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Sample records for a375 human melanoma

  1. Synergistic Apoptosis-Inducing Effects on A375 Human Melanoma Cells of Natural Borneol and Curcumin

    PubMed Central

    Chen, Jianping; Li, Lin; Su, Jianyu; Li, Bing; Chen, Tianfeng; Wong, Yum-Shing

    2014-01-01

    This study was to investigate the synergistic effect of NB/Cur on growth and apoptosis in A375 human melanoma cell line by MTT assay, flow cytometry and Western blotting. Our results demonstrated that NB effectively synergized with Cur to enhance its antiproliferative activity on A375 human melanoma cells by induction of apoptosis, as evidenced by an increase in sub-G1 cell population, DNA fragmentation, PARP cleavage and caspase activation. Further mechanistic studies by Western blotting showed that after treatment of the cells with NB/Cur, up-regulation of the expression level of phosphorylated JNK and down-regulation of the expression level of phosphorylated ERK and Akt contributed to A375 cells apoptosis. Moreover, NB also potentiated Cur to trigger intracellular ROS overproduction and the DNA damage with up-regulation of the expression level of phosphorylated ATM, phosphorylated Brca1 and phosphorylated p53. The results indicate the combinational application potential of NB and Cur in treatments of cancers. PMID:24971451

  2. Anti-Proliferative Effect of Rosmarinus officinalis L. Extract on Human Melanoma A375 Cells

    PubMed Central

    Cattaneo, Lucia; Cicconi, Rosella; Mignogna, Giuseppina; Giorgi, Alessandra; Mattei, Maurizio; Graziani, Giulia; Ferracane, Rosalia; Grosso, Alessandro; Aducci, Patrizia; Schininà, M. Eugenia; Marra, Mauro

    2015-01-01

    Rosemary (Rosmarinus officinalis L.) has been used since ancient times in traditional medicine, while nowadays various rosemary formulations are increasingly exploited by alternative medicine to cure or prevent a wide range of health disorders. Rosemary’s bioproperties have prompted scientific investigation, which allowed us to ascertain antioxidant, anti-inflammatory, cytostatic, and cytotoxic activities of crude extracts or of pure components. Although there is a growing body of experimental work, information about rosemary’s anticancer properties, such as chemoprotective or anti-proliferative effects on cancer cells, is very poor, especially concerning the mechanism of action. Melanoma is a skin tumor whose diffusion is rapidly increasing in the world and whose malignancy is reinforced by its high resistance to cytotoxic agents; hence the availability of new cytotoxic drugs would be very helpful to improve melanoma prognosis. Here we report on the effect of a rosemary hydroalcoholic extract on the viability of the human melanoma A375 cell line. Main components of rosemary extract were identified by liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) and the effect of the crude extract or of pure components on the proliferation of cancer cells was tested by MTT and Trypan blue assays. The effect on cell cycle was investigated by using flow cytometry, and the alteration of the cellular redox state was evaluated by intracellular ROS levels and protein carbonylation analysis. Furthermore, in order to get information about the molecular mechanisms of cytotoxicity, a comparative proteomic investigation was performed. PMID:26176704

  3. Anti-Proliferative Effect of Rosmarinus officinalis L. Extract on Human Melanoma A375 Cells.

    PubMed

    Cattaneo, Lucia; Cicconi, Rosella; Mignogna, Giuseppina; Giorgi, Alessandra; Mattei, Maurizio; Graziani, Giulia; Ferracane, Rosalia; Grosso, Alessandro; Aducci, Patrizia; Schininà, M Eugenia; Marra, Mauro

    2015-01-01

    Rosemary (Rosmarinus officinalis L.) has been used since ancient times in traditional medicine, while nowadays various rosemary formulations are increasingly exploited by alternative medicine to cure or prevent a wide range of health disorders. Rosemary's bioproperties have prompted scientific investigation, which allowed us to ascertain antioxidant, anti-inflammatory, cytostatic, and cytotoxic activities of crude extracts or of pure components. Although there is a growing body of experimental work, information about rosemary's anticancer properties, such as chemoprotective or anti-proliferative effects on cancer cells, is very poor, especially concerning the mechanism of action. Melanoma is a skin tumor whose diffusion is rapidly increasing in the world and whose malignancy is reinforced by its high resistance to cytotoxic agents; hence the availability of new cytotoxic drugs would be very helpful to improve melanoma prognosis. Here we report on the effect of a rosemary hydroalcoholic extract on the viability of the human melanoma A375 cell line. Main components of rosemary extract were identified by liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) and the effect of the crude extract or of pure components on the proliferation of cancer cells was tested by MTT and Trypan blue assays. The effect on cell cycle was investigated by using flow cytometry, and the alteration of the cellular redox state was evaluated by intracellular ROS levels and protein carbonylation analysis. Furthermore, in order to get information about the molecular mechanisms of cytotoxicity, a comparative proteomic investigation was performed. PMID:26176704

  4. AM251 induces apoptosis and G2/M cell cycle arrest in A375 human melanoma cells.

    PubMed

    Carpi, Sara; Fogli, Stefano; Romanini, Antonella; Pellegrino, Mario; Adinolfi, Barbara; Podestà, Adriano; Costa, Barbara; Da Pozzo, Eleonora; Martini, Claudia; Breschi, Maria Cristina; Nieri, Paola

    2015-08-01

    Human cutaneous melanoma is an aggressive and chemotherapy-resistant type of cancer. AM251 is a cannabinoid type 1 (CB1) receptor antagonist/inverse agonist with off-target antitumor activity against pancreatic and colon cancer cells. The current study aimed to characterize the in-vitro antimelanoma activity of AM251. The BRAF V600E mutant melanoma cell line, A375, was used as an in-vitro model system. Characterization tools included a cell viability assay, nuclear morphology assessment, gene expression, western blot, flow cytometry with Annexin V-FITC/7-AAD double staining, cell cycle analyses, and measurements of changes in intracellular cAMP and calcium concentrations. AM251 exerted a marked cytotoxic effect against A375 human melanoma cells with potency comparable with that observed for cisplatin without significant changes in the human dermal fibroblasts viability. AM251, at a concentration that approximates the IC50, downregulated genes encoding antiapoptotic proteins (BCL2 and survivin) and increased transcription levels of proapoptotic BAX, induced alteration of Annexin V reactivity, DNA fragmentation, chromatin condensation in the cell nuclei, and G2/M phase arrest.AM251 also induced a 40% increase in the basal cAMP levels, but it did not affect intracellular calcium concentrations. The involvement of GPR55, TRPA1, and COX-2 in the AM251 mechanism of action was excluded. The combination of AM251 with celecoxib produced a synergistic antitumor activity, although the mechanism underlying this effect remains to be elucidated. This study provides the first evidence of a proapoptotic effect and G2/M cell cycle arrest of AM251 on A375 cells. This compound may be a potential prototype for the development of promising diarylpyrazole derivatives to be evaluated in human cutaneous melanoma. PMID:25974027

  5. Extracts of centipede Scolopendra subspinipes mutilans induce cell cycle arrest and apoptosis in A375 human melanoma cells.

    PubMed

    Ma, Weina; Liu, Rui; Qi, Junpeng; Zhang, Yanmin

    2014-07-01

    Extracts from the centipede Scolopendra genus, have been used in traditional medicine for the treatment of various diseases and have been found to exhibit anticancer activity in tumor cells. To investigate the potential and associated antitumor mechanism of alcohol extracts of the centipede Scolopendra subspinipes mutilans (AECS), cell viability, cell cycle and cell apoptosis were studied and the results revealed that AECS inhibits A375 cell proliferation in a dose- and time-dependent manner. In addition, AECS was found to arrest the cell cycle of A375 cells at the S phase, which was accompanied by a marked increase in the protein levels of cyclin E and a decrease in the protein levels of cyclin D1. In a cell culture system, AECS markedly induced the apoptosis of A375 cells, which was closely associated with the effects on the Bcl-2 family, whereby decreased Bcl-2 and increased Bak, Bax and Bad expression levels were observed. The underlying mechanism of AECS inhibiting A375 cell proliferation was associated with the induction of cell cycle arrest and apoptosis, indicating that AECS may present as a potential therapeutic agent for administration in human melanoma cancer intervention. PMID:24959287

  6. Antioxidant enzymes and the mechanism of the bystander effect induced by ultraviolet C irradiation of A375 human melanoma cells.

    PubMed

    Ghosh, Rita; Guha, Dipanjan; Bhowmik, Sudipta; Karmakar, Sayantani

    2013-09-18

    Irradiated cells generate dynamic responses in non-irradiated cells; this signaling phenomenon is known as the bystander effect (BE). Factors secreted by the irradiated cells communicate some of these signals. Conditioned medium from UVC-irradiated A375 human melanoma cells was used to study the BE. Exposure of cells to conditioned medium induce cell-cycle arrest at the G2/M transition. Although conditioned medium treatment, by itself, did not alter cell viability, treated cells were more resistant to the lethal action of UVC or H2O2. This protective effect of conditioned medium was lost within 8h. Apoptotic or autophagic cell death was not involved in this resistance. Exposure to conditioned medium did not influence the rate of DNA repair, as measured by NAD(+) depletion. The activities of catalase and superoxide dismutase were elevated in cells exposed to conditioned medium, but returned to normal levels by 8h post-treatment. These results indicate a close correlation between BE-stimulated antioxidant activity and cellular sensitivity. Cell-cycle arrest and stimulation of antioxidant activity may account for the resistance to killing that was observed in bystander cells exposed to UVC or H2O2 treatment and are consistent with the role of the BE as a natural defense function triggered by UVC irradiation. PMID:23845763

  7. Casticin Inhibits A375.S2 Human Melanoma Cell Migration/Invasion through Downregulating NF-κB and Matrix Metalloproteinase-2 and -1.

    PubMed

    Wu, Zih-Yun; Lien, Jin-Cherng; Huang, Yi-Ping; Liao, Ching-Lung; Lin, Jen-Jyh; Fan, Ming-Jen; Ko, Yang-Ching; Hsiao, Yu-Ping; Lu, Hsu-Feng; Chung, Jing-Gung

    2016-01-01

    Casticin is one of the main components from Fructus Viticis, which is widely used as an anti-inflammatory agent. The mechanism of how casticin affects melanoma cell migration and invasion is still not well known. Here we studied the anti-metastasis effects of casticin on A375.S2 melanoma cells by using a non-lethal concentration. First; we used an adhesion assay to test the A375.S2 cells' adhesion ability after treatment with casticin. We next investigated the cell migration ability after casticin treatment by using a wound healing assay to prove that the migration of A375.S2 cells can be inhibited by casticin and double checked the results using the transwell-migration assay. The suppressive effects on matrix metalloproteinase-2; and -9 (MMP-2; and -9) activities were examined by gelatin zymography. Furthermore, western blotting was used to investigate the protein level changes in A375.S2 cells. We found that p-EGFR; Ras and p-ERK1/2 are decreased by casticin, indicating that casticin can down-regulate the migration and invasion ability of A375.S2 cells via the p-EGFR/Ras/p-ERK pathway. The NF-κB p65 and p-ERK levels in nuclear proteins are also decreased by treatment with casticin. An EMSA assay also discovered that the NF-κB p65 and DNA interaction is decreased. NF-κB p65 protein level was examined by immunofluorescence staining and also decreased. Our findings suggest that casticin has anti-metastatic potential by decreasing the invasiveness of A375.S2 cells. We also found that casticin suppressed A375.S2 cell proliferation and cell adhesion ability, but did not affect cell death, as examined using cytometry and a collagen adhesion assay. Based on these observations, casticin could be used as an inhibitor of migration and invasion of human melanoma cells in the future. PMID:27007357

  8. Nitric oxide induces apoptosis and autophagy; autophagy down-regulates NO synthesis in physalin A-treated A375-S2 human melanoma cells.

    PubMed

    He, Hao; Feng, Yong-Sheng; Zang, Ling-He; Liu, Wei-Wei; Ding, Li-Qin; Chen, Li-Xia; Kang, Ning; Hayashi, Toshihiko; Tashiro, Shin-ichi; Onodera, Satoshi; Qiu, Feng; Ikejima, Takashi

    2014-09-01

    Physalin A is an active withanolide isolated from Physalis alkekengi var. franchetii, a traditional Chinese herbal medicine named Jindenglong, which has been used for the treatment of sore throat, hepatitis, eczema and tumors in China. Our previous study demonstrated that physalin A induced apoptosis and cyto-protective autophagy in A375-S2 human melanoma cells. Induction of reactive oxygen species (ROS) with physalin A triggered apoptosis. In this study, NO generated by physalin A induced apoptosis and autophagy in A375-S2 cells, since physalin A induced the expression of inducible nitric oxide synthase (iNOS) in the cells. Generation of NO partially promoted both apoptosis and autophagy in A375-S2 cells. NO suppressed mTOR expression, which led to autophagy induction. An autophagic inhibitor, 3-methyladenine (3MA) promoted NO production, while acceleration of autophagy with an autophagic agonist rapamycin repressed NO production, suggesting that autophagy and NO production form a negative feedback loop that eventually protects the cells from apoptosis. The results together with the previous study indicate apoptosis and autophagy induced by physalin A in A375-S2 cells; the autophagy, repressing production of reactive nitrogen species (RNS) and ROS, protects the cells from apoptosis. PMID:24952311

  9. 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

  10. 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.

  11. Acid Ceramidase Expression Modulates the Sensitivity of A375 Melanoma Cells to Dacarbazine*

    PubMed Central

    Bedia, Carmen; Casas, Josefina; Andrieu-Abadie, Nathalie; Fabriàs, Gemma; Levade, Thierry

    2011-01-01

    Dacarbazine (DTIC) is the treatment of choice for metastatic melanoma, but its response in patients remains very poor. Ceramide has been shown to be a death effector and to play an important role in regulating cancer cell growth upon chemotherapy. Among ceramidases, the enzymes that catabolize ceramide, acid ceramidase (aCDase) has been implicated in cancer progression. Here we show that DTIC elicits a time- and dose-dependent decrease of aCDase activity and an increase of intracellular ceramide levels in human A375 melanoma cells. The loss of enzyme activity occurred as a consequence of reactive oxygen species-dependent activation of cathepsin B-mediated degradation of aCDase. These events preceded autophagic features and loss of cell viability. Down-regulation of acid but not neutral or alkaline ceramidase 2 resulted in elevated levels of ceramide and sensitization to the toxic effects of DTIC. Conversely, inducible overexpression of acid but not neutral ceramidase reduced ceramide levels and conferred resistance to DTIC. In conclusion, we report that increased levels of ceramide, due to enhanced degradation of aCDase, are in part responsible for the cell death effects of DTIC. These results suggest that down-regulation of aCDase alone or in combination with DTIC may represent a useful tool in the treatment of metastatic melanoma. PMID:21700700

  12. Lipopeptide Biosurfactant Pseudofactin II Induced Apoptosis of Melanoma A 375 Cells by Specific Interaction with the Plasma Membrane

    PubMed Central

    Janek, Tomasz; Krasowska, Anna; Radwańska, Agata; Łukaszewicz, Marcin

    2013-01-01

    In the case of melanoma, advances in therapies are slow, which raises the need to evaluate new therapeutic strategies and natural products with potential cancer cell inhibiting effect. Pseudofactin II (PFII), a novel cyclic lipopeptide biosurfactant has been isolated from the Arctic strain of Pseudomonas fluorescens BD5. The aim of this study was to investigate the effect of PFII on A375 melanoma cells compared with the effect of PFII on Normal Human Dermis Fibroblast (NHDF) cells and elucidate the underlying mechanism of PFII cytotoxic activity. Melanoma A375 cells and NHDF cells were exposed to PFII or staurosporine and apoptotic death was assessed by monitoring caspase 3-like activity and DNA fragmentation. From time-dependent monitoring of lactate dehydrogenase (LDH) release, Ca2+ influx, and a correlation between Critical Micelle Concentration (CMC) we concluded that cell death is the consequence of plasma membrane permeabilisation by micelles. This finding suggests that pro-apoptotic mechanism of PFII is different from previously described cyclic lipopeptides. The mechanism of PFII specificity towards malignant cells remains to be discovered. The results of this study show that PFII could be a new promising anti-melanoma agent. PMID:23483962

  13. Extracellular S100A4 affects endothelial cell integrity and stimulates transmigration of A375 melanoma cells.

    PubMed

    Herwig, Nadine; Belter, Birgit; Pietzsch, Jens

    2016-09-01

    High extracellular S100A4 level proves a specific characteristic of some cancer cases, including malignant melanoma. Concerning the latter, extracellular S100A4 in an autocrine manner was shown to promote prometastatic activation of A375 cells by interaction with the receptor for advanced glycation endproducts (RAGE). We hypothesized that interaction of extracellular S100A4 with RAGE in a paracrine manner will affect endothelial cell (EC) integrity thus further promoting melanoma metastasis. We investigated the influence of recombinant and cell (A375)-derived S100A4 on junction protein expression and EC (hCMEC/D3) integrity by measuring transendothelial electrical resistance (TEER). Decrease of TEER and diminished expression of both occludin and VE-cadherin revealed the loss of EC integrity. Transmigration of transgenic A375 cells (A375-hS100A4/A375-hRAGE) through the EC monolayer was significantly higher compared to wild-type A375 cells, and was substantially decreased by sRAGE. A pilot study in mice, intracardially injected with A375-hS100A4 or A375-hRAGE cells, showed lower survival rates and a higher incidence of metastases compared to wild-type A375 cells. Tumor development was mostly located in the brain, bones, and ovaries. These findings provide further evidence on extracellular S100A4 as paracrine mediator of prometastatic endothelial dysfunction involving its interaction with RAGE. PMID:27387233

  14. HDAC-inhibitor (S)-8 disrupts HDAC6-PP1 complex prompting A375 melanoma cell growth arrest and apoptosis.

    PubMed

    Balliu, Manjola; Guandalini, Luca; Romanelli, Maria Novella; D'Amico, Massimo; Paoletti, Francesco

    2015-01-01

    Histone deacetylase inhibitors (HDACi) are agents capable of inducing growth arrest and apoptosis in different tumour cell types. Previously, we reported a series of novel HDACi obtained by hybridizing SAHA or oxamflatin with 1,4-benzodiazepines. Some of these hybrids proved effective against haematological and solid cancer cells and, above all, compound (S)-8 has emerged for its activities in various biological systems. Here, we describe the effectiveness of (S)-8 against highly metastatic human A375 melanoma cells by using normal PIG1 melanocytes as control. (S)-8 prompted: acetylation of histones H3/H4 and α-tubulin; G0 /G1 and G2 /M cell cycle arrest by rising p21 and hypophos-phorylated RB levels; apoptosis involving the cleavage of PARP and caspase 9, BAD protein augmentation and cytochrome c release; decrease in cell motility, invasiveness and pro-angiogenic potential as shown by results of wound-healing assay, down-regulation of MMP-2 and VEGF-A/VEGF-R2, besides TIMP-1/TIMP-2 up-regulation; and also intracellular accumulation of melanin and neutral lipids. The pan-caspase inhibitor Z-VAD-fmk, but not the antioxidant N-acetyl-cysteine, contrasted these events. Mechanistically, (S)-8 allows the disruption of cytoplasmic HDAC6-protein phosphatase 1 (PP1) complex in A375 cells thus releasing the active PP1 that dephosphorylates AKT and blocks its downstream pro-survival signalling. This view is consistent with results obtained by: inhibiting PP1 with Calyculin A; using PPP1R2-transfected cells with impaired PP1 activity; monitoring drug-induced HDAC6-PP1 complex re-shuffling; and, abrogating HDAC6 expression with specific siRNA. Altogether, (S)-8 proved very effective against melanoma A375 cells, but not normal melanocytes, and safe to normal mice thus offering attractive clinical prospects for treating this aggressive malignancy. PMID:25376115

  15. An Uncoordinated-5 Homolog B Receptor Monoclonal Antibody Regulates A375 Melanoma Cell Migration

    PubMed Central

    Zuo, Yuanling; He, Jun; Zhao, Yiming; He, Yang

    2014-01-01

    Uncoordinated-5 homolog B receptor (UNC5B) was first found to mediate neural chemorepulsive effects by binding to its ligand netrin-1 in the nervous system. Newer evidence indicated that UNC5B also has functions outside the nervous system. In this study, we report on the generation of a monoclonal antibody specific to the outer-membrane immunoglobulin-like domains of UNC5B using the hybridoma technique. Western blot, immunofluorescence, and flow cytometry analyses showed that the antibody specifically bound to UNC5B protein. Interestingly, the antibody blocked the Netrin-1-induced inhibitory effect on the mobility of melanoma A375 cells by wound healing assay and transwell migration assay, whereas it had no effects on cell proliferation measured by CCK-8 assay. Thus, the functional antibody may provide a useful tool for the study of UNC5B expression profiles and functions outside the nervous system. PMID:25171009

  16. Proteomic investigation of the sinulariolide-treated melanoma cells A375: effects on the cell apoptosis through mitochondrial-related pathway and activation of caspase cascade.

    PubMed

    Li, Hsing-Hui; Su, Jui-Hsin; Chiu, Chien-Chih; Lin, Jen-Jie; Yang, Zih-Yan; Hwang, Wen-Ing; Chen, Yu-Kuei; Lo, Yu-Hsuan; Wu, Yu-Jen

    2013-07-01

    Sinulariolide is an active compound isolated from the cultured soft coral Sinularia flexibilis. In this study, we investigated the effects of sinulariolide on A375 melanoma cell growth and protein expression. Sinulariolide suppressed the proliferation and migration of melanoma cells in a concentration-dependent manner and was found to induce both early and late apoptosis by flow cytometric analysis. Comparative proteomic analysis was conducted to investigate the effects of sinulariolide at the molecular level by comparison between the protein profiles of melanoma cells treated with sinulariolide and those without treatment. Two-dimensional gel electrophoresis (2-DE) master maps of control and treated A375 cells were generated by analysis with PDQuest software. Comparison between these maps showed up- and downregulation of 21 proteins, seven of which were upregulated and 14 were downregulated. The proteomics studies described here identify some proteins that are involved in mitochondrial dysfunction and apoptosis-associated proteins, including heat shock protein 60, heat shock protein beta-1, ubiquinol cytochrome c reductase complex core protein 1, isocitrate dehydrogenase (NAD) subunit alpha (down-regulated), and prohibitin (up-regulated), in A375 melanoma cells exposed to sinulariolide. Sinulariolide-induced apoptosis is relevant to mitochondrial-mediated apoptosis via caspase-dependent pathways, elucidated by the loss of mitochondrial membrane potential, release of cytochrome c, and activation of Bax, Bad and caspase-3/-9, as well as suppression of p-Bad, Bcl-xL and Bcl-2. Taken together, our results show that sinulariolide-induced apoptosis might be related to activation of the caspase cascade and mitochondria dysfunction pathways. Our results suggest that sinulariolide merits further evaluation as a chemotherapeutic agent for human melanoma. PMID:23880933

  17. Proteomic Investigation of the Sinulariolide-Treated Melanoma Cells A375: Effects on the Cell Apoptosis through Mitochondrial-Related Pathway and Activation of Caspase Cascade

    PubMed Central

    Li, Hsing-Hui; Su, Jui-Hsin; Chiu, Chien-Chih; Lin, Jen-Jie; Yang, Zih-Yan; Hwang, Wen-Ing; Chen, Yu-Kuei; Lo, Yu-Hsuan; Wu, Yu-Jen

    2013-01-01

    Sinulariolide is an active compound isolated from the cultured soft coral Sinularia flexibilis. In this study, we investigated the effects of sinulariolide on A375 melanoma cell growth and protein expression. Sinulariolide suppressed the proliferation and migration of melanoma cells in a concentration-dependent manner and was found to induce both early and late apoptosis by flow cytometric analysis. Comparative proteomic analysis was conducted to investigate the effects of sinulariolide at the molecular level by comparison between the protein profiles of melanoma cells treated with sinulariolide and those without treatment. Two-dimensional gel electrophoresis (2-DE) master maps of control and treated A375 cells were generated by analysis with PDQuest software. Comparison between these maps showed up- and downregulation of 21 proteins, seven of which were upregulated and 14 were downregulated. The proteomics studies described here identify some proteins that are involved in mitochondrial dysfunction and apoptosis-associated proteins, including heat shock protein 60, heat shock protein beta-1, ubiquinol cytochrome c reductase complex core protein 1, isocitrate dehydrogenase (NAD) subunit alpha (down-regulated), and prohibitin (up-regulated), in A375 melanoma cells exposed to sinulariolide. Sinulariolide-induced apoptosis is relevant to mitochondrial-mediated apoptosis via caspase-dependent pathways, elucidated by the loss of mitochondrial membrane potential, release of cytochrome c, and activation of Bax, Bad and caspase-3/-9, as well as suppression of p-Bad, Bcl-xL and Bcl-2. Taken together, our results show that sinulariolide-induced apoptosis might be related to activation of the caspase cascade and mitochondria dysfunction pathways. Our results suggest that sinulariolide merits further evaluation as a chemotherapeutic agent for human melanoma. PMID:23880933

  18. Fig latex (Ficus carica L. cultivar Dottato) in combination with UV irradiation decreases the viability of A375 melanoma cells in vitro.

    PubMed

    Menichini, Giulio; Alfano, Carmine; Provenzano, Eugenio; Marrelli, Mariangela; Statti, Giancarlo A; Somma, Francesco; Menichini, Francesco; Conforti, Filomena

    2012-10-01

    Melanoma and nonmelanoma skin cancers are among the most prevalent cancers in the human population. In the present work latex of Ficus carica cultivar Dottato from Italy collected from fruits and leaves was examined to assess its free radical-scavenging activity with 1,1-diphenyl-2 picrylhydrazyl (DPPH) and its phototoxicity on A375 human melanoma cells. The latex obtained from the fruits of Ficus carica cv. Dottato showed the best antiradical activity with an IC50 value of 0.05 mg/ml while the latex obtained from the leaves showed the best antiproliferative activity with an IC50 value of 1.5 μg/ml on the human tumor cell line A375 (melanoma) after irradiation at a specific UVA dose (1.08 J/cm2). Control experiments with UVA light or drugs alone were carried out without significant cytotoxic effects. Polyphenolic content of the samples was also evaluated. This is the first study comparing F. carica latex of leaves and fruits. Plant derived natural products have long been and will continue to be an important source for anticancer drug development. PMID:22339064

  19. A DC-81-indole conjugate agent suppresses melanoma A375 cell migration partially via interrupting VEGF production and stromal cell-derived factor-1{alpha}-mediated signaling

    SciTech Connect

    Hsieh, Ming-Chu; Hu, Wan-Ping; Yu, Hsin-Su; Wu, Wen-Chuan; Chang, Long-Sen; Kao, Ying-Hsien; Wang, Jeh-Jeng

    2011-09-01

    Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) chemicals are antitumor antibiotics inhibiting nucleic acid synthesis. An indole carboxylate-PBD hybrid with six-carbon spacer structure (IN6CPBD) has been previously demonstrated to induce melanoma cell apoptosis and reduce metastasis in mouse lungs. This study aimed at investigating the efficacy of the other hybrid compound with four-carbon spacer (IN4CPBD) and elucidating its anti-metastatic mechanism. Human melanoma A375 cells with IN4CPBD treatment underwent cytotoxicity and apoptosis-associated assays. Transwell migration assay, Western blotting, and ELISA were used for mechanistic study. IN4CPBD exhibited potent melanoma cytotoxicity through interrupting G1/S cell cycle progression, increasing DNA fragmentation and hypodipoidic DNA contents, and reducing mitochondrial membrane potential. Caspase activity elevation suggested that both intrinsic and extrinsic pathways were involved in IN4CPBD-induced melanoma apoptosis. IN4CPBD up-regulated p53 and p21, thereby concomitantly derailing the equilibrium between Bcl-2 and Bax levels. Transwell migration assay demonstrated that stromal cell-derived factor-1{alpha} (SDF-1{alpha}) stimulated A375 cell motility, while kinase inhibitors treatment confirmed that Rho/ROCK, Akt, ERK1/2, and p38 MAPK pathways were involved in SDF-1{alpha}-enhanced melanoma migration. IN4CPBD not only abolished the SDF-1{alpha}-enhanced chemotactic motility but also suppressed constitutive MMP-9 and VEGF expression. Mechanistically, IN4CPBD down-regulated Akt, ERK1/2, and p38 MAPK total proteins and MYPT1 phosphorylation. In conclusion, beyond the fact that IN4CPBD induces melanoma cell apoptosis at cytotoxic dose, the interruption in the VEGF expression and the SDF-1{alpha}-related signaling at cytostatic dose may partially constitute the rationale for its in vivo anti-metastatic potency. - Research Highlights: > A novel carboxylate-PBD hybrid as anti-melanoma drug. > IN4CPBD interrupts melanoma cell

  20. Mitotic arrest-associated apoptosis induced by sodium arsenite in A375 melanoma cells is BUBR1-dependent

    SciTech Connect

    McNeely, Samuel C.; Taylor, B. Frazier; States, J. Christopher

    2008-08-15

    A375 human malignant melanoma cells undergo mitotic arrest-associated apoptosis when treated with pharmacological concentrations of sodium arsenite, a chemotherapeutic for acute promyelocytic leukemia. Our previous studies indicated that decreased arsenite sensitivity correlated with reduced mitotic spindle checkpoint function and reduced expression of the checkpoint protein BUBR1. In the current study, arsenite induced securin and cyclin B stabilization, BUBR1 phosphorylation, and spindle checkpoint activation. Arsenite also increased activating cyclin dependent kinase 1 (CDK1) Thr{sup 161} phosphorylation but decreased inhibitory Tyr15 phosphorylation. Mitotic arrest resulted in apoptosis as indicated by colocalization of mitotic phospho-Histone H3 with active caspase 3. Apoptosis was associated with BCL-2 Ser70 phosphorylation. Inhibition of CDK1 with roscovitine in arsenite-treated mitotic cells inhibited spindle checkpoint maintenance as inferred from reduced BUBR1 phosphorylation, reduced cyclin B expression, and diminution of mitotic index. Roscovitine also reduced BCL-2 Ser70 phosphorylation and protected against apoptosis, suggesting mitotic arrest caused by hyperactivation of CDK1 directly or indirectly leads to BCL-2 phosphorylation and apoptosis. In addition, suppression of BUBR1 with siRNA prevented arsenite-induced mitotic arrest and apoptosis. These findings provide insight into the mechanism of arsenic's chemotherapeutic action and indicate a functional spindle checkpoint may be required for arsenic-sensitivity.

  1. Lipid raft regulates the initial spreading of melanoma A375 cells by modulating β1 integrin clustering.

    PubMed

    Wang, Ruifei; Bi, Jiajia; Ampah, Khamal Kwesi; Zhang, Chunmei; Li, Ziyi; Jiao, Yang; Wang, Xiaoru; Ba, Xueqing; Zeng, Xianlu

    2013-08-01

    Cell adhesion and spreading require integrins-mediated cell-extracellular matrix interaction. Integrins function through binding to extracellular matrix and subsequent clustering to initiate focal adhesion formation and actin cytoskeleton rearrangement. Lipid raft, a liquid ordered plasma membrane microdomain, has been reported to play major roles in membrane motility by regulating cell surface receptor function. Here, we identified that lipid raft integrity was required for β1 integrin-mediated initial spreading of melanoma A375 cells on fibronectin. We found that lipid raft disruption with methyl-β-cyclodextrin led to the inability of focal adhesion formation and actin cytoskeleton rearrangement by preventing β1 integrin clustering. Furthermore, we explored the possible mechanism by which lipid raft regulates β1 integrin clustering and demonstrated that intact lipid raft could recruit and modify some adaptor proteins, such as talin, α-actinin, vinculin, paxillin and FAK. Lipid raft could regulate the location of these proteins in lipid raft fractions and facilitate their binding to β1 integrin, which may be crucial for β1 integrin clustering. We also showed that lipid raft disruption impaired A375 cell migration in both transwell and wound healing models. Together, these findings provide a new insight for the relationship between lipid raft and the regulation of integrins. PMID:23665237

  2. Cell Internalization Studies of Gadofullerene-(ZME-018) Immunoconjugates into A375m Melanoma Cells12

    PubMed Central

    Berger, Christopher Scott; Marks, John W; Bolskar, Robert D; Rosenblum, Michael G; Wilson, Lon J

    2011-01-01

    Fullerene (C60)-monoclonal antibody (mAb) immunoconjugates have been determined to internalize into target cells using water-soluble Gd3+ ion-filled metallofullerenes (Gd@C60[OH]x). Two separate conjugations of Gd@C60(OH)x with the antibody ZME-018 and a murine antibody mixture (MuIgG) were performed in a 1:5 mAb/Gd@C60 ratio. Characterization of the immunoconjugates was established using inductively coupled plasma mass spectrometry (ICP-MS) for Gd3+ and UV-Vis spectrometry (for Gd@C60 + C60). Once conjugated, enzyme-linked immunosorbent assays showed little change in the specific binding of ZME-018. Each immunoconjugate was exposed to two cancer cell lines, A375m (antigen positive), and T24, bladder carcinoma (antigen negative). Internalization levels of the immunoconjugate were determined at various time points during 24 hours by harvesting and digesting the cells with 70% HNO3 for Gd3+ ion analysis by ICP-MS. These results are the first to demonstrate the practicality of a targeted cancer therapy based on fullerene immunotherapy. PMID:22190999

  3. CD147-targeted siRNA in A375 malignant melanoma cells induces the phosphorylation of EGFR and downregulates cdc25C and MEK phosphorylation

    PubMed Central

    HATANAKA, MIHO; HIGASHI, YUKO; KAWAI, KAZUHIRO; SU, JUAN; ZENG, WEIQI; CHEN, XIANG; KANEKURA, TAKURO

    2016-01-01

    The Raf-MEK-ERK signaling pathway is important during oncogenesis. An activating mutation of BRAF constitutively activates the Raf-MEK-ERK signaling cascade, and has been identified in ~70% of malignant melanomas (MMs). Cluster of differentiation 147 (CD147)/basigin is an integral plasma membrane protein belonging to the immunoglobulin superfamily. The protein is highly expressed on MM cells, and promotes cellular proliferation and tumor growth. The present study investigated the correlation between CD147 expression and Raf-MEK-ERK signaling in MM using the A375 human MM cell line, which harbors the activating mutation of BRAF. The phosphorylation of epidermal growth factor receptor (EGFR) was upregulated, and mitogen-activated protein kinase kinase (MEK) and cell division cycle 25C phosphorylation was downregulated by CD147 silencing in the A375 cells. Cell growth was inhibited by the EGFR inhibitor erlotinib and by CD147 silencing, and additive growth inhibition was observed when these techniques were combined. The results of the present study indicate that the combination of EGFR and CD147 inhibition may be useful in BRAF-mutated MM. PMID:27073491

  4. MK615, a Prunus mume Steb. Et Zucc ('Ume') extract, attenuates the growth of A375 melanoma cells by inhibiting the ERK1/2-Id-1 pathway.

    PubMed

    Tada, Ko-ichi; Kawahara, Ko-ichi; Matsushita, Shigeto; Hashiguchi, Teruto; Maruyama, Ikuro; Kanekura, Takuro

    2012-06-01

    The Japanese apricot, a commonly consumed food called 'Ume' in Japan, has been used for a traditional Japanese medicine for centuries. MK615, an extract of compounds from 'Ume', has strong antitumorigenic and antiinflammatory effects including the induction of apoptosis and autophagy, and inhibition of cytokine production mediated via the inhibition of MAPKs signaling including ERK-1/2, JNK and p38MAPK. The inhibitor of DNA binding 1 (Id-1), a basic helix-loop-helix (bHLH) transcription factor family, is essential for DNA binding and the transcriptional regulation of various proteins that play important roles in the development, progression and invasion of tumors. In melanoma, Id-1 is constitutively expressed in the late and early stages, suggesting it as a therapeutic target in patients with melanoma. This study reports that MK615 profoundly reduced both the mRNA- and protein expression levels of Id-1 and inhibited cell growth in A375 melanoma cells. MK615 markedly inhibited the phosphorylation of ERK1/2, which is associated with Id-1 protein expression in A375 cells. Id-1-specific RNAi induced the death of A375 cells. Moreover, the expression of Bcl-2 was decreased by both MK615 and Id-1-specific RNAi in A375 cells. The results suggest that MK615 is a potential therapeutic agent for treating malignant melanoma. PMID:22076920

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

    PubMed

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

    2016-09-01

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

  6. Induction of apoptosis in melanoma A375 cells by a chloroform fraction of Centratherum anthelminticum (L.) seeds involves NF-kappaB, p53 and Bcl-2-controlled mitochondrial signaling pathways

    PubMed Central

    2013-01-01

    Background Centratherum anthelminticum (L.) Kuntze (scientific synonyms: Vernonia anthelmintica; black cumin) is one of the ingredients of an Ayurvedic preparation, called “Kayakalp”, commonly applied to treat skin disorders in India and Southeast Asia. Despite its well known anti-inflammatory property on skin diseases, the anti-cancer effect of C. anthelminticum seeds on skin cancer is less documented. The present study aims to investigate the anti-cancer effect of Centratherum anthelminticum (L.) seeds chloroform fraction (CACF) on human melanoma cells and to elucidate the molecular mechanism involved. Methods A chloroform fraction was extracted from C. anthelminticum (CACF). Bioactive compounds of the CACF were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Human melanoma cell line A375 was treated with CACF in vitro. Effects of CACF on growth inhibition, morphology, stress and survival of the cell were examined with MTT, high content screening (HSC) array scan and flow cytometry analyses. Involvement of intrinsic or extrinsic pathways in the CACF-induced A375 cell death mechanism was examined using a caspase luminescence assay. The results were further verified with different caspase inhibitors. In addition, Western blot analysis was performed to elucidate the changes in apoptosis-associated molecules. Finally, the effect of CACF on the NF-κB nuclear translocation ability was assayed. Results The MTT assay showed that CACF dose-dependently inhibited cell growth of A375, while exerted less cytotoxic effect on normal primary epithelial melanocytes. We demonstrated that CACF induced cell growth inhibition through apoptosis, as evidenced by cell shrinkage, increased annexin V staining and formation of membrane blebs. CACF treatment also resulted in higher reactive oxygen species (ROS) production and lower Bcl-2 expression, leading to decrease mitochondrial membrane potential (MMP). Disruption of the MMP facilitated the release of

  7. Vitamin E δ-tocotrienol triggers endoplasmic reticulum stress-mediated apoptosis in human melanoma cells.

    PubMed

    Montagnani Marelli, Marina; Marzagalli, Monica; Moretti, Roberta M; Beretta, Giangiacomo; Casati, Lavinia; Comitato, Raffaella; Gravina, Giovanni L; Festuccia, Claudio; Limonta, Patrizia

    2016-01-01

    Malignant melanoma is the leading cause of death from skin cancer. Drug toxicity and resistance represent a serious challange for melanoma treatments. Evidence demonstrates that natural compounds may play a crucial role in cancer prevention, growth and progression. Vitamin E tocotrienols (TT) were shown to possess antitumor activity. Here, we analyzed the effects of δ-TT on melanoma cell growth and the involvement of the endoplasmic reticulum (ER) stress in this activity. The experiments were performed on human melanoma cell lines, BLM and A375. δ-TT exerted a significant proapoptotic effect on both cell lines, involving the intrinsic apoptosis pathway; importantly, this compound did not affect the viability of normal human melanocytes. In melanoma cells, δ-TT exerted its antitumor effect through activation of the PERK/p-eIF2α/ATF4/CHOP, IRE1α and caspase-4 ER stress-related branches. Salubrinal, an inhibitor of the ER stress, counteracted the cytotoxic activity of δ-TT. In vivo experiments performed in nude mice bearing A375 xenografts evidenced that δ-TT reduces tumor volume and tumor mass; importantly, tumor progression was significantly delayed by δ-TT treatment. In conclusion, δ-TT exerts a proapoptotic activity on melanoma cells, through activation of the ER stress-related pathways. δ-TT might represent an effective option for novel chemopreventive/therapeutic strategies for melanoma. PMID:27461002

  8. Vitamin E δ-tocotrienol triggers endoplasmic reticulum stress-mediated apoptosis in human melanoma cells

    PubMed Central

    Montagnani Marelli, Marina; Marzagalli, Monica; Moretti, Roberta M.; Beretta, Giangiacomo; Casati, Lavinia; Comitato, Raffaella; Gravina, Giovanni L.; Festuccia, Claudio; Limonta, Patrizia

    2016-01-01

    Malignant melanoma is the leading cause of death from skin cancer. Drug toxicity and resistance represent a serious challange for melanoma treatments. Evidence demonstrates that natural compounds may play a crucial role in cancer prevention, growth and progression. Vitamin E tocotrienols (TT) were shown to possess antitumor activity. Here, we analyzed the effects of δ-TT on melanoma cell growth and the involvement of the endoplasmic reticulum (ER) stress in this activity. The experiments were performed on human melanoma cell lines, BLM and A375. δ-TT exerted a significant proapoptotic effect on both cell lines, involving the intrinsic apoptosis pathway; importantly, this compound did not affect the viability of normal human melanocytes. In melanoma cells, δ-TT exerted its antitumor effect through activation of the PERK/p-eIF2α/ATF4/CHOP, IRE1α and caspase-4 ER stress-related branches. Salubrinal, an inhibitor of the ER stress, counteracted the cytotoxic activity of δ-TT. In vivo experiments performed in nude mice bearing A375 xenografts evidenced that δ-TT reduces tumor volume and tumor mass; importantly, tumor progression was significantly delayed by δ-TT treatment. In conclusion, δ-TT exerts a proapoptotic activity on melanoma cells, through activation of the ER stress-related pathways. δ-TT might represent an effective option for novel chemopreventive/therapeutic strategies for melanoma. PMID:27461002

  9. Predominance of the metastatic phenotype in hybrids formed by fusion of mouse and human melanoma clones.

    PubMed

    van Golen, K L; Risin, S; Staroselsky, A; Berger, D; Tainsky, M A; Pathak, S; Price, J E

    1996-03-01

    The fusion of mouse and human melanoma cells that were tumorigenic but had different metastatic capabilities resulted in hybrids that were metastatic when injected intravenously or subcutaneously into nude mice, regardless of whether it was the mouse or the human melanoma clone that was metastatic. The H7 hybrid line, formed by fusing murine nonmetastatic K1735 C19 cells with human metastatic A375 C15 cells retained high metastatic potential over more than 50 sub-culture passages, suggesting that the dominant metastatic phenotype in these hybrid cells was stable. Using fluorescent in situ hybridization (FISH), human chromosome 17 was consistently identified as the predominant human chromosome in the majority of H7 cells tested between passages 20 and 60. Western blot analysis showed that the hybrid cells expressed human nm23 protein, indicating that at least one gene on the human chromosome 17 was functional. Immunocytochemistry and immunoprecipitation showed that the metastatic A375 C15 and H7 cells expressed p53 protein, but that the nonmetastatic K1735 C19 melanoma cells did not. Sequencing the human p53 gene in A375 C15N and H7 showed mutations in exon 7. Using a bioassay technique, we showed that K1735 C19 cells can spread from subcutaneous tumors to the lungs of nude mice yet fail to form metastases. With the addition of human chromosome 17 from A375 C15 cells, which carries a mutant p53 gene, the cells readily formed lung metastases. In this melanoma hybrid, a mutant p53 gene appears to confer a survival advantage on cells arrested in the lungs of nude mice and thus contributes to the growth of metastatic cells. PMID:8605733

  10. Cross-species models of human melanoma.

    PubMed

    van der Weyden, Louise; Patton, E Elizabeth; Wood, Geoffrey A; Foote, Alastair K; Brenn, Thomas; Arends, Mark J; Adams, David J

    2016-01-01

    Although transformation of melanocytes to melanoma is rare, the rapid growth, systemic spread, as well as the chemoresistance of melanoma present significant challenges for patient care. Here we review animal models of melanoma, including murine, canine, equine, and zebrafish models, and detail the immense contribution these models have made to our knowledge of human melanoma development, and to melanocyte biology. We also highlight the opportunities for cross-species comparative genomic studies of melanoma to identify the key molecular events that drive this complex disease. PMID:26354726

  11. PRMT1 regulates tumor growth and metastasis of human melanoma via targeting ALCAM.

    PubMed

    Li, Lei; Zhang, Zhengwen; Ma, Tengxiao; Huo, Ran

    2016-07-01

    Overexpression of protein arginine methyltransferases (PRMTs) is associated with various types of cancer. The present study aimed to determine the expression level of PRMT1 in human melanoma and investigate its biological function. The clinical significance of PRMT1 was determined by screening the Oncomine database, and the increased expression of PRMT in melanoma was confirmed by western blot analysis. Furthermore, the current study demonstrated that PRMT1 was overexpressed in melanoma cell lines compared with human immortalized keratinocytes and PIG1 immortalized human melanocytes. Silencing PRMT1 in A375 and Hs294T cells significantly suppressed tumor growth and metastatic ability of the melanoma cell line compared with the negative control. These changes were in accordance with the upregulation of the cadherin 1 level and downregulation of several metastatic‑associated genes determined by a quantitative polymerase chain reaction array. Liquid chromatography‑mass spectrometry demonstrated that activated leukocyte cell adhesion molecule (ALCAM) may be a direct target of PRMT1, and the interaction was confirmed by co‑immunoprecipitation. Compared with negative controls, the protein level of ALCAM was decreased following the silencing of PRMT1, and re‑expression of ALCAM in A375/shPRMT1 or Hs294T/shPRMT1 cells using an expression vector restored the colony formation and metastatic ability of the cells. In conclusion, the current results indicated that PRMT1 is overexpressed in human melanoma, and may regulate tumor growth and metastasis via targeting ALCAM. PMID:27175582

  12. Variant G6PD levels promote tumor cell proliferation or apoptosis via the STAT3/5 pathway in the human melanoma xenograft mouse model

    PubMed Central

    2013-01-01

    Background Glucose-6-phosphate dehydrogenase (G6PD), elevated in tumor cells, catalyzes the first reaction in the pentose-phosphate pathway. The regulation mechanism of G6PD and pathological change in human melanoma growth remains unknown. Methods HEM (human epidermal melanocyte) cells and human melanoma cells with the wild-type G6PD gene (A375-WT), G6PD deficiency (A375-G6PD∆), G6PD cDNA overexpression (A375-G6PD∆-G6PD-WT), and mutant G6PD cDNA (A375-G6PD∆-G6PD-G487A) were subcutaneously injected into 5 groups of nude mice. Expressions of G6PD, STAT3, STAT5, cell cycle-related proteins, and apoptotic proteins as well as mechanistic exploration of STAT3/STAT5 were determined by quantitative real-time PCR (qRT-PCR), immunohistochemistry and western blot. Results Delayed formation and slowed growth were apparent in A375-G6PD∆ cells, compared to A375-WT cells. Significantly decreased G6PD expression and activity were observed in tumor tissues induced by A375-G6PD∆, along with down-regulated cell cycle proteins cyclin D1, cyclin E, p53, and S100A4. Apoptosis-inhibited factors Bcl-2 and Bcl-xl were up-regulated; however, apoptosis factor Fas was down-regulated, compared to A375-WT cells. Moderate protein expressions were observed in A375-G6PD∆-G6PD-WT and A375-G6PD∆-G6PD-G487A cells. Conclusions G6PD may regulate apoptosis and expression of cell cycle-related proteins through phosphorylation of transcription factors STAT3 and STAT5, thus mediating formation and growth of human melanoma cells. Further study will, however, be required to determine potential clinical applications. PMID:23693134

  13. Heterogeneity of cytokine and growth factor gene expression in human melanoma cells with different metastatic potentials.

    PubMed

    Singh, R K; Gutman, M; Radinsky, R

    1995-01-01

    The purpose of this study was to determine the mRNA expression level of multiple cytokine and growth factor genes in human malignant melanoma. Melanoma cells were isolated from several surgical specimens, adapted to growth in culture, characterized for their ability to produce experimental metastases in nude mice, and assessed for cytokine and growth factor steady-state gene expression. Highly metastatic in vivo- and in vitro-derived variants isolated from a single melanoma, A375, were also analyzed. Northern blot analyses revealed that all melanomas analyzed constitutively expressed steady-state mRNA transcripts for the growth and angiogenic factors, basic fibroblast growth factor (bFGF), and transforming growth factor alpha (TGF-alpha), which correlated with metastatic propensity. Only one highly metastatic melanoma, TXM-1, originally isolated from a lymph node metastasis, expressed mRNA transcripts specific for monocyte chemotactic and activating factor (MCAF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Similarly, of the nine melanomas examined, only TXM-1 expressed interleukin (IL)-1 alpha, IL-1 beta, and IL-6, important immunomodulatory cytokines. These data demonstrate the differential and heterogeneous expression of cytokine and growth factor genes in human malignant melanoma. PMID:7648437

  14. Enhancing the treatment effect on melanoma by heat shock protein 70-peptide complexes purified from human melanoma cell lines

    PubMed Central

    Gao, Yanwei; Gao, Weishi; Chen, Xia; Cha, Nier; Wang, Xiaoli; Jia, Xiangdong; Wang, Bingping; Ren, Meng; Ren, Jun

    2016-01-01

    Dendritic cell (DC) vaccines are currently one of the most effective approaches to treat melanoma. The immunogenicity of antigens loaded into DCs determines the treatment effects. Patients treated with autologous antigen-loaded DC vaccines achieve the best therapeutic effects. In China, most melanoma patients cannot access their autologous antigens because of formalin treatment of tumor tissue after surgery. In the present study, we purified heat shock protein 70 (HSP70)-peptide complexes (PCs) from human melanoma cell lines A375, A875, M21, M14, WM-35, and SK-HEL-1. We named the purified product as M-HSP70-PCs, and determined its immunological activities. Autologous HSP70-PCs purified from primary tumor cells of melanoma patients (nine cases) were used as controls. These two kinds of tumor antigenic complexes loaded into DCs were used to stimulate an antitumor response against tumor cells in the corresponding patients. Mature DCs pulsed with M-HSP70-PCs stimulated autologous T cells to secrete the same levels of type I cytokines compared with the autologous HSP70-PCs. Moreover, DCs pulsed with M-HSP70-PCs induced CD8+ T cells with an equal ability to kill melanoma cells from patients compared with autologous HSP70-PCs. Next, we used these PC-pulsed autologous DCs and induced autologous specific CD8+ T cells to treat one patient with melanoma of the nasal skin and lung metastasis. The treatment achieved a good effect after six cycles. These findings provide a new direction for DC-based immunotherapy for melanoma patients who cannot access autologous antigens. PMID:27431432

  15. Enhancing the treatment effect on melanoma by heat shock protein 70-peptide complexes purified from human melanoma cell lines.

    PubMed

    Gao, Yanwei; Gao, Weishi; Chen, Xia; Cha, Nier; Wang, Xiaoli; Jia, Xiangdong; Wang, Bingping; Ren, Meng; Ren, Jun

    2016-09-01

    Dendritic cell (DC) vaccines are currently one of the most effective approaches to treat melanoma. The immunogenicity of antigens loaded into DCs determines the treatment effects. Patients treated with autologous antigen-loaded DC vaccines achieve the best therapeutic effects. In China, most melanoma patients cannot access their autologous antigens because of formalin treatment of tumor tissue after surgery. In the present study, we purified heat shock protein 70 (HSP70)-peptide complexes (PCs) from human melanoma cell lines A375, A875, M21, M14, WM‑35, and SK‑HEL‑1. We named the purified product as M‑HSP70‑PCs, and determined its immunological activities. Autologous HSP70‑PCs purified from primary tumor cells of melanoma patients (nine cases) were used as controls. These two kinds of tumor antigenic complexes loaded into DCs were used to stimulate an antitumor response against tumor cells in the corresponding patients. Mature DCs pulsed with M‑HSP70‑PCs stimulated autologous T cells to secrete the same levels of type I cytokines compared with the autologous HSP70‑PCs. Moreover, DCs pulsed with M‑HSP70‑PCs induced CD8+ T cells with an equal ability to kill melanoma cells from patients compared with autologous HSP70‑PCs. Next, we used these PC‑pulsed autologous DCs and induced autologous specific CD8+ T cells to treat one patient with melanoma of the nasal skin and lung metastasis. The treatment achieved a good effect after six cycles. These findings provide a new direction for DC-based immunotherapy for melanoma patients who cannot access autologous antigens. PMID:27431432

  16. Knockdown of asparagine synthetase by RNAi suppresses cell growth in human melanoma cells and epidermoid carcinoma cells.

    PubMed

    Li, Hui; Zhou, Fusheng; Du, Wenhui; Dou, Jinfa; Xu, Yu; Gao, Wanwan; Chen, Gang; Zuo, Xianbo; Sun, Liangdan; Zhang, Xuejun; Yang, Sen

    2016-05-01

    Melanoma, the most aggressive form of skin cancer, causes more than 40,000 deaths each year worldwide. And epidermoid carcinoma is another major form of skin cancer, which could be studied together with melanoma in several aspects. Asparagine synthetase (ASNS) gene encodes an enzyme that catalyzes the glutamine- and ATP-dependent conversion of aspartic acid to asparagine, and its expression is associated with the chemotherapy resistance and prognosis in several human cancers. The present study aims to explore the potential role of ASNS in melanoma cells A375 and human epidermoid carcinoma cell line A431. We applied a lentivirus-mediated RNA interference (RNAi) system to study its function in cell growth of both cells. The results revealed that inhibition of ASNS expression by RNAi significantly suppressed the growth of melanoma cells and epidermoid carcinoma cells, and induced a G0/G1 cell cycle arrest in melanoma cells. Knockdown of ASNS in A375 cells remarkably downregulated the expression levels of CDK4, CDK6, and Cyclin D1, and upregulated the expression of p21. Therefore, our study provides evidence that ASNS may represent a potential therapeutic target for the treatment of melanoma. PMID:25858017

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

    PubMed

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

    2014-12-01

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

  18. 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

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

    PubMed Central

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

    2016-01-01

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

  20. Biology of Human Cutaneous Melanoma

    PubMed Central

    Elias, Elias G.; Hasskamp, Joanne H.; Sharma, Bhuvnesh K.

    2010-01-01

    A review of the natural behavior of cutaneous melanoma, clinical and pathological factors, prognostic indicators, some basic research and the present and possible futuristic strategies in the management of this disease are presented. While surgery remains to be the most effective therapeutic approach in the management of early primary lesions, there is no standard adjuvant therapy after surgical resection, or for metastatic disease. PMID:24281039

  1. Irradiation affects cellular properties and Eph receptor expression in human melanoma cells

    PubMed Central

    Mosch, Birgit; Pietzsch, Doreen; Pietzsch, Jens

    2012-01-01

    X-ray irradiation influences metastatic properties of tumor cells and, moreover, metastasis and cellular motility can be modified by members of the Eph receptor/ephrin family of receptor tyrosine kinases. We hypothesized that irradiation-induced changes in cellular properties relevant for metastasis in melanoma cells could be mediated by Eph receptor/ephrin signaling. In this pilot study, we analyzed one pre-metastatic (Mel-Juso) and three metastatic human melanoma (Mel-Juso-L3, A375, and A2058) cells lines and predominantly found anti-metastatic effects of X-ray irradiation with impaired cell growth, clonal growth and motility. Additionally, we observed an irradiation-induced increase in adhesion paralleled by a decrease in migration in Mel-Juso and Mel-Juso-L3 cells and, in part, also in A375 cells. We further demonstrate a decrease of EphA2 both in expression and activity at 7 d after irradiation paralleled by an upregulation of EphA3. Analyzing downstream signaling after irradiation, we detected decreased Src kinase phosphorylation, but unchanged focal adhesion kinase (FAK) phosphorylation, indicating, in part, irradiation-induced downregulation of signaling via the EphA2-Src-FAK axis in melanoma cells. However, to which extent this finding contributes to the modification of metastasis-relevant cellular properties remains to be elucidated. PMID:22568947

  2. Interleukin 1-induced augmentation of experimental metastases from a human melanoma in nude mice

    SciTech Connect

    Giavazzi, R.; Garofalo, A.; Bani, M.R.; Abbate, M.; Ghezzi, P.; Boraschi, D.; Mantovani, A.; Dejana, E. )

    1990-08-01

    This study has examined the effect of the cytokine interleukin 1 (IL-1) on metastasis formation by the human melanoma A375M in nude mice. We have found that human recombinant IL-1 beta (a single injection greater than 0.01 micrograms per mouse i.v. given before tumor cells) induced an augmentation of experimental lung metastases from the A375M tumor cells in nude mice. This effect was rapidly induced and reversible within 24 h after IL-1 injection. A similar effect was induced by human recombinant IL-1 alpha and human recombinant tumor necrosis factor, but not by human recombinant interleukin 6. 5-(125I)odo-2'-deoxyuridine-radiolabeled A375M tumor cells injected i.v. remained at a higher level in the lungs of nude mice receiving IL-1 than in control mice. In addition, IL-1 injected 1 h, but not 24 h, after tumor cells enhanced lung colonization as well, thus suggesting an effect of IL-1 on the vascular transit of tumor cells. These findings may explain the observation of enhanced secondary localization of tumor cells at inflammatory sites and suggest that modulation of secondary spread should be carefully considered when assessing the ability of this cytokine to complement cytoreductive therapies.

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

    PubMed

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

    2002-07-01

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

  4. IMMUNE-DEFICIENT MOUSE STRAINS DISPLAY MARKED VARIABILITY IN GROWTH OF HUMAN MELANOMA LUNG METASTASES

    PubMed Central

    Carreno, Beatriz M.; Garbow, Joel R.; Kolar, Grant R.; Jackson, Erin N.; Engelbach, John A.; Becker-Hapak, Michelle; Carayannopoulos, Leonidas N.; Piwnica-Worms, David; Linette, Gerald P.

    2009-01-01

    Purpose Immune-deficient mice serve as critical hosts for transplantation of xenogeneic cells for in vivo analysis of various biological processes. Since investigators typically select one or two immune-deficient mouse strains as recipients, no comprehensive study has been published documenting differences in human tumor engraftment. Taking advantage of the increased metastatic potential of RhoC-expressing human (A375) melanoma cells, we evaluate 4 immune-deficient mouse strains: scid, NOD-scid, NOD-scid β2mnull, and NOD-scid IL2Rγnull as xenograft tumor recipients. Experimental design Bioluminescence, magnetic resonance imaging and histopathology was employed to monitor serial tumor growth. NK cell function was examined in each mouse strain using standard 51 Chromium release assays. Results Melanoma metastases growth is delayed and variable in scid, and NOD-scid mice. In contrast, NOD-scid β2mnull and NOD-scid IL2Rγnull mice show rapid tumor engraftment, although tumor growth is variable in NOD-scid β2mnull mice. NK cells were detected in all strains except NOD-scid IL2Rγnull, and in vitro activated scid, NOD-scid and NOD-scid β2mnull NK cells kill human melanoma lines and primary melanoma cells. Expression of human NKG2D ligands MHC class I chain-related A and B molecules renders melanoma susceptible to murine NK cell-mediated cytotoxicity and killing is inhibited by antibody blockade of murine NKG2D. Conclusions Murine NKG2D recognition of MICA/B is an important receptor-ligand interaction employed by NK cells in immune-deficient strains to limit engraftment of human tumors. The absolute NK deficiency in NOD-scid IL2Rγnull animals makes this strain an excellent recipient of melanoma and potentially other human malignancies. PMID:19447870

  5. Raman spectroscopy detects melanoma and the tissue surrounding melanoma using tissue-engineered melanoma models

    PubMed Central

    Yorucu, Ceyla; Lau, Katherine; Mittar, Shweta; Green, Nicola H.; Raza, Ahtasham; Rehman, Ihtesham Ur; MacNeil, Sheila

    2016-01-01

    ABSTRACT Invasion of melanoma cells from the primary tumor involves interaction with adjacent tissues and extracellular matrix. The extent of this interaction is not fully understood. In this study Raman spectroscopy was applied to cryo-sections of established 3D models of melanoma in human skin. Principal component analysis was used to investigate differences between the tumor and normal tissue and between the peri-tumor area and the normal skin. Two human melanoma cells lines A375SM and C8161 were investigated and compared in 3D melanoma models. Changes were found in protein conformations and tryptophan configurations across the entire melanoma samples, in tyrosine orientation and in more fluid lipid packing only in tumor dense areas, and in increased glycogen content in the peri-tumor areas of melanoma. Raman spectroscopy revealed changes around the perimeter of a melanoma tumor as well as detecting differences between the tumor and the normal tissue. PMID:27158185

  6. Theranostic Properties of a Survivin-Directed Molecular Beacon in Human Melanoma Cells

    PubMed Central

    Carpi, Sara; Fogli, Stefano; Giannetti, Ambra; Adinolfi, Barbara; Tombelli, Sara; Da Pozzo, Eleonora; Vanni, Alessia; Martinotti, Enrica; Martini, Claudia; Breschi, Maria Cristina; Pellegrino, Mario

    2014-01-01

    Survivin is an inhibitor of apoptosis overexpressed in different types of tumors and undetectable in most terminally differentiated normal tissues. In the current study, we sought to evaluate the in vitro theranostic properties of a molecular beacon-oligodeoxynucleotide (MB) that targets survivin mRNA. We used laser scanning confocal microscopy to study MB delivery in living cells and real-time PCR and western blot to assess selective survivin-targeting in human malignant melanoma cells. We further assess the pro-apoptotic effect of MB by measuring internucleosomal DNA fragmentation, dissipation of mitochondrial membrane potential (MMP) and changes in nuclear morphology. Transfection of MB into A375 and 501 Mel cells generated high signal intensity from the cytoplasm, while no signal was detected in the extracellular environment and in survivin-negative cells (i.e., human melanocytes and monocytes). MB time dependently decreased survivin mRNA and protein expression in melanoma cells with the maximum effect reached at 72 h. Treatment of melanoma cells with MB induced apoptosis by significant changes in MMP, accumulation of histone-complexed DNA fragments in the cytoplasm and nuclear condensation. MB also enhanced the pro-apoptotic effect of standard chemotherapeutic drugs tested at clinically relevant concentrations. The MB tested in the current study conjugates the ability of imaging with the pharmacological silencing activity against survivin mRNA in human melanoma cells and may represent an innovative approach for cancer diagnosis and treatment. PMID:25501971

  7. Melanoma

    MedlinePlus

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

  8. Sporadic naturally occurring melanoma in dogs as a preclinical model for human melanoma

    PubMed Central

    Simpson, R Mark; Bastian, Boris C; Michael, Helen T; Webster, Joshua D; Prasad, Manju L; Conway, Catherine M; Prieto, Victor M; Gary, Joy M; Goldschmidt, Michael H; Esplin, D Glen; Smedley, Rebecca C; Piris, Adriano; Meuten, Donald J; Kiupel, Matti; Lee, Chyi-Chia R; Ward, Jerrold M; Dwyer, Jennifer E; Davis, Barbara J; Anver, Miriam R; Molinolo, Alfredo A; Hoover, Shelley B; Rodriguez-Canales, Jaime; Hewitt, Stephen M

    2014-01-01

    Melanoma represents a significant malignancy in humans and dogs. Different from genetically engineered models, sporadic canine melanocytic neoplasms share several characteristics with human disease that could make dogs a more relevant preclinical model. Canine melanomas rarely arise in sun-exposed sites. Most occur in the oral cavity, with a subset having intra-epithelial malignant melanocytes mimicking the in situ component of human mucosal melanoma. The spectrum of canine melanocytic neoplasia includes benign lesions with some analogy to nevi, as well as invasive primary melanoma, and widespread metastasis. Growing evidence of distinct subtypes in humans, differing in somatic and predisposing germ-line genetic alterations, cell of origin, epidemiology, relationship to ultraviolet radiation and progression from benign to malignant tumors, may also exist in dogs. Canine and human mucosal melanomas appear to harbor BRAF, NRAS, and c-kit mutations uncommonly, compared with human cutaneous melanomas, although both species share AKT and MAPK signaling activation. We conclude that there is significant overlap in the clinical and histopathological features of canine and human mucosal melanomas. This represents opportunity to explore canine oral cavity melanoma as a preclinical model. PMID:24128326

  9. Unfolding the mutational landscape of human melanoma.

    PubMed

    Davar, Diwakar; Lin, Yan; Kirkwood, John M

    2015-03-01

    Over the preceding two decades, sophisticated sequencing techniques have been used to characterize the genetic drivers of adult melanoma. However, our understanding of pediatric melanomas is still rudimentary. In this report, we comment on a thorough multi-platform analysis of common pediatric melanoma subsets, including pediatric conventional melanoma (CM), congenital nevus-derived melanoma (CNM), and Spitzoid melanoma (SM), contributed by Lu et al. PMID:25666674

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

    PubMed

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

    2016-02-01

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

  11. Effect of Three Centaurea Species Collected from Central Anatolia Region of Turkey on Human Melanoma Cells.

    PubMed

    Russo, Alessandra; Cardile, Venera; Graziano, Adriana C E; Rigano, Daniela; Aktumsek, Abdurrahman; Zengin, Gokhan; Senatore, Felice

    2016-03-01

    Centaurea is the largest genus within the Asteraceae family. Many members of this genus are used in traditional folk medicine, such as Centaurea pulchella used to treat skin problems such as to resolve the abscess. Although biological activities of many Centaurea species have been investigated in different countries and Turkey, cytotoxic effect of C. patula, C. pulchella and C. tchihatcheffii has not been studied yet. Melanoma is one of the most invasive and deadly forms of skin cancer. Therefore, in an ongoing effort to identify new natural anticancer products for the treatment and/or prevention of melanoma cancer, the present study was undertaken to investigate the effect of these Centaurea species, collected from Central Anatolia region of Turkey on cell growth and death in human melanoma cell line, A375.The results revealed that all extracts were able to inhibit, after 48 h of treatment, the growth of cancer cells, that could be related to an overall action of the phenolic compounds present. In fact, C. pulchella, with the highest level of phenolics, showed a major activity followed by C. patula and C. tchihatcheffii. Our data also demonstrate that these natural products induce apoptotic cell death. In conclusion, the study of plant extracts for their cytotoxic and apoptotic properties has shown that medicinal herbs from Centaurea species might have also importance in the prevention and treatment of melanoma. PMID:27169173

  12. Fas-mediated apoptosis of melanoma cells and infiltrating lymphocytes in human malignant melanomas.

    PubMed

    Shukuwa, Tetsuo; Katayama, Ichiro; Koji, Takehiko

    2002-04-01

    In a rodent system, melanoma cells expressing Fas ligand (FasL) could kill Fas-positive lymphocytes, suggesting that FasL expression was an essential factor for melanoma cell survival in vivo. These findings led us to investigate apoptosis, and to histochemically analyze involvement of Fas and FasL in the induction of apoptosis, in human malignant melanoma tissues. The percentages of terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end-labeling (TUNEL)-positive melanoma cells and of proliferating cell nuclear antigen (PCNA)-positive melanoma cells in melanoma tissues (n = 22) were greater than those in melanocytes in uninvolved skin (n = 6) and nevus cells in nevi tissues (n = 9). The infiltrating lymphocytes around melanomas were also TUNEL positive. Immunohistochemistry revealed expression of Fas and FasL in melanoma cells and lymphocytes, whereas no Fas or FasL expression was detected in normal skin melanocytes and nevus cells. There was significant correlation between Fas-positive indices and TUNEL indices in melanoma tissues. Moreover, TUNEL-, Fas-, and FasL-positive indices of melanoma cells from patients with Stage 3 melanomas were significantly lower than those with Stage 2 melanomas. The PCNA index of Stage 1 melanoma was significantly lower than that of the other stages, although the difference of PCNA index was insignificant among Stages 2 to 4. Among Stages 1 to 4, there was no difference in the PCNA, TUNEL-, and Fas-positive indices of lymphocytes, although the FasL-positive index of lymphocytes from Stage 3 melanomas was significantly lower than in that from Stage 2. These data reveal that melanoma cells and infiltrating lymphocytes have the potential to induce their own apoptosis regulated by Fas and FasL in an autocrine and/or paracrine fashion and that the decline of Fas-mediated apoptosis of melanoma cells, rather than the apoptosis of infiltrating lymphocytes, may affect the prognosis of melanoma patients, possibly through the

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

    PubMed

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

    2011-02-25

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

  14. Developing an irreversible inhibitor of human DDAH-1, an enzyme upregulated in melanoma.

    PubMed

    Wang, Yun; Hu, Shougang; Gabisi, Abdul M; Er, Joyce A V; Pope, Arthur; Burstein, Gayle; Schardon, Christopher L; Cardounel, Arturo J; Ekmekcioglu, Suhendan; Fast, Walter

    2014-04-01

    Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH-1) can raise endogenous levels of asymmetric dimethylarginine (ADMA) and lead to a subsequent inhibition of nitric oxide synthesis. In this study, N(5) -(1-imino-2-chloroethyl)-L-ornithine (Cl-NIO) is shown to be a potent time- and concentration-dependent inhibitor of purified human DDAH-1 (KI =1.3±0.6 μM; kinact =0.34±0.07 min(-1) ), with >500-fold selectivity against two arginine-handling enzymes in the same pathway. An activity probe is used to measure the "in cell" IC50 value (6.6±0.2 μM) for Cl-NIO inhibition of DDAH-1 artificially expressed within cultured HEK293T cells. A screen of diverse melanoma cell lines reveals that a striking 50/64 (78 %) of melanoma lines tested showed increased levels of DDAH-1 relative to normal melanocyte control lines. Treatment of the melanoma A375 cell line with Cl-NIO shows a subsequent decrease in cellular nitric oxide production. Cl-NIO is a promising tool for the study of methylarginine-mediated nitric oxide control and a potential therapeutic lead compound for other indications with elevated nitric oxide production, such as septic shock and idiopathic pulmonary fibrosis. PMID:24574257

  15. Developing an irreversible inhibitor of human DDAH-1, an enzyme upregulated in melanoma

    PubMed Central

    Wang, Yun; Hu, Shougang; Gabisi, Abdul M.; Er, Joyce A. V.; Pope, Arthur; Burstein, Gayle; Schardon, Christopher L.

