Capaldo, Brian; Bekiranov, Stefan; Mackey, Aaron J.; Conaway, Mark R.; Petricoin, Emanuel F.; Gioeli, Daniel; Weber, Michael J.

    2016-01-01

    Over half of BRAFV600E melanomas display intrinsic resistance to BRAF inhibitors, in part due to adaptive signaling responses. In this communication we ask whether BRAFV600E melanomas share common adaptive responses to BRAF inhibition that can provide clinically relevant targets for drug combinations. We screened a panel of 12 treatment-naïve BRAFV600E melanoma cell lines with MAP Kinase pathway inhibitors in pairwise combination with 58 signaling inhibitors, assaying for synergistic cytotoxicity. We found enormous diversity in the drug combinations that showed synergy, with no two cell lines having an identical profile. Although the 6 lines most resistant to BRAF inhibition showed synergistic benefit from combination with lapatinib, the signaling mechanisms by which this combination generated synergistic cytotoxicity differed between the cell lines. We conclude that adaptive responses to inhibition of the primary oncogenic driver (BRAFV600E) are determined not only by the primary oncogenic driver but also by diverse secondary genetic and epigenetic changes (“back-seat drivers”) and hence optimal drug combinations will be variable. Because upregulation of receptor tyrosine kinases is a major source of drug resistance arising from diverse adaptive responses, we propose that inhibitors of these receptors may have substantial clinical utility in combination with inhibitors of the MAP Kinase pathway. PMID:26673621

  8. Characterization and Inducing Melanoma Cell Apoptosis Activity of Mannosylerythritol Lipids-A Produced from Pseudozyma aphidis.

    PubMed

    Fan, Linlin; Li, Hongji; Niu, Yongwu; Chen, Qihe

    2016-01-01

    Mannosylerythritol lipids (MELs) are natural glycolipid biosurfactants which have potential applications in the fields of food, cosmetic and medicine. In this study, MELs were produced from vegetable oil by Pseudozyma aphidis. Their structural data through LC/MS, GC/MS and NMR analysis revealed that MEL-A with two acetyls was the major compound and the identified homologs of MEL-A contained a length of C8 to C14 fatty acid chains. This glycolipid exhibited a surface tension of 27.69 mN/m at a critical micelle concentration (CMC), self-assembling into particles in the water solution. It was observed to induce cell growth-inhibition and apoptosis of B16 melanoma cells in a dose-dependent manner, as well as cause cell cycle arrest at the S phase. Further quantitative RT-PCR analysis and western blotting revealed an increasing tendency of both mRNA and protein expressions of Caspase-12, CHOP, GRP78 and Caspase-3, and a down-regulation of protein Bcl-2. Combined with the up regulation of signaling IRE1 and ATF6, it can be speculated that MEL-A-induced B16 melanoma cell apoptosis was associated with the endoplasmic reticulum stress (ERS). PMID:26828792

  9. Characterization and Inducing Melanoma Cell Apoptosis Activity of Mannosylerythritol Lipids-A Produced from Pseudozyma aphidis

    PubMed Central

    Fan, Linlin; Li, Hongji; Niu, Yongwu; Chen, Qihe

    2016-01-01

    Mannosylerythritol lipids (MELs) are natural glycolipid biosurfactants which have potential applications in the fields of food, cosmetic and medicine. In this study, MELs were produced from vegetable oil by Pseudozyma aphidis. Their structural data through LC/MS, GC/MS and NMR analysis revealed that MEL-A with two acetyls was the major compound and the identified homologs of MEL-A contained a length of C8 to C14 fatty acid chains. This glycolipid exhibited a surface tension of 27.69 mN/m at a critical micelle concentration (CMC), self-assembling into particles in the water solution. It was observed to induce cell growth-inhibition and apoptosis of B16 melanoma cells in a dose-dependent manner, as well as cause cell cycle arrest at the S phase. Further quantitative RT-PCR analysis and western blotting revealed an increasing tendency of both mRNA and protein expressions of Caspase-12, CHOP, GRP78 and Caspase-3, and a down-regulation of protein Bcl-2. Combined with the up regulation of signaling IRE1 and ATF6, it can be speculated that MEL-A-induced B16 melanoma cell apoptosis was associated with the endoplasmic reticulum stress (ERS). PMID:26828792

  10. Monoclonal Antibodies Inhibit the Adhesion of Mouse B 16 Melanoma Cells in vitro and Block Lung Metastasis in vivo

    NASA Astrophysics Data System (ADS)

    Vollmers, H. Peter; Birchmeier, Walter

    1983-06-01

    Seven monoclonal antibodies against mouse B 16 melanoma cells (produced in syngeneic C57BL/6 mice) were selected that blocked the adhesion of melanoma cells to tissue culture dishes. These antibodies were found to be directed against antigens on the surface of mouse B 16 melanoma cells but not on normal mouse cells such as 3T3 fibroblasts. Similarly, the antigens could not be detected in normal mouse tissues (e.g., lung, kidney, liver) but were found in lungs colonized by B 16 melanoma cells. Significantly, three of these antibodies virtually abolished lung colonization of highly invasive B 16 sublines injected into the animals' bloodstream. They exerted their effect both when preabsorbed by the melanoma cell in vitro and when delivered to the animals prior to the tumor cells. It is suggested that monoclonal antibodies might be a promising tool for preventing metastasis.

  11. Evaluation of antioxidant and anti-melanogenic activities of different extracts from aerial parts of Nepeta binaludensis Jamzad in murine melanoma B16F10 cells

    PubMed Central

    Tayarani-Najaran, Zahra; Akaberi, Maryam; Vatani, Mohsen; Emami, Seyed Ahmad

    2016-01-01

    Objective(s): Nepeta binaludensis Jamzad (Lamiaceae) has been used in folk medicine of Iran to cure various diseases. The plant is an endemic species to the country that has recently been identified in Razavi Khorasan province. To evaluate the antioxidant and anti-melanogenesis of N. binaludensis, in this study the inhibitory activity of different extracts of N. binaludensis in murine melanoma B16F10 cells is investigated. Materials and Methods: The effects of petroleum ether, dichloromethane, ethyl acetate, and methanol extracts isolated from the plant on melanogenesis in B16 melanoma cells were investigated. To assess the inhibitory effects of this plant on melanogenesis, various assays were used including cytotoxicity, inhibition of mushroom tyrosinase and cellular tyrosinase, determination of melanin content, the effect of extracts on reactive oxygen species and western blot analysis of proteins involved in melanogenesis process. Results: The content of melanin and the activity of tyrosinase were significantly reduced with different extracts of N. binaludensis in cells. Reactive oxygen species was also significantly decreased following the treatment of cell with the mentioned extracts, while a resazurin assay showed no cytotoxicity. Furthermore, we have found that the plant decreased the amount of tyrosinase and microphthalmia-associated transcription factor proteins, which verify the role of suppression of microphthalmia-associated transcription factor protein in melanogenesis inhibition. Conclusion: Taken together the data indicate that N. binaludensis has inhibitory activity on melanin synthesis with no cytotoxic effects in B16 melanoma cells. Therefore, it merits future investigations to apply as whitening agent in hyperpigmentation. PMID:27482348

  12. Absent in Melanoma 2 (AIM2) limits pro-inflammatory cytokine transcription in cardiomyocytes by inhibiting STAT1 phosphorylation.

    PubMed

    Furrer, Antonia; Hottiger, Michael O; Valaperti, Alan

    2016-06-01

    Interferon (IFN)-γ is highly upregulated during heart inflammation and enhances the production of pro-inflammatory cytokines. Absent in Melanoma 2 (AIM2) is an IFN-inducible protein implicated as a component of the inflammasome. Here we seek to determine the role of AIM2 during inflammation in cardiac cells. We found that the presence of AIM2, but not of the other inflammasome components Nod-like receptor (NLR) NLRP3 or NLRC4, specifically limited the transcription of the pro-inflammatory cytokines interleukin (IL)-6, IP-10, and tumor necrosis factor (TNF)-α in HL-1 mouse cardiomyocytes stimulated with IFN-γ and lipopolysaccharides (LPS). Similarly, AIM2 reduced pro-inflammatory cytokine transcription in primary mouse neonatal cardiomyocytes (MNC), but not in primary mouse neonatal cardiac fibroblasts (MNF). Interestingly, AIM2-dependent reduction of pro-inflammatory cytokines in cardiomyocytes was independent of Caspase-1. Mechanistically, AIM2 reduced pro-inflammatory cytokine transcription in cardiomyocytes by interacting with and inhibiting the phosphorylation of STAT1. In AIM2-depleted cardiomyocytes, increased STAT1 phosphorylation enhanced the NF-κB pathway by promoting NF-κB p65 phosphorylation and acetylation. These results show for the first time that AIM2 plays an important anti-inflammatory, yet inflammasome-independent function in cardiomyocytes. Our findings will help to further understand how the various heart cell types differently react to inflammatory stimuli. PMID:27148820

  13. Priming Endothelial Cells With a Melanoma-Derived Extracellular Matrix Triggers the Activation of αvβ3/VEGFR2 Axis.

    PubMed

    Helal-Neto, Edward; Brandão-Costa, Renata M; Saldanha-Gama, Roberta; Ribeiro-Pereira, Cristiane; Midlej, Victor; Benchimol, Marlene; Morandi, Verônica; Barja-Fidalgo, Christina

    2016-11-01

    The unique composition of tumor-produced extracellular matrix (ECM) can be a determining factor in changing the profile of endothelial cells in the tumor microenvironment. As the main receptor for ECM proteins, integrins can activate a series of signaling pathways related to cell adhesion, migration, and differentiation of endothelial cells that interact with ECM proteins. We studied the direct impact of the decellularized ECM produced by a highly metastatic human melanoma cell line (MV3) on the activation of endothelial cells and identified the intracellular signaling pathways associated with cell differentiation. Our data show that compared to the ECM derived from a human melanocyte cell line (NGM-ECM), ECM produced by a melanoma cell line (MV3-ECM) is considerably different in ultrastructural organization and composition and possesses a higher content of tenascin-C and laminin and a lower expression of fibronectin. When cultured directly on MV3-ECM, endothelial cells change morphology and show increased adhesion, migration, proliferation, and tubulogenesis. Interaction of endothelial cells with MV3-ECM induces the activation of integrin signaling, increasing FAK phosphorylation and its association with Src, which activates VEGFR2, potentiating the receptor response to VEGF. The blockage of αvβ3 integrin inhibited the FAK-Src association and VEGFR activation, thus reducing tubulogenesis. Together, our data suggest that the interaction of endothelial cells with the melanoma-ECM triggers integrin-dependent signaling, leading to Src pathway activation that may potentiate VEGFR2 activation and up-regulate angiogenesis. J. Cell. Physiol. 231: 2464-2473, 2016. © 2016 Wiley Periodicals, Inc. PMID:27420801

  14. Quantitative Proteomics Identifies Activation of Hallmark Pathways of Cancer in Patient Melanoma

    PubMed Central

    Byrum, Stephanie D.; Larson, Signe K.; Avaritt, Nathan L.; Moreland, Linley E.; Mackintosh, Samuel G.; Cheung, Wang L.; Tackett, Alan J.

