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Sample records for human melanoma blood

  1. High-throughput miRNA profiling of human melanoma blood samples

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    Rass Knuth

    2010-06-01

    Full Text Available Abstract Background MicroRNA (miRNA signatures are not only found in cancer tissue but also in blood of cancer patients. Specifically, miRNA detection in blood offers the prospect of a non-invasive analysis tool. Methods Using a microarray based approach we screened almost 900 human miRNAs to detect miRNAs that are deregulated in their expression in blood cells of melanoma patients. We analyzed 55 blood samples, including 20 samples of healthy individuals, 24 samples of melanoma patients as test set, and 11 samples of melanoma patients as independent validation set. Results A hypothesis test based approch detected 51 differentially regulated miRNAs, including 21 miRNAs that were downregulated in blood cells of melanoma patients and 30 miRNAs that were upregulated in blood cells of melanoma patients as compared to blood cells of healthy controls. The tets set and the independent validation set of the melanoma samples showed a high correlation of fold changes (0.81. Applying hierarchical clustering and principal component analysis we found that blood samples of melanoma patients and healthy individuals can be well differentiated from each other based on miRNA expression analysis. Using a subset of 16 significant deregulated miRNAs, we were able to reach a classification accuracy of 97.4%, a specificity of 95% and a sensitivity of 98.9% by supervised analysis. MiRNA microarray data were validated by qRT-PCR. Conclusions Our study provides strong evidence for miRNA expression signatures of blood cells as useful biomarkers for melanoma.

  2. Transcriptome profiling of whole blood cells identifies PLEK2 and C1QB in human melanoma.

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    Yuchun Luo

    Full Text Available Developing analytical methodologies to identify biomarkers in easily accessible body fluids is highly valuable for the early diagnosis and management of cancer patients. Peripheral whole blood is a "nucleic acid-rich" and "inflammatory cell-rich" information reservoir and represents systemic processes altered by the presence of cancer cells.We conducted transcriptome profiling of whole blood cells from melanoma patients. To overcome challenges associated with blood-based transcriptome analysis, we used a PAXgene™ tube and NuGEN Ovation™ globin reduction system. The combined use of these systems in microarray resulted in the identification of 78 unique genes differentially expressed in the blood of melanoma patients. Of these, 68 genes were further analyzed by quantitative reverse transcriptase PCR using blood samples from 45 newly diagnosed melanoma patients (stage I to IV and 50 healthy control individuals. Thirty-nine genes were verified to be differentially expressed in blood samples from melanoma patients. A stepwise logit analysis selected eighteen 2-gene signatures that distinguish melanoma from healthy controls. Of these, a 2-gene signature consisting of PLEK2 and C1QB led to the best result that correctly classified 93.3% melanoma patients and 90% healthy controls. Both genes were upregulated in blood samples of melanoma patients from all stages. Further analysis using blood fractionation showed that CD45(- and CD45(+ populations were responsible for the altered expression levels of PLEK2 and C1QB, respectively.The current study provides the first analysis of whole blood-based transcriptome biomarkers for malignant melanoma. The expression of PLEK2, the strongest gene to classify melanoma patients, in CD45(- subsets illustrates the importance of analyzing whole blood cells for biomarker studies. The study suggests that transcriptome profiling of blood cells could be used for both early detection of melanoma and monitoring of patients

  3. Frequent MAGE mutations in human melanoma.

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    Otavia L Caballero

    Full Text Available BACKGROUND: Cancer/testis (CT genes are expressed only in the germ line and certain tumors and are most frequently located on the X-chromosome (the CT-X genes. Amongst the best studied CT-X genes are those encoding several MAGE protein families. The function of MAGE proteins is not well understood, but several have been shown to potentially influence the tumorigenic phenotype. METHODOLOGY/PRINCIPAL FINDINGS: We undertook a mutational analysis of coding regions of four CT-X MAGE genes, MAGEA1, MAGEA4, MAGEC1, MAGEC2 and the ubiquitously expressed MAGEE1 in human melanoma samples. We first examined cell lines established from tumors and matching blood samples from 27 melanoma patients. We found that melanoma cell lines from 37% of patients contained at least one mutated MAGE gene. The frequency of mutations in the coding regions of individual MAGE genes varied from 3.7% for MAGEA1 and MAGEA4 to 14.8% for MAGEC2. We also examined 111 fresh melanoma samples collected from 86 patients. In this case, samples from 32% of the patients exhibited mutations in one or more MAGE genes with the frequency of mutations in individual MAGE genes ranging from 6% in MAGEA1 to 16% in MAGEC1. SIGNIFICANCE: These results demonstrate for the first time that the MAGE gene family is frequently mutated in melanoma.

  4. Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors.

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    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.

  5. GLO1 Overexpression in Human Malignant Melanoma

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    Bair, Warner B; Cabello, Christopher M; Uchida, Koji; Bause, Alexandra S; Wondrak, Georg T

    2010-01-01

    Glyoxalase I [lactoylglutathione lyase (EC 4.4.1.5) encoded by GLO1] is a ubiquitous cellular defense enzyme involved in the detoxification of methylglyoxal, a cytotoxic byproduct of glycolysis. Accumulative evidence suggests an important role of GLO1 expression in protection against methylglyoxal-dependent protein adduction and cellular damage associated with diabetes, cancer, and chronological aging. Based on the hypothesis that GLO1 upregulation may play a functional role in glycolytic adaptations of cancer cells, we examined GLO1 expression status in human melanoma tissue. Quantitative RT-PCR analysis of a cDNA tissue array containing 40 human melanoma tissues (stages III and IV) and 13 healthy controls revealed pronounced upregulation of GLO1 expression at the mRNA level. Immunohistochemical analysis of a melanoma tissue microarray confirmed upregulation of glyoxalase 1 protein levels in malignant melanoma tissue versus healthy human skin. Consistent with an essential role of GLO1 in melanoma cell defense against methylglyoxal cytotoxicity, siRNA interference targeting GLO1-expression (siGLO1) sensitized A375 and G361 human metastatic melanoma cells towards the antiproliferative, apoptogenic, and oxidative stress-inducing activity of exogenous methylglyoxal. Protein adduction by methylglyoxal was increased in siGLO1-transfected cells as revealed by immunodetection using a monoclonal antibody directed against the major methylglyoxal-derived epitope argpyrimidine that detected a single band of methylglyoxal-adducted protein in human LOX, G361, and A375 total cell lysates. Using 2D-proteomics followed by mass spectrometry the methylglyoxal-adducted protein was identified as heat shock protein 27 (Hsp27; HSPB1). Taken together, our data suggest a function of GLO1 in the regulation of detoxification and target-adduction by the glycolytic byproduct methylglyoxal in malignant melanoma. PMID:20093988

  6. Mitochondrial DNA copy number in peripheral blood and melanoma risk.

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    Jie Shen

    Full Text Available Mitochondrial DNA (mtDNA copy number in peripheral blood has been suggested as risk modifier in various types of cancer. However, its influence on melanoma risk is unclear. We evaluated the association between mtDNA copy number in peripheral blood and melanoma risk in 500 melanoma cases and 500 healthy controls from an ongoing melanoma study. The mtDNA copy number was measured using real-time polymerase chain reaction. Overall, mean mtDNA copy number was significantly higher in cases than in controls (1.15 vs 0.99, P<0.001. Increased mtDNA copy number was associated with a 1.45-fold increased risk of melanoma (95% confidence interval: 1.12-1.97. Significant joint effects between mtDNA copy number and variables related to pigmentation and history of sunlight exposure were observed. This study supports an association between increased mtDNA copy number and melanoma risk that is independent on the known melanoma risk factors (pigmentation and history of sunlight exposure.

  7. Global methylation of blood leukocyte DNA and risk of melanoma.

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    Shen, Jie; Song, Renduo; Wan, Jie; Huff, Chad; Fang, Shenying; Lee, Jeffrey E; Zhao, Hua

    2017-04-01

    Global DNA methylation, possibly influenced by lifestyle and environmental factors, has been suggested to play an active role in carcinogenesis. However, its role in melanoma has rarely been explored. The aims of this study were to evaluate the relationship between melanoma risk and levels of 5-methylcytosine (5-mC), a marker for global DNA methylation, in blood leukocyte DNA, and to determine whether this 5-mC level is influenced by pigmentation and sun exposure. This case-control study included 540 melanoma cases and 540 healthy controls. Overall, melanoma cases had significantly lower levels of 5-mC% than healthy controls (median: 3.24 vs. 3.91, p melanoma (OR: 1.25; 95% CI: 1.08, 1.37). A significant dose-response relationship was observed in quartile analysis (p = 0.001). Our results suggest that global hypomethylation in blood leukocyte DNA is associated with increased risk of melanoma and that the level of methylation is influenced by pigmentation and sun exposure. © 2016 UICC.

  8. Melanoma cells treated with GGTI and IFN-gamma allow murine vaccination and enhance cytotoxic response against human melanoma cells.

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    Guillaume Sarrabayrouse

    Full Text Available BACKGROUND: Suboptimal activation of T lymphocytes by melanoma cells is often due to the defective expression of class I major histocompatibility antigens (MHC-I and costimulatory molecules. We have previously shown that geranylgeranyl transferase inhibition (done with GGTI-298 stimulates anti-melanoma immune response through MHC-I and costimulatory molecule expression in the B16F10 murine model [1]. METHODOLOGY/PRINCIPAL FINDINGS: In this study, it is shown that vaccination with mIFN-gand GGTI-298 pretreated B16F10 cells induces a protection against untreated tumor growth and pulmonary metastases implantation. Furthermore, using a human melanoma model (LB1319-MEL, we demonstrated that in vitro treatment with hIFN-gamma and GGTI-298 led to the up regulation of MHC-I and a costimulatory molecule CD86 and down regulation of an inhibitory molecule PD-1L. Co-culture experiments with peripheral blood mononuclear cells (PBMC revealed that modifications induced by hIFN-gamma and GGTI-298 on the selected melanoma cells, enables the stimulation of lymphocytes from HLA compatible healthy donors. Indeed, as compared with untreated melanoma cells, pretreatment with hIFN-gamma and GGTI-298 together rendered the melanoma cells more efficient at inducing the: i activation of CD8 T lymphocytes (CD8+/CD69+; ii proliferation of tumor-specific CD8 T cells (MelanA-MART1/TCR+; iii secretion of hIFN-gamma; and iv anti-melanoma specific cytotoxic cells. CONCLUSIONS/SIGNIFICANCE: These data indicate that pharmacological treatment of melanoma cell lines with IFN-gamma and GGTI-298 stimulates their immunogenicity and could be a novel approach to produce tumor cells suitable for vaccination and for stimulation of anti-melanoma effector cells.

  9. Melanoma

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    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 ...

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

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    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.

  11. K-type human endogenous retroviral elements in human melanoma

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    Rincon L

    2014-10-01

    Full Text Available Liliana Rincon,1 Michael P Sedrak,1 Huijian Sun,2 Gregorio Garza,3 Brent Kelly,4 Jianli Dong11Department of Pathology, 2School of Medicine, University of Texas Medical Branch, TX, USA; 3Ciencias de la Salud, Universidad de Monterrey, Nuevo León, Mexico; 4Department of Dermatology, University of Texas Medical Branch, TX, USAAbstract: Human endogenous retroviral elements (HERVs are thought to be germline-integrated genetic remnants of exogenous retroviral infections. HERVs comprise approximately 5%–8% of the human genome. Although all HERV genomes are highly defective, some, especially the K type (HERV-K, have the potential to be expressed and have biological activities. HERV-K expression has been detected in human melanomas. There are also reports on the regulation and potential activities of HERV-K in melanoma cells. Although a causal link between the activation of HERV-K and melanoma development has yet to be determined, existing data support the further research efforts in this area. In this review, we summarize the published studies on the expression, regulation, and activity of HERV-K in human melanoma.Keywords: HERV, HERV-K, melanoma development

  12. Mitochondrial DNA 4977-base pair common deletion in blood leukocytes and melanoma risk.

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    Shen, Jie; Wan, Jie; Huff, Chad; Fang, Shenying; Lee, Jeffrey E; Zhao, Hua

    2016-05-01

    The 4977-base pair common deletion DmtDNA4977 is the most frequently observed mitochondrial DNA mutation in human tissues. Because mitochondrial DNA mutations are mainly caused by reactive oxygen species (ROS), and given that oxidative stress plays an important role in melanoma carcinogenesis, the investigation of DmtDNA4977 may be particularly relevant to the development of melanoma. In this study, we compared DmtDNA4977 levels in blood leukocytes from 206 melanoma patients and 219 healthy controls. Overall, melanoma cases had significantly higher levels of DmtDNA4977 than healthy controls (median: 0.60 vs 0.20, P = 0.008). The difference was evident among individuals who were older than 47 yrs, women, and had pigmentation risk factors (e.g., blond or red hair, blue eye, fair skin, light, or none tanning ability after prolonged sun exposure, and freckling in the sun as a child). The difference was also evident among those who had at least one lifetime sunburn with blistering and had no reported use of a sunlamp. Interestingly, among controls, DmtDNA4977 levels differed by phenotypic index and reported use of a sunlamp. In the risk assessment, increased levels of DmtDNA4977 were associated with a 1.23-fold increased risk of melanoma (odds ratio (OR): 1.23, 95% confidence interval (90% CI): 1.01, 1.50). A significant dose-response relationship was observed in quartile analysis (P = 0.001). In summary, our study suggests that high levels of DmtDNA4977 in blood leukocytes are associated with increased risk of melanoma and that association is affected by both pigmentation and personal history of sun exposure.

  13. Intercellular crosstalk in human malignant melanoma.

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    Dvořánková, Barbora; Szabo, Pavol; Kodet, Ondřej; Strnad, Hynek; Kolář, Michal; Lacina, Lukáš; Krejčí, Eliška; Naňka, Ondřej; Šedo, Aleksi; Smetana, Karel

    2017-05-01

    Incidence of malignant melanoma is increasing globally. While the initial stages of tumors can be easily treated by a simple surgery, the therapy of advanced stages is rather limited. Melanoma cells spread rapidly through the body of a patient to form multiple metastases. Consequently, the survival rate is poor. Therefore, emphasis in melanoma research is given on early diagnosis and development of novel and more potent therapeutic options. The malignant melanoma is arising from melanocytes, cells protecting mitotically active keratinocytes against damage caused by UV light irradiation. The melanocytes originate in the neural crest and consequently migrate to the epidermis. The relationship between the melanoma cells, the melanocytes, and neural crest stem cells manifests when the melanoma cells are implanted to an early embryo: they use similar migratory routes as the normal neural crest cells. Moreover, malignant potential of these melanoma cells is overdriven in this experimental model, probably due to microenvironmental reprogramming. This observation demonstrates the crucial role of the microenvironment in melanoma biology. Indeed, malignant tumors in general represent complex ecosystems, where multiple cell types influence the growth of genetically mutated cancer cells. This concept is directly applicable to the malignant melanoma. Our review article focuses on possible strategies to modify the intercellular crosstalk in melanoma that can be employed for therapeutic purposes.

  14. Monitoring the systemic human memory B cell compartment of melanoma patients for anti-tumor IgG antibodies.

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    Amy E Gilbert

    Full Text Available Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10 to primary and metastatic melanoma cells compared to healthy volunteers (n = 10 (P<0.0001. Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21 (P<0.0001. Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800 compared to 2% of cultures from healthy controls (n = 600 produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer.

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

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    Suping Ren

    Full Text Available 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.

  16. Radiosensitivity of Human Melanoma Cell Lines

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    Bergoc, R. M.; Medina, V.; Cricco, G.; Mohamed, N.; Martin, G.; Nunez, M.; Croci, M.; Crescenti, E. J.; Rivera, E. S.

    2004-07-01

    Cutaneous melanoma is a skin cancer resulting from the malign transformation of skin-pigment cells, the melanocytes. The radiotherapy, alone or in combination with other treatment, is an important therapy for this disease. the objective of this paper was to determine in vitro the radiosensitivity of two human melanoma cell lines with different metastatic capability: WM35 and MI/15, and to study the effect of drugs on radiobiological parameters. The Survival Curves were adjusted to the mathematical Linear-quadratic model using GrapsPad Prism software. Cells were seeded in RPMI medium (3000-3500 cells/flask), in triplicate and irradiated 24 h later. The irradiation was performed using an IBL 437C H Type equipment (189 TBq, 7.7 Gy/min) calibrated with a TLD 700 dosimeter. The range of Doses covered from 0 to 10 Gy and the colonies formed were counted at day 7th post-irradiation. Results obtained were: for WM35, {alpha}=0.37{+-}0.07 Gy''-1 and {beta}=0.06{+-}0.02 Gy''-2, for M1/15m {alpha}=0.47{+-}0.03 Gy''-1 and {beta}=0.06{+-}0.01 Gy''-2. The {alpha}/{beta} values WM35: {alpha}/{beta} values WM35: {alpha}/{beta}=6.07 Gy and M1/15: {alpha}/{beta}{sub 7}.33 Gy were similar, independently of their metastatic capabillity and indicate that both lines exhibit high radioresistance. Microscopic observation of irradiated cells showed multinuclear cells with few morphologic changes non-compatible with apoptosis. By means of specific fluorescent dyes and flow cytometry analysis we determined the intracellular levels of the radicals superoxide and hydrogen peroxide and their modulation in response to ionizing radiation. The results showed a marked decreased in H{sub 2}O{sub 2} intracellular levels with a simultaneous increase in superoxide that will be part of a mechanism responsible for induction of cell radioresistance. This response triggered by irradiated cells could not be abrogated by different treatments like histamine or the

  17. Point of care assessment of melanoma tumor signaling and metastatic burden from μNMR analysis of tumor fine needle aspirates and peripheral blood.

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    Gee, Michael S; Ghazani, Arezou A; Haq, Rizwan; Wargo, Jennifer A; Sebas, Matthew; Sullivan, Ryan J; Lee, Hakho; Weissleder, Ralph

    2017-04-01

    This study evaluates μNMR technology for molecular profiling of tumor fine needle aspirates and peripheral blood of melanoma patients. In vitro assessment of melanocyte (MART-1, HMB45) and MAP kinase signaling (pERK, pS6K) molecule expression was performed in human cell lines, while clinical validation was performed in an IRB-approved study of melanoma patients undergoing biopsy and blood sampling. Tumor FNA and blood specimens were compared with BRAF genetic analysis and cross-sectional imaging. μNMR in vitro analysis showed increased expression of melanocyte markers in melanoma cells as well as increased expression of phosphorylated MAP kinase targets in BRAF-mutant melanoma cells. Melanoma patient FNA samples showed increased pERK and pS6K levels in BRAF mutant compared with BRAF WT melanomas, with μNMR blood circulating tumor cell level increased with higher metastatic burden visible on imaging. These results indicate that μNMR technology provides minimally invasive point-of-care evaluation of tumor signaling and metastatic burden in melanoma patients. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Animal models of melanoma: a somatic cell gene delivery mouse model allows rapid evaluation of genesimplicated in human melanoma%Animal models of melanoma: a somatic cell gene delivery mouse model allows rapid evaluation of genes implicated in human melanoma

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    Andrea J. McKinney; Sheri L. Holmen

    2011-01-01

    The increasing incidence and mortality associated with advanced stages of melanoma are cause for concern. Few treatment options are available for advanced melanoma and the 5-year survival rate is less than 15%. Targeted therapies may revolutionize melanoma treatment by providing less toxic and more effective strategies. However, maximizing effectiveness requires further understanding of the molecular alterations that drive tumor formation, progression, and maintenance, as well as elucidating the mechanisms of resistance. Several different genetic alterations identified in human melanoma have been recapitulated in mice. This review outlines recent progress made in the development of mouse models of melanoma and summarizes what these findings reveal about the human disease. We begin with a discussion of traditional models and conclude with the recently developed RCAS/TVA somatic cell gene delivery mouse model of melanoma.

  19. Engineering melanoma progression in a humanized environment in vivo.

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    Kiowski, Gregor; Biedermann, Thomas; Widmer, Daniel S; Civenni, Gianluca; Burger, Charlotte; Dummer, Reinhard; Sommer, Lukas; Reichmann, Ernst

    2012-01-01

    To overcome the lack of effective therapeutics for aggressive melanoma, new research models closely resembling the human disease are required. Here we report the development of a fully orthotopic, humanized in vivo model for melanoma, faithfully recapitulating human disease initiation and progression. To this end, human melanoma cells were seeded into engineered human dermo-epidermal skin substitutes. Transplantation onto the back of immunocompromised rats consistently resulted in the development of melanoma, displaying the hallmarks of their parental tumors. Importantly, all initial steps of disease progression were recapitulated, including the incorporation of the tumor cells into their physiological microenvironment, transition of radial to vertical growth, and establishment of highly vascularized, aggressive tumors with dermal involvement. Because all cellular components can be individually accessed using this approach, it allows manipulation of the tumor cells, as well as of the keratinocyte and stromal cell populations. Therefore, in one defined model system, tumor cell-autonomous and non-autonomous pathways regulating human disease progression can be investigated in a humanized, clinically relevant context.

  20. Effect of Genistein on vasculogenic mimicry formation by human uveal melanoma cells

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    Gu Haijuan

    2009-09-01

    Full Text Available Abstract Background Vasculogenic mimicry (VM was increasingly recognized as a form of aggressive melanoma acquiring blood supply. Genistein had attracted much attention as a potential anticancer agent. Therefore, we examined the effect of Genistein on VM in human uveal melanoma cells. Methods VM structure was detected by periodic acid-Schiff (PAS staining for uveal melanoma C918 cells cultured on the three-dimensional type I collagen gels after exposed to Genistein. We used reverse transcription polymerase chain reaction (RT-PCR and Western Blot analysis to examine the effect of Genistein on vascular endothelial cadherin (VE-cadherin mRNA and protein expression. The nude mice models of human uveal melanoma C918 cells were established to assess the number of VM using immunohistochemical and PAS double-staining. Results Genistein inhibited the survival of C918 cells in vitro. The ectopic model study showed that VM in tumor tissue sections were significantly reduced by Genistein in vivo. In vitro, the VM structure was found in control, 25 and 50 μM Genistein-treatment groups but not in 100 and 200 μM. RT-PCR and Western Blot showed that 100 and 200 μM concentration of Genistein could significantly decrease VE-cadherin mRNA and protein expression of C918 cells compared with control (P 0.05. Conclusion Genistein inhibits VM formation of uveal melanoma cells in vivo and in vitro. One possible underlying molecular mechanism by which Genistein could inhibit VM formation of uveal melanoma is related to down-regulation of VE-cadherin.

  1. Anti-proliferative and proapoptotic effects of (-)-epigallocatechin-3-gallate on human melanoma: possible implications for the chemoprevention of melanoma.

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    Nihal, Minakshi; Ahmad, Nihal; Mukhtar, Hasan; Wood, Gary S

    2005-04-20

    Melanoma accounts for only about 4% of all skin cancer cases but most of skin cancer-related deaths. Standard systemic therapies such as interferon (IFN) have not been adequately effective in the management of melanoma. Therefore, novel approaches are needed for prevention and treatment of this disease. Chemoprevention by naturally occurring agents present in food and beverages has shown benefits in certain cancers including nonmelanoma skin cancers. Here, employing 2 human melanoma cell lines (A-375 amelanotic malignant melanoma and Hs-294T metastatic melanoma) and normal human epidermal melanocytes (NHEM), we studied the antiproliferative effects of epigallocatechin-3-gallate (EGCG), the major polyphenolic antioxidant present in green tea. EGCG treatment was found to result in a dose-dependent decrease in the viability and growth of both melanoma cell lines. Interestingly, at similar EGCG concentrations, the normal melanocytes were not affected. EGCG treatment of the melanoma cell lines resulted in decreased cell proliferation (as assessed by Ki-67 and PCNA protein levels) and induction of apoptosis (as assessed cleavage of PARP, TUNEL assay and JC-1 assay). EGCG also significantly inhibited the colony formation ability of the melanoma cells studied. EGCG treatment of melanoma cells resulted in a downmodulation of anti-apoptotic protein Bcl2, upregulation of proapoptotic Bax and activation of caspases -3, -7 and -9. Furthermore, our data demonstrated that EGCG treatment resulted in a significant, dose-dependent decrease in cyclin D1 and cdk2 protein levels and induction of cyclin kinase inhibitors (ckis) p16INK4a, p21WAF1/CIP1 and p27KIP1. Our data suggest that EGCG causes significant induction of cell cycle arrest and apoptosis of melanoma cells that is mediated via modulations in the cki-cyclin-cdk network and Bcl2 family proteins. Thus, EGCG, alone or in conjunction with current therapies, could be useful for the management of melanoma.

  2. Melanoma

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    ... from generations ago. Back in your parents' and grandparents' day, most people (including doctors) thought it was safe and even ... it again somewhere else) Although it's less likely, people can still get melanoma even if they're dark skinned, young, and have no family history. Even for them, ...

  3. Procoagulant properties of human MV3 melanoma cells

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    D.L.A. Geaquinto

    2008-02-01

    Full Text Available A correlation between cancer and prothrombotic states has long been described. More recently, a number of studies have focused on the procoagulant mechanisms exhibited by tumor cells. In the present study, we dissected the molecular mechanisms responsible for the procoagulant activity of MV3, a highly aggressive human melanoma cell line. It was observed that tumor cells strongly accelerate plasma coagulation as a result of: i expression of the blood clotting initiator protein, a tissue factor, as shown by flow cytometry and functional assays (factor Xa formation in the presence of cells and factor VIIa, and ii direct activation of prothrombin to thrombin by cells, as evidenced by hydrolysis of the synthetic substrate, S-2238, and the natural substrate, fibrinogen. This ability was highly potentiated by the addition of exogenous factor Va, which functions as a co-factor for the enzyme factor Xa. In contrast, prothrombin activation was not observed when cells were previously incubated with DEGR-factor Xa, an inactive derivative of the enzyme. Moreover, a monoclonal antibody against bovine factor Xa reduced the prothrombin-converting activity of tumor cells. In conclusion, the data strongly suggest that MV3 cells recruit factor Xa from the culture medium, triggering an uncommon procoagulant mechanism.

  4. Recombinant anti-human melanoma antibodies are versatile molecules.

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    Neri, D; Natali, P G; Petrul, H; Soldani, P; Nicotra, M R; Vola, R; Rivella, A; Creighton, A M; Neri, P; Mariani, M

    1996-08-01

    The low cost, high versatility, and reliable production of bacterially produced recombinant antibody fragments speeds up the development of tumor-targeting agents. High-quality recombinant anti-melanoma antibodies are much sought after in the scientific community. We cloned the murine antibody 225.28S, currently used in radioimmunoimaging of human melanoma lesions, in single-chain Fv configuration (scFv) for soluble expression in bacteria. The recombinant antibody fragment conserved the binding specificity of the parental antibody. In order to arm the scFv(225.28S) with biologically useful effector functions, we developed vectors for soluble expression of scFv(225.28S) in bacteria that allow both covalent and noncovalent chemical antibody modification at positions that do not interfere with antigen binding. An expression vector was developed that appends a cysteine residue at the C-terminal extremity of the recombinant antibody, thus allowing reaction with thiol-specific reagents, including 99mTc labeling, at a position that does not interfere with antigen binding. The scFv(225.28S) was also successfully expressed with a casein kinase II substrate tag that enables efficient and stable 32P labeling. For noncovalent antibody modification, we developed an expression vector that appends the human calmodulin gene at the C-terminal extremity of scFv(225.28S). The calmodulin domain is poorly immunogenic and can be targeted with chemically modified high-affinity calmodulin ligands. The recombinant anti-human melanoma antibodies described in this article should prove useful "building blocks" for the development of anti-melanoma diagnostic and therapeutic strategies.

  5. Melanoma affects the composition of blood cell-derived extracellular vesicles

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    Nina Koliha

    2016-07-01

    Full Text Available Extracellular vesicles are specifically loaded with nucleic acids, lipids, and proteins from their parental cell. Therefore, the constitution of extracellular vesicles reflects the type and status of the originating cell and extracellular vesicles in melanoma patient’s plasma could be indicative for the tumor. Likewise, extracellular vesicles might influence tumor progression by regulating immune responses. We performed a broad protein characterization of extracellular vesicles from plasma of melanoma patients and healthy donors as well as from T cells, B cells, natural killer cells, monocytes, monocyte-derived dendritic cells and platelets using a multiplex bead-based platform. Using this method, we succeeded in analyzing 58 proteins that were differentially displayed on extracellular vesicles. Hierarchal clustering of protein intensity patterns grouped extracellular vesicles according to their originating cell type. The analysis of extracellular vesicles from stimulated B cells and monocyte-derived dendritic cells revealed the transfer of surface proteins to vesicles depending on the cell status. The protein profiles of plasma vesicles resembled the protein profiles of extracellular vesicles from platelets, antigen presenting cells and natural cells as shown by platelet markers, costimulatory proteins, and a natural killer cell subpopulation marker. In comparison to healthy plasma vesicles, melanoma plasma vesicles showed altered signals for platelet markers indicating a changed vesicle secretion or protein loading of extracellular vesicles by platelets and a lower CD8 signal that might be associated with a diminished activity of natural killer cells or T cells. As we hardly detected melanoma-derived vesicles in patient’s plasma, we concluded that blood cells induced the observed differences. In summary, our results question a direct effect of melanoma cells on the composition of extracellular vesicles in melanoma plasma, but rather argue

  6. Redirected lysis of human melanoma cells by a MCSP/CD3-bispecific BiTE antibody that engages patient-derived T cells.

    Science.gov (United States)

    Torisu-Itakura, Hitoe; Schoellhammer, Hans F; Sim, Myung-Shin; Irie, Reiko F; Hausmann, Susanne; Raum, Tobias; Baeuerle, Patrick A; Morton, Donald L

    2011-10-01

    Melanoma-associated chondroitin sulfate proteoglycan (MCSP; also called HMW-MAA, CSPG4, NG2, MSK16, MCSPG, MEL-CSPG, or gp240) is a well characterized melanoma cell-surface antigen. In this study, a new bispecific T-cell engaging (BiTE) antibody that binds to MCSP and human CD3 (MCSP-BiTE) was tested for its cytotoxic activity against human melanoma cell lines. When unstimulated peripheral mononuclear blood cells (PBMCs) derived from healthy donors were cocultured with melanoma cells at effector:target ratios of 1:1, 1:5, or 1:10, and treated with MCSP-BiTE antibody at doses of 10, 100, or 1000 ng/mL, all MCSP-expressing melanoma cell lines (n=23) were lysed in a dose-dependent and effector:target ratio-dependent manner, whereas there was no cytotoxic activity against MCSP-negative melanoma cell lines (n=2). To investigate whether T cells from melanoma patients could act as effector cells, we cocultured unstimulated PBMCs with allogeneic melanoma cells from 13 patients (4 stage I/II, 3 stage III, and 6 stage IV) or with autologous melanoma cells from 2 patients (stage IV). Although cytotoxic activity varied, all 15 PBMC samples mediated significant redirected lysis by the BiTE antibody. When PBMC or CD8 T cells were prestimulated by anti-CD3 antibody OKT-3 and interleukin-2, the MCSP-BiTE concentrations needed for melanoma cell lysis decreased up to 1000-fold. As MCSP is expressed on most human melanomas, immunotherapy with MCSP/CD3-bispecific antibodies merits clinical investigation.

  7. Centrosomal dysregulation in human metastatic melanoma cell lines.

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    Charters, Geoffrey A; Stones, Clare J; Shelling, Andrew N; Baguley, Bruce C; Finlay, Graeme J

    2011-09-01

    Correct partitioning of the replicated genome during mitosis is orchestrated by centrosomes, and chromosomal instability is a commonly reported feature of human cancer. Melanomas are notorious for their genetic instability and rapid clonal evolution that may be manifested as aggressive growth and facile generation of therapy-resistant variants. We characterized the centrosomal status, ploidy, and gene status (TP53, CDKN2A/B, BRAF, and NRAS) of 15 human metastatic melanoma cell lines. Cells were labelled for pericentrin (a centrosomal marker), DNA and α-tubulin, and scored for centrosome morphology, supernumerary centrosomes, and mitotic symmetry. The incidence of supernumerary centrosomes correlated with that of gross centrosomal abnormalities (r = 0.90), mitotic asymmetry (r = 0.90), and, surprisingly, increased content of G/M cells (r = 0.79). Centrosomal numerical dysregulation, observed in all cell lines, was found not to be specifically related to the status of any of the characterized gene mutations that were found in 13/15 cell lines. We conclude that centrosomal dysregulation may arise from multiple mechanisms and may drive the generation of genetic and phenotypic diversity in melanoma.

  8. Epigenetic impacts of ascorbate on human metastatic melanoma cells.

    Science.gov (United States)

    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

  9. Epigenetic impacts of ascorbate on human metastatic melanoma cells

    Directory of Open Access Journals (Sweden)

    Sascha eVenturelli

    2014-08-01

    Full Text Available 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 HIF-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 expression after 12 h. The results were analyzed with the MIRUMIR online tool that estimates the power of microRNA to serve as potential biomarkers to predict survival of cancer patients.FACS cell cycle analyses showed that 8 mM ascorbate shifted BLM melanoma cells towards 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 microRNAs was altered 12 h after ascorbate treatment of BLM cells in physiological or pharmacological doses. Pharmacological doses up-regulated 32 microRNAs (≥4-fold mainly involved in tumor suppression and drug resistance in our preliminary microRNA screening array. The most prominently up-regulated microRNAs correlated with a significantly increased overall survival of breast cancer- or nasopharyngeal carcinoma patients of the MIRUMIR database with high expression of the respective microRNA. 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 ascorbate

  10. Irreversible electroporation of human primary uveal melanoma in enucleated eyes.

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    Yossi Mandel

    Full Text Available Uveal melanoma (UM is the most common primary intraocular tumor in adults and is characterized by high rates of metastatic disease. Although brachytherapy is the most common globe-sparing treatment option for small- and medium-sized tumors, the treatment is associated with severe adverse reactions and does not lead to increased survival rates as compared to enucleation. The use of irreversible electroporation (IRE for tumor ablation has potential advantages in the treatment of tumors in complex organs such as the eye. Following previous theoretical work, herein we evaluate the use of IRE for uveal tumor ablation in human ex vivo eye model. Enucleated eyes of patients with uveal melanoma were treated with short electric pulses (50-100 µs, 1000-2000 V/cm using a customized electrode design. Tumor bioimpedance was measured before and after treatment and was followed by histopathological evaluation. We found that IRE caused tumor ablation characterized by cell membrane disruption while sparing the non-cellular sclera. Membrane disruption and loss of cellular capacitance were also associated with significant reduction in total tumor impedance and loss of impedance frequency dependence. The effect was more pronounced near the pulsing electrodes and was dependent on time from treatment to fixation. Future studies should further evaluate the potential of IRE as an alternative method of uveal melanoma treatment.

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

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    János Haskó

    2014-05-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  13. Aggressiveness of human melanoma xenograft models is promoted by aneuploidy-driven gene expression deregulation

    OpenAIRE

    Mathieu, Véronique; Pirker, Christine; Schmidt, Wolfgang M.; Spiegl-Kreinecker, Sabine; Lötsch, Daniela; Heffeter, Petra; Hegedus, Balazs; Grusch, Michael; Kiss, Robert; Berger, Walter

    2012-01-01

    Melanoma is a devastating skin cancer characterized by distinct biological subtypes. Besides frequent mutations in growth- and survival-promoting genes like BRAF and NRAS, melanomas additionally harbor complex non-random genomic alterations. Using an integrative approach, we have analysed genomic and gene expression changes in human melanoma cell lines (N=32) derived from primary tumors and various metastatic sites and investigated the relation to local growth aggressiveness as xenografts in ...

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

    OpenAIRE

    Syed, Deeba N.; Lall, Rahul K.; 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...

  15. Cellular radiosensitivity of primary and metastatic human uveal melanoma cell lines

    NARCIS (Netherlands)

    G.J.M.J. van den Aardweg (Gerard J. M.); N.C. Naus (Nicole); A.C. Verhoeven; J.E.M.M. de Klein (Annelies); G.P.M. Luyten (Gré)

    2002-01-01

    textabstractPURPOSE: To investigate the radiosensitivity of uveal melanoma cell lines by a clonogenic survival assay, to improve the efficiency of the radiation regimen. METHODS: Four primary and four metastatic human uveal melanoma cell lines were cultured in the presence of

  16. Cellular radiosensitivity of primary and metastatic human uveal melanoma cell lines

    NARCIS (Netherlands)

    G.J.M.J. van den Aardweg (Gerard J. M.); N.C. Naus (Nicole); A.C. Verhoeven; J.E.M.M. de Klein (Annelies); G.P.M. Luyten (Gré)

    2002-01-01

    textabstractPURPOSE: To investigate the radiosensitivity of uveal melanoma cell lines by a clonogenic survival assay, to improve the efficiency of the radiation regimen. METHODS: Four primary and four metastatic human uveal melanoma cell lines were cultured in the presence of condi

  17. Proteomic analysis of proton beam irradiated human melanoma cells.

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    Sylwia Kedracka-Krok

    Full Text Available 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.

  18. Isolation of tumorigenic circulating melanoma cells

    Science.gov (United States)

    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

  19. HOTAIR role in melanoma progression and its identification in the blood of patients with advanced disease.

    Science.gov (United States)

    Cantile, Monica; Scognamiglio, Giosuè; Marra, Laura; Aquino, Gabriella; Botti, Chiara; Falcone, Maria Rosaria; Malzone, Maria Gabriella; Liguori, Giuseppina; Di Bonito, Maurizio; Franco, Renato; Ascierto, Paolo Antonio; Botti, Gerardo

    2017-12-01

    The molecular mechanisms responsible for the metastatic progression of melanoma have not been fully defined yet. We have recently shown that an important role in this process is certainly played by HOX genes, whose regulation is under control of particular non-coding RNAs, some of which are present within the HOX locus. HOTAIR is the most studied among them, whose aberrant expression is associated with the metastatic progression of many malignancies. The aim of this study was to verify the role played by HOTAIR in metastatic progression of melanoma and to evaluate the circulating levels of HOTAIR in the blood of patients with metastatic melanoma. A series of melanocytic lesions were selected to evaluate the potential changes in the expression of HOTAIR during the evolution of the disease through in situ and molecular approaches. None of the benign melanocytic lesions showed the presence of HOTAIR. The staining of HOTAIR resulted very weak in the primary pT1 lesions, while it was very strong in all pairs of primary tissues and corresponding metastases. Surprisingly, we found the presence of HOTAIR in some intratumoral lymphocytes, while this positivity decreased in lymphocyte component further away from the tumor. HOTAIR was also detected in the serum of selected metastatic patients. These data allowed us to speculate on the fundamental role played by HOTAIR in tumor evolution of melanoma. Its presence in intratumoral lymphocytes might suggest that its involvement in the modulation of tumor microenvironment and the detection in the serum could be used in the management of melanoma patients. © 2017 Wiley Periodicals, Inc.

  20. Use of gene expression and pathway signatures to characterize the complexity of human melanoma.

    Science.gov (United States)

    Freedman, Jennifer A; Tyler, Douglas S; Nevins, Joseph R; Augustine, Christina K

    2011-06-01

    A defining characteristic of most human cancers is heterogeneity, resulting from the somatic acquisition of a complex array of genetic and genomic alterations. Dissecting this heterogeneity is critical to developing an understanding of the underlying mechanisms of disease and to paving the way toward personalized treatments of the disease. We used gene expression data sets from the analysis of primary and metastatic melanomas to develop a molecular description of the heterogeneity that characterizes this disease. Unsupervised hierarchical clustering, gene set enrichment analyses, and pathway activity analyses were used to describe the genetic heterogeneity of melanomas. Patterns of gene expression that revealed two distinct classes of primary melanoma, two distinct classes of in-transit melanoma, and at least three subgroups of metastatic melanoma were identified. Expression signatures developed to predict the status of oncogenic signaling pathways were used to explore the biological basis underlying these differential patterns of expression. This analysis of activities revealed unique pathways that distinguished the primary and metastatic subgroups of melanoma. Distinct patterns of gene expression across primary, in-transit, and metastatic melanomas underline the genetic heterogeneity of this disease. This heterogeneity can be described in terms of deregulation of signaling pathways, thus increasing the knowledge of the biological features underlying individual melanomas and potentially directing therapeutic opportunities to individual patients with melanoma.

  1. Distinct host cell fates for human malignant melanoma targeted by oncolytic rodent parvoviruses.

    Science.gov (United States)

    Vollmers, Ellen M; Tattersall, Peter

    2013-11-01

    The rodent parvoviruses are known to be oncoselective, and lytically infect many transformed human cells. Because current therapeutic regimens for metastatic melanoma have low response rates and have little effect on improving survival, this disease is a prime candidate for novel approaches to therapy, including oncolytic parvoviruses. Screening of low-passage, patient-derived melanoma cell lines for multiplicity-dependent killing by a panel of five rodent parvoviruses identified LuIII as the most melanoma-lytic. This property was mapped to the LuIII capsid gene, and an efficiently melanoma tropic chimeric virus shown to undergo three types of interaction with primary human melanoma cells: (1) complete lysis of cultures infected at very low multiplicities; (2) acute killing resulting from viral protein synthesis and DNA replication, without concomitant expansion of the infection, due to failure to export progeny virions efficiently; or (3) complete resistance that operates at an intracellular step following virion uptake, but preceding viral transcription.

  2. Serum anti-BPAG1 auto-antibody is a novel marker for human melanoma.

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    Takashi Shimbo

    Full Text Available Malignant melanoma is one of the most aggressive types of tumor. Because malignant melanoma is difficult to treat once it has metastasized, early detection and treatment are essential. The search for reliable biomarkers of early-stage melanoma, therefore, has received much attention. By using a novel method of screening tumor antigens and their auto-antibodies, we identified bullous pemphigoid antigen 1 (BPAG1 as a melanoma antigen recognized by its auto-antibody. BPAG1 is an auto-antigen in the skin disease bullous pemphigoid (BP and anti-BPAG1 auto-antibodies are detectable in sera from BP patients and are used for BP diagnosis. However, BPAG1 has been viewed as predominantly a keratinocyte-associated protein and a relationship between BPAG1 expression and melanoma has not been previously reported. In the present study, we show that bpag1 is expressed in the mouse F10 melanoma cell line in vitro and F10 melanoma tumors in vivo and that BPAG1 is expressed in human melanoma cell lines (A375 and G361 and normal human melanocytes. Moreover, the levels of anti-BPAG1 auto-antibodies in the sera of melanoma patients were significantly higher than in the sera of healthy volunteers (p<0.01. Furthermore, anti-BPAG1 auto-antibodies were detected in melanoma patients at both early and advanced stages of disease. Here, we report anti-BPAG1 auto-antibodies as a promising marker for the diagnosis of melanoma, and we discuss the significance of the detection of such auto-antibodies in cancer biology and patients.

  3. Effect of proteasome inhibitors on proliferation and apoptosis of human cutaneous melanoma-derived cell lines.

    Science.gov (United States)

    Sorolla, A; Yeramian, A; Dolcet, X; Pérez de Santos, A M; Llobet, D; Schoenenberger, J A; Casanova, J M; Soria, X; Egido, R; Llombart, A; Vilella, R; Matias-Guiu, X; Marti, R M

    2008-03-01

    Cutaneous malignant melanoma is an aggressive type of skin cancer which causes disproportionate mortality in young and middle-aged adults. Once disseminated, melanoma can be considered an incurable disease, highly resistant to standard antineoplastic treatment, such as chemotherapy or radiation therapy. The proteasome represents a novel target for cancer therapy that can potentially be used in melanoma. To assess the effect of four structurally different proteasome inhibitors on human cutaneous melanoma-derived cell lines. Sixteen human cutaneous melanoma-derived cell lines which are original were obtained from patients who were treated by two of the authors. Cells were cultured, exposed to proteasome inhibitors (bortezomib, ALLN, MG-132 and epoxomicin) and then assayed for cell cycle and cell death analyses. Proteasome inhibitors inhibited the in vitro growth of melanoma cells, and this effect was due to a reduction in cell proliferation rate and an induction of both caspase-dependent and caspase-independent cell death. Moreover, release of apoptosis-inducing factor was observed in the presence of the broad-specificity caspase inhibitor BAF (Boc-D-fmk). In addition, the four different proteasome inhibitors induced caspase 2 processing. This study provides information regarding the in vitro effects of proteasome inhibitors on melanoma cell lines, and the molecular mechanisms involved. It also gives support to the future use of such inhibitors in the treatment of patients with melanoma, either administered alone or in combination with other drugs.

  4. SC-535, a Novel Oral Multikinase Inhibitor, Showed Potent Antitumor Activity in Human Melanoma Models

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    Xin Chen

    2013-07-01

    Full Text Available Background: Melanoma is considered as one of the most aggressive and deadliest cancers and current targeted therapies of melanoma often suffer limited efficacy or drug resistance. Discovery of novel multikinase inhibitors as anti-melanoma drug candidates is still needed. Methods: In this investigation, we assessed the in vitro and in vivo anti-melanoma activities of SC-535, which is a novel small molecule multikinase inhibitor discovered by us recently. We analyzed inhibitory effects of SC-535 on various melanoma cell lines and human umbilical vascular endothelial cells (HUVEC in vitro. Tumor xenografts in athymic mice were used to examine the in vivo activity of SC-535. Results: SC-535 could efficiently inhibit vascular endothelial growth factor receptor (VEGFR 1/2/3, B-RAF, and C-RAF kinases. It showed significant antiangiogenic potencies both in vitro and in vivo and considerable anti-proliferative ability against several melanoma cell lines. Oral administration of SC-535 resulted in dose-dependent suppression of tumor growth in WM2664 and C32 xenograft mouse models. Studies of mechanisms of action indicated that SC-535 suppressed the tumor angiogenesis and induced G2/M phase cell cycle arrest in human melanoma cells. SC-535 possesses favorable pharmacokinetic properties. Conclusion: All of these results support SC-535 as a potential candidate for clinical studies in patients with melanoma.

  5. Growth Hormone Receptor Knockdown Sensitizes Human Melanoma Cells to Chemotherapy by Attenuating Expression of ABC Drug Efflux Pumps.

    Science.gov (United States)

    Basu, Reetobrata; Baumgaertel, Nicholas; Wu, Shiyong; Kopchick, John J

    2017-03-14

    Melanoma remains one of the most therapy-resistant forms of human cancer despite recent introductions of highly efficacious targeted therapies. The intrinsic therapy resistance of human melanoma is largely due to abundant expression of a repertoire of xenobiotic efflux pumps of the ATP-binding cassette (ABC) transporter family. Here, we report that GH action is a key mediator of chemotherapeutic resistance in human melanoma cells. We investigated multiple ABC efflux pumps (ABCB1, ABCB5, ABCB8, ABCC1, ABCC2, ABCG1, and ABCG2) reportedly associated with melanoma drug resistance in different human melanoma cells and tested the efficacy of five different anti-cancer compounds (cisplatin, doxorubicin, oridonin, paclitaxel, vemurafenib) with decreased GH action. We found that GH treatment of human melanoma cells upregulates expression of multiple ABC transporters and increases the EC50 of melanoma drug vemurafenib. Also, vemurafenib-resistant melanoma cells had upregulated levels of GH receptor (GHR) expression as well as ABC efflux pumps. GHR knockdown (KD) using siRNA in human melanoma cells treated with sub-EC50 doses of anti-tumor compounds resulted in significantly increased drug retention, decreased cell proliferation and increased drug efficacy, compared to mock-transfected controls. Our set of findings identify an unknown mechanism of GH regulation in mediating melanoma drug resistance and validates GHR as a unique therapeutic target for sensitizing highly therapy-resistant human melanoma cells to lower doses of anti-cancer drugs.

  6. Prognostic Value of RT-PCR Tyrosinase Detection in Peripheral Blood of Melanoma Patients

    Directory of Open Access Journals (Sweden)

    Esmeralda Carrillo

    2006-01-01

    Full Text Available Malignant melanoma (MM prognosis has been related to tumour thickness and clinical stage and metastasis risk has been associated with presence of tumour cells in peripheral blood. The aim of this study was to determine the relationship between presence of tyrosinase in peripheral blood of MM patients and their clinical prognosis. Blood samples from 58 MM patients (stage I–IV were analysed, using RT-PCR assay to detect tyrosinase mRNA. The results showed that positive RT-PCR assay for tyrosinase were significantly associated with clinical status and tumour thickness. After a median follow-up of 24 months, RT-PCR results were found to be significant correlated with recurrence (p < 0.05 and clinical stage III (p < 0.05. Separate analysis of stage III tumours to determine the prognostic value of tyrosinase presence in peripheral blood showed an overall 24-month survival rate of 70% in the RT-PCR negative group versus 10% in the positive group (p < 0.02. These results suggest that detection of circulating melanoma cells may be especially relevant in stage III patients, in whom RT-PCR positivity defines a subpopulation at high risk of recurrence.

  7. Functional expression of voltage-gated calcium channels in human melanoma.

    Science.gov (United States)

    Das, A; Pushparaj, C; Bahí, N; Sorolla, A; Herreros, J; Pamplona, R; Vilella, R; Matias-Guiu, X; Martí, R M; Cantí, C

    2012-03-01

    The expression of voltage-gated calcium channels (VGCCs) has not been reported previously in melanoma cells in spite of increasing evidence of a role of VGCCs in tumorigenesis and tumour progression. To address this issue we have performed an extensive RT-PCR analysis of VGCC expression in human melanocytes and a range of melanoma cell lines and biopsies. In addition, we have tested the functional expression of these channels using Ca(2+) imaging techniques and examined their relevance for the viability and proliferation of the melanoma cells. Our results show that control melanocytes and melanoma cells express channel isoforms belonging to the Ca(v) 1 and Ca(v) 2 gene families. Importantly, the expression of low voltage-activated Ca(v) 3 (T-type) channels is restricted to melanoma. We have confirmed the function of T-type channels as mediators of constitutive Ca(2+) influx in melanoma cells. Finally, pharmacological and gene silencing approaches demonstrate a role for T-type channels in melanoma viability and proliferation. These results encourage the analysis of T-type VGCCs as targets for therapeutic intervention in melanoma tumorigenesis and/or tumour progression. © 2012 John Wiley & Sons A/S.

  8. Dissection of T-cell antigen specificity in human melanoma

    DEFF Research Database (Denmark)

    Andersen, Rikke Sick; Albæk Thrue, Charlotte; Junker, Niels

    2012-01-01

    -associated antigens and applying a novel technology for high-throughput analysis of T-cell responses, we dissected the composition of melanoma-restricted T-cell responses in 63 TIL cultures. T-cell reactivity screens against 175 melanoma-associated epitopes detected 90 responses against 18 different epitopes...

  9. Laminin-dependent and laminin-independent adhesion of human melanoma cells to sulfatides

    DEFF Research Database (Denmark)

    Roberts, D D; Wewer, U M; Liotta, L A

    1988-01-01

    Sulfatides (galactosylceramide-I3-sulfate) but not neutral glycolipids or gangliosides adsorbed on plastic promote adhesion of the human melanoma cell line G361. Direct adhesion of G361 cells requires densities of sulfatide greater than 1 pmol/mm2. In the presence of laminin, however, specific...... by cross-linking receptors on the melanoma cell surface to sulfatide adsorbed on the plastic. Although thrombospondin binds to sulfatides and G361 cells, it does not enhance, but rather inhibits direct and laminin-dependent G361 cell adhesion to sulfatide. In contrast, C32 melanoma cells also adhere...

  10. Manipulation of radiobiological hypoxia in a human melanoma xenograft to exploit the bioreductive cytotoxicity of RSU-1069

    Energy Technology Data Exchange (ETDEWEB)

    Cole, S.; Stratford, I.J.; Adams, G.E. (Medical Research Council, Harwell (UK). Radiobiological Research Unit)

    1989-11-01

    RSU-1069 (a 2-nitroimidazole) is known to be an efficient hypoxic cell radiosensitizer and potent cytotoxin for hypoxic cells in murine tumours (Chaplin et al. 1986). The authors demonstrated that the compound is also able to kill human melanoma cells, growing as a xenograft, provided the tumour blood supply is occluded for an hour or more, thereby substantially increasing the proportion of susceptible, hypoxic cells. Pharmacokinetic effects may also contribute to this phenomenon, as the effectiveness of bioreductive cytotoxic agents may increase with prolonged contact times (Stratford et al. 1986b). Hydralazine increased radiobiological hypoxia in the HX118 xenograft but this did not enhance the susceptibility of clonogenic melanoma cells to killing by RSU-1069. (author).

  11. Interleukin-32α induces migration of human melanoma cells through downregulation of E-cadherin.

    Science.gov (United States)

    Lee, Joohyun; Kim, Kyung Eun; Cheon, Soyoung; Song, Ju Han; Houh, Younkyung; Kim, Tae Sung; Gil, Minchan; Lee, Kyung Jin; Kim, Seonghan; Kim, Daejin; Hur, Dae Young; Yang, Yoolhee; Bang, Sa Ik; Park, Hyun Jeong; Cho, Daeho

    2016-10-04

    Interleukin (IL)-32α, the shortest isoform of proinflammatory cytokine IL-32, is associated with various inflammatory diseases and cancers. However, its involvement in human melanoma is not understood. To determine the effect of IL-32α in melanoma, IL-32α levels were examined in human melanoma cell lines that exhibit different migratory abilities. IL-32α levels were higher in human melanoma cell lines with more migratory ability. An IL-32α-overexpressing G361 human melanoma cell line was generated to investigate the effect of IL-32α on melanoma migration. IL-32α-overexpressing G361 cells (G361-IL-32α) exhibit an increased migratory ability compared to vector control cells (G361-vector). To identify factors involved in IL-32α-induced migration, we compared expression of E-cadherin in G361-vector and G361-IL-32α cells. We observed decreased levels of E-cadherin in G361-IL-32α cells, resulting in F-actin polymerization. To further investigate signaling pathways related to IL-32α-induced migration, we treated G361-vector and G361-IL-32α cells with PD98059, a selective MEK inhibitor. Inhibition of Erk1/2 by PD98059 restored E-cadherin expression and decreased IL-32α-induced migration. In addition, cell invasiveness of G361-IL-32α cells was tested using an in vivo lung metastasis model. As results, lung metastasis was significantly increased by IL-32α overexpression. Taken together, these data indicate that IL-32α induced human melanoma migration via Erk1/2 activation, which repressed E-cadherin expression. Our findings suggest that IL-32α is a novel regulator of migration in melanoma.