    2015-01-01

    Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH-1) can raise endogenous levels of asymmetric dimethylarginine (ADMA) and lead to a subsequent inhibition of nitric oxide synthesis. Herein, N5-(1-imino-2-chloroethyl)-L-ornithine (Cl-NIO) is shown to be a potent time- and concentration-dependent inhibitor of purified human DDAH-1 (KI = 1.3 ± 0.6 μM; kinact = 0.34 ± 0.07 min−1), with > 500-fold selectivity against two arginine-handling enzymes in the same pathway. An activity probe is used to measure the “in cell” IC50 value (6.6 ± 0.2 μM) for Cl-NIO inhibition of DDAH-1 artificially expressed within cultured HEK293T cells. A screen of diverse melanoma cell lines reveals that a striking 50 / 64 (78 %) of melanoma lines tested showed increased levels of DDAH-1 in comparison to normal melanocyte control lines. Treatment of the melanoma A375 cell line with Cl-NIO shows a subsequent decrease in cellular nitric oxide production. Cl-NIO represents a promising tool for the study of methylarginine-mediated nitric oxide control and a potential therapeutic lead compound for other indications with elevated nitric oxide production such as septic shock and idiopathic pulmonary fibrosis. PMID:24574257

  16. Autocrine secretion of 15d-PGJ2 mediates simvastatin-induced apoptotic burst in human metastatic melanoma cells

    PubMed Central

    Wasinger, Christine; Künzl, Martin; Minichsdorfer, Christoph; Höller, Christoph; Zellner, Maria; Hohenegger, Martin

    2014-01-01

    Background and Purpose Despite new therapeutic approaches, metastatic melanomas still have a poor prognosis. Statins reduce low-density lipoprotein cholesterol and exert anti-inflammatory and anti-proliferative actions. We have recently shown that simvastatin triggers an apoptotic burst in human metastatic melanoma cells by the synthesis of an autocrine factor. Experimental Approach The current in vitro study was performed in human metastatic melanoma cell lines (A375, 518a2) and primary human melanocytes and melanoma cells. The secretome of simvastatin-stressed cells was analysed with two-dimensional difference gel electrophoresis and MS. The signalling pathways involved were analysed at the protein and mRNA level using pharmacological approaches and siRNA technology. Key Results Simvastatin was shown to activate a stress cascade, leading to the synthesis of 15-deoxy-12,14-PGJ2 (15d-PGJ2), in a p38- and COX-2-dependent manner. Significant concentrations of 15d-PGJ2 were reached in the medium of melanoma cells, which were sufficient to activate caspase 8 and the mitochondrial pathway of apoptosis. Inhibition of lipocalin-type PGD synthase, a key enzyme for 15d-PGJ2 synthesis, abolished the apoptotic effect of simvastatin. Moreover, 15d-PGJ2 was shown to bind to the fatty acid-binding protein 5 (FABP5), which was up-regulated and predominantly detected in the secretome of simvastatin-stressed cells. Knockdown of FABP5 abolished simvastatin-induced activation of PPAR-γ and amplified the apoptotic response. Conclusions and Implications We characterized simvastatin-induced activation of the 15d-PGJ2/FABP5 signalling cascades, which triggered an apoptotic burst in melanoma cells but did not affect primary human melanocytes. These data support the rationale for the pharmacological targeting of 15d-PGJ2 in metastatic melanoma. PMID:25091578

  17. TET2 Negatively Regulates Nestin Expression in Human Melanoma.

    PubMed

    Gomes, Camilla B F; Zechin, Karina G; Xu, Shuyun; Stelini, Rafael F; Nishimoto, Ines N; Zhan, Qian; Xu, Ting; Qin, Gungwei; Treister, Nathaniel S; Murphy, George F; Lian, Christine G

    2016-06-01

    Although melanoma is an aggressive cancer, the understanding of the virulence-conferring pathways involved remains incomplete. We have demonstrated that loss of ten-eleven translocation methylcytosine dioxygenase (TET2)-mediated 5-hydroxymethylcytosine (5-hmC) is an epigenetic driver of melanoma growth and a biomarker of clinical virulence. We also have determined that the intermediate filament protein nestin correlates with tumorigenic and invasive melanoma growth. Here we examine the relationships between these two biomarkers. Immunohistochemistry staining of nestin and 5-hmC in 53 clinically annotated primary and metastatic patient melanomas revealed a significant negative correlation. Restoration of 5-hmC, as assessed in a human melanoma cell line by introducing full-length TET2 and TET2-mutated constructs, decreased nestin gene and protein expression in vitro. Genome-wide mapping using hydroxymethylated DNA immunoprecipitation sequencing disclosed significantly less 5-hmC binding in the 3' untranslated region of the nestin gene in melanoma compared to nevi, and 5-hmC binding in this region was significantly increased after TET2 overexpression in human melanoma cells in vitro. Our findings provide evidence suggesting that nestin regulation is negatively controlled epigenetically by TET2 via 5-hmC binding at the 3' untranslated region of the nestin gene, providing one potential pathway for understanding melanoma growth characteristics. Studies are now indicated to further define the interplay between 5-hmC, nestin expression, and melanoma virulence. PMID:27102770

  18. Neutron irradiation of human melanoma cells.

    PubMed

    Brown, K; Mountford, M H; Allen, B J; Mishima, Y; Ichihashi, M; Parsons, P

    1989-01-01

    The biological characteristics and in vitro radiosensitivity of melanoma cells to thermal neutrons were investigated as a guide to the effectiveness of boron neutron capture therapy. Plateau phase cultures of three human malignant melanoma-established cell lines were examined for cell density at confluence, doubling time, cell cycle parameters, chromosome constitution, and melanin content. Cell survival dose-response curves, for cells preincubated in the presence or absence of p-boronophenylalanine. HCl (10B1-BPA), were measured over the dose range 0.6-8.0 Gy (N + gamma). The neutron fluence rate was 2.6 x 10(9) n/cm2/s and the total dose rate 3.7 Gy/h (31% gamma). Considerable differences were observed in the morphology and cellular properties of the cell lines. Two cell lines (96E and 96L) were amelanotic, and one was melanotic (418). An enhanced killing for neutron irradiation was found only for the melanotic cells after 20 h preincubation with 10 micrograms/ml 10B1-BPA. In view of the doubling times of the cell lines of about 23 h (96E and 96L) or of 36 h (418), it seems likely that an increased boron uptake, and hence increased radiosensitivity, might result if the preincubation period with 10B1-BPA is extended to several hours longer than the respective cell cycle times. PMID:2798324

  19. Use of a Tissue Engineered Human Skin Model to Investigate the Effects of Wounding and of an Anti-Inflammatory on Melanoma Cell Invasion

    PubMed Central

    Marques, Claudia Mirian de Godoy; MacNeil, Sheila

    2016-01-01

    An increasing number of studies suggest inflammation stimulates tumour invasion. In melanoma, despite recent advances in targeted therapy and immunomodulatory therapies, this cancer remains difficult to treat. Our previous studies show melanoma cells interact with skin cells in their invasion into tissue engineered skin and suggest inflammation stimulates invasion. The aim of this study was to investigate the use of an anti-inflammatory on melanoma invasion. To do this we developed a wounded and inflamed in vitro 3D melanoma model in which to investigate the use of an anti-inflammatory on melanoma invasion. The tissue engineered skin model was based on human de-epidermised acellular dermis to which keratinocytes, fibroblasts and three different melanoma cell lines were added in various combinations. A simple incisional wound was made in the model and TNF-α and fibrin were added to simulate conditions of inflammation. Topical ibuprofen in a hydrogel was added and the extent of melanoma invasion into the dermis was assessed under the various conditions. The results showed that penetration of two of the cell lines (HBL and A375SM) into the tissue engineered skin was exacerbated by wounding and ibuprofen significantly decreased invasion of A375SM cells and slightly reduced invasion of HBL cells. A third cell line, C8161, was aggressively invasive under all conditions to an extent that was not influenced by wounding, TNF-α or the addition of ibuprofen. In summary, the results for one these cell lines (and a trend for a second cell line) support the hypothesis that a wound environment is conducive to melanoma invasion but the local addition of an anti-inflammatory drug such as ibuprofen may attenuate invasion. PMID:27270229

  20. Ex Vivo and In Vivo Imaging and Biodistribution of Aptamers Targeting the Human Matrix MetalloProtease-9 in Melanomas

    PubMed Central

    Kryza, David; Debordeaux, Frédéric; Azéma, Laurent; Hassan, Aref; Paurelle, Olivier; Schulz, Jürgen; Savona-Baron, Catherine; Charignon, Elsa; Bonazza, Pauline; Taleb, Jacqueline; Fernandez, Philippe; Janier, Marc; Toulmé, Jean Jacques

    2016-01-01

    The human Matrix MetalloProtease-9 (hMMP-9) is overexpressed in tumors where it promotes the release of cancer cells thus contributing to tumor metastasis. We raised aptamers against hMMP-9, which constitutes a validated marker of malignant tumors, in order to design probes for imaging tumors in human beings. A chemically modified RNA aptamer (F3B), fully resistant to nucleases was previously described. This compound was subsequently used for the preparation of F3B-Cy5, F3B-S-acetylmercaptoacetyltriglycine (MAG) and F3B-DOTA. The binding properties of these derivatives were determined by surface plasmon resonance and electrophoretic mobility shift assay. Optical fluorescence imaging confirmed the binding to hMMP-9 in A375 melanoma bearing mice. Quantitative biodistribution studies were performed at 30 min, 1h and 2 h post injection of 99mTc-MAG-aptamer and 111In-DOTA-F3B. 99mTc radiolabeled aptamer specifically detected hMMP-9 in A375 melanoma tumors but accumulation in digestive tract was very high. Following i.v. injection of 111In-DOTA-F3B, high level of radioactivity was observed in kidneys and bladder but digestive tract uptake was very limited. Tumor uptake was significantly (student t test, p<0.05) higher for 111In-DOTA-F3B with 2.0%ID/g than for the 111In-DOTA-control oligonucleotide (0.7%ID/g) with tumor to muscle ratio of 4.0. Such difference in tumor accumulation has been confirmed by ex vivo scintigraphic images performed at 1h post injection and by autoradiography, which revealed the overexpression of hMMP-9 in sections of human melanomas. These results demonstrate that F3B aptamer is of interest for detecting hMMP-9 in melanoma tumor. PMID:26901393

  1. Ex Vivo and In Vivo Imaging and Biodistribution of Aptamers Targeting the Human Matrix MetalloProtease-9 in Melanomas.

    PubMed

    Kryza, David; Debordeaux, Frédéric; Azéma, Laurent; Hassan, Aref; Paurelle, Olivier; Schulz, Jürgen; Savona-Baron, Catherine; Charignon, Elsa; Bonazza, Pauline; Taleb, Jacqueline; Fernandez, Philippe; Janier, Marc; Toulmé, Jean Jacques

    2016-01-01

    The human Matrix MetalloProtease-9 (hMMP-9) is overexpressed in tumors where it promotes the release of cancer cells thus contributing to tumor metastasis. We raised aptamers against hMMP-9, which constitutes a validated marker of malignant tumors, in order to design probes for imaging tumors in human beings. A chemically modified RNA aptamer (F3B), fully resistant to nucleases was previously described. This compound was subsequently used for the preparation of F3B-Cy5, F3B-S-acetylmercaptoacetyltriglycine (MAG) and F3B-DOTA. The binding properties of these derivatives were determined by surface plasmon resonance and electrophoretic mobility shift assay. Optical fluorescence imaging confirmed the binding to hMMP-9 in A375 melanoma bearing mice. Quantitative biodistribution studies were performed at 30 min, 1h and 2 h post injection of 99mTc-MAG-aptamer and 111In-DOTA-F3B. 99mTc radiolabeled aptamer specifically detected hMMP-9 in A375 melanoma tumors but accumulation in digestive tract was very high. Following i.v. injection of 111In-DOTA-F3B, high level of radioactivity was observed in kidneys and bladder but digestive tract uptake was very limited. Tumor uptake was significantly (student t test, p<0.05) higher for 111In-DOTA-F3B with 2.0%ID/g than for the 111In-DOTA-control oligonucleotide (0.7%ID/g) with tumor to muscle ratio of 4.0. Such difference in tumor accumulation has been confirmed by ex vivo scintigraphic images performed at 1h post injection and by autoradiography, which revealed the overexpression of hMMP-9 in sections of human melanomas. These results demonstrate that F3B aptamer is of interest for detecting hMMP-9 in melanoma tumor. PMID:26901393

  2. Human malignant melanoma heterotransplanted to nude mice.

    PubMed

    Tropé, C; Johnsson, J E; Alm, P; Landberg, T; Olsson, H; Wennerberg, J

    1981-01-01

    Five different human malignant melanoma were heterotransplanted subcutaneously to nude mice. When small tissue pieces were used 3 out of 5 tumors grew. Subcutaneous injections of suspended tumor cells were also made, but all failed to take. Metastatic or infiltrative growth was never seen in the mice observed for up to 2.5 months. The successful grafts largely retained the original morphologicaL features. The three successfully transplanted tumors could all be serially transferred with 100% tumor take. In one case passage time was reduced from 40 days to 15 days. As measured with 3H-thymidine incorporation the proliferation rate increased during the passages. These changes might be due to a selection of more rapidly growing tumor cells in the nudes. PMID:7312076

  3. Melanoma

    MedlinePlus

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

  4. 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

  5. Synthesis and biological evaluation of Fotemustine analogues on human melanoma cell lines.

    PubMed

    Winum, Jean Yves; Bouissière, Jean Luc; Passagne, Isabelle; Evrard, Alexandre; Montero, Véronique; Cuq, Pierre; Montero, Jean Louis

    2003-03-01

    Two new analogues of Fotemustine have been synthesized and tested on two melanoma cell lines. Compounds 4 and 8 proved to be more potent than the reference compound on A375 cell line which express the MGMT enzyme involved in the chemoresistance of tumoral cells. PMID:12667699

  6. Human hemokinin-1 promotes migration of melanoma cells and increases MMP-2 and MT1-MMP expression by activating tumor cell NK1 receptors.

    PubMed

    Zhang, Yixin; Li, Xiaofang; Li, Jingyi; Hu, Hui; Miao, Xiaokang; Song, Xiaoyun; Yang, Wenle; Zeng, Qian; Mou, Lingyun; Wang, Rui

    2016-09-01

    Receptors and their regulatory peptides are aberrantly expressed in tumors, suggesting a potential tumor therapy target. Human hemokinin-1 (hHK-1) is a tachykinin peptide ligand of the neurokinin-1 (NK1) receptor which is overexpressed in melanoma and other tumor tissues. Here, we investigated the role of hHK-1 and the NK1 receptor in melanoma cell migration. NK1 receptor expression was associated with melanoma metastatic potential. Treatment with hHK-1 significantly enhanced A375 and B16F10 melanoma cell migration and an NK1 receptor antagonist L732138 blocked this effect. MMP-2 and MT1-MMP expression were up-regulated in hHK-1-treated melanoma cells and cell signaling data suggested that hHK-1 induced phosphorylation of ERK1/2, JNK and p38 by way of PKC or PKA. Kinase activation led to increased MMP-2 and MT1-MMP expression and melanoma cell migration induced by hHK-1. Thus, hHK-1 and the NK1 receptor are critical to melanoma cell migration and each may be a promising chemotherapeutic target. PMID:27458061

  7. [Melanoma and Human Papillomaviruses: Is There an Outlook for Study?].

    PubMed

    Volgareva, G M; Mikhaylova, I N; Golovina, D A

    2016-01-01

    Melanoma is one of the most aggressive human malignant tumors. Its incidence and mortality are growing steadily. Ultraviolet irradiation is the main risk factor for melanoma involved in melanomagenesis. The probability of viral etiology of melanoma has been discussed. Human papillomaviruses (HPV) have been mentioned among candidates for its etiologic agents because some HPV types are the powerful carcinogens causing cervical cancer and other cancers. The review analyses the literature data on the association of melanoma with HPV Several groupsfound HPVin skin melanomas as well as in mucosa; viruses of high oncogenic risk were detected in some cases. For some organs the etiological role of high-risk HPV as inducers of invasive carcinomas is confirmed. These organs require special mention: cervix uteri, vulva, vagina, penis, anal region, and oral cavity. However in the majority of the studies in which viral DNA-positive melanomas were found, testing for viral genome expression was not done while this is the fact of primary importance. HPVare found in normal skin and mucous membranes thus creating justifiable threat of tumor specimen contamination with viral DNA in vivo. There are limited data on aggravation of the disease prognosis in papillomavirus-positive melanomas. However, any systematic observation of a sizeable patient group distinguished by that tumor type has not been performed yet. Viral E6 and E7 oncogenes of high-risk papillomaviruses were shown to be able to transform normal human melanocytes in vitro experiments. Thus, we can assume the presence of the association of melanoma with oncogenic HPV. The clinical significance of this problem is indisputable under the conditions of the steady increase in melanoma incidence and mortality rates in Russia and abroad. The problem requires further study. PMID:27522713

  8. Melanin content of hamster tissues, human tissues, and various melanomas

    SciTech Connect

    Watts, K.P.; Fairchild, R.G.; Slatkin, D.N.; Greenberg, D.; Packer, S.; Atkins, H.L.; Hannon, S.J.

    1981-02-01

    Melanin content (percentage by weight) was determined in both pigmented and nonpigmented tissues of Syrian golden hamsters bearing Greene melanoma. Melanin content was also measured in various other melanoma models (B-16 in C57 mice, Harding-Passey in BALB/c mice, and KHDD in C3H mice) and in nine human melanomas, as well as in selected normal tissues. The purpose was to evaluate the possible efficacy of chlorpromazine, which is known to bind to melanin, as a vehicle for boron transport in neutron capture therapy. Successful therapy would depend upon selective uptake and absolute concentration of borated compounds in tumors; these parameters will in turn depend upon melanin concentration in melanomas and nonpigmented ''background'' tissues. Hamster whole eyes, hamster melanomas, and other well-pigmented animal melanomas were found to contain 0.3 to 0.8% melanin by weight, whereas human melanomas varied from 0.1 to 0.9% (average, 0.35%). Other tissues, with the exception of skin, were lower in content by a factor of greater than or equal to30. Melanin pigment was extracted from tissues, and the melanin content was determined spectrophotometrically. Measurements were found to be sensitive to the presence of other proteins. Previous procedures for isolating and quantifying melanin often neglected the importance of removing proteins and other interfering nonmelanic substances.

  9. Aloe-emodin exerts a potent anticancer and immunomodulatory activity on BRAF-mutated human melanoma cells.

    PubMed

    Tabolacci, Claudio; Cordella, Martina; Turcano, Lorenzo; Rossi, Stefania; Lentini, Alessandro; Mariotti, Sabrina; Nisini, Roberto; Sette, Giovanni; Eramo, Adriana; Piredda, Lucia; De Maria, Ruggero; Facchiano, Francesco; Beninati, Simone

    2015-09-01

    Aim of this study was to extend the knowledge on the antineoplastic effect of aloe-emodin (AE), a natural hydroxyanthraquinone compound, both in metastatic human melanoma cell lines and in primary stem-like cells (melanospheres). Treatment with AE caused reduction of cell proliferation and induction of SK-MEL-28 and A375 cells differentiation, characterized by a marked increase of transamidating activity of transglutaminase whose expression remained unmodified. In vitro antimetastatic property of AE was evaluated by adhesion and Boyden chamber invasion assays. The effect of AE on melanoma cytokines/chemokines production was determined by a multiplex assay: interestingly AE showed an immunomodulatory activity through GM-CSF and IFN-γ production. We report also that AE significantly reduced the proliferation, stemness and invasive potential of melanospheres. Moreover, AE treatment significantly enhanced dabrafenib (a BRAF inhibitor) antiproliferative activity in BRAF mutant cell lines. Our results confirm that AE possesses remarkable antineoplastic properties against melanoma cells, indicating this anthraquinone as a promising agent for differentiation therapy of cancer, or as adjuvant in chemotherapy and targeted therapy. Further, its mechanisms of action support a potential efficacy of AE treatment to counteract resistance of BRAF-mutated melanoma cells to target therapy. PMID:26048310

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

    PubMed

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

    2014-03-01

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

  11. Potential role of human growth hormone in melanoma growth promotion.

    PubMed

    Handler, Marc Z; Ross, Andrew L; Shiman, Michael I; Elgart, George W; Grichnik, James M

    2012-10-01

    BACKGROUND Human growth hormone (HGH) and insulin-like growth factor-1 (IGF-1) have been shown to play a role in the malignant transformation and progression of a variety of cancers. HGH is also known to upregulate molecular signaling pathways implicated in the pathogenesis of melanoma. Although HGH has previously been implicated in promoting the clinical growth of both benign and malignant melanocytic neoplasms, to our knowledge there are no conclusive studies demonstrating an increased risk of melanoma following HGH therapy. Nevertheless, there are reports of melanoma developing subsequent to HGH coadministered with either other hormones or following irradiation. OBSERVATION A 49-year-old white man presented with a new pigmented papule that was diagnosed as melanoma. The patient reported using HGH for 3 months prior to the diagnosis. His 51-year-old wife, who also was white, had also been using exogenous HGH for 3 months and had been diagnosed as having a melanoma 2 weeks prior. CONCLUSIONS Given the unlikelihood of 2 unrelated people developing melanoma within a short time span, it is reasonable to assume that a common environmental component (HGH or other shared exposure) contributed to the development of both melanomas. Because of the increased use of exogenous HGH as an antiaging agent, it is important to be aware of the growth-promoting effects of this hormone. Until better data are available that determines the true risk of exogenous HGH, its use as an antiaging agent merits increased surveillance. PMID:23069955

  12. Growth and invasion of human melanomas in human skin grafted to immunodeficient mice.

    PubMed Central

    Juhasz, I.; Albelda, S. M.; Elder, D. E.; Murphy, G. F.; Adachi, K.; Herlyn, D.; Valyi-Nagy, I. T.; Herlyn, M.

    1993-01-01

    An orthotopic model of human melanoma was developed in which malignant cells were injected into human skin grafted to nude and SCID mice. Melanoma cells proliferated and invaded the human skin grafts with characteristic patterns. Three of six melanomas grew as multiple nodules and infiltered the grafts without major architectural changes in the dermis, whereas the others invaded the dermis along collagen fibers with prominent endothelial vessels. By contrast, melanoma cells inoculated into mouse skin grew as diffusely expanding nodules that did not invade the murine dermis. In human skin grafts, human melanoma cells were angiogenic for human blood vessels, and murine vessels were only found at the periphery of grafts. Tumor cells invaded the human vessels, and four out of seven cell lines metastasized to lungs, suggesting that this model is useful to determine in vivo the interactions between normal and malignant human cells. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8342600

  13. Pre-clinical assessment of A-674563 as an anti-melanoma agent.

    PubMed

    Zou, Ying; Fan, Guobiao; Wang, Xuemin

    2016-08-12

    The present study aims to investigate the anti-melanoma activity by an Akt1 specific inhibitor A-674563. We showed that A-674563 was anti-proliferative and cytotoxic when added to human melanoma cells (A375, WM-115 and SK-Mel-2 lines). A-674563 induced caspase-dependent apoptotic death of human melanoma cells, and its cytotoxicity was inhibited with pre-treatment of caspase inhibitors. Further, A-674563 treatment blocked Akt and its downstream S6 Kinase 1 (S6K1) activation in A375 melanoma cells. Significantly, restoring Akt-S6K1 activation via introduction of constitutively-active Akt1 (ca-Akt1) only partially attenuated A-674563's cytotoxicity against A375 cells. Further, A-674563 induced pro-apoptotic ceramide production in A375 cells. Significantly, sphingosine-1-phosphate (S1P) inhibited A-674563-induced ceramide production and subsequent A375 cell apoptosis. On the other hand, co-treatment with the glucosylceramide synthase (GCS) inhibitor PDMP or the cell permeable short-chain ceramide (C6) potentiated A-674563's cytotoxicity against A375 cells. In vivo, A-674563 oral gavage inhibited A375 xenograft growth in severe combined immunodeficiency (scid) mice. Akt inactivation, caspase-3 activation and ceramide production were also observed in A-674563-treated A375 xenografts. Together, these results suggest that A-674563 exerts potent anti-melanoma activity, involving Akt-dependent and Akt-independent mechanisms. PMID:26970307

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

    PubMed

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

    2009-12-01

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

  15. 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

  16. Oxidative stress inhibits distant metastasis by human melanoma cells

    PubMed Central

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

    2015-01-01

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

  17. Biologic and therapeutic significance of MYB expression in human melanoma.

    PubMed Central

    Hijiya, N; Zhang, J; Ratajczak, M Z; Kant, J A; DeRiel, K; Herlyn, M; Zon, G; Gewirtz, A M

    1994-01-01

    We investigated the therapeutic potential of employing antisense oligodeoxynucleotides to target the disruption of MYB, a gene which has been postulated to play a pathogenetic role in cutaneous melanoma. We found that MYB was expressed at low levels in several human melanoma cell lines. Also, growth of representative lines in vitro was inhibited in a dose- and sequence-dependent manner by targeting the MYB gene with unmodified or phosphorothioate-modified antisense oligodeoxynucleotides. Inhibition of cell growth correlated with specific decrease of MYB mRNA. In SCID mice bearing human melanoma tumors, infusion of MYB antisense transiently suppressed MYB gene expression but effected long-term growth suppression of transplanted tumor cells. Toxicity of the oligodeoxynucleotides was minimal in mice, even when targeted to the murine Myb gene. These results suggest that the MYB gene may play an important, though undefined, role in the growth of at least some human melanomas. Inhibition of MYB expression might be of use in the treatment of this disease. Images PMID:8183937

  18. Biologic and Therapeutic Significance of MYB Expression in Human Melanoma

    NASA Astrophysics Data System (ADS)

    Hijiya, Nobuko; Zhang, Jin; Ratajczak, Mariusz Z.; Kant, Jeffrey A.; Deriel, Kim; Herlyn, Meenhard; Zon, Gerald; Gewirtz, Alan M.

    1994-05-01

    We investigated the therapeutic potential of employing antisense oligodeoxynucleotides to target the disruption of MYB, a gene which has been postulated to play a pathogenetic role in cutaneous melanoma. We found that MYB was expressed at low levels in several human melanoma cell lines. Also, growth of representative lines in vitro was inhibited in a dose- and sequence-dependent manner by targeting the MYB gene with unmodified or phosphorothioate-modified antisense oligodeoxynucleotides. Inhibition of cell growth correlated with specific decrease of MYB mRNA. In SCID mice bearing human melanoma tumors, infusion of MYB antisense transiently suppressed MYB gene expression but effected long-term growth suppression of transplanted tumor cells. Toxicity of the oligodeoxynucleotides was minimal in mice, even when targeted to the murine Myb gene. These results suggest that the MYB gene may play an important, though undefined, role in the growth of at least some human melanomas. Inhibition of MYB expression might be of use in the treatment of this disease.

  19. Thiostrepton is an Inducer of Oxidative and Proteotoxic Stress that Impairs Viability of Human Melanoma Cells but not Primary Melanocytes

    PubMed Central

    Qiao, Shuxi; Lamore, Sarah D.; Cabello, Christopher M.; Lesson, Jessica L.; Muñoz-Rodriguez, José L.; Wondrak, Georg T.

    2012-01-01

    Pharmacological induction of oxidative and proteotoxic stress has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Guided by a differential phenotypic drug screen for novel lead compounds that selectively induce melanoma cell apoptosis without compromising viability of primary human melanocytes, we have focused on the cyclic pyridinyl-polythiazolyl peptide-antimicrobial thiostrepton. Using comparative gene expression-array analysis, the early cellular stress response induced by thiostrepton was examined in human A375 metastatic melanoma cells and primary melanocytes. Thiostrepton displayed selective antimelanoma activity causing early induction of proteotoxic stress with massive upregulation of heat shock (HSPA6, HSPA1A, DNAJB4, HSPB1, HSPH1, HSPA1L, CRYAB, HSPA5, DNAJA1), oxidative stress (HMOX1, GSR, SOD1), and ER stress response (DDIT3) gene expression, confirmed by immunodetection (Hsp70, Hsp70B′, HO-1, phospho-eIF2α). Moreover, upregulation of p53, proapoptotic modulation of Bcl-2 family members (Bax, Noxa, Mcl-1, Bcl-2), and induction of apoptotic cell death were observed. Thiostrepton rapidly induced cellular oxidative stress followed by inactivation of chymotrypsin-like proteasomal activity and melanoma cell-directed accumulation of ubiquitinated proteins, not observed in melanocytes that were resistant to thiostrepton-induced apoptosis. Proteotoxic and apoptogenic effects were fully antagonized by antioxidant intervention. In RPMI 8226 multiple myeloma cells, known to be exquisitely sensitive to proteasome inhibition, early proteotoxic and apoptogenic effects of thiostrepton were confirmed by array analysis indicating pronounced upregulation of heat shock response gene expression. Our findings demonstrate that thiostrepton displays dual activity as a selective prooxidant and proteotoxic chemotherapeutic, suggesting feasibility of experimental intervention targeting metastatic melanoma and other

  20. Melanoma

    MedlinePlus

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

  1. Gastrin exerts pleiotropic effects on human melanoma cell biology.

    PubMed

    Mathieu, Véronique; Mijatovic, Tatjana; van Damme, Marc; Kiss, Robert

    2005-10-01

    The effects of gastrin (G17) on the growth and migration factors of four human melanoma cell lines (HT-144, C32, G-361, and SKMEL-28) were investigated. The expression patterns of cholecystokinin (CCK)(A), CCK(B), and CCK(C) gastrin receptors were investigated in these cells and in seven clinical samples by means of reverse transcription polymerase chain reaction. Melanoma cells appear to express mRNA for CCK(C) receptors, but not for CCK(A) or CCK(B) receptors. Although gastrin does not significantly modify the growth characteristics of the cell lines under study, it significantly modifies their cell migration characteristics. These modifications occur at adhesion level by modifying the expression levels of alpha(v) and beta3 integrins, at motility level by modifying the organization of the actin cytoskeleton, and at invasion level by modifying the expression levels of matrix metalloproteinase 14. We recently demonstrated the presence of CCK(B) receptors in mouse endothelial cells involved in glioblastoma neoangiogenesis. Chronic in vivo administration of a selective CCK(B) receptor antagonist to mice bearing xenografts of human C32 melanoma cells significantly decreased levels of neoangiogenesis, resulting in considerable delays in the growth of these C32 xenografts. In conclusion, our study identifies the pleiotropic effects of gastrin on melanoma cell biology. PMID:16242076

  2. UVB induces atypical melanocytic lesions and melanoma in human skin.

    PubMed Central

    Atillasoy, E. S.; Seykora, J. T.; Soballe, P. W.; Elenitsas, R.; Nesbit, M.; Elder, D. E.; Montone, K. T.; Sauter, E.; Herlyn, M.

    1998-01-01

    A direct causal relationship between ultraviolet (UV) light in the B range and melanoma development has not been demonstrated in humans; this study aims to establish causality. A total of 158 RAG-1 mice, grafted with human newborn foreskin, were separated into four groups and observed for a median of 10 months: 1) no treatment, 2) a single treatment with 7,12-dimethyl(a)benzanthracene (DMBA), 3) UVB irradiation at 500 J/m2 alone, three times weekly, and 4) a combination of DMBA and UVB. Twenty-three percent of 40 normal human skin grafts treated with UVB only and 38% of 48 grafts treated with the combination of DMBA and UVB developed solar lentigines within 5 to 10 months of treatment. Melanocytic hyperplasia was found in 73% of all UVB-treated xenografts. Histological melanocytic changes resembling lentigo and lentigo maligna were seen in several skin grafts treated with both DMBA and UVB. In one graft of an animal treated with a combination of DMBA and UVB, a human malignant melanoma, nodular type, developed. This experimental system demonstrates that chronic UVB irradiation with or without an initiating carcinogen can induce human melanocytic lesions, including melanoma. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:9588887

  3. Topical Delivery of 5-Fluorouracil from Pheroid™ Formulations and the In Vitro Efficacy Against Human Melanoma.

    PubMed

    Chinembiri, Tawona N; Gerber, Minja; du Plessis, Lissinda; du Preez, Jan; du Plessis, Jeanetta

    2015-12-01

    Drug delivery vehicles can influence the topical delivery and the efficacy of an active pharmaceutical ingredient (API). In this study, the influence of Pheroid™ technology, which is a unique colloidal drug delivery system, on the skin permeation and antimelanoma efficacy of 5-fluorouracil were investigated. Lotions containing Pheroid™ with different concentrations of 5-fluorouracil were formulated then used in Franz cell skin diffusion studies and tape stripping. The in vitro efficacy of 5-fluorouracil against human melanoma cells (A375) was investigated using a flow cytometric apoptosis assay. Statistically significant concentrations of 5-fluorouracil diffused into and through the skin with Pheroid™ formulations resulting in an enhanced in vitro skin permeation from the 4.0% 5-fluorouracil lotion (p < 0.05). The stratum corneum-epidermis and epidermis-dermis retained 5-fluorouracil concentrations of 2.31 and 6.69 μg/ml, respectively, after a diffusion study with the 4.0% Pheroid™ lotion. Subsequent to the apoptosis assay, significant differences were observed between the effect of 13.33 μg/ml 5-fluorouracil in Pheroid™ lotion and the effects of the controls. The results obtained suggest that the Pheroid™ drug delivery system possibly enhances the flux and delivery of 5-fluorouracil into the skin. Therefore, using Pheroid™ could possibly be advantageous with respect to topical delivery of 5-fluorouracil. PMID:25956486

  4. Identification of nucleolin as a lipid-raft-dependent β1-integrin-interacting protein in A375 cell migration.

    PubMed

    Bi, Jiajia; Wang, Ruifei; Zhang, Yue; Han, Xiaoqing; Ampah, Khamal Kwesi; Liu, Wenguang; Zeng, Xianlu

    2013-12-01

    Lipid rafts are related to cell surface receptor function. Integrin is a major surface receptor protein in cell adhesion and migration on the extracellular matrix (ECM). Here, we showed that lipid rafts played a critical role in human melanoma A375 cell spreading and migration on fibronectin; an important component of the ECM that interacts with β1 integrin. We found that the disruption of lipid rafts did not markedly inhibit the expression and activation of β1 integrin. By coimmunoprecipitation and mass spectrometry, we investigated the influence of lipid rafts on the β1 integrin complex and identified nucleolin as a potential lipid-raft-dependent β1-integrin-interacting protein. Upon confirmation of the interaction between β1 integrin and nucleolin, further studies revealed that nucleolin colocalized with β1 integrin in lipid rafts and raft disruption interrupted their association. In addition, knockdown of nucleolin markedly attenuated A375 cell spreading and migration on fibronectin. Taken together, we demonstrated that nucleolin is a critical lipid-raft-dependent β1-integrin-interacting protein in A375 cell spreading and migration on fibronectin. PMID:24292944

  5. Hibiscus sabdariffa leaf polyphenolic extract induces human melanoma cell death, apoptosis, and autophagy.

    PubMed

    Chiu, Chun-Tang; Hsuan, Shu-Wen; Lin, Hui-Hsuan; Hsu, Cheng-Chin; Chou, Fen-Pi; Chen, Jing-Hsien

    2015-03-01

    Melanoma is the least common but most fatal form of skin cancer. Previous studies have indicated that an aqueous extract of Hibiscus sabdariffa leaves possess hypoglycemic, hypolipidemic, and antioxidant effects. In this study, we want to investigate the anticancer activity of Hibiscus leaf polyphenolic (HLP) extract in melanoma cells. First, HLP was exhibited to be rich in epicatechin gallate (ECG) and other polyphenols. Apoptotic and autophagic activities of HLP and ECG were further evaluated by DAPI stain, cell-cycle analysis, and acidic vascular organelle (AVO) stain. Our results revealed that both HLP and ECG induced the caspases cleavages, Bcl-2 family proteins regulation, and Fas/FasL activation in A375 cells. In addition, we also revealed that the cells presented AVO-positive after HLP treatments. HLP could increase the expressions of autophagy-related proteins autophagy-related gene 5 (ATG5), Beclin1, and light chain 3-II (LC3-II), and induce autophagic cell death in A375 cells. These data indicated that the anticancer effect of HLP, partly contributed by ECG, in A375 cells. HLP potentially could be developed as an antimelanoma agent. PMID:25694272

  6. [Melanoma].

    PubMed

    Uhara, Hisashi

    2016-04-01

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

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

    PubMed

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

    2016-01-01

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

  8. 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

  9. Pentoxifylline triggers autophagy via ER stress response that interferes with Pentoxifylline induced apoptosis in human melanoma cells.

    PubMed

    Sharma, Kapil; Ishaq, Mohammad; Sharma, Gaurav; Khan, Mohammad Aslam; Dutta, Rajesh Kumar; Majumdar, Sekhar

    2016-03-01

    Pentoxifylline (PTX), a non-specific phosphodiesterase inhibitor is known to inhibit the growth of various cancer cells including melanoma. Here in this study, we have found that PTX induces autophagy in human melanoma cell lines (A375 and MeWo). Induction of autophagy is associated with the increase in Atg5 expression as knockdown of Atg5 effectively inhibited PTX mediated autophagy. A decrease in mTOR activation was also observed after PTX treatment. We observed that autophagy was activated as a downstream effector mechanism of ER stress induced by PTX. ER stress response was confirmed by upregulation of IRE-1α, GRP78 and CHOP expression. PTX treatment also resulted in an increase in intracellular calcium (Ca(2+)) level. Ca(2+) is the central player as blocking Ca(2+) by intracellular calcium chelator (BAPTA-AM) effectively inhibited the PTX induced ER stress response as well as autophagy. Moreover, silencing of CHOP also resulted in autophagy inhibition with a decrease in Atg5 expression. Collectively, PTX triggers ER stress response followed by induction of autophagy via involvement of Ca(2+)→CHOP→Atg5 signalling cascade. Interestingly, inhibition of intracellular calcium level by BAPTA-AM significantly increased PTX mediated cell death by augmenting intrinsic apoptotic pathway. Inhibition of autophagy by the ATG5 siRNA and pharmacological inhibitor, chloroquine also enhances PTX induced cell death. Taken together, our results clearly indicate that activation of ER stress response and autophagy provides resistance to PTX mediated apoptosis, and thus, interferes with the anticancer activity of PTX in human melanoma cells. PMID:26793997

  10. Epigenetic Impacts of Ascorbate on Human Metastatic Melanoma Cells

    PubMed Central

    Venturelli, Sascha; Sinnberg, Tobias W.; Berger, Alexander; Noor, Seema; Levesque, Mitchell Paul; Böcker, Alexander; Niessner, Heike; Lauer, Ulrich M.; Bitzer, Michael; Garbe, Claus; Busch, Christian

    2014-01-01

    In recent years, increasing evidence has emerged demonstrating that high-dose ascorbate bears cytotoxic effects on cancer cells in vitro and in vivo, making ascorbate a pro-oxidative drug that catalyzes hydrogen peroxide production in tissues instead of acting as a radical scavenger. This anticancer effect of ascorbate is hypoxia-inducible factor-1α- and O2-dependent. However, whether the intracellular mechanisms governing this effect are modulated by epigenetic phenomena remains unknown. We treated human melanoma cells with physiological (200 μM) or pharmacological (8 mM) ascorbate for 1 h to record the impact on DNA methyltransferase (DNMT)-activity, histone deacetylases (HDACs), and microRNA (miRNA) expression after 12 h. The results were analyzed with the MIRUMIR online tool that estimates the power of miRNA to serve as potential biomarkers to predict survival of cancer patients. FACS cell-cycle analyses showed that 8 mM ascorbate shifted BLM melanoma cells toward the sub-G1 fraction starting at 12 h after an initial primary G2/M arrest, indicative for secondary apoptosis induction. In pharmacological doses, ascorbate inhibited the DNMT activity in nuclear extracts of MeWo and BLM melanoma cells, but did not inhibit human HDAC enzymes of classes I, II, and IV. The expression of 151 miRNAs was altered 12 h after ascorbate treatment of BLM cells in physiological or pharmacological doses. Pharmacological doses up-regulated 32 miRNAs (≥4-fold) mainly involved in tumor suppression and drug resistance in our preliminary miRNA screening array. The most prominently up-regulated miRNAs correlated with a significantly increased overall survival of breast cancer or nasopharyngeal carcinoma patients of the MIRUMIR database with high expression of the respective miRNA. Our results suggest a possible epigenetic signature of pharmacological doses of ascorbate in human melanoma cells and support further pre-clinical and possibly even clinical evaluation of

  11. Different activities of unscheduled DNA synthesis in human melanoma and bone marrow cells

    SciTech Connect

    Lewensohn, R.; Ringborg, U.; Hansson, J.

    1982-01-01

    Unscheduled DNA synthesis (UDS) indicated by melphalan was studied in freshly collected tumor cells from human melanoma metastases. Comparative studies were done on human bone marrow blast cells. Significant levels of UDS comparable with those in myeloblasts were found in only two of eight melanoma cell populations. This difference between melanoma and blast cells was not related to different cellular uptake of melphalan. When UDS was induced by ultraviolet irradiation, significant levels of UDS were found in all melanoma and blast cell populations studied. Also, in a human melanoma cell line, high levels of UDS were found after exposure to ultraviolet irradiation, while treatment with melphalan did not result in detectable levels of UDS. Possible explanations for the divergent results of UDS in melphalan-exposed melanoma cells are discussed.

  12. Theranostics of Malignant Melanoma with 64CuCl2

    PubMed Central

    Qin, Chunxia; Liu, Hongguang; Chen, Kai; Hu, Xiang; Ma, Xiaowei; Lan, Xiaoli; Zhang, Yongxue; Cheng, Zhen

    2015-01-01

    Human copper transporter 1 (CTR1) is overexpressed in a variety of cancers. This study aimed to evaluate the use of 64CuCI2 as a theranostic agent for PET and radionuclide therapy of malignant melanoma. Methods CTR1 expression levels were detected by Western blot analysis of a group of tumor cell lines. Two melanoma cell lines (B16F10 and A375M) that highly expressed CTR1 were then selected to study the uptake and efflux of 64CuCI2. Mice bearing B16F10 or A375M tumors (n = 4 for each group) were subjected to 5 min of static whole-body PET scans at different time points after intravenous injection of 64CuCI2. Dynamic scans were also obtained for B16F10 tumor-bearing mice. All mice were sacrificed at 72 h after injection of 64CuCI2, and biodistribution studies were performed. Mice bearing B16F10 or A375M tumors were further subjected to 64CuCI2 radionuclide therapy. Specifically, when the tumor size reached 0.5–0.8 cm in diameter, tumor-bearing mice were systemically administered 64CuCI2 (~74 MBq) or phosphate-buffered saline, and tumor sizes were monitored over the treatment period. Results CTR1 was found to be overexpressed in the cancer cell lines tested at different levels, and high expression levels in melanoma cells and tissues were observed (melanotic B16F10 and amelanotic A375M). 64CuCI2 displayed high and specific uptake in B16F10 and A375M cells. In vivo 64CuCI2 PET imaging demonstrated that both B16F10 and A375M tumors were clearly visualized. Radionuclide treatment studies showed that the tumor growth in both the B16F10 and the A375M models under 64CuCI2 treatment were much slower than that of the control group. Conclusion Both melanotic and amelanotic melanomas (B16F10 and A375M) tested were found to overexpress CTR1. The tumors can be successfully visualized by 64CuCI2 PET and further treated by 64CuCI2, highlighting the high potential of using 64CuCI2 as a theranostic agent for the management of melanoma. PMID:24627435

  13. Detection of circulating melanoma cells in human blood using photoacoustic flowmetry.

    PubMed

    Weight, Ryan M; Dale, Paul S; Viator, John A

    2009-01-01

    Detection of circulating tumor cells (CTC's) in human blood and lymph systems has the potential to aid clinical decision making in the treatment of cancer. The presence of CTC's may signify the onset of metastasis, indicate relapse, or may be used to monitor disease progression. A photoacoustic flowmetry system was designed and tested for detecting circulating melanoma cells (CMC's) by exploiting the broadband absorption spectrum of melanin within CMC's. The device was tested on cultured melanoma cells in saline suspension and in a Stage IV melanoma patient. The device showed a detection threshold of a single melanotic melanoma cell from culture. Transient photoacoustic events were detected in a sample derived from a Stage IV melanoma patient that corresponded to particles passing through the laser beam path, indicating the presence of single melanoma cells in the human circulatory system. PMID:19965119

  14. Human Single-Chain Fv Immunoconjugates Targeted to a Melanoma-Associated Chondroitin Sulfate Proteoglycan Mediate Specific Lysis of Human Melanoma Cells by Natural Killer Cells and Complement

    NASA Astrophysics Data System (ADS)

    Wang, Baiyang; Chen, Yi-Bin; Ayalon, Oran; Bender, Jeffrey; Garen, Alan

    1999-02-01

    Two antimelanoma immunoconjugates containing a human single-chain Fv (scFv) targeting domain conjugated to the Fc effector domain of human IgG1 were synthesized as secreted two-chain molecules in Chinese hamster ovary and Drosophila S2 cells, and purified by affinity chromatography on protein A. The scFv targeting domains originally were isolated as melanoma-specific clones from a scFv fusion-phage library, derived from the antibody repertoire of a vaccinated melanoma patient. The purified immunoconjugates showed similar binding specificity as did the fusion-phage clones. Binding occurred to human melanoma cells but not to human melanocytes or to several other types of normal cells and tumor cells. A 250-kDa melanoma protein was immunoprecipitated by the immunoconjugates and analyzed by mass spectrometry, using two independent procedures. A screen of protein sequence databases showed an exact match of several peptide masses between the immunoprecipitated protein and the core protein of a chondroitin sulfate proteoglycan, which is expressed on the surface of most human melanoma cells. The Fc effector domain of the immunoconjugates binds natural killer (NK) cells and also the C1q protein that initiates the complement cascade; both NK cells and complement can activate powerful cytolytic responses against the targeted tumor cells. An in vitro cytolysis assay was used to test for an immunoconjugate-dependent specific cytolytic response against cultured human melanoma cells by NK cells and complement. The melanoma cells, but not the human fibroblast cells used as the control, were efficiently lysed by both NK cells and complement in the presence of the immunoconjugates. The in vitro results suggest that the immunoconjugates also could activate a specific cytolytic immune response against melanoma tumors in vivo.

  15. Photosensitized rose Bengal-induced phototoxicity on human melanoma cell line under natural sunlight exposure.

    PubMed

    Srivastav, Ajeet K; Mujtaba, Syed Faiz; Dwivedi, Ashish; Amar, Saroj K; Goyal, Shruti; Verma, Ankit; Kushwaha, Hari N; Chaturvedi, Rajnish K; Ray, Ratan Singh

    2016-03-01

    Rose Bengal (RB) is an anionic water-soluble xanthene dye, which used for many years to assess eye cornea and conjunctiva damage. RB showed strong absorption maxima (λmax) under visible light followed by UV-B and UV-A. RB under sunlight exposure showed a time-dependent photodegradation. Our results show that photosensitized RB generates (1)O2 via Type-II photodynamic pathway and induced DNA damage under sunlight/UV-R exposure. 2'dGuO degradation, micronuclei formation, and single- and double-strand breakage were the outcome of photogenotoxicity caused by RB. Quenching studies with NaN3 advocate the involvement of (1)O2 in RB photogenotoxicity. RB induced linoleic acid photoperoxidation, which was parallel to (1)O2-mediated DNA damage. Oxidative stress in A375 cell line (human melanoma cell line) was detected through DCF-DA assay. Photosensitized RB decreased maximum cellular viability under sunlight followed by UV-B and UV-A exposures. Apoptosis was detected as a pattern of cell death through the increased of caspase-3 activity, decreased mitochondrial membrane potential, and PS translocation through inner to outer plasma membrane. Increased cytosolic levels of Bax also advocate the apoptotic cell death. We propose a p53-mediated apoptosis via increased expression of Bax gene and protein. Thus, the exact mechanism behind RB phototoxicity was the involvement of (1)O2, which induced oxidative stress-mediated DNA and membrane damage, finally apoptotic cell death under natural sunlight exposure. The study suggests that after the use of RB, sunlight exposure may avoid to prevent from its harmful effects. PMID:26866294

  16. Endoplasmic reticulum stress-induced autophagy determines the susceptibility of melanoma cells to dabrafenib

    PubMed Central

    Ji, Chao; Zhang, Ziping; Chen, Lihong; Zhou, Kunli; Li, Dongjun; Wang, Ping; Huang, Shuying; Gong, Ting; Cheng, Bo

    2016-01-01

    Melanoma is one of the deadliest skin cancers and accounts for most skin-related deaths due to strong resistance to chemotherapy drugs. In the present study, we investigated the mechanisms of dabrafenib-induced drug resistance in human melanoma cell lines A375 and MEL624. Our studies support that both endoplasmic reticulum (ER) stress and autophagy were induced in the melanoma cells after the treatment with dabrafenib. In addition, ER stress-induced autophagy protects melanoma cells from the toxicity of dabrafenib. Moreover, inhibition of both ER stress and autophagy promote the sensitivity of melanoma cells to dabrafenib. Taken together, the data suggest that ER stress-induced autophagy determines the sensitivity of melanoma cells to dabrafenib. These results provide us with promising evidence that the inhibition of autophagy and ER stress could serve a therapeutic effect for the conventional dabrafenib chemotherapy. PMID:27536070

  17. Gamma-interferon-inducible lysosomal thiol reductase is upregulated in human melanoma.

    PubMed

    Nguyen, Jennifer; Bernert, Richard; In, Kevin; Kang, Paul; Sebastiao, Noemi; Hu, Chengcheng; Hastings, K Taraszka

    2016-04-01

    T-cell-mediated immunity has the ability to produce durable antimelanoma responses, resulting in improved survival of patients with advanced melanoma. Antigen presentation is a key determinant of T-cell responses. Gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of multiple melanoma antigens to CD4+ T cells. However, GILT expression in melanoma has not been defined. We evaluated GILT and MHC class II expression in human primary and metastatic melanomas and nevi using immunohistochemical analysis. GILT staining in melanocytes was observed in 70% of primary and 58% of metastatic melanomas versus 0% of nevi. When present, the GILT staining intensity in melanocytes was typically faint. Both GILT and MHC class II expression were increased in melanocytes of primary and metastatic melanomas compared with nevi. GILT staining in antigen-presenting cells (APCs) was detected in 100% of primary and metastatic melanomas versus 31% of nevi, and it was typically intense. GILT expression was increased in APCs of primary and metastatic melanomas compared with nevi, whereas MHC class II had equivalent high expression in APCs of all melanocytic lesions. GILT staining in keratinocytes was detected in 67% of primary melanomas versus 14% of nevi and 6% of metastatic melanomas. GILT, but not MHC class II, expression was increased in keratinocytes of primary melanomas compared with nevi and metastases. GILT expression is anticipated to result in improved presentation of melanoma antigens and more effective antimelanoma T-cell responses. GILT expression may be a biomarker of immune recognition of melanoma. PMID:26930048

  18. Sensitivity to sodium arsenite in human melanoma cells depends upon susceptibility to arsenite-induced mitotic arrest

    SciTech Connect

    McNeely, Samuel C.; Belshoff, Alex C.; Taylor, B. Frazier; Fan, Teresa W-M.; McCabe, Michael J.; Pinhas, Allan R.

    2008-06-01

    Arsenic induces clinical remission in patients with acute promyelocytic leukemia and has potential for treatment of other cancers. The current study examines factors influencing sensitivity to arsenic using human malignant melanoma cell lines. A375 and SK-Mel-2 cells were sensitive to clinically achievable concentrations of arsenite, whereas SK-Mel-3 and SK-Mel-28 cells required supratherapeutic levels for toxicity. Inhibition of glutathione synthesis, glutathione S-transferase (GST) activity, and multidrug resistance protein (MRP) transporter function attenuated arsenite resistance, consistent with studies suggesting that arsenite is extruded from the cell as a glutathione conjugate by MRP-1. However, MRP-1 was not overexpressed in resistant lines and GST-{pi} was only slightly elevated. ICP-MS analysis indicated that arsenite-resistant SK-Mel-28 cells did not accumulate less arsenic than arsenite-sensitive A375 cells, suggesting that resistance was not attributable to reduced arsenic accumulation but rather to intrinsic properties of resistant cell lines. The mode of arsenite-induced cell death was apoptosis. Arsenite-induced apoptosis is associated with cell cycle alterations. Cell cycle analysis revealed arsenite-sensitive cells arrested in mitosis whereas arsenite-resistant cells did not, suggesting that induction of mitotic arrest occurs at lower intracellular arsenic concentrations. Higher intracellular arsenic levels induced cell cycle arrest in the S-phase and G{sub 2}-phase in SK-Mel-3 and SK-Mel-28 cells, respectively. The lack of arsenite-induced mitotic arrest in resistant cell lines was associated with a weakened spindle checkpoint resulting from reduced expression of spindle checkpoint protein BUBR1. These data suggest that arsenite has potential for treatment of solid tumors but a functional spindle checkpoint is a prerequisite for a positive response to its clinical application.

  19. A Novel Fully Humanized 3D Skin Equivalent to Model Early Melanoma Invasion.

    PubMed

    Hill, David S; Robinson, Neil D P; Caley, Matthew P; Chen, Mei; O'Toole, Edel A; Armstrong, Jane L; Przyborski, Stefan; Lovat, Penny E

    2015-11-01

    Metastatic melanoma remains incurable, emphasizing the acute need for improved research models to investigate the underlying biologic mechanisms mediating tumor invasion and metastasis, and to develop more effective targeted therapies to improve clinical outcome. Available animal models of melanoma do not accurately reflect human disease and current in vitro human skin equivalent models incorporating melanoma cells are not fully representative of the human skin microenvironment. We have developed a robust and reproducible, fully humanized three-dimensional (3D) skin equivalent comprising a stratified, terminally differentiated epidermis and a dermal compartment consisting of fibroblast-generated extracellular matrix. Melanoma cells incorporated into the epidermis were able to invade through the basement membrane and into the dermis, mirroring early tumor invasion in vivo. Comparison of our novel 3D melanoma skin equivalent with melanoma in situ and metastatic melanoma indicates that this model accurately recreates features of disease pathology, making it a physiologically representative model of early radial and vertical growth-phase melanoma invasion. PMID:26330548

  20. Lymphatic vessels regulate immune microenvironments in human and murine melanoma.

    PubMed

    Lund, Amanda W; Wagner, Marek; Fankhauser, Manuel; Steinskog, Eli S; Broggi, Maria A; Spranger, Stefani; Gajewski, Thomas F; Alitalo, Kari; Eikesdal, Hans P; Wiig, Helge; Swartz, Melody A

    2016-09-01

    Lymphatic remodeling in tumor microenvironments correlates with progression and metastasis, and local lymphatic vessels play complex and poorly understood roles in tumor immunity. Tumor lymphangiogenesis is associated with increased immune suppression, yet lymphatic vessels are required for fluid drainage and immune cell trafficking to lymph nodes, where adaptive immune responses are mounted. Here, we examined the contribution of lymphatic drainage to tumor inflammation and immunity using a mouse model that lacks dermal lymphatic vessels (K14-VEGFR3-Ig mice). Melanomas implanted in these mice grew robustly, but exhibited drastically reduced cytokine expression and leukocyte infiltration compared with those implanted in control animals. In the absence of local immune suppression, transferred cytotoxic T cells more effectively controlled tumors in K14-VEGFR3-Ig mice than in control mice. Furthermore, gene expression analysis of human melanoma samples revealed that patient immune parameters are markedly stratified by levels of lymphatic markers. This work suggests that the establishment of tumor-associated inflammation and immunity critically depends on lymphatic vessel remodeling and drainage. Moreover, these results have implications for immunotherapies, the efficacies of which are regulated by the tumor immune microenvironment. PMID:27525437

  1. Proteomic Analysis of Proton Beam Irradiated Human Melanoma Cells

    PubMed Central

    Kedracka-Krok, Sylwia; Jankowska, Urszula; Elas, Martyna; Sowa, Urszula; Swakon, Jan; Cierniak, Agnieszka; Olko, Pawel; Romanowska-Dixon, Bozena; Urbanska, Krystyna

    2014-01-01

    Proton beam irradiation is a form of advanced radiotherapy providing superior distributions of a low LET radiation dose relative to that of photon therapy for the treatment of cancer. Even though this clinical treatment has been developing for several decades, the proton radiobiology critical to the optimization of proton radiotherapy is far from being understood. Proteomic changes were analyzed in human melanoma cells treated with a sublethal dose (3 Gy) of proton beam irradiation. The results were compared with untreated cells. Two-dimensional electrophoresis was performed with mass spectrometry to identify the proteins. At the dose of 3 Gy a minimal slowdown in proliferation rate was seen, as well as some DNA damage. After allowing time for damage repair, the proteomic analysis was performed. In total 17 protein levels were found to significantly (more than 1.5 times) change: 4 downregulated and 13 upregulated. Functionally, they represent four categories: (i) DNA repair and RNA regulation (VCP, MVP, STRAP, FAB-2, Lamine A/C, GAPDH), (ii) cell survival and stress response (STRAP, MCM7, Annexin 7, MVP, Caprin-1, PDCD6, VCP, HSP70), (iii) cell metabolism (TIM, GAPDH, VCP), and (iv) cytoskeleton and motility (Moesin, Actinin 4, FAB-2, Vimentin, Annexin 7, Lamine A/C, Lamine B). A substantial decrease (2.3 x) was seen in the level of vimentin, a marker of epithelial to mesenchymal transition and the metastatic properties of melanoma. PMID:24392146

  2. Human melanoma immunotherapy using tumor antigen-specific T cells generated in humanized mice

    PubMed Central

    Hu, Zheng; Xia, Jinxing; Fan, Wei; Wargo, Jennifer; Yang, Yong-Guang

    2016-01-01

    A major factor hindering the exploration of adoptive immunotherapy in preclinical settings is the limited availability of tumor-reactive human T cells. Here we developed a humanized mouse model that permits large-scale production of human T cells expressing the engineered melanoma antigen MART-1-specific TCR. Humanized mice, made by transplantation of human fetal thymic tissue and CD34+ cells virally-transduced with HLA class I-restricted melanoma antigen (MART-1)-specific TCR gene, showed efficient development of MART-1-TCR+ human T cells with predominantly CD8+ cells. Importantly, MART-1-TCR+CD8+ T cells developing in these mice were capable of mounting antigen-specific responses in vivo, as evidenced by their proliferation, phenotypic conversion and IFN-γ production following MART-1 peptide immunization. Moreover, these MART-1-TCR+CD8+ T cells mediated efficient killing of melanoma cells in an HLA/antigen-dependent manner. Adoptive transfer of in vitro expanded MART-1-TCR+CD8+ T cells induced potent antitumor responses that were further enhanced by IL-15 treatment in melanoma-bearing recipients. Finally, a short incubation of MART-1-specific T cells with rapamycin acted synergistically with IL-15, leading to significantly improved tumor-free survival in recipients with metastatic melanoma. These data demonstrate the practicality of using humanized mice to produce potentially unlimited source of tumor-specific human T cells for experimental and preclinical exploration of cancer immunotherapy. This study also suggests that pretreatment of tumor-reactive T cells with rapamycin in combination with IL-15 administration may be a novel strategy to improve the efficacy of adoptive T cell therapy. PMID:26824989

  3. Atypical signaling of metabotropic glutamate receptor 1 in human melanoma cells.

    PubMed

    Gelb, Tara; Pshenichkin, Sergey; Hathaway, Hannah A; Grajkowska, Ewa; Dalley, Carrie Bowman; Wolfe, Barry B; Wroblewski, Jarda T

    2015-11-01

    The metabotropic glutamate 1 (mGlu1) receptor has emerged as a novel target for the treatment of metastatic melanoma and various other cancers. Our laboratory has demonstrated that a selective, non-competitive mGlu1 receptor antagonist slows human melanoma growth in vitro and in vivo. In this study, we sought to determine if the activation of a canonical G protein-dependent signal transduction cascade, which is often used as an output of mGlu1 receptor activity in neuronal cells, correlated with mGlu1 receptor-mediated melanoma cell viability. Glutamate, the endogenous ligand of mGlu1 receptors, significantly increased melanoma cell viability, but did not stimulate phosphoinositide (PI) hydrolysis in several human melanoma cell lines. In contrast, melanoma cell viability was not increased by quisqualate, a highly potent mGlu1 receptor agonist, or DHPG, a selective group I mGlu receptor agonist. Similarly to glutamate, quisqualate also failed to stimulate PI hydrolysis in mGlu1 receptor-expressing melanoma cells. These results suggest that the canonical G protein-dependent signal transduction cascade is not coupled to mGlu1 receptors in all human melanoma cells. On the other hand, dynamin inhibition selectively decreased viability of mGlu1 receptor-expressing melanoma cells, suggesting that a mechanism requiring internalization may control melanoma cell viability. Taken together, these data demonstrate that the approaches commonly used to study mGlu1 receptor function and signaling in other systems may be inappropriate for studying mGlu1 receptor-mediated melanoma cell viability. PMID:26291396

  4. Zebrafish Melanoma.

    PubMed

    Kaufman, Charles K

    2016-01-01

    Melanoma skin cancer is a potentially deadly disease in humans and has remained extremely difficult to treat once it has metastasized. In just the last 10 years, a number of models of melanoma have been developed in the zebrafish that are biologically faithful to the human disease and have already yielded important insights into the fundamental biology of melanoma and offered new potential avenues for treatment. With the diversity and breadth of the molecular genetic tools available in the zebrafish, these melanoma models will continue to be refined and expanded upon to keep pace with the rapidly evolving field of melanoma biology. PMID:27165365

  5. Toxicity of oxidized phosphatidylcholines in cultured human melanoma cells.

    PubMed

    Ramprecht, Claudia; Jaritz, Hannah; Streith, Ingo; Zenzmaier, Elfriede; Köfeler, Harald; Hofmann-Wellenhof, Rainer; Schaider, Helmut; Hermetter, Albin

    2015-07-01

    The oxidized phospholipids (oxPL) 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) are generated from 1-palmitoyl-2-arachidonoyl-phosphatidylcholine under conditions of oxidative stress. These oxPL are components of oxidized low density lipoprotein. They are cytotoxic in cells of the arterial wall thus playing an important role in the development and progression of atherosclerosis. The toxic lipid effects include inflammation and under sustained exposure apoptosis. The aim of this study was to find out whether such toxic effects, especially apoptosis, are also elicited by oxPL in melanocytic cells in order to assess their potential for therapeutic intervention. FACS analysis after staining with fluorescent markers was performed to identify the mode of lipid-induced cell death. Activation of sphingomyelinase which generates apoptotic ceramide was measured using an established fluorescence assay. Ceramide profiles were determined by mass spectrometry. We found that 50μM POVPC induce cell death in human melanoma cells isolated from different stages of tumor progression but affect primary human melanocytes to a much lesser extent. In contrast, 50μM PGPC was only apoptotic in two out of four cell lines used in this study. The toxicity of both compounds was associated with efficient lipid uptake into the tumor cells and activation of acid sphingomyelinase. In several but not all melanoma cell lines used in this study, activation of the sphingomyelin degrading enzyme correlated with an increase in the concentration of the apoptotic mediator ceramide. The individual patterns of the newly formed ceramide species were also cell line-specific. PGPC and POVPC may be considered potential drug candidates for topical skin cancer treatment. They are toxic in malignant cells. The respective oxidized phospholipids are naturally formed in the body and resistance to these compounds is not likely to occur

  6. Interferons Induce CXCR3-cognate Chemokine Production by Human Metastatic Melanoma

    PubMed Central

    Dengel, Lynn T.; Norrod, Allison G.; Gregory, Briana L.; Clancy-Thompson, Eleanor; Burdick, Marie D.; Strieter, Robert M.; Slingluff, Craig L.; Mullins, David W.

    2010-01-01

    Immune-mediated cancer regression requires tumor infiltration by Ag-specific effector T cells, but lymphocytes are commonly sparse in melanoma metastases. Activated T cells express CXCR3, whose cognate chemokines are CXCL9/MIG, CXCL10/IP-10 and CXCL11/I-TAC. Little is known about expression of these chemokines in lymph node (LN) metastases of melanoma. We evaluated whether metastatic melanoma induces these CXCR3-cognate chemokines in human LN-derived tissues. Also, because these chemokines can be induced by interferon (IFN), we evaluated whether type I or II IFNs (IFN-α or IFN-γ, respectively) can modulate chemokine expression in an in vitro model of the human tumor microenvironment. Production of CXCL9-11 by melanoma-infiltrated nodes (MIN) was no different than tumor-free nodes (TFN); both produced less chemokine than activated LN (sentinel immunized nodes, SIN). These data suggest melanoma infiltration into LN neither induces nor reduces CXCL9-11. Stimulation with IFN-α or IFN-γ increased production of CXCL10-11 from MIN, but not TFN or SIN. IFN-γ also increased production of CXCL9 in MIN. In IFN-treated SIN, CD14+ cells were the primary source of CXCL9-11, whereas melanoma cells were the source of chemokine in MIN. Melanoma cells in MIN express IFN receptors. Consistent with these observations, multiple human melanoma lines expressed IFN receptors and produced CXCL9-11 in response to IFN treatment. Thus, melanoma infiltration of LN is insufficient to induce the production of CXCL9-11, but melanoma may be a significant source of IFN-induced chemokines. Collectively, these data suggest that IFN-α or IFN-γ may act in the tumor microenvironment to increase the chemotactic gradient for CXCR3+ T cells. PMID:20948440

  7. Plancitoxin I from the venom of crown-of-thorns starfish (Acanthaster planci) induces oxidative and endoplasmic reticulum stress associated cytotoxicity in A375.S2 cells.

    PubMed

    Lee, Chi-Chiu; Hsieh, Hernyi Justin; Hsieh, Cheng-Hong; Hwang, Deng-Fwu

    2015-08-01

    The crown-of-thorns starfish Acanthaster planci is a venomous starfish whose venom provokes strong cytotoxicity. In the present study, the purified cytotoxic toxin of A. planci venom (CAV) was identified as plancitoxin I protein by mass spectrum analyses. This study aims to investigate the molecular mechanism underlying the cytotoxicity function of plancitoxin I by focusing on the oxidative stress, mitochondrial dysfunction and endoplasmic reticulum (ER) stress pathway in human melanoma A375.S2 cells. The results indicated that after being treated with CAV toxin, A375.S2 cells significantly decreased viability in a dose-dependent manner. The CAV was found to reduce the cellular antioxidant enzymes such as SOD and CAT, and there was a significant decrease in total thiol level and mtDNA integrity, and it enhanced the lipid peroxidation. In addition, CAV increased cytosolic Ca(2+) concentration, and enhanced the expression of the ER molecular chaperones GRP78 and CHOP in a dose-dependent manner. CAV significantly elevated the activity of caspase-3, -8 and -9, and reduced the ratio of Bcl-2/Bax. The cells exhibited apoptosis were determined by using propidium iodide (PI) staining of DNA fragmentation (sub-G1 peak). In summary, the results demonstrated that plancitoxin I inhibits the proliferation of A375.S2 cells through induction of oxidative stress, mitochondrial dysfunction and ER stress associated apoptosis. PMID:25952364

  8. Naringin suppresses the metabolism of A375 cells by inhibiting the phosphorylation of c-Src.

    PubMed

    Guo, Bingyu; Zhang, Yu; Hui, Qiang; Wang, Hongyi; Tao, Kai

    2016-03-01

    Elevation of glycolysis, increase in lactic acid production, and enhancement of mitochondrial biogenesis are all the changes of energy metabolism of melanoma cells. Melanoma cells' metabolism and energy production networks play an important role in cancer proliferation, survival, motility, invasiveness, metastasis, and angiogenesis. Since the Warburg theory was put forward in the 1930s, more researchers focus on finding new ways for effectively eliminating cancer cells by targeting their energy metabolism. In this study, we found naringin has the inhibitory effects on the glucose metabolism of A375 cells, a melanoma cell line, in a concentration-dependent manner. We also found that naringin could significantly reduce the phosphorylation of c-Src. In summary, we demonstrated that naringin inhibits the malignant phenotype of A375 cells by suppressing c-Src and its downstream signaling pathway. More importantly, we provide the novel mechanism that, as a natural inhibitor of c-Src, naringin could be an effective candidate for the treatment of melanoma. PMID:26476533

  9. The oncolytic peptide LTX-315 induces cell death and DAMP release by mitochondria distortion in human melanoma cells.

    PubMed

    Eike, Liv-Marie; Yang, Nannan; Rekdal, Øystein; Sveinbjørnsson, Baldur

    2015-10-27

    Host defense peptides (HDPs) are naturally occurring molecules found in most species, in which they play a significant role in the first line defense against intruding pathogens, and several HDPs have been shown to possess anticancer activity. Structure-activity relationship studies on the HDP bovine lactoferricin revealed a de novo design of a nonamer peptide LTX-315, with oncolytic properties. In the present study, we investigated the oncolytic activity of LTX-315 in human melanoma cells (A375). LTX-315 induced a rapid plasma membrane disruption and cell death within 2 hours. At a low concentration, fluorescence-labeled LTX-315 was internalized and accumulated in cytoplasmic vacuoles in close proximity to the mitochondria. The mitochondrial membrane potential was shown to depolarize as a consequence of LTX-315 treatment and at ultrastructural level, the mitochondria morphology was significantly altered. Release of danger signals (DAMPs) such as ATP, Cytochrome C and HMGB1 into the cell supernatant of cultured cells was evident minutes after peptide treatment. The oncolytic effect of LTX-315 involving perturbation of both the cell membrane and the mitochondria with subsequent release of DAMPs may highlight the ability of LTX-315 to induce complete regression and long-term protective immune responses as previously reported in experimental animal models. PMID:26472184

  10. The oncolytic peptide LTX-315 induces cell death and DAMP release by mitochondria distortion in human melanoma cells

    PubMed Central

    Eike, Liv-Marie; Yang, Nannan; Rekdal, Øystein; Sveinbjørnsson, Baldur

    2015-01-01

    Host defense peptides (HDPs) are naturally occurring molecules found in most species, in which they play a significant role in the first line defense against intruding pathogens, and several HDPs have been shown to possess anticancer activity. Structure-activity relationship studies on the HDP bovine lactoferricin revealed a de novo design of a nonamer peptide LTX-315, with oncolytic properties. In the present study, we investigated the oncolytic activity of LTX-315 in human melanoma cells (A375). LTX-315 induced a rapid plasma membrane disruption and cell death within 2 hours. At a low concentration, fluorescence-labeled LTX-315 was internalized and accumulated in cytoplasmic vacuoles in close proximity to the mitochondria. The mitochondrial membrane potential was shown to depolarize as a consequence of LTX-315 treatment and at ultrastructural level, the mitochondria morphology was significantly altered. Release of danger signals (DAMPs) such as ATP, Cytochrome C and HMGB1 into the cell supernatant of cultured cells was evident minutes after peptide treatment. The oncolytic effect of LTX-315 involving perturbation of both the cell membrane and the mitochondria with subsequent release of DAMPs may highlight the ability of LTX-315 to induce complete regression and long-term protective immune responses as previously reported in experimental animal models. PMID:26472184

  11. Two new trifunctional antibodies for the therapy of human malignant melanoma.

    PubMed

    Ruf, Peter; Jäger, Michael; Ellwart, Joachim; Wosch, Susanne; Kusterer, Elisabeth; Lindhofer, Horst

    2004-02-20

    Trifunctional antibodies are able to redirect T cells and Fcgamma receptor(+) accessory immune cells to tumor targets. The simultaneous activation of these different classes of effector cells results in efficient killing of the tumor cells by different mechanisms such as phagocytosis and perforin-mediated cytotoxicity. Here, we introduce 2 new trifunctional antibodies specific for human melanoma. These trifunctional antibodies recognize with one binding arm CD3 on human T cells. The other binding arm is directed against melanoma-associated proteoglycans or melanoma-associated gangliosides (GD2 as well as GD3). They mediate specific lysis of various melanoma cell lines in correlation with the level of antigen expression in short-term cytotoxicity experiments. A combination of the 2 trifunctional antibodies was equally or even more efficient. Moreover, they induced a strong Th1 cytokine pattern with high amounts of IFN-gamma and low or no IL-4. Accordingly, CD4(+) and especially CD8(+) T cells expanded, whereas B cells, NK cells and monocytes decreased. The cytokine response was up to 16-fold higher when tumor cells were present. IFN-gamma reached cytotoxic concentrations for SK-MEL-23 melanoma cells. The induction of a T-cell-activatory and melanoma cell-inhibitory cytokine milieu together with the redirection of T-cell- and accessory cell-mediated cytotoxicity are interesting features of these trifunctional antibodies. They may be a new option for the therapy of human malignant melanoma. PMID:14696099

  12. Targeting human melanoma neoantigens by T cell receptor gene therapy.

    PubMed

    Leisegang, Matthias; Kammertoens, Thomas; Uckert, Wolfgang; Blankenstein, Thomas

    2016-03-01

    In successful cancer immunotherapy, T cell responses appear to be directed toward neoantigens created by somatic mutations; however, direct evidence that neoantigen-specific T cells cause regression of established cancer is lacking. Here, we generated T cells expressing a mutation-specific transgenic T cell receptor (TCR) to target different immunogenic mutations in cyclin-dependent kinase 4 (CDK4) that naturally occur in human melanoma. Two mutant CDK4 isoforms (R24C, R24L) similarly stimulated T cell responses in vitro and were analyzed as therapeutic targets for TCR gene therapy. In a syngeneic HLA-A2-transgenic mouse model of large established tumors, we found that both mutations differed dramatically as targets for TCR-modified T cells in vivo. While T cells expanded efficiently and produced IFN-γ in response to R24L, R24C failed to induce an effective antitumor response. Such differences in neoantigen quality might explain why cancer immunotherapy induces tumor regression in some individuals, while others do not respond, despite similar mutational load. We confirmed the validity of the in vivo model by showing that the melan-A-specific (MART-1-specific) TCR DMF5 induces rejection of tumors expressing analog, but not native, MART-1 epitopes. The described model allows identification of those neoantigens in human cancer that serve as suitable T cell targets and may help to predict clinical efficacy. PMID:26808500

  13. A novel orally active water-soluble inhibitor of human glutathione transferase exerts a potent and selective antitumor activity against human melanoma xenografts

    PubMed Central

    De Luca, Anastasia; Rotili, Dante; Carpanese, Debora; Lenoci, Alessia; Calderan, Laura; Scimeca, Manuel; Mai, Antonello; Bonanno, Elena; Rosato, Antonio; Geroni, Cristina; Quintieri, Luigi; Caccuri, Anna Maria

    2015-01-01

    We designed and synthesized two novel nitrobenzoxadiazole (NBD) analogues of the anticancer agent 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX). The new compounds, namely MC3165 and MC3181, bear one and two oxygen atoms within the hydroxy-containing alkyl chain at the C4 position of the NBD scaffold, respectively. This insertion did not alter the chemical reactivity with reduced glutathione, while it conferred a remarkable increase in water solubility. MC3181 was more selective than NBDHEX towards the target protein, glutathione transferase P1-1, and highly effective in vitro against a panel of human melanoma cell lines, with IC50 in the submicromolar-low micromolar range. Interestingly, the cellular response to MC3181 was cell-type-specific; the compound triggered a JNK-dependent apoptosis in the BRAF-V600E-mutated A375 cells, while it induced morphological changes together with an increase in melanogenesis in BRAF wild-type SK23-MEL cells. MC3181 exhibited a remarkable therapeutic activity against BRAF-V600E-mutant xenografts, both after intravenous and oral administration. Outstandingly, no treatment-related signs of toxicity were observed both in healthy and tumor-bearing mice after single and repeated administrations. Taken together, these results indicate that MC3181 may represent a potential novel therapeutic opportunity for BRAF-mutated human melanoma, while being safe and water-soluble and thus overcoming all the critical aspects of NBDHEX in vivo. PMID:25595904

  14. A novel orally active water-soluble inhibitor of human glutathione transferase exerts a potent and selective antitumor activity against human melanoma xenografts.