    2013-01-01

    Molecular pathways regulating melanoma initiation and progression are potential targets of therapeutic development for this aggressive cancer. Identification and molecular analysis of these pathways in patients has been primarily restricted to targeted studies on individual proteins. Here, we report the most comprehensive analysis of formalin-fixed paraffin-embedded human melanoma tissues using quantitative proteomics. From 61 patient samples, we identified 171 proteins varying in abundance among benign nevi, primary melanoma, and metastatic melanoma. Seventy-three percent of these proteins were validated by immunohistochemistry staining of malignant melanoma tissues from the Human Protein Atlas database. Our results reveal that molecular pathways involved with tumor cell proliferation, motility, and apoptosis are mis-regulated in melanoma. These data provide the most comprehensive proteome resource on patient melanoma and reveal insight into the molecular mechanisms driving melanoma progression. PMID:23976835

  15. Inducible but Not Constitutive Expression of PD-L1 in Human Melanoma Cells Is Dependent on Activation of NF-κB

    PubMed Central

    Gowrishankar, Kavitha; Gunatilake, Dilini; Gallagher, Stuart J.; Tiffen, Jessamy; Rizos, Helen; Hersey, Peter

    2015-01-01

    Monoclonal antibodies against immune checkpoint blockade have proven to be a major success in the treatment of melanoma. The programmed death receptor-1 ligand-1 (PD-L1) expression on melanoma cells is believed to have an inhibitory effect on T cell responses and to be an important escape mechanism from immune attack. Previous studies have shown that PD-L1 can be expressed constitutively or can be induced by IFN-γ secreted by infiltrating lymphocytes. In the present study we have investigated the mechanism underlying these two modes of PD-L1 expression in melanoma cells including cells that had acquired resistance to the BRAF inhibitor vemurafenib. PD-L1 expression was examined by flow cytometry and immunoblotting. Specific inhibitors and siRNA knockdown approaches were used to examine the roles of the RAF/ MEK, PI3K, NF-κB, STAT3 and AP1/ c-Jun pathways. IFN-γ inducible expression of PD-L1 was dependent on NF-κB as shown by inhibition with BMS-345541, an inhibitor of IκB and the BET protein inhibitor I-BET151, as well as by siRNA knockdown of NF-κB subunits. We were unable to implicate the BRAF/MEK pathway as major regulators in PD-L1 expression on vemurafenib resistant cells. Similarly the PI3K/AKT pathway and the transcription factors STAT3 and c-Jun had only minor roles in IFN-γ induced expression of PD-L1. The mechanism underlying constitutive expression remains unresolved. We suggest these results have significance in selection of treatments that can be used in combination with monoclonal antibodies against PD1, to enhance their effectiveness and to reduce inhibitory effects melanoma cells have against cytotoxic T cell activity. PMID:25844720

  16. Melanoma-derived factors alter the maturation and activation of differentiated tissue-resident dendritic cells.

    PubMed

    Hargadon, Kristian M; Bishop, Johnathan D; Brandt, John P; Hand, Zachary C; Ararso, Yonathan T; Forrest, Osric A

    2016-01-01

    Dendritic cells (DCs) are key regulators of host immunity that are capable of inducing either immune tolerance or activation. In addition to their well-characterized role in shaping immune responses to foreign pathogens, DCs are also known to be critical for the induction and maintenance of anti-tumor immune responses. Therefore, it is important to understand how tumors influence the function of DCs and the quality of immune responses they elicit. Although the majority of studies in this field to date have utilized either immortalized DC lines or DC populations that have been generated under artificial conditions from hematopoietic precursors in vitro, we wished to investigate how tumors impact the function of already differentiated, tissue-resident DCs. Therefore, we used both an ex vivo and in vivo model system to assess the influence of melanoma-derived factors on DC maturation and activation. In ex vivo studies with freshly isolated splenic DCs, we demonstrate that the extent to which DC maturation and activation are altered by these factors correlates with melanoma tumorigenicity, and we identify partial roles for tumor-derived transforming growth factor (TGF)β1 and vascular endothelial growth factor (VEGF)-A in the altered functionality of DCs. In vivo studies using a lung metastasis model of melanoma also demonstrate tumorigenicity-dependent alterations to the function of lung-resident DCs, and skewed production of proinflammatory cytokines and chemokines by these tumor-altered cells is associated with recruitment of an immune infiltrate that may ultimately favor tumor immune escape and outgrowth. PMID:26010746

  17. Valencene from the Rhizomes of Cyperus rotundus Inhibits Skin Photoaging-Related Ion Channels and UV-Induced Melanogenesis in B16F10 Melanoma Cells.

    PubMed

    Nam, Joo Hyun; Nam, Da-Yeong; Lee, Dong-Ung

    2016-04-22

    Ultraviolet (UV) radiation deeply penetrates skin and causes inflammation and pigmentary changes and triggers immune responses. Furthermore, accumulating evidence suggests that calcium ion channels, such as TRPV1 and ORAI1, mediate diverse dermatological processes including melanogenesis, skin wrinkling, and inflammation. The rhizomes of Cyperus rotundus have been used to treat inflammatory diseases including dermatitis. However, their effects on UV-induced photoaging-related ion channels remain unknown. Therefore, this study was undertaken to evaluate the antagonistic effects of C. rotundus extract and their constituents on TRPV1 and ORAI1 channels. Electrophysiological analysis revealed that valencene (1) isolated from the hexane fraction potently inhibited capsaicin-induced TRPV1 and ORAI1 currents at 90 μM (69 ± 15% and 97 ± 2% at -60 and -120 mV, respectively). The inhibitory effect of 1 on cytoplasmic Ca(2+) concentrations in response to ORAI1 activation (85 ± 2% at 50 μM) was also confirmed. Furthermore, 1 concentration-dependently decreased the melanin content after UVB irradiation in murine B16F10 melanoma cells by 82.66 ± 2.14% at 15 μg/mL. These results suggest that C. rotundus rhizomes have potential therapeutic effects on UV-induced photoaging and indicate that the therapeutic and cosmetic applications of 1 are worth further investigation. PMID:26967731

  18. Inhibition of cytokine-induced microvascular arrest of tumor cells by recombinant endostatin prevents experimental hepatic melanoma metastasis.

    PubMed

    Mendoza, Lorea; Valcárcel, María; Carrascal, Teresa; Egilegor, Eider; Salado, Clarisa; Sim, B Kim Lee; Vidal-Vanaclocha, Fernando

    2004-01-01

    We investigated effects of endostatin (ES) in the prometastatic microenvironment of inflammation occurring during the microvascular phase of cancer cell infiltration in the liver. We used a model of intrasplenic injection of B16 melanoma (B16M) cells leading to hepatic metastasis through vascular cell adhesion molecule-(VCAM-1)-mediated capillary arrest of cancer cells via interleukin-18 (IL-18)-dependent mechanism. We show that administration of 50 mg/kg recombinant human (rh) ES 30 min before B16M, plus repetition of same dose for 3 additional days decreased metastasis number by 60%. A single dose of rhES before B16M injection reduced hepatic microvascular retention of luciferase-transfected B16M by 40% and inhibited hepatic production of tumor necrosis factor alpha (TNF-alpha) and IL-18 and VCAM-1 expression by hepatic sinusoidal endothelia (HSE). Consistent with these data, rhES inhibited VCAM-1-dependent B16M cell adhesion to primary cultured HSE receiving B16M conditioned medium, and it abolished the HSE cell production of TNF-alpha and IL-18 induced by tumor-derived vascular endothelial cell growth factor (VEGF). rhES abrogated recombinant murine VEGF-induced tyrosine phosphorylation of KDR/flk-1 receptor in HSE cells, preventing the proinflammatory action of tumor-derived VEGF on HSE. rhES also abolished hepatic production of TNF-alpha, microvascular retention of luciferase-transfected B16M, and adhesion of B16M cells to isolated HSE cells, all of them induced in mice given 5 micro g/kg recombinant murine VEGF for 18 h. This capillary inflammation-deactivating capability constitutes a nonantiangiogenic antitumoral action of endostatin that decreases cancer cell arrest within liver microvasculature and prevents metastases promoted by proinflammatory cytokines induced by VEGF. PMID:14729638

  19. A novel temozolomide analog, NEO212, with enhanced activity against MGMT-positive melanoma in vitro and in vivo.

    PubMed

    Chen, Thomas C; Cho, Hee-Yeon; Wang, Weijun; Nguyen, Jenny; Jhaveri, Niyati; Rosenstein-Sisson, Rachel; Hofman, Florence M; Schönthal, Axel H

    2015-03-28

    The alkylating agent temozolomide (TMZ) represents an important component of current melanoma therapy, but overexpression of O6-methyl-guanine DNA methyltransferase (MGMT) in tumor cells confers resistance to TMZ and impairs therapeutic outcome. We investigated a novel perillyl alcohol (POH)-conjugated analog of TMZ, NEO212, for its ability to exert anticancer activity against MGMT-positive melanoma cells. Human melanoma cells with variable MGMT expression levels were treated with NEO212, TMZ, or perillyl alcohol in vitro and in vivo, and markers of DNA damage and apoptosis, and tumor cell growth were investigated. NEO212 displayed substantially greater anticancer activity than any of the other treatments. It reduced colony formation of MGMT-positive cells up to eight times more effectively than TMZ, and much more potently induced DNA damage and cell death. In a nude mouse tumor model, NEO212 showed significant activity against MGMT-positive melanoma, whereas TMZ, or a mix of TMZ plus POH, was ineffective. At the same time, NEO212 was well tolerated. NEO212 may have potential as a more effective therapy for advanced melanoma, and should become particularly suitable for the treatment of patients with MGMT-positive tumors. PMID:25524552

  20. Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma

    PubMed Central

    Jayachandran, Aparna; Anaka, Matthew; Prithviraj, Prashanth; Hudson, Christopher; McKeown, Sonja J; Lo, Pu-Han; Vella, Laura J; Goding, Colin R; Cebon, Jonathan; Behren, Andreas

    2014-01-01

    Epithelial-to-mesenchymal transition (EMT), in which epithelial cells loose their polarity and become motile mesenchymal cells, is a determinant of melanoma metastasis. We compared gene expression signatures of mesenchymal-like melanoma cells with those of epithelial-like melanoma cells, and identified Thrombospondin 1 (THBS1) as highly up-regulated in the mesenchymal phenotype. This study investigated whether THBS1, a major physiological activator of transforming growth factor (TGF)-beta, is involved in melanoma EMT-like process. We sought to examine expression patterns in distinct melanoma phenotypes including invasive, de-differentiated, label-retaining and drug resistant populations that are putatively associated with an EMT-like process. Here we show that THBS1 expression and secretion was elevated in melanoma cells exhibiting invasive, drug resistant, label retaining and mesenchymal phenotypes and correlated with reduced expression of genes involved in pigmentation. Elevated THBS1 levels were detected in Vemurafenib resistant melanoma cells and inhibition of THBS1 led to significantly reduced chemoresistance in melanoma cells. Notably, siRNA-mediated silencing of THBS1 and neutralizing antibody to THBS1 reduced invasion in mesenchymal-like melanoma cells, while ectopic THBS1 expression in epithelial-like melanoma cells enhanced invasion. Furthermore, the loss of THBS1 inhibited in vivo motility of melanoma cells within the embryonic chicken neural tube. In addition, we found aberrant THBS1 protein expression in metastatic melanoma tumor biopsies. These results implicate a role for THBS1 in EMT, and hence THBS1 may serve as a novel target for strategies aimed at the treatment of melanoma invasion and drug resistance. PMID:25051363

  1. Trp2 peptide vaccine adjuvanted with (R)-DOTAP inhibits tumor growth in an advanced melanoma model

    PubMed Central

    Vasievich, Elizabeth A.; Ramishetti, Srinivas; Zhang, Yuan; Huang, Leaf

    2012-01-01

    Previously we have shown cationic lipid (R)-DOTAP as the immunologically active enantiomer of the DOTAP racemic mixture, initiating complete tumor regression in an exogenous antigen model (murine cervical cancer model). Here, we investigate the use of (R)-DOTAP as an efficacious adjuvant delivering an endogenous antigen in an aggressive murine solid tumor melanoma model. (R)-DOTAP/Trp2 peptide complexes showed decreasing size and charge with increasing peptide concentration, taking a rod-shape at highest concentrations. The particles were stable for at 2 weeks at 4°C. A dose of 75nmol Trp2 (formulated in (R)-DOTAP) was able to show statistically significant tumor growth delay compared to lower doses of 5 and 25nmol which were no different than untreated tumors. (R)-DOTAP/Trp2 (75nmol) treated mice also showed increased T cell IFN-γ secretion after restimulation with Trp2, as well as CTL activity in vivo. This vaccination group also showed the highest population of functionally active tumor-infiltrating lymphocytes, indicated by IFN-γ secretion after restimulation with Trp2. Thus, (R)-DOTAP has shown the ability to break tolerance as an adjuvant. Its activity to enhance immunogenicity of other tumor associated antigens should be studied further. PMID:22142394

