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Sample records for activation inhibits melanoma

  1. Inhibition of mutated, activated BRAF in metastatic melanoma.

    PubMed

    Flaherty, Keith T; Puzanov, Igor; Kim, Kevin B; Ribas, Antoni; McArthur, Grant A; Sosman, Jeffrey A; O'Dwyer, Peter J; Lee, Richard J; Grippo, Joseph F; Nolop, Keith; Chapman, Paul B

    2010-08-26

    The identification of somatic mutations in the gene encoding the serine-threonine protein kinase B-RAF (BRAF) in the majority of melanomas offers an opportunity to test oncogene-targeted therapy for this disease. We conducted a multicenter, phase 1, dose-escalation trial of PLX4032 (also known as RG7204), an orally available inhibitor of mutated BRAF, followed by an extension phase involving the maximum dose that could be administered without adverse effects (the recommended phase 2 dose). Patients received PLX4032 twice daily until they had disease progression. Pharmacokinetic analysis and tumor-response assessments were conducted in all patients. In selected patients, tumor biopsy was performed before and during treatment to validate BRAF inhibition. A total of 55 patients (49 of whom had melanoma) were enrolled in the dose-escalation phase, and 32 additional patients with metastatic melanoma who had BRAF with the V600E mutation were enrolled in the extension phase. The recommended phase 2 dose was 960 mg twice daily, with increases in the dose limited by grade 2 or 3 rash, fatigue, and arthralgia. In the dose-escalation cohort, among the 16 patients with melanoma whose tumors carried the V600E BRAF mutation and who were receiving 240 mg or more of PLX4032 twice daily, 10 had a partial response and 1 had a complete response. Among the 32 patients in the extension cohort, 24 had a partial response and 2 had a complete response. The estimated median progression-free survival among all patients was more than 7 months. Treatment of metastatic melanoma with PLX4032 in patients with tumors that carry the V600E BRAF mutation resulted in complete or partial tumor regression in the majority of patients. (Funded by Plexxikon and Roche Pharmaceuticals.)

  2. Inhibition of Mutated, Activated BRAF in Metastatic Melanoma

    PubMed Central

    Flaherty, Keith T.; Puzanov, Igor; Kim, Kevin B.; Ribas, Antoni; McArthur, Grant A.; Sosman, Jeffrey A.; O'Dwyer, Peter J.; Lee, Richard J.; Grippo, Joseph F.; Nolop, Keith; Chapman, Paul B.

    2013-01-01

    Background The identification of somatic mutations in the gene encoding the serine–threonine protein kinase B-RAF (BRAF) in the majority of melanomas offers an opportunity to test oncogene-targeted therapy for this disease. Methods We conducted a multicenter, phase 1, dose-escalation trial of PLX4032 (also known as RG7204), an orally available inhibitor of mutated BRAF, followed by an extension phase involving the maximum dose that could be administered without adverse effects (the recommended phase 2 dose). Patients received PLX4032 twice daily until they had disease progression. Pharmacokinetic analysis and tumor-response assessments were conducted in all patients. In selected patients, tumor biopsy was performed before and during treatment to validate BRAF inhibition. Results A total of 55 patients (49 of whom had melanoma) were enrolled in the dose-escalation phase, and 32 additional patients with metastatic melanoma who had BRAF with the V600E mutation were enrolled in the extension phase. The recommended phase 2 dose was 960 mg twice daily, with increases in the dose limited by grade 2 or 3 rash, fatigue, and arthralgia. In the dose-escalation cohort, among the 16 patients with melanoma whose tumors carried the V600E BRAF mutation and who were receiving 240 mg or more of PLX4032 twice daily, 10 had a partial response and 1 had a complete response. Among the 32 patients in the extension cohort, 24 had a partial response and 2 had a complete response. The estimated median progression-free survival among all patients was more than 7 months. Conclusions Treatment of metastatic melanoma with PLX4032 in patients with tumors that carry the V600E BRAF mutation resulted in complete or partial tumor regression in the majority of patients. (Funded by Plexxikon and Roche Pharmaceuticals.) PMID:20818844

  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. Slit3 inhibits activator protein 1-mediated migration of malignant melanoma cells.

    PubMed

    Denk, Alexandra E; Braig, Simone; Schubert, Thomas; Bosserhoff, Anja K

    2011-11-01

    The repellent factor family of Slit molecules has been described to have repulsive function in the developing nervous system on growing axons expressing the Robo receptors. Alterations of the Slit/Robo system have been observed in various pathological conditions and in cancer. However, until today no detailed studies on Slit function on melanoma migration are available. Therefore, we analysed the mRNA expression in melanoma cells and found induction of Robo3 expression compared to normal melanocytes. Functional assays performed with melanoma cells revealed that treatment with Slit3 led to strong inhibition of migration. Interestingly, we observed down-regulation of AP-1 activity and target gene expression after Slit3 treatment contributing to the negative regulation of migration. Taken together, our data showed that Slit3 reduces the migratory activity of melanoma cells, potentially by repulsion of the cells in analogy to the neuronal system. Further studies will be necessary to prove Slit activity in vivo, but due to its function, Slit3 activity may be helpful in the treatment of melanoma.

  5. Melanoma cells inhibit natural killer cell function by modulating the expression of activating receptors and cytolytic activity.

    PubMed

    Pietra, Gabriella; Manzini, Claudia; Rivara, Silvia; Vitale, Massimo; Cantoni, Claudia; Petretto, Andrea; Balsamo, Mirna; Conte, Romana; Benelli, Roberto; Minghelli, Simona; Solari, Nicola; Gualco, Marina; Queirolo, Paola; Moretta, Lorenzo; Mingari, Maria Cristina

    2012-03-15

    Natural killer (NK) cells play a key role in tumor immune surveillance. However, adoptive immunotherapy protocols using NK cells have shown limited clinical efficacy to date, possibly due to tumor escape mechanisms that inhibit NK cell function. In this study, we analyzed the effect of coculturing melanoma cells and NK cells on their phenotype and function. We found that melanoma cells inhibited the expression of major NK receptors that trigger their immune function, including NKp30, NKp44, and NKG2D, with consequent impairment of NK cell-mediated cytolytic activity against various melanoma cell lines. This inhibitory effect was primarily mediated by indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2). Together, our findings suggest that immunosuppressive barriers erected by tumors greatly hamper the antitumor activity of human NK cells, thereby favoring tumor outgrowth and progression.

  6. [6]-Shogaol inhibits melanogenesis in B16 mouse melanoma cells through activation of the ERK pathway

    PubMed Central

    Yao, Cheng; Oh, Jang-hee; Oh, Inn Gyung; Park, Chi-hyun; Chung, Jin Ho

    2013-01-01

    Aim: To investigate the effect of [6]-shogaol, an active ingredient in ginger, on melanogenesis and the underlying mechanisms. Methods: B16F10 mouse melanoma cells were tested. Cell viability was determined with the MTT assay. Melanin content and tyrosinase activity were analyzed with a spectrophotometer. The protein expression of tyrosinase and microphthalmia associated transcription factor (MITF), as well as phosphorylated or total ERK1/2 and Akt were measured using Western blot. Results: Treatment of the cells with [6]-shogaol (1, 5, 10 μmol/L) reduced the melanin content in a concentration-dependent manner. [6]-Shogaol (5 and 10 μmol/L) significantly decreased the intracellular tyrosinase activity, and markedly suppressed the expression levels of tyrosinase and MITF proteins in the cells. Furthermore, [6]-shogaol (10 μmol/L) activated ERK, which was known to negatively regulate melanin synthesis in these cells. Pretreatment with the specific ERK pathway inhibitor PD98059 (20 μmol/L) greatly attenuated the inhibition of melanin synthesis by [6]-shogaol (10 μmol/L). Conclusion: The results demonstrate that [6]-shogaol inhibits melanogenesis in B16F10 mouse melanoma cells via activating the ERK pathway. PMID:23123645

  7. Hmgb1 inhibits Klotho expression and malignant phenotype in melanoma cells by activating NF-κB.

    PubMed

    Xie, Biao; Cao, Ke; Li, Jinjin; Chen, Jia; Tang, Jintian; Chen, Xiang; Xia, Kun; Zhou, Xiao; Cheng, Yan; Zhou, Jianda; Xie, Huiqing

    2016-12-06

    The molecular and cellular mechanisms behind the involvement of inflammation in melanoma have not been fully elucidated. In this study, knockdown of Hmgb1 expression increased apoptosis, reduced invasion and p-NF-κB expression, but increased Klotho protein level in melanoma tumor cells. The effect of Hmgb1 knockdown was overcome by LPS. Introduction of exogenous Hmgb1 significantly decreased apoptosis, increased invasion, elevated p-NF-κB, but lowered Klotho protein level in melanoma cells. The effect of exogenous Hmgb1 was agonized by NF-κB inhibitor CAPE. Hmgb1 knockdown activated, but exogenous Hmgb1 inactivated, p-IGF1R/p-PI3K p-85/p-Akt/p-mTOR signaling. Knockdown of Klotho gene expression significantly decreased apoptosis, increased invasion in melanoma cells, and inhibited xenograft A375 tumor growth. A significantly high percentage of cells stained positive for p-NF-κB, but negative for Klotho, in melanoma tissues compared to normal and benign skin tissues. The positive p-NF-κB and negative Klotho protein expression correlated with poor prognosis in melanoma patients. Multivariate analysis revealed an independent association between p-NF-κB / Klotho protein level and overall survival. In conclusion, Hmgb1 can inhibit Klotho gene expression and malignant phenotype in melanoma cells through activation of NF-κB signaling.

  8. Selecting patients for KIT inhibition in melanoma.

    PubMed

    Carvajal, Richard D; Hamid, Omid; Antonescu, Cristina R

    2014-01-01

    For many years, melanoma has been regarded as a single disease in terms of therapeutic considerations. The more recent identification of multiple molecular mechanisms underlying the development, progression, and prognosis of melanoma has led to a new paradigm for the management of this disease, has created new therapeutic opportunities, and has led to improved clinical outcomes. Such advances, however, are dependent upon methods that can reproducibly identify key molecular alterations within an individual tumor, define clinically relevant genetic subgroups of disease, and permit improved patient selection for targeted therapies.Melanomas harboring genetic alterations of KIT have been demonstrated to constitute one such molecular subgroup of disease. In this chapter, we will discuss the biology of KIT in melanoma, review the rationale for and clinical data regarding KIT inhibition in melanomas harboring activating alterations of KIT, propose guidelines for the selection of patients for KIT inhibitor therapy, and, finally, present laboratory methods for KIT assessment in melanoma.

  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.

  10. Potent anti-tumour activity of a novel conditionally replicating adenovirus for melanoma via inhibition of migration and invasion

    PubMed Central

    Jiang, G; Yang, C-S; Xu, D; Sun, C; Zheng, J-N; Lei, T-C; Liu, Y-Q

    2014-01-01

    Background: Conditionally replicating adenoviruses (CRAds) represent a novel class of oncological therapeutic agents. One strategy to ensure tumour targeting is to place the essential viral genes under the control of tumour-specific promoters. Ki67 has been selected as a cancer gene therapy target, as it is expressed in most malignant cells but is barely detectable in most normal cells. This study aimed to investigate the effects of a Ki67 promoter-controlled CRAd (Ki67-ZD55-IL-24) on the proliferation and apoptosis of melanoma cells. Methods: Melanoma cells were independently treated with Ki67-ZD55-IL-24, ZD55-IL-24, Ki67-ZD55, and ZD55-EGFP. The cytotoxic potential of each treatment was assessed using cell viability measurements. Cell migration and invasion were assayed using cell migration and invasion assays. Apoptosis was assayed using the annexin V-FITC assay, western blotting, reverse transcriptase PCR (RT–PCR), haematoxylin and eosin (H&E) staining, and the TUNEL assay. Results: Our results showed that Ki67-ZD55-IL-24 had significantly enhanced anti-tumour activity as it more effectively induced apoptosis in melanoma cells than the other agents. Ki67-ZD55-IL-24 also caused the most significant inhibition of cell migration and invasion of melanoma cells. Furthermore, apoptosis was induced more effectively in melanoma xenografts in nude mice. Conclusions: This strategy holds promising potential for the further development of an effective approach to treat malignant melanoma. PMID:24714752

  11. Targeting Sphingosine Kinase-1 To Inhibit Melanoma

    PubMed Central

    Madhunapantula, SubbaRao V.; Hengst, Jeremy; Gowda, Raghavendra; Fox, Todd E.; Yun, Jong K; Robertson, Gavin P.

    2012-01-01

    SUMMARY Resistance to therapies develops rapidly for melanoma leading to more aggressive disease. Therefore, agents are needed that specifically inhibit proteins or pathways controlling the development of this disease, which can be combined, dependent on genes deregulated in a particular patient’s tumors. This study shows that elevated sphingosine-1-phosphate (S-1-P) levels resulting from increased activity of sphingosine kinase-1 (SPHK1) occur in advanced melanomas. Targeting SPHK1 using siRNA decreased anchorage dependent and independent growth as well as sensitized melanoma cells to apoptosis inducing agents. Pharmacological SPHK1 inhibitors SKI-I but not SKI-II decreased S-1-P content, elevated ceramide levels, caused a G2-M block and induced apoptotic cell death in melanomas. Targeting SPHK1 using siRNA or the pharmacological agent called SKI-I, decreased the levels of pAKT. Furthermore, SKI-I inhibited the expression of CYCLIN D1 protein and increased the activity of caspase-3/7, which in turn led to the degradation of PARP. In animals, SKI-I but not SKI-II retarded melanoma growth by 25-40%. Thus, targeting SPHK1 using siRNAs or SKI-I has therapeutic potential for melanoma treatment either alone or in combination with other targeted agents. PMID:22236408

  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. 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

  14. 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 (p22(phox) and p47(phox)) of the NADPH oxidase complex. Treatment of Hs294t and SK-Mel28 cells with honokiol resulted in accumulation of the cytosolic p47(phox) protein and decreased levels of the membrane-bound p22(phox) 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.

  15. The BRAF inhibitor vemurafenib activates mitochondrial metabolism and inhibits hyperpolarized pyruvate-lactate exchange in BRAF mutant human melanoma cells

    PubMed Central

    Delgado-Goni, Teresa; Falck Miniotis, Maria; Wantuch, Slawomir; Parkes, Harold G.; Marais, Richard; Workman, Paul; Leach, Martin O.; Beloueche-Babari, Mounia

    2016-01-01

    Understanding the impact of BRAF signaling inhibition in human melanoma on key disease mechanisms is important for developing biomarkers of therapeutic response and combination strategies to improve long term disease control. This work investigates the downstream metabolic consequences of BRAF inhibition with vemurafenib, the molecular and biochemical processes that underpin them, their significance for antineoplastic activity and potential as non-invasive imaging response biomarkers.1H NMR spectroscopy showed that vemurafenib decreases the glycolytic activity of BRAF mutant (WM266.4 and SKMEL28) but not BRAFWT (CHL-1 and D04) human melanoma cells. In WM266.4 cells, this was associated with increased acetate, glycine and myo-inositol levels and decreased fatty acyl signals, while the bioenergetic status was maintained. 13C NMR metabolic flux analysis of treated WM266.4 cells revealed inhibition of de novo lactate synthesis and glucose utilization, associated with increased oxidative and anaplerotic pyruvate carboxylase mitochondrial metabolism and decreased lipid synthesis. This metabolic shift was associated with depletion of HKII, acyl-CoA dehydrogenase 9, 3-phosphoglycerate dehydrogenase and monocarboxylate transporter (MCT) 1 and 4 in BRAF mutant but not BRAFWT cells and, interestingly, decreased BRAF mutant cell dependency on glucose and glutamine for growth. Further, the reduction in MCT1 expression observed led to inhibition of hyperpolarized 13C-pyruvate-lactate exchange, a parameter that is translatable to in vivo imaging studies, in live WM266.4 cells. In conclusion, our data provide new insights into the molecular and metabolic consequences of BRAF inhibition in BRAF-driven human melanoma cells that may have potential for combinatorial therapeutic targeting as well as non-invasive imaging of response. PMID:27765851

  16. Modulation of cartilage differentiation by melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP).

    PubMed

    Schubert, Thomas; Schlegel, Jacqueline; Schmid, Rainer; Opolka, Alfred; Grassel, Susanne; Humphries, Martin; Bosserhoff, Anja-Katrin

    2010-03-31

    Melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP) is a small soluble protein secreted from malignant melanoma cells and from chondrocytes. Recently, we revealed that MIA/CD-RAP can modulate bone morphogenetic protein (BMP)2-induced osteogenic differentiation into a chondrogenic direction. In the current study we aimed to find the molecular details of this MIA/CD-RAP function. Direct influence of MIA on BMP2 by protein-protein-interaction or modulating SMAD signaling was ruled out experimentally. Instead, we revealed inhibition of ERK signaling by MIA/CD-RAP. This inhibition is regulated via binding of MIA/CD-RAP to integrin alpha5 and abolishing its activity. Active ERK signaling is known to block chondrogenic differentiation and we revealed induction of aggrecan expression in chondrocytes by treatment with MIA/CD-RAP or PD098059, an ERK inhibitor. In in vivo models we could support the role of MIA/CD-RAP in influencing osteogenic differentiation negatively. Further, MIA/CD-RAP-deficient mice revealed an enhanced calcified cartilage layer of the articular cartilage of the knee joint and disordered arrangement of chondrocytes. Taken together, our data indicate that MIA/CD-RAP stabilizes cartilage differentiation and inhibits differentiation into bone potentially by regulating signaling processes during differentiation.

  17. The plasma membrane Ca(2+) pump PMCA4b inhibits the migratory and metastatic activity of BRAF mutant melanoma cells.

    PubMed

    Hegedũs, Luca; Garay, Tamás; Molnár, Eszter; Varga, Karolina; Bilecz, Ágnes; Török, Szilvia; Padányi, Rita; Pászty, Katalin; Wolf, Matthias; Grusch, Michael; Kállay, Enikõ; Döme, Balázs; Berger, Walter; Hegedũs, Balázs; Enyedi, Agnes

    2017-06-15

    Oncogenic mutations of BRAF lead to constitutive ERK activity that supports melanoma cell growth and survival. While Ca(2+) signaling is a well-known regulator of tumor progression, the crosstalk between Ca(2+) signaling and the Ras-BRAF-MEK-ERK pathway is much less explored. Here we show that in BRAF mutant melanoma cells the abundance of the plasma membrane Ca(2+) ATPase isoform 4b (PMCA4b, ATP2B4) is low at baseline but markedly elevated by treatment with the mutant BRAF specific inhibitor vemurafenib. In line with these findings gene expression microarray data also shows decreased PMCA4b expression in cutaneous melanoma when compared to benign nevi. The MEK inhibitor selumetinib-similarly to that of the BRAF-specific inhibitor-also increases PMCA4b levels in both BRAF and NRAS mutant melanoma cells suggesting that the MAPK pathway is involved in the regulation of PMCA4b expression. The increased abundance of PMCA4b in the plasma membrane enhances [Ca(2+) ]i clearance from cells after Ca(2+) entry. Moreover we show that both vemurafenib treatment and PMCA4b overexpression induce marked inhibition of migration of BRAF mutant melanoma cells. Importantly, reduced migration of PMCA4b expressing BRAF mutant cells is associated with a marked decrease in their metastatic potential in vivo. Taken together, our data reveal an important crosstalk between Ca(2+) signaling and the MAPK pathway through the regulation of PMCA4b expression and suggest that PMCA4b is a previously unrecognized metastasis suppressor. © 2016 UICC.

  18. 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.

  19. α-Solanine inhibits human melanoma cell migration and invasion by reducing matrix metalloproteinase-2/9 activities.

    PubMed

    Lu, Ming-Kun; Shih, Yuan-Wei; Chang Chien, Tzu-Tsung; Fang, Li-Heng; Huang, Hsiang-Ching; Chen, Pin-Shern

    2010-01-01

    α-Solanine, a naturally occurring steroidal glycoalkaloid in potato sprouts, was found to possess anti-carcinogenic properties, such as inhibiting proliferation and inducing apoptosis of tumor cells. However, the effect of α-solanine on cancer metastasis remains unclear. In the present study, we examined the effect of α-solanine on metastasis in vitro. Data demonstrated that α-solanine inhibited proliferation of human melanoma cell line A2058 in a dose-dependent manner. When treated with non-toxic doses of α-solanine, cell migration and invasion were markedly suppressed. Furthermore, α-solanine reduced the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9, which are involved in the migration and invasion of cancer cells. Our biochemical assays indicated that α-solanine potently suppressed the phosphorylation of c-Jun N-terminal kinase (JNK), phosphatidylinositide-3 kinase (PI3K) and Akt, while it did not affect phosphorylation of extracellular signal regulating kinase (ERK). In addition, α-solanine significantly decreased the nuclear level of nuclear factor kappa B (NF-κB), suggesting that α-solanine inhibited NF-κB activity. Taken together, the results suggested that α-solanine inhibited migration and invasion of A2058 cells by reducing MMP-2/9 activities. It also inhibited JNK and PI3K/Akt signaling pathways as well as NF-κB activity. These findings reveal new therapeutic potential for α-solanine in anti-metastatic therapy.

  20. Plumbagin Suppresses α-MSH-Induced Melanogenesis in B16F10 Mouse Melanoma Cells by Inhibiting Tyrosinase Activity

    PubMed Central

    Oh, Taek-In; Yun, Jeong-Mi; Park, Eun-Ji; Kim, Young-Seon; Lee, Yoon-Mi; Lim, Ji-Hong

    2017-01-01

    Recent studies have shown that plumbagin has anti-inflammatory, anti-allergic, antibacterial, and anti-cancer activities; however, it has not yet been shown whether plumbagin suppresses alpha-melanocyte stimulating hormone (α-MSH)-induced melanin synthesis to prevent hyperpigmentation. In this study, we demonstrated that plumbagin significantly suppresses α-MSH-stimulated melanin synthesis in B16F10 mouse melanoma cells. To understand the inhibitory mechanism of plumbagin on melanin synthesis, we performed cellular or cell-free tyrosinase activity assays and analyzed melanogenesis-related gene expression. We demonstrated that plumbagin directly suppresses tyrosinase activity independent of the transcriptional machinery associated with melanogenesis, which includes micropthalmia-associated transcription factor (MITF), tyrosinase (TYR), and tyrosinase-related protein 1 (TYRP1). We also investigated whether plumbagin was toxic to normal human keratinocytes (HaCaT) and lens epithelial cells (B3) that may be injured by using skin-care cosmetics. Surprisingly, lower plumbagin concentrations (0.5–1 μM) effectively inhibited melanin synthesis and tyrosinase activity but do not cause toxicity in keratinocytes, lens epithelial cells, and B16F10 mouse melanoma cells, suggesting that plumbagin is safe for dermal application. Taken together, these results suggest that the inhibitory effect of plumbagin to pigmentation may make it an acceptable and safe component for use in skin-care cosmetic formulations used for skin whitening. PMID:28165370

  1. The activation of the G protein-coupled estrogen receptor (GPER) inhibits the proliferation of mouse melanoma K1735-M2 cells.

    PubMed

    Ribeiro, Mariana P C; Santos, Armanda E; Custódio, José B A

    2017-09-22

    The activation of the G protein-coupled estrogen receptor (GPER) by its specific agonist G-1 inhibits prostate cancer and 17β-estradiol-stimulated breast cancer cell proliferation. Tamoxifen (TAM), which also activates the GPER, decreases melanoma cell proliferation, but its action mechanism remains controversial. Here we investigated the expression and the effects of GPER activation by G-1, TAM and its key metabolite endoxifen (EDX) on melanoma cells. Mouse melanoma K1735-M2 cells expressed GPER and G-1 reduced cell biomass, and the number of viable cells, without increasing cell death. Rather, G-1 decreased cell division by blocking cell cycle progression in G2. Likewise, TAM and EDX exhibited an antiproliferative activity in melanoma cells due to decreased cell division. Both G-1 and the antiestrogens showed a trend to decrease the levels of phosphorylated ERK 1/2 after 1 h treatment, although only EDX, the most potent antiproliferative antiestrogen, induced significant effects. Importantly, the targeting of GPER with siRNA abolished the cytostatic activity of both G-1 and antiestrogens, suggesting that the antitumor actions of antiestrogens in melanoma cells involve GPER activation. Our results unveil a new target for melanoma therapy and identify GPER as a key mediator of antiestrogen antiproliferative effects, which may contribute to select the patients that benefit from an antiestrogen-containing regimen. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Oncogenic BRAF fusions in mucosal melanomas activate the MAPK pathway and are sensitive to MEK/PI3K inhibition or MEK/CDK4/6 inhibition.

    PubMed

    Kim, H S; Jung, M; Kang, H N; Kim, H; Park, C-W; Kim, S-M; Shin, S J; Kim, S H; Kim, S G; Kim, E K; Yun, M R; Zheng, Z; Chung, K Y; Greenbowe, J; Ali, S M; Kim, T-M; Cho, B C

    2017-01-16

    Despite remarkable progress in cutaneous melanoma genomic profiling, the mutational landscape of primary mucosal melanomas (PMM) remains unclear. Forty-six PMMs underwent targeted exome sequencing of 111 cancer-associated genes. Seventy-six somatic nonsynonymous mutations in 42 genes were observed, and recurrent mutations were noted on eight genes, including TP53 (13%), NRAS (13%), SNX31 (9%), NF1 (9%), KIT (7%) and APC (7%). Mitogen-activated protein kinase (MAPK; 37%), cell cycle (20%) and phosphatidylinositol 3-kinase (PI3K)-mTOR (15%) pathways were frequently mutated. We biologically characterized a novel ZNF767-BRAF fusion found in a vemurafenib-refractory respiratory tract PMM, from which cell line harboring ZNF767-BRAF fusion were established for further molecular analyses. In an independent data set, NFIC-BRAF fusion was identified in an oral PMM case and TMEM178B-BRAF fusion and DGKI-BRAF fusion were identified in two malignant melanomas with a low mutational burden (number of mutation per megabase, 0.8 and 4, respectively). Subsequent analyses revealed that the ZNF767-BRAF fusion protein promotes RAF dimerization and activation of the MAPK pathway. We next tested the in vitro and in vivo efficacy of vemurafenib, trametinib, BKM120 or LEE011 alone and in combination. Trametinib effectively inhibited tumor cell growth in vitro, but the combination of trametinib and BKM120 or LEE011 yielded more than additive anti-tumor effects both in vitro and in vivo in a melanoma cells harboring the BRAF fusion. In conclusion, BRAF fusions define a new molecular subset of PMM that can be targeted therapeutically by the combination of a MEK inhibitor with PI3K or cyclin-dependent kinase 4/6 inhibitors.Oncogene advance online publication,16 January 2017; doi:10.1038/onc.2016.486.

  3. Inhibition of activated receptor tyrosine kinases by Sunitinib induces growth arrest and sensitizes melanoma cells to Bortezomib by blocking Akt pathway.

    PubMed

    Yeramian, Andree; Sorolla, Anabel; Velasco, Ana; Santacana, Maria; Dolcet, Xavier; Valls, Joan; Abal, Leandre; Moreno, Sara; Egido, Ramón; Casanova, Josep M; Puig, Susana; Vilella, Ramón; Llombart-Cussac, Antonio; Matias-Guiu, Xavier; Martí, Rosa M

    2012-02-15

    Despite the use of multiple therapeutic strategies, metastatic melanoma remains a challenge for oncologists. Thus, new approaches using combinational treatment may be used to try to improve the prognosis of this disease. In this report, we have analyzed the expression of receptor tyrosine kinases (RTKs) in melanoma specimens and in four metastatic melanoma cell lines. Both melanoma specimens and cell lines expressed RTKs, suggesting that they may represent eventual targets for multitargeted tyrosine kinase inhibitor, Suntinib. Sunitinib reduced the proliferation of two melanoma cell lines (M16 and M17) and increased apoptosis in one of them (M16). Moreover, the two metastatic melanoma cell lines harbored an activated receptor (PDGFRα and VEGFR, respectively), and Sunitinib suppressed the phosphorylation of the RTKs and their downstream targets Akt and ribosomal protein S6, in these two cell lines. Similar results were obtained when either PDGFRα or VEGFR2 expression was silenced by lentiviral-mediated short-hairpin RNA delivery in M16 and M17, respectively. To evaluate the interaction between Sunitinib and Bortezomib, median dose effect analysis using MTT assay was performed, and combination index was calculated. Bortezomib synergistically enhanced the Sunitinib-induced growth arrest in Sunitinib-sensitive cells (combination index < 1). Moreover, LY294002, a PI3K inhibitor, sensitized melanoma cells to Bortezomib treatment, suggesting that downregulation of phospho-Akt by Sunitinib mediates the synergy obtained by Bortezomib + Sunitinib cotreatment. Altogether, our results suggest that melanoma cells harboring an activated RTK may be clinically responsive to pharmacologic RTK inhibition by Sunitinib, and a strategy combining Sunitinib and Bortezomib, may provide therapeutic benefit. Copyright © 2011 UICC.

  4. Inhibition of Oncogenic BRAF Activity by Indole-3-Carbinol Disrupts Microphthalmia-Associated Transcription Factor Expression and Arrests Melanoma Cell Proliferation

    PubMed Central

    Kundu, Aishwarya; Quirit, Jeanne G.; Khouri, Michelle G.; Firestone, Gary L.

    2016-01-01

    Indole-3-carbinol (I3C), an anti-cancer phytochemical derived from cruciferous vegetables, strongly inhibited proliferation and down-regulated protein levels of the melanocyte master regulator micropthalmia-associated transcription factor (MITF-M) in oncogenic BRAF-V600E expressing melanoma cells in culture as well as in vivo in tumor xenografted athymic nude mice. In contrast, wild type BRAF-expressing melanoma cells remained relatively insensitive to I3C anti-proliferative signaling. In BRAF-V600E-expressing melanoma cells, I3C treatment inhibited phosphorylation of MEK and ERK/MAPK, the down stream effectors of BRAF. The I3C anti-proliferative arrest was concomitant with the down-regulation of MITF-M transcripts and promoter activity, loss of endogenous BRN-2 binding to the MITF-M promoter, and was strongly attenuated by expression of exogenous MITF-M. Importantly, in vitro kinase assays using immunoprecipitated BRAF-V600E and wild type BRAF demonstrated that I3C selectively inhibited the enzymatic activity of the oncogenic BRAF-V600E but not of the wild type protein. In silico modeling predicted an I3C interaction site in the BRAF-V600E protomer distinct from where the clinically used BRAF-V600E inhibitor Vemurafenib binds to BRAF-V600E. Consistent with this prediction, combinations of I3C and Vemurafenib more potently inhibited melanoma cell proliferation and reduced MITF-M levels in BRAF-V600E expressing melanoma cells compared to the effects of each compound alone. Thus, our results demonstrate that oncogenic BRAF-V600E is a new cellular target of I3C that implicate this indolecarbinol compound as a potential candidate for novel single or combination therapies for melanoma. PMID:26878440

  5. Melanoma inhibitory activity in Brazilian patients with cutaneous melanoma*

    PubMed Central

    Odashiro, Macanori; Hans Filho, Gunter; Pereira, Patricia Rusa; Castro, Ana Rita Coimbra Motta; Stief, Alcione Cavalheiro; Pontes, Elenir Rose Jardim Cury; Odashiro, Alexandre Nakao

    2015-01-01

    BACKGROUND: Melanoma inhibitory activity is a protein secreted by melanoma cells and has been used as a tumor marker. Increased Melanoma inhibitory activity serum levels are related to metastatic disease or tumor recurrence. Currently there are no studies on Melanoma inhibitory activity and cutaneous melanoma involving Brazilian patients. OBJECTIVE: To evaluate the performance and feasibility of measuring Melanoma inhibitory activity levels in Brazilian patients with cutaneous melanoma. METHODS: Blood was obtained from ten patients with proved metastatic cutaneous melanoma (Group 1), 15 patients resected for cutaneous melanoma without metastasis (Group 2) and 5 healthy donors (Group 3). Melanoma inhibitory activity was measured using a commercially available ELISA kit. RESULTS: There was a statistically significant difference of Melanoma inhibitory activity levels between patients with and without metastasis (p=0.002), and between patients with metastasis and healthy donors (p=0.002). There was no difference between patients without metastasis and healthy donors (p=0.443). CONCLUSION: Melanoma inhibitory activity is a tumor marker for cutaneous melanoma and the Melanoma inhibitory activity-ELISA test can be easily performed. Patients with metastasis have increased Melanoma inhibitory activity serum levels when compared to patients without metastasis and healthy donors. PMID:26131861

  6. A novel 7-bromoindirubin with potent anticancer activity suppresses survival of human melanoma cells associated with inhibition of STAT3 and Akt signaling.

    PubMed

    Liu, Lucy; Kritsanida, Marina; Magiatis, Prokopios; Gaboriaud, Nicolas; Wang, Yan; Wu, Jun; Buettner, Ralf; Yang, Fan; Nam, Sangkil; Skaltsounis, Leandros; Jove, Richard

    2012-11-01

    STAT3 and Akt signaling have been validated as potential molecular targets for treatment of cancers including melanoma. These small molecule inhibitors of STAT3 or Akt signaling are promising for developing anti-melanoma therapeutic agents. MLS-2438, a novel 7-bromoindirubin, a derivative of the natural product indirubin, was synthesized with a bromo-group at the 7-position on one indole ring and a hydrophilic group at the 3'-position on the other indole ring. We tested the anticancer activity of MLS-2438 and investigated its mechanism of action in human melanoma cell lines. Here, we show that MLS-2438 inhibits viability and induces apoptosis of human melanoma cells associated with inhibition of STAT3 and Akt signaling. Several pro-apoptotic Bcl-2 family proteins are involved in the MLS-2438 mediated apoptosis. MLS-2438 inhibits Src kinase activity in vitro and phosphorylation of JAK2, Src, STAT3 and Akt in cultured cancer cells. In contrast to the decreased phosphorylation levels of JAK2, Src, STAT3 and Akt, phosphorylation levels of the MAPK (Erk1/2) signaling protein were not reduced in cells treated with MLS-2438. These results demonstrate that MLS-2438, a novel natural product derivative, is a Src inhibitor and potentially regulates kinase activity of JAK2 and Akt in cancer cells. Importantly, MLS-2438 suppressed tumor growth with low toxicity in a mouse xenograft model of human melanoma. Our findings support further development of MLS-2438 as a potential small-molecule therapeutic agent that targets both STAT3 and Akt signaling in human melanoma cells.

  7. A novel 7-bromoindirubin with potent anticancer activity suppresses survival of human melanoma cells associated with inhibition of STAT3 and Akt signaling

    PubMed Central

    Liu, Lucy; Kritsanida, Marina; Magiatis, Prokopios; Gaboriaud, Nicolas; Wang, Yan; Wu, Jun; Buettner, Ralf; Yang, Fan; Nam, Sangkil; Skaltsounis, Leandros; Jove, Richard

    2012-01-01

    STAT3 and Akt signaling have been validated as potential molecular targets for treatment of cancers including melanoma. These small molecule inhibitors of STAT3 or Akt signaling are promising for developing anti-melanoma therapeutic agents. MLS-2438, a novel 7-bromoindirubin, a derivative of the natural product indirubin, was synthesized with a bromo-group at the 7-position on one indole ring and a hydrophilic group at the 3′-position on the other indole ring. We tested the anticancer activity of MLS-2438 and investigated its mechanism of action in human melanoma cell lines. Here, we show that MLS-2438 inhibits viability and induces apoptosis of human melanoma cells associated with inhibition of STAT3 and Akt signaling. Several pro-apoptotic Bcl-2 family proteins are involved in the MLS-2438 mediated apoptosis. MLS-2438 inhibits Src kinase activity in vitro and phosphorylation of JAK2, Src, STAT3 and Akt in cultured cancer cells. In contrast to the decreased phosphorylation levels of JAK2, Src, STAT3 and Akt, phosphorylation levels of the MAPK (Erk1/2) signaling protein were not reduced in cells treated with MLS-2438. These results demonstrate that MLS-2438, a novel natural product derivative, is a Src inhibitor and potentially regulates kinase activity of JAK2 and Akt in cancer cells. Importantly, MLS-2438 suppressed tumor growth with low toxicity in a mouse xenograft model of human melanoma. Our findings support further development of MLS-2438 as a potential small-molecule therapeutic agent that targets both STAT3 and Akt signaling in human melanoma cells. PMID:22895078

  8. Plitidepsin has a dual effect inhibiting cell cycle and inducing apoptosis via Rac1/c-Jun NH2-terminal kinase activation in human melanoma cells.

    PubMed

    Muñoz-Alonso, María J; González-Santiago, Laura; Zarich, Natasha; Martínez, Teresa; Alvarez, Enrique; Rojas, José María; Muñoz, Alberto

    2008-03-01

    Melanoma is the most aggressive skin cancer and a serious health problem worldwide because of its increasing incidence and the lack of satisfactory chemotherapy for late stages of the disease. The marine depsipeptide Aplidin (plitidepsin) is an antitumoral agent under phase II clinical development against several neoplasias, including melanoma. We report that plitidepsin has a dual effect on the human SK-MEL-28 and UACC-257 melanoma cell lines; at low concentrations (inhibits the cell cycle by inducing G(1) and G(2)/M arrest, whereas at higher concentrations it induces apoptosis as assessed by poly-(ADP-ribose) polymerase cleavage and the appearance of a hypodiploid peak in flow cytometry analyses. Plitidepsin activates Rac1 GTPase and c-Jun NH(2)-terminal kinase (JNK). In addition, it induces AKT and p38 mitogen-activated protein kinase (MAPK) phosphorylation. By using inhibitors, we found that JNK and p38 MAPK activation depends on Rac1 but not on phosphatidylinositol 3-kinase (PI3K), whereas AKT activation is independent of Rac1 but requires PI3K activity. Plitidepsin cytotoxicity diminishes by Rac1 inhibition or by the blockage of JNK and p38 MAPK using 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), but not by PI3K inhibition using wortmannin or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). It is remarkable that plitidepsin and dacarbazine, the alkylating agent most active for treating metastatic melanoma, show a synergistic antiproliferative effect that was paralleled at the level of JNK activation. These results indicate that Rac1/JNK activation is critical for cell cycle arrest and apoptosis induction by plitidepsin in melanoma cells. They also support the combined use of plitidepsin and dacarbazine in in vivo studies.

  9. 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

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

    PubMed

    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

    2015-09-08

    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.

  11. MITF suppression by CH5552074 inhibits cell growth in melanoma cells.

    PubMed

    Aida, Satoshi; Sonobe, Yukiko; Yuhki, Munehiro; Sakata, Kiyoaki; Fujii, Toshihiko; Sakamoto, Hiroshi; Mizuno, Takakazu

    2017-06-01

    Although treatment of melanoma with BRAF inhibitors and immune checkpoint inhibitors achieves a high response rate, a subset of melanoma patients with intrinsic and acquired resistance are insensitive to these therapeutics, so to improve melanoma therapy other target molecules need to be found. Here, we screened our chemical library to identify an anti-melanoma agent and examined its action mechanisms to show cell growth inhibition activity. We screened a chemical library against multiple skin cancer cell lines and conducted ingenuity pathway analysis (IPA) to investigate the mechanisms of CH5552074 activity. Suppression of microphthalmia-associated transcription factor (MITF) expression levels was determined in melanoma cells treated with CH5552074. Cell growth inhibition activity of CH5552074 was evaluated in MITF-dependent melanoma cell lines. We identified an anti-melanoma compound, CH5552074, which showed remarkable cell growth inhibition activity in melanoma cell lines. The IPA results suggested that CH5552074-sensitive cell lines had activated MITF. In further in vitro studies in the melanoma cell lines, a knockdown of MITF with siRNA resulted in cell growth inhibition, which showed that CH5552074 inhibited cell growth by reducing the expression level of MITF protein. These results suggest that CH5552074 can inhibit cell growth in melanoma cells by reducing the protein level of MITF. MITF inhibition by CH5552074 would be an attractive option for melanoma treatment.

  12. Cambodian Phellinus linteus inhibits experimental metastasis of melanoma cells in mice via regulation of urokinase type plasminogen activator.

    PubMed

    Lee, Hyo-Jung; Lee, Hyo-Jeong; Lim, Eu-Soo; Ahn, Kyoo-Seok; Shim, Beom-Sang; Kim, Hyung-Min; Gong, Soo-Ja; Kim, Dae-Keun; Kim, Sung-Hoon

    2005-01-01

    Phellinus linteus (PL) is a fungus mainly found in tropical America, Africa and Asian countries including Korea, Japan and China. PL has been traditionally used for the treatment of arthritis, liver damage and cancer. However, little was known on the biological activity and characterization of Phellinus species in Cambodia. Thus, in the present study, the anti-metastatic mechanism of aqueous extract of Cambodian Phellinus linteus (CPL) was evaluated. Cambodian mushroom was identified as a Phellinus species with 99% homology of Phellinus linteus by DNA sequence analysis and comparison by the National Center for Biotechnology Information. CPL did not exhibit any significant cytotoxicity against B16BL6 cells, invasive melanoma cells at 1 mg/ml. However, CPL inhibited platelet aggregation induced by B16BL6 cells and also disrupted the adhesion to gelatin and invasion of B16BL6 cells in a concentration dependent manner. Similarly, CPL dose-dependently inhibited the pulmonary metastatic colonies in C57BL/6 mice intravenously injected by B16BL6 cells up to 55.5% at a dose of 50 mg/kg compared with untreated control. CPL also down-regulated the expression of urokinase type plasminogen activator (uPA), one of key proteins associated with invasion and metastasis of tumor cells in a concentration dependent fashion, while CPL didn't significantly affect the expression of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) by reverse transcriptase-polymerase chain reaction (RT-PCR). Taken together, these findings indicate that Cambodian Phellinus linteus may inhibit metastasis at least partly via regulation of uPA associated with tumor cell induced platelet aggregation (TCIPA) and also suggest a further study for isolation of active ingredients and the involvement of adhesion molecule signaling pathway.

  13. 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

  14. The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition.

    PubMed

    Jiang, Xiaofeng; Zhou, Jun; Giobbie-Hurder, Anita; Wargo, Jennifer; Hodi, F Stephen

    2013-02-01

    Selective BRAF inhibition (BRAFi) provides a paradigm shift for melanoma treatment. The duration of benefit is typically limited before resistance develops. Interest remains in combining targeted and immune therapies to overcome resistance and improve durability of clinical benefit. One mechanism of evading immune destruction is programmed death-1-ligand 1 (PD-L1) expression by tumors that results in potent antitumor immune suppression. BRAFi-resistant melanoma cells were examined for changes in PD-L1 expression by immunoblot and flow cytometry. Signaling pathways involved in altering PD-L1 expression were examined. Strategies to maximize the effect of the BRAFi therapy were studied including MEKi, MEKi combinations, and additional pathways including phosphoinositide-3 kinase (PI3K). Melanoma cells resistant to BRAFi exhibit increased MAPK signaling and promotion of PD-L1 expression. PD-L1 expression is transcriptionally modulated by c-Jun and augmented by STAT3. MEK inhibition (MEKi) regains downregulation of MAPK signaling and suppresses the production of PD-L1. MEKi in melanoma cells shows dual therapeutic effects with simultaneous suppression of PD-L1 expression and induction of apoptosis. By combining MEKi with BRAFi, an additive effect on the inhibition of PD-L1 expression results. We report a novel mechanism that suppresses preexisting immune responses in patients with melanoma receiving BRAFi therapy. BRAFi resistance leads to increased expression of PD-L1 in melanoma cells, mediated by c-Jun and STAT3. MEKi may be feasible to counteract BRAFi resistance of MAPK reactivation and also for the additive effect of PD-L1 suppression. Potential therapeutic benefits of combining targeted inhibitors and immune modulation to improve patient outcomes should be investigated.

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

    PubMed

    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

    2015-05-01

    (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.

  16. The Hydrogen Sulfide Releasing Molecule Acetyl Deacylasadisulfide Inhibits Metastatic Melanoma.

    PubMed

    De Cicco, Paola; Panza, Elisabetta; Armogida, Chiara; Ercolano, Giuseppe; Taglialatela-Scafati, Orazio; Shokoohinia, Yalda; Camerlingo, Rosa; Pirozzi, Giuseppe; Calderone, Vincenzo; Cirino, Giuseppe; Ianaro, Angela

    2017-01-01

    Melanoma is the most common form of skin cancer. Given its high mortality, the interest in the search of preventive measures, such as dietary factors, is growing significantly. In this study we tested, in vitro and in vivo, the potential anti-cancer effect of the acetyl deacylasadisulfide (ADA), a vinyl disulfide compound, isolated and purified from asafoetida a foul-smelling oleo gum-resin of dietary and medicinal relevance. ADA markedly suppressed proliferation of human melanoma cell lines by inducing apoptosis. Moreover, treatment of melanoma cells with ADA reduced nuclear translocation and activation of NF-κB, decreased the expression of the anti-apoptotic proteins c-FLIP, XIAP, and Bcl-2 and inhibited the phosphorylation and activation of both AKT and ERK proteins, two of the most frequently deregulated pathways in melanoma. Finally, the results obtained in vitro were substantiated by the findings that ADA significantly and dose-dependently reduced lung metastatic foci formation in C57BL/6 mice. In conclusion, our findings suggest that ADA significantly inhibits melanoma progression in vivo and could represent an important lead compound for the development of new anti-metastatic agents.

  17. The Hydrogen Sulfide Releasing Molecule Acetyl Deacylasadisulfide Inhibits Metastatic Melanoma

    PubMed Central

    De Cicco, Paola; Panza, Elisabetta; Armogida, Chiara; Ercolano, Giuseppe; Taglialatela-Scafati, Orazio; Shokoohinia, Yalda; Camerlingo, Rosa; Pirozzi, Giuseppe; Calderone, Vincenzo; Cirino, Giuseppe; Ianaro, Angela

    2017-01-01

    Melanoma is the most common form of skin cancer. Given its high mortality, the interest in the search of preventive measures, such as dietary factors, is growing significantly. In this study we tested, in vitro and in vivo, the potential anti-cancer effect of the acetyl deacylasadisulfide (ADA), a vinyl disulfide compound, isolated and purified from asafoetida a foul-smelling oleo gum-resin of dietary and medicinal relevance. ADA markedly suppressed proliferation of human melanoma cell lines by inducing apoptosis. Moreover, treatment of melanoma cells with ADA reduced nuclear translocation and activation of NF-κB, decreased the expression of the anti-apoptotic proteins c-FLIP, XIAP, and Bcl-2 and inhibited the phosphorylation and activation of both AKT and ERK proteins, two of the most frequently deregulated pathways in melanoma. Finally, the results obtained in vitro were substantiated by the findings that ADA significantly and dose-dependently reduced lung metastatic foci formation in C57BL/6 mice. In conclusion, our findings suggest that ADA significantly inhibits melanoma progression in vivo and could represent an important lead compound for the development of new anti-metastatic agents. PMID:28289382

  18. 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.

  19. Differential Inhibition of Ex-Vivo Tumor Kinase Activity by Vemurafenib in BRAF(V600E) and BRAF Wild-Type Metastatic Malignant Melanoma

    PubMed Central

    Tahiri, Andliena; Røe, Kathrine; Ree, Anne H.; de Wijn, Rik; Risberg, Karianne; Busch, Christian; Lønning, Per E.; Kristensen, Vessela; Geisler, Jürgen

    2013-01-01

    Background Treatment of metastatic malignant melanoma patients harboring BRAF(V600E) has improved drastically after the discovery of the BRAF inhibitor, vemurafenib. However, drug resistance is a recurring problem, and prognoses are still very bad for patients harboring BRAF wild-type. Better markers for targeted therapy are therefore urgently needed. Methodology In this study, we assessed the individual kinase activity profiles in 26 tumor samples obtained from patients with metastatic malignant melanoma using peptide arrays with 144 kinase substrates. In addition, we studied the overall ex-vivo inhibitory effects of vemurafenib and sunitinib on kinase activity status. Results Overall kinase activity was significantly higher in lysates from melanoma tumors compared to normal skin tissue. Furthermore, ex-vivo incubation with both vemurafenib and sunitinib caused significant decrease in phosphorylation of kinase substrates, i.e kinase activity. While basal phosphorylation profiles were similar in BRAF wild-type and BRAF(V600E) tumors, analysis with ex-vivo vemurafenib treatment identified a subset of 40 kinase substrates showing stronger inhibition in BRAF(V600E) tumor lysates, distinguishing the BRAF wild-type and BRAF(V600E) tumors. Interestingly, a few BRAF wild-type tumors showed inhibition profiles similar to BRAF(V600E) tumors. The kinase inhibitory effect of vemurafenib was subsequently analyzed in cell lines harboring different BRAF mutational status with various vemurafenib sensitivity in-vitro. Conclusions Our findings suggest that multiplex kinase substrate array analysis give valuable information about overall tumor kinase activity. Furthermore, intra-assay exposure to kinase inhibiting drugs may provide a useful tool to study mechanisms of resistance, as well as to identify predictive markers. PMID:24023633

  20. 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.

  1. Honokiol Inhibits Melanoma Stem Cells by Targeting Notch Signaling

    PubMed Central

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

    2016-01-01

    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. Targeting macrophage anti-tumor activity to suppress melanoma progression

    PubMed Central

    Yang, Luhong; Liu, Chengfang; Zhang, Qi; Zhang, Linjing

    2017-01-01

    By phagocytosing cancer cells and their cellular debris, macrophages play a critical role in nonspecific defense (innate immunity) and, as antigen presenters, they help initiate specific defense mechanisms (adaptive immunity). Malignant melanoma is a lethal disease due to its aggressive capacity for metastasis and resistance to therapy. For decades, considerable effort has gone into development of an effective immunotherapy for treatment of metastatic melanoma. In this review, we focus on the anti-tumor activities of macrophages in melanoma and their potential as therapeutic targets in melanoma. Although macrophages can be re-educated through intercellular signaling to promote tumor survival owing to their plasticity, we expect that targeting the anti-tumor activity of macrophages remains a promising strategy for melanoma inhibition. The combination of tumoricidal macrophage activation and other treatments such as surgery, chemotherapy, and radiotherapy, may provide an effective and comprehensive anti-melanoma strategy. PMID:28060744

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

    PubMed

    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.

  4. 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

  5. Effect of inhibition of aloe-emodin on N-acetyltransferase activity and gene expression in human malignant melanoma cells (A375.S2).

    PubMed

    Lin, Shuw-Yuan; Yang, Jen-Hung; Hsia, Te-Chun; Lee, Jau-Hong; Chiu, Tsan-Hung; Wei, Yau-Huei; Chung, Jing-Gung

    2005-12-01

    Arylamine carcinogens and drugs are N-acetylated by cytosolic N-acetyltransferase (NAT), which uses acetyl-coenzyme A as a cofactor. NAT plays an initial role in the metabolism of these arylamine compounds. 2-Aminofluorene is one of the arylamine carcinogens which have been demonstrated to undergo N-acetylation in laboratory animals and humans. Our previous study showed that human cancer cell lines (colon cancer, colo 205; liver cancer, Hep G2; bladder cancer, T24; leukemia, HL-60; prostate cancer, LNCaP; osteogenic sarcoma, U-2 OS; malignant melanoma, A375.S2) displayed NAT activity, which was affected by aloe-emodin in human leukemia cells. The purpose of this study was to determine whether aloe-emodin could affect the enzyme activity and gene expression of NAT at the mRNA and protein levels in malignant human melanoma A375.S2 cells. The results showed that aloe-emodin inhibited NAT1 activity (decreased N-acetylation of 2-aminofluorene) in intact cells in a dose-dependent manner. The effect of aloe-emodin on NAT1 at the protein level was determined by Western blotting and the mRNA levels were examined by polymerase chain reaction (PCR) and cDNA microarray. These results clearly indicate that aloe-emodin inhibits the mRNA expression and enzyme activity of NAT1 in A375.S2 cells.

  6. 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.

  7. Vanillin Analogues o-Vanillin and 2,4,6-Trihydroxybenzaldehyde Inhibit NFĸB Activation and Suppress Growth of A375 Human Melanoma.

    PubMed

    Marton, Annamária; Kúsz, Erzsébet; Kolozsi, Csongor; Tubak, Vilmos; Zagotto, Giuseppe; Buzás, Krisztina; Quintieri, Luigi; Vizler, Csaba

    2016-11-01

    Constitutive activation of nuclear factor kappa-B (NFĸB) is a hallmark of various cancer types, including melanoma. Chemotherapy may further increase tumour NFĸB activity, a phenomenon that, in turn, exacerbates drug resistance. This study aimed at preliminary screening of a panel of aromatic aldehydes, including vanillin, for cytotoxicity and suppression of tumour cell NFĸB activity. The cytotoxic and NFĸB-inhibitory effects of 10 aromatic aldehydes, including vanillin, were investigated in cultured A375 human melanoma cells. Each compound was assayed alone and in combination with the model NFĸB-activating drug doxorubicin. The most promising analogues were then tested alone and in combination with 4-hydroperoxycyclophosphamide in vitro, and with cyclophosphamide in mice bearing A375 xenografts. The vanillin analogues o-vanillin and 2,4,6-trihydroxybenzaldehyde exhibited cytotoxicity against cultured A375 cells, and inhibited doxorubicin- and 4-hydroperoxycyclophosphamide-induced NFĸB activation. They also suppressed A375 cell growth in mice. o-vanillin and 2,4,6-trihydroxybenzaldehyde deserve further evaluation as potential anticancer drugs. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

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

    PubMed

    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.

  9. Beta-catenin inhibits melanocyte migration but induces melanoma metastasis

    PubMed Central

    Gallagher, Stuart J.; Rambow, Florian; Kumasaka, Mayuko; Champeval, Delphine; Bellacosa, Alfonso; Delmas, Véronique; Larue, Lionel

    2013-01-01

    The canonical Wnt signalling pathway induces the β-catenin/LEF transcription factors. It is activated in various cancers, most characteristically carcinomas, in which it promotes metastatic spread by increasing migration and/or invasion. The Wnt/β-catenin signalling pathway is frequently activated in melanoma, but the presence of β-catenin in the nucleus does not seem to be a sign of aggressiveness in these tumours. We found that, unlike its positive role in stimulating migration and invasion of carcinoma cells, β-catenin signalling decreased the migration of melanocytes and melanoma cell lines. In vivo, β-catenin signalling in melanoblasts reduced the migration of these cells, causing a white belly-spot phenotype. The inhibition, by β-catenin of migration was dependent on MITF-M, a key transcription factor of the melanocyte lineage, and CSK, a Src-inhibitor. Despite reducing migration, β-catenin signalling promoted lung metastasis in the Nras-driven melanoma murine model. Thus, β-catenin may play conflicting roles in the metastatic spread of melanoma, repressing migration while promoting metastasis. These results highlight that metastasis formation requires a series of successful cellular processes, any one of which may not be optimally efficient. PMID:22665063

  10. 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.

  11. 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

  12. 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.

  13. 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

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

    SciTech Connect

    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. - Highlights: • miR-26b is downregulated in human melanomas. • miR-26b suppressed melanoma cell proliferation and enhanced cell apoptosis. • TRAF5 is a direct target of miR-26b and inversely correlates with miR-26b expression. • miR-26b modulated MAPK signaling pathway by targeting TRAF5.

  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. IGFBP‐3 inhibits Wnt signaling in metastatic melanoma cells

    PubMed Central

    Zingariello, Maria; Sancillo, Laura; Panasiti, Vincenzo; Polinari, Dorina; Martella, Marianna; Rosa Alba, Rana; Londei, Paola

    2016-01-01

    In previous works, we have shown that insulin‐like growth factor‐binding protein‐3 (IGFBP‐3), a tissue and circulating protein able to bind to IGFs, decreases drastically in the blood serum of patients with diffuse metastatic melanoma. In agreement with the clinical data, recombinant IGFBP‐3 was found to inhibit the motility and invasiveness of cultured metastatic melanoma cells and to prevent growth of grafted melanomas in mice. The present work was aimed at identifying the signal transduction pathways underlying the anti‐tumoral effects of IGFBP‐3. We show that the anti‐tumoral effect of IGFBP‐3 is due to inhibition of the Wnt pathway and depends upon the presence of CD44, a receptor protein known to modulate Wnt signaling. Once it has entered the cell, IGFBP‐3 binds the Wnt signalosome interacting specifically with its component GSK‐3β. As a consequence, the β‐catenin destruction complex dissociates from the LRP6 Wnt receptor and GSK‐3β is activated through dephosphorylation, becoming free to target cytoplasmic β‐catenin which is degraded by the proteasomal pathway. Altogether, the results suggest that IGFBP‐3 is a novel and effective inhibitor of Wnt signaling. As IGFBP‐3 is a physiological protein which has no detectable toxic effects either on cultured cells or live mice, it might qualify as an interesting new therapeutic agent in melanoma, and potentially many other cancers with a hyperactive Wnt signaling. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc. PMID:27377812

  17. Andrographolide inhibits melanoma tumor growth by inactivating the TLR4/NF-κB signaling pathway.

    PubMed

    Zhang, Qian-Qian; Zhou, Da-Lei; Ding, Yi; Liu, Hong-Ying; Lei, Yan; Fang, Hai-Yan; Gu, Qu-Liang; He, Xiao-Dong; Qi, Cui-Ling; Yang, Yi; Lan, Tian; Li, Jiang-Chao; Gong, Ping; Wu, Xiao-Yun; Yang, Xuesong; Li, Wei-Dong; Wang, Li-Jing

    2014-12-01

    The TLR4/NF-κB signaling pathway plays a critical role in tumor progression. Andrographolide (Andro) has been reported to have anticancer activity in multiple types of cancer. However, the pharmacological activities of Andro in melanoma are not completely understood. In this study, we defined the anticancer effects of Andro in melanoma and elucidated its potential mechanisms of action. Our experiments showed that Andro significantly inhibited melanoma tumor growth and metastasis by inducing cell cycle arrest and apoptosis. In addition, Andro significantly inhibited the TLR4/NF-κB signaling pathway. Furthermore, the inactivation of TLR4/NF-κB signaling inhibited the mRNA and protein expression of CXCR4 and Bcl-6, which are antitumor genes. This work provides evidence that the TLR4/NF-κB signaling pathway is a potential therapeutic target and may also be indispensable in the Andro-mediated anticancer effect in melanoma.