  12. Tanapoxvirus lacking a neuregulin-like gene regresses human melanoma tumors in nude mice.

    Science.gov (United States)

    Zhang, Tiantian; Suryawanshi, Yogesh R; Kordish, Dennis H; Woyczesczyk, Helene M; Jeng, David; Essani, Karim

    2017-02-01

    Neuregulin (NRG), an epidermal growth factor is known to promote the growth of various cell types, including human melanoma cells through ErbB family of tyrosine kinases receptors. Tanapoxvirus (TPV)-encoded protein TPV-15L, a functional mimic of NRG, also acts through ErbB receptors. Here, we show that the TPV-15L protein promotes melanoma proliferation. TPV recombinant generated by deleting the 15L gene (TPVΔ15L) showed replication ability similar to that of wild-type TPV (wtTPV) in owl monkey kidney cells, human lung fibroblast (WI-38) cells, and human melanoma (SK-MEL-3) cells. However, a TPV recombinant with both 15L and the thymidine kinase (TK) gene 66R ablated (TPVΔ15LΔ66R) replicated less efficiently compared to TPVΔ15L and the parental virus. TPVΔ15L exhibited more robust tumor regression in the melanoma-bearing nude mice compared to other TPV recombinants. Our results indicate that deletion of TPV-15L gene product which facilitates the growth of human melanoma cells can be an effective strategy to enhance the oncolytic potential of TPV for the treatment of melanoma.

  13. Aggressiveness of human melanoma xenograft models is promoted by aneuploidy-driven gene expression deregulation.

    Science.gov (United States)

    Mathieu, Véronique; Pirker, Christine; Schmidt, Wolfgang M; Spiegl-Kreinecker, Sabine; Lötsch, Daniela; Heffeter, Petra; Hegedus, Balazs; Grusch, Michael; Kiss, Robert; Berger, Walter

    2012-04-01

    Melanoma is a devastating skin cancer characterized by distinct biological subtypes. Besides frequent mutations in growth- and survival-promoting genes like BRAF and NRAS, melanomas additionally harbor complex non-random genomic alterations. Using an integrative approach, we have analysed genomic and gene expression changes in human melanoma cell lines (N=32) derived from primary tumors and various metastatic sites and investigated the relation to local growth aggressiveness as xenografts in immuno-compromised mice (N=22). Although the vast majority >90% of melanoma models harbored mutations in either BRAF or NRAS, significant differences in subcutaneous growth aggressiveness became obvious. Unsupervised clustering revealed that genomic alterations rather than gene expression data reflected this aggressive phenotype, while no association with histology, stage or metastatic site of the original melanoma was found. Genomic clustering allowed separation of melanoma models into two subgroups with differing local growth aggressiveness in vivo. Regarding genes expressed at significantly altered levels between these subgroups, a surprising correlation with the respective gene doses (>85% accordance) was found. Genes deregulated at the DNA and mRNA level included well-known cancer genes partly already linked to melanoma (RAS genes, PTEN, AURKA, MAPK inhibitors Sprouty/Spred), but also novel candidates like SIPA1 (a Rap1GAP). Pathway mining further supported deregulation of Rap1 signaling in the aggressive subgroup e.g. by additional repression of two Rap1GEFs. Accordingly, siRNA-mediated down-regulation of SIPA1 exerted significant effects on clonogenicity, adherence and migration in aggressive melanoma models. Together our data suggest that an aneuploidy-driven gene expression deregulation drives local aggressiveness in human melanoma.

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

    Science.gov (United States)

    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.

  15. Inhibitors of 5-lipoxygenase inhibit expression of intercellular adhesion molecule-1 in human melanoma cells

    Institute of Scientific and Technical Information of China (English)

    Yin WANG; Bin ZHOU; Ji LI; Yong-bing CAO; Xin-sheng CHEN; Ming-he CHENG; Ming YIN

    2004-01-01

    AIM: To study the effect of 5-lipoxygenase inhibitors on the expression of intercellular adhesion molecule-1 (ICAM-1) in melanoma cells. METHODS: ICAM-1 protein of human melanoma cell a375 was detected by enzyme-linked immunosorbent, flow cytometry and Western blot analysis. Level of ICAM-1 mRNA in a375 was evaluated by Northern blot analysis. Adhesion of a375 to endothelial cell EC304 was analyzed by isotopic tracing. RESULTS:5-Lipoxygenase inhibitors nordihydroguaiaretic acid, AA861 and MK886, could suppress the expression of ICAM-1 protein as well as of its mRNA in a375 cells and reduce the adhesion of a375 to EC304. CONCLUSION:5-Lipoxygenase inhibitors can inhibit the expression of ICAM-1 in human melanoma cells and may be valuable for treatment of melanoma metastasis.

  16. The efficacy of dandelion root extract in inducing apoptosis in drug-resistant human melanoma cells.

    Science.gov (United States)

    Chatterjee, S J; Ovadje, P; Mousa, M; Hamm, C; Pandey, S

    2011-01-01

    Notoriously chemoresistant melanoma has become the most prevalent form of cancer for the 25-29 North American age demographic. Standard treatment after early detection involves surgical excision (recurrence is possible), and metastatic melanoma is refractory to immuno-, radio-, and most harmful chemotherapies. Various natural compounds have shown efficacy in killing different cancers, albeit not always specifically. In this study, we show that dandelion root extract (DRE) specifically and effectively induces apoptosis in human melanoma cells without inducing toxicity in noncancerous cells. Characteristic apoptotic morphology of nuclear condensation and phosphatidylserine flipping to the outer leaflet of the plasma membrane of A375 human melanoma cells was observed within 48 hours. DRE-induced apoptosis activates caspase-8 in A375 cells early on, demonstrating employment of an extrinsic apoptotic pathway to kill A375 cells. Reactive Oxygen Species (ROS) generated from DRE-treated isolated mitochondria indicates that natural compounds in DRE can also directly target mitochondria. Interestingly, the relatively resistant G361 human melanoma cell line responded to DRE when combined with the metabolism interfering antitype II diabetic drug metformin. Therefore, treatment with this common, yet potent extract of natural compounds has proven novel in specifically inducing apoptosis in chemoresistant melanoma, without toxicity to healthy cells.

  17. The Efficacy of Dandelion Root Extract in Inducing Apoptosis in Drug-Resistant Human Melanoma Cells

    Directory of Open Access Journals (Sweden)

    S. J. Chatterjee

    2011-01-01

    Full Text Available Notoriously chemoresistant melanoma has become the most prevalent form of cancer for the 25–29 North American age demographic. Standard treatment after early detection involves surgical excision (recurrence is possible, and metastatic melanoma is refractory to immuno-, radio-, and most harmful chemotherapies. Various natural compounds have shown efficacy in killing different cancers, albeit not always specifically. In this study, we show that dandelion root extract (DRE specifically and effectively induces apoptosis in human melanoma cells without inducing toxicity in noncancerous cells. Characteristic apoptotic morphology of nuclear condensation and phosphatidylserine flipping to the outer leaflet of the plasma membrane of A375 human melanoma cells was observed within 48 hours. DRE-induced apoptosis activates caspase-8 in A375 cells early on, demonstrating employment of an extrinsic apoptotic pathway to kill A375 cells. Reactive Oxygen Species (ROS generated from DRE-treated isolated mitochondria indicates that natural compounds in DRE can also directly target mitochondria. Interestingly, the relatively resistant G361 human melanoma cell line responded to DRE when combined with the metabolism interfering antitype II diabetic drug metformin. Therefore, treatment with this common, yet potent extract of natural compounds has proven novel in specifically inducing apoptosis in chemoresistant melanoma, without toxicity to healthy cells.

  18. Tumor-promoting effects of cannabinoid receptor type 1 in human melanoma cells.

    Science.gov (United States)

    Carpi, Sara; Fogli, Stefano; Polini, Beatrice; Montagnani, Valentina; Podestà, Adriano; Breschi, Maria Cristina; Romanini, Antonella; Stecca, Barbara; Nieri, Paola

    2017-04-01

    The role of endocannabinoid system in melanoma development and progression is actually not fully understood. This study was aimed at clarifying whether cannabinoid-type 1 (CB1) receptor may function as tumor-promoting or -suppressing signal in human cutaneous melanoma. CB1 receptor expression was measured in human melanoma cell lines by real-time PCR. A genetic deletion of CB1 receptors in selected melanoma cells was carried out by using three different short hairpin RNAs (shRNAs). Performance of target gene silencing was verified by real-time PCR and Western blot. The effects of CB1 receptor silencing on cell growth, clonogenicity, migration capability, cell cycle progression, and activation of mitogenic signals was tested. Lentiviral shRNAs vectors targeting different regions of the human CB1 gene led to a significant reduction in CB1 receptor mRNA and a near complete loss of CB1 receptor protein, compared to control vector (LV-c). The number of viable cells, the colony-forming ability and cell migration were significantly reduced in cells transduced with CB1 lentiviral shRNAs compared to LV-c. Cell cycle analyses showed arrest at G1/S phase. p-Akt and p-ERK expression were decreased in transduced versus control cells. Findings of this study suggest that CB1 receptor might function as tumor-promoting signal in human cutaneous melanoma. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. In vitro chemosensitivity tests on xenografted human melanomas.

    OpenAIRE

    1980-01-01

    An in vitro chemosensitivity test has been applied to malignant melanoma cells from 5 patients. The tumour cells were first grown as xenografts in immune-suppressed mice, so that the results of the in vitro test could be compared with precise measurements of the sensitivity of the melanoma cells when exposed to chemotherapeutic drugs in vivo in the mouse. The in vitro assay involved exposing the tumour cells to each of 8 drugs, after which cell survival was determined by colony assay in soft ...

  20. ANTIPROLIFERATIVE EFFECT OF INOSITOL HEXAPHOSPHATE ON HUMAN SKIN MELANOMA CELLS IN VITRO.

    Science.gov (United States)

    Wawszczyk, Joanna; Kapral, Małgorzata; Lodowska, Jolanta; Jesse, Katarzyna; Hollek, Andrzej; Węglarz, Ludmiła

    2015-01-01

    Human malignant melanoma is a highly metastatic tumor with poor prognosis. The majority of metastatic melanomas are resistant to diverse chemotherapeutic agents. Consequently, the search for novel antimelanoma agents continues. In recent years, the interest in plants and their biologically active constituents as a source of novel potential drugs significantly increased. Inositol hexaphosphate (IP6) is a naturally occurring compound that has been shown to inhibit the growth of a wide variety of tumor cells in multiple experimental model systems. The aim of this study was to evaluate the antiproliferative and cytotoxic influence of IP6 on melanotic melanoma cells in vitro. The A2058 cells used as a model of human skin melanoma malignum were exposed to different concentrations of IP6 (0.1-5 mM) for a various period of time and their growth was determined by sulforhodamine B assay after 24, 48 and 72 h. The cytotoxicity of IP6 was measured at 24 and 72 h by XTT assay. IP6 has been found to cause dose-dependent growth suppression of A2058 melanoma cells. At low concentrations (0.1 and 0.5 mM) it did not exert any influence on the cell proliferation as compared to control cultures. Higher concentrations of IP6 (from 1 to 5 mM) had a statistically significant, suppressive effect on cell proliferation after 24 h incubation. When the experimental time period was increased up to 72 h, statistically significant inhibition of cell proliferation was monitored at all IP6 concentrations used. Data obtained from XTT assay indicated that IP6 had dose- and time-dependent cytotoxic effect on melanoma cells. The results demonstrate the antiproliferative and cytotoxic properties of IP6 in a wide range of concentrations on human A2058 melanoma cells. Hence, it can be suggested that IP6 could have a promising therapeutic significance in treating cancer.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  2. Detection of Melanoma Metastases in Resected Human Lymph Nodes by Noninvasive Multispectral Photoacoustic Imaging

    Directory of Open Access Journals (Sweden)

    Gerrit Cornelis Langhout

    2014-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  4. Combination therapy targeting the tumor microenvironment is effective in a model of human ocular melanoma

    Directory of Open Access Journals (Sweden)

    Schafer Peter H

    2007-07-01

    Full Text Available Abstract Background Ocular melanoma is the leading intraocular malignancy. There is no effective treatment for metastatic ocular melanoma. We sought a treatment targeting the tumor microenvironment as well as the tumor cells. Methods Migration of HUVEC cells, the ability of HUVEC cells to form tubes, and proliferative capacity of a human ocular melanoma cell line were tested in the presence of lenalidomide and sorafenib alone and in combination. The compounds were also tested in a rat aortic ring assay and were tested in a highly aggressive human ocular melanoma xenograft model. Results Lenalidomide and Sorafenib inhibit HUVEC ability to migrate and form tubes and when used in combination the inhibition is increased. The agents alone and in combination inhibit outgrowth in the rat aortic ring model. The combination of the agents improved the inhibition over either single agent. In a xenograft model, combination therapy inhibited tumor growth over inhibition by single agent alone in a significant fashion (p Conclusion Lenalidomide and sorafenib are effective at targeting endothelial cells, inhibiting growth of ocular melanoma cells and can inhibit growth of tumors in a xenograft model as well as inhibit development of metastases. Combining these agents works in an additive to synergistic way to inhibit the growth of tumors and development of metastases.

  5. Isolation and Molecular Characterization of Circulating Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Xi Luo

    2014-05-01

    Full Text Available Melanoma is an invasive malignancy with a high frequency of blood-borne metastases, but circulating tumor cells (CTCs have not been readily isolated. We adapted microfluidic CTC capture to a tamoxifen-driven B-RAF/PTEN mouse melanoma model. CTCs were detected in all tumor-bearing mice and rapidly declined after B-RAF inhibitor treatment. CTCs were shed early from localized tumors, and a short course of B-RAF inhibition following surgical resection was sufficient to dramatically suppress distant metastases. The large number of CTCs in melanoma-bearing mice enabled a comparison of RNA-sequencing profiles with matched primary tumors. A mouse melanoma CTC-derived signature correlated with invasiveness and cellular motility in human melanoma. CTCs were detected in smaller numbers in patients with metastatic melanoma and declined with successful B-RAF-targeted therapy. Together, the capture and molecular characterization of CTCs provide insight into the hematogenous spread of melanoma.

  6. Osmotic stress affects functional properties of human melanoma cell lines

    CERN Document Server

    La Porta, Caterina A M; Pasini, Maria; Laurson, Lasse; Alava, Mikko J; Zapperi, Stefano; Amar, Martine Ben

    2015-01-01

    Understanding the role of microenvironment in cancer growth and metastasis is a key issue for cancer research. Here, we study the effect of osmotic pressure on the functional properties of primary and metastatic melanoma cell lines. In particular, we experimentally quantify individual cell motility and transmigration capability. We then perform a circular scratch assay to study how a cancer cell front invades an empty space. Our results show that primary melanoma cells are sensitive to a low osmotic pressure, while metastatic cells are less. To better understand the experimental results, we introduce and study a continuous model for the dynamics of a cell layer and a stochastic discrete model for cell proliferation and diffusion. The two models capture essential features of the experimental results and allow to make predictions for a wide range of experimentally measurable parameters.

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

    Science.gov (United States)

    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.

  8. Estrogen Receptor β Agonists Differentially Affect the Growth of Human Melanoma Cell Lines.

    Directory of Open Access Journals (Sweden)

    Monica Marzagalli

    Full Text Available Cutaneous melanoma is an aggressive malignancy; its incidence is increasing worldwide and its prognosis remains poor. Clinical observations indicate that estrogen receptor β (ERβ is expressed in melanoma tissues and its expression decreases with tumor progression, suggesting its tumor suppressive function. These experiments were performed to investigate the effects of ERβ activation on melanoma cell growth.Protein expression was analyzed by Western blot and immunofluorescence assays. Cell proliferation was assessed by counting the cells by hemocytometer. ERβ transcriptional activity was evaluated by gene reporter assay. Global DNA methylation was analyzed by restriction enzyme assay and ERβ isoforms were identified by qRT-PCR. We demonstrated that ERβ is expressed in a panel of human melanoma cell lines (BLM, WM115, A375, WM1552. In BLM (NRAS-mutant cells, ERβ agonists significantly and specifically inhibited cell proliferation. ERβ activation triggered its cytoplasmic-to-nuclear translocation and transcriptional activity. Moreover, the antiproliferative activity of ERβ agonists was associated with an altered expression of G1-S transition-related proteins. In these cells, global DNA was found to be hypomethylated when compared to normal melanocytes; this DNA hypomethylation status was reverted by ERβ activation. ERβ agonists also decreased the proliferation of WM115 (BRAF V600D-mutant cells, while they failed to reduce the growth of A375 and WM1552 (BRAF V600E-mutant cells. Finally, we could observe that ERβ isoforms are expressed at different levels in the various cell lines. Specific oncogenic mutations or differential expression of receptor isoforms might be responsible for the different responses of cell lines to ERβ agonists.Our results demonstrate that ERβ is expressed in melanoma cell lines and that ERβ agonists differentially regulate the proliferation of these cells. These data confirm the notion that melanoma is a

  9. 跨膜型血型A抗原模拟肽疫苗诱导黑色素瘤细胞凋亡的实验研究%Research on the apoptosis of malignant melanoma cell induced by transmembrane form of human blood group A mimotope vaccine

    Institute of Scientific and Technical Information of China (English)

    岑东芝; 李许锋; 邹建军; 罗敏; 张积仁

    2011-01-01

    Objective To investigate the apoptotic effect of the transmembrane form vaccine of human blood group A mimotope on malignant melanoma cell line B16. Methods B16 cells were transfected with different recombinant plasmid through Lipofectamine 2000 and incubated with different concentration of monoclonal anti-A antibody at 2.5 μg/ml, 5 μg/ml,10 μg/ml and 20 μg/ml. Apoptosis rate of cells was determined with Annexin Ⅴ/PI double staining by flow cytometry. Results Apoptosis rate to P/F-M-pIRES group B16 cells was 74.74% when anti-A monoclonal antibody concentration was 10 μg/ml; apoptosis rate of plasmids carrying peptide/Fas fusion gene such as P/F-M-pIRES group and P/F-pIRES group were significantly higher than M-pIRES group and pIRES group. The apoptosis rate was statistically significantly different between different recombinated plasmid groups (F=669.707,P<0.01). The apoptosis rate was statistically significantly different between different antibody groups (F=106.596,P<0.01). The interaction between recombinated plasmid groups and antibody groups was statistically significant (F=34.806,P<0.01). Conclusions The transmembrane form vaccine of human blood group A mimotope could induce B16 cell apoptosis in vitro. This vaccine may be a promising candidate for potential malignant melanoma therapy.%目的 研究跨膜型血型A抗原模拟肽疫苗诱导恶性黑色素瘤细胞B16凋亡的作用.方法 采用脂质体转染法分别将前期构建的模拟肽疫苗稳定转染B16细胞,加入终浓度为2.5 μg/ml、5 μg/ml、10 μg/ml及20 μg/ml的抗血型A单克隆抗体培养72 h,Annexin Ⅴ/PI双染法检测细胞凋亡率.结果 P/F-M-pIRES组的B16细胞经10 μg/ml抗血型A单克隆抗体诱导后凋亡率为74.74%,转染有模拟肽/Fas融合基因的P/F-M-pIRES组和P/F-pIRES组凋亡率明显高于未转染的M-pIRES组和pIRES组.析因设计的方差分析显示不同实验分组之间的差异主效应差异有统计学意义(F=669

  10. Nutrition and melanoma prevention.

    Science.gov (United States)

    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.

  11. Induction of G2/M arrest and apoptosis by sesquiterpene lactones in human melanoma cell lines.

    Science.gov (United States)

    Rozenblat, Sharon; Grossman, Shlomo; Bergman, Margalit; Gottlieb, Hugo; Cohen, Yigal; Dovrat, Sara

    2008-01-15

    Malignant melanoma is a highly aggressive tumor which frequently resists chemotherapy, therefore, the search for new agents for its treatment is of great importance. In this study, we purified the sesquiterpene lactones (SLs), Tomentosin and Inuviscolide from Inula viscosa (Compositae) leaves and studied their anti-cancer potency against human melanoma cell lines in order to develop new agents for melanoma treatment. SLs inhibited the proliferation of three human melanoma cell lines: SK-28, 624 mel and 1363 mel in a dose-dependent manner. We further investigated SLs mechanism of action using SK-28 as a representative cell line model. SLs caused cell-cycle arrest at G(2)/M, accompanied by the appearance of a sub-G0 fraction, indicative of apoptotic cell death. Induction of apoptosis was further confirmed by changes in membrane phospholipids, changes in mitochondrial membrane potential (DeltaPsi) and by detection of Caspase-3 activity. Rapid inhibitory phosphorylation of Cdc2 (Thr14 and Tyr15) was seen early after treatment, followed by a later decrease in the expression level of both Cyclin b1 and Cdc2. Induction of p53 and p21(waf1) proteins and phosphorylation of p53 at Ser15 were also detected early after treatment. The anti-apoptotic proteins, p65 subunit of nuclear factor kappaB (NF-kappaB), and Survivin were reduced in a dose-dependent manner. Taken together, these changes partially explain the ability of the SLs to induce G(2)/M arrest and apoptosis. Induction of apoptosis by Tomentosin and Inuviscolide in human aggressive melanoma cell lines has high pharmacological value and implies that SLs might be developed as new agents for melanoma treatment.

  12. Impedance Spectroscopy of Human Blood

    Science.gov (United States)

    Mesa, Francisco; Bernal, José J.; Sosa, Modesto A.; Villagómez, Julio C.; Palomares, Pascual

    2004-09-01

    The blood is one of the corporal fluids more used with analytical purposes. When the blood is extracted, immediately it is affected by agents that act on it, producing transformations in its elements. Among the effects of these transformations the hemolysis phenomenon stands out, which consists of the membrane rupture and possible death of the red blood cells. The main purpose of this investigation was the quantification of this phenomenon. A Solartron SI-1260 Impedance Spectrometer was used, which covers a frequency range of work from 1 μHz to 10 MHz, and its accuracy has been tested in the accomplishment of several applications. Measurements were performed on 3 mL human blood samples, from healthy donors. Reactive strips for sugar test of 2 μL, from Bayer, were used as electrodes, which allow gathering a portion of the sample, to be analyzed by the spectrometer. Preliminary results of these measurements are presented.

  13. Glucosaminylmuramyldipeptide-induced changes in phenotype of melanoma cells result in their increased lysis by peripheral blood cells.

    Science.gov (United States)

    Valyakina, T; Malakhov, A; Malakhova, N; Petrova, E; Bykovskaya, S; Revazova, E; Nesmeyanov, V

    1996-11-01

    Flow cytometry was used to show that biologically active N-acetylglucosamine-containing muramylpeptides (GMPs) induced in vitro dose-dependent increase in the expression of tumor-associated antigens (TAAs) characteristic for colon and mammary gland carcinomas, melanoma and lung adenocarcinoma. Forty to two hundred percent enhancement in TAA-expressing cells was observed after 18-48 h incubation with GMPs. In contrast, MHC class I antigen expression was not altered. Using MTT and chromium-release assays, melanoma cells treated in vitro with GMDP were shown to be more susceptible to killing by peripheral blood cells of healthy donors than non-treated cells. Fractionation of blood cells revealed that platelets were responsible for this effect.

  14. Selective cytotoxic effect of 1-O-undecylglycerol in human melanoma cells

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    Marian Hernández-Colina

    2016-04-01

    Full Text Available Context: 1-O-alkylglycerols are ether-linked glycerols derived from shark liver oil and found in small amounts in human milk. Previous studies showed antineoplastic activity for this family of compounds, structurally related to alkylphospholipids, but the activity of linear chain synthetic alkylglycerols in cancer cell lines is less documented. Melanoma is a high incidence cancer, highly resistant to potential treatments. Finding new anti-cancer compounds to improve melanoma prognosis is a relevant research issue. Aims: To study the cytotoxic effect of 1-O-undecylglycerol in primary cultured normal fibroblasts and A375 human melanoma cell line. Methods: Cells were treated with different concentrations of 1-O-undecylglycerol and viability assessed by MTT assay. Morphological changes were visualized by DAPI and acridine orange-ethidium bromide staining. Mitochondrial membrane potential was evaluated, and gene expression of P53 and BcL-2 was semi-quantified. Results: 1-O-undecylglycerol decreased viability of A375 cells and exerted very low cytotoxicity on primary cultured normal fibroblasts. Necrosis appeared in A375 cells but not in fibroblasts, and no apoptotic changes were visualized in DAPI staining experiments. After 24 h fibroblasts and melanoma cells developed mitochondrial potential changes similar to valinomycin. The gene expression of P53 and BcL-2 decreased in treated cells. Conclusions: 1-O-undecylglycerol exhibited selective cytotoxic activity in A375 melanoma cells when compared with primary cultured fibroblast. Its toxicity is mediated by necrosis that may be related with mitochondrial events and decrease in P53 and BcL-2 expression. The results suggest that UDG could be a useful strategy to combine with other chemotherapeutic agents in melanoma treatment.

  15. Role of the cystathionine γ lyase/hydrogen sulfide pathway in human melanoma progression.

    Science.gov (United States)

    Panza, Elisabetta; De Cicco, Paola; Armogida, Chiara; Scognamiglio, Giosuè; Gigantino, Vincenzo; Botti, Gerardo; Germano, Domenico; Napolitano, Maria; Papapetropoulos, Andreas; Bucci, Mariarosaria; Cirino, Giuseppe; Ianaro, Angela

    2015-01-01

    In humans, two main metabolic enzymes synthesize hydrogen sulfide (H2 S): cystathionine γ lyase (CSE) and cystathionine β synthase (CBS). A third enzyme, 3-mercaptopyruvate sulfurtransferase (3-MST), synthesizes H2 S in the presence of the substrate 3-mercaptopyruvate (3-MP). The immunohistochemistry analysis performed on human melanoma samples demonstrated that CSE expression was highest in primary tumors, decreased in the metastatic lesions and was almost silent in non-lymph node metastases. The primary role played by CSE was confirmed by the finding that the overexpression of CSE induced spontaneous apoptosis of human melanoma cells. The same effect was achieved using different H2 S donors, the most active of which was diallyl trisulfide (DATS). The main pro-apoptotic mechanisms involved were suppression of nuclear factor-κB activity and inhibition of AKT and extracellular signal-regulated kinase pathways. A proof of concept was obtained in vivo using a murine melanoma model. In fact, either l-cysteine, the CSE substrate, or DATS inhibited tumor growth in mice. In conclusion, we have determined that the l-cysteine/CSE/H2 S pathway is involved in melanoma progression.

  16. Lansoprazole induces sensitivity to suboptimal doses of paclitaxel in human melanoma.

    Science.gov (United States)

    Azzarito, Tommaso; Venturi, Giulietta; Cesolini, Albino; Fais, Stefano

    2015-01-28

    Tumor acidity is now considered an important determinant of drug-resistance and tumor progression, and anti-acidic approaches, such as Proton Pump inhibitors (PPIs), have demonstrated promising antitumor and chemo-sensitizing efficacy. The main purpose of the present study was to evaluate the possible PPI-induced sensitization of human melanoma cells to Paclitaxel (PTX). Our results show that PTX and the PPI Lansoprazole (LAN) combination was extremely efficient against metastatic melanoma cells, as compared to the single treatments, both in vitro and in vivo. We also showed that acidity plays an important role on the anti-tumor activity of these drugs, being detrimental for PTX activity, while crucial for the synergistic effect of PTX following pretreatment with LAN, due to its nature of pro-drug needing protonation for a full activation. We obtained straightforward results in a human melanoma xenograft model combining well tolerated LAN doses with suboptimal and poorly toxic doses of PTX. With this study we provide a clear evidence that the PPI LAN may be included in new combined therapy of human melanoma together with low doses of PTX.

  17. Theranostic properties of a survivin-directed molecular beacon in human melanoma cells.

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    Sara Carpi

    Full Text Available 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.

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

    DEFF Research Database (Denmark)

    Verdegaal, Els M. E.; De Miranda, Noel F. C. C.; Visser, Marten

    2016-01-01

    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...... 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...

  19. Evaluation of microphthalmia associated transcription factor (MITF expression in peripheral blood of a population with malign melanoma and control population and cell lines

    Directory of Open Access Journals (Sweden)

    Nelson Rangel

    2009-03-01

    Full Text Available Background: The incidence of malign melanoma tumours has increased more rapidly than any other type of cancer; this has intensified the searching for tools that facilitate early detection of melanoma. Microphthalmia associated transcription factor (MITF is currently known as being a master melanocyte regulator. The article analyses MITF gene expression in peripheral blood of individuals suffering from melanoma, compared to people without any type of cancer and some cell lines.Materials and methods: Thirty one samples of peripheral blood were used: 19 from patients having melanoma and 12 from healthy people. Then RNA was extracted from these samples. MITF and housekeeping genes (b2M and GAPDH expression levels were then quantified by real-time PCR. Five cell lines were also used to determine the MITF expression.Results: MITF gene expression could be observed in all individuals, though no statistical significant differences were found among expression levels in the groups studied (p=0.09. Even so, MITF expression in the group of patients suffering from melanoma was much more variable than that observed in the group of cancer-free people. Expression was detected in the cell line AGS (gastric adenocarcinoma, not yet described.Conclusions: MITF gene expression levels were detected in the peripheral blood from both people suffering from melanoma and people without any type of cancer. However, variability in the number of molecules in MITF gene expression was observed in people with melanoma, this suggests the presence of tumour cells in circulation.

  20. A Web-based data warehouse on gene expression in human malignant melanoma.

    Science.gov (United States)

    Györffy, Balazs; Lage, Hermann

    2007-02-01

    The identification of melanoma-specific dysregulated genes could identify new molecular markers. By applying bioinformatic tools for screening of biomedical databases, a melanoma-specific gene expression profile "data warehouse" was constructed. Utilizable data sets of global gene expression analyses were available from nine studies that applied different technology platforms. A single study used cell lines, five investigations analyzed cell lines and tissues obtained from patients, two studies used exclusively specimens obtained from patients, and one study analyzed blood cells prepared from patients. The total number of investigated patients was 116. From 815 differential-regulated genes, 772 (95%) were identified merely in a single study, 37 in at least two studies, five (RAB33A, ERBB3, ADRB2, MERTK, SNF1LK, and ITPKB) in at least three studies, and a single gene, RAB33A, in four studies. These data show that the accuracy, reproducibility, and comparability among different gene expression profile studies are low in melanoma. In conclusion, the study demonstrates the high diversity of gene expression profiles associated with melanoma, the necessity to include a sufficient number of samples regarding clinical standards, for the design of standardized sample collecting and preparation, for the development of common standards for microarray data processing, and for developing standardized bioinformatic tools.

  1. Identification of melanoma biomarkers based on network modules by integrating the human signaling network with microarrays

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    Chunyun Huang

    2014-01-01

    Full Text Available Background: Melanoma is a leading cause of cancer death. Thus, accurate prognostic biomarkers that will assist rational treatment planning need to be identified. Methods: Microarray analysis of melanoma and normal tissue samples was performed to identify differentially expressed modules (DEMs from the signaling network and ultimately detect molecular markers to support histological examination. Network motifs were extracted from the human signaling network. Then, significant expression-correlation differential modules were identified by comparing the network module expression-correlation differential scores under normal and disease conditions using the gene expression datasets. Finally, we obtained DEMs by the Wilcoxon rank test and considered the average gene expression level in these modules as the classification features for diagnosing melanoma. Results: In total, 99 functional DEMs were identified from the signaling network and gene expression profiles. The area under the curve scores for cancer module genes, melanoma module genes, and whole network modules are 92.4%, 90.44%, and 88.45%, respectively. The classification efficiency rates for nonmodule features are 71.04% and 79.38%, which correspond to the features of cancer genes and melanoma cancer genes, respectively. Finally, we acquired six significant molecular biomarkers, namely, module 10 (CALM3, Ca 2+ , PKC, PDGFRA, phospholipase-g, PIB5PA, and phosphatidylinositol-3-kinase, module 14 (SRC, Src homology 2 domain-containing [SHC], SAM68, GIT1, transcription factor-4, CBLB, GRB2, VAV2, LCK, YES, PTCH2, downstream of tyrosine kinase [DOK], and KIT, module 16 (ELK3, p85beta, SHC, ZFYVE9, TGFBR1, TGFBR2, CITED1, SH3KBP1, HCK, DOK, and KIT, module 45 (RB, CCND3, CCNA2, CDK4, and CDK6, module 75 (PCNA, CDK4, and CCND1, and module 114 (PSD93, NMDAR, and FYN. Conclusion: We explored the gene expression profile and signaling network in a global view and identified DEMs that can be used as

  2. Expression and functions of galectin-7 in human and murine melanomas.

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    Katherine Biron-Pain

    Full Text Available The identification of galectin-7 as a p53-induced gene and its ability to induce apoptosis in many cell types support the hypothesis that galectin-7 has strong antitumor activity. This has been well documented in colon cancer. However, in some cases, such as breast cancer and lymphoma, its high expression level correlates with aggressive subtypes of cancer, suggesting that galectin-7 may have a dual role in cancer progression. In fact, in breast cancer, overexpression of galectin-7 alone is sufficient to promote metastasis to the bone and lung. In the present work, we investigated the expression and function of galectin-7 in melanoma. An analysis of datasets obtained from whole-genome profiling of human melanoma tissues revealed that galectin-7 mRNA was detected in more than 90% of biopsies of patients with nevi while its expression was more rarely found in biopsies collected from patients with malignant melanoma. This frequency, however, was likely due to the presence of normal epidermis tissues in biopsies, as shown our studies at the protein level by immunohistochemical analysis. Using the experimental melanoma B16F1 cell line, we found that melanoma cells can express galectin-7 at the primary tumor site and in lung metastasis. Moreover, we found that overexpression of galectin-7 increased the resistance of melanoma cells to apoptosis while inducing de novo egr-1 expression. Overexpression of galectin-7, however, was insufficient to modulate the growth of tumors induced by the subcutaneous injection of B16F1 cells. It also failed to modulate the dissemination of B16F1 cells to the lung.

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

    Science.gov (United States)

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

    2016-02-01

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

  4. Computational modeling of photoacoustic signals from mixtures of melanoma and red blood cells.

    Science.gov (United States)

    Saha, Ratan K

    2014-10-01

    A theoretical approach to model photoacoustic (PA) signals from mixtures of melanoma cells (MCs) and red blood cells (RBCs) is discussed. The PA signal from a cell approximated as a fluid sphere was evaluated using a frequency domain method. The tiny signals from individual cells were summed up obtaining the resultant PA signal. The local signal to noise ratio for a MC was about 5.32 and 5.40 for 639 and 822 nm illuminations, respectively. The PA amplitude exhibited a monotonic rise with increasing number of MCs for each incident radiation. The power spectral lines also demonstrated similar variations over a large frequency range (5-200 MHz). For instance, spectral intensity was observed to be 5.5 and 4.0 dB greater at 7.5 MHz for a diseased sample containing 1 MC and 22,952 RBCs than a normal sample composed of 22,958 RBCs at those irradiations, respectively. The envelope histograms generated from PA signals for mixtures of small numbers of MCs and large numbers of RBCs seemed to obey pre-Rayleigh statistics. The generalized gamma distribution found to facilitate better fits to the histograms than the Rayleigh and Nakagami distributions. The model provides a means to study PAs from mixtures of different populations of absorbers.

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

    Science.gov (United States)

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

    2017-04-01

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

  6. Programmed death-1 blockade enhances expansion and functional capacity of human melanoma antigen-specific CTLs.

    Science.gov (United States)

    Wong, Raymond M; Scotland, Ron R; Lau, Roy L; Wang, Changyu; Korman, Alan J; Kast, W M; Weber, Jeffrey S

    2007-10-01

    Negative co-stimulatory signaling mediated via cell surface programmed death (PD)-1 expression modulates T and B cell activation and is involved in maintaining peripheral tolerance. In this study, we examined the effects of a fully human PD-1-abrogating antibody on the in vitro expansion and function of human vaccine-induced CD8+ T cells (CTLs) specific for the melanoma-associated antigens glycoprotein 100 (gp100) and melanoma antigen recognized by T cells (MART)-1. PD-1 blockade during peptide stimulation augmented the absolute numbers of CD3+, CD4+, CD8+ and gp100/MART-1 MHC:peptide tetramer+ CTLs. This correlated with increased frequencies of IFN-gamma-secreting antigen-specific cells and augmented lysis of gp100+/MART-1+ melanoma targets. PD-1 blockade also increased the fraction of antigen-specific CTLs that recognized melanoma targets by degranulation, suggesting increased recognition efficiency for cognate peptide. The increased frequencies and absolute numbers of antigen-specific CTLs by PD-1 blockade resulted from augmented proliferation, not decreased apoptosis. Kinetic analysis of cytokine secretion demonstrated that PD-1 blockade increased both type-1 and type-2 cytokine accumulation in culture without any apparent skewing of the cytokine repertoire. These findings have implications for developing new cancer immunotherapy strategies.

  7. Laboratory blood group examination of proteolysis degradation human blood

    OpenAIRE

    Beta Ahlam Gizela, Beta Ahlam Gizela

    2015-01-01

    Background: Blood group examination has many purposes and one of them is identification. In several forensic cases there is incompatibility of blood group in corpse and in other evidences usually used blood group examination is serum agglutination method. From the previous study, it was found that there was increasing osmotic fragility of red cell. For that reason, we need to know how the result of blood group tests in degradation human blood.Objective: The purpose of this study is to know bl...

  8. The Non-Coding RNA Llme23 Drives the Malignant Property of Human Melanoma Cells

    Institute of Scientific and Technical Information of China (English)

    Chuan-Fang Wu; Guang-Hong Tan; Cheng-Chuan Ma; Ling Li

    2013-01-01

    Several lines of evidence support the notion that increased RNA-binding ability of polypyrimidine tract-binding (PTB) proteinassociated splicing factor (PSF) and aberrant expression of long non-coding RNAs (lncRNAs) are associated with mouse and human tumors.To identify the PSF-binding IncRNA involved in human oncogenesis,we screened a nuclear RNA repertoire of human melanoma cell line,YUSAC,through RNA-SELEX affinity chromatography.A previously unreported lncRNA,termed as Lime23,was found to bind immobilized PSF resin.The specific binding of Llme23 to both recombinant and native PSF protein was confirmed in vitro and in vivo.The expression of PSF-binding Llme23 is exclusively detected in human melanoma lines.Knocking down Lime23 remarkably suppressed the malignant property of YUSAC cells,accompanied by the repressed expression of proto-oncogene Rab23.These results may indicate that Llme23 can function as an oncogenic RNA and directly associate the PSF-binding IncRNA with human melanoma.

  9. HER2 as a promising target for cytotoxicity T cells in human melanoma therapy.

    Directory of Open Access Journals (Sweden)

    Juan Ma

    Full Text Available Anti-HER2/neu antibody therapy has been reported to mediate tumor regression of HER2/ neu(+ tumors. Here we demonstrated the expression of HER2 in a wide range of human melanoma cells including a primary culture and seven cell lines, and we further investigated whether HER2 could be served as a target for T cell mediated immunotherapy of human melanoma. Specific cytolytic activity of activated T cells (ATC armed with anti-CD3 x anti-HER2 bispecific antibody (HER2Bi-Ab against Malme-3M-luc cells was evaluated by bioluminescent signal generated by luciferase reporter which did not alter HER2 expression or proliferation ability of Malme-3M cells. Contrast with unarmed ATC, increased cytotoxic activity of HER2Bi-armed ATC against Malme-3M-luc cells was observed at effector/target (E/T ratios of 1:1, 5:1, and 20:1. Moreover, HER2Bi-armed ATC expressed higher level of activation marker CD69 and secreted significantly higher level of IFN-γ than unarmed ATC counterpart at the E/T ratio of 20:1. In addition, compared with anti-HER2 mAb (Herceptin® or unarmed ATC, HER2Bi-armed ATC showed remarkable suppression effect on Malme-3M-luc tumor cells. Furthermore, in melanoma tumor cell xenograft mice, infusion of HER2Bi-armed ATC successfully inhibited the growth of melanoma tumors. The anti-tumor effect of HER2Bi-armed ATC may provide a promising immunotherapy for melanoma in the future.

  10. V3 versican isoform expression has a dual role in human melanoma tumor growth and metastasis.

    Science.gov (United States)

    Miquel-Serra, Laia; Serra, Montserrat; Hernández, Daniel; Domenzain, Clelia; Docampo, María José; Rabanal, Rosa M; de Torres, Inés; Wight, Thomas N; Fabra, Angels; Bassols, Anna

    2006-09-01

    Versican is a large chondroitin sulfate proteoglycan produced by several tumor cell types, including malignant melanoma, which exists as four different splice variants. The presence of versican in the extracellular matrix plays a role in tumor cell growth, adhesion and migration, which could be altered by altering the ratio between versican isoforms. We have previously shown that overexpression of the V3 isoform of versican in human melanoma cell lines markedly reduces cell growth in vitro and in vivo, since V3-overexpressing (LV3SN) cultured cells as well as primary tumors arising from these cells grow slower than their vector-only counterparts (LXSN). In the present work, we have extended these observations to demonstrate that the delayed cell growth is due to multiple events since differences in proliferative index as well as in apoptosis are observed in LV3SN cells and tumors compared to LXSN. For example, LV3SN melanoma cells exhibit delayed activation of MAPK in response to EGF, we have also characterized further the primary tumors originated in nude mice from V3-transduced melanoma cells to determine if other events affect the V3 tumor phenotype. For example, hyaluronan content of LV3SN tumors was higher than in LXSN tumors, whereas other related matrix components and vascularization were unaffected. Furthermore, lung metastasis in nude mice occurred only in animals carrying LV3SN tumors, indicating a dual role for this molecule, both as an inhibitor of tumor growth and a metastasis inductor.

  11. Nitric oxide donor augments antineoplastic effects of arginine deprivation in human melanoma cells.

    Science.gov (United States)

    Mayevska, Oksana; Chen, Oleh; Karatsai, Olena; Bobak, Yaroslav; Barska, Maryna; Lyniv, Liliana; Pavlyk, Iuliia; Rzhepetskyy, Yuriy; Igumentseva, Natalia; Redowicz, Maria Jolanta; Stasyk, Oleh

    2017-06-15

    Anticancer therapy based on recombinant arginine-degrading enzymes has been proposed for the treatment of several types of malignant cells deficient in arginine biosynthesis. One of the predicted side effects of such therapy is restricted bioavailability of nitric oxide as arginine catabolic product. Prolonged NO limitation may lead to unwanted disturbances in NO-dependent vasodilation, cardiovascular and immune systems. This problem can be overcome by co-supplementation with exogenous NO donor. However, NO may potentially counteract anticancer effects of therapy based on arginine deprivation. In this study, we evaluate for the first time the effects of an exogenous NO donor, sodium nitroprusside, on viability and metastatic properties of two human melanoma cell lines SK-MEL-28 and WM793 under arginine-deprived conditions. It was revealed that NO did not rescue melanoma cells from specific effects evoked by arginine deprivation, namely decreased viability and induction of apoptosis, dramatically reduced motility, invasiveness and clonogenic potential. Moreover, sodium nitroprusside co-treatment augmented several of these antineoplastic effects. We report that a combination of NO-donor and arginine deprivation strongly and specifically impaired metastatic behavior of melanoma cells. Thus, sodium nitroprusside can be considered as an adjuvant for the more efficient treatment of malignant melanoma and possibly other tumors with arginine-degrading enzymes. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Silymarin targets β-catenin signaling in blocking migration/invasion of human melanoma cells.

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    Mudit Vaid

    Full Text Available Metastatic melanoma is a leading cause of death from skin diseases, and is often associated with activation of Wnt/β-catenin signaling pathway. We have examined the inhibitory effect of silymarin, a plant flavanoid from Silybum marianum, on cell migration of metastasis-specific human melanoma cell lines (A375 and Hs294t and assessed whether Wnt/β-catenin signaling is the target of silymarin. Using an in vitro invasion assay, we found that treatment of human melanoma cell lines with silymarin resulted in concentration-dependent inhibition of cell migration, which was associated with accumulation of cytosolic β-catenin, while reducing the nuclear accumulation of β-catenin (i.e., β-catenin inactivation and reducing the levels of matrix metalloproteinase (MMP -2 and MMP-9 which are the down-stream targets of β-catenin. Silymarin enhanced: (i the levels of casein kinase 1α, glycogen synthase kinase-3β and phosphorylated-β-catenin on critical residues Ser(45, Ser(33/37 and Thr(41, and (ii the binding of β-transducin repeat-containing proteins (β-TrCP with phospho forms of β-catenin in melanoma cells. These events play important roles in degradation or inactivation of β-catenin. To verify whether β-catenin is a potent molecular target of silymarin, the effect of silymarin was determined on β-catenin-activated (Mel 1241 and β-catenin-inactivated (Mel 1011 melanoma cells. Treatment of Mel 1241 cells with silymarin or FH535, an inhibitor of Wnt/β-catenin pathway, significantly inhibited cell migration of Mel 1241 cells, which was associated with the elevated levels of casein kinase 1α and glycogen synthase kinase-3β, and decreased accumulation of nuclear β-catenin and inhibition of MMP-2 and MMP-9 levels. However, this effect of silymarin and FH535 was not found in Mel 1011 melanoma cells. These results indicate for the first time that silymarin inhibits melanoma cell migration by targeting β-catenin signaling pathway.

  13. Tissue-based microarray expression of genes predictive of metastasis in uveal melanoma and differentially expressed in metastatic uveal melanoma.

    Science.gov (United States)

    Demirci, Hakan; Reed, David; Elner, Victor M

    2013-10-01

    To screen the microarray expression of CDH1, ECM1, EIF1B, FXR1, HTR2B, ID2, LMCD1, LTA4H, MTUS1, RAB31, ROBO1, and SATB1 genes which are predictive of primary uveal melanoma metastasis, and NFKB2, PTPN18, MTSS1, GADD45B, SNCG, HHIP, IL12B, CDK4, RPLP0, RPS17, RPS12 genes that are differentially expressed in metastatic uveal melanoma in normal whole human blood and tissues prone to metastatic involvement by uveal melanoma. We screened the GeneNote and GNF BioGPS databases for microarray analysis of genes predictive of primary uveal melanoma metastasis and those differentially expressed in metastatic uveal melanoma in normal whole blood, liver, lung and skin. Microarray analysis showed expression of all 22 genes in normal whole blood, liver, lung and skin, which are the most common sites of metastases. In the GNF BioGPS database, data for expression of the HHIP gene in normal whole blood and skin was not complete. Microarray analysis of genes predicting systemic metastasis of uveal melanoma and genes differentially expressed in metastatic uveal melanoma may not be used as a biomarker for metastasis in whole blood, liver, lung, and skin. Their expression in tissues prone to metastasis may suggest that they play a role in tropism of uveal melanoma metastasis to these tissues.

  14. Tissue-Based Microarray Expression of Genes Predictive of Metastasis in Uveal Melanoma and Differentially Expressed in Metastatic Uveal Melanoma

    Directory of Open Access Journals (Sweden)

    Hakan Demirci

    2013-01-01

    Full Text Available Purpose: To screen the microarray expression of CDH1, ECM1, EIF1B, FXR1, HTR2B, ID2, LMCD1, LTA4H, MTUS1, RAB31, ROBO1, and SATB1 genes which are predictive of primary uveal melanoma metastasis, and NFKB2, PTPN18, MTSS1, GADD45B, SNCG, HHIP, IL12B, CDK4, RPLP0, RPS17, RPS12 genes that are differentially expressed in metastatic uveal melanoma in normal whole human blood and tissues prone to metastatic involvement by uveal melanoma. Methods: We screened the GeneNote and GNF BioGPS databases for microarray analysis of genes predictive of primary uveal melanoma metastasis and those differentially expressed in metastatic uveal melanoma in normal whole blood, liver, lung and skin. Results: Microarray analysis showed expression of all 22 genes in normal whole blood, liver, lung and skin, which are the most common sites of metastases. In the GNF BioGPS database, data for expression of the HHIP gene in normal whole blood and skin was not complete. Conclusions: Microarray analysis of genes predicting systemic metastasis of uveal melanoma and genes differentially expressed in metastatic uveal melanoma may not be used as a biomarker for metastasis in whole blood, liver, lung, and skin. Their expression in tissues prone to metastasis may suggest that they play a role in tropism of uveal melanoma metastasis to these tissues.

  15. Molecular mechanism implicated in Pemetrexed-induced apoptosis in human melanoma cells

    Directory of Open Access Journals (Sweden)

    Buqué Aitziber

    2012-04-01

    Full Text Available Abstract Background Metastatic melanoma is a lethal skin cancer and its incidence is rising every year. It represents a challenge for oncologist, as the current treatment options are non-curative in the majority of cases; therefore, the effort to find and/or develop novel compounds is mandatory. Pemetrexed (Alimta®, MTA is a multitarget antifolate that inhibits folate-dependent enzymes: thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyltransferase, required for de novo synthesis of nucleotides for DNA replication. It is currently used in the treatment of mesothelioma and non-small cell lung cancer (NSCLC, and has shown clinical activity in other tumors such as breast, colorectal, bladder, cervical, gastric and pancreatic cancer. However, its effect in human melanoma has not been studied yet. Results In the current work we studied the effect of MTA on four human melanoma cell lines A375, Hs294T, HT144 and MeWo and in two NSCLC cell lines H1299 and Calu-3. We have found that MTA induces DNA damage, S-phase cell cycle arrest, and caspase- dependent and –independent apoptosis. We show that an increment of the intracellular reactive oxygen species (ROS and p53 is required for MTA-induced cytotoxicity by utilizing N-Acetyl-L-Cysteine (NAC to blockage of ROS and p53-defective H1299 NSCLC cell line. Pretreatment of melanoma cells with NAC significantly decreased the DNA damage, p53 up-regulation and cytotoxic effect of MTA. MTA was able to induce p53 expression leading to up-regulation of p53-dependent genes Mcl-1 and PIDD, followed by a postranscriptional regulation of Mcl-1 improving apoptosis. Conclusions We found that MTA induced DNA damage and mitochondrial-mediated apoptosis in human melanoma cells in vitro and that the associated apoptosis was both caspase-dependent and –independent and p53-mediated. Our data suggest that MTA may be of therapeutic relevance for the future treatment of human malignant melanoma.

  16. Ocular Melanoma

    Science.gov (United States)

    ... Español Eye Health / Eye Health A-Z Ocular Melanoma Sections What is Ocular Melanoma? Ocular Melanoma Causes ... Melanoma Diagnosis Ocular Melanoma Treatment What is Ocular Melanoma? Leer en Español: ¿Qué Es el Melanoma Ocular? ...

  17. [Cytotoxic effects of local anesthesia through lidocaine/ropivacaine on human melanoma cell lines].

    Science.gov (United States)

    Kang, Ding-Kun; Zhao, Li-Yan; Wang, Hong-Li

    Local anesthetics (LAs) are generally considered as safe, but cytotoxicity has been reported for several local anesthetics used in humans, which is not well investigated. In the present study, the cytotoxicity of lidocaine, ropivacaine and the combination of lidocaine and ropivacaine were evaluated on human melanoma cell lines. Melphalan, a nitrogen mustard alkylating agent, was used as a control agent for comparison of cytotoxic activity. Melanoma cell lines, A375 and Hs294T, were exposed to 1h to different concentrations of above agents. Cell-viability after exposure was determined by flow cytometry. Investigated LAs showed detrimental cytotoxicity on studied melanoma cell lines in time- (p<0.001), concentration- (p<0.001), and agent dependant. In both A375 and Hs294T cell lines, minimum cell viability rates were found after 72h of exposure to these agents. Lidocaine 2% caused a reduction of vital cells to 10%±2% and 14%±2% in A375 and Hs294T, respectively after 72h of exposure. Ropivacaine 0.75% after 72h reduced viable cells to 15%±3% and 25%±3% in A375 and Hs294T, respectively. Minimum cell viability after 72h exposure to the combination was 10%±2% and 18%±2% in A375 and Hs294T, respectively. Minimum cell viability after 72h exposure to melphalan was 8%±1% and 12%±2%, in A375 and Hs294T, respectively. LAs have cytotoxic activity on human melanoma cell lines in a time-, concentration- and agent-dependant manner. Apoptosis in the cell lines was mediated through activity of caspases-3 and caspases-8. Copyright © 2016 Sociedade Brasileira de Anestesiologia. Publicado por Elsevier Editora Ltda. All rights reserved.

  18. Cytotoxic effects of local anesthesia through lidocaine/ropivacaine on human melanoma cell lines.

    Science.gov (United States)

    Kang, Ding-Kun; Zhao, Li-Yan; Wang, Hong-Li

    Local anesthetics (LAs) are generally considered as safe, but cytotoxicity has been reported for several local anesthetics used in humans, which is not well investigated. In the present study, the cytotoxicity of lidocaine, ropivacaine and the combination of lidocaine and ropivacaine were evaluated on human melanoma cell lines. Melphalan, a nitrogen mustard alkylating agent, was used as a control agent for comparison of cytotoxic activity. Melanoma cell lines, A375 and Hs294T, were exposed to 1h to different concentrations of above agents. Cell-viability after exposure was determined by flow cytometry. Investigated LAs showed detrimental cytotoxicity on studied melanoma cell lines in time- (p<0.001), concentration- (p<0.001), and agent dependant. In both A375 and Hs294T cell lines, minimum cell viability rates were found after 72h of exposure to these agents. Lidocaine 2% caused a reduction of vital cells to 10%±2% and 14%±2% in A375 and Hs294T, respectively after 72h of exposure. Ropivacaine 0.75% after 72h reduced viable cells to 15%±3% and 25%±3% in A375 and Hs294T, respectively. Minimum cell viability after 72h exposure to the combination was 10%±2% and 18%±2% in A375 and Hs294T, respectively. Minimum cell viability after 72h exposure to melphalan was 8%±1% and 12%±2%, in A375 and Hs294T, respectively. LAs have cytotoxic activity on human melanoma cell lines in a time-, concentration- and agent-dependant manner. Apoptosis in the cell lines was mediated through activity of caspases-3 and caspases-8. Copyright © 2016 Sociedade Brasileira de Anestesiologia. Published by Elsevier Editora Ltda. All rights reserved.