  2. Ubiquitin ligase UBE3C promotes melanoma progression by increasing epithelial-mesenchymal transition in melanoma cells

    PubMed Central

    Tang, Li; Yi, Xue-Mei; Chen, Jia; Chen, Fu-Juan; Lou, Wei; Gao, Yun-Lu; Zhou, Jing; Su, Li-Na; Xu, Xin; Lu, Jia-Qing; Ma, Jun; Yu, Ning; Ding, Yang-Feng

    2016-01-01

    Melanoma is the most aggressive type of skin cancer, exhibiting extensive local invasion and early distant metastasis. Aberrant expression of ubiquitin-protein ligase E3C (UBE3C) plays a key role in tumor development and progression. In the present study, we analyzed UBE3C expression in samples of cancerous and normal skin tissue. Levels of UBE3C expression were much higher in primary and metastatic melanoma tissues than in normal skin, cutaneous squamous cell carcinoma or basal cell carcinoma. Melanoma cells overexpressing UBE3C frequently exhibited a mesenchymal phenotype, including reduced expression of the epithelial marker E-cadherin and expression of the mesenchymal marker vimentin. Knockdown of UBE3C expression in melanoma cells significantly suppressed melanoma growth and progression. Furthermore, silencing UBE3C led to increased E-cadherin expression and decreased vimentin and Snail1 expression. Thus UBE3C promotes melanoma progression, possibly by inducing epithelial-mesenchymal transition in melanoma cells. Inhibiting UBE3C activity may suppress melanoma invasion and metastasis and may represent a targeted therapeutic approach. PMID:26894856

  3. Antitumor activities of interferon alpha, beta, and gamma and their combinations on human melanoma cells in vitro: changes of proliferation, melanin synthesis, and immunophenotype.

    PubMed

    Garbe, C; Krasagakis, K; Zouboulis, C C; Schröder, K; Krüger, S; Stadler, R; Orfanos, C E

    1990-12-01

    The antitumor activities of human interferon (IFN) alpha, beta, and gamma alone or in combination were studied on four human melanoma cell lines (StML-11, StML-12, StML-14, and SKMel-28) in various concentrations (1-50,000 IU/ml IFN alpha, 0.1-1000 IU/ml IFN beta, 1-10,000 IU/ml IFN gamma) in vitro. In all experiments IFN beta exhibited the most potent antiproliferative effect of all IFN tested. After 3 d of incubation a 50% growth inhibition was achieved with 20-40 IU/ml for natural IFN beta and with 600-1200 U/ml for recombinant IFN gamma. Substantially higher doses (7,000 to more than 50,000 IU/ml) of recombinant IFN alpha 2a were required to achieve a 50% growth inhibition. A strong synergistic antiproliferative activity resulted from the combination of IFN alpha with IFN gamma and IFN beta with IFN gamma. None of the IFN tested induced terminal differentiation of melanoma cells in vitro. The formation of dendrites was inhibited, and the portion of differentiated cells in vitro was reduced after treatment with IFN in comparison to the untreated controls (untreated controls: 100%; portion of differentiated cells after treatment with IFN alpha: 58%-74%, IFN beta: 48%-96%, IFN gamma: 10%-33%). The melanin synthesis was slightly elevated after treatment with IFN alpha (untreated controls: 100%; after treatment with IFN alpha: 103%-157%, ns.) and decreased significantly after treatment with IFN beta (49%-71%, p less than 0.05) as well as with IFN gamma (80%-88%, ns.). Cell surface markers were modulated varyingly by the IFN: HLA-I antigens were enhanced by all IFN, with IFN beta emerging as the most potent inducer. Only IFN gamma, however, induced a de novo expression of HLA-DR and -DQ antigens and increased the expression of the ICAM-1 molecule and of the melanoma progression marker A.1.43. Possibly, these findings indicate a biologically more aggressive phenotype of melanoma cells. PMID:2124247

  4. Genotype-Selective Combination Therapies for Melanoma Identified by High Throughput Drug Screening

    PubMed Central

    Held, Matthew A.; Langdon, Casey G.; Platt, James T.; Graham-Steed, Tisheeka; Liu, Zongzhi; Chakraborty, Ashok; Bacchiocchi, Antonella; Koo, Andrew; Bosenberg, Marcus W.; Stern, David F.

    2012-01-01

    Resistance and partial responses to targeted monotherapy are major obstacles in cancer treatment. Systematic approaches to identify efficacious drug combinations for cancer are not well established, especially in the context of genotype. To address this, we have tested pairwise combinations of an array of small molecule inhibitors on early passage melanoma cultures using combinatorial drug screening. Results reveal several inhibitor combinations effective for melanomas with activating RAS or BRAF mutations, including mutant BRAF melanomas with intrinsic or acquired resistance to vemurafenib. Inhibition of both EGFR and AKT sensitized treatment-resistant BRAF-mutant melanoma cultures to vemurafenib. Melanomas with RAS mutations were more resistant to combination therapies relative to BRAF mutants, but were sensitive to combinations of statins and cyclin-dependent kinase inhibitors in vitro and in vivo. These results demonstrate the utility of combinatorial drug screening for discovering unique treatment regimens that overcome resistance phenotypes of mutant BRAF and RAS driven melanomas. PMID:23239741

  5. Pulse infusion interleukin-2 with famotidine and cyclophosphamide has activity in previously treated metastatic melanoma.

    PubMed

    Quan, Walter; Knupp, Charles; Quan, Francine; Walker, Paul

    2010-04-01

    There is no established systemic therapy for patients with stage IV melanoma refractory to prior systemic treatment. Interleukin-2 (IL-2) is capable of inducing T-lymphocyte cytotoxicity against melanoma in vitro and in vivo. Famotidine may enhance the activity of T-cells further by allowing for increased IL-2 internalization by the IL-2 receptor on lymphocytes. Cyclophosphamide may decrease the immunosuppressive effects of regulatory T-cells. Daily short intravenous (i.v.) infusions (pulses) of IL-2 were used to treat 14 patients with metastatic melanoma, all of whom had experienced disease progression despite prior systemic therapy. The patients received 21.6 million IU/m(2) of pulse IL-2 i.v. for 15-30 minutes, preceded by 20 mg of famotidine i.v. (13) patients received 350 mg/m(2) of cyclophosphamide i.v. on day 1 (1 patient did not). Eight (8) patients were treated in an oncology inpatient unit while, most recently, 6 patients have received therapy on an outpatient basis. The cycles were repeated every 3 weeks until disease progression occurred. The patients included 10 males with a median age of 56 (range 31-87) with an Eastern Cooperative Oncology Group performance status of -1 (range 0 - -1). Common metastatice sites included lymph nodes (13), lungs (8), liver (4), and subcutaneous (4). Prior systemic therapy included IL-2 (11), interferon (7), and chemotherapy (7). The median number of cycles the patients underwent was 3 with a range of 1-7. The most common toxic reactions were fever, rigors, nausea/emesis, hypomagnesemia, and hypophosphatemia. One complete response and four partial responses were observed (response rate, 36%; 95% confidence interval: 14%-64%). Responses occurred in the lungs, liver, lymph nodes, and subcutaneous sites. The median response duration was 3.4 months, with a median survival of 8.3 months for the entire group. Six (6) patients remain alive with a median survival of 10.3 months. Pulse IL-2 with famotidine and cyclophosphamide

  6. PDK1 and SGK3 contribute to the growth of BRAF mutant melanomas and are potential therapeutic targets

    PubMed Central

    Scortegagna, Marzia; Lau, Eric; Zhang, Tongwu; Feng, Yongmei; Sereduk, Chris; Yin, Hongwei; De, Surya K.; Meeth, Katrina; Platt, James T.; Langdon, Casey G.; Halaban, Ruth; Pellecchia, Maurizio; Davies, Michael A.; Brown, Kevin; Stern, David F.; Bosenberg, Marcus; Ronai, Ze’ev A.

    2015-01-01

    Melanoma development involves members of the AGC kinase family including AKT, PKC and, most recently, PDK1, as elucidated recently in studies of Braf::Pten mutant melanomas. Here we report that PDK1 contributes functionally to skin pigmentation and to the development of melanomas harboring a wild-type PTEN genotype, which occurs in ~70% of human melanomas. The PDK1 substrate SGK3 was determined to be is an important mediator of PDK1 activities in melanoma cells. Genetic or pharmacological inhibition of PDK1 and SGK3 attenuated melanoma growth by inducing G1 phase cell cycle arrest. In a synthetic lethal screen, pan-PI3K inhibition synergized with PDK1 inhibition to suppress melanoma growth, suggesting that focused blockade of PDK1/PI3K signaling might offer a new therapeutic modality for wild-type PTEN tumors. We also noted that responsiveness to PDK1 inhibition associated with decreased expression of pigmentation genes and increased expression of cytokines and inflammatory genes, suggesting a method to stratify melanoma patients for PDK1-based therapies. Overall, our work highlights the potential significance of PDK1 as a therapeutic target to improve melanoma treatment. PMID:25712345

  7. Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

    SciTech Connect

    Krauthammer, Michael; Kong, Yong; Ha, Byung Hak; Evans, Perry; Bacchiocchi, Antonella; McCusker, James P.; Cheng, Elaine; Davis, Matthew J.; Goh, Gerald; Choi, Murim; Ariyan, Stephan; Narayan, Deepak; Dutton-Regester, Ken; Capatana, Ana; Holman, Edna C.; Bosenberg, Marcus; Sznol, Mario; Kluger, Harriet M.; Brash, Douglas E.; Stern, David F.; Materin, Miguel A.; Lo, Roger S.; Mane, Shrikant; Ma, Shuangge; Kidd, Kenneth K.; Hayward, Nicholas K.; Lifton, Richard P.; Schlessinger, Joseph; Boggon, Titus J.; Halaban, Ruth

    2012-10-11

    We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1{sup P29S}) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1{sup P29S} showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.

  8. Secretome from senescent melanoma engages the STAT3 pathway to favor reprogramming of naive melanoma towards a tumor-initiating cell phenotype

    PubMed Central

    Bonet, Caroline; Bonazzi, Vanessa F; Allegra, Marylin; Giuliano, Sandy; Bille, Karine; Bahadoran, Philippe; Giacchero, Damien; Lacour, Jean Philippe; Boyle, Glen M; Hayward, Nicholas F

    2013-01-01

    Here, we showed that the secretome of senescent melanoma cells drives basal melanoma cells towards a mesenchymal phenotype, with characteristic of stems illustrated by increased level of the prototype genes FN1, SNAIL, OCT4 and NANOG. This molecular reprogramming leads to an increase in the low-MITF and slow-growing cell population endowed with melanoma-initiating cell features. The secretome of senescent melanoma cells induces a panel of 52 genes, involved in cell movement and cell/cell interaction, among which AXL and ALDH1A3 have been implicated in melanoma development. We found that the secretome of senescent melanoma cells activates the STAT3 pathway and STAT3 inhibition prevents secretome effects, including the acquisition of tumorigenic properties. Collectively, the findings provide insights into how the secretome of melanoma cells entering senescence upon chemotherapy treatments increases the tumorigenicity of naïve melanoma cells by inducing, through STAT3 activation, a melanoma-initiating cell phenotype that could favor chemotherapy resistance and relapse. PMID:24344100

  9. Exome sequencing of desmoplastic melanoma identifies recurrent NFKBIE promoter mutations and diverse activating mutations in the MAPK pathway.