  18. Cuprous oxide nanoparticle-inhibited melanoma progress by targeting melanoma stem cells

    PubMed Central

    Yu, Bin; Wang, Ye; Yu, Xinlu; Zhang, Hongxia; Zhu, Ji; Wang, Chen; Chen, Fei; Liu, Changcheng; Wang, Jingqiang; Zhu, Haiying

    2017-01-01

    Recent studies have shown that metal and metal oxide have a potential function in antitumor therapy. Our previous studies demonstrated that cuprous oxide nanoparticles (CONPs) not only selectively induce apoptosis of tumor cells in vitro but also inhibit the growth and metastasis of melanoma by targeting mitochondria with little hepatic and renal toxicities in mice. As a further study, our current research revealed that CONPs induced apoptosis of human melanoma stem cells (CD271+/high cells) in A375 and WM266-4 melanoma cell lines and could significantly suppress the expression of MITF, SOX10 and CD271 involved in the stemness maintenance and tumorigenesis of melanoma stem cells. CD271+/high cells could accumulate more CONPs than CD271−/low through clathrin-mediated endocytosis. In addition, lower dosage of CONPs exhibited good anti-melanoma effect by decreasing the cell viability, stemness and tumorigenesis of A375 and WM266-4 cells through reducing the expression of SOX10, MITF, CD271 and genes in MAPK pathway involved in tumor progression. Finally, CONPs obviously suppressed the growth of human melanoma in tumor-bearing nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice, accompanied with tumors structural necrosis and fibrosis remarkably and decreased expression of CD271, SOX10 and MITF. These results above proved the effectiveness of CONPs in inhibiting melanoma progress through multiple pathways, especially through targeting melanoma stem cells. PMID:28435246

  19. Cuprous oxide nanoparticle-inhibited melanoma progress by targeting melanoma stem cells.

    PubMed

    Yu, Bin; Wang, Ye; Yu, Xinlu; Zhang, Hongxia; Zhu, Ji; Wang, Chen; Chen, Fei; Liu, Changcheng; Wang, Jingqiang; Zhu, Haiying

    2017-01-01

    Recent studies have shown that metal and metal oxide have a potential function in antitumor therapy. Our previous studies demonstrated that cuprous oxide nanoparticles (CONPs) not only selectively induce apoptosis of tumor cells in vitro but also inhibit the growth and metastasis of melanoma by targeting mitochondria with little hepatic and renal toxicities in mice. As a further study, our current research revealed that CONPs induced apoptosis of human melanoma stem cells (CD271(+/high) cells) in A375 and WM266-4 melanoma cell lines and could significantly suppress the expression of MITF, SOX10 and CD271 involved in the stemness maintenance and tumorigenesis of melanoma stem cells. CD271(+/high) cells could accumulate more CONPs than CD271(-/low) through clathrin-mediated endocytosis. In addition, lower dosage of CONPs exhibited good anti-melanoma effect by decreasing the cell viability, stemness and tumorigenesis of A375 and WM266-4 cells through reducing the expression of SOX10, MITF, CD271 and genes in MAPK pathway involved in tumor progression. Finally, CONPs obviously suppressed the growth of human melanoma in tumor-bearing nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice, accompanied with tumors structural necrosis and fibrosis remarkably and decreased expression of CD271, SOX10 and MITF. These results above proved the effectiveness of CONPs in inhibiting melanoma progress through multiple pathways, especially through targeting melanoma stem cells.

  20. Modulation of T Cell Activation by Malignant Melanoma Initiating Cells

    PubMed Central

    Schatton, Tobias; Schütte, Ute; Frank, Natasha Y.; Zhan, Qian; Hoerning, André; Robles, Susanne C.; Zhou, Jun; Hodi, F. Stephen; Spagnoli, Giulio C.; Murphy, George F.; Frank, Markus H.

    2010-01-01

    Highly immunogenic cancers such as malignant melanoma are capable of inexorable tumor growth despite the presence of antitumor immunity. This raises the possibility that only a restricted minority of tumorigenic malignant cells might possess the phenotypic and functional characteristics to modulate tumor-directed immune activation. Here we provide evidence supporting this hypothesis, by demonstrating that tumorigenic ABCB5+ malignant melanoma-initiating cells (MMICs) possess the capacity to preferentially inhibit interleukin (IL)-2-dependent T cell activation and to support, in a B7.2-dependent manner, regulatory T (Treg) cell induction. Compared to melanoma bulk populations, ABCB5+ MMICs expressed lower levels of the major histocompatibility complex (MHC) class I, showed aberrant positivity for MHC class II, and exhibited lower expression levels of the melanoma-associated antigens (MAAs) MART-1, ML-IAP, NY-ESO-1, and MAGE-A. In addition, tumorigenic ABCB5+ subpopulations preferentially expressed the costimulatory molecules B7.2 and PD-1 in both established melanoma xenografts and clinical tumor specimens in vivo. In immune activation assays, ABCB5+ melanoma cells inhibited mitogen-dependent human peripheral blood mononuclear cell (PBMC) proliferation and IL-2 production more efficiently than ABCB5− populations. Moreover, coculture with ABCB5+ MMICs increased, in a B7.2 signalling-dependent manner, CD4+CD25+FoxP3+ Treg cell abundance and IL-10 production by mitogen-activated PBMCs. Consistent with these findings, ABCB5+ melanoma subsets also preferentially inhibited IL-2 production and induced IL-10 secretion by cocultured patient-derived, syngeneic PBMCs. Our findings identify novel T cell-modulatory functions of ABCB5+ melanoma subpopulations and suggest specific roles for MMICs in the evasion of antitumor immunity and in cancer immunotherapeutic resistance. PMID:20068175

  1. Telomerase activity in melanoma and non-melanoma skin cancer

    PubMed Central

    Parris, C N; Jezzard, S; Silver, A; MacKie, R; McGregor, J M; Newbold, R F

    1999-01-01

    Telomeres are specialized structures consisting of repeat arrays of TTAGGGn located at the ends of chromosomes. They are essential for chromosome stability and, in the majority of normal somatic cells, telomeres shorten with each cell division. Most immortalized cell lines and tumours reactivate telomerase to stabilize the shortening chromosomes. Telomerase activation is regarded as a central step in carcinogenesis and, here, we demonstrate telomerase activation in premalignant skin lesions and also in all forms of skin cancer. Telomerase activation in normal skin was a rare event, and among 16 samples of normal skin (one with a history of chronic sun exposure) 12.5% (2 out of 16) exhibited telomerase activity. One out of 16 (6.25%) benign proliferative lesions, including viral and seborrhoeic wart samples, had telomerase activity. In premalignant actinic keratoses and Bowen's disease, 42% (11 out of 26) of samples exhibited telomerase activity. In the basal cell carcinoma and cutaneous malignant melanoma (CMM) lesions, telomerase was activated in 77% (10 out of 13) and 69% (22 out of 32) respectively. However, only 25% (3 out of 12) of squamous cell carcinomas (SCC) had telomerase activity. With the exception of one SCC sample, telomerase activity in a positive control cell line derived from a fibrosarcoma (HT1080) was not inhibited when mixed with the telomerase-negative SCC or CMM extracts, indicating that, overall, Taq polymerase and telomerase inhibitors were not responsible for the negative results. Mean telomere hybridizing restriction fragment (TRF) analysis was performed in a number of telomerase-positive and -negative samples and, although a broad range of TRF sizes ranging from 3.6 to 17 kb was observed, a relationship between telomerase status and TRF size was not found. © 1999 Cancer Research Campaign PMID:10408692

  2. 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

  3. Phytochemical potential of Eruca sativa for inhibition of melanoma tumor growth.

    PubMed

    Khoobchandani, M; Ganesh, N; Gabbanini, S; Valgimigli, L; Srivastava, M M

    2011-06-01

    Solvent extracts from the aerial and root parts and seed oil from E. sativa (rocket salad) were assayed for anticancer activity against melanoma cells. The seed oil (isothiocyanates rich) significantly (p<0.01) reduced the tumor growth comparable to the control. Remarkably, the seed oil inhibited melanoma growth and angiogenesis in mice without any major toxicity. The findings qualify seed oil for further investigations in the real of cancer prevention and treatment.

  4. 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.

  5. Inhibition of glutathione S-transferase activity in human melanoma cells by alpha,beta-unsaturated carbonyl derivatives. Effects of acrolein, cinnamaldehyde, citral, crotonaldehyde, curcumin, ethacrynic acid, and trans-2-hexenal.

    PubMed

    Iersel, M L; Ploemen, J P; Struik, I; van Amersfoort, C; Keyzer, A E; Schefferlie, J G; van Bladeren, P J

    1996-10-21

    The glutathione S-transferase (GST) activity towards 1-chloro-2,4-dinitrobenzene in intact human IGR-39 melanoma cells was determined by the quantification by HPLC-analysis of the excreted glutathione (GSH) conjugate (S-(2,4-dinitrophenyl)glutathione; DNPSG). The major GST subunit expressed in these melanoma cells is the pi-class GST subunit P1. Using this system, the effect of exposure for 1 h to a series of alpha, beta-unsaturated carbonyl compounds at non-toxic concentrations was studied. Curcumin was the most potent inhibitor (96% inhibition at 25 microM), while 67 and 61% inhibition at 25 microM was observed for ethacrynic acid and trans-2-hexenal, respectively. Moderate inhibition was observed for cinnamaldehyde and crotonaldehyde, while no inhibition was found for citral. The reactive acrolein did not inhibit the DNPSG-excretion at 2.5 microM, the highest non-toxic concentration. Up to about 50% GSH-depletion was found after treatment with crotonaldehyde, curcumin and ethacrynic acid, however the consequences for GST conjugation are presumably small. Reversible inhibition of GST was the major mechanism of inhibition of DNPSG-excretion in melanoma cells, except in the cases of curcumin and ethacrynic acid, which compounds also inactivated GSTP1-1 by covalent modification. This was clear from the fact that depending on the dose between 30 and 80% inhibition was still observed after lysis of the cells, under which conditions reversible inhibition was is absent. Intracellular levels of DNPSG remained relatively high in the case of ethacrynic acid. It is possible that ethacrynic acid also inhibits the transport of DNPSG by inhibition of the multidrug resistance-associated protein gene encoding glutathione conjugate export pump (MRP/GS-X pump) in some way.

  6. Melanoma.

    PubMed

    Schadendorf, Dirk; Fisher, David E; Garbe, Claus; Gershenwald, Jeffrey E; Grob, Jean-Jacques; Halpern, Allan; Herlyn, Meenhard; Marchetti, Michael A; McArthur, Grant; Ribas, Antoni; Roesch, Alexander; Hauschild, Axel

    2015-04-23

    Melanoma is a common cancer in the Western world with an increasing incidence. Sun exposure is still considered to be the major risk factor for melanoma. The prognosis of patients with malignant (advanced-stage) melanoma differs widely between countries, but public campaigns advocating early detection have led to significant reductions in mortality rates. As well as sun exposure, distinct genetic alterations have been identified as associated with melanoma. For example, families with melanoma who have germline mutations in CDKN2A are well known, whereas the vast majority of sporadic melanomas have mutations in the mitogen-activated protein kinase cascade, which is the pathway with the highest oncogenic and therapeutic relevance for this disease. BRAF and NRAS mutations are typically found in cutaneous melanomas, whereas KIT mutations are predominantly observed in mucosal and acral melanomas. GNAQ and GNA11 mutations prevail in uveal melanomas. Additionally, the PI3K-AKT-PTEN pathway and the immune checkpoint pathways are important. The finding that programmed cell death protein 1 ligand 1 (PDL1) and PDL2 are expressed by melanoma cells, T cells, B cells and natural killer cells led to the recent development of programmed cell death protein 1 (PD1)-specific antibodies (for example, nivolumab and pembrolizumab). Alongside other new drugs - namely, BRAF inhibitors (vemurafenib and dabrafenib) and MEK inhibitors (trametinib and cobimetinib) - these agents are very promising and have been shown to significantly improve prognosis for patients with advanced-stage metastatic disease. Early signs are apparent that these new treatment modalities are also improving long-term clinical benefit and the quality of life of patients. This Primer summarizes the current understanding of melanoma, from mechanistic insights to clinical progress. For an illustrated summary of this Primer, visit: http://go.nature.com/vX2N9s.

  7. Fermented Viola mandshurica inhibits melanogenesis in B16 melanoma cells.

    PubMed

    Kwak, Yeon-Joo; Kim, Kyoung-Sook; Kim, Kyung-Mi; Yu, Hai Yang; Chung, Eunsook; Kim, Seok-Jo; Cha, Jae-Young; Lee, Young-Choon; Lee, Jai-Heon

    2011-01-01

    We assessed the effects of chloroform extract of fermented Viola mandshurica (CEFV) on melanogenesis B16 melanoma cells. CEFV treatment significantly decreased melanin content and tyrosinase activity in dose-dependent manners. To elucidate the mechanism of the inhibitory effects of CEFV on melanogenesis, we performed RT-PCR and Western blotting for melanogenesis-related genes such as tyrosinase, tyrosinase-related protein-1 (TRP-1), TRP-2, and microphthalmia-associated transcription factor (MITF). CEFV strongly inhibited mRNA as well as the protein expression of tyrosinase and MITF, but had no significant effect on TRP-1 or TRP-2 expressions. It markedly decreased the phosphorylation of cAMP responsive element binding protein (CREB), and induced the duration of extracellular signal-regulated kinase (ERK) activation, leading to reduction of MITF expression and subsequently that of tyrosinase. Therefore, we suggest that CEFV induces downregulation of melanogenesis through decreased CREB phosphorylation and ERK activation.

  8. SIRT1 promotes proliferation and inhibits the senescence-like phenotype in human melanoma cells

    PubMed Central

    Ohanna, Mickaël; Bonet, Caroline; Bille, Karine; Allegra, Maryline; Davidson, Irwin; Bahadoran, Philippe; Lacour, Jean-Philippe; Ballotti, Robert; Bertolotto, Corine

    2014-01-01

    SIRT1 operates as both a tumor suppressor and oncogenic factor depending on the cell context. Whether SIRT1 plays a role in melanoma biology remained poorly elucidated. Here, we demonstrate that SIRT1 is a critical regulator of melanoma cell proliferation. SIRT1 suppression by genetic or pharmacological approaches induces cell cycle arrest and a senescence-like phenotype. Gain and loss of function experiments show that M-MITF regulates SIRT1 expression, thereby revealing a melanocyte-specific control of SIRT1. SIRT1 over-expression relieves the senescence-like phenotype and the proliferation arrest caused by MITF suppression, demonstrating that SIRT1 is an effector of MITF-induced proliferation in melanoma cells. Interestingly, SIRT1 level and activity are enhanced in the PLX4032-resistant BRAFV600E-mutated melanoma cells compared with their sensitive counterpart. SIRT1 inhibition decreases melanoma cell growth and rescues the sensibility to PLX4032 of PLX4032-resistant BRAFV600E-mutated melanoma cells. In conclusion, we provide the first evidence that inhibition of SIRT1 warrants consideration as an anti-melanoma therapeutic option. PMID:24742694

  9. 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.

  10. 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

  11. 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

    2015-01-09

    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.

  12. 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

  13. 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

    2016-05-03

    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.

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

    PubMed Central

    Chen, Xu; Wu, Qiuxia; Tan, Lujian; Porter, Dale; Jager, Martine J.; Emery, Caroline; Bastian, Boris C.

    2015-01-01

    Uveal melanoma (UM) is a genetically and biologically distinct type of melanoma, and once metastatic there is no effective treatment currently available. 80% 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 GNAQQ209L 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 PD0325901 or 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 uveal melanoma xenograft model. Our data identifies PKC as a rational therapeutic target for melanoma patients with GNAQ or GNA11 mutations, and demonstrates combined MEK and PKC inhibition is synergistic, with superior efficacy compared to treatment with either approach alone. PMID:24141786

  15. MILI, a PIWI family protein, inhibits melanoma cell migration through methylation of LINE1.

    PubMed

    Wang, Xiuxing; Jiang, Chen; Fu, Bingyuan; Zhu, Ruilou; Diao, Fan; Xu, Na; Chen, Zhong; Tao, Weiwei; Li, Chao-Jun

    2015-02-20

    MILI, a member of the PIWI/AGO gene family, has been well documented to maintain genome integrity by transposon silencing in animal germ cells. It has been reported to be selectively expressed in precancerous stem cells (pCSCs), tumor cell lines and various malignancies. However, the underlying mechanism remains largely unclear. Here, we found that MILI is expressed in the melanoma cell line B16 but not in the highly metastatic mouse melanoma model B16BL6. Interestingly, the knockdown of MILI in B16 could activate MAGEA expression and increase the cell migration ability, whereas the overexpression of MILI in B16BL6 could inhibit MAGEA expression and decrease the cell migration ability. Further investigations showed that MILI can methylate LINE1, which is crucial for MAGEA expression and melanoma cell migration. Our results provide a novel function of MILI in melanoma metastasis and tumor progression.

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

    PubMed

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

    2016-02-01

    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.

  17. Inhibition of Src family kinases with dasatinib blocks migration and invasion of human melanoma cells.

    PubMed

    Buettner, Ralf; Mesa, Tania; Vultur, Adina; Lee, Frank; Jove, Richard

    2008-11-01

    Src family kinases (SFK) are involved in regulating a multitude of biological processes, including cell adhesion, migration, proliferation, and survival, depending on the cellular context. Therefore, although SFKs are currently being investigated as potential targets for treatment strategies in various cancers, the biological responses to inhibition of SFK signaling in any given tumor type are not predictable. Dasatinib (BMS-354825) is a dual Src/Abl kinase inhibitor with potent antiproliferative activity against hematologic malignancies harboring activated BCR-ABL. In this study, we show that dasatinib blocks migration and invasion of human melanoma cells without affecting proliferation and survival. Moreover, dasatinib completely inhibits SFK kinase activity at low nanomolar concentrations in all eight human melanoma cell lines investigated. In addition, two known downstream targets of SFKs, focal adhesion kinase and Crk-associated substrate (p130(CAS)), are inhibited with similar concentrations and kinetics. Consistent with inhibition of these signaling pathways and invasion, dasatinib down-regulates expression of matrix metalloproteinase-9. We also provide evidence that dasatinib directly inhibits kinase activity of the EphA2 receptor tyrosine kinase, which is overexpressed and/or overactive in many solid tumors, including melanoma. Thus, SFKs and downstream signaling are implicated as having key roles in migration and invasion of melanoma cells.

  18. 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

  19. Fucose-Containing Sulfated Polysaccharides from Brown Seaweeds Inhibit Proliferation of Melanoma Cells and Induce Apoptosis by Activation of Caspase-3 in Vitro

    PubMed Central

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

    2011-01-01

    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 Sargassum henslowianum 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

  20. 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.

  1. Response of BRAF-mutant melanoma to BRAF inhibition is mediated by a network of transcriptional regulators of glycolysis.

    PubMed

    Parmenter, Tiffany J; Kleinschmidt, Margarete; Kinross, Kathryn M; Bond, Simon T; Li, Jason; Kaadige, Mohan R; Rao, Aparna; Sheppard, Karen E; Hugo, Willy; Pupo, Gulietta M; Pearson, Richard B; McGee, Sean L; Long, Georgina V; Scolyer, Richard A; Rizos, Helen; Lo, Roger S; Cullinane, Carleen; Ayer, Donald E; Ribas, Antoni; Johnstone, Ricky W; Hicks, Rodney J; McArthur, Grant A

    2014-04-01

    Deregulated glucose metabolism fulfills the energetic and biosynthetic requirements for tumor growth driven by oncogenes. Because inhibition of oncogenic BRAF causes profound reductions in glucose uptake and a strong clinical benefit in BRAF-mutant melanoma, we examined the role of energy metabolism in responses to BRAF inhibition. We observed pronounced and consistent decreases in glycolytic activity in BRAF-mutant melanoma cells. Moreover, we identified a network of BRAF-regulated transcription factors that control glycolysis in melanoma cells. Remarkably, this network of transcription factors, including hypoxia-inducible factor-1α, MYC, and MONDOA (MLXIP), drives glycolysis downstream of BRAF(V600), is critical for responses to BRAF inhibition, and is modulated by BRAF inhibition in clinical melanoma specimens. Furthermore, we show that concurrent inhibition of BRAF and glycolysis induces cell death in BRAF inhibitor (BRAFi)-resistant melanoma cells. Thus, we provide a proof-of-principle for treatment of melanoma with combinations of BRAFis and glycolysis inhibitors. BRAF is suppress glycolysis and provide strong clinical benefi t in BRAF V600 melanoma. We show that BRAF inhibition suppresses glycolysis via a network of transcription factors that are critical for complete BRAFi responses. Furthermore, we provide evidence for the clinical potential of therapies that combine BRAFis with glycolysis inhibitors.

  2. 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.

  3. 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.

  4. Selective histone deacetylase 6 inhibitors bearing substituted urea linkers inhibit melanoma cell growth.

    PubMed

    Bergman, Joel A; Woan, Karrune; Perez-Villarroel, Patricio; Villagra, Alejandro; Sotomayor, Eduardo M; Kozikowski, Alan P

    2012-11-26

    The incidence of malignant melanoma has dramatically increased in recent years thus requiring the need for improved therapeutic strategies. In our efforts to design selective histone deactylase inhibitors (HDACI), we discovered that the aryl urea 1 is a modestly potent yet nonselective inhibitor. Structure-activity relationship studies revealed that adding substituents to the nitrogen atom of the urea so as to generate compounds bearing a branched linker group results in increased potency and selectivity for HDAC6. Compound 5 g shows low nanomolar inhibitory potency against HDAC6 and a selectivity of ∼600-fold relative to the inhibition of HDAC1. These HDACIs were evaluated for their ability to inhibit the growth of B16 melanoma cells with the most potent and selective HDAC6I being found to decrease tumor cell growth. To the best of our knowledge, this work constitutes the first report of HDAC6-selective inhibitors that possess antiproliferative effects against melanoma cells.

  5. Selective histone deacetylase 6 inhibitors bearing substituted urea linkers inhibit melanoma cell growth

    PubMed Central

    Bergman, Joel A.; Woan, Karrune; Perez-Villarroel, Patricio; Villagra, Alejandro; Sotomayor, Eduardo M.; Kozikowski, Alan P.

    2012-01-01

    The incidence of malignant melanoma has dramatically increased in recent years thus requiring the need for improved therapeutic strategies. In our efforts to design selective histone deactylase inhibitors (HDACI), we discovered that the aryl urea 1 is a modestly potent yet non-selective inhibitor. Structure activity relationship studies revealed that adding substituents to the nitrogen atom of the urea so as to generate compounds bearing a branched linker group results in increased potency and selectivity for HDAC6. Compound 5g shows low nanomolar inhibitory potency against HDAC6 and a selectivity of ~600-fold relative to the inhibition of HDAC1. These HDACIs were evaluated for their ability to inhibit the growth of B16 melanoma cells with the most potent and selective HDAC6I being found to decrease tumor cell growth. To the best of our knowledge, this work constitutes the first report of HDAC6 selective inhibitors that possess antiproliferative effects against melanoma cells. PMID:23009203

  6. Inhibition of Peroxisome Proliferator-Activated Receptor Gamma Prevents the Melanogenesis in Murine B16/F10 Melanoma Cells

    PubMed Central

    Chang, Junn-Liang; Tang, Shih-Tsang; Pan, Shwu-Fen; Lin, Tzer-Bin; Chen, Kang-Hua

    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

  7. 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.

  8. 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 ...

  9. 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.

  10. Acetylsalicylic acid inhibits the growth of melanoma tumors via SOX2-dependent-PAF-R-independent signaling pathway

    PubMed Central

    Thyagarajan, Anita; Saylae, Jeremiah; Sahu, Ravi P.

    2017-01-01

    Acquired resistance to standard therapies remains a serious challenge, requiring novel therapeutic approaches that incorporate potential factors involved in tumor resistance. As cancers including melanoma express inflammatory cyclooxygenases generating prostaglandins implicated in tumor growth, we investigated mechanism of anti-inflammatory drug, acetylsalicylic acid (ASA) which has been shown to inhibit various tumor types, however, its effects against highly aggressive melanoma model are unclear. Given our reports that an activation of platelet-activating factor-receptor (PAF-R) augments the growth and impede efficacies of therapeutic agents in experimental melanoma, we also sought to determine if PAF-R mediates anti-melanoma activity of ASA. The current studies using stably PAF-R-positive (B16-PAFR) and negative (B16-MSCV) murine melanoma cells and PAF-R-expressing and deficient mice, demonstrate that ASA inhibits the in-vitro and in-vivo growth of highly aggressive B16F10 melanoma via bypassing tumoral or stromal PAF-R signaling. Similar ASA-induced effects in-vitro were seen in human melanoma and nasopharyngeal carcinoma cells positive or negative in PAF-R. Mechanistically, the ASA-induced decrease in cell survival and increase in apoptosis were significantly blocked by prostaglandin F2 alpha (PGF2α) agonists. Importantly, PCR array and qRT-PCR analysis of B16-tumors revealed significant downregulation of sry-related high-mobility-box-2 (SOX2) oncogene by ASA treatment. Interestingly, modulation of SOX2 expression by PGF2α agonists and upregulation by fibroblast growth factor 1 (FGF-1) rescued melanoma cells from ASA-induced decreased survival and increased apoptosis. Moreover, PGF2α-receptor antagonist, AL8810 mimics ASA-induced decreased melanoma cells survival which was significantly blocked by PGF2α and FGF-1. These findings indicate that ASA inhibits the growth of aggressive melanoma via SOX2-dependent-PAF-R-indepedent pathway. PMID:28636992

  11. Metapristone (RU486 derivative) inhibits cell proliferation and migration as melanoma metastatic chemopreventive agent.

    PubMed

    Zheng, Ning; Chen, Jiahang; Liu, Weiqun; Wang, Jichuang; Liu, Jian; Jia, Lee

    2017-04-01

    Uncontrolled cell proliferation and metastasis are the two well-known manifestations of melanoma. We hypothesized that metapristone, a potential cancer metastatic chemopreventive agent derived from mifepristone (RU486), had a dual function to fight cancer. In the present study, our findings clearly demonstrated that metapristone had modest cytostatic effect in melanoma cells. Metapristone inhibited cell viability and induced both early and late apoptosis in B16F10 and A375 cells in a time- and concentrate-dependent manner. Metapristone-treatment caused the cell arrest at the G0/G1 stage, and the inhibition of colony formation in B16F10 cells. Western blot analysis further revealed that metapristone treatment elicited a decline of Akt and ERK phosphorylation and Bcl-2, and facilitated expression of total P53 and Bax in A375 cells. In addition, cell migration and invasion were significantly suppressed by metapristone through down-regulating the expression of MMP-2, MMP-9, N-cadherin and vimentin, whereas up-regulating E-cadherin expression. Notably, metapristone exhibited anti-metastatic activity in melanoma B16F10 cells in vivo. Our results reveal metapristone, having the dual function of anti-proliferation and anti-migration for melanoma cell lines, may be a useful chemopreventive agent to reduce the risk of melanoma cancer metastasis.

  12. 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

  13. 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.

  14. 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

  15. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. NF-κB inhibition is associated with OPN/MMP-9 downregulation in cutaneous melanoma

    PubMed Central

    Guarneri, Claudio; Bevelacqua, Valentina; Polesel, Jerry; Falzone, Luca; Cannavò, Patrizia S.; Spandidos, Demetrios A.; Malaponte, Grazia; Libra, Massimo

    2017-01-01

    The development of cutaneous melanoma is influenced by genetic factors, including BRAF mutations and environmental factors, such as ultraviolet exposure. Its progression has been also associated with the involvement of several tumour microenvironmental molecules. Among these, nuclear factor-κB (NF-κB) has been indicated as a key player of osteopontin (OPN) and matrix metalloproteinase-9 (MMP-9) activation. However, whether NF-κB plays a role in the development and progression of melanoma in association with the OPN/MMP-9 axis according to the BRAFV600E mutation status has not been investigated in detail to date. Thus, in the present study, in order to shed light on this matter, 148 patients with melanoma and 53 healthy donors were recruited for the analysis of OPN, MMP-9 and NF-κB. Significantly higher circulating levels of OPN and MMP-9 were observed in the patients with melanoma when compared to the healthy donors. Similar data were obtained for NF-κB p65 activity. The OPN levels did not differ significantly between melanomas with or without BRAFV600E mutation. However, as regards NF-κB and MMP-9, significant differences were observed between the melanomas with or without BRAFV600E mutation. To determine whether NF-κB inhibition is associated with a decrease in the levels of OPN and MMP-9, peripheral blood mononuclear cells from 29 patients with melanoma were treated with the NF-κB inhibitor, dehydroxymethylepoxyquinomycin (DHMEQ), with or without OPN. As expected, the inhibition of NF-κB induced a marked decrease in both the OPN and MMP-9 levels. Furthermore, the decrease in MMP-9 levels was higher among melanomas harbouring the BRAFV600E mutation. Overall, our data suggest that the activation of MMP-9 is associated with the BRAFV600E mutation status. Furthermore, such an activation is mediated by NF-κB, suggesting its role as therapeutic target in patients with melanoma. PMID:28075446

  17. NF‑κB inhibition is associated with OPN/MMP‑9 downregulation in cutaneous melanoma.

    PubMed

    Guarneri, Claudio; Bevelacqua, Valentina; Polesel, Jerry; Falzone, Luca; Cannavò, Patrizia S; Spandidos, Demetrios A; Malaponte, Grazia; Libra, Massimo

    2017-02-01

    The development of cutaneous melanoma is influenced by genetic factors, including BRAF mutations and environmental factors, such as ultraviolet exposure. Its progression has been also associated with the involvement of several tumour microenvironmental molecules. Among these, nuclear factor‑κB (NF‑κB) has been indicated as a key player of osteopontin (OPN) and matrix metalloproteinase‑9 (MMP‑9) activation. However, whether NF‑κB plays a role in the development and progression of melanoma in association with the OPN/MMP‑9 axis according to the BRAFV600E mutation status has not been investigated in detail to date. Thus, in the present study, in order to shed light on this matter, 148 patients with melanoma and 53 healthy donors were recruited for the analysis of OPN, MMP‑9 and NF‑κB. Significantly higher circulating levels of OPN and MMP‑9 were observed in the patients with melanoma when compared to the healthy donors. Similar data were obtained for NF‑κB p65 activity. The OPN levels did not differ significantly between melanomas with or without BRAFV600E mutation. However, as regards NF‑κB and MMP‑9, significant differences were observed between the melanomas with or without BRAFV600E mutation. To determine whether NF‑κB inhibition is associated with a decrease in the levels of OPN and MMP‑9, peripheral blood mononuclear cells from 29 patients with melanoma were treated with the NF‑κB inhibitor, dehydroxymethylepoxyquinomycin (DHMEQ), with or without OPN. As expected, the inhibition of NF‑κB induced a marked decrease in both the OPN and MMP‑9 levels. Furthermore, the decrease in MMP‑9 levels was higher among melanomas harbouring the BRAFV600E mutation. Overall, our data suggest that the activation of MMP‑9 is associated with the BRAFV600E mutation status. Furthermore, such an activation is mediated by NF‑κB, suggesting its role as therapeutic target in patients with melanoma.

  18. Parthenolide enhances dacarbazine activity against melanoma cells.

    PubMed

    Koprowska, Kamila; Hartman, Mariusz L; Sztiller-Sikorska, Malgorzata; Czyz, Malgorzata E

    2013-09-01

    Dacarbazine induces a clinical response only in 15% of melanoma patients. New treatment strategies may involve combinations of drugs with different modes of action to target the tumor heterogeneity. We aimed to determine whether the combined treatment of heterogeneous melanoma cell populations in vitro with the alkylating agent dacarbazine and the nuclear factor-κB inhibitor parthenolide could be more effective than either drug alone. A panel of melanoma cell lines, including highly heterogeneous populations derived from surgical specimens, was treated with dacarbazine and parthenolide. The effect of drugs on the viable cell number was examined using an acid phosphatase activity assay, and the combination effect was determined by median-effect analysis. Cell death and cell-cycle arrest were assessed by flow cytometry. Gene expression was measured by real-time PCR and changes in the protein levels were evaluated by western blotting. Secretion of vascular endothelial growth factor and interleukin-8 was determined using an enzyme-linked immunosorbent assay. The self-renewing capacity was assessed using a clonogenic assay. Dacarbazine was less effective in heterogeneous melanoma populations than in the A375 cell line. Parthenolide and dacarbazine synergistically reduced the viable cell numbers. Both drugs induced cell-cycle arrest and apoptotic cell death. Importantly, parthenolide abrogated the baseline and dacarbazine-induced vascular endothelial growth factor secretion from melanoma cells in heterogeneous populations, whereas interleukin-8 secretion was not significantly affected by either drug. Parthenolide eradicated melanoma cells with self-renewing capacity also in cultures simultaneously treated with dacarbazine. The combination of parthenolide and dacarbazine might be considered as a new therapeutic modality against metastatic melanoma.

  19. 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.

  20. Retinoic acid decreases ATF-2 phosphorylation and sensitizes melanoma cells to taxol-mediated growth inhibition.

    PubMed

    Huang, Ying; Minigh, Jennifer; Miles, Sarah; Niles, Richard M

    2008-02-12

    Cutaneous melanoma is often resistant to chemo- and radiotherapy. This resistance has recently been demonstrated to be due, at least in part, to high activating transcription factor 2 (ATF-2) activity in these tumors. In concordance with these reports, we found that B16 mouse melanoma cells had higher levels of ATF-2 than immortalized, but non-malignant mouse melanocytes. In addition, the melanoma cells had a much higher amount of phosphorylated (active) ATF-2 than the immortalized melanocytes. In the course of determining how retinoic acid (RA) stimulates activating protein-1 (AP-1) activity in B16 melanoma, we discovered that this retinoid decreased the phosphorylation of ATF-2. It appears that this effect is mediated through p38 MAPK, because RA decreased p38 phosphorylation, and a selective inhibitor of p38 MAPK (SB203580) also inhibited the phosphorylation of ATF-2. Since ATF-2 activity appears to be involved in resistance of melanoma to chemotherapy, we tested the hypothesis that treatment of the melanoma cells with RA would sensitize them to the growth-inhibitory effect of taxol. We found that pretreatment of B16 cells with RA decreased the IC50 from 50 nM to 1 nM taxol. On the basis of these findings and our previous work on AP-1, we propose a model in which treatment of B16 cells with RA decreases the phosphorylation of ATF-2, which results in less dimer formation with Jun. The "freed-up" Jun can then form a heterodimer with Fos, resulting in the increased AP-1 activity observed in RA-treated B16 cells. Shifting the balance from predominantly ATF-2:Jun dimers to a higher amount of Jun:Fos dimers could lead a change in target gene expression that reduces resistance to chemotherapeutic drugs and contributes to the pathway by which RA arrests proliferation and induces differentiation.

  1. Yessotoxin, a Marine Toxin, Exhibits Anti-Allergic and Anti-Tumoural Activities Inhibiting Melanoma Tumour Growth in a Preclinical Model

    PubMed Central

    Tobío, Araceli; Alfonso, Amparo; Madera-Salcedo, Iris; Botana, Luis M.

    2016-01-01

    Yessotoxins (YTXs) are a group of marine toxins produced by the dinoflagellates Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulax spinifera. They may have medical interest due to their potential role as anti-allergic but also anti-cancer compounds. However, their biological activities remain poorly characterized. Here, we show that the small molecular compound YTX causes a slight but significant reduction of the ability of mast cells to degranulate. Strikingly, further examination revealed that YTX had a marked and selective cytotoxicity for the RBL-2H3 mast cell line inducing apoptosis, while primary bone marrow derived mast cells were highly resistant. In addition, YTX exhibited strong cytotoxicity against the human B-chronic lymphocytic leukaemia cell line MEC1 and the murine melanoma cell line B16F10. To analyse the potential role of YTX as an anti-cancer drug in vivo we used the well-established B16F10 melanoma preclinical mouse model. Our results demonstrate that a few local application of YTX around established tumours dramatically diminished tumour growth in the absence of any significant toxicity as determined by the absence of weight loss and haematological alterations. Our data support that YTX may have a minor role as an anti-allergic drug, but reveals an important potential for its use as an anti-cancer drug. PMID:27973568

  2. Yessotoxin, a Marine Toxin, Exhibits Anti-Allergic and Anti-Tumoural Activities Inhibiting Melanoma Tumour Growth in a Preclinical Model.

    PubMed

    Tobío, Araceli; Alfonso, Amparo; Madera-Salcedo, Iris; Botana, Luis M; Blank, Ulrich

    2016-01-01

    Yessotoxins (YTXs) are a group of marine toxins produced by the dinoflagellates Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulax spinifera. They may have medical interest due to their potential role as anti-allergic but also anti-cancer compounds. However, their biological activities remain poorly characterized. Here, we show that the small molecular compound YTX causes a slight but significant reduction of the ability of mast cells to degranulate. Strikingly, further examination revealed that YTX had a marked and selective cytotoxicity for the RBL-2H3 mast cell line inducing apoptosis, while primary bone marrow derived mast cells were highly resistant. In addition, YTX exhibited strong cytotoxicity against the human B-chronic lymphocytic leukaemia cell line MEC1 and the murine melanoma cell line B16F10. To analyse the potential role of YTX as an anti-cancer drug in vivo we used the well-established B16F10 melanoma preclinical mouse model. Our results demonstrate that a few local application of YTX around established tumours dramatically diminished tumour growth in the absence of any significant toxicity as determined by the absence of weight loss and haematological alterations. Our data support that YTX may have a minor role as an anti-allergic drug, but reveals an important potential for its use as an anti-cancer drug.

  3. Galangin inhibits tumor growth and metastasis of B16F10 melanoma.

    PubMed

    Zhang, Wenjing; Tang, Bo; Huang, Qilai; Hua, Zichun

    2013-01-01

    Galangin, an active flavonoid extracted from the root of the Alpinia officinarum Hance, showed a cytotoxic effect on several cancer cell lines in vitro. However, there is no information available concerning its antimetastatic effect. Focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, is involved in many aspects of cellular processes such as proliferation, adhesion, and invasion. Studies have shown that FAK is a promising target for therapeutic intervention in melanoma. In the present study, proliferation of B16F10 cells was suppressed when exposed to various doses of galangin. Inhibition on proliferation by galangin was also detected by clonogenic survival assay. The capabilities of cell adhesion, cell spreading, and cell motility were impaired by galangin, reinforced by F-actin rearrangement. Molecular data showed that both FAK mRNA level and protein level were reduced dose-dependently. Additionally, galangin reduced phosphorylation of FAK (Tyr397) protein. Transient transfection reporter assays showed that galangin suppressed the transcription of FAK gene, indicating FAK expression is a candidate target of galangin. The antimetastatic function of galangin is further supported by the fact that it could inhibit the formation of tumor colonies in the lung tissue on C57BL/6J mouse lung metastatic model using B16F10 melanoma cells. Immunochemical analyses showed that galangin decreased FAK expression in vivo. These data add to our new understanding that galangin can inhibit B16F10 melanoma metastasis both in vivo and in vitro, and that FAK is a valid therapeutic target against melanoma.

  4. Enhanced anti-melanoma efficacy of interferon alfa-2b via inhibition of Shp2.

    PubMed

    Win-Piazza, Hla; Schneeberger, Valentina E; Chen, Liwei; Pernazza, Daniele; Lawrence, Harshani R; Sebti, Said M; Lawrence, Nicholas J; Wu, Jie

    2012-07-01

    Interferon-α2b (IFN-α2b) is used to treat melanoma but there is a need to improve its efficacy. IFN-α2b signaling requires STAT1/STAT2 tyrosine phosphorylation and is subject to negative regulation by phosphatases. In this study, we determined whether inhibition of the protein tyrosine phosphatase Shp2 could enhance IFN-α2b responses in human melanoma cells. Shp2 knockdown increased IFN-α2b-stimulated STAT1 Tyr-701 phosphorylation and ISRE-luciferase activity even though it did not affect STAT2 Tyr-690 phosphorylation in A375 cells. In A375 tumor xenografts, Shp2 knockdown enhanced the anti-melanoma effect of IFN-α2b. Furthermore, the Shp2 inhibitor SPI-112Me increased the IFN-α2b-induced STAT1 activation and anti-proliferative response in A375 and SK-MEL-2 cells. These results demonstrate that inhibition of Shp2 can enhance the anti-melanoma activity of IFN-α2b.

  5. 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

    2016-02-02

    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.

  6. 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

  7. Acetylsalicylic acid regulates MMP-2 activity and inhibits colorectal invasion of murine B16F0 melanoma cells in C57BL/6J mice: effects of prostaglandin F(2)alpha.

    PubMed

    Tsai, Chin-Shaw Stella; Luo, Shue-Fen; Ning, Chung-Chu; Lin, Chien-Liang; Jiang, Ming-Chung; Liao, Ching-Fong

    2009-08-01

    Epidemiological studies indicate that acetylsalicylic acid may reduce the risk of mortality due to colon cancers. Metastasis is the major cause of cancer death. Matrix metalloproteinases (MMPs) play important roles in tumor invasion regulation, and prostaglandin F(2)alpha (PGF(2)alpha) is a key stimulator of MMP production. Thus, we investigated whether acetylsalicylic acid regulated MMP activity and the invasion of cancer cells and whether PGF(2)alpha attenuated acetylsalicylic acid-inhibited invasion of cancer cells. Gelatin-based zymography assays showed that acetylsalicylic acid inhibited the MMP-2 activity of B16F0 melanoma cells. Matrigel-based chemoinvasion assays showed that acetylsalicylic acid inhibited the invasion of B16F0 cells. Acetylsalicylic acid can inhibit PGF(2)alpha synthesis and PGF(2)alpha is a key stimulator of MMP-2 production. Our data showed that PGF(2)alpha treatment attenuated the acetylsalicylic acid-inhibited invasion of B16F0 cells. In animal experiments, acetylsalicylic acid reduced colorectal metastasis of B16F0 cells in C57BL/6J mice by 44%. Our results suggest that PGF(2)alpha is a therapeutic target for metastasis inhibition and acetylsalicylic acid may possess anti-metastasis ability.

  8. Activation of an early feedback survival loop involving phospho-ErbB3 is a general response of melanoma cells to RAF/MEK inhibition and is abrogated by anti-ErbB3 antibodies

    PubMed Central

    2013-01-01

    Background Treatment of advanced melanoma has been improved with the advent of the BRAF inhibitors. However, a limitation to such treatment is the occurrence of resistance. Several mechanisms have been identified to be responsible for the development of resistance, either MEK-dependent or MEK-independent. In order to overcome resistance due to reactivation of MEK signaling, MEK inhibitors are being clinically developed with promising results. However, also in this case resistance inevitably occurs. It has been recently reported that ErbB3, a member of the EGFR receptor family, may be involved in the establishment of drug resistance. Methods Three melanoma cell lines were tested: LOX IMVI (BRAF V600E), MST-L (BRAF V600R) and WM266 (BRAF V600D). Phosphorylation of Receptor Tyrosine Kinases (RTKs) was assessed by an RTK array. Western blot analysis was performed on total protein extracts using anti-ErbB3, anti-AKT and anti-ERK 1/2 antibodies. The expression of neuregulin after vemurafenib treatment was assessed by Real Time PCR and Western blotting. The growth inhibitory effects of vemurafenib, GSK1120212b and/or anti-ErbB3 mAbs were evaluated by in vitro colony formation assays. Results In the present study we demonstrate that ErbB3 is the main RTK undergoing rapidly hyperphosphorylation upon either treatment with a BRAF inhibitor or with a MEK inhibitor in a panel of melanoma cell lines harboring a variety of V600BRAF mutations and that this results in a strong activation of phospho-AKT. Importantly, ErbB3 activation is fully abrogated by the simultaneous use of anti-ErbB3 monoclonal antibodies, which are also shown to potently synergize with BRAF inhibitors in the inactivation of both AKT and ERK pathways and in the inhibition of melanoma cell growth. We show that upregulation of phospho-ErbB3 is due to an autocrine loop involving increased transcription and production of neuregulin by melanoma cells. Conclusions On the basis of these results, we propose that

  9. Inhibition of melanoma cell motility by the snake venom disintegrin eristostatin

    PubMed Central

    Tian, Jing; Paquette-Straub, Carrie; Sage, E. Helene; Funk, Sarah E.; Patel, Vivek; Galileo, Deni; McLane, Mary Ann

    2007-01-01

    Eristostatin, an RGD-containing disintegrin isolated from the venom of Eristicophis macmahoni, inhibits lung or liver colonization of melanoma cells in a mouse model. In this study, transwell migration and in vitro wound closure assays were used to determine the effect of eristostatin on the migration of melanoma cells. Eristostatin significantly impaired the migration of 5 human melanoma cell lines. Furthermore, it specifically inhibited cell migration on fibronectin in a concentration-dependent manner, but not that on collagen IV or laminin. In contrast, eristostatin was found to have no effect on cell proliferation or angiogenesis. These results indicate that the interaction between eristostatin and melanoma cells may involve fibronectin-binding integrins that mediate cell migration. Mutations to alanine of seven residues within the RGD loop of eristostatin and four residues outside the RGD loop of eristostatin resulted in significantly less potency in both platelet aggregation and wound closure assays. For six of the mutations, however, decreased activity was found only in the latter assay. We conclude that a different mechanism and/or integrin is involved in these two cell activities. PMID:17316731

  10. Ginsenoside Rg3-induced EGFR/MAPK pathway deactivation inhibits melanoma cell proliferation by decreasing FUT4/LeY expression.

    PubMed

    Shan, Xiu; Aziz, Faisal; Tian, Li Li; Wang, Xiao Qi; Yan, Qiu; Liu, Ji Wei

    2015-04-01

    Malignant melanoma is a destructive and lethal form of skin cancer with poor prognosis. An effective treatment for melanoma is greatly needed. Ginsenoside Rg3 is a herbal medicine with high antitumor activity. It is reported that abnormal glycosylation is correlated with the tumor cell growth. However, the antitumor effect of Rg3 on melanoma and its mechanism on regulating glycosylation are unknown. We found that Rg3 did not only inhibit A375 melanoma cell proliferation in a dose-dependent manner, but also decreased the expression of fucosyltransferase IV (FUT4) and its synthetic product Lewis Y (LeY), a tumor-associated carbohydrate antigen (TACA). Knocking down FUT4 expression by siRNA dramatically reduced FUT4/LeY level and inhibited cell proliferation through preventing the activation of EGFR/MAPK pathway. Consistently, the inhibitory effect of the Rg3 and FUT4 knockdown on melanoma growth was also seen in a xenograft melanoma mouse model. In conclusion, Rg3 effectively inhibited melanoma cell growth by downregulating FUT4 both in vitro and in vivo. Targeting FUT4/LeY mediated fucosylation by Rg3 inhibited the activation of EGFR/MAPK pathway and prevented melanoma growth. Results from this study suggest Rg3 is a potential novel therapy agent for melanoma treatment.

  11. Inositol hexaphosphate (IP6) inhibits cellular proliferation in melanoma.

    PubMed

    Rizvi, Irfan; Riggs, Dale R; Jackson, Barbara J; Ng, Alex; Cunningham, Cynthia; McFadden, David W

    2006-06-01

    Inositol Hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate found in food sources high in fiber content. We have previously reported IP6 to have significant inhibitory effects against pancreatic cancer in vitro. We hypothesized that the IP6 would significantly inhibit cell growth of cutaneous melanoma in vitro. The melanoma line HTB68 was cultured using standard techniques and treated with IP6 at doses ranging from 0.2 to 1.0 mM/well. Cell viability was measured by MTT at 72 h. VEGF production was measured in the cell supernatants by ELISA. Apoptosis was evaluated by Annexin V-FITC and results calculated using FACS analysis. Statistical analysis was performed by ANOVA. Significant reductions (P < 0.001) in cellular proliferation were observed with IP6. Overall, IP6 exhibited a mean inhibition of cell growth of 52.1 +/- 11.5% (range, 1.6-83.0%) at 72 h of incubation. VEGF production was significantly reduced (P < 0.001) by the addition of IP6 (7.5 pg/ml) compared to control (40.9 pg/ml). IP6 significantly increased (P = 0.029) late apoptosis from 5.3 to 7.0% gated events. No changes in necrosis or early apoptosis were observed. Adjuvant treatment of melanoma continues to challenge clinicians and patients. Our findings that IP6 significantly decreased cellular growth, VEGF production and increased late apoptosis in melanoma suggest its potential therapeutic value. Further in vivo studies are planned to evaluate safety and clinical utility of this agent.

  12. The natural compound fucoidan from New Zealand Undaria pinnatifida synergizes with the ERBB inhibitor lapatinib enhancing melanoma growth inhibition.

    PubMed

    Thakur, Varsha; Lu, Jun; Roscilli, Giuseppe; Aurisicchio, Luigi; Cappelletti, Manuela; Pavoni, Emiliano; White, William Lindsey; Bedogni, Barbara

    2017-03-14

    Melanoma remains one of the most aggressive and therapy-resistant cancers. Finding new treatments to improve patient outcomes is an ongoing effort. We previously demonstrated that melanoma relies on the activation of ERBB signaling, specifically of the ERBB3/ERBB2 cascade. Here we show that melanoma tumor growth is inhibited by 60% over controls when treated with lapatinib, a clinically approved inhibitor of ERBB2/EGFR. Importantly, tumor growth is further inhibited to 85% when the natural compound fucoidan from New Zealand U. pinnatifida is integrated into the treatment regimen. Fucoidan not only enhances tumor growth inhibition, it counteracts the morbidity associated with prolonged lapatinib treatment. Fucoidan doubles the cell killing capacity of lapatinib. These effects are associated with a further decrease in AKT and NFκB signaling, two key pathways involved in melanoma cell survival. Importantly, the enhancing cell killing effects of fucoidan can be recapitulated by inhibiting ERBB3 by either a specific shRNA or a novel, selective ERBB3 neutralizing antibody, reiterating the key roles played by this receptor in melanoma. We therefore propose the use of lapatinib or specific ERBB inhibitors, in combination with fucoidan as a new treatment of melanoma that potentiates the effects of the inhibitors while protecting from their potential side effects.

  13. 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. © 2015 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  14. 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

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

    PubMed

    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

    2016-02-25

    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.

  16. 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

  17. Simultaneous Targeting of COX-2 and AKT Using Selenocoxib-1-GSH to Inhibit Melanoma

    PubMed Central

    Gowda, Raghavendra; Madhunapantula, SubbaRao V.; Desai, Dhimant; Amin, Shantu; Robertson, Gavin P.

    2012-01-01

    Melanoma is a highly metastatic and deadly disease. An agent simultaneously targeting COX-2, PI3K/Akt and MAPK signaling pathways that are deregulated in up to 70% of sporadic melanoma might be an effective treatment but no agent of this type exists. To develop a single drug inhibiting COX-2 and PI3K/Akt signaling (and increasing MAPK pathway activity to inhibitory levels as a result of Akt inhibition), a selenium-containing glutathione (GSH) analog of celecoxib, called selenocoxib-1-GSH was synthesized. It killed melanoma cells with an average IC50 of 7.66 µmol/L compared to control celecoxib at 55.6 µmol/L. The IC50 range for normal cells was 36.3–41.2 µmol/L compared to 7.66 µmol/L for cancer cells. Selenocoxib-1-GSH reduced xenografted tumor development by ~70% with negligible toxicity by targeting COX-2, like celecoxib, and having new inhibitory properties acting as a PI3K/Akt inhibitor (and MAPK pathway activator to inhibitory levels due to Akt inhibition). The consequence of this inhibitory activity was an ~80% decrease in cultured cell proliferation and a ~200% increase in apoptosis following 24 hours treatment with 15.5 µmol/L of drug. Thus, this study details development of selenocoxib-1-GSH, which is a non-toxic agent that targets the COX-2 and PI3K/Akt signaling pathways in melanomas to inhibit tumor development. PMID:23112250

  18. Spirooxindole derivative SOID-8 induces apoptosis associated with inhibition of JAK2/STAT3 signaling in melanoma cells.

    PubMed

    Tian, Yan; Nam, Sangkil; Liu, Lucy; Yakushijin, Fumiko; Yakushijin, Kenichi; Buettner, Ralf; Liang, Wei; Yang, Fan; Ma, Yuelong; Horne, David; Jove, Richard

    2012-01-01

    Melanoma is generally refractory to current chemotherapy, thus new treatment strategies are needed. In this study, we synthesized a series of spirooxindole derivatives (SOID-1 to SOID-12) and evaluated their antitumor effects on melanoma. Among the 12 spirooxindole derivatives, SOID-8 showed the strongest antitumor activity by viability screening. SOID-8 inhibited viability of A2058, A375, SK-MEL-5 and SK-MEL-28 human melanoma cells in a dose- and time-dependent manner. SOID-8 also induced apoptosis of these tumor cells, which was confirmed by positive Annexin V staining and an increase of poly(ADP-ribose) polymerase cleavage. The antiapoptotic protein Mcl-1, a member of the Bcl-2 family, was downregulated and correlated with SOID-8 induced apoptosis. In addition, SOID-8 reduced tyrosine phosphorylation of Signal Tansducer and Activator of Transcription 3 (STAT3) in both dose- and time-dependent manners. This inhibition was associated with decreased levels of phosphorylation of Janus-activated kinase-2 (JAK2), an upstream kinase that mediates STAT3 phosphorylation at Tyr705. Accordingly, SOID-8 inhibited IL-6-induced activation of STAT3 and JAK2 in melanoma cells. Finally, SOID-8 suppressed melanoma tumor growth in a mouse xenograft model, accompanied with a decrease of phosphorylation of JAK2 and STAT3. Our results indicate that the antitumor activity of SOID-8 is at least partially due to inhibition of JAK2/STAT3 signaling in melanoma cells. These findings suggest that the spirooxindole derivative SOID-8 is a promising lead compound for further development of new preventive and therapeutic agents for melanoma.

  19. c-Abl and Arg are activated in human primary melanomas, promote melanoma cell invasion via distinct pathways, and drive metastatic progression.

    PubMed

    Ganguly, S S; Fiore, L S; Sims, J T; Friend, J W; Srinivasan, D; Thacker, M A; Cibull, M L; Wang, C; Novak, M; Kaetzel, D M; Plattner, R

    2012-04-05

    Despite 35 years of clinical trials, there is little improvement in 1-year survival rates for patients with metastatic melanoma, and the disease is essentially untreatable if not cured surgically. The paucity of chemotherapeutic agents that are effective for treating metastatic melanoma indicates a dire need to develop new therapies. Here, we found a previously unrecognized role for c-Abl and Arg in melanoma progression. We demonstrate that the kinase activities of c-Abl and Arg are elevated in primary melanomas (60%), in a subset of benign nevi (33%) and in some human melanoma cell lines. Using siRNA and pharmacological approaches, we show that c-Abl/Arg activation is functionally relevant because it is requiredfor melanoma cell proliferation, survival and invasion. Significantly, we identify the mechanism by which activated c-Abl promotes melanoma invasion by showing that it transcriptionally upregulates matrix metalloproteinase-1 (MMP-1), and using rescue approaches we demonstrate that c-Abl promotes invasion through a STAT3 → MMP-1 pathway. Additionally, we show that c-Abl and Arg are not merely redundant, as active Arg drives invasion in a STAT3-independent manner, and upregulates MMP-3 and MT1-MMP, in addition to MMP-1. Most importantly, c-Abl and Arg not only promote in vitro processes important for melanoma progression, but also promote metastasis in vivo, as inhibition of c-Abl/Arg kinase activity with the c-Abl/Arg inhibitor, nilotinib, dramatically inhibits metastasis in a mouse model. Taken together, these data identify c-Abl and Arg as critical, novel, drug targets in metastatic melanoma, and indicate that nilotinib may be useful in preventing metastasis in patients with melanomas harboring active c-Abl and Arg.