  19. Expression and cytotoxic effect of transmembrane form of human blood group A antigen mimotope vaccine in a malignant melanoma cell line B16%跨膜型血型A抗原模拟肽疫苗在黑素瘤细胞B16中表达及细胞毒效应检测

    Institute of Scientific and Technical Information of China (English)

    岑东芝; 孟辉; 张积仁

    2011-01-01

    Objective To establish a stable cell line expressing transmembrane form of human blood group A antigen mimotope vaccine by transfecting malignant melanoma cell line B16, and to detect the cytotoxicity of the vaccine against melanoma cells. Methods Cultured B16 cells were classified into 4 groups, i.e.,P/F-M-pIRES group [transfected with the recombinant plasmid mimotope peptide/Fas-macrophage inflammatory protein (Mip)-pIRES], P/F-pIRES group (transfected with the recombinant plasmid mimotope peptide/FaspIRES), M-pIRES group (transfected with the recombinant plasmid Mip-pIRES), and pIRES group (transfected with the empty plasmid pIRES). B 16 cells were transfected through Lipofectamine 2000. Subsequently, RT-PCR and Western blotting were performed to detect the mRNA and protein expressions of the mimotope peptide/Fas fusion gene and Mip3β in transfected B16 cells. Cell counting kit-8 (CCK-8) was used to evaluate the vaccinemediated complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)against B16 cells. Results RT-PCR yielded specific DNA fragments with expected size. Western blotting revealed the anti-A antibody-binding activity of the recombinant mimotope peptide/Fas fusion protein. Factor analysis indicated significant differences in CDC (F = 244.522, P < 0.01 ) and ADCC (F = 71.593, P < 0.01 )against B16 cells between the 4 groups. Group comparisons demonstrated more intense CDC and ADCC in P/FM-pIRES and P/F-pIRES groups compared with M-plRES and pIRES groups, stronger ADCC in P/F-M-pIRES group in comparison with P/F-pIRES group (F = 15.42, P < 0.05), but no significant difference in CDC was observed between M-pIRES and pIRES group. Conclusions The transmembrane form of human blood group A antigen mimotope vaccine could be stably expressed in B16 cells, and mediate ADCC and CDC against B16 cells in vitro.%目的 建立稳定表达跨膜型血型A抗原模拟肽疫苗的恶性黑素瘤细胞株B16,并检

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

    Energy Technology Data Exchange (ETDEWEB)

    Siqueira-Moura, Marigilson P. [Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP), Universidade de São Paulo, Ribeirão Preto-SP (Brazil); Departamento de Química, Laboratório de Fotobiologia e Fotomedicina, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto (FFCLRP), Universidade de São Paulo, Ribeirão Preto-SP (Brazil); Primo, Fernando L. [Departamento de Química, Laboratório de Fotobiologia e Fotomedicina, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto (FFCLRP), Universidade de São Paulo, Ribeirão Preto-SP (Brazil); Espreafico, Enilza M. [Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto (FMRP), Universidade de São Paulo, Ribeirão Preto-SP (Brazil); Tedesco, Antonio C., E-mail: atedesco@usp.br [Departamento de Química, Laboratório de Fotobiologia e Fotomedicina, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto (FFCLRP), Universidade de São Paulo, Ribeirão Preto-SP (Brazil)

    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{sup 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{sup −1}) under light irradiation at 20 mJ cm{sup −2}. On the other hand, the cell survival percentage for all the melanoma cell lines treated with the highest light dose (150 mJ cm{sup −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. - Highlights: ► Nanocapsules containing a hydrophobic metallophthalocyanine (ClAlPc) were developed. ► The colloidal formulations were characterized by their physicochemical parameters

  1. The prognostic role of blood lymphocyte subset distribution in patients with resected high-risk primary or regionally metastatic melanoma

    DEFF Research Database (Denmark)

    Hernberg, Micaela; Mattila, Petri S; Rissanen, Marjo;

    2007-01-01

    Cooperative Group adjuvant interferon study. The frequencies of peripheral blood lymphocyte subsets were monitored by flow cytometry using CD3, CD4, CD8, CD56, and CD69 monoclonal antibodies. Patients with low proportions of CD3+CD4+CD69+ cells and of CD3+CD56+ cells before treatment had an improved disease...... independent prognostic factors for overall survival. Our data show that both the proportions of CD3+CD4+CD69+ cells and of CD3+CD56+ cells seem to have a prognostic potential in the natural course of melanoma. These results need to be confirmed in larger studies. Udgivelsesdato: 2007-Oct...

  2. Chondroitin sulfate proteoglycan-4: a biomarker and a potential immunotherapeutic target for canine malignant melanoma.

    Science.gov (United States)

    Mayayo, Saray Lorda; Prestigio, Simone; Maniscalco, Lorella; La Rosa, Giuseppe; Aricò, Arianna; De Maria, Raffaella; Cavallo, Federica; Ferrone, Soldano; Buracco, Paolo; Iussich, Selina

    2011-11-01

    Chondroitin sulfate proteoglycan-4 (CSPG4), also known as high molecular weight-melanoma associated antigen (HMW-MAA), is a membrane-bound chondroitin sulfate proteoglycan highly expressed by human melanoma cells. This phylogenetically conserved tumour antigen plays an important biological role in human melanoma, where it is used as a marker to diagnose forms with unusual characteristics, such as desmoplastic melanoma, and to detect melanoma cells in lymph nodes and peripheral blood, and as a target for immunotherapy because of its restricted distribution in normal tissues. To identify suitable targets to develop novel approaches of treating canine melanoma, CSPG4 was studies to see whether it is expressed in canine malignant melanomas. Immunohistochemical staining of 65 canine malignant melanomas with an anti-human CSPG4-specific antibody detected CSPG4 in 37 cases (56.9%). Positive staining was more frequent, albeit not significantly, in amelanotic compared to melanotic tumours and was statistically associated with tumours having both melanin and the epithelioid histotype. The frequency of CSPG4 expression was similar to that of other melanoma antigens used as diagnostic markers for canine malignant melanoma, such as Melan A and the protein recognized by the PNL2 monoclonal antibody. The results suggest that CSPG4 constitutes a new potential immunohistochemical marker of canine malignant melanoma and may represent an immunotherapeutic target as in humans.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation, differentiation, and maintenance of hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Stopp, Sabine; Bornhäuser, Martin; Ugarte, Fernando; Wobus, Manja; Kuhn, Matthias; Brenner, Sebastian; Thieme, Sebastian

    2013-04-01

    The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo. The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal stromal cells to evaluate the effect of the melanoma cell adhesion molecule on their proliferation and differentiation as well as its influence on co-cultivated hematopoietic stem and progenitor cells. Knockdown and overexpression of the melanoma cell adhesion molecule affected several characteristics of human mesenchymal stromal cells related to osteogenic differentiation, proliferation, and migration. Furthermore, knockdown of the melanoma cell adhesion molecule in human mesenchymal stromal cells stimulated the proliferation of hematopoietic stem and progenitor cells, and strongly reduced the formation of long-term culture-initiating cells. In contrast, melanoma cell adhesion molecule-overexpressing human mesenchymal stromal cells provided a supportive microenvironment for hematopoietic stem and progenitor cells. Expression of the melanoma cell adhesion molecule increased the adhesion of hematopoietic stem and progenitor cells to human mesenchymal stromal cells and their migration beneath the monolayer of human mesenchymal stromal cells. Our results demonstrate that the expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells determines their fate and regulates the maintenance of hematopoietic stem and progenitor cells through direct cell-cell contact.

  5. Physalin B from Physalis angulata triggers the NOXA-related apoptosis pathway of human melanoma A375 cells.

    Science.gov (United States)

    Hsu, Chia-Chun; Wu, Yang-Chang; Farh, Lynn; Du, Ying-Chi; Tseng, Wei-Kung; Wu, Chau-Chung; Chang, Fang-Rong

    2012-03-01

    Melanoma is a lethal form of skin cancer that can metastasize rapidly. While surgery and radiation therapy provide palliative therapy for local tumor growth, systemic therapy is the mainstay of treatment for metastatic melanoma. However, limited chemotherapeutic agents are available for melanoma treatment. In this study, we investigated the anti-melanoma effect of physalin B, the major active compound from a widely used herb medicine, Physalis angulata L. This study demonstrated that physalin B exhibits cytotoxicity towards v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-mutated melanoma A375 and A2058 cells (the IC50 values are lower than 4.6 μg/ml). Cytotoxicity is likely resulted from apoptosis since the apoptotic marker phosphatidylserine are detected immediately under physalin B treatment and apoptotic cells formation. Further examination revealed that physalin B induces expression of the proapoptotic protein NOXA within 2 h and later triggers the expression of Bax and caspase-3 in A375 cells. These results indicate that physalin B can induce apoptosis of melanoma cancer cells via the NOXA, caspase-3, and mitochondria-mediated pathways, but not of human skin fibroblast cells and myoblastic cells. Thus, physalin B has the potential to be developed as an effective chemotherapeutic lead compound for the treatment of malignant melanoma.

  6. H Ferritin Gene Silencing in a Human Metastatic Melanoma Cell Line: A Proteomic Analysis

    DEFF Research Database (Denmark)

    Di Sanzo, Maddalena; Gaspari, Marco; Misaggi, Roberta

    2011-01-01

    and pathologic states (i.e., neurodegeneration and cancer). This study is aimed at investigating the whole-cell proteome of FHC-expressing and sh-RNA-silenced human metastatic melanoma cells (MM07(m)) in the attempt to identify and classify the highest number of proteins directly or indirectly controlled...... by the FHC. We identified about 200 differentially expressed proteins and classified them in clusters on the basis of their functions, as proteins involved in metabolic processes, cell adhesion, migration, and proliferation processes. Some of them have captured our attention because of their involvement...... of H ferritin signaling pathways and lend support to the hypothesis that specific targeting of this gene might be an attractive and potentially effective strategy for the management of metastatic melanoma....

  7. Basic and clinical aspects of malignant melanoma

    Energy Technology Data Exchange (ETDEWEB)

    Nathanson, L. (Health Sciences Center, State Univ. of New York at Stony Brook, Stony Brook, NY (US))

    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.

  8. Two types of human malignant melanoma cell lines revealed by expression patterns of mitochondrial and survival-apoptosis genes: implications for malignant melanoma therapy.

    Science.gov (United States)

    Su, David M; Zhang, Qiuyang; Wang, Xuexi; He, Ping; Zhu, Yuelin Jack; Zhao, Jianxiong; Rennert, Owen M; Su, Yan A

    2009-05-01

    Human malignant melanoma has poor prognosis because of resistance to apoptosis and therapy. We describe identification of the expression profile of 1,037 mitochondria-focused genes and 84 survival-apoptosis genes in 21 malignant melanoma cell lines and 3 normal melanocyte controls using recently developed hMitChip3 cDNA microarrays. Unsupervised hierarchical clustering analysis of 1,037 informative genes, and 84 survival-apoptosis genes, classified these malignant melanoma cell lines into type A (n = 12) and type B (n = 9). Three hundred fifty-five of 1,037 (34.2%) genes displayed significant (P ≤ 0.030; false discovery rate ≤ 3.68%) differences (± ≥ 2.0-fold) in average expression, with 197 genes higher and 158 genes lower in type A than in type B. Of 84 genes with known survival-apoptosis functions, 38 (45.2%) displayed the significant (P genes expressed at higher levels in type A than in type B, whereas the different set of antiapoptotic (PSEN1, PPP2CA, API5, PPP2R1B, PPP2R1A, and FIS1), antioxidant (HSPD1, GSS, SOD1, ATOX1, and CAT), and proapoptotic (ENDOG, BAK1, CASP2, CASP4, PDCD5, HTRA2, SEPT4, TNFSF10, and PRODH) genes expressed at lower levels in type A than in type B. Microarray data were validated by quantitative reverse transcription-PCR. These results showed the presence of two types of malignant melanoma, each with a specific set of dysregulated survival-apoptosis genes, which may prove useful for development of new molecular targets for therapeutic intervention and novel diagnostic biomarkers for treatment and prognosis of malignant melanoma.

  9. Analysis of the Antitumor Activity of Clotrimazole on A375 Human Melanoma Cells

    DEFF Research Database (Denmark)

    Adinolfi, Barbara; Carpi, Sara; Romanini, Antonella

    2015-01-01

    analyses of cell viability, gene expression, cell-cycle progression, annexin V reactivity and internucleosomal DNA fragmentation. RESULTS: Clotrimazole induced cytotoxicity in A375 human melanoma cells without significant changes of human keratinocyte cell viability. Clotrimazole, at a concentration...... that approximates the inhibitory concentration 50% (IC50) value (i.e. 10 μM), reduced the expression of hexokinase type-II, induced cell-cycle arrest at G1-S phase transition, altered annexin V reactivity and induced DNA fragmentation without evidence of necrosis. CONCLUSION: The current study provides evidence...

  10. Effect of kappa elastin on melanogenesis in A375 human melanoma cells and its related mechanism

    Institute of Scientific and Technical Information of China (English)

    TIAN Shan; HE Pei-ying; ZHANG Jian-zhong; CHEN Zhou

    2012-01-01

    Background Elastin derived peptides can regulate melanocyte precursor development.Ultraviolet irradiation,infrared radiation and heat can increase the synthesis of tropoelastin in human skin epidermis.The aim of this study was to investigate whether the over expressed tropoelastin in epidermis has some role in melanogenesis of melanocytes.Methods A375 human melanoma cells were treated with different concentrations of kappa elastin for 24 hours.A375 human melanoma cells were randomly assigned to control,kappa elastin,and lactose pre-incubated groups.The cell viabilities were detected by the methyl thiazoleterazolium assay.Melanin content and tyrosinase activity in A375 melanoma cells were measured.The expressions of endothelin B receptor(ETBR)mRNA and c-kit mRNA in A375 melanoma cells were measured by quantative reverse transcription polymerase chain reaction.Results Fifty μg/ml of kappa elastin significantly increased the melanin content by 56.64% compared with the control(P<0.05).Kappa elastin increased cellular tyrosinase activity by 46.73% compared with the control at 24 hours(P<0.05).Kappa elastin increased the expressions of ETBR and c-kit mRNA levels by 2.13-fold and 2.47-fold compared with the controls,respectively.When pre-incubating cells with a lactose solution(10 mmol/L),the inhibition on melanin production was 34.96% compared with the kappa elastin group(P<0.05),tyrosinase activity was inhibited by 29.93% compared with kappa elastin group(P<0.05),and the expressions of ETBR mRNA and c-kit mRNA were decreased by 1.56-fold and 0.82-fold compared with kappa elastin group,respectively.Conclusion Kappa elastin increased the melanogenesis in A375 melanoma cells via the stimulation of tyrosinase activity and the expression of ETBR and c-kit.The over expressed tropoelastin produced by keratinocytes might play a role in melanogenesis of epidermal melanocytes.

  11. Mechanism of retinoid receptors in inhibiting proliferation and inducing apoptosis of human melanoma cell line A375

    Institute of Scientific and Technical Information of China (English)

    NIU Xin-wu; PENG Zhen-hui; FENG Jie; MA Hui-qun; LIU Chao; YUAN Jing-yi

    2005-01-01

    @@ Malignant melanoma is a common cancer of skin. Its incidence is growing rapidly in recent years,1 however, there is no effective therapy for this cancer. Retinoids are metabolites or derivatives of vitamin A. They are essential for growth, differentiation, and maintenance of epithelial tissues.2 Previous studies showed that retinoids could inhibit growth of many kinds of malignant tumor cell lines and induce its apoptosis,3,4 including malignant melanoma cell lines.5 Some retinoids have therapeutic action to malignant melanoma, such as all-trans retinoic acid (ATRA) and 13-cis-RA.6,7 Retinoids take effects mainly through two kinds of nuclear receptors, retinoic acid receptor (RAR) and retinoic acid X receptor (RXR). In this study, we have investigated the effects of diverse retinoids and receptor agonists in inhibiting proliferation and inducing apoptosis of human melanoma cell line A375.

  12. A melanoma immune response signature including Human Leukocyte Antigen-E.

    Science.gov (United States)

    Tremante, Elisa; Ginebri, Agnese; Lo Monaco, Elisa; Benassi, Barbara; Frascione, Pasquale; Grammatico, Paola; Cappellacci, Sandra; Catricalà, Caterina; Arcelli, Diego; Natali, Pier Giorgio; Di Filippo, Franco; Mottolese, Marcella; Visca, Paolo; Benevolo, Maria; Giacomini, Patrizio

    2014-01-01

    Paired cultures of early-passage melanoma cells and melanocytes were established from metastatic lesions and the uninvolved skin of five patients. In this stringent autologous setting, cDNA profiling was used to analyze a subset of 1477 genes selected by the Gene Ontology term 'immune response'. Human Leukocyte Antigen E (HLA-E) was ranked 19th among melanoma-overexpressed genes and was embedded in a transformation signature including its preferred peptide ligand donors HLA-A, HLA-B, HLA-C, and HLA-G. Mostly undetectable in normal skin and 39 nevi (including rare and atypical lesions), HLA-E was detected by immunohistochemistry in 17/30 (57%) and 32/48 (67%) primary and metastatic lesions, respectively. Accordingly, surface HLA-E was higher on melanoma cells than on melanocytes and protected the former (6/6 cell lines) from lysis by natural killer (NK) cells, functionally counteracting co-expressed triggering ligands. Although lacking HLA-E, melanocytes (4/4 cultures) were nevertheless (and surprisingly) fully protected from NK cell lysis.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sustarsic, Elahu G. [Edison Biotechnology Institute, 1 Watertower Drive, Athens, OH (United States); Department of Biological Sciences, Ohio University, Athens, OH (United States); Junnila, Riia K. [Edison Biotechnology Institute, 1 Watertower Drive, Athens, OH (United States); Kopchick, John J., E-mail: kopchick@ohio.edu [Edison Biotechnology Institute, 1 Watertower Drive, Athens, OH (United States); Department of Biological Sciences, Ohio University, Athens, OH (United States); Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH (United States)

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

    Energy Technology Data Exchange (ETDEWEB)

    Rappa, Germana [Cancer Research Program, Roseman University of Health Sciences, 10530 Discovery Drive. Las Vegas, NV 89135 (United States); College of Pharmacy, Roseman University of Health Sciences, Henderson, NV 89104 (United States); Mercapide, Javier; Anzanello, Fabio [Cancer Research Program, Roseman University of Health Sciences, 10530 Discovery Drive. Las Vegas, NV 89135 (United States); Le, Thuc T. [Nevada Cancer Institute, Las Vegas, NV 89135 (United States); Johlfs, Mary G. [Cancer Research Program, Roseman University of Health Sciences, 10530 Discovery Drive. Las Vegas, NV 89135 (United States); Center for Diabetes and Obesity Prevention, Treatment, Research and Education, Roseman University of Health Sciences, Henderson, NV 89104 (United States); Fiscus, Ronald R. [Cancer Research Program, Roseman University of Health Sciences, 10530 Discovery Drive. Las Vegas, NV 89135 (United States); College of Pharmacy, Roseman University of Health Sciences, Henderson, NV 89104 (United States); Center for Diabetes and Obesity Prevention, Treatment, Research and Education, Roseman University of Health Sciences, Henderson, NV 89104 (United States); Wilsch-Bräuninger, Michaela [Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden (Germany); Corbeil, Denis [Tissue Engineering Laboratories (BIOTEC) and DFG Research Center and Cluster of Excellence for Regenerative Therapies Dresden (CRTD), Technische Universität Dresden, Tatzberg 47–49, 01307 Dresden, Germany Technische Universitat Dresden, Dresden (Germany); Lorico, Aurelio, E-mail: alorico@roseman.edu [Cancer Research Program, Roseman University of Health Sciences, 10530 Discovery Drive. Las Vegas, NV 89135 (United States); College of Pharmacy, Roseman University of Health Sciences, Henderson, NV 89104 (United States)

    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

  16. The redox antimalarial dihydroartemisinin targets human metastatic melanoma cells but not primary melanocytes with induction of NOXA-dependent apoptosis.

    Science.gov (United States)

    Cabello, Christopher M; Lamore, Sarah D; Bair, Warner B; Qiao, Shuxi; Azimian, Sara; Lesson, Jessica L; Wondrak, Georg T

    2012-08-01

    Recent research suggests that altered redox control of melanoma cell survival, proliferation, and invasiveness represents a chemical vulnerability that can be targeted by pharmacological modulation of cellular oxidative stress. The endoperoxide artemisinin and semisynthetic artemisinin-derivatives including dihydroartemisinin (DHA) constitute a major class of antimalarials that kill plasmodium parasites through induction of iron-dependent oxidative stress. Here, we demonstrate that DHA may serve as a redox chemotherapeutic that selectively induces melanoma cell apoptosis without compromising viability of primary human melanocytes. Cultured human metastatic melanoma cells (A375, G361, LOX) were sensitive to DHA-induced apoptosis with upregulation of cellular oxidative stress, phosphatidylserine externalization, and activational cleavage of procaspase 3. Expression array analysis revealed DHA-induced upregulation of oxidative and genotoxic stress response genes (GADD45A, GADD153, CDKN1A, PMAIP1, HMOX1, EGR1) in A375 cells. DHA exposure caused early upregulation of the BH3-only protein NOXA, a proapototic member of the Bcl2 family encoded by PMAIP1, and genetic antagonism (siRNA targeting PMAIP1) rescued melanoma cells from apoptosis indicating a causative role of NOXA-upregulation in DHA-induced melanoma cell death. Comet analysis revealed early DHA-induction of genotoxic stress accompanied by p53 activational phosphorylation (Ser 15). In primary human epidermal melanocytes, viability was not compromised by DHA, and oxidative stress, comet tail moment, and PMAIP1 (NOXA) expression remained unaltered. Taken together, these data demonstrate that metastatic melanoma cells display a specific vulnerability to DHA-induced NOXA-dependent apoptosis and suggest feasibility of future anti-melanoma intervention using artemisinin-derived clinical redox antimalarials.

  17. MIF inhibition reverts the gene expression profile of human melanoma cell line-induced MDSCs to normal monocytes

    Directory of Open Access Journals (Sweden)

    Sabine Waigel

    2016-03-01

    Full Text Available Myeloid-derived suppressor cells (MDSCs are potently immunosuppressive innate immune cells that accumulate in advanced cancer patients and actively inhibit anti-tumor T lymphocyte responses [1]. Increased numbers of circulating MDSCs directly correlate with melanoma patient morbidity and reduced anti-tumor immune responses [2,3]. Previous studies have revealed that monocyte-derived macrophage migration inhibitory factor (MIF is necessary for the immune suppressive function of MDSCs in mouse models of melanoma [4,5]. To investigate whether MIF participates in human melanoma-induced MDSC differentiation and/or suppressive function, we have established an in vitro MDSC induction model using primary, normal human monocytes co-cultured with human melanoma cell lines in the presence or absence of the MIF antagonist—4-IPP [4,6–9]. To identify potential mechanistic effectors, we have performed transcriptome analyses on cultured monocytes and on melanoma-induced MDSCs obtained from either untreated or 4-IPP-treated A375:monocyte co-cultures. Here, we present a detailed protocol, which can facilitate easy reproduction of the microarray results (NCBI GEO accession number GSE73333 published by Yaddanapudi et al. (2015 in Cancer Immunology Research [10].

  18. Gene expression of panaxydol-treated human melanoma cells using radioactive cDNA microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Joong Youn; Yu, Su Jin; Soh, Jeong Won; Kim, Meyoung Kon [College of Medicine, Korea Univ., Seoul (Korea, Republic of)

    2001-07-01

    Polyacetylenic alcohols derived from Panax ginseng have been studied to be an anticancer reagent previously. One of the Panax ginseng polyacetylenic alcohols, i.e., panaxydol, has been studied to possess an antiproliferative effect on human melanoma cell line (SK-MEL-1). In ths study, radioactive cDNA microarrays enabled an efficient approach to analyze the pattern of gene expression (3.194 genes in a total) simultaneously. The bioinformatics selection of human cDNAs, which is specifically designed for immunology, apoptosis and signal transduction, were arrayed on nylon membranes. Using with {sup 33}P labeled probes, this method provided highly sensitive gene expression profiles of our interest including apoptosis, cell proliferation, cell cycle, and signal transduction. Gene expression profiles were also classified into several categories in accordance with the duration of panaxydol treatment. Consequently, the gene profiles of our interest were significantly up (199 genes, > 2.0 of Z-ratio) or down-(196 genes, < 2.0 of Z-ratio) regulated in panaxydol-treated human melanoma cells.

  19. Human survivin and Trypanosoma cruzi calreticulin act in synergy against a murine melanoma in vivo.

    Directory of Open Access Journals (Sweden)

    Lorena Aguilar-Guzmán

    Full Text Available Immune-based anti-tumor or anti-angiogenic therapies hold considerable promise for the treatment of cancer. The first approach seeks to activate tumor antigen-specific T lymphocytes while, the second, delays tumor growth by interfering with blood supply. Tumor Associated Antigens are often employed to target tumors with therapeutic drugs, but some are also essential for tumor viability. Survivin (Surv is a member of the inhibitor of apoptosis protein family that is considered a Tumor Associated Antigen important for cancer cell viability and proliferation. On the other hand, Trypanosoma cruzi (the agent of Chagas' disease calreticulin (TcCRT displays remarkable anti-angiogenic properties. Because these molecules are associated with different tumor targets, we reasoned that immunization with a Surv-encoding plasmid (pSurv and concomitant TcCRT administration should generate a stronger anti-tumor response than application of either treatment separately. To evaluate this possibility, C57BL/6 mice were immunized with pSurv and challenged with an isogenic melanoma cell line that had been pre-incubated with recombinant TcCRT (rTcCRT. Following tumor cell inoculation, mice were injected with additional doses of rTcCRT. For the combined regimen we observed in mice that: i. Tumor growth was impaired, ii. Humoral anti-rTcCRT immunity was induced and, iii. In vitro rTcCRT bound to melanocytes, thereby promoting the incorporation of human C1q and subsequent macrophage phagocytosis of tumor cells. These observations are interpreted to reflect the consequence of the following sequence of events: rTcCRT anti-angiogenic activity leads to stress in tumor cells. Murine CRT is then translocated to the external membrane where, together with rTcCRT, complement C1 is captured, thus promoting tumor phagocytosis. Presentation of the Tumor Associated Antigen Surv induces the adaptive anti-tumor immunity and, independently, mediates anti-endothelial cell immunity leading

  20. Differential capacity of chaperone-rich lysates in cross-presenting human endogenous and exogenous melanoma differentiation antigens.

    Science.gov (United States)

    Bleifuss, Elke; Bendz, Henriette; Sirch, Birgit; Thompson, Sylvia; Brandl, Anna; Milani, Valeria; Graner, Michael W; Drexler, Ingo; Kuppner, Maria; Katsanis, Emmanuel; Noessner, Elfriede; Issels, Rolf-Dieter

    2008-12-01

    The goal of immune-based tumor therapies is the activation of immune cells reactive against a broad spectrum of tumor-expressed antigens. Vaccines based on chaperone proteins appear promising as these proteins naturally exist as complexes with various protein fragments including those derived from tumor-associated antigens. Multi-chaperone systems are expected to have highest polyvalency as different chaperones can carry distinct sets of antigenic fragments. A free-solution isoelectric focusing (FS-IEF) technique was established to generate chaperone-rich cell lysates (CRCL). Results from murine systems support the contention that CRCL induce superior anti-tumor responses than single chaperone vaccines. We established an in vitro model for human melanoma to evaluate the capacity of CRCL to transfer endogenously expressed tumor antigens to the cross-presentation pathway of dendritic cells (DC) for antigen-specific T cell stimulation. CRCL prepared from human melanoma lines contained the four major chaperone proteins Hsp/Hsc70, Hsp90, Grp94/gp96 and calreticulin. The chaperones within the melanoma cell-derived CRCL were functionally active in that they enhanced cross-presentation of exogenous peptides mixed into the CRCL preparation. Superior activity was observed for Hsp70-rich CRCL obtained from heat-stressed melanoma cells. Despite the presence of active chaperones, melanoma cell-derived CRCL failed to transfer endogenously expressed melanoma-associated antigens to DC for cross-presentation and cytotoxic T cell (CTL) recognition, even after increasing intracellular protein levels of tumor antigen or chaperones. These findings reveal limitations of the CRCL approach regarding cross-presentation of endogenously expressed melanoma-associated antigens. Yet, CRCL may be utilized as vehicles to enhance the delivery of exogenous antigens for DC-mediated cross-presentation and T cell stimulation.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    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...... 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....

  2. c-RET molecule in malignant melanoma from oncogenic RET-carrying transgenic mice and human cell lines.

    Directory of Open Access Journals (Sweden)

    Yuichiro Ohshima

    Full Text Available Malignant melanoma is one of the most aggressive cancers and its incidence worldwide has been increasing at a greater rate than that of any other cancer. We previously reported that constitutively activated RFP-RET-carrying transgenic mice (RET-mice spontaneously develop malignant melanoma. In this study, we showed that expression levels of intrinsic c-Ret, glial cell line-derived neurotrophic factor (Gdnf and Gdnf receptor alpha 1 (Gfra1 transcripts in malignant melanomas from RET-transgenic mice were significantly upregulated compared with those in benign melanocytic tumors. These results suggest that not only introduced oncogenic RET but also intrinsic c-Ret/Gdnf are involved in murine melanomagenesis in RET-mice. We then showed that c-RET and GDNF transcript expression levels in human malignant melanoma cell lines (HM3KO and MNT-1 were higher than those in primary cultured normal human epithelial melanocytes (NHEM, while GFRa1 transcript expression levels were comparable among NHEM, HM3KO and MNT-1. We next showed c-RET and GFRa1 protein expression in HM3KO cells and GDNF-mediated increased levels of their phosphorylated c-RET tyrosine kinase and signal transduction molecules (ERK and AKT sited potentially downstream of c-RET. Taken together with the finding of augmented proliferation of HM3KO cells after GDNF stimulation, our results suggest that GDNF-mediated c-RET kinase activation is associated with the pathogenesis of malignant melanoma.

  3. Targeting tumor-associated macrophages and inhibition of MCP-1 reduce angiogenesis and tumor growth in a human melanoma xenograft.

    Science.gov (United States)

    Gazzaniga, Silvina; Bravo, Alicia I; Guglielmotti, Angelo; van Rooijen, Nico; Maschi, Fabricio; Vecchi, Annunciata; Mantovani, Alberto; Mordoh, José; Wainstok, Rosa

    2007-08-01

    Chemokines such as monocyte chemoattractant protein (MCP)-1 are key agonists that attract macrophages to tumors. In melanoma, it has been previously shown that variable levels of MCP-1/CCL2 appear to correlate with infiltrating macrophages and tumor fate, with low to intermediate levels of the chemokine contributing to melanoma development. To work under such conditions, a poorly tumorigenic human melanoma cell line was transfected with an expression vector encoding MCP-1. We found that M2 macrophages are associated to MCP-1+ tumors, triggering a profuse vascular network. To target the protumoral macrophages recruitment and reverting tumor growth promotion, clodronate-laden liposomes (Clod-Lip) or bindarit were administered to melanoma-bearing mice. Macrophage depletion after Clod-Lip treatment induced development of smaller tumors than in untreated mice. Immunohistochemical analysis with an anti-CD31 antibody revealed scarce vascular structures mainly characterized by narrow vascular lights. Pharmacological inhibition of MCP-1 with bindarit also reduced tumor growth and macrophage recruitment, rendering necrotic tumor masses. We suggest that bindarit or Clod-Lip abrogates protumoral-associated macrophages in human melanoma xenografts and could be considered as complementary approaches to antiangiogenic therapy.

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

    Science.gov (United States)

    Morita, Hiroshi; Murata, Taku; Shimizu, Kasumi; Okumura, Kenya; Inui, Madoka; Tagawa, Toshiro

    2013-04-01

    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.

  5. Withania somnifera Root Extract Has Potent Cytotoxic Effect against Human Malignant Melanoma Cells.

    Science.gov (United States)

    Halder, Babli; Singh, Shruti; Thakur, Suman S

    2015-01-01

    In Ayurveda, Withania somnifera is commonly known as Ashwagandha, its roots are specifically used in medicinal and clinical applications. It possesses numerous therapeutic actions which include anti-inflammatory, sedative, hypnotic and narcotic. Extracts from this plant have been reported for its anticancer properties. In this study we evaluated for the first time, the cytotoxic effect of Withania root extract on human malignant melanoma A375 cells. The crude extract of Withania was tested for cytotoxicity against A375 cells by MTT assay. Cell morphology of treated A375 cells was visualized through phase contrast as well as fluorescence microscopy. Agarose gel electrophoresis was used to check DNA fragmentation of the crude extract treated cells. Crude extract of Withania root has the potency to reduce viable cell count in dose as well as time dependent manner. Morphological change of the A375 cells was also observed in treated groups in comparison to untreated or vehicle treated control. Apoptotic body and nuclear blebbing were observed in DAPI stained treated cells under fluorescence microscope. A ladder of fragmented DNA was noticed in treated cells. Thus it might be said that the crude water extract of Withania somnifera has potent cytotoxic effect on human malignant melanoma A375 cells.

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

    Institute of Scientific and Technical Information of China (English)

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

    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:250,1000,1750,2500 V/cm;50 μs by5 impulses for every case. The viability of cells after EP was estimated byMTT assay. The ultrastructural analysis was observed by transmission electron microscope (ZeissEM900). Results:In the current study we observed the intracellular effect followingEP 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 byEP. Conversely, we showed thatEP in some conditions can stimulate cells to proliferation. Some changes induced byEP were only visible in electron microscopy. In fibroblast cells we observed significant changes in lower parameters ofEP (250 and1000 V/cm). After applying higher electric field intensities (2500 V/cm) we detected many vacuoles, myelin-like bodies and swallowed endoplasmic reticulum. In melanoma cells such strong pathological modifications afterEP were not observed, in comparison with control cells. The ultrastructure of both treated cell lines was changed according to the applied parameters ofEP.Conclusions:We can claim thatEP 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 thatEP 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.

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

    Science.gov (United States)

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

    2011-04-01

    To estimate electroporation (EP) influence on malignant and normal cells. 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). 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. 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.

  8. MicroRNA miR-125b induces senescence in human melanoma cells

    DEFF Research Database (Denmark)

    Glud, Martin; Manfé, Valentina; Biskup, Edyta

    2011-01-01

    in malignant melanoma producing lymph node micrometastases than in nonmetastasizing tumors. To get further insight into the functional role of miR-125b, we assessed whether its overexpression or silencing affects apoptosis, proliferation, or senescence in melanoma cell lines. We showed that overexpression...... in an early cutaneous malignant melanoma can contribute to the increased metastatic capability of this tumor....

  9. A combination of photodynamic therapy and chemotherapy displays a differential cytotoxic effect on human metastatic melanoma cells.

    Science.gov (United States)

    Biteghe, Fa Nsole; Davids, L M

    2017-01-01

    Cutaneous melanoma represents the most lethal form of skin cancer and remains refractory to current therapies. Failure of treatment has been attributed to the over-expression of ABC transporters which efflux the drugs, below their cytotoxic threshold within cells. Therefore, this study set to investigate; the efficacy of a combinatorial approach comprising chemotherapy (Dacarbazine) and photodynamic therapy (PDT) to overcome resistance in pigmented and unpigmented metastatic melanoma and potentially identify resistant mechanisms. The cytotoxic effect of the chemotherapy, PDT and combination therapy treatment (Dacarbazine+PDT) was determined using a cell viability XTT assay. Thereafter, melanoma cells morphology, self-renewal capacity and ABCG2 protein expression, were determined using fluorescence microscopy, clonogenic assay, western blot and flow cytometry. All results were analyzed by t-test and ANOVA, followed by individual comparisons with post-tests. This study describes possible synergism of PDT+DTIC in reducing melanoma cell viability in vitro. At 24h post-treatment, only the unpigmented melanomas were sensitive to DTIC treatment (20-25% death at 1.25mM). At 48h, a lethal dose of 50% was reached in these cells in contrast to the pigmented melanoma (20% at 48h). The same trend was observed with the combination therapy (DTIC+PDT) at both time points. Furthermore, complete morphological disruption could be observed upon PDT only and PDT+DTIC treatments. Moreover, PDT and DTIC+PDT suppressed the self-renewal capacity of both melanoma cell lines. No significant differences in ABCG2 protein expression was found at 24h post-treatment. Overall, these results suggest that human melanomas remain heterogeneous in their phenotypes. Moreover, in our metastatic melanoma cells, ABCG2 transporters did not seem to be involved in resistance to therapies. Significantly though, a combinatorial approach of PDT and chemotherapy significantly decreases the self-renewal capacity

  10. Expression of somatostatin receptors in human melanoma cell lines: effect of two different somatostatin analogues, octreotide and SOM230, on cell proliferation.

    Science.gov (United States)

    Martinez-Alonso, M; Llecha, N; Mayorga, M E; Sorolla, A; Dolcet, X; Sanmartin, V; Abal, L; Casanova, J M; Baradad, M; Yeramian, A; Egido, R; Puig, S; Vilella, R; Matias-Guiu, X; Marti, R M

    2009-01-01

    Somatostatin analogues (SAs) are potential anticancer agents. This study was designed to investigate the expression of somatostatin receptors (SSTRs) in melanoma cells and the effect of two SAs on cell proliferation and viability. Eighteen primary and metastatic human cutaneous melanoma cell lines were treated with octreotide and SOM230. Expression of SSTR1, SSTR2, SSTR3 and SSTR5 was assessed by real-time polymerase chain reaction. Proliferation, viability and cell death were assessed using standard assays. Inhibition was modelled by mixed-effect regression. Melanoma cells expressed one or more SSTR. Both SAs inhibited proliferation of most melanoma cell lines, but inhibition was < 50%. Neither SA affected cell viability or induced cell death. The results suggest that melanoma cell lines express SSTRs. The SAs investigated, under the conditions used in this study, did not, however, significantly inhibit melanoma growth or induce cell death. Novel SAs, combination therapy with SAs and their anti-angiogenic properties should be further investigated.

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

    Science.gov (United States)

    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.

  12. Analysis of in vitro activity of high dilutions of Euphorbia tirucalli L. in human melanoma cells

    Directory of Open Access Journals (Sweden)

    Teresa Cristina Andrade Leitao Aguiar

    2011-09-01

    Full Text Available Aveloz (firestick cactus; Euphorbia tirucalli L. belongs to Euphorbiaceae family, characterized by the production of a toxic latex that has corrosive effects on the skin and mucous membranes. Continual topic use of the latex is recommended by popular medicine to treat warts, and epitheliomas. To validate this indication, ultra diluted latex and homeopathic medicine Euphorbia tirucalli were tested in vitro on the proliferation of melanoma cells. Ultra diluted latex was prepared in homeopathic dilutions 5cH, 15cH and 30cH by dilution and agitation (trituration for solid and sucussion for liquid phases using 70º GL (Gay Lussac ethylic alcohol (70º GL EtOH 70ºGL as inert medium according to the guidelines in Farmacopéia Homeopática Brasileira (FHB. Homeopathic medicine Euphorbia tirucalli was prepared from mother-tincture according to the centesimal Hahnemannian method. Solutions 0.5% and 5% of 70ºGL EtOH were succussed and used as control. Human melanoma cells were cultured, treated and monitored by method MTT for 24 to 72 hours. It was observed that 0.5% 70ºGL EtOH solution had little or no effect on the proliferation of melanoma cells (5.1% maximal inhibition in dilution 30cH. Positive correlation was observed in most groups between inhibition of proliferation and diluted preparations, maximal increase (9% was seen in with 5% latex. Moreover, mother-tincture proved to be more active than latex; treatment with 0.5% solution of latex 30cH exhibited 19.7% inhibition, whereas treatment with 0.5% solution of Euphorbia tirucalli 30cH exhibited 32.1% inhibition of cell proliferation (p<0.05. These results suggest that high dilutions of firestick cactus (especially dilution 30cH might have a therapeutic indication in melanoma, further studies are needed in this regard.

  13. The NK-1 Receptor Antagonist L-732,138 Induces Apoptosis and Counteracts Substance P-Related Mitogenesis in Human Melanoma Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Muñoz, Miguel, E-mail: mmunoz@cica.es; Rosso, Marisa; González-Ortega, Ana [Research Laboratory on Neuropeptides, Virgen del Rocío University Hospital, Sevilla (Spain); Coveñas, Rafael [Institute of Neurosciences of Castilla y León (INCYL), Laboratory of Neuroanatomy of the Peptidergic Systems (Laboratory 14), Salamanca (Spain)

    2010-04-20

    It has been recently demonstrated that substance P (SP) and neurokinin-1 (NK-1) receptor antagonists induce cell proliferation and cell inhibition in human melanoma cells, respectively. However, the antitumor action of the NK-1 receptor antagonist L-732,138 on such cells is unknown. The aim of this study was to demonstrate an antitumor action of L-732,138 against three human melanoma cell lines (COLO 858, MEL HO, COLO 679). We found that L-732,138 elicits cell growth inhibition in a concentration dependent manner in the melanoma cells studied. Moreover, L-732,138 blocks SP mitogen stimulation. The specific antitumor action of L-732,138 occurred through the NK-1 receptor and melanoma cell death was by apoptosis. These findings indicate that the NK-1 receptor antagonist L-732,138 could be a new antitumor agent in the treatment of human melanoma.

  14. Correlation of TGF-β1 and oxidative stress in the blood of patients with melanoma: a clue to understanding melanoma progression?

    Science.gov (United States)

    Santos Bernardes, Sara; de Souza-Neto, Fernando Pinheiro; Pasqual Melo, Gabriella; Guarnier, Flávia Alessandra; Marinello, Poliana Camila; Cecchini, Rubens; Cecchini, Alessandra L

    2016-08-01

    TGF-β1 and oxidative stress are involved in cancer progression, but in melanoma, their role is still controversial. Our aim was to correlate plasma TGF-β1 levels and systemic oxidative stress biomarkers in patients with melanoma, with or without disease metastasis, to understand their participation in melanoma progression. Thirty patients were recruited for melanoma surveillance, together with 30 healthy volunteers. Patients were divided into two groups: Non-metastasis, comprising patients with tumor removal and no metastatic episode for 3 years; and Metastasis, comprising patients with a metastatic episode. The plasmatic cytokines TGF-β1, IL-1 β, and TNF-α were analyzed by ELISA. For oxidative stress, the following assays were performed: malondialdehyde (MDA), advanced oxidation protein products (AOPP) levels, total radical-trapping antioxidant parameter (TRAP) and thiol in plasma, and lipid peroxidation, SOD and catalase activity and GSH in erythrocytes. Patients with a metastatic episode had less circulating TGF-β1 and increased TRAP, thiol, AOPP and lipid peroxidation levels. MDA was increased in both melanoma groups, while catalase, GSH, and IL-1β was decreased in Non-metastasis patients. Significant negative correlations were observed between TGF-β1 levels and systemic MDA, and TGF-β1 levels and systemic AOPP, while a positive correlation was observed between TGF-β1 levels and erythrocyte GSH. Lower levels of TGF-β1 were related to increased oxidative stress in Metastasis patients, reinforcing new evidence that in melanoma TGF-β1 acts as a tumor suppressor, inhibiting tumor relapse. These findings provide new knowledge concerning this cancer pathophysiology, extending the possibilities of investigating new therapies based on this evidence.

  15. Human melanoma cells express FGFR/Src/Rho signaling that entails an adhesion-independent caveolin-1 membrane association.

    Science.gov (United States)

    Fecchi, Katia; Travaglione, Sara; Spadaro, Francesca; Quattrini, Adriano; Parolini, Isabella; Piccaro, Giovanni; Raggi, Carla; Fabbri, Alessia; Felicetti, Federica; Carè, Alessandra; Fiorentini, Carla; Sargiacomo, Massimo

    2012-03-15

    Caveolae have been indicated as a center of cytoskeleton regulation for Src kinase/Rho GTPase signaling. In addition, Src recruitment on intact cortical actin cytoskeleton appears to be required for bFGF/FGFR signal activation. Recently, we established a relationship between caveolin-1 (Cav-1) expression and cell migration in human malignant melanoma, constitutively activated by a bFGF autoregulatory loop. This work intends to investigate whether caveolae's asset, through bFGF/FGFR/c-Src/Rho signaling, could be related to melanoma cell anchorage. Accordingly, we revealed the existence of a FGFR/Src kinase pathway in Cav-1 enriched detergent-resistant membranes (DRMs) of Me665/1 metastatic melanoma cells, as confirmed by FGFR silencing. Moreover, we determined the expression and phosphorylation levels of Cav-1/Src/Erk signal pathway as a function of FGFR activation and cell density. A sucrose density gradient ultracentrifugation was employed to monitor Cav-1 membrane association and buoyancy in Me665/1 cells treated for actin fragmentation or for altered phosphorylation signals. As a result, melanoma cells show remarkable resistance to Cav-1 disassembly, together with persisting cell signal activity, being Src and Cav-1 crucial modulators of Rho GTPases. In conclusion, our study primarily highlights, in a metastatic melanoma cell line expressing caveolin, the circumstances whereby caveola structural and functional endurance enables the FGFR/Src/Rho GTPases pathway to keep on cell progression.

  16. Immunohistochemical expression of the glucose transporters Glut-1 and Glut-3 in human malignant melanomas and benign melanocytic lesions

    Directory of Open Access Journals (Sweden)

    Parente Paola

    2008-09-01

    Full Text Available Abstract Background Reported data indicate that cancer cells have increased rates of glucose metabolism, as determined by 18FDG-PET imaging in patients with malignancies. The results of many studies have demonstrated that the expression of glucose transporters, especially Glut-1, is increased in a variety of malignancies. This study was undertaken to assess the differential expression of Glut-1 and Glut-3 by benign and malignant melanocytic lesions. Methods Immunohistochemical staining for Glut-1 and Glut-3 was performed on paraffin-embedded tissue sections prepared from melanocytic nevi (12 cases, Spitz nevi (12 cases and primary cutaneous malignant melanomas (20 cases. Results We observed immunoreactivity for Glut-1 in all melanocytic nevi, 9 of the 12 Spitz nevi and in 9 of the 20 malignant melanomas, whereas Glut-3 was expressed in all the melanocytic lesions, both benign and malignant. Conclusion These findings indicate that the glucose transporters Glut-1 and Glut-3 play a role in the glucose metabolism of melanocytic cells. Glut-1 was present in the majority of benign nevi, whereas its expression was downregulated in 55% of malignant melanomas. Our results suggest that glucose transporter Glut-1 expression can significantly discriminate between human malignant melanoma and benign melanocytic nevi, and support the idea that additional mechanisms other than Glut-1 may contribute to glucose uptake in melanomas.

  17. Effect and mechanism of tacrolimus on melanogenesis on A375 human melanoma cells

    Institute of Scientific and Technical Information of China (English)

    Huang Haiyan; Wang Xiaoyan; Ding Xiaolan; Xu Qianxi; Sonia Kay Hwang; Wang Fang; Du Juan

    2014-01-01

    Background Topical tacrolimus has been used for vitiligo as a common treatment option for more than ten years while the underlying mechanism is still uncertain.The aim of this study was to investigate the direct effects of tacrolimus on the melanogenesis and migration on human A375 melanoma cells.The expression of c-KIT mRNA and protein of human A375 cells were also investigated.Methods The cultured A375 human melanoma cells were randomly assigned to control and tacrolimus treatment groups (10,102,103and 104 nmol/L).The cell proliferation was measured with Cell Counting Kit-8 assays.Melanin content was measured with NaOH method.Transwell migration assay was used to measure cell migration.The expression of c-KIT mRNA and protein were measured with real-time fluorescence quantitative polymerase chain reaction and immunohistochemistry respectively.Results The cell proliferation of the 103 and 104 nmol/L tacrolimus groups were significantly lower (0.666±0.062 and 0.496±0.038) as compared with the control (0.841±0.110,P <0.05).The mean melanin content in all groups treated with different concentration of tacrolimus (10,102,103,104 nmol/L) increased compared with the control group (P <0.05).Dosedependent increase in cell migration were seen in all tacrolimus-treated groups (P <0.01).The expression of c-KIT mRNA level in A375 cells exposed to tacrolimus (103and 104 nmol/L) had significantly increased by 3.03-fold and 3.19-fold respectively compared with the control (P <0.05).Conclusions Although tacrolimus had no effects on cell proliferation on A375 human melanoma cells,it could increase the melanin content and cell migration.The expression of c-KIT mRNA and protein increased dose-dependently in tacrolimus-treated groups as compared with the control.Our study demonstrated that tacrolimus could enhance the melanogenesis and cell migration on A375 cells.

  18. Human placental lipid induces mitogenesis and melanogenesis in B16F10 melanoma cells.

    Science.gov (United States)

    Mallick, Shampa; Mandal, Samir Kumar; Bhadra, Ranjan

    2002-06-01

    A hydroalcoholic extract of fresh term human placenta was found to be mitogenic as well as melanogenic on B16F10 mouse melanoma in an in vitro culture. The extract, a reservoir of a large number of bioactive molecules, was resolved to get the lipid fraction. Its activity was evaluated on B16F10 mouse melanoma by assessing the change in cellular morphology, growth and melanin induction. The lipid fraction, placental total lipid fraction (PTLF) tested in the study employed doses of 0 01 to 200 microg/ml; optimum growth and melanization accompanied by morphological changes were recorded at 10 and 100 microg/ml respectively. At intermediate doses growth and melanization were found to show a pattern of change over between growth and melanization and finally reached at an inverse relation at the respective optimal dose of response. Compared with defined sphingolipids, C(2) ceramide and sphingosine-1-phosphate, the results were mostly corroborative. The duality of biological response of sphingolipids as reported in numerous studies was comparable for the PTLF suggesting that its active component is a sphingolipid and showing its use for pigment recovery in vitiligo.

  19. Human placental lipid induces mitogenesis and melanogenesis in B16F10 melanoma cells

    Indian Academy of Sciences (India)

    Shampa Mallick; Samir Kumar Mandal; Ranjan Bhadra

    2002-06-01

    A hydroalcoholic extract of fresh term human placenta was found to be mitogenic as well as melanogenic on B16F10 mouse melanoma in an in vitro culture. The extract, a reservoir of a large number of bioactive molecules, was resolved to get the lipid fraction. Its activity was evaluated on B16F10 mouse melanoma by assessing the change in cellular morphology, growth and melanin induction. The lipid fraction, placental total lipid fraction (PTLF) tested in the study employed doses of 0.01 to 200 g/ml; optimum growth and melanization accompanied by morphological changes were recorded at 10 and 100 g/ml respectively. At intermediate doses growth and melanization were found to show a pattern of change over between growth and melanization and finally reached at an inverse relation at the respective optimal dose of response. Compared with defined sphingolipids, C2 ceramide and sphingosine-1-phosphate, the results were mostly corroborative. The duality of biological response of sphingolipids as reported in numerous studies was comparable for the PTLF suggesting that its active component is a sphingolipid and showing its use for pigment recovery in vitiligo.

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

    Science.gov (United States)

    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.

  1. Stimulation of differentiated functions in human melanoma cells by tumor-promoting agents and dimethyl sulfoxide

    Energy Technology Data Exchange (ETDEWEB)

    Huberman, E.; Heckman, C.; Langenbach, R.

    1979-07-01

    Treatment of cultured human HO melanoma cells with the mouse skin tumor promoter phorbol-12-myristate-13-acetate (PMA) at 5 x 10/sup -10/ to 5 x 10/sup -7/ M resulted in a dose-related inhibition of growth and a stimulation of differentiated functions. These included melanin synthesis and formation of dendrite-like structues. Higher doses of phorbol dibutyrate, a less potent tumor promoter, were required to produce an effect comparable to that of PMA for dendrite induction. Phorbol and two other phorbal esters, which lack tumor-promoting activity, were either inactive or elicited a poor response. In addition to morphological changes, treatment with PMA altered glucosamine incorporation into membrane gangliosides. After PMA treatment, glucosamine incorporation increased 8- to 10 fold in the G/sub m3/ ganglioside and decreased 2-fold in the G/sub m1/ ganglioside, as compared to phorbol or untreated control. Inhibition of cell growth and stimulation of melanin synthesis were also observed after treatment of the HO cells with dimethyl sulfoxide. Unlike the tumor-promoting agents, dimethyl sulfoxide did not induce the formation of dendrite-like structures in the cells. These findings indicate that HO melanoma cells can be stimulated into terminally differentiated cells after treatment with tumor-promoting agents such as phorbol diesters.

  2. Apoptotic effect and mechanisms of AHPN on human skin malignant melanoma cell A375

    Institute of Scientific and Technical Information of China (English)

    Min Pan; Zhenhui Peng; Shengxiang Xiao; Jianwen Ren; Yan Liu; Xiaoli Li; Zhengxiao Li

    2008-01-01

    Objective: To study apoptotic effects of synthetic retinoic acid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid(AHPN) on human skin malignant melanoma A375 cells in comparison with the natural iigand all-trans-retinoic acid(ATRA) in vitro and the mechanisms related to the actions of AHPN. Methods:MTT assay was used to determine the anti-proliferative effects of AHPN and ATRA on A375 cells. Flow cytometry was performed to investigate the influence of AHPN and ATRA on cell cycle and cell apoptosis. In addition, transfection and luciferase activity assays were employed to explore the mechanisms of how AHPN executes its proapoptotic function. Results:Firstly, AHPN promoted apoptosis and G1 arrest in A375 cells compared with ATRA. Secondly, the activity of NF-kB in A375 cells treated with AHPN increased 2-3 times compared with solvent DMSO treatment. Conelusion:AHPN,in comparison with ATRA, is a more effective alternative for therapy of malignant melanoma. The potentially proapoptotic function of AHPN requires activation of NF-kB.

  3. The influence of photodynamic therapy on apoptosis in human melanoma cell line

    Directory of Open Access Journals (Sweden)

    T. Banas´

    2011-08-01

    Full Text Available Melanoma is the most severe of all skin cancers as it may grow rapidly and metastasize. The application of photodynamic therapy (PDT opens new perspectives in treatment of this cancer. Numerous studies suggest that the exposure of tumor cells to PDT can lead to cell death via two separate processes: apoptosis or necrosis. The aim of this study was to assess in vitro photodynamic therapy which induces apoptosis in the human Beidegröm Melanoma (BM cell line, using neutral comet assay. The cells were incubated with Photofrin II (15 μg/ml and 30 μg/ml 4 h before and 3 h after irradiation for 5 or 10 min with the light intensity of 10 mW/cm2, using a lamp with red filter (632.8 nm. The percentage of apoptotic cells was significantly higher after PDT comparing to control cells. We observed 25% and 70% of apoptotic cells after shorter irradiation and treatment with 15 μg/ml and 30 μg/ml of Ph II, respectively. After longer irradiation, the respective values were 71.9% and 90%. The results suggest that induction of apoptosis is an important determinant of photodynamic sensitivity in the studied cell line and that some types of DNA damage are dependent on photosensitizer concentration and time of irradiation.