    PubMed

    Shain, A Hunter; Garrido, Maria; Botton, Thomas; Talevich, Eric; Yeh, Iwei; Sanborn, J Zachary; Chung, Jongsuk; Wang, Nicholas J; Kakavand, Hojabr; Mann, Graham J; Thompson, John F; Wiesner, Thomas; Roy, Ritu; Olshen, Adam B; Gagnon, Alexander; Gray, Joe W; Huh, Nam; Hur, Joe S; Busam, Klaus J; Scolyer, Richard A; Cho, Raymond J; Murali, Rajmohan; Bastian, Boris C

    2015-10-01

    Desmoplastic melanoma is an uncommon variant of melanoma with sarcomatous histology, distinct clinical behavior and unknown pathogenesis. We performed low-coverage genome and high-coverage exome sequencing of 20 desmoplastic melanomas, followed by targeted sequencing of 293 genes in a validation cohort of 42 cases. A high mutation burden (median of 62 mutations/Mb) ranked desmoplastic melanoma among the most highly mutated cancers. Mutation patterns strongly implicate ultraviolet radiation as the dominant mutagen, indicating a superficially located cell of origin. Newly identified alterations included recurrent promoter mutations of NFKBIE, encoding NF-κB inhibitor ɛ (IκBɛ), in 14.5% of samples. Common oncogenic mutations in melanomas, in particular in BRAF (encoding p.Val600Glu) and NRAS (encoding p.Gln61Lys or p.Gln61Arg), were absent. Instead, other genetic alterations known to activate the MAPK and PI3K signaling cascades were identified in 73% of samples, affecting NF1, CBL, ERBB2, MAP2K1, MAP3K1, BRAF, EGFR, PTPN11, MET, RAC1, SOS2, NRAS and PIK3CA, some of which are candidates for targeted therapies. PMID:26343386

  10. Melanoma Treatments: Advances and Mechanisms.

    PubMed

    Marzuka, Alexander; Huang, Laura; Theodosakis, Nicholas; Bosenberg, Marcus

    2015-11-01

    Advances in the understanding of the molecular pathogenesis of melanoma and in cancer immunology have led to the rational design and recent clinical implementation of a variety of novel therapies for metastatic melanoma. BRAF and MEK inhibitors that target the MAPK pathway have high rates of clinical response in BRAF-mutant melanoma. Therapies that modulate the immune response to melanoma, including monoclonal antibodies that interfere with pathways that inhibit T-cell function, have been shown to be effective in melanoma. Lessons learned from these encouraging results are driving the development of new treatments for melanoma and other cancers. This review will focus on the science and clinical findings related to targeted therapies that inhibit BRAF or MEK as well as the immunotherapies that block the CTLA-4 or PD-1 pathways. Other experimental and combinatorial therapeutic approaches will also be discussed. PMID:25899612

  11. Narrow-band UVB radiation promotes dendrite formation by activating Rac1 in B16 melanoma cells.

    PubMed

    Wang, Wu-Qing; Wu, Jin-Feng; Xiao, Xiao-Qing; Xiao, Qin; Wang, Jing; Zuo, Fu-Guo

    2013-09-01

    Melanocytes are found scattered throughout the basal layer of the epidermis. Following hormone or ultraviolet (UV) light stimulation, the melanin pigments contained in melanocytes are transferred through the dendrites to the surrounding keratinocytes to protect against UV light damage or carcinogenesis. This has been considered as a morphological indicator of melanocytes and melanoma cells. Small GTPases of the Rho family have been implicated in the regulation of actin reorganization underlying dendrite formation in melanocytes and melanoma cells. It has been proven that ultraviolet light plays a pivotal role in melanocyte dendrite formation; however, the molecular mechanism underlying this process has not been fully elucidated. The effect of small GTPases, such as Rac1 and RhoA, on the morphology of B16 melanoma cells treated with narrow-band UVB radiation was investigated. The morphological changes were observed under a phase contrast microscope and the F-actin microfilament of the cytoskeleton was observed under a laser scanning confocal microscope. The pull-down assay was performed to detect the activity of the small GTPases Rac1 and RhoA. The morphological changes were evident, with globular cell bodies and increased numbers of tree branch-like dendrites. The cytoskeletal F-actin appeared disassembled following narrow-band UVB irradiation of B16 melanoma cells. Treatment of B16 melanoma cells with narrow-band UVB radiation resulted in the activation of Rac1 in a time-dependent manner. In conclusion, the present study may provide a novel method through which narrow-band UVB radiation may be used to promote dendrite formation by activating the Rac1 signaling pathway, resulting in F-actin rearrangement in B16 melanoma cells. PMID:24649261

  12. Resistance to selective BRAF inhibition can be mediated by modest upstream pathway activation.

    PubMed

    Su, Fei; Bradley, William D; Wang, Qiongqing; Yang, Hong; Xu, Lizhong; Higgins, Brian; Kolinsky, Kenneth; Packman, Kathryn; Kim, Min Jung; Trunzer, Kerstin; Lee, Richard J; Schostack, Kathleen; Carter, Jade; Albert, Thomas; Germer, Soren; Rosinski, Jim; Martin, Mitchell; Simcox, Mary Ellen; Lestini, Brian; Heimbrook, David; Bollag, Gideon

    2012-02-15

    A high percentage of patients with BRAF(V600E) mutant melanomas respond to the selective RAF inhibitor vemurafenib (RG7204, PLX4032) but resistance eventually emerges. To better understand the mechanisms of resistance, we used chronic selection to establish BRAF(V600E) melanoma clones with acquired resistance to vemurafenib. These clones retained the V600E mutation and no second-site mutations were identified in the BRAF coding sequence. Further characterization showed that vemurafenib was not able to inhibit extracellular signal-regulated kinase phosphorylation, suggesting pathway reactivation. Importantly, resistance also correlated with increased levels of RAS-GTP, and sequencing of RAS genes revealed a rare activating mutation in KRAS, resulting in a K117N change in the KRAS protein. Elevated levels of CRAF and phosphorylated AKT were also observed. In addition, combination treatment with vemurafenib and either a MAP/ERK kinase (MEK) inhibitor or an AKT inhibitor synergistically inhibited proliferation of resistant cells. These findings suggest that resistance to BRAF(V600E) inhibition could occur through several mechanisms, including elevated RAS-GTP levels and increased levels of AKT phosphorylation. Together, our data implicate reactivation of the RAS/RAF pathway by upstream signaling activation as a key mechanism of acquired resistance to vemurafenib, in support of clinical studies in which combination therapy with other targeted agents are being strategized to combat resistance. PMID:22205714

  13. Clinical activity and safety of Pembrolizumab in Ipilimumab pre-treated patients with uveal melanoma

    PubMed Central

    Karydis, Ioannis; Chan, Pui Ying; Wheater, Matthew; Arriola, Edurne; Szlosarek, Peter W.; Ottensmeier, Christian H.

    2016-01-01

    ABSTRACT Background: Untreated metastatic uveal melanoma (UM) carries a grave prognosis. Unlike cutaneous melanoma (CM), there are no established treatments known to significantly improve outcomes for a meaningful proportion of patients. Inhibition of the PD1–PDL1 axis has shown promise in the management of CM and we here report a two center experience of UM patients receiving pembrolizumab. Methods: To assess the efficacy and safety of pembrolizumab, we retrospectively analyzed outcome data of 25 consecutive UM patients participating in the MK3475 expanded access program (EAP) who received pembrolizumab at 2 mg/kg 3 weekly. Tumor assessment was evaluated using RECIST 1.1 and immune-related Response Criteria (irRC) by CT scanning. Toxicity was recorded utilizing Common Terminology Criteria for Adverse Events (“CTCAE”) v4.03. Results: Twenty-five patients were identified receiving a median of six cycles of treatment. Two patients achieved a partial response and six patients stable disease. After a median follow-up of 225 d median progression free survival (PFS) was 91 d and overall survival (OS) was not reached. There was a significant trend for improved outcomes in patients with extrahepatic disease progression as opposed to liver only progression at the outset. Five patients experienced grade 3 or 4 adverse events (AEs); there were no treatment related deaths. Conclusions: Pembrolizumab 2mg/kg q3w is a safe option in UM patients. Disease control rates, particularly in the subgroup of patients without progressive liver disease at the outset are promising; these results merit further investigation in clinical trials possibly incorporating liver targeted treatment modalities.

  14. Biologically active monoiodinated alpha-MSH derivatives for receptor binding studies using human melanoma cells

    SciTech Connect

    Eberle, A.N.; Verin, V.J.; Solca, F.; Siegrist, W.; Kueenlin, C.B.; Bagutti, C.; Stutz, S.; Girard, J. , University Hospital, Basel )

    1991-01-01

    Three different monoiodinated radioligands of alpha-MSH (alpha-melanocyte-stimulating hormone) were compared in a binding assay with human D10 melanoma cells: (Tyr(125I)2)-alpha-MSH, (Tyr(125I)2,NIe4)-alpha-MSH, and (Tyr(125I)2,NIe4,D-Phe7)-alpha-MSH. They were prepared either by the classical chloramine T method or by the Enzymobead method. A simple and rapid purification scheme was developed consisting of a primary separation on reversed-phase C18 silica cartridges immediately after the iodination, followed by HPLC purification before each binding experiment. Biological testing of the three radioligands showed that they all retained high melanotropic activity in the B16 melanin assay and the Anolis melanophore assay. However, in human D10 melanoma cells, (Tyr(125I)2,NIe4)-alpha-MSH led to a high degree of non-specific binding to the cells which could not be displaced by excess alpha-MSH and only partially by (NIe4)-alpha-MSH. The (Tyr(125I)2,NIe4,D-Phe7)-alpha-MSH tracer gave similar results but with a much lower proportion of non-specific binding. On the other hand, (Tyr(125I)2)-alpha-MSH proved to be an excellent radioligand whose non-specific binding to the D10 cells was not higher than 20% of the total binding.

  15. Evaluation of Depigmenting Activity by 8-Hydroxydaidzein in Mouse B16 Melanoma Cells and Human Volunteers

    PubMed Central

    Tai, Sorgan Shou-Ku; Lin, Ching-Gong; Wu, Mon-Han; Chang, Te-Sheng

    2009-01-01

    In our previous study, 8-hydroxydaidzein (8-OHDe) was demonstrated to be a potent and unique suicide substrate of mushroom tyrosinase. In this study, the compound was evaluated for in vitro cellular tyrosinase and melanogenesis inhibitory activities in mouse B16 melanoma cells and for in vivo skin-whitening activity in human volunteers. Tyrosinase activity and melanogenesis in the cell culture incubated with 10 μM of 8-OHDe were decreased to 20.1% and 51.8% of control, respectively, while no obvious cytotoxicity was observed in this concentration. In contrast, a standard tyrosinase inhibitor, kojic acid, showed 69.9% and 71.3% of control in cellular tyrosinase and melanogenesis activity, respectively, at a concentration as high as 100 μM. Hence, 8-OHDe exhibited more than an inhibitory effects on melanin production in B16 cells 10-fold stronger than kojic acid. In addition, when a cream containing 4% 8-OHDe was applied to human skin in an in vivo study, significant increases in the dL*-values were observed after three weeks. Moreover, the increase in the dL*-values after 8-week treatment with 4% 8-OHDe (from −0.57 to 1.94) is stronger than those of 2% 8-OHDe treatment (from 0.26 to 0.94) and 2% ascorbic acid-2-glucoside treatment (from 0.07 to 1.54). From the results of the study, it was concluded that 8-OHDe, the potent suicide substrate of mushroom tyrosinase, has depigmenting activities in both mouse melanoma cells and in human volunteers. Thus, the compound has significant potential for use in cosmetics as a skin-whitening ingredient. PMID:20057943