  20. 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

    2016-01-05

    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.

  1. 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

  2. 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.

  3. The gallium complex KP46 exerts strong activity against primary explanted melanoma cells and induces apoptosis in melanoma cell lines

    PubMed Central

    Valiahdi, Seied Mojtaba; Heffeter, Petra; Jakupec, Michael A.; Marculescu, Rodrig; Berger, Walter; Rappersberger, Klemens; Keppler, Bernhard K.

    2012-01-01

    The antineoplastic properties of gallium are well documented. Owing to their robust accumulation of gallium, melanoma cells should be amenable to gallium-based anticancer drugs. With the aim of improving the disappointingly low activity of inorganic gallium salts, we have developed the orally bioavailable gallium complex KP46 [tris(8-quinolinolato)gallium(III)] that was already successfully studied in a phase I clinical trial. To assess its therapeutic potential in malignant melanoma, its antiproliferative effects were investigated in series of human cell lines and primary explanted melanoma samples by means of the MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay and the Human Tumor Cloning Assay, respectively. When compared with other cell lines, the majority of melanoma cells rank among the KP46-sensitive cell lines (50% inhibitory concentration values: 0.8–3.7 μmol/l). Clinically achievable concentrations of KP46 proved to be highly effective in melanoma cells from primary explants of cutaneous and lymph node metastases. Colony growth was inhibited in 10 of 10 specimens by 5 lmol/l KP46 (corresponding to the steady-state plasma concentration measured earlier in a study patient) and in four of 10 specimens by 0.5 μmol/l KP46. In-vitro potency of KP46 is higher than that of dacarbazine or fotemustine and comparable with that of cisplatin. The effects induced by KP46 in melanoma cell lines involve cell cycle perturbations (S-phase arrest) and apoptosis (activation of caspase-9, PARP [poly(ADP-ribose) polymerase] cleavage, formation of apoptotic bodies). No effects on DNA secondary structure could be observed in an electrophoretic mobility shift assay using double-stranded plasmid DNA. Thus, further studies on the therapeutic applicability of KP46 in malignant melanoma are warranted. PMID:19584767

  4. 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

  5. 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.

  6. 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.

  7. Apigenin inhibits proliferation and invasion, and induces apoptosis and cell cycle arrest in human melanoma cells.

    PubMed

    Zhao, Guangming; Han, Xiaodong; Cheng, Wei; Ni, Jing; Zhang, Yunfei; Lin, Jingrong; Song, Zhiqi

    2017-04-01

    Malignant melanoma is the most invasive and fatal form of cutaneous cancer. Moreover it is extremely resistant to conventional chemotherapy and radiotherapy. Apigenin, a non-mutagenic flavonoid, has been found to exhibit chemopreventive and/or anticancerogenic properties in many different types of human cancer cells. Therefore, apigenin may have particular relevance for development as a chemotherapeutic agent for cancer treatment. In the present study, we investigated the effects of apigenin on the viability, migration and invasion potential, dendrite morphology, cell cycle distribution, apoptosis, phosphorylation of the extracellular signal-regulated protein kinase (ERK) and the AKT/mTOR signaling pathway in human melanoma A375 and C8161 cell lines in vitro. Apigenin effectively suppressed the proliferation of melanoma cells in vitro. Moreover, it inhibited cell migration and invasion, lengthened the dendrites, and induced G2/M phase arrest and apoptosis. Furthermore, apigenin promoted the activation of cleaved caspase-3 and cleaved PARP proteins and decreased the expression of phosphorylated (p)‑ERK1/2 proteins, p-AKT and p-mTOR. Consequently, apigenin is a novel therapeutic candidate for melanoma.

  8. 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

  9. Antitumor Activity of BRAF Inhibitor and IFNα Combination in BRAF-Mutant Melanoma

    PubMed Central

    Sabbatino, Francesco; Wang, Yangyang; Scognamiglio, Giosuè; Favoino, Elvira; Feldman, Steven A.; Villani, Vincenzo; Flaherty, Keith T.; Nota, Sjoerd; Giannarelli, Diana; Simeone, Ester; Anniciello, Anna M.; Palmieri, Giuseppe; Pepe, Stefano; Botti, Gerardo; Ascierto, Paolo A.; Ferrone, Cristina R.

    2016-01-01

    Background: BRAFV600E-mediated MAPK pathway activation is associated in melanoma cells with IFNAR1 downregulation. IFNAR1 regulates melanoma cell sensitivity to IFNα, a cytokine used for the adjuvant treatment of melanoma. These findings and the limited therapeutic efficacy of BRAF-I prompted us to examine whether the efficacy of IFNα therapy of BRAFV600E melanoma can be increased by its combination with BRAF-I. Methods: BRAF/NRAS genotype, ERK activation, IFNAR1, and HLA class I expression were tested in 60 primary melanoma tumors from treatment-naive patients. The effect of BRAF-I on IFNAR1 expression was assessed in three melanoma cell lines and in four biopsies of BRAFV600E metastases. The antiproliferative, pro-apoptotic and immunomodulatory activity of BRAF-I and IFNα combination was tested in vitro and in vivo utilizing three melanoma cell lines, HLA class I-MA peptide complex-specific T-cells and immunodeficient mice (5 per group for survival and 10 per group for tumor growth inhibition). All statistical tests were two-sided. Differences were considered statistically significant when the P value was less than .05. Results: The IFNAR1 level was statistically significantly (P < .001) lower in BRAFV600E primary melanoma tumors than in BRAF wild-type tumors. IFNAR1 downregulation was reversed by BRAF-I treatment in the three melanoma cell lines (P ≤ .02) and in three out of four metastases. The IFNAR1 level in the melanoma tumors analyzed was increased as early as 10 to 14 days following the beginning of the treatment. These changes were associated with: 1) an increased susceptibility in vitro of melanoma cells to the antiproliferative (P ≤ .04), pro-apoptotic (P ≤ .009) and immunomodulatory activity, including upregulation of HLA class I antigen APM component (P ≤ .04) and MA expression as well as recognition by cognate T-cells (P < .001), of BRAF-I and IFNα combination and 2) an increased survival (P < .001) and inhibition of tumor growth of

  10. Myc down-regulation sensitizes melanoma cells to radiotherapy by inhibiting MLH1 and MSH2 mismatch repair proteins.

    PubMed

    Bucci, Barbara; D'Agnano, Igea; Amendola, Donatella; Citti, Arianna; Raza, Giorgio H; Miceli, Roberto; De Paula, Ugo; Marchese, Rodolfo; Albini, Sonia; Felsani, Armando; Brunetti, Ercole; Vecchione, Aldo

    2005-04-01

    Melanoma patients have a very poor prognosis with a response rate of <1% due to advanced diagnosis. This type of tumor is particularly resistant to conventional chemotherapy and radiotherapy, and the surgery remains the principal treatment for patients with localized melanoma. For this reason, there is particular interest in the melanoma biological therapy. Using two p53 mutant melanoma models stably expressing an inducible c-myc antisense RNA, we have investigated whether Myc protein down-regulation could render melanoma cells more susceptible to radiotherapy, reestablishing apoptotic p53-independent pathway. In addition to address the role of p53 in the activation of apoptosis, we studied the effect of Myc down-regulation on radiotherapy sensitivity also in a p53 wild-type melanoma cell line. Myc down-regulation is able per se to induce apoptosis in a fraction of the cell population (approximately 40% at 72 hours) and in combination with gamma radiation efficiently enhances the death process. In fact, approximately 80% of apoptotic cells are evident in Myc down-regulated cells exposed to gamma radiation for 72 hours compared with approximately 13% observed after only gamma radiation treatment. Consistent with the enhanced apoptosis is the inhibition of the MLH1 and MSH2 mismatch repair proteins, which, preventing the correction of ionizing radiation mismatches occurring during DNA replication, renders the cells more prone to radiation-induced apoptosis. Data herein reported show that Myc down-regulation lowers the apoptotic threshold in melanoma cells by inhibiting MLH1 and MSH2 proteins, thus increasing cell sensitivity to gamma radiation in a p53-independent fashion. Our results indicate the basis for developing new antitumoral therapeutic strategy, improving the management of melanoma patients.

  11. Flavonoids inhibit cell growth and induce apoptosis in B16 melanoma 4A5 cells.

    PubMed

    Iwashita, K; Kobori, M; Yamaki, K; Tsushida, T

    2000-09-01

    We investigated the growth inhibitory activity of several flavonoids, including apigenin, luteolin, kaempherol, quercetin, butein, isoliquiritigenin, naringenin, genistein, and daizein against B16 mouse melanoma 4A5 cells. Isoliquiritigenin and butein, belonging to the chalcone group, markedly suppressed the growth of B16 melanoma cells and induced cell death. The other flavonoids tested showed little growth inhibitory activity and scarcely caused cell death. In cells treated with isoliquiritigenin or butein, condensation of nuclei and fragmentation of nuclear DNA, which are typical phenomena of apoptosis, were observed by Hoechst 33258 staining and by agarose gel electrophoresis of DNA. Flowcytometric analysis showed that isoliquiritigenin and butein increased the proportion of hypodiploid cells in the population of B16 melanoma cells. These results demonstrate that isoliquiritigenin and butein inhibit cell proliferation and induce apoptosis in B16 melanoma cells. Extracellular glucose decreased the proportion of hypodiploid cells that appeared as a result of isoliquiritigenin treatment. p53 was not detected in cells treated with either of these chalcones, however, protein of the Bcl-2 family were detected. The level of expression of Bax in cells treated with either of these chalcones was markedly elevated and the level of Bcl-XL decreased slightly. Isoliquiritigenin did not affect Bcl-2 expression, but butein down-regulated Bcl-2 expression. From these results, it seems that the pathway by which the chalcones induce apoptosis may be independent of p53 and dependent on proteins of the Bcl-2 family. It was supposed that isoliquiritigenin induces apoptosis in B16 cells by a mechanism involving inhibition of glucose transmembrane transport and promotion of Bax expression. On the other hand, it was suggested that butein induces apoptosis via down-regulation of Bcl-2 expression and promotion of Bax expression. This mechanism differs from the isoliquiritigenin induction

  12. Lipid-Soluble Ginseng Extract Inhibits Invasion and Metastasis of B16F10 Melanoma Cells

    PubMed Central

    Yun, Jieun; Kim, Bo Geun; Kang, Jong Soon; Park, Song-Kyu; Lee, Kiho; Hyun, Dong-Hoon; In, Man-Jin

    2015-01-01

    Abstract This study was performed to elucidate the effect of a lipid-soluble ginseng extract (LSGE) on cancer invasion and metastasis. The LSGE, even at noncytotoxic concentrations, potently inhibited invasion and migration of B16F10 mouse melanoma cells in a dose-dependent manner. In the presence of 3 μg/mL of LSGE, the invasion and migration of B16F10 cells were significantly inhibited by 98.1% and 71.4%, respectively. Furthermore, the LSGE decreased mRNA and protein levels of matrix metalloproteinase (MMP)-2 in B16F10 cells, leading to a decrease in MMP-2 activity. After B16F10 cells were intravenously injected in the tail vein of C57BL/6 mice, 1000 mg/kg/day of LSGE was orally administered for 13 days, after which lung metastasis of cancer cells was inhibited by 59.3%. These findings indicate that LSGE inhibits cancer cell invasion and migration in vitro and lung metastasis of melanoma cells in vivo by inhibiting MMP-2 expression. PMID:25354136

  13. Statins improve survival by inhibiting spontaneous metastasis and tumor growth in a mouse melanoma model

    PubMed Central

    Tsubaki, Masanobu; Takeda, Tomoya; Kino, Toshiki; Obata, Naoya; Itoh, Tatsuki; Imano, Motohiro; Mashimo, Kenji; Fujiwara, Daichiro; Sakaguchi, Katsuhiko; Satou, Takao; Nishida, Shozo

    2015-01-01

    Metastatic melanoma is a life-threatening disease for which no effective treatment is currently available. In melanoma cells, Rho overexpression promotes invasion and metastasis. However, the effect of statins on spontaneous metastasis and tumor growth remains unclear. In the present study, we investigated the mechanism of statin-mediated tumor growth and metastasis inhibition in an in vivo model. We found that statins significantly inhibited spontaneous metastasis and tumor growth. Statins inhibited the mRNA expression and enzymatic activities of matrix metalloproteinases (MMPs) in vivo and also suppressed the mRNA and protein expression of very late antigens (VLAs). Moreover, statins inhibited the prenylation of Rho as well as the phosphorylation of LIM kinase, serum response factor (SRF), and c-Fos downstream of the Rho signaling pathway. In addition, statins enhanced p53, p21, and p27 expression and reduced phosphorylation of cyclin-dependent kinase and expression of cyclin D1 and E2. These results indicate that statins suppress Rho signaling pathways, thereby inhibiting tumor metastasis and growth. Furthermore, statins markedly improved the survival rate in a metastasis model, suggesting that statins have potential clinical applications for the treatment of metastatic cancers. PMID:26693069

  14. Parallel in vivo and in vitro melanoma RNAi dropout screens reveal synthetic lethality between hypoxia and DNA damage response inhibition.

    PubMed

    Possik, Patricia A; Müller, Judith; Gerlach, Carmen; Kenski, Juliana C N; Huang, Xinyao; Shahrabi, Aida; Krijgsman, Oscar; Song, Ji-Ying; Smit, Marjon A; Gerritsen, Bram; Lieftink, Cor; Kemper, Kristel; Michaut, Magali; Beijersbergen, Roderick L; Wessels, Lodewyk; Schumacher, Ton N; Peeper, Daniel S

    2014-11-20

    To identify factors preferentially necessary for driving tumor expansion, we performed parallel in vitro and in vivo negative-selection short hairpin RNA (shRNA) screens. Melanoma cells harboring shRNAs targeting several DNA damage response (DDR) kinases had a greater selective disadvantage in vivo than in vitro, indicating an essential contribution of these factors during tumor expansion. In growing tumors, DDR kinases were activated following hypoxia. Correspondingly, depletion or pharmacologic inhibition of DDR kinases was toxic to melanoma cells, including those that were resistant to BRAF inhibitor, and this could be enhanced by angiogenesis blockade. These results reveal that hypoxia sensitizes melanomas to targeted inhibition of the DDR and illustrate the utility of in vivo shRNA dropout screens for the identification of pharmacologically tractable targets. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  15. [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.

  16. Prolongation of survival in metastatic melanoma after active specific immunotherapy with a new polyvalent melanoma vaccine.

    PubMed Central

    Morton, D L; Foshag, L J; Hoon, D S; Nizze, J A; Famatiga, E; Wanek, L A; Chang, C; Davtyan, D G; Gupta, R K; Elashoff, R

    1992-01-01

    A new polyvalent melanoma cell vaccine (MCV) was administered to 136 stage IIIA and IV (American Joint Committee on Cancer) melanoma patients. Induction of cell-mediated and humoral immune responses to common melanoma-associated antigens present on autologous melanoma cells was observed in patients receiving the new MCV. This was accompanied by increased activation of tumor-infiltrating lymphocytes. Survival correlated significantly with delayed cutaneous hypersensitivity (p = 0.0066) and antibody responses to MCV (p = 0.0117). Of 40 patients with evaluable disease, nine (23%) had regressions (three complete). From our historical database of 126 stage IIIA and 1275 stage IV melanoma patients, there were no significant changes in the natural history of metastatic melanoma during the past 20 years. Univariate and multivariate analyses demonstrated prognostic significance for site of metastases (p = 0.0001) and immunotherapy with the new MCV (p = 0.0001). Overall our new MCV increased the median and 5-year survival of stage IIIA melanoma patients with regional soft tissue metastases twofold (p = 0.00024), and stage IV patients threefold (p = 0.0001) compared with previous immunotherapy and other treatments. Images FIG. 5. FIG. 5. FIG. 6. FIG. 18. FIG. 18. FIG. 19. PMID:1417196

  17. Dimethylthiourea inhibition of B16 melanoma growth and induction of phenotypic alterations; relationship to ATP levels.

    PubMed Central

    Fux, A.; Sidi, Y.; Kessler-Icekson, G.; Wasserman, L.; Novogrodsky, A.; Nordenberg, J.

    1991-01-01

    1,3 Dimethylthiourea (DMTU) has previously been shown by us to inhibit the growth of melanoma cells and to induce phenotypic alterations in these cells, including ultrastructural alterations of mitochondria. These findings raised the possibility that impaired mitochondrial function might be involved in mediating the effect of DMTU on cell growth and phenotypic expression. The present study indicates that DMTU as well as another growth inhibitory methylurea derivative, tetramethylurea (TMU) significantly decrease ATP content in the B16 melanoma cell line. 1,3 Dimethylurea (1,3DMU) and 1,1 dimethylurea (1,1DMU) which are poor growth inhibitors, do not reduce ATP content significantly. Altered energy metabolism in the DMTU-treated cells is reflected by inhibition of the activity of cytochrome c oxidase and by increased lactate levels. A cell line selected for resistance to growth inhibition by DMTU was shown to be completely resistant to induction of phenotypic alterations by DMTU. These cells possess high lactate levels, high ATP content and a somewhat decreased Na/K ATPase activity as compared to wild type B16 F10 cells. 1,3 DMTU treatment of the resistant cells leads to a decrease in the activity of the mitochondrial enzyme cytochrome c oxidase, similar to its effect on the wild type B16 F10 cells. DMTU also reduces ATP content moderately in the resistant cells. However, the levels of ATP do not decrease beyond those found in untreated B16 F10 wild type cells. Taken together the results suggest that decreased ATP content might be involved, at least partially, in mediating the effects of DMTU on B16 melanoma cell growth and phenotypic expression. PMID:1850608

  18. Gβγ subunits inhibit Epac-induced melanoma cell migration

    PubMed Central

    2011-01-01

    Background Recently we reported that activation of Epac1, an exchange protein activated by cAMP, increases melanoma cell migration via Ca 2+ release from the endoplasmic reticulum (ER). G-protein βγ subunits (Gβγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gβγ in cell migration and Ca 2+ signaling in melanoma has not been well studied. Here we report that there is crosstalk of Ca 2+ signaling between Gβγ and Epac in melanoma, which plays a role in regulation of cell migration. Methods SK-Mel-2 cells, a human metastatic melanoma cell line, were mainly used in this study. Intracellular Ca 2+ was measured with Fluo-4AM fluorescent dyes. Cell migration was examined using the Boyden chambers. Results The effect of Gβγ on Epac-induced cell migration was first examined. Epac-induced cell migration was inhibited by mSIRK, a Gβγ -activating peptide, but not its inactive analog, L9A, in SK-Mel-2 cells. Guanosine 5', α-β-methylene triphosphate (Gp(CH2)pp), a constitutively active GTP analogue that activates Gβγ, also inhibited Epac-induced cell migration. In addition, co-overexpression of β1 and γ2, which is the major combination of Gβγ, inhibited Epac1-induced cell migration. By contrast, when the C-terminus of β adrenergic receptor kinase (βARK-CT), an endogenous inhibitor for Gβγ, was overexpressed, mSIRK's inhibitory effect on Epac-induced cell migration was negated, suggesting the specificity of mSIRK for Gβγ. We next examined the effect of mSIRK on Epac-induced Ca 2+ response. When cells were pretreated with mSIRK, but not with L9A, 8-(4-Methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT), an Epac-specific agonist, failed to increase Ca 2+ signal. Co-overexpression of β1 and γ2 subunits inhibited 8-pMeOPT-induced Ca 2+ elevation. Inhibition of Gβγ with βARK-CT or guanosine 5'-O-(2-thiodiphosphate) (GDPβS), a GDP analogue that

  19. Gβγ subunits inhibit Epac-induced melanoma cell migration.

    PubMed

    Baljinnyam, Erdene; Umemura, Masanari; De Lorenzo, Mariana S; Xie, Lai-Hua; Nowycky, Martha; Iwatsubo, Mizuka; Chen, Suzie; Goydos, James S; Iwatsubo, Kousaku

    2011-06-17

    Recently we reported that activation of Epac1, an exchange protein activated by cAMP, increases melanoma cell migration via Ca 2+ release from the endoplasmic reticulum (ER). G-protein βγ subunits (Gβγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gβγ in cell migration and Ca 2+ signaling in melanoma has not been well studied. Here we report that there is crosstalk of Ca 2+ signaling between Gβγ and Epac in melanoma, which plays a role in regulation of cell migration. SK-Mel-2 cells, a human metastatic melanoma cell line, were mainly used in this study. Intracellular Ca 2+ was measured with Fluo-4AM fluorescent dyes. Cell migration was examined using the Boyden chambers. The effect of Gβγ on Epac-induced cell migration was first examined. Epac-induced cell migration was inhibited by mSIRK, a Gβγ -activating peptide, but not its inactive analog, L9A, in SK-Mel-2 cells. Guanosine 5', α-β-methylene triphosphate (Gp(CH2)pp), a constitutively active GTP analogue that activates Gβγ, also inhibited Epac-induced cell migration. In addition, co-overexpression of β1 and γ2, which is the major combination of Gβγ, inhibited Epac1-induced cell migration. By contrast, when the C-terminus of β adrenergic receptor kinase (βARK-CT), an endogenous inhibitor for Gβγ, was overexpressed, mSIRK's inhibitory effect on Epac-induced cell migration was negated, suggesting the specificity of mSIRK for Gβγ. We next examined the effect of mSIRK on Epac-induced Ca 2+ response. When cells were pretreated with mSIRK, but not with L9A, 8-(4-Methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT), an Epac-specific agonist, failed to increase Ca 2+ signal. Co-overexpression of β1 and γ2 subunits inhibited 8-pMeOPT-induced Ca 2+ elevation. Inhibition of Gβγ with βARK-CT or guanosine 5'-O-(2-thiodiphosphate) (GDPβS), a GDP analogue that inactivates Gβγ, restored 8-p

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

    PubMed

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

    2016-03-15

    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.

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

    SciTech Connect

    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. - Highlights: • MAGE-A1 promotes proliferation and clone formation in melanoma cell lines. • MAGE-A1 enhances tumor cell migration and invasion in melanoma cell lines. • Network including C-JUN, IL8, and ARHGAP29 play critical role in malignant melanoma. • Oncogenic MAGE-A1 increases p-C-JUN levels, possibly via ERK-MAPK signaling pathway.

  2. 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

  3. 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

  4. Exchange protein directly activated by cyclic AMP increases melanoma cell migration by a Ca2+-dependent mechanism.

    PubMed

    Baljinnyam, Erdene; De Lorenzo, Mariana S; Xie, Lai-Hua; Iwatsubo, Mizuka; Chen, Suzie; Goydos, James S; Nowycky, Martha C; Iwatsubo, Kousaku

    2010-07-01

    Melanoma has a poor prognosis due to its strong metastatic ability. Although Ca(2+) plays a major role in cell migration, little is known about the role of Ca(2+) in melanoma cell migration. We recently found that the exchange protein directly activated by cyclic AMP (Epac) increases melanoma cell migration via a heparan sulfate-related mechanism. In addition to this mechanism, we also found that Epac regulates melanoma cell migration by a Ca(2+)-dependent mechanism. An Epac agonist increased Ca(2+) in several different melanoma cell lines but not in melanocytes. Ablation of Epac1 with short hairpin RNA inhibited the Epac agonist-induced Ca(2+) elevation, suggesting the critical role of Epac1 in Ca(2+) homeostasis in melanoma cells. Epac-induced Ca(2+) elevation was negated by the inhibition of phospholipase C (PLC) and inositol triphosphate (IP(3)) receptor. Furthermore, Epac-induced cell migration was reduced by the inhibition of PLC or IP(3) receptor. These data suggest that Epac activates Ca(2+) release from the endoplasmic reticulum via the PLC/IP(3) receptor pathway, and this Ca(2+) elevation is involved in Epac-induced cell migration. Actin assembly was increased by Epac-induced Ca(2+), suggesting the involvement of actin in Epac-induced cell migration. In human melanoma specimens, mRNA expression of Epac1 was higher in metastatic melanoma than in primary melanoma, suggesting a role for Epac1 in melanoma metastasis. In conclusion, our findings reveal that Epac is a potential target for the suppression of melanoma cell migration, and, thus, the development of metastasis. Copyright 2010 AACR.

  5. An adaptive signaling network in melanoma inflammatory niches confers tolerance to MAPK signaling inhibition

    PubMed Central

    Luheshi, Nadia; Arozarena, Imanol; Acosta, Juan-Carlos; Kamarashev, Jivko; Frederick, Dennie T.; Cooper, Zachary A.; Flaherty, Keith T.; Smith, Michael P.

    2017-01-01

    Mitogen-activated protein kinase (MAPK) pathway antagonists induce profound clinical responses in advanced cutaneous melanoma, but complete remissions are frustrated by the development of acquired resistance. Before resistance emerges, adaptive responses establish a mutation-independent drug tolerance. Antagonizing these adaptive responses could improve drug effects, thereby thwarting the emergence of acquired resistance. In this study, we reveal that inflammatory niches consisting of tumor-associated macrophages and fibroblasts contribute to treatment tolerance through a cytokine-signaling network that involves macrophage-derived IL-1β and fibroblast-derived CXCR2 ligands. Fibroblasts require IL-1β to produce CXCR2 ligands, and loss of host IL-1R signaling in vivo reduces melanoma growth. In tumors from patients on treatment, signaling from inflammatory niches is amplified in the presence of MAPK inhibitors. Signaling from inflammatory niches counteracts combined BRAF/MEK (MAPK/extracellular signal–regulated kinase kinase) inhibitor treatment, and consequently, inhibiting IL-1R or CXCR2 signaling in vivo enhanced the efficacy of MAPK inhibitors. We conclude that melanoma inflammatory niches adapt to and confer drug tolerance toward BRAF and MEK inhibitors early during treatment. PMID:28450382

  6. Vaccination with a human high molecular weight melanoma-associated antigen mimotope induces a humoral response inhibiting melanoma cell growth in vitro.

    PubMed

    Wagner, Stefan; Hafner, Christine; Allwardt, Dorothee; Jasinska, Joanna; Ferrone, Soldano; Zielinski, Christoph C; Scheiner, Otto; Wiedermann, Ursula; Pehamberger, Hubert; Breiteneder, Heimo

    2005-01-15

    Peptide mimics of a conformational epitope that is recognized by a mAb with antitumor activity are promising candidates for formulations of anticancer vaccines. These mimotope vaccines are able to induce a polyclonal Ab response focused to the determinant of the mAb. Such attempts at cancer immunotherapy are of special interest for malignant melanoma that is highly resistant to chemotherapy and radiotherapy. In this study, we describe for the first time the design and immunogenicity of a vaccine containing a mimotope of the human high m.w. melanoma-associated Ag (HMW-MAA) and the biological potential of the induced Abs. Mimotopes were selected from a pVIII-9mer phage display peptide library with the anti-HMW-MAA mAb 225.28S. The mimotope vaccine was then generated by coupling the most suitable candidate mimotope to tetanus toxoid as an immunogenic carrier. Immunization of rabbits with this vaccine induced a specific humoral immune response directed toward the epitope recognized by the mAb 225.28S on the native HMW-MAA. The induced Abs inhibited the in vitro growth of the melanoma cell line 518A2 up to 62%. In addition, the Abs mediated 26% lysis of 518A2 cells in Ab-dependent cellular cytotoxicity. Our results indicate a possible application of this mimotope vaccine as a novel immunotherapeutic agent for the treatment of malignant melanoma.

  7. 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.

  8. Sarcophine-Diol, a Skin Cancer Chemopreventive Agent, Inhibits Proliferation and Stimulates Apoptosis in Mouse Melanoma B16F10 Cell Line

    PubMed Central

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

    2011-01-01

    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

  9. Inhibition of uPAR-TGFβ crosstalk blocks MSC-dependent EMT in melanoma cells.

    PubMed

    Laurenzana, Anna; Biagioni, Alessio; Bianchini, Francesca; Peppicelli, Silvia; Chillà, Anastasia; Margheri, Francesca; Luciani, Cristina; Pimpinelli, Nicola; Del Rosso, Mario; Calorini, Lido; Fibbi, Gabriella

    2015-07-01

    The capacity of cancer cells to undergo epithelial-to-mesenchymal transition (EMT) is now considered a hallmark of tumor progression, and it is known that interactions between cancer cells and mesenchymal stem cells (MSCs) of tumor microenvironment may promote this program. Herein, we demonstrate that MSC-conditioned medium (MSC-CM) is a potent inducer of EMT in melanoma cells. The EMT profile acquired by MSC-CM-exposed melanoma cells is characterized by an enhanced level of mesenchymal markers, including TGFβ/TGFβ-receptors system upregulation, by increased invasiveness and uPAR expression, and in vivo tumor growth. Silencing TGFβ in MSC is found to abrogate ability of MSC to promote EMT characteristics in melanoma cells, together with uPAR expression, and this finding is strengthened using an antagonist peptide of TGFβRIII, the so-called P17. Finally, we demonstrate that the uPAR antisense oligonucleotide (uPAR aODN) may inhibit EMT of melanoma cells either stimulated by exogenous TGFβ or MSC-CM. Thus, uPAR upregulation in melanoma cells exposed to MSC-medium drives TGFβ-mediated EMT. On the whole, TGFβ/uPAR dangerous liaison in cancer cell/MSC interactions may disclose a new strategy to abrogate melanoma progression. Mesenchymal stem cell (MSC)-conditioned medium induces EMT-like profile in melanoma. MSC-derived TGFβ promotes uPAR and TGFβ/TGFβ-receptor upregulation in melanoma. TGFβ gene silencing in MSCs downregulates uPAR expression and EMT in melanoma. uPAR downregulation prevents MSC-induced EMT-like profile in melanoma cells. Inhibition of the dangerous TGFβ/uPAR relationship might abrogate melanoma progression.

  10. Roscovitine inhibits differentiation and invasion in a three-dimensional skin reconstruction model of metastatic melanoma.

    PubMed

    Mohapatra, Subhra; Coppola, Domenico; Riker, Adam I; Pledger, W Jack

    2007-02-01

    The aim of this study was to investigate the therapeutic potential of a cyclin-dependent kinase inhibitor, roscovitine, in cultured melanoma cells and a three-dimensional skin reconstruction model of metastatic melanoma. The modulatory effects of roscovitine on the growth and survival of normal melanocytes and cultured melanoma cell lines were tested. Additionally, we investigated the potential of roscovitine to regulate the growth and differentiation of a metastatic melanoma cell line (A375) in a three-dimensional skin reconstruction culture consisting of A375 cells admixed with normal human keratinocytes embedded within a collagen-constricted fibroblast matrix. We show that roscovitine is able to induce apoptosis in the melanoma cell lines A375, 888, and 624 but not in normal human cultured epithelial melanocytes. The degree of apoptosis within these cell lines correlated with the accumulation of p53 protein and concomitant reduction of X-linked inhibitor of apoptosis protein, with no change in the proteins Bcl-2 and survivin. We also found that roscovitine inhibited the growth and differentiation of A375 melanoma cells within the dermal layer of the skin. The results of this study show that roscovitine has the potential to inhibit the differentiation and invasion of metastatic melanoma and may be useful as a therapy for the treatment of patients with metastatic melanoma.

  11. Dimethylfumarate inhibits melanoma cell proliferation via p21 and p53 induction and bcl-2 and cyclin B1 downregulation.

    PubMed

    Kaluzki, Irina; Hrgovic, Igor; Hailemariam-Jahn, Tsige; Doll, Monika; Kleemann, Johannes; Valesky, Eva Maria; Kippenberger, Stefan; Kaufmann, Roland; Zoeller, Nadja; Meissner, Markus

    2016-10-01

    Recent evidence suggests that dimethylfumarate (DMF), known as a highly potent anti-psoriatic agent, might have anti-tumorigenic properties in melanoma. It has recently been demonstrated that DMF inhibits melanoma proliferation by apoptosis and cell cycle inhibition and therefore inhibits melanoma metastasis. Nonetheless, the underlying mechanisms remain to be evaluated. To elucidate the effects of DMF on melanoma cell lines (A375, SK-Mel), we first performed cytotoxicity assays. No significant lactatedehydogenase (LDH) release could be found. In further analysis, we showed that DMF suppresses melanoma cell proliferation in a concentration-dependent manner. To examine whether these effects are conveyed by apoptotic mechanisms, we studied the amount of apoptotic nucleosomes and caspase 3/7 activity using ELISA analysis. Significant apoptosis was induced by DMF in both cell lines, and this could be paralleled with bcl-2 downregulation and PARP-1 cleavage. We also performed cell cycle analysis and found that DMF induced concentration-dependent arrests of G0/G1 as well as G2/M. To examine the underlying mechanisms of cell cycle arrest, we analyzed the expression profiles of important cell cycle regulator proteins such as p53, p21, cyclins A, B1, and D1, and CDKs 3, 4, and 6. Interestingly, DMF induced p53 and p21 yet inhibited cyclin B1 expression in a concentration-dependent manner. Other cell cycle regulators were not influenced by DMF. The knockdown of DMF induced p53 via siRNA led to significantly reduced apoptosis but had no influence on cell cycle arrest. We examined the adhesion of melanoma cells on lymphendothelial cells during DMF treatment and found a significant reduction in interaction. These data provide evidence that DMF inhibits melanoma proliferation by reinduction of important cell cycle inhibitors leading to a concentration-dependent G0/G1 or G2/M cell cycle arrest and induction of apoptosis via downregulation of bcl-2 and induction of p53 and PARP-1

  12. Different immunology mechanisms of Phellinus igniarius in inhibiting growth of liver cancer and melanoma cells.

    PubMed

    Zhou, Cui; Jiang, Song-Song; Wang, Cui-Yan; Li, Rong; Che, Hui-Lian

    2014-01-01

    To assess inhibition mechanisms of a Phellinus igniarius (PI) extract on cancer, C57BL/6 mice were orally treated with PI extractive after or before implanting H22 (hepatocellular carcinoma ) or B16 (melanoma) cells. Mice were orally gavaged with different doses of PI for 36 days 24h after introduction of H22 or B16 cells. Mice in another group were orally treated as above daily for 42 days and implanted with H22 cells on day 7. Then the T lymphocyte, antibody, cytokine, LAK, NK cell activity in spleen, tumor cell apoptosis status and tumor inhibition in related organs, as well as the expression of iNOS and PCNA in tumor tissue were examined. The PI extract could improve animal immunity as well as inhibit cancer cell growth and metastasis with a dose-response relationship. Notably, PI's regulation with the two kinds of tumor appeared to occur in different ways, since the antibody profile and tumor metastasis demonstrated variation between animals implanted with hepatocellular carcinoma and melanoma cells.

  13. Combination small molecule MEK and PI3K inhibition enhances uveal melanoma cell death in a mutant GNAQ- and GNA11-dependent manner.

    PubMed

    Khalili, Jahan S; Yu, Xiaoxing; Wang, Ji; Hayes, Brendan C; Davies, Michael A; Lizee, Gregory; Esmaeli, Bita; Woodman, Scott E

    2012-08-15

    Activating Q209L/P mutations in GNAQ or GNA11 (GNAQ/11) are present in approximately 80% of uveal melanomas. Mutant GNAQ/11 are not currently therapeutically targetable. Inhibiting key down-stream effectors of GNAQ/11 represents a rational therapeutic approach for uveal melanomas that harbor these mutations. The mitogen-activated protein/extracellular signal-regulated kinase/mitogen-activated protein kinase (MEK/MAPK) and PI3K/AKT pathways are activated in uveal melanoma. In this study, we test the effect of the clinically relevant small molecule inhibitors GSK1120212 (MEK inhibitor) and GSK2126458 (pan class I PI3K inhibitor) on uveal melanoma cells with different GNAQ/11 mutation backgrounds. We use the largest set of genetically annotated uveal melanoma cell lines to date to carry out in vitro cellular signaling, cell-cycle regulation, growth, and apoptosis analyses. RNA interference and small molecule MEK and/or PI3K inhibitor treatment were used to determine the dependency of uveal melanoma cells with different GNAQ/11 mutation backgrounds on MEK/MAPK and/or PI3K/AKT signaling. Proteomic network analysis was done to unveil signaling alterations in response to MEK and/or PI3K small molecule inhibition. GNAQ/11 mutation status was not a determinant of whether cells would undergo cell-cycle arrest or growth inhibition to MEK and/or phosphoinositide 3-kinase (PI3K) inhibition. A reverse correlation was observed between MAPK and AKT phosphorylation after MEK or PI3K inhibition, respectively. Neither MEK nor PI3K inhibition alone was sufficient to induce apoptosis in the majority of cell lines; however, the combination of MEK + PI3K inhibitor treatment resulted in the marked induction of apoptosis in a GNAQ/11 mutant-dependent manner. MEK + PI3K inhibition may be an effective combination therapy in uveal melanoma, given the inherent reciprocal activation of these pathways within these cells.

  14. Noxa upregulation by oncogenic activation of MEK/ERK through CREB promotes autophagy in human melanoma cells

    PubMed Central

    Wilmott, James S.; Yan, Xu Guang; Liu, Xiao Ying; Luan, Qi; Guo, Su Tang; Jiang, Chen Chen; Tseng, Hsin-Yi; Scolyer, Richard A.; Jin, Lei; Zhang, Xu Dong

    2014-01-01

    Reduction in the expression of the anti-survival BH3-only proteins PUMA and Bim is associated with the pathogenesis of melanoma. However, we have found that the expression of the other BH3-only protein Noxa is commonly upregulated in melanoma cells, and that this is driven by oncogenic activation of MEK/ERK. Immunohistochemistry studies showed that Noxa was expressed at higher levels in melanomas than nevi. Moreover, the expression of Noxa was increased in metastatic compared to primary melanomas, and in thick primaries compared to thin primaries. Inhibition of oncogenic BRAFV600E or MEK downregulated Noxa, whereas activation of MEK/ERK caused its upregulation. In addition, introduction of BRAFV600E increased Noxa expression in melanocytes. Upregulation of Noxa was due to a transcriptional increase mediated by cAMP responsive element binding protein, activation of which was also increased by MEK/ERK signaling in melanoma cells. Significantly, Noxa appeared necessary for constitutive activation of autophagy, albeit at low levels, by MEK/ERK in melanoma cells. Furthermore, it was required for autophagy activation that delayed apoptosis in melanoma cells undergoing nutrient deprivation. These results reveal that oncogenic activation of MEK/ERK drives Noxa expression to promote autophagy, and suggest that Noxa has an indirect anti-apoptosis role in melanoma cells under nutrient starvation conditions. PMID:25365078

  15. Structural Modifications of (Z)-3-(2-aminoethyl)-5-(4-ethoxybenzylidene)thiazolidine-2,4-dione that Improve Selectivity for the Inhibition of Melanoma Cells Containing Active ERK Signaling

    PubMed Central

    Jung, Kwan-Young; Samadani, Ramin; Chauhan, Jay; Nevels, Kerrick; Yap, Jeremy L.; Zhang, Jun; Worlikar, Shilpa; Lanning, Maryanna E.; Chen, Lijia; Ensey, Mary; Shukla, Sagar; Salmo, Rosene; Heinzl, Geoffrey; Gordon, Caryn; Dukes, Troy; MacKerell, Alexander D.; Shapiro, Paul; Fletcher, Steven

    2013-01-01

    Towards the development of potent and selective inhibitors of melanoma cells containing active ERK signaling, we herein report on the pharmacophore determination and optimization of the ERK docking domain inhibitor (Z)-3-(2-aminoethyl)-5-(4-ethoxybenzylidene)thiazolidine-2,4-dione. PMID:23624850

  16. Topical treatment of all-trans retinoic acid inhibits murine melanoma partly by promoting CD8(+) T-cell immunity.

    PubMed

    Yin, Wei; Song, Yan; Liu, Qing; Wu, Yunyun; He, Rui

    2017-10-01

    All-trans retinoic acid (atRA), the main biologically active metabolite of vitamin A, has been implicated in immunoregulation and anti-cancer. A recent finding that vitamin A could decrease the risk of melanoma in humans indicates the beneficial role of atRA in melanoma. However, it remains unknown whether topical application of atRA could inhibit melanoma growth by influencing tumour immunity. We demonstrate topical application of tretinoin ointment (atRA as the active ingredient) effectively inhibited B16F10 melanoma growth. This is accompanied by markedly enhanced CD8(+) T-cell responses, as evidenced by significantly increased proportions of effector CD8(+) T cells expressing granzyme B, tumour necrosis factor-α, or interferon-γ, and Ki67(+) proliferating CD8(+) T cells in atRA-treated tumours compared with vaseline controls. Furthermore, topical atRA treatment promoted the differentiation of effector CD8(+) T cells in draining lymph nodes (DLN) of tumour-bearing mice. Interestingly, atRA did not affect tumoral CD4(+) T-cell response, and even inhibited the differentiation of interferon-γ-expressing T helper type 1 cells in DLN. Importantly, we demonstrated that the tumour-inhibitory effect of atRA was partly dependent on CD8(+) T cells, as CD8(+) T-cell depletion restored tumour volumes in atRA-treated mice, which, however, was still significantly smaller than those in vaseline-treated mice. Finally, we demonstrated that atRA up-regulated MHCI expression in B16F10 cells, and DLN cells from tumour-bearing mice had a significantly higher killing rate when culturing with atRA-treated B16F10 cells. Hence, our study demonstrates that topical atRA treatment effectively inhibits melanoma growth partly by promoting the differentiation and the cytotoxic function of effector CD8(+) T cells. © 2017 John Wiley & Sons Ltd.

  17. 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

  18. 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.

  19. Selective growth inhibition of human malignant melanoma cells by syringic acid-derived proteasome inhibitors

    PubMed Central

    2013-01-01

    Background It has been shown that proteasome inhibition leads to growth arrest in the G1 phase of the cell cycle and/or induction of apoptosis. However, it was found that some of these inhibitors do not induce apoptosis in several human normal cell lines. This selective activity makes proteasome inhibition a promising target for new generation of anticancer drugs. Clinical validation of the proteasome, as a therapeutic target in oncology, has been provided by the dipeptide boronic acid derivative; bortezomib. Bortezomib has proven to be effective as a single agent in multiple myeloma and some forms of non-Hodgkin’s lymphoma. Syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid, 1), a known phenolic acid, was isolated from the methanol extract of Tamarix aucheriana and was shown to possess proteasome inhibitory activity. Methods Using Surflex-Dock program interfaced with SYBYL, the docking affinities of syringic acid and its proposed derivatives to 20S proteasome were studied. Several derivatives were virtually proposed, however, five derivatives: benzyl 4-hydroxy-3,5-dimethoxybenzoate (2), benzyl 4-(benzyloxy)-3,5-dimethoxybenzoate (3), 3'-methoxybenzyl 3,5-dimethoxy-4-(3'-methoxybenzyloxy)benzoate (4), 3'-methoxybenzyl 4-hydroxy-3,5-dimethoxybenzoate (5) and 3',5'-dimethoxybenzyl 4-hydroxy-3,5-dimethoxybenzoate (6), were selected based on high docking scores, synthesized, and tested for their anti-mitogenic activity against human colorectal, breast and malignant melanoma cells as well as normal human fibroblast cells. Results Derivatives 2, 5, and 6 showed selective dose-dependent anti-mitogenic effect against human malignant melanoma cell lines HTB66 and HTB68 with minimal cytotoxicity on colorectal and breast cancer cells as well as normal human fibroblast cells. Derivatives 2, 5 and 6 significantly (p ≤ 0.0001) inhibited the various proteasomal chymotrypsin, PGPH, and trypsin like activities. They growth arrested the growth of HTB66 cells at G1 and G2

  20. Melanoma

    MedlinePlus

    ... flat or raised, large or small, light or dark, and can appear anywhere on our bodies. Sometimes, ... can still get melanoma even if they're dark skinned, young, and have no family history. Even ...

  1. Melanoma

    MedlinePlus

    ... the most important contributors to melanoma is ultraviolet (UV) sun damage. Cells that have been damaged — particularly ... red hair) multiple moles (typically, more than 25) UV exposure (whether from the sun or a tanning ...

  2. Melanoma

    MedlinePlus

    ... gls/pdf/melanoma.pdf . Accessed March 17, 2016. Review Date 1/31/2016 Updated by: Kevin Berman, ... PhD, Atlanta Center for Dermatologic Disease, Atlanta, GA. Review provided by VeriMed Healthcare Network. Also reviewed by ...

  3. Inhibition of L-tyrosine-induced micronuclei production by phenylthiourea in human melanoma cells.

    PubMed

    Poma, A; Bianchini, S; Miranda, M

    1999-12-13

    It was previously found that L-tyrosine oxidation product(s) are cytotoxic, genotoxic and increase the sister chromatid exchange (SCE) levels in human melanoma cells. In this work, the micronucleus assay has been performed on human melanotic and amelanotic melanoma cell lines (Carl-1 MEL and AMEL) in the presence of 1.0, 0.5 and 0.1 mM L-tyrosine concentrations to investigate if melanin synthesis intermediate(s) increase micronuclei production. L-Tyrosine oxidation product(s) increased the frequency of micronuclei in melanoma cells; 0.1 mM phenylthiourea (PTU), an inhibitor of L-tyrosine oxidation by tyrosinase, lowered the micronucleus production to the control levels. The culture of melanoma cells with high L-tyrosine in the culture medium resulted in a positive response to an ELISA-based apoptotic test. For comparison the effect of L-tyrosine on micronuclei production in human amelanotic melanoma cells was also investigated; the micronucleus production in the presence of 1 mM L-tyrosine in the culture medium was lower than that found with melanotic melanoma cells of the same cell line. The data suggest that melanin synthesis intermediates arising from L-tyrosine oxidation may cause micronuclei production in Carl-1 human melanoma cells; the addition of PTU in the presence of L-tyrosine decreased the frequency of micronuclei to about the control values thus the inhibition of melanogenesis may have some clinical implication in melanotic melanoma.

  4. Long non-coding RNA GAS5 inhibits tumorigenesis via miR-137 in melanoma

    PubMed Central

    Bian, Donghui; Shi, Wen; Shao, Yang; Li, Peilong; Song, Guodong

    2017-01-01

    Melanoma is the leading cause of death in patients with skin cancer. In the present study, we aimed to prove the functions and molecular mechanisms of lncRNA-GAS5 in melanoma. Herein, we found that the expression of GAS5 was down-regulated in melanoma tissues compared to adjacent normal tissues. GAS5 was significantly associated with distal metastasis and TNM stage in melanoma. Furthermore, we found that GAS5 suppressed melanoma cell proliferation, migration and invasion. Then, we found thatmiR-137 was decreased in melanoma tissues compared to adjacent normal tissues and was correlated with GAS5. Using a luciferase reporter gene assay, we also demonstrated that GAS5 positively regulated miR-137 transcription. Finally, we suggested that GAS5 inhibited the growth of melanoma through miR-137 in vivo. Therefore, our research demonstrated that the GAS5/miR-137 axis could be a potential therapeutic target for the treatment of melanoma. PMID:28386376

  5. Hypericum perforatum L. subsp. perforatum induces inhibition of free radicals and enhanced phototoxicity in human melanoma cells under ultraviolet light.

    PubMed

    Menichini, G; Alfano, C; Marrelli, M; Toniolo, C; Provenzano, E; Statti, G A; Nicoletti, M; Menichini, F; Conforti, F

    2013-04-01

    Our interest continues in discovering phytocomplexes from medicinal plants with phototoxic activity against human melanoma cells; thus the aim of the present study was to assess antioxidant, anti-inflammatory and phototoxic activity of Hypericum perforatum L. subsp. perforatum, and relate these properties to the plant's chemical composition. Components of H. perforatum subsp. perforatum were extracted by hydroalcoholic solution and chemical profiles of preparations (HyTE-3) performed by HPTLC. Linoleic acid peroxidation and DPPH tests were used to assess antioxidant activity, while MTT assay allowed evaluation of anti-proliferative activity with respect to A375 human melanoma cells after irradiation with UVA dose, 1.8 J/cm(2) . Inhibition of nitric oxide production of macrophages was also investigated. HyTE-3 indicated better antioxidant activity with β-carotene bleaching test in comparison to DPPH assay (IC50 = 0.89 μg/ml); significant phototoxicity in A375 cells at 78 μg/ml concentration resulted in cell destruction of 50%. HyTE-3 caused significant dose-related inhibition of nitric oxide production in murine monocytic macrophage cell line RAW 264.7 with IC50 value of 342 μg/ml. The H. perforatum subsp. perforatum-derived product was able to suppress proliferation of human malignant melanoma A375 cells; extract together with UVA irradiation enhanced phototoxicity. This biological activity of antioxidant effects was combined with inhibition of nitric oxide production. © 2013 Blackwell Publishing Ltd.

  6. 6-Bromoindirubin-3'-oxime inhibits JAK/STAT3 signaling and induces apoptosis of human melanoma cells.

    PubMed

    Liu, Lucy; Nam, Sangkil; Tian, Yan; Yang, Fan; Wu, Jun; Wang, Yan; Scuto, Anna; Polychronopoulos, Panos; Magiatis, Prokopios; Skaltsounis, Leandros; Jove, Richard

    2011-06-01

    STAT3 is persistently activated and contributes to malignant progression in various cancers. Janus activated kinases (JAK) phosphorylate STAT3 in response to stimulation by cytokines or growth factors. The STAT3 signaling pathway has been validated as a promising target for development of anticancer therapeutics. Small-molecule inhibitors of JAK/STAT3 signaling represent potential molecular-targeted cancer therapeutic agents. In this study, we investigated the role of JAK/STAT3 signaling in 6-bromoindirubin-3'-oxime (6BIO)-mediated growth inhibition of human melanoma cells and assessed 6BIO as a potential anticancer drug candidate. We found that 6BIO is a pan-JAK inhibitor that induces apoptosis of human melanoma cells. 6BIO directly inhibited JAK-family kinase activity, both in vitro and in cancer cells. Apoptosis of human melanoma cells induced by 6BIO was associated with reduced phosphorylation of JAKs and STAT3 in both dose- and time-dependent manners. Consistent with inhibition of STAT3 signaling, expression of the antiapoptotic protein Mcl-1 was downregulated. In contrast to the decreased levels of phosphorylation of JAKs and STAT3, phosphorylation levels of the Akt and mitogen-activated protein kinase (MAPK) signaling proteins were not inhibited in cells treated with 6BIO. Importantly, 6BIO suppressed tumor growth in vivo with low toxicity in a mouse xenograft model of melanoma. Taken together, these results show that 6BIO is a novel pan-JAK inhibitor that can selectively inhibit STAT3 signaling and induces tumor cell apoptosis. Our findings support further development of 6BIO as a potential anticancer therapeutic agent that targets JAK/STAT3 signaling in tumor cells.

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

    PubMed Central

    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

    2016-01-01

    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 BRAFV600E, the driver oncogene in the majority of melanomas, and in extrapolation of the correlation data, interference with\\ Pin1 in BRAFV600E-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 BRAFV600E-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

  8. Apigenin Attenuates Melanoma Cell Migration by Inducing Anoikis through Integrin and Focal Adhesion Kinase Inhibition.

    PubMed

    Hasnat, Md Abul; Pervin, Mehnaz; Lim, Ji Hong; Lim, Beong Ou

    2015-11-27

    Apigenin, a nonmutagenic flavonoid, has been found to have antitumor properties and is therefore particularly relevant for the development of chemotherapeutic agents for cancers. In this study, time- and dose-dependent cell viability and cytotoxicity were assessed to determine the effects of apigenin on A2058 and A375 melanoma cells. Melanoma cells were pretreated with different concentrations of apigenin and analyzed for morphological changes, anoikis induction, cell migration, and levels of proteins associated with apoptosis. Apigenin reduced integrin protein levels and inhibited the phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK1/2), which induce anoikis in human cutaneous melanoma cells. Apigenin exhibited dose-dependent inhibition of melanoma cell migration, unlike untreated controls. Furthermore, apigenin treatment increased apoptotic factors such as caspase-3 and cleaved poly(ADP-ribose) polymerase in a dose-dependent manner, demonstrating the metastasis of melanoma cells. Our results provide a new insight into the mechanisms by which apigenin prevents melanoma metastasis by sensitizing anoikis induced by the loss of integrin proteins in the FAK/ERK1/2 signaling pathway. These findings elucidate the related mechanisms and suggest the potential of apigenin in developing clinical treatment strategies against malignant melanoma.

  9. Human adipose tissue-derived mesenchymal stem cells inhibit melanoma growth in vitro and in vivo.

    PubMed

    Ahn, Jin-Ok; Coh, Ye-Rin; Lee, Hee-Woo; Shin, Il-Seob; Kang, Sung-Keun; Youn, Hwa-Young

    2015-01-01

    The effects of adipose tissue-derived mesenchymal stem cells (AT-MSCs) on the growth of human malignancies, including melanoma, are controversial and the underlying mechanisms are not yet-well understood. The aim of the present study was to investigate the in vitro and in vivo anti-tumor effects of human AT-MSCs on human melanoma. The inhibitory effect of AT-MSC-conditioned medium (AT-MSC-CM) on the growth of A375SM and A375P (human melanoma) cells was evaluated using a cell viability assay. Cell-cycle arrest and apoptosis in melanoma cells were investigated by flow cytometry and western blot analysis. To evaluate the in vivo anti-tumor effect of AT-MSCs, CM-DiI-labeled AT-MSCs were circumtumorally injected in tumor-bearing athymic mice and tumor size was measured. AT-MSC-CM inhibited melanoma growth by altering cell-cycle distribution and inducing apoptosis in vitro. AT-MSCs suppressed tumor growth in tumor-bearing athymic mice and fluorescence analysis showed that AT-MSCs migrated efficiently to tumor tissues. AT-MSCs inhibit the growth of melanoma suggesting promise as a novel therapeutic agent for melanoma. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  10. Anticancer activity of cationic porphyrins in melanoma tumour-bearing mice and mechanistic in vitro studies

    PubMed Central

    2014-01-01

    Background Porphyrin TMPyP4 (P4) and its C14H28-alkyl derivative (C14) are G-quadruplex binders and singlet oxygen (1O2) generators. In contrast, TMPyP2 (P2) produces 1O2 but it is not a G-quadruplex binder. As their photosensitizing activity is currently undefined, we report in this study their efficacy against a melanoma skin tumour and describe an in vitro mechanistic study which gives insights into their anticancer activity. Methods Uptake and antiproliferative activity of photoactivated P2, P4 and C14 have been investigated in murine melanoma B78-H1 cells by FACS, clonogenic and migration assays. Apoptosis was investigated by PARP-1 cleavage and annexin-propidium iodide assays. Biodistribution and in vivo anticancer activity were tested in melanoma tumour-bearing mice. Porphyrin binding and photocleavage of G-rich mRNA regions were investigated by electrophoresis and RT-PCR. Porphyrin effect on ERK pathway was explored by Western blots. Results Thanks to its higher lipophylicity C14 was taken up by murine melanoma B78-H1 cells up to 30-fold more efficiently than P4. When photoactivated (7.2 J/cm2) in B78-H1 melanoma cells, P4 and C14, but not control P2, caused a strong inhibition of metabolic activity, clonogenic growth and cell migration. Biodistribution studies on melanoma tumour-bearing mice showed that P4 and C14 localize in the tumour. Upon irradiation (660 nm, 193 J/cm2), P4 and C14 retarded tumour growth and increased the median survival time of the treated mice by ~50% (P <0.01 by ANOVA), whereas porphyrin P2 did not. The light-dependent mechanism mediated by P4 and C14 is likely due to the binding to and photocleavage of G-rich quadruplex-forming sequences within the 5′-untranslated regions of the mitogenic ras genes. This causes a decrease of RAS protein and inhibition of downstream ERK pathway, which stimulates proliferation. Annexin V/propidium iodide and PARP-1 cleavage assays showed that the porphyrins arrested tumour growth by apoptosis

  11. Fisetin inhibits human melanoma cell invasion through promotion of mesenchymal to epithelial transition and by targeting MAPK and NFκB signaling pathways.