    PubMed

    De Luca, Anastasia; Rotili, Dante; Carpanese, Debora; Lenoci, Alessia; Calderan, Laura; Scimeca, Manuel; Mai, Antonello; Bonanno, Elena; Rosato, Antonio; Geroni, Cristina; Quintieri, Luigi; Caccuri, Anna Maria

    2015-02-28

    We designed and synthesized two novel nitrobenzoxadiazole (NBD) analogues of the anticancer agent 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX). The new compounds, namely MC3165 and MC3181, bear one and two oxygen atoms within the hydroxy-containing alkyl chain at the C4 position of the NBD scaffold, respectively. This insertion did not alter the chemical reactivity with reduced glutathione, while it conferred a remarkable increase in water solubility. MC3181 was more selective than NBDHEX towards the target protein, glutathione transferase P1-1, and highly effective in vitro against a panel of human melanoma cell lines, with IC50 in the submicromolar-low micromolar range. Interestingly, the cellular response to MC3181 was cell-type-specific; the compound triggered a JNK-dependent apoptosis in the BRAF-V600E-mutated A375 cells, while it induced morphological changes together with an increase in melanogenesis in BRAF wild-type SK23-MEL cells. MC3181 exhibited a remarkable therapeutic activity against BRAF-V600E-mutant xenografts, both after intravenous and oral administration. Outstandingly, no treatment-related signs of toxicity were observed both in healthy and tumor-bearing mice after single and repeated administrations. Taken together, these results indicate that MC3181 may represent a potential novel therapeutic opportunity for BRAF-mutated human melanoma, while being safe and water-soluble and thus overcoming all the critical aspects of NBDHEX in vivo. PMID:25595904

  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. PMID:27102177

  16. The Cinnamon-derived Michael Acceptor Cinnamic Aldehyde Impairs Melanoma Cell Proliferation, Invasiveness, and Tumor Growth

    PubMed Central

    Cabello, Christopher M.; Bair, Warner B.; Lamore, Sarah D.; Ley, Stephanie; Bause, Alexandra S.; Azimian, Sara; Wondrak, Georg T.

    2009-01-01

    Redox dysregulation in cancer cells represents a chemical vulnerability that can be targeted by prooxidant redox intervention. Dietary constituents that contain an electrophilic Michael acceptor pharmacophore may therefore display promising chemopreventive and chemotherapeutic anti-cancer activity. Here, we demonstrate that the cinnamon-derived dietary Michael acceptor trans-cinnamic aldehyde (CA) impairs melanoma cell proliferation and tumor growth. Feasibility of therapeutic intervention using high doses of CA (120 mg/kg, p.o., q.d., 10 days) was demonstrated in a human A375 melanoma SCID-mouse xenograft model. Low micromolar concentrations (IC50 < 10 μM) of CA, but not closely related CA-derivatives devoid of Michael acceptor activity, suppressed proliferation of human metastatic melanoma cell lines (A375, G361, LOX) with G1 cell cycle arrest, elevated intracellular ROS, and impaired invasiveness. Expression array analysis revealed that CA induced an oxidative stress response in A375 cells, up-regulating heme oxygenase-1 (HMOX1), sulfiredoxin 1 homolog (SRXN1), thioredoxin reductase 1 (TXNRD1), and other genes including the cell cycle regulator and stress-responsive tumor suppressor gene cyclin-dependent kinase inhibitor 1A (CDKN1A), a key mediator of G1 phase arrest. CA, but not Michael-inactive derivatives, inhibited NFκB transcriptional activity and TNFα-induced IL-8 production in A375 cells. These findings support a previously unrecognized role of CA as a dietary Michael acceptor with potential anticancer activity. PMID:19000754

  17. [Molecular biology of familial cases of human melanoma].

    PubMed

    Kit, O I; Vodolazhsky, D L; Timoshkina, N N; Przhedetsky, Yu V; Khokhlova, O V

    2015-01-01

    Skin melanoma is an etiologically heterogeneous disease, the development of which is related to a complex interaction of environmental factors and individual genetic characteristics. This article provides current molecular-genetic aspects of familial cases of melanoma and polymorphism of genes directly related to the risk of developing this hereditary disease. The studies of hereditary cancer cases add our knowledge of mechanisms oncotransformation, genetic changes in signaling pathways, which are responsible for invasiveness, metastasis and drug resistance of melanoma cells of the skin. PMID:26995975

  18. Plasmonic enhanced fs-laser optoporation of human melanoma cells

    NASA Astrophysics Data System (ADS)

    Baumgart, J.; Humbert, L.; St.-Louis Lalonde, B.; Lebrun, J.-J.; Meunier, M.

    2011-03-01

    In this paper, we present the results of in vitro gene transfer by plasmonic enhanced optoporation of human melanoma cells. The fs-laser based optoporation is a gentle and efficient method for transfection. An optimum perforation rate with efficient dye or DNA uptake and high viability of the cells (~90%) was found for different types of nanostructures, spherical and rod shaped. The technique offers a very high selectivity and the low damage induced to the cell leads to a high transfection efficiency. The cell selectivity of this technique on the one hand is realized by using bioconjugated nanostructures, that couple selectively to a special cell type, and on the other hand, the spatial selectivity is due to the fact that only irradiated cells are perforated. In many biological applications a virus free and efficient transfection method is needed, especially in terms of its use in vivo. In cancer cells, the aggressiveness of the cells is shown in the migration and invasion velocity. The laser based and nanostructure enhanced transfection of cells offers the possibility to directly compare the treated and untreated cells. The treatment for migration and invasion assays can be performed by laser-scraping and laser transfection, resulting in a fully non-contact and therefore sterile method where the shape and the size of the scrape is well defined and reproducible. The laser based scrape test therefore offers less uncertainty due to scrape variations, high transfection efficiency, as well as direct comparison of treated and control cells in the same dish.

  19. Neoantigen landscape dynamics during human melanoma-T cell interactions.

    PubMed

    Verdegaal, Els M E; de Miranda, Noel F C C; Visser, Marten; Harryvan, Tom; van Buuren, Marit M; Andersen, Rikke S; Hadrup, Sine R; van der Minne, Caroline E; Schotte, Remko; Spits, Hergen; Haanen, John B A G; Kapiteijn, Ellen H W; Schumacher, Ton N; van der Burg, Sjoerd H

    2016-08-01

    Recognition of neoantigens that are formed as a consequence of DNA damage is likely to form a major driving force behind the clinical activity of cancer immunotherapies such as T-cell checkpoint blockade and adoptive T-cell therapy. Therefore, strategies to selectively enhance T-cell reactivity against genetically defined neoantigens are currently under development. In mouse models, T-cell pressure can sculpt the antigenicity of tumours, resulting in the emergence of tumours that lack defined mutant antigens. However, whether the T-cell-recognized neoantigen repertoire in human cancers is constant over time is unclear. Here we analyse the stability of neoantigen-specific T-cell responses and the antigens they recognize in two patients with stage IV melanoma treated by adoptive T-cell transfer. The T-cell-recognized neoantigens can be selectively lost from the tumour cell population, either by overall reduced expression of the genes or loss of the mutant alleles. Notably, loss of expression of T-cell-recognized neoantigens was accompanied by development of neoantigen-specific T-cell reactivity in tumour-infiltrating lymphocytes. These data demonstrate the dynamic interactions between cancer cells and T cells, which suggest that T cells mediate neoantigen immunoediting, and indicate that the therapeutic induction of broad neoantigen-specific T-cell responses should be used to avoid tumour resistance. PMID:27350335

  20. Cell-type dependent response of melanoma cells to aloe emodin.

    PubMed

    Radovic, J; Maksimovic-Ivanic, D; Timotijevic, G; Popadic, S; Ramic, Z; Trajkovic, V; Miljkovic, D; Stosic-Grujicic, S; Mijatovic, S

    2012-09-01

    Intrinsic characteristics of melanoma cells such as expression of inducible nitric oxide synthase (iNOS), redox status, and activity of signaling pathways involved in proliferation, differentiation and cell death define the response of the cells to the diverse treatments. In this context we compared the effectiveness of herbal antaquinone aloe emodin (AE) against mouse B16 melanoma and human A375, different in initial activity of ERK1/2, constitutive iNOS expression and basal level of reactive oxygen species (ROS). Both cell lines are sensitive to AE treatment. However, while the agent induces differentiation of B16 cells toward melanocytes, in A375 cells promoted massive apoptosis. Differentiation of B16 cells, characterized by enhanced melanin production and tyrosinase activity, was mediated by H(2)O(2) production synchronized with rapid p53 accumulation and enhanced expression of cyclins D1 and D3. Caspase mediated apoptosis triggered in A375 cells was accompanied with Bcl-2 but not iNOS down-regulation. In addition, opposite regulation of Akt-ERK1/2 axis in AE treated B16 and A375 cells correlated with different outcome of the treatment. However, AE in a dose-dependent manner rescued both B16 and A375 cells from doxorubicin- or paclitaxel-induced killing. These data indicate that caution is warranted when AE is administrated to the patients with conventional chemotherapy. PMID:22683487

  1. Biflorin induces cytotoxicity by DNA interaction in genetically different human melanoma cell lines.

    PubMed

    Ralph, Ana Carolina Lima; Calcagno, Danielle Queiroz; da Silva Souza, Luciana Gregório; de Lemos, Telma Leda Gomes; Montenegro, Raquel Carvalho; de Arruda Cardoso Smith, Marília; de Vasconcellos, Marne Carvalho

    2016-08-01

    Cancer is a public health problem and the second leading cause of death worldwide. The incidence of cutaneous melanoma has been notably increasing, resulting in high aggressiveness and poor survival rates. Taking into account the antitumor activity of biflorin, a substance isolated from Capraria biflora L. roots that is cytotoxic in vitro and in vivo, this study aimed to demonstrate the action of biflorin against three established human melanoma cell lines that recapitulate the molecular landscape of the disease in terms of genetic alterations and mutations, such as the TP53, NRAS and BRAF genes. The results presented here indicate that biflorin reduces the viability of melanoma cell lines by DNA interactions. Biflorin causes single and double DNA strand breaks, consequently inhibiting cell cycle progression, replication and DNA repair and promoting apoptosis. Our data suggest that biflorin could be considered as a future therapeutic option for managing melanoma. PMID:27079618

  2. Antiproliferative effect of linalool on RPMI 7932 human melanoma cell line: ultrastructural studies.

    PubMed

    Cerchiara, Teresa; Straface, Serafina Vittoria; Brunelli, Elvira; Tripepi, Sandro; Gallucci, Maria Caterina; Chidichimo, Giuseppe

    2015-04-01

    Linalool, a small monoterpene molecule, is used widely for its flavoring and fragrant properties in many cosmetic products. In this work, we investigated the antiproliferative effect of two different linalool solutions on RPMI 7932 human melanoma and NCTC 2544 normal keratinocites cell lines using the trypan blue method. Morphological changes in cells were investigated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In addition, apoptosis was evaluated using caspase 3-antibody. Linalool showed a selective inhibitory effect on the growth of melanoma cells in a concentrationdependent manner, inducing several morphological changes, as revealed by SEM and TEM analysis. Moreover, the labelling for caspase-3 is abundant in the melanoma cells and almost absent in the normal keratinocites cells. The results suggest that linalool could be used as drug and/or as model drug for developing potential therapeutic agents for melanoma. PMID:25973472

  3. Human malignant melanoma-derived progestagen-associated endometrial protein immunosuppresses T lymphocytes in vitro.

    PubMed

    Ren, Suping; Chai, Lina; Wang, Chunyan; Li, Changlan; Ren, Qiquan; Yang, Lihua; Wang, Fumei; Qiao, Zhixin; Li, Weijing; He, Min; Riker, Adam I; Han, Ying; Yu, Qun

    2015-01-01

    Progestagen-associated endometrial protein (PAEP) is a glycoprotein of the lipocalin family that acts as a negative regulator of T cell receptor-mediated activation. However, the function of tumor-derived PAEP on the human immune system in the tumor microenvironment is unknown. PAEP is highly expressed in intermediate and thick primary melanomas (Breslow's 2.5mm or greater) and metastatic melanomas, correlating with its expression in daughter cell lines established in vitro. The current study investigates the role of melanoma cell-secreted PAEP protein in regulating T cell function. Upon the enrichment of CD3+, CD4+ and CD8+ T cells from human peripheral blood mononuclear cells, each subset was then mixed with either melanoma-derived PAEP protein or PAEP-poor supernatant of gene-silenced tumor cells. IL-2 and IFN-γ secretion of CD4+ T cells significantly decreased with the addition of PAEP-rich supernatant. And the addition of PAEP-positive cell supernatant to activated lymphocytes significantly inhibited lymphocyte proliferation and cytotoxic T cell activity, while increasing lymphocyte apoptosis. Our result suggests that melanoma cell-secreted PAEP protein immunosuppresses the activation, proliferation and cytotoxicity of T lymphocytes, which might partially explain the mechanism of immune tolerance induced by melanoma cells within the tumor microenvironment. PMID:25785839

  4. Cannibalism of live lymphocytes by human metastatic but not primary melanoma cells.

    PubMed

    Lugini, Luana; Matarrese, Paola; Tinari, Antonella; Lozupone, Francesco; Federici, Cristina; Iessi, Elisabetta; Gentile, Massimo; Luciani, Francesca; Parmiani, Giorgio; Rivoltini, Licia; Malorni, Walter; Fais, Stefano

    2006-04-01

    The phenomenon of cell cannibalism, which generally refers to the engulfment of cells within other cells, was described in malignant tumors, but its biological significance is still largely unknown. In the present study, we investigated the occurrence, the in vivo relevance, and the underlying mechanisms of cannibalism in human melanoma. As first evidence, we observed that tumor cannibalism was clearly detectable in vivo in metastatic lesions of melanoma and often involved T cells, which could be found in a degraded state within tumor cells. Then, in vitro experiments confirmed that cannibalism of T cells was a property of metastatic melanoma cells but not of primary melanoma cells. In particular, morphologic analyses, including time-lapse cinematography and electron microscopy, revealed a sequence of events, in which metastatic melanoma cells were able to engulf and digest live autologous melanoma-specific CD8(+) T cells. Importantly, this cannibalistic activity significantly increased metastatic melanoma cell survival, particularly under starvation condition, supporting the evidence that tumor cells may use the eating of live lymphocytes as a way to "feed" in condition of low nutrient supply. The mechanism underlying cannibalism involved a complex framework, including lysosomal protease cathepsin B activity, caveolae formation, and ezrin cytoskeleton integrity and function. In conclusion, our study shows that human metastatic melanoma cells may eat live T cells, which are instead programmed to kill them, suggesting a novel mechanism of tumor immune escape. Moreover, our data suggest that cannibalism may represent a sort of "feeding" activity aimed at sustaining survival and progression of malignant tumor cells in an unfavorable microenvironment. PMID:16585188

  5. The new face of nucleolin in human melanoma.

    PubMed

    Hoja-Łukowicz, Dorota; Przybyło, Małgorzata; Pocheć, Ewa; Drabik, Anna; Silberring, Jerzy; Kremser, Marcelina; Schadendorf, Dirk; Laidler, Piotr; Lityńska, Anna

    2009-09-01

    Nucleolin is multifunctional protein mainly present in nucleoli but also detected in cytoplasm and plasma membranes. Extranuclear nucleolin differs from the nuclear form by its glycosylation. Studies on expression of nucleolin in breast cancer suggest a possible association to the metastatic cascade. In the present study, Vicia villosa lectin (VVL) precipitation followed by subsequent polyacrylamide gel electrophoresis and mass spectrometry analysis demonstrates nucleolin as a VVL-positive glycoprotein expressed in melanoma. The presence of VVL-positive nucleolin in the melanoma cell membrane and cytoplasm was confirmed by confocal microscopy. Using bioinformatic peptide prediction programs, nucleolin was shown to contain multiple possible MHC class-I binding peptides in its sequence which makes nucleolin an interesting melanoma marker and target for immunodiagnostic and possibly therapeutic purposes. PMID:19363676

  6. Pro-Proliferative Function of Mitochondrial Sirtuin Deacetylase SIRT3 in Human Melanoma.

    PubMed

    George, Jasmine; Nihal, Minakshi; Singh, Chandra K; Zhong, Weixiong; Liu, Xiaoqi; Ahmad, Nihal

    2016-04-01

    Melanoma, the most aggressive form of skin cancer, is often fatal if not treated early. Therefore, novel target-based strategies are required to combat this neoplasm. The objective of this study was to determine the role and functional significance of the mitochondrial sirtuin 3 (SIRT3) in melanoma. We found that compared with normal primary and immortalized human melanocytes, SIRT3 is significantly overexpressed in multiple human melanoma cells at mRNA and protein levels. Further, employing human tissue microarray, we found that SIRT3 is significantly upregulated in clinical melanoma tissues, compared with melanocytic nevi tissues. Furthermore, a short hairpin RNA-mediated knockdown of SIRT3 in human melanoma cells resulted in (i) a decrease in cellular proliferation, colony formation, and cellular migration; (ii) induction of senescence as shown by an increase in senescence-associated beta-galactosidase activity and formation of senescence-associated heterochromatin foci as well as an increase in mRNA and protein levels of p16(INK4a) and p21(Waf1); (iii) G1-phase arrest of the cell cycle; and (iv) decreases in mRNA and protein levels of cyclins (D1, E1) and cyclin-dependent kinases (2, 4, and 6). Conversely, forced exogenous overexpression of SIRT3 promoted an increase in proliferative potential of Hs294T melanoma cells and normal immortalized Mel-ST melanocytes. Finally, we found that SIRT3 knockdown significantly inhibited tumorigenesis in a xenograft model in vivo. To our knowledge, this is the first study supporting the pro-proliferative function of SIRT3 in melanoma. PMID:26743598

  7. A new water soluble MAPK activator exerts antitumor activity in melanoma cells resistant to the BRAF inhibitor vemurafenib.

    PubMed

    Graziani, Grazia; Artuso, Simona; De Luca, Anastasia; Muzi, Alessia; Rotili, Dante; Scimeca, Manuel; Atzori, Maria Grazia; Ceci, Claudia; Mai, Antonello; Leonetti, Carlo; Levati, Lauretta; Bonanno, Elena; Tentori, Lucio; Caccuri, Anna Maria

    2015-05-01

    Recovery of mitogen activated protein kinase (MAPK) or activation of alternative pathways, such as the PI3K/AKT/mTOR, are involved in acquired resistance to BRAF inhibitors which represent the first-line treatment of BRAF-mutated metastatic melanoma. We recently demonstrated that 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and its water soluble analog 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)ethoxy)ethoxy)ethanol (MC3181) trigger apoptosis in BRAF V600E mutated melanoma cells through activation of the MAPK c-Jun N-terminal kinase (JNK). Herein, we investigated whether NBDHEX and MC3181 might exert antitumor activity against BRAF V600E mutated human melanoma cells rendered resistant to the BRAF inhibitor vemurafenib. To this aim we generated a subline of A375 melanoma resistant in vitro and in vivo to vemurafenib (A375-VR8) and characterized by NRAS G13R mutation, high basal levels of CRAF protein and phospho-activation of AKT. In these cells ERK phosphorylation was not significantly down-modulated by vemurafenib concentrations capable of abrogating ERK phosphorylation in sensitive A375 cells. Both NBDHEX and MC3181 induced marked antiproliferative and apoptotic effects in A375-VR8 cells and, at equitoxic concentrations, caused a strong phosphorylation of JNK, p38, and of the downstream mediators of apoptosis ATF2 and p53. Drug treatment further increased ERK phosphorylation, which was required for the cellular response to the NBD derivatives, as apoptosis was antagonized by the ERK inhibitor FR180204. Finally, in vivo administration of MC3181 provoked JNK activation at the tumor site and markedly reduced A375-VR8 growth. These evidences strongly suggest that the activation of multiple pro-apoptotic MAPK pathways by MC3181 might represent a new strategy for the treatment of melanoma resistant to BRAF inhibitors. PMID:25795251

  8. Fluorescence in situ detection of human cutaneous melanoma: study of diagnostic parameters of the method.

    PubMed

    Chwirot, B W; Chwirot, S; Sypniewska, N; Michniewicz, Z; Redzinski, J; Kurzawski, G; Ruka, W

    2001-12-01

    Multicenter study of the diagnostic parameters was conducted by three groups in Poland to determine if in situ fluorescence detection of human cutaneous melanoma based on digital imaging of spectrally resolved autofluorescence can be used as a tool for a preliminary selection of patients at increased risk of the disease. Fluorescence examinations were performed for 7228 pigmented lesions in 4079 subjects. Histopathologic examinations showed 56 cases of melanoma. A sensitivity of fluorescence detection of melanoma was 82.7% in agreement with 82.5% found in earlier work. Using as a reference only the results of histopathologic examinations obtained for 568 cases we found a specificity of 59.9% and a positive predictive value of 17.5% (melanomas versus all pigmented lesions) or 24% (melanomas versus common and dysplastic naevi). The specificity and positive predictive value found in this work are significantly lower than reported earlier but still comparable with those reported for typical screening programs. In conclusion, the fluorescence method of in situ detection of melanoma can be used in screening large populations of patients for a selection of patients who should be examined by specialists. PMID:11886507

  9. Effects of Zeaxanthin on Growth and Invasion of Human Uveal Melanoma in Nude Mouse Model

    PubMed Central

    Xu, Xiaoliang L.; Iacob, Codrin; Jordan, Adrienne; Gandhi, Sandipkumar; Gierhart, Dennis L.; Rosen, Richard

    2015-01-01

    Uveal melanoma cells were inoculated into the choroid of nude mice and treated with or without intraocular injection of zeaxanthin. After 21 days, mice were sacrificed and the eyes enucleated. Histopathological analysis was performed in hematoxylin and eosin stained frozen sections. Melanoma developed rapidly in the control group (without treatment of zeaxanthin). Tumor-bearing eye mass and tumor mass in the control group were significantly greater than those in zeaxanthin treated group. Melanoma in the controlled eyes occupied a large part of the eye, was epithelioid in morphology, and was with numerous mitotic figures. Scleral perforation and extraocular extension were observed in half of the eyes. Melanomas in zeaxanthin treated eyes were significantly smaller with many necrosis and apoptosis areas and no extraocular extension could be found. Quantitative image analysis revealed that the tumor size was reduced by 56% in eyes treated with low dosages of zeaxanthin and 92% in eyes treatment with high dosages of zeaxanthin, as compared to the controls. This study demonstrated that zeaxanthin significantly inhibits the growth and invasion of human uveal melanoma in nude mice, suggesting that zeaxanthin may be a promising agent to be explored for the prevention and treatment of uveal melanoma. PMID:26682063

  10. In vitro efficiency and mechanistic role of indocyanine green as photodynamic therapy agent for human melanoma

    SciTech Connect

    Mamoon, A.M.; Miller, L.; Gamal-Eldeen, A. M.; Ruppel, M. E.; Smith, R. J.; Tsang, T.; Miller, L. M.

    2009-05-02

    Photodynamic therapy (PDT) is a promising treatment for superficial cancer. However, poor therapeutic results have been reported for melanoma, due to the high melanin content. Indocyanine green (ICG) has near infrared absorption (700-800 nm) and melanins do not absorb strongly in this area. This study explores the efficiency of ICG as a PDT agent for human melanoma, and its mechanistic role in the cell death pathway. Human skin melanoma cells (Sk-Mel-28) were incubated with ICG and exposed to a low power Ti:Sapphire laser. Synchrotron-assisted Fourier transform infrared microspectroscopy and hierarchical cluster analysis were used to assess the cell damage and changes in lipid, protein, and nucleic acids. The cell death pathway was determined by analysis of cell viability and apoptosis and necrosis markers. In the cell death pathway, {sup 1}O{sub 2} generation evoked rapid multiple consequences that trigger apoptosis after laser exposure for only 15min including the release of cytochrome c, the activation of total caspases, caspase-3, and caspase-9, the inhibition of NF-{Kappa}B P65, and the enhancement of DNA fragmentation, and histone acetylation. ICG/PDT can efficiently and rapidly induce apoptosis in human melanoma cells and it can be considered as a new therapeutic approach for topical treatment of melanoma.

  11. Radiation survival of murine and human melanoma cells utilizing two assay systems: monolayer and soft agar.

    PubMed Central

    Yohem, K. H.; Slymen, D. J.; Bregman, M. D.; Meyskens, F. L.

    1988-01-01

    The radiation response of murine and human melanoma cells assayed in bilayer soft agar and monolayer was examined. Cells from the murine melanoma Cloudman S91 CCL 53.1 cell line and three human melanoma cell strains (C8146C, C8161, and R83-4) developed in our laboratory were irradiated by single dose X-rays and plated either in agar or on plastic. D0 values were the same within 95% confidence intervals for cells from the human melanoma cell strains C8146C, C8161, and R83-4 but were dissimilar for the murine cell line CCL 53.1 Dq values were different for all cells studied. The shape of the survival curve for all four melanomas was not identical for cells assayed in soft agar versus cells grown on plastic. This would indicate that apparent radiosensitivity was influenced by the method of assay although there were no apparent consistent differences between the curves generated by monolayer or bilayer soft agar assays. PMID:3348949

  12. Sensitization of human melanoma cells by tamoxifen to apoptosis induction by pancratistatin, a nongenotoxic natural compound.

    PubMed

    Chatterjee, Sudipa June; McNulty, James; Pandey, Siyaram

    2011-02-01

    The objective of this study was to determine the efficacy of the natural compound pancratistatin (PST), isolated from the Hymenocallis littoralis, in human melanoma cells. Melanoma is an aggressive form of skin cancer that is commonly fatal if not diagnosed in its early stage of development. Melanoma is resistant to many treatments, thus drastically limiting chemotherapy options for this cancer. We have shown that exposure to PST induces apoptosis in human melanoma within 72 h using Hoechst staining. Interestingly tamoxifen (TAM), an estrogen receptor antagonist, sensitizes these cells to apoptosis induction by PST as observed with Hoechst and annexin-V staining. This cotreatment did not affect the viability of normal noncancerous human fibroblasts. Both of these compounds have been shown to target the mitochondria synergistically, as indicated by higher levels of reactive oxygen species generation from isolated mitochondria. PST alone and in combination with TAM shows depolarization of the mitochondrial membrane potential as shown by JC-1 staining. Melanoma drug resistance was not observed after posttreatment recuperation, as cells displayed apoptotic morphology up to 96 h after drug-free media replacement. Our results indicate that TAM alone does not induce apoptosis in this cell line, but sensitizes the mitochondria, thereby enhancing the effect of PST exposure. In conclusion, combination of two nongenotoxic compounds offers a novel treatment regime for this notoriously resilient form of skin cancer. PMID:20300039

  13. Initial results of imaging melanoma metastasis in resected human lymph nodes using photoacoustic computed tomography

    NASA Astrophysics Data System (ADS)

    Jose, Jithin; Grootendorst, Diederik J.; Vijn, Thomas W.; Wouters, Michel W.; van Boven, Hester; van Leeuwen, Ton G.; Steenbergen, Wiendelt; Ruers, Theo J. M.; Manohar, Srirang

    2011-09-01

    The pathological status of the sentinel lymph node is important for accurate melanoma staging, ascertaining prognosis and planning treatment. The standard procedure involves biopsy of the node and histopathological assessment of its status. Drawbacks of this examination include a finite sampling of the node with the likelihood of missing metastases, and a significant time-lag before histopathological results are available to the surgeon. We studied the applicability of photoacoustic computed tomographic imaging as an intraoperative modality for examining the status of resected human sentinel lymph nodes. We first applied the technique to image ex vivo pig lymph nodes carrying metastases-simulating melanoma cells using multiple wavelengths. The experience gained was applied to image a suspect human lymph node. We validated the photoacoustic imaging results by comparing a reconstructed slice with a histopathological section through the node. Our results suggest that photoacoustics has the potential to develop into an intraoperative imaging method to detect melanoma metastases in sentinel lymph nodes.

  14. Zinc Induces Apoptosis of Human Melanoma Cells, Increasing Reactive Oxygen Species, p53 and FAS Ligand.

    PubMed

    Provinciali, Mauro; Pierpaoli, Elisa; Bartozzi, Beatrice; Bernardini, Giovanni

    2015-10-01

    The aim of this study was to examine the in vitro effect of zinc on the apoptosis of human melanoma cells, by studying the zinc-dependent modulation of intracellular levels of reactive oxygen species (ROS) and of p53 and FAS ligand proteins. We showed that zinc concentrations ranging from 33.7 μM to 75 μM Zn(2+) induced apoptosis in the human melanoma cell line WM 266-4. This apoptosis was associated with an increased production of intracellular ROS, and of p53 and FAS ligand protein. Treatment of tumor cells with the antioxidant N-acetylcysteine was able to prevent Zn(2+)-induced apoptosis, as well as the increase of p53 and FAS ligand protein induced by zinc. Zinc induces apoptosis in melanoma cells by increasing ROS and this effect may be mediated by the ROS-dependent induction of p53 and FAS/FAS ligand. PMID:26408691

  15. Induction of monocyte chemoattractant protein-1 and interleukin-10 by TGFbeta1 in melanoma enhances tumor infiltration and immunosuppression.

    PubMed

    Díaz-Valdés, Nancy; Basagoiti, María; Dotor, Javier; Aranda, Fernando; Monreal, Iñaki; Riezu-Boj, José Ignacio; Borrás-Cuesta, Francisco; Sarobe, Pablo; Feijoó, Esperanza

    2011-02-01

    Melanoma progression is associated with the expression of different growth factors, cytokines, and chemokines. Because TGFβ1 is a pleiotropic cytokine involved not only in physiologic processes but also in cancer development, we analyzed in A375 human melanoma cells, the effect of TGFβ1 on monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10) expression, two known factors responsible for melanoma progression. TGFβ1 increased the expression of MCP-1 and IL-10 in A375 cells, an effect mediated by the cross-talk between Smad, PI3K (phosphoinositide 3-kinase)/AKT, and BRAF-MAPK (mitogen activated protein kinase) signaling pathways. Supernatants from TGFβ1-treated A375 cells enhanced MCP-1-dependent migration of monocytes, which, in turn, expressed high levels of TGF,β1, bFGF, and VEGF mRNA. Moreover, these supernatants also inhibited functional properties of dendritic cells through IL-10-dependent mechanisms. When using in vitro, the TGFβ1-blocking peptide P144, TGFβ1-dependent Smad3 phosphorylation, and expression of MCP-1 and IL-10 were inhibited. In vivo, treatment of A375 tumor-bearing athymic mice with P144 significantly reduced tumor growth, associated with a lower macrophage infiltrate and decreased intratumor MCP-1 and VEGF levels, as well as angiogenesis. Finally, in C57BL/6 mice with B16-OVA melanoma tumors, when administered with immunotherapy, P144 decreased tumor growth and intratumor IL-10 levels, linked to enhanced activation of dendritic cells and natural killer cells, as well as anti-OVA T-cell responses. These results show new effects of TGFβ1 on melanoma cells, which promote tumor progression and immunosuppression, strongly reinforcing the relevance of this cytokine as a molecular target in melanoma. PMID:21159663

  16. The human homologue of Dictyostelium discoideum phg1A is expressed by human metastatic melanoma cells.

    PubMed

    Lozupone, Francesco; Perdicchio, Maurizio; Brambilla, Daria; Borghi, Martina; Meschini, Stefania; Barca, Stefano; Marino, Maria Lucia; Logozzi, Mariantonia; Federici, Cristina; Iessi, Elisabetta; de Milito, Angelo; Fais, Stefano

    2009-12-01

    Tumour cannibalism is a characteristic of malignancy and metastatic behaviour. This atypical phagocytic activity is a crucial survival option for tumours in conditions of low nutrient supply, and has some similarities to the phagocytic activity of unicellular microorganisms. In fact, Dictyostelium discoideum has been used widely as a model to study phagocytosis. Recently, phg1A has been described as a protein that is primarily involved in the phagocytic process of this microorganism. The closest human homologue to phg1A is transmembrane 9 superfamily protein member 4 (TM9SF4). Here, we report that TM9SF4 is highly expressed in human malignant melanoma cells deriving from metastatic lesions, whereas it is undetectable in healthy human tissues and cells. TM9SF4 is predominantly expressed in acidic vesicles of melanoma cells, in which it co-localizes with the early endosome antigens Rab5 and early endosome antigen 1. TM9SF4 silencing induced marked inhibition of cannibal activity, which is consistent with a derangement of intracellular pH gradients, with alkalinization of acidic vesicles and acidification of the cell cytosol. We propose TM9SF4 as a new marker of malignancy, representing a potential new target for anti-tumour strategies with a specific role in tumour cannibalism and in the establishment of a metastatic phenotype. PMID:19893578

  17. Detection of melanoma metastases in resected human lymph nodes by noninvasive multispectral photoacoustic imaging.

    PubMed

    Langhout, Gerrit Cornelis; Grootendorst, Diederik Johannes; Nieweg, Omgo Edo; Wouters, Michel Wilhelmus Jacobus Maria; van der Hage, Jos Alexander; Jose, Jithin; van Boven, Hester; Steenbergen, Wiendelt; Manohar, Srirang; Ruers, Theodoor Jacques Marie

    2014-01-01

    Objective. Sentinel node biopsy in patients with cutaneous melanoma improves staging, provides prognostic information, and leads to an increased survival in node-positive patients. However, frozen section analysis of the sentinel node is not reliable and definitive histopathology evaluation requires days, preventing intraoperative decision-making and immediate therapy. Photoacoustic imaging can evaluate intact lymph nodes, but specificity can be hampered by other absorbers such as hemoglobin. Near infrared multispectral photoacoustic imaging is a new approach that has the potential to selectively detect melanin. The purpose of the present study is to examine the potential of multispectral photoacoustic imaging to identify melanoma metastasis in human lymph nodes. Methods. Three metastatic and nine benign lymph nodes from eight melanoma patients were scanned ex vivo using a Vevo LAZR(©) multispectral photoacoustic imager and were spectrally analyzed per pixel. The results were compared to histopathology as gold standard. Results. The nodal volume could be scanned within 20 minutes. An unmixing procedure was proposed to identify melanoma metastases with multispectral photoacoustic imaging. Ultrasound overlay enabled anatomical correlation. The penetration depth of the photoacoustic signal was up to 2 cm. Conclusion. Multispectral three-dimensional photoacoustic imaging allowed for selective identification of melanoma metastases in human lymph nodes. PMID:25028587

  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. [Characterization of genetic alterations in primary human melanomas carrying BRAF or NRAS mutation].

    PubMed

    Lázár, Viktória

    2013-06-01

    Human malignant melanoma is one of the most aggressive forms of skin cancer with an exceptionally bad prognosis. Melanoma often displays constitutively activated MAPK pathway through BRAF or NRAS mutations. It is also known that these mutations are almost never simultaneously present and that they appear at early stages and preserved throughout tumor progression, although it is proved that these alterations alone are insufficient to cause tumor progression. Therefore the first aim of our study was to evaluate those distinct genetic alterations which can properly differentiate the three important molecular subtypes of primary melanomas with a) BRAF, b) NRAS mutation and c) WT (wild type for both loci). High-resolution array comparative genomic hybridization (array CGH) was used to assess genome-wide analysis of DNA copy number alterations. Primary melanomas with BRAF mutation more frequently exhibited losses on 10q23-10q26 and gains on chromosome 7 and 1q23-1q25 compared to melanomas with NRAS mutation. Loss on the 11q23-11q25 sequence was found mainly in conjunction with NRAS mutation. Based on these results, we proved the existence of marked differences in the genetic pattern of the BRAF and NRAS mutated melanoma subgroups, which might suggest that these mutations contribute to the development of malignant melanoma in conjunction with distinct cooperating oncogenic events. In general, it is an interesting phenomenon suggesting that these mutations provide probably the "guiding force" for these tumors and it also suggests that there are alternative genetic pathways to melanoma. These additional oncogenic events which are associated with BRAF or NRAS mutations can provide rational additional targets for a combination therapy with kinase inhibitors. In this study we also investigated the specific dynamic activities among different signalling pathways highlighting the frequent alterations of genes involved in the signalling interactions between the MAPK-JAK pathways

  20. Serological survey of normal humans for natural antibody to cell surface antigens of melanoma.

    PubMed Central

    Houghton, A N; Taormina, M C; Ikeda, H; Watanabe, T; Oettgen, H F; Old, L J

    1980-01-01

    Sera of 106 normal adult men were tested for antibodies reacting with cell surface antigens of three established lines of cultured malignant melanoma. Positive reactions with a protein A assay for IgG antibodies were extremely rare (1-2%). The frequency of positive reactions with assays for IgM antibodies was higher: 5-15% in immune adherence assays and 55-82% in anti-C3 mixed hemadsorption assays. After low-titered sera and sera reacting with fetal calf serum components, conventional alloantigens, and widely distributed class 3 antigens were excluded, sera from seven individuals (one with IgG antibody and six with IgM antibodies) were selected for detailed analysis. The serum containing the IgG antibody came from a healthy 65-year-old Caucasian man; titers of antibody in his serum ranged from < 1/10 to 1/40,000 in tests with different melanoma cell lines. This IgG antibody identifies a differentiation antigen of melanocytes, provisionally designated Mel 1, that distinguishes two classes of melanomas: 22 melanoma cell lines typed Mel 1+ and 17 types Mel 1-. Mel 1 is expressed by fetal fibroblasts but not adult fibroblasts and can be found on a proportion of cultured epithelial cancer cell lines (5 out of 23) but not on glioma or B-cell lines. The melanoma antigens detected by the naturally occurring IgM antibodies are serologically unrelated to Mel 1 but, like Mel 1, appear to be differentiation antigens that distinguish subsets of melanoma. These IgM antibodies detect antigens that are identical or closely related to the AH antigen, a melanoma surface antigen that was initially defined by autologous antibody in a patient with melanoma. In view of the immunogenicity of both Mel 1 and the AH antigens in humans and their occurrence on more than 50% of melanomas, it remains to be seen whether antibody to these antigens can be elicited by specific vaccination of seronegative melanoma patients and whether this will have an influence on the clinical course of the disease

  1. Simulation study of melanoma detection in human skin tissues by laser-generated surface acoustic waves.

    PubMed

    Chen, Kun; Fu, Xing; Dorantes-Gonzalez, Dante J; Lu, Zimo; Li, Tingting; Li, Yanning; Wu, Sen; Hu, Xiaotang

    2014-01-01

    Air pollution has been correlated to an increasing number of cases of human skin diseases in recent years. However, the investigation of human skin tissues has received only limited attention, to the point that there are not yet satisfactory modern detection technologies to accurately, noninvasively, and rapidly diagnose human skin at epidermis and dermis levels. In order to detect and analyze severe skin diseases such as melanoma, a finite element method (FEM) simulation study of the application of the laser-generated surface acoustic wave (LSAW) technique is developed. A three-layer human skin model is built, where LSAW’s are generated and propagated, and their effects in the skin medium with melanoma are analyzed. Frequency domain analysis is used as a main tool to investigate such issues as minimum detectable size of melanoma, filtering spectra from noise and from computational irregularities, as well as on how the FEM model meshing size and computational capabilities influence the accuracy of the results. Based on the aforementioned aspects, the analysis of the signals under the scrutiny of the phase velocity dispersion curve is verified to be a reliable, a sensitive, and a promising approach for detecting and characterizing melanoma in human skin. PMID:25057963

  2. Simulation study of melanoma detection in human skin tissues by laser-generated surface acoustic waves

    NASA Astrophysics Data System (ADS)

    Chen, Kun; Fu, Xing; Dorantes-Gonzalez, Dante J.; Lu, Zimo; Li, Tingting; Li, Yanning; Wu, Sen; Hu, Xiaotang

    2014-07-01

    Air pollution has been correlated to an increasing number of cases of human skin diseases in recent years. However, the investigation of human skin tissues has received only limited attention, to the point that there are not yet satisfactory modern detection technologies to accurately, noninvasively, and rapidly diagnose human skin at epidermis and dermis levels. In order to detect and analyze severe skin diseases such as melanoma, a finite element method (FEM) simulation study of the application of the laser-generated surface acoustic wave (LSAW) technique is developed. A three-layer human skin model is built, where LSAW's are generated and propagated, and their effects in the skin medium with melanoma are analyzed. Frequency domain analysis is used as a main tool to investigate such issues as minimum detectable size of melanoma, filtering spectra from noise and from computational irregularities, as well as on how the FEM model meshing size and computational capabilities influence the accuracy of the results. Based on the aforementioned aspects, the analysis of the signals under the scrutiny of the phase velocity dispersion curve is verified to be a reliable, a sensitive, and a promising approach for detecting and characterizing melanoma in human skin.

  3. Substantial expression of luteinizing hormone-releasing hormone (LHRH) receptor type I in human uveal melanoma

    PubMed Central

    Schally, Andrew V.; Block, Norman L; Dezso, Balazs; Olah, Gabor; Rozsa, Bernadett; Fodor, Klara; Buglyo, Armin; Gardi, Janos; Berta, Andras; Halmos, Gabor

    2013-01-01

    Uveal melanoma is the most common primary intraocular malignancy in adults, with a very high mortality rate due to frequent liver metastases. Consequently, the therapy of uveal melanoma remains a major clinical challenge and new treatment approaches are needed. For improving diagnosis and designing a rational and effective therapy, it is essential to elucidate molecular characteristics of this malignancy. The aim of this study therefore was to evaluate as a potential therapeutic target the expression of luteinizing hormone-releasing hormone (LHRH) receptor in human uveal melanoma. The expression of LHRH ligand and LHRH receptor transcript forms was studied in 39 human uveal melanoma specimens by RT-PCR using gene specific primers. The binding charachteristics of receptors for LHRH on 10 samples were determined by ligand competition assays. The presence of LHRH receptor protein was further evaluated by immunohistochemistry. The expression of mRNA for type I LHRH receptor was detected in 18 of 39 (46%) of tissue specimens. mRNA for LHRH-I ligand could be detected in 27 of 39 (69%) of the samples. Seven of 10 samples investigated showed high affinity LHRH-I receptors. The specific presence of full length LHRH receptor protein was further confirmed by immunohistochemistry. A high percentage of uveal melanomas express mRNA and protein for type-I LHRH receptors. Our results support the merit of further investigation of LHRH receptors in human ophthalmological tumors. Since diverse analogs of LHRH are in clinical trials or are already used for the treatment of various cancers, these analogs could be considered for the LHRH receptor-based treatment of uveal melanoma. PMID:24077773

  4. Melanotransferrin induces human melanoma SK-Mel-28 cell invasion in vivo

    SciTech Connect

    Bertrand, Yanick . E-mail: oncomol@nobel.si.uqam.ca

    2007-02-09

    The expression of melanotransferrin (MTf), a membrane-bound glycoprotein highly expressed in melanomas, is correlated with tumor vascularization and progression, suggesting a proinvasive function associated with MTf in malignant tumors. To test this hypothesis, we silenced MTf in human melanoma SK-MEL-28 cells using small interfering RNA (siRNA) and examined the plasmin activity and invasiveness of MTf-silenced melanoma. In vitro, the siRNA-mediated MTf knockdown inhibited by 58% the cell surface activation of plasminogen into plasmin. In addition, decreased expression of MTf in melanoma cells reduced cell migration. In vivo, we used a nude mice invasion model in which tissue factor (TF) induces vascular [{sup 125}I]-fibrin deposition following injection. Using this metastasis model, the invasive potential of MTf-silenced cells into the lungs was reduced by fivefold. Altogether, these findings strongly suggest that MTf overexpression in melanoma cells contributes to tumor progession by stimulating plasmin generation as well as cell migration and invasion.

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

    PubMed

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

    2007-06-01

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

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

    PubMed

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

    2007-02-01

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

  7. Nutrition and melanoma prevention.

    PubMed

    Jensen, J Daniel; Wing, Gregory J; Dellavalle, Robert P

    2010-01-01

    Melanoma has continued to rise in incidence despite public efforts to promote sun protection behaviors. Because sunscreen use does not completely prevent skin cancer induced by ultraviolet radiation, additional chemopreventive methods for protecting against and reversing the effects of ultraviolet photodamage need evaluation. Recent years have brought increased interest in dietary factors, such as natural botanicals and vitamins, for the prevention of melanoma. This contribution provides a narrative review of the relevant, nutrition-related literature found by searching the keywords "melanoma chemoprevention," "nutrition and melanoma," "dietary botanicals and melanoma prevention," "green tea and melanoma," "vitamin D and melanoma," and "vitamin E and melanoma" in the PubMed database. Although randomized controlled trials of humans are lacking, basic science and epidemiologic studies show promising benefits of many natural products in chemoprevention for melanoma. Future studies, hopefully, will yield concrete answers and clarify the role of commonly available dietary nutrients in melanoma chemoprevention. PMID:21034988

  8. Modulation of tumor growth by inhibitory Fcγ receptor expressed by human melanoma cells

    PubMed Central

    Cassard, Lydie; Cohen-Solal, Joël F.G.; Galinha, Annie; Sastre-Garau, Xavier; Mathiot, Claire; Galon, Jérôme; Dorval, Thierry; Bernheim, Alain; Fridman, Wolf H.; Sautès-Fridman, Catherine

    2002-01-01

    The efficacy of anti-tumor IgG reflects the balance between opposing signals mediated by activating and inhibitory Fcγ receptors (FcγRs) expressed by effector cells. Here, we show that human malignant melanoma cells express the inhibitory low-affinity Fcγ receptor FcγRIIB1 in 40% of tested metastases. When melanoma cells were grafted in nude mice, a profound inhibition of FcγRIIB1 tumor growth that required the intracytoplasmic region of the receptor was observed. IgG immune complexes (ICs) may be required for this inhibition, since sera from nude mice bearing tumors contained IgG that decreased the proliferation of FcγRIIB1-positive cells in vitro, and tumor development of FcγRIIB1-positive melanoma lines was not inhibited in antibody-defective severe combined immunodeficiency (SCID) mice. Passive immunization of SCID mice with anti–ganglioside GD2 antibody resulted in significant inhibition of growth of FcγRIIB1-positive tumors in an intracytoplasmic-dependent manner. Altogether, these data suggest that human melanoma cells express biologically active inhibitory FcγRIIB1, which regulates their development upon direct interaction with anti-tumor antibodies. Therefore, FcγR expression on human tumors may be one component of the efficacy of antibody-mediated therapies, and FcγR-positive tumors could be the most sensitive candidates for such treatments. PMID:12438452

  9. Orthotopic Human Choroidal Melanoma Xenografts in Nude Rats with Aggressive and Nonaggressive PAS Staining Patterns

    PubMed Central

    Braun, Rod D.; Abbas, Asad

    2007-01-01

    PURPOSE Choroidal melanoma is the most common primary ocular cancer among the adult population. Patient survival has been linked to the periodic acid-Schiff base (PAS)–positive vascular patterns in the tumors. The presence of PAS-positive loops or cross-linking parallel channels is a marker of an aggressive tumor. The purpose of this study was to develop new xenograft models of human choroidal melanoma that predictably demonstrate the PAS staining patterns associated with nonaggressive and aggressive tumors in humans. METHODS Three human choroidal melanoma cell lines (C918, M619, and OCM-1) were used. C918 and M619 are considered aggressive, based on their ability to form PAS-positive channels in vitro. The nonaggressive OCM-1 cells do not form these channels. C918, M619, and OCM-1 spheroids were grown and implanted in the suprachoroidal space of 20, 17, and 16 WAG/RijHs-rnu nude rats, respectively. Tumors were grown for 1 to >4 weeks, and histology was performed to evaluate tumor growth and determine PAS labeling patterns. RESULTS Growth of C918, M619, and OCM-1 xenografts were histologically verified in 20/20, 15/17, and 16/16 rats, respectively. PAS staining revealed loops and cross-linking parallel channels, typical of aggressive tumors in patients, in 90% of C918 and 100% of M619 xenografts. Only 4 of 16 OCM-1 xenografts showed PAS-positive loops. The rest showed no PAS staining or only perivascular staining, indicative of nonaggressive tumors. CONCLUSIONS It is possible to grow human choroidal melanoma orthotopic xenografts in nude rats that reproduce the PAS staining patterns associated with aggressive and nonaggressive choroidal melanomas in patients. PMID:16384938

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2016-05-01

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

  12. Clinically proven markers of metastasis predict metastatic spread of human melanoma cells engrafted in scid mice

    PubMed Central

    Thies, A; Mauer, S; Fodstad, O; Schumacher, U

    2007-01-01

    Metastasis formation is a complex process and as such can only be modelled in vivo. As markers indicating metastatic spread in syngenic mouse models differ significantly from those in man, this study aimed to develop a human melanoma xenograft mouse model that reflects the clinical situation. Six human melanoma cell lines (LOX, MV3, FEMX-1, G361, MeWo and UISO-Mel6) were xenografted into severe combined immunodeficient mice and tumour growth, metastatic behaviour and number of lung metastases were assessed. Tumours and metastases were analysed for HPA binding and expression of CEACAM-1 and L1, all markers indicative of metastasis in clinical studies. Development of primary tumour nodules ranged from 3 weeks (MV3) to 3 months (MeWo). Whereas G361 and FEMX-1 rarely formed lung metastases, MeWo, MV3 and LOX were moderately and UISO-Mel6 was highly metastatic. Similar to clinical studies, HPA, CEACAM1 and L1 indicated metastatic spread in the xenograft melanoma model, but were not all simultaneously expressed in all cell lines. Considering the strongest expression of one marker combined with an absent or low expression of the other two markers, we conclude that LOX is the cell line of choice for analyses of the functional role of HPA-binding glycoconjugates, UISO-Mel6 is ideally suited to study CEACAM1 function in melanoma spread and L1 function can best be modelled using MeWo. PMID:17262079

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-11-24

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

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

    PubMed Central

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

    2015-01-01

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

  16. Realgar (As4S4) nanoparticles and arsenic trioxide (As2O3) induced autophagy and apoptosis in human melanoma cells in vitro.

    PubMed

    Pastorek, M; Gronesova, P; Cholujova, D; Hunakova, L; Bujnakova, Z; Balaz, P; Duraj, J; Lee, T C; Sedlak, J

    2014-01-01

    The aim of the present study was to compare the effect of realgar nanoparticles and arsenic trioxide (ATO) on viability, DNA damage, proliferation, autophagy and apoptosis in the human melanoma cell lines BOWES and A375. The application of various flow cytometric methods for measurements cell viability, DNA cell cycle, mitochondrial potential, lysosomal activity, and intracellular content of glutathione was used. In addition, quantitative PCR, western blotting and multiplex bead array analyses were applied for evaluation of redox stress, autophagic flux, and cell signaling alterations.The results showed that realgar treatment of studied cells caused modulation of cell proliferation, induced a block in G2/M phase of the cell cycle and altered phosphorylation of IκB, Akt, ERK1/2, p38, and JNK kinases, as well as decreased mitochondrial membrane potential. Additionally, it appeared that induction of cell death by both realgar and ATO was dose-dependent, when lower (0.3 µM) dosage increased lysosomal activity and induced autophagy and higher (1.25 µM) concentration resulted in the appearance of apoptosis, while pan-caspase inhibitor attenuated more efficiently realgar- than ATO-induced cell death. Furthermore, low concentrations of ATO and realgar nanoparticles increased the content of intracellular glutathione and elevated γ-H2AX expression confirmed DNA damage preferentially at higher concentrations of both drugs used. Further analysis revealed slight differences in time-dependent phosphorylation pattern due to both realgar and ATO treatments, while significant differences were noticed between cell lines. In conclusion, realgar nanoparticles and ATO treatment induced dose-dependent activation of autophagy and apoptosis in both melanoma cell lines, when autophagy flux was determined at lower drug concentrations and the switch to apoptosis occurred at higher concentrations of both arsenic forms. PMID:25150315

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

    PubMed

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

    2015-11-01

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

  18. Gene expression profiles of human melanoma cells with different invasive potential reveal TSPAN8 as a novel mediator of invasion

    PubMed Central

    Berthier-Vergnes, O; Kharbili, M El; de la Fouchardière, A; Pointecouteau, T; Verrando, P; Wierinckx, A; Lachuer, J; Le Naour, F; Lamartine, J

    2011-01-01

    Background: Metastatic melanoma requires early detection, being treatment resistant. However, the earliest events of melanoma metastasis, and especially of dermal invasion, remain ill defined. Results and methods: Gene expression profiles of two clonal subpopulations, selected from the same human melanoma cell line, but differing in ability to cross the dermal–epidermal junction in skin reconstructs, were compared by oligonucleotide microarray. Of 26 496 cDNA probes, 461 were differentially expressed (>2-fold; P< 0.001), only 71 genes being upregulated in invasive cells. Among them, TSPAN8, a tetraspanin not yet described in melanoma, was upregulated at mRNA and protein levels in melanoma cells from the invasive clone, as assessed by RT–PCR, flow cytometry and western blot analysis. Interestingly, TSPAN8 was the only tetraspanin in which overexpression correlated with invasive phenotype. Flow cytometry of well-defined melanoma cell lines confirmed that TSPAN8 was exclusively expressed by invasive, but not non-invasive melanoma cells or normal melanocytes. Immunohistochemistry revealed that TSPAN8 was expressed by melanoma cells in primary melanomas and metastases, but not epidermal cells in healthy skin. The functional role of TSPAN8 was demonstrated by silencing endogenous TSPAN8 with siRNA, reducing invasive outgrowth from tumour spheroids within matrigel without affecting cell proliferation or survival. Conclusion: TSPAN8 expression may enable melanoma cells to cross the cutaneous basement membrane, leading to dermal invasion and progression to metastasis. TSPAN8 could be a promising target in early detection and treatment of melanoma. PMID:21081927

  19. Identification of a Cell Surface Protein, p97, in Human Melanomas and Certain Other Neoplasms

    NASA Astrophysics Data System (ADS)

    Woodbury, Richard G.; Brown, Joseph P.; Yeh, Ming-Yang; Hellstrom, Ingegerd; Hellstrom, Karl Erik

    1980-04-01

    BALB/c mice were immunized with a human melanoma cell line, SK-MEL 28, and their spleen cells were fused with mouse NS-1 myeloma cells. Hybrid cells were tested in an indirect 125I-labeled protein A assay for production of antibodies that bound to surface antigens of SK-MEL 28 melanoma cells but not to autologous skin fibroblasts. One hybridoma, designated 4.1, had the required specificity. It was cloned and grown in mice as an ascites tumor. The monoclonal IgG1 antibody produced by the hybridoma was purified from the ascites fluid and labeled with 125I. The labeled antibody bound, at significant levels, to approximately 90% of the melanomas tested and to approximately 55% of other tumor cells, but not to three B-lymphoblastoid cell lines or to cultivated fibroblasts from 15 donors. Immunoprecipitation and sodium dodecyl sulfate gel electrophoresis were used to detect the target antigen in 125I-labeled cell membranes of both cultivated cells and tumor biopsy samples. A protein with a molecular weight of 97,000 was identified. This protein, designated p97, was present in both cultured cells and biopsy material from melanomas and certain other tumors, but it was not detected in eight different samples of normal adult epithelial or mesenchymal tissues obtained from five donors.

  20. Spectrophotometric Method for Differentiation of Human Skin Melanoma. I. Optical Diffuse Reflection Coefficient

    NASA Astrophysics Data System (ADS)

    Petruk, V. G.; Ivanov, A. P.; Kvaternyuk, S. M.; Barun, V. V.

    2016-03-01

    We have designed an experimental setup, based on two integrating spheres, that lets us measure the optical diffuse reflectance spectra (diffuse reflection coefficient vs. wavelength) of human skin quickly under clinical conditions in vivo. For the wavelength interval 520-1100 nm, we give the values of the diffuse reflection coefficient for healthy tissue, skin with a benign nevus, and skin with a malignant melanoma for a large group of test subjects. We experimentally established a number of wavelengths in the red-near IR region of the spectrum which can be used for early differential diagnosis of nevi and melanoma in patient cancer screening. According to the Kramer-Welch test, the probability of the diffuse reflection coefficient for skin with melanoma and a nevus having different distributions is >0.94, and at many wavelengths it is >0.999. By solving the inverse problem, we estimated the changes in a number of structural and biophysical parameters of the tissue on going from healthy skin to nevus and melanoma. The results obtained can provide a basis for developing a clinical approach to identifying the risk of malignant transformation of the skin before surgery and histological analysis of the tissue.

  1. Sex-dependent liver colonization of human melanoma in SCID mice--role of host defense mechanisms.

    PubMed

    Dobos, Judit; Mohos, Anita; Tóvári, József; Rásó, Erzsébet; Lőrincz, Tamás; Zádori, Gergely; Tímár, József; Ladányi, Andrea

    2013-04-01

    The possibility that endocrine factors may influence the clinical course of malignant melanoma is suggested by the superior survival data of women. In preclinical models we observed a higher rate of colony formation by human melanoma cells in male compared to female SCID mice, but only in the case of the liver and not in other organs. The gender difference could be seen at an early phase of colony formation. On the other hand, in our human melanoma cell lines we failed to detect steroid receptor protein expression, and treatment with sex hormones did not considerably influence their in vitro behavior. Investigating the possible contribution of host cells to the observed gender difference, we performed in vivo blocking experiments applying pretreatment of the animals with Kupffer cell inhibitor gadolinium chloride and the NK cell inhibitor anti-asialo GM1 antibody. While Kupffer cell blockade enhanced melanoma liver colonization equally in the two sexes, a more prominent increase was observed in female than in male mice in the case of NK cell inhibition. Further supporting the importance of NK cells in the lower liver colonization efficiency of melanoma cells in females, gender difference in colony formation was lost in NSG mice lacking NK activity. Although in humans no organ selectivity of gender difference in melanoma progression has been observed according to data in the literature, our results possibly indicate a contribution of natural host defense mechanisms to gender difference in survival of patients with melanoma or other tumor types as well. PMID:23203681

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

    PubMed

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

    2014-08-01

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

  3. Ursolic acid and resveratrol synergize with chloroquine to reduce melanoma cell viability.

    PubMed

    Junco, Jacob J; Mancha-Ramirez, Anna; Malik, Gunjan; Wei, Sung-Jen; Kim, Dae Joon; Liang, Huiyun; Slaga, Thomas J

    2015-04-01

    Malignant melanoma is associated with a 5-year survival rate of less than 20% once metastasized. Malignant melanoma cells exhibit increased levels of autophagy, a process of intracellular digestion that allows cells to survive various stresses including chemotherapies, resulting in reduced patient survival. Autophagy can be inhibited by chemicals like chloroquine (CQ), which prevents fusion of autophagosomes to lysosomes, resulting in autophagosome accumulation in most systems. Here, we describe how tested CQ to see whether it could sensitize B16F10 metastatic mouse melanoma cells to the anticancer activities of the natural compounds ursolic acid (UA) and resveratrol (RES). CQ with UA or RES strongly and synergistically reduced the viability of B16F10 mouse melanoma and A375 human melanoma cells. Surprisingly, flow cytometry of acridine orange-stained cells showed that UA or RES in combination with CQ significantly reduced autophagosome levels. Western blotting analysis revealed that CQ plus UA or RES paradoxically increased LC3II, indicative of autophagosome accumulation. In addition, CQ plus RES synergistically decreased the levels of both autophagy initiator beclin-1 and autophagy supporter p62. These results indicate that CQ with UA or RES strongly and synergistically reduces the viability of B16F10 and A375 melanoma cells. However, studies on B16F10 cells have shown that the synergistic effect was not mediated by inhibition of autophagy induced by UA or RES. These compounds are well-tolerated in humans, and CQ has shown promise as an adjuvant therapy. These combinations may be valuable treatment strategies for melanoma. PMID:25647735

  4. An Endogenous Electron Spin Resonance (ESR) Signal Discriminates Nevi from Melanomas in Human Specimens: A Step Forward in Its Diagnostic Application

    PubMed Central

    Cesareo, Eleonora; Korkina, Liudmila; D’Errico, Gerardino; Vitiello, Giuseppe; Aguzzi, Maria Simona; Passarelli, Francesca; Pedersen, Jens Z.; Facchiano, Antonio

    2012-01-01

    Given the specific melanin-associated paramagnetic features, the Electron Spin Resonance (ESR, called also Electron Paramagnetic Resonance, EPR) analysis has been proposed as a potential tool for non-invasive melanoma diagnosis. However, studies comparing human melanoma tissues to the most appropriate physiological counterpart (nevi) have not been performed, and ESR direct correlation with melanoma clinical features has never been investigated. ESR spectrum was obtained from melanoma and non-melanoma cell-cultures as well as mouse melanoma and non-melanoma tissues and an endogenous ESR signal (g = 2.005) was found in human melanoma cells and in primary melanoma tissues explanted from mice, while it was always absent in non-melanoma samples. These characteristics of the measured ESR signal strongly suggested its connection with melanin. Quantitative analyses were then performed on paraffin-embedded human melanoma and nevus sections, and validated on an independent larger validation set, for a total of 112 sections (52 melanomas, 60 nevi). The ESR signal was significantly higher in melanomas (p = 0.0002) and was significantly different between “Low Breslow’s and “High Breslow’s” depth melanomas (p<0.0001). A direct correlation between ESR signal and Breslow’s depth, expressed in millimetres, was found (R = 0.57; p<0.0001). The eu/pheomelanin ratio was found to be significantly different in melanomas “Low Breslow’s” vs melanomas “High Breslow’s” depth and in nevi vs melanomas “High Breslow’s depth”. Finally, ROC analysis using ESR data discriminated melanomas sections from nevi sections with up to 90% accuracy and p<0.0002. In the present study we report for the first time that ESR signal in human paraffin-embedded nevi is significantly lower than signal in human melanomas suggesting that spectrum variations may be related to qualitative melanin differences specifically occurring in melanoma cells. We therefore conclude that

  5. Altered Splicing of JARID1B in Development of Human Cutaneous Melanoma?

    PubMed

    Kuźbicki, Łukasz; Lange, Dariusz; Strączyńska-Niemiec, Anita; Chwirot, Barbara W

    2016-03-01

    Upregulated expression of histone H3K4 demethylase JARID1B has been found in several types of human cancer, but the expression pattern of this protein in benign naevi and human cutaneous melanomas seems to differ from that described for other tumors. We demonstrate that the apparent contradiction may be because of the fact that the malignant transformation of melanocytes is associated not so much with a general enhancement of a total expression of JARID1B but rather with a change in relative expression levels of individual splicing variants of the protein. Our data indicate that parallel immunohistochemical assays of the expression levels of all the isoforms and of the RBP2-H1 variant of JARID1B may be an efficient technique of differentiating between benign naevi and melanomas. PMID:25789538

  6. Development, characterization, and photocytotoxicity assessment on human melanoma of chloroaluminum phthalocyanine nanocapsules.

    PubMed

    Siqueira-Moura, Marigilson P; Primo, Fernando L; Espreafico, Enilza M; Tedesco, Antonio C

    2013-04-01

    In this work we have developed nanocapsules containing chloroaluminum phthalocyanine (ClAlPc) and assessed their phototoxic action on WM1552C, WM278, and WM1617 human melanoma cell lines. The ClAlPc-loaded nanocapsules were prepared by the nanoprecipitation method and optimized by means of a 2(3) full factorial design. The ClAlPc nanocapsules were characterized by particle size and distribution, zeta potential, morphology, encapsulation efficiency, singlet oxygen production, stability, and phototoxic action on melanoma cells. Both the development and optimization studies revealed that stable colloidal formulations could be obtained by using 1.75% (w/v) soybean lecithin, 1.25% (w/v) Poloxamer 188, 2.5% (v/v) soybean oil, and 0.75% (w/v) poly(D,L-lactide-co-glycolide). The nanocapsules had a mean diameter of 230 nm, homogeneous size distribution (polydispersity index<0.3), and negative zeta potential (about -30 mV). Their morphology was spherical, with evident polymer membrane coating droplet. The encapsulation efficiency was 70%, as expected for hydrophobic drugs, and the nanoencapsulated ClAlPc was able to produce high singlet oxygen quantum yield. ClAlPc nanocapsules exhibited good physical stability over a 12-month period. WM1552C primary melanoma cells were more sensitive (p<0.05) to the phototoxic effect elicited by ClAlPc nanocapsules (0.3 μg ml(-1)) under light irradiation at 20 mJ cm(-2). On the other hand, the cell survival percentage for all the melanoma cell lines treated with the highest light dose (150 mJ cm(-2)) was lower than 10%. In summary, ClAlPc nanoencapsulation could enable application of this hydrophobic photosensitizer in the treatment of malignant melanoma with the use of both low sensitizer drug concentration and light dose. PMID:23827632

  7. Analysis and significance of the malignant 'eclipse' during the progression of primary cutaneous human melanomas.

    PubMed

    Kerbel, R S; Kobayashi, H; Graham, C H; Lu, C

    1996-04-01

    Why is it that primary melanomas which are less than 0.76 mm in thickness are almost always curable by surgery whereas thicker lesions are associated with a worse prognosis? Put in another way, why is it that such small increases in tumor thickness beyond 0.76 mm are often associated with the eventual formation of distant metastases and death? Part of the answer lies in the dramatic qualitative changes which can accompany small increases in the size of primary human melanomas. Thus, primary melanomas less than 0.76 mm in thickness usually contain very low proportions of metastatically competent tumor cells, whereas slightly thicker lesions can contain very high proportions of such cells, resulting from a selective growth advantage of the latter in the dermal mesenchyme. This overgrowth process is akin to a 'malignant eclipse' phenomenon (by analogy with a solar eclipse). We have been studying the causes of the malignant eclipse in melanoma, for which there are at least four possibilities: 1) an increase in autocrine, mitogenic growth factors by melanoma cells; 2) a decreased rate of apoptosis in the same population; 3) an acquired resistance to paracrine growth inhibitory factors; and 4) an increased ability to induce an angiogenic response. Evidence exists for all four possibilities. Our experimental approach to studying this problem has relied heavily on the use of cell lines obtained from early stage radial growth phase or vertical growth phase lesions which have a clinical-like inability to grow progressively in nude mice, and variants obtained from such lines which are aggressively tumorigenic. Using such paired lines, and other experimental systems, we have obtained evidence that shows early stage melanoma cell lines may be deficient in inducing angiogenesis, are highly sensitive to the growth inhibitory effects of a plethora of cytokines, including transforming growth factor beta, interleukin-6, and oncostatin M, and are more sensitive to undergoing

  8. The activation of human endogenous retrovirus K (HERV-K) is implicated in melanoma cell malignant transformation

    SciTech Connect

    Serafino, A. Balestrieri, E.; Pierimarchi, P.; Matteucci, C.; Moroni, G.; Oricchio, E.; Rasi, G.; Mastino, A.; Spadafora, C.; Garaci, E.; Vallebona, P. Sinibaldi

    2009-03-10

    Melanoma development is a multi-step process arising from a series of genetic and epigenetic events. Although the sequential stages involved in progression from melanocytes to malignant melanoma are clearly defined, our current understanding of the mechanisms leading to melanoma onset is still incomplete. Growing evidence show that the activation of endogenous retroviral sequences might be involved in transformation of melanocytes as well as in the increased ability of melanoma cells to escape immune surveillance. Here we show that human melanoma cells in vitro undergo a transition from adherent to a more malignant, non-adherent phenotype when exposed to stress conditions. Melanoma-derived non-adherent cells are characterized by an increased proliferative potential and a decreased expression of both HLA class I molecules and Melan-A/MART-1 antigen, similarly to highly malignant cells. These phenotypic and functional modifications are accompanied by the activation of human endogenous retrovirus K expression (HERV-K) and massive production of viral-like particles. Down-regulation of HERV-K expression by RNA interference prevents the transition from the adherent to the non-adherent growth phenotype in low serum. These results implicate HERV-K in at least some critical steps of melanoma progression.