  16. Evaluation of depigmenting activity by 8-hydroxydaidzein in mouse B16 melanoma cells and human volunteers.

    PubMed

    Tai, Sorgan Shou-Ku; Lin, Ching-Gong; Wu, Mon-Han; Chang, Te-Sheng

    2009-10-01

    In our previous study, 8-hydroxydaidzein (8-OHDe) was demonstrated to be a potent and unique suicide substrate of mushroom tyrosinase. In this study, the compound was evaluated for in vitro cellular tyrosinase and melanogenesis inhibitory activities in mouse B16 melanoma cells and for in vivo skin-whitening activity in human volunteers. Tyrosinase activity and melanogenesis in the cell culture incubated with 10 microM of 8-OHDe were decreased to 20.1% and 51.8% of control, respectively, while no obvious cytotoxicity was observed in this concentration. In contrast, a standard tyrosinase inhibitor, kojic acid, showed 69.9% and 71.3% of control in cellular tyrosinase and melanogenesis activity, respectively, at a concentration as high as 100 microM. Hence, 8-OHDe exhibited more than an inhibitory effects on melanin production in B16 cells 10-fold stronger than kojic acid. In addition, when a cream containing 4% 8-OHDe was applied to human skin in an in vivo study, significant increases in the dL*-values were observed after three weeks. Moreover, the increase in the dL*-values after 8-week treatment with 4% 8-OHDe (from -0.57 to 1.94) is stronger than those of 2% 8-OHDe treatment (from 0.26 to 0.94) and 2% ascorbic acid-2-glucoside treatment (from 0.07 to 1.54). From the results of the study, it was concluded that 8-OHDe, the potent suicide substrate of mushroom tyrosinase, has depigmenting activities in both mouse melanoma cells and in human volunteers. Thus, the compound has significant potential for use in cosmetics as a skin-whitening ingredient. PMID:20057943

  17. Monoclonal antibody OKM5 inhibits the in vitro binding of Plasmodium falciparum-infected erythrocytes to monocytes, endothelial, and C32 melanoma cells

    SciTech Connect

    Barnwell, J.W.; Ockenhouse, C.F.; Knowles, D.M. II

    1985-11-01

    Plasmodium falciparum-infected erythrocytes bind in vitro to human endothelial cells, monocytes, and a certain melanoma cell line. Evidence suggests that this interaction is mediated by similar mechanisms which lead to the sequestration of parasitized erythrocytes in vivo through their attachment to endothelial cells of small blood vessels. They show here the monoclonal antibody OKM5, previously shown to react with the membranes of endothelial cells, monocyte,s and platelets, also reacts with the C32 melanoma cell line which also binds P. falciparum-infected erythrocytes. At relatively low concentrations, OKM5 inhibits and reverses the in vitro adherence of infected erythrocytes to target cells. As with monocytes, OKM5 antibody recognizes an /sup 125/I-labeled protein of approximately 88 Kd on the surface of C32 melanoma cells. It seems likely, therefore, that the 88 Kd polypeptide plays a role in cytoadherence, possibly as the receptor or part of a receptor for a ligand on the surface of infected erythrocytes.

  18. Antiangiogenesis, Loss of Cell Adhesion and Apoptosis Are Involved in the Antitumoral Activity of Proteases from V. cundinamarcensis (C. candamarcensis) in Murine Melanoma B16F1

    PubMed Central

    Dittz, Dalton; Figueiredo, Cinthia; Lemos, Fernanda O.; Viana, Celso T. R.; Andrade, Silvia P.; Souza-Fagundes, Elaine M.; Fujiwara, Ricardo T.; Salas, Carlos E.; Lopes, Miriam T. P.

    2015-01-01

    The proteolytic enzymes from V. cundinamarcensis latex, (P1G10), display healing activity in animal models following various types of lesions. P1G10 or the purified isoforms act as mitogens on fibroblast and epithelial cells by stimulating angiogenesis and wound healing in gastric and cutaneous ulcers models. Based on evidence that plant proteinases act as antitumorals, we verified this effect on a murine melanoma model. The antitumoral effect analyzed mice survival and tumor development after subcutaneous administration of P1G10 into C57BL/6J mice bearing B16F1 low metastatic melanoma. Possible factors involved in the antitumoral action were assessed, i.e., cytotoxicity, cell adhesion and apoptosis in vitro, haemoglobin (Hb), vascular endothelial growth factor (VEGF), tumor growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α) content and N-acetyl-glucosaminidase (NAG) activity. We observed that P1G10 inhibited angiogenesis measured by the decline of Hb and VEGF within the tumor, and TGF-β displayed a non-significant increase and TNF-α showed a minor non-significant reduction. On the other hand, there was an increase in NAG activity. In treated B16F1 cells, apoptosis was induced along with decreased cell binding to extracellular matrix components (ECM) and anchorage, without impairing viability. PMID:25826531

  19. l-Tyrosine-loaded nanoparticles increase the antitumoral activity of direct electric current in a metastatic melanoma cell model

    PubMed Central

    de Campos, Vânia Emerich Bucco; Teixeira, Cesar Augusto Antunes; da Veiga, Venicio Feo; Júnior, Eduardo Ricci; Holandino, Carla

    2010-01-01

    Inhibition of tumor growth induced by treatment with direct electric current (DC) has been reported in several models. One of the mechanisms responsible for the antitumoral activity of DC is the generation of oxidative species, known as chloramines. With the aim of increasing chloramine production in the electrolytic medium and optimizing the antitumoral effects of DC, poly(ɛ-caprolactone) (PCL) nanoparticles (NPs) loaded with the amino acid tyrosine were obtained. The physical–chemical characterization showed that the NPs presented size in nanometric range and monomodal distribution. A slightly negative electrokinetic potential was also found in both blank NPs and l-tyrosine-loaded PCL NPs. The yield of the loading process was approximately 50%. Within 3 h of dissolution assay, a burst release of about 80% l-tyrosine was obtained. The in vitro cytotoxicity of DC was significantly increased when associated with l-tyrosine-loaded NPs, using a murine multidrug-resistant melanoma cell line model. This study showed that the use of the combination of nanotechnology and DC has a promising antineoplastic potential and opens a new perspective in cancer therapy. PMID:21187948

  20. ARMS depletion facilitates UV irradiation induced apoptotic cell death in melanoma.

    PubMed

    Liao, Yi-Hua; Hsu, Su-Ming; Huang, Pei-Hsin

    2007-12-15

    Tumor cells often aberrantly reexpress molecules that mediate proper embryonic development for advantageous growth or survival. Here, we report that ankyrin repeat-rich membrane spanning (ARMS), a transmembrane protein abundant in the developing and adult neural tissues, is overexpressed in melanoma, a tumor ontogenetically originating from neural crest. Immunohistochemical study of 79 melanocytic lesions showed significantly increased expression of ARMS in primary malignant melanomas (92.9%) and metastatic melanoma (60.0%) in comparison with benign nevocellular nevi (26.7%). To investigate the role of ARMS in melanoma formation, murine B16F0 melanoma cells with stable knockdown of ARMS were established by RNA interference. Down-regulation of ARMS resulted in significant inhibition of anchorage-independent growth in soft agar and restrictive growth of melanoma in severe combined immunodeficient mice. Importantly, depletion of ARMS facilitated UVB-induced apoptosis in melanoma cells through inactivation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK. Addition of MEK inhibitor PD98059 further sensitized ARMS-depleted melanoma cells to UVB-induced apoptosis, whereas constitutively active MEK rescued ARMS-depleted cells from apoptosis. We further showed that BRAF, a downstream signaling molecule of ARMS in ERK pathway, is not mutated as a constitutively active form in acral lentiginous melanoma; in contrast, BRAF(T1799A) mutation, which leads to constitutive activation of ERK signaling, was detected in 57.1% of superficial spreading melanoma. Our study suggests that overexpression of ARMS per se serves as one mechanism to promote melanoma formation by preventing stress-induced apoptotic death mediated by the MEK/ERK signaling pathway, especially in acral lentiginous melanoma, most of which does not harbor BRAF mutation. PMID:18089783

  1. Different activities of unscheduled DNA synthesis in human melanoma and bone marrow cells

    SciTech Connect

    Lewensohn, R.; Ringborg, U.; Hansson, J.

    1982-01-01

    Unscheduled DNA synthesis (UDS) indicated by melphalan was studied in freshly collected tumor cells from human melanoma metastases. Comparative studies were done on human bone marrow blast cells. Significant levels of UDS comparable with those in myeloblasts were found in only two of eight melanoma cell populations. This difference between melanoma and blast cells was not related to different cellular uptake of melphalan. When UDS was induced by ultraviolet irradiation, significant levels of UDS were found in all melanoma and blast cell populations studied. Also, in a human melanoma cell line, high levels of UDS were found after exposure to ultraviolet irradiation, while treatment with melphalan did not result in detectable levels of UDS. Possible explanations for the divergent results of UDS in melphalan-exposed melanoma cells are discussed.

  2. Activin inhibits telomerase activity in cancer

    SciTech Connect

    Katik, Indzi; Mackenzie-Kludas, Charley; Nicholls, Craig; Jiang, Fang-Xu; Zhou, Shufeng; Li, He; Liu, Jun-Ping

    2009-11-27

    Activin is a pleiotropic cytokine with broad tissue distributions. Recent studies demonstrate that activin-A inhibits cancer cell proliferation with unknown mechanisms. In this report, we demonstrate that recombinant activin-A induces telomerase inhibition in cancer cells. In breast and cervical cancer cells, activin-A resulted in telomerase activity in a concentration-dependent manner. Significant inhibition was observed at 10 ng/ml of activin-A, with a near complete inhibition at 80 ng/ml. Consistently, activin-A induced repression of the telomerase reverse transcriptase (hTERT) gene, with the hTERT gene to be suppressed by 60-80% within 24 h. In addition, activin-A induced a concomitant increase in Smad3 signaling and decrease of the hTERT gene promoter activity in a concentration-dependent fashion. These data suggest that activin-A triggered telomerase inhibition by down-regulating hTERT gene expression is involved in activin-A-induced inhibition of cancer cell proliferation.

  3. [Endocrine factors influencing melanoma progression].

    PubMed

    Dobos, Judit

    2009-03-01

    According to recent findings that beside cancers traditionally considered as hormone-dependent, several other tumor types show different behavior in the two sexes, indicating the possible role of endocrine factors in the course of these diseases. The possibility that endocrine factors may influence the clinical course of human malignant melanoma is suggested by the higher survival rate in premenopausal vs. postmenopausal women or men of any ages. However, investigations on the sex hormone receptor status of human cutaneous melanomas and experiments attempting to support the epidemiological results yielded conflicting results. In our human melanoma cell lines we failed to detect steroid receptors at protein level, while quantitative PCR demonstrated that their mRNA expression level was orders of magnitude lower compared to the positive control cell lines. Sex hormones did not influence the in vitro features of the human melanoma cells considerably. On the other hand, glucocorticoid receptor was present both at mRNA and protein level, although dexamethasone was effective in vitro only at high doses. Our previous experiments showed that intrasplenic injection of human melanoma cells resulted in a significantly higher number of liver colonies in male than in female SCID mice. We now show that this difference evolves during the first day. After injection into the tail vein we did not observe gender-dependent difference in the efficiency of pulmonary colonization. Examining the pattern of metastasis formation after intracardiac injection, we have found differences between the two sexes in the incidence or number of colonies only in the case of the liver but not in other organs. We concluded that the observed phenomenon is specific to the liver; therefore we investigated the effects of 2-methoxyestradiol, an endogenous metabolite of estradiol produced mainly in the liver, with an estrogen receptor-independent antitumor activity. 2ME2 effectively inhibited melanoma cell

  4. BFD-22 a new potential inhibitor of BRAF inhibits the metastasis of B16F10 melanoma cells and simultaneously increased the tumor immunogenicity.