  4. Extracellular protonation modulates cell-cell interaction mechanics and tissue invasion in human melanoma cells

    Science.gov (United States)

    Hofschröer, Verena; Koch, Kevin Alexander; Ludwig, Florian Timo; Friedl, Peter; Oberleithner, Hans; Stock, Christian; Schwab, Albrecht

    2017-01-01

    Detachment of cells from the primary tumour precedes metastatic progression by facilitating cell release into the tissue. Solid tumours exhibit altered pH homeostasis with extracellular acidification. In human melanoma, the Na+/H+ exchanger NHE1 is an important modifier of the tumour nanoenvironment. Here we tested the modulation of cell-cell-adhesion by extracellular pH and NHE1. MV3 tumour spheroids embedded in a collagen matrix unravelled the efficacy of cell-cell contact loosening and 3D emigration into an environment mimicking physiological confinement. Adhesive interaction strength between individual MV3 cells was quantified using atomic force microscopy and validated by multicellular aggregation assays. Extracellular acidification from pHe7.4 to 6.4 decreases cell migration and invasion but increases single cell detachment from the spheroids. Acidification and NHE1 overexpression both reduce cell-cell adhesion strength, indicated by reduced maximum pulling forces and adhesion energies. Multicellular aggregation and spheroid formation are strongly impaired under acidification or NHE1 overexpression. We show a clear dependence of melanoma cell-cell adhesion on pHe and NHE1 as a modulator. These effects are opposite to cell-matrix interactions that are strengthened by protons extruded via NHE1. We conclude that these opposite effects of NHE1 act synergistically during the metastatic cascade. PMID:28205573

  5. Changes of proliferation kinetics after X-irradiation of a human malignant melanoma grown in nude mice

    DEFF Research Database (Denmark)

    Spang-Thomsen, M; Vindeløv, L L

    1984-01-01

    A human malignant melanoma grown in nude mice was exposed to single-dose X-irradiation and the effect on the proliferation kinetics was investigated by two methods. Flow cytometric DNA analysis was performed on tumour tissue obtained by sequential fine-needle aspirations after the treatment to mo......-related increasing proportion of radiation-inactivated tumour cells....

  6. Changes in peripheral blood level of regulatory T cells in patients with malignant melanoma during treatment with dendritic cell vaccination and low-dose IL-2

    DEFF Research Database (Denmark)

    Bjoern, J; Brimnes, M K; Andersen, M H

    2011-01-01

    In this study, changes in peripheral blood regulatory T cell (Treg) levels were evaluated in 46 progressive patients with melanoma treated with a dendritic cell-based vaccine and concomitant low-dose IFN-a and IL-2. The regulatory subset of CD4 T cells, characterized by CD25(high) , was prospecti......In this study, changes in peripheral blood regulatory T cell (Treg) levels were evaluated in 46 progressive patients with melanoma treated with a dendritic cell-based vaccine and concomitant low-dose IFN-a and IL-2. The regulatory subset of CD4 T cells, characterized by CD25(high......) , was prospectively analysed in fresh blood, and treatment-associated quantitative and qualitative changes were analysed. By the 4th vaccine, patients showed a marked increase in CD4+ CD25(high) T cell subset from 6% to 22% (P...

  7. Changes in peripheral blood level of regulatory T cells in patients with malignant melanoma during treatment with dendritic cell vaccination and low-dose IL-2

    DEFF Research Database (Denmark)

    Bjoern, J; Brimnes, M K; Andersen, M H

    2011-01-01

    In this study, changes in peripheral blood regulatory T cell (Treg) levels were evaluated in 46 progressive patients with melanoma treated with a dendritic cell-based vaccine and concomitant low-dose IFN-α and IL-2. The regulatory subset of CD4 T cells, characterized by CD25(high) , was prospecti......In this study, changes in peripheral blood regulatory T cell (Treg) levels were evaluated in 46 progressive patients with melanoma treated with a dendritic cell-based vaccine and concomitant low-dose IFN-α and IL-2. The regulatory subset of CD4 T cells, characterized by CD25(high......) , was prospectively analysed in fresh blood, and treatment-associated quantitative and qualitative changes were analysed. By the 4th vaccine, patients showed a marked increase in CD4+ CD25(high) T cell subset from 6% to 22% (P...

  8. The induction of apoptosis in human melanoma, breast and ovarian cancer cell lines using an essential oil extract from the conifer Tetraclinis articulata.

    Science.gov (United States)

    Buhagiar, J A; Podesta, M T; Wilson, A P; Micallef, M J; Ali, S

    1999-01-01

    The cytotoxic effect of conifer Tetraclinis articulata essential oil (TAEO) on a number of human cancer cell lines and peripheral blood lymphocytes was assessed at various concentrations and time exposures. The cytotoxic effect showed the hallmarks of apoptosis confirmed by a variety of techniques including flow cytometry, an apoptosis- specific marker combined to fluorescent staining and DNA laddering. All cell lines tested were inhibited in a dose-dependent fashion and within a contact time of less than eight hours for the higher concentrations. Melanoma, breast and ovarian cancer cells gave IC50s of around 80 micrograms/ml whilst the IC50s on peripheral blood lymphocytes was almost double this value. We conclude that the essential oil contains components that are effective at inducing apoptosis. The advantages of using a mixture of monoterpenes (C10) as present in an EO over a single component, are discussed.

  9. Compact NMR relaxometry of human blood and blood components.

    Science.gov (United States)

    Cistola, David P; Robinson, Michelle D

    2016-11-01

    Nuclear magnetic resonance relaxometry is a uniquely practical and versatile implementation of NMR technology. Because it does not depend on chemical shift resolution, it can be performed using low-field compact instruments deployed in atypical settings. Early relaxometry studies of human blood were focused on developing a diagnostic test for cancer. Those efforts were misplaced, as the measurements were not specific to cancer. However, important lessons were learned about the factors that drive the water longitudinal (T1) and transverse (T2) relaxation times. One key factor is the overall distribution of proteins and lipoproteins. Plasma water T2 can detect shifts in the blood proteome resulting from inflammation, insulin resistance and dyslipidemia. In whole blood, T2 is sensitive to hemoglobin content and oxygenation, although the latter can be suppressed by manipulating the static and applied magnetic fields. Current applications of compact NMR relaxometry include blood tests for candidiasis, hemostasis, malaria and insulin resistance.

  10. Specific sialyltransferase is responsible for the synthesis of GD/sup 3/, a ganglioside preferentially expressed on human metastatic melanoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Rosenberg, J.M.; Reisfeld, R.A.; Sander, D.J.; Winfield, J.B.; Cheresh, D.A.

    1986-05-01

    A number of studies have been directed toward defining surface structures that may be preferentially expressed on human tumor cells of the metastatic phenotype. Human melanoma was used as a tumor model to study the molecular events associated with metastasis. Using monoclonal antibodies directed to a variety of human melanoma associated antigens, they demonstrate that the disialoganglioside GD/sub 3/ is preferentially expressed on human melanoma cells derived from metastatic foci, whereas cells derived from primary lesions as well as melanocytes express minimal levels of this antigen. The enhanced expression of GD/sub 3/ on metastatic melanoma cells is due to an increased biosynthetic rate as shown by intrinsic labeling with (/sup 3/H)-glucosamine. Moreover, they demonstrate the presence of a specific sialyltransferase (GD/sub 3/ synthetase) responsible for the synthesis of GD/sub 3/. This enzyme activity is associated with a membrane fraction of human melanoma cells and converts the monosialylated precursor GM/sub 3/ to GD/sub 3/. In fact, a cultured human melanoma cell line derived from a metastatic foci was shown to contain a five-fold increase in GD/sub 3/ synthetase specific activity as compared to that observed for a cell line derived from a primary lesion of the same patient. The elucidation of the mechanism regulating the expression of this enzyme may lead to a more complete understanding of the metastatic phenotype of human melanoma.

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

    Energy Technology Data Exchange (ETDEWEB)

    Ungerer, Christopher; Doberstein, Kai [Pharmazentrum Frankfurt/ZAFES, University Hospital Goethe University Frankfurt, Frankfurt am Main (Germany); Buerger, Claudia; Hardt, Katja; Boehncke, Wolf-Henning [Department of Dermatology, Clinic of the Goethe-University, Theodor-Stern-Kai, Frankfurt (Germany); Boehm, Beate [Division of Rheumatology, Goethe University, Frankfurt am Main (Germany); Pfeilschifter, Josef [Pharmazentrum Frankfurt/ZAFES, University Hospital Goethe University Frankfurt, Frankfurt am Main (Germany); Dummer, Reinhard [Department of Pathology, Institute of Surgical Pathology, University Hospital, Zurich (Switzerland); Mihic-Probst, Daniela [Department of Dermatology, University Hospital Zurich (Switzerland); Gutwein, Paul, E-mail: p.gutwein@med.uni-frankfurt.de [Pharmazentrum Frankfurt/ZAFES, University Hospital Goethe University Frankfurt, Frankfurt am Main (Germany)

    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.

  12. Ionizing radiation modulates the surface expression of human leukocyte antigen-G in a human melanoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Michelin, S.; Gallegos, C.E.; Dubner, D. [Radiopathology Laboratory, Nuclear Regulatory Authority, Buenos Aires (Argentina); Favier, B.; Carosella, E.D. [CEA, I2BM, Hopital Saint-Louis, IUH, Service de Recherches en Hemato-Immunologie, Paris (France)

    2009-07-01

    Human leukocyte antigen G (HLA-G) is a nonclassical HLA class I molecule involved in fetus protection from the maternal immune system, transplant tolerance, and viral and tumoral immune escape. Tumor-specific HLA-G expression has been described for a wide variety of malignancies, including melanomas. The aim of this study was to evaluate whether ionizing radiation (IR) could modulate the surface expression of HLA-G1 in a human melanoma cell line that expresses endogenously membrane-bound HLA-G1. For this purpose, cells were exposed to increasing doses of {gamma}-irradiation (0-20 Gy) and HLA-G1 levels at the plasma membrane were analyzed at different times postirradiation by flow cytometry. HLA-G total expression and the presence of the soluble form of HLA-G1 (sHLA-G1) in the culture medium of irradiated cells were also evaluated. IR was capable of down regulating cell surface and total HLA-G levels, with a concomitant increase of sHLA-G1 in the medium. These results could indicate that {gamma}-irradiation decreases HLA-G1 surface levels by enhancing the proteolytic cleavage of this molecule. (authors)

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  15. Comparison of the lectin-binding pattern in different human melanoma cell lines.

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    Lityńska, A; Przybyło, M; Pocheć, E; Hoja-Łukowicz, D; Ciołczyk, D; Laidler, P; Gil, D

    2001-06-01

    Glycosylation is generally altered in tumour cells in comparison with their normal counterparts. These alterations are thought to be important because they contribute to the abnormal behaviour of cancer cells. Therefore, we have comparatively analysed the glycoproteins in cell extracts from human melanoma (primary site--WM35; metastatic sites-- WM239, WM9 and A375) cell lines using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and lectin staining. The glycoprotein pattern of the WM35 line differed from that of the other cell lines in having less proteins that reacted with Sambucus nigra, Maackia amurensis and Phaseolus vulgaris agglutinins. A glycoprotein of about 70 kDa had a significantly increased reaction with Sambucus nigra agglutinin in all the cell lines from metastatic sites. In the WM9, WM239 and A375 cell lines, additional bands (160-100 kDa) were stained with Phaseolus vulgaris agglutinin, suggesting that cells from metastatic sites contain more glycoproteins with beta1-6 branches. On the other hand, only minor changes in the reaction with Galanthus nivalis agglutinin, a mannose-specific lectin, were detected. Among the proteins showing different lectin staining, one, with an apparent molecular weight of 133 kDa, was recognized by antibodies as N-cadherin. The present results suggest that in human melanoma the expression of branched and sialylated complex type N-oligosaccharides consistently increased in cells from metastatic sites, and support the view that carbohydrates are associated with the acquisition of the metastatic potential of tumour cells.

  16. Mechanism of apoptosis of triptolide induced human choroidal melanoma cell line OCM-1

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    Chao LI

    2015-04-01

    Full Text Available Objective To investigate the effect of triptolide (TPL on proliferation of human choroidal melanoma cell line OCM-1, and analyze the mechanism of apoptosis induction. Methods OCM-1 cells were divided into control and experimental groups. 20μl TPL (final concentration was 5, 10, 20, 40, 80 and 160 nmol/L respectively was used for experimental group, and an equal volume of serum-free RPMI 1640 medium for the control group. The culture time was 24, 48 and 72 hours, respectively. The effect of TPL on OCM-1 cell proliferation was detected by MTT assay. The morphological change of apoptotic cells was observed by Switzerland-Giemsa staining. The expressions of TPL-induced OCM-1 apoptosis-associated proteins Bcl-2, Bax, survivin and caspase-3 were determined by Western blotting. Results TPL suppressed OCM-1 cell proliferation, at 48 and 72h, and IC50 concentration was 56.14±6.72 and 15.57±4.28 nmol/L respectively. After OCM-1 cells were cultured with TPL, apoptotic bodies were observed with Switzerland-Giemsa staining and subdiploid apoptotic peaks by flow cytometry. The expression of protein Bax was up-regulated (P<0.01, while Bcl-2 and survivin down-regulated (P<0.01, and caspase-3 was activated during apoptosis induced by TPL. Conclusion TPL could inhibit proliferation and induce apoptosis of human choroidal melanoma cell line OCM-1, and the proteins survivin, Bcl-2, Bax and caspase-3 play an important role in the induction of OCM-1 apoptosis. DOI: 10.11855/j.issn.0577-7402.2015.02.06

  17. Transport and expression in human melanomas of a transferrin-like glycosylphosphatidylinositol-anchored protein.

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    Food, M R; Rothenberger, S; Gabathuler, R; Haidl, I D; Reid, G; Jefferies, W A

    1994-01-28

    Melanotransferrin, also called p97, is a cell surface glycoprotein which was first described as a marker antigen for human melanoma cells. Although p97 has a striking structural similarity to human serum transferrin and lactoferrin, its function has not yet been determined. One feature that distinguishes p97 from the other members of the transferrin family is the presence of a stretch of 24 hydrophobic amino acids at the C terminus, previously assumed to form a proteinacious transmembrane domain. In this study, sensitivity to bacterial phosphatidylinositol-specific phospholipase C, biosynthetic labeling with [3H]ethanolamine, and partitioning in Triton X-114 are used to establish that p97 is expressed at the cell surface as a glycosylphosphatidylinositol-anchored protein. In addition, to gain insight into the intracellular transport of p97, biosynthetic transport studies were performed on a melanoma cell line. These studies resulted in the identification of an additional form of p97 which is found in the medium and which likely does not originate from an alternatively spliced form of the p97 mRNA. These findings, together with our recent observation of the co-localization of p97 and the transferrin receptor in brain capillary endothelium (W. A. Jefferies, M. R. Food, R. Gabathuler, S. Rothenberger, T. Yamada, and P. L. McGeer, manuscript submitted) raise important questions about the function of the two forms of p97 detected and the possible involvement of this protein in a cellular iron uptake mechanism that is independent from the transferrin/transferrin receptor system.

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

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

    2014-01-01

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

  19. Biological activity and binding of estradiol to SK-Mel 23 human melanoma cells

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    Sarti M.S.M.V.

    2004-01-01

    Full Text Available Patients expressing estradiol receptors in melanoma cells have been reported to have a better prognosis. We therefore decided to investigate the in vitro effects of ß-estradiol and tamoxifen on the growth and tyrosinase activity of SK-Mel 23 human melanoma cells. Twenty-four-hour treatment with 0.4 nM ß-estradiol inhibited cell proliferation in 30% (0.70 ± 0.03 x 10(5 cells and increased tyrosinase activity in 50% (7130.5 ± 376.5 cpm/10(5 cells, as compared to untreated cells (1.0 ± 0.05 x 10(5 cells and 4769 ± 25.5 cpm/10(5 cells, respectively. Both responses were completely (100% blocked by 1 µM tamoxifen. Higher concentrations (up to 1.6 nM or longer treatments (up to 72 h did not result in a larger effect of the hormone on proliferation or tyrosinase activity. Competition binding assays demonstrated the presence of binding sites to [2,4,6,7-³H]-ß-estradiol, and that the tritiated analogue was displaced by the unlabeled hormone (1 nM to 100 µM, Kd = 0.14 µM, maximal displacement of 93% or by 10 µM tamoxifen (displacement of 60%. ß-estradiol also increased the phosphorylated state of two proteins of 16 and 46 kDa, after 4-h treatment, as determined by Western blot. The absorbance of each band was 1.9- and 4-fold the controls, respectively, as determined with Image-Pro Plus software. Shorter incubation periods with ß-estradiol did not enhance phosporylation; after 6-h treatment with the hormone, the two proteins returned to the control phosphorylation levels. The growth inhibition promoted by estradiol may explain the better prognosis of melanoma-bearing women as compared to men, and open new perspectives for drug therapy.

  20. Expression and function of hypoxia inducible factor-1 alpha in human melanoma under non-hypoxic conditions

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    Joshi Sandeep S

    2009-11-01

    Full Text Available Abstract Background Hypoxia inducible factor-1 alpha (HIF-1α protein is rapidly degraded under normoxic conditions. When oxygen tensions fall HIF-1α protein stabilizes and transactivates genes involved in adaptation to hypoxic conditions. We have examined the normoxic expression of HIF-1α RNA and protein in normal human melanocytes and a series of human melanoma cell lines isolated from radial growth phase (RGP, vertical growth phase (VGP and metastatic (MET melanomas. Results HIF-1α mRNA and protein was increased in RGP vs melanocytes, VGP vs RGP and MET vs VGP melanoma cell lines. We also detected expression of a HIF-1α mRNA splice variant that lacks part of the oxygen-dependent regulation domain in WM1366 and WM9 melanoma cells. Over-expression of HIF-1α and its splice variant in the RGP cell line SbCl2 resulted in a small increase in soft agar colony formation and a large increase in matrigel invasion relative to control transfected cells. Knockdown of HIF-1α expression by siRNA in the MET WM9 melanoma cell line resulted in a large decrease in both soft agar colony formation and matrigel invasion relative to cells treated with non-specific siRNA. There is a high level of ERK1/2 phosphorylation in WM9 cells, indicating an activated Ras-Raf-MEK-ERK1/2 MAPK pathway. Treatment of WM9 cells with 30 μM U0126 MEK inhibitor, decreased ERK1/2 phosphorylation and resulted in a decrease in HIF-1α expression. However, a 24 h treatment with 10 μM U0126 totally eliminated Erk1/2 phosphorylation, but did not change HIF-1alpha levels. Furthermore, siRNA knockdown of MEK siRNA did not change HIF-1alpha levels. Conclusion We speculate that metabolic products of U0126 decrease HIF-1alpha expression through "off target" effects. Overall our data suggest that increased HIF-1α expression under normoxic conditions contributes to some of the malignant phenotypes exhibited by human melanoma cells. The expanded role of HIF-1α in melanoma biology increases

  1. Human spleen and red blood cells

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    Pivkin, Igor; Peng, Zhangli; Karniadakis, George; Buffet, Pierre; Dao, Ming

    2016-11-01

    Spleen plays multiple roles in the human body. Among them is removal of old and altered red blood cells (RBCs), which is done by filtering cells through the endothelial slits, small micron-sized openings. There is currently no experimental technique available that allows us to observe RBC passage through the slits. It was previously noticed that people without a spleen have less deformable red blood cells, indicating that the spleen may play a role in defining the size and shape of red blood cells. We used detailed RBC model implemented within the Dissipative Particle Dynamics (DPD) simulation framework to study the filter function of the spleen. Our results demonstrate that spleen indeed plays major role in defining the size and shape of the healthy human red blood cells.

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

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    Matthew A Bill

    Full Text Available 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.

  3. Glutathione and Bcl-2 targeting facilitates elimination by chemoradiotherapy of human A375 melanoma xenografts overexpressing bcl-xl, bcl-2, and mcl-1

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    Mena Salvador

    2012-01-01

    Full Text Available Abstract Background Bcl-2 is believed to contribute to melanoma chemoresistance. However, expression of Bcl-2 proteins may be different among melanomas. Thus correlations among expression of Bcl-2-related proteins and in vivo melanoma progression, and resistance to combination therapies, was investigated. Methods Human A375 melanoma was injected s.c. into immunodeficient nude mice. Protein expression was studied in tumor samples obtained by laser microdisection. Transfection of siRNA or ectopic overexpression were applied to manipulate proteins which are up- or down-regulated, preferentially, during melanoma progression. Anti-bcl-2 antisense oligonucleotides and chemoradiotherapy (glutathione-depleting agents, paclitaxel protein-binding particles, daunorubicin, X rays were administered in combination. Results In vivo A375 cells down-regulated pro-apoptotic bax expression; and up-regulated anti-apoptotic bcl-2, bcl-xl, and mcl-1, however only Bcl-2 appeared critical for long-term tumor cell survival and progression in vivo. Reduction of Bcl-2, combined with partial therapies, decreased melanoma growth. But only Bcl-2 targeting plus the full combination of chemoradiotherapy eradicated A375 melanoma, and led to long-term survival (> 120 days without recurrence in 80% of mice. Tumor regression was not due to immune stimulation. Hematology and clinical chemistry data were within accepted clinical toxicities. Conclusion Strategies to target Bcl-2, may increase the effectiveness of antitumor therapies against melanomas overexpressing Bcl-2 and likely other Bcl-2-related antiapoptotic proteins.

  4. Patient-derived xenografts recapitulate molecular features of human uveal melanomas.

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    Laurent, Cécile; Gentien, David; Piperno-Neumann, Sophie; Némati, Fariba; Nicolas, André; Tesson, Bruno; Desjardins, Laurence; Mariani, Pascale; Rapinat, Audrey; Sastre-Garau, Xavier; Couturier, Jérôme; Hupé, Philippe; de Koning, Leanne; Dubois, Thierry; Roman-Roman, Sergio; Stern, Marc-Henri; Barillot, Emmanuel; Harbour, J William; Saule, Simon; Decaudin, Didier

    2013-06-01

    We have previously developed a new method for the development and maintenance of uveal melanoma (UM) xenografts in immunodeficient mice. Here, we compare the genetic profiles of the primary tumors to their corresponding xenografts that have been passaged over time. The study included sixteen primary UMs and corresponding xenografts at very early (P1), early (P4), and late (P9) in vivo passages. The tumors were analyzed for mutation status of GNAQ, GNA11, GNAS, GNA15, BAP1, and BRAF, chromosomal copy number alterations using Affymetrix GeneChip(®) Genome-Wide Human SNP6.0 arrays, gene expression profiles using GeneChip(®) Human Exon 1.0 ST arrays, BAP1 mRNA and protein expression, and MAPK pathway status using Reverse Phase Protein Arrays (RPPA). The UM xenografts accurately recapitulated the genetic features of primary human UMs and they exhibited genetic stability over the course of their in vivo maintenance. Our technique for establishing and maintaining primary UMs as xenograft tumors in immunodeficient mice exhibit a high degree of genetic conservation between the primary tumors and the xenograft tumors over multiple passages in vivo. These models therefore constitute valuable preclinical tool for drug screening in UM.

  5. DMEM enhances tyrosinase activity in B16 mouse melanoma cells and human melanocytes

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    Panpen Diawpanich

    2008-07-01

    Full Text Available Media components may affect the activities of cultured cells. In this study, tyrosinase activity was evaluated by using B16-F10 mouse melanoma cell lines (B16-F10 and primary human melanocytes cultured in different media. An optical density measurement and a L-dopa reaction assay were used as the determination of the tyrosinase activity. The study of B16-F10 found the optical density to be 2010, 2246 and 2961 in cells cultured in RPMI Medium 1640 (RPMI1640,Minimum Essential Medium (MEM and Dulbecco’s Modified Eagle Medium (DMEM, respectively. Moreover, compared to RPMI 1640 and MEM, DMEM showed the darkest color of melanin formation in culture media and in cells after the L-dopa reaction assay. Addition of kojic acid showed a significant inhibitory effect on tyrosinase activity in all media.Whereas MCDB153 showed no significant effect on human melanocytes, DMEM caused a dramatic increase in tyrosinase activity after 4 days of cultivation. Addition of kojic acid showed a significant tyrosinase inhibitory effect in DMEM only. Furthermore, an active ingredient in green tea, epigallocathechin gallate (EGCG could inhibit tyrosinase activity in both B16-F10 and human melanocytes cultured in DMEM. In summary, these results suggest that DMEM is a suitable medium that provides high detection sensitivity in a tyrosinase inhibition assay.

  6. Blood type biochemistry and human disease.

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    Ewald, D Rose; Sumner, Susan C J

    2016-11-01

    Associations between blood type and disease have been studied since the early 1900s when researchers determined that antibodies and antigens are inherited. In the 1950s, the chemical identification of the carbohydrate structure of surface antigens led to the understanding of biosynthetic pathways. The blood type is defined by oligosaccharide structures, which are specific to the antigens, thus, blood group antigens are secondary gene products, while the primary gene products are various glycosyltransferase enzymes that attach the sugar molecules to the oligosaccharide chain. Blood group antigens are found on red blood cells, platelets, leukocytes, plasma proteins, certain tissues, and various cell surface enzymes, and also exist in soluble form in body secretions such as breast milk, seminal fluid, saliva, sweat, gastric secretions, urine, and amniotic fluid. Recent advances in technology, biochemistry, and genetics have clarified the functional classifications of human blood group antigens, the structure of the A, B, H, and Lewis determinants and the enzymes that produce them, and the association of blood group antigens with disease risks. Further research to identify differences in the biochemical composition of blood group antigens, and the relationship to risks for disease, can be important for the identification of targets for the development of nutritional intervention strategies, or the identification of druggable targets. WIREs Syst Biol Med 2016, 8:517-535. doi: 10.1002/wsbm.1355 For further resources related to this article, please visit the WIREs website.

  7. First-in-Human Proof-of-Concept Study: Intralesional Administration of BQ788, an Endothelin Receptor B Antagonist, to Melanoma Skin Metastases

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    Hunger, Robert E.; Garrod, Terence; Dubuis, Benoit; Hunziker, Thomas; van den Oord, Joost J.; Lahav-le Coutre, Ronit

    2015-01-01

    Lessons Learned Local tolerance of intralesional treatment of melanoma skin metastases with BQ788 is excellent. Hints of efficacy are observed, consisting of both direct effects (decreased expression of endothelin receptor B [EDNRB] and of survival factors, reduced proliferation) and indirect effects (enhanced immune cell infiltration and angiogenesis). Patients in future trials should be screened for EDNRB expression level prior to treatment because only patients with high EDNRB-expressing melanomas (four of five) responded to BQ788. Future studies should be performed for at least 2 weeks because reduced lesion growth was observed in the only patient that was treated for longer than 1 week. Background. This first-in-human proof-of-concept study aimed to check whether safety and preclinical results obtained by intratumoral administration of BQ788, an endothelin receptor B (EDNRB) antagonist, can be repeated in human melanoma patients. Methods. Three patients received a single intralesional BQ788 application of 3 mg. After 3–7 days, the lesions were measured and removed for analysis. The administered dose was increased to a cumulative dosage of 8 mg in patient 4 (4 × 2.0 mg, days 0–3; lesion removed on day 4) and to 10 mg in patient 5 (3 × 3.3 mg, days 0, 3, and 10; lesion removed after 14 days). Control lesions were simultaneously treated with phosphate-buffered saline (PBS). All samples were processed and analyzed without knowledge of the clinical findings. Results. No statistical evaluation was possible because of the number of patients (n = 5) and the variability in the mode of administration. No adverse events were observed, regardless of administered dose. All observations were in accordance with results obtained in preclinical studies. Accordingly, no difference in degree of tumor necrosis was detected between BQ788- and PBS-treated samples. In addition, both EDNRB and Ki67 showed decreased expression in patients 2 and 5 and, to a lesser extent, in

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

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    Rizos Helen

    2011-05-01

    Full Text Available Abstract 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

  9. Antiproliferative and anti-invasive effects of inorganic and organic arsenic compounds on human and murine melanoma cells in vitro.

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    Hiwatashi, Yoko; Tadokoro, Hiroko; Henmi, Kayo; Arai, Mariko; Kaise, Toshikazu; Tanaka, Sachiko; Hirano, Toshihiko

    2011-09-01

    For patients with advanced melanoma, no treatment options are available at present that provide either sufficient response rates or a significant prolongation of overall survival. The present study examines the effects of two inorganic and six organic arsenic compounds on cell proliferation and cell invasion of melanoma cells in vitro. The effects of arsenic compounds on proliferation of human melanoma A375 cells and murine melanoma B16F10 cells were examined by MTT assay and 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, and the effects of the compounds on cell invasion were examined by the Boyden chamber invasion assay. The amounts of active matrix metalloproteinase (MMP)-2 and pro-MMP-2 in the culture supernatant of A375 cells were determined by an MMP-2 activity assay system. Arsenate and arsenic trioxide (As(2) O(3) ) inhibited the proliferation of A375 and B16F10 cells significantly at concentration ranges of 0.1-20µg/ml (Porganic compounds arsenobetaine, arsenocholine, dimethylarsinic acid, methylarsonic acid, tetramethylarsonium and trimethylarsine oxide did not show any inhibitory effects even at 20µg/ml. Cell invasion of A375 and B16F10 cells through a layer of collagen IV was significantly inhibited by 0.1-20 µg/ml of arsenate or As(2) O(3) (Porganic compounds did not inhibit cell invasion. Arsenate or As(2) O(3) at 0.2-10µg/ml significantly inhibited the amount of active MMP-2 and pro-MMP-2 secreted into the A375 cell culture supernatant (Pinorganic arsenic compounds arsenate and As(2) O(3) inhibit cell proliferation and prevent the invasive properties of melanoma cells, possibly by decreasing MMP-2 production from the cells. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

  10. Deep-proteome mapping of WM-266-4 human metastatic melanoma cells: From oncogenic addiction to druggable targets

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    Litou, Zoi I.; Konstandi, Ourania A.; Giannopoulou, Aikaterini F.; Anastasiadou, Ema; Voutsinas, Gerassimos E.; Tsangaris, George Th.; Stravopodis, Dimitrios J.

    2017-01-01

    Cutaneous melanoma is a malignant tumor of skin melanocytes that are pigment-producing cells located in the basal layer (stratum basale) of epidermis. Accumulation of genetic mutations within their oncogenes or tumor-suppressor genes compels melanocytes to aberrant proliferation and spread to distant organs of the body, thereby resulting in severe and/or lethal malignancy. Metastatic melanoma’s heavy mutational load, molecular heterogeneity and resistance to therapy necessitate the development of novel biomarkers and drug-based protocols that target key proteins involved in perpetuation of the disease. To this direction, we have herein employed a nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS) proteomics technology to profile the deep-proteome landscape of WM-266-4 human metastatic melanoma cells. Our advanced melanoma-specific catalogue proved to contain 6,681 unique proteins, which likely constitute the hitherto largest single cell-line-derived proteomic collection of the disease. Through engagement of UNIPROT, DAVID, KEGG, PANTHER, INTACT, CYTOSCAPE, dbEMT and GAD bioinformatics resources, WM-266-4 melanoma proteins were categorized according to their sub-cellular compartmentalization, function and tumorigenicity, and successfully reassembled in molecular networks and interactomes. The obtained data dictate the presence of plastically inter-converted sub-populations of non-cancer and cancer stem cells, and also indicate the oncoproteomic resemblance of melanoma to glioma and lung cancer. Intriguingly, WM-266-4 cells seem to be subjected to both epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial (MET) programs, with 1433G and ADT3 proteins being identified in the EMT/MET molecular interface. Oncogenic addiction of WM-266-4 cells to autocrine/paracrine signaling of IL17-, DLL3-, FGF(2/13)- and OSTP-dependent sub-routines suggests their critical contribution to the metastatic melanoma chemotherapeutic refractoriness. Interestingly, the

  11. Expression and migratory analysis of 5 human uveal melanoma cell lines for CXCL12, CXCL8, CXCL1, and HGF

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    Di Cesare Sebastian

    2007-01-01

    Full Text Available Abstract Background The aim of this study was to characterize the presence and roles of CXCL12, CXCL8, CXCL1, and HGF in five human uveal melanoma cell lines, using different methods, in order to ascertain their significance in this disease. Methods Five human uveal melanoma cell lines (92.1, SP6.5, MKT-BR, OCM-1, and UW-1 of known proliferative, invasive, and metastatic potential were used in this experiment. A migration assay was used in order to assess the responsiveness of each cell line towards the four chosen chemotactic factors. Immunohistochemistry was then performed for all five cell lines (cytospins using antibodies directed toward CXCL1, CXCL8 and their receptors CXCR2 and CXCR1 respectively. Quantitative real-time PCR was then performed on all five cell lines in order to establish the presence of these four chemotactic factors. Results All five human uveal melanoma cell lines migrated towards the four chosen chemotactic factors at a level greater than that of the negative control. Chemokines CXCL1 and CXCL8 resulted in the greatest number of migrating cells in all five of our cell lines. Immunohistochemistry confirmed the expression of CXCL1, CXCL8, and their receptors CXCR2 and CXCR1 in all five of the cell lines. Quantitative real-time PCR results established expression of CXCL8, CXCL1, and HGF in all 5 cell lines tested. CXCL1 and CXCL8 are highly expressed in SP6.5 and UW-1. None of the five cell lines expressed any detectable levels of CXCL12. Conclusion The migratory ability of the 5 human uveal melanoma cell lines was positively influenced by the four chemotactic factors tested, namely CXCL12, CXCL8, CXCL1, and HGF. Self-expression of chemotactic factors CXCL8, CXCL1, and HGF may indicate an autocrine system, which perhaps contributes to the cells' metastatic ability in vivo.

  12. Activation of the Long Terminal Repeat of Human Endogenous Retrovirus K by Melanoma-Specific Transcription Factor MITF-M

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    Iyoko Katoh

    2011-11-01

    Full Text Available The human and Old World primate genomes possess conserved endogenous retrovirus sequences that have been implicated in evolution, reproduction, and carcinogenesis. Human endogenous retrovirus (HERV-K with 5′LTR-gag-pro-pol-env-rec/np9-3′LTR sequences represents the newest retrovirus family that integrated into the human genome 1 to 5 million years ago. Although a high-level expression of HERV-K in melanomas, breast cancers, and terato-carcinomas has been demonstrated, the mechanism of the lineage-specific activation of the long terminal repeat (LTR remains obscure. We studied chromosomal HERV-K expression in MeWo melanoma cells in comparison with the basal expression in human embryonic kidney 293 (HEK293 cells. Cloned LTR of HERV-K (HML-2.HOM was also characterized by mutation and transactivation experiments. We detected multiple transcriptional initiator (Inr sites in the LTR by rapid amplification of complementary DNA ends (5′ RACE. HEK293 and MeWo showed different Inr usage. The most potent Inr was associated with a TATA box and three binding motifs of microphthalmia-associated transcription factor (MITF. Both chromosomal HERV-K expression and the cloned LTR function were strongly activated in HEK293 by transfection with MITF-M, a melanocyte/melanoma–specific isoform of MITF. Coexpression of MITF and the HERV-K core antigen was detected in retinal pigmented epithelium by an immunofluorescence analysis. Although malignant melanoma lines MeWo, G361, and SK-MEL-28 showed enhanced HERV-K transcription compared with normal melanocytes, the level of MITF-M messenger RNA persisted from normal to transformed melanocytes. Thus, MITF-M may be a prerequisite for the pigmented cell lineage–specific function of HERV-K LTR, leading to the high-level expression in malignant melanomas.

  13. Fibroblast growth factor receptors as therapeutic targets in human melanoma: synergism with BRAF inhibition.

    Science.gov (United States)

    Metzner, Thomas; Bedeir, Alexandra; Held, Gerlinde; Peter-Vörösmarty, Barbara; Ghassemi, Sara; Heinzle, Christine; Spiegl-Kreinecker, Sabine; Marian, Brigitte; Holzmann, Klaus; Grasl-Kraupp, Bettina; Pirker, Christine; Micksche, Michael; Berger, Walter; Heffeter, Petra; Grusch, Michael

    2011-10-01

    Cutaneous melanoma is a tumor with rising incidence and a very poor prognosis at the disseminated stage. Melanomas are characterized by frequent mutations in BRAF and also by overexpression of fibroblast growth factor 2 (FGF2), offering opportunities for therapeutic intervention. We investigated inhibition of FGF signaling and its combination with dacarbazine or BRAF inhibitors as an antitumor strategy in melanoma. The majority of melanoma cell lines displayed overexpression of FGF2 but also FGF5 and FGF18 together with different isoforms of FGF receptors (FGFRs) 1-4. Blockade of FGF signals with dominant-negative receptor constructs (dnFGFR1, 3, or 4) or small-molecule inhibitors (SU5402 and PD166866) reduced melanoma cell proliferation, colony formation, as well as anchorage-independent growth, and increased apoptosis. DnFGFR constructs also significantly inhibited tumor growth in vivo. Combination of FGF inhibitors with dacarbazine showed additive or antagonistic effects, whereas synergistic drug interaction was observed when combining FGFR inhibition with the multikinase/BRAF inhibitor sorafenib or the V600E mutant-specific BRAF inhibitor RG7204. In conclusion, FGFR inhibition has antitumor effects against melanoma cells in vitro and in vivo. Combination with BRAF inhibition offers a potential for synergistic antimelanoma effects and represents a promising therapeutic strategy against advanced melanoma.

  14. MicroRNA-155 targets the SKI gene in human melanoma cell lines.

    Science.gov (United States)

    Levati, Lauretta; Pagani, Elena; Romani, Sveva; Castiglia, Daniele; Piccinni, Eugenia; Covaciu, Claudia; Caporaso, Patrizia; Bondanza, Sergio; Antonetti, Francesca R; Bonmassar, Enzo; Martelli, Fabio; Alvino, Ester; D'Atri, Stefania

    2011-06-01

    The SKI protein is a transcriptional coregulator over-expressed in melanoma. Experimentally induced down-regulation of SKI inhibits melanoma cell growth in vitro and in vivo. MicroRNAs (miRNAs) negatively modulate gene expression and have been implicated in oncogenesis. We previously showed that microRNA-155 (miR-155) is down-regulated in melanoma cells as compared with normal melanocytes and that its ectopic expression impairs proliferation and induces apoptosis. Here, we investigated whether miR-155 could mediate melanoma growth inhibition via SKI gene silencing. Luciferase reporter assays demonstrated that miR-155 interacted with SKI 3'UTR and impaired gene expression. Transfection of melanoma cells with miR-155 reduced SKI levels, while inhibition of endogenous miR-155 up-regulated SKI expression. Specifically designed small interfering RNAs reduced SKI expression and inhibited proliferation. However, melanoma cells over-expressing a 3'UTR-deleted SKI were still susceptible to the antiproliferative effect of miR-155. Our data demonstrate for the first time that SKI is a target of miR-155 in melanoma. However, impairment of SKI expression is not the leading mechanism involved in the growth-suppressive effect of miR-155 found in this malignancy.

  15. Characterization of human γδ T lymphocytes infiltrating primary malignant melanomas.

    Directory of Open Access Journals (Sweden)

    Adriana Cordova

    Full Text Available T lymphocytes are often induced naturally in melanoma patients and infiltrate tumors. Given that γδ T cells mediate antigen-specific killing of tumor cells, we studied the representation and the in vitro cytokine production and cytotoxic activity of tumor infiltrating γδ T cells from 74 patients with primary melanoma. We found that γδ T cells represent the major lymphocyte population infiltrating melanoma, and both Vδ1(+ and Vδ2(+ cells are involved. The majority of melanoma-infiltrating γδ cells showed effector memory and terminally-differentiated phenotypes and, accordingly, polyclonal γδ T cell lines obtained from tumor-infiltrating immune cells produced IFN-γ and TNF-α and were capable of killing melanoma cell lines in vitro. The cytotoxic capability of Vδ2 cell lines was further improved by pre-treatment of tumor target cells with zoledronate. Moreover, higher rate of γδ T cells isolation and percentages of Vδ2 cells correlate with early stage of development of melanoma and absence of metastasis. Altogether, our results suggest that a natural immune response mediated by γδ T lymphocytes may contribute to the immunosurveillance of melanoma.

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

    Science.gov (United States)

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

    2014-01-01

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

  17. Uveal melanoma: Estimating prognosis

    Directory of Open Access Journals (Sweden)

    Swathi Kaliki

    2015-01-01

    Full Text Available 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.

  18. Different dose rate-dependent responses of human melanoma cells and fibroblasts to low dose fast neutrons.

    Science.gov (United States)

    Dionet, Claude; Müller-Barthélémy, Melanie; Marceau, Geoffroy; Denis, Jean-Marc; Averbeck, Dietrich; Gueulette, John; Sapin, Vincent; Pereira, Bruno; Tchirkov, Andrei; Chautard, Emmanuel; Verrelle, Pierre

    2016-09-01

    To analyze the dose rate influence in hyper-radiosensitivity (HRS) of human melanoma cells to very low doses of fast neutrons and to compare to the behaviour of normal human skin fibroblasts. We explored different neutron dose rates as well as possible implication of DNA double-strand breaks (DSB), apoptosis, and energy-provider adenosine-triphosphate (ATP) levels during HRS. HRS in melanoma cells appears only at a very low dose rate (VLDR), while a high dose rate (HDR) induces an initial cell-radioresistance (ICRR). HRS does not seem to be due either to DSB or to apoptosis. Both phenomena (HRS and ICRR) appear to be related to ATP availability for triggering cell repair. Fibroblast survival after neutron irradiation is also dose rate-dependent but without HRS. Melanoma cells or fibroblasts exert their own survival behaviour at very low doses of neutrons, suggesting that in some cases there is a differential between cancer and normal cells radiation responses. Only the survival of fibroblasts at HDR fits the linear no-threshold model. This new insight into human cell responses to very low doses of neutrons, concerns natural radiations, surroundings of accelerators, proton-therapy devices, flights at high altitude. Furthermore, ATP inhibitors could increase HRS during high-linear energy transfer (high-LET) irradiation.

  19. Staphylococcus aureus survival in human blood.

    Science.gov (United States)

    Malachowa, Natalia; DeLeo, Frank R

    2011-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is abundant in hospitals and in the United States is a leading cause of mortality due to infectious agents. Community-associated MRSA (CA-MRSA) strains such as USA300, which typically cause disease outside of healthcare settings, are also prevalent in the United States. Although most CA-MRSA infections affect skin and soft tissue, the pathogen can enter the bloodstream and ultimately cause severe disease. In a recent paper, we used USA300-specific microarrays to generate a comprehensive view of the molecules that facilitate S. aureus immune evasion and survival in human blood. Notably, genes encoding proteins involved in iron-uptake and utilization and gamma-hemolysin (hlgABC) are highly up-regulated by USA300 during culture in human blood. Here we discuss the potential implication of these findings and the possible role of gamma-hemolysin in the success of S. aureus as a human pathogen.

  20. Broadband Dielectric Spectroscopy on Human Blood

    CERN Document Server

    Wolf, M; Lunkenheimer, P; Loidl, A

    2011-01-01

    Dielectric spectra of human blood reveal a rich variety of dynamic processes. Achieving a better characterization and understanding of these processes not only is of academic interest but also of high relevance for medical applications as, e.g., the determination of absorption rates of electromagnetic radiation by the human body. The dielectric properties of human blood are studied using broadband dielectric spectroscopy, systematically investigating the dependence on temperature and hematocrit value. By covering a frequency range from 1 Hz to 40 GHz, information on all the typical dispersion regions of biological matter is obtained. We find no evidence for a low-frequency relaxation (alpha-relaxation) caused, e.g., by counterion diffusion effects as reported for some types of biological matter. The analysis of a strong Maxwell-Wagner relaxation arising from the polarization of the cell membranes in the 1-100 MHz region (beta-relaxation) allows for the test of model predictions and the determination of variou...

  1. Intracranial Tumor Cell Migration and the Development of Multiple Brain Metastases in Malignant Melanoma

    Directory of Open Access Journals (Sweden)

    Trude G. Simonsen

    2016-06-01

    Full Text Available INTRODUCTION: A majority of patients with melanoma brain metastases develop multiple lesions, and these patients show particularly poor prognosis. To develop improved treatment strategies, detailed insights into the biology of melanoma brain metastases, and particularly the development of multiple lesions, are needed. The purpose of this preclinical investigation was to study melanoma cell migration within the brain after cell injection into a well-defined intracerebral site. METHODS: A-07, D-12, R-18, and U-25 human melanoma cells transfected with green fluorescent protein were injected stereotactically into the right cerebral hemisphere of nude mice. Moribund mice were killed and autopsied, and the brain was evaluated by fluorescence imaging or histological examination. RESULTS: Intracerebral inoculation of melanoma cells produced multiple lesions involving all regions of the brain, suggesting that the cells were able to migrate over substantial distances within the brain. Multiple modes of transport were identified, and all transport modes were observed in all four melanoma lines. Thus, the melanoma cells were passively transported via the flow of cerebrospinal fluid in the meninges and ventricles, they migrated actively along leptomeningeal and brain parenchymal blood vessels, and they migrated actively along the surfaces separating different brain compartments. CONCLUSION: Migration of melanoma cells after initial arrest, extravasation, and growth at a single location within the brain may contribute significantly to the development of multiple melanoma brain metastases.

  2. Cisplatin-induced apoptosis inhibits autophagy, which acts as a pro-survival mechanism in human melanoma cells.

    Directory of Open Access Journals (Sweden)

    Barbara Del Bello

    Full Text Available The interplay between a non-lethal autophagic response and apoptotic cell death is still a matter of debate in cancer cell biology. In the present study performed on human melanoma cells, we investigate the role of basal or stimulated autophagy in cisplatin-induced cytotoxicity, as well as the contribution of cisplatin-induced activation of caspases 3/7 and conventional calpains. The results show that, while down-regulating Beclin-1, Atg14 and LC3-II, cisplatin treatment inhibits the basal autophagic response, impairing a physiological pro-survival response. Consistently, exogenously stimulated autophagy, obtained with trehalose or calpains inhibitors (MDL-28170 and calpeptin, protects from cisplatin-induced apoptosis, and such a protection is reverted by inhibiting autophagy with 3-methyladenine or ATG5 silencing. In addition, during trehalose-stimulated autophagy, the cisplatin-induced activation of calpains is abrogated, suggesting the existence of a feedback loop between the autophagic process and calpains. On the whole, our results demonstrate that in human melanoma cells autophagy may function as a beneficial stress response, hindered by cisplatin-induced death mechanisms. In a therapeutic perspective, these findings suggest that the efficacy of cisplatin-based polychemotherapies for melanoma could be potentiated by inhibitors of autophagy.

  3. Lichen metabolites prevent UV light and nitric oxide-mediated plasmid DNA damage and induce apoptosis in human melanoma cells.

    Science.gov (United States)

    Russo, A; Piovano, M; Lombardo, L; Garbarino, J; Cardile, V

    2008-09-26

    In humans both UV-A and UV-B can cause gene mutations and suppress immunity, which leads to skin cancer, including melanoma. Inhibition of reactive oxygen species (ROS) and reactive nitrogen species (RNS) appears particularly promising as ROS and RNS production by both UV-A and UV-B contributes to inflammation, immunosuppression, gene mutation and carcinogenesis. We evaluated the effect of two lichen compounds, sphaerophorin (depside) and pannarin (depsidone) on pBR322 DNA cleavage induced by hydroxyl radicals (()OH), and by nitric oxide (NO), and their superoxide anion (O(2)(-)) scavenging capacity. In addition, we investigated the growth inhibitory activity of these compounds against human melanoma cells (M14 cell line). Sphaerophorin and pannarin showed a protective effect on plasmid DNA and exhibited a superoxide dismutase like effect. The data obtained in cell culture show that these lichen metabolites inhibit the growth of melanoma cells, inducing an apoptotic cell death, demonstrated by the fragmentation of genomic DNA (COMET and TUNEL Assays) and by a significant increase of caspase-3 activity, and correlated, at least in part, to the increase of ROS generation, These results confirm the promising biological properties of sphaerophorin and pannarin and encourage further investigations on their molecular mechanisms.

  4. Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma.

    Science.gov (United States)

    Calero, R; Morchon, E; Martinez-Argudo, I; Serrano, R

    2017-10-10

    Drug resistance by MAPK signaling recovery or activation of alternative signaling pathways, such as PI3K/AKT/mTOR, is an important factor that limits the long-term efficacy of targeted therapies in melanoma patients. In the present study, we investigated the phospho-proteomic profile of RTKs and its correlation with downstream signaling pathways in human melanoma. We found that tyrosine kinase receptors expression correlated with the expression of pivotal downstream components of the RAS/RAF/MAPK and PI3K/AKT/mTOR pathways in melanoma cell lines and tumors. We also found high expression of HSP90 and the PI3K/AKT/mTOR pathway proteins, 4EBP1 and AKT compared with healthy tissue and this correlated with poor overall survival of melanoma patients. The combination of the HSP90 inhibitor 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 showed a synergistic activity decreasing melanoma cell growth, inducing apoptosis and targeting simultaneously the MAPK and PI3K/AKT/mTOR pathways. These results demonstrate that the combination of HSP90 and PI3K/mTOR inhibitors could be an effective therapeutic strategy that target the main survival pathways in melanoma and must be considered to overcome resistance to BRAF inhibitors in melanoma patients. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. The Efficacy of Dandelion Root Extract in Inducing Apoptosis in Drug-Resistant Human Melanoma Cells

    OpenAIRE

    Chatterjee, S. J.; P. Ovadje; M. Mousa; Hamm, C.; Pandey, S.

    2010-01-01

    Notoriously chemoresistant melanoma has become the most prevalent form of cancer for the 25–29 North American age demographic. Standard treatment after early detection involves surgical excision (recurrence is possible), and metastatic melanoma is refractory to immuno-, radio-, and most harmful chemotherapies. Various natural compounds have shown efficacy in killing different cancers, albeit not always specifically. In this study, we show that dandelion root extract (DRE) specifically and eff...

  6. Radiosensitivity of human ovarian carcinoma and melanoma cells to γ-rays and protons

    Science.gov (United States)

    Keta, Otilija; Todorović, Danijela; Popović, Nataša; Korićanac, Lela; Cuttone, Giacomo; Petrović, Ivan

    2014-01-01

    Introduction Proton radiation offers physical advantages over conventional radiation. Radiosensitivity of human 59M ovarian cancer and HTB140 melanoma cells was investigated after exposure to γ-rays and protons. Material and methods Irradiations were performed in the middle of a 62 MeV therapeutic proton spread out Bragg peak with doses ranging from 2 to 16 Gy. The mean energy of protons was 34.88 ±2.15 MeV, corresponding to the linear energy transfer of 4.7 ±0.2 keV/µm. Irradiations with γ-rays were performed using the same doses. Viability, proliferation and survival were assessed 7 days after both types of irradiation while analyses of cell cycle and apoptosis were performed 48 h after irradiation. Results Results showed that γ-rays and protons reduced the number of viable cells for both cell lines, with stronger inactivation achieved after irradiation with protons. Surviving fractions for 59M were 0.91 ±0.01 for γ-rays and 0.81 ±0.01 for protons, while those for HTB140 cells were 0.93 ±0.01 for γ-rays and 0.86 ±0.01 for protons. Relative biological effectiveness of protons, being 2.47 ±0.22 for 59M and 2.08 ±0.36 for HTB140, indicated that protons provoked better cell elimination than γ-rays. After proton irradiation proliferation capacity of the two cell lines was slightly higher as compared to γ-rays. Proliferation was higher for 59M than for HTB140 cells after both types of irradiation. Induction of apoptosis and G2 arrest detected after proton irradiation were more prominent in 59M cells. Conclusions The obtained results suggest that protons exert better antitumour effects on ovarian carcinoma and melanoma cells than γ-rays. The dissimilar response of these cells to radiation is related to their different features. PMID:25097591

  7. Effects of histamine on growth and apoptosis of human melanoma cells A375

    Institute of Scientific and Technical Information of China (English)

    RAN Li-wei; TAN Sheng-shun; XU Xin-ling; ZHANG Jiang-an; WANG Wan-juan

    2005-01-01

    Objective: To investigate the effects of histamine on growth and apoptosis of human melanoma cells A375. Methods: The effect of histamine on growth of A375 cells in vitro was examined by MTT assay and Trypan blue exclusion assay. Cell cycle analysis, early apoptosis analysis by double staining with Annexin V-FITC and PI, and active caspase-3 analysis by staining FITC-conjugated monoclonal rabbit anti-active caspase-3 antibody were made by flow cytometer. StreptAvidin-Biotin Complex (SABC)immunocytochemical assays were adopted to detect Bax/Bcl-2 protein expressions. Results: Histamine inhibited proliferation of A375 cells in a dose- and time-dependent manner, and altered cell cycle distribution of A375 cells revealing an increase in G0/G1-phase population, a decrease in S-phase population and the inhibition of G1/S switching. Histamine induced apoptosis of A375 cells (P<0. 05), elevated the cells population with detectable active caspase-3 (P<0. 05), increased the number of cells forming Bax and decreased the number of cells forming Bcl-2 significantly (P<0.05). Conclusion: That histamine inhibits cell cycle progress of A375 cells is one of the possible mechanisms of proliferation arrest of A375 cells elicited by histamine. Histamine mediates apoptosis in A375 cells that may be caspase-dependent through mitochondria routine. Histamine with high concentration inhibits growth of A375 cells in vitro by interfering proliferation and inducing apoptosis of cells.

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

    Energy Technology Data Exchange (ETDEWEB)

    Mahdipour, Seyed Ali [Physics Department, Hakim Sabzevari University, Sabzevar (Iran, Islamic Republic of); Mowlavi, Ali Asghar, E-mail: amowlavi@hsu.ac.ir [Physics Department, Hakim Sabzevari University, Sabzevar (Iran, Islamic Republic of); ICTP, Associate Federation Scheme, Medical Physics Field, Trieste (Italy)

    2016-07-01

    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 47.8 MeV proton, 190.1 MeV alpha, and 1060 MeV 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.