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

    SciTech Connect

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

    1987-08-01

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

  10. Human papillomavirus and the development of non-melanoma skin cancer.

    PubMed Central

    Harwood, C A; McGregor, J M; Proby, C M; Breuer, J

    1999-01-01

    Human papillomaviruses (HPV) are increasingly recognised as important human carcinogens. The best established association with human malignancy is that of high-risk mucosal HPV types and anogenital cancer. HPV-induced transformation of anogenital epithelia has been the subject of intense research which has identified the cellular tumour suppressor gene products, p53 and pRB, as important targets for the viral oncoproteins E6 and E7 respectively. Certain HPV types are also strongly associated with the development of non-melanoma skin cancer in the inherited disorder epidermodysplasia verruciformis (EV). However, in contrast with anogenital malignancy the oncogenic mechanisms of EV-HPV types remain uncertain, and there appears to be a crucial additional requirement for ultraviolet radiation. Cutaneous HPV types in the general population are predominantly associated with benign viral warts, but a role in non-melanoma skin cancer has recently been postulated. Polymerase chain reaction based HPV detection techniques have shown a high prevalence of HPV DNA, particularly in skin cancers from immunosuppressed patients and to a lesser extent in malignancies from otherwise immunocompetent individuals. No particular HPV type has yet emerged as predominant, and the role of HPV in cutaneous malignancy is unclear at present. It remains to be established whether HPV plays an active or purely a passenger role in the evolution of non-melanoma skin cancer. PMID:10474513

  11. Inhibitor of DNA Binding 4 (ID4) Is Highly Expressed in Human Melanoma Tissues and May Function to Restrict Normal Differentiation of Melanoma Cells

    PubMed Central

    Peretz, Yuval; Wu, Hong; Patel, Shayan; Bellacosa, Alfonso; Katz, Richard A.

    2015-01-01

    Melanoma tissues and cell lines are heterogeneous, and include cells with invasive, proliferative, stem cell-like, and differentiated properties. Such heterogeneity likely contributes to the aggressiveness of the disease and resistance to therapy. One model suggests that heterogeneity arises from rare cancer stem cells (CSCs) that produce distinct cancer cell lineages. Another model suggests that heterogeneity arises through reversible cellular plasticity, or phenotype-switching. Recent work indicates that phenotype-switching may include the ability of cancer cells to dedifferentiate to a stem cell-like state. We set out to investigate the phenotype-switching capabilities of melanoma cells, and used unbiased methods to identify genes that may control such switching. We developed a system to reversibly synchronize melanoma cells between 2D-monolayer and 3D-stem cell-like growth states. Melanoma cells maintained in the stem cell-like state showed a striking upregulation of a gene set related to development and neural stem cell biology, which included SRY-box 2 (SOX2) and Inhibitor of DNA Binding 4 (ID4). A gene set related to cancer cell motility and invasiveness was concomitantly downregulated. Intense and pervasive ID4 protein expression was detected in human melanoma tissue samples, suggesting disease relevance for this protein. SiRNA knockdown of ID4 inhibited switching from monolayer to 3D-stem cell-like growth, and instead promoted switching to a highly differentiated, neuronal-like morphology. We suggest that ID4 is upregulated in melanoma as part of a stem cell-like program that facilitates further adaptive plasticity. ID4 may contribute to disease by preventing stem cell-like melanoma cells from progressing to a normal differentiated state. This interpretation is guided by the known role of ID4 as a differentiation inhibitor during normal development. The melanoma stem cell-like state may be protected by factors such as ID4, thereby potentially identifying a

  12. Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment.

    PubMed

    Sustarsic, Elahu G; Junnila, Riia K; Kopchick, John J

    2013-11-01

    Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute's NCI60 panel includes 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on this data, GH could be a new therapeutic target in melanoma. PMID:24134847

  13. Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

    PubMed Central

    Sustarsic, Elahu G.; Junnila, Riia K.; Kopchick, John J.

    2013-01-01

    Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute’s NCI60 panel includes 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on this data, GH could be a new therapeutic target in melanoma. PMID:24134847

  14. Overcoming MITF-conferred drug resistance through dual AURKA/MAPK targeting in human melanoma cells

    PubMed Central

    Pathria, G; Garg, B; Borgdorff, V; Garg, K; Wagner, C; Superti-Furga, G; Wagner, S N

    2016-01-01

    MITF (microphthalmia-associated transcription factor) is a frequently amplified lineage-specific oncogene in human melanoma, whose role in intrinsic drug resistance has not been systematically investigated. Utilizing chemical inhibitors for major signaling pathways/cellular processes, we witness MITF as an elicitor of intrinsic drug resistance. To search kinase(s) targets able to bypass MITF-conferred drug resistance, we employed a multi-kinase inhibitor-directed chemical proteomics-based differential affinity screen in human melanocytes carrying ectopic MITF overexpression. A subsequent methodical interrogation informed mitotic Ser/Thr kinase Aurora Kinase A (AURKA) as a crucial regulator of melanoma cell proliferation and migration, independent of the underlying molecular alterations, including TP53 functional status and MITF levels. Crucially, assessing the efficacy of investigational AURKA inhibitor MLN8237, we pre-emptively witness the procurement of a molecular program consistent with acquired drug resistance. This involved induction of multiple MAPK (mitogen-activated protein kinase) signaling pathway components and their downstream proliferation effectors (Cyclin D1 and c-JUN) and apoptotic regulators (MITF and Bcl-2). A concomitant AURKA/BRAF and AURKA/MEK targeting overcame MAPK signaling activation-associated resistance signature in BRAF- and NRAS-mutated melanomas, respectively, and elicited heightened anti-proliferative activity and apoptotic cell death. These findings reveal a previously unreported MAPK signaling-mediated mechanism of immediate resistance to AURKA inhibitors. These findings could bear significant implications for the application and the success of anti-AURKA approaches that have already entered phase-II clinical trials for human melanoma. PMID:26962685

  15. Overcoming MITF-conferred drug resistance through dual AURKA/MAPK targeting in human melanoma cells.

    PubMed

    Pathria, G; Garg, B; Borgdorff, V; Garg, K; Wagner, C; Superti-Furga, G; Wagner, S N

    2016-01-01

    MITF (microphthalmia-associated transcription factor) is a frequently amplified lineage-specific oncogene in human melanoma, whose role in intrinsic drug resistance has not been systematically investigated. Utilizing chemical inhibitors for major signaling pathways/cellular processes, we witness MITF as an elicitor of intrinsic drug resistance. To search kinase(s) targets able to bypass MITF-conferred drug resistance, we employed a multi-kinase inhibitor-directed chemical proteomics-based differential affinity screen in human melanocytes carrying ectopic MITF overexpression. A subsequent methodical interrogation informed mitotic Ser/Thr kinase Aurora Kinase A (AURKA) as a crucial regulator of melanoma cell proliferation and migration, independent of the underlying molecular alterations, including TP53 functional status and MITF levels. Crucially, assessing the efficacy of investigational AURKA inhibitor MLN8237, we pre-emptively witness the procurement of a molecular program consistent with acquired drug resistance. This involved induction of multiple MAPK (mitogen-activated protein kinase) signaling pathway components and their downstream proliferation effectors (Cyclin D1 and c-JUN) and apoptotic regulators (MITF and Bcl-2). A concomitant AURKA/BRAF and AURKA/MEK targeting overcame MAPK signaling activation-associated resistance signature in BRAF- and NRAS-mutated melanomas, respectively, and elicited heightened anti-proliferative activity and apoptotic cell death. These findings reveal a previously unreported MAPK signaling-mediated mechanism of immediate resistance to AURKA inhibitors. These findings could bear significant implications for the application and the success of anti-AURKA approaches that have already entered phase-II clinical trials for human melanoma. PMID:26962685

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  18. In-vivo xenograft murine human uveal melanoma model develops hepatic micrometastases

    PubMed Central

    Yang, Hua; Fang, Guofu; Huang, Xinping; Yu, Jie; Hsieh, Chia-Ling; Grossniklaus, Hans E.

    2009-01-01

    The purpose of the study is to develop a mouse ocular melanoma model with human uveal melanoma cells that forms hepatic micrometastases. Human uveal melanoma Mel290 cells were transfected with a lentiviral-enhanced green fluorescent protein (EGFP) expression vector. Proliferation assays were performed by comparing Mel290-EGFP and Mel290 cells. After stable expression of EGFP and proliferation was ascertained, 1 × 106 Mel290-EGFP cells were introduced into NU/NU mice by posterior compartment (PC) inoculation or tail vein injection. Control groups were inoculated or injected with Mel290 cells. Ocular and hepatic frozen sections were examined by fluorescence microscopy, and the number of hepatic micrometastases was determined. EGFP expression was observed at 24 h after transfection. At 72 h after transfection, more than 70% of Mel290 cells expressed EGFP. At 45 days (six passages), 90% of Mel290 cells stably expressed EGFP. Histologic examination showed that Mel290-EGFP cells formed hepatic micrometastases after either PC inoculation or tail vein injection. A significant difference in the number of hepatic micrometastases between PC inoculation and tail vein injection (P<0.01) was observed. Mel290-EGFP cells stably expressed green fluorescent protein in vitro at 45 days (six passages). These cells formed hepatic micrometastases in NU/NU mice after PC inoculation or tail vein injection, with significantly more micrometastases developing in the PC inoculation model than after tail vein injection. PMID:18337645

  19. In Vitro Efficacy and Mechanistic Role of Indocyanine Green as a Photodynamic Therapy Agent for Human Melanoma

    SciTech Connect

    Mamoon, A.; Gamal-Eldeen, A; Ruppel, M; Smith, R; Tsang, T; Miller, L

    2009-01-01

    Photodynamic therapy (PDT) is a promising treatment for superficial cancer. However, poor therapeutic results have been reported for melanoma, due to the high melanin content. Indocyanine green (ICG) has near infrared absorption (700-800nm) and melanins do not absorb strongly in this area. This study explores the efficiency of ICG as a PDT agent for human melanoma, and its mechanistic role in the cell death pathway.

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

    PubMed

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

    2015-09-01

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

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

    PubMed

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

    2016-01-01

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

  2. 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

  3. Functional Classification of Cellular Proteome Profiles Support the Identification of Drug Resistance Signatures in Melanoma Cells

    PubMed Central

    2013-01-01

    Drug resistance is a major obstacle in melanoma treatment. Recognition of specific resistance patterns, the understanding of the patho-physiology of drug resistance, and identification of remaining options for individual melanoma treatment would greatly improve therapeutic success. We performed mass spectrometry-based proteome profiling of A375 melanoma cells and HeLa cells characterized as sensitive to cisplatin in comparison to cisplatin resistant M24met and TMFI melanoma cells. Cells were fractionated into cytoplasm, nuclei and secretome and the proteome profiles classified according to Gene Ontology. The cisplatin resistant cells displayed increased expression of lysosomal as well as Ca2+ ion binding and cell adherence proteins. These findings were confirmed using Lysotracker Red staining and cell adhesion assays with a panel of extracellular matrix proteins. To discriminate specific survival proteins, we selected constitutively expressed proteins of resistant M24met cells which were found expressed upon challenging the sensitive A375 cells. Using the CPL/MUW proteome database, the selected lysosomal, cell adherence and survival proteins apparently specifying resistant cells were narrowed down to 47 proteins representing a potential resistance signature. These were tested against our proteomics database comprising more than 200 different cell types/cell states for its predictive power. We provide evidence that this signature enables the automated assignment of resistance features as readout from proteome profiles of any human cell type. Proteome profiling and bioinformatic processing may thus support the understanding of drug resistance mechanism, eventually guiding patient tailored therapy. PMID:23713901

  4. Basic and clinical aspects of malignant melanoma

    SciTech Connect

    Nathanson, L. )

    1987-01-01

    This book contains the following 10 chapters: The role of oncogenes in the pathogenesis of malignant melanoma; Laminin and fibronectin modulate the metastatic activity of melanoma cells; Structure, function and biosynthesis of ganglioside antigens associated with human tumors derived from the neuroectoderm; Epidemiology of ocular melanoma; Malignant melanoma: Prognostic factors; Endocrine influences on the natural history of human malignant melanoma; Psychosocial factors associated with prognostic indicators, progression, psychophysiology, and tumor-host response in cutaneous malignant melanoma; Central nervous system metastases in malignant melanoma; Interferon trials in the management of malignant melanoma and other neoplasms: an overview; and The treatment of malignant melanoma by fast neutrons.

  5. Targeting the PI3K/AKT/mTOR pathway overcomes the stimulating effect of dabrafenib on the invasive behavior of melanoma cells with acquired resistance to the BRAF inhibitor.

    PubMed

    Caporali, Simona; Alvino, Ester; Lacal, Pedro Miguel; Levati, Lauretta; Giurato, Giorgio; Memoli, Domenico; Caprini, Elisabetta; Antonini Cappellini, Gian Carlo; D'Atri, Stefania

    2016-09-01

    BRAF inhibitors (BRAFi) have proven clinical benefits in patients with BRAF-mutant melanoma. However, acquired resistance eventually arises. The effects of BRAFi on melanoma cell proliferation and survival have been extensively studied, and several mechanisms involved in acquired resistance to the growth suppressive activity of these drugs have been identified. Much less is known about the impact of BRAFi, and in particular of dabrafenib, on the invasive potential of melanoma cells. In the present study, the BRAF-mutant human melanoma cell line A375 and its dabrafenib-resistant subline A375R were analyzed for invasive capacity, expression of vascular endothelial growth factor receptor (VEGFR)-2, and secretion of VEGF-A and matrix metalloproteinase (MMP)-9, under basal conditions or in response to dabrafenib. The consequences of inhibiting the PI3K/AKT/mTOR pathway on A375R cell responses to dabrafenib were also evaluated. We found that A375R cells were more invasive and secreted higher levels of VEGF-A and MMP-9 as compared with A375 cells. Dabrafenib reduced invasiveness, VEGFR-2 expression and VEGF-A secretion in A375 cells, whereas it increased invasiveness, VEGF-A and MMP-9 release in A375R cells. In these latter cells, the stimulating effects of dabrafenib on the invasive capacity were markedly impaired by the anti-VEGF‑A antibody bevacizumab, or by AKT1 silencing. A375R cells were not cross-resistant to the PI3K/mTOR inhibitor GSK2126458A. Moreover, this inhibitor given in combination with dabrafenib efficiently counteracted the stimulating effects of the BRAFi on invasiveness and VEGF-A and MMP-9 secretion. Our data demonstrate that melanoma cells with acquired resistance to dabrafenib possess a more invasive phenotype which is further stimulated by exposure to the drug. Substantial evidence indicates that continuing BRAFi therapy beyond progression produces a clinical benefit. Our results suggest that after the development of resistance, a regimen

  6. Orthotopic xenografts of human melanoma and colonic and ovarian carcinoma in sheep to evaluate radioimmunotherapy.

    PubMed Central

    Turner, J. H.; Rose, A. H.; Glancy, R. J.; Penhale, W. J.

    1998-01-01

    Extrapolation to humans from experimental radioimmunotherapy in nude mouse xenograft models is confounded by large relative tumour size and small volume of distribution in mice allowing tumour uptake of radiolabelled antibodies unattainable in patients. Our large animal model of human tumours in cyclosporin-immunosuppressed sheep demonstrated tumour uptake of targeted radiolabelled monoclonal antibodies comparable with uptakes reported in clinical trials. Sheep immunosuppression with daily intravenous cyclosporin augmented by oral ketoconazole maintained trough blood levels of cyclosporin within the range 1000-1500 ng ml(-1). Human tumour cells were transplanted orthotopically by inoculation of 10(7) cells: SKMEL melanoma subcutaneously; LS174T and HT29 colon carcinoma into bowel, peritoneum and liver; and JAM ovarian carcinoma into ovary and peritoneum. Tumour xenografts grew at all sites within 3 weeks of inoculation, preserving characteristic morphology without evidence of necrosis or host rejection. Lymphatic metastasis was demonstrated in regional nodes draining xenografts of melanoma and ovarian carcinoma. Colonic LS1 74T xenografts produced mucin and carcinoembryonic antigen (CEA). The anti-CEA IgG1 monoclonal antibody A5B7 was radiolabelled with iodine-131 and administered intravenously to sheep. Peak uptake at 5 days in orthotopic human tumour transplants in gut was 0.027% DI g(-1) (percentage of injected dose per gram) and 0.034% DI g(-1) in hepatic metastases with tumour to blood ratios of 2-2.5. Non-specific tumour uptake in melanoma was 0.003% DI g(-1). Uptake of radiolabelled monoclonal antibody in human tumours in our large animal model is comparable with that observed in patients and may be more realistic than nude mice xenografts for prediction of clinical efficacy of radioimmunotherapy. Images Figure 1 Figure 2 Figure 3 PMID:9716032

  7. PGC1α Expression Defines a Subset of Human Melanoma Tumors with Increased Mitochondrial Capacity and Resistance to Oxidative Stress

    PubMed Central

    Vazquez, Francisca; Lim, Ji-Hong; Chim, Helen; Bhalla, Kavita; Girnun, Geoff; Pierce, Kerry; Clish, Clary B.; Granter, Scott R.; Widlund, Hans R.; Spiegelman, Bruce M.; Puigserver, Pere

    2013-01-01

    SUMMARY Cancer cells reprogram their metabolism using different strategies to meet energy and anabolic demands to maintain growth and survival. Understanding the molecular and genetic determinants of these metabolic programs is critical to successfully exploit them for therapy. Here, we report that the oncogenic melanocyte lineage-specification transcription factor MITF drives PGC1α (PPARGC1A) overexpression in a subset of human melanomas and derived cell lines. Functionally, PGC1α positive melanoma cells exhibit increased mitochondrial energy metabolism and ROS detoxification capacities that enables survival under oxidative stress conditions. Conversely, PGC1α negative melanoma cells are more glycolytic and sensitive to ROS-inducing drugs. These results demonstrate that differences in PGC1α levels in melanoma tumors have a profound impact in their metabolism, biology and drug sensitivity. PMID:23416000

  8. 7-Hydroxydehydronuciferine induces human melanoma death via triggering autophagy and apoptosis.

    PubMed

    Wu, Pei-Fang; Chiu, Chien-Chih; Chen, Chung-Yi; Wang, Hui-Min David

    2015-12-01

    Melanoma is the deadliest cancer. We identified 7-hydroxydehydronuciferine (7-HDNF) isolated from the leaves of Nelumbo nucifera Gaertn cv. Rosa-plena to be a bio-active agent that antagonizes melanoma tumor growth in mice xenograft model in vivo. Cell proliferation assay demonstrated strong anticancer effects of 7-HDNF to exhibit a dose-dependent behaviour and displayed minor cytotoxicities on normal human skin cells, including epidermal keratinocytes and melanocytes, and dermal fibroblasts. With acridine orange (AO) staining and flow analysis, we found 7-HDNF induced the formation of intracellular vacuoles and the augmentation of acidic vesicular organelles (AVO). The apoptotic cell death ratio was measured via two-dimensional flow cytometry by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double stained to confirm the cellular membrane asymmetry lost. One-dimensional flow cytometric analysis showed 7-HDNF increased the cellular arrest in cell cycle at the G2/M phase. Through Western blot examinations, protein expressions were discovered to verify autophagy and apoptosis response mechanisms sharing the associated pathways. Finally, 7-HDNF presented a high-quality antimigratory activity in wound-healing assay. Overall, 7-HDNF presented high-quality anticancer bio-functions and inhibited melanoma tumor growth in vivo and in vitro. PMID:26174122

  9. Mutational and Functional Analysis of the Tumor-Suppressor PTPRD in Human Melanoma

    PubMed Central

    Walia, Vijay; Prickett, Todd D.; Kim, Jung-Sik; Gartner, Jared J.; Lin, Jimmy C.; Zhou, Ming; Rosenberg, Steven A.; Elble, Randolph C.; Solomon, David A.; Waldman, Todd; Samuels, Yardena

    2015-01-01

    Protein tyrosine phosphatases (PTPs) tightly regulate tyrosine phosphorylation essential for cell growth, adhesion, migration, and survival. We performed a mutational analysis of the PTP gene family in cutaneous metastatic melanoma and identified 23 phosphatase genes harboring somatic mutations. Among these, receptor-type tyrosine–protein phosphatase delta (PTPRD) was one of the most highly mutated genes, harboring 17 somatic mutations in 79 samples, a prevalence of 21.5%. Functional evaluation of six PTPRD mutations revealed enhanced anchorage-dependent and anchorage-independent growth. Interestingly, melanoma cells expressing mutant PTPRD were significantly more migratory than cells expressing wild-type PTPRD or vector alone, indicating a novel gain-of-function associated with mutant PTPRD. To understand the molecular mechanisms of PTPRD mutations, we searched for its binding partners by converting the active PTPRD enzyme into a “substrate trap” form. Using mass spectrometry and coimmunoprecipitation, we report desmoplakin, a desmosomal protein that is implicated in cell–cell adhesion, as a novel PTPRD substrate. Further analysis showed reduced phosphatase activity of mutant PTPRD against desmoplakin. Our findings identify an essential signaling cascade that is disrupted in melanoma. Moreover, because PTPRD is also mutated in glioblastomas and adenocarcinoma of the colon and lung, our data might be applicable to a large number of human cancers. PMID:25113440

  10. Response of human neuroblastoma and melanoma multicellular tumor spheroids (MTS) to single dose irradiation

    SciTech Connect

    Evans, S.M.; Labs, L.M.; Yuhas, J.M.

    1986-06-01

    The growth characteristics of 6 human cell line derived multicellular tumor spheroids (MTS) were studied. Melanoma MTS (C32, HML-A, HML-B) were slow growing with baseline growth rates of 13.9 to 27.3 microns diameter/day. Neuroblastoma MTS (Lan-1, NB-100, NB-134) grew rapidly, with baseline growth rates of 32.1 to 40.3 microns diameter/day, that is, 1.2 to 2.9 times as fast as the melanomas. Delay constants were calculated for all six lines. The neuroblastomas were more sensitive to radiation than melanomas, as reflected in a greater value for the radiation-induced growth delay constant. One neuroblastoma line, Lan-1, was highly radioresponsive; that is, after a subcurative dose of radiation, the MTS diameter decreased beyond the original diameter, which was followed by recovery and regrowth. Irrespective of these initial changes in diameter, growth delay sensitivity (value of delay constant) was the same for Lan-1 and NB-100, an MTS line that did not show the responsive pattern.

  11. A non-canonical adenosinergic pathway led by CD38 in human melanoma cells induces suppression of T cell proliferation

    PubMed Central

    Chillemi, Antonella; Quarona, Valeria; Zaccarello, Gianluca; Carrega, Paolo; Ferlazzo, Guido; Mingari, Maria Cristina; Moretta, Lorenzo

    2015-01-01

    Nucleotide-metabolizing ectoenzymes are endowed with an extracellular catalytic domain, which is involved in regulating the extracellular nucleotide/nucleoside balance. The tumor microenvironment contains high levels of adenosine (ADO) generated by this enzymatic network, thus promoting tumor growth by inhibiting anti-tumor immune responses. ADO inhibition in melanoma murine models limits tumor metastases and restores anti-tumor immune responses. This work investigates the expression and function of ectoenzymes in primary human melanoma cell lines. All of latter cells expressed CD38, CD39, CD73, and CD203a/PC-1, and produced ADO from AMP and NAD+. Melanoma cells inhibited T cell proliferation through an ADO-dependent mechanism, since such inhibition was reverted using CD38/CD73 specific inhibitors. Melanoma cells abolished the function of effector memory, central memory and reduced naïve CD4+ T cell proliferation. Accordingly, phosphorylation of S6 ribosomal protein, p38 and Stat1 was lower in activated memory cells than in naïve CD4+ T lymphocytes. Melanoma cells also inhibited proliferation of naïve, memory and -to a lesser extent- of effector CD8+ T cells. These different inhibitory effects correlated with distinct patterns of expression of the ADO receptor A2a and A2b. These results show that primary human melanoma cell lines suppress in vitro T cell proliferation through an adenosinergic pathway in which CD38 and CD73 play a prominent role. PMID:26329660

  12. Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor.

    PubMed Central

    Kawakami, Y; Eliyahu, S; Delgado, C H; Robbins, P F; Rivoltini, L; Topalian, S L; Miki, T; Rosenberg, S A

    1994-01-01

    By cDNA expression cloning we have isolated a gene encoding a shared human melanoma antigen recognized by HLA-A2 restricted autologous and allogenic tumor-infiltrating lymphocytes (TILs) from patients with metastatic melanoma. By using both transient and stable expression systems, transfection of this gene into non-antigen-expressing HLA-A2+ cell lines resulted in recognition by the antigen-specific TILs. The sequence of this cDNA revealed a previously undescribed putative transmembrane protein whose expression was restricted to melanoma and melanocyte cell lines and human retina but no other fresh or cultured normal tissues tested or other tumor histologies. Thus, we have identified a gene encoding a melanocyte lineage-specific protein (MART-1; melanoma antigen recognized by T cells 1) that is a widely shared melanoma antigen recognized by the T lymphocytes of patients with established malignancy. Identification of this gene opens possibilities for the development of immunotherapies for patients with melanoma. PMID:8170938

  13. Wnt interaction and extracellular release of prominin-1/CD133 in human malignant melanoma cells.

    PubMed

    Rappa, Germana; Mercapide, Javier; Anzanello, Fabio; Le, Thuc T; Johlfs, Mary G; Fiscus, Ronald R; Wilsch-Bräuninger, Michaela; Corbeil, Denis; Lorico, Aurelio

    2013-04-01

    Prominin-1 (CD133) is the first identified gene of a novel class of pentaspan membrane glycoproteins. It is expressed by various epithelial and non-epithelial cells, and notably by stem and cancer stem cells. In non-cancerous cells such as neuro-epithelial and hematopoietic stem cells, prominin-1 is selectively concentrated in plasma membrane protrusions, and released into the extracellular milieu in association with small vesicles. Previously, we demonstrated that prominin-1 contributes to melanoma cells pro-metastatic properties and suggested that it may constitute a molecular target to prevent prominin-1-expressing melanomas from colonizing and growing in lymph nodes and distant organs. Here, we report that three distinct pools of prominin-1 co-exist in cultures of human FEMX-I metastatic melanoma. Morphologically, in addition to the plasma membrane localization, prominin-1 is found within the intracellular compartments, (e.g., Golgi apparatus) and in association with extracellular membrane vesicles. The latter prominin-1-positive structures appeared in three sizes (small, ≤40 nm; intermediates ~40-80 nm, and large, >80 nm). Functionally, the down-regulation of prominin-1 in FEMX-I cells resulted in a significant reduction of number of lipid droplets as observed by coherent anti-Stokes Raman scattering image analysis and Oil red O staining, and surprisingly in a decrease in the nuclear localization of beta-catenin, a surrogate marker of Wnt activation. Moreover, the T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter activity was 2 to 4 times higher in parental than in prominin-1-knockdown cells. Collectively, our results point to Wnt signaling and/or release of prominin-1-containing membrane vesicles as mediators of the pro-metastatic activity of prominin-1 in FEMX-I melanoma. PMID:23318676

  14. Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

    SciTech Connect

    Sustarsic, Elahu G.; Junnila, Riia K.; Kopchick, John J.

    2013-11-08

    Highlights: •Most cancer types of the NCI60 have sub-sets of cell lines with high GHR expression. •GHR is highly expressed in melanoma cell lines. •GHR is elevated in advanced stage IV metastatic tumors vs. stage III. •GH treatment of metastatic melanoma cell lines alters growth and cell signaling. -- Abstract: Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute’s NCI60 panel includes 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on

  15. Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish.

    PubMed

    Haldi, Maryann; Ton, Christopher; Seng, Wen Lin; McGrath, Patricia

    2006-01-01

    In this research, we optimized parameters for xenotransplanting WM-266-4, a metastatic melanoma cell line, including zebrafish site and stage for transplantation, number of cells, injection method, and zebrafish incubation temperature. Melanoma cells proliferated, migrated and formed masses in vivo. We transplanted two additional cancer cell lines, SW620, a colorectal cancer cell line, and FG CAS/Crk, a pancreatic cancer cell line and these human cancers also formed masses in zebrafish. We also transplanted CCD-1092Sk, a human fibroblast cell line established from normal foreskin and this cell line migrated, but did not proliferate or form masses. We quantified the number of proliferating melanoma and normal skin fibroblasts by dissociating xenotransplant zebrafish, dispensing an aliquot of CM-DiI labeled human cells from each zebrafish onto a hemocytometer slide and then visually counting the number of fluorescently labeled cancer cells. Since zebrafish are transparent until approximately 30 dpf, the interaction of labeled melanoma cells and zebrafish endothelial cells (EC) can be visualized by whole-mount immunochemical staining. After staining with Phy-V, a mouse anti-zebrafish monoclonal antibody (mAb) that specifically labels activated EC and angioblasts, using immunohistology and 2-photon microscopy, we observed activated zebrafish EC embedded in human melanoma cell masses. The zebrafish model offers a rapid efficient approach for assessing human cancer cells at various stages of tumorigenesis. PMID:17051341

  16. T cells in the human metastatic melanoma microenvironment express site-specific homing receptors and retention integrins.

    PubMed

    Salerno, Elise P; Olson, Walter C; McSkimming, Chantel; Shea, Sofia; Slingluff, Craig L

    2014-02-01

    T-cell infiltration into the metastatic melanoma microenvironment (MME) correlates with improved patient survival. However, diffuse infiltration into tumor occurs in only 8% of melanoma metastases. Little is known about mechanisms governing T-cell infiltration into human melanoma metastases or about how those mechanisms may be altered therapeutically. We hypothesized that T cells in the MME would be enriched for chemokine receptors CCR4, CCR5, CXCR3 and homing receptors relevant to the tissue site. Viably cryopreserved single cell suspensions from nineteen melanoma metastases representing three metastatic sites (tumor-infiltrated lymph node, skin and small bowel) were evaluated by multiparameter flow cytometry and compared to benign lymph nodes and peripheral blood mononuclear cells from patients with Stage IIB-IV melanoma. T cells in the melanoma metastases contained large effector memory populations, high proportions of activated, moderately differentiated cells and few regulatory T cells. Site-specific homing was suggested in bowel, with high expression of CCR9. We neither encounter the anticipated enrichment of integrin α4β7 in bowel, cutaneous leukocyte antigen (CLA) in skin, nor integrin α4β1 or receptor CXCR3 in metastatic sites. Retention integrins αEβ7, α1β1 and α2β1 were significantly elevated in metastases. These data suggest limited tissue site-specific homing to human melanoma metastases, but a significant role for retention integrins in maintaining intratumoral T cells. Our findings also raise the possibility that T-cell homing, infiltration, and retention in melanoma metastases may be increased by increasing expression of ligands for CLA, α4β1 and CXCR3 on intratumoral endothelium. PMID:23873187

  17. Optimization of radioimmunotherapy using human malignant melanoma multicell spheroids as a model

    SciTech Connect

    Kwok, C.S.; Crivici, A.; MacGregor, W.D.; Unger, M.W. )

    1989-06-15

    In vitro multicell spheroids from a human melanoma cell line and the human colon cancer cell line HT29, used as control, have been established as a model of poorly vascularized micrometastases in vivo. The antimelanoma monoclonal antibody 96.5 was radiolabeled with 131I at specific radioactivities from 1.85 to 3.96 GBq/mg. Cytotoxicity of 131I-96.5 to the spheroids, at an initial size of 300 microns in diameter, was investigated as a function of concentration of 131I-96.5 in the incubation medium, specific radioactivity, and treatment time. Spheroid growth delay and clonogenic survival of cells disaggregated from the spheroids at various times after treatment were used as end points. Therapeutic effects increased with the concentration of 131I-96.5 within the range 0.2 to 2 mg/liter (0.34 to 3.4 GBq/liter) at a fixed specific radioactivity. The effects increased with specific radioactivity at a fixed concentration of 131I-96.5. Difference in therapeutic effect was also observed between treatment times of 8 and 24 h. Radiation doses to the melanoma spheroids varied from 10 to 16 Gy. Unlabeled 96.5 at 2 mg/liter or 131I-iodide at 1.7 GBq/liter did not affect the growth of the melanoma spheroids. The HT29 spheroids, however, only suffered slight cytotoxicity at 1 or 2 mg/liter of 131I-96.5 and for a treatment time of 24 h despite comparable radiosensitivity of HT29 cells and melanoma cells to high-dose-rate radiation. Similar cytotoxicity was observed in the HT29 group treated with 131I-iodide at 1.7 GBq/liter. Present findings therefore demonstrate preferential and adequate killing of the melanoma spheroids by 131I-96.5 at 0.5 mg/liter and 3.96 GBq/mg in 8 h.

  18. TGF-β induces SOX2 expression in a time-dependent manner in human melanoma cells.

    PubMed

    Weina, Kasia; Wu, Huizi; Knappe, Nathalie; Orouji, Elias; Novak, Daniel; Bernhardt, Mathias; Hüser, Laura; Larribère, Lionel; Umansky, Viktor; Gebhardt, Christoffer; Utikal, Jochen

    2016-07-01

    The sry-related high-mobility box (SOX)-2 protein has recently been proven to play a significant role in progression, metastasis, and clinical prognosis spanning several cancer types. Research on the role of SOX2 in melanoma is limited and currently little is known about the mechanistic function of this gene in this context. Here, we observed high expression of SOX2 in both human melanoma cell lines and primary melanomas in contrast to melanocytic nevi. This overexpression in melanoma can, in part, be explained by extra gene copy numbers of SOX2 in primary samples. Interestingly, we were able to induce SOX2 expression, mediated by SOX4, via TGF-β1 stimulation in a time-dependent manner. Moreover, the knockdown of SOX2 impaired TGF-β-induced invasiveness. This phenotype switch can be explained by SOX2-mediated cross talk between TGF-β and non-canonical Wnt signaling. Thus, we propose that SOX2 is involved in the critical TGF-β signaling pathway, which has been shown to correlate with melanoma aggressiveness and metastasis. In conclusion, we have identified a novel downstream factor of TGF-β signaling in melanoma, which may have further implications in the clinic. PMID:27105574

  19. Cell surface molecules of human melanoma. Immunohistochemical analysis of the gp57, GD3, and mel-CSPG antigenic systems.

    PubMed Central

    Garin-Chesa, P.; Beresford, H. R.; Carrato-Mena, A.; Oettgen, H. F.; Old, L. J.; Melamed, M. R.; Rettig, W. J.

    1989-01-01

    The rapidly expanding list of monoclonal antibodies (MAbs) to human cell surface antigens provides reagents to probe the biology of malignant melanoma and to develop new diagnostic and therapeutic approaches to this disease. The criteria used to select MAb-defined antigens as targets for passive immunotherapy or immunolocalization of melanoma include: 1) consistent antigen expression in melanomas, 2) restricted antigen distribution in normal tissues and nonmelanocytic tumors, and 3) cytotoxic activity of the MAb or MAb conjugates. The present study examined the tissue distribution of three prototype melanoma cell surface antigens, the Mr 57,000 glycoprotein (gp57) recognized by MAb A42, the GD3 ganglioside, and the mel-CSPG chondroitin sulfate proteoglycan. The avidin-biotin immunoperoxidase method was used to examine a large panel of normal tissues and over 150 malignant tumors. It was found that A42 has a highly restricted distribution in normal tissues and is expressed in subsets of melanomas and nonmelanocytic tumors. It was also found that GD3 and mel-CSPG are more widely distributed in normal tissues and among tumors than was thought previously. These immunohistochemical patterns provide an essential data base to evaluate the ongoing clinical trials employing MAbs to GD3 and mel-CSPG for the therapy and immunolocalization of melanomas, and they identify gp57 as a potential marker for subsets of normal and transformed melanocytic cells. Images Figure 1 Figure 2 Figure 3 PMID:2916650

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

    PubMed Central

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

    2016-01-01

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

  1. Wnt interaction and extracellular release of prominin-1/CD133 in human malignant melanoma cells

    SciTech Connect

    Rappa, Germana; Mercapide, Javier; Anzanello, Fabio; Le, Thuc T.; Johlfs, Mary G.; Fiscus, Ronald R.; Wilsch-Bräuninger, Michaela; Corbeil, Denis; Lorico, Aurelio

    2013-04-01

    Prominin-1 (CD133) is the first identified gene of a novel class of pentaspan membrane glycoproteins. It is expressed by various epithelial and non-epithelial cells, and notably by stem and cancer stem cells. In non-cancerous cells such as neuro-epithelial and hematopoietic stem cells, prominin-1 is selectively concentrated in plasma membrane protrusions, and released into the extracellular milieu in association with small vesicles. Previously, we demonstrated that prominin-1 contributes to melanoma cells pro-metastatic properties and suggested that it may constitute a molecular target to prevent prominin-1-expressing melanomas from colonizing and growing in lymph nodes and distant organs. Here, we report that three distinct pools of prominin-1 co-exist in cultures of human FEMX-I metastatic melanoma. Morphologically, in addition to the plasma membrane localization, prominin-1 is found within the intracellular compartments, (e.g., Golgi apparatus) and in association with extracellular membrane vesicles. The latter prominin-1–positive structures appeared in three sizes (small, ≤40 nm; intermediates ∼40–80 nm, and large, >80 nm). Functionally, the down-regulation of prominin-1 in FEMX-I cells resulted in a significant reduction of number of lipid droplets as observed by coherent anti-Stokes Raman scattering image analysis and Oil red O staining, and surprisingly in a decrease in the nuclear localization of beta-catenin, a surrogate marker of Wnt activation. Moreover, the T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter activity was 2 to 4 times higher in parental than in prominin-1-knockdown cells. Collectively, our results point to Wnt signaling and/or release of prominin-1–containing membrane vesicles as mediators of the pro-metastatic activity of prominin-1 in FEMX-I melanoma. - Highlights: ► First report of release of prominin-1–containing microvesicles from cancer cells. ► Pro-metastatic role of prominin-1–containing microvesicles in

  2. Investigation of the phototoxic effect of ZnO nanorods on fibroblasts and melanoma human cells

    NASA Astrophysics Data System (ADS)

    Kishwar, S.; Siddique, M.; Israr-Qadir, M.; Nur, O.; Willander, M.; Öllinger, K.

    2014-11-01

    Photocytotoxic effects of as-grown and zinc oxide (ZnO) nanorods coated with 5-aminolevulinic acid (ALA) have been studied on human cells, i.e. melanoma and foreskin fibroblast, under dark and ultraviolet light exposures. Zinc oxide nanorods have been grown on the very sharp tip (diameter = 700 nm) of borosilicate glass pipettes and then were coated by the photosensitizer for targeted investigations inside human cells. The coated glass pipette’s tip with photosensitizer has been inserted inside the cells with the help of a micro-manipulator and irradiated through ultraviolet light (UVA), which reduces the membrane potential of the mitochondria leading to cell death. Cell viability loss has been detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay when exposed to the dissolved ZnO nanorods and the production of the reactive oxygen species (ROS) has been detected along with the enhanced cytotoxic effect under UVA irradiation. Additionally, the influence of the lipid soluble antioxidant vitamin E and water-soluble N-acetyl-cysteine toward the enhancement or reduction of the toxicity has been investigated. A comparative analysis of the toxic nature of ZnO nanorods has been drawn between normal human fibroblast and melanoma cells, which can be favorable for understanding the clinical setting for killing tumor cells.

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

    PubMed

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

    2003-10-01

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

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

    PubMed

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

    2014-07-01

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

  5. A nude rat model for neutron capture therapy of human intracerebral melanoma

    SciTech Connect

    Barth, R.F.; Matalka, K.Z.; Bailey, M.Q.; Staubus, A.E.; Soloway, A.H.; Moeschberger, M.L. ); Coderre, J.A. ); Rofstad, E.K. )

    1994-03-30

    The present study was carried out to determine the efficacy of Boron Neutron Capture Therapy (BNCT) for intracerebral melanoma using nude rats, the human melanoma cell line MRA 27, and boronophenylalanine as the capture agent. MRA 27 cells (2 [times] 10[sup 5]) were implanted intracerebrally, and 30 days later, 120 mg of [sup 10]B-L-BPA were injected intraperitoneally into nude rats. Thirty days following implantation, tumor bearing rats were irradiated at the Brookhaven Medical Research Reactor. Six hours following administration of BPA, tumor, blood, and normal brain boron-10 levels were 23.7, 9.4, and 8.4 [mu]g/g respectively. Median survival time of untreated rats was 44 days compared to 76 days and 93 days for those receiving physical doses of 2.73 Gy and 3.64 Gy, respectively. Rats that have received both [sup 10]B-BPA and physical doses of 1.82, 2.73, or 3.64 Gy had median survival times of 170, 182, and 262 days, respectively. Forty percent of rats that had received the highest tumor dose (10.1 Gy) survived for > 300 days and in a replicate experiment 21% of the rats were longterm survivors (>220 days). Animals that received 12 Gy in a single dose or 18 Gy fractionated (2 Gy [times] 9) of gamma photons from a [sup 137]Cs source had median survival times of 86 and 79 days, respectively, compared to 47 days for untreated animals. Histopathologic examination of the brains of longterm surviving rats, euthanized at 8 or 16 months following BNCT, showed no residual tumor, but dense accumulations of melanin laden macrophages and minimal gliosis were observed. Significant prolongations in median survival time were noted in nude rats with intracerebral human melanoma that had received BNCT, thereby suggesting therapeutic efficacy. Large animal studies should be carried out to further assess BNCT of intracerebral melanoma before any human trials are contemplated. 49 refs., 7 figs., 2 tabs.

  6. Potentiation of cytotoxicity of paclitaxel in combination with Cl-IB-MECA in human C32 metastatic melanoma cells: A new possible therapeutic strategy for melanoma.

    PubMed

    Soares, Ana S; Costa, Vera M; Diniz, Carmen; Fresco, Paula

    2013-10-01

    Metastatic melanoma monotherapies with drugs such as dacarbazine, cisplatin or paclitaxel (PXT) are associated with significant toxicity and low efficacy rates. These facts reinforce the need for development of novel agents or combinatory strategies. Cl-IB-MECA is a small molecule, orally bioavailable, well tolerated and currently under clinical trials as an anticancer agent. Our aim was to investigate a possible combinatory therapeutic strategy using PXT and Cl-IB-MECA on human C32 melanoma cells and its underlying mechanisms. Cytotoxicity was evaluated using MTT reduction, lactate dehydrogenase leakage and neutral red uptake assays, for different concentrations and combinations of both agents, at 24 and 48 h. Apoptosis was also assessed using fluorescence microscopy and through the evaluation of caspases 8, 9, and 3 activities. We demonstrated, for the first time, that combination of PXT and Cl-IB-MECA significantly increases cytotoxicity for clinically relevant concentrations. This combination seems to act synergistically in disrupting membrane integrity, but also causing lysosomal and mitochondrial dysfunction. When using the lowest PTX concentration (10 ng/mL), co-incubation with CI-IB-MECA (micromolar concentrations) potentiated overall cytotoxic effects and morphological signs of apoptosis. All combinations studied enhanced caspase 8, 9, and 3 activities, suggesting the involvement of both intrinsic and extrinsic apoptotic pathways. The possibility that cytotoxicity elicited by Cl-IB-MECA, alone or in combination with PXT, involves adenosine receptor activation was discarded and results confirmed that oxidative stress is only involved in cytotoxicity after treatment with PXT, alone. Being melanoma a very apoptosis-resistance cancer, this combination seems to hold promise as a new therapeutic strategy for melanoma. PMID:24035253

  7. Adipose-derived mesenchymal stem cell-facilitated TRAIL expression in melanoma treatment in vitro

    PubMed Central

    JING, HAI XIA; DUAN, DE JIAN; ZHOU, HUI; HU, QING MEI; LEI, TIE CHI

    2016-01-01

    Adipose-derived stem cells (ADSCs) may be useful as an efficient vehicle in cell-based gene therapy of human diseases due to their ability to migrate to disease lesions. This study investigated the ability of ADSC-harbored human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cDNA to facilitate TRAIL expression and induce A375 melanoma cell apoptosis as observed using a Transwell co-culture system. A cell migration assay was used to observe ADSC migration ability. In addition, TRAIL protein expression was successfully detected by western blot analysis in ADSCs after stable transfection of TRAIL cDNA. The Transwell co-culture system data showed that TRAIL-ADSCs could induce A375 cell apoptosis in a dose-dependent manner. At the gene level, the killing activity of TRAIL-ADSCs was associated with activation of caspase-4 and caspase-8. Collectively, the data from the current study provides preclinical support of ADSC-facilitated TRAIL expression in the treatment of melanoma. Further investigation is required to evaluate and confirm the in vivo ability of TRAIL-ADSCs in therapy of melanoma in animal models. PMID:27177242

  8. ETM study of electroporation influence on cell morphology in human malignant melanoma and human primary gingival fibroblast cells

    PubMed Central

    Skolucka, Nina; Daczewska, Malgorzata; Saczko, Jolanta; Chwilkowska, Agnieszka; Choromanska, Anna; Kotulska, Malgorzata; Kaminska, Iwona; Kulbacka, Julita

    2011-01-01

    Objective To estimate electroporation (EP) influence on malignant and normal cells. Methods Two cell lines including human malignant melanoma (Me-45) and normal human gingival fibroblast (HGFs) were used. EP parameters were the following: 250, 1 000, 1 750, 2 500 V/cm; 50 µs by 5 impulses for every case. The viability of cells after EP was estimated by MTT assay. The ultrastructural analysis was observed by transmission electron microscope (Zeiss EM 900). Results In the current study we observed the intracellular effect following EP on Me-45 and HGF cells. At the conditions applied, we did not observe any significant damage of mitochondrial activity in both cell lines treated by EP. Conversely, we showed that EP in some conditions can stimulate cells to proliferation. Some changes induced by EP were only visible in electron microscopy. In fibroblast cells we observed significant changes in lower parameters of EP (250 and 1 000 V/cm). After applying higher electric field intensities (2 500 V/cm) we detected many vacuoles, myelin-like bodies and swallowed endoplasmic reticulum. In melanoma cells such strong pathological modifications after EP were not observed, in comparison with control cells. The ultrastructure of both treated cell lines was changed according to the applied parameters of EP. Conclusions We can claim that EP conditions are cell line dependent. In terms of the intracellular morphology, human fibroblasts are more sensitive to electric field as compared with melanoma cells. Optimal conditions should be determined for each cell line. Summarizing our study, we can conclude that EP is not an invasive method for human normal and malignant cells. This technique can be safely applied in chemotherapy for delivering drugs into tumor cells. PMID:23569735

  9. Involvement of vacuolar H(+)-ATPase in killing of human melanoma cells by the sphingosine kinase analogue FTY720.

    PubMed

    Tay, Kwang Hong; Liu, Xiaoying; Chi, Mengna; Jin, Lei; Jiang, Chen Chen; Guo, Su Tang; Verrills, Nicole M; Tseng, Hsin-Yi; Zhang, Xu Dong

    2015-03-01

    Targeting the sphingosine 1-phosphate (S1P)/S1P receptor (S1PR) signalling axis is emerging as a promising strategy in the treatment of cancer. However, the effect of such an approach on survival of human melanoma cells remains less understood. Here, we show that the sphingosine analogue FTY720 that functionally antagonises S1PRs kills human melanoma cells through a mechanism involving the vacuolar H(+) -ATPase activity. Moreover, we demonstrate that FTY720-triggered cell death is characterized by features of necrosis and is not dependent on receptor-interacting protein kinase 1 or lysosome cathepsins, nor was it associated with the activation of protein phosphatase 2A. Instead, it is mediated by increased production of reactive oxygen species and is antagonized by activation of autophagy. Collectively, these results suggest that FTY720 and its analogues are promising candidates for further development as new therapeutic agents in the treatment of melanoma. PMID:25358761

  10. Characterization of phosphodiesterase 2A in human malignant melanoma PMP cells

    PubMed Central

    MORITA, HIROSHI; MURATA, TAKU; SHIMIZU, KASUMI; OKUMURA, KENYA; INUI, MADOKA; TAGAWA, TOSHIRO

    2013-01-01

    The prognosis for malignant melanoma is poor; therefore, new diagnostic methods and treatment strategies are urgently needed. Phosphodiesterase 2 (PDE2) is one of 21 phosphodiesterases, which are divided into 11 families (PDE1-PDE11). PDE2 hydrolyzes cyclic AMP (cAMP) and cyclic GMP (cGMP), and its binding to cGMP enhances the hydrolysis of cAMP. We previously reported the expression of PDE1, PDE3 and PDE5 in human malignant melanoma cells. However, the expression of PDE2 in these cells has not been investigated. Herein, we examined the expression of PDE2A and its role in human oral malignant melanoma PMP cells. Sequencing of RT-PCR products revealed that PDE2A2 was the only variant expressed in PMP cells. Four point mutations were detected; one missense mutation at nucleotide position 734 (from C to T) resulted in the substitution of threonine with isoleucine at amino acid position 214. The other three were silent mutations. An in vitro migration assay and a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay revealed that suppressing PDE2 activity with its specific inhibitor, erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), had no impact on cell motility or apoptosis. Furthermore, the cytotoxicity of EHNA, assessed using a trypan blue exclusion assay, was negligible. On the other hand, assessment of cell proliferation by BrdU incorporation and cell cycle analysis by flow cytometry revealed that EHNA treatment inhibited DNA synthesis and increased the percentage of G2/M-arrested cells. Furthermore, cyclin A mRNA expression was downregulated, while cyclin E mRNA expression was upregulated in EHNA-treated cells. Our results demonstrated that the PDE2A2 variant carrying point mutations is expressed in PMP cells and may affect cell cycle progression by modulating cyclin A expression. Thus, PDE2A2 is a possible new molecular target for the treatment of malignant melanoma. PMID:23381931

  11. Biologically active monoiodinated alpha-MSH derivatives for receptor binding studies using human melanoma cells

    SciTech Connect

    Eberle, A.N.; Verin, V.J.; Solca, F.; Siegrist, W.; Kueenlin, C.B.; Bagutti, C.; Stutz, S.; Girard, J. , University Hospital, Basel )

    1991-01-01

    Three different monoiodinated radioligands of alpha-MSH (alpha-melanocyte-stimulating hormone) were compared in a binding assay with human D10 melanoma cells: (Tyr(125I)2)-alpha-MSH, (Tyr(125I)2,NIe4)-alpha-MSH, and (Tyr(125I)2,NIe4,D-Phe7)-alpha-MSH. They were prepared either by the classical chloramine T method or by the Enzymobead method. A simple and rapid purification scheme was developed consisting of a primary separation on reversed-phase C18 silica cartridges immediately after the iodination, followed by HPLC purification before each binding experiment. Biological testing of the three radioligands showed that they all retained high melanotropic activity in the B16 melanin assay and the Anolis melanophore assay. However, in human D10 melanoma cells, (Tyr(125I)2,NIe4)-alpha-MSH led to a high degree of non-specific binding to the cells which could not be displaced by excess alpha-MSH and only partially by (NIe4)-alpha-MSH. The (Tyr(125I)2,NIe4,D-Phe7)-alpha-MSH tracer gave similar results but with a much lower proportion of non-specific binding. On the other hand, (Tyr(125I)2)-alpha-MSH proved to be an excellent radioligand whose non-specific binding to the D10 cells was not higher than 20% of the total binding.

  12. [Human recombinant leukocyte interferon alpha-2-A in 22 cases of metastatic malignant melanoma].

    PubMed

    Maral, J; Steinberg, M; Weil, M; Chleq, C; Khayat, D; Banzet, P; Jacquillat, C

    1987-06-01

    Twenty-two patients with metastatic malignant melanoma received either 36 X 10(6) U (15 patients) or 18 X 10(6) U (7 patients) of human recombinant interferon alpha-2-A daily for 3 months by the intramuscular route, with progressive increase of dosage. This was followed in responders by a maintenance treatment consisting of 3 intramuscular injections per week in the same doses as those received at the end of the induction treatment. Out of 18 patients assessable for effectiveness, 1 had complete remission (7 months +) and 3 had partial response (52,61 and 82 days respectively), an overall improvement rate of 22%. The main side-effects observed were: pseudoinfluenza syndrome (100%), fatigue (100%), somnolence (95%), anorexia (90%) and haematological disorders. Dosage reduction was necessary in 13 of the 15 patients receiving 36 MU. This study shows that human recombinant interferon alpha-2-A has antitumoral activity in metastatic malignant melanoma. Other studies, notably with therapeutic combinations, are needed to determine the optimal dosage regimen of the drug and to increase its effectiveness. PMID:2955323

  13. Differential expression of TYRP1 in adult human retinal pigment epithelium and uveal melanoma cells

    PubMed Central

    QIU, CHUN; LI, PENG; BI, JIANJUN; WU, QING; LU, LINNA; QIAN, GUANXIANG; JIA, RENBING; JIA, RONG

    2016-01-01

    Uveal melanoma (UM) is the most frequently occurring primary intraocular malignancy in adults. Tyrosinase (TYR) is a copper-containing enzyme and a type I membrane protein that is involved in the generation of melanin, the main pigment in vertebrates. TYR-related protein 1 (TYRP1) is regarded to have a crucial role in the immunotherapy of melanoma. As biomarkers, the TYR-related proteins, TYRP1 and TYRP2, exhibit specific expression in melanocytes, while also contributing to melanin synthesis within melanosomes. In the present study, the differential expression of TYRP1 was investigated at the mRNA, protein and morphological levels in four human UM cell lines (SP6.5, OM431, OCM1 and OCM290) and the human retinal pigment epithelium (RPE) cell line, using polymerase chain reaction, western blotting, immunocytochemistry and immunofluorescence staining. It was found that SP6.5 cells expressed the highest level of TYRP1, in comparison to SP6.5 OCM1 and OM431 cells, which produced less TYRP1, and OCM290 cells, which produced almost no TYRP1. No TYRP1 protein expression was identified in the RPE cell line. These findings indicate the potential use of TYRP1 in the development of therapy for UM. PMID:27073483

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

    PubMed

    Sahu, Ravi Prakash

    2015-07-01

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

  15. Autophagy Is a Protective Mechanism for Human Melanoma Cells under Acidic Stress*

    PubMed Central

    Marino, Maria Lucia; Pellegrini, Paola; Di Lernia, Giuseppe; Djavaheri-Mergny, Mojgan; Brnjic, Slavica; Zhang, Xiaonan; Hägg, Maria; Linder, Stig; Fais, Stefano; Codogno, Patrice; De Milito, Angelo

    2012-01-01

    Cyclic hypoxia and alterations in oncogenic signaling contribute to switch cancer cell metabolism from oxidative phosphorylation to aerobic glycolysis. A major consequence of up-regulated glycolysis is the increased production of metabolic acids responsible for the presence of acidic areas within solid tumors. Tumor acidosis is an important determinant of tumor progression and tumor pH regulation is being investigated as a therapeutic target. Autophagy is a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, currently considered an important survival mechanism in cancer cells under metabolic stress or subjected to chemotherapy. We investigated the response of human melanoma cells cultured in acidic conditions in terms of survival and autophagy regulation. Melanoma cells exposed to acidic culture conditions (7.0 < pH < 6.2) promptly accumulated LC3+ autophagic vesicles. Immunoblot analysis showed a consistent increase of LC3-II in acidic culture conditions as compared with cells at normal pH. Inhibition of lysosomal acidification by bafilomycin A1 further increased LC3-II accumulation, suggesting an active autophagic flux in cells under acidic stress. Acute exposure to acidic stress induced rapid inhibition of the mammalian target of rapamycin signaling pathway detected by decreased phosphorylation of p70S6K and increased phosphorylation of AMP-activated protein kinase, associated with decreased ATP content and reduced glucose and leucine uptake. Inhibition of autophagy by knockdown of the autophagic gene ATG5 consistently reduced melanoma cell survival in low pH conditions. These observations indicate that induction of autophagy may represent an adaptation mechanism for cancer cells exposed to an acidic environment. Our data strengthen the validity of therapeutic strategies targeting tumor pH regulation and autophagy in progressive malignancies. PMID:22761435

  16. Malignant potential of cells isolated from lymph node or brain metastases of melanoma patients and implications for prognosis.

    PubMed

    Zhang, R D; Price, J E; Schackert, G; Itoh, K; Fidler, I J

    1991-04-15

    We studied the correlation between the formation of brain metastasis and the malignant growth potential of seven human melanoma cell lines, isolated from lymph node metastases (A375-SM, TXM-1, DM-4) or from brain metastases (TXM-13, TXM-18, TXM-34, TXM-40), and the potential of three variants of the mouse K-1735 melanoma. Growth rates in different concentrations of fetal bovine serum and colony-forming efficiency in semisolid agarose were measured, and the tumorigenicity and metastatic ability were determined in nude mice (for the human melanoma cell lines) or in C3H/HeN mice (for the K-1735 variants). The ability to form brain metastasis was tested by injection of cells into the carotid artery. A high colony-forming efficiency in agarose, especially at concentrations of agarose greater than 0.6%, corresponded with high tumor take rates, rapid tumor growth rates, and metastatic colonization of the lungs of the recipient mice. For the human melanomas, the lymph node metastasis-derived cells were more tumorigenic and metastatic than the brain metastasis-derived cells. In the K-1735 mouse melanoma, the tumorigenic and metastatic behavior of the cells after i.v. and s.c. injection corresponded with growth in agarose cultures. However, for growth in the brain after intracarotid injection, the different melanoma cell lines showed similar frequencies of tumor take, regardless of tumorigenicity in other sites of the recipient mice, although mice given injections of brain metastasis-derived cells survived longer than mice given injections of lymph node metastasis (human melanoma) or lung metastasis (K-1735 M-2)-derived cell lines. The results from the human and mouse melanoma cell lines show that the brain metastasis-derived cell lines were not more malignant than the lymph node or lung metastasis-derived cells. These data imply that the production of brain metastasis is not always the final stage of a metastatic cascade. PMID:1826230

  17. Immunotherapy against Metastatic Melanoma with Human iPS Cell-Derived Myeloid Cell Lines Producing Type I Interferons.

    PubMed

    Miyashita, Azusa; Fukushima, Satoshi; Nakahara, Satoshi; Kubo, Yosuke; Tokuzumi, Aki; Yamashita, Junji; Aoi, Jun; Haruta, Miwa; Senju, Satoru; Nishimura, Yasuharu; Jinnin, Masatoshi; Ihn, Hironobu

    2016-03-01

    In recent years, immunotherapy for advanced melanoma has been gaining increased attention. The efficacy of anti-cytotoxic T-lymphocyte antigen 4 antibodies, anti-programmed cell death 1 antibodies, and the BRAF(V600E) kinase inhibitor has been proven in metastatic melanoma. At the same time, adoptive cell transfer has significant effects against metastatic melanoma; however, it is difficult to apply on a broad scale because of the problems related to cell preparation. To overcome these problems, we developed immune cell therapy using induced pluripotent stem (iPS) cells. The benefit of our method is that a large number of cells can be readily obtained. We focused on macrophages for immune cell therapy because macrophage infiltration is frequently observed in solid cancers. In this study, the efficacy of human iPS cell-derived myeloid cell lines (iPS-ML) genetically modified to express type I IFNs against human melanoma cells was examined. The morphology, phagocytic ability, and surface markers of iPS-ML were similar to those of macrophages. The iPS-ML that express type I IFNs (iPS-ML-IFN) showed significant effects in inhibiting the growth of disseminated human melanoma cells in SCID mice. The infiltration of iPS-ML into the tumor nests was confirmed immunohistologically. The iPS-ML-IFNs increased the expression of CD169, a marker of M1 macrophages that can activate antitumor immunity. The iPS-ML-IFNs could infiltrate into tumor tissue and exert anticancer effects in the local tumor tissue. In conclusion, this method will provide a new therapeutic modality for metastatic melanoma. Cancer Immunol Res; 4(3); 248-58. ©2015 AACR. PMID:26714554

  18. Comparative transforming potential of different human papillomaviruses associated with non-melanoma skin cancer

    SciTech Connect

    Massimi, Paola; Thomas, Miranda; Bouvard, Veronique; Ruberto, Irene; Campo, M. Saveria; Tommasino, Massimo; Banks, Lawrence

    2008-02-20

    It is well established that high-risk human papillomaviruses (HPVs) that infect mucosal epithelia are the causative agents of cervical cancer. In contrast, the association of cutaneo-tropic HPV types with the development of non-melanoma skin cancer (NMSC) is less well defined. In this study, we have analysed the in vitro transforming potential of various cutaneous HPV types. Using oncogene cooperation assays with activated ras, we have shown that diverse cutaneous types, including 12, 14, 15, 24, 36 and 49, have significant transforming potential. Interestingly, most of this activity appears to be encoded by the E6 gene product. In contrast, the common HPV-10 exhibits no significant transforming potential in these assays. This difference may be a reflection of different patterns of cellular localization, with transforming E6s being nuclear and non-transforming being cytoplasmic. These results provide molecular support for a role of these viruses in the development of certain human malignancies.

  19. Hormone Conjugated with Antibody to CD3 Mediates Cytotoxic T Cell Lysis of Human Melanoma Cells

    NASA Astrophysics Data System (ADS)

    Liu, Margaret Ann; Nussbaum, Samuel R.; Eisen, Herman N.

    1988-01-01

    Cytotoxic T lymphocytes can be activated by antibodies to their antigen-specific receptor complex (TCR-CD3) to destroy target cells, regardless of the specificity of the cytotoxic T cells. A novel hormone-antibody conjugate, consisting of an analog of melanocyte-stimulating hormone chemically coupled to a monoclonal antibody to CD3, the invariant component of the T cell receptor complex, was used to target human melanoma cells for destruction by human cytotoxic T lymphocytes that bear no specificity for the tumor cells. As targeting components of such anti-CD3 conjugates, hormones or growth factors are expected to prove more effective than antibodies to tumor-associated antigens in focusing the destructive activity of cytotoxic T cells on tumor target cells.

  20. Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

    PubMed Central

    Hallberg, Andrea R; Vorrink, Sabine U; Hudachek, Danielle R; Cramer-Morales, Kimberly; Milhem, Mohammed M; Cornell, Robert A; Domann, Frederick E

    2014-01-01

    Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs. PMID:25625848

  1. Characterization of a new human melanoma cell line with CD133 expression.

    PubMed

    Gil-Benso, Rosario; Monteagudo, Carlos; Cerdá-Nicolás, Miguel; Callaghan, Robert C; Pinto, Sandra; Martínez-Romero, Alicia; Pellín-Carcelén, Ana; San-Miguel, Teresa; Cigudosa, Juan C; López-Ginés, Concha

    2012-06-01

    A novel human malignant melanoma cell line, designated MEL-RC08, was established from a pericranial metastasis of a malignant melanoma of the skin. The cell line has been subcultured for more than 150 passages and is tumorigenic in nude mice. Growth kinetics, cytogenetics, flow cytometry, and molecular techniques for analysis of the genes implicated in cell cycle control; mutations in BRAF, NRAS, C-KiT, RB, and TP53 genes; and amplification of MDM2, CDK4, and cyclin D1 have been studied. Cytogenetically, the tumor and the cell line showed a hypertriploid karyotype with many clonal numeric and structural abnormalities. DNA flow cytometry showed an aneuploid peak with a DNA index value of 1.5. Mutations in TP53 and BRAF genes were demonstrated in both tumor and cell line. Furthermore, stem cell marker CD133 expression was detected in most cells, together with other stem cell markers, suggesting the presence of cells with tumor-initiating potential in this cell line. PMID:22529031

  2. Tocilizumab unmasks a stage-dependent interleukin-6 component in statin-induced apoptosis of metastatic melanoma cells.

    PubMed

    Minichsdorfer, Christoph; Wasinger, Christine; Sieczkowski, Evelyn; Atil, Bihter; Hohenegger, Martin

    2015-08-01

    The interleukin (IL)-6 inhibits the growth of early-stage melanoma cells, but not metastatic cells. Metastatic melanoma cells are susceptible to statin-induced apoptosis, but this is not clear for early-stage melanoma cells. This study aimed to investigate the IL-6 susceptibility of melanoma cells from different stages in the presence of simvastatin to overcome loss of growth arrest. ELISA was used to detect secreted IL-6 in human melanoma cells. The effects of IL-6 were measured by western blots for STAT3 and Bcl-2 family proteins. Apoptosis and proliferation were measured by caspase 3 activity, Annexin V staining, cell cycle analysis, and a wound-healing assay. Human metastatic melanoma cells A375 and 518A2 secrete high amounts of IL-6, in contrast to early-stage WM35 cells. Canonical IL-6 signaling is intact in these cells, documented by transient phosphorylation of STAT3. Although WM35 cells are highly resistant to simvastatin-induced apoptosis, coadministration with IL-6 enhanced the susceptibility to undergo apoptosis. This proapoptotic effect of IL-6 might be explained by a downregulation of Bcl-XL, observed only in WM35 cells. Furthermore, the IL-6 receptor blocking antibody tocilizumab was coadministered and unmasked an IL-6-sensitive proportion in the simvastatin-induced caspase 3 activity of metastatic melanoma cells. These results confirm that simvastatin facilitates apoptosis in combination with IL-6. Although endogenous IL-6 secretion is sufficient in metastatic melanoma cells, exogenously added IL-6 is needed for WM35 cells. This effect may explain the failure of simvastatin to reduce melanoma incidence in clinical trials and meta-analyses. PMID:26020489

  3. NF-YA promotes invasion and angiogenesis by upregulating EZH2-STAT3 signaling in human melanoma cells.

    PubMed

    Xu, Zihan; Sun, Yaowen; Guo, Yadong; Qin, Gaoping; Mu, Shengzhi; Fan, Ronghui; Wang, Benfeng; Gao, Wenjie; Wu, Hangli; Wang, Guodong; Zhang, Zhenxin

    2016-06-01

    The process of angiogenesis is essential for tumor development and metastasis. Vascular endothelial growth factor (VEGF), which is overexpressed in most human cancers, has been demonstrated to be a major modulator of angiogenesis. Thus, inhibition of VEGF signaling has the potential for tumor anti-angiogenic therapy. Signal transducer and activator of transcription-3 (STAT3) is a key regulator for angiogenesis by directly binding to the VEGF promoter to upregulate its transcription. Several factors can enhance STAT3 activity to affect angiogenesis. Here, we found that overexpression of nuclear transcription factor-Y alpha (NF-YA) gene could promote cell invasion and angiogenesis accompanying the increase of STAT3 signaling in human melanoma cells. Moreover, the expression and secretion of VEGF was also found to be upregulated by the overexpression of NF-YA gene in melanoma cells. The STAT3 inhibitor was able to attenuate the upregulation of VEGF induced by NF-YA overexpression. Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of the Polycomb repressive complex 2, enhances STAT3 activity by mediating its lysine methylation. We also showed that NF-YA upregulated the expression of EZH2 and NF-YA‑induced angiogenesis could be inhibited by EZH2 knockdown. Taken together, these findings indicate that overexpression of NF-YA contributes to tumor angiogenesis through EZH2-STAT3 signaling in human melanoma cells, highlighting NF-YA as a potential therapeutic target in human melanoma. PMID:27109360

  4. Evaluation of Depigmenting Activity by 8-Hydroxydaidzein in Mouse B16 Melanoma Cells and Human Volunteers

    PubMed Central

    Tai, Sorgan Shou-Ku; Lin, Ching-Gong; Wu, Mon-Han; Chang, Te-Sheng

    2009-01-01

    In our previous study, 8-hydroxydaidzein (8-OHDe) was demonstrated to be a potent and unique suicide substrate of mushroom tyrosinase. In this study, the compound was evaluated for in vitro cellular tyrosinase and melanogenesis inhibitory activities in mouse B16 melanoma cells and for in vivo skin-whitening activity in human volunteers. Tyrosinase activity and melanogenesis in the cell culture incubated with 10 μM of 8-OHDe were decreased to 20.1% and 51.8% of control, respectively, while no obvious cytotoxicity was observed in this concentration. In contrast, a standard tyrosinase inhibitor, kojic acid, showed 69.9% and 71.3% of control in cellular tyrosinase and melanogenesis activity, respectively, at a concentration as high as 100 μM. Hence, 8-OHDe exhibited more than an inhibitory effects on melanin production in B16 cells 10-fold stronger than kojic acid. In addition, when a cream containing 4% 8-OHDe was applied to human skin in an in vivo study, significant increases in the dL*-values were observed after three weeks. Moreover, the increase in the dL*-values after 8-week treatment with 4% 8-OHDe (from −0.57 to 1.94) is stronger than those of 2% 8-OHDe treatment (from 0.26 to 0.94) and 2% ascorbic acid-2-glucoside treatment (from 0.07 to 1.54). From the results of the study, it was concluded that 8-OHDe, the potent suicide substrate of mushroom tyrosinase, has depigmenting activities in both mouse melanoma cells and in human volunteers. Thus, the compound has significant potential for use in cosmetics as a skin-whitening ingredient. PMID:20057943

  5. Evaluation of depigmenting activity by 8-hydroxydaidzein in mouse B16 melanoma cells and human volunteers.

    PubMed

    Tai, Sorgan Shou-Ku; Lin, Ching-Gong; Wu, Mon-Han; Chang, Te-Sheng

    2009-10-01

    In our previous study, 8-hydroxydaidzein (8-OHDe) was demonstrated to be a potent and unique suicide substrate of mushroom tyrosinase. In this study, the compound was evaluated for in vitro cellular tyrosinase and melanogenesis inhibitory activities in mouse B16 melanoma cells and for in vivo skin-whitening activity in human volunteers. Tyrosinase activity and melanogenesis in the cell culture incubated with 10 microM of 8-OHDe were decreased to 20.1% and 51.8% of control, respectively, while no obvious cytotoxicity was observed in this concentration. In contrast, a standard tyrosinase inhibitor, kojic acid, showed 69.9% and 71.3% of control in cellular tyrosinase and melanogenesis activity, respectively, at a concentration as high as 100 microM. Hence, 8-OHDe exhibited more than an inhibitory effects on melanin production in B16 cells 10-fold stronger than kojic acid. In addition, when a cream containing 4% 8-OHDe was applied to human skin in an in vivo study, significant increases in the dL*-values were observed after three weeks. Moreover, the increase in the dL*-values after 8-week treatment with 4% 8-OHDe (from -0.57 to 1.94) is stronger than those of 2% 8-OHDe treatment (from 0.26 to 0.94) and 2% ascorbic acid-2-glucoside treatment (from 0.07 to 1.54). From the results of the study, it was concluded that 8-OHDe, the potent suicide substrate of mushroom tyrosinase, has depigmenting activities in both mouse melanoma cells and in human volunteers. Thus, the compound has significant potential for use in cosmetics as a skin-whitening ingredient. PMID:20057943

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

    PubMed

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

    1994-12-01

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

  7. Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma

    PubMed Central

    Jayachandran, Aparna; Anaka, Matthew; Prithviraj, Prashanth; Hudson, Christopher; McKeown, Sonja J; Lo, Pu-Han; Vella, Laura J; Goding, Colin R; Cebon, Jonathan; Behren, Andreas

    2014-01-01

    Epithelial-to-mesenchymal transition (EMT), in which epithelial cells loose their polarity and become motile mesenchymal cells, is a determinant of melanoma metastasis. We compared gene expression signatures of mesenchymal-like melanoma cells with those of epithelial-like melanoma cells, and identified Thrombospondin 1 (THBS1) as highly up-regulated in the mesenchymal phenotype. This study investigated whether THBS1, a major physiological activator of transforming growth factor (TGF)-beta, is involved in melanoma EMT-like process. We sought to examine expression patterns in distinct melanoma phenotypes including invasive, de-differentiated, label-retaining and drug resistant populations that are putatively associated with an EMT-like process. Here we show that THBS1 expression and secretion was elevated in melanoma cells exhibiting invasive, drug resistant, label retaining and mesenchymal phenotypes and correlated with reduced expression of genes involved in pigmentation. Elevated THBS1 levels were detected in Vemurafenib resistant melanoma cells and inhibition of THBS1 led to significantly reduced chemoresistance in melanoma cells. Notably, siRNA-mediated silencing of THBS1 and neutralizing antibody to THBS1 reduced invasion in mesenchymal-like melanoma cells, while ectopic THBS1 expression in epithelial-like melanoma cells enhanced invasion. Furthermore, the loss of THBS1 inhibited in vivo motility of melanoma cells within the embryonic chicken neural tube. In addition, we found aberrant THBS1 protein expression in metastatic melanoma tumor biopsies. These results implicate a role for THBS1 in EMT, and hence THBS1 may serve as a novel target for strategies aimed at the treatment of melanoma invasion and drug resistance. PMID:25051363

  8. Silencing cAMP-response element-binding protein (CREB) identifies CYR61 as a tumor suppressor gene in melanoma.

    PubMed

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

    2009-09-18

    Metastatic progression of melanoma is associated with overexpression and activity of cAMP-response element-binding protein (CREB). However, the mechanism by which CREB contributes to tumor progression and metastasis remains unclear. Here, we demonstrate that stably silencing CREB expression in two human metastatic melanoma cell lines, A375SM and C8161-c9, suppresses tumor growth and experimental metastasis. Analysis of cDNA microarrays revealed that CREB silencing leads to increased expression of cysteine-rich protein 61 (CCN1/CYR61) known to mediate adhesion, chemostasis, survival, and angiogenesis. Promoter analysis and chromatin immunoprecipitation assays demonstrated that CREB acts as a negative regulator of CCN1/CYR61 transcription by directly binding to its promoter. Re-expression of CREB in CREB-silenced cells rescued the low CCN1/CYR61 expression phenotype. CCN1/CYR61 overexpression resulted in reduced tumor growth and metastasis and inhibited the activity of matrix metalloproteinase-2. Furthermore, its overexpression decreased melanoma cell motility and invasion through Matrigel, which was abrogated by silencing CCN1/CYR61 in low metastatic melanoma cells. Moreover, a significant decrease in angiogenesis as well as an increase in apoptosis was seen in tumors overexpressing CCN1/CYR61. Our results demonstrate that CREB promotes melanoma growth and metastasis by down-regulating CCN1/CYR61 expression, which acts as a suppressor of melanoma cell motility, invasion and angiogenesis. PMID:19632997

  9. Silencing cAMP-response Element-binding Protein (CREB) Identifies CYR61 as a Tumor Suppressor Gene in Melanoma*

    PubMed Central

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

    2009-01-01

    Metastatic progression of melanoma is associated with overexpression and activity of cAMP-response element-binding protein (CREB). However, the mechanism by which CREB contributes to tumor progression and metastasis remains unclear. Here, we demonstrate that stably silencing CREB expression in two human metastatic melanoma cell lines, A375SM and C8161-c9, suppresses tumor growth and experimental metastasis. Analysis of cDNA microarrays revealed that CREB silencing leads to increased expression of cysteine-rich protein 61 (CCN1/CYR61) known to mediate adhesion, chemostasis, survival, and angiogenesis. Promoter analysis and chromatin immunoprecipitation assays demonstrated that CREB acts as a negative regulator of CCN1/CYR61 transcription by directly binding to its promoter. Re-expression of CREB in CREB-silenced cells rescued the low CCN1/CYR61 expression phenotype. CCN1/CYR61 overexpression resulted in reduced tumor growth and metastasis and inhibited the activity of matrix metalloproteinase-2. Furthermore, its overexpression decreased melanoma cell motility and invasion through Matrigel, which was abrogated by silencing CCN1/CYR61 in low metastatic melanoma cells. Moreover, a significant decrease in angiogenesis as well as an increase in apoptosis was seen in tumors overexpressing CCN1/CYR61. Our results demonstrate that CREB promotes melanoma growth and metastasis by down-regulating CCN1/CYR61 expression, which acts as a suppressor of melanoma cell motility, invasion and angiogenesis. PMID:19632997

  10. Primary malignant melanoma

    PubMed Central

    Mısır, A. Ferhat; Durmuşlar, Mustafa C.; Zerener, Tamer; Gün, Banu D.