    PubMed

    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

  12. 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

  13. Targeting BMK1 Impairs the Drug Resistance to Combined Inhibition of BRAF and MEK1/2 in Melanoma

    PubMed Central

    Song, Chengli; Wang, Lina; Xu, Qiang; Wang, Kai; Xie, Dan; Yu, Zhe; Jiang, Kui; Liao, Lujian; Yates, John R.; Lee, Jiing-Dwan; Yang, Qingkai

    2017-01-01

    Combined inhibition of BRAF and MEK1/2 (CIBM) improves therapeutic efficacy of BRAF-mutant melanoma. However, drug resistance to CIBM is inevitable and the drug resistance mechanisms still remain to be elucidated. Here, we show that BMK1 pathway contributes to the drug resistance to CIBM. Considering that ERK1/2 pathway regulates cellular processes by phosphorylating, we first performed a SILAC phosphoproteomic profiling of CIBM. Phosphorylation of 239 proteins was identified to be downregulated, while phosphorylation of 47 proteins was upregulated. Following siRNA screening of 47 upregulated proteins indicated that the knockdown of BMK1 showed the most significant ability to inhibit the proliferation of CIBM resistant cells. It was found that phosphorylation of BMK1 was enhanced in resistant cells, which suggested an association of BMK1 with drug resistance. Further study indicated that phospho-activation of BMK1 by MEK5D enhanced the resistance to CIBM. Conversely, inhibition of BMK1 by shRNAi or BMK1 inhibitor (XMD8-92) impaired not only the acquirement of resistance to CIBM, but also the proliferation of CIBM resistant cells. Further kinome-scale siRNA screening demonstrated that SRC\\MEK5 cascade promotes the phospho-activation of BMK1 in response to CIBM. Our study not only provides a global phosphoproteomic view of CIBM in melanoma, but also demonstrates that inhibition of BMK1 has therapeutic potential for the treatment of melanoma. PMID:28387310

  14. Small molecule inhibition of polo-like kinase 1 by volasertib (BI 6727) causes significant melanoma growth delay and regression in vivo.

    PubMed

    Cholewa, Brian D; Ndiaye, Mary A; Huang, Wei; Liu, Xiaoqi; Ahmad, Nihal

    2017-01-28

    The objective of this study was to determine the therapeutic potential of polo-like kinase 1 (Plk1) inhibition in melanoma, in vivo. Employing Vectra technology, we assessed the Plk1 expression profile in benign nevi, malignant (stages I-IV) and metastatic melanomas. We found a significant elevation of Plk1 immunostaining in melanoma tissues. Further, a second generation small molecule Plk1 inhibitor, BI 6727, resulted in reductions in growth, viability and clonogenic survival, as well as an increase in apoptosis of A375 and Hs 294T melanoma cells. BI 6727 treatment also resulted in a G2/M-as well as S-phase cell cycle arrest in melanoma cells. Importantly, BI 6727 (intravenous injection; 10 and 25 mg/kg body weight) treatment resulted in significant tumor growth delay and regression in vivo in A375-and Hs 294T-implanted xenografts in athymic nude mice. These anti-melanoma effects were accompanied with a decreased cellular proliferation (Ki-67 staining) and induction of apoptosis (caspase 3 activation). In addition, BI 6727 treatment caused a marked induction of p53 and p21 in vitro as well as in vivo. Overall, we suggest that Plk1 inhibition may be a useful approach as a monotherapy as well as in combination with other existing therapeutics, for melanoma management. Published by Elsevier Ireland Ltd.

  15. Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinct mechanisms

    PubMed Central

    Yang, Jianbo; Price, Matthew A.; Neudauer, Cheryl L.; Wilson, Christopher; Ferrone, Soldano; Xia, Hong; Iida, Joji; Simpson, Melanie A.; McCarthy, James B.

    2004-01-01

    Melanoma chondroitin sulfate proteoglycan (MCSP) is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. Focal adhesion kinase (FAK) plays a pivotal role in integrating growth factor and adhesion-related signaling pathways, facilitating cell spreading and migration. Extracellular signal–regulated kinase (ERK) 1 and 2, implicated in tumor growth and survival, has also been linked to clinical melanoma progression. We have cloned the MCSP core protein and expressed it in the MCSP-negative melanoma cell line WM1552C. Expression of MCSP enhances integrin-mediated cell spreading, FAK phosphorylation, and activation of ERK1/2. MCSP transfectants exhibit extensive MCSP-rich microspikes on adherent cells, where it also colocalizes with α4 integrin. Enhanced activation of FAK and ERK1/2 by MCSP appears to involve independent mechanisms because inhibition of FAK activation had no effect on ERK1/2 phosphorylation. These results indicate that MCSP may facilitate primary melanoma progression by enhancing the activation of key signaling pathways important for tumor invasion and growth. PMID:15210734

  16. Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinct mechanisms.

    PubMed

    Yang, Jianbo; Price, Matthew A; Neudauer, Cheryl L; Wilson, Christopher; Ferrone, Soldano; Xia, Hong; Iida, Joji; Simpson, Melanie A; McCarthy, James B

    2004-06-21

    Melanoma chondroitin sulfate proteoglycan (MCSP) is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. Focal adhesion kinase (FAK) plays a pivotal role in integrating growth factor and adhesion-related signaling pathways, facilitating cell spreading and migration. Extracellular signal-regulated kinase (ERK) 1 and 2, implicated in tumor growth and survival, has also been linked to clinical melanoma progression. We have cloned the MCSP core protein and expressed it in the MCSP-negative melanoma cell line WM1552C. Expression of MCSP enhances integrin-mediated cell spreading, FAK phosphorylation, and activation of ERK1/2. MCSP transfectants exhibit extensive MCSP-rich microspikes on adherent cells, where it also colocalizes with alpha4 integrin. Enhanced activation of FAK and ERK1/2 by MCSP appears to involve independent mechanisms because inhibition of FAK activation had no effect on ERK1/2 phosphorylation. These results indicate that MCSP may facilitate primary melanoma progression by enhancing the activation of key signaling pathways important for tumor invasion and growth.

  17. Melanoma.

    PubMed

    Gershenwald, J E

    2001-01-01

    The presentations at the American Society of Clinical Oncology 2001 meeting reported or updated the results of phase I, II, and III randomized trials and also reported important meta-analyses and retrospective studies impacting on the management of patients with melanoma. In the treatment of early stage melanoma, the prognostic significance of pathologic status of sentinel lymph nodes was affirmed. With respect to regional nodal involvement (American Joint Committee on Cancer [AJCC] stage III), investigators presented the interim results of the United Kingdom randomized low-dose interferon (IFN) trial, and up-to-date meta-analyses of several IFN trials including a pooled analysis of the Eastern Cooperative Oncology Group trials evaluating interferon in the adjuvant setting. In the advanced disease setting (AJCC stage IV), several studies elucidated the pros and cons of biochemotherapy in patients with metastatic melanoma, with an emphasis on seeking to improve response in the central nervous system and durability of response in general. Thought provoking was new data regarding the potential for lovastatin to act as a chemopreventive agent for melanoma. Translational studies were presented, one supporting the importance of HLA-typing in developing targeted vaccine therapy. Finally, the results of a novel experimental melanoma vaccine were presented using autologous tumor-derived heat-shock protein peptide complex-96 (HSPPC-96).

  18. 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

  19. The NC1 domain of type XIX collagen inhibits in vivo melanoma growth.

    PubMed

    Ramont, Laurent; Brassart-Pasco, Sylvie; Thevenard, Jessica; Deshorgue, Aurélie; Venteo, Lydie; Laronze, Jean Yves; Pluot, Michel; Monboisse, Jean-Claude; Maquart, François-Xavier

    2007-02-01

    Type XIX collagen is a minor collagen that localizes to basement membrane zones, together with types IV, XV, and XVIII collagens. Because several NC1 COOH-terminal domains of other chains from basement membrane collagens were reported to exhibit antitumor activity, we decided to study the effects of the NC1(XIX) collagen domain on tumor progression using an experimental in vivo model of mouse melanoma. We observed a 70% reduction in tumor volume in NC1(XIX)-treated mice compared with the corresponding controls. Histologic examination of the tumors showed a strong decrease in tumor vascularization in treated mice. In vitro, NC1(XIX) inhibited the migrating capacity of tumor cells and their capacity to invade Matrigel. It also inhibited the capacity of human microvascular endothelial cells to form pseudotubes in Matrigel. This effect was accompanied by a strong inhibition of membrane type-1 matrix metalloproteinase (matrix metalloproteinase-14) and vascular endothelial growth factor expression. Collectively, our data indicate that the NC1 domain of type XIX collagen exerts antitumor activity. This effect is mediated by a strong inhibition of the invasive capacities of tumor cells and antiangiogenic effects. NC1(XIX) should now be considered as a new member of the basement membrane collagen-derived matrikine family with antitumor and antiangiogenic activity.

  20. 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

  1. 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.

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

    PubMed Central

    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

    2015-01-01

    Abstract 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

  3. Tumor heterogeneity and plasticity as elusive drivers for resistance to MAPK pathway inhibition in melanoma.

    PubMed

    Roesch, A

    2015-06-04

    Despite the recent success of MAPK signaling-targeted drugs in melanoma, the majority of patients with metastatic melanoma still undergo disease progression after initial tumor shrinkage indicating gradually developing therapy resistance. This review will give an overview on currently suggested concepts of resistance to MAPK pathway inhibitors in melanoma with particular focus on inter- and intraindividual as well as intratumor heterogeneity. The high plasticity of melanoma cells promotes both the clonal evolution of genetic resistance, for example, because of mutations in the MAPK or PI3K/AKT/PTEN pathways, and the emergence of cell phenotypes that functionally and metabolically overcome MAPK inhibition. Like a 'moving target', melanoma cells are shifting between different metabolic, cell cycle and differentiation states reflecting a highly dynamic potential to adapt to exogenous stressors including drugs. The introduction of MAPK inhibitors into the clinics has tremendously pushed the field of melanoma research not just because of the historic therapeutic success but also by providing a new tool to study human melanoma in its natural microenvironment.

  4. Depletion of p42.3 gene inhibits proliferation and invasion in melanoma cells.

    PubMed

    Liu, Hui; Zhu, Min; Li, Zhongwu; Wang, Yan; Xing, Rui; Lu, Youyong; Xue, Weicheng

    2017-04-01

    The p42.3 gene is identified recently, and the upregulated expression has been characterized in a variety of human cancers and embryonic tissues but not yet in malignant melanoma. In this study, we explored the role of p42.3 gene in the development of melanoma. The expression of p42.3 was detected by immunohistochemistry staining of 261 cases of patient lesions, including nevi and melanoma, and its correlation with clinical pathological characteristics and prognosis was analyzed. Furthermore, a series of in vitro assays were used to investigate the biological function of p42.3 in melanoma cells. Immunohistochemistry staining showed an elevated expression level of p42.3 in melanoma compared to nevi (P = 0.001). Statistical analysis indicated that this high level was well correlated with patients' clinical stage (P = 0.045), but not with gender, age, clinical type, mitotic rate, and overall survival (P > 0.05). Moreover, in vitro assays showed knockdown p42.3 gene expression could inhibit the biological profiling, including proliferation, migration, and invasion of melanoma cells, and also affect PI3K/Akt pathway, MAPK pathway, and β-catenin. This study suggests that p42.3, acting like an oncogene, is involved in the malignant transformation process of melanoma and may serve as a biomarker for diagnostic and treatment purposes.

  5. 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

  6. Stochastic model of contact inhibition and the proliferation of melanoma in situ.

    PubMed

    Morais, Mauro César Cafundó; Stuhl, Izabella; Sabino, Alan U; Lautenschlager, Willian W; Queiroga, Alexandre S; Tortelli, Tharcisio Citrangulo; Chammas, Roger; Suhov, Yuri; Ramos, Alexandre F

    2017-08-14

    Contact inhibition is a central feature orchestrating cell proliferation in culture experiments; its loss is associated with malignant transformation and tumorigenesis. We performed a co-culture experiment with human metastatic melanoma cell line (SKMEL- 147) and immortalized keratinocyte cells (HaCaT). After 8 days a spatial pattern was detected, characterized by the formation of clusters of melanoma cells surrounded by keratinocytes constraining their proliferation. In addition, we observed that the proportion of melanoma cells within the total population has increased. To explain our results we propose a spatial stochastic model (following a philosophy of the Widom-Rowlinson model from Statistical Physics and Molecular Chemistry) which considers cell proliferation, death, migration, and cell-to-cell interaction through contact inhibition. Our numerical simulations demonstrate that loss of contact inhibition is a sufficient mechanism, appropriate for an explanation of the increase in the proportion of tumor cells and generation of spatial patterns established in the conducted experiments.

  7. 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.

  8. 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.

  9. 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.

  10. Activated CD11b+ CD15+ Granulocytes Increase in the Blood of Patients with Uveal Melanoma

    PubMed Central

    McKenna, Kyle C.; Beatty, Kelly M.; Bilonick, Richard A.; Schoenfield, Lynn; Lathrop, Kira L.; Singh, Arun D.

    2017-01-01

    Purpose To determine whether activated CD11b+ CD15+ granulocytes increase in the blood of patients with uveal melanoma. Methods Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation from the blood of patients with primary choroidal/ciliochoroidal uveal melanomas (six women, four men; age range, 46–91 years) and healthy control donors (14 women, 10 men; age range, 50 – 81 years). The expression of CD15 and CD68 on CD11b+ myeloid cells within PBMCs and primary uveal melanomas was evaluated by flow cytometry. CD3ζ chain expression by CD3ε+ T cells in PBMCs and within primary uveal melanomas was measured as an indirect indication of T-cell function. Results The percentage of CD11b+ cells in PBMCs of patients with uveal melanoma increased 1.8-fold in comparison to healthy donors and comprised three subsets: CD68 negative CD15+ granulocytes, which increased 4.1-fold; CD68− CD15− cells, which increased threefold; and CD68+ CD15low cells, which were unchanged. A significant (2.7-fold) reduction in CD3ζ chain expression on CD3ε+ T cells, a marker of T-cell dysfunction, was observed in PBMCs of patients with uveal melanoma in comparison with healthy control subjects and correlated significantly with the percentage of CD11b+ cells in PBMCs. CD3ζ chain expression on T cells within primary tumors was equivalent to CD3ζ expression in PBMCs of the same patient in four of five patients analyzed. Conclusions Activated CD11b+ CD15+ granulocytes expand in the blood of patients with uveal melanoma and may contribute to immune evasion by ocular tumors by inhibiting T-cell function via decreasing CD3ζ chain expression. PMID:19369244

  11. 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.

  12. 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.

  13. P53 and MITF/Bcl-2 identified as key pathways in the acquired resistance of NRAS-mutant melanoma to MEK inhibition.

    PubMed

    Najem, Ahmad; Krayem, Mohammad; Salès, François; Hussein, Nader; Badran, Bassam; Robert, Caroline; Awada, Ahmad; Journe, Fabrice; Ghanem, Ghanem E

    2017-09-01

    Activating mutations in Neuroblastoma RAS viral oncogene homolog (NRAS) are found in 15-30% of melanomas and are associated with a poor prognosis. Although MAP kinase kinase (MEK) inhibitors used as single agents showed a limited clinical benefit in patients with NRAS-mutant melanoma due to their rather cytostatic effect and high toxicity, their combination with other inhibitors of pathways known to cooperate with MEK inhibition may maximise their antitumour activity. Similarly, in a context where p53 is largely inactivated in melanoma, hyperexpression of Microphthalmia associated transcription factor (MITF) and its downstream anti-apoptotic targets may be the cause of the restraint cytotoxic effects of MEK inhibitors. Indeed, drug combinations targeting both mutant BRAF and MITF or one of its important targets Bcl-2 were effective in mutant BRAF melanoma but had no effect on acquired resistance. Therefore, we aimed to further investigate the downstream MITF targets that can explain this anti-apoptotic effect and to evaluate in parallel the effect of p53 reactivation on the promotion of apoptosis under MEK inhibition in a panel of (Q61)NRAS-mutant melanoma cells. First, we showed that MEK inhibition (pimasertib) led to a significant inhibition of cell proliferation but with a limited effect on apoptosis that could be explained by the systematic MITF upregulation. Mimicking the MITF effect via cyclic adenosine monophosphate activation conferred resistance to MEK inhibition and upregulated Bcl-2 expression. In addition, acquired resistance to MEK inhibition was associated with a strong activation of the anti-apoptotic signalling MITF/Bcl-2. More importantly, selective Bcl-2 inhibition by ABT-199 or Bcl-2 knockout using CRISPR/Cas9 system annihilated the acquired resistance and restored the sensitivity of NRAS-mutant melanoma cells to MEK inhibition. Strikingly and similarly, direct p53 reactivation (PRIMA-1(Met), APR-246) also broke resistance and synergised with

  14. Resveratrol Is Rapidly Metabolized in Athymic (Nu/Nu) Mice and Does Not Inhibit Human Melanoma Xenograft Tumor Growth1

    PubMed Central

    Niles, Richard M.; Cook, Carla P.; Meadows, Gary G.; Fu, Ya-Min; McLaughlin, Jerry L.; Rankin, Gary O.

    2006-01-01

    Resveratrol has been shown to have anticarcinogenic activity. We previously found that resveratrol inhibited growth and induced apoptosis in 2 human melanoma cell lines. In this study we determined whether resveratrol would inhibit human melanoma xenograft growth. Athymic mice received control diets or diets containing 110 μmol/L or 263 μmol/L resveratrol, 2 wk prior to subcutaneous injection of the tumor cells. Tumor growth was measured during a 3-wk period. Metabolism of resveratrol was assayed by bolus gavage of 75 mg/kg resveratrol in tumor-bearing and nontumor-bearing mice. Pellets containing 10–100 mg resveratrol were implanted into the mice, next to newly palpated tumors, and tumor growth determined. We also determined the effect of a major resveratrol metabolite, piceatannol, on experimental lung metastasis. Resveratrol, at any concentration tested, did not have a statistically significant effect on tumor growth. The higher levels of resveratrol tested (0.006% in food or 100 mg in slow-release pellets) tended to stimulate tumor growth (P = 0.08–0.09). Resveratrol and its major metabolites, resveratrol glucuronide and piceatannol, were found in serum, liver, skin, and tumor tissue. Piceatannol did not affect the in vitro growth of a murine melanoma cell line, but significantly stimulated the number of lung metastases when these melanoma cells were directly injected into the tail vein of the mouse. These results suggest that resveratrol is not likely to be useful in the treatment of melanoma and that the effects of phytochemicals on cell cultures may not translate to the whole animal system. PMID:16988123

  15. 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.

  16. 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.

  17. Inhibition of mouse B16 melanoma by sodium butyrate correlated to tumor associated macrophages differentiation suppression

    PubMed Central

    Xiong, Fen; Mou, Yun-Zhu; Xiang, Xiao-Yan

    2015-01-01

    Objective: As one member of the histone deacetylase inhibitor (HDACi) family, Sodium butyrate (NaB) was found out that could be used as a differentiation inducer of much cancer cell. But its effects on tumor microenvironment cells are not well recognized. The goal of this research is to investigate the effect of NaB on B16 melanoma and analysis its relevant mechanism. Methods: We observed the effect of sodium butyrate on B16 melanoma in vivo and in vitro. MTT method was performed to detect cell apoptosis rate after treatment. Tumor associated macrophage infiltration condition was detected by flow cytometry. Western-blotting and immunohistochemical method were used to detect the expression of tumor associated macrophage cytokines. Results: A certain concentration of sodium butyrate could effectively inhibit B16 melanoma growth in vivo and in vitro, and this inhibition effects related to the suppression of tumor associated macrophage differentiation. At the same time we observed the relevant macrophage factors were down-regulated compared to the control. Conclusion: Sodium butyrate could effectively inhibit B16 melanoma growth through suppressing tumor associated macrophage proliferation and reduce relevant pro-tumor macrophage factors expression, which may help to promote the clinical study of melanoma epigenetic therapy. PMID:26064327

  18. Effect of low frequency magnetic fields on melanoma: tumor inhibition and immune modulation

    PubMed Central

    2013-01-01

    Background We previously found that the low frequency magnetic fields (LF-MF) inhibited gastric and lung cancer cell growth. We suppose that exposure to LF-MF may modulate immune function so as to inhibit tumor. We here investigated whether LF-MF can inhibit the proliferation and metastasis of melanoma and influence immune function. Methods The effect of MF on the proliferation, cell cycle and ultrastracture of B16-F10 in vitro was detected by cell counting Kit-8 assay, flow cytometry, and transmission electron microscopy. Lung metastasis mice were prepared by injection of 2 × 105 B16-F10 melanoma cells into the tail vein in C57BL/6 mice. The mice were then exposed to an LF-MF (0.4 T, 7.5 Hz) for 43 days. Survival rate, tumor markers and the innate and adaptive immune parameters were measured. Results The growth of B16-F10 cells was inhibited after exposure to the LF-MF. The inhibition was related to induction of cell cycle arrest and decomposition of chromatins. Moreover, the LF-MF prolonged the mouse survival rate and inhibited the proliferation of B16-F10 in melanoma metastasis mice model. Furthermore, the LF-MF modulated the immune response via regulation of immune cells and cytokine production. In addition, the number of Treg cells was decreased in mice with the LF-MF exposure, while the numbers of T cells as well as dendritic cells were significantly increased. Conclusion LF-MF inhibited the growth and metastasis of melanoma cancer cells and improved immune function of tumor-bearing mice. This suggests that the inhibition may be attributed to modulation of LF-MF on immune function and LF-MF may be a potential therapy for treatment of melanoma. PMID:24314291

  19. 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

    2014-08-08

    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. Copyright © 2014. Published by Elsevier Inc.

  20. CD147 silencing inhibits tumor growth by suppressing glucose transport in melanoma

    PubMed Central

    Su, Juan; Gao, Tianyuan; Jiang, Minghao; Wu, Lisha; Zeng, Weiqi; Zhao, Shuang; Peng, Cong; Chen, Xiang

    2016-01-01

    Melanoma is a very malignant disease and there are still no effective treatments. CD147 participates in the carcinogenesis of multiple human cancers and GLUT-1, as a glucose transporter, is associated with tumor growth. However, the function of CD147 and GLUT-1 in melanoma have not been completely understood. Thus, in this study we investigated the expression of CD147 and GLUT-1 in melanoma tissue, which were overexpressed compared with that in nevus tissue. In addition, CD147 and GLUT-1 were co-localized in the cytoplasm of human melanoma A375 cells. Immunoprecipitation proved that CD147 interacted with GLUT-1 at D105-199. Silencing CD147 by specific siRNA could downregulate GLUT-1 level via inhibiting PI3K/Akt signaling and decrease glucose uptake in A375 cells. In vivo experiments also supported that CD147 knockdown suppressed the tumor growth in melanoma subcutaneous mice model, observed by micro PET/CT. Our results could help validate CD147 as a new therapeutic target for treating melanoma. PMID:27556188

  1. Anti-melanoma activity of the 9.2.27PE immunotoxin in dacarbazine resistant cells.

    PubMed

    Risberg, Karianne; Fodstad, Oystein; Andersson, Yvonne

    2010-04-01

    We have earlier shown that the 9.2.27 Pseudomonas Exotoxin A (PE) immunotoxin (IT) efficiently kills melanoma cells through inhibition of protein synthesis followed by some morphologic and biochemical features of apoptosis, a different cell killing mechanism than the one caused by Dacarbazine (DTIC), a chemotherapeutic drug used to treat malignant melanoma. To examine whether induced DTIC resistance also is a determining factor for the effectiveness of 9.2.27PE IT, we developed a DTIC resistant subline, FEMX-200DR, from the DTIC sensitive cell line FEMX. The cell variants were treated with 9.2.27PE, an IT binding to the high molecular weight-melanoma associated antigen (HMW-MAA) expressed on most malignant melanoma cells. The IT was equally effective in killing the FEMX-200DR and the FEMX cells, and the cell death was primarily caused by inhibition of protein synthesis. The DNA repair enzyme and apoptotic marker PARP, a substrate of caspase-3, was inactivated, although we observed only a minor activation of caspase-3 and caspase-8, intracellular proteases involved in apoptosis. In addition to being DTIC resistant, the FEMX-200DR cells were also more resistant to apoptosis than the parent cells as a 3 times higher concentration of the apoptotic inducer Staurosporine was needed to obtain IC50. Furthermore, in early passage malignant melanoma cell lines established from lymph node metastases, the 9.2.27PE caused a time-dependent and dose-dependent decrease in cell viability independent of their DTIC sensitivity. These findings show that the 9.2.27PE IT efficiently can cause cell death in malignant melanoma cells independent of their level of resistance to apoptosis and DTIC.

  2. Parthenolide, a sesquiterpene lactone from the medical herb feverfew, shows anticancer activity against human melanoma cells in vitro.

    PubMed

    Lesiak, Karolina; Koprowska, Kamila; Zalesna, Izabela; Nejc, Dariusz; Düchler, Markus; Czyz, Malgorzata

    2010-02-01

    Metastatic melanoma is a highly life-threatening disease. The lack of response to radiotherapy and chemotherapy highlights the critical need for novel treatments. Parthenolide, an active component of feverfew (Tanacetum parthenium), inhibits proliferation and kills various cancer cells mainly by inducing apoptosis. The aim of the study was to examine anticancer effects of parthenolide in melanoma cells in vitro. The cytotoxicity of parthenolide was tested in melanoma cell lines and melanocytes, as well as melanoma cells directly derived from a surgical excision. Adherent cell proliferation was measured by tetrazolium derivative reduction assay. Loss of the plasma membrane integrity, hypodiploid events, reactive oxygen species generation, mitochondrial membrane potential dissipation, and caspase-3 activity were assessed by flow cytometric analysis. Microscopy was used to observe morphological changes and cell detachment. Parthenolide reduced the number of viable adherent cells in melanoma cultures. Half maximal inhibitory concentration values around 4 mumol/l were determined. Cell death accompanied by mitochondrial membrane depolarization and caspase-3 activation was observed as the result of parthenolide application. Interestingly, the melanoma cells from vertical growth phase and melanocytes were less susceptible to parthenolide-induced cell death than metastatic cells when drug concentration was at least 6 mumol/l. Reactive oxygen species level was not significantly increased in melanoma cells. However, preincubation of parthenolide with the thiol nucleophile N-acetyl-cysteine protected melanoma cells from parthenolide-induced cell death suggesting the reaction with intracellular thiols as the mechanism responsible for parthenolide activity. In conclusion, the observed anticancer activity makes parthenolide an attractive drug candidate for further testing in melanoma therapy.

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

    PubMed

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

    2014-06-01

    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. Published by Elsevier Inc.

  4. 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

  5. Identification of MET and SRC Activation in Melanoma Cell Lines Showing Primary Resistance to PLX403212

    PubMed Central

    Vergani, Elisabetta; Vallacchi, Viviana; Frigerio, Simona; Deho, Paola; Mondellini, Piera; Perego, Paola; Cassinelli, Giuliana; Lanzi, Cinzia; Testi, Maria Adele; Rivoltini, Licia; Bongarzone, Italia; Rodolfo, Monica

    2011-01-01

    PLX4032/vemurafenib is a first-in-class small-molecule BRAFV600E inhibitor with clinical activity in patients with BRAF mutant melanoma. Nevertheless, drug resistance develops in treated patients, and strategies to overcome primary and acquired resistance are required. To explore the molecular mechanisms involved in primary resistance to PLX4032, we investigated its effects on cell proliferation and signaling in a panel of 27 genetically characterized patient-derived melanoma cell lines. Cell sensitivity to PLX4032 was dependent on BRAFV600E and independent from other gene alterations that commonly occur in melanoma such as PTEN loss, BRAF, and MITF gene amplification. Two cell lines lacking sensitivity to PLX4032 and harboring a different set of genetic alterations were studied as models of primary resistance. Treatment with the MEK inhibitor UO126 but not with PLX4032 inhibited cell growth and ERK activation. Resistance to PLX4032 was maintained after CRAF down-regulation by siRNA indicating alternative activation of MEK-ERK signaling. Genetic characterization by multiplex ligation-dependent probe amplification and analysis of phosphotyrosine signaling by MALDI-TOF mass spectrometry analysis revealed the activation of MET and SRC signaling, associated with the amplification of MET and of CTNNB1 and CCND1 genes, respectively. The combination of PLX4032 with drugs or siRNA targeting MET was effective in inhibiting cell growth and reducing cell invasion and migration in melanoma cells with MET amplification; similar effects were observed after targeting SRC in the other cell line, indicating a role for MET and SRC signaling in primary resistance to PLX4032. Our results support the development of classification of melanoma in molecular subtypes for more effective therapies. PMID:22241959

  6. Activation of the glutamate receptor GRM1 enhances angiogenic signaling to drive melanoma progression.

    PubMed

    Wen, Yu; Li, Jiadong; Koo, Jasmine; Shin, Seung-Shick; Lin, Yong; Jeong, Byeong-Seon; Mehnert, Janice M; Chen, Suzie; Cohen-Sola, Karine A; Goydos, James S

    2014-05-01

    Glutamate-triggered signal transduction is thought to contribute widely to cancer pathogenesis. In melanoma, overexpression of the metabotropic glutamate receptor (GRM)-1 occurs frequently and its ectopic expression in melanocytes is sufficient for neoplastic transformation. Clinical evaluation of the GRM1 signaling inhibitor riluzole in patients with advanced melanoma has demonstrated tumor regressions that are associated with a suppression of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathways. Together, these results prompted us to investigate the downstream consequences of GRM1 signaling and its disruption in more detail. We found that melanoma cells with enhanced GRM1 expression generated larger tumors in vivo marked by more abundant blood vessels. Media conditioned by these cells in vitro contained relatively higher concentrations of interleukin-8 and VEGF due to GRM1-mediated activation of the AKT-mTOR-HIF1 pathway. In clinical specimens from patients receiving riluzole, we confirmed an inhibition of MAPK and PI3K/AKT activation in posttreatment as compared with pretreatment tumor specimens, which exhibited a decreased density of blood vessels. Together, our results demonstrate that GRM1 activation triggers proangiogenic signaling in melanoma, offering a mechanistic rationale to design treatment strategies for the most suitable combinatorial use of GRM1 inhibitors in patients. ©2014 AACR.

  7. TERT promoter mutations in melanoma render TERT expression dependent on MAPK pathway activation.

    PubMed

    Vallarelli, Andrelou F; Rachakonda, P Sivaramakrishna; André, Jocelyne; Heidenreich, Barbara; Riffaud, Laurence; Bensussan, Armand; Kumar, Rajiv; Dumaz, Nicolas

    2016-08-16

    The mechanism of telomerase re-activation in cancer had remained elusive until the discovery of frequent mutations in the promoter of the TERT gene that encodes the catalytic reverse transcriptase subunit of telomerase. We investigated the regulation of TERT expression in melanoma cell lines and our results show that promoter mutations render TERT expression dependent on MAPK activation due to oncogenic BRAF or NRAS mutations. Mutations in the TERT promoter create binding sites for ETS transcription factors. ETS1, expressed in melanoma cell lines, undergoes activating phosphorylation by ERK at Thr38 residue as a consequence of constitutively activated MAPK pathway. We demonstrate that ETS1 binds on the mutated TERT promoter leading to the re-expression of the gene. The inhibition of ETS1 resulted in reduced TERT expression. We provide evidence that the TERT promoter mutations provide a direct link between TERT expression and MAPK pathway activation due to BRAF or NRAS mutations via the transcription factor ETS1.

  8. 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.

  9. PDGFR-alpha inhibits melanoma growth via CXCL10/IP-10: a multi-omics approach

    PubMed Central

    D'Arcangelo, Daniela; Facchiano, Francesco; Nassa, Giovanni; Stancato, Andrea; Antonini, Annalisa; Rossi, Stefania; Senatore, Cinzia; Cordella, Martina; Tabolacci, Claudio; Salvati, Annamaria; Tarallo, Roberta; Weisz, Alessandro; Facchiano, Angelo M.; Facchiano, Antonio

    2016-01-01

    Melanoma is the most aggressive skin-cancer, showing high mortality at advanced stages. Platelet Derived Growth Factor Receptor-alpha (PDGFR-alpha) potently inhibits melanoma- and endothelium-proliferation and its expression is significantly reduced in melanoma-biopsies, suggesting that melanoma progression eliminates cells expressing PDGFR-alpha. In the present study transient overexpression of PDGFR-alpha in endothelial (HUVEC) and melanoma (SKMel-28, A375, Preyer) human-cells shows strong anti-proliferative effects, with profound transcriptome and miRNome deregulation. PDGFR-alpha overexpression strongly affects expression of 82 genes in HUVEC (41 up-, 41 down-regulated), and 52 genes in SKMel-28 (43 up-, 9 down-regulated). CXCL10/IP-10 transcript showed up to 20 fold-increase, with similar changes detectable at the protein level. miRNA expression profiling in cells overexpressing PDGFR-alpha identified 14 miRNAs up- and 40 down-regulated, with miR-503 being the most down-regulated (6.4 fold-reduction). miR-503, miR-630 and miR-424 deregulation was confirmed by qRT-PCR. Interestingly, the most upregulated transcript (i.e., CXCL10/IP-10) was a validated miR-503 target and CXCL10/IP-10 neutralization significantly reverted the anti-proliferative action of PDGFR-alpha, and PDGFR-alpha inhibition by Dasatinb totally reverted the CXCL10/IP10 induction, further supporting a functional interplay of these factors. Finally, integration of transcriptomics and miRNomics data highlighted several pathways affected by PDGFR-alpha. This study demonstrates for the first time that PDGFR-alpha strongly inhibits endothelial and melanoma cells proliferation in a CXCL10/IP-10 dependent way, via miR-503 down-regulation. PMID:27764787

  10. 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.

  11. 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

  12. 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.

  13. Melanoma: the intersection of molecular targeted therapy and immune checkpoint inhibition.

    PubMed

    Lau, Peter Kar Han; Ascierto, Paolo A; McArthur, Grant

    2016-04-01

    Melanoma is at the forefront of development of systemic therapeutics with both molecular targeted therapies and immune checkpoint inhibitors as cornerstones of treatment. Although responses to molecularly targeted therapy is largely from blockade of oncogenic pathways, evidence is emerging of the immunomodulatory effects from BRAF inhibition. Additionally programmed-death-1 (PD-1) inhibitors have revolutionized the treatment of melanoma and are set to pave future improvements in other solid tumors. Combinations of PD-1 inhibitors with novel immune checkpoints or with molecularly targeted therapies are under investigation and may improve on the considerable progress made. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. The Brn-2 transcription factor links activated BRAF to melanoma proliferation.

    PubMed

    Goodall, Jane; Wellbrock, Claudia; Dexter, Timothy J; Roberts, Karen; Marais, Richard; Goding, Colin R

    2004-04-01

    Malignant melanoma, an aggressive and increasingly common cancer, is characterized by a strikingly high rate (70%) of mutations in BRAF, a key component of the mitogen-activated protein (MAP) kinase signaling pathway. How signaling events downstream from BRAF affect the underlying program of gene expression is poorly understood. We show that the Brn-2 POU domain transcription factor is highly expressed in melanoma cell lines but not in melanocytes or melanoblasts and that overexpression of Brn-2 in melanocytes results in increased proliferation. Expression of Brn-2 is strongly upregulated by Ras and MAP kinase signaling. Importantly, the Brn-2 promoter is stimulated by kinase-activating BRAF mutants and endogenous Brn-2 expression is inhibited by RNA interference-mediated downregulation of BRAF. Moreover, silent interfering RNA-mediated depletion of Brn-2 in melanoma cells expressing activated BRAF leads to decreased proliferation. The results suggest that the high levels of Brn-2 expression observed in melanomas link BRAF signaling to increased proliferation.

  15. Activation of the Glutamate Receptor GRM1 Enhances Angiogenic Signaling to Drive Melanoma Progression

    PubMed Central

    Wen, Yu; Li, Jiadong; Koo, Jasmine; Shin, Seung-Shick; Lin, Yong; Jeong, Byeong-Seon; Mehnert, Janice; Chen, Suzie; Cohen-Solal, Karine; Goydos, James S

    2014-01-01

    Glutamate-triggered signal transduction is thought to contribute widely to cancer pathogenesis. In melanoma, over-expression of the metabotropic glutamate receptor GRM1 occurs frequently and its ectopic expression in melanocytes is sufficient for neoplastic transformation. Clinical evaluation of the GRM1 signaling inhibitor riluzole in patients with advanced melanoma has demonstrated tumor regressions that are associated with a suppression of the MAPK and PI3K/AKT pathways. Together, these results prompted us to investigate the downstream consequences of GRM1 signaling and its disruption in more detail. We found that melanoma cells with enhanced GRM1 expression generated larger tumors in vivo marked by more abundant blood vessels. Media conditioned by these cells in vitro contained relatively higher concentrations of IL-8 and VEGF, due to GRM1-mediated activation of the AKT-mTOR-HIF1 pathway. In clinical specimens from patients receiving riluzole, we confirmed an inhibition of MAPK and PI3K/AKT activation in post-treatment as compared to pre-treatment tumor specimens, which exhibited a decreased density of blood vessels. Together, our results demonstrate that GRM1 activation triggers pro-angiogenic signaling in melanoma, offering a mechanistic rationale to design treatment strategies for the most suitable combinatorial use of GRM1 inhibitors in patients. PMID:24491800

  16. Vanillin Suppresses Cell Motility by Inhibiting STAT3-Mediated HIF-1α mRNA Expression in Malignant Melanoma Cells.

    PubMed

    Park, Eun-Ji; Lee, Yoon-Mi; Oh, Taek-In; Kim, Byeong Mo; Lim, Beong-Ou; Lim, Ji-Hong

    2017-03-01

    Recent studies have shown that vanillin has anti-cancer, anti-mutagenic, and anti-metastatic activity; however, the precise molecular mechanism whereby vanillin inhibits metastasis and cancer progression is not fully elucidated. In this study, we examined whether vanillin has anti-cancer and anti-metastatic activities via inhibition of hypoxia-inducible factor-1α (HIF-1α) in A2058 and A375 human malignant melanoma cells. Immunoblotting and quantitative real time (RT)-PCR analysis revealed that vanillin down-regulates HIF-1α protein accumulation and the transcripts of HIF-1α target genes related to cancer metastasis including fibronectin 1 (FN1), lysyl oxidase-like 2 (LOXL2), and urokinase plasminogen activator receptor (uPAR). It was also found that vanillin significantly suppresses HIF-1α mRNA expression and de novo HIF-1α protein synthesis. To understand the suppressive mechanism of vanillin on HIF-1α expression, chromatin immunoprecipitation was performed. Consequently, it was found that vanillin causes inhibition of promoter occupancy by signal transducer and activator of transcription 3 (STAT3), but not nuclear factor-κB (NF-κB), on HIF1A. Furthermore, an in vitro migration assay revealed that the motility of melanoma cells stimulated by hypoxia was attenuated by vanillin treatment. In conclusion, we demonstrate that vanillin might be a potential anti-metastatic agent that suppresses metastatic gene expression and migration activity under hypoxia via the STAT3-HIF-1α signaling pathway.

  17. Vanillin Suppresses Cell Motility by Inhibiting STAT3-Mediated HIF-1α mRNA Expression in Malignant Melanoma Cells

    PubMed Central

    Park, Eun-Ji; Lee, Yoon-Mi; Oh, Taek-In; Kim, Byeong Mo; Lim, Beong-Ou; Lim, Ji-Hong

    2017-01-01

    Recent studies have shown that vanillin has anti-cancer, anti-mutagenic, and anti-metastatic activity; however, the precise molecular mechanism whereby vanillin inhibits metastasis and cancer progression is not fully elucidated. In this study, we examined whether vanillin has anti-cancer and anti-metastatic activities via inhibition of hypoxia-inducible factor-1α (HIF-1α) in A2058 and A375 human malignant melanoma cells. Immunoblotting and quantitative real time (RT)-PCR analysis revealed that vanillin down-regulates HIF-1α protein accumulation and the transcripts of HIF-1α target genes related to cancer metastasis including fibronectin 1 (FN1), lysyl oxidase-like 2 (LOXL2), and urokinase plasminogen activator receptor (uPAR). It was also found that vanillin significantly suppresses HIF-1α mRNA expression and de novo HIF-1α protein synthesis. To understand the suppressive mechanism of vanillin on HIF-1α expression, chromatin immunoprecipitation was performed. Consequently, it was found that vanillin causes inhibition of promoter occupancy by signal transducer and activator of transcription 3 (STAT3), but not nuclear factor-κB (NF-κB), on HIF1A. Furthermore, an in vitro migration assay revealed that the motility of melanoma cells stimulated by hypoxia was attenuated by vanillin treatment. In conclusion, we demonstrate that vanillin might be a potential anti-metastatic agent that suppresses metastatic gene expression and migration activity under hypoxia via the STAT3-HIF-1α signaling pathway. PMID:28257048

  18. Androgen receptor exon 1 mutation causes androgen insensitivity by creating phosphorylation site and inhibiting melanoma antigen-A11 activation of NH2- and carboxyl-terminal interaction-dependent transactivation.

    PubMed

    Lagarde, William H; Blackwelder, Amanda J; Minges, John T; Hnat, Andrew T; French, Frank S; Wilson, Elizabeth M

    2012-03-30

    Naturally occurring germ line mutations in the X-linked human androgen receptor (AR) gene cause incomplete masculinization of the external genitalia by disrupting AR function in males with androgen insensitivity syndrome. Almost all AR missense mutations that cause androgen insensitivity syndrome are located in the highly structured DNA and ligand binding domains. In this report we investigate the functional defect associated with an AR exon 1 missense mutation, R405S, that caused partial androgen insensitivity. The 46,XX heterozygous maternal carrier had a wild-type Arg-405 CGC allele but transmitted an AGC mutant allele coding for Ser-405. At birth, the 46,XY proband had a bifid scrotum, hypospadias, and micropenis consistent with clinical stage 3 partial androgen insensitivity. Androgen-dependent transcriptional activity of AR-R405S expressed in CV1 cells was less than wild-type AR and refractory in androgen-dependent AR NH(2)- and carboxyl interaction transcription assays that depend on the coregulator effects of melanoma antigen-A11. This mutation created a Ser-405 phosphorylation site evident by the gel migration of an AR-R405S NH(2)-terminal fragment as a double band that converted to the wild-type single band after treatment with λ-phosphatase. Detrimental effects of the R405S mutation were related to the proximity of the AR WXXLF motif (433)WHTLF(437) required for melanoma antigen-A11 and p300 to stimulate transcriptional activity associated with the AR NH(2)- and carboxyl-terminal interaction. We conclude that the coregulator effects of melanoma antigen-A11 on the AR NH(2)- and carboxyl-terminal interaction amplify the androgen-dependent transcriptional response to p300 required for normal human male sex development in utero.

  19. 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

  20. Inhibition of mTORC1/2 overcomes resistance to MAPK pathway inhibitors mediated by PGC1α and Oxidative Phosphorylation in melanoma

    PubMed Central

    Gopal, Y.N. Vashisht; Rizos, Helen; Chen, Guo; Deng, Wanleng; Frederick, Dennie T.; Cooper, Zachary A.; Scolyer, Richard A; Pupo, Gulietta; Komurov, Kakajan; Sehgal, Vasudha; Zhang, Jiexin; Patel, Lalit; Pereira, Cristiano G.; Broom, Bradley M.; Mills, Gordon B.; Ram, Prahlad; Smith, Paul D.; Wargo, Jennifer A.; Long, Georgina V.; Davies, Michael A.

    2014-01-01

    Metabolic heterogeneity is a key factor in cancer pathogenesis. We found that a subset of BRAF and NRAS mutant human melanomas resistant to the MEK inhibitor selumetinib displayed increased oxidative phosphorylation (OxPhos) mediated by the transcriptional co-activator PGC1α. Notably, all selumetinib-resistant cells with elevated OxPhos could be re-sensitized by co-treatment with the mTORC1/2 inhibitor AZD8055, whereas this combination was ineffective in resistant cell lines with low OxPhos. In both BRAF- and NRAS-mutant melanoma cells, MEK inhibition increased MITF expression which in turn elevated levels of PGC1α. In contrast, mTORC1/2 inhibition triggered cytoplasmic localization of MITF, decreasing PGC1α expression and inhibiting OxPhos. Analysis of tumor biopsies from BRAF-mutant melanoma patients progressing on BRAF inhibitor {plus minus} MEK inhibitor revealed that PGC1α levels were elevated in approximately half of the resistant tumors. Overall, our findings highlight the significance of OxPhos in melanoma and suggest that combined targeting of the MAPK and mTORC pathways may offer an effective therapeutic strategy to treat melanomas with this metabolic phenotype. PMID:25297634

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

    PubMed

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

    2015-08-07

    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.

  2. Inhalation therapy with M1 inhibits experimental melanoma development and metastases in mice.

    PubMed

    Ferrari de Andrade, Lucas; Mozeleski, Brian; Leck, Aline Raquell; Rossi, Gustavo; da Costa, Cleber Rafael Vieira; de Souza Fonseca Guimarães, Fernando; Zotz, Rafael; Fialho do Nascimento, Katia; Camargo de Oliveira, Carolina; de Freitas Buchi, Dorly; da Silva Trindade, Edvaldo

    2016-02-01

    M1 is a homeopathic medicine with immunostimulatory properties used mainly by cancer patients to complement current therapies. Metastatic melanoma is a skin-originated form of cancer without a single therapy able to produce high rate and sustained responses, which attracts the use of complementary therapies such as M1. However, M1's anti-melanoma effects remain to be pre-clinically demonstrated. Therefore in the present work, we utilized a pulmonary metastatic melanoma model and a subcutaneous melanoma growth model to investigate the potential benefits of treatment with M1. C57BL/6 mice were injected intravenously or subcutaneously with B16F10 mouse melanoma cells. After 24 h, mice were treated with either M1 or vehicle (water) for 14 days, euthanized and harvested for multi-parameter pulmonary and tumor analyses. Mice treated with M1 had significantly lower tumor burden in the lungs and subcutaneous tissue than control mice. Furthermore, tumors were impaired in proliferation and tumor related angiogenesis by the inhibition of myeloid derived suppressor cells (MDSC) positive for angiotensin II type 1 receptor (AT1R). Altogether these data suggest M1 is an efficient candidate for melanoma therapy to be considered for future clinic studies as this study is the first supporting the idea that melanoma patients may benefit with the treatment. The treatment with M1 provides advantages considering the highly-diluted properties and a cost effective alternative to costly chemotherapeutic approaches with, if any, lower toxicity. Copyright © 2015 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved.

  3. In vivo overexpression of tumstatin domains by tumor cells inhibits their invasive properties in a mouse melanoma model.

    PubMed

    Pasco, Sylvie; Ramont, Laurent; Venteo, Lydie; Pluot, Michel; Maquart, François-Xavier; Monboisse, Jean-Claude

    2004-12-10

    Our previous studies demonstrated that a synthetic peptide encompassing residues 185-203 of the noncollagenous (NC1) domain of the alpha3 chain of type IV collagen, named tumstatin, inhibits in vitro melanoma cell proliferation and migration. In the present study, B16F1 melanoma cells were stably transfected to overexpress the complete tumstatin domain (Tum 1-232) or its C-terminal part, encompassing residues 185-203 (Tum 183-232). Tumstatin domain overexpression inhibited B16F1 in vitro cell proliferation, anchorage-independent growth, and invasive properties. For studying the in vivo effect of overexpression, representative clones were subcutaneously injected into the left side of C57BL6 mice. In vivo tumor growth was decreased by -60% and -56%, respectively, with B16F1 cells overexpressing Tum 1-232 or Tum 183-232 compared to control cells. This inhibitory effect was associated with a decrease of in vivo cyclin D1 expression. We also demonstrated that the overexpression of Tum 1-232 or Tum 183-232 induced an in vivo down-regulation of proteolytic cascades involving matrix metalloproteinases (MMPs), especially the production or activation of MMP-2, MMP-9, MMP-13, as well as MMP-14. The plasminogen activation system was also altered in tumors with a decrease of urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) and a strong increase of plasminogen activator inhibitor-1 (PAI-1). Collectively, our results demonstrate that tumstatin or its C-terminal antitumor fragment, Tum 183-232, inhibits in vivo melanoma progression by triggering an intracellular transduction pathway, which involves a cyclic AMP (cAMP)-dependent mechanism.

  4. Active specific immunotherapy with polyvalent melanoma cell vaccine for patients with in-transit melanoma metastases.

    PubMed

    Hsueh, E C; Nathanson, L; Foshag, L J; Essner, R; Nizze, J A; Stern, S L; Morton, D L

    1999-05-15

    This study was conducted to document the rate, duration, and type of objective response to active specific immunotherapy with a polyvalent melanoma cell vaccine (PMCV) for patients with in-transit melanoma metastases and to identify any acute or chronic toxic effects of PMCV treatment. An analysis was conducted of all in-transit melanoma patients seen at the John Wayne Cancer Institute in Santa Monica, California, during the period 1985-1997 who were enrolled in prospective PMCV protocols in the absence of other therapies with possible antitumor activity (n = 54). Clinical response to PMCV was assessed by standard criteria. Survival curves were estimated by the Kaplan-Meier method. Toxicity was graded according to the Eastern Cooperative Oncology Group standard. PMCV produced a 17% (9 of 54 patients) objective response rate with a 13% rate (7 of 54 patients) of complete remission (CR). The median duration of CR was >22 months. Complete response lasting more than 1 year was observed in 4 patients (7.2%); 1 patient remained in remission over 9 years. Median survival was >53 months (i.e., not reached) for responders, 42 months for nonresponders, and 53 months overall. Salvage interventions allowed reinduction with PMCV in 23 of 25 patients, who subsequently remained clinically free of disease for a median of 14 months. Overall toxicity was mild, easily tolerable, and did not significantly change the quality of life. There were no toxic deaths. PMCV can cause objective complete regression of measurable intransit metastatic melanoma with minimal toxicity, and may prolong patients' median survival.

  5. Sensitization of melanoma cells to alkylating agent-induced DNA damage and cell death via orchestrating oxidative stress and IKKβ inhibition.

    PubMed

    Tse, Anfernee Kai-Wing; Chen, Ying-Jie; Fu, Xiu-Qiong; Su, Tao; Li, Ting; Guo, Hui; Zhu, Pei-Li; Kwan, Hiu-Yee; Cheng, Brian Chi-Yan; Cao, Hui-Hui; Lee, Sally Kin-Wah; Fong, Wang-Fun; Yu, Zhi-Ling

    2017-04-01

    Nitrosourea represents one of the most active classes of chemotherapeutic alkylating agents for metastatic melanoma. Treatment with nitrosoureas caused severe systemic side effects which hamper its clinical use. Here, we provide pharmacological evidence that reactive oxygen species (ROS) induction and IKKβ inhibition cooperatively enhance nitrosourea-induced cytotoxicity in melanoma cells. We identified SC-514 as a ROS-inducing IKKβ inhibitor which enhanced the function of nitrosoureas. Elevated ROS level results in increased DNA crosslink efficiency triggered by nitrosoureas and IKKβ inhibition enhances DNA damage signals and sensitizes nitrosourea-induced cell death. Using xenograft mouse model, we confirm that ROS-inducing IKKβ inhibitor cooperates with nitrosourea to reduce tumor size and malignancy in vivo. Taken together, our results illustrate a new direction in nitrosourea treatment, and reveal that the combination of ROS-inducing IKKβ inhibitors with nitrosoureas can be potentially exploited for melanoma therapy.

  6. Components in aqueous Hibiscus rosa-sinensis flower extract inhibit in vitro melanoma cell growth.

    PubMed

    Goldberg, Karina H; Yin, Ariel C; Mupparapu, Archana; Retzbach, Edward P; Goldberg, Gary S; Yang, Catherine F

    2017-01-01

    Skin cancer is extremely common, and melanoma causes about 80% of skin cancer deaths. In fact, melanoma kills over 50 thousand people around the world each year, and these numbers are rising. Clearly, standard treatments are not effectively treating melanoma, and alternative therapies are needed to address this problem. Hibiscus tea has been noted to have medicinal properties, including anticancer effects. Extracts from Hibiscus have been shown to inhibit the growth of a variety of cancer cells. In particular, recent studies found that polyphenols extracted from Hibiscus sabdariffa by organic solvents can inhibit melanoma cell growth. However, effects of aqueous extracts from Hibiscus rosa-sinesis flowers, which are commonly used to make traditional medicinal beverages, have not been examined on melanoma cells. Here, we report that aqueous H. rosa-sinesis flower extract contains compounds that inhibit melanoma cell growth in a dose dependent manner at concentrations that did not affect the growth of nontransformed cells. In addition, these extracts contain low molecular weight growth inhibitory compounds below 3 kD in size that combine with larger compounds to more effectively inhibit melanoma cell growth. Future work should identify these compounds, and evaluate their potential to prevent and treat melanoma and other cancers.

  7. Monoclonal antibody-induced ErbB3 receptor internalization and degradation inhibits growth and migration of human melanoma cells.

    PubMed

    Belleudi, Francesca; Marra, Emanuele; Mazzetta, Francesca; Fattore, Luigi; Giovagnoli, Maria Rosaria; Mancini, Rita; Aurisicchio, Luigi; Torrisi, Maria Rosaria; Ciliberto, Gennaro

    2012-04-01

    Members of the ErbB receptor family are targets of a growing numbers of small molecules and monoclonal antibodies inhibitors currently under development for the treatment of cancer. Although historical efforts have been directed against ErbB1 (EGFR) and ErbB2 (HER2/neu), emerging evidences have pointed to ErbB3 as a key node in the activation of proliferation/survival pathways from the ErbB receptor family and have fueled enthusiasm toward the clinical development of anti-ErbB3 agents. In this study, we have evaluated the potential therapeutic efficacy of a set of three recently generated anti-human ErbB3 monoclonals, A2, A3 and A4, in human primary melanoma cells. We show that in melanoma cells expressing ErbB1, ErbB3 and ErbB4 but not ErbB2 receptor ligands activate the PI3K/AKT pathway, and this leads to increased cell proliferation and migration. While antibodies A3 and A4 are able to potently inhibit ligand-induced signaling, proliferation and migration, antibody A2 is unable to exert this effect. In attempt to understand the mechanism of action and the basis of this different behavior, we demonstrate, through a series of combined approaches, that antibody efficacy strongly correlates with antibody-induced receptor internalization, degradation and inhibition of receptor recycling to the cell surface. Finally, fine epitope mapping studies through a peptide array show that inhibiting vs. non-inhibiting antibodies have a dramatically different mode of binding to the to the receptor extracellular domain. Our study confirms the key role of ErbB3 and points to exploitation of novel combination therapies for treatment of malignant melanoma.

  8. Inducible nitric oxide synthase (iNOS) drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2

    PubMed Central

    Lopez-Rivera, Esther; Jayaraman, Padmini; Parikh, Falguni; Davies, Michael A.; Ekmekcioglu, Suhendan; Izadmehr, Sudeh; Milton, Denái R.; Chipuk, Jerry E.; Grimm, Elizabeth A.; Estrada, Yeriel; Aguirre-Ghiso, Julio; Sikora, Andrew G.

    2014-01-01

    Melanoma is one of the cancers of fastest-rising incidence in the world. iNOS is overexpressed in melanoma and other cancers, and previous data suggest that iNOS and nitric oxide (NO) drive survival and proliferation of human melanoma cells. However, specific mechanisms through which this occurs are poorly defined. One candidate is the PI3K/AKT/mTOR pathway, which plays a major role in proliferation, angiogenesis, and metastasis of melanoma and other cancers. We used the chick embryo chorioallantoic membrane (CAM) assay to test the hypothesis that melanoma growth is regulated by iNOS-dependent mTOR pathway activation. Both pharmacologic inhibition and siRNA-mediated gene silencing of iNOS suppressed melanoma proliferation and in vivo growth on the CAM in human melanoma models. This was associated with strong downregulation of mTOR pathway activation by Western blot analysis of p-mTOR, p-P70S6K, p-S6RP, and p-4EBP1. iNOS expression and NO were associated with reversible nitrosylation of TSC2, and inhibited dimerization of TSC2 with its inhibitory partner TSC1, enhancing GTPase activity of its target Rheb, a critical activator of mTOR signaling. Immunohistochemical analysis of tumor specimens from stage III melanoma patients showed a significant correlation between iNOS expression levels and expression of mTOR pathway members. Exogenously-supplied NO was also sufficient to reverse mTOR pathway inhibition by the B-Raf inhibitor Vemurafenib. In summary, covalent modification of TSC2 by iNOS-derived NO is associated with impaired TSC2/TSC1 dimerization, mTOR pathway activation, and proliferation of human melanoma. This model is consistent with the known association of iNOS overexpression and poor prognosis in melanoma and other cancers. PMID:24398473

  9. 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. © 2014 Wiley Periodicals, Inc.

  10. 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.

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

    PubMed Central

    Kansal, Rita G.; McCravy, Matthew S.; Basham, Jacob H.; Earl, Joshua A.; McMurray, Stacy L.; Starner, Chelsey J.

    2016-01-01

    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

  12. Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade.