  9. Response of human HTB140 melanoma cells to conventional radiation and hadrons.

    Science.gov (United States)

    Ristić-Fira, A; Todorović, D; Zakula, J; Keta, O; Cirrone, P; Cuttone, G; Petrović, I

    2011-01-01

    Conventional radiotherapy with X- and gamma-rays is one of the common and effective treatments of cancer. High energy hadrons, i.e., charged particles like protons and (12)C ions, due to their specific physics and radiobiological advantages are increasingly used. In this study, effectiveness of different radiation types is evaluated on the radio-resistant human HTB140 melanoma cells. The cells were irradiated with gamma-rays, the 62 MeV protons at the Bragg peak and in the middle of the spread-out Bragg peak (SOBP), as well as with the 62 MeV/u (12)C ions. The doses ranged from 2 to 24 Gy. Cell survival and proliferation were assessed 7 days after irradiation, whereas apoptosis was evaluated after 48 h. The acquired results confirmed the high radio-resistance of cells, showing better effectiveness of protons than gamma-rays. The best efficiency was obtained with (12)C ions due to higher linear energy transfer. All analyzed radiation qualities reduced cell proliferation. The highest proliferation was detected for (12)C ions because of their large killing capacity followed by small induction of reparable lesions. This enabled unharmed cells to preserve proliferative activity. Irradiations with protons and (12)C ions revealed similar moderate pro-apoptotic ability that is in agreement with the level of cellular radio-resistance.

  10. Selective growth inhibition of a human malignant melanoma cell line by sesame oil in vitro.

    Science.gov (United States)

    Smith, D E; Salerno, J W

    1992-06-01

    Ayurveda, an ancient and comprehensive system of natural medicine, recommends regular topical application to the skin of sesame oil, above all other oils, as a health-promoting procedure. We examined the effect of sesame oil and several other vegetable oils and their major component fatty acids on the proliferation rate of human normal and malignant melanocytes growing at similar rates in serum-free media. We found that sesame and safflower oils, both of which contain large amounts of linoleate in triglyceride form, selectively inhibited malignant melanoma growth over normal melanocytes whereas coconut, olive and mineral oils, which contain little or no linoleate as triglyceride, did not. These oils were tested at a range of 10-300 micrograms/ml. We found that of the fatty acids tested, only linoleic acid was selectively inhibitory while palmitic and oleic were not. These fatty acids were tested in the range of 3-100 micrograms/ml. These results suggest that certain vegetable oils rich in linoleic acid, such as the sesame oil, recommended for topical use by Ayurveda, may contain selective antineoplastic properties which are similar to those demonstrated for essential polyunsaturated fatty acids and their metabolites. This suggests that whole vegetable oils may have potential clinical usefulness.

  11. Role of melanoma chondroitin sulphate proteoglycan in patterning stem cells in human interfollicular epidermis.

    Science.gov (United States)

    Legg, James; Jensen, Uffe B; Broad, Simon; Leigh, Irene; Watt, Fiona M

    2003-12-01

    Human interfollicular epidermis is renewed by stem cells that are clustered in the basal layer in a patterned, non-random distribution. Stem cells can be distinguished from other keratinocytes by high expression of beta1 integrins and lack of expression of terminal differentiation markers; they divide infrequently in vivo but form actively growing colonies in culture. In a search for additional stem cell markers, we observed heterogeneous epidermal expression of melanoma chondroitin sulphate proteoglycan (MCSP). MCSP was expressed by those keratinocytes with the highest beta1 integrin levels. In interfollicular epidermis, expression was confined to non-cycling cells and, in culture, to self-renewing clones. However, fluorescence-activated cell sorting on the basis of MCSP and beta1 integrin expression gave no more enrichment for clonogenic keratinocytes than sorting for beta1 integrins alone. To interfere with endogenous MCSP, we retrovirally infected keratinocytes with a chimera of the CD8 extracellular domain and the MCSP cytoplasmic domain. CD8/MCSP did not affect keratinocyte proliferation or differentiation but the cohesiveness of keratinocytes in isolated clones or reconstituted epidermal sheets was greatly reduced. CD8/MCSP caused stem cell progeny to scatter without differentiating. CD8/MCSP did not alter keratinocyte motility but disturbed cadherin-mediated cell-cell adhesion and the cortical actin cytoskeleton, effects that could be mimicked by inhibiting Rho. We conclude that MCSP is a novel marker for epidermal stem cells that contributes to their patterned distribution by promoting stem cell clustering.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  14. Melanoma genetics

    DEFF Research Database (Denmark)

    Read, Jazlyn; Wadt, Karin A W; Hayward, Nicholas K

    2016-01-01

    Approximately 10% of melanoma cases report a relative affected with melanoma, and a positive family history is associated with an increased risk of developing melanoma. Although the majority of genetic alterations associated with melanoma development are somatic, the underlying presence...... of heritable melanoma risk genes is an important component of disease occurrence. Susceptibility for some families is due to mutation in one of the known high penetrance melanoma predisposition genes: CDKN2A, CDK4, BAP1, POT1, ACD, TERF2IP and TERT. However, despite such mutations being implicated...... in a combined total of approximately 50% of familial melanoma cases, the underlying genetic basis is unexplained for the remainder of high-density melanoma families. Aside from the possibility of extremely rare mutations in a few additional high penetrance genes yet to be discovered, this suggests a likely...

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

    Directory of Open Access Journals (Sweden)

    Anna Piotrowska

    2016-01-01

    Full Text Available 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(OH2D2 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(OH2D2 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(OH2D3. 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.

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

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    Harish Chandra Pal

    Full Text Available 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

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

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    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

  18. Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle.

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    Katrin Spiesberger

    Full Text Available 30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested-mostly from the milk-of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4.With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody's activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M.Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma.

  19. Effects of antimetabolites on adenovirus replication in sensitive and resistant human melanoma cell lines.

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    Musk, P; Stowers, A; Parsons, P G

    1990-02-15

    Methotrexate (MTX), 6-thioguanine (6-TG) and cytosine arabinoside (ara-C) inhibited the replication of adenovirus (viral capacity) more in drug-sensitive than in resistant human melanoma cell lines. By comparison, inhibition of cellular DNA and RNA synthesis after short treatment periods (less than 48 hr) was not a good predictor of cellular sensitivity. MTX, an inhibitor of de novo nucleotide synthesis, was most effective when added to cells just before infection with virus and inhibited viral capacity at doses 10-1000-fold lower than those required to affect cell survival. The MTX-sensitive cell lines, members of a DNA repair deficient group sensitive also to killing by methylating agents (the Mer- phenotype), were not deficient in dihydrofolate reductase but exhibited DNA fragmentation after treatment with MTX for 48 hr. 6-TG and ara-C, inhibitors of purine and pyrimidine salvage, were most inhibitory to viral capacity when added greater than 36 hr before virus infection and were less effective than MTX (doses 5-7-fold and 4-24-fold higher than for cell survival respectively). No correlation was found between MTX sensitivity and sensitivity to 6-TG or ara-C. These results indicate that (i) inhibition of viral capacity is a more comprehensive test of antimetabolite cytotoxicity than inhibition of cellular DNA or RNA synthesis; (ii) the viral capacity assay correctly predicts cellular sensitivity to MTX, 6-TG and ara-C and therefore has potential for application to primary cultures of human tumours; and (iii) MTX-sensitive cell lines and adenovirus replication rely heavily on de novo nucleotide synthesis, which in Mer- cells appears to be linked to a DNA repair defect as yet undefined.

  20. Discrepancy Between Tumor Antigen Distribution and Radiolabeled Antibody Binding in a Nude Mouse Xenograft Model of Human Melanoma.

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    Kim, Yong-Il; Paeng, Jin Chul; Cheon, Gi Jeong; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key

    2017-04-01

    Biodistribution of antibodies is vital to successful immunoscintigraphy/immunotherapy, and it is assumed to be similar to antigen distribution. We measured and compared the binding pattern of radiolabeled antibody to tissue antigen distribution in a nude mouse xenograft model of human melanoma. We transplanted 10(7) FEM-XII human melanoma cells into the right flank of five nude mice. For the control, we transplanted 5 × 10(6) LS174T human colon cancer cells into the left flank. Two weeks later, 10 μCi of (131)I-labeled melanoma-associated 96.5 monoclonal antibody (targeting p97 antigen) was intravenously injected. Three days later, we sacrificed the mice and evaluated 96.5 antibody binding and concentration in the tumors by ex vivo quantitative autoradiography (QAR). Two months later, we incubated adjacent tumor tissue slices in various concentrations of (125)I-labeled 96.5 MoAb and evaluated the distribution/concentration of p97 antigen by in vitro QAR. p97 antigen distribution was homogeneous in the tumors (total antigen concentration [Bmax] = 17.36-38.36 pmol/g). In contrast, radiolabeled 96.5 antibody binding was heterogenous between location within the tumor (estimated bound antigen concentration = 0.7-6.6 pmol/g). No quantifiable parameters were found to be related with radiolabeled antibody binding and tumor antigen distribution. Antibody-bound tumor antigen to total antigen ratios ranged between 2% and 38%. Heterogeneous features of target antibody binding were observed in contrast to relatively homogenous feature of tumor antigen. We did not identify any correlations between p97 antigen distribution and 96.5 antibody binding in melanoma tissue. Radiolabeled 96.5 antibody binding patterns within melanoma cannot be predicted based on p97 antigen distribution in the tumor, which needs to be further studied with several other methods and more subjects in the future.

  1. Detailed analysis of immunologic effects of the cytotoxic T lymphocyte-associated antigen 4-blocking monoclonal antibody tremelimumab in peripheral blood of patients with melanoma

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    Gualberto Antonio

    2008-05-01

    Full Text Available Abstract Background CTLA4-blocking antibodies induce tumor regression in a subset of patients with melanoma. Analysis of immune parameters in peripheral blood may help define how responses are mediated. Methods Peripheral blood from HLA-A*0201-positive patients with advanced melanoma receiving tremelimumab (formerly CP-675,206 at 10 mg/kg monthly was repeatedly sampled during the first 4 cycles. Samples were analyzed by 1 tetramer and ELISPOT assays for reactivity to CMV, EBV, MART1, gp100, and tyrosinase; 2 activation HLA-DR and memory CD45RO markers on CD4+/CD8+ cells; and 3 real-time quantitative PCR of mRNA for FoxP3 transcription factor, preferentially expressed by T regulatory cells. The primary endpoint was difference in MART1-specific T cells by tetramer assay. Immunological data were explored for significant trends using clustering analysis. Results Three of 12 patients eligible for immune monitoring had tumor regression lasting > 2 years without relapse. There was no significant change in percent of MART1-specific T cells by tetramer assay. Additionally, there was no generalized trend toward postdosing changes in other antigen-specific CD8+ cell populations, FoxP3 transcripts, or overall changes in surface expression of T-cell activation or memory markers. Unsupervised hierarchical clustering based on immune monitoring data segregated patients randomly. However, clustering according to T-cell activation or memory markers separated patients with clinical response and most patients with inflammatory toxicity into a common subgroup. Conclusion Administration of CTLA4-blocking antibody tremelimumab to patients with advanced melanoma results in a subset of patients with long-lived tumor responses. T-cell activation and memory markers served as the only readout of the pharmacodynamic effects of this antibody in peripheral blood. Clinical trial registration number NCT00086489

  2. Varicella-zoster virus: isolation and propagation in human melanoma cells at 36 and 32 degrees C.

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    Grose, C; Brunel, P A

    1978-01-01

    Cell lines derived from human malignant melanoma tumors are susceptible to infection with varicella-zoster virus (VZV). Within 5 days after inoculation of vesicular fluid, cytopathic changes appeared in melanoma cell monolayer cultures that were incubated at either 36 or 32 degrees C. The VZV isolates at the two temperatures were serially propagated by passage of trypsin-dispersed infected cells. A plaque assay was developed utilizing melanoma cell monolayers overlaid with nutrient medium containing carboxymethylcellulose. By this assay method, the growth cycle of a VZV isolate propagated at 36 degrees C was studied and compared with that of another VZV isolate grown at 32 degrees C. With equivalent infected-cell inocula at a ratio on one inoculum cell to eight uninfected cells, the yield of cell-free virus at an incubation temperature of 32 degrees C was slightly higher than at 36 degrees C, although the peak occurred 60 h, rather than 36 h, postinfection. It was also found that the titer of low-passage VZV propagated at 36 degrees C was 0.5 to 1 log higher when assayed at 32 degrees C rather than at 36 degrees C.

  3. The glycocalyx maintains a cell surface pH nanoenvironment crucial for integrin-mediated migration of human melanoma cells.

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    Krähling, Hermann; Mally, Sabine; Eble, Johannes A; Noël, Josette; Schwab, Albrecht; Stock, Christian

    2009-10-01

    The glycocalyx consists of proteoglycans, glycoproteins, glycosaminoglycans, associated plasma proteins, and soluble glycosaminoglycans and covers the surface of all eukaryotic cells. It mediates specific recognition events, modulates biological processes such as ligand-receptor interactions, and has been proposed to affect tumor metastasis. Here, we studied whether the glycocalyx is required for melanoma cell migration. We diminished the glycocalyx of human melanoma cells by inhibiting posttranslational N-glycosylation or by enzymatic digestion of the N-glycosides. This partial destruction of the glycocalyx reduced melanoma cell migration by up to 60%. It was accompanied by the disintegration of a characteristic pH nanoenvironment typically surrounding migrating cells. Restoring this pH profile by stimulating the activity of the Na(+)/H(+) exchanger NHE1 rescued cell migration even in the absence of an intact glycocalyx. The effects of partially removing the glycocalyx compared to those of knocking down beta(1)-integrin expression points to a close functional correlation between glycocalyx, integrins, and cell surface pH nanoenvironment. We conclude that the glycocalyx is required for tumor cell migration. It stabilizes the cell surface pH nanoenvironment allowing a concerted pH-dependent interaction of adhesion receptors and extracellular matrix.

  4. The effects of a cyclooxygenase-2 (COX-2 expression and inhibition on human uveal melanoma cell proliferation and macrophage nitric oxide production

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    Marshall Jean-Claude

    2007-01-01

    Full Text Available Abstract Background Cyclooxygenase-2 (COX-2 expression has previously been identified in uveal melanoma although the biological role of COX-2 in this intraocular malignancy has not been elucidated. This study aimed to investigate the effect of a COX-2 inhibitor on the proliferation rate of human uveal melanoma cells, as well as its effect on the cytotoxic response of macrophages. Methods Human uveal melanoma cell lines were transfected to constitutively express COX-2 and the proliferative rate of these cells using two different methods, with and without the addition of Amfenac, was measured. Nitric oxide production by macrophages was measured after exposure to melanoma-conditioned medium from both groups of cells as well as with and without Amfenac, the active metabolite of Nepafenac. Results Cells transfected to express COX-2 had a higher proliferation rate than those that did not. The addition of Amfenac significantly decreased the proliferation rate of all cell lines. Nitric oxide production by macrophages was inhibited by the addition of melanoma conditioned medium, the addition of Amfenac partially overcame this inhibition. Conclusion Amfenac affected both COX-2 transfected and non-transfected uveal melanoma cells in terms of their proliferation rates as well as their suppressive effects on macrophage cytotoxic activity.

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

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    Manel B. Hammouda

    2016-07-01

    Full Text Available 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.

  6. Oncolysis of malignant human melanoma tumors by Coxsackieviruses A13, A15 and A18

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    Barry Richard D

    2011-01-01

    Full Text Available Abstract Many RNA viruses are displaying great promise in the field of oncolytic virotherapy. Previously, we reported that the picornavirus Coxsackievirus A21 (CVA21 possessed potent oncolytic activity against cultured malignant melanoma cells and melanoma xenografts in mice. In the present study, we demonstrate that three additional Group A Coxsackieviruses; Coxsackievirus A13 (CVA13, Coxsackievirus A15 (CVA15 and Coxsackievirus A18 (CVA18, also have similar oncolytic activity against malignant melanoma. Each of the viruses grew quickly to high titers in cancer cells expressing ICAM-1 and intratumoral injection of preformed subcutaneous SK-Mel-28 xenografts in mice with CVA13, CVA15 and CVA18 resulted in significant tumor volume reduction. As preexisting immunity could potentially hinder oncolytic virotherapy, sera from stage IV melanoma patients and normal controls were tested for levels of protective antibody against the panel of oncolytic Coxsackieviruses. Serum neutralization assays revealed that 3 of 21 subjects possessed low levels of anti-CVA21 antibodies, while protective antibodies for CVA13, CVA15 and CVA18 were not detected in any sample. Serum from individuals who were seropositive for CVA21 failed to exhibit cross-neutralization of CVA13, CVA15 and CVA18. From these studies it can be concluded that the administration of CVA13, CVA15 or CVA18 could be employed as a potential multivalent oncolytic therapy against malignant melanoma.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  9. T-cell receptor gene therapy targeting melanoma-associated antigen-A4 inhibits human tumor growth in non-obese diabetic/SCID/γcnull mice.

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    Shirakura, Yoshitaka; Mizuno, Yukari; Wang, Linan; Imai, Naoko; Amaike, Chisaki; Sato, Eiichi; Ito, Mamoru; Nukaya, Ikuei; Mineno, Junichi; Takesako, Kazutoh; Ikeda, Hiroaki; Shiku, Hiroshi

    2012-01-01

    Adoptive cell therapy with lymphocytes that have been genetically engineered to express tumor-reactive T-cell receptors (TCR) is a promising approach for cancer immunotherapy. We have been exploring the development of TCR gene therapy targeting cancer/testis antigens, including melanoma-associated antigen (MAGE) family antigens, that are ideal targets for adoptive T-cell therapy. The efficacy of TCR gene therapy targeting MAGE family antigens, however, has not yet been evaluated in vivo. Here, we demonstrate the in vivo antitumor activity in immunodeficient non-obese diabetic/SCID/γc(null) (NOG) mice of human lymphocytes genetically engineered to express TCR specific for the MAGE-A4 antigen. Polyclonal T cells derived from human peripheral blood mononuclear cells were transduced with the αβ TCR genes specific for MAGE-A4, then adoptively transferred into NOG mice inoculated with MAGE-A4 expressing human tumor cell lines. The transferred T cells maintained their effector function in vivo, infiltrated into tumors, and inhibited tumor growth in an antigen-specific manner. The combination of adoptive cell therapy with antigen peptide vaccination enhanced antitumor activity, with improved multifunctionality of the transferred cells. These data suggest that TCR gene therapy with MAGE-A4-specific TCR is a promising strategy to treat patients with MAGE-A4-expressing tumors; in addition, the acquisition of multifunctionality in vivo is an important factor to predict the quality of the T-cell response during adoptive therapy with human lymphocytes.

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

    Science.gov (United States)

    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. Copyright © 2015. Published by Elsevier B.V.

  11. RIPK1 regulates survival of human melanoma cells upon endoplasmic reticulum stress through autophagy.

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    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.

  12. Construction of Ang2-siRNA chitosan magnetic nanoparticles and the effect on Ang2 gene expression in human malignant melanoma cells

    Science.gov (United States)

    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

  13. Construction of Ang2-siRNA chitosan magnetic nanoparticles and the effect on Ang2 gene expression in human malignant melanoma cells.

    Science.gov (United States)

    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

    2016-06-01

    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% (Pconstructed. 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.

  14. Roles of different IRES-dependent FGF2 isoforms in the acquisition of the major aggressive features of human metastatic melanoma.

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    Andreucci, Elena; Bianchini, Francesca; Biagioni, Alessio; Del Rosso, Mario; Papucci, Laura; Schiavone, Nicola; Magnelli, Lucia

    2017-01-01

    Fibroblast growth factor 2 (FGF2) is involved in many physiological and pathological processes. Fgf2 deregulation contributes to the acquisition of malignant features of melanoma and other cancers. FGF2 is an alternative translation product expressed as five isoforms, a low-molecular-weight (18 KDa) and four high-molecular-weight (22, 22.5, 24, 34 KDa) isoforms, with different subcellular distributions. An internal ribosomal entry site (IRES) in its mRNA controls the translation of all the isoforms with the exception for the cap-dependent 34 KDa. The 18-KDa isoform has been extensively studied, while very few is known about the roles of high molecular weight isoforms. FGF2 is known to promote melanoma development and progression. To disclose the differential contribution of FGF2 isoforms in melanoma, we forced the expression of IRES-dependent low-molecular-weight (LMW, 18 KDa) and high-molecular-weight (HMW, 22, 22.5, 24 KDa) isoforms in a human metastatic melanoma cell line. This comparative study highlights that, while LMW isoform confers stem-like features to melanoma cells and promotes angiogenesis, HMW isoforms induce higher migratory ability and contribute to tumor perfusion by promoting vasculogenic mimicry (VM) when endothelial cell-driven angiogenesis is lacking. To conclude, FGF2 isoforms mainly behave in specific, antithetical manners, but can cooperate in different steps of tumor progression, providing melanoma cells with major malignant features.

  15. Epigenetic regulation of microRNA genes and the role of miR-34b in cell invasion and motility in human melanoma.

    Directory of Open Access Journals (Sweden)

    Joseph Mazar

    Full Text Available Invasive melanoma is the most lethal form of skin cancer. The treatment of melanoma-derived cell lines with 5-aza-2'-deoxycytidine (5-Aza-dC markedly increases the expression of several miRNAs, suggesting that the miRNA-encoding genes might be epigenetically regulated, either directly or indirectly, by DNA methylation. We have identified a group of epigenetically regulated miRNA genes in melanoma cells, and have confirmed that the upstream CpG island sequences of several such miRNA genes are hypermethylated in cell lines derived from different stages of melanoma, but not in melanocytes and keratinocytes. We used direct DNA bisulfite and immunoprecipitated DNA (Methyl-DIP to identify changes in CpG island methylation in distinct melanoma patient samples classified as primary in situ, regional metastatic, and distant metastatic. Two melanoma cell lines (WM1552C and A375 derived from stage 3 and stage 4 human melanoma, respectively were engineered to ectopically express one of the epigenetically modified miRNA: miR-34b. Expression of miR-34b reduced cell invasion and motility rates of both WM1552C and A375, suggesting that the enhanced cell invasiveness and motility observed in metastatic melanoma cells may be related to their reduced expression of miR-34b. Total RNA isolated from control or miR-34b-expressing WM1552C cells was subjected to deep sequencing to identify gene networks around miR-34b. We identified network modules that are potentially regulated by miR-34b, and which suggest a mechanism for the role of miR-34b in regulating normal cell motility and cytokinesis.

  16. COX-2 expression positively correlates with PD-L1 expression in human melanoma cells.

    Science.gov (United States)

    Botti, Gerardo; Fratangelo, Federica; Cerrone, Margherita; Liguori, Giuseppina; Cantile, Monica; Anniciello, Anna Maria; Scala, Stefania; D'Alterio, Crescenzo; Trimarco, Chiara; Ianaro, Angela; Cirino, Giuseppe; Caracò, Corrado; Colombino, Maria; Palmieri, Giuseppe; Pepe, Stefano; Ascierto, Paolo Antonio; Sabbatino, Francesco; Scognamiglio, Giosuè

    2017-02-23

    The resistance to PD-1/PD-L1 inhibitors for the treatment of melanoma have prompted investigators to implement novel clinical trials which combine immunotherapy with different treatment modalities. Moreover is also important to investigate the mechanisms which regulate the dynamic expression of PD-L1 on tumor cells and PD-1 on T cells in order to identify predictive biomarkers of response. COX-2 is currently investigated as a major player of tumor progression in several type of malignancies including melanoma. In the present study we investigated the potential relationship between COX-2 and PD-L1 expression in melanoma. Tumor samples obtained from primary melanoma lesions and not matched lymph node metastases were analyzed for both PD-L1 and COX-2 expression by IHC analysis. Status of BRAF and NRAS mutations was analyzed by sequencing and PCR. Co-localization of PD-L1 and COX-2 expression was analyzed by double fluorescence staining. Lastly the BRAF(V600E) A375 and NRAS(Q61R) SK-MEL-2 melanoma cell lines were used to evaluate the effect of COX-2 inhibition by celecoxib on expression of PD-L1 in vitro. BRAF(V600E/V600K) and NRAS(Q61R/Q61L) were detected in 57.8 and 8.9% of the metastatic lesions, and in 65.9 and 6.8% of the primary tumors, respectively. PD-L1 and COX-2 expression were heterogeneously expressed in both primary melanoma lesions and not matched lymph node metastases. A significantly lower number of PD-L1 negative lesions was found in primary tumors as compared to not matched metastatic lesions (P = 0.002). COX-2 expression significantly correlated with PD-L1 expression in both primary (P = 0.001) and not matched metastatic (P = 0.048) lesions. Furthermore, in melanoma tumors, cancer cells expressing a higher levels of COX-2 also co-expressed a higher level of PD-L1. Lastly, inhibition of COX-2 activity by celecoxib down-regulated the expression of PD-L1 in both BRAF(V600E) A375 and NRAS(Q61R) SK-MEL-2 melanoma cell lines. COX-2 expression

  17. Plumbagin Enhances TRAIL-mediated Apoptosis through Up-regulation of Death Receptor in Human Melanoma A375 Cells

    Institute of Scientific and Technical Information of China (English)

    李家文; 沈琴; 彭锐; 陈嵘袆; 蒋苹; 李艳秋; 张丽; 卢静静

    2010-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent. However, emergence of drug resistance limits its potential use. Plumbagin is a natural quinonoid compound isolated from plant. In this study, induced apoptosis effect of the combined treatment with plumbagin and TRAIL on human melanoma A375 cell line was examined and possible mechanism was investigated. The cells were divided into four groups: control group, plumbagin group (plumbagin, 5 or 10 μmol/L), TRAIL gr...

  18. DMSO Represses Inflammatory Cytokine Production from Human Blood Cells and Reduces Autoimmune Arthritis

    Science.gov (United States)

    Elisia, Ingrid; Nakamura, Hisae; Lam, Vivian; Hofs, Elyse; Cederberg, Rachel; Cait, Jessica; Hughes, Michael R.; Lee, Leora; Jia, William; Adomat, Hans H.; Guns, Emma S.; McNagny, Kelly M.; Samudio, Ismael; Krystal, Gerald

    2016-01-01

    Dimethyl sulfoxide (DMSO) is currently used as an alternative treatment for various inflammatory conditions as well as for cancer. Despite its widespread use, there is a paucity of data regarding its safety and efficacy as well as its mechanism of action in human cells. Herein, we demonstrate that DMSO has ex-vivo anti-inflammatory activity using Escherichia coli- (E. coli) and herpes simplex virus-1 (HSV-1)-stimulated whole human blood. Specifically, we found that between 0.5%– 2%, DMSO significantly suppressed the expression of many pro-inflammatory cytokines/chemokines and prostaglandin E2 (PGE2). However, a significant reduction in monocyte viability was also observed at 2% DMSO, suggesting a narrow window of efficacy. Anti-inflammatory concentrations of DMSO suppressed E. coli-induced ERK1/2, p38, JNK and Akt phosphorylation, suggesting DMSO acts on these signaling pathways to suppress inflammatory cytokine/chemokine production. Although DMSO induces the differentiation of B16/F10 melanoma cells in vitro, topical administration of DMSO to mice subcutaneously implanted with B16 melanoma cells was ineffective at reducing tumor growth, DMSO was also found to block mouse macrophages from polarizing to either an M1- or an M2-phenotype, which may contribute to its inability to slow tumor growth. Topical administration of DMSO, however, significantly mitigated K/BxN serum-induced arthritis in mice, and this was associated with reduced levels of pro-inflammatory cytokines in the joints and white blood cell levels in the blood. Thus, while we cannot confirm the efficacy of DMSO as an anti-cancer agent, the use of DMSO in arthritis warrants further investigation to ascertain its therapeutic potential. PMID:27031833

  19. Effective intra-S checkpoint responses to UVC in primary human melanocytes and melanoma cell lines.

    Science.gov (United States)

    Cordeiro-Stone, Marila; McNulty, John J; Sproul, Christopher D; Chastain, Paul D; Gibbs-Flournoy, Eugene; Zhou, Yingchun; Carson, Craig; Rao, Shangbang; Mitchell, David L; Simpson, Dennis A; Thomas, Nancy E; Ibrahim, Joseph G; Kaufmann, William K

    2016-01-01

    The objective of this study was to assess potential functional attenuation or inactivation of the intra-S checkpoint during melanoma development. Proliferating cultures of skin melanocytes, fibroblasts, and melanoma cell lines were exposed to increasing fluences of UVC and intra-S checkpoint responses were quantified. Melanocytes displayed stereotypic intra-S checkpoint responses to UVC qualitatively and quantitatively equivalent to those previously demonstrated in skin fibroblasts. In comparison with fibroblasts, primary melanocytes displayed reduced UVC-induced inhibition of DNA strand growth and enhanced degradation of p21Waf1 after UVC, suggestive of enhanced bypass of UVC-induced DNA photoproducts. All nine melanoma cell lines examined, including those with activating mutations in BRAF or NRAS oncogenes, also displayed proficiency in activation of the intra-S checkpoint in response to UVC irradiation. The results indicate that bypass of oncogene-induced senescence during melanoma development was not associated with inactivation of the intra-S checkpoint response to UVC-induced DNA replication stress.

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

    NARCIS (Netherlands)

    Jose, J.; Grootendorst, D.J.; Vijn, T.W.; Wouters, M.W.; van Boven, H.; van Leeuwen, T.G.; van Steenbergen, W.; Ruers, T.J.M.; Manohar, S.

    2011-01-01

    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

  1. Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines

    DEFF Research Database (Denmark)

    Mohammed, M Z; Vyjayanti, V N; Laughton, C A

    2011-01-01

    Modulation of DNA base excision repair (BER) has the potential to enhance response to chemotherapy and improve outcomes in tumours such as melanoma and glioma. APE1, a critical protein in BER that processes potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer. In the...

  2. Computational Analysis of Human Blood Flow

    Science.gov (United States)

    Panta, Yogendra; Marie, Hazel; Harvey, Mark

    2009-11-01

    Fluid flow modeling with commercially available computational fluid dynamics (CFD) software is widely used to visualize and predict physical phenomena related to various biological systems. In this presentation, a typical human aorta model was analyzed assuming the blood flow as laminar with complaint cardiac muscle wall boundaries. FLUENT, a commercially available finite volume software, coupled with Solidworks, a modeling software, was employed for the preprocessing, simulation and postprocessing of all the models.The analysis mainly consists of a fluid-dynamics analysis including a calculation of the velocity field and pressure distribution in the blood and a mechanical analysis of the deformation of the tissue and artery in terms of wall shear stress. A number of other models e.g. T branches, angle shaped were previously analyzed and compared their results for consistency for similar boundary conditions. The velocities, pressures and wall shear stress distributions achieved in all models were as expected given the similar boundary conditions. The three dimensional time dependent analysis of blood flow accounting the effect of body forces with a complaint boundary was also performed.

  3. Downregulation of discoidin domain receptor 2 in A375 human melanoma cells reduces its experimental liver metastasis ability.

    Science.gov (United States)

    Badiola, Iker; Villacé, Patricia; Basaldua, Iratxe; Olaso, Elvira

    2011-10-01

    Discoidin domain receptors (DDR1 and DDR2) are tyrosine kinase receptors for fibrillar collagen implicated in postnatal development, tissue repair, and primary and metastatic cancer progression. While DDR1 has been described in tumor cells, DDR2 has been localized in the tumor stroma, but its presence in the tumor cells remains unknown. The aim of this study was to elucidate the role of DDR2 signaling in tumor cells during hepatic metastasis progression. DDR2 expression and phosphorylation in cultured human A375 melanoma cells was documented by Western blot analysis. A375 cells were stably transfected with a small interfering RNA (siRNA) against DDR2 and two clones were selected: A375R2-70 and A375R2-40, with 70 and 40% of the DDR2 protein expression respectively, compared to mock-transfected cells (A375R2-100). Development of experimental liver metastasis by intrasplenic inoculation of A375R2-70 and A37R2-40 clones was reduced by 60 and 75%, respectively, measured as tumor volume, compared to livers injected with A375R2-100 cells. Accordingly, A375R2-70 and A37R2-40 clones showed reduced in vitro gelatinase activity and JNK phosphorylation, compared to mock transfected cells, with maximal inhibition in A375R2-40. Additionally, A375 melanoma, SK-HEP hepatoma and HT-29 colon carcinoma human cell lines transiently transfected with siRNA against DDR2 also showed reduced proliferation and migration rates compared to mock-transfected ones. In conclusion, DDR2 promotes A375 melanoma metastasis to the liver and the underlying mechanism implicates regulation of metalloproteinase release, cell growth and chemotactic invasion of the host tissue.

  4. EPR studies of free radicals in A-2058 human melanoma cells treated by valproic acid and 5,7-dimethoxycoumarin.

    Science.gov (United States)

    Zdybel, Magdalena; Chodurek, Ewa; Pilawa, Barbara

    2014-01-01

    Free radicals in A-2058 human melanoma cells were studied by the use of electron paramagnetic resonance (EPR) spectroscopy. The aim of this work was to determine the changes in relative free radical concentrations in tumor A-2058 cells after treatment by valproic acid (VPA) and 5,7-dimethoxycoumarin (DMC). The influences of VPA and DMC on free radicals in A-2058 cells were compared with those for human melanoma malignum A-375 and G-361 cells, which were tested by us earlier. Human malignant melanoma A-2058 cells were exposed to interactions with VPA, DMC, and both VPA and DMC. The tumor cells A-2058 were purchased from LGC Standards (Lomianki, Poland), and they were grown in the standard conditions: at 37°C and in an atmosphere containing 95% air and 5% CO2, in the Minimum Essential Medium Eagle (MEM, Sigma-Aldrich). The A-2058 cells were incubated with VPA (1 mM) and DMC (10 μM) for 4 days. The first-derivative EPR spectra of the control A-2058 cells, and the cells treated with VPA, DMC, and both VPA and DMC, were measured by the electron paramagnetic resonance spectrometer of Radiopan (Poznań, Poland) with microwaves from an X-band (9.3 GHz). The parameters of the EPR lines: amplitudes (A), integral intensities (I), line widths (ΔBpp), and g-factors, were analyzed. The changes of amplitudes and line widths with microwave power increasing from 2.2 to 70 mW were drawn evaluated, o-Semiquinone free radicals of melanin biopolymer are mainly responsible for the EPR lines of A-2058 melanoma malignum cells. The amounts of free radicals in A-2058 cells treated with VPA, and both VPA and DMC, were lower than in the untreated control cells. Application of the tested substances (VPA, and both VPA and DMC) as the antitumor compounds was discussed. DMC without VPA did not decrease free radicals concentration in A-2058 cells. The studies con-firmed that EPR spectroscopy may be used to examine interactions of free radicals with antitumor compounds.

  5. The Human Blood Metabolome-Transcriptome Interface

    Science.gov (United States)

    Schramm, Katharina; Adamski, Jerzy; Gieger, Christian; Herder, Christian; Carstensen, Maren; Peters, Annette; Rathmann, Wolfgang; Roden, Michael; Strauch, Konstantin; Suhre, Karsten; Kastenmüller, Gabi; Prokisch, Holger; Theis, Fabian J.

    2015-01-01

    Biological systems consist of multiple organizational levels all densely interacting with each other to ensure function and flexibility of the system. Simultaneous analysis of cross-sectional multi-omics data from large population studies is a powerful tool to comprehensively characterize the underlying molecular mechanisms on a physiological scale. In this study, we systematically analyzed the relationship between fasting serum metabolomics and whole blood transcriptomics data from 712 individuals of the German KORA F4 cohort. Correlation-based analysis identified 1,109 significant associations between 522 transcripts and 114 metabolites summarized in an integrated network, the ‘human blood metabolome-transcriptome interface’ (BMTI). Bidirectional causality analysis using Mendelian randomization did not yield any statistically significant causal associations between transcripts and metabolites. A knowledge-based interpretation and integration with a genome-scale human metabolic reconstruction revealed systematic signatures of signaling, transport and metabolic processes, i.e. metabolic reactions mainly belonging to lipid, energy and amino acid metabolism. Moreover, the construction of a network based on functional categories illustrated the cross-talk between the biological layers at a pathway level. Using a transcription factor binding site enrichment analysis, this pathway cross-talk was further confirmed at a regulatory level. Finally, we demonstrated how the constructed networks can be used to gain novel insights into molecular mechanisms associated to intermediate clinical traits. Overall, our results demonstrate the utility of a multi-omics integrative approach to understand the molecular mechanisms underlying both normal physiology and disease. PMID:26086077

  6. The Human Blood Metabolome-Transcriptome Interface.

    Directory of Open Access Journals (Sweden)

    Jörg Bartel

    2015-06-01

    Full Text Available Biological systems consist of multiple organizational levels all densely interacting with each other to ensure function and flexibility of the system. Simultaneous analysis of cross-sectional multi-omics data from large population studies is a powerful tool to comprehensively characterize the underlying molecular mechanisms on a physiological scale. In this study, we systematically analyzed the relationship between fasting serum metabolomics and whole blood transcriptomics data from 712 individuals of the German KORA F4 cohort. Correlation-based analysis identified 1,109 significant associations between 522 transcripts and 114 metabolites summarized in an integrated network, the 'human blood metabolome-transcriptome interface' (BMTI. Bidirectional causality analysis using Mendelian randomization did not yield any statistically significant causal associations between transcripts and metabolites. A knowledge-based interpretation and integration with a genome-scale human metabolic reconstruction revealed systematic signatures of signaling, transport and metabolic processes, i.e. metabolic reactions mainly belonging to lipid, energy and amino acid metabolism. Moreover, the construction of a network based on functional categories illustrated the cross-talk between the biological layers at a pathway level. Using a transcription factor binding site enrichment analysis, this pathway cross-talk was further confirmed at a regulatory level. Finally, we demonstrated how the constructed networks can be used to gain novel insights into molecular mechanisms associated to intermediate clinical traits. Overall, our results demonstrate the utility of a multi-omics integrative approach to understand the molecular mechanisms underlying both normal physiology and disease.

  7. Noncontact discrimination of animal and human blood with vacuum blood vessel and factors affect the discrimination

    Science.gov (United States)

    Zhang, Linna; Zhang, Shengzhao; Sun, Meixiu; Li, Hongxiao; Li, Yingxin; Fu, Zhigang; Guan, Yang; Li, Gang; Lin, Ling

    2017-03-01

    Discrimination of human and nonhuman blood is crucial for import-export ports and inspection and quarantine departments. Current methods are usually destructive, complicated and time-consuming. We had previously demonstrated that visible diffuse reflectance spectroscopy combining PLS-DA method can successfully realize human blood discrimination. In that research, the spectra were measured with the fiber probe under the surface of blood samples. However, open sampling may pollute the blood samples. Virulence factors in blood samples can also endanger inspectors. In this paper, we explored the classification effect with the blood samples measured in the original containers-vacuum blood vessel. Furthermore, we studied the impact of different conditions of blood samples, such as coagulation and hemolysis, on the prediction ability of the discrimination model. The calibration model built with blood samples in different conditions displayed a satisfactory prediction result. This research demonstrated that visible and near-infrared diffuse reflectance spectroscopy method was potential for noncontact discrimination of human blood.

  8. Genetics of Melanoma

    Directory of Open Access Journals (Sweden)

    Janet eWangari-Talbot

    2013-01-01

    Full Text Available 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 predisposed 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 MAPK 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 personalized route

  9. Simvastatin rises reactive oxygen species levels and induces senescence in human melanoma cells by activation of p53/p21 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Guterres, Fernanda Augusta de Lima Barbosa; Martinez, Glaucia Regina; Rocha, Maria Eliane Merlin; Winnischofer, Sheila Maria Brochado, E-mail: sheilambw@ufpr.br

    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.

  10. Involvement of mitochondrial and B-RAF/ERK signaling pathways in berberine-induced apoptosis in human melanoma cells.

    Science.gov (United States)

    Burgeiro, Ana; Gajate, Consuelo; Dakir, El Habib; Villa-Pulgarín, Janny A; Oliveira, Paulo J; Mollinedo, Faustino

    2011-07-01

    The natural isoquinoline alkaloid berberine exhibits a wide spectrum of biological activities including antitumor activity, but its mechanism of action remains to be fully elucidated. Here, we report that berberine induced apoptosis in human melanoma cells, through a process that involved mitochondria and caspase activation. Berberine-induced activation of a number of caspases, including caspases 3, 4, 7, 8, and 9. Pan-caspase inhibitor, z-VAD-fmk, and caspase-8 and caspase-9 inhibitors prevented apoptosis. Berberine also led to the generation of the p20 cleavage fragment of BAP31, involved in directing proapoptotic signals between the endoplasmic reticulum and the mitochondria. Treatment of SK-MEL-2 melanoma cells with berberine induced disruption of the mitochondrial transmembrane potential, release of cytochrome c and apoptosis-inducing factor from the mitochondria to the cytosol, generation of reactive oxygen species (ROS), and a decreased ATP/ADP ratio. Overexpression of bcl-xL by gene transfer prevented berberine-induced cell death, mitochondrial transmembrane potential loss, and cytochrome c and apoptosis-inducing factor release, but not ROS generation. N-acetyl-L-cysteine inhibited the production of ROS, but did not abrogate the berberine-induced apoptosis. Inhibition of extracellular signal-regulated kinase (ERK) phosphorylation, by using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059, and reduction of B-RAF levels by silencing RNA induced cell death of SK-MEL-2 cells, and diminished the berberine concentration required to promote apoptosis. These data show that berberine-induced apoptosis in melanoma cells involves mitochondria and caspase activation, but ROS generation was not essential. Our results indicate that inhibition of B-RAF/ERK survival signaling facilitates the cell death response triggered by berberine.

  11. Specific targeting of Wnt/β-catenin signaling in human melanoma cells by a dietary triterpene lupeol.

    Science.gov (United States)

    Tarapore, Rohinton S; Siddiqui, Imtiaz A; Saleem, Mohammad; Adhami, Vaqar M; Spiegelman, Vladimir S; Mukhtar, Hasan

    2010-10-01

    Wingless (Wnt) signaling pathway regulates a variety of cellular processes including proliferation, differentiation, survival, apoptosis and cell motility. Aberrant activation of Wnt/β-catenin pathway has been observed in approximately one-third of melanomas and this subset has very poor prognosis suggesting that targeting Wnt signaling could be a promising strategy against this subtype. Mel 928 and Mel 1241 melanoma cells representative of cells with constitutive activation of Wnt/β-catenin signaling pathway and Mel 1011 representative of cells that lack this pathway were treated with a dietary triterpene lupeol and its effects on growth, proliferation, β-catenin transcriptional activity and Wnt target genes were determined both in vitro and in vivo. Lupeol treatment to Mel 928 and Mel 1241 but not Mel 1011 cells resulted in a dose-dependent (i) decrease in cell viability, (ii) induction of apoptosis, (iii) decrease in colonogenic potential, (iv) decrease in β-catenin transcriptional activity and (v) decrease in the expression of Wnt target genes. Most importantly, lupeol restricted the translocation of β-catenin from the cytoplasm to the nucleus. Lupeol also decreased the growth of Mel 928 but not Mel 1011-derived tumors implanted in the athymic nude mice. The decrease in Mel 928-derived tumor growth was associated with a decrease in the expression of Wnt target genes c-myc, cyclin D1, proliferation markers proliferating cell nuclear antigen and Ki-67 and invasion marker osteopontin. We suggest that lupeol alone or as an adjuvant to current therapies could be developed as an agent for the management of human melanomas harboring constitutive Wnt/β-catenin signaling.

  12. Comparison of responses of human melanoma cell lines to MEK and BRAF inhibitors.

    Science.gov (United States)

    Stones, Clare J; Kim, Ji Eun; Joseph, Wayne R; Leung, Euphemia; Marshall, Elaine S; Finlay, Graeme J; Shelling, Andrew N; Baguley, Bruce C

    2013-01-01

    The NRAS and BRAF genes are frequently mutated in melanoma, suggesting that the NRAS-BRAF-MEK-ERK signaling pathway is an important target for therapy. Two classes of drugs, one targeting activated BRAF and one targeting MEK, are currently undergoing clinical evaluation. We have analysed the NRAS and BRAF mutational status of a series of 44 early passage lines developed from New Zealand patients with metastatic melanoma. 41% of the lines analysed had BRAF mutations, 23% had NRAS mutations, and 36% had neither. We then determined IC50 values (drug concentrations for 50% growth inhibition) for CI-1040, a commonly used inhibitor of MEK kinase; trametinib, a clinical agent targeting MEK kinase; and vemurafenib, an inhibitor of mutant BRAF kinase. Cell lines with activating BRAF mutations were significantly more sensitive to vemurafenib than lines with NRAS mutations or lines lacking either mutation (p < 0.001). IC50 values for CI-1040 and trametinib were strongly correlated (r = 0.98) with trametinib showing ~100-fold greater potency. Cell lines sensitive to vemurafenib were also sensitive to CI-1040 and trametinib, but there was no relationship between IC50 values and NRAS mutation status. A small number of lines lacking a BRAF mutation were sensitive to CI-1040 but resistant to vemurafenib. We used western blotting to investigate the effect on ERK phosphorylation of CI-1040 in four lines, of vemurafenib in two lines and of trametinib in two lines. The results support the view that MEK inhibitors might be combined with BRAF inhibitors in the treatment of melanomas with activated BRAF. The high sensitivity to trametinib of some lines with wildtype BRAF status also suggests that MEK inhibitors could have a therapeutic effect against some melanomas as single agents.

  13. Comparison of responses of human melanoma cell lines to MEK and BRAF inhibitors

    Directory of Open Access Journals (Sweden)

    Clare Judith Stones

    2013-05-01

    Full Text Available The NRAS and BRAF genes are frequently mutated in melanoma, suggesting that the NRAS-BRAF-MEK-ERK signalling pathway is an important target for therapy. Two classes of drugs, one targeting activated BRAF and one targeting MEK, are currently undergoing clinical evaluation. We have analysed the NRAS and BRAF mutational status of a series of 44 early passage lines developed from New Zealand patients with metastatic melanoma. 41% of the lines analysed had BRAF mutations, 23% had NRAS mutations and 36% had neither. We then determined IC50 values (drug concentrations for 50% growth inhibition for CI-1040, a commonly used inhibitor of MEK kinase; trametinib, a clinical agent targeting MEK kinase; and vemurafenib, an inhibitor of mutant BRAF kinase. Cell lines with activating BRAF mutations were significantly more sensitive to vemurafenib than lines with NRAS mutations or lines lacking either mutation (p < 0.001. IC50 values for CI-1040 and trametinib were strongly correlated (r = 0.98 with trametinib showing ~100-fold greater potency. Cell lines sensitive to vemurafenib were also sensitive to CI-1040 and trametinib, but there was no relationship between IC50 values and NRAS mutation status. A small number of lines lacking a BRAF mutation were sensitive to CI-1040 but resistant to vemurafenib. We used western blotting to investigate the effect on ERK phosphorylation of CI-1040 in four lines, of vemurafenib in two lines and of trametinib in two lines. The results support the view that MEK inhibitors might be combined with BRAF inhibitors in the treatment melanomas of with activated BRAF. The high sensitivity to trametinib of some lines with wild-type BRAF status also suggests that MEK inhibitors could have a therapeutic effect against some melanomas as single agents.

  14. Selective BRAF inhibition decreases tumor-resident lymphocyte frequencies in a mouse model of human melanoma.

    Science.gov (United States)

    Hooijkaas, Anna; Gadiot, Jules; Morrow, Michelle; Stewart, Ross; Schumacher, Ton; Blank, Christian U

    2012-08-01

    The development of targeted therapies and immunotherapies has markedly advanced the treatment of metastasized melanoma. While treatment with selective BRAF(V600E) inhibitors (like vemurafenib or dabrafenib) leads to high response rates but short response duration, CTLA-4 blocking therapies induce sustained responses, but only in a limited number of patients. The combination of these diametric treatment approaches may further improve survival, but pre-clinical data concerning this approach is limited. We investigated, using Tyr::CreER(T2)PTEN(F-/-)BRAF(F-V600E/+) inducible melanoma mice, whether BRAF(V600E) inhibition can synergize with anti-CTLA-4 mAb treatment, focusing on the interaction between the BRAF(V600E) inhibitor PLX4720 and the immune system. While PLX4720 treatment strongly decreased tumor growth, it did not induce cell death in BRAF(V600E)/PTEN(-/-) melanomas. More strikingly, PLX4720 treatment led to a decreased frequency of tumor-resident T cells, NK-cells, MDSCs and macrophages, which could not be restored by the addition of anti-CTLA-4 mAb. As this effect was not observed upon treatment of BRAF wild-type B16F10 tumors, we conclude that the decreased frequency of immune cells correlates to BRAF(V600E) inhibition in tumor cells and is not due to an off-target effect of PLX4720 on immune cells. Furthermore, anti-CTLA-4 mAb treatment of inducible melanoma mice treated with PLX4720 did not result in enhanced tumor control, while anti-CTLA-4 mAb treatment did improve the effect of tumor-vaccination in B16F10-inoculated mice. Our data suggest that vemurafenib may negatively affect the immune activity within the tumor. Therefore, the potential effect of targeted therapy on the tumor-microenvironment should be taken into consideration in the design of clinical trials combining targeted and immunotherapy.

  15. CXCR6, a newly defined biomarker of tissue-specific stem cell asymmetric self-renewal, identifies more aggressive human melanoma cancer stem cells.

    Directory of Open Access Journals (Sweden)

    Rouzbeh Taghizadeh

    Full Text Available A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells. Recent investigations of a variety of tumor types have shown that phenotypically identifiable and isolable subfractions of cells possess the tumor-forming ability. In the present paper, using two lineage-related human melanoma cell lines, primary melanoma line IGR39 and its metastatic derivative line IGR37, two main observations are reported. The first one is the first phenotypic evidence to support the origin of melanoma cancer stem cells (CSCs from mutated tissue-specific stem cells; and the second one is the identification of a more aggressive subpopulation of CSCs in melanoma that are CXCR6+.We defined CXCR6 as a new biomarker for tissue-specific stem cell asymmetric self-renewal. Thus, the relationship between melanoma formation and ABCG2 and CXCR6 expression was investigated. Consistent with their non-metastatic character, unsorted IGR39 cells formed significantly smaller tumors than unsorted IGR37 cells. In addition, ABCG2+ cells produced tumors that had a 2-fold greater mass than tumors produced by unsorted cells or ABCG2- cells. CXCR6+ cells produced more aggressive tumors. CXCR6 identifies a more discrete subpopulation of cultured human melanoma cells with a more aggressive MCSC phenotype than cells selected on the basis of the ABCG2+ phenotype alone.The association of a more aggressive tumor phenotype with asymmetric self-renewal phenotype reveals a previously unrecognized aspect of tumor cell physiology. Namely, the retention of some tissue-specific stem cell attributes, like the ability to asymmetrically self-renew, impacts the natural history of human tumor development. Knowledge of this new aspect of tumor development and progression may provide new targets for cancer prevention and treatment.

  16. Technetium-99m-labeled Arg-Gly-Asp-conjugated alpha-melanocyte stimulating hormone hybrid peptides for human melanoma imaging

    Energy Technology Data Exchange (ETDEWEB)

    Yang Jianquan; Guo Haixun [College of Pharmacy, University of New Mexico, Albuquerque, NM 87131 (United States); Miao Yubin, E-mail: ymiao@salud.unm.ed [College of Pharmacy, University of New Mexico, Albuquerque, NM 87131 (United States); Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM 87131 (United States); Department of Dermatology, University of New Mexico, Albuquerque, NM 87131 (United States)

    2010-11-15

    Introduction: The purpose of this study was to examine whether {sup 99m}Tc-labeled Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone ({alpha}-MSH) hybrid peptide targeting both melanocortin-1 (MC1) and {alpha}{sub v{beta}3} integrin receptors was superior in melanoma targeting to {sup 99m}Tc-labeled {alpha}-MSH or RGD peptide targeting only the MC1 or {alpha}{sub v{beta}3} integrin receptor. Methods: RGD-Lys-(Arg{sup 11})CCMSH, RAD-Lys-(Arg{sup 11})CCMSH and RGD-Lys-(Arg{sup 11})CCMSHscramble were designed to target both MC1 and {alpha}{sub v{beta}3} integrin receptors, MC1 receptor only and {alpha}{sub v{beta}3} integrin receptor only, respectively. The MC1 or {alpha}{sub v{beta}3} integrin receptor binding affinities of three peptides were determined in M21 human melanoma cells. The melanoma targeting properties of {sup 99m}Tc-labeled RGD-Lys-(Arg{sup 11})CCMSH, RAD-Lys-(Arg{sup 11})CCMSH and RGD-Lys-(Arg{sup 11})CCMSHscramble were determined in M21 human melanoma-xenografted nude mice. Meanwhile, the melanoma uptake of {sup 99m}Tc-RGD-Lys-(Arg{sup 11})CCMSH was blocked with various non-radiolabeled peptides in M21 melanoma xenografts. Results: RGD-Lys-(Arg{sup 11})CCMSH displayed 2.0 and 403 nM binding affinities to both MC1 and {alpha}{sub v{beta}3} integrin receptors, whereas RAD-Lys-(Arg{sup 11})CCMSH or RGD-Lys-(Arg{sup 11})CCMSHscramble lost their {alpha}{sub v{beta}3} integrin receptor binding affinity by greater than 248-fold or MC1 receptor binding affinity by more than 100-fold, respectively. The melanoma uptake of {sup 99m}Tc-RGD-Lys-(Arg{sup 11})CCMSH was 2.49 and 2.24 times (P < .05) the melanoma uptakes of {sup 99m}Tc-RAD-Lys-(Arg{sup 11})CCMSH and {sup 99m}Tc-RGD-Lys-(Arg{sup 11})CCMSHscramble at 2 h post-injection, respectively. Either RGD or (Arg{sup 11})CCMSH peptide co-injection could block 42% and 57% of the tumor uptake of {sup 99m}Tc-RGD-Lys-(Arg{sup 11})CCMSH, whereas the coinjection of RGD+(Arg{sup 11})CCMSH peptide mixture

  17. Cytotoxic effect of xanthones from pericarp of the tropical fruit mangosteen (Garcinia mangostana Linn.) on human melanoma cells.