    PubMed

    Ferreira, Adilson Kleber; Pasqualoto, Kerly Fernanda Mesquita; Kruyt, Frank A E; Palace-Berl, Fanny; Azevedo, Ricardo Alexandre; Turra, Kely Medeiros; Rodrigues, Cecilia Pessoa; Ferreira, Ana Carolina Franco; Salomón, Maria Alejandra Clavijo; de Sá Junior, Paulo Luiz; Farias, Camyla Fernandes; Figueiredo, Carlos Rogerio; Tavares, Leoberto Costa; Barbuto, José Alexandre Marzagão; Jorge, Salomão Dória

    2016-03-15

    Benzofuroxan is an interesting ring system, which has shown a wide spectrum of biological responses against tumor cell lines. We investigated, herein, the antitumor effects of benzofuroxan derivatives (BFDs) in vitro and in a melanoma mouse model. Cytotoxic effects of twenty-two BFDs were determined by MTT assay. Effects of BFD-22 in apoptosis and cell proliferation were evaluated using Annexin V-FITC/PI and CFSE staining. In addition, the effects in the cell cycle were assessed. Flow cytometry, western blot, and fluorescence microscopy analysis were employed to investigate the apoptosis-related proteins and the BRAF signaling. Cell motility was also exploited through cell invasion and migration assays. Molecular docking approach was performed in order to verify the BFD-22 binding mode into the ATP catalytic site of BRAF kinase. Moreover, the BFD-22 antitumor effects were evaluated in a melanoma murine model using B16F10. BFD-22 was identified as a potential hit against melanoma cells. BFD-22 induced apoptosis and inhibited cell proliferation of B16F10 cells. BFD-22 has suppressed, indeed, the migratory and invasive behavior of B16F10 cells. Cyclin D1 and CDK4 expression were reduced leading to cell cycle arrest at G0/G1 phase. Of note, phosphorylation of BRAF at Ser338 was strongly down-regulated by BFD-22 in B16F10 cells. The accommodation/orientation into the binding site of BRAF was similar of BAY43-9006 (co-crystallized inhibitor of BRAF, sorafenib). Importantly, BFD-22 presented in vivo antimetastatic effects and showed better therapeutic efficacy than sorafenib and taxol. BFD-22 can be considered as a new lead compound and, then, can be helpful for the designing of novel drug candidates to treat melanoma. PMID:26876618

  5. Tumor immunotherapy in melanoma: strategies for overcoming mechanisms of resistance and escape.

    PubMed

    Zigler, Maya; Villares, Gabriel J; Lev, Dina C; Melnikova, Vladislava O; Bar-Eli, Menashe

    2008-01-01

    The incidence of melanoma has been steadily increasing over the last 3 decades. Currently, there are several approved treatments for metastatic melanoma, including chemotherapy and biologic therapy as both single treatments and in combination, but none is associated with a significant increase in survival. The chemotherapeutic agent dacarbazine is the standard treatment for metastatic melanoma, with a response rate of 15-20%, although most responses are not sustained. One of the main problems with melanoma treatment is chemotherapeutic resistance. The mechanisms of resistance of melanoma cells to chemotherapy have yet to be elucidated. Following treatment with dacarbazine, melanoma cells activate the extracellular signal-regulated kinase pathway, which results in over-expression and secretion of interleukin (IL)-8 and vascular endothelial growth factor. Melanoma cells utilize this mechanism to escape from the cytotoxic effect of the drug. We have previously reported on the development of fully human neutralizing antibodies against IL-8 (anti-IL-8-monoclonal-antibody [ABX-IL8]). In preclinical studies, ABX-IL8 inhibited tumor growth, angiogenesis, and metastasis of human melanoma in vivo. We propose that combination treatment with dacarbazine and IL-8 will potentiate the cytotoxic effect of the drug. Furthermore, formation of metastasis is a multistep process that includes melanoma cell adhesion to endothelial cells. Melanoma cell adhesion molecule (MUC18) mediates these processes in melanoma and is therefore a good target for eliminating metastasis. We have developed a fully human antibody against MUC18 that has shown promising results in preclinical studies. Since resistance is one of the major obstacles in the treatment of melanoma, we propose that utilization of antibodies against IL-8 or MUC18 alone, or as part of a 'cocktail' in combination with dacarbazine, may be a new treatment modality for metastatic melanoma that overcomes resistance of the disease to

  6. A Role for Poly(ADP-ribose) Polymerase in the Transcriptional Regulation of the Melanoma Growth Stimulatory Activity (CXCL1) Gene Expression*

    PubMed Central

    Nirodi, Chaitanya; NagDas, Subir; Gygi, Steven P.; Olson, Gary; Aebersold, Ruedi; Richmond, Ann

    2012-01-01

    The melanoma growth stimulatory activity/growth-regulated protein, CXCL1, is constitutively expressed at high levels during inflammation and progression of melanocytes into malignant melanoma. It has been shown previously that CXCL1 overexpression in melanoma cells is due to increased transcription as well as stability of the CXCL1 message. The transcription of CXCL1 is regulated through several cis-acting elements including Sp1, NF-κB, HMGI(Y), and the immediate upstream region (IUR) element (nucleotides −94 to −78), which lies immediately upstream to the nuclear factor κB (NF-κB) element. Previously, it has been shown that the IUR is necessary for basal and cytokine-induced transcription of the CXCL1 gene. UV cross-linking and Southwestern blot analyses indicate that the IUR oligonucleotide probe selectively binds a 115-kDa protein. In this study, the IUR element has been further characterized. We show here that proximity of the IUR element to the adjacent NF-κB element is critical to its function as a positive regulatory element. Using binding site oligonucleotide affinity chromatography, we have selectively purified the 115-kDa IUR-F. Mass spectrometry/mass spectrometry/matrix-assisted laser desorption ionization/time of flight spectroscopy and amino acid analysis as well as microcapillary reverse phase chromatography electrospray ionization tandem mass spectrometry identified this protein as the 114-kDa poly(ADP-ribose) polymerase (PARP1). Furthermore, 3-aminobenzamide, an inhibitor of PARP-specific ADP-ribosylation, inhibits CXCL1 promoter activity and reduces levels of CXCL1 mRNA. The data point to the possibility that PARP may be a coactivator of CXCL1 transcription. PMID:11112786

  7. Cell proliferation activity in posterior uveal melanoma after Ru-106 brachytherapy: an EORTC ocular oncology group study

    PubMed Central

    Pe'er, J.; Stefani, F.; Seregard, S.; Kivela, T.; Lommatzsch, P.; Prause, J.; Sobottka, B.; Damato, B.; Chowers, I.

    2001-01-01

    AIM—To evaluate the cell proliferation activity in posterior uveal melanomas after Ru-106 brachytherapy.
METHODS—Eyes containing choroidal or ciliary body melanoma from seven ocular oncology centres, which were enucleated after first being treated by Ru-106 brachytherapy and which had enough melanoma tissue to enable histological assessment, were included. The 57 eligible specimens were divided into a group of 44 eyes that were enucleated because of tumour regrowth, and a non-recurrent group of 13 eyes that were enucleated because of complications such as neovascular glaucoma. 46 non-irradiated eyes harbouring uveal melanoma served as a control group. All specimens underwent routine processing. They were cut into 5 µm sections, and were stained with two main cell proliferation markers: PC-10 for PCNA and MIB-1 for Ki-67. The stained sections were assessed, and the cells that were positive in the immunostaining were counted in each section. The results were evaluated by various statistical methods.
RESULTS—The PC-10 score showed a statistically significant difference across the three groups (p = 0.002). The control group showed the highest PC-10 score (median 31.0 PCC/HPF) followed by the tumour regrowth group (median 4.9 PCC/HPF). The lowest PC-10 scores were found in the non-recurrent tumours (median 0.05 PCC/HPF). The MIB-1 score in the control group (median 5.77 PCC/HPF) was similar to the regrowth group (median 5.4 PCC/HPF). In contrast, the MIB-1 score in the non-recurrent tumours was statistically significantly lower (median 0.42 PCC/HPF). The PC-10 and MIB-1 scores were similar in tumours composed of either spindle cells or epithelioid cells in all groups.
CONCLUSIONS—The non-recurrent melanomas demonstrate significantly lower cellular proliferation activity than melanomas that showed regrowth or that were not irradiated at all. In our hands, PCNA gave more meaningful information than Ki-67. Our findings strongly support the need

  8. Honokiol affects melanoma cell growth by targeting the AMPK signaling pathway

    PubMed Central

    Kaushik, Gaurav; Kwatra, Deep; Subramaniam, Dharmalingam; Jensen, Roy A.; Anant, Shrikant; Mammen, Joshua M.V.

    2015-01-01

    Background Malignant melanoma is an aggressive form of skin cancer with limited effective therapeutic options. Melanoma research concentrates on maximizing the effect on cancer cells with minimal toxicity to normal cells. AMP-activated protein kinase (AMPK) is an important regulator of cellular energy homeostasis and has been shown to control tumor progression regulating the cell cycle, protein synthesis and cell growth and/or survival. Honokiol (HNK) is a biphenolic compound derived from Magnolia officianalis, a plant that has been used in traditional Chinese and Japanese medicine for the treatment of various pathological conditions. Recent studies have shown that HNK has antitumor activity with relatively low toxicity. In this study we demonstrated that the growth inhibitory effects of HNK on melanoma and melanoma cancer stem cells (CSCs) was mediated through the activation of AMPK and hence AMPK signaling in melanoma cells. Methods We determined the effects of HNK treatment on various melanoma cell lines. HNK induced cell growth inhibitory effects were determined using hexosaminidase assay. Protein expression studies were done by immunoblotting. Primary spheroid assay was used to assess stemness by growing single suspension cells in ultra-low attachment plates. Results HNK is highly effective in inhibiting melanoma cells by attenuating AKT/mammalian target of rapamycin and AMPK signaling. HNK showed significant inhibition of the spheroid forming capacity of melanoma cells and, hence, stemness. HNK significantly decreased the number and size of melanospheres in a dose dependent manner. Western blot analyses showed enhanced phosphorylation of AMPK in melanoma cells. Furthermore, HNK decreased the cellular ATP pool in a dose-dependent manner with maximum effects observed at 48 h. Conclusion The results suggest that HNK can target melanoma cells and mark them for cell death through AMPK signaling. Further studies are warranted for developing HNK as an effective

  9. Aloe-emodin exerts a potent anticancer and immunomodulatory activity on BRAF-mutated human melanoma cells.

    PubMed

    Tabolacci, Claudio; Cordella, Martina; Turcano, Lorenzo; Rossi, Stefania; Lentini, Alessandro; Mariotti, Sabrina; Nisini, Roberto; Sette, Giovanni; Eramo, Adriana; Piredda, Lucia; De Maria, Ruggero; Facchiano, Francesco; Beninati, Simone

    2015-09-01

    Aim of this study was to extend the knowledge on the antineoplastic effect of aloe-emodin (AE), a natural hydroxyanthraquinone compound, both in metastatic human melanoma cell lines and in primary stem-like cells (melanospheres). Treatment with AE caused reduction of cell proliferation and induction of SK-MEL-28 and A375 cells differentiation, characterized by a marked increase of transamidating activity of transglutaminase whose expression remained unmodified. In vitro antimetastatic property of AE was evaluated by adhesion and Boyden chamber invasion assays. The effect of AE on melanoma cytokines/chemokines production was determined by a multiplex assay: interestingly AE showed an immunomodulatory activity through GM-CSF and IFN-γ production. We report also that AE significantly reduced the proliferation, stemness and invasive potential of melanospheres. Moreover, AE treatment significantly enhanced dabrafenib (a BRAF inhibitor) antiproliferative activity in BRAF mutant cell lines. Our results confirm that AE possesses remarkable antineoplastic properties against melanoma cells, indicating this anthraquinone as a promising agent for differentiation therapy of cancer, or as adjuvant in chemotherapy and targeted therapy. Further, its mechanisms of action support a potential efficacy of AE treatment to counteract resistance of BRAF-mutated melanoma cells to target therapy. PMID:26048310

