Science.gov

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

    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 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

  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

    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

  17. 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

  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. 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

  4. 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

  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

    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 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

  7. 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

  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

    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

  16. 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

  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. 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

  20. 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

  1. 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

  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. 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.

  7. 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

  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. 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

  12. 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

  13. 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 ...

  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 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

  14. 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

  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 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

  18. 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

  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. 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

  5. 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

  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 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

  16. 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

  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. 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

  1. 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.

  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. 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

  4. 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

  5. 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

  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