    2016-01-01

    Malignant melanomas (MM) of the oral cavity are extremely rare, accounting for 0.2% to 8.0% of all malignant melanomas. Malignant melanomas is more frequently seen at the level of the hard palate and gingiva. Early diagnosis and treatment are important for reducing morbidity. Malignant melanoma cells stain positively with antibodies to human melanoma black 45, S-100 protein, and vimentin; therefore, immunohistochemistry can play an important role in evaluating the depth of invasion and the location of metastases. A 76-year-old man developed an oral malignant melanoma, which was originally diagnosed as a bluish reactive denture hyperplasia caused by an ill-fitting lower denture. The tumor was removed surgically, and histopathological examination revealed a nodular-type MM. There was no evidence of recurrence over a 4-year follow-up period. PMID:27052289

  11. Monoclonal antibody imaging of human melanoma. Radioimmunodetection by subcutaneous or systemic injection.

    PubMed Central

    Lotze, M T; Carrasquillo, J A; Weinstein, J N; Bryant, G J; Perentesis, P; Reynolds, J C; Matis, L A; Eger, R R; Keenan, A M; Hellström, I

    1986-01-01

    Fab fragments of monoclonal antibodies (MoAb) to melanoma, radiolabeled with 131I, were evaluated as diagnostic reagents to determine their ability to localize systemic--MoAb injected intravenously (IV)--or nodal metastatic disease--injected subcutaneously (SQ) at a site proximal to draining lymph nodes. Sixty-one scans were performed (40 IV, 21 SQ) in 59 patients who had injections of 0.2-50 mg of 131I coupled (0.2-12 mCi) antibody. These included 48.7, which identifies a high molecular weight antigen (HMW), or 96.5, which identifies a transferrin like molecule, p97. 125I coupled nonspecific Fab 1.4, reacting with murine leukemia virus, or the whole antibody BL3, reactive with a human B cell idiotypic determinant, was generally used in tandem with the patients injected SQ as a nonspecific control. All patients had immunohistochemical studies performed on biopsied lesions and demonstrated binding to the antibodies injected. Of the IV patients, 22/38 (58%) had (+) scans, 13 at SQ or nodal sites, four at visceral sites, and five at visceral and SQ sites. Patients with clinical stage II disease had SQ injection of MoAb, including 11 additional patients injected with the whole antibody 9.2.27 (anti-HMW) labeled with 111In (6 patients) or 131I (5 patients). Nodal dissection was performed 2-4 days later. All 111In coupled antibodies demonstrated excellent nodal delineation without specific identification of tumor deposits. Of the 21 patients injected SQ with MoAb, 17 had confirmed tumor in nodes. Of patients injected with Fab fragments, 4/8 (50%) had specific uptake of MoAb, although only two were successfully imaged. Increased uptake of antimelanoma antibodies was observed in some patients in lymph nodes not containing tumor and was possibly related to antigen shedding. Clearance of labeled antibody from the injection site occurred with a half life of 16-50 hours. Toxicity was limited to local discomfort at the site of SQ injection. Melanoma metastases can be identified

  12. Antibody-drug conjugates: targeting melanoma with cisplatin encapsulated in protein-cage nanoparticles based on human ferritin

    NASA Astrophysics Data System (ADS)

    Falvo, Elisabetta; Tremante, Elisa; Fraioli, Rocco; Leonetti, Carlo; Zamparelli, Carlotta; Boffi, Alberto; Morea, Veronica; Ceci, Pierpaolo; Giacomini, Patrizio

    2013-11-01

    A novel antibody-drug conjugate (ADC) was synthesized incorporating ferritin-based nanoparticles. An average of three molecules of monoclonal antibody (mAb) Ep1 to the human melanoma-specific antigen CSPG4 were conjugated to a single ferritin cage encapsulating about 50 cisplatin molecules (HFt-Pt-Ep1). The HFt-Pt-Ep1 nanoparticle had an estimated molecular size of about 900 kD and 33 nm, and flow cytometry demonstrated specific binding to a CSPG4+ melanoma cell line, but not to a CSPG4- breast carcinoma cell line. As compared to the cisplatin-containing ferritin nanoparticle alone (HFt-Pt), which inhibited thymidine incorporation more efficiently in breast carcinoma than melanoma cells, the mAb-derivatized HFt-Pt-Ep1 nanoparticle had a 25-fold preference for the latter. A similar preference for melanoma was observed upon systemic intravenous administration of HFt-Pt-Ep1 to nude mice xenotransplanted with pre-established, palpable melanoma and breast carcinoma tumors. Thus, we have been able to determine precise combinations and stoichiometric relationships between mAbs and nanoparticle protein cages, whereby the latter lose their tropism for ubiquitously distributed cellular receptors, and acquire instead remarkably lineage-selective binding. HFt-Pt-Ep1 is therefore an interesting model to improve the therapeutic index of antiblastic therapy in a tumor such as melanoma, which at its advanced stages is totally refractory to mono- and combination-chemotherapy.A novel antibody-drug conjugate (ADC) was synthesized incorporating ferritin-based nanoparticles. An average of three molecules of monoclonal antibody (mAb) Ep1 to the human melanoma-specific antigen CSPG4 were conjugated to a single ferritin cage encapsulating about 50 cisplatin molecules (HFt-Pt-Ep1). The HFt-Pt-Ep1 nanoparticle had an estimated molecular size of about 900 kD and 33 nm, and flow cytometry demonstrated specific binding to a CSPG4+ melanoma cell line, but not to a CSPG4- breast carcinoma cell

  13. Further characterization of a melanoma-specific protein from human urine.

    PubMed Central

    Bennett, C.; Cooke, K. B.

    1980-01-01

    Isolation of a melanoma-specific protein (MSP) from human urine has been achieved using antibody affinity chromatography. MSP migrates as a single homogeneous protein on SDS PAGE and comparison of these data and ultracentrifuge analyses indicates that MSP contains a single polypeptide chain. MSP, however, shows considerable charge heterogeneity on isoelectric focusing. The desialo form, alpha 2 MSP, is found predominantly in patients with advanced metastatic disease, whilst only the sialo form alpha 1 MSP, is obtained from the urine of patients with early-stage disease. MSP does not react with antisera raised to alpha 1 foetoprotein (AFP) or carcinoembryonic antigen (CEA) and hence is immunologically distinct from these other tumour-associated glycoproteins. Antisera raised to MSP do not react with normal skin melanocytes nor with any foetal tissue tested, and hence the origin of MSP remains unresolved. Images Fig. 1 Fig. 2 PMID:6158965

  14. Thrombospondin-1 domain-containing peptide properdistatin improves vascular function in human melanoma xenografts.

    PubMed

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

    2015-03-01

    Properdistatin is a novel peptide derived from the thrombospondin-1 domain of the plasma protein properdin. The purpose of this study was to investigate the effect of properdistatin treatment on the morphology and function of tumor vasculature. A-07 human melanoma xenografts grown in dorsal window chambers were used as preclinical model. Tumors were treated with 80 mg/kg/day properdistatin or vehicle for 4 days. Morphologic parameters of tumor vascular networks were assessed from high-resolution transillumination images, and tumor blood supply time and plasma velocities were assessed from first-pass imaging movies recorded after a bolus of 155 kDa tetramethylrhodamine isothiocyanate-labeled dextran had been administered intravenously. Properdistatin-treated tumors showed reduced density of small-diameter vessels, reduced blood supply time, and increased plasma velocities. In conclusion, properdistatin treatment inhibited angiogenesis and improved vascular function in A-07 tumors. PMID:24555949

  15. Tumour procurement, DNA extraction, coverage analysis and optimisation of mutation-detection algorithms for human melanoma genomes.

    PubMed

    Wilmott, James S; Field, Matthew A; Johansson, Peter A; Kakavand, Hojabr; Shang, Ping; De Paoli-Iseppi, Ricardo; Vilain, Ricardo E; Pupo, Gulietta M; Tembe, Varsha; Jakrot, Valerie; Shang, Catherine A; Cebon, Jonathan; Shackleton, Mark; Fitzgerald, Anna; Thompson, John F; Hayward, Nicholas K; Mann, Graham J; Scolyer, Richard A

    2015-12-01

    Whole genome sequencing (WGS) of cancer patients' tumours offers the most comprehensive method of identifying both novel and known clinically-actionable genomic targets. However, the practicalities of performing WGS on clinical samples are poorly defined.This study was designed to test sample preparation, sequencing specifications and bioinformatic algorithms for their effect on accuracy and cost-efficiency in a large WGS analysis of human melanoma samples.WGS was performed on melanoma cell lines (n = 15) and melanoma fresh frozen tumours (n = 222). The appropriate level of coverage and the optimal mutation detection algorithm for the project pipeline were determined.An incremental increase in sequencing coverage from 36X to 132X in melanoma tissue samples and 30X to 103X for cell lines only resulted in a small increase (1-2%) in the number of mutations detected, and the quality scores of the additional mutations indicated a low probability that the mutations were real. The results suggest that 60X coverage for melanoma tissue and 40X for melanoma cell lines empower the detection of 98-99% of informative single nucleotide variants (SNVs), a sensitivity level at which clinical decision making or landscape research projects can be carried out with a high degree of confidence in the results. Likewise the bioinformatic mutation analysis methodology strongly influenced the number and quality of SNVs detected. Detecting mutations in the blood genomes separate to the tumour genomes generated 41% more SNVs than if the blood and melanoma tissue genomes were analysed simultaneously. Therefore, simultaneous analysis should be employed on matched melanoma tissue and blood genomes to reduce errors in mutation detection.This study provided valuable insights into the accuracy of SNV with WGS at various coverage levels in human clinical cancer specimens. Additionally, we investigated the accuracy of the publicly available mutation detection algorithms to detect cancer

  16. Role of Phosphodiesterase 2 in Growth and Invasion of Human Malignant Melanoma Cells

    PubMed Central

    Hiramoto, Kenichi; Murata, Taku; Shimizu, Kasumi; Morita, Hiroshi; Inui, Madoka; Manganiello, Vincent C.; Tagawa, Toshiro; Arai, Naoya

    2014-01-01

    Cyclic nucleotide phosphodiesterases (PDEs) regulate the intracellular concentrations and effects of adenosine 3’,5’-cyclic monophosphate (cAMP) and guanosine 3’,5’-cyclic monophosphate (cGMP). The role of PDEs in malignant tumor cells is still uncertain. The role of PDEs, especially PDE2, in human malignant melanoma PMP cell line was examined in this study. In PMP cells, 8-bromo-cAMP, a cAMP analog, inhibited cell growth and invasion. However, 8-bromo-cGMP, a cGMP analog, had little or no effect. PDE2 and PDE4, but not PDE3, were expressed in PMP cells. Growth and invasion of PMP cells were inhibited by erythro-9-(2-Hydroxy-3-nonyl) adenine (EHNA), a specific PDE2 inhibitor, but not by rolipram, a specific PDE4 inhibitor. Moreover, cell growth and invasion were inhibited by transfection of small interfering RNAs (siRNAs) specific for PDE2A and a catalytically-dead mutant of PDE2A. After treating cells with EHNA or rolipram, intracellular cAMP concentrations were increased. Growth and invasion were stimulated by PKA14-22, a PKA inhibitor, and inhibited by N6-benzoyl-c AMP, a PKA specific cAMP analogue, whereas 8-(4-chlorophenylthio)-2’-O-methyl-cAMP, an Epac specific cAMP analogue, did not. Invasion, but not growth, was stimulated by A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide. Based on these results, PDE2 appears to play an important role in growth and invasion of the human malignant melanoma PMP cell line. Selectively suppressing PDE2 might possibly inhibit growth and invasion of other malignant tumor cell lines. PMID:24705027

  17. Role of phosphodiesterase 2 in growth and invasion of human malignant melanoma cells.

    PubMed

    Hiramoto, Kenichi; Murata, Taku; Shimizu, Kasumi; Morita, Hiroshi; Inui, Madoka; Manganiello, Vincent C; Tagawa, Toshiro; Arai, Naoya

    2014-09-01

    Cyclic nucleotide phosphodiesterases (PDEs) regulate the intracellular concentrations and effects of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP). The role of PDEs in malignant tumor cells is still uncertain. The role of PDEs, especially PDE2, in human malignant melanoma PMP cell line was examined in this study. In PMP cells, 8-bromo-cAMP, a cAMP analog, inhibited cell growth and invasion. However, 8-bromo-cGMP, a cGMP analog, had little or no effect. PDE2 and PDE4, but not PDE3, were expressed in PMP cells. Growth and invasion of PMP cells were inhibited by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a specific PDE2 inhibitor, but not by rolipram, a specific PDE4 inhibitor. Moreover, cell growth and invasion were inhibited by transfection of small interfering RNAs (siRNAs) specific for PDE2A and a catalytically-dead mutant of PDE2A. After treating cells with EHNA or rolipram, intracellular cAMP concentrations were increased. Growth and invasion were stimulated by PKA14-22, a PKA inhibitor, and inhibited by N(6)-benzoyl-c AMP, a PKA specific cAMP analog, whereas 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, an Epac specific cAMP analog, did not. Invasion, but not growth, was stimulated by A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide. Based on these results, PDE2 appears to play an important role in growth and invasion of the human malignant melanoma PMP cell line. Selectively suppressing PDE2 might possibly inhibit growth and invasion of other malignant tumor cell lines. PMID:24705027

  18. Cinnamic acid induces apoptotic cell death and cytoskeleton disruption in human melanoma cells

    PubMed Central

    2013-01-01

    Anticancer activities of cinnamic acid derivatives include induction of apoptosis by irreversible DNA damage leading to cell death. The present work aimed to compare the cytotoxic and genotoxic potential of cinnamic acid in human melanoma cell line (HT-144) and human melanocyte cell line derived from blue nevus (NGM). Viability assay showed that the IC50 for HT-144 cells was 2.4 mM, while NGM cells were more resistant to the treatment. The growth inhibition was probably associated with DNA damage leading to DNA synthesis inhibition, as shown by BrdU incorporation assay, induction of nuclear aberrations and then apoptosis. The frequency of cell death caused by cinnamic acid was higher in HT-144 cells. Activated-caspase 3 staining showed apoptosis after 24 hours of treatment with cinnamic acid 3.2 mM in HT-144 cells, but not in NGM. We observed microtubules disorganization after cinnamic acid exposure, but this event and cell death seem to be independent according to M30 and tubulin labeling. The frequency of micronucleated HT-144 cells was higher after treatment with cinnamic acid (0.4 and 3.2 mM) when compared to the controls. Cinnamic acid 3.2 mM also increased the frequency of micronucleated NGM cells indicating genotoxic activity of the compound, but the effects were milder. Binucleation and multinucleation counting showed similar results. We conclude that cinnamic acid has effective antiproliferative activity against melanoma cells. However, the increased frequency of micronucleation in NGM cells warrants the possibility of genotoxicity and needs further investigation. PMID:23701745

  19. Treatment of melanoma with a serotype 5/3 chimeric oncolytic adenovirus coding for GM-CSF: Results in vitro, in rodents and in humans.

    PubMed

    Bramante, Simona; Kaufmann, Johanna K; Veckman, Ville; Liikanen, Ilkka; Nettelbeck, Dirk M; Hemminki, Otto; Vassilev, Lotta; Cerullo, Vincenzo; Oksanen, Minna; Heiskanen, Raita; Joensuu, Timo; Kanerva, Anna; Pesonen, Sari; Matikainen, Sampsa; Vähä-Koskela, Markus; Koski, Anniina; Hemminki, Akseli

    2015-10-01

    Metastatic melanoma is refractory to irradiation and chemotherapy, but amenable to immunological approaches such as immune-checkpoint-inhibiting antibodies or adoptive cell therapies. Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules. Therefore, oncolytic immuno-virotherapy of malignant melanoma is an appealing approach, which was recently validated by a positive phase 3 trial. We investigated the potency of oncolytic adenovirus Ad5/3-D24-GMCSF on a panel of melanoma cell lines and animal models, and summarized the melanoma-specific human data from the Advanced Therapy Access Program (ATAP). The virus effectively eradicated human melanoma cells in vitro and subcutaneous SK-MEL-28 melanoma xenografts in nude mice when combined with low-dose cyclophosphamide. Furthermore, virally-expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated the differentiation of human monocytes into macrophages. In contrast to human cells, RPMI 1846 hamster melanoma cells exhibited no response to oncolytic viruses and the chimeric 5/3 fiber failed to increase the efficacy of transduction, suggesting limited utility of the hamster model in the context of viruses with this capsid. In ATAP, treatments appeared safe and well-tolerated. Four out of nine melanoma patients treated were evaluable for possible therapy benefit with modified RECIST criteria: one patient had minor response, two had stable disease, and one had progressive disease. Two patients were alive at 559 and 2,149 days after treatment. Ad5/3-D24-GMCSF showed promising efficacy in preclinical studies and possible antitumor activity in melanoma patients refractory to other forms of therapy. This data supports continuing the clinical development of oncolytic adenoviruses for treatment of malignant melanoma. PMID:25821063

  20. 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.

  1. A non-canonical adenosinergic pathway led by CD38 in human melanoma cells induces suppression of T cell proliferation.

    PubMed

    Morandi, Fabio; Morandi, Barbara; Horenstein, Alberto L; Chillemi, Antonella; Quarona, Valeria; Zaccarello, Gianluca; Carrega, Paolo; Ferlazzo, Guido; Mingari, Maria Cristina; Moretta, Lorenzo; Pistoia, Vito; Malavasi, Fabio

    2015-09-22

    Nucleotide-metabolizing ectoenzymes are endowed with an extracellular catalytic domain, which is involved in regulating the extracellular nucleotide/nucleoside balance. The tumor microenvironment contains high levels of adenosine (ADO) generated by this enzymatic network, thus promoting tumor growth by inhibiting anti-tumor immune responses. ADO inhibition in melanoma murine models limits tumor metastases and restores anti-tumor immune responses. This work investigates the expression and function of ectoenzymes in primary human melanoma cell lines. All of latter cells expressed CD38, CD39, CD73, and CD203a/PC-1, and produced ADO from AMP and NAD(+ )T cell proliferation. Accordingly, phosphorylation of S6 ribosomal protein, p38 and Stat1 was lower in activated memory cells than in naïve CD4(+) T lymphocytes. Melanoma cells also inhibited proliferation of naïve, memory and -to a lesser extent- of effector CD8(+) T cells. These different inhibitory effects correlated with distinct patterns of expression of the ADO receptor A2a and A2b. These results show that primary human melanoma cell lines suppress in vitro T cell proliferation through an adenosinergic pathway in which CD38 and CD73 play a prominent role. PMID:26329660

  2. Serological analysis of Melan-A(MART-1), a melanocyte-specific protein homogeneously expressed in human melanomas.

    PubMed Central

    Chen, Y T; Stockert, E; Jungbluth, A; Tsang, S; Coplan, K A; Scanlan, M J; Old, L J

    1996-01-01

    Recent progress in the structural identification of human melanoma antigens recognized by autologous cytotoxic T cells has led to the recognition of a new melanocyte differentiation antigen, Melan-A(MART-1). To determine the properties of the Melan-A gene product, Melan-A recombinant protein was produced in Escherichia coli and used to generate mouse monoclonal antibodies (mAbs). Two prototype mAbs, A103 and A355, were selected for detailed study. Immunoblotting results with A103 showed a 20-22-kDa doublet In Melan-A mRNA positive melanoma cell lines and no reactivity with Melan-A mRNA-negative cell lines. A355, in addition to the 20-22-kDa doublet, recognized several other protein species in Melan-A mRNA-positive cell lines. Immunocytochemical assays on cultured melanoma cells showed specific and uniform cytoplasmic staining in Melan-A mRNA-positive cell lines. Immunohistochemical analysis of normal human tissues with both mAbs showed staining of adult melanocytes and no reactivity with the other normal tissues tested. Analysis of 21 melanoma specimens showed homogenous staining of tumor cell cytoplasm in 16 of 17 Melan-A mRNA-positive cases and no reactivity with the three Melan-A mRNA-negative cases. Images Fig. 1 Fig. 2 Fig. 3 PMID:8650193

  3. ADAM15 expression is downregulated in melanoma metastasis compared to primary melanoma

    SciTech Connect

    Ungerer, Christopher; Doberstein, Kai; Boehm, Beate; Pfeilschifter, Josef; Mihic-Probst, Daniela; Gutwein, Paul

    2010-10-22

    Research highlights: {yields} Strong ADAM15 expression is found in normal melanocytes. {yields} ADAM15 expression is significantly downregulated in patients with melanoma metastasis. {yields} TGF-{beta} can downregulate ADAM15 expression in melanoma cells. {yields} Overexpression of ADAM15 in melanoma cells inhibits migration, proliferation and invasion of melanoma cells. {yields} Conclusion: ADAM15 represents an tumor suppressor protein in melanoma. -- Abstract: In a mouse melanoma metastasis model it has been recently shown that ADAM15 overexpression in melanoma cells significantly reduced the number of metastatic nodules on the lung. Unfortunately, the expression of ADAM15 in human melanoma tissue has not been determined so far. In our study, we characterized the expression of ADAM15 in tissue micro-arrays of patients with primary melanoma with melanoma metastasis. ADAM15 was expressed in melanocytes and endothelial cells of benign nevi and melanoma tissue. Importantly, ADAM15 was significantly downregulated in melanoma metastasis compared to primary melanoma. We further demonstrate that IFN-{gamma} and TGF-{beta} downregulate ADAM15 protein levels in melanoma cells. To investigate the role of ADAM15 in melanoma progression, we overexpressed ADAM15 in melanoma cells. Importantly, overexpression of ADAM15 in melanoma cells reduced the migration, invasion and the anchorage dependent and independent cell growth of melanoma cells. In summary, the downregulation of ADAM15 plays an important role in melanoma progression and ADAM15 act as a tumorsuppressor in melanoma.

  4. The antimalarial amodiaquine causes autophagic-lysosomal and proliferative blockade sensitizing human melanoma cells to starvation- and chemotherapy-induced cell death

    PubMed Central

    Qiao, Shuxi; Tao, Shasha; Rojo de la Vega, Montserrat; Park, Sophia L; Vonderfecht, Amanda A; Jacobs, Suesan L; Zhang, Donna D; Wondrak, Georg T

    2013-01-01

    Pharmacological inhibition of autophagic-lysosomal function has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Repurposing approved and abandoned non-oncological drugs is an alternative approach to the identification and development of anticancer therapeutics, and antimalarials that target autophagic-lysosomal functions have recently attracted considerable attention as candidates for oncological repurposing. Since cumulative research suggests that dependence on autophagy represents a specific vulnerability of malignant melanoma cells, we screened a focused compound library of antimalarials for antimelanoma activity. Here we report for the first time that amodiaquine (AQ), a clinical 4-aminoquinoline antimalarial with unexplored cancer-directed chemotherapeutic potential, causes autophagic-lysosomal and proliferative blockade in melanoma cells that surpasses that of its parent compound chloroquine. Monitoring an established set of protein markers (LAMP1, LC3-II, SQSTM1) and cell ultrastructural changes detected by electron microscopy, we observed that AQ treatment caused autophagic-lysosomal blockade in malignant A375 melanoma cells, a finding substantiated by detection of rapid inactivation of lysosomal cathepsins (CTSB, CTSL, CTSD). AQ-treatment was associated with early induction of energy crisis (ATP depletion) and sensitized melanoma cells to either starvation- or chemotherapeutic agent-induced cell death. AQ displayed potent antiproliferative effects, and gene expression array analysis revealed changes at the mRNA (CDKN1A, E2F1) and protein level (TP53, CDKN1A, CCND1, phospho-RB1 [Ser 780]/[Ser 807/811], E2F1) consistent with the observed proliferative blockade in S-phase. Taken together, our data suggest that the clinical antimalarial AQ is a promising candidate for repurposing efforts that aim at targeting autophagic-lysosomal function and proliferative control in malignant melanoma cells. PMID:24113242

  5. Ectopic G-CSF expression in human melanoma lines marks a trans-dominant pathway of tumor progression.

    PubMed Central

    Safarians, S.; Rivera, S. P.; Sternlicht, M. D.; Naeim, F.; Barsky, S. H.

    1997-01-01

    Using a human melanoma/Scid xenograft model with the C8161, M24-met, LD-1 and other human melanoma lines to investigate spontaneous metastasis, we made the observation of marked splenomegaly (up to five times normal weight and size) in only those xenografts exhibiting high degrees of spontaneous metastasis. Evaluation of this revealed the cause to be massive myelopoiesis due to ectopic granulocyte/ colony-stimulating factor (G-CSF) production by the melanoma cells. Because of these observations linking G-CSF expression with metastasis of human melanoma, we decided to investigate the mechanism of this ectopic production. No gross amplification or rearrangement of the G-CSF gene could be detected as the basis for the increased transcriptional activity in any of these lines. Human-human somatic cell hybridization studies carried out between the metastatic C8161 and several different nonmetastatic non-G-CSF-expressing lines revealed, in addition to metastatic dominance, 3- to 10-fold enhancement of G-CSF transcription and expression in the fusions compared with C8161 itself. The suggestion of a trans-dominant mechanism was further supported by transfection studies with a human G-CSF promoter-CAT-reporter construct, which revealed 3- to 5-fold increased reporter activity in only those melanoma lines and hybrids expressing G-CSF. Furthermore, no obvious autocrine or paracrine effects of this ectopic G-CSF expression on the melanoma lines' growth or metastasis were apparent, as all of the G-CSF-expressing lines lacked the G-CSF receptor and injections of purified recombinant G-CSF exerted no stimulatory effects on their tumorigenicity, latency, growth, or metastasis in Scid mice. Thus, we advance the hypothesis that G-CSF expression is serving as a marker of a more generalized trans-dominant pathway linked to tumor progression and metastasis. This hypothesis has direct relevance to many human cancers where ectopic hormone or growth factor production occurs with no obvious

  6. Synthesis and Preclinical Characterization of [18F]FPBZA: A Novel PET Probe for Melanoma

    PubMed Central

    Huang, Shih-Pin; Lo, Yen-Chen; Liu, Ren-Shyan; Shen, Chih-Chieh

    2014-01-01

    Introduction. Benzamide can specifically bind to melanoma cells. A 18F-labeled benzamide derivative, [18F]N-(2-diethylaminoethyl)-4-[2-(2-(2-fluoroethoxy) ethoxy)ethoxy]benzamide ([18F]FPBZA), was developed as a promising PET probe for primary and metastatic melanoma. Methods. [18F]FPBZA was synthesized via a one-step radiofluorination in this study. The specific uptake of [18F]FPBZA was studied in B16F0 melanoma cells, A375 amelanotic melanoma cells, and NB-DNJ-pretreated B16F0 melanoma cells. The biological characterization of [18F]FPBZA was performed on mice bearing B16F0 melanoma, A375 amelanotic melanoma, or inflammation lesion. Results. [18F]FPBZA can be prepared efficiently with a yield of 40–50%. The uptake of [18F]FPBZA by B16F0 melanoma cells was significantly higher than those by A375 tumor cells and NB-DNJ-pretreated B16F0 melanoma cells. B16F0 melanoma displayed prominent uptake of [18F]FPBZA at 2 h (7.81 ± 0.82 %ID/g), compared with A375 tumor and inflammation lesion (3.00 ± 0.71 and 1.67 ± 0.56 %ID/g, resp.). [18F]FPBZA microPET scan clearly delineated B16F0 melanoma but not A375 tumor and inflammation lesion. In mice bearing pulmonary metastases, the lung radioactivity reached 4.77 ± 0.36 %ID/g at 2 h (versus 1.16 ± 0.23 %ID/g in normal mice). Conclusions. Our results suggested that [18F]FPBZA PET would provide a promising and specific approach for the detection of primary and metastatic melanoma lesions. PMID:25254219

  7. Adipose‑derived mesenchymal stem cell‑facilitated TRAIL expression in melanoma treatment in vitro.

    PubMed

    Jing, Hai Xia; Duan, De Jian; Zhou, Hui; Hu, Qing Mei; Lei, Tie Chi

    2016-07-01

    Adipose-derived stem cells (ADSCs) may be useful as an efficient vehicle in cell-based gene therapy of human diseases due to their ability to migrate to disease lesions. This study investigated the ability of ADSC‑harbored human tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL) cDNA to facilitate TRAIL expression and induce A375 melanoma cell apoptosis as observed using a Transwell co‑culture system. A cell migration assay was used to observe ADSC migration ability. In addition, TRAIL protein expression was successfully detected by western blot analysis in ADSCs after stable transfection of TRAIL cDNA. The Transwell co‑culture system data showed that TRAIL-ADSCs could induce A375 cell apoptosis in a dose‑dependent manner. At the gene level, the killing activity of TRAIL-ADSCs was associated with activation of caspase‑4 and caspase‑8. Collectively, the data from the current study provides preclinical support of ADSC‑facilitated TRAIL expression in the treatment of melanoma. Further investigation is required to evaluate and confirm the in vivo ability of TRAIL-ADSCs in therapy of melanoma in animal models. PMID:27177242

  8. sup 211 At-methylene blue for targeted radiotherapy of human melanoma xenografts: Treatment of micrometastases

    SciTech Connect

    Link, E.M.; Carpenter, R.N. )

    1990-05-15

    Treatment of micrometastases of HX34 human melanoma grown as xenografts in nude mice represents an advanced stage of preclinical investigations concerning targeted radiotherapy of this neoplasm using 3,7-(dimethylamino)phenazathionium chloride methylene blue (MTB) labeled with astatine-211 (211At) (alpha-particle emitter). The therapeutic effectiveness of 211At-MTB administered i.v. was determined by a lung colony assay combined with a search for metastases to organs other than the lungs. A single dose of 211At-MTB lowered the HX34 cell surviving fraction in lungs to below 10% almost independently of the time interval between cell inoculation and radioisotope injection and of 211At-MTB radioactivity within its investigated range. Radiation dose and the time of its administration did, however, influence the size of lung colonies. In contrast, the efficacy of 211At-MTB treatment as assessed by both surviving fraction and colony size was significantly dependent on a number of HX34 cells inoculated initially into mice. These results are explained by a short range of alpha-particles emitted by 211At and a mechanism of growth of lung colonies from tumor cells circulating with blood and blocking lung capillaries. Metastases in organs other than lungs and characteristic of control animals were not found in mice treated with 211At-MTB. The high therapeutic efficacy achieved proved that 211At-MTB is a very efficient scavenger of single melanoma cells distributed through blood and micrometastases with sizes below the limit of clinical detection.

  9. Melanoma in Immunosuppressed Patients

    PubMed Central

    Kubica, Agnieszka W.; Brewer, Jerry D.

    2012-01-01

    The immunogenic characteristics of malignant melanoma are intriguing. To date, multiple studies exist regarding the immunogenicity of melanoma. In this article, we summarize data in the literature on the role of immunosuppression in melanoma and discuss several immunocompromised patient populations in detail. A comprehensive PubMed search was conducted with no date limitation. The following search terms were used: melanoma in combination with immunosuppression, immunocompromised, genetics, antigen processing, UV radiation, organ transplantation, organ transplant recipients, lymphoproliferative disease, lymphoma, CLL, NHL, radiation, and HIV/AIDS. Although no formal criteria were used for inclusion of studies, most pertinent studies on the topic were reviewed, with the exception of smaller case reports and case series. The included studies were generally large (≥1000 patients in organ transplant recipient studies; ≥500 patients in lymphoma studies), with a focus on institutional experiences, or population-based national or international epidemiologic studies. Melanoma-induced immunosuppression, the role of UV radiation in melanoma development, and the epidemiology, clinical course, and prognosis of melanoma in immunocompromised patients are highlighted. Organ transplant recipients, patients with lymphoproliferative disorders, patients with iatrogenic immunosuppression, and patients with human immunodeficiency virus infection/AIDS are also highlighted. Recommendations are proposed for the care and monitoring of immunosuppressed patients with melanoma. With better understanding of the molecular microenvironment and clinical course of melanoma in immunosuppressed patients, novel therapies could be developed and outcomes potentially affected in these patients. PMID:23036673

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

    PubMed

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

    2016-03-11

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

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

  13. IDH1(R132) mutation identified in one human melanoma metastasis, but not correlated with metastases to the brain.

    PubMed

    Lopez, Giselle Y; Reitman, Zachary J; Solomon, David; Waldman, Todd; Bigner, Darell D; McLendon, Roger E; Rosenberg, Steven A; Samuels, Yardena; Yan, Hai

    2010-07-30

    Isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) are enzymes which convert isocitrate to alpha-ketoglutarate while reducing nicotinamide adenine dinucleotide phosphate (NADP+to NADPH). IDH1/2 were recently identified as mutated in a large percentage of progressive gliomas. These mutations occur at IDH1(R132) or the homologous IDH2(R172). Melanomas share some genetic features with IDH1/2-mutated gliomas, such as frequent TP53 mutation. We sought to test whether melanoma is associated with IDH1/2 mutations. Seventy-eight human melanoma samples were analyzed for IDH1(R132) and IDH2(R172) mutation status. A somatic, heterozygous IDH1 c.C394T (p.R132C) mutation was identified in one human melanoma metastasis to the lung. Having identified this mutation in one metastasis, we sought to test the hypothesis that certain selective pressures in the brain environment may specifically favor the cell growth or survival of tumor cells with mutations in IDH1/2, regardless of primary tumor site. To address this, we analyzed IDH1(R132) and IDH2(R172) mutation status 53 metastatic brain tumors, including nine melanoma metastases. Results revealed no mutations in any samples. This lack of mutations would suggest that mutations in IDH1(R132) or IDH2(R172) may be necessary for the formation of tumors in a cell-lineage dependent manner, with a particularly strong selective pressure for mutations in progressive gliomas; this also suggests the lack of a particular selective pressure for growth in brain tissue in general. Studies on the cell-lineages of tumors with IDH1/2 mutations may help clarify the role of these mutations in the development of brain tumors. PMID:20603105

  14. A Molecular Switch Abrogates Glycoprotein 100 (gp100) T-cell Receptor (TCR) Targeting of a Human Melanoma Antigen*

    PubMed Central

    Bianchi, Valentina; Bulek, Anna; Fuller, Anna; Lloyd, Angharad; Attaf, Meriem; Rizkallah, Pierre J.; Dolton, Garry; Sewell, Andrew K.; Cole, David K.

    2016-01-01

    Human CD8+ cytotoxic T lymphocytes can mediate tumor regression in melanoma through the specific recognition of HLA-restricted peptides. Because of the relatively weak affinity of most anti-cancer T-cell receptors (TCRs), there is growing emphasis on immunizing melanoma patients with altered peptide ligands in order to induce strong anti-tumor immunity capable of breaking tolerance toward these self-antigens. However, previous studies have shown that these immunogenic designer peptides are not always effective. The melanocyte differentiation protein, glycoprotein 100 (gp100), encodes a naturally processed epitope that is an attractive target for melanoma immunotherapies, in particular peptide-based vaccines. Previous studies have shown that substitutions at peptide residue Glu3 have a broad negative impact on polyclonal T-cell responses. Here, we describe the first atomic structure of a natural cognate TCR in complex with this gp100 epitope and highlight the relatively high affinity of the interaction. Alanine scan mutagenesis performed across the gp100280–288 peptide showed that Glu3 was critically important for TCR binding. Unexpectedly, structural analysis demonstrated that the Glu3 → Ala substitution resulted in a molecular switch that was transmitted to adjacent residues, abrogating TCR binding and T-cell recognition. These findings help to clarify the mechanism of T-cell recognition of gp100 during melanoma responses and could direct the development of altered peptides for vaccination. PMID:26917722

  15. Double Positive CD4CD8 αβ T Cells: A New Tumor-Reactive Population in Human Melanomas

    PubMed Central

    Desfrançois, Juliette; Moreau-Aubry, Agnès; Vignard, Virginie; Godet, Yann; Khammari, Amir; Dréno, Brigitte; Jotereau, Francine; Gervois, Nadine

    2010-01-01

    Background Double positive (DP) CD4CD8 Tαβ cells have been reported in normal individuals as well as in different pathological conditions including inflammatory diseases, viral infections and cancer, but their function remains to be elucidated. We recently reported the increased frequency of DP Tαβ cells in human breast pleural effusions. This manuscript addresses the question of the existence and above all the role of this non-conventional DP sub-population among tumor associated lymphocytes in melanomas. Methodology/Principal Findings We analyzed the intratumoral cell infiltrate in solid metastasis (n = 6) and tumor invaded lymph nodes (n = 26) samples from melanomas patients by multiparametric cytometry. Here we documented for the first time significant increased frequency of DP T cells in about 60% of melanoma tumors compared to blood samples. Interestingly, a high proportion of these cells produced TNF-α in response to autologous melanoma cell lines. Besides, they are characterized by a unique cytokine profile corresponding to higher secretion of IL-13, IL-4 and IL-5 than simple positive T cells. In deep analysis, we derived a representative tumor-reactive DP T cell clone from a melanoma patient's invaded lymph node. This clone was restricted by HLA-A*2402 and recognized both autologous and allogeneic tumor cells of various origins as well as normal cells, suggesting that the target antigen was a ubiquitous self antigen. However, this DP T cell clone failed to kill HLA-A*2402 EBV-transformed B cells, probably due to the constitutive expression of immunoproteasome by these cells. Conclusions/Significance In conclusion, we can postulate that, according to their broad tumor reactivity and to their original cytokine profile, the tumor associated DP T cells could participate in immune responses to tumors in vivo. Therefore, the presence of these cells and their role will be crucial to address in cancer patients, especially in the context of

  16. P53 in human melanoma fails to regulate target genes associated with apoptosis and the cell cycle and may contribute to proliferation

    PubMed Central

    2011-01-01

    Background Metastatic melanoma represents a major clinical problem. Its incidence continues to rise in western countries and there are currently no curative treatments. While mutation of the P53 tumour suppressor gene is a common feature of many types of cancer, mutational inactivation of P53 in melanoma is uncommon; however, its function often appears abnormal. Methods In this study whole genome bead arrays were used to examine the transcript expression of P53 target genes in extracts from 82 melanoma metastases and 6 melanoma cell lines, to provide a global assessment of aberrant P53 function. The expression of these genes was also examined in extracts derived from diploid human melanocytes and fibroblasts. Results The results indicated that P53 target transcripts involved in apoptosis were under-expressed in melanoma metastases and melanoma cell lines, while those involved in the cell cycle were over-expressed in melanoma cell lines. There was little difference in the transcript expression of P53 target genes between cell lines with null/mutant P53 compared to those with wild-type P53, suggesting that altered expression in melanoma was not related to P53 status. Similarly, down-regulation of P53 by short-hairpin RNA (shRNA) had limited effect on P53 target gene expression in melanoma cells, whereas there were a large number of P53 target genes whose mRNA expression was significantly altered by P53 inhibition in melanocytes. Analysis of whole genome gene expression profiles indicated that the ability of P53 to regulate genes involved in the cell cycle was significantly reduced in melanoma cells. Moreover, inhibition of P53 in melanocytes induced changes in gene expression profiles that were characteristic of melanoma cells and resulted in increased proliferation. Conversely, knockdown of P53 in melanoma cells resulted in decreased proliferation. Conclusions These results indicate that P53 target genes involved in apoptosis and cell cycle regulation are aberrantly

  17. Recurrent inactivating RASA2 mutations in melanoma

    PubMed Central

    Arafeh, Rand; Qutob, Nouar; Emmanuel, Rafi; Keren-Paz, Alona; Madore, Jason; Elkahloun, Abdel; Wilmott, James S.; Gartner, Jared J.; Di Pizio, Antonella; Winograd-Katz, Sabina; Sindiri, Sivasish; Rotkopf, Ron; Dutton-Regester, Ken; Johansson, Peter; Pritchard, Antonia; Waddell, Nicola; Hill, Victoria K.; Lin, Jimmy C.; Hevroni, Yael; Rosenberg, Steven A.; Khan, Javed; Ben-Dor, Shifra; Niv, Masha Y.; Ulitsky, Igor; Mann, Graham J; Scolyer, Richard A.; Hayward, Nicholas K.; Samuels, Yardena

    2016-01-01

    Analysis of 501 melanoma exomes revealed RASA2, encoding a RasGAP, as a tumor-suppressor gene mutated in 5% of melanomas. Recurrent loss-of-function mutations in RASA2 were found to increase RAS activation, melanoma cell growth and migration. RASA2 expression was lost in ≥30% of human melanomas and was associated with reduced patient survival. These findings reveal RASA2 inactivation as a melanoma driver and highlight the importance of Ras GAPs in cancer. PMID:26502337

  18. Arginine-glycine-aspartic acid-conjugated dendrimer-modified quantum dots for targeting and imaging melanoma.

    PubMed

    Li, Zhiming; Huang, Peng; Lin, Jing; He, Rong; Liu, Bing; Zhang, Xiaomin; Yang, Sen; Xi, Peng; Zhang, Xuejun; Ren, Qiushi; Cui, Daxiang

    2010-08-01

    Angiogenesis is essential for the development of malignant tumors and provides important targets for tumor diagnosis and therapy. Quantum dots have been broadly investigated for their potential application in cancer molecular imaging. In present work, CdSe quantum dots were synthesized, polyamidoamine dendrimers were used to modify surface of quantum dots and improve their solubility in water solution. Then, dendrimer-modified CdSe quantum dots were conjugated with arginine-glycine-aspartic acid (RGD) peptides. These prepared nanoprobes were injected into nude mice loaded with melanoma (A375) tumor xenografts via tail vessels, IVIS imaging system was used to image the targeting and bio-distribution of as-prepared nanoprobes. The dendrimer-modified quantum dots exhibit water-soluble, high quantum yield, and good biocompatibility. RGD-conjugated quantum dots can specifically target human umbilical vein endothelial cells (HUVEC) and A375 melanoma cells, as well as nude mice loaded with A735 melanoma cells. High-performance RGD-conjugated dendrimers modified quantum dot-based nanoprobes have great potential in application such as tumor diagnosis and therapy. PMID:21125820

  19. Targeting Nitric Oxide Signaling with nNOS Inhibitors As a Novel Strategy for the Therapy and Prevention of Human Melanoma

    PubMed Central

    Yang, Zhen; Misner, Bobbye; Ji, Haitao; Poulos, Thomas L.; Silverman, Richard B.; Meyskens, Frank L.

    2013-01-01

    Abstract Aims: Our previous studies have shown that nitric oxide (NO) plays an important role in increasing the invasion and proliferation of human melanoma cells, suggesting that targeting NO signaling may facilitate therapy and prevention. Neuronal nitric oxide synthase (nNOS) is present in melanocytes, a cell type that originates from the neural crest. The aims of this study were to determine the role of nNOS in melanoma progression and the potential antitumor effects of novel synthesized nNOS inhibitors. Results: In vitro studies demonstrated abundant expression of nNOS in melanoma compared to melanocytes, which was inducible by ultraviolet radiation and was associated with increased NO generation. nNOS was also detected in melanoma biopsies that increased with disease stage. Knockdown of nNOS in melanoma cells diminished L-arginine-induced NO production; the metastatic capacity was also reduced as well as the levels of MMP-1, Bcl-2, JunD, and APE/Ref-1. Similar inhibition of NO and invasion potential was observed utilizing novel, highly selective nNOS inhibitors. In three-dimensional human skin reconstructs, the nNOS inhibitor cpd8 effectively reversed the melanoma overgrowth stimulated by NO stress. Innovation: Our work lays the foundation for development of clinical “drug-like” nNOS inhibitors as a new and promising strategy for the chemoprevention of early melanoma progression and the inhibition of secondary melanoma in high-risk individuals. Conclusion: Based on our observations, we propose that nNOS in melanoma results in constitutive overproduction of NO, which stimulates proliferation and increases invasion potential, leading to subsequent development of metastases. Antioxid. Redox Signal. 19, 433–447. PMID:23199242

  20. Uveal melanoma: estimating prognosis.

    PubMed

    Kaliki, Swathi; Shields, Carol L; Shields, Jerry A

    2015-02-01

    Uveal melanoma is the most common primary malignant tumor of the eye in adults, predominantly found in Caucasians. Local tumor control of uveal melanoma is excellent, yet this malignancy is associated with relatively high mortality secondary to metastasis. Various clinical, histopathological, cytogenetic features and gene expression features help in estimating the prognosis of uveal melanoma. The clinical features associated with poor prognosis in patients with uveal melanoma include older age at presentation, male gender, larger tumor basal diameter and thickness, ciliary body location, diffuse tumor configuration, association with ocular/oculodermal melanocytosis, extraocular tumor extension, and advanced tumor staging by American Joint Committee on Cancer classification. Histopathological features suggestive of poor prognosis include epithelioid cell type, high mitotic activity, higher values of mean diameter of ten largest nucleoli, higher microvascular density, extravascular matrix patterns, tumor-infiltrating lymphocytes, tumor-infiltrating macrophages, higher expression of insulin-like growth factor-1 receptor, and higher expression of human leukocyte antigen Class I and II. Monosomy 3, 1p loss, 6q loss, and 8q and those classified as Class II by gene expression are predictive of poor prognosis of uveal melanoma. In this review, we discuss the prognostic factors of uveal melanoma. A database search was performed on PubMed, using the terms "uvea," "iris," "ciliary body," "choroid," "melanoma," "uveal melanoma" and "prognosis," "metastasis," "genetic testing," "gene expression profiling." Relevant English language articles were extracted, reviewed, and referenced appropriately. PMID:25827538

  1. Identification of unique sensitizing targets for anti-inflammatory CDDO-Me in metastatic melanoma by a large-scale synthetic lethal RNAi screening

    PubMed Central

    Ekmekcioglu, Suhendan; Grimm, Elizabeth A.

    2013-01-01

    Summary CDDO-Me has been shown to exert potent anti-inflammatory activity for chronic kidney disease and antitumor activity for several tumors, including melanoma, in early clinical trials. To improve CDDO-Me response in melanoma, we utilized a large-scale synthetic lethal RNAi screen targeting 6,000 human druggable genes to identify targets that would sensitize melanoma cells to CDDO-Me. Based on screening results, five unique genes (GNPAT, SUMO1, SPINT2, FLI1, and SSX1) significantly potentiated the growth-inhibitory effects of CDDO-Me and induced apoptosis in A375, a BRAF mutated melanoma line (P<0.001). These five genes were then individually validated as targets to potentiate CDDO-Me activity, and related downstream signaling pathways of these genes were analyzed. In addition, the levels of phosphorylated Erk1/2, Akt, GSK-2, and PRAS40 were dramatically decreased by downregulating each of these five genes separately, suggesting a set of common mediators. Our findings indicate that GNPAT, SUMO1, SPINT2, FLI1, and SSX1 play critical roles in synergy with inflammation pathways in modulating melanoma cell survival, and could serve as sensitizing targets to enhance CDDO-Me efficacy in melanoma growth control. PMID:23020131

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

    PubMed

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

    2015-04-28

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

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

    PubMed

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

    2013-01-01

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

  4. Orthotopic human melanoma xenograft model systems for studies of tumour angiogenesis, pathophysiology, treatment sensitivity and metastatic pattern.

    PubMed Central

    Rofstad, E. K.

    1994-01-01

    Adequate tumour models are a prerequisite in experimental cancer research. The purpose of the present work was to establish and assess the validity of four new orthotopic human melanoma xenograft model systems (A-07, D-12, R-18, U-25). Permanent cell lines were established in monolayer culture from subcutaneous metastases of four different melanoma patients by using an in vivo-in vitro procedure, and cells from these lines were inoculated intradermally in Balb/c nu/nu mice to form tumours. Individual xenografted tumours of the same line differed substantially in growth and pathophysiological parameters, probably as a consequence of differences between inoculation sites in host factors which influence tumour angiogenesis. Nevertheless, xenografted tumours of different lines showed distinctly different biological characteristics. Several biological characteristics of the donor patients' tumours were retained in the xenografted tumours, including angiogenic potential; growth, histopathological and pathophysiological parameters; and sensitivity to radiation, heat and dacarbazine treatment. Moreover, the organ-specific metastatic pattern of the xenografted tumours reflected the pattern of distant metastases in the donor patients. The organs of preference for distant metastases were lungs (A-07, D-12), lymph nodes (R-18) and brain (U-25). R-18 lymph node metastases and U-25 brain metastases developed in the absence of lung involvement. The four orthotopic human melanoma xenograft model systems show great promise for future studies of tumour angiogenesis, pathophysiology, treatment sensitivity and metastatic pattern. Images Figure 1 Figure 4 Figure 5 Figure 7 PMID:7947084

  5. On the composition and function of the carbohydrate moiety of tissue-type plasminogen activator from human melanoma cells.

    PubMed

    Rijken, D C; Emeis, J J; Gerwig, G J

    1985-12-17

    Two variants (I and II) of tissue-type plasminogen activator (t-PA) from human melanoma cells were separated by Lysine Sepharose chromatography. The carbohydrate compositions of the forms were determined by gas-liquid chromatography. Variant I contained 12.8 g and variant II 7.1 g of carbohydrate per 100 g protein. Both variants contained N-acetylgalactosamine, suggesting O-glycosylation in addition to N-glycosylation. The possible role of N-linked oligosaccharides for the biological activity of t-PA was studied using t-PA secreted by melanoma cells in the presence of tunicamycin, an inhibitor of N-glycosylation. The latter t-PA showed the same plasminogen activating and fibrin binding properties as normally glycosylated t-PA, indicating that N-linked carbohydrate is not involved in the fibrinolytic activity of t-PA. PMID:3937276

  6. Histamine elevates the expression of Ets-1, a protooncogen in human melanoma cell lines through H2 receptor.

    PubMed

    Hegyesi, Hargita; Horváth, Barnabás; Pállinger, Eva; Pós, Zoltán; Molnár, Viktor; Falus, András

    2005-04-25

    Histamine is known to act, at least in part, as a growth factor for several cell types, and as production of this biogen amine has been found to accelerate the rate of tissue proliferation in wound repair, embryogenesis and malignant growth. Abundant experimental and clinical data suggest that histamine augments in vivo tumour cell proliferation via histamine H2 receptors (H2R). Here, we report that exogenously added histamine stimulates Ets-1 (v-ets erythroblastosis virus E26 oncogene homolog 1) synthesis in human melanoma cells. Involvement of histamine receptors in the histamine induced ets-1 expression has been also studied. Our data show that these newly recognized actions of histamine are mediated by the H2R. Modification of local protooncogen Ets-1 level is likely being involved in the regulation of melanoma growth. PMID:15848191

  7. Embryonic Zebrafish: Different Phenotypes after Injection of Human Uveal Melanoma Cells.

    PubMed

    van der Ent, Wietske; Burrello, Claudia; de Lange, Mark J; van der Velden, Pieter A; Jochemsen, Aart G; Jager, Martine J; Snaar-Jagalska, B Ewa

    2015-04-01

    Although murine xenograft models for human uveal melanoma (UM) are available, they are of limited utility for screening large compound libraries for the discovery of new drugs. We need new preclinical models which can efficiently evaluate drugs that can treat UM metastases. The zebrafish embryonic model is ideal for drug screening purposes because it allows the investigation of potential antitumor properties of drugs within 1 week. The optical transparency of the zebrafish provides unique possibilities for live imaging of fluorescence-labelled cancer cells and their behavior. In addition, the adaptive immune response, which is responsible for the rejection of transplanted material, is not yet present in the early stages of fish development, and systemic immunosuppression is therefore not required to allow growth of tumor cells. We studied the behavior of UM cells following injection into zebrafish embryos and observed different phenotypes. We also analyzed cell migration, proliferation, formation of micrometastasis and interaction with the host microenvironment. Significant differences were noted between cell lines: cells derived from metastases showed more migration and proliferation than cells derived from the primary tumors. The addition of the c-Met inhibitor crizotinib to the water in which the larvae were kept reduced the migration and proliferation of UM cells expressing c-Met. This indicates the applicability of the zebrafish xenografts for testing novel inhibitory compounds and provides a fast and sensitive in vivo vertebrate model for preclinical drug screening to combat UM. PMID:27171126

  8. Internalization and re-expression of antigens of human melanoma cells following exposure to monoclonal antibody

    SciTech Connect

    Wang, B.S.; Lumanglas, A.L.; Silva, J.; Ruszala-Mallon, V.; Durr, F.E.

    1987-04-15

    Modulation of the surface membrane of human Sk-Mel-28 melanoma cells by monoclonal antibody (MoAb) 96.5 recognizing p97 determinants was examined using direct radioimmunoassay and indirect fluorescent antibody-staining techniques. It was determined that the majority of /sup 111/In-labeled antibody that remained associated with cells after a 24-hr incubation at 37 degrees C had been internalized because MoAb 96.5 was no longer visible on the cell surface. A second treatment of these cells with the same antibody 24 hr later not only increased the cell-associated radioactivity, reflecting an increase of total antibody bound, but also rendered these cells membrane immunofluorescent again, indicating the re-expression of surface antigens. Autoradiographs of the electrophoretically analyzed membrane components of Sk-Mel-28 cells further demonstrated the appearance of newly synthesized 97-kDa proteins that were immunoprecipitable with MoAb 96.5. Taken together, the present findings suggest that p97 antigens undergo endocytosis in Sk-Mel-28 cells following exposure to MoAb 96.5. However, the same antigens were regenerated and expressed on the cell surface within a period of 24 hr. The re-expression of tumor cell surface antigen following initial internalization of the MoAb-antigen complex may have implications for diagnosis and therapy.

  9. Radiolabeled porphyrin versus gallium-67 citrate for the detection of human melanoma in athymic mice

    SciTech Connect

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

    1987-01-01

    We performed the biodistribution and imaging studies of /sup 111/In and /sup 67/Ga labeled tetra(4-N-methylpyridyl) porphine, (T4NMPYP), and compared it to that of /sup 67/Ga citrate in athymic mice bearing a human melanoma xenograft. The biodistribution results of both /sup 111/In and /sup 67/Ga labeled T4NMPYP (3, 6, 24, and 48 hours) were similar but differed from that of /sup 67/Ga citrate (48 hours). The optimum tumor uptake of both radiolabeled porphyrins was at 6 hours postinjection and was lower than the tumor uptake of /sup 67/Ga citrate at 48 hours postinjection. Kidney was the only organ showing higher uptake of radiolabeled porphyrin compared to that of /sup 67/Ga citrate. The imaging studies performed with /sup 111/In T4NMPYP and /sup 67/Ga citrate correspond to the biodistribution results. Osteomyelitis present in one mouse showed good localization of /sup 111/In T4NMPYP. 15 refs., 3 figs., 5 tabs.

  10. β-lapachone suppresses the proliferation of human malignant melanoma cells by targeting specificity protein 1.

    PubMed

    Bang, Woong; Jeon, Young-Joo; Cho, Jin Hyoung; Lee, Ra Ham; Park, Seon-Min; Shin, Jae-Cheon; Choi, Nag-Jin; Choi, Yung Hyun; Cho, Jung-Jae; Seo, Jae-Min; Lee, Seung-Yeop; Shim, Jung-Hyun; Chae, Jung-Il

    2016-02-01

    β-lapachone (β-lap), a novel natural quinone derived from the bark of the Pink trumpet tree (Tabebuia avellanedae) has been demonstrated to have anticancer activity. In this study, we investigated whether β-lap exhibits anti-proliferative effects on two human malignant melanoma (HMM) cell lines, G361 and SK-MEL-28. The effects of β-lap on the HMM cell lines were investigated using 3-(4,5-dimethylthiazol-2-yl)‑5-(3-carboxymethoxyphenyl)‑2-(4-sulfophenyl-2H-tetrazolium (MTS) assay, 4',6-diamidino-2-phenylindole (DAPI) staining, Annexin V and Dead cell assay, mitochondrial membrane potential (MMP) assay and western blot analysis. We demonstrated that β-lap significantly induced apoptosis and suppressed cell viability in the HMM cells. Intriguingly, the transcription factor specificity protein 1 (Sp1) was significantly downregulated by β-lap in a dose- and time-dependent manner. Furthermore, β-lap modulated the protein expression level of the Sp1 regulatory genes including cell cycle regulatory proteins and apoptosis-associated proteins. Taken together, our findings indicated that β-lap modulates Sp1 transactivation and induces apoptotic cell death through the regulation of cell cycle- and apoptosis-associated proteins. Thus, β-lap may be used as a promising anticancer drug for cancer prevention and may improve the clinical outcome of patients with cancer. PMID:26718788

  11. Ion therapy for uveal melanoma in new human eye phantom based on GEANT4 toolkit.

    PubMed

    Mahdipour, Seyed Ali; Mowlavi, Ali Asghar

    2016-01-01

    Radiotherapy with ion beams like proton and carbon has been used for treatment of eye uveal melanoma for many years. In this research, we have developed a new phantom of human eye for Monte Carlo simulation of tumors treatment to use in GEANT4 toolkit. Total depth-dose profiles for the proton, alpha, and carbon incident beams with the same ranges have been calculated in the phantom. Moreover, the deposited energy of the secondary particles for each of the primary beams is calculated. The dose curves are compared for 47.8MeV proton, 190.1MeV alpha, and 1060MeV carbon ions that have the same range in the target region reaching to the center of tumor. The passively scattered spread-out Bragg peak (SOBP) for each incident beam as well as the flux curves of the secondary particles including neutron, gamma, and positron has been calculated and compared for the primary beams. The high sharpness of carbon beam׳s Bragg peak with low lateral broadening is the benefit of this beam in hadrontherapy but it has disadvantages of dose leakage in the tail after its Bragg peak and high intensity of neutron production. However, proton beam, which has a good conformation with tumor shape owing to the beam broadening caused by scattering, can be a good choice for the large-size tumors. PMID:26831752

  12. Alterations in transcription factor binding in radioresistant human melanoma cells after ionizing radiation

    SciTech Connect

    Sahijdak, W.M.; Yang, Chin-Rang; Zuckerman, J.S.; Meyers, M.; Boothman, D.A.

    1994-04-01

    We analyzed alterations in transcription factor binding to specific, known promoter DNA consensus sequences between irradiated and unirradiated radioresistant human melanoma (U1-Mel) cells. The goal of this study was to begin to investigate which transcription factors and DNA-binding sites are responsible for the induction of specific transcripts and proteins after ionizing radiation. Transcription factor binding was observed using DNA band-shift assays and oligonucleotide competition analyses. Confluence-arrested U1-Mel cells were irradiated (4.5 Gy) and harvested at 4 h. Double-stranded oligonucleotides containing known DNA-binding consensus sites for specific transcription factors were used. Increased DNA binding activity after ionizing radiation was noted with oligonucleotides containing the CREB, NF-kB and Sp1 consensus sites. No changes in protein binding to AP-1, AP-2, AP-3, or CTF/NF1, GRE or Oct-1 consensus sequences were noted. X-ray activation of select transcription factors, which bind certain consensus sites in promoters, may cause specific induction or repression of gene transcription. 22 refs., 2 figs.

  13. A Novel Nanoprobe for Multimodal Imaging Is Effectively Incorporated into Human Melanoma Metastatic Cell Lines

    PubMed Central

    Aasen, Synnøve Nymark; Pospisilova, Aneta; Eichler, Tilo Wolf; Panek, Jiri; Hruby, Martin; Stepanek, Petr; Spriet, Endy; Jirak, Daniel; Skaftnesmo, Kai Ove; Thorsen, Frits

    2015-01-01

    To facilitate efficient drug delivery to tumor tissue, several nanomaterials have been designed, with combined diagnostic and therapeutic properties. In this work, we carried out fundamental in vitro and in vivo experiments to assess the labeling efficacy of our novel theranostic nanoprobe, consisting of glycogen conjugated with a red fluorescent probe and gadolinium. Microscopy and resazurin viability assays were used to study cell labeling and cell viability in human metastatic melanoma cell lines. Fluorescence lifetime correlation spectroscopy (FLCS) was done to investigate nanoprobe stability. Magnetic resonance imaging (MRI) was performed to study T1 relaxivity in vitro, and contrast enhancement in a subcutaneous in vivo tumor model. Efficient cell labeling was demonstrated, while cell viability, cell migration, and cell growth was not affected. FLCS showed that the nanoprobe did not degrade in blood plasma. MRI demonstrated that down to 750 cells/μL of labeled cells in agar phantoms could be detected. In vivo MRI showed that contrast enhancement in tumors was comparable between Omniscan contrast agent and the nanoprobe. In conclusion, we demonstrate for the first time that a non-toxic glycogen-based nanoprobe may effectively visualize tumor cells and tissue, and, in future experiments, we will investigate its therapeutic potential by conjugating therapeutic compounds to the nanoprobe. PMID:26370983

  14. Tumourigenic phenotypes of human melanoma cell lines in nude mice determined by an active antitumour mechanism.

    PubMed Central

    Jacubovich, R.; Cabrillat, H.; Gerlier, D.; Bailly, M.; Doré, J. F.

    1985-01-01

    Ten human melanoma cell lines (HMCL) were tested for their ability to grow subcutaneously in nude mice. Using a standard inoculum, the HMCL could be characterized by their highly, fairly or poorly xenografting phenotype. These phenotypes were stable and the phenotype of one HMCL was recovered within cell clones derived from it. The role of nude mice natural defences in the expression of HMCL xenografting phenotypes was studied. Sublethal whole body irradiation and silica pretreatment of recipients enabled poorly tumourigenic HMCL to grow in most animals without affecting their splenic NK activity. Admixture of BCG or MDP encapsulated in liposomes with highly tumourigenic HMCL resulted in the abrogation of tumour growth in naive nude mice. The long lasting abrogating of NK activity in vivo by treatment with anti-asialo-GM1 anti-serum did not enhance the growth of a poorly tumourigenic HMCL. The HMCL were found to be resistant to in vitro murine NK activity. These results showed that the expression of the HMCL xenografting phenotypes could be controlled by the nude mice natural defences. NK cells did not seem to be largely involved whereas macrophages might be good candidates as anti-xenograft effectors. PMID:3882112

  15. Lack of lymphangiogenesis in human primary cutaneous melanoma. Consequences for the mechanism of lymphatic dissemination.

    PubMed Central

    de Waal, R. M.; van Altena, M. C.; Erhard, H.; Weidle, U. H.; Nooijen, P. T.; Ruiter, D. J.

    1997-01-01

    Cutaneous melanoma has an initial preference for lymphatic spread. Remarkably, melanoma progression toward this metastasizing phenotype is accompanied by intense blood vessel angiogenesis (hemangiogenesis), but lymphangiogenesis, the formation of new lymph vessels in the tumor, has never been reported. To investigate how primary melanoma cells interact with the existing lymphatic microvasculature, and whether lymphangiogenesis occurs, an immunostaining was developed that differentially decorates blood and lymph vessels in frozen tissue sections. The density and distribution of both these vessel types in and around thin (< or = 1.5 mm) and thick (> or = 1.5 mm) primary melanoma lesions and in normal and uninvolved skin were determined. Although especially in thick melanoma lesions a significant increase in blood vessel density was observed, lymphatic density remained unaltered, showing that lymphangiogenesis did not occur. Morphological analysis indicated, however, that melanoma progression is accompanied by a sequence of events that involves hemangiogenesis supporting tumor expansion, especially in the vertical growth phase. Often, stromal sepia are formed around the blood capillaries in the tumor neovasculature protecting them from invasion. Lymph vessels inside the tumor were infrequently observed. However, subepidermal lymph vessels often seemed to be entrapped and penetrated by the expanding tumor mass. In this way, hemangiogenesis, as the driving force behind tumor expansion, might indirectly increase the chance of lymphatic invasion in the absence of lymphangiogenesis. This model explains the paradox that, although melanoma metastasis seems to require angiogenesis, a consistent relation of prognosis with blood capillary density in primary cutaneous melanoma is lacking. Images Figure 1 Figure 2 Figure 3 PMID:9176389

  16. Synthesis and evaluation of ¹²³/¹³¹I-Iochlonicotinamide as a novel SPECT probe for malignant melanoma.

    PubMed

    Chang, Chih-Chao; Chang, Chih-Hsien; Shen, Chih-Chieh; Chen, Chuan-Lin; Liu, Ren-Shyan; Lin, Ming-Hsien; Wang, Hsin-Ell

    2015-05-01

    Malignant melanoma expresses a highly aggressive metastasis. Early diagnosis of malignant melanoma is important for patient survival. Radiolabeled benzamides and nicotinamides have been reported to be attractive candidates for malignant melanoma diagnosis as they bind to melanin, a characteristic substance that displays in malignant melanoma, and show high tumor accumulation and retention. Herein, we designed and synthesized a novel (123/131)I-labeled nicotinamide derivative that specifically binds to melanin. (123/131)I-Iochlonicotinamide was prepared with good radiochemical yield (50-70%, decay corrected) and high specific radioactivity (50-80 GBq/μmol). (131)I-Iochlonicotinamide exhibited good in vitro stability (radiochemical purity >95% after a 24-h incubation) in human serum. High uptake of (123/131)I-Iochlonicotinamide in B16F0 melanoma cells compared to that in A375 amelanotic cells demonstrated its selective binding to melanin. Intravenous administration of (123/131)I-Iochlonicotinamide in a melanoma-bearing mouse model revealed high uptake in melanotic melanoma and high tumor-to-muscle ratio. MicroSPECT scan of (123/131)I-Iochlonicotinamide injected mice also displayed high contrast tumor imaging as compared with normal organs. The radiation-absorbed dose projection for the administration of (131)I-Iochlonicotinamide to human was based on the results of biodistribution study. The effective dose appears to be approximately 0.44 mSv/MBq(-1). The specific binding of (123/131)I-Iochlonicotinamide to melanin along with a prolonged tumor retention and acceptable projected human dosimetry suggest that it may be a promising theranostic agent for treating malignant melanoma. PMID:25800432

  17. Expression of the RNase III enzyme DROSHA is reduced during progression of human cutaneous melanoma

    PubMed Central

    Jafarnejad, Seyed Mehdi; Sjoestroem, Cecilia; Martinka, Magdalena; Li, Gang

    2016-01-01

    Aberrant expression of miRNAs and their biogenesis factors has been frequently observed in different types of cancer. We recently reported that expression of DICER1 is reduced in metastatic melanoma. Nevertheless, so far very little is known about the expression pattern of other miRNA biogenesis factors in this type of malignancy. Here, we investigated the expression pattern of DROSHA in a large set of melanocytic lesions by tissue microarray and immunohistochemistry (n = 409). We found that nuclear expression of DROSHA is markedly reduced in the early stages of melanoma progression (P = 0.0001) and is inversely correlated with melanoma thickness (P = 0.0001), AJCC stages (P = 0.0001), and ulceration status (P = 0.002). We also confirmed the reduced expression of nuclear DROSHA by a second specific antibody raised against a different region of the DROSHA protein. In addition, we observed that the reduced nuclear expression of DROSHA during melanoma progression is accompanied by an increased cytoplasmic expression of this protein (P = 0.0001). Finally, we found that expression pattern of DROSHA varies from that of DICER1 and concomitant loss of expression of both DICER1 and DROSHA confers the worse outcome for melanoma patients. Our results demonstrate a reduced nuclear expression of DROSHA which further highlights a perturbed miRNA biogenesis pathway in melanoma. In addition, the aberrant subcellular localization of DROSHA indicates possible deregulation in the mechanisms responsible for its proper localization in the nucleus. PMID:23370771

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

    PubMed

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

    1996-05-01

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

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

    PubMed Central

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

    2011-01-01

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

  20. Targeting Tumor Vasculature Endothelial Cells and Tumor Cells for Immunotherapy of Human Melanoma in a Mouse Xenograft Model

    NASA Astrophysics Data System (ADS)

    Hu, Zhiwei; Sun, Ying; Garen, Alan

    1999-07-01

    An immunotherapy treatment for cancer that targets both the tumor vasculature and tumor cells has shown promising results in a severe combined immunodeficient mouse xenograft model of human melanoma. The treatment involves systemic delivery of an immunoconjugate molecule composed of a tumor-targeting domain conjugated to the Fc effector domain of human IgG1. The effector domain induces a cytolytic immune response against the targeted cells by natural killer cells and complement. Two types of targeting domains were used. One targeting domain is a human single-chain Fv molecule that binds to a chondroitin sulfate proteoglycan expressed on the surface of most human melanoma cells. Another targeting domain is factor VII (fVII), a zymogen that binds with high specificity and affinity to the transmembrane receptor tissue factor (TF) to initiate the blood coagulation cascade. TF is expressed by endothelial cells lining the tumor vasculature but not the normal vasculature, and also by many types of tumor cells including melanoma. Because the binding of a fVII immunoconjugate to TF might cause disseminated intravascular coagulation, the active site of fVII was mutated to inhibit coagulation without affecting the affinity for TF. The immunoconjugates were encoded as secreted molecules in a replication-defective adenovirus vector, which was injected into the tail vein of severe combined immunodeficient mice. The results demonstrate that a mutated fVII immunoconjugate, administered separately or together with a single-chain Fv immunoconjugate that binds to the tumor cells, can inhibit the growth or cause regression of an established human tumor xenograft. This procedure could be effective in treating a broad spectrum of human solid tumors that express TF on vascular endothelial cells and tumor cells.