    PubMed

    Cooper, Zachary A; Juneja, Vikram R; Sage, Peter T; Frederick, Dennie T; Piris, Adriano; Mitra, Devarati; Lo, Jennifer A; Hodi, F Stephen; Freeman, Gordon J; Bosenberg, Marcus W; McMahon, Martin; Flaherty, Keith T; Fisher, David E; Sharpe, Arlene H; Wargo, Jennifer A

    2014-07-01

    BRAF-targeted therapy results in objective responses in the majority of patients; however, the responses are short lived (∼6 months). In contrast, treatment with immune checkpoint inhibitors results in a lower response rate, but the responses tend to be more durable. BRAF inhibition results in a more favorable tumor microenvironment in patients, with an increase in CD8(+) T-cell infiltrate and a decrease in immunosuppressive cytokines. There is also increased expression of the immunomodulatory molecule PDL1, which may contribute to the resistance. On the basis of these findings, we hypothesized that BRAF-targeted therapy may synergize with the PD1 pathway blockade to enhance antitumor immunity. To test this hypothesis, we developed a BRAF(V600E)/Pten(-/-) syngeneic tumor graft immunocompetent mouse model in which BRAF inhibition leads to a significant increase in the intratumoral CD8(+) T-cell density and cytokine production, similar to the effects of BRAF inhibition in patients. In this model, CD8(+) T cells were found to play a critical role in the therapeutic effect of BRAF inhibition. Administration of anti-PD1 or anti-PDL1 together with a BRAF inhibitor led to an enhanced response, significantly prolonging survival and slowing tumor growth, as well as significantly increasing the number and activity of tumor-infiltrating lymphocytes. These results demonstrate synergy between combined BRAF-targeted therapy and immune checkpoint blockade. Although clinical trials combining these two strategies are ongoing, important questions still remain unanswered. Further studies using this new melanoma mouse model may provide therapeutic insights, including optimal timing and sequence of therapy.

  13. 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

  14. 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. Copyright © 2014. Published by Elsevier Masson SAS.

  15. Icaritin, a novel FASN inhibitor, exerts anti-melanoma activities through IGF-1R/STAT3 signaling

    PubMed Central

    Wu, Jinfeng; Du, Juan; Fu, Xiuqiong; Liu, Bin; Cao, Huihui; Li, Ting; Su, Tao; Xu, Jinhua; Tse, Anfernee Kai-Wing; Yu, Zhi-Ling

    2016-01-01

    Icaritin (IT) is a flavonoid isolated from Herba Epimedii. In this study, we evaluated the anti-melanoma activities of IT, and determined its cytotoxic mechanism. We found that IT exerted cytotoxicity to melanoma cells. Furthermore, IT induced melanoma cell apoptosis, which was accompanied with PARP cleavage. Mechanistically, IT suppressed p-STAT3 (tyr705) level in parallel with increases of p-STAT3 (ser727), p-ERK and p-AKT. IT significantly inhibited STAT3 nuclear translocation and reduced the levels of STAT3 -targeted genes. IT also inhibited IGF-1-induced STAT3 activation through down-regulation of total IGF-1R level. No dramatic changes in IGF-1R mRNA levels were observed in IT-treated cells, suggesting that IT acted primarily at a post-transcriptional level. Using molecular docking analysis, IT was identified as a novel fatty acid synthase (FASN) inhibitor. We found that IT reduced the level of total IGF-1R via FASN inhibition. In summary, we reported that IT exerted anti-melanoma activities, and these effects were partially due to inhibition of FASN/IGF-1R/STAT3 signaling. PMID:27323414

  16. Oncolytic adenovirus expressing interleukin-18 improves antitumor activity of dacarbazine for malignant melanoma

    PubMed Central

    Yang, Chunhua; Cao, Hang; Liu, Ning; Xu, Kai; Ding, Meng; Mao, Li-jun

    2016-01-01

    Conditionally replicating adenoviruses have emerged as novel therapeutic agents for cancer. This study aimed to evaluate synergistic antitumor activity of replication-competent adenovirus armed with interleukin (IL)-18 (ZD55-IL-18) and dacarbazine (DTIC) against melanoma. Melanoma A375 cells or nude mouse tumor xenografts were treated with ZD55-IL-18 alone or together with DTIC. The results showed that ZD55-IL-18 competently replicated in A375 cells and expressed IL-18, and these were not affected by DTIC. ZD55-IL-18 enhanced the cytotoxicity of DTIC accompanied by increased apoptosis. Moreover, ZD55-IL-18 and DTIC synergistically inhibited the growth but promoted the apoptosis of A375 xenografts and inhibited vascular endothelial growth factor expression and lung metastasis in xenografts of nude mice. In conclusion, this is the first study to show synergistic anticancer activity of ZD55-IL-18 and DTIC for malignant melanoma. Our results provide evidence that chemo-gene-viro therapeutic approach has greater potential for malignant cancers than conventional chemotherapy or gene therapy. PMID:27895465

  17. Oncolytic adenovirus expressing interleukin-18 improves antitumor activity of dacarbazine for malignant melanoma.

    PubMed

    Yang, Chunhua; Cao, Hang; Liu, Ning; Xu, Kai; Ding, Meng; Mao, Li-Jun

    2016-01-01

    Conditionally replicating adenoviruses have emerged as novel therapeutic agents for cancer. This study aimed to evaluate synergistic antitumor activity of replication-competent adenovirus armed with interleukin (IL)-18 (ZD55-IL-18) and dacarbazine (DTIC) against melanoma. Melanoma A375 cells or nude mouse tumor xenografts were treated with ZD55-IL-18 alone or together with DTIC. The results showed that ZD55-IL-18 competently replicated in A375 cells and expressed IL-18, and these were not affected by DTIC. ZD55-IL-18 enhanced the cytotoxicity of DTIC accompanied by increased apoptosis. Moreover, ZD55-IL-18 and DTIC synergistically inhibited the growth but promoted the apoptosis of A375 xenografts and inhibited vascular endothelial growth factor expression and lung metastasis in xenografts of nude mice. In conclusion, this is the first study to show synergistic anticancer activity of ZD55-IL-18 and DTIC for malignant melanoma. Our results provide evidence that chemo-gene-viro therapeutic approach has greater potential for malignant cancers than conventional chemotherapy or gene therapy.

  18. Biochemical characterization of three hamster melanoma variants--I. Tyrosinase activity and melanin content.

    PubMed

    Słomiński, A; Scisłowski, P W; Bomirski, A

    1984-01-01

    Tyrosinase activity in the soluble fraction of the cells and melanin content in the whole cells of the black-melanotic (Ma), brown-melanotic (MI) and amelanotic (Ab) hamster melanomas were studied. The activity of the soluble tyrosinase was highest in MI lower in Ma, and very low in Ab melanoma. Melanin content was greatest in the Ma, lower in MI, and none in Ab melanoma. Acrylamide gel electrophoretic pattern of the soluble tyrosinase consisted of 2 bands in Ma and MI melanomas, and of 1 band in Ab melanoma.

  19. Combination Small Molecule MEK and PI3K Inhibition Enhances Uveal Melanoma Cell Death in a Mutant GNAQ and GNA11 Dependent Manner

    PubMed Central

    Khalili, Jahan S.; Yu, Xiaoxing; Wang, Ji; Hayes, Brendan C.; Davies, Michael A.; Lizee, Gregory; Esmaeli, Bita; Woodman, Scott E.

    2014-01-01

    Purpose Activating Q209L/P mutations in GNAQ or GNA11 (GNAQ/11) are present in ∼ 80% of uveal melanomas (UM). Mutant GNAQ/11 are not currently therapeutically targetable. Inhibiting key downstream effectors of GNAQ/11 represents a rational therapeutic approach for UMs that harbor these mutations. The MEK/MAPK and PI3K/AKT pathways are activated in UM. In this study, we test the effect of the clinically relevant small molecule inhibitors GSK1120212 (MEK inhibitor) and GSK2126458 (pan class I PI3K inhibitor) on UM cells with different GNAQ/11 mutation backgrounds. Experimental Design We use the largest set of genetically annotated uveal melanoma cell lines to-date to perform in vitro cellular signaling, cell cycle regulation, growth and apoptosis analyses. RNA interference and small molecule MEK and/or PI3K inhibitor treatment were employed to determine the dependency of uveal melanoma cells with different GNAQ/11 mutation backgrounds on MEK/MAPK and/or PI3K/AKT signaling. Proteomic network analysis was performed to unveil signaling alterations in response to MEK and/or PI3K small molecule inhibition. Results GNAQ/11 mutation status was not a determinant of whether cells would undergo cell cycle arrest or growth inhibition to MEK and/or PI3K inhibition. A reverse correlation was observed between MAPK and AKT phosphorylation after MEK or PI3K inhibition, respectively. Neither MEK nor PI3K inhibition alone was sufficient to induce apoptosis in the majority of cell lines; however, the combination of MEK + PI3K inhibitor treatment resulted in the marked induction of apoptosis in a GNAQ/11 mutant-dependent manner. Conclusions MEK + PI3K inhibition may be an effective combination therapy in uveal melanoma given the inherent reciprocal activation of these pathways within these cells. PMID:22733540

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

    PubMed Central

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

    2014-01-01

    Summary 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. Significance Approximately 30% of all cutaneous melanomas are wild-type for both BRAF and NRAS. As yet, no targeted therapy strategies exist for this sub-set of tumors. Constitutive signaling through the PI3K/AKT pathway is a common occurrence in cutaneous melanoma, irrespective of the driver mutation. Here we report durable responses to the AKT inhibitor MK-2206 in combination with carboplatin and paclitaxel in two patients with BRAF wild-type melanoma. Through mechanistic studies, we demonstrate a role for autophagy induction in the response to the AKT inhibitor/chemotherapy combination and suggest that autophagy inhibitors may be one strategy to enhance efficacy in the clinical setting. PMID:24490764

  1. 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.

  2. Mitogen-activated protein kinase pathway-dependent tumor-specific survival signaling in melanoma cells through inactivation of the proapoptotic protein bad.

    PubMed

    Eisenmann, Kathryn M; VanBrocklin, Matthew W; Staffend, Nancy A; Kitchen, Susan M; Koo, Han-Mo

    2003-12-01

    Mitogen-activated protein kinase (MAPK) signaling regulates fundamental cellular functions including proliferation, differentiation, and survival. We have demonstrated previously that inhibiting MAPK signaling induces apoptosis in melanoma cells but not in normal melanocytes, suggesting that the MAPK pathway propagates essential survival signals in melanoma cells. Here, we report that the 90-kDa ribosomal S6 kinase (RSK), a downstream effector in the MAPK signaling cascade, phosphorylates and inactivates the Bcl-2 homology 3-only proapoptotic protein Bad, thereby mediating a MAPK-dependent tumor-specific survival signal in melanoma cells. The MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK)/RSK MAPK signaling module is constitutively hyperactivated, and Bad is maintained in its inactive state by phosphorylation at Ser(75) in a MEK/ERK/RSK-dependent manner in melanoma cells. In contrast, in normal melanocytes, Bad is highly phosphorylated at multiple residues (Ser(75), Ser(99), and Ser(118)) in a MAPK pathway-independent manner. Importantly, ectopic expression of a constitutively activated RSK mutant abrogates Bad activation and renders melanoma cells resistant to apoptosis induced by a MEK inhibitor. Furthermore, overexpressing alanine-substituted (S75A) Bad further sensitizes melanoma cells to MEK inhibitor-induced apoptosis. Our results suggest that the MAPK pathway mediates melanoma-specific survival signaling by differentially regulating RSK-mediated phosphorylation of the proapoptotic protein Bad and may present potentially selective therapeutic targets for the treatment of melanomas.

  3. uPA/uPAR system activation drives a glycolytic phenotype in melanoma cells.

    PubMed

    Laurenzana, Anna; Chillà, Anastasia; Luciani, Cristina; Peppicelli, Silvia; Biagioni, Alessio; Bianchini, Francesca; Tenedini, Elena; Torre, Eugenio; Mocali, Alessandra; Calorini, Lido; Margheri, Francesca; Fibbi, Gabriella; Del Rosso, Mario

    2017-09-15

    In this manuscript, we show the involvement of the uPA/uPAR system in the regulation of aerobic glycolysis of melanoma cells. uPAR over-expression in human melanoma cells controls an invasive and glycolytic phenotype in normoxic conditions. uPAR down-regulation by siRNA or its uncoupling from integrins, and hence from integrin-linked tyrosine kinase receptors (IL-TKRs), by an antagonist peptide induced a striking inhibition of the PI3K/AKT/mTOR/HIF1α pathway, resulting into impairment of glucose uptake, decrease of several glycolytic enzymes and of PKM2, a checkpoint that controls metabolism of cancer cells. Further, binding of uPA to uPAR regulates expression of molecules that govern cell invasion, including extracellular matrix metallo-proteinases inducer (EMPPRIN) and enolase, a glycolytyc enzyme that also serves as a plasminogen receptor, thus providing a common denominator between tumor metabolism and phenotypic invasive features. Such effects depend on the α5β1-integrin-mediated uPAR connection with EGFR in melanoma cells with engagement of the PI3K-mTOR-HIFα pathway. HIF-1α trans-activates genes whose products mediate tumor invasion and glycolysis, thus providing the common denominator between melanoma metabolism and its invasive features. These findings unveil a unrecognized interaction between the invasion-related uPAR and IL-TKRs in the control of glycolysis and disclose a new pharmacological target (i.e., uPAR/IL-TKRs axis) for the therapy of melanoma. © 2017 UICC.

  4. Modulation of Brahma expression by the mitogen-activated protein kinase/extracellular signal regulated kinase pathway is associated with changes in melanoma proliferation.

    PubMed

    Mehrotra, Aanchal; Saladi, Srinivas Vinod; Trivedi, Archit R; Aras, Shweta; Qi, Huiling; Jayanthy, Ashika; Setaluri, Vijayasaradhi; de la Serna, Ivana L

    2014-12-01

    Brahma (BRM) and Brahma-related gene 1(BRG1) are catalytic subunits of SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes. BRM is epigenetically silenced in a wide-range of tumors. Mutations in the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene occur frequently in melanoma and lead to constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK1/2) pathway. We tested the hypothesis that BRM expression is modulated by oncogenic BRAF and phosphorylation of ERK1/2 in melanocytes and melanoma cells. Expression of oncogenic BRAF in melanocytes and melanoma cells that are wild-type for BRAF decreased BRM expression and increased BRG1 expression. Inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) or selective inhibition of BRAF in melanoma cells that harbor oncogenic BRAF increased BRM expression and decreased BRG1 expression. Increased BRM expression was associated with increased histone acetylation on the BRM promoter. Over-expression of BRM in melanoma cells that harbor oncogenic BRAF promoted changes in cell cycle progression and apoptosis consistent with a tumor suppressive role. Upon inhibition of BRAF(V600E) with PLX4032, BRM promoted survival. PLX4032 induced changes in BRM function were correlated with increased acetylation of the BRM protein. This study provides insights into the epigenetic consequences of inhibiting oncogenic BRAF in melanoma through modulation of SWI/SNF subunit expression and function.

  5. 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

  6. CCR5 Blockade Suppresses Melanoma Development Through Inhibition of IL-6-Stat3 Pathway via Upregulation of SOCS3.

    PubMed

    Tang, Qiu; Jiang, Jun; Liu, Jian

    2015-12-01

    In order to understand how tumor cells can escape immune surveillance mechanisms and thus develop antitumor therapies, it is critically important to investigate the mechanisms by which the immune system interacts with the tumor microenvironment. In our current study, we found that chemokine receptor 5 (CCR5) neutralization resulted in reduced melanoma tumor size, decreased percentage of CD11b+ Gr-1(+) myeloid-derived suppressor cells (MDSCs), and increased proportion of cluster of differentiation (CD)3+ T cells in tumor tissues. Suppressive activity of MDSCs on CD4+ T cells and CD8+ T cell proliferation is significantly inhibited by anti-CCR5 antibody. CCR5 blockade also suppresses interleukin (IL)-6 induction, which in turn deactivates signal transducer and activator of transcription 3 (Stat3) in tumors. Furthermore, the suppressed B16 tumor growth induced by CCR5 blockade is abolished with additional administration of recombinant IL-6. CCR5 blockade also induces suppressor of cytokine signaling 3 (SOCS3) upregulations, and anti-CCR5 antibody fails to suppress expression of phospho-Stat3 (p-Stat3), matrix metallopeptidase 9 (MMP9), and IL-6 in cells transfected with SOCS3 short-interfering RNA (SiRNA). All these data suggest that CCR5 blockade suppresses melanoma development through inhibition of IL-6-Stat3 pathway via upregulation of SOCS3.

  7. ANTIPROLIFERATIVE ACTIVITY OF NOVEL ACETYLENIC DERIVATIVES OF BETULIN AGAINST G-361 HUMAN MELANOMA CELLS.

    PubMed

    Bębenek, Ewa; Chodurek, Ewa; Orchel, Arkadiusz; Dzierżewicz, Zofia; Boryczka, Stanisław

    2015-01-01

    Acetylenic derivatives of betulin were tested in vitro for their antiproliferative activity against G-361 human melanoma cells. Two types of betulin derivatives were studied: monoesters, obtained by modification of the hydroxyl group at C-28 position, and diesters modified at both C-28 and C-3 positions. To assess cell proliferation, a colorimetric sulforhodamine B based method was used. All the tested monoesters inhibited cellular growth and 28-O-propynoylbetulin showed the strongest cytotoxic effect. Esterification of the C-3 hydroxyl group of the molecule abolished its growth inhibitory activity.

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

    PubMed

    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.

  9. Targeting the protein ubiquitination machinery in melanoma by the NEDD8-activating enzyme inhibitor pevonedistat (MLN4924).

    PubMed

    Wong, Kit Man; Micel, Lindsey N; Selby, Heather M; Tan, Aik Choon; Pitts, Todd M; Bagby, Stacey M; Spreafico, Anna; Klauck, Peter J; Blakemore, Stephen J; Smith, Peter F; McDonald, Alice; Berger, Allison; Tentler, John J; Eckhardt, S Gail

    2017-02-01

    Background The neddylation pathway conjugates NEDD8 to cullin-RING ligases and controls the proteasomal degradation of specific proteins involved in essential cell processes. Pevonedistat (MLN4924) is a selective small molecule targeting the NEDD8-activating enzyme (NAE) and inhibits an early step in neddylation, resulting in DNA re-replication, cell cycle arrest and death. We investigated the anti-tumor potential of pevonedistat in preclinical models of melanoma. Methods Melanoma cell lines and patient-derived tumor xenografts (PDTX) treated with pevonedistat were assessed for viability/apoptosis and tumor growth, respectively, to identify sensitive/resistant models. Gene expression microarray and gene set enrichment analyses were performed in cell lines to determine the expression profiles and pathways of sensitivity/resistance. Pharmacodynamic changes in treated-PDTX were also characterized. Results Pevonedistat effectively inhibited cell viability (IC50 < 0.3 μM) and induced apoptosis in a subset of melanoma cell lines. Sensitive and resistant cell lines exhibited distinct gene expression profiles; sensitive models were enriched for genes involved in DNA repair, replication and cell cycle regulation, while immune response and cell adhesion pathways were upregulated in resistant models. Pevonedistat also reduced tumor growth in melanoma cell line xenografts and PDTX with variable responses. An accumulation of pevonedistat-NEDD8 adduct and CDT1 was observed in sensitive tumors consistent with its mechanism of action. Conclusions This study provided preclinical evidence that NAE inhibition by pevonedistat has anti-tumor activity in melanoma and supports the clinical benefits observed in recent Phase 1 trials of this drug in melanoma patients. Further investigations are warranted to develop rational combinations and determine predictive biomarkers of pevonedistat.

  10. 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.

  11. Inhibition of vemurafenib-resistant melanoma by interference with pre-mRNA splicing

    PubMed Central

    Salton, Maayan; Kasprzak, Wojciech K.; Voss, Ty; Shapiro, Bruce A.; Poulikakos, Poulikos I.; Misteli, Tom

    2015-01-01

    Mutations in the serine/threonine kinase BRAF are found in more than 60% of melanomas. The most prevalent melanoma mutation is BRAF(V600E), which constitutively activates downstream MAPK signaling. Vemurafenib is a potent RAF kinase inhibitor with remarkable clinical activity in BRAF(V600E)-positive melanoma tumors. However, patients rapidly develop resistance to vemurafenib treatment. One resistance mechanism is the emergence of BRAF alternative splicing isoforms leading to elimination of the RAS-binding domain. Here we identify interference with pre-mRNA splicing as a mechanism to combat vemurafenib resistance. We find that small molecule pre-mRNA splicing modulators reduce BRAF3-9 production and limit in-vitro cell growth of vemurafenib-resistant cells. In xenograft models, interference with pre-mRNA splicing prevents tumor formation and slows growth of vemurafenib-resistant tumors. Our results identify an intronic mutation as a molecular basis for RNA splicing-mediated RAF inhibitor resistance and we identify pre-mRNA splicing interference as a potential therapeutic strategy for drug resistance in BRAF melanoma. PMID:25971842

  12. Inhibition of vemurafenib-resistant melanoma by interference with pre-mRNA splicing.

    PubMed

    Salton, Maayan; Kasprzak, Wojciech K; Voss, Ty; Shapiro, Bruce A; Poulikakos, Poulikos I; Misteli, Tom

    2015-05-14

    Mutations in the serine/threonine kinase BRAF are found in more than 60% of melanomas. The most prevalent melanoma mutation is BRAF(V600E), which constitutively activates downstream MAPK signalling. Vemurafenib is a potent RAF kinase inhibitor with remarkable clinical activity in BRAF(V600E)-positive melanoma tumours. However, patients rapidly develop resistance to vemurafenib treatment. One resistance mechanism is the emergence of BRAF alternative splicing isoforms leading to elimination of the RAS-binding domain. Here we identify interference with pre-mRNA splicing as a mechanism to combat vemurafenib resistance. We find that small-molecule pre-mRNA splicing modulators reduce BRAF3-9 production and limit in-vitro cell growth of vemurafenib-resistant cells. In xenograft models, interference with pre-mRNA splicing prevents tumour formation and slows growth of vemurafenib-resistant tumours. Our results identify an intronic mutation as the molecular basis for a RNA splicing-mediated RAF inhibitor resistance mechanism and we identify pre-mRNA splicing interference as a potential therapeutic strategy for drug resistance in BRAF melanoma.

  13. Inhibition of aquaporin-1 dependent angiogenesis impairs tumour growth in a mouse model of melanoma.

    PubMed

    Nicchia, Grazia P; Stigliano, Cinzia; Sparaneo, Angelo; Rossi, Andrea; Frigeri, Antonio; Svelto, Maria

    2013-05-01

    Prohibiting angiogenesis is an important therapeutic approach for fighting cancer and other angiogenic related diseases. Research focused on proteins that regulate abnormal angiogenesis has attracted intense interest in both academia and industry. Such proteins are able to target several angiogenic factors concurrently, thereby increasing the possibility of therapeutic success. Aquaporin-1 (AQP1) is a water channel membrane protein that promotes tumour angiogenesis by allowing faster endothelial cell migration. In this study we test the hypothesis that AQP1 inhibition impairs tumour growth in a mouse model of melanoma. After validating the inhibitor efficacy of two different AQP1 specific siRNAs in cell cultures, RNA interference experiments were performed by intratumoural injections of AQP1 siRNAs in mice. After 6 days of treatment, AQP1 siRNA treated tumours showed a 75 % reduction in volume when compared to controls. AQP1 protein level, in AQP1 knockdown tumours, was around 75 % that of the controls and was associated with a significant 40 % reduced expression of the endothelial marker, Factor VIII. Immunofluorescence analysis of AQP1 siRNA treated tumours showed a significantly lower microvessel density. Time course experiments demonstrated that repeated injections of AQP1 siRNA over time are effective in sustaining the inhibition of tumour growth. Finally, we have confirmed the role of AQP1 in sustaining an active endothelium during angiogenesis and we have shown that AQP1 reduction causes an increase in VEGF levels. In conclusion, this study validates AQP1 as a pro-angiogenic protein, relevant for the therapy of cancer and other angiogenic-related diseases such as psoriasis, endometriosis, arthritis and atherosclerosis.

  14. Acid Ceramidase in Melanoma

    PubMed Central

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

    2016-01-01

    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

  15. Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells.

    PubMed

    Basit, Farhan; van Oppen, Lisanne Mpe; Schöckel, Laura; Bossenbroek, Hasse M; van Emst-de Vries, Sjenet E; Hermeling, Johannes Cw; Grefte, Sander; Kopitz, Charlotte; Heroult, Melanie; Hgm Willems, Peter; Koopman, Werner Jh

    2017-03-30

    Inhibition of complex I (CI) of the mitochondrial respiratory chain by BAY 87-2243 ('BAY') triggers death of BRAF(V600E) melanoma cell lines and inhibits in vivo tumor growth. Here we studied the mechanism by which this inhibition induces melanoma cell death. BAY treatment depolarized the mitochondrial membrane potential (Δψ), increased cellular ROS levels, stimulated lipid peroxidation and reduced glutathione levels. These effects were paralleled by increased opening of the mitochondrial permeability transition pore (mPTP) and stimulation of autophagosome formation and mitophagy. BAY-induced cell death was not due to glucose shortage and inhibited by the antioxidant α-tocopherol and the mPTP inhibitor cyclosporin A. Tumor necrosis factor receptor-associated protein 1 (TRAP1) overexpression in BAY-treated cells lowered ROS levels and inhibited mPTP opening and cell death, whereas the latter was potentiated by TRAP1 knockdown. Knockdown of autophagy-related 5 (ATG5) inhibited the BAY-stimulated autophagosome formation, cellular ROS increase and cell death. Knockdown of phosphatase and tensin homolog-induced putative kinase 1 (PINK1) inhibited the BAY-induced Δψ depolarization, mitophagy stimulation, ROS increase and cell death. Dynamin-related protein 1 (Drp1) knockdown induced mitochondrial filamentation and inhibited BAY-induced cell death. The latter was insensitive to the pancaspase inhibitor z-VAD-FMK, but reduced by necroptosis inhibitors (necrostatin-1, necrostatin-1s)) and knockdown of key necroptosis proteins (receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and mixed lineage kinase domain-like (MLKL)). BAY-induced cell death was also reduced by the ferroptosis inhibitor ferrostatin-1 and overexpression of the ferroptosis-inhibiting protein glutathione peroxidase 4 (GPX4). This overexpression also inhibited the BAY-induced ROS increase and lipid peroxidation. Conversely, GPX4 knockdown potentiated BAY-induced cell death. We propose a

  16. The combination of vemurafenib and procaspase-3 activation is synergistic in mutant BRAF melanomas

    PubMed Central

    Peh, Jessie; Fan, Timothy M.; Wycislo, Kathryn L.; Roth, Howard S.; Hergenrother, Paul J.

    2016-01-01

    The development of vemurafenib resistance limits the long-term efficacy of this drug for treatment of metastatic melanomas with the V600EBRAF mutation. Inhibition of downstream MAPK signaling with vemurafenib induces apoptotic cell death mediated by caspase-3, suggesting that addition of a procaspase-3 activator could enhance anticancer effects. Here we show that the combination of PAC-1, a procaspase-activating compound, and vemurafenib is highly synergistic in enhancing caspase-3 activity and apoptotic cell death in melanoma cell lines harboring the V600EBRAF mutation. In vivo, the combination displays a favorable safety profile in mice, and exerts significant antitumor effects. We further demonstrate that addition of PAC-1 to the clinically useful combination of vemurafenib and a MEK inhibitor, trametinib, starkly enhances the caspase-3 activity and proapoptotic effect of the combination. Moreover, addition of low concentration PAC-1 also delays the regrowth of cells following treatment with vemurafenib. Finally, PAC-1 remains potent against vemurafenib-resistant A375VR cells in cell culture and synergizes with vemurafenib to exert antitumor effects on A375VR cell growth in vivo. Collectively, our data suggest that inhibition of MAPK signaling combined with concurrent procaspase-3 activation is an effective strategy to enhance the antitumor activity of vemurafenib and mitigate the development of resistance. PMID:27297867

  17. Harmine activates intrinsic and extrinsic pathways of apoptosis in B16F-10 melanoma

    PubMed Central

    2011-01-01

    Background Harmine is a beta-carboline alkaloid from the plant Peganum harmala. Previous studies found that harmine inhibited metastasis of B16F-10 melanoma cells. This study aims to elucidate the role of harmine in apoptosis of B16F-10 cells. Methods B16F-10 melanoma cells were treated in the presence and absence of harmine in vitro. Morphological changes, cell cycle and expression of various pro and anti- apoptotic genes were analyzed for the study of apoptosis. Results Morphological observation and DNA laddering assay showed that harmine treated cells displayed marked apoptotic characteristics, such as nuclear fragmentation, appearance of apoptotic bodies and DNA laddering fragment. TUNEL assay and flow cytometric analysis also confirmed apoptosis. Furthermore, RT-PCR analysis showed that harmine induced apoptosis in B16F-10 melanoma cells by up-regulating Bax and activating Caspase-3, 9 and p53 and down-regulating Bcl-2. Harmine also up-regulated Caspase-8 and Bid, indicating that harmine affected both extrinsic and intrinsic pathways of apoptosis. This study also showed inhibitory effects of harmine on some transcription factors and pro- inflammatory cytokines that protect cell from apoptosis. Conclusion Harmine activates both intrinsic and extrinsic pathways of apoptosis and regulates some transcription factors and pro-inflammatory cytokines. PMID:21429205

  18. 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

  19. Activation of the Kinin B1 Receptor Attenuates Melanoma Tumor Growth and Metastasis

    PubMed Central

    Dillenburg-Pilla, Patricia; Maria, Andrea G.; Reis, Rosana I.; Floriano, Elaine Medeiros; Pereira, Cacilda Dias; De Lucca, Fernando Luiz; Ramos, Simone Gusmão; Pesquero, João B.; Jasiulionis, Miriam G.; Costa-Neto, Claudio M.

    2013-01-01

    Melanoma is a very aggressive tumor that does not respond well to standard therapeutic approaches, such as radio- and chemotherapies. Furthermore, acquiring the ability to metastasize in melanoma and many other tumor types is directly related to incurable disease. The B1 kinin receptor participates in a variety of cancer-related pathophysiological events, such as inflammation and angiogenesis. Therefore, we investigated whether this G protein-coupled receptor plays a role in tumor progression. We used a murine melanoma cell line that expresses the kinin B1 receptor and does not express the kinin B2 receptor to investigate the precise contribution of activation of the B1 receptor in tumor progression and correlated events using various in vitro and in vivo approaches. Activation of the kinin B1 receptor in the absence of B2 receptor inhibits cell migration in vitro and decreases tumor formation in vivo. Moreover, tumors formed from cells stimulated with B1-specific agonist showed several features of decreased aggressiveness, such as smaller size and infiltration of inflammatory cells within the tumor area, higher levels of pro-inflammatory cytokines implicated in the host anti-tumor immune response, lower number of cells undergoing mitosis, a poorer vascular network, no signs of invasion of surrounding tissues or metastasis and increased animal survival. Our findings reveal that activation of the kinin B1 receptor has a host protective role during murine melanoma tumor progression, suggesting that the B1 receptor could be a new anti-tumor GPCR and provide new opportunities for therapeutic targeting. PMID:23691222

  20. Activation of the kinin B1 receptor attenuates melanoma tumor growth and metastasis.

    PubMed

    Dillenburg-Pilla, Patricia; Maria, Andrea G; Reis, Rosana I; Floriano, Elaine Medeiros; Pereira, Cacilda Dias; De Lucca, Fernando Luiz; Ramos, Simone Gusmão; Pesquero, João B; Jasiulionis, Miriam G; Costa-Neto, Claudio M

    2013-01-01

    Melanoma is a very aggressive tumor that does not respond well to standard therapeutic approaches, such as radio- and chemotherapies. Furthermore, acquiring the ability to metastasize in melanoma and many other tumor types is directly related to incurable disease. The B1 kinin receptor participates in a variety of cancer-related pathophysiological events, such as inflammation and angiogenesis. Therefore, we investigated whether this G protein-coupled receptor plays a role in tumor progression. We used a murine melanoma cell line that expresses the kinin B1 receptor and does not express the kinin B2 receptor to investigate the precise contribution of activation of the B1 receptor in tumor progression and correlated events using various in vitro and in vivo approaches. Activation of the kinin B1 receptor in the absence of B2 receptor inhibits cell migration in vitro and decreases tumor formation in vivo. Moreover, tumors formed from cells stimulated with B1-specific agonist showed several features of decreased aggressiveness, such as smaller size and infiltration of inflammatory cells within the tumor area, higher levels of pro-inflammatory cytokines implicated in the host anti-tumor immune response, lower number of cells undergoing mitosis, a poorer vascular network, no signs of invasion of surrounding tissues or metastasis and increased animal survival. Our findings reveal that activation of the kinin B1 receptor has a host protective role during murine melanoma tumor progression, suggesting that the B1 receptor could be a new anti-tumor GPCR and provide new opportunities for therapeutic targeting.

  1. NLRC4 suppresses melanoma tumor progression independently of inflammasome activation

    PubMed Central

    Janowski, Ann M.; Colegio, Oscar R.; Hornick, Emma E.; McNiff, Jennifer M.; Martin, Matthew D.; Badovinac, Vladimir P.; Norian, Lyse A.; Zhang, Weizhou; Cassel, Suzanne L.

    2016-01-01

    Members of the NLR family can assemble inflammasome complexes with the adaptor protein ASC and caspase-1 that result in the activation of caspase-1 and the release of IL-1β and IL-18. Although the NLRC4 inflammasome is known to have a protective role in tumorigenesis, there is an increased appreciation for the inflammasome-independent actions of NLRC4. Here, we utilized a syngeneic subcutaneous murine model of B16F10 melanoma to explore the role of NLRC4 in tumor suppression. We found that NLRC4-deficient mice exhibited enhanced tumor growth that was independent of the inflammasome components ASC and caspase-1. Nlrc4 expression was critical for cytokine and chemokine production in tumor-associated macrophages and was necessary for the generation of protective IFN-γ–producing CD4+ and CD8+ T cells. Tumor progression was diminished when WT or caspase-1–deficient, but not NLRC4-deficient, macrophages were coinjected with B16F10 tumor cells in NLRC4-deficient mice. Finally, examination of human primary melanomas revealed the extensive presence of NLRC4+ tumor-associated macrophages. In contrast, there was a paucity of NLRC4+ tumor-associated macrophages observed in human metastatic melanoma, supporting the concept that NLRC4 expression controls tumor growth. These results reveal a critical role for NLRC4 in suppressing tumor growth in an inflammasome-independent manner. PMID:27617861

  2. HERV-K activation is strictly required to sustain CD133+ melanoma cells with stemness features.

    PubMed

    Argaw-Denboba, Ayele; Balestrieri, Emanuela; Serafino, Annalucia; Cipriani, Chiara; Bucci, Ilaria; Sorrentino, Roberta; Sciamanna, Ilaria; Gambacurta, Alessandra; Sinibaldi-Vallebona, Paola; Matteucci, Claudia

    2017-01-26

    Melanoma is a heterogeneous tumor in which phenotype-switching and CD133 marker have been associated with metastasis promotion and chemotherapy resistance. CD133 positive (CD133+) subpopulation has also been suggested as putative cancer stem cell (CSC) of melanoma tumor. Human endogenous retrovirus type K (HERV-K) has been described to be aberrantly activated during melanoma progression and implicated in the etiopathogenesis of disease. Earlier, we reported that stress-induced HERV-K activation promotes cell malignant transformation and reduces the immunogenicity of melanoma cells. Herein, we investigated the correlation between HERV-K and the CD133+ melanoma cells during microenvironmental modifications. TVM-A12 cell line, isolated in our laboratory from a primary human melanoma lesion, and other commercial melanoma cell lines (G-361, WM-115, WM-266-4 and A375) were grown and maintained in the standard and stem cell media. RNA interference, Real-time PCR, flow cytometry analysis, self-renewal and migration/invasion assays were performed to characterize cell behavior and HERV-K expression. Melanoma cells, exposed to stem cell media, undergo phenotype-switching and expansion of CD133+ melanoma cells, concomitantly promoted by HERV-K activation. Notably, the sorted CD133+ subpopulation showed stemness features, characterized by higher self-renewal ability, embryonic genes expression, migration and invasion capacities compared to the parental cell line. RNA interference-mediated downregulation experiments showed that HERV-K has a decisive role to expand and maintain the CD133+ melanoma subpopulation during microenvironmental modifications. Similarly, non nucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine were effective to restrain the activation of HERV-K in melanoma cells, to antagonize CD133+ subpopulation expansion and to induce selective high level apoptosis in CD133+ cells. HERV-K activation promotes melanoma cells phenotype

  3. microRNA-7-5p inhibits melanoma cell proliferation and metastasis by suppressing RelA/NF-κB.

    PubMed

    Giles, Keith M; Brown, Rikki A M; Ganda, Clarissa; Podgorny, Melissa J; Candy, Patrick A; Wintle, Larissa C; Richardson, Kirsty L; Kalinowski, Felicity C; Stuart, Lisa M; Epis, Michael R; Haass, Nikolas K; Herlyn, Meenhard; Leedman, Peter J

    2016-05-31

    microRNA-7-5p (miR-7-5p) is a tumor suppressor in multiple cancer types and inhibits growth and invasion by suppressing expression and activity of the epidermal growth factor receptor (EGFR) signaling pathway. While melanoma is not typically EGFR-driven, expression of miR-7-5p is reduced in metastatic tumors compared to primary melanoma. Here, we investigated the biological and clinical significance of miR-7-5p in melanoma. We found that augmenting miR-7-5p expression in vitro markedly reduced tumor cell viability, colony formation and induced cell cycle arrest. Furthermore, ectopic expression of miR-7-5p reduced migration and invasion of melanoma cells in vitro and reduced metastasis in vivo. We used cDNA microarray analysis to identify a subset of putative miR-7-5p target genes associated with melanoma and metastasis. Of these, we confirmed nuclear factor kappa B (NF-κB) subunit RelA, as a novel direct target of miR-7-5p in melanoma cells, such that miR-7-5p suppresses NF-κB activity to decrease expression of canonical NF-κB target genes, including IL-1β, IL-6 and IL-8. Importantly, the effects of miR-7-5p on melanoma cell growth, cell cycle, migration and invasion were recapitulated by RelA knockdown. Finally, analysis of gene array datasets from multiple melanoma patient cohorts revealed an association between elevated RelA expression and poor survival, further emphasizing the clinical significance of RelA and its downstream signaling effectors. Taken together, our data show that miR-7-5p is a potent inhibitor of melanoma growth and metastasis, in part through its inactivation of RelA/NF-κB signaling. Furthermore, miR-7-5p replacement therapy could have a role in the treatment of this disease.

  4. 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.

  5. Dependence On Glycolysis Sensitizes BRAF-mutated Melanomas For Increased Response To Targeted BRAF Inhibition

    PubMed Central

    Hardeman, Keisha N.; Peng, Chengwei; Paudel, Bishal B.; Meyer, Christian T.; Luong, Thong; Tyson, Darren R.; Young, Jamey D.; Quaranta, Vito; Fessel, Joshua P.

    2017-01-01

    Dysregulated metabolism can broadly affect therapy resistance by influencing compensatory signaling and expanding proliferation. Given many BRAF-mutated melanoma patients experience disease progression with targeted BRAF inhibitors, we hypothesized therapeutic response is related to tumor metabolic phenotype, and that altering tumor metabolism could change therapeutic outcome. We demonstrated the proliferative kinetics of BRAF-mutated melanoma cells treated with the BRAF inhibitor PLX4720 fall along a spectrum of sensitivity, providing a model system to study the interplay of metabolism and drug sensitivity. We discovered an inverse relationship between glucose availability and sensitivity to BRAF inhibition through characterization of metabolic phenotypes using nearly a dozen metabolic parameters in Principle Component Analysis. Subsequently, we generated rho0 variants that lacked functional mitochondrial respiration and increased glycolytic metabolism. The rho0 cell lines exhibited increased sensitivity to PLX4720 compared to the respiration-competent parental lines. Finally, we utilized the FDA-approved antiretroviral drug zalcitabine to suppress mitochondrial respiration and to force glycolysis in our cell line panel, resulting in increased PLX4720 sensitivity via shifts in EC50 and Hill slope metrics. Our data suggest that forcing tumor glycolysis in melanoma using zalcitabine or other similar approaches may be an adjunct to increase the efficacy of targeted BRAF therapy. PMID:28205616

  6. 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

  7. Epigallocatechin-3-gallate(EGCG) suppresses melanoma cell growth and metastasis by targeting TRAF6 activity

    PubMed Central

    Zhang, Xu; Zhou, Youyou; Luo, Zhongling; Zeng, Weiqi; Su, Juan; Peng, Cong; Chen, Xiang

    2016-01-01

    TRAF6 (TNF Receptor-Associated Factor 6) is an E3 ubiquitin ligase that contains a Ring domain, induces K63-linked polyubiquitination, and plays a critical role in signaling transduction. Our previous results demonstrated that TRAF6 is overexpressed in melanoma and that TRAF6 knockdown dramatically attenuates tumor cell growth and metastasis. In this study, we found that EGCG can directly bind to TRAF6, and a computational model of the interaction between EGCG and TRAF6 revealed that EGCG probably interacts with TRAF6 at the residues of Gln54, Gly55, Asp57 ILe72, Cys73 and Lys96. Among these amino acids, mutation of Gln54, Asp57, ILe72 in TRAF6 could destroy EGCG bound to TRAF6, furthermore, our results demonstrated that EGCG significantly attenuates interaction between TRAF6 and UBC13(E2) and suppresses TRAF6 E3 ubiquitin ligase activity in vivo and in vitro. Additionally, the phosphorylation of IκBα, p-TAK1 expression are decreased and the nuclear translocation of p65 and p50 is blocked by treatment with EGCG, leading to inactivation of the NF-κB pathway. Moreover, EGCG significantly inhibits cell growth as well as the migration and invasion of melanoma cells. Taken together, these findings show that EGCG is a novel E3 ubiquitin ligase inhibitor that could be used to target TRAF6 for chemotherapy or the prevention of melanoma. PMID:27791197

  8. 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

  9. Novel Targeted Therapies for Metastatic Melanoma.

    PubMed

    Iams, Wade T; Sosman, Jeffrey A; Chandra, Sunandana

    Oncogene-targeted therapy is a major component of precision oncology, and although patients with metastatic melanoma have experienced improved outcomes with this strategy, there are a number of potential therapeutic targets currently under study that may further increase the drug armamentarium for this patient population. In this review, we discuss the landscape of targeted therapies for patients with advanced melanoma, focusing on oncogene mutation-specific targets. In patients with typical BRAF V600-mutant melanoma, combination BRAF and MEK inhibition has surpassed outcomes compared with monotherapy with BRAF or MEK inhibition alone, and current strategies seek to address inevitable resistance mechanisms. For patients with NRAS-mutant melanoma, MEK inhibitor monotherapy and combined MEK and CDK4/6 inhibition are burgeoning strategies; for patients with KIT-mutant melanoma, tyrosine kinase inhibition is being leveraged, and for NF-1-mutant melanoma, mTOR and MEK inhibition is being actively evaluated. In patients with atypical, non-V600 BRAF-mutant melanoma, MEK inhibitor monotherapy is the potential novel targeted approach on the horizon. For advanced uveal melanoma, novel targets such as IMCgp100 and glembatumumab have shown activity in early studies. We review additional strategies that remain in the preclinical and early clinical pipeline, so there is much hope for the future of targeted agents for distinct molecular cohorts of patients with advanced melanoma.

  10. Effect of melanoma cells on proliferation and migration of activated hepatic stellate cells in vitro.

    PubMed

    Meyer, Theresa; Koch, Andreas; Ebert, Eva-Vanessa; Czech, Barbara; Mueller, Martina; Bosserhoff, Anja; Lang, Sven Arke; Hellerbrand, Claus

    2017-04-01

    Melanoma is a highly aggressive tumor of the skin. The clinical outcome is determined by the presence or absence of metastases, and the liver is a common site of distant metastases. Hepatic metastasis is causing activation of hepatic stellate cells (HSC), which form the stroma of hepatic metastases and are increasingly recognized as a crucial component of the pro- metastatic liver microenvironment. Most studies have focused on the effects of HSC on (metastasizing) tumor cells. Here, we aimed to analyze functional in vitro effects of conditioned medium (CM) of twelve different human melanoma cell lines on LX2 cells and HSC(htert) cells, two well established human activated HSC cell lines. CM from melanoma cells significantly induced HSC proliferation and acted as chemoattractant for HSC in Boyden chamber assays. The CM effects significantly varied between different HSC as well as melanoma cells. Interestingly, CM from melanoma cell lines derived from melanoma metastases (WM239A, WM9, WM1158, WM1232, 451Lu and 1205Lu) had a stronger effect on proliferation of HSC(htert) cells than CM derived from primary melanoma tumors (SbCl2, WM3211, WM35, WM278, WM1366 and WM793). Moreover, we observed a significant correlation between the chemoattractive effects of CM from the different melanoma cells on HSC(htert) and LX2 cells. In contrast, the melanoma CM effects on the proliferation of the two HSC lines did not show a significant correlation. In summary, our data indicate that melanoma cells metastasizing to the liver have the potential to attract HSC and to induce HSC proliferation, respectively. Still, it appears that melanoma effects on HSC migration and proliferation are mediated via different soluble factors indicating the complexity of melanoma-HSC interaction. Furthermore, the intensity of at least some functional effects varies between different human tumor cells and HSC which may point to mechanisms explaining diverse hepatic metastasis in melanoma patients

  11. Disulfiram induces copper-dependent stimulation of reactive oxygen species and activation of the extrinsic apoptotic pathway in melanoma.

    PubMed

    Morrison, Brian W; Doudican, Nicole A; Patel, Kirtesh R; Orlow, Seth J

    2010-02-01

    Melanoma is the most aggressive and deadly form of skin cancer. The current standard of care produces response rates of less than 20%, underscoring the critical need for identification of new effective, nontoxic therapies. Disulfiram (DSF) was identified using a drug screen as one of the several compounds that preferentially decreased proliferation in multiple melanoma subtypes compared with benign melanocytes. DSF, a member of the dithiocarbamate family, is a copper (Cu) chelator, and Cu has been shown previously to enhance DSF-mediated growth inhibition and apoptosis in cancer cells. Here, we report that in the presence of free Cu, DSF inhibits cellular proliferation and induces apoptosis in a panel of cell lines representing primary and metastatic nodular and superficial spreading melanoma. Both decreased cellular proliferation and increased apoptosis were seen at 50-500 nmol/l DSF concentrations that are achievable through oral dosing of the medication. In the presence of Cu, DSF caused activation of the extrinsic pathway of apoptosis as measured by caspase-8 cleavage. The addition of Z-IETD-FMK, a selective caspase-8 inhibitor, was protective against DSF-Cu-induced apoptosis. Production of reactive oxygen species (ROS) in response to DSF-Cu treatment preceded the induction of apoptosis. Both ROS production and apoptosis were prevented by coincubation of N-acetyl cysteine, a free radical scavenger. Our study shows that DSF might be used to target both nodular and superficial spreading melanoma through ROS production and activation of the extrinsic pathway of apoptosis.

  12. Curcumin analog DM-1 in monotherapy or combinatory treatment with dacarbazine as a strategy to inhibit in vivo melanoma progression.

    PubMed

    Faião-Flores, Fernanda; Quincoces Suarez, José Agustín; Fruet, Andréa Costa; Maria-Engler, Silvya Stuchi; Pardi, Paulo Celso; Maria, Durvanei Augusto

    2015-01-01

    Malignant melanoma is a highly aggressive form of skin cancer with a high mortality rate if not discovered in early stages. Although a limited number of treatment options for melanoma currently exist, patients with a more aggressive form of this cancer frequently decline treatment. DM-1 is a sodium phenolate and curcumin analog with proven anticancer, anti-proliferative and anti-metastatic properties. In this paper, the DM-1 compound showed in vivo antitumor activity alone or in combination with chemotherapeutic DTIC in B16F10 melanoma-bearing mice. Beneficial effects such as melanoma tumor burden reduction with pyknotic nuclei, decreased nuclei/cytoplasmic ratio and nuclear degradation occurred after DM-1 treatment. No toxicological changes were observed in the liver, kidneys, spleen and lungs after DM-1 monotherapy or DTIC combined therapy. DTIC+DM-1 treatment induced the recovery of anemia arising from melanoma and immunomodulation. Both DM-1 treatment alone and in combination with DTIC induced apoptosis with the cleavage of caspase-3, -8 and -9. Furthermore, melanoma tumors treated with DM-1 showed a preferential apoptotic intrinsic pathway by decreasing Bcl-2/Bax ratio. Considering the chemoresistance exhibited by melanoma towards conventional chemotherapy drugs, DM-1 compound in monotherapy or in combination therapy provides a promising improvement in melanoma treatment with a reduction of side effects.

  13. Curcumin Analog DM-1 in Monotherapy or Combinatory Treatment with Dacarbazine as a Strategy to Inhibit In Vivo Melanoma Progression

    PubMed Central

    Faião-Flores, Fernanda; Quincoces Suarez, José Agustín; Fruet, Andréa Costa; Maria-Engler, Silvya Stuchi; Pardi, Paulo Celso; Maria, Durvanei Augusto

    2015-01-01

    Malignant melanoma is a highly aggressive form of skin cancer with a high mortality rate if not discovered in early stages. Although a limited number of treatment options for melanoma currently exist, patients with a more aggressive form of this cancer frequently decline treatment. DM-1 is a sodium phenolate and curcumin analog with proven anticancer, anti-proliferative and anti-metastatic properties. In this paper, the DM-1 compound showed in vivo antitumor activity alone or in combination with chemotherapeutic DTIC in B16F10 melanoma-bearing mice. Beneficial effects such as melanoma tumor burden reduction with pyknotic nuclei, decreased nuclei/cytoplasmic ratio and nuclear degradation occurred after DM-1 treatment. No toxicological changes were observed in the liver, kidneys, spleen and lungs after DM-1 monotherapy or DTIC combined therapy. DTIC+DM-1 treatment induced the recovery of anemia arising from melanoma and immunomodulation. Both DM-1 treatment alone and in combination with DTIC induced apoptosis with the cleavage of caspase-3, -8 and -9. Furthermore, melanoma tumors treated with DM-1 showed a preferential apoptotic intrinsic pathway by decreasing Bcl-2/Bax ratio. Considering the chemoresistance exhibited by melanoma towards conventional chemotherapy drugs, DM-1 compound in monotherapy or in combination therapy provides a promising improvement in melanoma treatment with a reduction of side effects. PMID:25742310

  14. Induction of arginosuccinate synthetase (ASS) expression affects the antiproliferative activity of arginine deiminase (ADI) in melanoma cells.

    PubMed

    Manca, Antonella; Sini, Maria Cristina; Izzo, Francesco; Ascierto, Paolo A; Tatangelo, Fabiana; Botti, Gerardo; Gentilcore, Giusy; Capone, Marilena; Mozzillo, Nicola; Rozzo, Carla; Cossu, Antonio; Tanda, Francesco; Palmieri, Giuseppe

    2011-06-01

    Arginine deiminase (ADI), an arginine-degrading enzyme, has been used in the treatment of tumours sensitive to arginine deprivation, such as malignant melanoma (MM) and hepatocellular carcinoma (HCC). Endogenous production of arginine is mainly dependent on activity of ornithine transcarbamylase (OTC) and argininosuccinate synthetase (ASS) enzymes. We evaluated the effect of ADI treatment on OTC and ASS expression in a series of melanoma cell lines. Twenty-five primary melanoma cell lines and normal fibroblasts as controls underwent cell proliferation assays and Western blot analyses in the presence or absence of ADI. Tissue sections from primary MMs (N = 20) and HCCs (N = 20) were investigated by immunohistochemistry for ASS expression. Overall, 21/25 (84%) MM cell lines presented a cell growth inhibition by ADI treatment; none of them presented constitutive detectable levels of the ASS protein. However, 7/21 (33%) ADI-sensitive melanoma cell lines presented markedly increased expression levels of the ASS protein following ADI treatment, with a significantly higher IC50 median value. Growth was not inhibited and the IC50 was not reached among the remaining 4/25 (16%) MM cell lines; all of them showed constitutive ASS expression. The OTC protein was found expressed in all melanoma cell lines before and after the ADI treatment. Lack of ASS immunostaining was observed in all analyzed in vivo specimens. Our findings suggest that response to ADI treatment in melanoma is significantly correlated with the ability of cells to express ASS either constitutively at basal level (inducing drug resistance) or after the treatment (reducing sensitivity to ADI).

  15. 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.

  16. 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

  17. Subcutaneous adipocytes promote melanoma cell growth by activating the Akt signaling pathway: role of palmitic acid.

    PubMed

    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

    2014-10-31

    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.

  18. PD-1 inhibition and treatment of advanced melanoma-role of pembrolizumab.

    PubMed

    Jazirehi, Ali R; Lim, Alexandra; Dinh, Tam

    2016-01-01

    Remarkable clinical responses have been seen in patients with metastatic melanoma with targeted therapy (BRAFi vemurafenib, MEKi) and with modern immune cell-based approaches such as TCR engineered adoptive cell transfer (ACT) and earlier experiences with high-dose IL-2. The proximal mediators of these immune therapies are tumor-reactive CTL. Various mechanisms of resistance to immune-mediated apoptotic signals have been described, including phenotypic changes, effector cell exhaustion, functional tolerance, deficiencies in Ag processing and presentation, and mutation or down-regulation of antigenic epitopes. The immune system and drugs eradicate tumors via apoptosis. Therefore, tumors' resistance to apoptosis may be a determining factor that limits the efficacy of immunotherapies. It is predicted that these therapies have limited efficacy in patients whose melanomas have developed resistance to targeted therapy such as vemurafenib. Upregulation of the immune checkpoint molecule CTLA-4 on activated T cells and its interaction with CD80/86 blocks T cell activation. The fully humanized mAb ipilimumab blocks this interaction, resulting in sustained T cell stimulation. Likewise, the programmed death receptor 1 (PD-1) is another member of the B7:CD28 family of costimulatory molecules that regulates T cell activation, whose ligand (PD-L1) is expressed on melanomas. The human anti-PD-1 mAb, Pembrolizumab, overcomes tolerance, has a favorable pharmacokinetics profile with minimal undesired toxic side effects and has shown remarkable improvement in melanoma therapy. This review focuses on recent advances in the development of various anti-PD-1 checkpoint blockade antibodies and will summarize recent clinical data using immune checkpoint blocking antibodies.

  19. miR-7 reverses the resistance to BRAFi in melanoma by targeting EGFR/IGF-1R/CRAF and inhibiting the MAPK and PI3K/AKT signaling pathways.

    PubMed

    Sun, Xiaoyan; Li, Jun; Sun, Yanhong; Zhang, Yi; Dong, Liyun; Shen, Chen; Yang, Liu; Yang, Ming; Li, Yan; Shen, Guanxin; Tu, Yating; Tao, Juan

    2016-08-16

    MicroRNAs (miRNAs) are attractive therapeutic targets for various therapy-resistant tumors. However, the association between miRNA and BRAF inhibitor resistance in melanoma remains to be elucidated. We used microarray analysis to comprehensively study the miRNA expression profiling of vemurafenib resistant (VemR) A375 melanoma cells in relation to parental A375 melanoma cells. MicroRNA-7 (miR-7) was identified to be the most significantly down-regulated miRNA in VemR A375 melanoma cells. We also found that miR-7 was down-regulated in Mel-CVR cells (vemurafenib resistant Mel-CV melanoma cells). Reestablishment of miR-7 expression could reverse the resistance of both cells to vemurafenib. We showed that epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R) and CRAF were over-expressed in VemR A375 melanoma cells. Introduction of miR-7 mimics could markedly decrease the expressions of EGFR, IGF-1R and CRAF and further suppressed the activation of MAPK and PI3K/AKT pathway in VemR A375 melanoma cells. Furthermore, tumor growth was inhibited in an in vivo murine VemR A375 melanoma tumor model transfected with miR-7 mimics. Collectively, our study demonstrated that miR-7 could reverse the resistance to BRAF inhibitors in certain vemurafenib resistant melanoma cell lines. It could advance the field and provide the basis for further studies in BRAF inhibitor resistance in melanoma.

  20. L576P KIT mutation in anal melanomas correlates with KIT protein expression and is sensitive to specific kinase inhibition.

    PubMed

    Antonescu, Cristina R; Busam, Klaus J; Francone, Todd D; Wong, Grace C; Guo, Tianhua; Agaram, Narasimhan P; Besmer, Peter; Jungbluth, Achim; Gimbel, Mark; Chen, Chin-Tung; Veach, Darren; Clarkson, Bayard D; Paty, Philip B; Weiser, Martin R

    2007-07-15

    Activating mutations in either BRAF or NRAS are seen in a significant number of malignant melanomas, but their incidence appears to be dependent to ultraviolet light exposure. Thus, BRAF mutations have the highest incidence in non-chronic sun damaged (CSD), and are uncommon in acral, mucosal and CSD melanomas. More recently, activating KIT mutations have been described in rare cases of metastatic melanoma, without further reference to their clinical phenotypes. This finding is intriguing since KIT expression is downregulated in most melanomas progressing to more aggressive lesions. In this study, we investigated a group of anal melanomas for the presence of BRAF, NRAS, KIT and PDGFRA mutations. A heterozygous KIT exon 11 L576P substitution was identified in 3 of 20 cases tested. The 3 KIT mutation-carrying tumors were strongly immunopositive for KIT protein. No KIT mutations were identified in tumors with less than 4+ KIT immunostaining. NRAS mutation was identified in one tumor. No BRAF or PDGFRA mutations were identified in either KIT positive or negative anal melanomas. In vitro drug testing of stable transformant Ba/F3 KIT(L576P) mutant cells showed sensitivity for dasatinib (previously known as BMS-354825), a dual SRC/ABL kinase inhibitor, and imatinib. However, compared to an imatinib-sensitive KIT mutant, dasatinib was potent at lower doses than imatinib in the KIT(L576P) mutant. These results suggest that a subset of anal melanomas show activating KIT mutations, which are susceptible for therapy with specific kinase inhibitors.