  10. Inhibition of CDK1 activity by sumoylation.

    PubMed

    Xiao, Yuxuan; Lucas, Benjamin; Molcho, Elana; Schiff, Tania; Vigodner, Margarita

    2016-09-16

    Sumoylation (a covalent modification by Small Ubiquitin-like Modifiers or SUMO proteins) has been implicated in the regulation of various cellular events including cell cycle progression. We have recently identified CDK1, a master regulator of mitosis and meiosis, as a SUMO target both in vivo and in vitro, supporting growing evidence concerning a close cross talk between sumoylation and phosphorylation during cell cycle progression. However, any data regarding the effect of sumoylation upon CDK1 activity have been missing. In this study, we performed a series of in vitro experiments to inhibit sumoylation by three different means (ginkgolic acid, physiological levels of oxidative stress, and using an siRNA approach) and assessed the changes in CDK1 activity using specific antibodies and a kinase assay. We have also tested for an interaction between SUMO and active and/or inactive CDK1 isoforms in addition to having assessed the status of CDK1-interacting sumoylated proteins upon inhibition of sumoylation. Our data suggest that inhibition of sumoylation increases the activity of CDK1 probably through changes in sumoylated status and/or the ability of specific proteins to bind CDK1 and inhibit its activity. PMID:27520372

  11. Hypoxia inhibits abdominal expiratory nerve activity.

    PubMed

    Fregosi, R F; Knuth, S L; Ward, D K; Bartlett, D

    1987-07-01

    Our purpose was to examine the influence of steady-state changes in chemical stimuli, as well as discrete peripheral chemoreceptor stimulation, on abdominal expiratory motor activity. In decerebrate, paralyzed, vagotomized, and ventilated cats that had bilateral pneumothoraces, we recorded efferent activity from a phrenic nerve and from an abdominal nerve (cranial iliohypogastric nerve, L1). All cats showed phasic expiratory abdominal nerve discharge at normocapnia [end-tidal PCO2 38 +/- 2 Torr], but small doses (2-6 mg/kg) of pentobarbital sodium markedly depressed this activity. Hyperoxic hypercapnia consistently enhanced abdominal expiratory activity and shortened the burst duration. Isocapnic hypoxia caused inhibition of abdominal nerve discharge in 11 of 13 cats. Carotid sinus nerve denervation (3 cats) exacerbated the hypoxic depression of abdominal nerve activity and depressed phrenic motor output. Stimulation of peripheral chemoreceptors with NaCN increased abdominal nerve discharge in 7 of 10 cats, although 2 cats exhibited marked inhibition. Four cats with intact neuraxis, but anesthetized with ketamine, yielded qualitatively similar results. We conclude that when cats are subjected to steady-state chemical stimuli in isolation (no interference from proprioceptive inputs), hypercapnia potentiates, but hypoxia attenuates, abdominal expiratory nerve activity. Mechanisms to explain the selective inhibition of expiratory motor activity by hypoxia are proposed, and physiological implications are discussed. PMID:3624126

  12. Recurrent inactivating RASA2 mutations in melanoma

    PubMed Central

    Arafeh, Rand; Qutob, Nouar; Emmanuel, Rafi; Keren-Paz, Alona; Madore, Jason; Elkahloun, Abdel; Wilmott, James S.; Gartner, Jared J.; Di Pizio, Antonella; Winograd-Katz, Sabina; Sindiri, Sivasish; Rotkopf, Ron; Dutton-Regester, Ken; Johansson, Peter; Pritchard, Antonia; Waddell, Nicola; Hill, Victoria K.; Lin, Jimmy C.; Hevroni, Yael; Rosenberg, Steven A.; Khan, Javed; Ben-Dor, Shifra; Niv, Masha Y.; Ulitsky, Igor; Mann, Graham J; Scolyer, Richard A.; Hayward, Nicholas K.; Samuels, Yardena

    2016-01-01

    Analysis of 501 melanoma exomes revealed RASA2, encoding a RasGAP, as a tumor-suppressor gene mutated in 5% of melanomas. Recurrent loss-of-function mutations in RASA2 were found to increase RAS activation, melanoma cell growth and migration. RASA2 expression was lost in ≥30% of human melanomas and was associated with reduced patient survival. These findings reveal RASA2 inactivation as a melanoma driver and highlight the importance of Ras GAPs in cancer. PMID:26502337

  13. Alkaloid constituents from flower buds and leaves of sacred lotus (Nelumbo nucifera, Nymphaeaceae) with melanogenesis inhibitory activity in B16 melanoma cells.

    PubMed

    Nakamura, Seikou; Nakashima, Souichi; Tanabe, Genzo; Oda, Yoshimi; Yokota, Nami; Fujimoto, Katsuyoshi; Matsumoto, Takahiro; Sakuma, Rika; Ohta, Tomoe; Ogawa, Keiko; Nishida, Shino; Miki, Hisako; Matsuda, Hisashi; Muraoka, Osamu; Yoshikawa, Masayuki

    2013-02-01

    Methanolic extracts from the flower buds and leaves of sacred lotus (Nelumbo nucifera, Nymphaeaceae) were found to show inhibitory effects on melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells. From the methanolic extracts, a new alkaloid, N-methylasimilobine N-oxide, was isolated together with eleven benzylisoquinoline alkaloids. The absolute stereostructure of the new alkaloid was determined from chemical and physicochemical evidence. Among the constituents isolated, nuciferine, N-methylasimilobine, (-)-lirinidine, and 2-hydroxy-1-methoxy-6a,7-dehydroaporphine showed potent inhibition of melanogenesis. Comparison of the inhibitory activities of synthetic related alkaloids facilitated characterization of the structure-activity relationships of aporphine- and benzylisoquinoline-type alkaloids. In addition, 3-30 μM nuciferine and N-methylasimilobine inhibited the expression of tyrosinase mRNA, 3-30 μM N-methylasimilobine inhibited the expression of TRP-1 mRNA, and 10-30 μM nuciferine inhibited the expression of TRP-2 mRNA. PMID:23270663

  14. Uveal melanoma: estimating prognosis.

    PubMed

    Kaliki, Swathi; Shields, Carol L; Shields, Jerry A

    2015-02-01

    Uveal melanoma is the most common primary malignant tumor of the eye in adults, predominantly found in Caucasians. Local tumor control of uveal melanoma is excellent, yet this malignancy is associated with relatively high mortality secondary to metastasis. Various clinical, histopathological, cytogenetic features and gene expression features help in estimating the prognosis of uveal melanoma. The clinical features associated with poor prognosis in patients with uveal melanoma include older age at presentation, male gender, larger tumor basal diameter and thickness, ciliary body location, diffuse tumor configuration, association with ocular/oculodermal melanocytosis, extraocular tumor extension, and advanced tumor staging by American Joint Committee on Cancer classification. Histopathological features suggestive of poor prognosis include epithelioid cell type, high mitotic activity, higher values of mean diameter of ten largest nucleoli, higher microvascular density, extravascular matrix patterns, tumor-infiltrating lymphocytes, tumor-infiltrating macrophages, higher expression of insulin-like growth factor-1 receptor, and higher expression of human leukocyte antigen Class I and II. Monosomy 3, 1p loss, 6q loss, and 8q and those classified as Class II by gene expression are predictive of poor prognosis of uveal melanoma. In this review, we discuss the prognostic factors of uveal melanoma. A database search was performed on PubMed, using the terms "uvea," "iris," "ciliary body," "choroid," "melanoma," "uveal melanoma" and "prognosis," "metastasis," "genetic testing," "gene expression profiling." Relevant English language articles were extracted, reviewed, and referenced appropriately. PMID:25827538

  15. Senescence evasion in melanoma progression: uncoupling of DNA-damage signaling from p53 activation and p21 expression.

    PubMed

    Mackenzie Ross, Alastair D; Cook, Martin G; Chong, Heung; Hossain, Mehnaz; Pandha, Hardev S; Bennett, Dorothy C

    2013-03-01

    The best-established function of the melanoma-suppressor p16 is mediation of cell senescence, a permanent arrest following cell proliferation or certain stresses. The importance of p16 in melanoma suggests indolence of the other major senescence pathway through p53. Little or no p53 is expressed in senescent normal human melanocytes, but p16-deficient melanocytes can undergo p53-mediated senescence. As p16 expression occurs in nevi but falls with progression toward melanoma, we here investigated whether p53-dependent senescence occurs at some stage and, if not, what defects were detectable in this pathway, using immunohistochemistry. Phosphorylated checkpoint kinase 2 (CHEK2) can mediate DNA-damage signaling, and under some conditions senescence, by phosphorylating and activating p53. Remarkably, we detected no prevalent p53-mediated senescence in any of six classes of lesions. Two separate defects in p53 signaling appeared common: in nevi, lack of p53 phosphorylation by activated CHEK2, and in melanomas, defective p21 upregulation by p53 even when phosphorylated. PMID:23253087

  16. Senescence evasion in melanoma progression: uncoupling of DNA-damage signaling from p53 activation and p21 expression

    PubMed Central

    MacKenzie Ross, Alastair D; Cook, Martin G; Chong, Heung; Hossain, Mehnaz; Pandha, Hardev S; Bennett, Dorothy C

    2013-01-01

    The best-established function of the melanoma-suppressor p16 is mediation of cell senescence, a permanent arrest following cell proliferation or certain stresses. The importance of p16 in melanoma suggests indolence of the other major senescence pathway through p53. Little or no p53 is expressed in senescent normal human melanocytes, but p16-deficient melanocytes can undergo p53-mediated senescence. As p16 expression occurs in nevi but falls with progression toward melanoma, we here investigated whether p53-dependent senescence occurs at some stage and, if not, what defects were detectable in this pathway, using immunohistochemistry. Phosphorylated checkpoint kinase 2 (CHEK2) can mediate DNA-damage signaling, and under some conditions senescence, by phosphorylating and activating p53. Remarkably, we detected no prevalent p53-mediated senescence in any of six classes of lesions. Two separate defects in p53 signaling appeared common: in nevi, lack of p53 phosphorylation by activated CHEK2, and in melanomas, defective p21 upregulation by p53 even when phosphorylated. PMID:23253087

  17. Melanoma (image)

    MedlinePlus

    ... tumor that involves the skin cells that produce pigment (melanin). The risk of melanoma increases with age, but frequently effects young, otherwise healthy people. Melanoma is an aggressive type of cancer that can spread very rapidly.

  18. CUB-domain–containing protein 1 (CDCP1) activates Src to promote melanoma metastasis

    PubMed Central

    Liu, Hui; Ong, Shao-En; Badu-Nkansah, Kwabena; Schindler, Jeffrey; White, Forest M.; Hynes, Richard O.