    Science.gov (United States)

    Wang, Jing J; Sanderson, Barbara J S; Zhang, Wei

    2011-09-01

    Mangosteen (Garcinia mangostana Linn.) is a tropical tree from South East Asia and its fruit pericarp is a well-known traditional medicine. In this study, the cytotoxic effect of three xanthone compounds (α-mangostin, γ-mangostin, and 8-deoxygartanin) from mangosteen pericarp was investigated using the human melanoma SK-MEL-28 cell line. Significant dose-dependent reduction in % cell viability was induced. γ-Mangostin and 8-deoxygartanine at 5 μg/ml increased the cell cycle arrest in G(1) phase (90% and 92%) compared with untreated cells (78%). All compounds induced apoptosis, of the highest being α-mangostin at 7.5 μg/ml that induced 59.6% early apoptosis, compared to 1.7% in untreated cells. The apoptotic effect of α-mangostin was via caspase activation and disruption of mitochondrial membrane pathways as evidenced by 25-fold increased caspase-3 activity and 9-fold decreased mitochondrial membrane potential when compared to untreated cells. In conclusion, these xanthones, especially α-mangostin, are potential candidates as anti-melanoma agents.

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

    Science.gov (United States)

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

    2016-01-01

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

  19. Endogenous HLA-DR-restricted presentation of the cartilage antigens human cartilage gp-39 and melanoma inhibitory activity in the inflamed rheumatoid joint

    NARCIS (Netherlands)

    van Lierop, M. J. C.; den Hoed, L.; Houbiers, J.; Vencovsky, J.; Ruzickova, S.; Krystufkova, O.; van Schaardenburg, M.; van den Hoogen, F.; Vandooren, B.; Baeten, D.; De Keyser, F.; Sonderstrup, G.; Bos, E.; Boots, A. M.

    2007-01-01

    Objective. The cartilage proteins melanoma inhibitory activity (MIA) and human cartilage gp-39 (HC gp-39) are candidate autoantigens in rheumatoid arthritis (RA). The present study was undertaken to investigate the endogenous HLA-DR4-restricted presentation of these self proteins, in order to seek i

  20. Apurinic/apyrimidinic endonuclease/redox effector factor-1(APE/Ref-1): a unique target for the prevention and treatment of human melanoma.

    Science.gov (United States)

    Yang, Sun; Meyskens, Frank L

    2009-03-01

    Management of melanoma is a growing and challenging public health issue requiring novel and multidisciplinary approaches to achieve more efficient prevention and therapeutic benefits. The aim of this article is to show the critical role of APE/Ref-1 on melanomagenesis and progression. APE/Ref-1 serves as a redox-sensitive node of convergence of various signals as well as a DNA-repair enzyme, and its activation protects melanocytes and melanoma cells from chronic oxidative stress and promotes cell survival via mediation of downstream pathways. APE/Ref-1 is a strong candidate as a potential drug-treatable target for the prevention and treatment of human melanoma. Lead compounds exhibiting inhibitory effects on APE/Ref-1 are also reviewed. We anticipate potential clinical benefit in the future through inhibition of APE/Ref-1 and/or Ref-1-mediated signaling.

  1. U1 Adaptor Oligonucleotides Targeting BCL2 and GRM1 Suppress Growth of Human Melanoma Xenografts In Vivo

    Directory of Open Access Journals (Sweden)

    Rafal Goraczniak

    2013-01-01

    Full Text Available U1 Adaptor is a recently discovered oligonucleotide-based gene-silencing technology with a unique mechanism of action that targets nuclear pre-mRNA processing. U1 Adaptors have two distinct functional domains, both of which must be present on the same oligonucleotide to exert their gene-silencing function. Here, we present the first in vivo use of U1 Adaptors by targeting two different human genes implicated in melanomagenesis, B-cell lymphoma 2 (BCL2 and metabotropic glutamate receptor 1 (GRM1, in a human melanoma cell xenograft mouse model system. Using a newly developed dendrimer delivery system, anti-BCL2 U1 Adaptors were very potent and suppressed tumor growth at doses as low as 34 µg/kg with twice weekly intravenous (iv administration. Anti-GRM1 U1 Adaptors suppressed tumor xenograft growth with similar potency. Mechanism of action was demonstrated by showing target gene suppression in tumors and by observing that negative control U1 Adaptors with just one functional domain show no tumor suppression activity. The anti-BCL2 and anti-GRM1 treatments were equally effective against cell lines harboring either wild-type or a mutant V600E B-RAF allele, the most common mutation in melanoma. Treatment of normal immune-competent mice (C57BL6 indicated no organ toxicity or immune stimulation. These proof-of-concept studies represent an in-depth (over 800 mice in ~108 treatment groups validation that U1 Adaptors are a highly potent gene-silencing therapeutic and open the way for their further development to treat other human diseases.

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

    Science.gov (United States)

    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.

  3. Toxicity and cytopathogenic properties toward human melanoma cells of activated cancer therapeutics in zebra fish.

    Science.gov (United States)

    Lewis, Thomas J

    2010-03-01

    There is an increasing body of data showing that activated cancer therapy--the synergistic effect of "preloaded" molecules and a tuned energy source to produce cytopathogenic moieties--is a promising new modality for cancer treatment. The key activated therapies are photodynamic therapy (PDT), which involves the synergy between light and photosensitizer molecules, and ultrasound activated therapy (USAT; also referred to as sonodynamic therapy), which involves the synergy between ultrasound and sonosensitizer molecules. PDT is a well-known activated therapy with roots dating back to 1900. However, minimal data exist on USAT. One reason is the lack of suitable sonosensitizers for clinical USAT use. The authors present both LC(50) toxicity and cancer cell cytotoxicity studies on 2 dual activation agents. These compounds function as both sonosensitizers and photosensitizers, and are referred to as SonneLux agents, designated SF1 and SF2. The sensitizers are derived from chlorophyll and are metal centered porphyrins known to specifically accumulate in hyperproliferating tissue. LC(50) studies on both SF1 and SF2 as determined in zebra fish reveal that both are essentially nontoxic to zebra fish. In the worst case, 5% zebra fish death is noted at the maximum soluble concentration of the sensitizer. In the cytotoxicity studies, melanoma cell line WM-266-4, derived from a metastatic site of a malignant melanoma, was tested against SF1 and SF2. Both sensitizer systems showed marked efficacy in the destruction of the implanted melanoma cells. They show great promise for clinical use in the future.

  4. Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines.

    Science.gov (United States)

    Mohammed, M Z; Vyjayanti, V N; Laughton, C A; Dekker, L V; Fischer, P M; Wilson, D M; Abbotts, R; Shah, S; Patel, P M; Hickson, I D; Madhusudan, S

    2011-02-15

    Modulation of DNA base excision repair (BER) has the potential to enhance response to chemotherapy and improve outcomes in tumours such as melanoma and glioma. APE1, a critical protein in BER that processes potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer. In the current study, we aimed to develop small molecule inhibitors of APE1 for cancer therapy. An industry-standard high throughput virtual screening strategy was adopted. The Sybyl8.0 (Tripos, St Louis, MO, USA) molecular modelling software suite was used to build inhibitor templates. Similarity searching strategies were then applied using ROCS 2.3 (Open Eye Scientific, Santa Fe, NM, USA) to extract pharmacophorically related subsets of compounds from a chemically diverse database of 2.6 million compounds. The compounds in these subsets were subjected to docking against the active site of the APE1 model, using the genetic algorithm-based programme GOLD2.7 (CCDC, Cambridge, UK). Predicted ligand poses were ranked on the basis of several scoring functions. The top virtual hits with promising pharmaceutical properties underwent detailed in vitro analyses using fluorescence-based APE1 cleavage assays and counter screened using endonuclease IV cleavage assays, fluorescence quenching assays and radiolabelled oligonucleotide assays. Biochemical APE1 inhibitors were then subjected to detailed cytotoxicity analyses. Several specific APE1 inhibitors were isolated by this approach. The IC(50) for APE1 inhibition ranged between 30 nM and 50 μM. We demonstrated that APE1 inhibitors lead to accumulation of AP sites in genomic DNA and potentiated the cytotoxicity of alkylating agents in melanoma and glioma cell lines. Our study provides evidence that APE1 is an emerging drug target and could have therapeutic application in patients with melanoma and glioma.

  5. A Melanoma Brain Metastasis with a Donor-Patient Hybrid Genome following Bone Marrow Transplantation: First Evidence for Fusion in Human Cancer.

    Directory of Open Access Journals (Sweden)

    Rossitza Lazova

    Full Text Available Tumor cell fusion with motile bone marrow-derived cells (BMDCs has long been posited as a mechanism for cancer metastasis. While there is much support for this from cell culture and animal studies, it has yet to be confirmed in human cancer, as tumor and marrow-derived cells from the same patient cannot be easily distinguished genetically.We carried out genotyping of a metastatic melanoma to the brain that arose following allogeneic bone-marrow transplantation (BMT, using forensic short tandem repeat (STR length-polymorphisms to distinguish donor and patient genomes. Tumor cells were isolated free of leucocytes by laser microdissection, and tumor and pre-transplant blood lymphocyte DNAs were analyzed for donor and patient alleles at 14 autosomal STR loci and the sex chromosomes.All alleles in the donor and patient pre-BMT lymphocytes were found in tumor cells. The alleles showed disproportionate relative abundances in similar patterns throughout the tumor, indicating the tumor was initiated by a clonal fusion event.Our results strongly support fusion between a BMDC and a tumor cell playing a role in the origin of this metastasis. Depending on the frequency of such events, the findings could have important implications for understanding the generation of metastases, including the origins of tumor initiating cells and the cancer epigenome.

  6. Comparative Aspects of Canine Melanoma

    Directory of Open Access Journals (Sweden)

    Adriana Tomoko Nishiya

    2016-02-01

    Full Text Available Melanomas are malignant neoplasms originating from melanocytes. They occur in most animal species, but the dog is considered the best animal model for the disease. Melanomas in dogs are most frequently found in the buccal cavity, but the skin, eyes, and digits are other common locations for these neoplasms. The aim of this review is to report etiological, epidemiological, pathological, and molecular aspects of melanomas in dogs. Furthermore, the particular biological behaviors of these tumors in the different body locations are shown. Insights into the therapeutic approaches are described. Surgery, chemotherapy, radiotherapy, immunotherapy, and the outcomes after these treatments are presented. New therapeutic perspectives are also depicted. All efforts are geared toward better characterization and control of malignant melanomas in dogs, for the benefit of these companion animals, and also in an attempt to benefit the treatment of human melanomas.

  7. Blood pressure estimation in the human fetal descending aorta.

    NARCIS (Netherlands)

    Struijk, P.C.; Mathews, V.J.; Loupas, T.; Stewart, P.A.; Clark, E.B.; Steegers, E.A.P.; Wladimiroff, J.W.

    2008-01-01

    OBJECTIVES: The objectives of this study were to estimate fetal blood pressure non-invasively from two-dimensional color Doppler-derived aortic blood flow and diameter waveforms, and to compare the results with invasively derived human fetal blood pressures available from the literature. METHODS:

  8. Blood pressure estimation in the human fetal descending aorta

    NARCIS (Netherlands)

    P.C. Struijk (Pieter); V.J. Mathews; T. Loupas; P.A. Stewart (Patricia); E.B. Clark; R.P.M. Steegers-Theunissen (Régine); J.W. Wladimiroff (Juriy)

    2008-01-01

    textabstractObjectives: The objectives of this study were to estimate fetal blood pressure non-invasively from two-dimensional color Doppler-derived aortic blood flow and diameter waveforms, and to compare the results with invasively derived human fetal blood pressures available from the literature.

  9. Human Blood Typing: A Forensic Science Approach: Part II. Experiments.

    Science.gov (United States)

    Kobilinsky, Lawrence; Sheehan, Francis X.

    1988-01-01

    Describes several experiments that explore the methodology available to the forensic serologist for typing a human bloodstain in the ABH grouping system. Presents ABO blood group of wet blood, Lattes Crust test procedure, and the absorption-elution procedure. Uses outdated blood; equipment requirements are minimal. (ML)

  10. Human Blood Typing: A Forensic Science Approach: Part II. Experiments.

    Science.gov (United States)

    Kobilinsky, Lawrence; Sheehan, Francis X.

    1988-01-01

    Describes several experiments that explore the methodology available to the forensic serologist for typing a human bloodstain in the ABH grouping system. Presents ABO blood group of wet blood, Lattes Crust test procedure, and the absorption-elution procedure. Uses outdated blood; equipment requirements are minimal. (ML)

  11. Polychelated cryogels: hemoglobin adsorption from human blood.

    Science.gov (United States)

    Erol, Kadir

    2017-02-01

    The separation and purification methods are extremely important for the hemoglobin (Hb) which is a crucial biomolecule. The adsorption technique is popular among these methods and the cryogels have been used quite much due to their macropores and interconnected flow channels. In this study, the Hb adsorption onto the Cu(II) immobilized poly(2-hydroxyethyl methacrylate-glycidyl methacrylate), poly(HEMA-GMA)-Cu(II), cryogels was investigated under different conditions (pH, interaction time, initial Hb concentration, temperature and ionic strength) to optimize adsorption conditions. The swelling test, Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscope (SEM), surface area (BET), elemental and ICP-OES analysis were performed for the characterization of cryogels. Polyethyleneimine (PEI) molecule was used as a Cu(II)-chelating ligand. The Hb adsorption capacity of cryogels was determined as 193.8 mg Hb/g cryogel. The isolation of Hb from human blood was also studied under optimum adsorption conditions determined and the Hb (124.5 mg/g cryogel) was isolated. The adsorption model was investigated in the light of Langmuir and Freundlich adsorption isotherm models and it was determined to be more appropriate to the Langmuir adsorption isotherm model.

  12. Pediatric melanoma.

    Science.gov (United States)

    Tracy, Elisabeth T; Aldrink, Jennifer H

    2016-10-01

    Childhood melanoma is a rare pediatric malignancy, with fewer than 500 new diagnoses annually. The incidence is increasing, particularly in the adolescent population. This review highlights the epidemiology, clinical presentation, and histopathologic challenges of pediatric melanoma. Surgical resection remains the cornerstone for localized and regionally advanced disease. Adjuvant therapies, including current options and potential novel therapeutics for this unique population will be discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Cutaneous melanoma.

    Science.gov (United States)

    Eggermont, Alexander M M; Spatz, Alan; Robert, Caroline

    2014-03-01

    In the past decade, major advances have been made in the understanding of melanoma. New predisposition genes have been reported and key somatic events, such as BRAF mutation, directly translated into therapeutic management. Surgery for localised melanoma and regional lymph node metastases is the standard of care. Sentinel-node biopsy provides precise staging, but has not been reported to affect survival. The effect of lymph-node dissection on survival is a topic of investigation. Two distinct approaches have emerged to try to extend survival in patients with metastatic melanoma: immunomodulation with anti-CTLA4 monoclonal antibodies, and targeted therapy with BRAF inhibitors or MEK inhibitors for BRAF-mutated melanoma. The combination of BRAF inhibitors and MEK inhibitors might improve progression-free survival further and, possibly, increase overall survival. Response patterns differ substantially-anti-CTLA4 immunotherapy can induce long-term responses, but only in a few patients, whereas targeted drugs induce responses in most patients, but nearly all of them relapse because of pre-existing or acquired resistance. Thus, the long-term prognosis of metastatic melanoma remains poor. Anti-PD1 and anti-PDL1 antibodies have emerged as breakthrough drugs for melanoma that have high response rates and long durability. Biomarkers that have predictive value remain elusive in melanoma, although emerging data for adjuvant therapy indicate that interferon sensitivity is associated with ulceration of the primary melanoma. Intense investigation continues for clinical and biological markers that predict clinical benefit of immunotherapeutic drugs, such as interferon alfa or anti-CTLA4 antibodies, and the mechanisms that lead to resistance of targeted drugs.

  14. Phenotyping of human melanoma cells reveals a unique composition of receptor targets and a subpopulation co-expressing ErbB4, EPO-R and NGF-R.

    Science.gov (United States)

    Mirkina, Irina; Hadzijusufovic, Emir; Krepler, Clemens; Mikula, Mario; Mechtcheriakova, Diana; Strommer, Sabine; Stella, Alexander; Jensen-Jarolim, Erika; Höller, Christoph; Wacheck, Volker; Pehamberger, Hubert; Valent, Peter

    2014-01-01

    Malignant melanoma is a life-threatening skin cancer increasingly diagnosed in the western world. In advanced disease the prognosis is grave. Growth and metastasis formation in melanomas are regulated by a network of cytokines, cytokine-receptors, and adhesion molecules. However, little is known about surface antigens and target expression profiles in human melanomas. We examined the cell surface antigen profile of human skin melanoma cells by multicolor flow cytometry, and compared their phenotype with 4 melanoma cell lines (A375, 607B, Mel-Juso, SK-Mel28). Melanoma cells were defined as CD45-/CD31- cells co-expressing one or more melanoma-related antigens (CD63, CD146, CD166). In most patients, melanoma cells exhibited ErbB3/Her3, CD44/Pgp-1, ICAM-1/CD54 and IGF-1-R/CD221, but did not express CD20, ErbB2/Her2, KIT/CD117, AC133/CD133 or MDR-1/CD243. Melanoma cell lines were found to display a similar phenotype. In most patients, a distinct subpopulation of melanoma cells (4-40%) expressed the erythropoietin receptor (EPO-R) and ErbB4 together with PD-1 and NGF-R/CD271. Both the EPO-R+ and EPO-R- subpopulations produced melanoma lesions in NOD/SCID IL-2Rgamma(null) (NSG) mice in first and secondary recipients. Normal skin melanocytes did not express ErbB4 or EPO-R, but expressed a functional KIT receptor (CD117) as well as NGF-R, ErbB3/Her3, IGF-1-R and CD44. In conclusion, melanoma cells display a unique composition of surface target antigens and cytokine receptors. Malignant transformation of melanomas is accompanied by loss of KIT and acquisition of EPO-R and ErbB4, both of which are co-expressed with NGF-R and PD-1 in distinct subfractions of melanoma cells. However, expression of EPO-R/ErbB4/PD-1 is not indicative of a selective melanoma-initiating potential.

  15. Nocturnal variations in peripheral blood flow, systemic blood pressure, and heart rate in humans

    DEFF Research Database (Denmark)

    Sindrup, J H; Kastrup, J; Christensen, H

    1991-01-01

    was associated with a 30-40% increase in blood flow rate and a highly significant decrease in mean arterial blood pressure and heart rate (P less than 0.001 for all). Approximately 100 min after the subjects went to sleep an additional blood flow rate increment (mean 56%) and a simultaneous significant decrease......Subcutaneous adipose tissue blood flow rate, together with systemic arterial blood pressure and heart rate under ambulatory conditions, was measured in the lower legs of 15 normal human subjects for 12-20 h. The 133Xe-washout technique, portable CdTe(Cl) detectors, and a portable data storage unit...... were used for measurement of blood flow rates. An automatic portable blood pressure recorder and processor unit was used for measurement of systolic blood pressure, diastolic blood pressure, and heart rate every 15 min. The change from upright to supine position at the beginning of the night period...

  16. UVB-Stimulated TNFα Release from Human Melanocyte and Melanoma Cells Is Mediated by p38 MAPK

    Directory of Open Access Journals (Sweden)

    Visalini Muthusamy

    2013-08-01

    Full Text Available 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.

  17. The Effect of Bevacizumab on Human Malignant Melanoma Cells with Functional VEGF/VEGFR2 Autocrine and Intracrine Signaling Loops

    Directory of Open Access Journals (Sweden)

    Una Adamcic

    2012-07-01

    Full Text Available Receptors for the angiogenic factor VEGF are expressed by tumor cancer cells including melanoma, although their functionality remains unclear. Paired human melanoma cell lines WM115 and WM239 were used to investigate differences in expression and functionality of VEGF and VEGFR2 in vitro and in vivo with the anti-VEGF antibody bevacizumab. Both WM115 and WM239 cells expressed VEGF and VEGFR2, the levels of which were modulated by hypoxia. Detection of native and phosphorylated VEGFR2 in subcellular fractions under serum-free conditions showed the presence of a functional autocrine as well as intracrine VEGF/VEGFR2 signaling loops. Interestingly, treatment of WM115 and WM239 cells with increasing doses of bevacizumab (0–300 µg/ml in vitro did not show any significant inhibition of VEGFR2 phosphorylation. Small-molecule tyrosine kinase inhibitor, sunitinib, caused an inhibition of VEGFR2 phosphorylation in WM239 but not in WM115 cells. An increase in cell proliferation was observed in WM115 cells treated with bevacizumab, whereas sunitinib inhibited proliferation. When xenografted to immune-deficient mice, we found bevacizumab to be an effective antiangiogenic but not antitumorigenic agent for both cell lines. Because bevacizumab is unable to neutralize murine VEGF, this supports a paracrine angiogenic response. We propose that the failure of bevacizumab to generate an antitumorigenic effect may be related to its generation of enhanced autocrine/intracrine signaling in the cancer cells themselves. Collectively, these results suggest that, for cancers with intracrine VEGF/ VEGFR2 signaling loops, small-molecule inhibitors of VEGFR2 may be more effective than neutralizing antibodies at disease control.

  18. Bone blood flow and metabolism in humans

    DEFF Research Database (Denmark)

    Heinonen, Ilkka; Kemppainen, Jukka; Kaskinoro, Kimmo

    2012-01-01

    in femoral bone at rest and during one leg intermittent isometric exercise with increasing exercise intensities. In nine men, blood flow in femur was determined at rest and during dynamic one leg exercise, and two other physiological perturbations: moderate systemic hypoxia (14 O(2) ) at rest and during...... leg. In conclusion, resting femoral bone blood flow increases by physical exercise, but appears to level off with increasing exercise intensities. Moreover, while moderate systemic hypoxia does not change bone blood flow at rest or during exercise, intra-arterially administered adenosine during...

  19. Concomitant targeting of programmed death-1 (PD-1) and CD137 improves the efficacy of radiotherapy in a mouse model of human BRAFV600-mutant melanoma.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2014-11-30

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

  1. Characterisation of Human Keratinocytes by Measuring Cellular Repair Capacity of UVB-Induced DNA Damage and Monitoring of Cytogenetic Changes in Melanoma Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Greinert, R.; Breibart, E.W.; Mitchell, D.; Smida, J.; Volkmer, B

    2000-07-01

    The molecular mechanisms for UV-induced photocarcinogenesis are far from being understood in detail, especially in the case of malignant melanoma of the skin. Nevertheless, it is known that deficiencies in cellular repair processes of UV-induced DNA damage (e.g. in the case of Xeroderma pigmentosum) represent important aetiological factors in the multistep development of skin cancer. The repair kinetics have therefore been studied of an established skin cell line (HaCaT), primary human keratinocytes, melanocytes and melanoma cell lines, using fluorescence microscopy and flow cytometry. Our data show a high degree of interindividual variability in cellular repair capacity for UV-induced DNA lesions, which might be due to individual differences in the degree of tolerable damage and/or the onsets of saturation of the enzymatic repair system. The cytogenetic analysis of melanoma cell lines, using spectral karyotyping (SKY) furthermore proves that malignant melanoma of the skin are characterised by high numbers of chromosomal aberrations. (author)

  2. Metagenomic Shotgun Sequencing and Unbiased Metabolomic Profiling Identify Specific Human Gut Microbiota and Metabolites Associated with Immune Checkpoint Therapy Efficacy in Melanoma Patients

    Directory of Open Access Journals (Sweden)

    Arthur E. Frankel

    2017-10-01

    Full Text Available This is the first prospective study of the effects of human gut microbiota and metabolites on immune checkpoint inhibitor (ICT response in metastatic melanoma patients. Whereas many melanoma patients exhibit profound response to ICT, there are fewer options for patients failing ICT—particularly with BRAF-wild-type disease. In preclinical studies, specific gut microbiota promotes regression of melanoma in mice. We therefore conducted a study of the effects of pretreatment gut microbiota and metabolites on ICT Response Evaluation Criteria in Solid Tumors response in 39 metastatic melanoma patients treated with ipilimumab, nivolumab, ipilimumab plus nivolumab (IN, or pembrolizumab (P. IN yielded 67% responses and 8% stable disease; P achieved 23% responses and 23% stable disease. ICT responders for all types of therapies were enriched for Bacteroides caccae. Among IN responders, the gut microbiome was enriched for Faecalibacterium prausnitzii, Bacteroides thetaiotamicron, and Holdemania filiformis. Among P responders, the microbiome was enriched for Dorea formicogenerans. Unbiased shotgun metabolomics revealed high levels of anacardic acid in ICT responders. Based on these pilot studies, both additional confirmatory clinical studies and preclinical testing of these bacterial species and metabolites are warranted to confirm their ICT enhancing activity.

  3. Acid Ceramidase in Melanoma

    DEFF Research Database (Denmark)

    Realini, Natalia; Palese, Francesca; Pizzirani, Daniela

    2016-01-01

    Acid ceramidase (AC) is a lysosomal cysteine amidase that controls sphingolipid signaling by lowering the levels of ceramides and concomitantly increasing those of sphingosine and its bioactive metabolite, sphingosine 1-phosphate. In the present study, we evaluated the role of AC-regulated sphing......Acid ceramidase (AC) is a lysosomal cysteine amidase that controls sphingolipid signaling by lowering the levels of ceramides and concomitantly increasing those of sphingosine and its bioactive metabolite, sphingosine 1-phosphate. In the present study, we evaluated the role of AC......-regulated sphingolipid signaling in melanoma. We found that AC expression is markedly elevated in normal human melanocytes and proliferative melanoma cell lines, compared with other skin cells (keratinocytes and fibroblasts) and non-melanoma cancer cells. High AC expression was also observed in biopsies from human...... generate lower amounts of ceramides than normal melanocytes do. This down-regulation in ceramide production appears to result from suppression of the de novo biosynthesis pathway. To test whether AC might contribute to melanoma cell proliferation, we blocked AC activity using a new potent (IC50 = 12 n...

  4. Immunotherapy of metastatic melanoma by reversal of immune suppression

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. Malignant Melanoma

    Directory of Open Access Journals (Sweden)

    Eshini Perera

    2013-12-01

    Full Text Available 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.

  6. Photosensitized inactivation of infectious blood-borne human parasites

    Science.gov (United States)

    Judy, Millard M.; Sogandares-Bernal, Franklin M.; Matthews, James Lester

    1995-05-01

    Blood-borne viruses and protozoan parasites that are infectious to humans pose risk world-wide of infection transmission through blood and blood product transfusion. Blood-borne infectious viruses include human immunodeficiency virus (HIV-I), which causes AIDS; hepatitis C virus, which can cause chronic hepatitis; and cytomegalovirus, which can be dangerous to immunocompromised patients, e.g., the newborn, transplant recipients, and AIDS patients. Infectious blood-borne protozoan parasites include Trypanosoma cruzi, which causes Chagas' disease, endemic throughout Central and South America; the Trypanosoma species causing African sleeping sickness endemic in Central Africa; and Plasmodium falciparum, which causes malignant and increasingly drug- resistant human malaria prevalent throughout the tropics. Some researchers have focused on using photosensitizers to inactivate HIV-I and other viruses in whole blood, packed red cells, and platelet concentrates without compromising blood product function. Our group previously has reported photosensitized in vitro inactivation of P. falciparum and the mouse malaria organism Plasmodium berghei in whole blood using hematoporphyrin derivative (HPD) and of T. cruzi using benzoporphyrin derivatives BPDMA and BPDDA, dihematoporphyrin ether (DHE), and hydroxyethylvinyldeuteroporphyrin (HEVD). These results suggest that continued investigation is warranted to evaluate the potential for photosensitized inactivation of blood-borne parasites in blood banking.

  7. Mechanisms contributing to differential regulation of PAX3 downstream target genes in normal human epidermal melanocytes versus melanoma cells.

    Science.gov (United States)

    Bartlett, Danielle; Boyle, Glen M; Ziman, Mel; Medic, Sandra

    2015-01-01

    Melanoma is a highly aggressive and drug resistant form of skin cancer. It arises from melanocytes, the pigment producing cells of the skin. The formation of these melanocytes is driven by the transcription factor PAX3 early during embryonic development. As a result of alternative splicing, the PAX3 gene gives rise to eight different transcripts which encode isoforms that have different structures and activate different downstream target genes involved in pathways of cell proliferation, migration, differentiation and survival. Furthermore, post-translational modifications have also been shown to alter the functions of PAX3. We previously identified PAX3 downstream target genes in melanocytes and melanoma cells. Here we assessed the effects of PAX3 down-regulation on this panel of target genes in primary melanocytes versus melanoma cells. We show that PAX3 differentially regulates various downstream target genes involved in cell proliferation in melanoma cells compared to melanocytes. To determine mechanisms behind this differential downstream target gene regulation, we performed immunoprecipitation to assess post-translational modifications of the PAX3 protein as well as RNAseq to determine PAX3 transcript expression profiles in melanocytes compared to melanoma cells. Although PAX3 was found to be post-translationally modified, there was no qualitative difference in phosphorylation and ubiquitination between melanocytes and melanoma cells, while acetylation of PAX3 was reduced in melanoma cells. Additionally, there were differences in PAX3 transcript expression profiles between melanocytes and melanoma cells. In particular the PAX3E transcript, responsible for reducing melanocyte proliferation and increasing apoptosis, was found to be down-regulated in melanoma cells compared to melanocytes. These results suggest that alternate transcript expression profiles activate different downstream target genes leading to the melanoma phenotype.

  8. Primate-specific Melanoma Antigen-A11 Regulates Isoform-specific Human Progesterone Receptor-B Transactivation*

    Science.gov (United States)

    Su, Shifeng; Blackwelder, Amanda J.; Grossman, Gail; Minges, John T.; Yuan, Lingwen; Young, Steven L.; Wilson, Elizabeth M.

    2012-01-01

    Progesterone acting through the progesterone receptor (PR) and its coregulators prepares the human endometrium for receptivity to embryo implantation and maintains pregnancy. The menstrual cycle-dependent expression of melanoma antigen-A11 (MAGE-11) in the mid-secretory human endometrium suggested a novel function in human PR signaling. Here we show that MAGE-11 is an isoform-specific coregulator responsible for the greater transcriptional activity of human PR-B relative to PR-A. PR was recruited to progesterone response regions of progesterone-regulated FK506-binding protein 5 (FKBP5) immunophilin and small Ras family G protein cell growth inhibitor RASD1 genes. Expression of MAGE-11 lentivirus shRNA in human endometrial Ishikawa cells expressing PR-B showed that MAGE-11 is required for isoform-specific PR-B up-regulation of FKBP5. In contrast, MAGE-11 was not required for progesterone up-regulation of RASD1 in endometrial cells expressing the PR-A/B heterodimer. Target gene specificity of PR-B depended on the synergistic actions of MAGE-11 and p300 mediated by the unique PR-B NH2-terminal 110LLXXVLXXLL119 motif that interacts with the MAGE-11 F-box region in a phosphorylation- and ubiquitinylation-dependent manner. A progesterone-dependent mechanism is proposed in which MAGE-11 and p300 increase PR-B up-regulation of the FKBP5 gene. MAGE-11 down-regulates PR-B, similar to the effects of progesterone, and interacts with FKBP5 to stabilize a complex with PR-B. We conclude that the coregulator function of MAGE-11 extends to isoform-specific regulation of PR-B during the cyclic development of the human endometrium. PMID:22891251

  9. Phenotype and functions of memory Tfh cells in human blood.

    Science.gov (United States)

    Schmitt, Nathalie; Bentebibel, Salah-Eddine; Ueno, Hideki

    2014-09-01

    Our understanding of the origin and functions of human blood CXCR5(+) CD4(+) T cells found in human blood has changed dramatically in the past years. These cells are currently considered to represent a circulating memory compartment of T follicular helper (Tfh) lineage cells. Recent studies have shown that blood memory Tfh cells are composed of phenotypically and functionally distinct subsets. Here, we review the current understanding of human blood memory Tfh cells and the subsets within this compartment. We present a strategy to define these subsets based on cell surface profiles. Finally, we discuss how increased understanding of the biology of blood memory Tfh cells may contribute insight into the pathogenesis of autoimmune diseases and the mode of action of vaccines.

  10. Direct determination of internal radiation dose in human blood

    OpenAIRE

    Tanır, Ayse Güneş; Güleç, Özge

    2014-01-01

    The purpose of this study is to measure the internal radiation dose using a human blood sample. In the literature, there is no process that allows the direct measurement of the internal radiation dose received by a person. The luminescence counts from a blood sample having a laboratory-injected radiation dose and the waste blood of the patient injected with a radiopharmaceutical for diagnostic purposes were both measured. The decay and dose-response curves were plotted for the different doses...

  11. Cooperative antiproliferative signaling by aspirin and indole-3-carbinol targets microphthalmia-associated transcription factor gene expression and promoter activity in human melanoma cells.

    Science.gov (United States)

    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.

  12. Regulation of apoptosis in human melanoma and neuroblastoma cells by statins, sodium arsenite and TRAIL: a role of combined treatment versus monotherapy

    Science.gov (United States)

    Ivanov, Vladimir N.; Hei, Tom K.

    2015-01-01

    Treatment of melanoma cells by sodium arsenite or statins (simvastatin and lovastatin) dramatically modified activities of the main cell signaling pathways resulting in the induction of heme oxygenase-1 (HO-1) and in a downregulation of cyclooxygenase-2 (COX-2) protein levels. Through heme degradation and the production of carbon monoxide and biliverdin, HO-1 plays a protective role in different scenario of oxidative stress followed by mitochondrial apoptosis. Both sodium arsenite and statins could be efficient inducers of apoptosis in some melanoma cell lines, but often exhibited only modest proapoptotic activity in others, due to numerous protective mechanisms. We demonstrated in the present study that treatment by sodium arsenite or statins with an additional inhibition of HO-1 expression (or activation) caused a substantial upregulation of apoptosis in melanoma cells. Sodium arsenite- or statin-induced apoptosis was independent of BRAF status (wild type versus V600E) in melanoma lines. Monotreatment required high doses of statins (20–40 μM) for effective induction of apoptosis. As an alternative approach, pretreatment of melanoma cells with statin at decreased doses (5–20 μM) dramatically enhanced TRAIL-induced apoptosis, due to suppression of the NF-κB and STAT3-transcriptional targets (including COX-2) and downregulation of cFLIP-L (a caspase-8 inhibitor) protein levels. Furthermore, combined treatment with sodium arsenite and TRAIL or simvastatin and TRAIL efficiently induced apoptotic commitment in human neuroblastoma cells. In summary, our findings on enhancing effects of combined treatment of cancer cells using statin and TRAIL provide the rationale for further preclinical evaluation. PMID:21910007

  13. Repression of microRNA-768-3p by MEK/ERK signalling contributes to enhanced mRNA translation in human melanoma.

    Science.gov (United States)

    Jiang, C C; Croft, A; Tseng, H-Y; Guo, S T; Jin, L; Hersey, P; Zhang, X D

    2014-05-15

    Increased global protein synthesis and selective translation of mRNAs encoding proteins contributing to malignancy is common in cancer cells. This is often associated with elevated expression of eukaryotic translation initiation factor 4 (eIF4E), the rate-limiting factor of cap-dependent translation initiation. We report here that in human melanoma downregulation of miR-768-3p as a result of activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway has an important role in the upregulation of eIF4E and enhancement in protein synthesis. Melanoma cells displayed increased nascent protein production and elevated eIF4E expression, which was associated with the downregulation of miR-768-3p that was predicted to target the 3'-untranslated region of the eIF4E mRNA. Overexpression of miR-768-3p led to the downregulation of the endogenous eIF4E protein, reduction in nascent protein synthesis and inhibition of cell survival and proliferation. These effects were efficiently reversed when eIF4E was co-overexpressed in melanoma cells. On the other hand, introduction of anti-miR-768-3p into melanocytes upregulated endogenous eIF4E protein expression and increased global protein synthesis. Downregulation of miR-768-3p appeared to be mediated by activation of the MEK/ERK pathway, in that treatment of BRAF(V600E) melanoma cells with the mutant BRAF inhibitor PLX4720 or exposure of either BRAF(V600E) or wild-type BRAF melanoma cells to the MEK inhibitor U0126 resulted in the upregulation of miR-768-3p and inhibition of nascent protein synthesis. This inhibition was partially blocked in cells cointroduced with anti-miR-768-3p. Significantly, miR-768-3p was similarly downregulated, which was inversely associated with the expression levels of eIF4E in fresh melanoma isolates. Taken together, these results identify downregulation of miR-768-3p and subsequent upregulation of eIF4E as an important mechanism in addition to

  14. Characterization of CD8+ T-Cell Responses in the Peripheral Blood and Skin Injection Sites of Melanoma Patients Treated with mRNA Electroporated Autologous Dendritic Cells (TriMixDC-MEL

    Directory of Open Access Journals (Sweden)

    Daphné Benteyn

    2013-01-01

    Full Text Available Treatment of melanoma patients with mRNA electroporated dendritic cells (TriMixDC-MEL stimulates T-cell responses against the presented tumor-associated antigens (TAAs. In the current clinical trials, melanoma patients with systemic metastases are treated, requiring priming and/or expansion of preexisting TAA-specific T cells that are able to migrate to both the skin and internal organs. We monitored the presence of TAA-specific CD8+ T cells infiltrating the skin at sites of intradermal TriMixDC-MEL injection (SKILs and within the circulation of melanoma patients treated in two clinical trials. In 10 out of fourteen (71% patients screened, CD8+ T cells recognizing any of the four TAA presented by TriMixDC-MEL cellular vaccine were found in both compartments. In total, 30 TAA-specific T-cell responses were detected among the SKILs and 29 among peripheral blood T cells, of which 24 in common. A detailed characterization of the antigen specificity of CD8+ T-cell populations in four patients indicates that the majority of the epitopes detected were only recognized by CD8+ T cells derived from either skin biopsies or peripheral blood, indicating that some compartmentalization occurs after TriMix-DC therapy. To conclude, functional TAA-specific CD8+ T cells distribute both to the skin and peripheral blood of patients after TriMixDC-MEL therapy.

  15. Evaluation of the effect of horse blood supplemented with human ...

    African Journals Online (AJOL)

    The study was conducted to evaluate the effect of horse blood supplemented with ..... proportion of lighter pupae (<22 mg) produced by the flies fed human blood .... Vreysen, M.J., Saleh, K.M., Ali, M.Y., Abdulla, A.M., Zhu, Z.R., Juma, K.G., ...

  16. Tracking blood vessels in human forearms using visual servoing

    DEFF Research Database (Denmark)

    Savarimuthu, Thiusius Rajeeth; Ellekilde, Lars-Peter; Hansen, Morten

    compensation. By using images taken with near-infrared light to locate the blood vessels in a human forearm and using the same images to detects movements of the arm, this paper shows that it is possible make a robot arm, potentially equipped with a needle for drawing the blood, compensate for the movements...

  17. [How human sciences can help to understand blood donation?].

    Science.gov (United States)

    Danic, B

    2003-06-01

    In France, the necessity of managing sanitary risks associated with blood transfusion has induced a strong medicalization of blood collection. Practicing this activity of transfusion medicine confronts the professional with problems which the medical science is not sufficient to answer: curbs and motivations for donating, seasonal and geographic fluctuations in blood collection, sense of pre-donation interview, individual or collective contestings of guidelines for blood collection. From recent sociological works on donation in France, especially on blood donation, this paper suggests reporting the complexity of blood donation thought process and the necessity to refer to human and social sciences to approach it in a best way. Understanding the gift should help professionals to communicate not only on the promotion of blood donation, but also on the risk and its management.

  18. PD-1 Blockade Augments Th1 and Th17 and Suppresses Th2 Responses in Peripheral Blood From Patients With Prostate and Advanced Melanoma Cancer

    NARCIS (Netherlands)

    Dulos, John; Carven, Gregory J.; van Boxtel, Susan J.; Evers, Sabine; Driessen-Engels, Lilian J. A.; Hobo, Willemijn; Gorecka, Monika A.; de Haan, Anton F. J.; Mulders, Peter; Punt, Cornelis J. A.; Jacobs, Joannes F. M.; Schalken, Jack A.; Oosterwijk, Egbert; van Eenennaam, Hans; Boots, Annemieke M.

    2012-01-01

    Negative costimulation on T cells is exploited by both prostate cancer and melanoma to evade antitumor immunity. Blocking such mechanisms restores antitumor immunity as was demonstrated by the improved survival of patients with metastatic melanoma after treatment with an antibody blocking the CTLA-4

  19. PD-1 blockade augments Th1 and Th17 and suppresses Th2 responses in peripheral blood from patients with prostate and advanced melanoma cancer.

    NARCIS (Netherlands)

    Dulos, J.; Carven, G.J.; Boxtel, S.J. van; Evers, S.; Driessen-Engels, L.J.; Hobo, W.A.; Gorecka, M.A.; Haan, A.F.J. de; Mulders, P.; Punt, C.J.A.; Jacobs, J.F.M.; Schalken, J.A.; Oosterwijk, E.; Eenennaam, H. van; Boots, A.M.H.

    2012-01-01

    Negative costimulation on T cells is exploited by both prostate cancer and melanoma to evade antitumor immunity. Blocking such mechanisms restores antitumor immunity as was demonstrated by the improved survival of patients with metastatic melanoma after treatment with an antibody blocking the CTLA-4

  20. Inhibition of uptake of adenosine into human blood platelets

    NARCIS (Netherlands)

    Lips, J.P.M.; Sixma, J.J.; Trieschnigg, A.C.

    1980-01-01

    Adenosine transport into human blood platelets is mediated by two independent systems with different affinities. Both systems transport only purine nucleosides and no pyrimidine nucleosides. In experiments with differently substituted purine nucleosides, purines and analogues, differences in carrier

  1. Bystander apoptosis in human cells mediated by irradiated blood plasma

    Energy Technology Data Exchange (ETDEWEB)

    Vinnikov, Volodymyr, E-mail: vlad.vinnikov@mail.ru [Grigoriev Institute for Medical Radiology of the National Academy of Medical Science of Ukraine (Ukraine); Lloyd, David; Finnon, Paul [Centre for Radiation, Chemical and Environmental Hazards of the Health Protection Agency of the United Kingdom (United Kingdom)

    2012-03-01

    Following exposure to high doses of ionizing radiation, due to an accident or during radiotherapy, bystander signalling poses a potential hazard to unirradiated cells and tissues. This process can be mediated by factors circulating in blood plasma. Thus, we assessed the ability of plasma taken from in vitro irradiated human blood to produce a direct cytotoxic effect, by inducing apoptosis in primary human peripheral blood mononuclear cells (PBM), which mainly comprised G{sub 0}-stage lymphocytes. Plasma was collected from healthy donors' blood irradiated in vitro to 0-40 Gy acute {gamma}-rays. Reporter PBM were separated from unirradiated blood with Histopaque and held in medium with the test plasma for 24 h at 37 Degree-Sign C. Additionally, plasma from in vitro irradiated and unirradiated blood was tested against PBM collected from blood given 4 Gy. Apoptosis in reporter PBM was measured by the Annexin V test using flow cytometry. Plasma collected from unirradiated and irradiated blood did not produce any apoptotic response above the control level in unirradiated reporter PBM. Surprisingly, plasma from irradiated blood caused a dose-dependent reduction of apoptosis in irradiated reporter PBM. The yields of radiation-induced cell death in irradiated reporter PBM (after subtracting the respective values in unirradiated reporter PBM) were 22.2 {+-} 1.8% in plasma-free cultures, 21.6 {+-} 1.1% in cultures treated with plasma from unirradiated blood, 20.2 {+-} 1.4% in cultures with plasma from blood given 2-4 Gy and 16.7 {+-} 3.2% in cultures with plasma from blood given 6-10 Gy. These results suggested that irradiated blood plasma did not cause a radiation-induced bystander cell-killing effect. Instead, a reduction of apoptosis in irradiated reporter cells cultured with irradiated blood plasma has implications concerning oncogenic risk from mutated cells surviving after high dose in vivo irradiation (e.g. radiotherapy) and requires further study.

  2. What Is Melanoma Skin Cancer?

    Science.gov (United States)

    ... Z About Melanoma Skin Cancer What Is Melanoma Skin Cancer? Key Statistics for Melanoma Skin Cancer What’s New in Melanoma ... Policy . About Melanoma Skin Cancer What Is Melanoma Skin Cancer? Key Statistics for Melanoma Skin Cancer What’s New in Melanoma ...

  3. Cutavirus in Cutaneous Malignant Melanoma

    DEFF Research Database (Denmark)

    Mollerup, Sarah; Fridholm, Helena; Vinner, Lasse

    2017-01-01

    A novel human protoparvovirus related to human bufavirus and preliminarily named cutavirus has been discovered. We detected cutavirus in a sample of cutaneous malignant melanoma by using viral enrichment and high-throughput sequencing. The role of cutaviruses in cutaneous cancers remains to be in......A novel human protoparvovirus related to human bufavirus and preliminarily named cutavirus has been discovered. We detected cutavirus in a sample of cutaneous malignant melanoma by using viral enrichment and high-throughput sequencing. The role of cutaviruses in cutaneous cancers remains...

  4. Cutavirus in Cutaneous Malignant Melanoma

    DEFF Research Database (Denmark)

    Mollerup, Sarah; Fridholm, Helena; Vinner, Lasse

    2017-01-01

    A novel human protoparvovirus related to human bufavirus and preliminarily named cutavirus has been discovered. We detected cutavirus in a sample of cutaneous malignant melanoma by using viral enrichment and high-throughput sequencing. The role of cutaviruses in cutaneous cancers remains to be in......A novel human protoparvovirus related to human bufavirus and preliminarily named cutavirus has been discovered. We detected cutavirus in a sample of cutaneous malignant melanoma by using viral enrichment and high-throughput sequencing. The role of cutaviruses in cutaneous cancers remains...

  5. Ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma

    Science.gov (United States)

    Bald, Tobias; Quast, Thomas; Landsberg, Jennifer; Rogava, Meri; Glodde, Nicole; Lopez-Ramos, Dorys; Kohlmeyer, Judith; Riesenberg, Stefanie; van den Boorn-Konijnenberg, Debby; Hömig-Hölzel, Cornelia; Reuten, Raphael; Schadow, Benjamin; Weighardt, Heike; Wenzel, Daniela; Helfrich, Iris; Schadendorf, Dirk; Bloch, Wilhelm; Bianchi, Marco E.; Lugassy, Claire; Barnhill, Raymond L.; Koch, Manuel; Fleischmann, Bernd K.; Förster, Irmgard; Kastenmüller, Wolfgang; Kolanus, Waldemar; Hölzel, Michael; Gaffal, Evelyn; Tüting, Thomas

    2014-03-01

    Intermittent intense ultraviolet (UV) exposure represents an important aetiological factor in the development of malignant melanoma. The ability of UV radiation to cause tumour-initiating DNA mutations in melanocytes is now firmly established, but how the microenvironmental effects of UV radiation influence melanoma pathogenesis is not fully understood. Here we report that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model promotes metastatic progression, independent of its tumour-initiating effects. UV irradiation enhanced the expansion of tumour cells along abluminal blood vessel surfaces and increased the number of lung metastases. This effect depended on the recruitment and activation of neutrophils, initiated by the release of high mobility group box 1 (HMGB1) from UV-damaged epidermal keratinocytes and driven by Toll-like receptor 4 (TLR4). The UV-induced neutrophilic inflammatory response stimulated angiogenesis and promoted the ability of melanoma cells to migrate towards endothelial cells and use selective motility cues on their surfaces. Our results not only reveal how UV irradiation of epidermal keratinocytes is sensed by the innate immune system, but also show that the resulting inflammatory response catalyses reciprocal melanoma-endothelial cell interactions leading to perivascular invasion, a phenomenon originally described as angiotropism in human melanomas by histopathologists. Angiotropism represents a hitherto underappreciated mechanism of metastasis that also increases the likelihood of intravasation and haematogenous dissemination. Consistent with our findings, ulcerated primary human melanomas with abundant neutrophils and reactive angiogenesis frequently show angiotropism and a high risk for metastases. Our work indicates that targeting the inflammation-induced phenotypic plasticity of melanoma cells and their association with endothelial cells represent rational strategies to specifically interfere

  6. The full blood count as a biomarker of outcome and toxicity in ipilimumab-treated cutaneous metastatic melanoma.

    Science.gov (United States)

    Khoja, Leila; Atenafu, Eshetu G; Templeton, Arnoud; Qye, Ye; Chappell, Mary Anne; Saibil, Sam; Hogg, David; Butler, Marcus O; Joshua, Anthony M

    2016-10-01

    Ipilimumab produces durable responses in some metastatic melanoma patients. Neutrophil, platelet, and eosinophil to lymphocyte ratios (NLR, PLR, and ELR) may be associated with the immune response in cancer thereby acting as biomarkers of toxicity and efficacy in ipilimumab-treated patients. Data were collected on clinical characteristics and lactate dehydrogenase (LDH), NLR, PLR, and ELR at baseline, post cycle 2 and at the end of treatment for 183 patients treated with ipilimumab between 2008 and 2015 at the Princess Margaret Cancer Centre. Associations between clinical characteristics, LDH, NLR, PLR, and ELR with toxicity or survival outcomes of progression-free (PFS) and overall survival (OS) were assessed using univariable and multivariable analysis. Prognostic models of outcome at each time point were determined. Of the 183 patients included, the median age was 58, 85% had M1c disease, 58% were performance status 1, and 64% received ipilimumab as second line therapy. Median follow up was 7.5 months (range: 0.3-49.5), median PFS was 2.8 months (95% confidence intervals (CI): 2.8-3.2), and median OS was 9.6 months (95% CI: 7.9-13.2). Prognostic factors for OS by multivariable analysis were LDH and NLR at all-time points. Prognostic models using LDH (× 2 upper limit of normal) and NLR 4) differentiated patients into high, moderate, and low risk of death prior to or on ipilimumab treatment (P < 0.0001 for each model). No factors were associated with toxicity. Prognostic models based on NLR and LDH values at baseline and on treatment differentiate patients into good, intermediate, and poor prognostic groups and may be relevant in patient management.

  7. [Vulvar melanoma].

    Science.gov (United States)

    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 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.

  8. Melanoma immunotherapy.

    Science.gov (United States)

    Sivendran, Shanthi; Glodny, Bradley; Pan, Michael; Merad, Miriam; Saenger, Yvonne

    2010-01-01

    Melanoma immunotherapy has been an area of intense research for decades, and this work is now yielding more tangible results for patients. Work has focused on 4 main areas: cytokine therapy, administration of immune-modulating antibodies, adoptive T-cell therapy, and vaccines. Cytokine therapy is an established treatment for advanced melanoma, and immune-modulating antibodies have recently emerged as an exciting new area of drug development with efficacy now established in a phase III trial. Adoptive T-cell therapy provides the proof of principle that T cells can attack and eliminate tumors. It has been challenging, however, to adapt this treatment for widespread use. Vaccines have generally yielded poor results, but intratumor pathogen-based strategies have shown encouraging results in recent trials, perhaps due to stronger immune stimulation. A review of the field of melanoma immunotherapy is provided here, with emphasis on those agents that have reached clinical testing. Novel strategies to induce the immune system to attack melanomas are reviewed. In the future, it is envisioned that immunotherapy will have further application in combination with cytotoxic and targeted therapies.

  9. Skin blood perfusion and oxygenation colour affect perceived human health.

    Directory of Open Access Journals (Sweden)

    Ian D Stephen

    Full Text Available Skin blood perfusion and oxygenation depends upon cardiovascular, hormonal and circulatory health in humans and provides socio-sexual signals of underlying physiology, dominance and reproductive status in some primates. We allowed participants to manipulate colour calibrated facial photographs along empirically-measured oxygenated and deoxygenated blood colour axes both separately and simultaneously, to optimise healthy appearance. Participants increased skin blood colour, particularly oxygenated, above basal levels to optimise healthy appearance. We show, therefore, that skin blood perfusion and oxygenation influence perceived health in a way that may be important to mate choice.

  10. Good manufacturing practices (GMP) utilized on human blood irradiation process

    OpenAIRE

    Cláudio Boghi; Dorlivete M. Shitsuka; Marcos Alexandruk; Ricardo Shitsuka; Paulo Roberto Rela

    2008-01-01

    Irradiation of human blood is used to avoid the TA-GVHD (transfusion-associated graft-versus-host-disease), a rare but devastating adverse effect of leukocytes present in blood components for immunocompetent transfusion recipients. Usually this irradiation practice is performed to a physical elimination of lymphocytes. The implementation of the GMP will assure that the properly dose in a range of 25Gy to 50Gy will be delivered to the blood in the bag collected in a blood tissue bank. The stud...

  11. Generation of induced pluripotent stem cells from human blood.

    Science.gov (United States)

    Loh, Yuin-Han; Agarwal, Suneet; Park, In-Hyun; Urbach, Achia; Huo, Hongguang; Heffner, Garrett C; Kim, Kitai; Miller, Justine D; Ng, Kitwa; Daley, George Q

    2009-05-28

    Human dermal fibroblasts obtained by skin biopsy can be reprogrammed directly to pluripotency by the ectopic expression of defined transcription factors. Here, we describe the derivation of induced pluripotent stem cells from CD34+ mobilized human peripheral blood cells using retroviral transduction of OCT4/SOX2/KLF4/MYC. Blood-derived human induced pluripotent stem cells are indistinguishable from human embryonic stem cells with respect to morphology, expression of surface antigens, and pluripotency-associated transcription factors, DNA methylation status at pluripotent cell-specific genes, and the capacity to differentiate in vitro and in teratomas. The ability to reprogram cells from human blood will allow the generation of patient-specific stem cells for diseases in which the disease-causing somatic mutations are restricted to cells of the hematopoietic lineage.

  12. What Does Melanoma Look Like?

    Science.gov (United States)

    ... Skin Cancer Skin Cancer Screening Research What Does Melanoma Look Like? Melanoma is a type of cancer ... melanoma is itchy, tender, or painful. Photos of Melanoma A large, asymmetrical melanoma with an uneven color ...