  1. Baicalein Inhibits the Migration and Invasion of B16F10 Mouse Melanoma Cells through Inactivation of the PI3K/Akt Signaling Pathway

    PubMed Central

    Choi, Eun-Ok; Cho, Eun-Ju; Jeong, Jin-Woo; Park, Cheol; Hong, Su-Hyun; Hwang, Hye-Jin; Moon, Sung-Kwon; Son, Chang Gue; Kim, Wun-Jae; Choi, Yung Hyun

    2017-01-01

    Baicalein, a natural flavonoid obtained from the rhizome of Scutellaria baicalensis Georgi, has been reported to have anticancer activities in several human cancer cell lines. However, its antimetastatic effects and associated mechanisms in melanoma cells have not been extensively studied. The current study examined the effects of baicalein on cell motility and anti-invasive activity using mouse melanoma B16F10 cells. Within the noncytotoxic concentration range, baicalein significantly inhibited the cell motility and invasiveness of B16F10 cells in a concentration-dependent manner. Baicalein also reduced the activity and expression of matrix metalloproteinase (MMP)-2 and -9; however, the levels of tissue inhibitor of metalloproteinase-1 and -2 were concomitantly increased. The inhibitory effects of baicalein on cell motility and invasiveness were found to be associated with its tightening of tight junction (TJ), which was demonstrated by an increase in transepithelial electrical resistance and downregulation of the claudin family of proteins. Additionally, treatment with baicalein markedly reduced the expression levels of lipopolysaccharide-induced phosphorylated Akt and the invasive activity in B16F10 cells. Taken together, these results suggest that baicalein inhibits B16F10 melanoma cell migration and invasion by reducing the expression of MMPs and tightening TJ through the suppression of claudin expression, possibly in association with a suppression of the phosphoinositide 3-kinase/Akt signaling pathway. PMID:27530117

  2. BRAF Inhibitors Amplify the Proapoptotic Activity of MEK Inhibitors by Inducing ER Stress in NRAS-Mutant Melanoma.

    PubMed

    Niessner, Heike; Sinnberg, Tobias; Kosnopfel, Corinna; Smalley, Keiran S M; Beck, Daniela; Praetorius, Christian; Mai, Marion; Beissert, Stefan; Kulms, Dagmar; Schaller, Martin; Garbe, Claus; Flaherty, Keith T; Westphal, Dana; Wanke, Ines; Meier, Friedegund

    2017-07-19

    Purpose: NRAS mutations in malignant melanoma are associated with aggressive disease requiring rapid antitumor intervention, but there is no approved targeted therapy for this subset of patients. In clinical trials, the MEK inhibitor (MEKi) binimetinib displayed modest antitumor activity, making combinations a requisite. In a previous study, the BRAF inhibitor (BRAFi) vemurafenib was shown to induce endoplasmic reticulum (ER) stress that together with inhibition of the RAF-MEK-ERK (MAPK) pathway amplified its proapoptotic activity in BRAF-mutant melanoma. The present study investigated whether this effect might extent to NRAS-mutant melanoma, in which MAPK activation would be expected.Experimental Design and Results: BRAFi increased pERK, but also significantly increased growth inhibition and apoptosis induced by the MEKi in monolayer, spheroids, organotypic, and patient-derived tissue slice cultures of NRAS-mutant melanoma. BRAFi such as encorafenib induced an ER stress response via the PERK pathway, as detected by phosphorylation of eIF2α and upregulation of the ER stress-related factors ATF4, CHOP, and NUPR1 and the proapoptotic protein PUMA. MEKi such as binimetinib induced the expression of the proapoptotic protein BIM and activation of the mitochondrial pathway of apoptosis, the latter of which was enhanced by combination with encorafenib. The increased apoptotic rates caused by the combination treatment were significantly reduced through siRNA knockdown of ATF4 and BIM, confirming its critical roles in this process.Conclusion: The data presented herein encourage further advanced in vivo and clinical studies to evaluate MEKi in combination with ER stress inducing BRAFi as a strategy to treat rapidly progressing NRAS-mutant melanoma. Clin Cancer Res; 1-12. ©2017 AACR. ©2017 American Association for Cancer Research.

  3. TERT promoter mutations in melanoma render TERT expression dependent on MAPK pathway activation

    PubMed Central

    Vallarelli, Andrelou F.; Rachakonda, P. Sivaramakrishna; André, Jocelyne; Heidenreich, Barbara; Riffaud, Laurence; Bensussan, Armand; Kumar, Rajiv; Dumaz, Nicolas

    2016-01-01

    The mechanism of telomerase re-activation in cancer had remained elusive until the discovery of frequent mutations in the promoter of the TERT gene that encodes the catalytic reverse transcriptase subunit of telomerase. We investigated the regulation of TERT expression in melanoma cell lines and our results show that promoter mutations render TERT expression dependent on MAPK activation due to oncogenic BRAF or NRAS mutations. Mutations in the TERT promoter create binding sites for ETS transcription factors. ETS1, expressed in melanoma cell lines, undergoes activating phosphorylation by ERK at Thr38 residue as a consequence of constitutively activated MAPK pathway. We demonstrate that ETS1 binds on the mutated TERT promoter leading to the re-expression of the gene. The inhibition of ETS1 resulted in reduced TERT expression. We provide evidence that the TERT promoter mutations provide a direct link between TERT expression and MAPK pathway activation due to BRAF or NRAS mutations via the transcription factor ETS1. PMID:27449293

  4. 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.

  5. Non-Canonical Smads Phosphorylation Induced by the Glutamate Release Inhibitor, Riluzole, through GSK3 Activation in Melanoma

    PubMed Central

    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

  6. Expression of microphthalmia-associated transcription factor (MITF), which is critical for melanoma progression, is inhibited by both transcription factor GLI2 and transforming growth factor-β.

    PubMed

    Pierrat, Marie-Jeanne; Marsaud, Véronique; Mauviel, Alain; Javelaud, Delphine

    2012-05-25

    The melanocyte-specific transcription factor M-MITF is involved in numerous aspects of melanoblast lineage biology including pigmentation, survival, and migration. It plays complex roles at all stages of melanoma progression and metastasis. We established previously that GLI2, a Kruppel-like transcription factor that acts downstream of Hedgehog signaling, is a direct transcriptional target of the TGF-β/SMAD pathway and contributes to melanoma progression, exerting antagonistic activities against M-MITF to control melanoma cell invasiveness. Herein, we dissected the molecular mechanisms underlying both TGF-β and GLI2-driven M-MITF gene repression. Using transient cell transfection experiments with M-MITF promoter constructs, chromatin immunoprecipitation, site-directed mutagenesis, and electrophoretic mobility shift assays, we identified a GLI2 binding site within the -334/-296 region of the M-MITF promoter, critical for GLI2-driven transcriptional repression. This region is, however, not needed for inhibition of M-MITF promoter activity by TGF-β. We determined that TGF-β rapidly repressed protein kinase A activity, thus reducing both phospho-cAMP-response element-binding protein (CREB) levels and CREB-dependent transcription of the M-MITF promoter. Increased GLI2 binding to its cognate cis-element, associated with reduced CREB-dependent transcription, allowed maximal inhibition of the M-MITF promoter via two distinct mechanisms.

  7. 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

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

    PubMed

    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

    2015-05-15

    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 are unknown. We conducted a phase II study of nilotinib 400 mg twice a day 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 (TTP), and overall survival (OS). A Simon two-stage and a single-stage design was planned to assess for the primary endpoint in cohorts A and B, respectively. Twenty patients were enrolled and 19 treated (11 in cohort A; 8 in cohort B). Three patients on cohort A [27%; 95% confidence interval (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 TTP and OS 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. 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. ©2015 American Association for Cancer Research.

  9. [Advances in clinical treatment of malignant melanoma: B-RAF kinase inhibition].

    PubMed

    Heneberg, P

    2011-01-01

    Malignant melanoma is an aggressive cancer of pigment-producing cells, derivates of the neural crest. Surgical resection is the most effective form of treatment during initial phases of the disease. Advanced stages are usually treated by adjuvant immunotherapy (interferon alpha) or dacarbazine + multiferon. Response and survival rates are extremely poor. The emerging approach of personalized medicine brings about significant advances in the treatment of melanoma. Apart from administration of imatinib for a small subgroup of melanomas harbouring KIT mutations, the most promising approach is the use of B-RAF kinase inhibitors. The previously tested RAF inhibitors (e.g. sorafenib) did not perform better compared to conventional chemotherapy or immunotherapy. However, the results are much more promising with the recently developed inhibitor PLX4032 (Plexxikon; RG7204, Roche Pharmaceuticals; vemurafenib). This inhibitor targets tumours harbouring B-RAF(V600E) of B-RAF(V600K) activating mutations, which are present in 40-70% of malignant melanomas. An absence of the above mentioned activating mutations or parallel presence of activating RAS mutations (e.g. RAS(G12D)) should be used as contraindications. The use of PLX4032 provides better outcome than any of the currently used therapies, including partial or complete response recorded in 81% of patients, and prolonged median survival. Currently, this drug is being tested in phase II and III trials. The incidence of PLX4032-related adverse effects is relatively high; acquired resistance repeatedly occurring within several months of treatment may also represent a significant problem. Combined therapy is probably needed to further increase the complete response rate and to prolong survival. This should either include some of the currently used chemotherapeutics, or alternatively it may employ inhibitors of some of the kinases capable of stimulating the MEK and ERK kinases independently of B-RAF (e.g. COT). Nevertheless, even

  10. Melanoma cell metastasis via P-selectin-mediated activation of acid sphingomyelinase in platelets.

    PubMed

    Becker, Katrin Anne; Beckmann, Nadine; Adams, Constantin; Hessler, Gabriele; Kramer, Melanie; Gulbins, Erich; Carpinteiro, Alexander

    2017-01-01

    Metastatic dissemination of cancer cells is one of the hallmarks of malignancy and accounts for approximately 90 % of human cancer deaths. Within the blood vasculature, tumor cells may aggregate with platelets to form clots, adhere to and spread onto endothelial cells, and finally extravasate to form metastatic colonies. We have previously shown that sphingolipids play a central role in the interaction of tumor cells with platelets; this interaction is a prerequisite for hematogenous tumor metastasis in at least some tumor models. Here we show that the interaction between melanoma cells and platelets results in rapid and transient activation and secretion of acid sphingomyelinase (Asm) in WT but not in P-selectin-deficient platelets. Stimulation of P-selectin resulted in activation of p38 MAPK, and inhibition of p38 MAPK in platelets prevented the secretion of Asm after interaction with tumor cells. Intravenous injection of melanoma cells into WT mice resulted in multiple lung metastases, while in P-selectin-deficient mice pulmonary tumor metastasis and trapping of tumor cells in the lung was significantly reduced. Pre-incubation of tumor cells with recombinant ASM restored trapping of B16F10 melanoma cells in the lung in P-selectin-deficient mice. These findings indicate a novel pathway in tumor metastasis, i.e., tumor cell mediated activation of P-selectin in platelets, followed by activation and secretion of Asm and in turn release of ceramide and tumor metastasis. The data suggest that p38 MAPK acts downstream from P-selectin and is necessary for the secretion of Asm.

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

    SciTech Connect

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

    2016-09-02

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

  12. Targeting antisense mitochondrial ncRNAs inhibits murine melanoma tumor growth and metastasis through reduction in survival and invasion factors

    PubMed Central

    Lobos-González, Lorena; Silva, Verónica; Araya, Mariela; Restovic, Franko; Echenique, Javiera; Oliveira-Cruz, Luciana; Fitzpatrick, Christopher; Briones, Macarena; Villegas, Jaime; Villota, Claudio; Vidaurre, Soledad; Borgna, Vincenzo; Socias, Miguel; Valenzuela, Sebastián; Lopez, Constanza; Socias, Teresa; Varas, Manuel; Díaz, Jorge; Burzio, Luis O.; Burzio, Verónica A.

    2016-01-01

    We reported that knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptotic death of several human tumor cell lines, but not normal cells, suggesting this approach for selective therapy against different types of cancer. In order to translate these results to a preclinical scenario, we characterized the murine noncoding mitochondrial RNAs (ncmtRNAs) and performed in vivo knockdown in syngeneic murine melanoma models. Mouse ncmtRNAs display structures similar to the human counterparts, including long double-stranded regions arising from the presence of inverted repeats. Knockdown of ASncmtRNAs with specific antisense oligonucleotides (ASO) reduces murine melanoma B16F10 cell proliferation and induces apoptosis in vitro through downregulation of pro-survival and metastasis markers, particularly survivin. For in vivo studies, subcutaneous B16F10 melanoma tumors in C57BL/6 mice were treated systemically with specific and control antisense oligonucleotides (ASO). For metastasis studies, tumors were resected, followed by systemic administration of ASOs and the presence of metastatic nodules in lungs and liver was assessed. Treatment with specific ASO inhibited tumor growth and metastasis after primary tumor resection. In a metastasis-only assay, mice inoculated intravenously with cells and treated with the same ASO displayed reduced number and size of melanoma nodules in the lungs, compared to controls. Our results suggest that ASncmtRNAs could be potent targets for melanoma therapy. To our knowledge, the ASncmtRNAs are the first potential non-nuclear targets for melanoma therapy. PMID:27507060

  13. 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.

  14. 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.

  15. 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.

  16. Targeting antisense mitochondrial ncRNAs inhibits murine melanoma tumor growth and metastasis through reduction in survival and invasion factors.

    PubMed

    Lobos-González, Lorena; Silva, Verónica; Araya, Mariela; Restovic, Franko; Echenique, Javiera; Oliveira-Cruz, Luciana; Fitzpatrick, Christopher; Briones, Macarena; Villegas, Jaime; Villota, Claudio; Vidaurre, Soledad; Borgna, Vincenzo; Socias, Miguel; Valenzuela, Sebastián; Lopez, Constanza; Socias, Teresa; Varas, Manuel; Díaz, Jorge; Burzio, Luis O; Burzio, Verónica A

    2016-09-06

    We reported that knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptotic death of several human tumor cell lines, but not normal cells, suggesting this approach for selective therapy against different types of cancer. In order to translate these results to a preclinical scenario, we characterized the murine noncoding mitochondrial RNAs (ncmtRNAs) and performed in vivo knockdown in syngeneic murine melanoma models. Mouse ncmtRNAs display structures similar to the human counterparts, including long double-stranded regions arising from the presence of inverted repeats. Knockdown of ASncmtRNAs with specific antisense oligonucleotides (ASO) reduces murine melanoma B16F10 cell proliferation and induces apoptosis in vitro through downregulation of pro-survival and metastasis markers, particularly survivin. For in vivo studies, subcutaneous B16F10 melanoma tumors in C57BL/6 mice were treated systemically with specific and control antisense oligonucleotides (ASO). For metastasis studies, tumors were resected, followed by systemic administration of ASOs and the presence of metastatic nodules in lungs and liver was assessed. Treatment with specific ASO inhibited tumor growth and metastasis after primary tumor resection. In a metastasis-only assay, mice inoculated intravenously with cells and treated with the same ASO displayed reduced number and size of melanoma nodules in the lungs, compared to controls. Our results suggest that ASncmtRNAs could be potent targets for melanoma therapy. To our knowledge, the ASncmtRNAs are the first potential non-nuclear targets for melanoma therapy.

  17. Constitutive Smad linker phosphorylation in melanoma: A mechanism of resistance to Transforming Growth Factor-β-mediated growth inhibition

    PubMed Central

    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

    2011-01-01

    SUMMARY 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 in 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 p15INK4B and p21WAF1, as compared with cells transfected with wild-type Smad3. In addition, the cell numbers of EPSM Smad3-expressing melanoma cells were significantly reduced compared to wild-type 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

  18. Antitumor Activity of miR-1280 in Melanoma by Regulation of Src

    PubMed Central

    Sun, Vera; Zhou, Wen B; Nosrati, Mehdi; Majid, Shahana; Thummala, Suresh; de Semir, David; Bezrookove, Vladimir; de Feraudy, Sebastien; Chun, Liane; Schadendorf, Dirk; Debs, Robert; Kashani-Sabet, Mohammed; Dar, Altaf A

    2015-01-01

    MicroRNAs (miRNAs) play a key role in cancer progression by coordinately repressing target genes involved in cell proliferation, migration, and invasion. miRNAs regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report that expression of miR-1280 is significantly suppressed in human melanoma specimens when compared with nevi, and in human melanoma cell lines when compared with cultured normal human melanocytes. The proto-oncogene Src was identified as a target of miR-1280 action. Levels of Src expression were significantly higher in melanoma samples and cell lines than in nevi and normal melanocytes. miR-1280 overexpression significantly suppressed the luciferase activity of reporter plasmids containing the full-length 3′ untranslated region of Src. miR-1280-mediated suppression of Src led to substantial decreases in melanoma cell proliferation, cell cycle progression, invasion, as well as induced melanoma cell apoptosis. The effects of miR-1280 overexpression on melanoma cell proliferation and growth were reversed by Src overexpression. Intratumoral delivery of miR-1280 significantly suppressed melanoma cell growth in vivo. Our results demonstrate a novel role for miR-1280 as a tumor suppressor in melanoma, identify the Src signaling pathway as a target of miR-1280 action, and suggest a potential therapeutic role for miR-1280 in melanoma. PMID:25195599

  19. Vascular endothelial growth factor expression and inhibition in uveal melanoma cell lines

    PubMed Central

    Logan, Patrick; Burnier, Julia; Burnier, Miguel N.

    2013-01-01

    Background: Uveal melanoma (UM) is a disease that affects approximately five people per million in the United States. This disease metastasises predominantly to the liver, and treatment options following the clinical detection of these sequelae are limited. Vascular endothelial growth factor-A (VEGF-A) is the primary activator of tumour angiogenesis and functions by binding to VEGF-Receptor 2 (VEGF-R2) and is often required for tumour growth beyond 2–3 mm. The purpose of this study was to investigate the expression of VEGF-A and the primary VEGF-R2 in three UM cell lines. Furthermore, we investigated the effects of VEGF-A inhibition on receptor activation and production of other cytokines. Finally, the effects of VEGF-A inhibition on the proliferation, migration, and invasion in the cell lines were ascertained. Materials: Three UM cell lines (92.1, OCM-1, and UW-1) were incubated with and without the addition of 100 μg/mL of bevacizumab. VEGF-A expression under both conditions was determined by sandwich enzyme-linked immunosorbent assay (ELISA), and phosphorylated VEGF-R2 expression was determined using western blot. The effects of VEGF-A inhibition on 20 cytokines (IL-1a, IL-2, IL-5, IL-8, IL-12p70, GM-CSF, IFNy, CCL3, MMP-9, TNF-a, IL-1b, IL-4, IL-6, IL-10, IL-13, GRO, MCP-1, MIP-1b, and RANTES) were determined using a multiplex sandwich ELISA. Proliferation rates before and after treatment were evaluated via sulforhodamine B assay, and migration and invasion assays implementing the Boyden chamber technique, the latter with artificial extracellular matrix, were used to assess their respective abilities. The Student’s t-test was used to compare changes in cytokine expression following VEGF-A inhibition. Analysis of variance was used to compare changes in the functional abilities of three uveal melanoma cell lines following VEGF-A inhibition. A P-value < 0.05 was considered statistically significant. Results: All three cell lines produced copious amounts of

  20. HDAC Inhibition Upregulates PD-1 Ligands in Melanoma and Augments Immunotherapy with PD-1 Blockade.

    PubMed

    Woods, David M; Sodré, Andressa L; Villagra, Alejandro; Sarnaik, Amod; Sotomayor, Eduardo M; Weber, Jeffrey

    2015-12-01

    Expression of PD-1 ligands by tumors and interaction with PD-1-expressing T cells in the tumor microenvironment can result in tolerance. Therapies targeting this coinhibitory axis have proven clinically successful in the treatment of metastatic melanoma, non-small cell lung cancer, and other malignancies. Therapeutic agents targeting the epigenetic regulatory family of histone deacetylases (HDAC) have shown clinical success in the treatment of some hematologic malignancies. Beyond direct tumor cell cytotoxicity, HDAC inhibitors have also been shown to alter the immunogenicity and enhance antitumor immune responses. Here, we show that class I HDAC inhibitors upregulated the expression of PD-L1 and, to a lesser degree, PD-L2 in melanomas. Evaluation of human and murine cell lines and patient tumors treated with a variety of HDAC inhibitors in vitro displayed upregulation of these ligands. This upregulation was robust and durable, with enhanced expression lasting past 96 hours. These results were validated in vivo in a B16F10 syngeneic murine model. Mechanistically, HDAC inhibitor treatment resulted in rapid upregulation of histone acetylation of the PD-L1 gene leading to enhanced and durable gene expression. The efficacy of combining HDAC inhibition with PD-1 blockade for treatment of melanoma was also explored in a murine B16F10 model. Mice receiving combination therapy had a slower tumor progression and increased survival compared with control and single-agent treatments. These results highlight the ability of epigenetic modifiers to augment immunotherapies, providing a rationale for combining HDAC inhibitors with PD-1 blockade.

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

    SciTech Connect

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

    2015-01-02

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

  2. Inhibition of proliferation and invasion in 2D and 3D models by 2-methoxyestradiol in human melanoma cells.

    PubMed

    Massaro, R R; Faião-Flores, F; Rebecca, V W; Sandri, S; Alves-Fernandes, D K; Pennacchi, P C; Smalley, K S M; Maria-Engler, S S

    2017-05-01

    Despite the recent advances in the clinical management of melanoma, there remains a need for new pharmacological approaches to treat this cancer. 2-methoxyestradiol (2ME) is a metabolite of estrogen that has shown anti-tumor effects in many cancer types. In this study we show that 2ME treatment leads to growth inhibition in melanoma cells, an effect associated with entry into senescence, decreased pRb and Cyclin B1 expression, increased p21/Cip1 expression and G2/M cell cycle arrest. 2ME treatment also inhibits melanoma cell growth in colony formation assay, including cell lines with acquired resistance to BRAF and BRAF+MEK inhibitors. We further show that 2ME is effective against melanoma with different BRAF and NRAS mutational status. Moreover, 2ME induced the retraction of cytoplasmic projections in a 3D spheroid model and significantly decreased cell proliferation in a 3D skin reconstruct model. Together our studies bring new insights into the mechanism of action of 2ME allowing melanoma targeted therapy to be further refined. Continued progress in this area is expected to lead to improved anti-cancer treatments and the development of new and more effective clinical analogues. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Inducible nitric oxide synthase drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2.

    PubMed

    Lopez-Rivera, Esther; Jayaraman, Padmini; Parikh, Falguni; Davies, Michael A; Ekmekcioglu, Suhendan; Izadmehr, Sudeh; Milton, Denái R; Chipuk, Jerry E; Grimm, Elizabeth A; Estrada, Yeriel; Aguirre-Ghiso, Julio; Sikora, Andrew G

    2014-02-15

    Melanoma is one of the cancers of fastest-rising incidence in the world. Inducible nitric oxide synthase (iNOS) is overexpressed in melanoma and other cancers, and previous data suggest that iNOS and nitric oxide (NO) drive survival and proliferation of human melanoma cells. However, specific mechanisms through which this occurs are poorly defined. One candidate is the PI3K-AKT-mTOR pathway, which plays a major role in proliferation, angiogenesis, and metastasis of melanoma and other cancers. We used the chick embryo chorioallantoic membrane (CAM) assay to test the hypothesis that melanoma growth is regulated by iNOS-dependent mTOR pathway activation. Both pharmacologic inhibition and siRNA-mediated gene silencing of iNOS suppressed melanoma proliferation and in vivo growth on the CAM in human melanoma models. This was associated with strong downregulation of mTOR pathway activation by Western blot analysis of p-mTOR, p70 ribosomal S6 kinase (p-P70S6K), p-S6RP, and p-4EBP1. iNOS expression and NO were associated with reversible nitrosylation of tuberous sclerosis complex (TSC) 2, and inhibited dimerization of TSC2 with its inhibitory partner TSC1, enhancing GTPase activity of its target Ras homolog enriched in brain (Rheb), a critical activator of mTOR signaling. Immunohistochemical analysis of tumor specimens from stage III melanoma patients showed a significant correlation between iNOS expression levels and expression of the mTOR pathway members. Exogenously supplied NO was also sufficient to reverse the mTOR pathway inhibition by the B-Raf inhibitor vemurafenib. In summary, covalent modification of TSC2 by iNOS-derived NO is associated with impaired TSC2/TSC1 dimerization, mTOR pathway activation, and proliferation of human melanoma. This model is consistent with the known association of iNOS overexpression and poor prognosis in melanoma and other cancers.

  4. Effects of AKT inhibitor therapy in response and resistance to BRAF inhibition in melanoma

    PubMed Central

    2014-01-01

    Background The clinical use of BRAF inhibitors for treatment of metastatic melanoma is limited by the development of drug resistance. In this study we investigated whether co-targeting the MAPK and the PI3K-AKT pathway can prevent emergence of resistance or provide additional growth inhibitory effects in vitro. Methods Anti-tumor effects of the combination of the BRAF inhibitor (BRAFi) dabrafenib and GSK2141795B (AKTi) in a panel of 23 BRAF mutated melanoma cell lines were evaluated on growth inhibition by an ATP-based luminescent assay, on cell cycle and apoptosis by flow cytometry and on cell signaling by western blot. Moreover, we investigated the possibilities of delaying or reversing resistance or achieving further growth inhibition by combining AKTi with dabrafenib and/or the MEK inhibitor (MEKi) trametinib by using long term cultures. Results More than 40% of the cell lines, including PTEN-/- and AKT mutants showed sensitivity to AKTi (IC50 < 1.5 μM). The combination of dabrafenib and AKTi synergistically potentiated growth inhibition in the majority of cell lines with IC50 > 5 nM dabrafenib. Combinatorial treatment induced apoptosis only in cell lines sensitive to AKTi. In long term cultures of a PTEN-/- cell line, combinatorial treatment with the MAPK inhibitors, dabrafenib and trametinib, and AKTi markedly delayed the emergence of drug resistance. Moreover, combining AKTi with the MAPK inhibitors from the beginning provided superior growth inhibitory effects compared to addition of AKTi upon development of resistance to MAPK inhibitors in this particular cell line. Conclusions AKTi combined with BRAFi-based therapy may benefit patients with tumors harboring BRAF mutations and particularly PTEN deletions or AKT mutations. PMID:24735930

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

    PubMed

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

    2016-01-01

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

  6. Melanoma Cell Galectin-1 Ligands Functionally Correlate with Malignant Potential.

    PubMed

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

    2015-07-01

    Galectin-1 (Gal-1)-binding to Gal-1 ligands on immune and endothelial cells can influence melanoma development through dampening antitumor immune responses and promoting angiogenesis. However, whether Gal-1 ligands are functionally expressed on melanoma cells to help control intrinsic malignant features remains poorly understood. Here, we analyzed expression, identity, and function of Gal-1 ligands in melanoma progression. Immunofluorescent analysis of benign and malignant human melanocytic neoplasms revealed that Gal-1 ligands were abundant in severely dysplastic nevi, as well as in primary and metastatic melanomas. Biochemical assessments indicated that melanoma cell adhesion molecule (MCAM) was a major Gal-1 ligand on melanoma cells that was largely dependent on its N-glycans. Other melanoma cell Gal-1 ligand activity conferred by O-glycans was negatively regulated by α2,6 sialyltransferase ST6GalNAc2. In Gal-1-deficient mice, MCAM-silenced (MCAM(KD)) or ST6GalNAc2-overexpressing (ST6(O/E)) melanoma cells exhibited slower growth rates, underscoring a key role for melanoma cell Gal-1 ligands and host Gal-1 in melanoma growth. Further analysis of MCAM(KD) or ST6(O/E) melanoma cells in cell migration assays indicated that Gal-1 ligand-dependent melanoma cell migration was severely inhibited. These findings provide a refined perspective on Gal-1/melanoma cell Gal-1 ligand interactions as contributors to melanoma malignancy.

  7. The neural guidance receptor Plexin C1 delays melanoma progression

    PubMed Central

    Chen, Y; Soong, J; Mohanty, S; Xu, L; Scott, G

    2013-01-01

    Plexin C1 is a type I transmembrane receptor with intrinsic R-Ras GTPase activity, which regulates cytoskeletal remodeling and adhesion in normal human melanocytes. Melanocytes are pigment-producing cells of the epidermis, precursors for melanoma, and express high levels of Plexin C1, which is lost in melanoma in vitro and in vivo. To determine if Plexin C1 is a tumor suppressor for melanoma, we introduced Plexin C1 into a primary human melanoma cell line, and phenotypes including migration, apoptosis, proliferation and tumor growth in mice were analyzed. Complimentary studies in which Plexin C1 was silenced in human melanocytes were performed. Plexin C1 significantly inhibited migration and proliferation in melanoma, whereas in melanocytes, loss of Plexin C1 increased migration and proliferation. In mouse xenografts, Plexin C1 delayed tumor growth of melanoma at early time points, but tumors eventually escaped the suppressive effects of Plexin C1, due to Plexin C1-dependent activation of the pro-survival protein Akt. R-Ras activation stimulates melanoma migration. Plexin C1 lowered R-Ras activity in melanoma and melanocytes, consistent with inhibitory effects of Plexin C1 on migration of melanocytes and melanoma. To determine if R-Ras is expressed in melanocytic lesions in vivo, staining of tissue microarrays of nevi and melanoma were performed. R-Ras expression was highly limited in melanocytic lesions, being essentially confined to primary melanoma, and almost completely absent in nevi and metastatic melanoma. These data suggest that loss of Plexin C1 in melanoma may promote early steps in melanoma progression through suppression of migration and proliferation, but pro-survival effects of Plexin C1 ultimately abrogate the tumor suppressive effects of Plexin C1. In primary melanoma, loss of Plexin C1 may function in early steps of melanoma progression by releasing inhibition of R-Ras activation, and stimulating migration. PMID:23160370

  8. The neural guidance receptor Plexin C1 delays melanoma progression.

    PubMed

    Chen, Y; Soong, J; Mohanty, S; Xu, L; Scott, G

    2013-10-10

    Plexin C1 is a type I transmembrane receptor with intrinsic R-Ras GTPase activity, which regulates cytoskeletal remodeling and adhesion in normal human melanocytes. Melanocytes are pigment-producing cells of the epidermis, precursors for melanoma, and express high levels of Plexin C1, which is lost in melanoma in vitro and in vivo. To determine if Plexin C1 is a tumor suppressor for melanoma, we introduced Plexin C1 into a primary human melanoma cell line, and phenotypes including migration, apoptosis, proliferation and tumor growth in mice were analyzed. Complimentary studies in which Plexin C1 was silenced in human melanocytes were performed. Plexin C1 significantly inhibited migration and proliferation in melanoma, whereas in melanocytes, loss of Plexin C1 increased migration and proliferation. In mouse xenografts, Plexin C1 delayed tumor growth of melanoma at early time points, but tumors eventually escaped the suppressive effects of Plexin C1, due to Plexin C1-dependent activation of the pro-survival protein Akt. R-Ras activation stimulates melanoma migration. Plexin C1 lowered R-Ras activity in melanoma and melanocytes, consistent with inhibitory effects of Plexin C1 on migration of melanocytes and melanoma. To determine if R-Ras is expressed in melanocytic lesions in vivo, staining of tissue microarrays of nevi and melanoma were performed. R-Ras expression was highly limited in melanocytic lesions, being essentially confined to primary melanoma, and almost completely absent in nevi and metastatic melanoma. These data suggest that loss of Plexin C1 in melanoma may promote early steps in melanoma progression through suppression of migration and proliferation, but pro-survival effects of Plexin C1 ultimately abrogate the tumor suppressive effects of Plexin C1. In primary melanoma, loss of Plexin C1 may function in early steps of melanoma progression by releasing inhibition of R-Ras activation, and stimulating migration.

  9. Constitutive ERK activity induces downregulation of tristetraprolin, a major protein controlling interleukin8/CXCL8 mRNA stability in melanoma cells.

    PubMed

    Bourcier, Christine; Griseri, Paola; Grépin, Renaud; Bertolotto, Corinne; Mazure, Nathalie; Pagès, Gilles

    2011-09-01

    Most melanoma cells are characterized by the V600E mutation in B-Raf kinase. This mutation leads to increased expression of interleukin (CXCL8), which plays a key role in cell growth and angiogenesis. Thus CXCL8 appears to be an interesting therapeutic target. Hence, we performed vaccination of mice with GST-CXCL8, which results in a reduced incidence of syngenic B16 melanoma cell xenograft tumors. We next addressed the molecular mechanisms responsible for aberrant CXCL8 expression in melanoma. The CXCL8 mRNA contains multiples AU-rich sequences (AREs) that modulate mRNA stability through the binding of tristetraprolin (TTP). Melanoma cell lines express very low TTP levels. We therefore hypothesized that the very low endogenous levels of TTP present in different melanoma cell lines might be responsible for the relative stability of CXCL8 mRNAs. We show that TTP is actively degraded by the proteasome and that extracellular-regulated kinase inhibition results in TTP accumulation. Conditional expression of TTP in A375 melanoma cells leads to CXCL8 mRNA destabilization via its 3' untranslated regions (3'-UTR), and TTP overexpression reduces its production. In contrast, downregulation of TTP by short hairpin RNA results in upregulation of CXCL8 mRNA. Maintaining high TTP levels in melanoma cells decreases cell proliferation and autophagy and induces apoptosis. Sorafenib, a therapeutic agent targeting Raf kinases, decreases CXCL8 expression in melanoma cells through reexpression of TTP. We conclude that loss of TTP represents a key event in the establishment of melanomas through constitutive expression of CXCL8, which constitutes a potent therapeutic target.

  10. Chronic inflammation promotes myeloid-derived suppressor cell activation blocking antitumor immunity in transgenic mouse melanoma model

    PubMed Central

    Meyer, Christiane; Sevko, Alexandra; Ramacher, Marcel; Bazhin, Alexandr V.; Falk, Christine S.; Osen, Wolfram; Borrello, Ivan; Kato, Masashi; Schadendorf, Dirk; Baniyash, Michal; Umansky, Viktor

    2011-01-01

    Tumor microenvironment is characterized by chronic inflammation represented by infiltrating leukocytes and soluble mediators, which lead to a local and systemic immunosuppression associated with cancer progression. Here, we used the ret transgenic spontaneous murine melanoma model that mimics human melanoma. Skin tumors and metastatic lymph nodes showed increased levels of inflammatory factors such as IL-1β, GM-CSF, and IFN-γ, which correlated with tumor progression. Moreover, Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs), known to inhibit tumor reactive T cells, were enriched in melanoma lesions and lymphatic organs during tumor progression. MDSC infiltration was associated with a strong TCR ζ-chain down-regulation in all T cells. Coculturing normal splenocytes with tumor-derived MDSC induced a decreased T-cell proliferation and ζ-chain expression, verifying the MDSC immunosuppressive function and suggesting that the tumor inflammatory microenvironment supports MDSC recruitment and immunosuppressive activity. Indeed, upon manipulation of the melanoma microenvironment with the phosphodiesterase-5 inhibitor sildenafil, we observed reduced levels of numerous inflammatory mediators (e.g., IL-1β, IL-6, VEGF, S100A9) in association with decreased MDSC amounts and immunosuppressive function, indicating an antiinflammatory effect of sildenafil. This led to a partial restoration of ζ-chain expression in T cells and to a significantly increased survival of tumor-bearing mice. CD8 T-cell depletion resulted in an abrogation of sildenafil beneficial outcome, suggesting the involvement of MDSC and CD8 T cells in the observed therapeutic effects. Our data imply that inhibition of chronic inflammation in the tumor microenvironment should be applied in conjunction with melanoma immunotherapies to increase their efficacy. PMID:21969559

  11. NLRP1 promotes tumor growth by enhancing inflammasome activation and suppressing apoptosis in metastatic melanoma.

    PubMed

    Zhai, Z; Liu, W; Kaur, M; Luo, Y; Domenico, J; Samson, J M; Shellman, Y G; Norris, D A; Dinarello, C A; Spritz, R A; Fujita, M

    2017-03-06

    Inflammasomes are mediators of inflammation, and constitutively activated NLRP3 inflammasomes have been linked to interleukin-1β (IL-1β)-mediated tumorigenesis in human melanoma. Whereas NLRP3 regulation of caspase-1 activation requires the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD (caspase recruitment domain)), caspase-1 activation by another danger-signaling sensor NLRP1 does not require ASC because NLRP1 contains a C-terminal CARD domain that facilitates direct caspase-1 activation via CARD-CARD interaction. We hypothesized that NLRP1 has additional biological activities besides IL-1β maturation and investigated its role in melanoma tumorigenesis. NLRP1 expression in melanoma was confirmed by analysis of 216 melanoma tumors and 13 human melanoma cell lines. Unlike monocytic THP-1 cells with prominent nuclear localization of NLRP1, melanoma cells expressed NLRP1 mainly in the cytoplasm. Knocking down NLRP1 revealed a tumor-promoting property of NLRP1 both in vitro and in vivo. Mechanistic studies showed that caspase-1 activity, IL-1β production, IL-1β secretion and nuclear factor-kB activity were reduced by knocking down of NLRP1 in human metastatic melanoma cell lines 1205Lu and HS294T, indicating that NLRP1 inflammasomes are active in metastatic melanoma. However, unlike previous reports showing that NLRP1 enhances pyroptosis in macrophages, NLRP1 in melanoma behaved differently in the context of cell death. Knocking down NLRP1 increased caspase-2, -9 and -3/7 activities and promoted apoptosis in human melanoma cells. Immunoprecipitation revealed interaction of NLRP1 with CARD-containing caspase-2 and -9, whereas NLRP3 lacking a CARD motif did not interact with the caspases. Consistent with these findings, NLRP1 activation but not NLRP3 activation reduced caspase-2, -9 and -3/7 activities and provided protection against apoptosis in human melanoma cells, suggesting a suppressive role of NLRP1 in caspase-3/7 activation

  12. The long non-coding RNA NKILA inhibits the invasion-metastasis cascade of malignant melanoma via the regulation of NF-ĸB

    PubMed Central

    Bian, Donghui; Gao, Cong; Bao, Kai; Song, Guodong

    2017-01-01

    The long non-coding RNA (lncRNA) NKILA has been reported to participate in the development of human cancers. The purpose of this study was to explore the potential role of lncRNA-NKILA, which acts through NF-ĸB, in the process of melanoma development. Real-time PCR (qRT-PCR) showed that NKILA was expressed at low levels in human melanoma tissues. The area under the ROC curve of NKILA was 0.875, which indicated that NKILA may be a potential diagnostic biomarker of melanoma. Our results also indicated that NKILA inhibited the progression of cell proliferation, migration, and invasion, and promoted apoptosis of melanoma cells. Furthermore, qRT-PCR showed that NF-κB, which was negatively correlated with NKILA, was highly expressed in human melanoma tissues. Moreover, our results indicated that NKILA inhibited the carcinogenesis of melanoma cells through the inhibition of NF-ĸB in vitro. More importantly, we found that NKILA suppressed the growth of melanoma tumors via NF-ĸB in vivo. In conclusion, NKILA suppressed the development of malignant melanoma via the regulation of NF-ĸB and may be a potential therapeutic target in patients with melanoma. PMID:28123845

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

    PubMed

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

    2012-10-01

    Persistent Jak/Stat3 signal transduction plays a crucial role in tumorigenesis and immune development. Activated Jak/Stat3 signaling has been validated as a promising molecular target for cancer therapeutics discovery and development. Berbamine (BBM), a natural bis-benzylisoquinoline alkaloid, was identified from the traditional Chinese herbal medicine Berberis amurensis used for treatment of cancer patients. While BBM has been shown to have potent antitumor activities with low toxicity in various cancer types, the molecular mechanism of action of BBM remains largely unknown. Here, we determine the antitumor activities of 13 synthetic berbamine derivatives (BBMDs) against human solid tumor cells. BBMD3, which is the most potent in this series of novel BBMDs, exhibits over 6-fold increase in biological activity compared to natural BBM. Moreover, BBMD3, directly inhibits Jak2 autophosphorylation kinase activity in vitro with IC(50)0.69 μM. Autophosphorylation of Jak2 kinase at Tyr1007/1008 sites also was strongly inhibited in the range of 15 μM of BBMD3 in human melanoma cells at 4h after treatment. Following inhibition of autophosphorylation of Jak2, BBMD3 blocked constitutive activation of downstream Stat3 signaling in melanoma cells. BBMD3 also down-regulated expression of the Stat3 target proteins Mcl-1and Bcl-x(L), associated with induction of apoptosis. In sum, our findings demonstrate that the novel berbamine derivative BBMD3 is an inhibitor of the Jak2/Stat3 signaling pathway, providing evidence for a molecular mechanism whereby BBMD3 exerts at least in part the apoptosis of human melanoma cells. In addition, BBMD3 represents a promising lead compound for development of new therapeutics for cancer treatment.

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

    PubMed

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

    2016-09-02

    Selective induction of cell death or growth inhibition of cancer cells is the future of chemotherapy. Clinical trials have found that cancer tissues are enriched with copper. Based on this finding, many copper-containing compounds and complexes have been designed to "copper" cancer cells using copper as bait. However, recent studies have demonstrated that copper boosts tumor development, and copper deprivation from serum was shown to effectively inhibit the promotion of cancer. Mechanistically, copper is an essential cofactor for mitogen-activated protein kinase (MAPK)/extracellular activating kinase (ERK) kinase (MEK), a central molecule in the BRAF/MEK/ERK pathway. Therefore, depleting copper from cancer cells by directly sequestering copper has a wider field for research and potential for combination therapy. Based on the affinity between sulfur and copper, we therefore designed sulfur nanoparticles (Nano-S) that detain copper, achieving tumor growth restriction. We found that spherical Nano-S could effectively bind copper and form a tighter surficial structure. Moreover, this Nano-S detention of copper effectively inhibited the proliferation of A375 melanoma and MCF-7 breast cancer cells with minimum toxicity to normal cells. Mechanistic studies revealed that Nano-S triggered inactivation of the MEK-ERK pathway followed by inhibition of the proliferation of the A375 and MCF-7 cells. In addition, lower Nano-S concentrations and shorter exposure stimulated the expression of a copper transporter as compensation, which further increased the cellular uptake and anticancer activities of cisplatin. Collectively, our results highlight the potential of Nano-S as an anticancer agent or adjuvant through its detention of copper. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Active immunotherapy with ultraviolet B-irradiated autologous whole melanoma cells plus DETOX in patients with metastatic melanoma.

    PubMed

    Eton, O; Kharkevitch, D D; Gianan, M A; Ross, M I; Itoh, K; Pride, M W; Donawho, C; Buzaid, A C; Mansfield, P F; Lee, J E; Legha, S S; Plager, C; Papadopoulos, N E; Bedikian, A Y; Benjamin, R S; Balch, C M

    1998-03-01

    Our objective was to determine the clinical activity, toxicity, and immunological effects of active immunotherapy using UVB-irradiated (UVR) autologous tumor (AT) cells plus adjuvant DETOX in metastatic melanoma patients. Eligibility included nonanergic patients fully recovered after resection of 5 or more grams of metastatic melanoma. Treatment consisted of intradermal injections of 10(7) UVR-AT plus 0.25 ml of DETOX every 2 weeks x 6, then monthly. Peripheral blood mononuclear cells (PBMCs) were harvested for cytotoxicity assays, and skin testing was performed for delayed-type hypersensitivity (DTH) determinations before the first, fourth, seventh, and subsequent treatments. Forty-two patients were treated, 18 in the adjuvant setting and 24 with measurable disease. Among the latter group, there were two durable responses in soft-tissue sites and in a bone metastasis. Treatment was well tolerated. Thirty-five patients were assessable for immunological parameters; 10 of these patients, including the 2 responders, demonstrated early induction of PBMC cytotoxicity against AT cells that persisted up to 10 months on treatment before falling to background levels. In five of seven patients, the fall-off heralded progressive disease. Late induction of a weak DTH reaction to AT cells was observed in eight patients. Active immunotherapy with UVR-AT + DETOX had modest but definite clinical activity in advanced melanoma. The induction of both PBMC cytotoxicity and DTH reactivity to AT cells supported a specific systemic immune effect of treatment, although the former more closely followed disease course in this study.

  16. Melanogenesis Inhibitory Activity of Two Generic Drugs: Cinnarizine and Trazodone in Mouse B16 Melanoma Cells

    PubMed Central

    Chang, Te-Sheng; Lin, Victor Chia-Hsiang

    2011-01-01

    More than 200 generic drugs were screened to identify the inhibitory activity on melanogenesis in mouse B16 melanoma cells. Cinnarizine and trazodone were identified as melanogenesis inhibitors. The inhibitory effects of the two drugs on cell survival, melanogenesis, and tyrosinase activity were investigated. The results showed that both cinnarizine and trazodone inhibited melanogenesis in B16 cells by a dose-dependent manner at the non-cytotoxic concentrations. Based on the results of the present study, seeking new melanogenesis inhibitors from generic drugs is an alternative approach to developing new depigmenting agents in cosmeceuticals. Moreover, cinnarizine and trazodone were proven to be good candidates as skin-whitening agents for treatment of skin hyperpigmentation. PMID:22272104

  17. Melanogenesis inhibitory activity of two generic drugs: cinnarizine and trazodone in mouse B16 melanoma cells.

    PubMed

    Chang, Te-Sheng; Lin, Victor Chia-Hsiang

    2011-01-01

    More than 200 generic drugs were screened to identify the inhibitory activity on melanogenesis in mouse B16 melanoma cells. Cinnarizine and trazodone were identified as melanogenesis inhibitors. The inhibitory effects of the two drugs on cell survival, melanogenesis, and tyrosinase activity were investigated. The results showed that both cinnarizine and trazodone inhibited melanogenesis in B16 cells by a dose-dependent manner at the non-cytotoxic concentrations. Based on the results of the present study, seeking new melanogenesis inhibitors from generic drugs is an alternative approach to developing new depigmenting agents in cosmeceuticals. Moreover, cinnarizine and trazodone were proven to be good candidates as skin-whitening agents for treatment of skin hyperpigmentation.

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

    PubMed

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

    2015-10-01

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

  19. Inhibition of mitochondrial protein translation sensitizes melanoma cells to arsenic trioxide cytotoxicity via a reactive oxygen species dependent mechanism

    PubMed Central

    Bowling, Benjamin D.; Doudican, Nicole; Manga, Prashiela; Orlow, Seth J.

    2009-01-01

    Purpose Current standard chemotherapeutic regimens for malignant melanoma are unsatisfactory. Although in vitro studies of arsenic trioxide (ATO) have demonstrated promise against melanoma, recent phase II clinical trials have failed to show any significant clinical benefit when used as a single agent. To enhance the efficacy of ATO in the treatment of melanoma, we sought to identify compounds that potentiate the cytotoxic effects of ATO in melanoma cells. Through a screen of 2000 marketed drugs and naturally occurring compounds, a variety of antibiotic inhibitors of mitochondrial protein translation were identified. Methods The mechanism of action for the most effective agent identified, thiostrepton, was examined in a panel of melanoma cells. Effects of combinatorial ATO and thiostrepton treatment on cytotoxicity, apoptosis, mitochondrial protein content, and reactive oxygen species (ROS) were assessed. Results Thiostrepton (1μM) sensitized 3 out of 5 melanoma cell lines to ATO-mediated growth inhibition. Treatment with thiostrepton resulted in reduced levels of the mitochondrial-encoded protein cytochrome oxidase I (COX1). Exposure to thiostrepton in combination with ATO resulted in increased levels of cleaved poly (ADP-ribose) polymerase and cellular ROS. The growth inhibitory and pro-apototic effects of addition of the ATO/thiostrepton combination were reversed by the free radical scavenger N-acetyl-l-cysteine. Conculsions Our data suggest that thiostrepton enhances the cytotoxic effects of ATO through a ROS-dependent mechanism. Co-administration of oxidative stress-inducing drugs such as thiostrepton in order to enhance the efficacy of ATO in the treatment of melanoma warrants further investigation. PMID:18297286

  20. Quercetin derivatives regulate melanosome transportation via EPI64 inhibition and elongate the cell shape of B16 melanoma cells.

    PubMed

    Yamauchi, Kosei; Mitsunaga, Tohru; Inagaki, Mizuho; Suzuki, Tohru

    2015-03-01

    4'-O-β-D-glucopyranosyl-quercetin-3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranoside (3C4'GQ), first isolated from Helminthostachys zeylanica root extract, was synthesized as a compound that stimulates intracellular melanogenesis. 3-O-methylquercetin (3MQ) and 3,4',7-O-trimethylquercetin (34'7TMQ) were synthesized as compounds that enhance extracellular melanin formation. The formation of dendrites and the expression of EBP50-PDZ interactor of 64 kDa (EPI64) relating to melanin transportation were investigated using B16 melanoma cells treated with 3C4'GQ, 3MQ, or 34'7TMQ in order to understand the mechanism underlying the observed activities. The influence of 3C4'GQ on the increase of intracellular melanin contents enhanced the expression of EPI64, exhibited no dendrite elongation activity, and inhibited melanin transportation. On the other hand, the increase of extracellular melanin content by 3MQ and 34'7TMQ inhibited the expression of EPI64 and formed elongated cells to stimulate melanin transportation.

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

    PubMed

    Park, Hye-Jin

    2014-09-12

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

  2. The tumour suppressor, miR-137, inhibits malignant melanoma migration by targetting the TBX3 transcription factor.

    PubMed

    Peres, Jade; Kwesi-Maliepaard, Eliza M; Rambow, Florian; Larue, Lionel; Prince, Sharon

    2017-10-01

    The transcription factor, TBX3, is a key driver of malignant melanoma and any drug that impacts its expression is likely to have an impact on the treatment of this highly aggressive and treatment resistant cancer. Replacement of miRNAs that target oncogenes has gained much attention as a therapy because it is anticipated to be effective with little side-effects since miRNAs are naturally occurring and often target large set of genes in the same oncogenic pathway. Here we show that miR-137 levels correlate inversely with TBX3 mRNA levels in a panel of melanoma cell lines and in a cohort of patients with primary melanoma. Low levels of miR-137 and high levels of TBX3 are shown to be associated with poor patient survival. We show that miR-137 binds a conserved site in the TBX3 3' untranslated region and that a miR-137 mimic significantly reduces endogenous levels of TBX3 and inhibits anchorage independent growth and migration of malignant melanoma cells. Novel data are provided that the miR-137/TBX3/E-cadherin axis plays an important role in melanomagenesis and that miR-137 replacement is a potential therapeutic approach for treating melanomas. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Loss of cohesin complex components STAG2 or STAG3 confers resistance to BRAF inhibition in melanoma

    PubMed Central

    Shen, Che-Hung; Kim, Sun Hye; Trousil, Sebastian; Frederick, Dennie T.; Piris, Adriano; Yuan, Ping; Cai, Li; Gu, Lei; Li, Man; Lee, Jung Hyun; Mitra, Devarati; Fisher, David E.; Sullivan, Ryan J.; Flaherty, Keith T.; Zheng, Bin

    2016-01-01

    The protein kinase V-Raf murine sarcoma viral oncogene homolog B (BRAF) is an oncogenic driver and therapeutic target in melanoma. Inhibitors of BRAF (BRAFi) have shown high response rates and extended survival in melanoma patients bearing tumors that express BRAF Val600 mutations, but a vast majority of these patients develop drug resistance. Here we show that loss of Stromal antigen 2 or 3 (STAG2 or STAG3), which encode subunits of the cohesin complex, in melanoma cells results in resistance to BRAFi. We identified loss-of-function mutations in STAG2 as well as decreased expression of STAG2 or STAG3 proteins in several tumor samples from patients with acquired resistance to BRAFi and in BRAFi-resistant melanoma cell lines. Knockdown of STAG2 or STAG3 decreased sensitivity of Val600Glu BRAF-mutant melanoma cells and xenograft tumors to BRAFi. Loss of STAG2 inhibited CCCTC-binding factor (CTCF)-mediated expression of dual specificity phosphatase 6 (DUSP6), leading to reactivation of ERK signaling. Our studies unveil a previously unknown genetic mechanism of BRAFi resistance and provide new insights into the tumor suppressor function of STAG2 and STAG3. PMID:27500726

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

    PubMed

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

    2016-02-01

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

  5. Upregulation of miR-124 by physcion 8-O-β-glucopyranoside inhibits proliferation and invasion of malignant melanoma cells via repressing RLIP76.

    PubMed

    Zhang, Di; Han, Yantao; Xu, Luo

    2016-12-01

    Melanoma is the most malignant type of skin cancer. In recent years, mounting studies have evidenced the involvement of miRNAs in melanoma. One of these miRNAs, miR-124 has been found aberrantly downregulated in a variety of human malignancies. In this study, our results showed that the expression of miR-124 was significantly lower in malignant melanoma tissues and cell lines and miR-124 functioned as a tumor suppressor in melanoma. Moreover, our findings showed that miR-124 exerted anti-tumor effect by directly targeting RLIP76, a stress-inducible non-ABC transporter that plays a crucial role in the development of melanoma. Furthermore, our study also showed that physcion 8-O-β-glucopyranoside, a natural compound from medicinal plant, could inhibit the proliferation and invasion of melanoma cells by targeting miR-124/RLIP76 signaling.

  6. Long noncoding RNA HEIH promotes melanoma cell proliferation, migration and invasion via inhibition of miR-200b/a/429

    PubMed Central

    Zhao, Haiying; Xing, Guoping; Wang, Yingying; Liu, Guoyan; Meng, Huijuan

    2017-01-01

    Long noncoding RNAs (lncRNAs) are frequently dysregulated and have important roles in many diseases, particularly cancers. lncRNA-HEIH was first identified in hepatocellular carcinoma (HCC). The expression, clinical significance and roles of lncRNA-HEIH in melanoma are still unknown. In the present study, we found that lncRNA-HEIH is highly expressed in melanoma tissues and cell lines, associated with advanced clinical stages, and predicts poor outcomes in melanoma patients. Functional assays showed that ectopic expression of lncRNA-HEIH promotes melanoma cell proliferation, migration and invasion. Knockdown of lncRNA-HEIH inhibits melanoma cell proliferation, migration and invasion. Mechanistically, we revealed that lncRNA-HEIH directly binds to miR-200b/a/429 promoter and represses miR-200b/a/429 transcription. The expression of miR-200b is inversely associated with lncRNA-HEIH in melanoma tissues. Furthermore, overexpression of miR-200b/a/429 abrogates melanoma cell proliferation, migration and invasion enhanced by lncRNA-HEIH. In conclusion, we identified lncRNA-HEIH as a key oncogene in melanoma via transcriptional inhibition of miR-200b/a/429. Our data suggested that lncRNA-HEIH may serve as a promising prognostic biomarker and therapeutic target for melanoma. PMID:28487474

  7. Anti-tumor activity of N-trimethyl chitosan-encapsulated camptothecin in a mouse melanoma model

    PubMed Central

    2010-01-01

    Background Camptothecin (CPT) has recently attracted increasing attention as a promising anticancer agent for a variety of tumors. But the clinical application is largely hampered by its extreme water insolubility and unpredictable side effect. It is essential to establish an efficient and safe protocol for the administration of CPT versus melanoma. Methods Camptothecin was encapsulated with N-trimethyl chitosan (CPT-TMC) through microprecipitation and sonication. Its inhibition effect on B16-F10 cell proliferation and induction of apoptosis was evaluated by MTT assay and flow cytometric analysis in vitro. The anti-tumor activity of CPT-TMC was evaluated in C57BL/6 mice bearing B16-F10 melanoma. Tumor volume, tumor weight and survival time were recorded. Assessment of apoptotic cells within tumor tissue was performed by TUNEL assay. Antiangiogenesis and antiproliferation effects of CPT-TMC in vivo were conducted via CD31 and PCNA immunohistochemistry, respectively. Results CPT-TMC efficiently inhibited B16-F10 cells proliferation and increased apoptosis in vitro. Experiment group showed significant inhibition compared with free CPT-treated group (81.3% vs. 56.9%) in the growth of B16-F10 melanoma xenografts and prolonged the survival time of the treated mice (P < 0.05). Decreased cell proliferation, increased tumor apoptosis as well as a reduction in angiogenesis were observed. Conclusions Our data suggest that N-trimethyl chitosan-encapsulated camptothecin is superior to free CPT by overcoming its insolubility and finally raises the potential of its application in melanoma therapy. PMID:20565783

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

    PubMed

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

    1999-01-01

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

  9. Alisol B, a triterpene from Alismatis rhizoma (dried rhizome of Alisma orientale), inhibits melanin production in murine B16 melanoma cells.