    2011-01-01

    We report the application of quantitative mass spectrometry to identify plasma membrane proteins differentially expressed in melanoma cells with high vs. low metastatic abilities. Using stable isotope labeling with amino acids in culture (SILAC) coupled with nanospray tandem mass spectrometry, we identified CUB-domain–containing protein 1 (CDCP1) as one such differentially expressed transmembrane protein. CDCP1 is not only a surface marker for cells with higher metastatic potential, but also functionally involved in enhancing tumor metastasis. Overexpression of CDCP1 also correlates with activation of Src. Pharmacological reagents, PP2 and Dasatinib, which block Src family kinase activation, blocked scattered growth of CDCP1-overexpressing cells in 3D Matrigel culture, suggesting that CDCP1 might function through the activation of Src-family kinases (SFKs). This hypothesis was further supported by mutational studies of CDCP1. Whereas wild-type CDCP1 enhances Src activation, point mutation Y734F abolishes in vitro dispersive growth in 3D culture and in vivo metastasis-enhancing activities of CDCP1. In addition, the Y734F mutation also eliminated enhanced Src activation. Thus, this work provides molecular mechanisms for the metastasis-enhancing functions of CDCP1. PMID:21220330

  19. Statins Reduce Melanoma Development and Metastasis through MICA Overexpression.

    PubMed

    Pich, Christine; Teiti, Iotefa; Rochaix, Philippe; Mariamé, Bernard; Couderc, Bettina; Favre, Gilles; Tilkin-Mariamé, Anne-Françoise

    2013-01-01

    Survival of melanoma patients after metastases detection remains short. Several clinical trials have shown moderate efficiency in improving patient survival, and the search for pharmacological agents to enhance the immune response and reduce melanoma metastases is still necessary. Statins block the mevalonate pathway, which leads to decreases in GTPase isoprenylation and activity, particularly those of the Ras superfamily. They are widely used as hypocholesterolemic agents in cardiovascular diseases and several studies have shown that they also have protective effects against cancers. Furthermore, we have previously demonstrated that treatment of melanoma cells with inhibitors of the mevalonate pathway, such as statins, favor the development of specific adaptive immune responses against these tumors. In the present study, we tested statin impact on the innate immune response against human metastatic melanoma cells. Our data shows that treatment of two human melanoma cell lines with statins induced a weak but significant increase of MHC class I Chain-related protein A (MICA) membrane expression. Peroxisome Proliferator-Activated Receptor gamma is involved in this statin-induced MICA overexpression, which is independent of Ras and Rho GTPase signaling pathways. Interestingly, this MICA overexpression makes melanoma cells more sensitive to in vitro lysis by NK cells. The impact of statin treatment on in vivo development of melanoma tumors and metastases was investigated in nude mice, because murine NK cells, which express NKG2D receptors, are able to recognize and kill human tumor cells expressing MICA. The results demonstrated that both local tumor growth and pulmonary metastases were strongly inhibited in nude mice injected with statin-treated melanoma cells. These results suggest that statins could be effective in melanoma immunotherapy treatments. PMID:23493799

  20. Inhibition of radiation-enhanced expression of integrin and metastatic potential in B16 melanoma cells by a lipoxygenase inhibitor

    SciTech Connect

    Onoda, J.M.; Honn, K.V. |; Kantak, S.S.; Piechocki, M.P.; Awad, W.; Chea, R.; Liu, B.

    1994-12-01

    Low-dose {gamma} radiation stimulates expression of phenotypic characteristics in B16 melanoma cells which regulate metastatic potential. A transient increase in the expression of an integrin receptor ({alpha}{sub IIb}{beta}{sub 3}) was observed after exposure of B16 melanoma cells to 0.25 to 2.0 Gy of {gamma} radiation. This increased receptor expression resulted in enhanced adhesion of tumor cells to fibronectin in vitro and increased experimentally induced metastasis in vivo. In this report, we determined a role for the 12-lipoxygenase metabolite, 12-HETE, in radiation-enhanced metastasis. A significant increase in biosynthesis of 12-HETE in B16 melanoma cells was detected <5 min after exposure to 0.5 Gy {gamma} radiation. We then determined that radiation-enhanced expression of {alpha}{sub IIb}{beta}{sub 3} integrin and adhesion of B16 melanoma cells to fibronectin in vitro and metastasis in vivo were reduced by treatment of the cells with the lipoxygenase inhibitor NDGA prior to irradiation. These findings suggest that low-dose radiation, at levels comparable to those used in fractionated or hyperfractionated radiotherapy, increases the metastatic potential of surviving tumor cells via a rapid and transient alteration in lipoxygenase metabolism of arachidonic acid and surface expression of an integrin receptor. 30 refs., 5 figs., 1 tab.

  1. On the composition and function of the carbohydrate moiety of tissue-type plasminogen activator from human melanoma cells.

    PubMed

    Rijken, D C; Emeis, J J; Gerwig, G J

    1985-12-17

    Two variants (I and II) of tissue-type plasminogen activator (t-PA) from human melanoma cells were separated by Lysine Sepharose chromatography. The carbohydrate compositions of the forms were determined by gas-liquid chromatography. Variant I contained 12.8 g and variant II 7.1 g of carbohydrate per 100 g protein. Both variants contained N-acetylgalactosamine, suggesting O-glycosylation in addition to N-glycosylation. The possible role of N-linked oligosaccharides for the biological activity of t-PA was studied using t-PA secreted by melanoma cells in the presence of tunicamycin, an inhibitor of N-glycosylation. The latter t-PA showed the same plasminogen activating and fibrin binding properties as normally glycosylated t-PA, indicating that N-linked carbohydrate is not involved in the fibrinolytic activity of t-PA. PMID:3937276

  2. Mitochondrial oxidative stress is the achille's heel of melanoma cells resistant to Braf-mutant inhibitor

    PubMed Central

    André, Fanny; Jonneaux, Aurélie; Scalbert, Camille; Garçon, Guillaume; Malet-Martino, Myriam; Balayssac, Stéphane; Rocchi, Stephane; Savina, Ariel; Formstecher, Pierre; Mortier, Laurent; Kluza, Jérome; Marchetti, Philippe

    2013-01-01

    Vemurafenib/PLX4032, a selective inhibitor of mutant BRAFV600E, constitutes a paradigm shift in melanoma therapy. Unfortunately, acquired resistance, which unavoidably occurs, represents one major limitation to clinical responses. Recent studies have highlighted that vemurafenib activated oxidative metabolism in BRAFV600E melanomas expressing PGC1α. However, the oxidative state of melanoma resistant to BRAF inhibitors is unknown. We established representative in vitro and in vivo models of human melanoma resistant to vemurafenib including primary specimens derived from melanoma patients. Firstly, our study reveals that vemurafenib increased mitochondrial respiration and ROS production in BRAFV600E melanoma cell lines regardless the expression of PGC1α. Secondly, melanoma cells that have acquired resistance to vemurafenib displayed intrinsically high rates of mitochondrial respiration associated with elevated mitochondrial oxidative stress irrespective of the presence of vemurafenib. Thirdly, the elevated ROS level rendered vemurafenib-resistant melanoma cells prone to cell death induced by pro-oxidants including the clinical trial drug, elesclomol. Based on these observations, we propose that the mitochondrial oxidative signature of resistant melanoma constitutes a novel opportunity to overcome resistance to BRAF inhibition. PMID:24161908

  3. Methylation-mediated loss of SFRP2 enhances melanoma cell invasion via Wnt signaling.

    PubMed

    Luo, Xiaoji; Wei, Bin; Chen, Aijun; Zhao, Hengguang; Huang, Kun; Chen, Jin

    2016-01-01

    Wnt signaling plays an essential role in the initiation and progression of melanoma tumors. The Secreted Frizzled Related Proteins (SFRPs) are a family of proteins that suppress Wnt signaling. The methylation of SFRPs reduces their activity, and hence augments Wnt signaling. However, whether the methylation of SFRP2, a member of SFRPs, may be involved in the pathogenesis of melanoma is not known. Here we investigated the expression levels of SFRP2 in melanoma specimens. We found that SFRP2 mRNA wassignificantly decreased and methylation of SFRP2 gene was significantly increased in malignant melanoma tumors ascompared to the paired adjacent non-tumor tissue. Moreover, SFRP2 expression was significantly decreased in the malignant melanoma celllines, HTB63, A2058 and A375, but not in the non-transformed melanocyte cell line, Hermes 3A. The demethylation of SFRP2 gene by 5'-aza-deoxycytidine (5-aza-dCyd) in melanoma cell lines restored SFRP2 expression, at both mRNA and protein levels, and suppressed cell invasion. Furthermore, the demethylation of SFRP2 geneappeared to inhibit nuclear retention of a key Wnt signaling factor, β-catenin, in melanoma cell lines. Together, these data suggest that SFRP2may function as a melanoma invasion suppressor byinterfering with Wnt signaling, and the methylation of SFRP2 gene may promote pathogenesis of melanoma. PMID:27186276

  4. Methylation-mediated loss of SFRP2 enhances melanoma cell invasion via Wnt signaling

    PubMed Central

    Luo, Xiaoji; Wei, Bin; Chen, Aijun; Zhao, Hengguang; Huang, Kun; Chen, Jin

    2016-01-01

    Wnt signaling plays an essential role in the initiation and progression of melanoma tumors. The Secreted Frizzled Related Proteins (SFRPs) are a family of proteins that suppress Wnt signaling. The methylation of SFRPs reduces their activity, and hence augments Wnt signaling. However, whether the methylation of SFRP2, a member of SFRPs, may be involved in the pathogenesis of melanoma is not known. Here we investigated the expression levels of SFRP2 in melanoma specimens. We found that SFRP2 mRNA wassignificantly decreased and methylation of SFRP2 gene was significantly increased in malignant melanoma tumors ascompared to the paired adjacent non-tumor tissue. Moreover, SFRP2 expression was significantly decreased in the malignant melanoma celllines, HTB63, A2058 and A375, but not in the non-transformed melanocyte cell line, Hermes 3A. The demethylation of SFRP2 gene by 5’-aza-deoxycytidine (5-aza-dCyd) in melanoma cell lines restored SFRP2 expression, at both mRNA and protein levels, and suppressed cell invasion. Furthermore, the demethylation of SFRP2 geneappeared to inhibit nuclear retention of a key Wnt signaling factor, β-catenin, in melanoma cell lines. Together, these data suggest that SFRP2may function as a melanoma invasion suppressor byinterfering with Wnt signaling, and the methylation of SFRP2 gene may promote pathogenesis of melanoma. PMID:27186276

  5. Targeting melanoma growth and metastasis with systemic delivery of liposome-incorporated protease-activated receptor-1 small interfering RNA.

    PubMed

    Villares, Gabriel J; Zigler, Maya; Wang, Hua; Melnikova, Vladislava O; Wu, Hong; Friedman, Ran; Leslie, Michael C; Vivas-Mejia, Pablo E; Lopez-Berestein, Gabriel; Sood, Anil K; Bar-Eli, Menashe

    2008-11-01

    The thrombin receptor [protease-activated receptor-1 (PAR-1)] is overexpressed in highly metastatic melanoma cell lines and in patients with metastatic lesions. Activation of PAR-1 leads to cell signaling and up-regulation of genes involved in adhesion, invasion, and angiogenesis. Herein, we stably silence PAR-1 through the use of lentiviral short hairpin RNA and found significant decreases in both tumor growth (P < 0.01) and metastasis (P < 0.001) of highly metastatic melanoma cell lines in vivo. The use of viruses for therapy is not ideal as it can induce toxic immune responses and possible gene alterations following viral integration. Therefore, we also used systemic delivery of PAR-1 small interfering RNA (siRNA) incorporated into neutral liposomes [1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)] to decrease melanoma growth and metastasis in vivo. Significant decreases in tumor growth, weight, and metastatic lung colonies (P < 0.001 for all) were found in mice treated with PAR-1 siRNA-DOPC. The in vivo effects of PAR-1 on invasion and angiogenesis were analyzed via immunohistochemistry. Concomitant decreases in vascular endothelial growth factor, interleukin-8, and matrix metalloproteinase-2 expression levels, as well as decreased blood vessel density (CD31), were found in tumor samples from PAR-1 siRNA-treated mice, suggesting that PAR-1 is a regulator of melanoma cell growth and metastasis by affecting angiogenic and invasive factors. We propose that siRNA incorporated into DOPC nanoparticles could be delivered systemically and used as a new modality for melanoma treatment. PMID:18974154

  6. Chemoprevention of skin melanoma: facts and myths.

    PubMed

    Uzarska, Małgorzata; Czajkowski, Rafał; Schwartz, Robert A; Bajek, Anna; Zegarska, Barbara; Drewa, Tomasz

    2013-12-01

    Melanoma is the most dangerous type of skin cancer. Despite the rise of public awareness, the incidence rate among the white population has been rising constantly for several decades. Systematic improvement in knowledge about the biology of pigment cells and molecular mechanisms of their neoplastic transformation has enhanced the possibility of melanoma chemoprevention. Hence, chemopreventive agents that prevent, inhibit, or reverse melanoma development are being investigated intensively. Among synthetic compounds, especially well studied are lipid-lowering drugs and cyclooxygenase inhibitors. Substances found in everyday diet, such as genistein, apigenin, quercetin, resveratrol, and curcumin may also have potential chemopreventive qualities. However, s