  13. Distribution and survival of Borrelia miyamotoi in human blood components.

    Science.gov (United States)

    Thorp, Aaron M; Tonnetti, Laura

    2016-03-01

    Borrelia miyamotoi, the agent of relapsing fever, is a tick-borne spirochete first isolated in Japan in 1994. Since then, the spirochete has been detected in ticks globally, generally in the same vectors as the Lyme disease agent. Human infection has been reported in Russia, Europe, Japan, and the United States, as influenza-like febrile illness. In addition, two cases of meningoencephalitis caused by B. miyamotoi have also been reported in immunocompromised patients. Here we evaluate the ability of the spirochete to survive in human blood components stored under standard blood bank conditions. Freshly collected human whole blood was spiked with in vitro cultured B. miyamotoi or B. miyamotoi-infected mouse plasma and separated into red blood cells (RBCs), plasma, and platelets. Components were either injected into immunocompromised (SCID) or wild-type immunocompetent mice or cultured in vitro, right after separation and after storage at the appropriate conditions. Infection was monitored by microscopic observation, blood smears, and polymerase chain reaction. In vivo, all the SCID mice challenged with the components before storage and the RBCs stored for up to 42 days developed the infection. Wild-type mice also developed the infection when injected with prestorage samples from all components, while a lower number of mice were infected by RBCs stored for 42 days. In vitro, spirochetes grew in all samples but frozen plasma. This study demonstrated that B. miyamotoi can survive standard storage conditions of most human blood components, suggesting the possibility of transmission by blood transfusion. © 2015 AABB.

  14. Radiopharmaceuticals targeting melanoma

    Energy Technology Data Exchange (ETDEWEB)

    Pham, T.Q.; Berghofer, P.; Liu, X.; Greguric, I.; Dikic, B.; Ballantyne, P.; Mattner, F.; Nguyen, V.; Loc' h, C.; Katsifis, A. [Radiopharmaceuticals Research Institute, Australian Nuclear Science and Technology Organisation, Menai, N.S.W., Sydney (Australia)

    2008-02-15

    Melanoma is one of the most aggressive cancers known with a high rate of mortality and increasing global incidence. So, the development of radiopharmaceuticals for either diagnostic or therapeutic purposes could make enormous contributions to melanoma patient health care. We have been studying melanoma tumours through several targeting mechanisms including melanin or specific receptor based radiopharmaceuticals Structure activity studies indicate that the substitution patterns on radioiodinated benzamides significantly influence the uptake mechanism from melanin to sigma-receptor binding. Furthermore, the position of the iodine as well as the presence of key functional groups and substituents has resulted in compounds with varying degrees of activity uptake and retention in tumours. From these results, a novel molecule 2-(2-(4-(4-iodo benzyl)piperazin-1-yl)-2-oxo-ethyl)isoindoline- 1,3-dione (M.E.L.037) was synthesized, labelled with iodine-123 and evaluated for application in melanoma tumour scintigraphy and radiotherapy. The tumour imaging potential of {sup 123}IM.E.L.037 was studied in vivo in C.57 B.L./ 6 J female mice bearing the B.16 F.0. murine melanoma tumour and in BALB/c nude mice bearing the A.375 human amelanotic melanoma tumour by biodistribution, competition studies and by SPECT imaging. {sup 123}I-M.E.L.037 exhibited high and rapid uptake in the B.16 F.0 melanoma tumour at 1 h (13 % I.D./g) increasing with time to reach 25 % I.D./g at 6 h. A significant uptake was also observed in the eyes (2% I.D., at 3-6 h p.i.) of black mice. No uptake was observed in the tumour or in the eyes of nude mice bearing the A.375 tumour. Due to high uptake and long retention in the tumour and rapid body clearance, standardized uptake values(S.U.V.) of {sup 123}I-M.E.L.037 were 30 and 60, at 24 and 48 h p.i.,respectively. SPECT imaging of mice bearing the B.16 melanoma indicated the radioactivity was predominately located in the tumour followed by the eyes, while no

  15. Developmental validation of a novel lateral flow strip test for rapid identification of human blood (Rapid Stain Identification--Blood).

    Science.gov (United States)

    Schweers, Brett A; Old, Jennifer; Boonlayangoor, P W; Reich, Karl A

    2008-06-01

    Human blood is the body fluid most commonly encountered at crime scenes, and blood detection may aid investigators in reconstructing what occurred during a crime. In addition, blood detection can help determine which items of evidence should be processed for DNA-STR testing. Unfortunately, many common substances can cause red-brown stains that resemble blood. Furthermore, many current human blood detection methods are presumptive and prone to false positive results. Here, the developmental validation of a new blood identification test, Rapid Stain Identification--Blood (RSID--Blood), is described. RSID--Blood utilizes two anti-glycophorin A (red blood cell membrane specific protein) monoclonal antibodies in a lateral flow strip test format to detect human blood. We present evidence demonstrating that this test is accurate, reproducible, easy to use, and highly specific for human blood. Importantly, RSID--Blood does not cross-react with ferret, skunk, or primate blood and exhibits no high-dose hook effect. Also, we describe studies on the sensitivity, body fluid specificity, and species specificity of RSID--Blood. In addition, we show that the test can detect blood from a variety of forensic exhibits prior to processing for DNA-STR analysis. In conclusion, we suggest that RSID--Blood is effective and useful for the detection of human blood on forensic exhibits, and offers improved blood detection when compared to other currently used methods.

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

    Science.gov (United States)

    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.

  17. Immunomodulatory Effectiveness of Fish Oil and omega-3 Fatty Acids in Human Non-melanoma Skin Carcinoma Cells.

    Science.gov (United States)

    Rehman, Khurram; Mohd Amin, Mohd Cairul Iqbal; Yuen, Ng Pei; Zulfakar, Mohd Hanif

    2016-01-01

    Fish oil is composed of various fatty acids among which omega-3 fatty acids are considered as most beneficial. The effects of fish oil on the activity of a topical anticancer drug, imiquimod, and the immunomodulatory activity of omega-3 fatty acids was investigated in human basal and squamous cell carcinoma cell lines. Imiquimod-fish oil mixture exhibited higher carcinoma cell growth inhibition and immunomodulatory activity than imiquimod alone, especially against squamous cell carcinoma cells. Omega-3 fatty acids exhibited growth inhibition of both basal cell and squamous cell carcinoma cell lines and modulated the immune response. Omega-3 fatty acids of fish oil serve as inducers of interleukin-10, an anti-inflammatory cytokine, and as suppressors of interleukin-6 and tumor necrosis factor-alpha, which not only depress tumor growth but also adequately control the inflammatory side effects of imiquimod. Thus, imiquimod administration with fish oil could be beneficial for inhibition of non-melanoma skin carcinoma cells but further in vivo studies are needed to understand their role in skin cancer.

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

    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. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Achievements and challenges of adoptive T cell therapy with tumor-infiltrating or blood-derived lymphocytes for metastatic melanoma

    DEFF Research Database (Denmark)

    Svane, Inge Marie; Verdegaal, Els M

    2014-01-01

    Adoptive cell therapy (ACT) based on autologous T cell derived either from tumor as tumor-infiltrating lymphocytes (TILs) or from peripheral blood is developing as a key area of future personalized cancer therapy. TIL-based ACT is defined as the infusion of T cells harvested from autologous fresh...... review....

  1. Direct determination of radiation dose in human blood

    CERN Document Server

    Tanir, Ayse Gunes; Sahiner, Eren; Bolukdemir, Mustafa Hicabi; Koc, Kemal; Meric, Niyazi; Kelec, Sule Kaya

    2014-01-01

    Our purpose is to measure the internal radiation dose (ID) using human blood sample. In the literature, there is no process that allows the direct measurement of ID received by a person. This study has shown that it is possible to determine ID in human blood exposed to internal or external ionizing radiation treatment both directly and retrospectively. OSL technique was used to measure the total dose from the blood sample. OSL counts from the waste blood of the patient injected with a radiopharmaceutical for diagnostic or treatment purposes and from a blood sample having a laboratory-injected radiation dose were both used for measurements. The decay and dose-response curves (DRC) were plotted for different doses. The doses received by different blood aliquots have been determined by interpolating the natural luminescence counts to DRC. In addition, OSL counts from a healthy blood sample exposed to an external radiation source were measured. The blood aliquots were given different 0-200Gy beta doses and their ...

  2. Mesenteric, coeliac and splanchnic blood flow in humans during exercise

    DEFF Research Database (Denmark)

    Perko, M J; Nielsen, H B; Skak, C

    1998-01-01

    blood flow as assessed by the Indocyanine Green dye-elimination technique. 3. Cycling increased arterial pressure, heart rate and cardiac output, while it reduced total vascular resistance. These responses were not altered in the postprandial state. During fasting, cycling increased mesenteric, coeliac......1. Exercise reduces splanchnic blood flow, but the mesenteric contribution to this response is uncertain. 2. In nineteen humans, superior mesenteric and coeliac artery flows were determined by duplex ultrasonography during fasting and postprandial submaximal cycling and compared with the splanchnic...... decreased by 51 and 31 % (0.49 +/- 0.07 and 0.96 +/- 0.28 l min-1). Splanchnic blood flow values assessed by duplex ultrasound and by dye-elimination techniques were correlated (r = 0.70; P exercise in humans, splanchnic resistance increases and blood flow is reduced following...

  3. Characterization of thermoplastic microfiltration chip for the separation of blood plasma from human blood.

    Science.gov (United States)

    Chen, Pin-Chuan; Chen, Chih-Chun; Young, Kung-Chia

    2016-09-01

    In this study, we developed a fully thermoplastic microfiltration chip for the separation of blood plasma from human blood. Spiral microchannels were manufactured on a PMMA substrate using a micromilling machine, and a commercial polycarbonate membrane was bonded between two thermoplastic substrates. To achieve an excellent bonding between the commercial membrane and the thermoplastic substrates, we used a two-step injection and curing procedure of UV adhesive into a ring-shaped structure around the microchannel to efficiently prevent leakage during blood filtration. We performed multiple filtration experiments using human blood to compare the influence of three factors on separation efficiency: hematocrit level (40%, 23.2%, and 10.9%), membrane pore size (5 μm, 2 μm, and 1 μm), and flow rate (0.02 ml/min, 0.06 ml/min, 0.1 ml/min). To prevent hemolysis, the pressure within the microchannel was kept below 0.5 bars throughout all filtration experiments. The experimental results clearly demonstrated the following: (1) The proposed microfiltration chip is able to separate white blood cells and red blood cells from whole human blood with a separation efficiency that exceeds 95%; (2) no leakage occurred during any of the experiments, thereby demonstrating the effectiveness of bonding a commercial membrane with a thermoplastic substrate using UV adhesive in a ring-shaped structure; (3) separation efficiency can be increased by using a membrane with smaller pore size, by using diluted blood with lower hematocrit, or by injecting blood into the microfiltration chip at a lower flow rate.

  4. Ultraviolet B radiation-stimulated urocortin 1 is involved in tyrosinase-related protein 1 production in human melanoma HMV-II cells.

    Science.gov (United States)

    Watanuki, Yutaka; Kageyama, Kazunori; Takayasu, Shinobu; Matsuzaki, Yasushi; Iwasaki, Yasumasa; Daimon, Makoto

    2014-11-01

    Ultraviolet B (UVB) radiation stimulates cutaneous melanin pigmentation. The melanosomal enzyme tyrosinase-related protein 1 (TRP1) is involved in the modulation of pigment production in response to this stressor. Recent molecular and biochemical analyses have revealed the presence of corticotropin-releasing factor (CRF) and urocortin 1 (Ucn1), together with their corresponding receptors, in mammalian skin. Although CRF and Ucn1 are thought to have potent effects on the skin system, their possible roles and regulations have yet to be determined fully. Our previous findings in human melanoma HMV-II cells suggest that both CRF and Ucn1 regulate TRP1 gene expression via Nurr-1/Nur77, transcription factors that constitute the nuclear receptor 4a subgroup of orphan nuclear receptors. HMV-II cells were found to express mainly Ucn1 mRNA. This study aimed to explore the effects of UVB on Ucn1 mRNA and TRP1 protein levels in HMV-II cells. UVB (30 mJ/cm(2)) increased Nurr-1, Nur77, and Ucn1 mRNA levels. UVB also increased TRP1 protein levels. Ucn1 knockdown inhibited the UVB-induced increases in TRP1 protein levels. These data suggest that UVB-stimulated Ucn1 contributes to TRP1 production via the transcription of both Nurr-1 and Nur77. Ucn1, produced in melanoma cells, acts on melanoma cells themselves in an autocrine manner.

  5. Rheology of human blood plasma: Viscoelastic versus Newtonian behavior

    CERN Document Server

    Brust, M; Pan, L; Garcia, M; Arratia, P E; Wagner, C; 10.1103/PhysRevLett.110.078305

    2013-01-01

    We investigate the rheological characteristics of human blood plasma in shear and elongational flows. While we can confirm a Newtonian behavior in shear flow within experimental resolution, we find a viscoelastic behavior of blood plasma in the pure extensional flow of a capillary break-up rheometer. The influence of the viscoelasticity of blood plasma on capillary blood flow is tested in a microfluidic device with a contraction-expansion geometry. Differential pressure measurements revealed that the plasma has a pronounced flow resistance compared to that of pure water. Supplementary measurements indicate that the viscoelasticity of the plasma might even lead to viscoelastic instabilities under certain conditions. Our findings show that the viscoelastic properties of plasma should not be ignored in future studies on blood flow.

  6. Direct determination of internal radiation dose in human blood

    CERN Document Server

    Tanır, Ayse Güneş

    2014-01-01

    The purpose of this study is to measure the internal radiation dose using a human blood sample. In the literature, there is no process that allows the direct measurement of the internal radiation dose received by a person. The luminescence counts from a blood sample having a laboratory-injected radiation dose and the waste blood of the patient injected with a radiopharmaceutical for diagnostic purposes were both measured. The decay and dose-response curves were plotted for the different doses. The doses received by the different blood aliquots can be determined by interpolating the luminescence counts to the dose-response curve. This study shows that the dose received by a person can be measured directly, simply and retrospectively by using only a very small amount of blood sample. The results will have important ramifications for the medicine and healthcare fields in particular. This will also be very important in cases of suspicion of radiation poisoning, malpractice and so on.

  7. The use of gamma-irradiation and ultraviolet-irradiation in the preparation of human melanoma cells for use in autologous whole-cell vaccines

    Directory of Open Access Journals (Sweden)

    Denlinger Chadrick E

    2008-12-01

    Full Text Available Abstract Background Human cancer vaccines incorporating autologous tumor cells carry a risk of implantation and subsequent metastasis of viable tumor cells into the patient who is being treated. Despite the fact that the melanoma cell preparations used in a recent vaccine trial (Mel37 were gamma-irradiated (200 Gy, approximately 25% of the preparations failed quality control release criteria which required that the irradiated cells incorporate 3H-thymidine at no more than 5% the level seen in the non-irradiated cells. We have, therefore, investigated ultraviolet (UV-irradiation as a possible adjunct to, or replacement for gamma-irradiation. Methods Melanoma cells were gamma- and/or UV-irradiated. 3H-thymidine uptake was used to assess proliferation of the treated and untreated cells. Caspase-3 activity and DNA fragmentation were measured as indicators of apoptosis. Immunohistochemistry and Western blot analysis was used to assess antigen expression. Results UV-irradiation, either alone or in combination with gamma-irradiation, proved to be extremely effective in controlling the proliferation of melanoma cells. In contrast to gamma-irradiation, UV-irradiation was also capable of inducing significant levels of apoptosis. UV-irradiation, but not gamma-irradiation, was associated with the loss of tyrosinase expression. Neither form of radiation affected the expression of gp100, MART-1/MelanA, or S100. Conclusion These results indicate that UV-irradiation may increase the safety of autologous melanoma vaccines, although it may do so at the expense of altering the antigenic profile of the irradiated tumor cells.

  8. Effect of Electrohydraulic Discharge on Viscosity of Human Blood

    Directory of Open Access Journals (Sweden)

    G. M. El-Aragi

    2013-01-01

    Full Text Available Electrohydraulic plasma discharge is a novel technology with high efficiency and high speed and can generate chemically active species like free radicals, ions, atoms, and metastables, accompanied by ultraviolet light emission and shock pressure waves. The aim of this work is to examine the effect of electrohydraulic discharge (EHD system on viscosity of the human blood after different exposure time. The voltage pulsation introduces electric field and temperature jump and at the same time leads to haemolysis of the blood cells. The ratio of blood viscosity under the influence of magnetic field to the viscosity in the absence of magnetic field is directly proportional to the applied magnetic field .

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

    Science.gov (United States)

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

    2017-05-01

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

  10. Morphological alterations and G0/G1 cell cycle arrest induced by curcumin in human SK-MEL-37 melanoma cells

    Directory of Open Access Journals (Sweden)

    Marcella Lemos Brettas Carneiro

    2010-04-01

    Full Text Available The aim of this work was to study the effect of curcumin on cell cycle in the human SK-MEL-37 melanoma cell line. In addition, morphological and structural analyses were also performed. Flow cytometric analysis showed a G0/G1 arrest at 5 µM after 24 h exposure and a concentration-dependent increase in the proportion of sub-G0 hypodiploid cells. Typical apoptotic events were also observed by the fluorescence microscopy, transmission and scanning electronic microscopy. Loss of mitochondrial membrane potential was not detected. Results suggested that curcumin could arrest human melanoma cells at G0/G1 phase and induce a mitochondrial-independent apoptotic pathway.O melanoma é um tipo agressivo de câncer cujo tratamento culmina com o estabelecimento de resistência aos quimioterápicos empregados. Portanto, é importante o desenvolvimento de novos agentes farmacológicos que sejam menos tóxicos e que não provoquem quimiorresistência. As inúmeras propriedades terapêuticas da curcumina vêm sendo confirmadas através de estudos sobre o seu mecanismo de ação em células cultivadas. No presente estudo, empregamos células de melanoma humano da linhagem SK-MEL-37, que desenvolveram resistência in vitro à doxorubicina e cisplatina, drogas normalmente utilizadas na clínica. Investigamos o efeito da curcumina sobre o ciclo celular através de citometria de fluxo. Além disso, análises morfológicas e estruturais também foram realizadas. Os resultados demonstraram que o tratamento com uma concentração de 5 ?M de curcumina provocou uma parada na subfase G0/G1. Além disso, observou-se um aumento dose-dependente na proporção de células hipodiplóides em sub-G0. Eventos apoptóticos típicos foram observados por microscopia de fluorescência, microscopia eletrônica de transmissão e microscopia eletrônica de varredura. Não foi detectada alteração no potencial de membrana mitocondrial. Os resultados indicam que futuros estudos poder

  11. Sympathetic reflex control of blood flow in human peripheral tissues

    DEFF Research Database (Denmark)

    Henriksen, O

    1991-01-01

    Sympathetic vasoconstrictor reflexes are essential for the maintenance of arterial blood pressure in upright position. It has been generally believed that supraspinal sympathetic vasoconstrictor reflexes elicited by changes in baroreceptor activity play an important role. Recent studies on human...... sympathetic vasoconstrictor reflexes are blocked. Blood flow has been measure by the local 133Xe-technique. The results indicate the presence of spinal as well as supraspinal sympathetic vasoconstrictor reflexes to human peripheral tissues. Especially is emphasized the presence of a local sympathetic veno...

  12. Photodynamic activities of silicon phthalocyanines against achromic M6 melanoma cells and healthy human melanocytes and keratinocytes.

    Science.gov (United States)

    Decreau, R; Richard, M J; Verrando, P; Chanon, M; Julliard, M

    1999-01-01

    Dichlorosilicon phthalocyanine (Cl2SiPc) and bis(tri-n-hexylsiloxy) silicon phthalocyanine (HexSiPc) have been evaluated in vitro as potential photosensitizers for photodynamic therapy (PDT) against the human amelanotic melanoma cell line M6. Each photosensitizer is dissolved in a solvent-PBS mixture, or entrapped in egg-yolk lecithin liposomes or in Cremophor EL micelles. The cells are incubated for 1 h with the sensitizer and then irradiated for 20 min, 1 h or 2 h (lambda > 480 nm, 10 mW cm-2). The photocytotoxic effect is dependent on the photosensitizer concentration and the light dose. Higher phototoxicity is observed after an irradiation of 2 h: treatment with a solution of photosensitizer (2 x 10(-9) M) leads to 10% (HexSiPc in egg-yolk lecithin liposomes) or 20% (Cl2SiPc in DMF-PBS solution) cell viability. After 1 h incubation and 20 min of light exposure, the photodynamic effect is connected with the type of delivery system used. For HexSiPc, lower cell viability is found when this photosensitizer is entrapped in egg-yolk lecithin instead of solvent-PBS or for Cremophor EL micelles with Cl2SiPc. Liposome-delivered HexSiPc leads to lipid damage in M6 cells, illustrated by an increase of thiobarbituric acid-reacting substances (TBARs), but the change is not significant with Cremophor EL. The same is observed for the antioxidative defences after photodynamic stress. The cells irradiated with HexSiPc entrapped in liposomes display an increase of superoxide dismutase (SOD) activity and a decrease of glutathione (GSH) level, glutathione peroxidase (GSHPx) and catalase (Cat) activities.

  13. Human Melanoma Cells under Endoplasmic Reticulum Stress Are More Susceptible to Apoptosis Induced by the BH3 Mimetic Obatoclax

    Directory of Open Access Journals (Sweden)

    Chen Chen Jiang

    2009-09-01

    Full Text Available Past studies have shown that melanoma cells have largely adapted to endoplasmic reticulum (ER stress, and this is associated with up-regulation of the antiapoptotic proteins Bcl-2 and Mcl-1. In this report, we show that the BH3 mimetic obatoclax potently overcomes resistance of melanoma cells to apoptosis induced by ER stress. Obatoclax, as a single agent at nanomolar concentrations, was relatively ineffective in the induction of apoptosis in melanoma cells, but treatment with obatoclax at these concentrations in combination with the ER stress inducer tunicamycin (TM or thapsigargin markedly enhanced apoptotic cell death. This was primarily because of the inhibition of Mcl-1 by obatoclax, in that cotreatment with TM and another BH3 mimetic ABT737, which does not antagonize Mcl-1, caused only minimal increases in apoptosis. Moreover, overexpression of Mcl-1 inhibited apoptosis to greater degrees than overexpression of Bcl-2. In addition to direct inhibition of Mcl-1 by obatoclax, the combination of obatoclax and TM caused strong up-regulation of the BH3-only protein Noxa. Small RNA interference knockdown of Noxa partially inhibited apoptosis induced by cotreatment with obatoclax and TM. Similarly, knockdown of Bak also blocked induction of apoptosis by the compounds. The Mcl-1/Bak interaction seemed to be disrupted more efficiently in melanoma cells cotreated with obatoclax and TM. Taken together, these results identify obatoclax as a potent agent that overcomes resistance of melanoma cells to ER stress-induced apoptosis and seem to have important implications in the use of BH3 mimetics in the treatment of melanoma.

  14. Cytotoxicity of new duplex drugs linking 3'-C-ethynylcytidine and 5-fluor-2'-deoxyuridine against human melanoma cells.

    Science.gov (United States)

    Schott, Sarah; Niessner, Heike; Sinnberg, Tobias; Venturelli, Sascha; Berger, Alexander; Ikenberg, Kristian; Villanueva, Jessie; Meier, Friedegund; Garbe, Claus; Busch, Christian

    2012-11-01

    Melanoma is an increasingly common and potentially fatal malignancy of the skin and some mucous membranes. As no cure exists for metastatic disease, there is an urgent need for novel drugs. 2'-Deoxy-5-fluorouridylyl-(3'-5')-3'-C-ethynylcytidine [5-FdU(3'-5')ECyd] and 3'-C-ethynylcytidinylyl-(5' → 1-O)-2-O-octadecyl-sn-glycerylyl-(3-O → 5')-2'-deoxy-5-fluorouridine [ECyd-lipid-5-FdU] represent cytostatic active duplex drugs, which can be metabolized into various active antimetabolites. We evaluated the cytotoxicity of these heterodinucleoside phosphate analogs, their corresponding monomers ECyd and 5-FdU and combinations thereof on six metastatic melanoma cell lines and six ex vivo patient-derived melanoma cells in comparison to current standard cytostatic agents and the BRAF V600E inhibitor Vemurafenib. In vitro (real-time)-proliferation assays demonstrated that 5-FdU(3'-5')ECyd and ECyd-lipid-5-FdU had a high cytotoxic efficacy causing 75% melanoma cell death at concentrations in the nanomolar and micromolar range. Cytotoxicity was conducted by induction of DNA cleavage indicating apoptotic cells. Chicken embryotoxicity demonstrated that the duplex drugs were less toxic than 5-FdU at 0.01 μM. In vivo the duplex drug 5-FdU(3'-5')ECyd was efficacious in the murine LOX IMVI melanoma xenograph model on administration of 11.2 mg/kg/injection every fourth day. Both duplex drugs are promising novel cytostatic agents for the treatment of malignant melanoma meriting clinical evaluation. Copyright © 2012 UICC.

  15. The DNA methylome of human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Li, Yingrui; Zhu, Jingde; Tian, Geng

    2010-01-01

    DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per...... strand), we report a comprehensive (92.62%) methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC) from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found...... that 68.4% of CpG sites and 80% displayed allele-specific expression (ASE). These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic...

  16. Distribution of TCDD in blood constituents of rats and humans

    Science.gov (United States)

    Wirsing, J. M.; Weber, L. W.; Kettrup, A. A.; Rozman, Karl K.

    1995-10-01

    Whole blood or plasma from male human volunteers or pooled from male Sprague-Dawley rats was incubated with varying amounts of 3H-2,3,7,8-tetrachlorodibenzo-p-dioxin (3H-TCDD). Blood was separated into cellular, protein and lipoprotein fractions by centrifugation. The distribution of 3H-TCDD between lipoproteins and plasma proteins was independent of 3H-TCDD concentration in the range of 65 fmol-1 nmol/ml plasma. The distribution of 3H-TCDD between the various lipoprotein fractions depended only on their relative content of total cholesterol plus triglycerides. The partitioning of 3H- TCDD between lipoproteins and plasma proteins was inversely proportional, whereas the distribution between the cellular fraction and the lipoproteins was directly proportional to the total plasma cholesterol plus triglyceride content. As a consequence of species differences in blood composition, the major part of 3H-TCDD-associated radioactivity was recovered from lipoproteins in human blood but from erythrocytes in rat blood. A mathematical description of the distribution of TCDD between blood components is presented.

  17. Generation of red blood cells from human embryonic/induced pluripotent stem cells for blood transfusion.

    Science.gov (United States)

    Ebihara, Yasuhiro; Ma, Feng; Tsuji, Kohichiro

    2012-06-01

    Red blood cell (RBC) transfusion is necessary for many patients with emergency or hematological disorders. However, to date the supply of RBCs remains labile and dependent on voluntary donations. In addition, the transmission of infectious disease via blood transfusion from unspecified donors remains a risk. Establishing a large quantity of safe RBCs would help to address this issue. Human embryonic stem (hES) cells and the recently established human induced pluripotent stem (hiPS) cells represent potentially unlimited sources of donor-free RBCs for blood transfusion, as they can proliferate indefinitely in vitro. Extensive research has been done to efficiently generate transfusable RBCs from hES/iPS cells. Nevertheless, a number of challenges must be overcome before the clinical usage of hES/iPS cell-derived RBCs can become a reality.

  18. Deep coverage mouse red blood cell proteome: a first comparison with the human red blood cell

    DEFF Research Database (Denmark)

    Pasini, Erica M; Kirkegaard, Morten; Salerno, Doris

    2008-01-01

    Mice have close genetic/physiological relationships to humans, breed rapidly, and can be genetically modified, making them the most used mammal in biomedical research. Because the red blood cell (RBC) is the sole gas transporter in vertebrates, diseases of the RBC are frequently severe; much...

  19. Does Every Cell Get Blood? Young Students' Discussions about Illustrations of Human Blood Circulation

    Science.gov (United States)

    Westman, Anna-Karin; Karlsson, Karl-Goran

    2016-01-01

    This article presents a study of how groups of young students discuss illustrations of human blood circulation. Transparency is not an innate quality of illustrations, visual information is always coded and interpretations are always related to culture and context. Results of this study are discussed with reference to Kress and van Leeuwens'…

  20. Mesenteric, coeliac and splanchnic blood flow in humans during exercise

    DEFF Research Database (Denmark)

    Perko, M J; Nielsen, H B; Skak, C;

    1998-01-01

    1. Exercise reduces splanchnic blood flow, but the mesenteric contribution to this response is uncertain. 2. In nineteen humans, superior mesenteric and coeliac artery flows were determined by duplex ultrasonography during fasting and postprandial submaximal cycling and compared with the splanchnic...... blood flow as assessed by the Indocyanine Green dye-elimination technique. 3. Cycling increased arterial pressure, heart rate and cardiac output, while it reduced total vascular resistance. These responses were not altered in the postprandial state. During fasting, cycling increased mesenteric, coeliac...... and splanchnic resistances by 76, 165 and 126 %, respectively, and it reduced corresponding blood flows by 32, 50 and 43 % (by 0.18 +/- 0.04, 0.42 +/- 0.03 and 0.60 +/- 0.04 l min-1). Postprandially, mesenteric and splanchnic vascular resistances decreased, thereby elevating regional blood flow, while...

  1. Laser-photophoretic migration and fractionation of human blood cells.

    Science.gov (United States)

    Monjushiro, Hideaki; Tanahashi, Yuko; Watarai, Hitoshi

    2013-05-13

    Laser photophoretic migration behavior of human blood cells in saline solution was investigated under the irradiation of Nd:YAG laser beam (532 nm) in the absence and the presence of the flow in a fused silica capillary. Red blood cells (RBC) were migrated faster than white blood cells (WBC) and blood pellets to the direction of propagation of laser light. The observed photophoretic velocity of RBC was about 11 times faster than those of others. This was understood from the larger photophoretic efficiency of RBC than that of WBC, which was simulated based on the Mie scattering theory. Furthermore, it was found that, during the photophoretic migration, RBCs spontaneously orientated parallel to the migration direction so as to reduce the drag force. Finally, it was demonstrated that RBC and WBC were separated in a micro-channel flow system by the laser photophoresis.

  2. Direct determination of external radiation dose in human blood

    CERN Document Server

    Tanir, AG; Sahiner, E; Bolukdemir, MH; Koc, K; Meric, N; Keles, SK; Kucuk, O

    2014-01-01

    In this study it was shown that it is possible to determine radiation doses from external beam therapy both directly and retrospectively from a human blood sample. To the best of our knowledge no other studies exist on the direct measurement of doses received by a person from external beam therapy. Optically stimulated luminescence counts from a healthy blood sample exposed to an external radiation source were measured. Blood aliquots were given 0, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 100 and 200Gy beta doses and their decay and dose-response curves were plotted. While the luminescence intensities were found to be relatively low for the doses smaller than 10Gy, they were measured considerably higher for doses greater than 10Gy. The dose received by the blood aliquots was determined by interpolating the luminescence counts of 10Gy to the dose-response curve. This study has important ramifications for healthcare, medicine and radiation protection

  3. Evolutionary aspects of ABO blood group in humans.

    Science.gov (United States)

    Franchini, Massimo; Bonfanti, Carlo

    2015-04-15

    The antigens of the ABO blood group system (A, B and H determinants) are complex carbohydrate molecules expressed on red blood cells and on a variety of other cell lines and tissues. Growing evidence is accumulating that ABO antigens, beyond their key role in transfusion medicine, may interplay with the pathogenesis of many human disorders, including infectious, cardiovascular and neoplastic diseases. In this narrative review, after succinct description of the current knowledge on the association between ABO blood groups and the most severe diseases, we aim to elucidate the particularly intriguing issue of the possible role of ABO system in successful aging. In particular, focus will be placed on studies evaluating the ABO phenotype in centenarians, the best human model of longevity.

  4. ACTIVATION OF HUMAN BLOOD MONONUCLEARS BY LIPOPOLYSACCHARIDE OF DIFFERENT COMPOSITION

    Directory of Open Access Journals (Sweden)

    S. V. Zubova

    2010-01-01

    Full Text Available Influence of lipopolysaccharide (LPS composition upon activation of human blood mononuclears was investigated, by measuring levels of pro-inflammatory TNFα and IL-6 cytokines released by the cells. It is shown that LPS from Rhodobacter capsulatus PG, in contrast to E. coli LPS, did not activate the target cells for synthesis of the cytokines.

  5. Expansion of human cord blood hematopoietic stem cells for transplantation.

    Science.gov (United States)

    Chou, Song; Chu, Pat; Hwang, William; Lodish, Harvey

    2010-10-08

    A recent Science paper reported a purine derivative that expands human cord blood hematopoietic stem cells in culture (Boitano et al., 2010) by antagonizing the aryl hydrocarbon receptor. Major problems need to be overcome before ex vivo HSC expansion can be used clinically.

  6. Detecting multiple DNA human profile from a mosquito blood meal.

    Science.gov (United States)

    Rabêlo, K C N; Albuquerque, C M R; Tavares, V B; Santos, S M; Souza, C A; Oliveira, T C; Moura, R R; Brandão, L A C; Crovella, S

    2016-08-26

    Criminal traces commonly found at crime scenes may present mixtures from two or more individuals. The scene of the crime is important for the collection of various types of traces in order to find the perpetrator of the crime. Thus, we propose that hematophagous mosquitoes found at crime scenes can be used to perform genetic testing of human blood and aid in suspect investigation. The aim of the study was to obtain a single Aedes aegypti mosquito profile from a human DNA mixture containing genetic materials of four individuals. We also determined the effect of blood acquisition time by setting time intervals of 24, 48, and 72 h after the blood meal. STR loci and amelogenin were analyzed, and the results showed that human DNA profiles could be obtained from hematophagous mosquitos at 24 h following the blood meal. It is possible that hematophagous mosquitoes can be used as biological remains at the scene of the crime, and can be used to detect human DNA profiles of up to four individuals.

  7. Sox2 is not required for melanomagenesis, melanoma growth and melanoma metastasis in vivo.

    Science.gov (United States)

    Cesarini, V; Guida, E; Todaro, F; Di Agostino, S; Tassinari, V; Nicolis, S; Favaro, R; Caporali, S; Lacal, P M; Botti, E; Costanzo, A; Rossi, P; Jannini, E A; Dolci, S

    2017-08-01

    Melanoma is a dangerous form of skin cancer derived from the malignant transformation of melanocytes. The transcription factor SOX2 is not expressed in melanocytes, however, it has been shown to be differentially expressed between benign nevi and malignant melanomas and to be essential for melanoma stem cell maintenance and expansion in vitro and in xenograft models. By using a mouse model in which BRaf(V600E) mutation cooperates with Pten loss to induce the development of metastatic melanoma, we investigated if Sox2 is required during the process of melanomagenesis, melanoma growth and metastasis and in the acquisition of resistance to BRAF inhibitors (BRAFi) treatments. We found that deletion of Sox2 specifically in Pten null and BRafV600E-expressing melanocytes did not prevent tumor formation and did not modify the temporal kinetics of melanoma occurrence compared to Sox2 wt mice. In addition, tumor growth was similar between Sox2 wt and Sox2 deleted (del) melanomas. By querying publicly available databases, we did not find statistically significant differences in SOX2 expression levels between benign nevi and melanomas, and analysis on two melanoma patient cohorts confirmed that Sox2 levels did not significantly change between primary and metastatic melanomas. Melanoma cell lines derived from both Sox2 genotypes showed a similar sensitivity to vemurafenib treatment and the same ability to develop vemurafenib resistance in long-term cultures. Development of vemurafenib resistance was not dependent on SOX2 expression also in human melanoma cell lines in vitro. Our findings exclude an oncogenic function for Sox2 during melanoma development and do not support a role for this transcription factor in the acquisition of resistance to BRAFi treatments.

  8. Recombinant Interferon Alfa-2b in Treating Patients With Melanoma

    Science.gov (United States)

    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

  9. Basic Fibroblast Growth Factor-Mediated Overexpression of Vascular Endothelial Growth Factor in 1F6 Human Melanoma Cells is Regulated by Activation of PI-3K and p38 MAPK

    Directory of Open Access Journals (Sweden)

    Dennis Fontijn

    2009-01-01

    Full Text Available Background: 1F6 human melanoma xenografts overexpressing either the 18 kD (18kD form or all (ALL forms of human basic fibroblast growth factor (bFGF demonstrate an abundant number of microvessels and accelerated growth. We now examined whether bFGF mediates vascular endothelial growth factor (VEGF expression.

  10. Optoacoustic mapping of cerebral blood oxygenation in humans

    Science.gov (United States)

    Petrov, Yuriy; Prough, Donald S.; Petrov, Irene Y.; Richardson, C. Joan; Fonseca, Rafael A.; Robertson, Claudia S.; Esenaliev, Rinat O.

    2017-03-01

    Noninvasive, transcranial mapping, monitoring, and imaging are highly important for detection and management of cerebral abnormalities and neuroscience research. Mapping, imaging, and monitoring of cerebral blood oxygenation are necessary for diagnostics and management of patients with traumatic brain injury, stroke, and other neurological conditions. We proposed to use optoacoustic technology for noninvasive, transcranial monitoring and imaging. In this work, we developed optoacoustic systems for mapping of cerebral blood oxygenation in humans and tested them in adults and neonates. The systems provide noninvasive, transcranial optoacoustic measurements in the transmission (forward) and reflection (backward) modes in the near infrared spectral range. Novel, ultra-sensitive probes were built for detection of optoacoustic signals and measurement of blood oxygenation in neonates and adults. Cerebral oxygenation was measured at different lateral sites from the superior sagittal sinus (SSS), a large central cerebral vein, located immediately beneath the midline of the human skull. In neonates, cerebral oxygenation was measured through open anterior and posterior fontanelles. Optoacoustic signal detection at different locations allowed for mapping of cerebral blood oxygenation. Our future studies will be focused on 3D mapping of cerebral blood oxygenation.

  11. Pleomorphic Structures in Human Blood Are Red Blood Cell-Derived Microparticles, Not Bacteria

    Science.gov (United States)

    Mitchell, Adam J.; Gray, Warren D.; Schroeder, Max; Yi, Hong; Taylor, Jeannette V.; Dillard, Rebecca S.; Ke, Zunlong; Wright, Elizabeth R.; Stephens, David; Roback, John D.; Searles, Charles D.

    2016-01-01

    Background Red blood cell (RBC) transfusions are a common, life-saving therapy for many patients, but they have also been associated with poor clinical outcomes. We identified unusual, pleomorphic structures in human RBC transfusion units by negative-stain electron microscopy that appeared identical to those previously reported to be bacteria in healthy human blood samples. The presence of viable, replicating bacteria in stored blood could explain poor outcomes in transfusion recipients and have major implications for transfusion medicine. Here, we investigated the possibility that these structures were bacteria. Results Flow cytometry, miRNA analysis, protein analysis, and additional electron microscopy studies strongly indicated that the pleomorphic structures in the supernatant of stored RBCs were RBC-derived microparticles (RMPs). Bacterial 16S rDNA PCR amplified from these samples were sequenced and was found to be highly similar to species that are known to commonly contaminate laboratory reagents. Conclusions These studies suggest that pleomorphic structures identified in human blood are RMPs and not bacteria, and they provide an example in which laboratory contaminants may can mislead investigators. PMID:27760197

  12. Identification of plasma microRNAs as new potential biomarkers with high diagnostic power in human cutaneous melanoma.

    Science.gov (United States)

    Fogli, Stefano; Polini, Beatrice; Carpi, Sara; Pardini, Barbara; Naccarati, Alessio; Dubbini, Nevio; Lanza, Maria; Breschi, Maria Cristina; Romanini, Antonella; Nieri, Paola

    2017-05-01

    Melanoma is a devastating disease with few therapeutic options in the advanced stage and with the urgent need of reliable biomarkers for early detection. In this context, circulating microRNAs are raising great interest as diagnostic biomarkers. We analyzed the expression profiles of 21 selected microRNAs in plasma samples from melanoma patients and healthy donors to identify potential diagnostic biomarkers. Data analysis was performed using global mean normalization and NormFinder algorithm. Linear regression followed by receiver operating characteristic analyses was carried out to evaluate whether selected plasma miRNAs were able to discriminate between cases and controls. We found five microRNAs that were differently expressed among cases and controls after Bonferroni correction for multiple testing. Specifically, miR-15b-5p, miR-149-3p, and miR-150-5p were up-regulated in plasma of melanoma patients compared with healthy controls, while miR-193a-3p and miR-524-5p were down-regulated. Receiver operating characteristic analyses of these selected microRNAs provided area under the receiver operating characteristic curve values ranging from 0.80 to 0.95. Diagnostic value of microRNAs is improved when considering the combination of miR-149-3p, miR-150-5p, and miR-193a-3p. The triple classifier had a high capacity to discriminate between melanoma patients and healthy controls, making it suitable to be used in early melanoma diagnosis.

  13. Efficient ferrocifen anticancer drug and Bcl-2 gene therapy using lipid nanocapsules on human melanoma xenograft in mouse.

    Science.gov (United States)

    Resnier, Pauline; Galopin, Natacha; Sibiril, Yann; Clavreul, Anne; Cayon, Jérôme; Briganti, Alessandro; Legras, Pierre; Vessières, Anne; Montier, Tristan; Jaouen, Gérard; Benoit, Jean-Pierre; Passirani, Catherine

    2017-01-31

    Metastatic melanoma has been described as a highly aggressive cancer with low sensibility to chemotherapeutic agents. New types of drug, such as metal-based drugs (ferrocifens) have emerged and could represent an alternative for melanoma treatment since they show interesting anticancer potential. Furthermore, molecular analysis has evidenced the role of apoptosis in the low sensibility of melanomas and especially of the key regulator, Bcl-2. The objective of this study was to combine two strategies in the same lipid nanocapsules (LNCs): i) gene therapy to modulate anti-apoptotic proteins by the use of Bcl-2 siRNA, and ii) ferrocifens as a new type of anticancer agent. The efficient gene silencing with LNCs was verified by the specific extinction of Bcl-2 in melanoma cells. The cellular toxicity of ferrocifens (ferrociphenol (FcDiOH) or Ansa-FcDiOH) was demonstrated, showing higher efficacy than dacarbazine. Interestingly, the association of siBcl-2 LNCs with Ansa-FcDiOH demonstrated a significant effect on melanoma cell viability. Moreover, the co-encapsulation of siRNA and ferrocifens was successfully performed into LNCs for animal experiments. A reduction of tumor volume and mass was proved after siBcl-2 LNC treatment and Ansa-FcDiOH LNC treatment, individually (around 25%). Finally, the association of both components into the same LNCs increased the reduction of tumor volume to about 50% compared to the control group. In conclusion, LNCs appeared to provide a promising tool for the co-encapsulation of a metal-based drug and siRNA.

  14. Sb(V) reactivity with human blood components: redox effects.

    Science.gov (United States)

    López, Silvana; Aguilar, Luis; Mercado, Luis; Bravo, Manuel; Quiroz, Waldo

    2015-01-01

    We assessed the reactivity of Sb(V) in human blood. Sb(V) reactivity was determined using an HPLC-HG-AFS hyphenated system. Sb(V) was partially reduced to Sb(III) in blood incubation experiments; however, Sb(III) was a highly unstable species. The addition of 0.1 mol L(-1) EDTA prevented Sb(III) oxidation, thus enabling the detection of the reduction of Sb(V) to Sb(III). The transformation of Sb(V) to Sb(III) in human whole blood was assessed because the reduction of Sb(V) in human blood may likely generate redox side effects. Our results indicate that glutathione was the reducing agent in this reaction and that Sb(V) significantly decreased the GSH/GSSG ratio from 0.32 ± 0.09 to 0.07 ± 0.03. Moreover, the presence of 200 ng mL(-1) of Sb(V) increased the activity of superoxide dismutase from 4.4 ± 0.1 to 7.0 ± 0.4 U mL(-1) and decreased the activity of glutathione peroxidase from 62 ± 1 to 34 ± 2 nmol min(-1) mL(-1).

  15. Sb(V reactivity with human blood components: redox effects.

    Directory of Open Access Journals (Sweden)

    Silvana López

    Full Text Available We assessed the reactivity of Sb(V in human blood. Sb(V reactivity was determined using an HPLC-HG-AFS hyphenated system. Sb(V was partially reduced to Sb(III in blood incubation experiments; however, Sb(III was a highly unstable species. The addition of 0.1 mol L(-1 EDTA prevented Sb(III oxidation, thus enabling the detection of the reduction of Sb(V to Sb(III. The transformation of Sb(V to Sb(III in human whole blood was assessed because the reduction of Sb(V in human blood may likely generate redox side effects. Our results indicate that glutathione was the reducing agent in this reaction and that Sb(V significantly decreased the GSH/GSSG ratio from 0.32 ± 0.09 to 0.07 ± 0.03. Moreover, the presence of 200 ng mL(-1 of Sb(V increased the activity of superoxide dismutase from 4.4 ± 0.1 to 7.0 ± 0.4 U mL(-1 and decreased the activity of glutathione peroxidase from 62 ± 1 to 34 ± 2 nmol min(-1 mL(-1.

  16. [A Nude Mouse Model for Human Umbilical Cord Blood Transplantation

    Science.gov (United States)

    Lan, Jiongcai; Liu, Hongyu; Chen, Qiang; Yang, Chongli; Zhang, Zhimei

    2000-03-01

    To evaluate the hematopoietic potentiality and the migration and homing routine of separated as well as cryopreserved umbilical cord blood hematopoietic cells, the BALB/cnu(+) mice were used to establish a murine model. This can prepare for the clinical transplantation and the establishment of a large-scale cord blood bank. The result indicated that the hydroxyethyl starch (HES) sedimentation and DMSO step-by-step cryopreservation procedure resulted in only less losses of hematopoietic progenitor cells and also unharmful to the hematopietic potentiality. We can found evidence for successful transplantation in each mouse which received (1.0 - 2.0) x 10(7) separated or cryopresered hematopoietic cells from cord blood, which lasted for about fifty days. The results demonstrated that (1) HES sedimentation and DMSO cryopreservation procedure can keep the hematopoietic potentiality of cord blood, and so can be used to clinical transplantation or establishment of a cord blood bank; (2) Rich hematopoietic stem cells in human cord blood can cross the xenogenetic barriers and successfully engraft mice; (3) The hematopoietic cells migrated among bone marrow, liver, spleen, lung and kidney in the mice and homed to bone marrow by the end. Cryopreservation may influence the adhesion molecule on the hematopoietic cells and the homing behaviour, but not influence their hematopoietic potentiality.

  17. Splanchnic blood flow and hepatic glucose production in exercising humans

    DEFF Research Database (Denmark)

    Bergeron, R; Kjaer, M; Simonsen, L

    2001-01-01

    The study examined the implication of the renin-angiotensin system (RAS) in regulation of splanchnic blood flow and glucose production in exercising humans. Subjects cycled for 40 min at 50% maximal O(2) consumption (VO(2 max)) followed by 30 min at 70% VO(2 max) either with [angiotensin......-blockade group vs. the control group, hormones, metabolites, VO(2), and RER followed the same pattern of changes in ACE-blockade and control groups during exercise. Splanchnic blood flow (at rest: 1.67 +/- 0.12, ACE blockade; 1.59 +/- 0.18 l/min, control) decreased during moderate exercise (0.78 +/- 0.07, ACE...

  18. Up-regulation of hepatoma-derived growth factor facilitates tumor progression in malignant melanoma [corrected].

    Directory of Open Access Journals (Sweden)

    Han-En Tsai

    Full Text Available Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression in developing melanoma remains unclear. In this study, human melanoma cell lines (A375, A2058, MEL-RM and MM200 showed higher levels of HDGF gene expression, whereas human epidermal melanocytes (HEMn expressed less. Exogenous application of HDGF stimulated colony formation and invasion of human melanoma cells. Moreover, HDGF overexpression stimulated the degree of invasion and colony formation of B16-F10 melanoma cells whereas HDGF knockdown exerted opposite effects in vitro. To evaluate the effects of HDGF on tumour growth and metastasis in vivo, syngeneic mouse melanoma and metastatic melanoma models were performed by manipulating the gene expression of HDGF in melanoma cells. It was found that mice injected with HDGF-overexpressing melanoma cells had greater tumour growth and higher metastatic capability. In contrast, mice implanted with HDGF-depleted melanoma cells exhibited reduced tumor burden and lung metastasis. Histological analysis of excised tumors revealed higher degree of cell proliferation and neovascularization in HDGF-overexpressing melanoma. The present study provides evidence that HDGF promotes tumor progression of melanoma and targeting HDGF may constitute a novel strategy for the treatment of melanoma.

  19. Alterations in cell surface area and deformability of individual human red blood cells in stored blood

    CERN Document Server

    Park, HyunJoo; Lee, SangYun; Kim, Kyoohyun; Sohn, Yong-Hak; Jang, Seongsoo; Park, YongKeun

    2015-01-01

    The functionality and viability of stored human red blood cells (RBCs) is an important clinical issue in transfusion. To systematically investigate changes in stored whole blood, the hematological properties of individual RBCs were quantified in blood samples stored for various periods with and without a preservation solution called CPDA-1. With 3-D quantitative phase imaging techniques, the optical measurements of the 3-D refractive index (RI) distributions and membrane fluctuations were done at the individual cell level. From the optical measurements, the morphological (volume, surface area and sphericity), biochemical (hemoglobin content and concentration), and mechanical parameters (dynamic membrane fluctuation) were simultaneously quantified to investigate the functionalities and their progressive alterations in stored RBCs. Our results show that the stored RBCs without CPDA-1 had a dramatic morphological transformation from discocytes to spherocytes within 2 weeks which was accompanied with significant ...

  20. Moringa oleifera fruit induce apoptosis via reactive oxygen species-dependent activation of mitogen-activated protein kinases in human melanoma A2058 cells.

    Science.gov (United States)

    Guon, Tae Eun; Chung, Ha Sook

    2017-08-01

    The present study was performed to determine the effect of Moringa oleifera fruit extract on the apoptosis of human melanoma A2058 cells. A2058 cells were treated for 72 h with Moringa oleifera fruit extract at 50-100 µg/ml, and cell viability with apoptotic changes was examined. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) was examined. It was revealed that Moringa oleifera fruit extract significantly inhibited the cell viability and promoted apoptosis of A2058 cells in a concentration-dependent manner. Moringa oleifera fruit extract-treated A2058 cells exhibited increased activities of cleaved caspase-9 and caspase-3. It also caused an enhancement of MAPK phosphorylation and ROS production. The pro-apoptotic activity of Moringa oleifera fruit extract was significantly reversed by pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125, extracellular-signal-regulated kinase (ERK) inhibitor PD98058 or ROS inhibitor N-acetyl-L-cysteine. Taken together, Moringa oleifera fruit extract is effective in inducing mitochondrial apoptosis of A2058 cells, which is mediated through induction of ROS formation, and JNK and ERK activation. Moringa oleifera fruit extract may thus have therapeutic benefits for human melanoma A2058 cells.

  1. Novel ZnO:Ag nanocomposites induce significant oxidative stress in human fibroblast malignant melanoma (Ht144 cells

    Directory of Open Access Journals (Sweden)

    Syeda Arooj

    2015-02-01

    Full Text Available 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.

  2. Bioaccumulation of metals in human blood in industrially contaminated area

    Institute of Scientific and Technical Information of China (English)

    F, Akbar Jan; M. Ishaq; S. Khan; M. Shakirullah; S. M. Asim; I. Ahmad; Fazal Mabood

    2011-01-01

    Heavy metals were analyzed in different foods crops,milk,meat and blood samples collected from different age group subjects such as children (1-12 years),adolescent (12-18 years),adults (18-45 years) and old age (above 45 and 55 years for males and females,respectively) from polluted and relatively less polluted areas.The results revealed that the consumption of contaminatel food crops,meat and milk have significantly increased the concentrations of selected metals in the human blood.Cu,Zn and Mn concentrations were significantly higher (p < 0.05) in the blood samples collected from the polluted area as compared to control area.Old people had accumulated high concentrations of metals as compared to the younger ones within the same area.Males accumulated higher concentrations of metals as compared to females.

  3. Blood flow and microdialysis in the human femoral head

    DEFF Research Database (Denmark)

    Bøgehøj, Morten; Emmeluth, Claus; Overgaard, Søren

    2007-01-01

    BACKGROUND: If it would be possible to detect lack of flow and/or the development of ischemia in bone, we might have a way of predicting whether a broken bone will heal. We established microdialysis (MD) and laser Doppler (LD) flow measurement in the human femoral head in order to be able to detect...... ischemia and measure changes in blood flow. MATERIAL AND METHODS: In 9 patients undergoing total hip arthroplasty for primary osteoarthrosis, two MD catheters were inserted into the femoral head through two drill holes after the blood flow had been visualized by LD. Then primary samples were collected...... detected within 2 h of cessation of blood flow in most patients....

  4. Pregnancy and melanoma.

    Science.gov (United States)

    Driscoll, Marcia S; Martires, Kathryn; Bieber, Amy Kalowitz; Pomeranz, Miriam Keltz; Grant-Kels, Jane M; Stein, Jennifer A

    2016-10-01

    Malignant melanoma is the most common malignancy during pregnancy, and is diagnosed during childbearing age in approximately one-third of women diagnosed with melanoma. The impact of hormonal changes during pregnancy and from iatrogenic hormones on melanoma is controversial. Women undergo immunologic changes during pregnancy that may decrease tumor surveillance. In addition, hormone receptors are found on some melanomas. In spite of these observations, the preponderance of evidence does not support a poorer prognosis for pregnancy-associated melanomas. There is also a lack of evidence that oral contraceptives or hormone replacement therapy worsens melanoma prognosis.

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

    Science.gov (United States)

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

    2016-03-19

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

  6. CD147 interacts with NDUFS6 in regulating mitochondrial complex I activity and the mitochondrial apoptotic pathway in human malignant melanoma cells.

    Science.gov (United States)

    Luo, Z; Zeng, W; Tang, W; Long, T; Zhang, J; Xie, X; Kuang, Y; Chen, M; Su, J; Chen, X

    2014-01-01

    Malignant melanoma (MM) is one of the most lethal tumors and is characterized by high invasiveness, frequent metastasis, and resistance to chemotherapy. The risk of metastatic MM is accompanied by disordered energy metabolism involving the oxidative phosphorylation (OXPHOS) process, which is largely carried out in mitochondrial complexes. Complex I is the first and largest mitochondrial enzyme complex associated with this process. CD147 is a transmembrane glycoprotein mainly expressed on the cell surface, and also appears in the cytoplasm in some tumors. We found that CD147 is often translocated to the cytoplasm in metastatic MM specimens as compared to primary MM. We also demonstrated high expression of CD147 in isolated mitochondrial fractions of A375 cells. The yeast two-hybrid (Y2H) assay identified NDUFS6 (which encodes a subunit of mitochondrial respiratory chain complex I) as a candidate that interacts with CD147 and depletion of CD147 in A375 cells significantly decreased complex I enzyme activity. We also showed that CD147 increased the viability of A375 cells exposed to berberine-induced mitochondrial damage, and protected them from apoptosis through a mitochondrial-dependent pathway. This finding was confirmed by adding exogenous Bcl-2 to A375 cell cultures. In summary, our results identify the existence of CD147 in human melanoma cell mitochondria. They indicate that CD147 appears to regulate complex I activity and apoptosis in MM by interacting with mitochondrial NDUFS6. Our findings provide new insight into the function of CD147 and identify it as a promising therapeutic target in melanoma through disruption of the energy metabolism.