    PubMed

    Yoshida, Ichiro; Ito, Chihiro; Matsuda, Shinya; Tsuji, Akihiko; Yanaka, Noriyuki; Yuasa, Keizo

    2017-03-01

    To develop new whitening agents from natural products, we screened 80 compounds derived from crude drugs in Kampo medicine in a melanin synthesis inhibition assay using murine B16 melanoma cells. The screen revealed that treatment with alisol B, a triterpene from Alismatis rhizoma, significantly decreased both melanin content and cellular tyrosinase activity in B16 cells. However, alisol B did not directly inhibit mushroom tyrosinase activity in vitro. Therefore, we investigated the mechanism underlying the inhibitory effect of alisol B on melanogenesis. Alisol B suppressed mRNA induction of tyrosinase and its transcription factor, microphthalmia-associated transcription factor (MITF). Furthermore, alisol B reduced the phosphorylation of CREB and maintained the activation of ERK1/2. These results suggest that the reduction in melanin production by alisol B is due to the downregulation of MITF through the suppression of CREB and activation of ERK and that alisol B may be useful as a new whitening agent.

  10. Identification of TDP-43 as an oncogene in melanoma and its function during melanoma pathogenesis.

    PubMed

    Zeng, Qinghai; Cao, Ke; Liu, Rui; Huang, Jinhua; Xia, Kun; Tang, Jintian; Chen, Xiang; Zhou, Ming; Xie, Huiqing; Zhou, Jianda

    2017-01-02

    Although recent studies have revealed TAR (trans-activating response region) DNA binding protein (TDP-43) as a potential therapeutic target for cancers, its role and clinical association with melanoma have not been explored. To identify the role and function of TDP-43 during melanoma pathogenesis. Firstly, the relationship between TDP-43 expression and patient survival was explored. Then TDP-43 expression level in melanoma tissue and different melanoma cell lines was measured. After silencing TDP-43 expression in melanoma cells, the impacts of TDP-43 on cellular proliferation, metastasis, glucose uptake, and glucose transporters levels were studied. In the end, effect of TDP-43 depletion on tumorigenicity of melanoma cells was tested in vivo. Our results showed that TDP-43 was overexpressed in melanoma paraffin samples compared with that in nevi tissues. The high expression level of TDP-43 was associated with poor patient survival. By silencing TDP-43, we saw significant inhibition of cell proliferation and metastasis in A375 and WM451 cells. TDP-43 knockdown could suppress glucose transporter type-4 (GLUT4) expression and reduce glucose uptake. And downregulation of GLUT4 in melanoma cells induced inhibition of cell proliferation and metastasis. TDP-43 knockdown significantly slowed down tumor growth and decreased GLUT4 expression in vivo. TDP-43 is a novel oncogene in melanoma and regulates melanoma proliferation and metastasis potentially through modulation of glucose metabolism.

  11. [Simulated weightlessness inhibits antitumor immunity of T lymphocytes in melanoma-bearing mice].

    PubMed

    Si, Shaoyan; Song, Shujun; Shi, Liang; Liu, Junli; Xu, Bingxin; Yi, Yong; Tan, Xiaoqing; Zhang, Jianzhong

    2013-02-01

    To investigate the effects of simulated weightlessness on antitumor immunity of T lymphocytes in mice. The malignant melanoma was xenografted by subcutaneous injection of B16 cells into the right hind limb of every mouse. The mice suspended by tail at a -15 degree to 20 degree head-down tilt were used as simulated weightlessness models. The effects of simulated weightlessness on tumor volume and survival time were observed. T the numbers of leucocytes, lymphocytes and T lymphocyte subsets in peripheral blood of tumor-bearing mice under simulated weightlessness were monitored by an automatic hemacytometer and a flow cytometer. The effects of simulated weightlessness on the production of IL-2, TNF-α and IFN-γ in T lymphocytes and the cytotoxicities of tumor-specific CTLs to tumor cells were analyzed by ELISA and LDH release. Compared with control group, the tumors grew faster, the survival times were shorter, the number of lymphocytes, the ratio of lymphocytes, CD3(+);, CD4(+);/CD3(+); and CD8(+);/CD3(+); T lymphocytes in peripheral blood dropped, and the proliferation of splenic T lymphocytes induced by mitogen was reduced (P<0.05 or P<0.01) in the simulated weightlessness group. The production of IL-2, TNF-α and IFN-γ induced by tumor cells and cytotoxicities of tumor-specific CTLs to tumor cells were inhibited in mice under simulated weightlessness (P<0.05 or P<0.01). Simulated weightlessness inhibits antitumor immunity of T lymphocytes.

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    2005-04-01

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

  14. UVA radiation augments cytotoxic activity of psoralens in melanoma cells.

    PubMed

    Wrześniok, Dorota; Beberok, Artur; Rok, Jakub; Delijewski, Marcin; Hechmann, Anna; Oprzondek, Martyna; Rzepka, Zuzanna; Bacler-Żbikowska, Barbara; Buszman, Ewa

    2017-07-01

    Melanoma is an aggressive form of skin cancer. The aim of the study was to evaluate the influence of UVA radiation and psoralens: 5-methoxypsoralen (5-MOP) or 8-methoxypsoralen (8-MOP) on melanoma cells viability. The amelanotic C32 and melanotic COLO829 human melanoma cell lines were exposed to increasing concentrations of psoralens (0.1-100 μM) in the presence or absence of UVA radiation. Cell viability was evaluated by the WST-1 assay. We demonstrated that 8-MOP, in contrast to 5-MOP, has no cytotoxic effect on both melanoma cell lines. Simultaneous exposure of cells to 8-MOP and UVA radiation caused significant cytotoxic response in C32 cells where the EC50 value was estimated to be 131.0 μM (UVA dose: 1.3 J/cm(2)) and 105.3 μM (UVA dose: 2.6 J/cm(2)). The cytotoxicity of 5-MOP on both C32 and COLO829 cells was significantly augmented by UVA radiation - the EC50 was estimated to be 22.7 or 7.9 μM (UVA dose: 1.3 J/cm(2)) and 24.2 or 7.0 μM (UVA dose: 2.6 J/cm(2)), respectively. The demonstrated high cytotoxic response after simultaneous exposure of melanoma cells to psoralens and UVA radiation in vitro suggests the usefulness of PUVA therapy to treat melanoma in vivo.

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

    PubMed Central

    Figueiredo, Carlos R.; Matsuo, Alisson L.; Azevedo, Ricardo A.; Massaoka, Mariana H.; Girola, Natalia; Polonelli, Luciano; Travassos, Luiz R.

    2015-01-01

    Short peptide sequences from complementarity-determining regions (CDRs) of different immunoglobulins may exert anti-infective, immunomodulatory and antitumor activities regardless of the specificity of the original monoclonal antibody (mAb). In this sense, they resemble early molecules of innate immunity. C36L1 was identified as a bioactive light-chain CDR1 peptide by screening 19 conserved CDR sequences targeting murine B16F10-Nex2 melanoma. The 17-amino acid peptide is readily taken up by melanoma cells and acts on microtubules causing depolymerization, stress of the endoplasmic reticulum and intrinsic apoptosis. At low concentrations, C36L1 inhibited migration, invasion and proliferation of B16F10-Nex2 cells with cell cycle arrest at G2/M phase, by regulating the PI3K/Akt signaling axis involving Rho-GTPase and PTEN mediation. Peritumor injection of the peptide delayed growth of subcutaneously grafted melanoma cells. Intraperitoneal administration of C36L1 induced a significant immune-response dependent anti-tumor protection in a syngeneic metastatic melanoma model. Dendritic cells stimulated ex-vivo by the peptide and transferred to animals challenged with tumor cells were equally effective. The C36 VL CDR1 peptide is a promising microtubule-interacting drug that induces tumor cell death by apoptosis and inhibits metastases of highly aggressive melanoma cells. PMID:26391685

  16. Knockout of MDA-9/Syntenin (SDCBP) expression in the microenvironment dampens tumor-supporting inflammation and inhibits melanoma metastasis

    PubMed Central

    Das, Swadesh K.; Guo, Chunqing; Pradhan, Anjan K.; Bhoopathi, Praveen; Talukdar, Sarmistha; Shen, Xue-Ning; Emdad, Luni; Subler, Mark A.; Windle, Jolene J.; Sarkar, Devanand; Wang, Xiang-Yang; Fisher, Paul B.

    2016-01-01

    Cancer development and progression to metastasis is a complex process, which largely depends on bidirectional communication between tumor cells and their microenvironment. Melanoma differentiation associated gene-9 (mda-9, also known as Syntenin-1, SDCBP), a gene first cloned by our group, is robustly expressed in multiple cancers including melanoma and contributes to invasion and metastasis in a tumor cell-intrinsic manner. However, the role of MDA-9/Syntenin in the tumor cell-extrinsic microenvironment remains unclear even though MDA-9/Syntenin is ubiquitously expressed in most organs that are active metastatic sites for melanoma, e.g., lung, lymph node, brain, and liver. In this study, we explored the effect of environmental mda-9/syntenin expression on melanoma growth and metastasis using multiple immunocompetent animal models, syngeneic B16 xenograft and intravenous B16 mouse model and a genetically engineered mouse (GEM) model of melanoma. Host-deficient expression of mda-9/syntenin in mice negatively impacted on subcutaneously implanted B16 tumor growth and lung metastasis. Absence of MDA-9/Syntenin in the lung microenvironment suppressed tumor growth by modulating in situ Interleukin 17A (IL17A) expression and impaired the recruitment of myeloid derived suppressor cells (MDSCs) and Th17 cells as compared to genetically wild type animals. Additionally, loss of mda-9/syntenin expression in a spontaneous melanoma model (melanocyte-specific pten loss and BrafV600E mutation) significantly delayed tumor initiation and suppressed metastasis to the lymph nodes and lungs. The present study highlights a novel role of mda-9/syntenin in tumor-promoting inflammation and immune suppression. These observations along with other documented roles of MDA-9/Syntenin in cancer and metastasis support the potential relevance of MDA-9/Syntenin in the carcinogenic process and as a target for developing improved therapies by using either genetic or pharmacologic approaches to treat

  17. TRIM16 inhibits proliferation and migration through regulation of interferon beta 1 in melanoma cells

    PubMed Central

    Sutton, Selina K.; Koach, Jessica; Tan, Owen; Liu, Bing; Carter, Daniel R.; Wilmott, James S.; Yosufi, Benafsha; Haydu, Lauren E.; Mann, Graham J.; Thompson, John F.; Long, Georgina V.; Liu, Tao; McArthur, Grant; Zhang, Xu Dong; Scolyer, Richard A.; Cheung, Belamy B.; Marshall, Glenn M.

    2014-01-01

    High basal or induced expression of the tripartite motif protein, TRIM16, leads to reduce cell growth and migration of neuroblastoma and skin squamous cell carcinoma cells. However, the role of TRIM16 in melanoma is currently unknown. TRIM16 protein levels were markedly reduced in human melanoma cell lines, compared with normal human epidermal melanocytes due to both DNA methylation and reduced protein stability. TRIM16 knockdown strongly increased cell migration in normal human epidermal melanocytes, while TRIM16 overexpression reduced cell migration and proliferation of melanoma cells in an interferon beta 1 (IFNβ1)-dependent manner. Chromatin immunoprecipitation assays revealed TRIM16 directly bound the IFNβ1 gene promoter. Low level TRIM16 expression in 91 melanoma patient samples, strongly correlated with lymph node metastasis, and, predicted poor patient prognosis in a separate cohort of 170 melanoma patients with lymph node metastasis. The BRAF inhibitor, vemurafenib, increased TRIM16 protein levels in melanoma cells in vitro, and induced growth arrest in BRAF-mutant melanoma cells in a TRIM16-dependent manner. High levels of TRIM16 in melanoma tissues from patients treated with Vemurafenib correlated with clinical response. Our data, for the first time, demonstrates TRIM16 is a marker of cell migration and metastasis, and a novel treatment target in melanoma. PMID:25333256

  18. Anti-DR5 monoclonal antibody-mediated DTIC-loaded nanoparticles combining chemotherapy and immunotherapy for malignant melanoma: target formulation development and in vitro anticancer activity

    PubMed Central

    Ding, Baoyue; Wu, Xin; Fan, Wei; Wu, Zhaoyong; Gao, Jing; Zhang, Wei; Ma, Lulu; Xiang, Wang; Zhu, Quangang; Liu, Jiyong; Ding, Xueying; Gao, Shen

    2011-01-01

    Background The increased incidence of malignant melanoma in recent decades, along with its high mortality rate and pronounced resistance to therapy pose an enormous challenge. Novel therapeutic strategies, such as immunotherapy and targeted therapy, are urgently needed for melanoma. In this study, a new active targeting drug delivery system was constructed to combine chemotherapy and active specific immunotherapy. Methods The chemotherapeutic drug, dacarbazine (DTIC), that induces apoptosis through the intrinsic pathway which typically responds to severe DNA damage, was used as a model drug to prepare DTIC-loaded polylactic acid (PLA) nanoparticles (DTIC-NPs), which were covalently conjugated to a highly specific targeting functional TRAIL-receptor 2 (DR5) monoclonal antibody (mAb) that can contribute directly to cancer cell apoptosis or growth inhibition through the extrinsic pathway. Results Our in vitro experiments demonstrated that DTIC-PLA-DR5 mAb nanoparticles (DTIC-NPs-DR5 mAb) are an active targeting drug delivery system which can specifically target DR5-overexpressing malignant melanoma cells and become efficiently internalized. Most strikingly, compared with conventional DTIC-NPs, DTIC-NPs-DR5 mAb showed significantly enhanced cytotoxicity and increased cell apoptosis in DR5-positive malignant melanoma cells. Conclusion The DTIC-NPs-DR5 mAb described in this paper might be a potential formulation for targeting chemotherapy and immunotherapy to DR5-overexpressing metastatic melanoma. PMID:21976975

  19. Anti-DR5 monoclonal antibody-mediated DTIC-loaded nanoparticles combining chemotherapy and immunotherapy for malignant melanoma: target formulation development and in vitro anticancer activity.

    PubMed

    Ding, Baoyue; Wu, Xin; Fan, Wei; Wu, Zhaoyong; Gao, Jing; Zhang, Wei; Ma, Lulu; Xiang, Wang; Zhu, Quangang; Liu, Jiyong; Ding, Xueying; Gao, Shen

    2011-01-01

    The increased incidence of malignant melanoma in recent decades, along with its high mortality rate and pronounced resistance to therapy pose an enormous challenge. Novel therapeutic strategies, such as immunotherapy and targeted therapy, are urgently needed for melanoma. In this study, a new active targeting drug delivery system was constructed to combine chemotherapy and active specific immunotherapy. The chemotherapeutic drug, dacarbazine (DTIC), that induces apoptosis through the intrinsic pathway which typically responds to severe DNA damage, was used as a model drug to prepare DTIC-loaded polylactic acid (PLA) nanoparticles (DTIC-NPs), which were covalently conjugated to a highly specific targeting functional TRAIL-receptor 2 (DR5) monoclonal antibody (mAb) that can contribute directly to cancer cell apoptosis or growth inhibition through the extrinsic pathway. Our in vitro experiments demonstrated that DTIC-PLA-DR5 mAb nanoparticles (DTIC-NPs-DR5 mAb) are an active targeting drug delivery system which can specifically target DR5-overexpressing malignant melanoma cells and become efficiently internalized. Most strikingly, compared with conventional DTIC-NPs, DTIC-NPs-DR5 mAb showed significantly enhanced cytotoxicity and increased cell apoptosis in DR5-positive malignant melanoma cells. The DTIC-NPs-DR5 mAb described in this paper might be a potential formulation for targeting chemotherapy and immunotherapy to DR5-overexpressing metastatic melanoma.

  20. Activation of Vav/Rho GTPase Signaling by CXCL12 Controls Membrane-Type Matrix Metalloproteinase–Dependent Melanoma Cell Invasion

    PubMed Central

    Bartolomé, Rubén A.; Molina-Ortiz, Isabel; Samaniego, Rafael; Sánchez-Mateos, Paloma; Bustelo, Xosé R.; Teixidó, Joaquin

    2007-01-01

    Melanoma cells express the chemokine receptor CXCR4, which confers invasive signals on binding to its ligand CXCL12. We show here that knocking down membrane-type matrix metal-loproteinase (MT1-MMP) expression translates into a blockade of invasion across reconstituted basement membranes and type I collagen gels in response to CXCL12, which is the result of lack of MMP-2 activation. Interference with MMP-2 expression further confirms its important role during this invasion. Vav proteins are guanine-nucleotide exchange factors for Rho GTPases that regulate actin dynamics and gene expression. We show that melanoma cells express Vav1 and Vav2, which are activated by CXCL12 involving Jak activity. Blocking Vav expression by RNA interference results in impaired activation of Rac and Rho by CXCL12 and in a remarkable inhibition of CXCL12-promoted invasion. Importantly, up-regulation of MT1-MMP expression by CXCL12, a mechanism contributing to melanoma cell invasion, is blocked by knocking down Vav expression or by inhibiting Jak. Together, these data indicate that activation of Jak/Vav/Rho GTPase pathway by CXCL12 is a key signaling event for MT1-MMP/MMP-2–dependent melanoma cell invasion. PMID:16397238

  1. Stimulation of lymphocyte anti-melanoma activity by co-cultured macrophages activated by complex homeopathic medication

    PubMed Central

    2009-01-01

    Background Melanoma is the most aggressive form of skin cancer, and the most rapidly expanding cancer in terms of worldwide incidence. Chemotherapeutic approaches to treat melanoma have been uniformly disappointing. A Brazilian complex homeopathic medication (CHM), used as an immune modulator, has been recommended for patients with depressed immune systems. Previous studies in mice have demonstrated that the CHM activates macrophages, induces an increase in the number of leukocytes and improves the murine response against Sarcoma-180. Methods Here we studied the interaction of mouse lymph node lymphocytes, co-cultured in vitro with macrophages in the presence or absence of the CHM, with B16F10 melanoma cells. Results Lymphocytes co-cultured with macrophages in the presence of the CHM had greater anti-melanoma activity, reducing melanoma cell density and increasing the number of lysed tumor cells. There was also a higher proportion of activated (CD25+) lymphocytes with increased viability. Overall, lymphocytes activated by treatment destroyed growing cancer cells more effectively than control lymphocytes. Conclusion Co-culture of macrophages with lymphocytes in the presence of the CHM enhanced the anti-cancer performance of lymphocytes against a very aggressive lineage of melanoma cells. These results suggest that non-toxic therapies using CHMs are a promising alternative approach to the treatment of melanomas. In addition, they are attractive combination-therapy candidates, which may enhance the efficacy of conventional medicines by improving the immune response against tumor cells. PMID:19698142

  2. [Mesenchymal stem cells expressing cytosine deaminase inhibit growth of murine melanoma B16F10 in vivo].

    PubMed

    Krasikova, L S; Karshieva, S S; Cheglakov, I B; Belyavsky, A V

    2015-01-01

    The aim of this study was to estimate the efficacy of mesenchymal stem cell-based suicide gene therapy in mice bearing murine melanoma B16F10. Adipose mesenchymal stem cells (MSCs) were transfected with plasmid constructs expressing cytosine deaminase fused with uracil phosphoribosyltransferase (CDA/UPRT) or CDA/UPRT fused with HSV-1 tegument protein VP22 (CDA/UPRT/VP22). In this study, we demonstrate that direct intratumoral transplantation of MSCs expressing CDA/UPRT or CDA/UPRT/VP22 followed by systemic administration of 5-fluorocytosine (5-FC) results in a significant inhibition of tumor growth. There was a 53% reduction in tumor volume in mice treated with CDA/UPRT-MSCs and 58% reduction in tumor volume in mice treated with CDA/UPRT/VP22-MSCs as compared with control animals transplanted with B16F10 melanoma alone. Injection of CDA/UPRT-MSC and CDA/UPRT/VP22-MSC prolonged the life span of mice bearing B16F10 melanoma by 15 and 26%, respectively. The data indicate that in murine B16F10 melanoma model, MSCs encoding CDA/UPRT suicide gene have a significant antitumor effect.

  3. Comparison of the Serum Tumor Markers S100 and Melanoma-inhibitory Activity (MIA) in the Monitoring of Patients with Metastatic Melanoma Receiving Vaccination Immunotherapy with Dendritic Cells.

    PubMed

    Uslu, Ugur; Schliep, Stefan; Schliep, Klaus; Erdmann, Michael; Koch, Hans-Uwe; Parsch, Hans; Rosenheinrich, Stina; Anzengruber, Doris; Bosserhoff, Anja Katrin; Schuler, Gerold; Schuler-Thurner, Beatrice

    2017-09-01

    In patients with melanoma, early dissemination via lymphatic and hematogenous routes is frequently seen. Thus, besides clinical follow-up examination and imaging, reliable melanoma-specific serological tumor markers are needed. We retrospectively compared two serum markers for melanoma, S100 and melanoma-inhibitory activity (MIA), for monitoring of patients with metastatic melanoma under either adjuvant or therapeutic vaccination immunotherapy with dendritic cells (DC). Serum was obtained from a total of 100 patients (28 patients in stage III and 72 patients in stage IV, according to the American Joint Committee on Cancer 2002) at regular intervals during therapy, accompanied by follow-up imaging. When relapse was detected, both markers often remained within normal range. In contrast, in patients with metastatic measurable disease receiving therapeutic and not adjuvant DC vaccination, an increase of both markers was a strong indicator for disease progression. When comparing both markers in the whole study population, MIA showed a superior sensitivity to detect disease progression. S100 and MIA are highly sensitive tumor markers for monitoring of patients with melanoma with current metastases, but less sensitive for monitoring of tumor-free patients. In the current study, MIA had a slightly superior sensitivity to detect progressive disease compared to S100 and seems to be more useful in monitoring of patients with metastatic melanoma receiving immunotherapy. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  4. Improved antitumor activity of immunotherapy with BRAF and MEK inhibitors in BRAFV600E melanoma

    PubMed Central

    Hu-Lieskovan, Siwen; Mok, Stephen; Moreno, Blanca Homet; Tsoi, Jennifer; Faja, Lidia Robert; Goedert, Lucas; Pinheiro, Elaine M.; Koya, Richard C.; Graeber, Thomas; Comin-Anduix, Begoña; Ribas, Antoni

    2016-01-01

    Combining immunotherapy and BRAF targeted therapy may result in improved antitumor activity with the high response rates of targeted therapy and the durability of responses with immunotherapy. However, the first clinical trial testing the combination of the BRAF inhibitor vemurafenib and the CTLA-4 antibody ipilimumab was terminated early due to substantial liver toxicities. MEK inhibitors can potentiate the MAPK inhibition in BRAF mutant cells, while potentially alleviating the unwanted paradoxical MAPK activation in BRAF wild type cells that lead to side effects when using BRAF inhibitors alone. However, there is the concern of MEK inhibitors being detrimental to T cell functionality. Using a mouse model of syngeneic BRAFV600E driven melanoma, we tested whether addition of the MEK inhibitor trametinib would enhance the antitumor activity of combined immunotherapy with the BRAF inhibitor dabrafenib. Combination of dabrafenib and trametinib with pmel-1 adoptive cell transfer (ACT) showed complete tumor regression, increased T cell infiltration into tumors and improved in vivo cytotoxicity. Single agent dabrafenib increased tumor-associated macrophages and T regulatory cells (Tregs) in tumors, which decreased with the addition of trametinib. The triple combination therapy resulted in increased melanosomal antigen and MHC expression, and global immune-related gene up-regulation. Given the up-regulation of PD-L1 seen with dabrafenib and/or trametinib combined with antigen-specific ACT, we tested combination of dabrafenib, trametinib with anti-PD1 therapy in SM1 tumors, and observed superior anti-tumor effect. Our findings support the testing of triple combination therapy of BRAF and MEK inhibitors with immunotherapy in patients with BRAFV600E mutant metastatic melanoma. PMID:25787767

  5. Stress-responsive JNK mitogen-activated protein kinase mediates aspirin-induced suppression of B16 melanoma cellular proliferation

    PubMed Central

    Ordan, Orly; Rotem, Ronit; Jaspers, Ilona; Flescher, Eliezer

    2003-01-01

    Available anticancer drugs do not seem to modify the prognosis of metastatic melanoma. Salicylate and acetyl salicylic acid (aspirin) were found to suppress growth in a number of transformed cells, that is, prostate and colon. Therefore, we studied the direct effects of aspirin on metastatic B16 melanoma cells. Aspirin at a plasma-attainable and nontoxic level suppressed the proliferation of B16 cells. Aspirin induced the activation of p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases. Inhibition of JNK, but not p38, decreased the suppressive effect of aspirin upon the proliferation of B16 cells. The aspirin-induced reduction in B16 proliferation was cumulative over time. Aspirin and the chemotherapeutic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) induced B16 cell death synergistically. In addition to the murine B16 cell line, the proliferation of SK-28 human melanoma cells was also suppressed by aspirin. In conclusion, aspirin suppresses the proliferation of metastatic B16 cells in a JNK-dependent mechanism. PMID:12684272

  6. Growth inhibition of human melanoma cells by a recombinant arginine deiminase expressed in Escherichia coli.

    PubMed

    Kozai, Megumi; Sasamori, Eriko; Fujihara, Masatoshi; Yamashita, Tetsuro; Taira, Hideharu; Harasawa, Ryô

    2009-10-01

    We have cloned the arginine deiminase (ADI) gene from Mycoplasma hominis PG21 genomic DNA by polymerase chain reaction, and changed four TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The recombinant ADI (rADI) was purified to apparent homogeneity by Ni-affinity chromatography after extraction from inclusion bodies followed by refolding. The rADI expressed in E. coli was estimated to be 50 kDa. Dimeric forms of rADI exerted enzymatic activity. We found that high concentration of potassium dihydrogenphosphate (PDP) and L-arginine addition in refolding reaction increases the enzyme activity. The specific activity of rADl was calculated as 0.618 U/mg. In addition, the enzyme activity of purified rADI remained for at least one month in 100 mM PDP solution (pH 6.5), but diminished within one week in 100 mM PDP solution (pH 7.4). Anti-tumor activity of the purified rADI was estimated to be 0.036 U/ml as 50% growth inhibitory activity against human melanoma cell line G-361.

  7. Activation of the A2B adenosine receptor in B16 melanomas induces CXCL12 expression in FAP-positive tumor stromal cells, enhancing tumor progression

    PubMed Central

    Sorrentino, Claudia; Miele, Lucio; Porta, Amalia; Pinto, Aldo; Morello, Silvana

    2016-01-01

    The A2B receptor (A2BR) can mediate adenosine-induced tumor proliferation, immunosuppression and angiogenesis. Targeting the A2BR has proved to be therapeutically effective in some murine tumor models, but the mechanisms of these effects are still incompletely understood. Here, we report that pharmacologic inhibition of A2BR with PSB1115, which inhibits tumor growth, decreased the number of fibroblast activation protein (FAP)-expressing cells in tumors in a mouse model of melanoma. This effect was associated with reduced expression of fibroblast growth factor (FGF)-2. Treatment of melanoma-associated fibroblasts with the A2BR agonist Bay60-6583 enhanced CXCL12 and FGF2 expression. This effect was abrogated by PSB1115. The A2AR agonist CGS21680 did not induce CXCL12 or FGF2 expression in tumor associated fibroblasts. Similar results were obtained under hypoxic conditions in skin-derived fibroblasts, which responded to Bay60-6583 in an A2BR-dependent manner, by stimulating pERK1/2. FGF2 produced by Bay60-6583-treated fibroblasts directly enhanced the proliferation of melanoma cells. This effect could be reversed by PSB1115 or an anti-FGF2 antibody. Interestingly, melanoma growth in mice receiving Bay60-6583 was attenuated by inhibition of the CXCL12/CXCR4 pathway with AMD3100. CXCL12 and its receptor CXCR4 are involved in angiogenesis and immune-suppression. Treatment of mice with AMD3100 reduced the number of CD31+ cells induced by Bay60-6583. Conversely, CXCR4 blockade did not affect the accumulation of tumor-infiltrating MDSCs or Tregs. Together, our data reveal an important role for A2BR in stimulating FGF2 and CXCL12 expression in melanoma-associated fibroblasts. These factors contribute to create a tumor-promoting microenvironment. Our findings support the therapeutic potential of PSB1115 for melanoma. PMID:27590504

  8. Anticancer Activity of Saponins from Allium chinense against the B16 Melanoma and 4T1 Breast Carcinoma Cell

    PubMed Central

    Yu, Zhihui; Zhang, Tong; Zhou, Fengjuan; Xiao, Xiuqing; Ding, Xuezhi; He, Hao; Rang, Jie; Quan, Meifang; Wang, Ting; Zuo, Mingxing; Xia, Liqiu

    2015-01-01

    The cytotoxic substance of A. chinense saponins (ACSs) was isolated using ethanol extraction and purified with the D101 macroporous adsorption resin approach. We investigated the anticancer activity of ACSs in the B16 melanoma and 4T1 breast carcinoma cell lines. Methylthioninium chloride and hematoxylin-eosin staining with Giemsa dyestuff were used when the cells were treated with ACSs. The results showed that the cells morphologies changed significantly; ACSs induced cell death in B16 and 4T1 cells based on acridine orange/ethidium bromide double fluorescence staining, with the number and degree of apoptotic tumor cells increasing as ACS concentration increased. ACSs inhibited the proliferation of B16 and 4T1 cells in a dose-dependent manner. They also inhibited cell migration and colony formation and exhibited a concentration-dependent effect. In addition, ACSs apparently inhibited the growth of melanoma in vivo. The preliminary antitumor in vivo assay revealed that early medication positively affected tumor inhibition action and effectively protected the liver and spleen of C57 BL/6 mice from injury. This study provides evidence for the cytotoxicity of ACSs and a strong foundation for further research to establish the theoretical basis for cell death and help in the design and development of new anticancer drugs. PMID:26146506

  9. PHA665752 inhibits the HGF-stimulated migration and invasion of cells by blocking PI3K/AKT pathway in human cell line uveal melanoma.

    PubMed

    Wang, Z; He, C; Liu, L; Ma, N; Chen, X; Zheng, D; Qiu, G H

    2017-03-03

    HGF/c-MET is frequently associated with tumor metastasis in many cancers, including uveal melanoma (UM). PHA665752, a selective c-MET inhibitor, exhibits anticancer activity through inhibiting cell motility in some cancers. In this study, we investigated the effects of PHA665752 on UM cell lines M17 and SP6.5. Our data show that HGF stimulated the motility of UM cells, and induced the activation of both c-MET and PI3K/AKT, but not ERK1/2. Moreover, consistent with the amount of c-MET within the nucleus, PHA665752 significantly inhibited HGF-promoted cell motility and suppressed the phosphorylation of c-MET and PI3K/AKT, but not ERK1/2 induced by HGF. Additionally, the effects of PHA665752 on both the inhibition of HGF-induced cell motility and the suppression of active AKT are similar to those of PI3K inhibitor LY294002. In xenograft models, PHA665752 significantly inhibited tumor growth in nude mice and similarly suppressed the phosphorylation of c-MET and PI3K/AKT. Our current findings, combined with previous results, demonstrate that PHA665752 inhibits HGF-induced motility via the inhibition of PI3K/AKT. This study suggests that targeting HGF/c-MET could be a promising therapeutic strategy for UM by preventing cell motility.

  10. Inhibition of HSP90 by AT13387 Delays the Emergence of Resistance to BRAF Inhibitors and Overcomes Resistance to dual BRAF and MEK Inhibition in Melanoma Models

    PubMed Central

    Hearn, Keisha; Rodriguez-Lopez, Ana M; Munck, Joanne M; Haarberg, H Eirik; Sondak, Vernon K; Thompson, Neil T; Azab, Mohammad; Lyons, John F; Smalley, Keiran S M; Wallis, Nicola G

    2014-01-01

    Emergence of clinical resistance to BRAF inhibitors, alone or in combination with MEK inhibitors, limits clinical responses in melanoma. Inhibiting HSP90 offers an approach to simultaneously interfere with multiple resistance mechanisms. Using the HSP90 inhibitor, AT13387, which is currently in clinical trials, we investigated the potential of HSP90 inhibition to overcome or delay the emergence of resistance to these kinase inhibitors in melanoma models. In vitro, treating vemurafenib-sensitive cells (A375 or SK-MEL-28) with a combination of AT13387 and vemurafenib prevented colony growth under conditions where vemurafenib treatment alone generated resistant colonies. In vivo, when AT13387 was combined with vemurafenib in a SK-MEL-28, vemurafenib-sensitive model, no regrowth of tumors was observed over 5 months, although 2 out of 7 tumors in the vemurafenib monotherapy group relapsed in this time. Together these data suggest that the combination of these agents can delay the emergence of resistance. Cell lines with acquired vemurafenib resistance, derived from these models (A375R, SK-MEL-28R) were also sensitive to HSP90 inhibitor treatment; key clients were depleted, apoptosis was induced and growth in 3D-culture was inhibited. Similar effects were observed in cell lines with acquired resistance to both BRAF and MEK inhibitors (SK-MEL-28RR, WM164RR, 1205LuRR). These data suggest that treatment with an HSP90 inhibitor, such as AT13387, is a potential approach for combatting resistance to BRAF and MEK inhibition in melanoma. Moreover, frontline combination of these agents with an HSP90 inhibitor could delay the emergence of resistance, providing a strong rationale for clinical investigation of such combinations in BRAF-mutated melanoma. PMID:25349308

  11. Properdistatin inhibits angiogenesis and improves vascular function in human melanoma xenografts with low thrombospondin-1 expression

    PubMed Central

    Gaustad, Jon-Vidar; Simonsen, Trude G.; Andersen, Lise Mari K.; Rofstad, Einar K.

    2016-01-01

    In this study, the effect of properdistatin, a novel peptide derived from the thrombospondin 1 (TSP-1) domain of properdin, was investigated in three melanoma xenograft models with different TSP-1 expression. The tumors were grown in dorsal window chambers and were treated with 80 mg/kg/day properdistatin or vehicle. Morphological parameters of the tumor vasculature were assessed from high resolution transillumination images. Blood supply time (i.e., the time required for arterial blood to flow from a supplying artery to downstream microvessels) and plasma velocities were assessed from first-pass imaging movies recorded after a bolus of fluorescence-labeled dextran had been administered intravenously. Gene and protein expression of TSP-1 were assessed with quantitative PCR and immunohistochemistry, respectively. Properdistatin treatment inhibited angiogenesis in low TSP-1 expressing tumors but did not alter the vasculature in high TSP-1 expressing tumors. In low TSP-1 expressing tumors, properdistatin selectively removed small-diameter capillaries, but did not change the morphology of tumor arterioles or tumor venules. Properdistatin also reduced blood supply times and increased plasma velocities, implying that the treatment reduced the geometric resistance to blood flow and improved vascular function. PMID:27756886

  12. Simultaneous inhibition of key growth pathways in melanoma cells and tumor regression by a designed bidentate constrained helical peptide.

    PubMed

    Dhar, Amlanjyoti; Mallick, Shampa; Ghosh, Piya; Maiti, Atanu; Ahmed, Israr; Bhattacharya, Seemana; Mandal, Tapashi; Manna, Asit; Roy, Koushik; Singh, Sandeep; Nayak, Dipak Kumar; Wilder, Paul T; Markowitz, Joseph; Weber, David; Ghosh, Mrinal K; Chattopadhyay, Samit; Guha, Rajdeep; Konar, Aditya; Bandyopadhyay, Santu; Roy, Siddhartha

    2014-07-01

    Protein-protein interactions are part of a large number of signaling networks and potential targets for drug development. However, discovering molecules that can specifically inhibit such interactions is a major challenge. S100B, a calcium-regulated protein, plays a crucial role in the proliferation of melanoma cells through protein-protein interactions. In this article, we report the design and development of a bidentate conformationally constrained peptide against dimeric S100B based on a natural tight-binding peptide, TRTK-12. The helical conformation of the peptide was constrained by the substitution of α-amino isobutyric acid--an amino acid having high helical propensity--in positions which do not interact with S100B. A branched bidentate version of the peptide was bound to S100B tightly with a dissociation constant of 8 nM. When conjugated to a cell-penetrating peptide, it caused growth inhibition and rapid apoptosis in melanoma cells. The molecule exerts antiproliferative action through simultaneous inhibition of key growth pathways, including reactivation of wild-type p53 and inhibition of Akt and STAT3 phosphorylation. The apoptosis induced by the bidentate constrained helix is caused by direct migration of p53 to mitochondria. At moderate intravenous dose, the peptide completely inhibits melanoma growth in a mouse model without any significant observable toxicity. The specificity was shown by lack of ability of a double mutant peptide to cause tumor regression at the same dose level. The methodology described here for direct protein-protein interaction inhibition may be effective for rapid development of inhibitors against relatively weak protein-protein interactions for de novo drug development.

  13. Forsythiae Fructus Inhibits B16 Melanoma Growth Involving MAPKs/Nrf2/HO-1 Mediated Anti-Oxidation and Anti-Inflammation.

    PubMed

    Bao, Jiaolin; Ding, Renbo; Zou, Lidi; Zhang, Chao; Wang, Kai; Liu, Fang; Li, Peng; Chen, Meiwan; Wan, Jian-Bo; Su, Huanxing; Wang, Yitao; He, Chengwei

    2016-01-01

    Forsythiae Fructus, the fruits of Forsythia suspensa (Thunb.) Vahl, Lianqiao in Chinese, is one of the most fundamental herbs in traditional Chinese medicine (TCM). It is a typical heat-clearing and detoxicating herb, according to TCM theory. In this study, we investigated the antitumor effect of Forsythiae Fructus aqueous extract (FAE) on B16-F10 melanoma cells in vivo. The transplanted B16-F10 melanoma in C57BL/6 mice was established and used for the evaluation of the in vivo antitumor effect of FAE. FAE strongly inhibited the growth of B16-F10 cells in vitro and the tumor in vivo. The survival time of tumor-bearing mice was significantly prolonged by FAE. FAE inhibited cancer cell proliferation and angiogenesis in the tumor, as indicated by the decreased expressions of Ki67 and CD31. The levels of ROS, MDA, TNF-[Formula: see text] and IL-6 decreased, while GSH increased in the FAE treatment group, indicating FAE possesses strong anti-oxidative and anti-inflammatory activity. The expression of anti-oxidant proteins Nrf-2 and HO-1, tumor suppressors P53 and p-PTEN, and the MAPK pathways in tumor tissues were upregulated by FAE treatment. These data demonstrated that FAE exhibited strong antitumor activity against B16-F10 murine melanoma both in vitro and in vivo. The antitumor effect of FAE involved decreases in oxidative stress and inflammation in the tumor, which is closely related to the heat-clearing and detoxicating properties of FAE.

  14. Melanoma Expressed-CD70 Is Regulated by RhoA and MAPK Pathways without Affecting Vemurafenib Treatment Activity

    PubMed Central

    Sarrabayrouse, Guillaume; Gallardo, Franck; Gence, Rémi; Tilkin-Mariamé, Anne-Françoise

    2016-01-01

    CD70 is a costimulatory molecule member of the Tumor Necrosis Factor family that is expressed on activated immune cells. Its ectopic expression has been described in several types of cancer cells including lymphomas, renal cell carcinomas and glioblastomas. We have recently described its expression in a part of tumor cells from the vast majority of melanoma biopsies and human melanoma cell lines, and found that CD70 expression decreased over time as the disease progressed. Here, we show that RhoA, BRAF and Mitogen Activating Protein Kinase pathways are involved in the positive transcriptional regulation of CD70 expression in melanomas. Interestingly, the clinical inhibitor of the common BRAF V600E/D variants, Vemurafenib (PLX-4032), which is currently used to treat melanoma patients with BRAF V600E/D-mutated metastatic melanomas, decreased CD70 expression in human CD70+ melanoma cell lines. This decrease was seen in melanoma cells both with and without the BRAFV600E/D mutation, although was less efficient in those lacking the mutation. But interestingly, by silencing CD70 in CD70+ melanoma cell lines we show that PLX-4032-induced melanoma cell killing and its inhibitory effect on MAPK pathway activation are unaffected by CD70 expression. Consequently, our work demonstrates that CD70 ectopic expression in melanomas is not a valuable biomarker to predict tumor cells sensitivity to BRAF V600 inhibitors. PMID:26828592

  15. Melanoma Expressed-CD70 Is Regulated by RhoA and MAPK Pathways without Affecting Vemurafenib Treatment Activity.

    PubMed

    Pich, Christine; Teiti, Iotefa; Sarrabayrouse, Guillaume; Gallardo, Franck; Gence, Rémi; Tilkin-Mariamé, Anne-Françoise

    2016-01-01

    CD70 is a costimulatory molecule member of the Tumor Necrosis Factor family that is expressed on activated immune cells. Its ectopic expression has been described in several types of cancer cells including lymphomas, renal cell carcinomas and glioblastomas. We have recently described its expression in a part of tumor cells from the vast majority of melanoma biopsies and human melanoma cell lines, and found that CD70 expression decreased over time as the disease progressed. Here, we show that RhoA, BRAF and Mitogen Activating Protein Kinase pathways are involved in the positive transcriptional regulation of CD70 expression in melanomas. Interestingly, the clinical inhibitor of the common BRAF V600E/D variants, Vemurafenib (PLX-4032), which is currently used to treat melanoma patients with BRAF V600E/D-mutated metastatic melanomas, decreased CD70 expression in human CD70+ melanoma cell lines. This decrease was seen in melanoma cells both with and without the BRAFV600E/D mutation, although was less efficient in those lacking the mutation. But interestingly, by silencing CD70 in CD70+ melanoma cell lines we show that PLX-4032-induced melanoma cell killing and its inhibitory effect on MAPK pathway activation are unaffected by CD70 expression. Consequently, our work demonstrates that CD70 ectopic expression in melanomas is not a valuable biomarker to predict tumor cells sensitivity to BRAF V600 inhibitors.

  16. Inhibition of metastasis of B16F-10 melanoma cells in C57BL/6 mice by an extract of Calendula officinalis L flowers.

    PubMed

    Preethi, Korangath C; Siveen, Kodappully S; Kuttan, Ramadasan; Kuttan, Girija

    2010-01-01

    To determine the effect of a Calendula officinalis flower extract on lung metastasis by B16F-10 melanoma cells in C57BL/6 mice. Male mice were injected with B16F-10 melanoma cells through the tail vein and simultaneously treated with C.officinalis flower extract. Parameters studied were lung tumor nodule count, life span of animals, gamma glutamyl transpeptidase activity, sialic acid, TNF-α, IL-1β, IL-6, IL-2, GM-CSF, VEGF and TIMP-1 levels in serum, and lung hydroxyproline, uronic acid and hexosamine levels, as well as histopathological features. Effects of C.officinalis on the expression of various genes involved in metastasis like matrix metalloproteases (MMPs), tissue inhibitor of metalloproteases (TIMPs), prolyl hydoxylase, lysyl oxidase, nm23, and proinflammatory cytokines were also investigated. Simultaneous administration of C. officinalis extract to tumor bearing C57BL/6 mice reduced the lung tumor nodules by 74% with 43.3% increase in life span. Elevated levels of hydroxyproline, uronic acid, hexosamine, serum sialic acid and γ-glutamyl transpeptidase in the metastatic controls were found to be significantly lowered in the C. officinalis treated animals. The extract also inhibited expression of MMP-2, MMP-9, prolyl hydroxylase and lysyl oxidase and activated TIMP-1 and TIMP-2 and downregulated proinflammatory cytokines. The present investigation indicated antimetastatic effects of Calendula officinalis flowers through the inhibition of key enzymes involved in processes of metastasis.

  17. Dopa oxidase activity and ceruloplasmin in the sera of hamsters with melanoma.

    PubMed

    Vachtenheim, J; Pavel, S; Duchon, J

    1981-01-01

    Two simple spectrophotometric assays have been employed for the measurement of dopa oxidase activity and ceruloplasmin polyphenol oxidase activity in the sera from normal hamsters and hamsters bearing melanotic melanoma. Both activities were found to be augmented in tumor animals, the dopa oxidase activity much more prominently. The levels of the enzymes tested increased proportionally to the tumor mass.

  18. Dehydroleucodine inhibits tumor growth in a preclinical melanoma model by inducing cell cycle arrest, senescence and apoptosis.

    PubMed

    Costantino, Valeria V; Lobos-Gonzalez, Lorena; Ibañez, Jorge; Fernandez, Dario; Cuello-Carrión, F Darío; Valenzuela, Manuel A; Barbieri, Manuel A; Semino, Silvana N; Jahn, Graciela A; Quest, Andrew F G; Lopez, Luis A

    2016-03-01

    Malignant melanoma represents the fastest growing public health risk of all cancer types worldwide. Several strategies and anti-cancer drugs have been used in an effort to improve treatments, but the development of resistance to anti-neoplastic drugs remains the major cause of chemotherapy failure in melanomas. Previously, we showed that the sesquiterpene lactone, dehydroleucodine (DhL), promotes the accumulation of DNA damage markers, such as H2AX and 53BP1, in human tumor cells. Also DhL was shown to trigger either cell senescence or apoptosis in a concentration-dependent manner in HeLa and MCF7 cells. Here, we evaluated the effects of DhL on B16F0 mouse melanoma cells in vitro and in a pre-clinical melanoma model. DhL inhibited the proliferation of B16F0 cells by inducing senescence or apoptosis in a concentration-dependent manner. Also, DhL reduced the expression of the cell cycle proteins cyclin D1 and B1 and the inhibitor of apoptosis protein, survivin. In melanomas generated by subcutaneous injection of B16F0 cells into C57/BL6 mice, the treatment with 20 mg DhL /Kg/day in preventive, simultaneous and therapeutic protocols reduced tumor volumes by 70%, 60% and 50%, respectively. DhL treatments reduced the number of proliferating, while increasing the number of senescent and apoptotic tumor cells. To estimate the long-term effects of DhL, a mathematical model was applied to fit experimental data. Extrapolation beyond experimental time points revealed that DhL administration following preventive and therapeutic protocols is predicted to be more effective than simultaneous treatments with DhL in restricting tumor growth. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  19. FOXP3 over-expression inhibits melanoma tumorigenesis via effects on proliferation and apoptosis.

    PubMed Central

    Tan, BeeShin; Anaka, Matthew; Deb, Siddhartha; Freyer, Claudia; Ebert, Lisa M.; Chueh, Anderly C.; Al-Obaidi, Sheren; Behren, Andreas; Jayachandran, Aparna; Cebon, Jonathan; Chen, Weisan; Mariadason, John M.

    2014-01-01

    The Forkhead box P3 (FOXP3) transcription factor is the key driver of regulatory T cell (Treg cells) differentiation and immunosuppressive function. In addition, FOXP3 has been reported to be expressed in many tumors, including melanoma. However, its role in tumorigenesis is conficting, with both tumor suppressive and tumor promoting functions described. The aim of the current study was to characterize the expression and function of FOXP3 in melanoma. FOXP3 expression was detected by immunohistochemistry (IHC) in 12% (18/146) of stage III and IV melanomas. However expression was confined to fewer than 1% of cells in these tumors. Stable over-expression of FOXP3 in the SK-MEL-28 melanoma cell line reduced cell proliferation and clonogenicity in vitro, and reduced xenograft growth in vivo. FOXP3 over-expression also increased pigmentation and the rate of apoptosis of SK-MEL-28 cells. Based on its infrequent expression in human melanoma, and its growth inhibitory and pro-apoptotic effect in over-expressing melanoma cells, we conclude that FOXP3 is not likely to be a key tumor suppressor or promoter in melanoma. PMID:24406338

  20. Platelet GPIIb supports initial pulmonary retention but inhibits subsequent proliferation of melanoma cells during hematogenic metastasis

    PubMed Central

    Echtler, Katrin; Konrad, Ildiko; Lorenz, Michael; Schneider, Simon; Hofmaier, Sebastian; Plenagl, Florian; Stark, Konstantin; Czermak, Thomas; Tirniceriu, Anca; Eichhorn, Martin; Walch, Axel; Enders, Georg; Massberg, Steffen; Schulz, Christian

    2017-01-01

    Platelets modulate the process of cancer metastasis. However, current knowledge on the direct interaction of platelets and tumor cells is mostly based on findings obtained in vitro. We addressed the role of the platelet fibrinogen receptor glycoprotein IIb (integrin αIIb) for experimental melanoma metastasis in vivo. Highly metastatic B16-D5 melanoma cells were injected intravenously into GPIIb-deficient (GPIIb-/-) or wildtype (WT) mice. Acute accumulation of tumor cells in the pulmonary vasculature was assessed in real-time by confocal videofluorescence microscopy. Arrest of tumor cells was dramatically reduced in GPIIb-/- mice as compared to WT. Importantly, we found that mainly multicellular aggregates accumulated in the pulmonary circulation of WT, instead B16-D5 aggregates were significantly smaller in GPIIb-/- mice. While pulmonary arrest of melanoma was clearly dependent on GPIIb in this early phase of metastasis, we also addressed tumor progression 10 days after injection. Inversely, and unexpectedly, we found that melanoma metastasis was now increased in GPIIb-/- mice. In contrast, GPIIb did not regulate local melanoma proliferation in a subcutaneous tumor model. Our data suggest that the platelet fibrinogen receptor has a differential role in the modulation of hematogenic melanoma metastasis. While platelets clearly support early steps in pulmonary metastasis via GPIIb-dependent formation of platelet-tumor-aggregates, at a later stage its absence is associated with an accelerated development of melanoma metastases. PMID:28253287

  1. c-Jun regulates phosphoinositide-dependent kinase 1 transcription: implication for Akt and protein kinase C activities and melanoma tumorigenesis.

    PubMed

    Lopez-Bergami, Pablo; Kim, Hyungsoo; Dewing, Antimone; Goydos, James; Aaronson, Stuart; Ronai, Ze'ev

    2010-01-08

    Mutations in N-RAS and B-RAF, which commonly occur in melanomas, result in constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) signaling. Active ERK increases expression and activity of the c-Jun transcription factor, linking ERK and Jun N-terminal kinase (JNK) cascades. Here, we show that c-Jun regulates transcription of phosphoinositide-dependent kinase 1 (PDK1) with a concomitant impact on Akt and protein kinase C (PKC) activity and related substrates. Inhibition of c-Jun reduces PDK1 expression and attenuates Akt and PKC activity, which can be restored by exogenous PDK1. c-Jun regulation of PDK1 in melanoma contributes to growth rate and the ability to form tumors in mice. Correspondingly, increased levels of c-Jun in melanoma cell lines coincide with up-regulation of PDK1 and phosphorylation of PKC and Akt. The identification of c-Jun as a transcriptional regulator of PDK1 expression highlights key mechanisms underlying c-Jun oncogenic activity, and provides new insight into the nature of up-regulated Akt and PKC in melanoma.

  2. Cellular Mechanisms Underlying Complete Hematological Response of Chronic Myeloid Leukemia to BRAF and MEK1/2 Inhibition in a Patient with Concomitant Metastatic Melanoma.

    PubMed

    Andrews, Miles C; Turner, Natalie; Boyd, Janis; Roberts, Andrew W; Grigg, Andrew P; Behren, Andreas; Cebon, Jonathan

    2015-12-01

    Targeted MEK inhibition is an emerging therapy in a number of solid tumors. It holds particular promise in BRAF V600E mutation-positive malignant melanoma, where constitutive activation and cell growth through the MAP kinase (MAPK) pathway is well established. In vitro and preclinical research indicates that MAPK pathway activation is important in chronic myeloid leukemia (CML) leukemogenesis; however, the potential of MEK inhibition has not yet been investigated clinically in the setting of such hematologic malignancies. We report a case of complete hematologic response of CML to MEK inhibition in a patient with synchronous metastatic melanoma, who received treatment with combination BRAF and MEK1/2 inhibitors. We studied the effects of these agents on proliferation and outgrowth of myeloid precursors, and longitudinal shifts in peripheral blood phenotyping during the course of treatment. A model cell line system was used to examine the effects of dabrafenib and trametinib on MAPK and BCR-ABL1 signaling. After 35 weeks on treatment with BRAF and MEK inhibitors, complete hematologic response was observed without recourse to BCR-ABL1-targeted therapy. MEK inhibition was principally responsible for impaired proliferation of both mature and primitive myeloid precursors, as well as growth and hemoglobinization of erythroid precursors. Paradoxical activation of the MAPK pathway was seen in response to BRAF inhibitor therapy but this was easily overcome by clinically relevant doses of concurrent MEK inhibitor. These studies suggest that further evaluation of the optimal MAPK targeting approach is warranted to extend therapeutic options in CML. ©2015 American Association for Cancer Research.

  3. DDR2 inhibition reduces migration and invasion of murine metastatic melanoma cells by suppressing MMP2/9 expression through ERK/NF-κB pathway.

    PubMed

    Poudel, Barun; Lee, Young-Mi; Kim, Dae-Ki

    2015-04-01

    Metastatic melanoma is one of the most deadly and evasive cancers. Collagen I in the extracellular matrix promotes the migration and invasion of tumor cells through the production of matrix metalloproteinase (MMP) 2 and 9. Discoidin domain receptor (DDR) 2 is a collagen receptor that is implicated in several cancer types including breast and prostate cancers. However, the role of DDR2 in the migration and invasion of murine melanoma cells is less studied. In the present study, we investigated the effects and underlying mechanisms of DDR2 in migration and invasion of B16BL6 melanoma cells in response to collagen I. Results demonstrated that DDR2 is expressed and is phosphorylated by collagen I in the cells. Upon down-regulation of DDR2 using small-interfering RNA (siRNA) approach, both of the cell migratory and invasive phenotypes were significantly attenuated when compared with the control cells. This effect was mediated via suppression of MMP2/9 upon DDR2 inhibition. Furthermore, inhibition of DDR2 by specific siRNA markedly reduced the activation of extracellular regulated kinase (ERK) 1 and 2 and nuclear factor of kappa B (NF-κB) in the cells when compared with the control cells. Overall, these data demonstrated that DDR2 siRNA-mediated suppression of ERK1/2 and NF-κB could down-regulate the expressions of MMP2/9 in response to collagen I to reduce the migratory and invasive phenotypes of the cells.

  4. Targeted inhibition of BRAF kinase: opportunities and challenges for therapeutics in melanoma.

    PubMed

    Pérez-Lorenzo, Rolando; Zheng, Bin

    2012-02-01

    Malignant melanoma is the most aggressive form of skin cancer and its incidence has increased dramatically in the last two decades. Even with a high rate of success in the treatment of early stages of this malignancy, currently there are no effective strategies for the treatment of advanced metastatic melanoma. Much effort has been put into the use of different target-specific drugs, among which BRAF kinase-specific small-molecule inhibitors have rendered promising results as therapeutic agents in metastatic melanoma. Nonetheless, some side effects, such as development of SCC (squamous cell carcinoma), as well as tumour resistance and recurrence, are common limitations of this therapeutic strategy. The use of combination treatments in which different regulatory pathways or the immunological response are targeted seems to be a promising tool for the future success of melanoma therapeutics.

  5. Antiproliferative and Pro-Apoptotic Effects of MiR-4286 Inhibition in Melanoma Cells

    PubMed Central

    Komina, Anna; Palkina, Nadezhda; Aksenenko, Mariya; Tsyrenzhapova, Seseg; Ruksha, Tatiana

    2016-01-01

    Introduction MicroRNAs are essential regulators of gene expression at the post-transcriptional level. Their expression is altered in cancer tissues, and evaluation of these alterations is considered a promising tool used to diagnose and identify prognostic markers. Materials and methods The microRNA expression profiles of formalin-fixed, paraffin-embedded melanoma and melanocytic nevi samples were estimated with a microarray and subsequently validated by real-time PCR. Melanoma cells were transfected with miR-4286 inhibitor to evaluate the influence of this microRNA on the viability, proliferation, apoptosis, migration, and invasion of melanoma cells. Results The microarray revealed that the expression of 1,171 microRNAs was altered in melanoma samples compared to melanocytic nevi. Real-time PCR validation experiments found the microRNA expression levels to correspond to the melanoma/melanocytic nevi microarray results. The pathway analysis identified 52 modulated pathways in melanoma. Moreover, the application of miR-4286 inhibitor to BRO melanoma cells resulted in a 2.6-fold increase in the apoptosis rate and a 1.7-fold decrease in the cell proliferation/viability but did not affect the invasiveness and migration of these cells. Furthermore, the use of miR-4286 inhibitor altered the mRNA expression of several miR-4286 gene targets: folylpolyglutamate synthase, RNA polymerase I-specific transcription initiation factor, apelin, G-protein-coupled receptor 55, and high-mobility group A1 protein, which have been implicated in cell proliferation/apoptosis regulation. Lastly, the transiently transfected SK-MEL-1 cells with miR-4286 inhibitor decreased proliferation rate and modulated folylpolyglutamate synthase rates of these cells. Conclusion Our results demonstrate that miR-4286 mediates proliferation and apoptosis in melanoma cells, these findings may represent a novel mechanism underlying these processes. PMID:28005927

  6. Angiostatic activity of obtustatin as α1β1 integrin inhibitor in experimental melanoma growth

    PubMed Central

    Brown, Meghan C.; Staniszewska, Izabela; Valle, Luis Del; Tuszynski, George P.; Marcinkiewicz, Cezary

    2008-01-01

    The presented results show the effect of targeting of collagen receptor, α1β1 integrin expressed on the endothelial cells on the development of experimental melanoma and pathological angiogenesis. Obtustatin, a snake venom KTS-disintegrin, was applied as a specific inhibitor of this integrin. This low molecular weight peptide revealed a potent therapeutic effect on melanoma progression in two animal systems, mouse and quail. Its oncostatic effect was related to the inhibition of angiogenesis. Obtustatin inhibited the neovascularization ratio on the CAM embryo of quail, which was pathologically induced by the developing tumor. The i.v. administration of obtustatin completely blocked cancer growth of MV3 human melanoma in nude mice. In B16F10 syngeneic mouse model treatment with the disintegrin revealed a lower effect, although the development of the tumor was significantly reduced for both dosages. The mechanism of obtustatin action is related to the blocking of microvascular endothelial cell proliferation, which undergoes apoptosis in caspase-dependent manner. Summarizing, we present studies of low molecular weight disintegrin, obtustatin as a potential therapeutic compound for treatment of melanoma that contain a high level of vascularization. PMID:18712720

  7. Inhibition of Hageman factor activation

    PubMed Central

    Nossel, H. L.; Rubin, H.; Drillings, M.; Hsieh, R.