  1. Evaluation of surrogate tissues as indicators of drug activity in a melanoma skin model.

    PubMed

    Parekh, Palak R; Choudhuri, Rohini; Weyemi, Urbain; Martin, Olga A; Bonner, William M; Redon, Christophe E

    2016-08-01

    The development of novel cancer treatments is a challenging task, partly because results from model systems often fail to predict drug efficacy in humans, and also tumors are often inaccessible for biochemical analysis, preventing effective monitoring of drug activity in vivo. Utilizing a model system, we evaluated the use of drug-induced DNA damage in surrogate tissues as indicators of drug efficacy. Samples of a commercially available melanoma skin model (Mattek MLNM-FT-A375) containing keratinocyte and fibroblast layers with melanoma nodules were subjected to various chemotherapeutic regimens for one, four, or eight days. At these times they were analyzed for DNA double-stranded breaks (γH2AX foci) and apoptosis (TUNEL). A wide range of drug responses in both tumor and normal tissues were observed and cataloged. For the melanoma, the most common drug response was apoptosis. The basal keratinocyte layer, which was the most reliable indicator of drug response in the melanoma skin model, responded with γH2AX foci formation that was abrupt and transient. The relationships between tumor and surrogate tissue drug responses are complex, indicating that while surrogate tissue drug responses may be useful clinical tools, careful control of variables such as the timing of sampling may be important in interpreting the results. PMID:27339860

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

    PubMed

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

    2016-07-01

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

  3. ³¹P-MRS studies of melanoma xenografts with different metastatic potential.

    PubMed

    Li, Lin Z; Zhou, Rong; Leeper, Dennis B; Glickson, Jerry D

    2011-01-01

    Previously we reported that three imaging methods, dynamic contrast enhanced magnetic resonance imaging (DCE-MRI), T1(ρ)-MRI, and ultralow temperature NADH/flavoprotein fluorescence imaging (redox scanning), could differentiate the less metastatic human melanoma cell line A375P from a more metastatic line C8161 growing as mouse xenografts in nude mice (Li LZ et al. Adv. Exp. Med. Biol., 2007, 599:67-78; PNAS, 2009, 106:6608-6613). The more metastatic C8161 tumor was characterized by less blood perfusion/permeability, a more oxidized mitochondrial redox state in the tumor core, and a smaller T1(ρ) relaxation time constant averaged across the entire tumor section. In the current study, we have further probed the bioenergetic status and tissue microenvironment of these tumors by applying whole tumor phosphorous magnetic resonance spectroscopy ((31)P-MRS) to these two xenografts in a vertical bore 9.4-T Varian magnet. The phosphomonoester (PME)/βNTP ratio and intracellular pH value (pHi) were determined. The phosphomonoester (PME)/βNTP was higher in the more metastatic C8161 tumors (n=4) than in the less metastatic A375P tumors (n=4) (p < 0.1). No significant difference between the pHi of C8161 and A375P was observed. PMID:21445771

  4. Calcium regulation by temperature-sensitive transient receptor potential channels in human uveal melanoma cells.

    PubMed

    Mergler, Stefan; Derckx, Raissa; Reinach, Peter S; Garreis, Fabian; Böhm, Arina; Schmelzer, Lisa; Skosyrski, Sergej; Ramesh, Niraja; Abdelmessih, Suzette; Polat, Onur Kerem; Khajavi, Noushafarin; Riechardt, Aline Isabel

    2014-01-01

    Uveal melanoma (UM) is both the most common and fatal intraocular cancer among adults worldwide. As with all types of neoplasia, changes in Ca(2+) channel regulation can contribute to the onset and progression of this pathological condition. Transient receptor potential channels (TRPs) and cannabinoid receptor type 1 (CB1) are two different types of Ca(2+) permeation pathways that can be dysregulated during neoplasia. We determined in malignant human UM and healthy uvea and four different UM cell lines whether there is gene and functional expression of TRP subtypes and CB1 since they could serve as drug targets to either prevent or inhibit initiation and progression of UM. RT-PCR, Ca(2+) transients, immunohistochemistry and planar patch-clamp analysis probed for their gene expression and functional activity, respectively. In UM cells, TRPV1 and TRPM8 gene expression was identified. Capsaicin (CAP), menthol or icilin induced Ca(2+) transients as well as changes in ion current behavior characteristic of TRPV1 and TRPM8 expression. Such effects were blocked with either La(3+), capsazepine (CPZ) or BCTC. TRPA1 and CB1 are highly expressed in human uvea, but TRPA1 is not expressed in all UM cell lines. In UM cells, the CB1 agonist, WIN 55,212-2, induced Ca(2+) transients, which were suppressed by La(3+) and CPZ whereas CAP-induced Ca(2+) transients could also be suppressed by CB1 activation. Identification of functional TRPV1, TRPM8, TRPA1 and CB1 expression in these tissues may provide novel drug targets for treatment of this aggressive neoplastic disease. PMID:24084605

  5. Melanoma (image)

    MedlinePlus

    ... tumor that involves the skin cells that produce pigment (melanin). The risk of melanoma increases with age, but frequently effects young, otherwise healthy people. Melanoma is an aggressive type of cancer that can spread very rapidly.

  6. Intraoral malignant melanoma

    PubMed Central

    Babburi, Suresh; Subramanyam, R. V.; Aparna, V.; Sowjanya, P.

    2013-01-01

    Primary oral mucosal melanoma is a rare aggressive neoplasm and accounts for only 0.2-8% of all reported melanomas. It is a malignant neoplasm of melanocytes that may arise from a benign melanocytic lesion or de novo from melanocytes within normal skin or mucosa. It is considered to be the most deadly and biologically unpredictable of all human neoplasms, having the worst prognosis. In this article, we report a case of oral melanoma in a 52-year-old female patient with a chief complaint of black discolouration of the maxillary gingiva and palate. PMID:24249959

  7. Marked Differences in Human Melanoma Antigen-Specific T Cell Responsiveness after Vaccination Using a Functional Microarray

    PubMed Central

    2005-01-01

    Background In contrast to many animal model studies, immunotherapeutic trials in humans suffering from cancer invariably result in a broad range of outcomes, from long-lasting remissions to no discernable effect. Methods and Findings In order to study the T cell responses in patients undergoing a melanoma-associated peptide vaccine trial, we have developed a high-throughput method using arrays of peptide-major histocompatibility complexes (pMHC) together with antibodies against secreted factors. T cells were specifically immobilized and activated by binding to particular pMHCs. The antibodies, spotted together with the pMHC, specifically capture cytokines secreted by the T cells. This technique allows rapid, simultaneous isolation and multiparametric functional characterization of antigen-specific T cells present in clinical samples. Analysis of CD8+ lymphocytes from ten melanoma patients after peptide vaccination revealed a diverse set of patient- and antigen-specific profiles of cytokine secretion, indicating surprising differences in their responsiveness. Four out of four patients who showed moderate or greater secretion of both interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) in response to a gp100 antigen remained free of melanoma recurrence, whereas only two of six patients who showed discordant secretion of IFNγ and TNFα did so. Conclusion Such multiparametric analysis of T cell antigen specificity and function provides a valuable tool with which to dissect the molecular underpinnings of immune responsiveness and how this information correlates with clinical outcome. PMID:16162034

  8. OBATOCLAX and ABT-737 Induce ER Stress Responses in Human Melanoma Cells that Limit Induction of Apoptosis

    PubMed Central

    Wroblewski, David; Jiang, Chen Chen; Croft, Amanda; Farrelly, Margaret L.; Zhang, Xu Dong; Hersey, Peter

    2013-01-01

    Anti-apoptotic Bcl-2 family proteins, in particular, Mcl-1, are known to play a critical role in resistance of human melanoma cells to induction of apoptosis by endoplasmic reticulum stress and other agents. The present study examined whether the BH3 mimetics, Obatoclax and ABT-737, which inhibit multiple anti-apoptotic Bcl-2 family proteins, would overcome resistance to apoptosis. We report that both agents induced a strong unfolded protein response (UPR) and that RNAi knockdown of UPR signalling proteins ATF6, IRE1α and XBP-1 inhibited Mcl-1 upregulation and increased sensitivity to the agents. These results demonstrate that inhibition of anti-apoptotic Bcl-2 proteins by Obatoclax and ABT-737 appears to elicit a protective feedback response in melanoma cells, by upregulation of Mcl-1 via induction of the UPR. We also report that Obatoclax, but not ABT-737, strongly induces autophagy, which appears to play a role in determining melanoma sensitivity to the agents. PMID:24367627

  9. Dual Suppression of the Cyclin-Dependent Kinase Inhibitors CDKN2C and CDKN1A in Human Melanoma

    PubMed Central

    2012-01-01

    Resistance to BRAFV600E inhibitors is associated with reactivation of mitogen-activated protein kinase (MAPK) signaling at different levels in melanoma. To identify downstream effectors of MAPK signaling that could be used as potential additional therapeutic targets for BRAFV600E inhibitors, we used hTERT/CDK4R24C/p53DD-immortalized primary human melanocytes genetically modified to ectopically express BRAF V600E or NRAS G12D and observed induction of the AP-1 transcription factor family member c-Jun. Using a dominant negative approach, in vitro cell proliferation assays, western blots, and flow cytometry showed that MAPK signaling via BRAFV600E promotes melanoma cell proliferation at G1 through AP-1-mediated negative regulation of the INK4 family member, cyclin-dependent kinase inhibitor 2C (CDKN2C), and the CIP/KIP family member, cyclin-dependent kinase inhibitor 1A (CDKN1A). These effects were antagonized by pharmacological inhibition of CDKN2C and CDKN1A targets CDK2 and CDK4 in vitro. In contrast to BRAF V600E or NRAS G12D-expressing melanocytes, melanoma cells have an inherent resistance to suppression of AP-1 activity by BRAFV600E- or MEK-inhibitors. Here, CDK2/4 inhibition statistically significantly augmented the effects of BRAFV600E- or MEK-inhibitors on melanoma cell viability in vitro and growth in athymic nude Foxn1 nu mice (P = .03 when mean tumor volume at day 13 was compared for BRAFV600E inhibitor vs BRAFV600E inhibitor plus CDK2/4 inhibition; P = .02 when mean tumor volume was compared for MEK inhibitor vs MEK inhibitor plus CDK2/4 inhibition; P values were calculated by a two-sided Welch t test; n = 4–8 mice per group). PMID:22997239

  10. Tyrosine transport in a human melanoma cell line as a basis for selective transport of cytotoxic analogues.

    PubMed Central

    Pankovich, J M; Jimbow, K

    1991-01-01

    Tyrosine is an essential amino acid for the initial step of melanin synthesis, yet little is known concerning its transport in melanocytes. As an important first step in the development of new anti-melanoma agents based upon chemical and pharmacological modifications of melanin synthesis, the present study characterized the transport mechanism of tyrosine in vitro using the human melanoma cell line SK-MEL 23. Several tyrosine transport systems may be involved in melanocytes: systems L and T, which transport neutral amino acids with branched or aromatic side chains, and systems A and ASC, which transport neutral amino acids with smaller side chains. In order to determine which system or combination of systems is involved in tyrosine transport in melanoma cells, studies of kinetics, Na(+)-dependence and competitive inhibition were undertaken. The Km and Vmax. for the Na(+)-independent transport system were found to be 0.164 +/- 0.016 mM and 21.6 +/- 1.1 nmol/min per mg of protein respectively. This transport was preferentially inhibited by the system L specific analogue, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, the system T substrate tryptophan, and the sulphur homologue of tyrosine, 4-S-cysteinylphenol. Sequential addition of these inhibitors at increasing concentrations indicated that they inhibit the same transporter. Our results suggest that tyrosine transport in SK-MEL 23 melanoma cells is similar to system L transport previously characterized in other cell types. This one transport system appears to supply all the tyrosine required for both cell growth and melanin synthesis. The transport system may be subject to manipulation by melanogenic stimulating factors, making the transport of cytotoxic tyrosine analogues an important area for further study. PMID:1764036

  11. Hypoxia-induced metastasis of human melanoma cells: involvement of vascular endothelial growth factor-mediated angiogenesis

    PubMed Central

    Rofstad, E K; Danielsen, T

    1999-01-01

    Tumour cells exposed to hypoxia have been shown to up-regulate the expression of vascular endothelial growth factor (VEGF). The purpose of the present work was to investigate whether hypoxia-induced VEGF up-regulation can result in increased metastatic efficiency of human melanoma cells. Two melanoma lines, one showing high (A-07) and the other showing low (D-12) VEGF secretion under aerobic conditions, were included in the study. Cell cultures were exposed to hypoxia (oxygen concentrations < 10 ppm) in vitro and metastatic efficiency, i.e. lung colonization efficiency, as well as transplantability and angiogenic potential were assessed in BALB/c-nu/nu mice. Both cell lines showed significantly increased VEGF secretion under hypoxic conditions as measured by enzyme-linked immunosorbent assay. The D-12 cells showed increased metastatic efficiency, transplantability and angiogenic potential following exposure to hypoxia. The metastatic efficiency increased with the duration of the hypoxia treatment and decreased with the time after reoxygenation. The A-07 cells on the other hand showed unchanged metastatic efficiency, transplantability and angiogenic potential following exposure to hypoxia. Both cell lines showed significantly decreased metastatic efficiency and angiogenic potential in mice treated with neutralizing antibody against VEGF. These results suggest that (a) VEGF is a limiting factor for the rate of angiogenesis in low but not in high VEGF-expressing melanomas under normoxic conditions and (b) transient hypoxia might promote the development of metastases in low VEGF-expressing melanomas by upregulating the expression of VEGF and hence enhancing the angiogenic potential of the tumour cells. © 1999 Cancer Research Campaign PMID:10468285

  12. Lebein, a Snake Venom Disintegrin, Induces Apoptosis in Human Melanoma Cells

    PubMed Central

    Hammouda, Manel B.; Montenegro, María F.; Sánchez-del-Campo, Luis; Zakraoui, Ons; Aloui, Zohra; Riahi-Chebbi, Ichrak; Karoui, Habib; Rodríguez-López, José Neptuno; Essafi-Benkhadir, Khadija

    2016-01-01

    Melanoma, the most threatening form of skin cancer, has a very poor prognosis and is characterized by its very invasive and chemoresistant properties. Despite the recent promising news from the field of immunotherapy, there is an urgent need for new therapeutic approaches that are free of resistance mechanisms and side effects. Anti-neoplasic properties have been highlighted for different disintegrins from snake venom including Lebein; however, the exact effect of Lebein on melanoma has not yet been defined. In this study, we showed that Lebein blocks melanoma cell proliferation and induces a more differentiated phenotype with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and microphthalmia-associated transcription factor (MITF) overexpression. Melanoma cells became detached but were less invasive with upregulation of E-cadherin after Lebein exposure. Lebein induced a caspase-independent apoptotic program with apoptosis inducing factor (AIF), BCL-2-associated X protein (BAX) and Bim overexpression together with downregulation of B-cell lymphoma-2 (BCL-2). It generated a distinct response in reactive oxygen species (ROS) generation and p53 levels depending on the p53 cell line status (wild type or mutant). Therefore, we propose Lebein as a new candidate for development of potential therapies for melanoma. PMID:27399772

  13. Lebein, a Snake Venom Disintegrin, Induces Apoptosis in Human Melanoma Cells.

    PubMed

    Hammouda, Manel B; Montenegro, María F; Sánchez-Del-Campo, Luis; Zakraoui, Ons; Aloui, Zohra; Riahi-Chebbi, Ichrak; Karoui, Habib; Rodríguez-López, José Neptuno; Essafi-Benkhadir, Khadija

    2016-01-01

    Melanoma, the most threatening form of skin cancer, has a very poor prognosis and is characterized by its very invasive and chemoresistant properties. Despite the recent promising news from the field of immunotherapy, there is an urgent need for new therapeutic approaches that are free of resistance mechanisms and side effects. Anti-neoplasic properties have been highlighted for different disintegrins from snake venom including Lebein; however, the exact effect of Lebein on melanoma has not yet been defined. In this study, we showed that Lebein blocks melanoma cell proliferation and induces a more differentiated phenotype with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and microphthalmia-associated transcription factor (MITF) overexpression. Melanoma cells became detached but were less invasive with upregulation of E-cadherin after Lebein exposure. Lebein induced a caspase-independent apoptotic program with apoptosis inducing factor (AIF), BCL-2-associated X protein (BAX) and Bim overexpression together with downregulation of B-cell lymphoma-2 (BCL-2). It generated a distinct response in reactive oxygen species (ROS) generation and p53 levels depending on the p53 cell line status (wild type or mutant). Therefore, we propose Lebein as a new candidate for development of potential therapies for melanoma. PMID:27399772

  14. Spectrophotometric Method for Differentiation of Human Skin Melanoma. II. Diagnostic Characteristics

    NASA Astrophysics Data System (ADS)

    Petruk, V. G.; Ivanov, A. P.; Kvaternyuk, S. M.; Barunb, V. V.

    2016-05-01

    Experimental data on the spectral dependences of the optical diffuse reflection coefficient for skin from different people with melanoma or nevus are presented in the form of the probability density of the diffuse reflection coefficient for the corresponding pigmented lesions. We propose a noninvasive technique for differentiating between malignant and benign tumors, based on measuring the diffuse reflection coefficient for a specific patient and comparing the value obtained with a pre-set threshold. If the experimental result is below the threshold, then it is concluded that the person has melanoma; otherwise, no melanoma is present. As an example, we consider the wavelength 870 nm. We determine the risk of malignant transformation of a nevus (its transition to melanoma) for different measured diffuse reflection coefficients. We have studied the errors in the method, its operating characteristics and probability characteristics as the threshold diffuse reflection coefficient is varied. We find that the diagnostic confidence, sensitivity, specificity, and effectiveness (accuracy) parameters are maximum (>0.82) for a threshold of 0.45-0.47. The operating characteristics for the proposed technique exceed the corresponding parameters for other familiar optical approaches to melanoma diagnosis. Its distinguishing feature is operation at only one wavelength, and consequently implementation of the experimental technique is simplified and made less expensive.

  15. Eradication of Human Hepatic and Pulmonary Melanoma Metastases in SCID Mice by Antibody--Interleukin 2 Fusion Proteins

    NASA Astrophysics Data System (ADS)

    Becker, Jurgen C.; Pancook, James D.; Gillies, Stephen D.; Mendelsohn, John; Reisfeld, Ralph A.

    1996-04-01

    Antibody--cytokine fusion proteins combine the unique targeting ability of antibodies with the multifunctional activity of cytokines. Here, we demonstrate the therapeutic efficacy of such constructs for the treatment of hepatic and pulmonary metastases of different melanoma cell lines. Two antibody--interleukin 2 (IL-2) fusion proteins, ch225-IL2 and ch14.18-IL2, constructed by fusion of a synthetic sequence coding for human IL-2 to the carboxyl end of the Cγ 1 gene of the corresponding antibodies, were tested for their therapeutic efficacy against xenografted human melanoma in vivo. Tumorspecific fusion proteins completely inhibited the growth of hepatic and pulmonary metastases in C.B-17 scid/scid mice previously reconstituted with human lymphokine-activated killer cells, whereas treatment with combinations of the corresponding antibodies plus recombinant IL-2 only reduced the tumor load. Even when treatment with fusion proteins was delayed up to 8 days after inoculation of tumor cells, it still resulted in complete eradication of micrometastases that were established at that time point. Selection of tumor cell lines expressing or lacking the targeted antigen of the administered fusion protein proved the specificity of the observed antitumor effect. Biodistribution analysis demonstrated that the tumorspecific fusion protein accumulated not only in subcutaneous tumors but also in lungs and livers affected with micrometastases. Survival times of animals treated with the fusion protein were more than doubled as compared to those treated with the combination of the corresponding antibody plus IL-2. Our data demonstrate that an immunotherapeutic approach using cytokines targeted by antibodies to tumor sites has potent effects against disseminated human melanoma.

  16. Genetics of melanoma predisposition.

    PubMed

    Lin, J; Hocker, T L; Singh, M; Tsao, H

    2008-08-01

    Over the past 10 years, our understanding of melanoma at the molecular level has blossomed with the advent of genomic technologies. The enormous enthusiasm for the Human Genome Project is slowly being replaced by an even greater excitement for the unravelling of disease genomes, including melanoma. In this review, we will consider some of the clinical implications of these genetic findings for both diagnostics and therapeutics. PMID:18547303

  17. Pericentric characterization of human chromosome 7 in a melanoma cell line

    SciTech Connect

    Fetni, R.; Lemieux, N.; Richer, C.L.

    1994-09-01

    Cytogenetic analyses of an established melanoma cell line show structural abnormalities involving mainly chromosome 7. Molecular cytogenetic examination of the different abnormalities (i(7q), i(7p), t(7;12)) was used to pinpoint the site of the break and to analyse the possible mechanisms by which isochromosomes 7 could be formed. Human chromosome 7 has been shown to contain two distinct alpha satellite arrays: D7Z1 and D7Z2 which are separated by 1Mb. We confirm the order to be short-arm- D7Z2 - D7Z1 - long arm. Both probes were then used to characterize two different types of isochromosomes 7 (one of the long arms and one of the short arms) and a translocation (7;12). Isochromosome 7q showed a single D7Z1 signal and loss of D7Z2. The unique centromeric structure of i(7q), with only the D7Z1 signal, suggests that a breakpoint occurred within D7Z1. Isochromosome 7p showed two distinct D7Z1 and D7Z2 hybridization signals. The distance observed between the two signals suggests that the breakpoint is in the proximal part of the long arms. This chromosome might be considered as a dicentric isochromosome 7p. Translocation (7;12) showed the two arrays of chromosome 7 {alpha} satellite DNA. The three derived chromosomes appeared to result from independent rearrangements. This observation shows that a variety of breaks may occur in the juxtacentromeric region of a given chromosome. It also shows that the functional centromere of chromosome 7 does not need the presence of D7Z2, since only D7Z1 was conserved in all cases, suggesting the importance of this sequence for the centromeric function.

  18. RIPK1 regulates survival of human melanoma cells upon endoplasmic reticulum stress through autophagy.

    PubMed

    Luan, Qi; Jin, Lei; Jiang, Chen Chen; Tay, Kwang Hong; Lai, Fritz; Liu, Xiao Ying; Liu, Yi Lun; Guo, Su Tang; Li, Chun Ying; Yan, Xu Guang; Tseng, Hsin-Yi; Zhang, Xu Dong

    2015-01-01

    Although RIPK1 (receptor [TNFRSF]-interacting protein kinase 1) is emerging as a critical determinant of cell fate in response to cellular stress resulting from activation of death receptors and DNA damage, its potential role in cell response to endoplasmic reticulum (ER) stress remains undefined. Here we report that RIPK1 functions as an important prosurvival mechanism in melanoma cells undergoing pharmacological ER stress induced by tunicamycin (TM) or thapsigargin (TG) through activation of autophagy. While treatment with TM or TG upregulated RIPK1 and triggered autophagy in melanoma cells, knockdown of RIPK1 inhibited autophagy and rendered the cells sensitive to killing by TM or TG, recapitulating the effect of inhibition of autophagy. Consistently, overexpression of RIPK1 enhanced induction of autophagy and conferred resistance of melanoma cells to TM- or TG-induced cell death. Activation of MAPK8/JNK1 or MAPK9/JNK2, which phosphorylated BCL2L11/BIM leading to its dissociation from BECN1/Beclin 1, was involved in TM- or TG-induced, RIPK1-mediated activation of autophagy; whereas, activation of the transcription factor HSF1 (heat shock factor protein 1) downstream of the ERN1/IRE1-XBP1 axis of the unfolded protein response was responsible for the increase in RIPK1 in melanoma cells undergoing pharmacological ER stress. Collectively, these results identify upregulation of RIPK1 as an important resistance mechanism of melanoma cells to TM- or TG-induced ER stress by protecting against cell death through activation of autophagy, and suggest that targeting the autophagy-activating mechanism of RIPK1 may be a useful strategy to enhance sensitivity of melanoma cells to therapeutic agents that induce ER stress. PMID:26018731

  19. Human recombinant RNASET2: A potential anti-cancer drug

    PubMed Central

    Roiz, Levava; Smirnoff, Patricia; Lewin, Iris; Shoseyov, Oded; Schwartz, Betty

    2016-01-01

    The roles of cell motility and angiogenetic processes in metastatic spread and tumor aggressiveness are well established and must be simultaneously targeted to maximize antitumor drug potency. This work evaluated the antitumorigenic capacities of human recombinant RNASET2 (hrRNASET2), a homologue of the Aspergillus niger T2RNase ACTIBIND, which has been shown to display both antitumorigenic and antiangiogenic activities. hrRNASET2 disrupted intracellular actin filament and actin-rich extracellular extrusion organization in both CT29 colon cancer and A375SM melanoma cells and induced a significant dose-dependent inhibition of A375SM cell migration. hrRNASET2 also induced full arrest of angiogenin-induced tube formation and brought to a three-fold lower relative HT29 colorectal and A375SM melanoma tumor volume, when compared to Avastin-treated animals. In parallel, mean blood vessel counts were 36.9% lower in hrRNASET2-vs. Avastin-treated mice and survival rates of hrRNASET2-treated mice were 50% at 73 days post-treatment, while the median survival time for untreated animals was 22 days. Moreover, a 60-day hrRNASET2 treatment period reduced mean A375SM lung metastasis foci counts by three-fold when compared to untreated animals. Taken together, the combined antiangiogenic and antitumorigenic capacities of hrRNASET2, seemingly arising from its direct interaction with intercellular and extracellular matrices, render it an attractive anticancer therapy candidate. PMID:27014725

  20. Human recombinant RNASET2: A potential anti-cancer drug.

    PubMed

    Roiz, Levava; Smirnoff, Patricia; Lewin, Iris; Shoseyov, Oded; Schwartz, Betty

    2016-01-01

    The roles of cell motility and angiogenetic processes in metastatic spread and tumor aggressiveness are well established and must be simultaneously targeted to maximize antitumor drug potency. This work evaluated the antitumorigenic capacities of human recombinant RNASET2 (hrRNASET2), a homologue of the Aspergillus niger T2RNase ACTIBIND, which has been shown to display both antitumorigenic and antiangiogenic activities. hrRNASET2 disrupted intracellular actin filament and actin-rich extracellular extrusion organization in both CT29 colon cancer and A375SM melanoma cells and induced a significant dose-dependent inhibition of A375SM cell migration. hrRNASET2 also induced full arrest of angiogenin-induced tube formation and brought to a three-fold lower relative HT29 colorectal and A375SM melanoma tumor volume, when compared to Avastin-treated animals. In parallel, mean blood vessel counts were 36.9% lower in hrRNASET2-vs. Avastin-treated mice and survival rates of hrRNASET2-treated mice were 50% at 73 days post-treatment, while the median survival time for untreated animals was 22 days. Moreover, a 60-day hrRNASET2 treatment period reduced mean A375SM lung metastasis foci counts by three-fold when compared to untreated animals. Taken together, the combined antiangiogenic and antitumorigenic capacities of hrRNASET2, seemingly arising from its direct interaction with intercellular and extracellular matrices, render it an attractive anticancer therapy candidate. PMID:27014725

  1. Construction of Ang2-siRNA chitosan magnetic nanoparticles and the effect on Ang2 gene expression in human malignant melanoma cells

    PubMed Central

    LIU, ZHAO-LIANG; YOU, CAI-LIAN; WANG, BIAO; LIN, JIAN-HONG; HU, XUE-FENG; SHAN, XIU-YING; WANG, MEI-SHUI; ZHENG, HOU-BING; ZHANG, YAN-DING

    2016-01-01

    The aim of the present study was to construct angiopoietin-2 (Ang2)-small interfering (si)RNA chitosan magnetic nanoparticles and to observe the interference effects of the nanoparticles on the expression of the Ang2 gene in human malignant melanoma cells. Ang2-siRNA chitosan magnetic nanoparticles were constructed and transfected into human malignant melanoma cells in vitro. Red fluorescent protein expression was observed, and the transfection efficiency was analyzed. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to assess the inhibition efficiency of Ang2 gene expression. Ang2-siRNA chitosan magnetic nanoparticles were successfully constructed, and at a mass ratio of plasmid to magnetic chitosan nanoparticles of 1:100, the transfection efficiency into human malignant melanoma cells was the highest of the ratios assessed, reaching 61.17%. RT-qPCR analysis showed that the magnetic chitosan nanoparticles effectively inhibited Ang2 gene expression in cells, and the inhibition efficiency reached 59.56% (P<0.05). Ang2-siRNA chitosan magnetic nanoparticles were successfully constructed. The in vitro studies showed that the nanoparticles inhibited Ang2 gene expression in human malignant melanoma tumor cells, which laid the foundation and provided experimental evidence for additional future in vivo studies of intervention targeting malignant melanoma tumor growth in nude mice. PMID:27313729

  2. Fisetin, a dietary flavonoid, augments the anti-invasive and anti-metastatic potential of sorafenib in melanoma

    PubMed Central

    Pal, Harish C.; Diamond, Ariana C.; Strickland, Leah R.; Kappes, John C.; Katiyar, Santosh K.; Elmets, Craig A.; Athar, Mohammad; Afaq, Farrukh

    2016-01-01

    Melanoma is the most aggressive and deadly form of cutaneous neoplasm due to its propensity to metastasize. Oncogenic BRAF drives sustained activation of the BRAF/MEK/ERK (MAPK) pathway and cooperates with PI3K/AKT/mTOR (PI3K) signaling to induce epithelial to mesenchymal transition (EMT), leading to cell invasion and metastasis. Therefore, targeting these pathways is a promising preventive/therapeutic strategy. We have shown that fisetin, a flavonoid, reduces human melanoma cell invasion by inhibiting EMT. In addition, fisetin inhibited melanoma cell proliferation and tumor growth by downregulating the PI3K pathway. In this investigation, we aimed to determine whether fisetin can potentiate the anti-invasive and anti-metastatic effects of sorafenib in BRAF-mutated melanoma. We found that combination treatment (fisetin + sorafenib) more effectively reduced the migration and invasion of BRAF-mutated melanoma cells both in vitro and in raft cultures compared to individual agents. Combination treatment also effectively inhibited EMT as observed by a decrease in N-cadherin, vimentin and fibronectin and an increase in E-cadherin both in vitro and in xenograft tumors. Furthermore, combination therapy effectively inhibited Snail1, Twist1, Slug and ZEB1 protein expression compared to monotherapy. The expression of MMP-2 and MMP-9 in xenograft tumors was further reduced in combination treatment compared to individual agents. Bioluminescent imaging of athymic mice, intravenously injected with stably transfected CMV-luciferase-ires-puromycin. T2A.EGFP-tagged A375 melanoma cells, demonstrated fewer lung metastases following combination treatment versus monotherapy. Our findings demonstrate that fisetin potentiates the anti-invasive and anti-metastatic effects of sorafenib. Our data suggest that fisetin may be a worthy adjuvant chemotherapy for the management of melanoma. PMID:26517521

  3. CD10-Equipped Melanoma Cells Acquire Highly Potent Tumorigenic Activity: A Plausible Explanation of Their Significance for a Poor Prognosis

    PubMed Central

    Hashimoto-Hachiya, Akiko; Liu, Min; Abe, Takeru; Hagihara, Akihito; Yokomizo, Takehiko; Furue, Masutaka

    2016-01-01

    CD10 has been widely used in cancer diagnosis. We previously demonstrated that its expression in melanoma increased with tumor progression and predicted poor patient survival. However, the mechanism by which CD10 promotes melanoma progression remains unclear. In order to elucidate the role of CD10 in melanoma, we established CD10-overexpressing A375 melanoma cells and performed DNA microarray and qRT–PCR analyses to identify changes in the gene expression profile. The microarray analysis revealed that up-regulated genes in CD10-A375 were mostly involved in cell proliferation, angiogenesis, and resistance to apoptosis; down-regulated genes mostly belonged to the categories associated with cell adhesion and migration. Accordingly, in functional experiments, CD10-A375 showed significantly greater cell proliferation in vitro and higher tumorigenicity in vivo; CD10 enzymatic inhibitors, thiorphan and phosphoramidon, significantly blocked the tumor growth of CD10-A375 in mice. In migration and invasion assays, CD10-A375 displayed lower migratory and invasive capacity than mock-A375. CD10 augmented melanoma cell resistance to apoptosis mediated by etoposide and gemcitabine. These findings indicate that CD10 may promote tumor progression by regulating the expression profiles of genes related to cell proliferation, angiogenesis, and resistance to apoptosis. PMID:26881775

  4. Effect of combined misonidazole and d(50)-Be neutrons on a human melanoma transplanted into nude mice

    SciTech Connect

    Guichard, M.; Gueulette, J.; Octave-Prignot, M.; Wambersie, A.; Malaise, E.P.

    1980-08-01

    This paper reports the effect of two different doses (0.1 and 1 mg/g) of misonidazole on the radiosensitivity of human Na 11 melanoma transplanted into athymic nude mice. The mice were irradiated with 50-MeV neutrons. The end point was the in vitro colony-forming assay. Repair of potentially lethal damage following neutron irradiation was comparable to that observed after ..gamma.. irradiation. This repair was no longer detectable when 1 mg/g of misonidazole was used. No toxic effect of the drug itself could be detected.

  5. Melanoma and genetics.

    PubMed

    Nelson, Andrew A; Tsao, Hensin

    2009-01-01

    As the incidence of malignant melanoma continues to increase and with the completion of the sequencing of the human genome, there have been increasing efforts to identify the "melanoma gene(s)." Although some patients and families have significantly increased risks due to genetic predisposition, most melanoma cases are sporadic and likely result from low to moderate risk genetic factors. This review focuses on the genes that cover the greatest risk of developing melanoma. It is important to remember that many--if not most--cases of melanoma are the result of undiscovered variants. The strongest genetic risk for the development of melanoma results from heritable alterations in cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, which encodes two separate but related proteins, p16/INK4a and p14/ARF. These proteins help regulate cell division and apoptosis, both of which are necessary to maintain cellular homeostasis. Other important genes include CDK4/6 and retinoblastoma (RB1), which encode downstream proteins in the same pathway as p16/INK4a and p14/ARF. Finally, we discuss the relative importance of the melanocortin 1 receptor (MC1R) gene as a moderate risk factor for melanoma. Although great advances have been made in understanding the molecular basis and genetic predisposition of melanoma, many questions still remain to be answered. Someday soon, it will be possible to predict a patient's risk of melanoma by DNA analysis; however, it is important to reconcile our tremendous technologic capabilities with documented clinical utility. PMID:19095153

  6. CD44/chondroitin sulfate proteoglycan and alpha 2 beta 1 integrin mediate human melanoma cell migration on type IV collagen and invasion of basement membranes.

    PubMed Central

    Knutson, J R; Iida, J; Fields, G B; McCarthy, J B

    1996-01-01

    Tumor cell invasion of basement membranes (BM) represents one of the critical steps in the metastatic process. Tumor cell recognition of individual BM matrix components may involve individual cell adhesion receptors, such as integrins or cell surface proteoglycans, or may involve a coordinate action of both types of receptors. In this study, we have focused on the identification of a cell surface CD44/chondroitin sulfate proteoglycan (CSPG) and alpha 2 beta 1 integrin on human melanoma cells that are both directly involved in the in vitro invasion of reconstituted BM via a type IV collagen-dependent mechanism. Interfering with cell surface expression of human melanoma CSPG with either p-nitro-phenyl-beta-D-xylopyranoside treatment or anti-CD44 monoclonal antibody (mAb) preincubation (mAb) preincubation inhibits melanoma cell invasion through reconstituted BM. These treatments also strongly inhibit melanoma cell migration on type IV collagen, however, they are ineffective at inhibiting cell adhesion to type IV collagen. Purified melanoma cell surface CD44/CSPG, or purified chondroitin sulfate, bind to type IV collagen affinity columns, consistent with a role for CD44/CSPG-type IV collagen interactions in mediating tumor cell invasion. In contrast, melanoma cell migration on laminin (LM) does not involve CD44/CSPG, nor does CD44/CSPG bind to LM, suggesting that CD44/CSPG-type IV collagen interactions are specific in nature. Additionally, anti-alpha 2 and anti-beta 1 integrin mAbs are capable of blocking melanoma cell invasion of reconstituted BM. Both of these anti-integrin mAbs inhibit melanoma cell adhesion and migration on type IV collagen, whereas only anti-beta 1 mAb inhibits cell adhesion to LM. Collectively, these results indicate that melanoma cell adhesion to type IV collagen is an important consideration in invasion of reconstituted BM in vitro, and suggest that CD44/CSPG and alpha 2 beta 1 integrin may collaborate to promote human melanoma cell adhesion

  7. The human antibody fragment DIATHIS1 specific for CEACAM1 enhances natural killer cell cytotoxicity against melanoma cell lines in vitro.

    PubMed

    Dupuis, Maria L; Fiori, Valentina; Soriani, Alessandra; Ricci, Biancamaria; Dominici, Sabrina; Moricoli, Diego; Ascione, Alessandro; Santoni, Angela; Magnani, Mauro; Cianfriglia, Maurizio

    2015-01-01

    Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1 malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1 melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1 melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro-expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell-mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin. PMID:26448580

  8. The Human Antibody Fragment DIATHIS1 Specific for CEACAM1 Enhances Natural Killer Cell Cytotoxicity Against Melanoma Cell Lines In Vitro

    PubMed Central

    Dupuis, Maria L.; Soriani, Alessandra; Ricci, Biancamaria; Dominici, Sabrina; Moricoli, Diego; Ascione, Alessandro; Santoni, Angela; Magnani, Mauro; Cianfriglia, Maurizio

    2015-01-01

    Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1+ malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1+ melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1+ melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro–expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell–mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin. PMID

  9. Glycogen Synthase Kinase-3 promotes cell survival, growth and PAX3 levels in human melanoma cells

    PubMed Central

    Kubic, Jennifer D.; Mascarenhas, Joseph B.; Iizuka, Takumi; Wolfgeher, Don; Lang, Deborah

    2012-01-01

    Glycogen Synthase Kinase-3 (GSK-3) is a serine/threonine kinase involved in a diverse range of cellular processes. GSK-3 exists in two isoforms, GSK-3α and GSK-3β, which possess some functional redundancy but also play distinct roles depending on developmental and cellular context. In this report we found that GSK-3 actively promoted cell growth and survival in melanoma cells, and blocking this activity with small molecule inhibitor SB216763 or gene-specific siRNA decreased proliferation, increased apoptosis and altered cellular morphology. These alterations coincided with loss of PAX3, a transcription factor implicated in proliferation, survival and migration of developing melanoblasts. We further found that PAX3 directly interacted with and was phosphorylated in vitro on a number of residues by GSK-3β. In melanoma cells, direct inhibition of PAX3 lead to cellular changes that paralleled the response to GSK-3 inhibition. Maintenance of PAX3 expression protected melanoma cells from the anti-tumor effects of SB216763. These data support a model wherein GSK-3 regulates proliferation and morphology of melanoma through phosphorylation and increased levels of PAX3. PMID:22679108

  10. Targeted delivery of CYP2E1 recombinant adenovirus to malignant melanoma by bone marrow-derived mesenchymal stem cells as vehicles.

    PubMed

    Wang, Jishi; Ma, Dan; Li, Yan; Yang, Yuan; Hu, Xiaoyan; Zhang, Wei; Fang, Qin

    2014-03-01

    The aim of this study was to explore the effects of bone marrow-derived mesenchymal stem cells (BMSCs) as intermediate carriers on targeting of P450 gene recombinant adenovirus to malignant melanoma in vitro and in vivo. BMSCs were transduced with pAd5-CMV-CYP2E1 recombinant adenovirus. BMSC migration was detected by Transwell plates in vitro and by superparamagnetic iron oxide particles in vivo. Growth-inhibitory effect and apoptosis were determined by MTT and immunity fluorescence staining. Anticancer effects were examined by a human melanoma nude mouse model in vivo. BMSCs moved toward A375 cells in Transwell plates. Numerous superparamagnetic MSCs labeled with iron oxide were identified in the peripheral areas of the tumor, but were detected in primary organs by Prussian blue staining. BMSC-CYP2E1 cells mediated a bystander killing effect on CYP2E1-negative A375 cells during coculture (IC50 values for A375 cells cocultured with BMSC-EGFP and BMSC-CYP2E1 were 4.08 and 2.68 mmol/l, respectively). Intravenously injecting CYP2E1 recombinant adenovirus-loaded BMSCs in mice with established human melanoma managed to target the tumor site, and BMSCs with forced expression of CYP2E1 inhibited the growth of malignant cells in vivo by activating 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide. BMSCs may serve as a platform of P450 gene-directed enzyme prodrug therapy for the delivery of chemotherapeutic prodrugs to tumors. PMID:24413391

  11. Molecular changes induced by the curcumin analogue D6 in human melanoma cells

    PubMed Central

    2013-01-01

    Background In a previous report, we described the in vitro and in vivo antiproliferative and proapoptotic activity of a hydroxylated biphenyl (D6), a structural analogue of curcumin, on malignant melanoma and neuroblastoma tumours. In this paper, we investigated the molecular changes induced by such a compound, underlying cell growth arrest and apoptosis in melanoma cells. Results To shed light on the mechanisms of action of D6, we firstly demonstrated its quick cellular uptake and subsequent block of cell cycle in G2/M phase transition. A gene expression profile analysis of D6-treated melanoma cells and fibroblasts was then carried out on high density microarrays, to assess gene expression changes induced by this compound. The expression profile study evidenced both an induction of stress response pathways and a modulation of cell growth regulation mechanisms. In particular, our data suggest that the antiproliferative and proapoptotic activities of D6 in melanoma could be partially driven by up-regulation of the p53 signalling pathways as well as by down-regulation of the PI3K/Akt and NF-kB pathways. Modulation of gene expression due to D6 treatment was verified by western blot analysis for single proteins of interest, confirming the results from the gene expression profile analysis. Conclusions Our findings contribute to the understanding of the mechanisms of action of D6, through a comprehensive description of the molecular changes induced by this compound at the gene expression level, in agreement with the previously reported anti-tumour effects on melanoma cells. PMID:23642048

  12. Therapeutic Efficacy of a {sup 188}Re-Labeled {alpha}-Melanocyte-Stimulating Hormone Peptide Analog in Murine and Human Melanoma-Bearing Mouse Models

    SciTech Connect

    Miao, Yubin; Owen, Nellie K.; Fisher, Darrell R.; Hoffman, Timothy J.; Quinn, Thomas P.

    2005-01-01

    The purpose of this study was to examine the therapeutic efficacy of {sup 188}Re-(Arg{sup 11})CCMSH in the B16/F1 murine melanoma and TXM13 human melanoma bearing mouse models. Method: (Arg11)CCMSH was synthesized and labeled with {sup 188}Re to form {sup 188}Re-(Agr{sup 11})CCMSH. B16/F1 melanoma tumor bearing mice were administrated with 200 Ci, 600 Ci and 2x400 Ci of {sup 188}Re-(Arg{sup 11})CCMSH via the tail vein, respectively. TXM13 melanoma tumor hearing mice were separately injected with 600 Ci, 2x400 Ci and 1000 Ci of 100Re-(Arg{sup 11})CCMSH through the tail vein. Two groups of 10 mice bearing either B16/F1 or TXM13 tumors were injected with saline as untreated controls. Results: In contrast to the untreated control group, {sup 188}Re(Arg11)CCMSH yielded rapid and lasting therapeutic effects in the treatment groups with either B16/F1 or TXM13 tumors. The tumor growth rate was reduced and the survival rate was prolonged in the treatment groups. Treatment with 2x400 Ci of {sup 188}Re-Arg{sup 11}CCMSH significantly extended the mean life of B16/F1 tumor mice (p<0.05), while the mean life of TXm13 tumor mice was significantly prolonged after treatment with 600 Ci and 1000 Ci doses of {sup 188}Re-(Arg{sup 11})CCMSH (p<0.05 High-dose {sup 188}Re-(Arg{sup 11}))CCMSH produced no observed normal-tissue toxicity. Conclusions: The therapy study results revealed that {sup 188}Re-Arg11 CCMSH yielded significant therapeutic effects in both B16/F1 murine melanoma and TXM13 human melanoma bearing mouse models. {sup 188}Re-(Arg{sup 11})CCMSH appears to be a promising radiolabeled peptide for targeted radionuclide therapy of melanoma.

  13. PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines.

    PubMed

    Katona, Éva; Juhász, Tamás; Somogyi, Csilla Szűcs; Hajdú, Tibor; Szász, Csaba; Rácz, Kálmán; Kókai, Endre; Gergely, Pál; Zákány, Róza

    2016-03-01

    Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment. PMID:26717964

  14. PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines

    PubMed Central

    KATONA, ÉVA; JUHÁSZ, TAMÁS; SOMOGYI, CSILLA SZŰCS; HAJDÚ, TIBOR; SZÁSZ, CSABA; RÁCZ, KÁLMÁN; KÓKAI, ENDRE; GERGELY, PÁL; ZÁKÁNY, RÓZA

    2016-01-01

    Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment. PMID:26717964

  15. Interferon-alpha and bortezomib overcome Bcl-2 and Mcl-1 over-expression in melanoma cells by stimulating the extrinsic pathway of apoptosis

    PubMed Central

    Lesinski, Gregory B.; Raig, Ene T.; Guenterberg, Kristan; Brown, Lloyd; Go, Michael R.; Shah, Nisha N.; Lewis, Adrian; Quimper, Megan; Hade, Erinn; Young, Gregory; Chaudhury, Abhik Ray; Ladner, Katherine J.; Guttridge, Denis C.; Bouchard, Page

    2008-01-01

    We hypothesized that interferon-alpha (IFN-α) would enhance the apoptotic activity of bortezomib on melanoma cells. Combined treatment with bortezomib and IFN-α induced synergistic apoptosis in melanoma and other solid tumor cell lines. Apoptosis was associated with processing of procaspases-3, -7, -8, -9, and with cleavage of Bid and PARP. Bortezomib plus IFN-α was effective at inducing apoptosis in melanoma cells that over-expressed Bcl-2 or Mcl-1, suggesting that this treatment combination can overcome mitochondrial pathways of cell survival and resistance to apoptosis. The pro-apoptotic effects of this treatment combination were abrogated by a caspase-8 inhibitor, led to increased association of Fas and FADD prior to the onset of cell death, and were significantly reduced in cells transfected with a dominant-negative FADD construct or siRNA targeting Fas. These data suggest that bortezomib and IFN-α act through the extrinsic pathway of apoptosis via FADD-induced caspase-8 activation to initiate cell death. Finally, bortezomib and IFN-α displayed statistically significant anti-tumor activity as compared to either agent alone in both the B16 murine model of melanoma and in athymic mice bearing human A375 xenografts. These data support the future clinical development of bortezomib and IFN-α for malignant melanoma. PMID:18922907

  16. Monoclonal antibody imaging of human melanoma. Radioimmunodetection by subcutaneous or systemic injection

    SciTech Connect

    Lotze, M.T.; Carrasquillo, J.A.; Weinstein, J.N.; Bryant, G.J.; Perentesis, P.; Reynolds, J.C.; Matis, L.A.; Eger, R.R.; Keenan, A.M.; Hellstroem, Ie.

    1986-09-01

    Fab fragments of monoclonal antibodies (MoAb) to melanoma, radiolabeled with /sup 131/I, were evaluated as diagnostic reagents to determine their ability to localize systemic--MoAb injected intravenously (IV)--or nodal metastatic disease--injected subcutaneously (SQ) at a site proximal to draining lymph nodes. Sixty-one scans were performed (40 IV, 21 SQ) in 59 patients who had injections of 0.2-50 mg of /sup 131/I coupled (0.2-12 mCi) antibody. These included 48.7, which identifies a high molecular weight antigen (HMW), or 96.5, which identifies a transferrin like molecule, p97. 125I coupled nonspecific Fab 1.4, reacting with murine leukemia virus, or the whole antibody BL3, reactive with a human B cell idiotypic determinant, was generally used in tandem with the patients injected SQ as a nonspecific control. All patients had immunohistochemical studies performed and demonstrated binding to the antibodies injected. Of the IV patients, 22/38 (58%) had (+) scans, 13 at SQ or nodal sites, four at visceral sites, and five at visceral and SQ sites. Patients with clinical stage II disease had SQ injection of MoAb, including 11 additional patients injected with the whole antibody 9.2.27 (anti-HMW) labeled with 111In (6 patients) or /sup 131/I (5 patients). Nodal dissection was performed 2-4 days later. All 111In coupled antibodies demonstrated excellent nodal delineation without specific identification of tumor deposits. Of the 21 patients injected SQ with MoAb, 17 had confirmed tumor in nodes. Of patients injected with Fab fragments, 50% had specific uptake of MoAb, although only two were successfully imaged. Increased uptake of antimelanoma antibodies was observed in some patients in lymph nodes not containing tumor. Clearance of labeled antibody from the injection site occurred with a half life of 16-50 hours. Toxicity was limited to local discomfort at the site of SQ injection.

  17. In vitro anticancer activity of Betulinic acid and derivatives thereof on equine melanoma cell lines from grey horses and in vivo safety assessment of the compound NVX-207 in two horses.

    PubMed

    Liebscher, G; Vanchangiri, K; Mueller, Th; Feige, K; Cavalleri, J-M V; Paschke, R

    2016-02-25

    Betulinic acid, a pentacyclic triterpene, and its derivatives are promising compounds for cancer treatment in humans. Melanoma is not only a problem for humans but also for grey horses as they have a high potential of developing melanoma lesions coupled to the mutation causing their phenotype. Current chemotherapeutic treatment carries the risk of adverse health effects for the horse owner or the treating veterinarian by exposure to antineoplastic compounds. Most treatments have low prospects for systemic tumor regression. Thus, a new therapy is needed. In this in vitro study, Betulinic acid and its two derivatives B10 and NVX-207, both with an improved water solubility compared to Betulinic acid, were tested on two equine melanoma cell lines (MelDuWi and MellJess/HoMelZh) and human melanoma (A375) cell line. We could demonstrate that all three compounds especially NVX-207 show high cytotoxicity on both equine melanoma cell lines. The treatment with these compounds lead to externalization of phosphatidylserines on the cell membrane (AnnexinV-staining), DNA-fragmentation (cell cycle analysis) and activation of initiator and effector caspases (Caspase assays). Our results indicate that the apoptosis is induced in the equine melanoma cells by all three compounds. Furthermore, we succeed in encapsulating the most active compound NVX-207 in 2-Hydroxyprolyl-β-cyclodextrine without a loss of its activity. This formulation can be used as a promising antitumor agent for treating grey horse melanoma. In a first tolerability evaluation in vivo the formulation was administered every one week for 19 consecutive weeks and well tolerated in two adult melanoma affected horses. PMID:26772157

  18. Primary structure of the human melanoma-associated antigen p97 (melanotransferrin) deduced from the mRNA sequence.

    PubMed Central

    Rose, T M; Plowman, G D; Teplow, D B; Dreyer, W J; Hellström, K E; Brown, J P

    1986-01-01

    p97 is a cell-surface glycoprotein that is present in most human melanomas but only in trace amounts in normal adult tissues. To determine the structure of this tumor-associated antigen and to identify its functional domains, we have purified and cloned p97 mRNA and determined its nucleotide sequence. The mRNA encodes a 738-residue precursor, which contains the previously determined N-terminal amino acid sequence of p97. After removal of a 19-residue signal peptide, the mature p97 molecule comprises extracellular domains of 342 and 352 residues and a C-terminal 25-residue stretch of predominantly uncharged and hydrophobic amino acids, which we believe acts as a membrane anchor. Each extracellular domain contains 14 cysteine residues, which form seven intradomain disulfide bridges, and one or two potential N-glycosylation sites. Protease digestion studies show that the three major antigenic determinants of p97 are present on the N-terminal domain. The domains are strikingly homologous to each other (46% amino acid sequence homology) and to the corresponding domains of human serum transferrin (39% homology). Conservation of disulfide bridges and of amino acids thought to compose the iron binding pockets suggests that p97 is also related to transferrin in tertiary structure and function. We propose that p97 be renamed melanotransferrin to denote its original identification in melanoma cells and its evolutionary relationship to serotransferrin and lactotransferrin, the other members of the transferrin superfamily. Images PMID:2419904

  19. The ginsenoside 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol induces autophagy and apoptosis in human melanoma via AMPK/JNK phosphorylation.

    PubMed

    Kang, Soouk; Kim, Jong-Eun; Song, Nu Ry; Jung, Sung Keun; Lee, Mee Hyun; Park, Jun Seong; Yeom, Myeong-Hun; Bode, Ann M; Dong, Zigang; Lee, Ki Won

    2014-01-01

    Studies have shown that a major metabolite of the red ginseng ginsenoside Rb1, called 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (GPD), exhibits anticancer properties. However, the chemotherapeutic effects and molecular mechanisms behind GPD action in human melanoma have not been previously investigated. Here we report the anticancer activity of GPD and its mechanism of action in melanoma cells. GPD, but not its parent compound Rb1, inhibited melanoma cell proliferation in a dose-dependent manner. Further investigation revealed that GPD treatment achieved this inhibition through the induction of autophagy and apoptosis, while Rb1 failed to show significant effect at the same concentrations. The inhibitory effect of GPD appears to be mediated through the induction of AMPK and the subsequent attenuation of mTOR phosphorylation. In addition, GPD activated c-Jun by inducing JNK phosphorylation. Our findings suggest that GPD suppresses melanoma growth by inducing autophagic cell death and apoptosis via AMPK/JNK pathway activation. GPD therefore has the potential to be developed as a chemotherapeutic agent for the treatment of human melanoma. PMID:25137374

  20. 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.

  1. Synthesis and characterization of a novel radioiodinated phenylacetamide and its homolog as theranostic agents for malignant melanoma.

    PubMed

    Chang, Chih-Chao; Chang, Chih-Hsien; Shen, Chih-Chieh; Chen, Chuan-Lin; Liu, Ren-Shyan; Lin, Ming-Hsien; Wang, Hsin-Ell

    2016-01-01

    Melanin is an attractive target for the diagnosis and treatment of malignant melanoma. This study reports the preparation and biological characterizations of N-(2-(diethylamino)ethyl)-2-(3-(123/131)I-iodo-4- hydroxyphenyl)acetamide and N-(2-(diethylamino)ethyl)-3-(3-(123/131)I-iodo-4-hydroxyphenyl)propanamide (123/131)I-IHPA and 123/131I-IHPP) as novel melanin-specific theranostic agents. These two tracers were hydrophilic, exhibited good serum stability and high binding affinity to melanin. In vitro and in vivo studies revealed rapid, high and tenacious uptakes of both 131I-IHPA and 131I-IHPP in melanotic B16F0 cell line and in C57BL/6 mice bearing B16F0 melanoma, but not in amelanonic A375 cell line and tumors. Small-animal SPECT imaging also clearly delineate B16F0 melanoma since 1 h postinjection of 123I-IHPA and 123I-IHPP in tumor-bearing mice. Owing to the favorable biodistribution of 131I-IHPA and 131I-IHPP after intravenous administration, the estimated absorption dose was low in most normal organs and relatively high in melanotic tumor. The melanin-specific binding ability, sustained tumor retention, fast normal tissues clearance and acceptable projected human dosimetry supported that these two tracers are promising theranostic agents for melanin-positive melanoma. PMID:26517961

  2. 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.

  3. Genetics of melanoma

    PubMed Central

    Wangari-Talbot, Janet; Chen, Suzie

    2013-01-01

    Genomic variation is a trend observed in various human diseases including cancer. Genetic studies have set out to understand how and why these variations result in cancer, why some populations are pre-disposed to the disease, and also how genetics affect drug responses. The melanoma incidence has been increasing at an alarming rate worldwide. The burden posed by melanoma has made it a necessity to understand the fundamental signaling pathways involved in this deadly disease. Signaling cascades such as mitogen-activated protein kinase and PI3K/AKT have been shown to be crucial in the regulation of processes that are commonly dysregulated during cancer development such as aberrant proliferation, loss of cell cycle control, impaired apoptosis, and altered drug metabolism. Understanding how these and other oncogenic pathways are regulated has been integral in our challenge to develop potent anti-melanoma drugs. With advances in technology and especially in next generation sequencing, we have been able to explore melanoma genomes and exomes leading to the identification of previously unknown genes with functions in melanomagenesis such as GRIN2A and PREX2. The therapeutic potential of these novel candidate genes is actively being pursued with some presenting as druggable targets while others serve as indicators of therapeutic responses. In addition, the analysis of the mutational signatures of melanoma tumors continues to cement the causative role of UV exposure in melanoma pathogenesis. It has become distinctly clear that melanomas from sun-exposed skin areas have distinct mutational signatures including C to T transitions indicative of UV-induced damage. It is thus necessary to continue spreading awareness on how to decrease the risk factors of developing the disease while at the same time working for a cure. Given the large amount of information gained from these sequencing studies, it is likely that in the future, treatment of melanoma will follow a highly

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

    PubMed

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

    2012-01-10

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

  5. Protein Kinase PKN1 Represses Wnt/β-Catenin Signaling in Human Melanoma Cells*

    PubMed Central

    James, Richard G.; Bosch, Katherine A.; Kulikauskas, Rima M.; Yang, Peitzu T.; Robin, Nick C.; Toroni, Rachel A.; Biechele, Travis L.; Berndt, Jason D.; von Haller, Priska D.; Eng, Jimmy K.; Wolf-Yadlin, Alejandro; Chien, Andy J.; Moon, Randall T.

    2013-01-01

    Advances in phosphoproteomics have made it possible to monitor changes in protein phosphorylation that occur at different steps in signal transduction and have aided the identification of new pathway components. In the present study, we applied this technology to advance our understanding of the responses of melanoma cells to signaling initiated by the secreted ligand WNT3A. We started by comparing the phosphopeptide patterns of cells treated with WNT3A for different periods of time. Next, we integrated these data sets with the results from a siRNA screen that targeted protein kinases. This integration of siRNA screening and proteomics enabled us to identify four kinases that exhibit altered phosphorylation in response to WNT3A and that regulate a luciferase reporter of β-catenin-responsive transcription (β-catenin-activated reporter). We focused on one of these kinases, an atypical PKC kinase, protein kinase N1 (PKN1). Reducing the levels of PKN1 with siRNAs significantly enhances activation of β-catenin-activated reporter and increases apoptosis in melanoma cell lines. Using affinity purification followed by mass spectrometry, we then found that PKN1 is present in a protein complex with a WNT3A receptor, Frizzled 7, as well as with proteins that co-purify with Frizzled 7. These data establish that the protein kinase PKN1 inhibits Wnt/β-catenin signaling and sensitizes melanoma cells to cell death stimulated by WNT3A. PMID:24114839

  6. Epigenetics of human cutaneous melanoma: setting the stage for new therapeutic strategies

    PubMed Central

    2010-01-01

    Cutaneous melanoma is a very aggressive neoplasia of melanocytic origin with constantly growing incidence and mortality rates world-wide. Epigenetic modifications (i.e., alterations of genomic DNA methylation patterns, of post-translational modifications of histones, and of microRNA profiles) have been recently identified as playing an important role in melanoma development and progression by affecting key cellular pathways such as cell cycle regulation, cell signalling, differentiation, DNA repair, apoptosis, invasion and immune recognition. In this scenario, pharmacologic inhibition of DNA methyltransferases and/or of histone deacetylases were demonstrated to efficiently restore the expression of aberrantly-silenced genes, thus re-establishing pathway functions. In light of the pleiotropic activities of epigenetic drugs, their use alone or in combination therapies is being strongly suggested, and a particular clinical benefit might be expected from their synergistic activities with chemo-, radio-, and immuno-therapeutic approaches in melanoma patients. On this path, an important improvement would possibly derive from the development of new generation epigenetic drugs characterized by much reduced systemic toxicities, higher bioavailability, and more specific epigenetic effects. PMID:20540720

  7. Melanoma cell surface-expressed phosphatidylserine as a therapeutic target for cationic anticancer peptide, temporin-1CEa.

    PubMed

    Wang, Che; Chen, Yin-Wang; Zhang, Liang; Gong, Xian-Ge; Zhou, Yang; Shang, De-Jing

    2016-07-01

    We have previously reported that temporin-1CEa, a cationic antimicrobial peptide, exerts preferential cytotoxicity toward cancer cells. However, the exact molecular mechanism for this cancer-selectivity is still largely unknown. Here, we found that the negatively charged phosphatidylserine (PS) expressed on cancer cell surface serves as a target for temporin-1CEa. Our results indicate that human A375 melanoma cells express 50-fold more PS than non-cancerous HaCaT cells. The expression of cell surface PS in various cancer cell lines closely correlated with their ability to be recognized, bound and killed by temporin-1CEa. Additionally, the cytotoxicity of temporin-1CEa against A375 cells can be ameliorated by annexin V, which binds to cell surface PS with high affinity. Moreover, the data of isothermal titration calorimetry assay further confirmed a direct binding of temporin-1CEa to PS, at a ratio of 1:5 (temporin-1CEa:PS). Interestingly, the circular dichroism spectra analysis using artificial biomembrane revealed that PS not only provides electrostatic attractive sites for temporin-1CEa but also confers the membrane-bound temporin-1CEa to form α-helical structure, therefore, enhances the affinity and membrane disrupting ability of temporin-1CEa. In summary, these findings suggested that the melanoma cells expressed PS may serve as a promising target for temporin-1CEa or other cationic anticancer peptides. PMID:26596643

  8. Melanoma Cancer Stem Cells: Markers and Functions

    PubMed Central

    Parmiani, Giorgio

    2016-01-01

    The discovery of cancer stem cells (CSCs) in human solid tumors has allowed a better understanding of the biology and neoplastic transformation of normal melanocytes, and the possible mechanisms by which melanoma cells acquire tumorigenicity. In this review I summarize the literature findings on the potential biomarkers of melanoma CSCs, their presence in the melanoma cell populations, the interaction with the immune system (with both T and NK cells) and the role of melanoma CSCs in the clinics. Given the extraordinary progress in the therapy of melanoma caused by immune checkpoint antibodies blockade, I discuss how these antibodies can work by the activation of melanoma infiltrating T cells specifically recognizing neo-antigens expressed even by melanoma CSCs. This is the mechanism that can induce a regression of the metastatic melanomas. PMID:26978405

  9. Interactions between ultraviolet light and interleukin-1 on MSH binding in both mouse melanoma and human squamous carcinoma cells.

    PubMed

    Birchall, N; Orlow, S J; Kupper, T; Pawelek, J

    1991-03-29

    Interactions between beta-melanotropin (MSH), interleukin 1-a (IL-1), and ultraviolet light (UV) were examined in Cloudman S91 mouse melanoma and RHEK human squamous carcinoma cell lines. The following points were established: 1) both cell lines produced IL-1 and their production was stimulated by exposure of the cells to UV; 2) both cell lines possessed high affinity binding sites for MSH, and their ability to bind MSH was modulated by IL-1; 3) IL-1 exhibited both stimulatory and inhibitory effects on MSH binding to Cloudman cells; and 4) the stimulatory effect of IL-1 on MSH binding to melanoma cells was reflected in enhanced cellular responsiveness to MSH regarding tyrosinase activity (E.C. 1.14.18.1) and melanin content. The findings raise the possibility that interactions between keratinocytes and melanocytes may be regulated by IL-1 and MSH, and suggest a possible mechanism for stimulation of cutaneous melanogenesis by solar radiation: enhancement of MSH receptor activity by induction of IL-1. PMID:2025257

  10. Interactions between ultraviolet light and interleukin-1 on MSH binding in both mouse melanoma and human squamous carcinoma cells

    SciTech Connect

    Birchall, N.; Orlow, S.J.; Kupper, T.; Pawelek, J. )

    1991-03-29

    Interactions between beta-melanotropin (MSH), interleukin 1-a (IL-1), and ultraviolet light (UV) were examined in Cloudman S91 mouse melanoma and RHEK human squamous carcinoma cell lines. The following points were established: (1) both cell lines produced IL-1 and their production was stimulated by exposure of the cells to UV; (2) both cell lines possessed high affinity binding sites for MSH, and their ability to bind MSH was modulated by IL-1; (3) IL-1 exhibited both stimulatory and inhibitory effects on MSH binding to Cloudman cells; and (4) the stimulatory effect of IL-1 on MSH binding to melanoma cells was reflected in enhanced cellular responsiveness to MSH regarding tyrosinase activity (E.C. 1.14.18.1) and melanin content. The findings raise the possibility that interactions between keratinocytes and melanocytes may be regulated by IL-1 and MSH, and suggest a possible mechanism for stimulation of cutaneous melanogenesis by solar radiation: enhancement of MSH receptor activity by induction of IL-1.

  11. Dual mechanisms of action of the RNA-binding protein human antigen R explains its regulatory effect on melanoma cell migration.

    PubMed

    Moradi, Farnaz; Berglund, Pontus; Linnskog, Rickard; Leandersson, Karin; Andersson, Tommy; Prasad, Chandra Prakash

    2016-06-01

    Overexpression of wingless-type MMTV integration site family 5A (WNT5A) plays a significant role in melanoma cancer progression; however, the mechanism(s) involved remains unknown. In breast cancer, the human antigen R (HuR) has been implicated in the regulation of WNT5A expression. Here, we demonstrate that endogenous expression of WNT5A correlates with levels of active HuR in HTB63 and WM852 melanoma cells and that HuR binds to WNT5A messenger RNA in both cell lines. Although the HuR inhibitor MS-444 significantly impaired migration in both melanoma cell lines, it reduced WNT5A expression only in HTB63 cells, as did small interfering RNA knockdown of HuR. Consistent with this finding, MS-444-induced inhibition of HTB63 cell migration was restored by the addition of recombinant WNT5A, whereas MS-444-induced inhibition of WM852 cell migration was restored by the addition of recombinant matrix metalloproteinase-9, another HuR-regulated protein. Clearly, HuR positively regulates melanoma cell migration via at least 2 distinct mechanisms making HuR an attractive therapeutic target for halting melanoma dissemination. PMID:26970271

  12. Genetic Engineering of T cells to Target HERV-K, an Ancient Retrovirus on Melanoma

    PubMed Central

    Krishnamurthy, Janani; Rabinovich, Brian A.; Mi, Tiejuan; Switzer, Kirsten C.; Olivares, Simon; Maiti, Sourindra N.; Plummer, Joshua B.; Singh, Harjeet; Kumaresan, Pappanaicken R.; Huls, Helen M.; Wang-Johanning, Feng; Cooper, Laurence J.N.

    2015-01-01

    Purpose The human endogenous retrovirus (HERV-K) envelope (env) protein is a tumor- associated antigen expressed on melanoma, but not normal cells. This study was designed to engineer a chimeric antigen receptor (CAR) on T cell surface, such that they target tumors in advanced stages of melanoma. Experimental Design Expression of HERV-K protein was analyzed in 220 melanoma samples (with various stages of disease) and 139 normal organ donor tissues using immuno-histochemical (IHC) analysis. HERV-K env-specific CAR derived from mouse monoclonal antibody was introduced into T cells using the transposon-based Sleeping Beauty (SB) system. HERV-K env-specific CAR+ T cells were expanded ex vivo on activating and propagating cells (AaPC), and characterized for CAR expression and specificity. This includes evaluating the HERV-K-specific CAR+ T cells for their ability to kill A375-SM metastasized tumors in a mouse xenograft model. Results We detected HERV-K env protein on melanoma, but not in normal tissues. After electroporation of T cells and selection on HERV-K+ AaPC, over 95% of genetically-modified T cells expressed the CAR with an effector memory phenotype and lysed HERV-K env+ tumor targets in an antigen specific manner. Even though there is apparent shedding of this TAA from tumor cells which can be recognized by HERV-K env-specific CAR+ T cells, we observed a significant anti-tumor effect. Conclusion Adoptive cellular immunotherapy with HERV-K env-specific CAR+ T cells represents a clinically-appealing treatment strategy for advanced-stage melanoma and provides an approach for targeting this TAA on other solid tumors. PMID:25829402

  13. Gossypin as a novel selective dual inhibitor of V-RAF murine sarcoma viral oncogene homolog B1 and cyclin-dependent kinase 4 for melanoma.

    PubMed

    Bhaskaran, Shylesh; Dileep, Kalarikkal V; Deepa, Sathyaseelan S; Sadasivan, Chittalakkottu; Klausner, Mitch; Krishnegowda, Naveen K; Tekmal, Rajeshwar R; VandeBerg, John L; Nair, Hareesh B

    2013-04-01

    Mutation in the BRAF gene (BRAFV600E) exists in nearly 70% of human melanomas. Targeted therapy against BRAFV600E kinase using a recently identified RAF-selective inhibitor, PLX4032, has been successful in early clinical trials. However, in patients with the normal BRAF allele (wild-type), PLX4032 is protumorigenic. This conundrum identifies the unmet need for novel therapeutic agents to target BRAFV600E kinase that are not counterproductive. We have identified gossypin, a pentahydroxy flavone, as a potent antimelanoma agent. Gossypin inhibited human melanoma cell proliferation, in vitro, in melanoma cell lines that harbor both BRAFV600E kinase and cyclin-dependent kinase 4 (CDK4) as well as in cells with BRAF wild-type allele. Gossypin inhibited kinase activities of BRAFV600E and CDK4, in vitro, possibly through direct binding of gossypin with these kinases, as confirmed by molecular docking studies. For cells harboring the BRAFV600E, gossypin inhibited cell proliferation through abrogation of the MEK-ERK-cyclin D1 pathway and in cells with BRAF wild-type allele, through attenuation of the retinoblastoma-cyclin D1 pathway. Furthermore, gossypin significantly inhibited melanoma growth in an organotypic three-dimensional skin culture mimicking human skin. Gossypin (10 and 100 mg/kg) treatment for 10 days in human melanoma (A375) cell xenograft tumors harboring BRAFV600E significantly reduced tumor volume through induction of apoptosis and increased survival rate in mice, and the effect was significantly superior to that of PLX4032 (10 mg/kg) or roscovitine 10 mg/kg. In summary, this study identified gossypin as a novel agent with dual inhibitory effects for BRAFV600E kinase and CDK4 for treatment of melanoma. PMID:23543365

  14. Cell Cycle Gene Networks Are Associated with Melanoma Prognosis

    PubMed Central

    Watkins, Wendy; Araki, Hiromitsu; Tamada, Yoshinori; Muthukaruppan, Anita; Ranjard, Louis; Derkac, Eliane; Imoto, Seiya; Miyano, Satoru; Crampin, Edmund J.; Print, Cristin G.

    2012-01-01

    Background Our understanding of the molecular pathways that underlie melanoma remains incomplete. Although several published microarray studies of clinical melanomas have provided valuable information, we found only limited concordance between these studies. Therefore, we took an in vitro functional genomics approach to understand melanoma molecular pathways. Methodology/Principal Findings Affymetrix microarray data were generated from A375 melanoma cells treated in vitro with siRNAs against 45 transcription factors and signaling molecules. Analysis of this data using unsupervised hierarchical clustering and Bayesian gene networks identified proliferation-association RNA clusters, which were co-ordinately expressed across the A375 cells and also across melanomas from patients. The abundance in metastatic melanomas of these cellular proliferation clusters and their putative upstream regulators was significantly associated with patient prognosis. An 8-gene classifier derived from gene network hub genes correctly classified the prognosis of 23/26 metastatic melanoma patients in a cross-validation study. Unlike the RNA clusters associated with cellular proliferation described above, co-ordinately expressed RNA clusters associated with immune response were clearly identified across melanoma tumours from patients but not across the siRNA-treated A375 cells, in which immune responses are not active. Three uncharacterised genes, which the gene networks predicted to be upstream of apoptosis- or cellular proliferation-associated RNAs, were found to significantly alter apoptosis and cell number when over-expressed in vitro. Conclusions/Significance This analysis identified co-expression of RNAs that encode functionally-related proteins, in particular, proliferation-associated RNA clusters that are linked to melanoma patient prognosis. Our analysis suggests that A375 cells in vitro may be valid models in which to study the gene expression modules that underlie some melanoma

  15. Isolation of tumorigenic circulating melanoma cells

    PubMed Central

    Ma, Jie; Lin, Jennifer Y.; Alloo, Allireza; Wilson, Brian J.; Schatton, Tobias; Zhan, Qian; Murphy, George F.; Waaga-Gasser, Ana-Maria; Gasser, Martin; Hodi, F. Stephen; Frank, Natasha Y.; Frank, Markus H.

    2010-01-01

    Circulating tumor cells (CTC) have been identified in several human malignancies, including malignant melanoma. However, whether melanoma CTC are tumorigenic and cause metastatic progression is currently unknown. Here we isolate for the first time viable tumorigenic melanoma CTC and demonstrate that this cell population is capable of metastasis formation in human-to-mouse xenotransplantation experiments. The presence of CTC among peripheral blood mononuclear cells (PBMC) of murine recipients of subcutaneous (s.c.) human melanoma xenografts could be detected based on mRNA expression for human GAPDH and/or ATP-binding cassette subfamily B member 5 (ABCB5), a marker of malignant melanoma-initiating cells previously shown to be associated with metastatic disease progression in human patients. ABCB5 expression could also be detected in PBMC preparations from human stage IV melanoma patients but not healthy controls. The detection of melanoma CTC in human-to-mouse s.c. tumor xenotransplantation models correlated significantly with pulmonary metastasis formation. Moreover, prospectively isolated CTC from murine recipients of s.c. melanoma xenografts were capable of primary tumor initiation and caused metastasis formation upon xenotransplantation to secondary murine NOD-scid IL2Rγnull recipients. Our results provide initial evidence that melanoma CTC are tumorigenic and demonstrate that CTC are capable of causing metastatic tumor progression. These findings suggest a need for CTC eradication to inhibit metastatic progression and provide a rationale for assessment of therapeutic responses of this tumorigenic cell population to promising emerging melanoma treatment modalities. PMID:20977885

  16. Targeting Syndecan-1, a molecule implicated in the process of vasculogenic mimicry, enhances the therapeutic efficacy of the L19-IL2 immunocytokine in human melanoma xenografts.

    PubMed

    Orecchia, Paola; Conte, Romana; Balza, Enrica; Pietra, Gabriella; Mingari, Maria Cristina; Carnemolla, Barbara

    2015-11-10

    Anti-angiogenic therapy of solid tumors has until now failed to produce the long lasting clinical benefits desired, possibly due to the complexity of the neoangiogenic process. Indeed, a prominent role is played by "vasculogenic" or "vascular" mimicry (VM), a phenomenon in which aggressive cancer cells form an alternative microvascular circulation, independently of endothelial cell angiogenesis. In this study we observed, in melanoma patient cell lines having vasculogenic/stem-cell like phenotype and in melanoma tumors, the syndecan-1 co-expression with VM markers, such as CD144 and VEGFR-2. We show that melanoma cells lose their ability to form tubule-like structures in vitro after blocking syndecan-1 activity by the specific human recombinant antibody, OC-46F2. Moreover, in a human melanoma xenograft model, the combined therapy using OC-46F2 and L19-IL2, an immunocytokine specific for the tumor angiogenic-associated B-fibronectin isoform(B-FN), led to a complete inhibition of tumor growth until day 90 from tumor implantation in 71% of treated mice, with statistically significant differences compared to groups treated with OC-46F2 or L19-IL2 as monotherapy. Furthermore, in the tumors recovered from mice treated with OC-46F2 either as monotherapy or in combination with L19-IL2, we observed a dramatic decrease of vascular density and loss of VM structures. These findings indicate for the first time a role of syndecan-1 in melanoma VM and that targeting syndecan-1, together with B-FN, could be promising in improving the treatment of metastatic melanoma. PMID:26460958

  17. Targeting Syndecan-1, a molecule implicated in the process of vasculogenic mimicry, enhances the therapeutic efficacy of the L19-IL2 immunocytokine in human melanoma xenografts

    PubMed Central

    Orecchia, Paola; Conte, Romana; Balza, Enrica; Pietra, Gabriella; Mingari, Maria Cristina; Carnemolla, Barbara

    2015-01-01

    Anti-angiogenic therapy of solid tumors has until now failed to produce the long lasting clinical benefits desired, possibly due to the complexity of the neoangiogenic process. Indeed, a prominent role is played by “vasculogenic” or “vascular” mimicry (VM), a phenomenon in which aggressive cancer cells form an alternative microvascular circulation, independently of endothelial cell angiogenesis. In this study we observed, in melanoma patient cell lines having vasculogenic/stem-cell like phenotype and in melanoma tumors, the syndecan-1 co-expression with VM markers, such as CD144 and VEGFR-2. We show that melanoma cells lose their ability to form tubule-like structures in vitro after blocking syndecan-1 activity by the specific human recombinant antibody, OC-46F2. Moreover, in a human melanoma xenograft model, the combined therapy using OC-46F2 and L19-IL2, an immunocytokine specific for the tumor angiogenic-associated B-fibronectin isoform(B-FN), led to a complete inhibition of tumor growth until day 90 from tumor implantation in 71% of treated mice, with statistically significant differences compared to groups treated with OC-46F2 or L19-IL2 as monotherapy. Furthermore, in the tumors recovered from mice treated with OC-46F2 either as monotherapy or in combination with L19-IL2, we observed a dramatic decrease of vascular density and loss of VM structures. These findings indicate for the first time a role of syndecan-1 in melanoma VM and that targeting syndecan-1, together with B-FN, could be promising in improving the treatment of metastatic melanoma. PMID:26460958

  18. Differential Growth Suppression of Human Melanoma Cells by Tea (Camellia sinensis) Epicatechins (ECG, EGC and EGCG)

    PubMed Central

    Ramasamy, Vaishali; Moon, Songeun; Ruiz, Carlos; Muthugounder, Sakunthala

    2009-01-01

    We previously reported that catechins of green tea have different antiproliferative effects on cell lines derived from gender-dependent cancers; epicatechin 3-gallate (ECG) had the strongest inhibitory effect. In the present study, we examined the effects of epigallocatechin (EGC), epicatechin-gallate (ECG) and EGC 3-gallate (EGCG) on the viability, density, doubling time and cycle number of cell lines derived from melanoma metastasized to lymph nodes (MB-1133 and SE-0154) or distant organs (CH-0356, JK-0346, SA-1171, GE-0208, NS-1176 and LF-0023). These catechins have been documented to have no growth suppressive or apoptotic effects on normal melanocytes (Nihal et al., Int J Cancer 2005;114:513–21). EGCG (50 μM) showed greater inhibitory potency than EGC (50 μM) in SE-0154, NS-1176, GE-0208 and LF-0023 cell lines but the two catechins produced similar inhibitory effects in CH-0356, JK-0346 and SA-1171 cell lines. The IC50 (50% inhibitory concentration) was lower for EGC than EGCG in MB-1133 and CH-0356 cells, higher for EGC than EGCG in GE-0208 cells and comparable (11–12 μM) for both the catechins in LF-0023 cells. When compared with EGC, the cytotoxic effect (% dead cell counts) and the suppression of the growth (change in cell number) of all melanoma cell lines tested were pronounced with EGCG. This investigation validates the hypothesis that anticancer action of the various catechins may vary with the type of malignancy and provides a model for tumor cell heterogeneity based on susceptibility and resistance of tumor cells to different green tea catechins. Therefore, this information is critical for undertaking chemopreventive or chemotherapeutic trials against melanoma and gender-based cancers. PMID:18955299

  19. Fisetin, a phytochemical, potentiates sorafenib-induced apoptosis and abrogates tumor growth in athymic nude mice implanted with BRAF-mutated melanoma cells

    PubMed Central

    Pal, Harish Chandra; Baxter, Ronald D.; Hunt, Katherine M.; Agarwal, Jyoti; Elmets, Craig A.; Athar, Mohammad; Afaq, Farrukh

    2015-01-01

    Melanoma is the most deadly form of cutaneous malignancy, and its incidence rates are rising worldwide. In melanoma, constitutive activation of the BRAF/MEK/ERK (MAPK) and PI3K/AKT/mTOR (PI3K) signaling pathways plays a pivotal role in cell proliferation, survival and tumorigenesis. A combination of compounds that lead to an optimal blockade of these critical signaling pathways may provide an effective strategy for prevention and treatment of melanoma. The phytochemical fisetin is known to possess anti-proliferative and pro-apoptotic activities. We found that fisetin treatment inhibited PI3K signaling pathway in melanoma cells. Therefore, we investigated the effect of fisetin and sorafenib (an RAF inhibitor) alone and in combination on cell proliferation, apoptosis and tumor growth. Combination treatment (fisetin + sorafenib) more effectively reduced the growth of BRAF-mutated human melanoma cells at lower doses when compared to individual agents. In addition, combination treatment resulted in enhanced (i) apoptosis, (ii) cleavage of caspase-3 and PARP, (iii) expression of Bax and Bak, (iv) inhibition of Bcl2 and Mcl-1, and (v) inhibition of expression of PI3K, phosphorylation of MEK1/2, ERK1/2, AKT and mTOR. In athymic nude mice subcutaneously implanted with melanoma cells (A375 and SK-MEL-28), we found that combination therapy resulted in greater reduction of tumor growth when compared to individual agents. Furthermore, combination therapy was more effective than monotherapy in: (i) inhibition of proliferation and angiogenesis, (ii) induction of apoptosis, and (iii) inhibition of the MAPK and PI3K pathways in xenograft tumors. These data suggest that simultaneous inhibition of both these signaling pathways using combination of fisetin and sorafenib may serve as a therapeutic option for the management of melanoma. PMID:26299806

  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

    2016-01-01

    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

  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. Cell cycle-dependent expression of Ki-67 antigen in human melanoma cells subjected to irradiation and/or hyperthermia

    SciTech Connect

    Zoelzer, F.; Streffer, C.

    1995-07-01

    The proliferation of human melanoma cells in vitro during the first 3 days after irradiation and/or hyperthermia was followed by two-parameter flow cytometry combining cell cycle analysis on the basis of DNA content with Ki-67 antibody labeling. It was found that cells arrested or delayed in the S and G{sub 2} phases of the cell cycle were Ki-67-positive in spite of the antigen`s very short half-life. Thus Ki-67 staining failed to reflect those changes in cell proliferation which typically occur in the course of a fractionated radiotherapy as well as those expected in the case of hyperthermia or a combined treatment. 24 refs., 3 figs., 1 tab.

  3. Uveal melanoma: Estimating prognosis

    PubMed Central

    Kaliki, Swathi; Shields, Carol L; Shields, Jerry A

    2015-01-01

    Uveal melanoma is the most common primary malignant tumor of the eye in adults, predominantly found in Caucasians. Local tumor control of uveal melanoma is excellent, yet this malignancy is associated with relatively high mortality secondary to metastasis. Various clinical, histopathological, cytogenetic features and gene expression features help in estimating the prognosis of uveal melanoma. The clinical features associated with poor prognosis in patients with uveal melanoma include older age at presentation, male gender, larger tumor basal diameter and thickness, ciliary body location, diffuse tumor configuration, association with ocular/oculodermal melanocytosis, extraocular tumor extension, and advanced tumor staging by American Joint Committee on Cancer classification. Histopathological features suggestive of poor prognosis include epithelioid cell type, high mitotic activity, higher values of mean diameter of ten largest nucleoli, higher microvascular density, extravascular matrix patterns, tumor-infiltrating lymphocytes, tumor-infiltrating macrophages, higher expression of insulin-like growth factor-1 receptor, and higher expression of human leukocyte antigen Class I and II. Monosomy 3, 1p loss, 6q loss, and 8q and those classified as Class II by gene expression are predictive of poor prognosis of uveal melanoma. In this review, we discuss the prognostic factors of uveal melanoma. A database search was performed on PubMed, using the terms “uvea,” “iris,” “ciliary body,” “choroid,” “melanoma,” “uveal melanoma” and “prognosis,” “metastasis,” “genetic testing,” “gene expression profiling.” Relevant English language articles were extracted, reviewed, and referenced appropriately. PMID:25827538

  4. 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

  5. Recurrent inactivating RASA2 mutations in melanoma.

    PubMed

    Arafeh, Rand; Qutob, Nouar; Emmanuel, Rafi; Keren-Paz, Alona; Madore, Jason; Elkahloun, Abdel; Wilmott, James S; Gartner, Jared J; Di Pizio, Antonella; Winograd-Katz, Sabina; Sindiri, Sivasish; Rotkopf, Ron; Dutton-Regester, Ken; Johansson, Peter; Pritchard, Antonia L; Waddell, Nicola; Hill, Victoria K; Lin, Jimmy C; Hevroni, Yael; Rosenberg, Steven A; Khan, Javed; Ben-Dor, Shifra; Niv, Masha Y; Ulitsky, Igor; Mann, Graham J; Scolyer, Richard A; Hayward, Nicholas K; Samuels, Yardena

    2015-12-01

    Analysis of 501 melanoma exomes identified RASA2, encoding a RasGAP, as a tumor-suppressor gene mutated in 5% of melanomas. Recurrent loss-of-function mutations in RASA2 were found to increase RAS activation, melanoma cell growth and migration. RASA2 expression was lost in ≥30% of human melanomas and was associated with reduced patient survival. These findings identify RASA2 inactivation as a melanoma driver and highlight the importance of RasGAPs in cancer. PMID:26502337

  6. Detection of melanomas by digital imaging of spectrally resolved ultraviolet light-induced autofluorescence of human skin.

    PubMed

    Chwirot, B W; Chwirot, S; Redziński, J; Michniewicz, Z

    1998-10-01

    Digital images of autofluorescence excited with 366 nm Hg line were recorded in a narrow 475 nm band for 408 pigmented lesions of the skin (90 melanomas, 205 common melanocytic and dysplastic naevi, 113 lesions of different kinds) and analysed photometrically with respect to spatial distribution of intensity to differentiate between melanomas and other melanocytic lesions. Earlier reports describing patterns of intensity distributions characteristic for melanomas have not been confirmed in this study. However, our evaluations showed that an algorithm based on ratios of maximum intensity recorded outside the lesions and minimum intensity found within them, allows melanomas to be detected with a sensitivity of 82.5%, a specificity of 78.6% and a positive predictive value of 58.9% (melanomas versus common and dysplastic naevi) or 76.7% (melanomas versus other pigmented lesions). The method is now being tested in a multicentre study involving three groups in three different cities in Poland. PMID:9893661

  7. In-vitro rescue and recovery studies of human melanoma (BLM) cell growth, adhesion and migration functions after treatment with progesterone.

    PubMed

    Leder, Douglas C; Brown, Jason R; Ramaraj, Pandurangan

    2015-01-01

    Treatment of human melanoma (BLM) cells for 48 hrs with progesterone resulted in a significant inhibition of cell growth. The mechanism of growth inhibition was due to autophagy and this action of progesterone was not mediated through progesterone receptor. As cells were floating during treatment, adhesion assay was performed, which showed complete loss of adhesion. When cells were allowed to recover after treatment by culturing in growth medium without progesterone, there was recovery in cell growth. Preliminary experiments on adhesion and recovery cell growth prompted us to suppress autophagic lysosomal degradation with 3-methyladenine (3-MA), which resulted in partial rescue of cell growth, adhesion and migration functions. The above experimental design gave rise to two experimental groups viz., progesterone treated and 3-MA rescued. Since, recovery studies also showed improvement in cell growth, progesterone treated and 3-MA rescued groups were allowed to recover on their own for first 48 hrs and then a second 48 hrs. Comparison of in-vitro cell growth, adhesion and migration functions of progesterone treated, 3-MA rescued and recovered human melanoma cells revealed that the recovery of 3-MA rescued cells was better than the recovery of progesterone treated cells in terms of cell growth and adhesion functions. These in-vitro experiments not only provided the scientific basis for epidemiological findings that menstruating females were better protected in melanoma, but also showed the potential of progesterone to act as an anti-cancer agent for melanoma treatment. PMID:26550137

  8. Angiogenesis and Melanoma

    PubMed Central

    Ribatti, Domenico; Annese, Tiziana; Longo, Vito

    2010-01-01

    Angiogenesis occurs in pathological conditions, such as tumors, where a specific critical point in tumor progression is the transition from the avascular to the vascular phase. Tumor angiogenesis depends mainly on the release by neoplastic cells of growth factors specific for endothelial cells, which are able to stimulate the growth of the host’s blood vessels. This article summarizes the literature concerning the relationship between angiogenesis and human melanoma progression. The recent applications of antiangiogenic agents which interfere with melanoma progression are also described. PMID:24281035

  9. Culturing Uveal Melanoma Cells.

    PubMed

    Angi, Martina; Versluis, Mieke; Kalirai, Helen

    2015-04-01

    A major challenge in cancer research is the use of appropriate models with which to study a specific biological question. Cell lines have long been used to study cellular processes and the effects of individual molecules because they are easy to use, grow rapidly, produce reproducible results and have a strong track record in research. In uveal melanoma in particular, the absence of animal models that faithfully replicate the behavior of the human disease has propagated the generation and use of numerous cell lines by individual research groups. This in itself, however, can be viewed as a problem due to the lack of standardization when characterizing these entities to determine how closely they reflect the genetic and phenotypic characteristics of this disease. The alternative is to use in vitro primary cultures of cells obtained directly from uveal melanoma patient samples, but this too has its difficulties. Primary cell cultures are difficult to use, hard to obtain and can show considerable heterogeneity. In this article, we review the following: (1) the uveal melanoma cell lines that are currently available, discussing the importance of establishing a bank of those that represent the molecular heterogeneity of uveal melanoma; (2) the methods used to isolate and perform short-term cultures of primary uveal melanoma cells, and (3) the establishment of 3D tissue culture models that bridge the gap between 2D in vitro systems and in vivo models with which to dissect cancer biology and perform therapeutic screens. PMID:27171555

  10. Amblyomin-X induces ER stress, mitochondrial dysfunction, and caspase activation in human melanoma and pancreatic tumor cell.

    PubMed

    Morais, Katia L P; Pacheco, Mario Thiego Fernandes; Berra, Carolina Maria; Bosch, Rosemary V; Sciani, Juliana Mozer; Chammas, Roger; de Freitas Saito, Renata; Iqbal, Asif; Chudzinski-Tavassi, Ana Marisa

    2016-04-01

    During the last two decades, new insights into proteasome function and its role in several human diseases made it a potential therapeutic target. In this context, Amblyomin-X is a Kunitz-type FXa inhibitor similar to endogenous tissue factor pathway inhibitor (TFPI) and is a novel proteasome inhibitor. Herein, we have demonstrated Amblyomin-X cytotoxicity to different tumor cells lines such as pancreatic (Panc1, AsPC1BxPC3) and melanoma (SK-MEL-5 and SK-MEL-28). Of note, Amblyomin-X was not cytotoxic to normal human fibroblast cells. In addition, Amblyomin-X promoted accumulation of ER stress markers (GRP78 and GADD153) in sensitive (SK-MEL-28) and bortezomib-resistant (Mia-PaCa-2) tumor cells. The intracellular calcium concentration [Ca(2+)] i was slightly modulated in human tumor cells (SK-MEL-28 and Mia-PaCa-2) after 24 h of Amblyomin-X treatment. Furthermore, Amblyomin-X induced mitochondrial dysfunction, cytochrome-c release, PARP cleavage, and activation of caspase cascade in both human tumor (SK-MEL-28 and Mia-PaCa-2) cells. These investigations might help in further understanding of the antitumor properties of Amblyomin-X. PMID:27015684

  11. Management of advanced melanoma

    SciTech Connect

    Nathanson, L. )

    1986-01-01

    This book presents papers on the subject of management of advanced melanoma. The topics covered are: non-investigational cytotoxic agents; high-dosage chemotherapy in antologous bone marrow transplantation; Radiotherapy of melanomas; hyperthermia; ureal melanoma; surgical treatment of recurrent a metastatic melanoma; role of interferons in management of melanoma and molecular genetics of melanoma.

  12. Estimation of absorbed dose in the covering skin of human melanoma treated by neutron capture therapy

    SciTech Connect

    Fukuda, H.; Kobayashi, T.; Hiratsuka, J.; Karashima, H.; Honda, C.; Yamamura, K.; Ichihashi, M.; Kanda, K.; Mishima, Y. )

    1989-07-01

    A patient with malignant melanoma was treated by thermal neutron capture therapy using 10B-paraboronophenylalanine. The compound was injected subcutaneously into ten locations in the tumor-surrounding skin, and the patient was then irradiated with thermal neutrons from the Musashi Reactor at reactor power of 100 KW and neutron flux of 1.2 X 10(9) n/cm{sup 2}/s. Total absorbed dose to the skin was 11.7-12.5 Gy in the radiation field. The dose equivalents of these doses were estimated as 21.5 and 24.4 Sv, respectively. Early skin reaction after irradiation was checked from day 1 to day 60. The maximum and mean skin scores were 2.0 and 1.5, respectively, and the therapy was safely completed as far as skin reaction was concerned. Some factors influencing the absorbed dose and dose equivalent to the skin are discussed.

  13. UVB-Stimulated TNFα Release from Human Melanocyte and Melanoma Cells Is Mediated by p38 MAPK

    PubMed Central

    Muthusamy, Visalini; Piva, Terrence J.

    2013-01-01

    Ultraviolet (UV) radiation activates cell signaling pathways in melanocytes. As a result of altered signaling pathways and UV-induced cellular damage, melanocytes can undergo oncogenesis and develop into melanomas. In this study, we investigated the effect of UV-radiation on p38 MAPK (mitogen-activated protein kinase), JNK and NFκB pathways to determine which plays a major role in stimulating TNFα secretion in human HEM (melanocytes) and MM96L (melanoma) cells. MM96L cells exhibited 3.5-fold higher p38 activity than HEM cells at 5 min following UVA + B radiation and 1.6-fold higher JNK activity at 15–30 min following UVB+A radiation, while NFκB was minimally activated in both cells. Irradiated HEM cells had the greatest fold of TNFα secretion (UVB: 109-fold, UVA + B: 103-fold & UVB+A: 130-fold) when co-exposed to IL1α. The p38 inhibitor, SB202190, inhibited TNFα release by 93% from UVB-irradiated HEM cells. In the UVB-irradiated MM96L cells, both SB202190 and sulfasalazine (NFκB inhibitor) inhibited TNFα release by 52%. Although, anisomycin was a p38 MAPK activator, it inhibited TNFα release in UV-irradiated cells. This suggests that UV-mediated TNFα release may occur via different p38 pathway intermediates compared to those stimulated by anisomycin. As such, further studies into the functional role p38 MAPK plays in regulating TNFα release in UV-irradiated melanocyte-derived cells are warranted. PMID:23965971

  14. The depth-dependent radiation response of human melanoma cells exposed to 65 MeV protons.

    PubMed

    Courdi, A; Brassart, N; Hérault, J; Chauvel, P

    1994-08-01

    Radiation therapy with positively charged particles implies that the Bragg peak be spread out to deliver a homogeneous dose to the tumour. The spread-out Bragg peak (SOBP) has a higher linear energy transfer (LET) than the entrance beam. In addition, there is an LET gradient from proximal to distal SOBP. The aim of this study is to find out whether these small LET variations lead to differences in radiation response. Human melanoma cells (CAL4) were exposed to 65 MeV proton beams produced by the cyclotron Medicyc at five different positions: 2 mm depth corresponding to the entrance, 15, 20, 25 and 26.8 mm depth corresponding to four different positions in the half-modulated SOBP. Survival curves were generated using the in vitro colony method and fitted with the linear-quadratic model. Survival differences were observed at high doses; they were statistically significant at a dose of 8 Gy. With respect to the entrance position (2 mm), the relative biological effectiveness (RBE) at 1% survival was 1.09, 1.12, 1.19 and 1.27 at 15, 20, 25 and 26.8 mm in the SOBP, respectively. Whereas RBE values in the SOBP greater than 1.0 relative to the entrance beam represent a small biological advantage to be added to the well-known physical advantage of high energy proton beams; the RBE gradient along the SOBP would imply that the distal end of the tumour would receive a higher biologically equivalent dose than the proximal end, despite a homogeneous physical dose, especially at the high doses per fraction given in ocular melanomas. Although the increase in effectiveness with depth is mild, it should be kept in mind during eye treatment planning, in case a critical target is present at the extreme end of the SOBP. PMID:8087487

  15. Histone Deacetylase Inhibitors Interact with Melanoma Differentiation Associated-7/Interleukin-24 to Kill Primary Human Glioblastoma Cells

    PubMed Central

    Hamed, Hossein A.; Yacoub, Adly; Park, Margaret A.; Archer, Kellie; Das, Swadesh K.; Sarkar, Devanand; Grant, Steven; Fisher, Paul B.

    2013-01-01

    We presently demonstrate that histone deacetylase inhibitors (HDACIs) enhance toxicity of melanoma differentiation-associated gene-7/interleukin 24 (mda-7/IL-24) in invasive primary human glioblastoma multiforme (GBM) cells. Additionally, a method is described to augment the efficacy of adenoviral delivery of mda-7/IL-24 in these cells. HDACIs synergized with melanoma differentiation-associated (MDA)-7/IL-24 killing GBM cells. Enhanced lethality correlated with increased autophagy that was dependent on the expression of ceramide synthase 6. HDACIs interacted with MDA-7/IL-24 prolonging generation of reactive oxygen species and Ca2+. Quenching of reactive oxygen species and Ca2+ blocked HDACI and MDA-7/IL-24 killing. In vivo MDA-7/IL-24 prolonged the survival of animals carrying orthotopic tumors, and HDACIs enhanced survival further. A serotype 5/3 adenovirus more effectively delivers mda-7/IL-24 to GBM tumors than a serotype 5 virus. Hence, we constructed a serotype 5/3 adenovirus that conditionally replicates in tumor cells expressing MDA-7/IL-24, in which the adenoviral early region 1A (E1A) gene was driven by the cancer-specific promoter progression elevated gene-3 [Ad.5/3 (INGN 241)-PEG-E1A-mda-7; also called Ad.5/3-CTV (cancer terminator virus)]. Ad.5/3-CTV increased the survival of mice carrying GBM tumors to a significantly greater extent than did a nonreplicative virus Ad.5/3-mda-7. Ad.5/3-CTV exhibited no toxicity in the brains of Syrian hamsters. Collectively our data demonstrate that HDACIs enhance MDA-7/IL-24 lethality, and adenoviral delivery of mda-7/IL-24 combined with tumor-specific viral replication is an effective preclinical GBM therapeutic. PMID:23661648

  16. Concomitant targeting of programmed death-1 (PD-1) and CD137 improves the efficacy of radiotherapy in a mouse model of human BRAFV600-mutant melanoma.

    PubMed

    Kroon, Paula; Gadiot, Jules; Peeters, Marlies; Gasparini, Alessia; Deken, Marcel A; Yagita, Hideo; Verheij, Marcel; Borst, Jannie; Blank, Christian U; Verbrugge, Inge

    2016-06-01

    T cell checkpoint blockade with antibodies targeting programmed cell death (ligand)-1 (PD-1/PD-L1) and/or cytotoxic T lymphocyte-antigen 4 (CTLA-4) has improved therapy outcome in melanoma patients. However, a considerable proportion of patients does not benefit even from combined α-CTLA-4 and α-PD-1 therapy. We therefore examined to which extent T cell (co)stimulation and/or stereotactic body radiation therapy (SBRT) could further enhance the therapeutic efficacy of T cell checkpoint blockade in a genetically engineered mouse melanoma model that is driven by PTEN-deficiency, and BRAFV600 mutation, as in human, but lacks the sporadic UV-induced mutations. Tumor-bearing mice were treated with different combinations of immunomodulatory antibodies (α-CTLA-4, α-PD-1, α-CD137) or interleukin-2 (IL-2) alone or in combination with SBRT. None of our immunotherapeutic approaches (alone or in combination) had any anti-tumor efficacy, while SBRT alone delayed melanoma outgrowth. However, α-CD137 combined with α-PD-1 antibodies significantly enhanced the anti-tumor effect of SBRT, while the anti-tumor effect of SBRT was not enhanced by interleukin-2, or the combination of α-CTLA-4 and α-PD-1. We conclude that α-CD137 and α-PD-1 antibodies were most effective in enhancing SBRT-induced tumor growth delay in this mouse melanoma model, outperforming the ability of IL-2, or the combination of α-CTLA-4 and α-PD-1 to synergize with SBRT. Given the high mutational load and increased immunogenicity of human melanoma with the same genotype, our findings encourage testing α-CD137 and α-PD-1 alone or in combination with SBRT clinically, particularly in patients refractory to α-CTLA-4 and/or α-PD-1 therapy. PMID:27160390

  17. Photodynamic therapy effect of zinc monoamino phthalocyanine-folic acid conjugate adsorbed on single walled carbon nanotubes on melanoma cells

    NASA Astrophysics Data System (ADS)

    Ogbodu, Racheal O.; Ndhundhuma, Ivy; Karsten, Aletta; Nyokong, Tebello

    2015-02-01

    This work reports on the photodynamic therapy effect of zinc monoamino phthalocyanine linked to folic acid represented as ZnMAPc-FA, which was further immobilized onto single walled carbon nanotube represented as ZnMAPc-FA-SWCNT on melanoma A375 cell line, the effect of SWCNT-FA (without ZnMAPc) was also examined. All the compounds were non-toxic to the melanoma A375 cell line in the absence of light. Upon irradiation of the melanoma A375 cell line with a 676 nm diode laser at a power density of 98 mW/cm2 at 5 J/cm2 about 60% and 63% cell death was observed in the presence of ZnMAPc-FA and ZnMAPc-FA-SWCNT respectively. SWCNT-FA had no significant photodynamic therapy or photothermal effect to the cell, only 23% of cell death was observed after irradiation.

  18. Transient receptor potential vanilloid 4 (TRPV4) is downregulated in keratinocytes in human non-melanoma skin cancer.

    PubMed

    Fusi, Camilla; Materazzi, Serena; Minocci, Daiana; Maio, Vincenza; Oranges, Teresa; Massi, Daniela; Nassini, Romina

    2014-09-01

    A subgroup of the transient receptor potential (TRP) channels, including vanilloid 1 (TRPV1), TRPV2, TRPV3, TRPV4, and TRP ankyrin 1 (TRPA1), is expressed in cutaneous peptidergic somatosensory neurons, and has been found in skin non-neuronal cells, such as keratinocytes. Different cancer cells express TRPs, where they may exert either pro- or antitumorigenic roles. Expression and function of TRPs in skin cancers have been, however, poorly investigated. Here, we have studied the distribution and expression of TRPs by immunohistochemistry and messenger RNA (mRNA) in human healthy skin and human keratinocytic tumors, including intraepidermal proliferative disorders (solar keratosis (SK) and Bowen's disease), and non-melanoma skin cancer (NMSC; basal cell and squamous cell carcinomas). Similar TRPV1, TRPV2, and TRPV3 staining was found in keratinocytes from healthy and tumor tissues. TRPA1 staining was increased solely in SK samples. However, the marked TRPV4 staining and TRPV4 mRNA expression, observed in healthy or inflamed skin, was abrogated both in premalignant lesions and NMSC. In a human keratinocyte cell line (HaCaT), TRPV4 stimulation released IL-8, which in turn downregulated TRPV4 expression. Selective reduction in TRPV4 expression could represent an early biomarker of skin carcinogenesis. Whether the cytokine-dependent, autocrine pathway that results in TRPV4 downregulation contributes to NMSC mechanism remains to be determined. PMID:24643128

  19. In Vivo Ultra-Fast Photoacoustic Flow Cytometry of Circulating Human Melanoma Cells Using Near-Ingrared High-Pulse Rate Lasers

    PubMed Central

    Nedosekin, Dmitry A.; Sarimollaoglu, Mustafa; Ye, John; Galanzha, Ekaterina I.; Zharov, Vladimir P.

    2011-01-01

    The circulating tumor cells (CTCs) appear to be a marker of metastasis development, especially, for highly aggressive and epidemically growing melanoma malignancy that is often metastatic at early stages. Recently, we introduced in vivo photoacoustic (PA) flow cytometry (PAFC) for label-free detection of mouse B16F10 CTCs in melanoma-bearing mice using melanin as an intrinsic marker. Here, we significantly improve the speed of PAFC by using a high pulse repetition rate laser operating at 820 and 1064 nm wavelengths. This platform was used in preclinical studies for label-free PA detection of low pigmented human CTCs. Demonstrated label-free PAFC detection, low level of background signals, and favorable safety standards for near infrared irradiation suggest that a fiber laser operating at 1064 nm at pulse repetition rates up to 0.5 MHz could be a promising source for portable clinical PAFC devices. The possible applications can include early diagnosis of melanoma at the parallel progression of primary tumor and CTCs, detection of cancer recurrence, residual disease, and real-time monitoring of therapy efficiency by counting CTCs before, during and after therapeutic intervention. Herewith, we also address sensitivity of label-free PAFC melanoma CTCs detection and introduce in vivo CTCs targeting by magnetic nanoparticles conjugated with specific antibody and magnetic cells enrichment. PMID:21786417

  20. In vivo ultra-fast photoacoustic flow cytometry of circulating human melanoma cells using near-infrared high-pulse rate lasers.

    PubMed

    Nedosekin, Dmitry A; Sarimollaoglu, Mustafa; Ye, Jian-Hui; Galanzha, Ekaterina I; Zharov, Vladimir P

    2011-10-01

    The circulating tumor cells (CTCs) appear to be a marker of metastasis development, especially, for highly aggressive and epidemically growing melanoma malignancy that is often metastatic at early stages. Recently, we introduced in vivo photoacoustic (PA) flow cytometry (PAFC) for label-free detection of mouse B16F10 CTCs in melanoma-bearing mice using melanin as an intrinsic marker. Here, we significantly improve the speed of PAFC by using a high-pulse repetition rate laser operating at 820 and 1064 nm wavelengths. This platform was used in preclinical studies for label-free PA detection of low-pigmented human CTCs. Demonstrated label-free PAFC detection, low level of background signals, and favorable safety standards for near-infrared irradiation suggest that a fiber laser operating at 1064 nm at pulse repetition rates up to 0.5 MHz could be a promising source for portable clinical PAFC devices. The possible applications can include early diagnosis of melanoma at the parallel progression of primary tumor and CTCs, detection of cancer recurrence, residual disease and real-time monitoring of therapy efficiency by counting CTCs before, during, and after therapeutic intervention. Herewith, we also address sensitivity of label-free detection of melanoma CTCs and introduce in vivo CTC targeting by magnetic nanoparticles conjugated with specific antibody and magnetic cells enrichment. PMID:21786417

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

    PubMed Central

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

    2015-01-01

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

  2. Resistance to ursolic acid-induced apoptosis through involvement of melanogenesis and COX-2/PGE2 pathways in human M4Beu melanoma cancer cells.

    PubMed

    Hassan, Lama; Pinon, Aline; Limami, Youness; Seeman, Josiane; Fidanzi-Dugas, Chloe; Martin, Frederique; Badran, Bassam; Simon, Alain; Liagre, Bertrand

    2016-07-01

    Melanoma is one of the most aggressive forms of cancer with a continuously growing incidence worldwide and is usually resistant to chemotherapy agents, which is due in part to a strong resistance to apoptosis. Previously, we had showed that B16-F0 murine melanoma cells undergoing apoptosis are able to delay their own death induced by ursolic acid (UA), a natural pentacyclic triterpenoid compound. We had demonstrated that tyrosinase and TRP-1 up-regulation in apoptotic cells and the subsequent production of melanin were implicated in an apoptosis resistance mechanism. Several resistance mechanisms to apoptosis have been characterized in melanoma such as hyperactivation of DNA repair mechanisms, drug efflux systems, and reinforcement of survival signals (PI3K/Akt, NF-κB and Raf/MAPK pathways). Otherwise, other mechanisms of apoptosis resistance involving different proteins, such as cyclooxygenase-2 (COX-2), have been described in many cancer types. By using a strategy of specific inhibition of each ways, we suggested that there was an interaction between melanogenesis and COX-2/PGE2 pathway. This was characterized by analyzing the COX-2 expression and activity, the expression of tyrosinase and melanin production. Furthermore, we showed that anti-proliferative and proapoptotic effects of UA were mediated through modulation of multiple signaling pathways including Akt and ERK-1/2 proteins. Our study not only uncovers underlying molecular mechanisms of UA action in human melanoma cancer cells but also suggest its great potential as an adjuvant in treatment and cancer prevention. PMID:27262506

  3. Promotion of melanoma growth by the metabolic hormone leptin.

    PubMed

    Ellerhorst, Julie A; Diwan, A H; Dang, Shyam M; Uffort, Deon G; Johnson, Marilyn K; Cooke, Carolyn P; Grimm, Elizabeth A

    2010-04-01

    We have previously shown that melanoma cells proliferate in response to the metabolic hormones TRH and TSH. The objective of the present study was to test the hypothesis that a third metabolic hormone, leptin, serves as a growth factor for melanoma. Using western blotting, indirect immunofluorescence, and RT-PCR, leptin receptors were found to be expressed by human melanoma cells. In contrast, cultured melanocytes expressed message for the receptor without detectable protein. Melanoma cells responded to treatment with leptin by activating the MAPK pathway and proliferating. Melanoma cells but not melanocytes, also expressed leptin protein, creating a potential autocrine loop. Examination of human melanoma tumors by immunohistochemistry revealed that melanomas and nevi expressed leptin at a high frequency. Melanomas also strongly expressed the leptin receptor, whereas nevi expressed this receptor to a much lesser degree. We conclude that leptin is a melanoma growth factor and that a leptin autocrine-loop may contribute to the uncontrolled proliferation of these cells. PMID:20204272

  4. Stimulation of the protein tyrosine kinase c-Yes but not c-Src by neurotrophins in human brain-metastatic melanoma cells.

    PubMed

    Marchetti, D; Parikh, N; Sudol, M; Gallick, G E

    1998-06-25

    The c-Yes proto-oncogene (pp62c-Yes) encodes a non-receptor-type protein tyrosine kinase (NRPTK) of the Src family. c-Yes activities and protein levels are elevated in human melanoma and melanocyte cell lines. Because the neurotrophins (NT) are important in the progression of melanoma to the brain-metastatic phenotype, we determined whether NT stimulate c-Yes activity in human MeWo melanoma cells and two variant sublines with opposite metastatic capabilities, 3 S 5 and 70W. The highly brain-metastatic 70W subline had an intrinsically higher c-Yes activity than parental MeWo or poorly metastatic 3 S 5 cells. c-Yes kinase was further induced by the prototypic human NT, nerve growth factor (NGF) in a dose and time-dependent manner. In contrast, c-Src activity (pp60-Src) was similar in all these cells and unaffected by NGF exposure. Additionally, human NGF and neurotrophin-3 stimulated c-Yes in brain-metastatic 70W cells. The magnitude of c-Yes activation correlated with the degree of invasion of 70 W cells following incubation of these neurotrophins. To further examine NT stimulation of c-Yes in melanoma cells, three additional cell lines were examined. Metastatic TXM-13 and TXM-18 increased c-Yes activity in response to NGF. In contrast, no increase was observed in low-metastatic TXM-40 cells. Together, these data suggest that altered c-Yes expression may play a role in the malignant progression of the human melanocyte towards the brain-metastatic phenotype and that NT enhance the activity of c-Yes in signaling penetration into the matrix of NT-rich stromal microenvironments such as the brain. PMID:9681823

  5. Effects of a novel Nodal-targeting monoclonal antibody in melanoma

    PubMed Central

    Margaryan, Naira V.; Focà, Annalia; Sanguigno, Luca; Bodenstine, Thomas M.; Chandler, Grace S.; Reed, David W.; Gilgur, Alina; Seftor, Elisabeth A.; Seftor, Richard E.B.; Khalkhali-Ellis, Zhila; Leonardi, Antonio; Ruvo, Menotti; Hendrix, Mary J.C.

    2015-01-01

    Nodal is highly expressed in various human malignancies, thus supporting the rationale for exploring Nodal as a therapeutic target. Here, we describe the effects of a novel monoclonal antibody (mAb), 3D1, raised against human Nodal. In vitro treatment of C8161 human melanoma cells with 3D1 mAb shows reductions in anchorage-independent growth and vasculogenic network formation. 3D1 treated cells also show decreases of Nodal and downstream signaling molecules, P-Smad2 and P-ERK and of P-H3 and CyclinB1, with an increase in p27. Similar effects were previously reported in human breast cancer cells where Nodal expression was generally down-regulated; following 3D1 mAb treatment, both Nodal and P-H3 levels are reduced. Noteworthy is the reduced growth of human melanoma xenografts in Nude mice treated with 3D1 mAb, where immunostaining of representative tumor sections show diminished P-Smad2 expression. Similar effects both in vitro and in vivo were observed in 3D1 treated A375SM melanoma cells harboring the active BRAF(V600E) mutation compared to treatments with IgG control or a BRAF inhibitor, dabrafenib. Finally, we describe a 3D1-based ELISA for the detection of Nodal in serum samples from cancer patients. These data suggest the potential of 3D1 mAb for selecting and targeting Nodal expressing cancers. PMID:26460952

  6. Immunotherapy of metastatic melanoma by reversal of immune suppression

    SciTech Connect

    Biggs, M.W.; Eiselein, J.E.

    1997-01-01

    Beginning with the observation that the human enteorvirus, Poliovirus Sabin 1, will lyse human melanoma cells in culture, clinical trials involving two patients with advance melanoma were performed. Parenteral injection of the viable Poliovirus into cutaneous melanoma metastases followed in 24 hours by oral administration of cyclophosphamide. The results of these two trials are described.

  7. MMP13 mediates cell cycle progression in melanocytes and melanoma cells: in vitro studies of migration and proliferation

    PubMed Central

    2010-01-01

    Background Melanoma cells are usually characterized by a strong proliferative potential and efficient invasive migration. Among the multiple molecular changes that are recorded during progression of this disease, aberrant activation of receptor tyrosine kinases (RTK) is often observed. Activation of matrix metalloproteases goes along with RTK activation and usually enhances RTK-driven migration. The purpose of this study was to examine RTK-driven three-dimensional migration of melanocytes and the pro-tumorigenic role of matrix metalloproteases for melanocytes and melanoma cells. Results Using experimental melanocyte dedifferentiation as a model for early melanomagenesis we show that an activated EGF receptor variant potentiates migration through three-dimensional fibrillar collagen. EGFR stimulation also resulted in a strong induction of matrix metalloproteases in a MAPK-dependent manner. However, neither MAPK nor MMP activity were required for migration, as the cells migrated in an entirely amoeboid mode. Instead, MMPs fulfilled a function in cell cycle regulation, as their inhibition resulted in strong growth inhibition of melanocytes. The same effect was observed in the human melanoma cell line A375 after stimulation with FCS. Using sh- and siRNA techniques, we could show that MMP13 is the protease responsible for this effect. Along with decreased proliferation, knockdown of MMP13 strongly enhanced pigmentation of melanocytes. Conclusions Our data show for the first time that growth stimuli are mediated via MMP13 in melanocytes and melanoma, suggesting an autocrine MMP13-driven loop. Given that MMP13-specific inhibitors are already developed, these results support the evaluation of these inhibitors in the treatment of melanoma. PMID:20667128

  8. Choroidal melanoma

    PubMed Central

    Singh, Parul; Singh, Abhishek

    2012-01-01

    Choroidal melanoma is the most common primary intra-ocular malignant tumor and second most common site of ten malignant melanoma sites in the body. Current diagnosis of choroidal melanoma is based on both the clinical experience of the specialist and modern diagnostic techniques such as indirect ophthalmoscopy, A- and B-ultrasonography scans, fundus fluorescein angiography, and transillumination. Invasive studies such as fine needle aspiration cytology can have significant morbidity and should only be considered if therapeutic intervention is indicated and diagnosis cannot be established by any other means. Several modes of treatment are available for choroidal melanoma. Multiple factors are taken into account when deciding one approach over other approaches, such as visual acuity of the affected eye, visual acuity of the contralateral eye, tumor size, location, ocular structures involved and presence of metastases. A comprehensive review of literature available in books and indexed journals was done. This article discusses in detail epidemiology, diagnosis, current available treatment options, and prognosis and survival of choroidal melanoma. PMID:22557869

  9. Melanoma Diagnosis

    NASA Astrophysics Data System (ADS)

    Horsch, Alexander

    The chapter deals with the diagnosis of the malignant melanoma of the skin. This aggressive type of cancer with steadily growing incidence in white populations can hundred percent be cured if it is detected in an early stage. Imaging techniques, in particular dermoscopy, have contributed significantly to improvement of diagnostic accuracy in clinical settings, achieving sensitivities for melanoma experts of beyond 95% at specificities of 90% and more. Automatic computer analysis of dermoscopy images has, in preliminary studies, achieved classification rates comparable to those of experts. However, the diagnosis of melanoma requires a lot of training and experience, and at the time being, average numbers of lesions excised per histology-proven melanoma are around 30, a number which clearly is too high. Further improvements in computer dermoscopy systems and their competent use in clinical settings certainly have the potential to support efforts of improving this situation. In the chapter, medical basics, current state of melanoma diagnosis, image analysis methods, commercial dermoscopy systems, evaluation of systems, and methods and future directions are presented.

  10. Malignant Melanoma

    PubMed Central

    Perera, Eshini; Gnaneswaran, Neiraja; Jennens, Ross; Sinclair, Rodney

    2013-01-01

    Melanomas are a major cause of premature death from cancer. The gradual decrease in rates of morbidity and mortality has occurred as a result of public health campaigns and improved rates of early diagnosis. Survival of melanoma has increased to over 90%. Management of melanoma involves a number of components: excision, tumor staging, re-excision with negative margins, adjuvant therapies (chemo, radiation or surgery), treatment of stage IV disease, follow-up examination for metastasis, lifestyle modification and counseling. Sentinel lymph node status is an important prognostic factor for survival in patients with a melanoma >1 mm. However, sentinel lymph node biopsies have received partial support due to the limited data regarding the survival advantage of complete lymph node dissection when a micrometastasis is detected in the lymph nodes. Functional mutations in the mitogen-activated pathways are commonly detected in melanomas and these influence the growth control. Therapies that target these pathways are rapidly emerging, and are being shown to increase survival rates in patients. Access to these newer agents can be gained by participation in clinical trials after referral to a multidisciplinary team for staging and re-excision of the scar. PMID:27429256

  11. Cooperative antiproliferative signaling by aspirin and indole-3-carbinol targets microphthalmia-associated transcription factor gene expression and promoter activity in human melanoma cells.

    PubMed

    Poindexter, Kevin M; Matthew, Susanne; Aronchik, Ida; Firestone, Gary L

    2016-04-01

    Antiproliferative signaling of combinations of the nonsteroidal anti-inflammatory drug acetylsalicylic acid (aspirin) and indole-3-carbinol (I3C), a natural indolecarbinol compound derived from cruciferous vegetables, was investigated in human melanoma cells. Melanoma cell lines with distinct mutational profiles were sensitive to different extents to the antiproliferative response of aspirin, with oncogenic BRAF-expressing G361 cells and wild-type BRAF-expressing SK-MEL-30 cells being the most responsive. I3C triggered a strong proliferative arrest of G361 melanoma cells and caused only a modest decrease in the proliferation of SK-MEL-30 cells. In both cell lines, combinations of aspirin and I3C cooperatively arrested cell proliferation and induced a G1 cell cycle arrest, and nearly ablated protein and transcript levels of the melanocyte master regulator microphthalmia-associated transcription factor isoform M (MITF-M). In melanoma cells transfected with a -333/+120-bp MITF-M promoter-luciferase reporter plasmid, treatment with aspirin and I3C cooperatively disrupted MITF-M promoter activity, which accounted for the loss of MITF-M gene products. Mutational analysis revealed that the aspirin required the LEF1 binding site, whereas I3C required the BRN2 binding site to mediate their combined and individual effects on MITF-M promoter activity. Consistent with LEF1 being a downstream effector of Wnt signaling, aspirin, but not I3C, downregulated protein levels of the Wnt co-receptor LDL receptor-related protein-6 and β-catenin and upregulated the β-catenin destruction complex component Axin. Taken together, our results demonstrate that aspirin-regulated Wnt signaling and I3C-targeted signaling pathways converge at distinct DNA elements in the MITF-M promoter to cooperatively disrupt MITF-M expression and melanoma cell proliferation. PMID:27055402

  12. Esculetin, a Coumarin Derivative, Exhibits Anti-proliferative and Pro-apoptotic Activity in G361 Human Malignant Melanoma

    PubMed Central

    Jeon, Young-Joo; Jang, Jeong-Yun; Shim, Jung-Hyun; Myung, Pyung Keun; Chae, Jung-Il

    2015-01-01

    Background: Although esculetin, a coumarin compound, is known to induce apoptosis in human cancer cells, the effects and molecular mechanisms on the apoptosis in human malignant melanoma (HMM) cells are not well understood yet. In this study, we investigated the anti-proliferative effects of esculetin on the G361 HMM cells. Methods: We analyzed the anti-proliferative effects and molecular mechanisms of esculetin on G361 cells by a 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, 4′,6-diamidino-2-phenylindole staining and Western blotting. Results: Esculetin exhibited significant anti-proliferative effects on the HMM cells in a dose-dependent manner. Interestingly, we found that esculetin induced nuclear shrinkage and fragmentation, typical apoptosis markers, by suppression of Sp1 transcription factor (Sp1). Notably, esculetin modulated Sp1 downstream target genes including p27, p21 and cyclin D1, resulted in activation of apoptosis signaling molecules such as caspase-3 and PARP in G361 HMM cells. Conclusions: Our results clearly demonstrated that esculetin induced apoptosis in the HMM cells by downregulating Sp1 protein levels. Thus, we suggest that esculetin may be a potential anti-proliferative agent that induces apoptotic cell death in G361 HMM cells. PMID:26151043

  13. von Willebrand factor fibers promote cancer-associated platelet aggregation in malignant melanoma of mice and humans.

    PubMed

    Bauer, Alexander T; Suckau, Jan; Frank, Kathrin; Desch, Anna; Goertz, Lukas; Wagner, Andreas H; Hecker, Markus; Goerge, Tobias; Umansky, Ludmila; Beckhove, Philipp; Utikal, Jochen; Gorzelanny, Christian; Diaz-Valdes, Nancy; Umansky, Viktor; Schneider, Stefan W

    2015-05-14

    Tumor-mediated procoagulatory activity leads to venous thromboembolism and supports metastasis in cancer patients. A prerequisite for metastasis formation is the interaction of cancer cells with endothelial cells (ECs) followed by their extravasation. Although it is known that activation of ECs and the release of the procoagulatory protein von Willebrand factor (VWF) is essential for malignancy, the underlying mechanisms remain poorly understood. We hypothesized that VWF fibers in tumor vessels promote tumor-associated thromboembolism and metastasis. Using in vitro settings, mouse models, and human tumor samples, we showed that melanoma cells activate ECs followed by the luminal release of VWF fibers and platelet aggregation in tumor microvessels. Analysis of human blood samples and tumor tissue revealed that a promoted VWF release combined with a local inhibition of proteolytic activity and protein expression of ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type I repeats 13) accounts for this procoagulatory milieu. Blocking endothelial cell activation by the low-molecular-weight heparin tinzaparin was accompanied by a lack of VWF networks and inhibited tumor progression in a transgenic mouse model. Our findings implicate a mechanism wherein tumor-derived vascular endothelial growth factor-A (VEGF-A) promotes tumor progression and angiogenesis. Thus, targeting EC activation envisions new therapeutic strategies attenuating tumor-related angiogenesis and coagulation. PMID:25977583

  14. Evaluation of cytotoxic activities of snake venoms toward breast (MCF-7) and skin cancer (A-375) cell lines.

    PubMed

    Bradshaw, Michael J; Saviola, Anthony J; Fesler, Elizabeth; Mackessy, Stephen P

    2014-11-19

    Snake venoms are mixtures of bioactive proteins and peptides that exhibit diverse biochemical activities. This wide array of pharmacologies associated with snake venoms has made them attractive sources for research into potentially novel therapeutics, and several venom-derived drugs are now in use. In the current study we performed a broad screen of a variety of venoms (61 taxa) from the major venomous snake families (Viperidae, Elapidae and "Colubridae") in order to examine cytotoxic effects toward MCF-7 breast cancer cells and A-375 melanoma cells. MTT cell viability assays of cancer cells incubated with crude venoms revealed that most venoms showed significant cytotoxicity. We further investigated venom from the Red-bellied Blacksnake (Pseudechis porphyriacus); venom was fractionated by ion exchange fast protein liquid chromatography and several cytotoxic components were isolated. SDS-PAGE and MALDI-TOF mass spectrometry were used to identify the compounds in this venom responsible for the cytotoxic effects. In general, viper venoms were potently cytotoxic, with MCF-7 cells showing greater sensitivity, while elapid and colubrid venoms were much less toxic; notable exceptions included the elapid genera Micrurus, Naja and Pseudechis, which were quite cytotoxic to both cell lines. However, venoms with the most potent cytotoxicity were often not those with low mouse LD50s, including some dangerously venomous viperids and Australian elapids. This study confirmed that many venoms contain cytotoxic compounds, including catalytic PLA2s, and several venoms also showed significant differential toxicity toward the two cancer cell lines. Our results indicate that several previously uncharacterized venoms could contain promising lead compounds for drug development. PMID:25407733

  15. Treatment of vemurafenib-resistant SKMEL-28 melanoma cells with paclitaxel.

    PubMed

    Thang, Nguyen Dinh; Nghia, Phan Tuan; Kumasaka, Mayuko Y; Yajima, Ichiro; Kato, Masashi

    2015-01-01

    Vemurafenib has recently been used as drug for treatment of melanomas with BRAFV600E mutation. Unfortunately, treatment with only vemurafenib has not been sufficiently effective, with recurrence after a short period. In this study, three vemurafenib-resistant BRAFV600E melanoma cell lines, A375PR, A375MR and SKMEL-28R, were established from the original A375P, A375M and SKMEL-28 cell lines. Examination of the molecular mechanisms showed that the phosphorylation levels of MEK and ERK, which play key roles in the RAS/RAF/MEK/ERK signaling pathway, were reduced in these three cell lines, with increased phosphorylation levels of pAKTs limited to SKMEL-28R cells. Treatment of SKMEL-28R cells with 100 nM paclitaxel resulted in increased apoptosis and decreased cellular proliferation, invasion and colony formation via reduction of expression levels of EGFR and pAKTs. Moreover, vemurafenib-induced pAKTs in SKMEL-28R were decreased by treatment with an AKT inhibitor, MK-2206. Taken together, our results revealed that resistance mechanisms of BRAFV600E-mutation melanoma cells to vemurafenib depended on the cell type. Our results suggested that paclitaxel should be considered as a drug in combination with vemurafenib to treat melanoma cells. PMID:25684511

  16. Effects of bavachin and its regulation of melanin synthesis in A375 cells

    PubMed Central

    WANG, JING-HUA; PEI, YUAN-YUAN; XU, HONG-DAN; LI, LI-JING; WANG, YE-QIU; LIU, GUO-LIANG; QU, YAN; ZHANG, NING

    2016-01-01

    The aim of the present study was to investigate the effect of bavachin treatment on A375 cells and the regulation of melanin synthesis. The cultured A375 cells in vitro were treated with bavachin; and the effect of bavachin on cell activity, tyrosinase (TYR) activity and melanin synthesis were respectively tested by the MTT assay, L-dopa oxidation assay and the NaOH lysis assay. The expression levels of TYR and c-Jun N-terminal kinases (JNK) proteins were tested by western blot analysis. The expression levels of TYR, tyrosinase-related protein-1 (TRP-1), TRP-2, extracellular signal-regulated kinase 1 (ERK1), ERK2 and JNK2 mRNA were tested by the reverse transcription-polymerase chain reaction assay. Simultaneously, the effect of estrogen receptor inhibitor (ICI182780) and ERK pathway inhibitor (U0126) was also tested on A375 cells following bavachin. The safe dose of bavachin significantly inhibited melanin synthesis and TYR activity. Bavachin (10 µmol/l) inhibited the expression of TYR and JNK proteins, and the expression of TYR, TRP-1, TRP-2, ERK1, ERK2 and JNK2 mRNA in A375 cells. ICI182780 and U0126 could significantly reverse the bavachin treatment on the protein expression levels and the mRNA expression of TYR, TRP-1, TRP-2, ERK1, ERK2 and JNK2. In conclusion, bavachin inhibited the synthesis of melanin on A375 cells by inhibiting the protein and mRNA expression of TYR, TRP-1, TRP-2, ERK1, ERK2 and JNK2. PMID:27347410

  17. Clinicopathological characterization of mouse models of melanoma.

    PubMed

    Ferguson, Blake; Soyer, H Peter; Walker, Graeme J

    2015-01-01

    Mouse models of melanoma have proven invaluable in the delineation of key molecular events involved in disease progression in humans and provide potential preclinical models for therapeutic testing (Damsky and Bosenberg, Pigment Cell Melanoma Res 25(4):404-405, 2012; Walker et al., Pigment Cell Melanoma Res 24(6):1158-1176, 2011). Here we concentrate on the clinicopathological analysis of melanocytic tumors. PMID:25636472

  18. RGD-conjugated gold nanorods induce radiosensitization in melanoma cancer cells by downregulating αvβ3 expression

    PubMed Central

    Xu, Wencai; Luo, Teng; Li, Ping; Zhou, Chuanqing; Cui, Daxiang; Pang, Bo; Ren, Qiushi; Fu, Shen

    2012-01-01

    Background Melanoma is known to be radioresistant and traditional treatments have been intractable. Therefore, novel approaches are required to improve the therapeutic efficacy of melanoma treatment. In our study, gold nanorods conjugated with Arg-Gly-Asp peptides (RGD-GNRs) were used as a sensitizer to enhance the response of melanoma cells to 6 mV radiation. Methods and materials A375 melanoma cells were treated by gold nanorods or RGD-GNRs with or without irradiation. The antiproliferative impact of the treatments was measured by MTT assay. Radiosensitizing effects were determined by colony formation assay. Apoptosis and cell cycle data were measured by flow cytometry. Integrin αvβ3 expression was also investigated by flow cytometry. Results Addition of RGD-GNRs enhanced the radiosensitivity of A375 cells with a dose-modifying factor of 1.35, and enhanced radiation-induced apoptosis. DNA flow cytometric analysis indicated that RGD-GNRs plus irradiation induced significant G2/M phase arrest in A375 cells. Both spontaneous and radiation-induced expressions of integrin αvβ3 were downregulated by RGD-GNRs. Conclusion Our study indicated that RGD-GNRs could sensitize melanoma A375 cells to irradiation. It was hypothesized that this was mainly through downregulation of radiation-induced αvβ3, in addition to induction of a higher proportion of cells within the G2/M phase. The combination of RGD-GNRs and radiation needs further investigation. PMID:22412298

  19. Ethanol induces upregulation of the nerve growth factor receptor CD271 in human melanoma cells via nuclear factor-κB activation

    PubMed Central

    RAPPA, GERMANA; ANZANELLO, FABIO; LORICO, AURELIO

    2015-01-01

    Alcohol consumption is one of the most important, and potentially avoidable, risk factors of human cancer, accounting for 3.6% of all types of cancer worldwide. In a recent meta-analysis, a 20% increased risk of melanoma was linked with regular alcohol consumption. In the present study, the effect of ethanol exposure on the expression of the nerve growth factor receptor, CD271, in human FEMX-I melanoma cells was investigated. Consistent with the derivation of melanocytes from the neural crest, the majority of melanomas express CD271, a protein that is crucial for maintaining the melanoma stem cell properties, including the capacity of self-renewal and resistance to chemotherapy and radiotherapy. Analysis of CD271-sorted subpopulations and clones of FEMX-I cells indicated no hierarchical organization of CD271+ and CD271− cells. In addition, CD271 expression was lost upon growth of FEMX-I melanoma cells in cancer stem cell-like conditions, while it was greatly increased upon CD133 knockdown or exposure to ethanol. After 24-h exposure to 100, 200 and 400 mM ethanol, the percentage of CD271+ cells increased from 14% in control cells to 24, 35 and 88%, respectively. An increase in the percentage of CD271+ cells was already evident 8 h after ethanol exposure and reached a maximum at 48 h. Ethanol-induced upregulation of CD271 was mediated by nuclear factor-κB (NF-κB). In fact, exposure of FEMX-I cells to 100–400 mM ethanol for 24 h resulted in a concentration- and time-dependent increase in NF-κB activity, up to 900% that of control cells. NF-κB activation was due to a decrease in p50 homodimers, which occupy the NF-κB binding site, blocking transactivation. No effects of ethanol on 9 additional signaling pathways of FEMX-I cells were observed. In the presence of CD271 blocking antibodies, NF-κB activation was not prevented, indicating that ethanol did not target CD271 directly. These data demonstrate that ethanol induces expression of CD271 in FEMX-I cells via

  20. Oncogenic role of microRNA-20a in human uveal melanoma

    PubMed Central

    Zhou, Jinzi; Jiang, Jian; Wang, Shuhong; Xia, Xiaobo

    2016-01-01

    As a member of the microRNA (miR)-17-92 cluster, miR-20a has been indicated to be involved in the regulation of the proliferation and invasion of various cancer cells. Previous studies have observed elevated plasma levels of miR-20a in patients with uveal melanoma (UM), compared with normal controls. In the present study, the potential function of miR-20a in UM was investigated. Reverse transcription-quantitative polymerase chain reaction analysis was performed to detect the expression levels of miR-20a in UM cells and tissues. The functions of miR-20a on cell proliferation, migration and invasion were determined in vitro using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide and Transwell assays, respectively. The expression levels of miR-20a were significantly increased in the UM cells and tissues (P<0.05). Subsequently, miR-20a mimics were transfected into UM cells, which led to increases in cell growth, migration and invasion activities. By contrast, miR-20a inhibition markedly suppressed the viability and motility of UM cells in vitro. These data provided convincing evidence that miR-20a may function as an oncogenic miRNA, and may be involved in promoting cell growth and motility in the molecular etiology of UM, suggesting its potential as a candidate therapeutic target for the treatment of patients with UM. PMID:27356499

  1. A therapeutic trial of human melanomas with combined small interfering RNAs targeting adaptor molecules p130Cas and paxillin activated under expression of ganglioside GD3.

    PubMed

    Makino, Yusuke; Hamamura, Kazunori; Takei, Yoshifumi; Bhuiyan, Robiul Hasan; Ohkawa, Yuki; Ohmi, Yuhsuke; Nakashima, Hideyuki; Furukawa, Keiko; Furukawa, Koichi

    2016-08-01

    We previously demonstrated that focal adhesion kinase (FAK), p130Cas and paxillin are crucially involved in the enhanced malignant properties under expression of ganglioside GD3 in melanoma cells. Therefore, molecules existing in the GD3-mediated signaling pathway could be considered as suitable targets for therapeutic intervention in malignant melanoma. The aim of this study was to determine whether blockade of p130Cas and/or paxillin by RNAi suppresses melanoma growth. We found a suitable dose (40 μM siRNA, 25 μl/tumor) of the siRNA to suppress p130Cas in the xenografts generated in nu/nu mice. Based on these results, we performed intratumoral (i.t.) treatment with anti-p130Cas and/or anti-paxillin siRNAs mixed with atelocollagen as a drug delivery system in a xenograft tumor of a human melanoma cell line, SK-MEL-28. Mixture of atelocollagen (1.75%) and an siRNA (500 or 1000 pmol/tumor) was injected into the tumors every 3 days after the first injection. An siRNA against human p130Cas markedly suppressed tumor growth of the xenograft in a dose-dependent manner, whereas siRNA against human paxillin slightly inhibited the tumor growth. A control siRNA against firefly luciferase showed no effect. To our surprise, siRNA against human p130Cas (500 or 1000 pmol/tumor) combined with siRNA against human paxillin dramatically suppressed tumor growth. In agreement with the tumor suppression effects of the anti-p130Cas siRNA, reduction in Ki-67 positive cell number as well as in p130Cas expression was demonstrated by immunohistostaining. These results suggested that blockade of GD3-mediated growth signaling pathways by siRNAs might be a novel and promising therapeutic strategy against malignant melanomas, provided signaling molecules such as p130Cas and paxillin are significantly expressed in individual cases. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc. PMID:27068854

  2. Treatment of malignant melanoma by selective thermal neutron capture therapy using melanoma-seeking compound

    SciTech Connect

    Mishima, Y.; Ichihashi, M.; Tsuji, M.; Hatta, S.; Ueda, M.; Honda, C.; Suzuki, T.

    1989-05-01

    As pigment cells undergo melanoma genesis, accentuated melanogenesis concurrently occurs in principle. Subsequent to the understanding of intrinsic factors controlling both processes, we found our selective melanoma neutron capture therapy (NCT) using 10B-dopa (melanin substrate) analogue, 10B1-p-boronophenylalanine (10B1-BPA), followed by 10B(n, alpha)7Li reaction, induced by essentially harmless thermal neutrons, which releases energy of 2.33 MeV to 14 mu, the diameter of melanoma cells. In vitro/in vivo radiobiological analysis revealed the highly enhanced melanoma killing effect of 10B1-BPA. Chemical and prompt gamma ray spectrometry assays of 10B accumulated within melanoma cells after 10B1-BPA administration in vitro and in vivo show high affinity, e.g., 10B melanoma/blood ratio of 11.5. After successfully eradicating melanoma transplanted into hamsters with NCT, we advanced to preclinical studies using spontaneously occurring melanoma in Duroc pig skin. We cured three melanoma cases, 4.6 to 12 cm in diameter, by single neutron capture treatment. Complete disappearance of melanoma was obtained without substantial side effects. Acute and subacute toxicity as well as pharmacodynamics of 10B1-BPA have been studied in relation to therapeutic dosage requirements. Clinical radiation dosimetry using human phantom has been carried out. Further preclinical studies using human melanoma transplanted into nude mouse have been a useful model for obtaining optimal results for each melanoma type. We recently treated the first human melanoma patient with our NCT, using essentially the method for Duroc pig melanoma, and obtained similar regression time course leading to cure.

  3. Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors.

    PubMed

    Devarapu, Satish Kumar; Mamidi, Srinivas; Plöger, Frank; Dill, Othmar; Blixt, Ola; Kirschfink, Michael; Schwartz-Albiez, Reinhard

    2016-06-15

    A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies. PMID:26830059

  4. Assessment of the chemical changes induced in human melanoma cells by boric acid treatment using infrared imaging

    SciTech Connect

    Acerbo, A.; Miller, L.

    2009-07-01

    Boron is found in everyday foods and drinking water in trace quantities. Boron exists as boric acid (BA) within plants and animals, where low levels have been linked to cancer incidence. However, this correlation is not well characterized. In this study, we examined the chemical and morphological effects of BA on human skin melanoma cells (SK-MEL28) using Fourier Transform InfraRed Imaging (FTIRI) with a Focal Plane Array (FPA) detector. Cells were grown under concentrations of BA ranging from 0 to 50 mM. Cell viability was determined after 1, 2, 3, 5, 7 and 10 days using trypan blue staining. With FTIRI, images of approximately twenty cells per time point per condition were collected. Principal components analysis (PCA) was used to evaluate changes in cell composition, with particular focus on the lipid, protein, and nucleic acid spectral components. Results from trypan blue staining revealed decreased cell viability as BA concentration increased. FTIRI data indicated that the protein and lipid contents (as indicated by the lipid/protein ratio) did not undergo substantial changes due to BA treatment. In contrast, the nucleic acid/protein ratio significantly decreased with BA treatment. PCA results showed an increase in {beta}-sheet protein at higher concentrations of BA (12.5, 25, and 50 mM). Together, these results suggest that high concentrations of BA have an anti-proliferative effect and show signs consistent with apoptosis.