    1968-01-01

    A method for studying inhibitors of the contact stages of blood coagulation is described. A number of positively charged substances were shown to inhibit the contact stages. The inhibitory substances include spermine, cytochrome c, ribonuclease, and lysozyme. The inhibitory effect of these substances was neutralized by the addition of an activated plasma thromboplastin antecedent, factor XI, (PTA) fraction. Other positively charged substances including protamine, hexadimethrine, polylysine, polyornithine, methylene blue, and ortho-toluidine blue also inhibited the contact stages of coagulation, but the inhibitory effect on coagulation was not neutralized by the activated PTA fraction. Negatively charged substances such as heparin and insulin did not inhibit the contact stages of coagulation. Cytochrome c inhibited Celite adsorption of a partially purified Hageman factor fraction, and cytochrome, ribonuclease, spermine, and lysozome inhibited the adsorption of Hageman factor from PTA-deficient plasma. Very much smaller quantities of Celite completely adsorbed Hageman factor from the fraction rather than from whole plasma, which suggested the possibility that plasma contains an inhibitor or inhibitors of Hageman factor adsorption. Furthermore cytochrome c, spermine, ribonuclease, and lysozyme inhibited the coagulant activity of the following activators of the Hageman and PTA factors: Celite, kaolin, sodium stearate, ellagic acid, and skin. It is suggested that negatively charged sites on these activators are critical for adsorption and activation and that inhibition results from neutralization of the negatively charged sites by the adsorbed inhibtor. Tests with polylysine polymers indicate that inhibitory activity is directly related to molecular size over the molecular weight range of 4000 to 100,000. PMID:5645860

  8. BRG1 promotes survival of UV-irradiated melanoma cells by cooperating with MITF to activate the melanoma inhibitor of apoptosis gene.

    PubMed

    Saladi, Srinivas V; Wong, Philip G; Trivedi, Archit R; Marathe, Himangi G; Keenen, Bridget; Aras, Shweta; Liew, Zi-Qi; Setaluri, Vijayasaradhi; de la Serna, Ivana L

    2013-05-01

    Microphthalmia-associated transcription factor (MITF) is a survival factor in melanocytes and melanoma cells. MITF regulates expression of antiapoptotic genes and promotes lineage-specific survival in response to ultraviolet (UV) radiation and to chemotherapeutics. SWI/SNF chromatin-remodeling enzymes interact with MITF to regulate MITF target gene expression. We determined that the catalytic subunit, BRG1, of the SWI/SNF complex protects melanoma cells against UV-induced death. BRG1 prevents apoptosis in UV-irradiated melanoma cells by activating expression of the melanoma inhibitor of apoptosis (ML-IAP). Down-regulation of ML-IAP compromises BRG1-mediated survival of melanoma cells in response to UV radiation. BRG1 regulates ML-IAP expression by cooperating with MITF to promote transcriptionally permissive chromatin structure on the ML-IAP promoter. The alternative catalytic subunit, BRM, and the BRG1-associated factor, BAF180, were found to be dispensable for elevated expression of ML-IAP in melanoma cells. Thus, we illuminate a lineage-specific mechanism by which a specific SWI/SNF subunit, BRG1, modulates the cellular response to DNA damage by regulating an antiapoptotic gene and implicate this subunit of the SWI/SNF complex in mediating the prosurvival function of MITF.

  9. Tea tree oil might combat melanoma.

    PubMed

    Bozzuto, Giuseppina; Colone, Marisa; Toccacieli, Laura; Stringaro, Annarita; Molinari, Agnese

    2011-01-01

    In this study we present new data from experiments focused on the antitumor activity of tea tree oil (TTO), an essential oil distilled from Melaleuca alternifolia. TTO proved to be capable of inhibiting the growth of melanoma cells and of overcoming multidrug resistance (MDR), as we reported in our previous study. Moreover, the survival role of the MDR-marker P-glycoprotein appears to be involved in the mechanism of invasion of melanoma cells. The results reported herein indicate that TTO and its main active component, terpinen-4-ol, can also interfere with the migration and invasion processes of drug-sensitive and drug-resistant melanoma cells.

  10. Vinculin activators target integrins from within the cell to increase melanoma sensitivity to chemotherapy

    PubMed Central

    Nelson, Elke S.; Folkmann, Andrew W.; Henry, Michael D.; DeMali, Kris A.

    2011-01-01

    Metastatic melanoma is an aggressive skin disease for which there are no effective therapies. Emerging evidence indicates that melanomas can be sensitized to chemotherapy by increasing integrin function. Current integrin therapies work by targeting the extracellular domain, resulting in complete gains or losses of integrin function that lead to mechanism-based toxicities. An attractive alternative approach is to target proteins, such as vinculin, that associate with the integrin cytoplasmic domains and regulate its ligand binding properties. Here we report that a novel reagent, denoted vinculin activating peptide or VAP, increases integrin activity from within the cell, as measured by elevated: (1) numbers of active integrins, (2) adhesion of cells to extracellular matrix ligands, (3) numbers of cell-matrix adhesions, and (4) downstream signaling. These effects are dependent on both integrins and a key regulatory residue A50 in the vinculin head domain. We further show that VAP dramatically increases the sensitivity of melanomas to chemotherapy in clonal growth assays and in vivo mouse models of melanoma. Finally, we demonstrate that the increase in chemosensitivity results from increases in DNA damage-induced apoptosis in a p53-dependent manner. Collectively these findings demonstrate for the first time that integrin function can be manipulated from within the cell and validate integrins as a new therapeutic target for the treatment of chemoresistant melanomas. PMID:21460181

  11. CD147 promotes melanoma progression through hypoxia-induced MMP2 activation.

    PubMed

    Zeng, W; Su, J; Wu, L; Yang, D; Long, T; Li, D; Kuang, Y; Li, J; Qi, M; Zhang, J; Chen, X

    2014-01-01

    Hypoxia enhances MMP2 expression and the invasion and metastatic potential of melanoma cells. CD147 has been shown to induce MMP2 in multiple cancers. To investigate the role of CD147 in hypoxiainduced MMP2 activation, we performed immunohistochemistry (IHC) staining in 206 normal and melanoma tissue samples, and analyzed the correlation between HIF1α and CD147. ChIP (chromosome Immunoprecipitation) in melanoma cell lines supports that HIF1α directly binds to CD147 promoter. Moreover, we made a series of deletion mutants of CD147 promoter, and identified a conserved HIF1α binding site. Point mutation in this site significantly decreased CD147 response to hypoxia. Importantly, knocking down CD147 attenuates MMP2 response to hypoxia in melanoma cell lines. MMP2 could not be efficiently activated by hypoxia in CD147 depletion cells. ELISA data showed that MMP2 secretion was reduced in CD147 depletion cells than control under hypoxia condition. To verify the data from cell culture model, we performed in vivo mouse xenograft experiment. IHC staining showed reduced MMP2 level in CD147 depleted xenografts compared to the control group, with the HIF1α level being comparable. Our study demonstrates a novel pathway mediated by CD147 to promote the MMP2 activation induced by hypoxia, and helps to understand the interplay between hypoxia and melanoma progression.

  12. Constitutive activation of the ERK pathway in melanoma and skin melanocytes in Grey horses.

    PubMed

    Jiang, Lin; Campagne, Cécile; Sundström, Elisabeth; Sousa, Pedro; Imran, Saima; Seltenhammer, Monika; Pielberg, Gerli; Olsson, Mats J; Egidy, Giorgia; Andersson, Leif; Golovko, Anna

    2014-11-21

    Constitutive activation of the ERK pathway, occurring in the vast majority of melanocytic neoplasms, has a pivotal role in melanoma development. Different mechanisms underlie this activation in different tumour settings. The Grey phenotype in horses, caused by a 4.6 kb duplication in intron 6 of Syntaxin 17 (STX17), is associated with a very high incidence of cutaneous melanoma, but the molecular mechanism behind the melanomagenesis remains unknown. Here, we investigated the involvement of the ERK pathway in melanoma development in Grey horses. Grey horse melanoma tumours, cell lines and normal skin melanocytes were analyzed with help of indirect immunofluorescence and immunoblotting for the expression of phospho-ERK1/2 in comparison to that in non-grey horse and human counterparts. The mutational status of BRAF, RAS, GNAQ, GNA11 and KIT genes in Grey horse melanomas was determined by direct sequencing. The effect of RAS, RAF and PI3K/AKT pathways on the activation of the ERK signaling in Grey horse melanoma cells was investigated with help of specific inhibitors and immunoblotting. Individual roles of RAF and RAS kinases on the ERK activation were examined using si-RNA based approach and immunoblotting. We found that the ERK pathway is constitutively activated in Grey horse melanoma tumours and cell lines in the absence of somatic activating mutations in BRAF, RAS, GNAQ, GNA11 and KIT genes or alterations in the expression of the main components of the pathway. The pathway is mitogenic and is mediated by BRAF, CRAF and KRAS kinases. Importantly, we found high activation of the ERK pathway also in epidermal melanocytes, suggesting a general predisposition to melanomagenesis in these horses. These findings demonstrate that the presence of the intronic 4.6 kb duplication in STX17 is strongly associated with constitutive activation of the ERK pathway in melanocytic cells in Grey horses in the absence of somatic mutations commonly linked to the activation of this

  13. Emerging targeted therapies for melanoma treatment (review).

    PubMed

    Russo, Angela; Ficili, Bartolomea; Candido, Saverio; Pezzino, Franca Maria; Guarneri, Claudio; Biondi, Antonio; Travali, Salvatore; McCubrey, James A; Spandidos, Demetrios A; Libra, Massimo

    2014-08-01

    Cutaneous melanoma is an aggressive cancer with a poor prognosis for patients with advanced disease. The identification of several key molecular pathways implicated in the pathogenesis of melanoma has led to the development of novel therapies for this devastating disease. In melanoma, both the Ras/Raf/MEK/ERK (MAPK) and the PI3K/AKT (AKT) signalling pathways are constitutively activated through multiple mechanisms. Targeting various effectors of these pathways with pharmacologic inhibitors may inhibit melanoma cell growth and angiogenesis. Ongoing clinical trials provide hope to improve progression-free survival of patients with advanced melanoma. This review summarizes the most relevant studies focused on the specific action of these new molecular targeted agents. Mechanisms of resistance to therapy are also discussed.

  14. Emerging targeted therapies for melanoma treatment (Review)

    PubMed Central

    RUSSO, ANGELA; FICILI, BARTOLOMEA; CANDIDO, SAVERIO; PEZZINO, FRANCA MARIA; GUARNERI, CLAUDIO; BIONDI, ANTONIO; TRAVALI, SALVATORE; McCUBREY, JAMES A.; SPANDIDOS, DEMETRIOS A.; LIBRA, MASSIMO

    2014-01-01

    Cutaneous melanoma is an aggressive cancer with a poor prognosis for patients with advanced disease. The identification of several key molecular pathways implicated in the pathogenesis of melanoma has led to the development of novel therapies for this devastating disease. In melanoma, both the Ras/Raf/MEK/ERK (MAPK) and the PI3K/AKT (AKT) signalling pathways are constitutively activated through multiple mechanisms. Targeting various effectors of these pathways with pharmacologic inhibitors may inhibit melanoma cell growth and angiogenesis. Ongoing clinical trials provide hope to improve progression-free survival of patients with advanced melanoma. This review summarizes the most relevant studies focused on the specific action of these new molecular targeted agents. Mechanisms of resistance to therapy are also discussed. PMID:24899250

  15. Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma

    PubMed Central

    Moore, Amanda R; Ceraudo, Emilie; Sher, Jessica J; Guan, Youxin; Shoushtari, Alexander N; Chang, Matthew T; Zhang, Jenny Q; Walczak, Edward G; Kazmi, Manija A; Taylor, Barry S; Huber, Thomas; Chi, Ping; Sakmar, Thomas P; Chen, Yu

    2016-01-01

    Uveal melanomas are molecularly distinct from cutaneous melanomas and lack mutations in BRAF, NRAS, KIT, and NF1. Instead, they are characterized by activating mutations in GNAQ and GNA11, two highly homologous α subunits of Gαq/11 heterotrimeric G proteins, and in PLCB4 (phospholipase C β4), the downstream effector of Gαq signaling 1–3. We analyzed genomics data from 136 uveal melanoma samples and found a recurrent mutation in CYSLTR2 (cysteinyl leukotriene receptor 2) encoding a p.Leu129Gln substitution in 4 of 9 samples that lacked mutations in GNAQ, GNA11, and PLCB4 but in 0 of 127 samples that harbored mutations in these genes. The Leu129Gln CysLT2R mutant protein constitutively activates endogenous Gαq and is unresponsive to stimulation by leukotriene. Expression of Leu129Gln CysLT2R in melanocytes enforces expression of a melanocyte-lineage signature, drives phorbol ester–independent growth in vitro, and promotes tumorigenesis in vivo. Our findings implicate CYSLTR2 as a uveal melanoma oncogene and highlight the critical role of Gαq signaling in uveal melanoma pathogenesis. PMID:27089179

  16. Selective in vitro anti-melanoma activity of ursolic and oleanolic acids.

    PubMed

    Oprean, Camelia; Ivan, Alexandra; Bojin, Florina; Cristea, Mirabela; Soica, Codruta; Drăghia, Lavinia; Caunii, Angela; Paunescu, Virgil; Tatu, Calin

    2017-09-29

    Products of natural origin have become important agents in the treatment of cancer, and the active principles of natural sources could be used in combination with chemotherapeutic agents to increase their effects and to minimize their toxicity. Oleanolic (OA) and ursolic (UA) acids are intensely studied for their promising anticancer potential. The aim of this study was focused on the in vitro toxicological effects induced by UA and OA human mesenchymal stem cells and on melanoma, one of the most frequent cancers whose incidence is increasing every year. The two compounds were tested for their cytotoxic, cell cycle arrest and pro-apoptotic effects on melanoma cells (A375 and B164A5) and mesenchymal stem cells. UA exerted a cytotoxic effect in a dose-dependent manner on melanoma cells, while OA's activity has been shown to be low or moderate. Both compounds produced alterations of the cell cycle, arresting cells in the G0/G1 phase. Furthermore, UA induced significant apoptosis through the bcl-2 genes family pathway, with the decrease of the bcl-2 gene expression. The two compounds exerted selective effects on melanoma cells with no effects on human mesenchymal stem cells. The presented results reveal the anticancer potential of UA on melanoma cells, with no detectable toxicity on the mesenchymal stem cells.

  17. Antibody therapies for melanoma: new and emerging opportunities to activate immunity (Review).

    PubMed

    Malas, Sadek; Harrasser, Micaela; Lacy, Katie E; Karagiannis, Sophia N

    2014-09-01

    The interface between malignant melanoma and patient immunity has long been recognised and efforts to treat this most lethal form of skin cancer by activating immune responses with cytokine, vaccine and also antibody immunotherapies have demonstrated promise in limited subsets of patients. In the present study, we discuss different antibody immunotherapy approaches evaluated in the context of melanoma, each designed to act on distinct targets and to employ different mechanisms to restrict tumour growth and spread. Monoclonal antibodies recognising melanoma-associated antigens such as CSPG4/MCSP and targeting elements of tumour-associated vasculature (VEGF) have constituted long-standing translational approaches aimed at reducing melanoma growth and metastasis. Recent insights into mechanisms of immune regulation and tumour-immune cell interactions have helped to identify checkpoint molecules on immune (CTLA4, PD-1) and tumour (PD-L1) cells as promising therapeutic targets. Checkpoint blockade with antibodies to activate immune responses and perhaps to counteract melanoma-associated immunomodulatory mechanisms led to the first clinical breakthrough in the form of an anti-CTLA4 monoclonal antibody. Novel modalities to target key mechanisms of immune suppression and to redirect potent effector cell subsets against tumours are expected to improve clinical outcomes and to provide previously unexplored avenues for therapeutic interventions.

  18. Antibody therapies for melanoma: New and emerging opportunities to activate immunity (Review)

    PubMed Central

    MALAS, SADEK; HARRASSER, MICAELA; LACY, KATIE E.; KARAGIANNIS, SOPHIA N.

    2014-01-01

    The interface between malignant melanoma and patient immunity has long been recognised and efforts to treat this most lethal form of skin cancer by activating immune responses with cytokine, vaccine and also antibody immunotherapies have demonstrated promise in limited subsets of patients. In the present study, we discuss different antibody immunotherapy approaches evaluated in the context of melanoma, each designed to act on distinct targets and to employ different mechanisms to restrict tumour growth and spread. Monoclonal antibodies recognising melanoma-associated antigens such as CSPG4/MCSP and targeting elements of tumour-associated vasculature (VEGF) have constituted long-standing translational approaches aimed at reducing melanoma growth and metastasis. Recent insights into mechanisms of immune regulation and tumour-immune cell interactions have helped to identify checkpoint molecules on immune (CTLA4, PD-1) and tumour (PD-L1) cells as promising therapeutic targets. Checkpoint blockade with antibodies to activate immune responses and perhaps to counteract melanoma-associated immunomodulatory mechanisms led to the first clinical breakthrough in the form of an anti-CTLA4 monoclonal antibody. Novel modalities to target key mechanisms of immune suppression and to redirect potent effector cell subsets against tumours are expected to improve clinical outcomes and to provide previously unexplored avenues for therapeutic interventions. PMID:24969320

  19. miR-146a promotes the initiation and progression of melanoma by activating Notch signaling

    PubMed Central

    Forloni, Matteo; Dogra, Shaillay Kumar; Dong, Yuying; Conte, Darryl; Ou, Jianhong; Zhu, Lihua Julie; Deng, April; Mahalingam, Meera; Green, Michael R; Wajapeyee, Narendra

    2014-01-01

    Oncogenic mutations in BRAF and NRAS occur in 70% of melanomas. In this study, we identify a microRNA, miR-146a, that is highly upregulated by oncogenic BRAF and NRAS. Expression of miR-146a increases the ability of human melanoma cells to proliferate in culture and form tumors in mice, whereas knockdown of miR-146a has the opposite effects. We show these oncogenic activities are due to miR-146a targeting the NUMB mRNA, a repressor of Notch signaling. Previous studies have shown that pre-miR-146a contains a single nucleotide polymorphism (C>G rs2910164). We find that the ability of pre-miR-146a/G to activate Notch signaling and promote oncogenesis is substantially higher than that of pre-miR-146a/C. Analysis of melanoma cell lines and matched patient samples indicates that during melanoma progression pre-miR-146a/G is enriched relative to pre-miR-146a/C, resulting from a C-to-G somatic mutation in pre-miR-146a/C. Collectively, our results reveal a central role for miR-146a in the initiation and progression of melanoma. DOI: http://dx.doi.org/10.7554/eLife.01460.001 PMID:24550252

  20. Stimulation of oncogenic metabotropic glutamate receptor 1 in melanoma cells activates ERK1/2 via PKCepsilon.

    PubMed

    Marín, Yarí E; Namkoong, Jin; Cohen-Solal, Karine; Shin, Seung-Shick; Martino, Jeffrey J; Oka, Masahiro; Chen, Suzie

    2006-08-01

    Metabotropic glutamate receptor 1 (Grm1, formerly mGluR1) is a G protein coupled receptor (GPCR) normally expressed and functional in the central nervous system. Studies of our transgenic mouse melanoma model (TG-3) revealed that ectopic expression of Grm1 in melanocytes is sufficient to induce melanoma development in vivo [P.M. Pollock, K. Cohen-Solal, R. Sood, J. Namkoong, J.J. Martino, A. Koganti, H. Zhu, C. Robbins, I. Makalowska, S.S. Shin, Y. Marin, K.G. Roberts, L.M. Yudt, A. Chen, J. Cheng, A. Incao, H.W. Pinkett, C.L. Graham, K. Dunn, S.M. Crespo-Carbone, K.R. Mackason, K.B. Ryan, D. Sinsimer, J. Goydos, K.R. Reuhl, M. Eckhaus, P.S. Meltzer, W.J. Pavan, J.M. Trent, S. Chen, Nat. Genet. 34 (2003) 108-112.]. We have established and characterized several cell lines in vitro from independent mouse melanoma tumors [Y.E. Marín, J. Namkoong, S.S. Shin, J. Raines, K. Degenhardt, E. White, S. Chen, Neuropharmacol. 49 (2005) 70-79.]. These cell lines are useful tools in the studies of signaling events that may be mediated by Grm1 in transformed melanocytes. Here we show that stimulation of Grm1 by l-quisqualate, a group I metabotropic glutamate receptor agonist, results in inositol triphosphate (IP3) accumulation, and the activation of ERK1/2 in these cell lines. IP3 accumulation and ERK1/2 activation were inhibited by pretreatment of the tumor cells with a Grm1-specific antagonist (LY367385) or by dominant negative mutants of Grm1, demonstrating the specificity of these events. We also show that ERK1/2 activation by Grm1 was PKC-dependent, but cAMP and PKA-independent. PKCepsilon was shown to play a pivotal role in Grm1-mediated ERK1/2 phosphorylation. Insights into the signaling cascades mediated by Grm1 in melanoma cells may aid in the identification of key molecular targets for the future design of combined therapies for melanoma.

  1. CCR5 in recruitment and activation of myeloid-derived suppressor cells in melanoma.

    PubMed

    Umansky, Viktor; Blattner, Carolin; Gebhardt, Christoffer; Utikal, Jochen

    2017-04-05

    Malignant melanoma is characterized by the development of chronic inflammation in the tumor microenvironment, leading to the accumulation of myeloid-derived suppressor cells (MDSCs). Using ret transgenic mouse melanoma model, we found a significant migration of MDSCs expressing C-C chemokine receptor (CCR)5 into primary tumors and metastatic lymph nodes, which was correlated with tumor progression. An increased CCR5 expression on MDSCs was associated with elevated concentrations of CCR5 ligands in melanoma microenvironment. In vitro experiments showed that the upregulation of CCR5 expression on CD11b(+)Gr1(+) immature myeloid cells was induced by CCR5 ligands, IL-6, GM-CSF, and other inflammatory factors. Furthermore, CCR5(+) MDSCs infiltrating melanoma lesions displayed a stronger immunosuppressive pattern than their CCR5(-) counterparts. Targeting CCR5/CCR5 ligand signaling via a fusion protein mCCR5-Ig, which selectively binds and neutralizes all three CCR5 ligands, increased the survival of tumor-bearing mice. This was associated with a reduced migration and immunosuppressive potential of tumor MDSCs. In melanoma patients, circulating CCR5(+) MDSCs were increased as compared to healthy donors. Like in melanoma-bearing mice, we observed an enrichment of these cells and CCR5 ligands in tumors as compared to the peripheral blood. Our findings define a critical role for CCR5 not only in the recruitment but also in the activation of MDSCs in tumor lesions, suggesting that novel strategies of melanoma treatment could be based on blocking CCR5/CCR5 ligand interactions.

  2. Proteasome inhibition blocks NF-κB and ERK1/2 pathways, restores antigen expression and sensitizes resistant human melanoma to TCR-engineered CTLs

    PubMed Central

    Jazirehi, Ali R.; Economou, James S.

    2012-01-01

    Adoptive cell transfer (ACT) of ex vivo engineered autologous lymphocytes encoding high-affinity MART-1/HLA-A*0201-specific T-cell receptor (TCR) α/β chains (F5 CTL), densely infiltrate into sites of metastatic disease, mediating dramatic but partial clinical responses in melanoma patients. We hypothesized that MART-1 down-modulation in addition to aberrant apoptotic/survival signaling could confer resistance to death signals delivered by transgenic CTLs. To explore this hypothesis, we established an in vitro model of resistant (R) lines from MART-1+/HLA-A*0201+ F5 CTL-sensitive parental (P) lines under serial F5 CTL-selective pressure. We have recently reported that several melanoma R lines, while retaining MART-1 expression, exhibited constitutive NF-κB activation and over-expression of NF-κB-dependent resistance factors. Another established melanoma cell line M244, otherwise sensitive to F5 CTL, yielded R lines after serial F5 CTL selective pressure which had both reduced MART-1 expression levels, thus, could not be recognized, and were resistant to CTL-delivered apoptotic death signals. The proteasome inhibitor bortezomib blocked NF-κB activity, decreased phopspho-ERK1/2, increased phospho-JNK levels, reduced expression of resistance-factors, restored MART-1 expression to sufficient levels, which in combination allowed M244R lines be sensitized to F5 CTL-killing. These findings suggest that proteasome inhibition in immune resistant tumors can restore proapoptotic signaling and improve tumor antigen expression. PMID:22532603

  3. Ophiobolin A Induces Autophagy and Activates the Mitochondrial Pathway of Apoptosis in Human Melanoma Cells

    PubMed Central

    Rodolfo, Carlo; Rocco, Mariapina; Cattaneo, Lucia; Tartaglia, Maria; Sassi, Mauro; Aducci, Patrizia; Scaloni, Andrea; Marra, Mauro

    2016-01-01

    Ophiobolin A, a fungal toxin from Bipolaris species known to affect different cellular processes in plants, has recently been shown to have anti-cancer activity in mammalian cells. In the present study, we investigated the anti-proliferative effect of Ophiobolin A on human melanoma A375 and CHL-1 cell lines. This cellular model was chosen because of the incidence of melanoma malignant tumor on human population and its resistance to chemical treatments. Ophyobolin A strongly reduced cell viability of melanoma cells by affecting mitochondrial functionality. The toxin induced depolarization of mitochondrial membrane potential, reactive oxygen species production and mitochondrial network fragmentation, leading to autophagy induction and ultimately resulting in cell death by activation of the mitochondrial pathway of apoptosis. Finally, a comparative proteomic investigation on A375 cells allowed to identify several Ophiobolin A down-regulated proteins, which are involved in fundamental processes for cell homeostasis and viability. PMID:27936075

  4. Cell surface chondroitin sulfate glycosaminoglycan in melanoma: role in the activation of pro-MMP-2 (pro-gelatinase A).

    PubMed

    Iida, Joji; Wilhelmson, Krista L; Ng, Janet; Lee, Peter; Morrison, Charlotte; Tam, Eric; Overall, Christopher M; McCarthy, James B

    2007-05-01

    We previously reported that CS (chondroitin sulfate) GAG (glycosaminoglycan), expressed on MCSP (melanoma-specific CS proteoglycan), is important for regulating MT3-MMP [membrane-type 3 MMP (matrix metalloproteinase)]-mediated human melanoma invasion and gelatinolytic activity in vitro. In the present study, we sought to determine if CS can directly enhance MT3-MMP-mediated activation of pro-MMP-2. Co-immunoprecipitation studies suggest that MCSP forms a complex with MT3-MMP and MMP-2 on melanoma cell surface. When melanoma cells were treated with betaDX (p-nitro-beta-D-xylopyranoside) to inhibit coupling of CS on the core protein, both active form and proform of MMP-2 were no longer co-immunoprecipitated with either MCSP or MT3-MMP, suggesting a model in which CS directly binds to MMP-2 and presents the gelatinase to MT3-MMP to be activated. By using recombinant proteins, we determined that MT3-MMP directly activates pro-MMP-2 and that this activation requires the interaction of the C-terminal domain of pro-MMP-2 with MT3-MMP. Activation of pro-MMP-2 by suboptimal concentrations of MT3-MMP is also significantly enhanced in the presence of excess C4S (chondroitin 4-sulfate), whereas C6S (chondroitin 6-sulfate) or low-molecular-mass hyaluronan was ineffective. Affinity chromatography studies using CS isolated from aggrecan indicate that the catalytic domain of MT3-MMP and the C-terminal domain of MMP-2 directly bind to the GAG. Thus the direct binding of pro-MMP-2 with CS through the C-domain would present the catalytic domain of pro-MMP-2 to MT3-MMP, which facilitates the generation of the active form of MMP-2. These results suggest that C4S, which is expressed on tumour cell surface, can function to bind to pro-MMP-2 and facilitate its activation by MT3-MMP-expressing tumour cells to enhance invasion and metastasis.

  5. Jolkinolide B induces apoptosis and inhibits tumor growth in mouse melanoma B16F10 cells by altering glycolysis

    PubMed Central

    Gao, Caixia; Yan, Xinyan; Wang, Bo; Yu, Lina; Han, Jichun; Li, Defang; Zheng, Qiusheng

    2016-01-01

    Most cancer cells preferentially rely on glycolysis to produce the energy (adenosine triphosphate, ATP) for growth and proliferation. Emerging evidence demonstrates that the apoptosis in cancer cells could be closely associated with the inhibition of glycolysis. In this study, we have found that jolkinolide B (JB), a bioactive diterpenoid extracted from the root of Euphorbia fischeriana Steud, induced tumor cells apoptosis and decreased the production of ATP and lactic acid in mouse melanoma B16F10 cells. Furthermore, we found that JB downregulated the mRNA expression of glucose transporter genes (Glut1, Glut3 and Glut4) and glycolysis-related kinase genes (Hk2 and Ldha) in B16F10 cells. Moreover, treatment with JB upregulated the mRNA expression of pro-apoptosis genes (Bax), downregulated the mRNA expression of anti-apoptosis genes (Bcl-2, Caspase-3 and Caspase-9), decreased the potential of mitochondrial membrane and increased reactive oxygen species (ROS) levels in B16F10 cells. Finally, intragastric administration of JB suppressed tumor growth and induced tumor apoptosis in mouse xenograft model of murine melanoma B16F10 cells. Taken together, these results suggest that JB could induce apoptosis through the mitochondrial pathway and inhibit tumor growth. The inhibition of glycolysis could play a crucial role in the induction of apoptosis in JB-treated B16F10 cells. PMID:27796318

  6. Jolkinolide B induces apoptosis and inhibits tumor growth in mouse melanoma B16F10 cells by altering glycolysis.

    PubMed

    Gao, Caixia; Yan, Xinyan; Wang, Bo; Yu, Lina; Han, Jichun; Li, Defang; Zheng, Qiusheng

    2016-10-31

    Most cancer cells preferentially rely on glycolysis to produce the energy (adenosine triphosphate, ATP) for growth and proliferation. Emerging evidence demonstrates that the apoptosis in cancer cells could be closely associated with the inhibition of glycolysis. In this study, we have found that jolkinolide B (JB), a bioactive diterpenoid extracted from the root of Euphorbia fischeriana Steud, induced tumor cells apoptosis and decreased the production of ATP and lactic acid in mouse melanoma B16F10 cells. Furthermore, we found that JB downregulated the mRNA expression of glucose transporter genes (Glut1, Glut3 and Glut4) and glycolysis-related kinase genes (Hk2 and Ldha) in B16F10 cells. Moreover, treatment with JB upregulated the mRNA expression of pro-apoptosis genes (Bax), downregulated the mRNA expression of anti-apoptosis genes (Bcl-2, Caspase-3 and Caspase-9), decreased the potential of mitochondrial membrane and increased reactive oxygen species (ROS) levels in B16F10 cells. Finally, intragastric administration of JB suppressed tumor growth and induced tumor apoptosis in mouse xenograft model of murine melanoma B16F10 cells. Taken together, these results suggest that JB could induce apoptosis through the mitochondrial pathway and inhibit tumor growth. The inhibition of glycolysis could play a crucial role in the induction of apoptosis in JB-treated B16F10 cells.

  7. Retinal vasculitis and ocular vitreous metastasis following complete response to PD-1 inhibition in a patient with metastatic cutaneous melanoma.

    PubMed

    Manusow, Joshua S; Khoja, Leila; Pesin, Nataly; Joshua, Anthony M; Mandelcorn, Efrem D

    2014-01-01

    We report on a 36-year-old woman treated with the anti PD-1 antibody Pembrolizumab for metastatic cutaneous melanoma in the first line setting. She achieved a complete response and then relapsed with metastases to the vitreous cavity with an associated angiographically determined retinal vasculitis. Vitreous metastasis without choroidal involvement is unusual and may be due to individual cell extravasation, vitreous hemorrhage containing malignant cells, or direct spread through the optic nerve. This finding highlights the need for immune sanctuary sites to be monitored in the presence of PD-1 inhibition and we hypothesize that the use of PD-1 inhibitor potentiated the patient's angiographically determined retinal vasculitis.

  8. PAK4 suppresses PDZ-RhoGEF activity to drive invadopodia maturation in melanoma cells

    PubMed Central

    Nicholas, Nicole S.; Pipili, Aikaterini; Lesjak, Michaela S.; Ameer, Simon M.; Geh, Jenny L. C.; Healy, Ciaran; Ross, Alistair D. MacKenzie; Parsons, Maddy; Nestle, Frank O.; Lacy, Katie E.; Wells, Claire M.

    2016-01-01

    Cancer cells are thought to use actin rich invadopodia to facilitate matrix degradation. Formation and maturation of invadopodia requires the co-ordained activity of Rho-GTPases, however the molecular mechanisms that underlie the invadopodia lifecycle are not fully elucidated. Previous work has suggested a formation and disassembly role for Rho family effector p-21 activated kinase 1 (PAK1) however, related family member PAK4 has not been explored. Systematic analysis of isoform specific depletion using in vitro and in vivo invasion assays revealed there are differential invadopodia-associated functions. We consolidated a role for PAK1 in the invadopodia formation phase and identified PAK4 as a novel invadopodia protein that is required for successful maturation. Furthermore, we find that PAK4 (but not PAK1) mediates invadopodia maturation likely via inhibition of PDZ-RhoGEF. Our work points to an essential role for both PAKs during melanoma invasion but provides a significant advance in our understanding of differential PAK function. PMID:27765920

  9. Constitutive activation of the ETS-1-miR-222 circuitry in metastatic melanoma

    PubMed Central

    Mattia, Gianfranco; Errico, M Cristina; Felicetti, Federica; Petrini, Marina; Bottero, Lisabianca; Tomasello, Luisa; Romania, Paolo; Boe, Alessandra; Segnalini, Patrizia; Di Virgilio, Antonio; Colombo, Mario P; Carè, Alessandra

    2011-01-01

    MicroRNAs-221 and -222 are highly upregulated in several solid tumors, including melanomas. We demonstrate that the proto-oncogene ETS-1, involved in the pathogenesis of cancers of different origin, is a transcriptional regulator of miR-222 by direct binding to its promoter region. Differently from 293FT cells or early stage melanomas, where unphosphorylated ETS-1 represses miR-222 transcription, in metastatic melanoma the constitutively Thr-38 phosphorylated fraction of ETS-1 induces miR-222. Despite its stepwise decreased expression along with melanoma progression, the oncogenic activity of ETS-1 relies on its RAS/RAF/ERK-dependent phosphorylation status more than on its total amount. To close the loop, we demonstrate ETS-1 as a direct target of miR-222, but not miR-221, showing the novel option of their uncoupled functions. In addition, a spatial redistribution of ETS-1 protein from the nucleus to the cytoplasm is also evidenced in advanced melanoma cells. Finally, in vivo studies confirmed the contribution of miR-222 to the increased invasive potential obtained by ETS- silencing. PMID:21711453

  10. Fluorescent peptide biosensor for monitoring CDK4/cyclin D kinase activity in melanoma cell extracts, mouse xenografts and skin biopsies.

    PubMed

    Prével, Camille; Pellerano, Morgan; González-Vera, Juan A; Henri, Pauline; Meunier, Laurent; Vollaire, Julien; Josserand, Véronique; Morris, May C

    2016-11-15

    Melanoma constitutes the most aggressive form of skin cancer, which further metastasizes into a deadly form of cancer. The p16(INK4a)-Cyclin D-CDK4/6-pRb pathway is dysregulated in 90% of melanomas. CDK4/Cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of Cyclin D or loss of p16(INK4a) leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker in melanoma and an emerging pharmacological target, however there are no tools enabling direct detection or quantification of its activity. Here we report on the design and application of a fluorescent peptide biosensor to quantify CDK4 activity in melanoma cell extracts, skin biopsies and melanoma xenografts. This biosensor provides sensitive means of comparing CDK4 activity between different melanoma cell lines and further responds to CDK4 downregulation by siRNA or small-molecule inhibitors. By affording means of monitoring CDK4 hyperactivity consequent to cancer-associated molecular alterations in upstream signaling pathways that converge upon this kinase, this biosensor offers an alternative to immunological identification of melanoma-specific biomarkers, thereby constituting an attractive tool for diagnostic purposes, providing complementary functional information to histological analysis, of particular utility for detection of melanoma onset in precancerous lesions. This is indeed the first fluorescent peptide biosensor which has been successfully implemented to monitor kinase activity in skin samples and melanoma tumour xenografts. Moreover by enabling to monitor response to CDK4 inhibitors, this biosensor constitutes an attractive companion assay to identify compounds of therapeutic relevance for melanoma.

  11. Chronic Resveratrol Treatment Inhibits MRC5 Fibroblast SASP-Related Protumoral Effects on Melanoma Cells.

    PubMed

    Menicacci, Beatrice; Laurenzana, Anna; Chillà, Anastasia; Margheri, Francesca; Peppicelli, Silvia; Tanganelli, Elisabetta; Fibbi, Gabriella; Giovannelli, Lisa; Del Rosso, Mario; Mocali, Alessandra

    2017-09-01

    Cellular senescence is related to organismal aging and is observed after DNA damaging cancer therapies, that induce tumor-suppressive modifications, but it is characterized by a strong increase in secreted factors, termed the "senescence-associated secretory phenotype" (SASP). Particularly, SASP from stroma senescent fibroblasts creates a cancer-favoring microenvironment, providing targets for anti-cancer interventions. In the present article, chronic treatment (5 weeks) with 5 µM resveratrol has been used to modulate senescence-related protumoral features of MRC5 fibroblasts, reducing SASP-related interleukins IL1α, IL1β, IL6, and IL8; transforming-growth-factor-β (TGFβ); matrix metallo-proteinases MMP3 and MMP2; urokinase plasminogen activator (uPA); receptor proteins uPAR, IL6R, insulin growth factor receptor-1 (IGF-1R), TGFβ-R2, and CXCR4. The cellular nuclear-factor-kB (NF-kB) protein level was also reduced, confirming its role in the induction of SASP. Resveratrol pretreated MRC5 fibroblasts were resistant to activation by TGFβ. Resveratrol treatment of senescent MRC5 induced the production of conditioned media (CM) which counteracted the protumoral effect of senescent CM on A375 and A375-M6 melanoma cell proliferation and invasiveness, and reduced the expression of epithelial-to-mesenchymal transition markers related to malignant features. This experimental approach proposes a treatment that targets the senescent stromal cell phenotype to induce an anti-tumor hosting microenvironment, which is suitable for both preventive and therapeutic purposes. © The Author 2017. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. Casticin Inhibits A375.S2 Human Melanoma Cell Migration/Invasion through Downregulating NF-κB and Matrix Metalloproteinase-2 and -1.

    PubMed

    Wu, Zih-Yun; Lien, Jin-Cherng; Huang, Yi-Ping; Liao, Ching-Lung; Lin, Jen-Jyh; Fan, Ming-Jen; Ko, Yang-Ching; Hsiao, Yu-Ping; Lu, Hsu-Feng; Chung, Jing-Gung

    2016-03-19

    Casticin is one of the main components from Fructus Viticis, which is widely used as an anti-inflammatory agent. The mechanism of how casticin affects melanoma cell migration and invasion is still not well known. Here we studied the anti-metastasis effects of casticin on A375.S2 melanoma cells by using a non-lethal concentration. First; we used an adhesion assay to test the A375.S2 cells' adhesion ability after treatment with casticin. We next investigated the cell migration ability after casticin treatment by using a wound healing assay to prove that the migration of A375.S2 cells can be inhibited by casticin and double checked the results using the transwell-migration assay. The suppressive effects on matrix metalloproteinase-2; and -9 (MMP-2; and -9) activities were examined by gelatin zymography. Furthermore, western blotting was used to investigate the protein level changes in A375.S2 cells. We found that p-EGFR; Ras and p-ERK1/2 are decreased by casticin, indicating that casticin can down-regulate the migration and invasion ability of A375.S2 cells via the p-EGFR/Ras/p-ERK pathway. The NF-κB p65 and p-ERK levels in nuclear proteins are also decreased by treatment with casticin. An EMSA assay also discovered that the NF-κB p65 and DNA interaction is decreased. NF-κB p65 protein level was examined by immunofluorescence staining and also decreased. Our findings suggest that casticin has anti-metastatic potential by decreasing the invasiveness of A375.S2 cells. We also found that casticin suppressed A375.S2 cell proliferation and cell adhesion ability, but did not affect cell death, as examined using cytometry and a collagen adhesion assay. Based on these observations, casticin could be used as an inhibitor of migration and invasion of human melanoma cells in the future.

  13. Targeted activation of innate immunity for therapeutic induction of autophagy and apoptosis in melanoma cells

    PubMed Central

    Tormo, Damià; Chęcińska, Agnieszka; Alonso-Curbelo, Direna; Pérez-Guijarro, Eva; Cañón, Estela; Riveiro-Falkenbach, Erica; Calvo, Tonantzin G.; Larribere, Lionel; Megías, Diego; Mulero, Francisca; Piris, Miguel A.; Dash, Rupesh; Barral, Paola M.; Rodríguez-Peralto, José L; Ortiz-Romero, Pablo; Tüting, Thomas; Fisher, Paul B.; Soengas, María S.

    2009-01-01

    Summary Inappropriate drug delivery, secondary toxicities and persistent chemo- and immuno-resistance have traditionally compromised treatment response in melanoma. Using cellular systems and genetically engineered mouse models, we show that melanoma cells retain an innate ability to recognize cytosolic dsRNA and mount persistent stress response programs able to block tumor growth, even in highly immunosuppressed backgrounds. The dsRNA mimic polyinosine-polycytidylic acid (pIC), coadministered with polyethyleneimine (PEI) as a carrier, was identified as an unanticipated inducer of autophagy downstream of an exacerbated endosomal maturation program. A concurrent activity of the dsRNA helicase MDA-5 driving the proapoptotic protein NOXA resulted in an efficient autodigestion of melanoma cells. These results reveal tractable links for therapeutic intervention among dsRNA helicases, endo/lysosomes and apoptotic factors. PMID:19647221

  14. Targeted activation of innate immunity for therapeutic induction of autophagy and apoptosis in melanoma cells.

    PubMed

    Tormo, Damià; Checińska, Agnieszka; Alonso-Curbelo, Direna; Pérez-Guijarro, Eva; Cañón, Estela; Riveiro-Falkenbach, Erica; Calvo, Tonantzin G; Larribere, Lionel; Megías, Diego; Mulero, Francisca; Piris, Miguel A; Dash, Rupesh; Barral, Paola M; Rodríguez-Peralto, José L; Ortiz-Romero, Pablo; Tüting, Thomas; Fisher, Paul B; Soengas, María S

    2009-08-04

    Inappropriate drug delivery, secondary toxicities, and persistent chemo- and immunoresistance have traditionally compromised treatment response in melanoma. Using cellular systems and genetically engineered mouse models, we show that melanoma cells retain an innate ability to recognize cytosolic double-stranded RNA (dsRNA) and mount persistent stress response programs able to block tumor growth, even in highly immunosuppressed backgrounds. The dsRNA mimic polyinosine-polycytidylic acid, coadministered with polyethyleneimine as carrier, was identified as an unanticipated inducer of autophagy downstream of an exacerbated endosomal maturation program. A concurrent activity of the dsRNA helicase MDA-5 driving the proapoptotic protein NOXA resulted in an efficient autodigestion of melanoma cells. These results reveal tractable links for therapeutic intervention among dsRNA helicases, endo/lysosomes, and apoptotic factors.

  15. Feruloylserotonin inhibits hydrogen peroxide-induced melanogenesis and apoptosis in B16F10 and SK-Mel-2 melanoma cells.

    PubMed

    Cho, Hyejoung; Kim, Okjoon; Lee, Younghee; Kang, Li-Jung; Nguyen, Cam Ngoc; Ishihara, Atsushi; Kim, Hye-Eun

    2017-09-30

    Feruloylserotonin (FS) is a major bioactive component of safflower seeds, with documented strong antibacterial, anti-inflammatory, and free radical scavenging activities. Reactive oxygen species (ROS) can strongly induce melanogenesis and cell apoptosis. The present study aimed to investigate the ability of FS in preventing hydrogen peroxide (H2O2)-induced melanogenesis and cell apoptosis. Melanogenesis and apoptotic cell death were induced by transient exposure to H2O2 in B16F10 and SK-Mel-2 melanoma cells. FS significantly inhibited melanogenesis and cell death in both cell lines. FS inhibited H2O2-induced melanin production by down-regulating CREB/MITF/TYR signaling via inhibited intracellular cAMP accumulation. Additionally, FS induced extracellular regulated kinase activation, which led to the degradation of MITF and consequently decreased TYR expression and melanin production in H2O2-stimulated cells. Furthermore, FS inhibited H2O2-induced apoptotic cell death by maintaining mitochondrial membrane potential. Therefore, FS might have potential use for cosmetic whitening and as a therapeutic agent for hyperpigmentation disorder. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Dual inhibition of (V600E)BRAF and the PI3K/AKT/mTOR pathway cooperates to induce apoptosis in melanoma cells through a MEK-independent mechanism.

    PubMed

    Sánchez-Hernández, Irene; Baquero, Pablo; Calleros, Laura; Chiloeches, Antonio

    2012-01-28

    BRAF is a main oncogene in human melanomas. Here, we show that BRAF depletion by siRNA or inhibition of its activity by treatment with RAF inhibitor Sorafenib induces apoptosis in NPA melanoma cells expressing oncogenic (V600E)BRAF. This effect is mediated through a MEK/ERK-independent mechanism, since treatment with the MEK inhibitor U0126 does not exert any effect. Moreover, we demonstrate that inhibition of the PI3K/AKT/mTOR cascade alone does not increase apoptosis in these cells. However, the blockage of this pathway in cells lacking either BRAF expression or activity cooperates to induce higher levels of apoptosis than those achieved by inhibition of BRAF alone. Consistently, we demonstrate that abrogation of BRAF expression increases AKT and mTOR phosphorylation, suggesting the existence of a compensatory pro-survival mechanism after BRAF depletion. Together, our data provide a rationale for dual targeting of BRAF and PI3K/AKT/mTOR signalling to effectively control melanoma disease.

  17. Beyond BRAF in melanoma.

    PubMed

    Daud, Adil; Bastian, Boris C

    2012-01-01

    Recent progress in the analysis of genetic alterations in melanoma has identified recurrent mutations that result in the activation of critical signaling pathways promoting growth and survival of tumors cells. Alterations in the RAS-RAF-MAP kinase and PI3-kinase signaling pathways are commonly altered in melanoma. Mutations in BRAF, NRAS, KIT, and GNAQ occur in a mutually exclusive pattern and lead to MAP-kinase activation. Loss of PTEN function, primarily by deletion, is the most common known genetic alteration in the PI3-kinase cascade, and is commonly associated with BRAF mutations (Curtin et al., N Engl J Med 353:2135-2147, 2005; Tsao et al., Cancer Res 60:1800-1804, 2000, J Investig Dermatol 122:337-341, 2004). The growth advantage conveyed by the constitutive activation of these pathways leads to positive selection of cells that have acquired the mutations and in many instances leads to critical dependency of the cancer cells on their activation. This creates opportunities for therapeutic interventions targeted at signaling components within these pathways that are amenable for pharmacological inhibition. This concept follows the paradigm established by the landmark discovery that inhibition of the fusion kinase BCR-ABL can be used to treat chronic myelogenous leukemia (Druker et al., N Engl J Med 344:1031-037, 2001). The review will focus primarily on kinases involved in signaling that are currently being evaluated for therapeutic intervention in melanoma.

  18. Simvastatin rises reactive oxygen species levels and induces senescence in human melanoma cells by activation of p53/p21 pathway

    SciTech Connect

    Guterres, Fernanda Augusta de Lima Barbosa; Martinez, Glaucia Regina; Rocha, Maria Eliane Merlin; Winnischofer, Sheila Maria Brochado

    2013-11-15

    Recent studies demonstrated that simvastatin has antitumor properties in several types of cancer cells, mainly by inducing apoptosis and inhibiting growth. The arrest of proliferation is a feature of cellular senescence; however, the occurrence of senescence in melanoma cells upon simvastatin treatment has not been investigated until now. Our results demonstrated that exposure of human metastatic melanoma cells (WM9) to simvastatin induces a senescent phenotype, characterized by G1 arrest, positive staining for senescence-associated β-galactosidase assay, and morphological changes. Also, the main pathways leading to cell senescence were examined in simvastatin-treated human melanoma cells, and the expression levels of phospho-p53 and p21 were upregulated by simvastatin, suggesting that cell cycle regulators and DNA damage pathways are involved in the onset of senescence. Since simvastatin can act as a pro-oxidant agent, and oxidative stress may be related to senescence, we measured the intracellular ROS levels in WM9 cells upon simvastatin treatment. Interestingly, we found an increased amount of intracellular ROS in these cells, which was accompanied by elevated expression of catalase and peroxiredoxin-1. Collectively, our results demonstrated that simvastatin can induce senescence in human melanoma cells by activation of p53/p21 pathway, and that oxidative stress may be related to this process. - Highlights: • Lower concentrations of simvastatin can induce senescent phenotype in melanoma cells. • Simvastatin induces senescence in human melanoma cells via p53/p21 pathway. • Senescent phenotype is related with increased intracellular ROS. • Partial detoxification of ROS by catalase/peroxiredoxin-1 could lead cells to senescence rather than apoptosis.

  19. Isoliquiritigenin Inhibits Proliferation and Induces Apoptosis via Alleviating Hypoxia and Reducing Glycolysis in Mouse Melanoma B16F10 Cells.

    PubMed

    Wang, Yanming; Ma, Jun; Yan, Xinyan; Chen, Xiaoyu; Si, Lingling; Liu, Ying; Han, Jichun; Hao, Wenjin; Zheng, Qiusheng

    2016-01-01

    Isoliquiritigenin (ISL) is a licorice chalcone. According to CN104758274, CN101658513 and US009089546, it is claimed that ISL has anti-inflammatory, anti-oxidative, and anti-tumoral effects. This study aimed to investigate the potential therapeutic effect of ISL in mouse melanoma B16F10 cells. Sulforhodamine B (SRB) colorimetric assay was used to test the effects of ISL on proliferation. Commercial assay kits were applied to assess glucose uptake, lactate production and ATP levels. Measurement of apoptosis was involved with Hoechst 33258, JC-1 and annexin V-FITC/PI staining. H2DCFDA probe was employed to detect ROS generation. Quantitative RT-PCR and western blot were utilized to measure the mRNA and protein levels. ISL abated hypoxia-inducible factor 1α (HIF-1α) stability and reduced a series of glycolysis-relevant enzymes expression, including glucose transporters 1/4 (GLUT 1/4), hexokinase 2 (HK2), pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA). Exposure to ISL induced the mitochondrial membrane potential depolarization and increased intracellular reactive oxygen species (ROS) level. ISL could effectively inhibit proliferation and alleviate hypoxia in mouse melanoma B16F10 cells via inducing apoptosis and reducing the expression of significant enzymes in the glycolysis. ISL significantly inhibited B16F10 cell proliferation via inducing apoptosis, and alleviated hypoxia by recovering mitochondrial function and reversing high glycolysis. Our findings propose that ISL can be a promising therapeutic agent for the melanoma via reliving hypoxia of microenvironment and targeting energy metabolism system of cancer cells. Consistent with WO2015079213 and WO2014084494, targeting glycolysis can be an effective means to anti-cancer.

  20. Selumetinib for the treatment of metastatic uveal melanoma: past and future perspectives.

    PubMed

    Komatsubara, Kimberly M; Manson, Daniel K; Carvajal, Richard D

    2016-06-01

    Uveal melanoma is a rare but aggressive subtype of melanoma. Nearly 50% of patients will develop metastatic disease despite primary enucleation or radiation therapy. There is currently no standard of care therapy for metastatic uveal melanoma, and no therapy that has been shown to prolong overall survival. Uveal melanoma is characterized by activation of signaling pathways including the MAPK pathway and the PI3K/AKT pathway, among others, via mutations in the G-α-proteins GNAQ and GNA11. MEK inhibition with selumetinib has been evaluated as a therapeutic strategy in metastatic uveal melanoma. This review will discuss preclinical and clinical studies evaluating selumetinib in metastatic uveal melanoma, as well as potential future perspectives on MEK inhibition in the management of metastatic uveal melanoma.

  1. Expression and activity of alcohol and aldehyde dehydrogenases in melanoma cells and in melanocytes.

    PubMed

    Amann, Philipp M; Hofmann, Claudia; Freudenberger, Muriel; Holland-Cunz, Stefan; Eichmüller, Stefan B; Bazhin, Alexandr V

    2012-03-01

    Disturbances in vitamin A metabolism are an important attribute of some cancer cells. Most evidence point that these disturbances lead to decreasing of the retinoic acid concentration in tumor cells. Up to now, in benign and malignant skin cells the features of vitamin A metabolism with its participating enzymes are not entirely understood. Alcohol and aldehyde dehydrogenases (ALDH) are involved in the retinol metabolism, oxidizing retinol, and retinal in retinoic acid or reducing retinal in retinol. In this work we investigated the expression and enzymatic activity of alcohol and ALDH in melanoma cells compared to their benign counterparts. We demonstrated that melanoma cell lines and melanocytes despite similar pattern of the enzyme expression, show different general ALDH activity. Retinal, the substrate of ALDH, could stimulate the ALDH activity through up-regulation of retinaldehyde dehydrogenase 1 and aldehyde dehydrogenase 6. Furthermore, we found that retinoids regulate alcohol dehydrogenase activity, probably via effects on alcohol dehydrogenase expression at the post-transcriptional level. We suggest that melanoma cells in contrast to melanocytes should favor the retinal reduction over its oxidation. The decreasing cellular amount of the precursor molecules of retinoic acid could result in a changed gene regulation in melanoma cells. Copyright © 2011 Wiley Periodicals, Inc.

  2. Intravenous Administration of Manuka Honey Inhibits Tumor Growth and Improves Host Survival When Used in Combination with Chemotherapy in a Melanoma Mouse Model

    PubMed Central

    Fernandez-Cabezudo, Maria J.; El-Kharrag, Rkia; Torab, Fawaz; Bashir, Ghada; George, Junu A.; El-Taji, Hakam; al-Ramadi, Basel K.

    2013-01-01

    Manuka honey has been recognized for its anti-bacterial and wound-healing activity but its potential antitumor effect is poorly studied despite the fact that it contains many antioxidant compounds. In this study, we investigated the antiproliferative activity of manuka honey on three different cancer cell lines, murine melanoma (B16.F1) and colorectal carcinoma (CT26) as well as human breast cancer (MCF-7) cells in vitro. The data demonstrate that manuka honey has potent anti-proliferative effect on all three cancer cell lines in a time- and dose-dependent manner, being effective at concentrations as low as 0.6% (w/v). This effect is mediated via the activation of a caspase 9-dependent apoptotic pathway, leading to the induction of caspase 3, reduced Bcl-2 expression, DNA fragmentation and cell death. Combination treatment of cancer cells with manuka and paclitaxel in vitro, however, revealed no evidence of a synergistic action on cancer cell proliferation. Furthermore, we utilized an in vivo syngeneic mouse melanoma model to assess the potential effect of intravenously-administered manuka honey, alone or in combination with paclitaxel, on the growth of established tumors. Our findings indicate that systemic administration of manuka honey was not associated with any alterations in haematological or clinical chemistry values in se