  5. The effects of perfusion conditions on melphalan distribution in the isolated perfused rat hindlimb bearing a human melanoma xenograft.

    PubMed

    Wu, Z Y; Smithers, B M; Parsons, P G; Roberts, M S

    1997-01-01

    An isolated rat hindlimb perfusion model carrying xenografts of the human melanoma cell line MM96 was used to study the effects of perfusion conditions on melphalan distribution. Krebs-Henseleit buffer and Hartmann's solution containing 4.7% bovine serum albumin (BSA) or 2.8% dextran 40 were used as perfusates. Melphalan concentrations in perfusate, tumour nodules and normal tissues were measured using high-performance liquid chromatography (HPLC). Increasing the perfusion flow rates (from 4 to 8 ml min(-1)) resulted in higher tissue blood flow (determined with 51Cr-labelled microspheres) and melphalan uptake by tumour and normal tissues. The distribution of melphalan within tumour nodules and normal tissues was similar for both Krebs-Henseleit buffer and Hartmann's solution; however, tissue concentrations of melphalan were significantly higher for a perfusate containing 2.8% dextran 40 than for one containing 4.7% BSA. The melphalan concentration in the tumour was one-third of that found in the skin if the perfusate contained 4.7% BSA. In conclusion, this study has shown that a high perfusion flow enhances the delivery of melphalan into implanted tumour nodules and normal tissues, and a perfusate with low melphalan binding (no albumin) is preferred for maximum uptake of drug by the tumour. PMID:9099965

  6. Different metastasis patterns of a human melanoma cell line in nude mice and rats: Influence of microenvironment

    SciTech Connect

    Kjonniksen, I.; Hoifodt, H.K.; Pihl, A.; Fodstad, O. )

    1991-07-17

    The metastatic capacity of intravenously injected human FEMX-I melanoma cells in athymic nude mice and rats was compared. Young rats given 1 {times} 10(6) ascites tumor cells all died of lung tumors with a life span of 50 {plus minus} 10 days (mean{plus minus} SD). In contrast, in accordance with previous findings, only extrapulmonary metastases developed in mice. This host-dependent difference in metastasis pattern permitted studies on the role of factors that may influence the organ specificity of metastases. The tissue distribution of 125I-labeled FEMX-I cells did not differ in the two nude species during the first 12 hours after cell injection. The plating efficiency of FEMX-I cells in soft agar was increased by the addition of conditioned medium prepared from rat lungs, resulting also in a significant increase in colony size. In contrast, conditioned medium prepared from mouse lungs reduced the clonogenic capacity of the FEMX-I cells in a dose-dependent manner. Conditioned media prepared from rat and mouse liver, kidney, and spleen tissues either inhibited or had no effect on colony formation. The results suggest that the unexpected differential metastatic patterns observed in vivo may reflect differences in the presence of growth-modulating paracrine factors in the host lungs.

  7. Local distribution and concentration of intravenously injected sup 131 I-9. 2. 27 monoclonal antibody in human malignant melanoma

    SciTech Connect

    Del Vecchio, S.; Reynolds, J.C.; Carrasquillo, J.A.; Blasberg, R.G.; Neumann, R.D.; Lotze, M.T.; Bryant, G.J.; Farkas, R.J.; Larson, S.M. )

    1989-05-15

    Regional measurements of {sup 131}I-9.2.27 distribution in human melanoma tumors were obtained using quantitative autoradiography. Tumors were removed from patients 72-96 h after they had received an i.v. injection of 9.15 mCi (100 mg) of {sup 131}I-9.2.27. The autoradiographic images showed that the radioactivity reaching the tumor was heterogeneously distributed. Areas of relative high and low uptake were selected in each tumor. Regions of high activity contained from 51 to 1371 nCi/g, while areas with low uptake had radioactivity ranging from 12 to 487 nCi/g. The reliability of the autoradiographic measurements was demonstrated by the strong positive correlation with direct tissue sample counting (r = 0.994 P less than 0.001). Since comparative immunocytochemistry showed a homogeneous and diffuse staining of target antigen on viable tumor cells, variability of monoclonal antibody uptake within individual tumors was not primarily due to heterogeneity of antigen expression in these cases. However, antigen levels accounted for some of the variation from tumor to tumor. When immunoperoxidase staining was repeated on adjacent sections without the addition of 9.2.27, it confirmed the nonuniform distribution of monoclonal antibody found at autoradiography. Thus, quantitative autoradiography gives information about the distribution and the local concentration of radioactive antibody in tumors allowing calculation of the radiation dose delivered to small regions within tumors.

  8. An Unusual Conformation of MSH Analogues Leads to a Selective Human Melanocortin 1 Receptor Antagonist for Targeting Melanoma Cells

    PubMed Central

    Cai, Minying; Stankova, Magda; Muthu, Dhanasekaran; Mayorov, Alexander; Yang, Zhehui; Trivedi, Devendra; Cabello, Christopher; Hruby, Victor J.

    2013-01-01

    γ-MSH (γ-melanocyte-stimulating hormone: H-Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH), with its exquisite specificity and potency, has recently created much excitement as a drug lead. However, this peptide like most peptides susceptible to proteolysis in vivo which potentially decreases its beneficial activities. In our continued effort to design a proteolytically stable with specific receptor binding ligand, we have engineered peptides by cyclizing γ-MSH using a thioether bridge. A number of novel cyclic truncated γ-MSH analogues were designed and synthesized, in which a thioether bridge was incorporated between a cysteine side chain and an N-terminal bromoacyl group. One of these peptides, cyclo-[(CH2)3CO-Gly1-His2-D-Phe3-Arg4-D-Trp5-Cys(S-)6]-Asp7-Arg8-Phe9-Gly10-NH2, demonstrated potent antagonist activity and receptor selectivity for the human melanocortin 1 receptor (hMC1R) (IC50 = 17 nM). This novel peptide is the most selective antagonist for the human hMC1R to date. Further pharmacological studies have shown that this peptide can specifically target melanoma cells. The NMR analysis of this peptide in a membrane–like environment revealed a new turn structure, specific to the hMC1R antagonist, at the C terminal, wherein the side chain and backbone conformation of D-Trp5 and Phe9 of the peptide are contributors to the hMC1R selectivity. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine. PMID:23276279

  9. AMP kinase-related kinase NUAK2 affects tumor growth, migration, and clinical outcome of human melanoma

    PubMed Central

    Namiki, Takeshi; Tanemura, Atsushi; Valencia, Julio C.; Coelho, Sergio G.; Passeron, Thierry; Kawaguchi, Masakazu; Vieira, Wilfred D.; Ishikawa, Masashi; Nishijima, Wataru; Izumo, Toshiyuki; Kaneko, Yasuhiko; Katayama, Ichiro; Yamaguchi, Yuji; Yin, Lanlan; Polley, Eric C.; Liu, Hongfang; Kawakami, Yutaka; Eishi, Yoshinobu; Takahashi, Eishi; Yokozeki, Hiroo; Hearing, Vincent J.

    2011-01-01

    The identification of genes that participate in melanomagenesis should suggest strategies for developing therapeutic modalities. We used a public array comparative genomic hybridization (CGH) database and real-time quantitative PCR (qPCR) analyses to identify the AMP kinase (AMPK)-related kinase NUAK2 as a candidate gene for melanomagenesis, and we analyzed its functions in melanoma cells. Our analyses had identified a locus at 1q32 where genomic gain is strongly associated with tumor thickness, and we used real-time qPCR analyses and regression analyses to identify NUAK2 as a candidate gene at that locus. Associations of relapse-free survival and overall survival of 92 primary melanoma patients with NUAK2 expression measured using immunohistochemistry were investigated using Kaplan–Meier curves, log rank tests, and Cox regression models. Knockdown of NUAK2 induces senescence and reduces S-phase, decreases migration, and down-regulates expression of mammalian target of rapamycin (mTOR). In vivo analysis demonstrated that knockdown of NUAK2 suppresses melanoma tumor growth in mice. Survival analysis showed that the risk of relapse is greater in acral melanoma patients with high levels of NUAK2 expression than in acral melanoma patients with low levels of NUAK2 expression (hazard ratio = 3.88; 95% confidence interval = 1.44–10.50; P = 0.0075). These data demonstrate that NUAK2 expression is significantly associated with the oncogenic features of melanoma cells and with the survival of acral melanoma patients. NUAK2 may provide a drug target to suppress melanoma progression. This study further supports the importance of NUAK2 in cancer development and tumor progression, while AMPK has antioncogenic properties. PMID:21460252

  10. [Vulvar melanoma].

    PubMed

    Chokoeva, A; Tchernev, G; Wollina, U

    2015-01-01

    Malignant melanoma of the vulva is a rare disease with aggressive behavior and poor prognosis. It consist < 5% of all cases of melanoma in females, as the ratio of its manifestation, compared with the cutaneous melanoma is 1:71. Higher risk of developing melanoma of the vulva is established in white women, as the peak of the incidence is between 60 and 70 years of age. Clinically, MM of the vulva manifests as asymptomatic pigmented, rarely a pigmented lesion, as the usual clinical form is superficial spreading MM and much less common nodular MM, which is associated with a poorer prognosis in. general. The diagnosis is confirmed by histological examination. Conduction of PCR and DNA analysis for detection of BRAF mutations, NRAS mutations and KIT amplification is also appropriate. Advanced age, black race, tumor size, tumor thickness, ulceration, presence of satellite lesions, involvement of adjacent organs (vagina, urethra), and the presence of regional or distant metastases are identified as the most important prognostic markers. Radical wide excision followed by bilateral lymphadenectomy id considered as the optimal therapeutic approach. PMID:25909143

  11. Malignant Melanoma of the Foot

    MedlinePlus

    ... Javascript in your browser. Malignant Melanoma of the Foot What is Malignant Melanoma? Melanoma is a cancer ... age groups, even the young. Melanoma in the Foot Melanoma that occurs in the foot or ankle ...

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

    PubMed Central

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

    2015-01-01

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

  13. Genetic and environmental melanoma models in fish

    PubMed Central

    Patton, E Elizabeth; Mitchell, David L; Nairn, Rodney S

    2010-01-01

    Experimental animal models are extremely valuable for the study of human diseases, especially those with underlying genetic components. The exploitation of various animal models, from fruitflies to mice, has led to major advances in our understanding of the etiologies of many diseases, including cancer. Cutaneous malignant melanoma is a form of cancer for which both environmental insult (i.e., UV) and hereditary predisposition are major causative factors. Fish melanoma models have been used in studies of both spontaneous and induced melanoma formation. Genetic hybrids between platyfish and swordtails, different species of the genus Xiphophorus, have been studied since the 1920s to identify genetic determinants of pigmentation and melanoma formation. Recently, transgenesis has been used to develop zebrafish and medaka models for melanoma research. This review will provide a historical perspective on the use of fish models in melanoma research, and an updated summary of current and prospective studies using these unique experimental systems. PMID:20230482

  14. Killing of melanoma cells and their metastases by human lactoferricin derivatives requires interaction with the cancer marker phosphatidylserine.

    PubMed

    Riedl, Sabrina; Rinner, Beate; Schaider, Helmut; Lohner, Karl; Zweytick, Dagmar

    2014-10-01

    Despite favorable advancements in therapy cancer is still not curative in many cases, which is often due to inadequate specificity for tumor cells. In this study derivatives of a short cationic peptide derived from the human host defense peptide lactoferricin were optimized in their selective toxicity towards cancer cells. We proved that the target of these peptides is the negatively charged membrane lipid phosphatidylserine (PS), specifically exposed on the surface of cancer cells. We have studied the membrane interaction of three peptides namely LF11-322, its N-acyl derivative 6-methyloctanoyl-LF11-322 and its retro repeat derivative R(etro)-DIM-P-LF11-322 with liposomes mimicking cancerous and non-cancerous cell membranes composed of PS and phosphatidylcholine (PC), respectively. Calorimetric and permeability studies showed that N-acylation and even more the repeat derivative of LF11-322 leads to strongly improved interaction with the cancer mimic PS, whereas only the N-acyl derivative also slightly affects PC. Tryptophan fluorescence of selective peptide R-DIM-P-LF11-322 revealed specific peptide penetration into the PS membrane interface and circular dichroism showed change of its secondary structure by increase of proportion of β-sheets just in the presence of the cancer mimic. Data correlated with in vitro studies with cell lines of human melanomas, their metastases and melanocytes, revealing R-DIM-P-LF11-322 to exhibit strongly increased specificity for cancer cells. This indicates the need of high affinity to the target PS, a minimum length and net positive charge, an adequate but moderate hydrophobicity, and capability of adoption of a defined structure exclusively in presence of the target membrane for high antitumor activity. PMID:24838743

  15. Identification of Genes Potentially Regulated by Human Polynucleotide Phosphorylase (hPNPaseold-35) Using Melanoma as a Model

    PubMed Central

    Sokhi, Upneet K.; Bacolod, Manny D.; Dasgupta, Santanu; Emdad, Luni; Das, Swadesh K.; Dumur, Catherine I.; Miles, Michael F.; Sarkar, Devanand; Fisher, Paul B.

    2013-01-01

    Human Polynucleotide Phosphorylase (hPNPaseold-35 or PNPT1) is an evolutionarily conserved 3′→5′ exoribonuclease implicated in the regulation of numerous physiological processes including maintenance of mitochondrial homeostasis, mtRNA import and aging-associated inflammation. From an RNase perspective, little is known about the RNA or miRNA species it targets for degradation or whose expression it regulates; except for c-myc and miR-221. To further elucidate the functional implications of hPNPaseold-35 in cellular physiology, we knocked-down and overexpressed hPNPaseold-35 in human melanoma cells and performed gene expression analyses to identify differentially expressed transcripts. Ingenuity Pathway Analysis indicated that knockdown of hPNPaseold-35 resulted in significant gene expression changes associated with mitochondrial dysfunction and cholesterol biosynthesis; whereas overexpression of hPNPaseold-35 caused global changes in cell-cycle related functions. Additionally, comparative gene expression analyses between our hPNPaseold-35 knockdown and overexpression datasets allowed us to identify 77 potential “direct” and 61 potential “indirect” targets of hPNPaseold-35 which formed correlated networks enriched for cell-cycle and wound healing functional association, respectively. These results provide a comprehensive database of genes responsive to hPNPaseold-35 expression levels; along with the identification new potential candidate genes offering fresh insight into cellular pathways regulated by PNPT1 and which may be used in the future for possible therapeutic intervention in mitochondrial- or inflammation-associated disease phenotypes. PMID:24143183

  16. In vitro effect of histamine and histamine H1 and H2 receptor antagonists on cellular proliferation of human malignant melanoma cell lines.

    PubMed

    Reynolds, J L; Akhter, J; Morris, D L

    1996-04-01

    Histamine is an established growth factor for gastric and colorectal cancer. Contradictory data for response of melanoma to histamine have been reported. Our aims were to determine the effect of histamine and H1 and H2 receptor antagonists on cell growth and cyclic AMP production. Four human melanoma cell lines were cultured with a range of concentrations of histamine, and with the H2 receptor antagonists cimetidine, ranitidine or famotidine, or the H1 receptor antagonist diphenhydramine. Cellular proliferation was measured by the uptake of [3H]-thymidine. Cyclic AMP production was also measured to determine the receptor status of the cell lines. Histamine significantly stimulated growth in two of four cell lines, with maximal stimulation at 1 x 10(-8) M. This effect was inhibited by all four antagonists in a dose-dependent manner. Histamine [10(-7) to 10(-4) M] also induced a dose-dependent increase in cyclic AMP production in the two histamine-responsive cell lines, suggesting that these cell lines express H2 receptors. We conclude that there may be a role for histamine receptor antagonists in melanoma treatment and that further investigation is warranted. PMID:8791266

  17. The constitutive level of vascular endothelial growth factor (VEGF) is more important than hypoxia-induced VEGF up-regulation in the angiogenesis of human melanoma xenografts

    PubMed Central

    Danielsen, T; Rofstad, E K

    2000-01-01

    Angiogenesis of tumours might develop as a result of environmental conditions, such as hypoxia, and/or as a result of genetic alterations specific for tumour cells. The relative contributions of these mechanisms were investigated by comparing the in vivo expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) to the hypoxic fraction, the angiogenic potential and the vascular density of four human melanoma lines (A-07, D-12, R-18, U-25) grown intradermally in Balb/c nu/nu mice. VEGF expression, bFGF expression and expression of pimonidazole, a marker of hypoxic cells, were investigated by immunohistochemistry. An association between high VEGF and bFGF expression and high angiogenic potential was detected, suggesting an important role for VEGF/bFGF in the angiogenesis of melanomas. High VEGF/bFGF expression was also related to low hypoxic fraction and high vascular density. Thus, the constitutive, genetically determined level of VEGF was probably more important than hypoxia-induced upregulation in the angiogenesis of the melanoma xenografts. © 2000 Cancer Research Campaign PMID:10789719

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

    PubMed

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

    2016-01-01

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

  19. Basal and treatment-induced activation of AKT mediates resistance to cell death by AZD6244 (ARRY-142886) in Braf-mutant human cutaneous melanoma cells

    PubMed Central

    Gopal, Y.N. Vashisht; Deng, Wanleng; Woodman, Scott E.; Komurov, Kakajan; Ram, Prahlad; Smith, Paul D.; Davies, Michael A.

    2014-01-01

    The majority of melanomas demonstrate constitutive activation of the RAS-RAF-MEK-MAPK pathway. AZD6244 is a selective MEK1/2 inhibitor which markedly reduces tumor P-MAPK levels, but it produced few clinical responses in melanoma patients. An improved understanding of the determinants of resistance to AZD6244 may lead to improved patient selection and effective combinatorial approaches. The effects of AZD6244 on cell growth and survival were tested in a total of 14 Braf-mutant and 3 wild-type human cutaneous melanoma cell lines. Quantitative assessment of phospho-protein levels in the Braf-mutant cell lines by reverse phase protein array (RPPA) analysis showed no significant association between P-MEK or P-MAPK levels and AZD6244 sensitivity, but activation-specific markers in the PI3K-AKT pathway correlated with resistance. We also identified resistant cell lines without basal activation of the PI3K-AKT pathway. RPPA characterization of the time-dependent changes in signaling pathways revealed that AZD6244 produced durable and potent inhibition of P-MAPK in sensitive and resistant Braf-mutant cell lines, but several resistant lines demonstrated AZD6244-induced activation of AKT. In contrast, sensitive cell lines demonstrated AZD6244 treatment-induced upregulation of PTEN protein and mRNA expression. Inhibition of AKT, TORC1/2, or IGF1R blocked AZD6244-induced activation of AKT and resulted in synergistic cell killing with AZD6244. These findings identify basal and treatment-induced regulation of the PI3K-AKT pathway as a critical regulator of AZD6244 sensitivity in Braf-mutant cutaneous melanoma cells, the novel regulation of PTEN expression by AZD6244 in sensitive cells, and suggest new combinatorial approaches for patients. PMID:20959481

  20. Inosine strongly enhances proliferation of human C32 melanoma cells through PLC-PKC-MEK1/2-ERK1/2 and PI3K pathways.

    PubMed

    Soares, Ana Sofia; Costa, Vera Marisa; Diniz, Carmen; Fresco, Paula

    2015-01-01

    Malignant melanoma is the most deadly type of skin cancer. The lack of effective pharmacological approaches for this tumour can be related to the incomplete understanding of the pathophysiological mechanisms involved in melanoma cell proliferation. Adenosine has growth-promoting and growth inhibitory effects on tumour cells. We aimed to investigate effects of adenosine and its metabolic product, inosine, on human C32 melanoma cells and the signalling pathways involved. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and bromodeoxyuridine (BrdU) proliferation assays were used to evaluate adenosine, adenosine deaminase and inosine effects, in the absence or presence of adenosine receptor (AR), A3 AR and P2Y1 R antagonists and PLC, PKC, MEK1/2 and PI3K inhibitors. ERK1/2 levels were determined using an ELISA kit. Adenosine and inosine levels were quantified using an enzyme-coupled assay. Adenosine caused cell proliferation through AR activation. Adenosine deaminase increased inosine levels (nanomolar concentrations) on the extracellular space, in a time-dependent manner, inducing proliferation through A3 AR activation. Micromolar concentrations of inosine enhanced proliferation through A3 AR activation, causing an increase in ERK1/2 levels, and P2Y1 R activation via ENT-dependent mechanisms. We propose the simultaneous activation of PLC-PKC-MEK1/2-ERK1/2 and PI3K pathways as the main mechanism responsible for the proliferative effect elicited by inosine and its significant role in melanoma cancer progression. PMID:24909096

  1. Human hepatocytes express absent in melanoma 2 and respond to hepatitis B virus with interleukin-18 expression.

    PubMed

    Pan, Xingfei; Xu, Haixia; Zheng, Changlong; Li, Mei; Zou, Xiaofang; Cao, Hong; Xu, Qihuan

    2016-08-01

    Absent in melanoma 2 (AIM2) is a recently recognized cytoplasmic receptor which could sense cytoplasmic double-stranded DNA (dsDNA). After AIM2 detects the presence of parasitic nucleic acids (dsDNA) derived from invasive bacteria or viral genomes (for example, vaccinia virus and cytomegalovirus) within infected cells, AIM2 inflammasome could be formed. The formed AIM2 inflammasome could induce innate immune response and increase expressions of IL-1β and IL-18. Hepatitis B virus (HBV) is a hepatotropic, non-cytopathic double-stranded DNA virus. The immune response to viral antigens or virus is thought to be responsible for both liver damage and viral clearance in patients with HBV infection. However, there are no reports about whether AIM2 inflammasome exists in hepatocytes. In the present study, we investigated the presence and activity of AIM2 inflammasome in human hepatocytes. We found that AIM2 was expressed in cytoplasm of hepatocytes, and IL-18 expression was increased after AIM2 sensed HBV in hepatocytes in vitro. These results showed that AIM2 inflammasome was active in hepatocytes. We also found that hepatic AIM2 expression of chronic hepatitis B (CHB) patients was higher than that of controls. Hepatic AIM2 expression levels were positively correlated to the severity of liver inflammation. IL-18 is already considered to be associated with hepatic injury during HBV infection. In conclusion, we, therefore, believe that AIM2 inflammasome in hepatocytes might play an important role in the development and maintenance of HBV-related hepatitis. PMID:27094165

  2. Bonded Cumomer Analysis of Human Melanoma Metabolism Monitored by 13C NMR Spectroscopy of Perfused Tumor Cells.

    PubMed

    Shestov, Alexander A; Mancuso, Anthony; Lee, Seung-Cheol; Guo, Lili; Nelson, David S; Roman, Jeffrey C; Henry, Pierre-Gilles; Leeper, Dennis B; Blair, Ian A; Glickson, Jerry D

    2016-03-01

    A network model for the determination of tumor metabolic fluxes from (13)C NMR kinetic isotopomer data has been developed and validated with perfused human DB-1 melanoma cells carrying the BRAF V600E mutation, which promotes oxidative metabolism. The model generated in the bonded cumomer formalism describes key pathways of tumor intermediary metabolism and yields dynamic curves for positional isotopic enrichment and spin-spin multiplets. Cells attached to microcarrier beads were perfused with 26 mm [1,6-(13)C2]glucose under normoxic conditions at 37 °C and monitored by (13)C NMR spectroscopy. Excellent agreement between model-predicted and experimentally measured values of the rates of oxygen and glucose consumption, lactate production, and glutamate pool size validated the model. ATP production by glycolytic and oxidative metabolism were compared under hyperglycemic normoxic conditions; 51% of the energy came from oxidative phosphorylation and 49% came from glycolysis. Even though the rate of glutamine uptake was ∼ 50% of the tricarboxylic acid cycle flux, the rate of ATP production from glutamine was essentially zero (no glutaminolysis). De novo fatty acid production was ∼ 6% of the tricarboxylic acid cycle flux. The oxidative pentose phosphate pathway flux was 3.6% of glycolysis, and three non-oxidative pentose phosphate pathway exchange fluxes were calculated. Mass spectrometry was then used to compare fluxes through various pathways under hyperglycemic (26 mm) and euglycemic (5 mm) conditions. Under euglycemic conditions glutamine uptake doubled, but ATP production from glutamine did not significantly change. A new parameter measuring the Warburg effect (the ratio of lactate production flux to pyruvate influx through the mitochondrial pyruvate carrier) was calculated to be 21, close to upper limit of oxidative metabolism. PMID:26703469

  3. Effects of verapamil and alcohol on blood flow, melphalan uptake and cytotoxicity, in murine fibrosarcomas and human melanoma xenografts.

    PubMed

    Robinson, B A; Clutterbuck, R D; Millar, J L; McElwain, T J

    1986-05-01

    Verapamil had previously been shown to increase cellular melphalan uptake and cytotoxicity in fibrosarcomas, and increased the area under the blood concentration versus time curve (AUC) for melphalan in CBA mice. Verapamil (10 mg kg-1 i.p.) had no effect on the fractional distribution of cardiac output (FDCO), measured with 86Rb-rubidium chloride, to subcutaneous fibrosarcomas. 14C-Melphalan uptake by FS13 fibrosarcomas was increased 60 min after verapamil (10 mg kg-1 i.p.), but not after lower doses which did not affect the AUC. Flunarizine (5 mg kg-1 i.p.) also had no effect on FDCO to FS13 fibrosarcomas, and tended to increase 14C-melphalan content of blood and the fibrosarcomas and to promote growth delay by melphalan. Alcohol increased FDCO to FS13 fibrosarcomas, maximally at a 1:20 dilution in saline, but had no effect on 14C-melphalan uptake or growth delay. Thus, melphalan cytotoxicity correlated with tumour melphalan uptake, and both followed changes in the AUC for melphalan but not changes in FDCO. In these murine fibrosarcomas melphalan uptake and cytotoxicity were not limited by blood flow. In subcutaneous human melanoma HX46 xenografts, verapamil had no effect on the FDCO, nor on 14C-melphalan uptake, and did not affect blood 14C-melphalan levels, suggesting absence of effects on the AUC and on cellular uptake. Alcohol did not increase the FDCO to HX46 xenografts, providing evidence for a different vascular supply. PMID:3718818

  4. Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection.

    PubMed Central

    Kawakami, Y; Eliyahu, S; Delgado, C H; Robbins, P F; Sakaguchi, K; Appella, E; Yannelli, J R; Adema, G J; Miki, T; Rosenberg, S A

    1994-01-01

    The cultured T-cell line TIL1200, established from the tumor-infiltrating lymphocytes (TILs) of a patient with advanced metastatic melanoma, recognized an antigen on most HLA-A2+ melanomas and on all HLA-A2+ cultured neonatal melanocytes in an HLA-A2 restricted manner but not on other types of tissues or cell lines tested. A cDNA encoding an antigen recognized by TIL1200 was isolated by screening an HLA-A2+ breast cancer cell line transfected with an expression cDNA library prepared from an HLA-A2+ melanoma cell line. The nucleotide and amino acid sequences of this cDNA were almost identical to the genes encoding glycoprotein gp100 or Pmel17 previously registered in the GenBank. Expression of this gene was restricted to melanoma and melanocyte cell lines and retina but was not expressed on other fresh or cultured normal tissues or other types of tumor tested. The cell line transfected with this cDNA also expressed antigen recognized by the melanoma-specific antibody HMB45 that bound to gp100. A synthetic 10-amino acid peptide derived from gp100 was recognized by TIL1200 in the context of HLA-A2.1. Since the administration of TIL1200 plus interleukin 2 resulted in regression of metastatic cancer in the autologous patient, gp100 is a possible tumor rejection antigen and may be useful for the development of immunotherapies for patients with melanoma. Images PMID:8022805

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2016-02-16

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

  7. 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

  8. Preparation and characterization of a novel Al(18)F-NOTA-BZA conjugate for melanin-targeted imaging of malignant melanoma.

    PubMed

    Chang, Chih-Chao; Chang, Chih-Hsien; Lo, Yi-Hsuan; Lin, Ming-Hsien; Shen, Chih-Chieh; Liu, Ren-Shyan; Wang, Hsin-Ell; Chen, Chuan-Lin

    2016-08-15

    Melanin is an attractive target for the diagnosis and treatment of malignant melanoma. Previous studies have demonstrated the specific binding ability of benzamide moiety to melanin. In this study, we developed a novel (18)F-labeled NOTA-benzamide conjugate, Al(18)F-NOTA-BZA, which can be synthesized in 30min with a radiochemical yield of 20-35% and a radiochemical purity of >95%. Al(18)F-NOTA-BZA is highly hydrophilic (logP=-1.96) and shows good in vitro stability. Intravenous administration of Al(18)F-NOTA-BZA in two melanoma-bearing mouse models revealed highly specific uptake in B16F0 melanotic melanoma (6.67±0.91 and 1.50±0.26%ID/g at 15 and 120min p.i., respectively), but not in A375 amelanotic melanoma (0.87±0.21 and 0.24±0.09%ID/g at 15 and 120min p.i., respectively). The clearance from most normal tissues was fast. A microPET scan of Al(18)F-NOTA-BZA-injected mice also displayed high-contrast tumor images as compared with normal organs. Owing to the favorable in vivo distribution of Al(18)F-NOTA-BZA after intravenous administration, the estimated absorption dose was low in all normal organs and tissues. The melanin-specific binding ability, sustained tumor retention, fast normal tissues clearance and thelow projected human dosimetry supported that Al(18)F-NOTA-BZA is a very promising melanin-specific PET probe for melanin-positive melanoma. PMID:27445169

  9. Transcutaneous delivery of leflunomide nanoemulgel: Mechanistic investigation into physicomechanical characteristics, in vitro anti-psoriatic and anti-melanoma activity.

    PubMed

    Pund, Swati; Pawar, Satish; Gangurde, Shashikant; Divate, Deepali

    2015-06-20

    The present study is a mechanistic validation of 'proof of concept' of effective topical delivery of leflunomide (LFD) nanoemulgel for localized efficient treatment of psoriatic lesions as well as melanoma affected skin regions. Hyperproliferation of keratinocytes in psoriasis and symbiotic relationship between keratinocytes and melanocytes, justifies the need of dual acting treatment. LFD is recently introduced significantly effective disease modifying anti-rheumatic drug and has been considered valuable for the treatment of psoriatic arthritis as well as melanoma. Current available treatments for psoriasis and melanoma are inefficient due to systemic side effects, poor transcutaneous permeation and thus present a challenge for development of novel colloidal carriers. We newly reformulated LFD as a nanoemulgel based on self nanoemulsifying technique using Capryol 90, Cremophor EL, Transcutol HP as nanoemulsifying components and Pluronic F127 as a gelling agent. This thermodynamically stable nanoemuslsifying preconcentrate after gelation showed mean globule size, 123.7 nm and viscosity 9620 ± 93 cp. Complete mechanical characterization was carried out using Texture Analyzer and hardness, adhesiveness and springiness index were found to be 523 gms, 431 gms and 1.02, respectively. Ex vivo permeation through rat abdominal skin revealed significant improvement in flux, apparent permeability coefficient, steady state diffusion coefficient and drug deposition in skin due to nanoemulsification of LFD. The in vitro cytoxicity of LFD nanoemulgel in human HaCaT, melanoma A375 and SK-MEL-2 cell lines showed significantly enhanced therapeutic response. In gist, LFD nanoemulgel for trancutaneous delivery will reduce the overall dose and drug consumption, by effectively localizing at the applied target site and will ultimately minimize systemic side effects. PMID:25869452

  10. Malicious masquerade: myxoid melanoma.

    PubMed

    Hitchcock, M G; White, W L

    1998-08-01

    The morphological spectrum of malignant melanoma is broad. Unusual stromal changes can distract pathologists from the correct diagnosis. Fibroblastic, chondroid, osteoid, and myxoid stroma have been documented in melanomas. Myxoid melanoma is problematic--introducing carcinomas and soft tissue sarcomas into the differential diagnosis. This review examines the clinicopathologic aspects of myxoid malignant melanoma, with emphasis on its differential diagnosis. PMID:9711669

  11. Endothelin-1 in the tumor microenvironment correlates with melanoma invasion.

    PubMed

    Chiriboga, Luis; Meehan, Shane; Osman, Iman; Glick, Michael; de la Cruz, Gelo; Howell, Brittny S; Friedman-Jiménez, George; Schneider, Robert J; Jamal, Sumayah

    2016-06-01

    Endothelin-1 (ET-1) is a vasoactive peptide that also plays a role in the tanning response of the skin. Animal and cell culture studies have also implicated ET-1 in melanoma progression, but no association studies have been performed to link ET-1 expression and melanoma in humans. Here, we present the first in-vivo study of ET-1 expression in pigmented lesions in humans: an ET-1 immunohistochemical screen of melanocytic nevi, melanoma in situ lesions, invasive melanomas, metastatic melanomas, and blue nevi was performed. Twenty-six percent of melanocytic nevi and 44% of melanoma in situ lesions demonstrate ET-1 expression in the perilesional microenvironment, whereas expression in nevus or melanoma cells was rare to absent. In striking contrast, 100% of moderately to highly pigmented invasive melanomas contained numerous ET-1-positive cells in the tumor microenvironment, with 79% containing ET-1-positive melanoma cells, confirmed by co-staining with melanoma tumor marker HMB45. Hypopigmented invasive melanomas had reduced ET-1 expression, suggesting a correlation between ET-1 expression and pigmented melanomas. ET-1-positive perilesional cells were CD68-positive, indicating macrophage origin. Sixty-two percent of highly pigmented metastatic melanomas demonstrated ET-1 expression in melanoma cells, in contrast to 28.2% of hypopigmented specimens. Eighty-nine percent of benign nevi, known as blue nevi, which have a dermal localization, were associated with numerous ET-1-positive macrophages in the perilesional microenvironment, but no ET-1 expression was detected in the melanocytes. We conclude that ET-1 expression in the microenvironment increases with advancing stages of melanocyte transformation, implicating a critical role for ET-1 in melanoma progression, and the importance of the tumor microenvironment in the melanoma phenotype. PMID:26825037

  12. Sema6A and Mical1 control cell growth and survival of BRAFV600E human melanoma cells

    PubMed Central

    Loria, Rossella; Bon, Giulia; Perotti, Valentina; Gallo, Enzo; Bersani, Ilaria; Baldassari, Paola; Porru, Manuela; Leonetti, Carlo; Di Carlo, Selene; Visca, Paolo; Brizzi, Maria Felice; Anichini, Andrea; Mortarini, Roberta; Falcioni, Rita

    2015-01-01

    We used whole genome microarray analysis to identify potential candidate genes with differential expression in BRAFV600E vs NRASQ61R melanoma cells. We selected, for comparison, a peculiar model based on melanoma clones, isolated from a single tumor characterized by mutually exclusive expression of BRAFV600E and NRASQ61R in different cells. This effort led us to identify two genes, SEMA6A and MICAL1, highly expressed in BRAF-mutant vs NRAS-mutant clones. Real-time PCR, Western blot and immunohistochemistry confirmed preferential expression of Sema6A and Mical1 in BRAFV600E melanoma. Sema6A is a member of the semaphorin family, and it complexes with the plexins to regulate actin cytoskeleton, motility and cell proliferation. Silencing of Sema6A in BRAF-mutant cells caused cytoskeletal remodeling, and loss of stress fibers, that in turn induced cell death. Furthermore, Sema6A depletion caused loss of anchorage-independent growth, inhibition of chemotaxis and invasion. Forced Sema6A overexpression, in NRASQ61R clones, induced anchorage-independent growth, and a significant increase of invasiveness. Mical1, that links Sema/PlexinA signaling, is also a negative regulator of apoptosis. Indeed, Mical-1 depletion in BRAF mutant cells restored MST-1-dependent NDR phosphorylation and promoted a rapid and massive NDR-dependent apoptosis. Overall, our data suggest that Sema6A and Mical1 may represent new potential therapeutic targets in BRAFV600E melanoma. PMID:25576923

  13. Erratum to “Comment on “Geometry effect on the magnetic ordering of geometrically frustrated rectangular and triangular magnets” [Phys. Lett. A 375 (13) (2011) 1548]” [Phys. Lett. A 375 (27) (2011) 2680-2681

    NASA Astrophysics Data System (ADS)

    Nascimento, F. S.; Mól, L. A. S.; Pereira, A. R.; Moura-Melo, W. A.

    2012-10-01

    In a recent comment [Phys. Lett. A 375 (2011) 2680] some of us argued that a misleading evaluation of dipolar interactions in spin ice systems studied by Li et al. [Phys. Lett. A 375 (2011) 1548], does not lead to the ground-state transitions that they observed. However, a bug found in our computational code showed that there is indeed the predicted transitions even for a proper evaluation of dipolar interactions.

  14. Heterogeneity in Melanoma.

    PubMed

    Shannan, Batool; Perego, Michela; Somasundaram, Rajasekharan; Herlyn, Meenhard

    2016-01-01

    Melanoma is among the most aggressive and therapy-resistant human cancers. While great strides in therapy have generated enthusiasm, many challenges remain. Heterogeneity is the most pressing issue for all types of therapy. This chapter summarizes the clinical classification of melanoma, of which the research community now adds additional layers of classifications for better diagnosis and prediction of therapy response. As the search for new biomarkers increases, we expect that biomarker analyses will be essential for all clinical trials to better select patient populations for optimal therapy. While individualized therapy that is based on extensive biomarker analyses is an option, we expect in the future genetic and biologic biomarkers will allow grouping of melanomas in such a way that we can predict therapy outcome. At this time, tumor heterogeneity continues to be the major challenge leading inevitably to relapse. To address heterogeneity therapeutically, we need to develop complex therapies that eliminate the bulk of the tumor and, at the same time, the critical subpopulations. PMID:26601857

  15. Doxorubicin conjugated with a monoclonal antibody directed to a human melanoma-associated proteoglycan suppresses the growth of established tumor xenografts in nude mice.

    PubMed Central

    Yang, H M; Reisfeld, R A

    1988-01-01

    Doxorubicin (DXR) was covalently conjugated to a monoclonal antibody (mAb), 9.2.27 (IgG2a), which recognizes a chondroitin sulfate proteoglycan expressed preferentially on the surface of human melanoma cells. Immunoconjugates with a molar ratio of DXR to mAb ranging from 2:1 to 10:1 were obtained by coupling the drug via an acid-sensitive linker, cis-aconitic anhydride. The immunoreactivity of mAb 9.2.27 was well retained after conjugation. DXR-mAb 9.2.27 conjugates were found to be 2 orders of magnitude more potent in killing tumor cells in vitro (IC50 = 0.1 microM) than free drug targeted to drug receptor(s). Most significantly, DXR-mAb 9.2.27 immunoconjugates specifically suppressed the growth of established tumors in vivo and prolonged the life-span of tumor-bearing nude mice. This suppression of melanoma growth achieved by the immunoconjugate was both tumor and antibody specific. A biodistribution study indicated that DXR-mAb 9.2.27 conjugates delivered at least 4 times more DXR (3.7% total injected dose per g of tumor) as compared to free DXR alone (0.8% total injected dose per g of tumor) in tumor-bearing nude mice 48 hr postinjection. The tumor-suppressive effects of DXR-mAb 9.2.27 conjugates are even more remarkable since free DXR did not suppress tumor growth in vivo and also because this drug per se is known to be quite ineffective for the treatment of human melanoma. Images PMID:3422487

  16. pO{sub 2} Fluctuation Pattern and Cycling Hypoxia in Human Cervical Carcinoma and Melanoma Xenografts

    SciTech Connect

    Ellingsen, Christine; Ovrebo, Kirsti Marie; Galappathi, Kanthi; Mathiesen, Berit; Rofstad, Einar K.

    2012-07-15

    Purpose: Blood perfusion in tumors is spatially and temporally heterogeneous, resulting in local fluctuations in tissue oxygen tension (pO{sub 2}) and tissue regions showing cycling hypoxia. In this study, we investigated whether the pO{sub 2} fluctuation pattern and the extent of cycling hypoxia differ between tumor types showing high (e.g., cervical carcinoma xenograft) and low (e.g., melanoma xenograft) fractions of connective tissue-associated blood vessels. Methods and Materials: Two cervical carcinoma lines (CK-160 and TS-415) and two melanoma lines (A-07 and R-18) transplanted into BALB/c nu/nu mice were included in the study. Tissue pO{sub 2} was measured simultaneously in two positions in each tumor by using a two-channel OxyLite fiber-optic oxygen-sensing device. The extent of acute and chronic hypoxia was assessed by combining a radiobiological and a pimonidazole-based immunohistochemical assay of tumor hypoxia. Results: The proportion of tumor regions showing pO{sub 2} fluctuations, the pO{sub 2} fluctuation frequency in these regions, and the relative amplitude of the pO{sub 2} fluctuations were significantly higher in the melanoma xenografts than in the cervical carcinoma xenografts. Cervical carcinoma and melanoma xenografts did not differ significantly in the fraction of acutely hypoxic cells or the fraction of chronically hypoxic cells. However, the ratio between fraction of acutely hypoxic cells and fraction of chronically hypoxic cells was significantly higher in melanoma than in cervical carcinoma xenografts. Conclusions: Temporal heterogeneity in blood flow and tissue pO{sub 2} in tumors may depend on tumor histology. Connective tissue surrounding microvessels may stabilize blood flow and pO{sub 2} and, thus, protect tumor tissue from cycling hypoxia.

  17. Methylation-mediated loss of SFRP2 enhances melanoma cell invasion via Wnt signaling.

    PubMed

    Luo, Xiaoji; Wei, Bin; Chen, Aijun; Zhao, Hengguang; Huang, Kun; Chen, Jin

    2016-01-01

    Wnt signaling plays an essential role in the initiation and progression of melanoma tumors. The Secreted Frizzled Related Proteins (SFRPs) are a family of proteins that suppress Wnt signaling. The methylation of SFRPs reduces their activity, and hence augments Wnt signaling. However, whether the methylation of SFRP2, a member of SFRPs, may be involved in the pathogenesis of melanoma is not known. Here we investigated the expression levels of SFRP2 in melanoma specimens. We found that SFRP2 mRNA wassignificantly decreased and methylation of SFRP2 gene was significantly increased in malignant melanoma tumors ascompared to the paired adjacent non-tumor tissue. Moreover, SFRP2 expression was significantly decreased in the malignant melanoma celllines, HTB63, A2058 and A375, but not in the non-transformed melanocyte cell line, Hermes 3A. The demethylation of SFRP2 gene by 5'-aza-deoxycytidine (5-aza-dCyd) in melanoma cell lines restored SFRP2 expression, at both mRNA and protein levels, and suppressed cell invasion. Furthermore, the demethylation of SFRP2 geneappeared to inhibit nuclear retention of a key Wnt signaling factor, β-catenin, in melanoma cell lines. Together, these data suggest that SFRP2may function as a melanoma invasion suppressor byinterfering with Wnt signaling, and the methylation of SFRP2 gene may promote pathogenesis of melanoma. PMID:27186276

  18. Methylation-mediated loss of SFRP2 enhances melanoma cell invasion via Wnt signaling

    PubMed Central

    Luo, Xiaoji; Wei, Bin; Chen, Aijun; Zhao, Hengguang; Huang, Kun; Chen, Jin

    2016-01-01

    Wnt signaling plays an essential role in the initiation and progression of melanoma tumors. The Secreted Frizzled Related Proteins (SFRPs) are a family of proteins that suppress Wnt signaling. The methylation of SFRPs reduces their activity, and hence augments Wnt signaling. However, whether the methylation of SFRP2, a member of SFRPs, may be involved in the pathogenesis of melanoma is not known. Here we investigated the expression levels of SFRP2 in melanoma specimens. We found that SFRP2 mRNA wassignificantly decreased and methylation of SFRP2 gene was significantly increased in malignant melanoma tumors ascompared to the paired adjacent non-tumor tissue. Moreover, SFRP2 expression was significantly decreased in the malignant melanoma celllines, HTB63, A2058 and A375, but not in the non-transformed melanocyte cell line, Hermes 3A. The demethylation of SFRP2 gene by 5’-aza-deoxycytidine (5-aza-dCyd) in melanoma cell lines restored SFRP2 expression, at both mRNA and protein levels, and suppressed cell invasion. Furthermore, the demethylation of SFRP2 geneappeared to inhibit nuclear retention of a key Wnt signaling factor, β-catenin, in melanoma cell lines. Together, these data suggest that SFRP2may function as a melanoma invasion suppressor byinterfering with Wnt signaling, and the methylation of SFRP2 gene may promote pathogenesis of melanoma. PMID:27186276

  19. Recombinant Interferon Alfa-2b in Treating Patients With Melanoma

    ClinicalTrials.gov

    2016-05-17

    Stage IA Skin Melanoma; Stage IB Skin Melanoma; Stage IIA Skin Melanoma; Stage IIB Skin Melanoma; Stage IIC Skin Melanoma; Stage IIIA Skin Melanoma; Stage IIIB Skin Melanoma; Stage IIIC Skin Melanoma; Stage IV Skin Melanoma

  20. The Quinone Methide Aurin Is a Heat Shock Response Inducer That Causes Proteotoxic Stress and Noxa-dependent Apoptosis in Malignant Melanoma Cells*

    PubMed Central

    Davis, Angela L.; Qiao, Shuxi; Lesson, Jessica L.; Rojo de la Vega, Montserrat; Park, Sophia L.; Seanez, Carol M.; Gokhale, Vijay; Cabello, Christopher M.; Wondrak, Georg T.

    2015-01-01

    Pharmacological induction of proteotoxic stress is rapidly emerging as a promising strategy for cancer cell-directed chemotherapeutic intervention. Here, we describe the identification of a novel drug-like heat shock response inducer for the therapeutic induction of proteotoxic stress targeting malignant human melanoma cells. Screening a focused library of compounds containing redox-directed electrophilic pharmacophores employing the Stress & Toxicity PathwayFinderTM PCR Array technology as a discovery tool, a drug-like triphenylmethane-derivative (aurin; 4-[bis(p-hydroxyphenyl)methylene]-2,5-cyclohexadien-1-one) was identified as an experimental cell stress modulator that causes (i) heat shock factor transcriptional activation, (ii) up-regulation of heat shock response gene expression (HSPA6, HSPA1A, DNAJB4, HMOX1), (iii) early unfolded protein response signaling (phospho-PERK, phospho-eIF2α, CHOP (CCAAT/enhancer-binding protein homologous protein)), (iv) proteasome impairment with increased protein-ubiquitination, and (v) oxidative stress with glutathione depletion. Fluorescence polarization-based experiments revealed that aurin displays activity as a geldanamycin-competitive Hsp90α-antagonist, a finding further substantiated by molecular docking and ATPase inhibition analysis. Aurin exposure caused caspase-dependent cell death in a panel of human malignant melanoma cells (A375, G361, LOX-IMVI) but not in non-malignant human skin cells (Hs27 fibroblasts, HaCaT keratinocytes, primary melanocytes) undergoing the aurin-induced heat shock response without impairment of viability. Aurin-induced melanoma cell apoptosis depends on Noxa up-regulation as confirmed by siRNA rescue experiments demonstrating that siPMAIP1-based target down-regulation suppresses aurin-induced cell death. Taken together, our data suggest feasibility of apoptotic elimination of malignant melanoma cells using the quinone methide-derived heat shock response inducer aurin. PMID:25477506

  1. Simulated microgravity reduces mRNA levels of multidrug resistance genes 4 and 5 in non-metastatic human melanoma cells

    NASA Astrophysics Data System (ADS)

    Eiermann, Peter; Tsiockas, Wasiliki; Hauslage, Jens; Hemmersbach, Ruth; Gerzer, Rupert; Ivanova, Krassimira

    Multidrug resistance proteins (MRP) are members of the ATP-binding cassette transporter superfamily that are able to export a large variety of substances into the extracellular space in-cluding nucleoside and nucleotide base analogs used in antiviral and anticancer therapy. MRP4 and 5 (MRP4/5) particularly transport cyclic nucleotides, e.g. guanosine 3',5'-cyclic monophos-phate (cGMP). The second messenger cGMP, which is synthesized by the catalytic activity of the guanylyl cyclase (GC), plays an import role in vasodilatation, smooth muscle relaxation, and nitric oxide (NO)-induced perturbation of melanocyte-extracellular matrix interactions. In previous studies we have reported that different GC isoforms are responsible for cGMP synthe-sis in melanocytic cells. Normal human melanocytes and non-metastatic melanoma cell lines predominantly express the NO-sensitive soluble GC isoform (sGC), a heterodimeric protein consisting of α and β subunits. Metastatic melanoma cells lack the expression of the β sub-unit and show up-regulated activities of the particulate isoforms. We have further found that long-term exposure to hypergravity (5 g for 24 h) induced an increased cGMP export in normal human melanocytes, and non-metastatic, but not in metastatic human melanoma cells as a re-sult of up-regulated MRP4/5 expression. The aim of the present study is to investigate whether simulated microgravity may also alter the expression of MRP4/5 in non-metastatic melanoma cells. Experiments were performed using a fast-rotating clinostat (60 rpm) with one rotation axis. The non-metastatic 1F6 melanoma cells were exposed to simulated microgravity (up to 1.21x10-2 g) for 24 h. The mRNA analyses were performed by a relative calibrator-normalized and efficiency corrected quantitative polymerase chain reaction (Light Cycler R , Roche). Our data show a reduced expression of approximately 35% for MRP4 and of 50% for MRP5 in simulated microgravity in comparison to 1 g controls. Also, the

  2. Whole Body Melanoma Transcriptome Response in Medaka

    PubMed Central

    Schartl, Manfred; Shen, Yingjia; Maurus, Katja; Walter, Ron; Tomlinson, Chad; Wilson, Richard K.; Postlethwait, John; Warren, Wesley C.

    2015-01-01

    The incidence of malignant melanoma continues to increase each year with poor prognosis for survival in many relapse cases. To reverse this trend, whole body response measures are needed to discover collaborative paths to primary and secondary malignancy. Several species of fish provide excellent melanoma models because fish and human melanocytes both appear in the epidermis, and fish and human pigment cell tumors share conserved gene expression signatures. For the first time, we have examined the whole body transcriptome response to invasive melanoma as a prelude to using transcriptome profiling to screen for drugs in a medaka (Oryzias latipes) model. We generated RNA-seq data from whole body RNA isolates for controls and melanoma fish. After testing for differential expression, 396 genes had significantly different expression (adjusted p-value <0.02) in the whole body transcriptome between melanoma and control fish; 379 of these genes were matched to human orthologs with 233 having annotated human gene symbols and 14 matched genes that contain putative deleterious variants in human melanoma at varying levels of recurrence. A detailed canonical pathway evaluation for significant enrichment showed the top scoring pathway to be antigen presentation but also included the expected melanocyte development and pigmentation signaling pathway. Results revealed a profound down-regulation of genes involved in the immune response, especially the innate immune system. We hypothesize that the developing melanoma actively suppresses the immune system responses of the body in reacting to the invasive malignancy, and that this mal-adaptive response contributes to disease progression, a result that suggests our whole-body transcriptomic approach merits further use. In these findings, we also observed novel genes not yet identified in human melanoma expression studies and uncovered known and new candidate drug targets for further testing in this malignant melanoma medaka model. PMID

  3. Whole Body Melanoma Transcriptome Response in Medaka.

    PubMed

    Schartl, Manfred; Shen, Yingjia; Maurus, Katja; Walter, Ron; Tomlinson, Chad; Wilson, Richard K; Postlethwait, John; Warren, Wesley C

    2015-01-01

    The incidence of malignant melanoma continues to increase each year with poor prognosis for survival in many relapse cases. To reverse this trend, whole body response measures are needed to discover collaborative paths to primary and secondary malignancy. Several species of fish provide excellent melanoma models because fish and human melanocytes both appear in the epidermis, and fish and human pigment cell tumors share conserved gene expression signatures. For the first time, we have examined the whole body transcriptome response to invasive melanoma as a prelude to using transcriptome profiling to screen for drugs in a medaka (Oryzias latipes) model. We generated RNA-seq data from whole body RNA isolates for controls and melanoma fish. After testing for differential expression, 396 genes had significantly different expression (adjusted p-value <0.02) in the whole body transcriptome between melanoma and control fish; 379 of these genes were matched to human orthologs with 233 having annotated human gene symbols and 14 matched genes that contain putative deleterious variants in human melanoma at varying levels of recurrence. A detailed canonical pathway evaluation for significant enrichment showed the top scoring pathway to be antigen presentation but also included the expected melanocyte development and pigmentation signaling pathway. Results revealed a profound down-regulation of genes involved in the immune response, especially the innate immune system. We hypothesize that the developing melanoma actively suppresses the immune system responses of the body in reacting to the invasive malignancy, and that this mal-adaptive response contributes to disease progression, a result that suggests our whole-body transcriptomic approach merits further use. In these findings, we also observed novel genes not yet identified in human melanoma expression studies and uncovered known and new candidate drug targets for further testing in this malignant melanoma medaka model. PMID

  4. Axitinib in Treating Patients With Melanoma That is Metastatic or Cannot Be Removed by Surgery

    ClinicalTrials.gov

    2016-05-31

    Extraocular Extension Melanoma; Metastatic Intraocular Melanoma; Recurrent Intraocular Melanoma; Recurrent Melanoma; Stage IIIA Intraocular Melanoma; Stage IIIA Melanoma; Stage IIIB Intraocular Melanoma; Stage IIIB Melanoma; Stage IIIC Intraocular Melanoma; Stage IIIC Melanoma; Stage IV Intraocular Melanoma; Stage IV Melanoma

  5. Gab2-Mediated Signaling Promotes Melanoma Metastasis

    PubMed Central

    Horst, Basil; Gruvberger-Saal, Sofia K.; Hopkins, Benjamin D.; Bordone, Lindsey; Yang, Ying; Chernoff, Karen A.; Uzoma, Ijeoma; Schwipper, Volker; Liebau, Jutta; Nowak, Norma J.; Brunner, Georg; Owens, David; Rimm, David L.; Parsons, Ramon; Celebi, Julide Tok

    2009-01-01

    Metastatic melanoma is a disease with a poor prognosis that currently lacks effective treatments. Critical biological features of metastasis include acquisition of migratory competence, growth factor independence, and invasive potential. In an attempt to identify genes that contribute to melanoma pathogenesis, a genome-wide search using bacterial artificial chromosome array comparative genomic hybridization and single nucleotide polymorphism arrays in a series of 64 metastatic melanoma samples and 20 melanoma cell lines identified increased copy numbers of Gab2 located on 11q14.1. Gab2 is an adaptor protein that potentiates the activation of the Ras-Erk and PI3K-Akt pathways and has recently been implicated in human cancer; however, its role in melanoma has not been explored. In this study, we found that Gab2 was either amplified (∼11%) and/or overexpressed (∼50%) in melanoma. Gab2 protein expression correlated with clinical melanoma progression, and higher levels of expression were seen in metastatic melanomas compared with primary melanoma and melanocytic nevi. We found that overexpression of Gab2 potentiates, whereas silencing of Gab2 reduces, migration and invasion of melanoma cells. Gab2 mediated the hyperactivation of Akt signaling in the absence of growth factors, whereas inhibition of the PI3K-Akt pathway decreased Gab2-mediated tumor cell migration and invasive potential. Gab2 overexpression resulted in enhanced tumor growth and metastatic potential in vivo. These studies demonstrate a previously undefined role for Gab2 in melanoma tumor progression and metastasis. PMID:19342374

  6. Determination of potential doubling times in human melanoma cell cultures subjected to irradiation and/or hyperthermia by flow cytometry

    SciTech Connect

    Zoelzer, F.; treffer, C.; Devi, P.U.

    1994-06-01

    The proliferation of human melanoma cells in vitro after irradiation and/or hyperthermia was studied by means of two-parameter flow cytometry. Cultures were incubated with BrdU for 30 min and fixed either immediately or after a delay of several hours. Cells having synthesized DNA were identified with the help of an antibody against BrdU. DNA was stained quantitatively with propidium iodide. In this way the distribution of cells in the phases of cell cycle could be determined and the movement of labeled cells through the phases of the cycle could be analyzed. Experiments in which the cell cycle distribution was studied at 4-h intervals after treatment showed the following: (1) Irradiation (4 Gy X rays) causes the expected G{sub 2} block with a maximum after 12-16 h. The proportion of S-phase cells decreases continually during the first 48 h after treatment. (2) Hyperthermia (1 h, 43{degrees}C) alone or in combination with irradiation causes a delay in S phase. The cells begin to move into G{sub 2} phase only after 12-16 h and accumulate there to some extent. From the progression of labeled cells through the cycle, the duration of S phase could be determined. Experiments and calculations of this kind were done 0, 24 and 48 h after treatment. The duration of S phase was increased only moderately (by 4 h) after irradiation, but a delay of about 30 h occurred after hyperthermia (alone or in combination with X rays). Smaller delays (up to 9 h) were observed 24 and 48 h after treatment. Two different methods were used to calculate potential doubling times. Both of them gave similar results, but a comparison with the actual population doubling times (determined by cell counting) showed that reasonable estimates could be achieved only for the untreated controls. With cultures subjected to irradiation and/or hyperthermia serious discrepancies were observed. 14 refs., 5 figs., 4 tabs.

  7. Novel ZnO:Ag nanocomposites induce significant oxidative stress in human fibroblast malignant melanoma (Ht144) cells

    PubMed Central

    Arooj, Syeda; Nazir, Samina; Nadhman, Akhtar; Ahmad, Nafees; Muhammad, Bakhtiar; Ahmad, Ishaq; Mazhar, Kehkashan

    2015-01-01

    Summary The use of photoactive nanoparticles (NPs) such as zinc oxide (ZnO) and its nanocomposites has become a promising anticancer strategy. However, ZnO has a low photocatalytic decomposition rate and the incorporation of metal ions such as silver (Ag) improves their activity. Here different formulations of ZnO:Ag (1, 3, 5, 10, 20 and 30% Ag) were synthesized by a simple co-precipitation method and characterized by powder X-ray diffraction, scanning electron microscopy, Rutherford back scattering and diffuse reflectance spectroscopy for their structure, morphology, composition and optical band gap. The NPs were investigated with regard to their different photocatalytic cytotoxic effects in human malignant melanoma (HT144) and normal (HCEC) cells. The ZnO:Ag nanocomposites killed cancer cells more efficiently than normal cells under daylight exposure. Nanocomposites having higher Ag content (10, 20 and 30%) were more toxic compared to low Ag content (1, 3 and 5%). For HT144, under daylight exposure, the IC50 values were ZnO:Ag (10%): 23.37 μg/mL, ZnO:Ag (20%): 19.95 μg/mL, and ZnO:Ag (30%): 15.78 μg/mL. ZnO:Ag (30%) was toxic to HT144 (IC50: 23.34 μg/mL) in dark as well. The three nanocomposites were further analyzed with regard to their ability to generate reactive oxygen species (ROS) and induce lipid peroxidation. The particles led to an increase in levels of ROS at cytotoxic concentrations, but only HT144 showed strongly induced MDA level. Finally, NPs were investigated for the ROS species they generated in vitro. A highly significant increase of 1O2 in the samples exposed to daylight was observed. Hydroxyl radical species, HO•, were also generated to a lesser extent. Thus, the incorporation of Ag into ZnO NPs significantly improves their photo-oxidation capabilities. ZnO:Ag nanocomposites could provide a new therapeutic option to selectively target cancer cells. PMID:25821698

  8. Novel ZnO:Ag nanocomposites induce significant oxidative stress in human fibroblast malignant melanoma (Ht144) cells.

    PubMed

    Arooj, Syeda; Nazir, Samina; Nadhman, Akhtar; Ahmad, Nafees; Muhammad, Bakhtiar; Ahmad, Ishaq; Mazhar, Kehkashan; Abbasi, Rashda

    2015-01-01

    The use of photoactive nanoparticles (NPs) such as zinc oxide (ZnO) and its nanocomposites has become a promising anticancer strategy. However, ZnO has a low photocatalytic decomposition rate and the incorporation of metal ions such as silver (Ag) improves their activity. Here different formulations of ZnO:Ag (1, 3, 5, 10, 20 and 30% Ag) were synthesized by a simple co-precipitation method and characterized by powder X-ray diffraction, scanning electron microscopy, Rutherford back scattering and diffuse reflectance spectroscopy for their structure, morphology, composition and optical band gap. The NPs were investigated with regard to their different photocatalytic cytotoxic effects in human malignant melanoma (HT144) and normal (HCEC) cells. The ZnO:Ag nanocomposites killed cancer cells more efficiently than normal cells under daylight exposure. Nanocomposites having higher Ag content (10, 20 and 30%) were more toxic compared to low Ag content (1, 3 and 5%). For HT144, under daylight exposure, the IC50 values were ZnO:Ag (10%): 23.37 μg/mL, ZnO:Ag (20%): 19.95 μg/mL, and ZnO:Ag (30%): 15.78 μg/mL. ZnO:Ag (30%) was toxic to HT144 (IC50: 23.34 μg/mL) in dark as well. The three nanocomposites were further analyzed with regard to their ability to generate reactive oxygen species (ROS) and induce lipid peroxidation. The particles led to an increase in levels of ROS at cytotoxic concentrations, but only HT144 showed strongly induced MDA level. Finally, NPs were investigated for the ROS species they generated in vitro. A highly significant increase of (1)O2 in the samples exposed to daylight was observed. Hydroxyl radical species, HO(•), were also generated to a lesser extent. Thus, the incorporation of Ag into ZnO NPs significantly improves their photo-oxidation capabilities. ZnO:Ag nanocomposites could provide a new therapeutic option to selectively target cancer cells. PMID:25821698

  9. Rad6 is a Potential Early Marker of Melanoma Development

    PubMed Central

    Rosner, Karli; Adsule, Shreelekha; Haynes, Brittany; Kirou, Evangelia; Kato, Ikuko; Mehregan, Darius R.; Shekhar, Malathy P.V.

    2014-01-01

    Melanoma is the leading cause of death from skin cancer in industrialized countries. Several melanoma-related biomarkers and signaling pathways have been identified; however, their relevance to melanoma development/progression or to clinical outcome remains to be established. Aberrant activation of Wnt/β-catenin pathway is implicated in various cancers including melanoma. We have previously demonstrated Rad6, an ubiquitin-conjugating enzyme, as an important mediator of β-catenin stability in breast cancer cells. Similar to breast cancer, β-catenin-activating mutations are rare in melanomas, and since β-catenin signaling is implicated in melanoma, we examined the relationship between β-catenin levels/activity and expression of β-catenin transcriptional targets Rad6 and microphthalmia-associated transcription factor-M (Mitf-M) in melanoma cell models, and expression of Rad6, β-catenin, and Melan-A in nevi and cutaneous melanoma tissue specimens. Our data show that Rad6 is only weakly expressed in normal human melanocytes but is overexpressed in melanoma lines. Unlike Mitf-M, Rad6 overexpression in melanoma lines is positively associated with high molecular weight β-catenin protein levels and β-catenin transcriptional activity. Double-immunofluorescence staining of Rad6 and Melan-A in melanoma tissue microarray showed that histological diagnosis of melanoma is significantly associated with Rad6/Melan-A dual positivity in the melanoma group compared to the nevi group (P = .0029). In contrast to strong β-catenin expression in normal and tumor areas of superficial spreading malignant melanoma (SSMM), Rad6 expression is undetectable in normal areas and Rad6 expression increases coincide with increased Melan-A in the transformed regions of SSMM. These data suggest a role for Rad6 in melanoma pathogenesis and that Rad6 expression status may serve as an early marker for melanoma development. PMID:24831578

  10. Rad6 is a Potential Early Marker of Melanoma Development.

    PubMed

    Rosner, Karli; Adsule, Shreelekha; Haynes, Brittany; Kirou, Evangelia; Kato, Ikuko; Mehregan, Darius R; Shekhar, Malathy P V

    2014-05-12

    Melanoma is the leading cause of death from skin cancer in industrialized countries. Several melanoma-related biomarkers and signaling pathways have been identified; however, their relevance to melanoma development/progression or to clinical outcome remains to be established. Aberrant activation of Wnt/β-catenin pathway is implicated in various cancers including melanoma. We have previously demonstrated Rad6, an ubiquitin-conjugating enzyme, as an important mediator of β-catenin stability in breast cancer cells. Similar to breast cancer, β-catenin-activating mutations are rare in melanomas, and since β-catenin signaling is implicated in melanoma, we examined the relationship between β-catenin levels/activity and expression of β-catenin transcriptional targets Rad6 and microphthalmia-associated transcription factor-M (Mitf-M) in melanoma cell models, and expression of Rad6, β-catenin, and Melan-A in nevi and cutaneous melanoma tissue specimens. Our data show that Rad6 is only weakly expressed in normal human melanocytes but is overexpressed in melanoma lines. Unlike Mitf-M, Rad6 overexpression in melanoma lines is positively associated with high molecular weight β-catenin protein levels and β-catenin transcriptional activity. Double-immunofluorescence staining of Rad6 and Melan-A in melanoma tissue microarray showed that histological diagnosis of melanoma is significantly associated with Rad6/Melan-A dual positivity in the melanoma group compared to the nevi group (P=.0029). In contrast to strong β-catenin expression in normal and tumor areas of superficial spreading malignant melanoma (SSMM), Rad6 expression is undetectable in normal areas and Rad6 expression increases coincide with increased Melan-A in the transformed regions of SSMM. These data suggest a role for Rad6 in melanoma pathogenesis and that Rad6 expression status may serve as an early marker for melanoma development. PMID:24831578

  11. Antitumor activities of interferon alpha, beta, and gamma and their combinations on human melanoma cells in vitro: changes of proliferation, melanin synthesis, and immunophenotype.

    PubMed

    Garbe, C; Krasagakis, K; Zouboulis, C C; Schröder, K; Krüger, S; Stadler, R; Orfanos, C E

    1990-12-01

    The antitumor activities of human interferon (IFN) alpha, beta, and gamma alone or in combination were studied on four human melanoma cell lines (StML-11, StML-12, StML-14, and SKMel-28) in various concentrations (1-50,000 IU/ml IFN alpha, 0.1-1000 IU/ml IFN beta, 1-10,000 IU/ml IFN gamma) in vitro. In all experiments IFN beta exhibited the most potent antiproliferative effect of all IFN tested. After 3 d of incubation a 50% growth inhibition was achieved with 20-40 IU/ml for natural IFN beta and with 600-1200 U/ml for recombinant IFN gamma. Substantially higher doses (7,000 to more than 50,000 IU/ml) of recombinant IFN alpha 2a were required to achieve a 50% growth inhibition. A strong synergistic antiproliferative activity resulted from the combination of IFN alpha with IFN gamma and IFN beta with IFN gamma. None of the IFN tested induced terminal differentiation of melanoma cells in vitro. The formation of dendrites was inhibited, and the portion of differentiated cells in vitro was reduced after treatment with IFN in comparison to the untreated controls (untreated controls: 100%; portion of differentiated cells after treatment with IFN alpha: 58%-74%, IFN beta: 48%-96%, IFN gamma: 10%-33%). The melanin synthesis was slightly elevated after treatment with IFN alpha (untreated controls: 100%; after treatment with IFN alpha: 103%-157%, ns.) and decreased significantly after treatment with IFN beta (49%-71%, p less than 0.05) as well as with IFN gamma (80%-88%, ns.). Cell surface markers were modulated varyingly by the IFN: HLA-I antigens were enhanced by all IFN, with IFN beta emerging as the most potent inducer. Only IFN gamma, however, induced a de novo expression of HLA-DR and -DQ antigens and increased the expression of the ICAM-1 molecule and of the melanoma progression marker A.1.43. Possibly, these findings indicate a biologically more aggressive phenotype of melanoma cells. PMID:2124247

  12. Isoangustone A, a novel licorice compound, inhibits cell proliferation by targeting PI3K, MKK4, and MKK7 in human melanoma.

    PubMed

    Song, Nu Ry; Lee, Eunjung; Byun, Sanguine; Kim, Jong-Eun; Mottamal, Madhusoodanan; Park, Jung Han Yoon; Lim, Soon Sung; Bode, Ann M; Lee, Hyong Joo; Lee, Ki Won; Dong, Zigang

    2013-12-01

    Licorice root is known to possess various bioactivities, including anti-inflammatory and anticancer effects. Glycyrrhizin, a triterpene compound, is the most abundant constituent of dried licorice root. However, high intake or long-term consumption of glycyrrhizin causes several side effects, such as hypertension, hypertensive encephalopathy, and hypokalemia. Therefore, finding additional active compounds other than glycyrrhizin in licorice that exhibit anticancer effects is worthwhile. We found that isoangustone A (IAA), a novel flavonoid from licorice root, suppressed proliferation of human melanoma cells. IAA significantly blocked cell-cycle progression at the G1-phase and inhibited the expression of G1-phase regulatory proteins, including cyclins D1 and E in the SK-MEL-28 human melanoma cell line. IAA suppressed the phosphorylation of Akt, GSK-3β, and JNK1/2. IAA also bound to phosphoinositide 3-kinase (PI3K), MKK4, and MKK7, strongly inhibiting their kinase activities in an ATP-competitive manner. Moreover, in a xenograft mouse model, IAA significantly decreased tumor growth, volume, and weight of SK-MEL-28 xenografts. Collectively, these results suggest that PI3K, MKK4, and MKK7 are the primary molecular targets of IAA in the suppression of cell proliferation. This insight into the biologic actions of IAA provides a molecular basis for the potential development of a new chemotherapeutic agent. PMID:24104352

  13. In Vitro Evaluation of the Antioxidant, 3,5-Dihydroxy-4-ethyl-trans-stilbene (DETS) Isolated from Bacillus cereus as a Potent Candidate against Malignant Melanoma

    PubMed Central

    Nath, Lekshmi R.; Kumar, S. N.; Das, Arya A.; Nambisan, Bala; Shabna, A.; Mohandas, Chellapan; Anto, Ruby John

    2016-01-01

    3,5-dihydroxy Q1 -4-ethyl-trans-stilbene (DETS) is a natural stilbene, which was first identified as bioactive bacterial secondary metabolite isolated from Bacillus cereus associated with a rhabditid entomopathogenic nematode. The present study was intended to investigate the antioxidant and anticancer activity of this compound in vitro. Antioxidant activity was investigated by assaying DPPH free radical scavenging, superoxide radical-(O2..) scavenging, hydroxyl radical scavenging and metal chelating activity, which proved that the compound is a powerful antioxidant. The metal chelating activity of DETS was higher than butylated hydroxyanisol (BHA) and gallic acid, two well-known antioxidants. As the molecule exhibited strong antioxidant potential, it was further evaluated for cytotoxic activity toward five cancer cells of various origins. Since the compound has a strong structural similarity with resveratrol (trans- 3,4,5-trihydroxystilbene), a well-studied chemopreventive polyphenolic antioxidant, its anticancer activity was compared with that of resveratrol. Among the five cancer cells studied, the compound showed maximum cytotoxicity toward the human melanoma cell line, [A375, IC50: 24.01 μM] followed by cervical [HeLa-46.17 μM], colon [SW480- 47.28 μM], liver [HepG2- 69.56 μM] and breast