  7. The Scent of Blood: A Driver of Human Behavior?

    Science.gov (United States)

    Moran, James K; Dietrich, Daniel R; Elbert, Thomas; Pause, Bettina M; Kübler, Lisa; Weierstall, Roland

    2015-01-01

    The scent of blood is potentially one of the most fundamental and survival-relevant olfactory cues in humans. This experiment tests the first human parameters of perceptual threshold and emotional ratings in men and women of an artificially simulated smell of fresh blood in contact with the skin. We hypothesize that this scent of blood, with its association with injury, danger, death, and nutrition will be a critical cue activating fundamental motivational systems relating to either predatory approach behavior or prey-like withdrawal behavior, or both. The results show that perceptual thresholds are unimodally distributed for both sexes, with women being more sensitive. Furthermore, both women and men's emotional responses to simulated blood scent divide strongly into positive and negative valence ratings, with negative ratings in women having a strong arousal component. For women, this split is related to the phase of their menstrual cycle and oral contraception (OC). Future research will investigate whether this split in both genders is context-dependent or trait-like.

  8. Correlation between blood adenosine metabolism and sleep in humans.

    Science.gov (United States)

    Díaz-Muñoz, M; Hernández-Muñoz, R; Suárez, J; Vidrio, S; Yááñez, L; Aguilar-Roblero, R; Rosenthal, L; Villalobos, L; Fernández-Cancino, F; Drucker-Colín, R; Chagoya De Sanchez, V

    1999-01-01

    Blood adenosine metabolism, including metabolites and metabolizing enzymes, was studied during the sleep period in human volunteers. Searching for significant correlations among biochemical parameters found: adenosine with state 1 of slow-wave sleep (SWS); activity of 5'-nucleotidase with state 2 of SWS; inosine and AMP with state 3-4 of SWS; and activity of 5'-nucleotidase and lactate with REM sleep. The correlations were detected in all of the subjects that presented normal hypnograms, but not in those who had fragmented sleep the night of the experiment. The data demonstrate that it is possible to obtain information of complex brain operations such as sleep by measuring biochemical parameters in blood. The results strengthen the notion of a role played by adenosine, its metabolites and metabolizing enzymes, during each of the stages that constitute the sleep process in humans.

  9. Implementation of good manufacturing practices (GMP) on human blood irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Boghi, Claudio; Napolitano, Celia M.; Ferreira, Danilo C.; Rela, Paulo Roberto [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)]. E-mails: cboghi@uol.com.br; cmnapoli@ipen.br; dancarde@ig.com.br; prela@ipen.br; Zarate, Herman S. [Comission Chilena de Energia Nuclear, Santiago (Chile)]. E-mail: hzarate@cchen.cl

    2007-07-01

    The irradiation of human blood is used to avoid the TA-GVHD (transfusion-associated graft-versus-host-disease), a rare but devastating adverse effect of leukocytes present in blood components for a immuno-competent transfusion recipients. Usually this irradiation practice is performed to a physical elimination of lymphocytes. The implementation of the GMP will assure that the properly dose in a range of 25 Gy to 50 Gy will be delivered to the blood in the bag collected in a blood tissue bank. The studies to establish the GMP were developed under the guidelines of the standard ISO 11137 - Sterilization of health care products - Requirements for validation and routine control - Radiation sterilization. In this work, two dosimetric systems were used for dose mapping during the studies of irradiator qualification, loading pattern, irradiation process validation and auditing. The CaSO{sub 4}: Dy dosimeter presented difficulties concerning to uncertainty on dose measurement, stability, trace ability and calibration system. The PMMA and gafchromic dosimetric systems have shown a better performance and were adopted on establishment of GMP procedures. The irradiation tests have been done using a Gammacell 220 Irradiator. The developed GMP can be adapted for different types of gamma irradiators, allowing to set up a quality assurance program for blood irradiation. (author)

  10. Good manufacturing practices (GMP utilized on human blood irradiation process

    Directory of Open Access Journals (Sweden)

    Cláudio Boghi

    2008-01-01

    Full Text Available Irradiation of human blood is used to avoid the TA-GVHD (transfusion-associated graft-versus-host-disease, a rare but devastating adverse effect of leukocytes present in blood components for immunocompetent transfusion recipients. Usually this irradiation practice is performed to a physical elimination of lymphocytes. The implementation of the GMP will assure that the properly dose in a range of 25Gy to 50Gy will be delivered to the blood in the bag collected in a blood tissue bank. The studies to establish the GMP were developed under the guidelines of the standard ISO 11137 - Sterilization of health care products - Requirements for validation and routine control - Radiation sterilization. In this work, two dosimetric systems were used for dose mapping during the studies of irradiator qualification, loading pattern, irradiation process validation and auditing. The CaSO4: Dy dosimeter presented difficulties concerning to uncertainty on dose measurement, stability, trace ability and calibration system. The PMMA and gafchromic dosimetric systems have shown a better performance and were adopted on establishment of GMP procedures. The irradiation tests have been done using a Gammacell 220 Irradiator. The developed GMP can be adapted for different types of gamma irradiators, allowing to set up a quality assurance program for blood irradiation.

  11. RNA-seq reveals more consistent reference genes for gene expression studies in human non-melanoma skin cancers

    Directory of Open Access Journals (Sweden)

    Van L.T. Hoang

    2017-08-01

    Full Text Available Identification of appropriate reference genes (RGs is critical to accurate data interpretation in quantitative real-time PCR (qPCR experiments. In this study, we have utilised next generation RNA sequencing (RNA-seq to analyse the transcriptome of a panel of non-melanoma skin cancer lesions, identifying genes that are consistently expressed across all samples. Genes encoding ribosomal proteins were amongst the most stable in this dataset. Validation of this RNA-seq data was examined using qPCR to confirm the suitability of a set of highly stable genes for use as qPCR RGs. These genes will provide a valuable resource for the normalisation of qPCR data for the analysis of non-melanoma skin cancer.

  12. Sympathetic reflex control of blood flow in human peripheral tissues

    DEFF Research Database (Denmark)

    Henriksen, O

    1991-01-01

    Sympathetic vasoconstrictor reflexes are essential for the maintenance of arterial blood pressure in upright position. It has been generally believed that supraspinal sympathetic vasoconstrictor reflexes elicited by changes in baroreceptor activity play an important role. Recent studies on human ...... to collision of normodromically and antidromically conducted impulses in efferent sympathetic vasoconstrictor fibers. The evidence obtained suggests that sympathetic vasoconstrictor reflexes to postural changes are complex and highly differentiated....

  13. Development of edge-activated liposomes for siRNA delivery to human basal epidermis for melanoma therapy.

    Science.gov (United States)

    Dorrani, Mania; Garbuzenko, Olga B; Minko, Tamara; Michniak-Kohn, Bozena

    2016-04-28

    Delivery of macromolecules such as siRNA into cells that reside in the basal epidermis of the skin is a major challenge due to the transport barriers that need to be overcome. siRNAs have potential therapeutic applications in various dermatological diseases such as psoriasis, atopic dermatitis, and cancer. Unfortunately, a low permeability of siRNA through the stratum corneum and epidermis has significantly limited its use for topical application. The objective of this study was to develop a topical siRNA delivery system that can permeate through the stratum corneum and viable epidermis and efficiently deposit therapeutic levels of siRNA to the basal epidermis/upper dermis where melanoma cells reside. To achieve this objective, a series of liposome compositions that contained various concentrations of edge activator in their structures were prepared and then complexed with siRNA at different ratios to generate a small library of liposome-siRNA complexes (lipoplexes) with different physicochemical properties. In this study we used melanoma as a disease model. Through use of quantitative imaging analysis, we identified the necessary design parameters for effective permeation of lipoplexes through the skin layers and deposition at the upper dermis. The ability of the formulated lipoplexes to internalize into melanoma cells, knockdown the expression of the BRAF protein and induce cell death in melanoma cells was studied by fluorescent microscopy, in-cell immunofluorescence assay and WST-1 cell proliferation assay. By providing direct quantitative and qualitative microscopy evidence, the results of this study demonstrate for the first time that the passive delivery of an edge-activated liposomal formulation can effectively carry siRNA through the stratum corneum and deposit it at the lower epidermis/upper dermis.

  14. Melanoma cell lysate induces CCR7 expression and in vivo migration to draining lymph nodes of therapeutic human dendritic cells.

    Science.gov (United States)

    González, Fermín E; Ortiz, Carolina; Reyes, Montserrat; Dutzan, Nicolás; Patel, Vyomesh; Pereda, Cristián; Gleisner, Maria A; López, Mercedes N; Gutkind, J Silvio; Salazar-Onfray, Flavio

    2014-07-01

    We have previously reported a novel method for the production of tumour-antigen-presenting cells (referred to as TAPCells) that are currently being used in cancer therapy, using an allogeneic melanoma-derived cell lysate (referred to as TRIMEL) as an antigen provider and activation factor. It was recently demonstrated that TAPCell-based immunotherapy induces T-cell-mediated immune responses resulting in improved long-term survival of stage IV melanoma patients. Clinically, dendritic cell (DC) migration from injected sites to lymph nodes is an important requirement for an effective anti-tumour immunization. This mobilization of DCs is mainly driven by the C-C chemokine receptor type 7 (CCR7), which is up-regulated on mature DCs. Using flow cytometry and immunohistochemistry, we investigated if TRIMEL was capable of inducing the expression of the CCR7 on TAPCells and enhancing their migration in vitro, as well as their in vivo relocation to lymph nodes in an ectopic xenograft animal model. Our results confirmed that TRIMEL induces a phenotypic maturation and increases the expression of surface CCR7 on melanoma patient-derived DCs, and also on the monocytic/macrophage cell line THP-1. Moreover, in vitro assays showed that TRIMEL-stimulated DCs and THP-1 cells were capable of migrating specifically in the presence of the CCR7 ligand CCL19. Finally, we demonstrated that TAPCells could migrate in vivo from the injection site into the draining lymph nodes. This work contributes to an increased understanding of the biology of DCs produced ex vivo allowing the design of new strategies for effective DC-based vaccines for treating aggressive melanomas.

  15. Melanoma - neck (image)

    Science.gov (United States)

    This melanoma on the neck is variously colored with a very darkly pigmented area found centrally. It has irregular ... be larger than 0.5 cm. Prognosis in melanoma is best defined by its depth on resection.

  16. Drugs Approved for Melanoma

    Science.gov (United States)

    ... Ask about Your Treatment Research Drugs Approved for Melanoma This page lists cancer drugs approved by the ... that are not listed here. Drugs Approved for Melanoma Aldesleukin Cobimetinib Cotellic (Cobimetinib) Dabrafenib Dacarbazine DTIC-Dome ( ...

  17. Molecular Classification of Melanoma

    Science.gov (United States)

    Tissue-based analyses of precursors, melanoma tumors and metastases within existing study populations to further understanding of the heterogeneity of melanoma and determine a predictive pattern of progression for dysplastic nevi.

  18. Bioactive Constituents of Zanthoxylum rhetsa Bark and Its Cytotoxic Potential against B16-F10 Melanoma Cancer and Normal Human Dermal Fibroblast (HDF) Cell Lines.

    Science.gov (United States)

    Santhanam, Ramesh Kumar; Ahmad, Syahida; Abas, Faridah; Safinar Ismail, Intan; Rukayadi, Yaya; Tayyab Akhtar, Muhammad; Shaari, Khozirah

    2016-05-24

    Zanthoxylum rhetsa is an aromatic tree, known vernacularly as "Indian Prickly Ash". It has been predominantly used by Indian tribes for the treatment of many infirmities like diabetes, inflammation, rheumatism, toothache and diarrhea. In this study, we identified major volatile constituents present in different solvent fractions of Z. rhetsa bark using GC-MS analysis and isolated two tetrahydrofuran lignans (yangambin and kobusin), a berberine alkaloid (columbamine) and a triterpenoid (lupeol) from the bioactive chloroform fraction. The solvent fractions and purified compounds were tested for their cytotoxic potential against human dermal fibroblasts (HDF) and mouse melanoma (B16-F10) cells, using the MTT assay. All the solvent fractions and purified compounds were found to be non-cytotoxic to HDF cells. However, the chloroform fraction and kobusin exhibited cytotoxic effect against B16-F10 melanoma cells. The presence of bioactive lignans and alkaloids were suggested to be responsible for the cytotoxic property of Z. rhetsa bark against B16-F10 cells.

  19. Bioactive Constituents of Zanthoxylum rhetsa Bark and Its Cytotoxic Potential against B16-F10 Melanoma Cancer and Normal Human Dermal Fibroblast (HDF Cell Lines

    Directory of Open Access Journals (Sweden)

    Ramesh Kumar Santhanam

    2016-05-01

    Full Text Available Zanthoxylum rhetsa is an aromatic tree, known vernacularly as “Indian Prickly Ash”. It has been predominantly used by Indian tribes for the treatment of many infirmities like diabetes, inflammation, rheumatism, toothache and diarrhea. In this study, we identified major volatile constituents present in different solvent fractions of Z. rhetsa bark using GC-MS analysis and isolated two tetrahydrofuran lignans (yangambin and kobusin, a berberine alkaloid (columbamine and a triterpenoid (lupeol from the bioactive chloroform fraction. The solvent fractions and purified compounds were tested for their cytotoxic potential against human dermal fibroblasts (HDF and mouse melanoma (B16-F10 cells, using the MTT assay. All the solvent fractions and purified compounds were found to be non-cytotoxic to HDF cells. However, the chloroform fraction and kobusin exhibited cytotoxic effect against B16-F10 melanoma cells. The presence of bioactive lignans and alkaloids were suggested to be responsible for the cytotoxic property of Z. rhetsa bark against B16-F10 cells.

  20. Pedunculated malignant melanoma

    Directory of Open Access Journals (Sweden)

    Bhat Ramesha

    1994-01-01

    Full Text Available Pedunculated malignant melanoma is a rare occurrence. A 29 year old woman presented with a pedunculated malignant melanoma on a congenital melanocytic naevus with halo. Pedunculated malignant melanoma is known to have a high incidence of metastasis. The absence of metastasis and the presence of halo, in the case presented, suggests, that the body′s immunological process may have arrested the spread of the melanoma.

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

    Science.gov (United States)

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

    2017-01-01

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

  2. Direct bony invasion of malignant melanoma

    Directory of Open Access Journals (Sweden)

    Mula Viswanath

    2009-01-01

    Full Text Available Malignant melanoma is known to spread by local extention, by the lymphatics by the blood stream. Direct invasion of the bone from a cutaneous melanoma is unknown. Hence, this case is presented in view of its rarity. A 75-year-old Caucasian lady presented with a small papillary lesion in the region of a recurrent chronic cellulitis on the lower third of the lateral aspect of the right leg. Histopathology diagnosed the lesion as locally advanced malignant melanoma. Radiological investigations by X-ray and magnetic resonance imaging revealed malignant infiltration of the tibia in its mid and lower third with two soft tissue metastatic masses adjacent. Histology following amputation confirmed malignant melanoma with cranial resection margin involvement. She underwent a further above-knee amputation followed by chemotherapy. The patient recovered from the amputation but subsequently died 6 months later due to bronchopneumonia from lung metastasis.

  3. Hemosideric heterochromia iridum in malignant melanoma of the choroid.

    Science.gov (United States)

    Awan, K J

    1975-08-01

    A case is reported in which hyperchromic heterochromia iridum developed due to blood staining of an eye with malignant melanoma of the choroid in which massive hemorrhage developed. It is suggested that a possibility of the malignant melanoma of the choroid be kept in mind where hemosiderin deposits are suspected to be the cause of heterochromia but no intraocular iron foreign body is present.

  4. The DNA methylome of human peripheral blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Yingrui Li

    Full Text Available DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per strand, we report a comprehensive (92.62% methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found that 68.4% of CpG sites and 80% displayed allele-specific expression (ASE. These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic research and confirms new sequencing technology as a paradigm for large-scale epigenomics studies.

  5. Are all melanomas dangerous?

    DEFF Research Database (Denmark)

    Nørgaard, Carsten; Glud, Martin; Gniadecki, Robert

    2011-01-01

    The increased incidence of cutaneous malignant melanoma, together with only minor changes in mortality, has brought into question the existence of a melanoma epidemic. The discrepancy between incidence and mortality suggests that most newly diagnosed melanomas have indolent behaviour. This review...

  6. Burden of Melanoma

    NARCIS (Netherlands)

    C. Holterhues (Cynthia)

    2011-01-01

    markdownabstract__Abstract__ Melanoma is a type of skin cancer that arises from melanocytes. More than 95% of all melanomas occur in the skin, but rarely in the pigmented cells of the eye, meninges or mucosa. This thesis will only regard the invasive cutaneous malignant melanomas.

  7. Drug effects on melanoma

    NARCIS (Netherlands)

    Koomen, Elsje Rosalie

    2010-01-01

    Cutaneous melanoma is the most aggressive form of skin cancer and its incidence among Caucasian populations has increased whereas mortality rates are stabilizing or decreasing. The total burden of melanoma is expected to be increasing. As effective treatment options for advanced melanoma are lackin

  8. Drug effects on melanoma

    NARCIS (Netherlands)

    Koomen, Elsje Rosalie

    2010-01-01

    Cutaneous melanoma is the most aggressive form of skin cancer and its incidence among Caucasian populations has increased whereas mortality rates are stabilizing or decreasing. The total burden of melanoma is expected to be increasing. As effective treatment options for advanced melanoma are

  9. Gene expression analysis in human breast cancer associated blood vessels.

    Directory of Open Access Journals (Sweden)

    Dylan T Jones

    Full Text Available Angiogenesis is essential for solid tumour growth, whilst the molecular profiles of tumour blood vessels have been reported to be different between cancer types. Although presently available anti-angiogenic strategies are providing some promise for the treatment of some cancers it is perhaps not surprisingly that, none of the anti-angiogenic agents available work on all tumours. Thus, the discovery of novel anti-angiogenic targets, relevant to individual cancer types, is required. Using Affymetrix microarray analysis of laser-captured, CD31-positive blood vessels we have identified 63 genes that are upregulated significantly (5-72 fold in angiogenic blood vessels associated with human invasive ductal carcinoma (IDC of the breast as compared with blood vessels in normal human breast. We tested the angiogenic capacity of a subset of these genes. Genes were selected based on either their known cellular functions, their enriched expression in endothelial cells and/or their sensitivity to anti-VEGF treatment; all features implicating their involvement in angiogenesis. For example, RRM2, a ribonucleotide reductase involved in DNA synthesis, was upregulated 32-fold in IDC-associated blood vessels; ATF1, a nuclear activating transcription factor involved in cellular growth and survival was upregulated 23-fold in IDC-associated blood vessels and HEX-B, a hexosaminidase involved in the breakdown of GM2 gangliosides, was upregulated 8-fold in IDC-associated blood vessels. Furthermore, in silico analysis confirmed that AFT1 and HEX-B also were enriched in endothelial cells when compared with non-endothelial cells. None of these genes have been reported previously to be involved in neovascularisation. However, our data establish that siRNA depletion of Rrm2, Atf1 or Hex-B had significant anti-angiogenic effects in VEGF-stimulated ex vivo mouse aortic ring assays. Overall, our results provide proof-of-principle that our approach can identify a cohort of

  10. Unexpected similarities between the Schizosaccharomyces and human blood metabolomes, and novel human metabolites.

    Science.gov (United States)

    Chaleckis, Romanas; Ebe, Masahiro; Pluskal, Tomáš; Murakami, Itsuo; Kondoh, Hiroshi; Yanagida, Mitsuhiro

    2014-10-01

    Metabolomics, a modern branch of chemical biology, provides qualitative and quantitative information about the metabolic states of organisms or cells at the molecular level. Here we report non-targeted, metabolomic analyses of human blood, using liquid chromatography-mass spectrometry (LC-MS). We compared the blood metabolome to the previously reported metabolome of the fission yeast, Schizosaccharomyces pombe. The two metabolomic datasets were highly similar: 101 of 133 compounds identified in human blood (75%) were also present in S. pombe, and 45 of 57 compounds enriched in red blood cells (RBCs) (78%) were also present in yeast. The most abundant metabolites were ATP, glutathione, and glutamine. Apart from these three, the next most abundant metabolites were also involved in energy metabolism, anti-oxidation, and amino acid metabolism. We identified fourteen new blood compounds, eight of which were enriched in RBCs: citramalate, GDP-glucose, trimethyl-histidine, trimethyl-phenylalanine, trimethyl-tryptophan, trimethyl-tyrosine, UDP-acetyl-glucosamine, UDP-glucuronate, dimethyl-lysine, glutamate methyl ester, N-acetyl-(iso)leucine, N-acetyl-glutamate, N2-acetyl-lysine, and N6-acetyl-lysine. Ten of the newly identified blood metabolites were also detected in S. pombe, and ten of the 14 newly identified blood metabolites were methylated or acetylated amino acids. Trimethylated or acetylated free amino acids were also abundant in white blood cells. It may be possible to investigate their physiological roles using yeast genetics.

  11. Polymorphism of the human vitronectin gene causes vitronectin blood type.

    Science.gov (United States)

    Kubota, K; Hayashi, M; Oishi, N; Sakaki, Y

    1990-03-30

    Human blood plasma/sera are classified into three distinct vitronectin types based on the relative amount of the 75 kDa polypeptide to its cleavage product of 65 kDa. We asked whether the vitronectin blood types correlated with the polymorphism of the vitronectin gene. A portion of the vitronectin gene was amplified by using polymerase chain reaction and digested with a restriction enzyme PmaC I which may distinguish the base sequence causing the polymorphic change at the amino acid position 381. Amplified DNAs of the blood type I (75 kDa-rich), II (75/65 kDa-even), and III (65 kDa-rich) were shown to be resistant, moderately sensitive and completely sensitive to PmaC I, respectively. These results suggest that Thr at position 381 is essential for the cleavage of the vitronectin 75 kDa polypeptide and that three possible combinations of two codominant alleles of vitronectin determine three vitronectin blood types.

  12. Human Elimination of Organochlorine Pesticides: Blood, Urine, and Sweat Study.

    Science.gov (United States)

    Genuis, Stephen J; Lane, Kevin; Birkholz, Detlef

    2016-01-01

    Background. Many individuals have been exposed to organochlorinated pesticides (OCPs) through food, water, air, dermal exposure, and/or vertical transmission. Due to enterohepatic reabsorption and affinity to adipose tissue, OCPs are not efficiently eliminated from the human body and may accrue in tissues. Many epidemiological studies demonstrate significant exposure-disease relationships suggesting OCPs can alter metabolic function and potentially lead to illness. There is limited study of interventions to facilitate OCP elimination from the human body. This study explored the efficacy of induced perspiration as a means to eliminate OCPs. Methods. Blood, urine, and sweat (BUS) were collected from 20 individuals. Analysis of 23 OCPs was performed using dual-column gas chromatography with electron-capture detectors. Results. Various OCPs and metabolites, including DDT, DDE, methoxychlor, endrin, and endosulfan sulfate, were excreted into perspiration. Generally, sweat samples showed more frequent OCP detection than serum or urine analysis. Many OCPs were not readily detected in blood testing while still being excreted and identified in sweat. No direct correlation was found among OCP concentrations in the blood, urine, or sweat compartments. Conclusions. Sweat analysis may be useful in detecting some accrued OCPs not found in regular serum testing. Induced perspiration may be a viable clinical tool for eliminating some OCPs.

  13. Human Elimination of Organochlorine Pesticides: Blood, Urine, and Sweat Study

    Directory of Open Access Journals (Sweden)

    Stephen J. Genuis

    2016-01-01

    Full Text Available Background. Many individuals have been exposed to organochlorinated pesticides (OCPs through food, water, air, dermal exposure, and/or vertical transmission. Due to enterohepatic reabsorption and affinity to adipose tissue, OCPs are not efficiently eliminated from the human body and may accrue in tissues. Many epidemiological studies demonstrate significant exposure-disease relationships suggesting OCPs can alter metabolic function and potentially lead to illness. There is limited study of interventions to facilitate OCP elimination from the human body. This study explored the efficacy of induced perspiration as a means to eliminate OCPs. Methods. Blood, urine, and sweat (BUS were collected from 20 individuals. Analysis of 23 OCPs was performed using dual-column gas chromatography with electron-capture detectors. Results. Various OCPs and metabolites, including DDT, DDE, methoxychlor, endrin, and endosulfan sulfate, were excreted into perspiration. Generally, sweat samples showed more frequent OCP detection than serum or urine analysis. Many OCPs were not readily detected in blood testing while still being excreted and identified in sweat. No direct correlation was found among OCP concentrations in the blood, urine, or sweat compartments. Conclusions. Sweat analysis may be useful in detecting some accrued OCPs not found in regular serum testing. Induced perspiration may be a viable clinical tool for eliminating some OCPs.

  14. Malignant uveal melanoma and similar lesions studied by computed tomography

    Energy Technology Data Exchange (ETDEWEB)

    Mafee, M.F.; Peyman, G.A.; McKusick, M.A.

    1985-08-01

    Forty-four patients with intraocular disease were studied by computed tomography (CT); in 19 cases malignant uveal melanoma was considered the likely diagnosis. CT proved to be accurate in determining the location and size of uveal melanomas, demonstrating scleral invasion, and differentiating melanoma from choroidal detachment or angioma, toxocariasis, and senile macular degeneration. On CT, uveal melanomas appeared as hyperdense lesions with slight to moderate contrast enhancement. Tumors thinner than 2 mm could not be seen. Using dynamic CT, the authors noted moderate peak amplitude, normal or delayed tissue transit time, and persistently elevated washout phase (downslope), indicating increased permeability as the result of an impaired tumor blood barrier. Histological types of uveal melanoma could not be differentiated on the basis of circulatory patterns. Dynamic CT may be useful in distinguishing uveal melanoma from choroidal hemangioma or hematoma.

  15. Deep coverage mouse red blood cell proteome: a first comparison with the human red blood cell.

    Science.gov (United States)

    Pasini, Erica M; Kirkegaard, Morten; Salerno, Doris; Mortensen, Peter; Mann, Matthias; Thomas, Alan W

    2008-07-01

    Mice have close genetic/physiological relationships to humans, breed rapidly, and can be genetically modified, making them the most used mammal in biomedical research. Because the red blood cell (RBC) is the sole gas transporter in vertebrates, diseases of the RBC are frequently severe; much research has therefore focused on RBC and cardiovascular disorders of mouse and humans. RBCs also host malaria parasites. Recently we presented an in-depth proteome for the human RBC. Here we present directly comparable data for the mouse RBC as membrane-only, soluble-only, and combined membrane-bound/soluble proteomes (comprising, respectively, 247, 232, and 165 proteins). All proteins were identified, validated, and categorized in terms of subcellular localization, protein family, and function, and in comparison with the human RBC, were classified as orthologs, family-related, or unique. Splice isoforms were identified, and polypeptides migrating with anomalous apparent molecular weights were grouped into putatively ubiquitinated or partially degraded complexes. Overall there was close concordance between mouse and human proteomes, confirming the unexpected RBC complexity. Several novel findings in the human proteome have been confirmed here. This comparison sheds light on several open issues in RBC biology and provides a departure point for more comprehensive understanding of RBC function.

  16. Expression of human papilloma virus type 16 E5 protein in amelanotic melanoma cells regulates endo-cellular pH and restores tyrosinase activity

    Directory of Open Access Journals (Sweden)

    Coccia Raffaella

    2009-01-01

    Full Text Available Abstract Background Melanin synthesis, the elective trait of melanocytes, is regulated by tyrosinase activity. In tyrosinase-positive amelanotic melanomas this rate limiting enzyme is inactive because of acidic endo-melanosomal pH. The E5 oncogene of the Human Papillomavirus Type 16 is a small transmembrane protein with a weak transforming activity and a role during the early steps of viral infections. E5 has been shown to interact with 16 kDa subunit C of the trans-membrane Vacuolar ATPase proton pump ultimately resulting in its functional suppressions. However, the cellular effects of such an interaction are still under debate. With this work we intended to explore whether the HPV16 E5 oncoprotein does indeed interact with the vacuolar ATPase proton pump once expressed in intact human cells and whether this interaction has functional consequences on cell metabolism and phenotype. Methods The expression of the HPV16-E5 oncoproteins was induced in two Tyrosinase-positive amelanotic melanomas (the cell lines FRM and M14 by a retroviral expression construct. Modulation of the intracellular pH was measured with Acridine orange and fluorescence microscopy. Expression of tyrosinase and its activity was followed by RT-PCR, Western Blot and enzyme assay. The anchorage-independence growth and the metabolic activity of E5 expressing cells were also monitored. Results We provide evidence that in the E5 expressing cells interaction between E5 and V-ATPase determines an increase of endo-cellular pH. The cellular alkalinisation in turn leads to the post-translational activation of tyrosinase, melanin synthesis and phenotype modulation. These effects are associated with an increased activation of tyrosine analogue anti-blastic drugs. Conclusion Once expressed within intact human cells the HPV16-E5 oncoprotein does actually interact with the vacuolar V-ATPase proton pump and this interaction induces a number of functional effects. In amelanotic melanomas these

  17. Optoelectronic investigation of nanodiamond interactions with human blood

    Science.gov (United States)

    Ficek, M.; Wróbel, M. S.; Wasowicz, M.; Jedrzejewska-Szczerska, M.

    2016-03-01

    We present optoelectronic investigation of in vitro interactions of whole human blood with different nanodiamond biomarkers. Plasmo-chemical modifications of detonation nanodiamond particles gives the possibility for controlling their surface for biological applications. Optical investigations reveal the biological activity of nanodiamonds in blood dependent on its surface termination. We compare different types of nanodiamonds: commercial non-modified detonation nanodiamonds, and nanodiamonds modified by MW PACVD method with H2-termination, and chemically modified nanodiamond with O2-termination. The absorption spectra, and optical microscope investigations were conducted. The results indicate haemocompatibility of non-modified detonation nanodiamond as well as modified nanodiamonds, which enables their application for drug delivery, as well as sensing applications.

  18. Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro.

    Science.gov (United States)

    Kemény, Lajos V; Kurgyis, Zsuzsanna; Buknicz, Tünde; Groma, Gergely; Jakab, Ádám; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B

    2016-06-02

    After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells' nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma-stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments.

  19. Ipilimumab til behandling af metastaserende melanoma

    DEFF Research Database (Denmark)

    Ghasemi, Habib; Schmidt, Henrik; Stolle, Lars Bjørn

    2015-01-01

    Until recently metastatic melanoma was a disease with limited treatment options and a poor prognosis. However, new promising products have been developed. Ipilimumab, a full human anti-cytotoxic T-lymphocyte antigen-4 antibody, has shown improved survival in several clinical trials and is now...... a part of the standard treatment options for this disease in Denmark. In this case report we present a 78-year-old man with metastatic melanoma who had complete remission after treatment with ipilimumab....

  20. Ecology of melanoma cell.

    Science.gov (United States)

    Lacina, Lukáš; Kodet, Ondřej; Dvořánková, Barbora; Szabo, Pavol; Smetana, Karel

    2017-08-29

    Melanoma represents a cancer with increasing incidence worldwide and limited curability of advanced stages of the disease. Similarly to other types of tumors, the microenvironment is an important factor that participates in the control of melanoma biological properties. This review summarizes data regarding the role of the microenvironment, namely fibroblasts, keratinocytes and infiltrating immune cells, on melanoma growth and spreading. The role of embryonic microenvironment on melanoma cell biological properties is also discussed. The potential of therapeutic targeting of the melanoma microenvironment is demonstrated.

  1. Distribution of Blood Groups(ABO between Symptomatic & Asymptomatic Human Leishmania Infantum Infection in Human

    Directory of Open Access Journals (Sweden)

    S Molaie

    2013-09-01

    Full Text Available Abstract Background & aim: According to the hypothesis that leishmania parasites can be escaped from immune system covered by blood group antigens (ABO to prevent its recognition by the immune system. The aim of this study was to show the associated blood groups with symptomatic or asymptomatic visceral leishmaniasis due to Leishmania infantum in human. Methods: In this cross-sectional study the population was divided into two groups. The first group included 54 patients with kala-azar (antibody against Leishmania titers ≥1:3200 by TDA with clinical specificity and the second group consisted of 45 subjects infected with Leishmania infantum (Leishmania antibody titers of1: 800 and 1:1600 by DAT method and non-specific symptoms. The distribution of the 4 main blood groups ABO type, sex, age, presence or absence of symptoms, clinical signs, and response to Glucantim therapy and DAT results were evaluated. Data were analyzed by chi-square test. Results: Most of the patients in group 1 were blood group A (37% and the lowest number of blood group were B (12.8%. In the second group, most of the ABO blood group A (42.2% and lowest in the ABO blood group AB (8.9%.There was no significant association between blood groups and clinical symptoms (p>0.05. Conclusion: This study showed that there is no association between blood group and incidence of symptomatic and asymptomatic kala-azar. Key words: Leishmania Infantum, Kala-azar, Blood Group, Human

  2. Up-regulation of Human Leukocyte Antigen G Expression in Primary Cutaneous Malignant Melanoma Associated with Host-vs-tumor Immune Response

    Institute of Scientific and Technical Information of China (English)

    Xianfeng FANG; Xuxin ZHANG; Jiawen LI

    2008-01-01

    Human leukocyte antigen G (HLA-G) is one of the molecules implicated in immunotolerance. To investigate the role of HLA-G in primary cutaneous malignant melanoma (CMM), a series of 47 skin melanocytic lesions were immunohistochemicaily evaluated. The correlation between HLA-G expression and CMM clinicohistopahtologicai data and Bcl-2 expression was also analyzed.HLA-G expression was detected in a variety of cell types. No significant difference in HLA-G expression was observed between malignant and non-malignant melanocytic lesions. HLA-G expres- sion was significantly correlated with the inflammatory infiltration and Bel-2 expression, whereas no significant correlation with ulceration, tumor thickness, clinical stage, histopathologicai subtypes were observed. HLA-G expression may be the result of host immune reaction in tumor microenvi- ronment rather than a malignant feature of CMM.

  3. A novel fully-humanised 3D skin equivalent to model early melanoma invasion

    Science.gov (United States)

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

    2015-01-01

    Metastatic melanoma remains incurable, emphasising the acute need for improved research models to investigate the underlying biological mechanisms mediating tumour 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-humanised 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 tumour invasion in vivo. Comparison of our novel 3D melanoma skin equivalent with melanoma in situ and metastatic melanoma indicates 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

  4. Malignant Melanoma of the Foot

    Science.gov (United States)

    ... page. Please enable Javascript in your browser. Malignant Melanoma of the Foot What Is Malignant Melanoma? Melanoma is a cancer that begins in the ... people of all age groups, even the young. Melanoma in the Foot Melanoma that occurs in the ...

  5. Peptidome profiling of human serum of uveal melanoma patients based on magnetic bead fractionation and mass spectrometry

    Directory of Open Access Journals (Sweden)

    Xiang-Yu Shi

    2017-06-01

    Full Text Available AIM: To find new biomarkers for uveal melanoma (UM by analyzing the serum peptidome profile. METHODS: Proteomic spectra in patients with UM before and after operation were analyzed and compared with those of healthy controls. Magnetic affinity beads were used to capture serum peptides and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF mass spectrometer were used to compile serum peptide profiles. RESULTS: A panel of 49 peptides were differentially expressed between UM patients and controls, of which 33 peptides were of higher intensities in patient group and 16 peptides were of higher intensities in control group. Based on combined use of these potential markers, peptides with mean molecular masses of 1467 and 9289.0 Da provide high sensitivity (83.3%, specificity (100% and accuracy rate (93.0% together to differentiate melanoma patients from healthy controls. At the time point of 6mo postoperatively, the levels of many peptides differentially expressed before surgery showed no more statistical difference between the patients and the control group. Fibrinogen α-chain precursors were identified as potential UM markers. CONCLUSION: We have shown that a convenient and fast proteomic technique, affinity bead separation and MALDI-TOF analysis combined with bioinformatic software, facilitates the identification of novel biomarkers for UM.

  6. Interaction of human malignant melanoma (ST-ML-12) tumor spheroids with endothelial cell monolayers. Damage to endothelium by oxygen-derived free radicals.

    Science.gov (United States)

    Offner, F. A.; Wirtz, H. C.; Schiefer, J.; Bigalke, I.; Klosterhalfen, B.; Bittinger, F.; Mittermayer, C.; Kirkpatrick, C. J.

    1992-01-01

    Clinical and experimental observations suggest that tumor-induced endothelial cell injury may be one of several initial events in the establishment of tumor metastases. To test this hypothesis, the authors have analyzed the interaction of malignant melanoma (ST-ML-12) multicenter tumor spheroids with endothelial cell monolayers in a three-dimensional coculture system. After 1.5 hours of interaction, the authors observed a toxic effect on endothelial cells in the perispheroid region. The latter was demonstrated by testing membrane integrity with the fluorescent probes acridine orange/ethidium bromide and resulted in sensitivity to shear stress of the damaged cells. The endothelium then underwent a regenerative cycle to replace the denuded halo. Addition of the oxygen radical-scavenging enzyme superoxide dismutase to the culture medium prevented this endothelial cell damage in a dose-dependent manner for up to 12 hours. By contrast, catalase, deferoxamine mesylate, allopurinol, and the proteinase inhibitors soybean trypsin inhibitor and aprotinin were not protective under the same conditions. The endothelial damage was dependent on the attachment of the spheroids. Medium conditioned by ST-ML-12-spheroids proved to be ineffective. A similar, but less prominent, deleterious effect was seen when human peritoneal mesothelial cells were used in place of the human umbilical vein endothelial cells. Spheroids of the uroepithelial cell line HU-609 were used as control. No toxicity was observed in these cocultures. Melanin biosynthesis is associated with the production of oxygen-derived free radicals. The results suggest a possible implication of these free radicals in metastasis formation of malignant melanoma. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:1519667

  7. Human Umbilical Cord Blood for Transplantation Therapy in Myocardial Infarction.

    Science.gov (United States)

    Acosta, Sandra A; Franzese, Nick; Staples, Meaghan; Weinbren, Nathan L; Babilonia, Monica; Patel, Jason; Merchant, Neil; Simancas, Alejandra Jacotte; Slakter, Adam; Caputo, Mathew; Patel, Milan; Franyuti, Giorgio; Franzblau, Max H; Suarez, Lyanne; Gonzales-Portillo, Chiara; Diamandis, Theo; Shinozuka, Kazutaka; Tajiri, Naoki; Sanberg, Paul R; Kaneko, Yuji; Miller, Leslie W; Borlongan, Cesar V

    2013-07-01

    Cell-based therapy is a promising therapy for myocardial infarction. Endogenous repair of the heart muscle after myocardial infarction is a challenge because adult cardiomyocytes have a limited capacity to proliferate and replace damaged cells. Pre-clinical and clinical evidence has shown that cell based therapy may promote revascularization and replacement of damaged myocytes after myocardial infarction. Adult stem cells can be harvested from different sources including bone marrow, skeletal myoblast, and human umbilical cord blood cells. The use of these cells for the repair of myocardial infarction presents various advantages over other sources of stem cells. Among these are easy harvesting, unlimited differentiation capability, and robust angiogenic potential. In this review, we discuss the milestone findings and the most recent evidence demonstrating the therapeutic efficacy and safety of the transplantation of human umbilical cord blood cells as a stand-alone therapy or in combination with gene therapy, highlighting the importance of optimizing the timing, dose and delivery methods, and a better understanding of the mechanisms of action that will guide the clinical entry of this innovative treatment for ischemic disorders, specifically myocardial infarction.

  8. Blood-Brain Barrier Breakdown in the Aging Human Hippocampus

    Science.gov (United States)

    Montagne, Axel; Barnes, Samuel R.; Sweeney, Melanie D.; Halliday, Matthew R.; Sagare, Abhay P.; Zhao, Zhen; Toga, Arthur W.; Jacobs, Russell E.; Liu, Collin Y.; Amezcua, Lilyana; Harrington, Michael G.; Chui, Helena C.; Law, Meng; Zlokovic, Berislav V.

    2014-01-01

    Summary The blood-brain barrier (BBB) limits entry of blood-derived products, pathogens and cells into the brain that is essential for normal neuronal functioning and information processing. Post-mortem tissue analysis indicates BBB damage in Alzheimer’s disease (AD). The timing of BBB breakdown remains, however, elusive. Using an advanced dynamic contrast-enhanced magnetic resonance imaging protocol with high spatial and temporal resolutions to quantify regional BBB permeability in the living human brain, we show an age-dependent BBB breakdown in the hippocampus, a region critical for learning and memory that is affected early in AD. The BBB breakdown in the hippocampus and its CA1 and dentate gyrus subdivisions worsened with mild cognitive impairment that correlated with injury to BBB-associated pericytes, as shown by the cerebrospinal fluid analysis. Our data suggest that BBB breakdown is an early event in the aging human brain that begins in the hippocampus and may contribute to cognitive impairment. PMID:25611508

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

    Science.gov (United States)

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

    2014-06-01

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

  10. Functional status and relationships of melanocortin 1 receptor signaling to the cAMP and extracellular signal-regulated protein kinases 1 and 2 pathways in human melanoma cells.

    Science.gov (United States)

    Herraiz, Cecilia; Journé, Fabrice; Ghanem, Ghanem; Jiménez-Cervantes, Celia; García-Borrón, José C

    2012-12-01

    Melanocortin 1 receptor (MC1R), a major determinant of skin phototype frequently mutated in melanoma, is a Gs protein-coupled receptor that regulates pigment production in melanocytes. MC1R stimulation activates cAMP synthesis and the extracellular signal-regulated (ERK) ERK1 and ERK2. In human melanocytes, ERK activation by MC1R relies on cAMP-independent transactivation of the c-KIT receptor. Thus MC1R functional coupling to the cAMP and ERK pathways may involve different structural requirements giving raise to biased effects of skin cancer-associated mutations. We evaluated the impact of MC1R mutations on ERK activation, cAMP production and agonist binding. We found that MC1R mutations impair cAMP production much more often than ERK activation, suggesting less stringent requirements for functional coupling to the ERK pathway. We examined the crosstalk of the cAMP and ERK pathways in HBL human melanoma cells (wild-type for MC1R, NRAS and BRAF). ERK activation by constitutively active upstream effectors or pharmacological inhibition had little effect on MC1R-stimulated cAMP synthesis. High cAMP levels were compatible with normal ERK activation but, surprisingly, the adenylyl cyclase activator forskolin abolished ERK activation by MC1R, most likely by a cAMP-independent mechanism. These results indicate little crosstalk of the cAMP and ERK pathways in HBL melanoma cells. Finally, we studied cAMP accumulation in a panel of 22 human melanoma cell lines stimulated with MC1R agonists or forskolin. cAMP synthesis was often inhibited, even in cells wild-type for MC1R and NRAS. Therefore, the cAMP pathway is more frequently impaired in melanoma than could be predicted by the MC1R or NRAS genotype.

  11. How Is Melanoma Skin Cancer Diagnosed?

    Science.gov (United States)

    ... Early Detection, Diagnosis, and Staging Tests for Melanoma Skin Cancer Most melanomas are brought to a doctor’s attention ... Melanoma Skin Cancer, by Stage More In Melanoma Skin Cancer About Melanoma Skin Cancer Causes, Risk Factors, and ...

  12. Importance of glycolysis and oxidative phosphorylation in advanced melanoma

    Directory of Open Access Journals (Sweden)

    Ho Jonhan

    2012-10-01

    Full Text Available Abstract Serum lactate dehydrogenase (LDH is a prognostic factor for patients with stage IV melanoma. To gain insights into the biology underlying this prognostic factor, we analyzed total serum LDH, serum LDH isoenzymes, and serum lactate in up to 49 patients with metastatic melanoma. Our data demonstrate that high serum LDH is associated with a significant increase in LDH isoenzymes 3 and 4, and a decrease in LDH isoenzymes 1 and 2. Since LDH isoenzymes play a role in both glycolysis and oxidative phosphorylation (OXPHOS, we subsequently determined using tissue microarray (TMA analysis that the levels of proteins associated with mitochondrial function, lactate metabolism, and regulators of glycolysis were all elevated in advanced melanomas compared with nevic melanocytes. To investigate whether in advanced melanoma, the glycolysis and OXPHOS pathways might be linked, we determined expression of the monocarboxylate transporters (MCT 1 and 4. Analysis of a nevus-to-melanoma progression TMA revealed that MCT4, and to a lesser extend MCT1, were elevated with progression to advanced melanoma. Further analysis of human melanoma specimens using the Seahorse XF24 extracellular flux analyzer indicated that metastatic melanoma tumors derived a large fraction of energy from OXPHOS. Taken together, these findings suggest that in stage IV melanomas with normal serum LDH, glycolysis and OXPHOS may provide metabolic symbiosis within the same tumor, whereas in stage IV melanomas with high serum LDH glycolysis is the principle source of energy.

  13. Copy number expansion of the STX17 duplication in melanoma tissue from Grey horses

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    Sundström Elisabeth

    2012-08-01

    Full Text Available Abstract Background Greying with age in horses is an autosomal dominant trait, associated with loss of hair pigmentation, melanoma and vitiligo-like depigmentation. We recently identified a 4.6 kb duplication in STX17 to be associated with the phenotype. The aims of this study were to investigate if the duplication in Grey horses shows copy number variation and to exclude that any other polymorphism is uniquely associated with the Grey mutation. Results We found little evidence for copy number expansion of the duplicated sequence in blood DNA from Grey horses. In contrast, clear evidence for copy number expansions was indicated in five out of eight tested melanoma tissues or melanoma cell lines. A tendency of a higher copy number in aggressive tumours was also found. Massively parallel resequencing of the ~350 kb Grey haplotype did not reveal any additional mutations perfectly associated with the phenotype, confirming the duplication as the true causative mutation. We identified three SNP alleles that were present in a subset of Grey haplotypes within the 350 kb region that shows complete linkage disequilibrium with the causative mutation. Thus, these three nucleotide substitutions must have occurred subsequent to the duplication, consistent with our interpretation that the Grey mutation arose more than 2,000 years before present. Conclusions These results suggest that the mutation acts as a melanoma-driving regulatory element. The elucidation of the mechanistic features of the duplication will be of considerable interest for the characterization of these horse melanomas as well as for the field of human melanoma research.

  14. Detection of ABCB5-tumour-antigen-specific CD8(+) T cells in Melanoma Patients and Implications for Immunotherapy.

    Science.gov (United States)

    Borchers, Sylvia; Masslo, Christoph; Müller, Christina Ann; Tahedl, Anika; Volkind, Jennifer; Nowak, Yvonne; Umansky, Viktor; Esterlechner, Jasmina; Frank, Markus Hermann; Ganss, Christoph; Kluth, Mark Andreas; Utikal, Jochen

    2017-09-23

    ABCB5 has been identified as a tumour initiating cell marker and is expressed in various malignancies, including melanoma. Moreover, treatment with anti-ABCB5 monoclonal antibodies has been shown to inhibit tumour growth in xenotransplantation models. Therefore, ABCB5 represents a potential target for cancer immunotherapy. However, cellular immune responses against ABCB5 in humans have not been described so far. Here, we investigated whether ABCB5-reactive T cells are present in human melanoma patients and tested the applicability of ABCB5-derived peptides for experimental induction of human T cell responses. Peripheral blood mononuclear cells (PBMNC) isolated from blood samples of melanoma patients (n=40) were stimulated with ABCB5 peptides, followed by intracellular cytokine staining (ICS) for IFN-γ and TNF-α. To evaluate immunogenicity of ABCB5 peptides in naïve healthy donors, CD8 T cells were co-cultured with ABCB5 antigen-loaded autologous dendritic cells (DC). ABCB5-reactivity in expanded T cells was likewise assessed by ICS. ABCB5-reactive CD8(+) T cells were detected ex vivo in 19 of 29 patients, MART-1-reactive CD8 T cells in 6 of 21 patients. In this small, heterogeneous cohort, reactivity against ABCB5 was significantly higher than against MART-1. It occurred significantly more often and independent of clinical characteristics. Reactivity against ABCB5 could be induced in 14 of 16 healthy donors in vitro by repeated stimulation with peptide-loaded autologous DC. Since ABCB5-reactive CD8 T cells can be found in the peripheral blood of melanoma patients and an ABCB5-specific response can be induced in vitro in naïve donors, ABCB5 could be a new target for immuno-therapies in melanoma. This article is protected by copyright. All rights reserved. © 2017 British Society for Immunology.

  15. Gamma-Secretase/Notch Signalling Pathway Inhibitor RO4929097 in Treating Patients With Stage IV Melanoma

    Science.gov (United States)

    2016-05-06

    Acral Lentiginous Malignant Melanoma; Lentigo Maligna Malignant Melanoma; Nodular Malignant Melanoma; Recurrent Melanoma; Solar Radiation-related Skin Melanoma; Stage IV Melanoma; Superficial Spreading Malignant Melanoma

  16. CDC Vital Signs: Preventing Melanoma

    Science.gov (United States)

    ... MMWR Science Clips Error processing SSI file Preventing Melanoma Communities Play a Vital Role Language: English Español ( ... and use of indoor tanning by minors. Problem Melanoma is increasing. Melanoma skin cancer is common and ...

  17. 78 FR 32668 - Draft Guidance for Industry: Changes to an Approved Application: Biological Products: Human Blood...

    Science.gov (United States)

    2013-05-31

    ...The Food and Drug Administration (FDA) is announcing the availability of a draft document entitled ``Guidance for Industry: Changes to an Approved Application: Biological Products: Human Blood and Blood Components Intended for Transfusion or for Further Manufacture'' dated June 2013. The draft guidance document provides manufacturers of licensed Whole Blood and blood components intended for......

  18. Melanoma immunotherapy: dendritic cell vaccines

    OpenAIRE

    Lozada-Requena, Ivan; Laboratorios de Inmunología #108, Laboratorio de investigación y Desarrollo, Facultad de Ciencieas y Filosofía, Universidad Cayetano Heredia. Lima, Perú Empresa de Investigación y Desarrollo en Cáncer (EMINDES) SAC. Lima, Perú.; Núñez, César; Empresa de Investigación y Desarrollo en Cáncer (EMINDES) SAC. Lima, Perú.; Aguilar, José Luis; Laboratorios de Inmunología #108, Laboratorio de investigación y Desarrollo, Facultad de Ciencieas y Filosofía, Universidad Cayetano Heredia. Lima, Perú.

    2015-01-01

    This is a narrative review that shows accessible information to the scientific community about melanoma and immunotherapy.Dendritic cells have the ability to participate in innate and adaptive immunity, but are not unfamiliar to the immune evasion oftumors. Knowing the biology and role has led to generate in vitro several prospects of autologous cell vaccines against diversetypes of cancer in humans and animal models. However, given the low efficiency they have shown, we must implementstrateg...

  19. Gold nanoparticles-based brachytherapy enhancement in choroidal melanoma using a full Monte Carlo modelling of human eye

    CERN Document Server

    Asadi, Somayeh; Masoudi, S Farhad; Rahmani, Faezeh

    2014-01-01

    Materials of high atomic number such as gold, can provide a high probability for photon interaction by photoelectric effects during radiation therapy. In cancer therapy, the object of brachytherapy as a kind of radiotherapy is to deliver adequate radiation dose to tumor while sparing surrounding healthy tissue. Several studies demonstrated that the preferential accumulation of gold nanoparticles within the tumor can enhance the absorbed dose by the tumor without increasing the radiation dose delivered externally. Accordingly, the required time for tumor irradiation decreases as the estimated adequate radiation dose for tumor is provided following this method. The dose delivered to healthy tissue is reduced when the time of irradiation is decreased. Hear, GNPs effects on choroidal Melanoma dosimetry is discussed by Monte Carlo study. Monte Carlo Ophthalmic brachytherapy dosimetry usually, is studied by simulation of water phantom. Considering the composition and density of eye material instead of water in thes...

  20. Biosynthesis and turnover of O-acetyl and N-acetyl groups in the gangliosides of human melanoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Manzi, A.E.; Sjoberg, E.R.; Diaz, S.; Varki, A.

    1990-08-05

    We and others previously described the melanoma-associated oncofetal glycosphingolipid antigen 9-O-acetyl-GD3, a disialoganglioside O-acetylated at the 9-position of the outer sialic acid residue. We have now developed methods to examine the biosynthesis and turnover of disialogangliosides in cultured melanoma cells and in Golgi-enriched vesicles from these cells. O-Acetylation was selectively expressed on di- and trisialogangliosides, but not on monosialogangliosides, nor on glycoprotein-bound sialic acids. Double-labeling of cells with (3H)acetate and (14C)glucosamine introduced easily detectable labels into each of the components of the ganglioside molecules. Pulse-chase studies of such doubly labeled molecules indicated that the O-acetyl groups turn over faster than the parent molecule. When Golgi-enriched vesicles from these cells were incubated with (acetyl-3H)acetyl-coenzyme A, the major labeled products were disialogangliosides. (Acetyl-3H)O-acetyl groups were found at both the 7- and the 9-positions, indicating that both 7-O-acetyl GD3 and 9-O-acetyl GD3 were synthesized by the action of O-acetyltransferase(s) on endogenous GD3. Analysis of the metabolically labeled molecules confirmed the existence of both 7- and 9-O-acetylated GD3 in the intact cells. Surprisingly, the major 3H-labeled product of the in vitro labeling reaction was not O-acetyl-GD3, but GD3, with the label exclusively in the sialic acid residues. Fragmentation of the labeled sialic acids by enzymatic and chemical methods showed that the 3H-label was exclusively in (3H)N-acetyl groups. Analyses of the double-labeled sialic acids from intact cells also showed that the 3H-label from (3H)acetate was exclusively in the form of (3H)N-acetyl groups, whereas the 14C-label was at the 4-position.

  1. Amyloid β levels in human red blood cells.

    Directory of Open Access Journals (Sweden)

    Takehiro Kiko

    Full Text Available UNLABELLED: Amyloid β-peptide (Aβ is hypothesized to play a key role by oxidatively impairing the capacity of red blood cells (RBCs to deliver oxygen to the brain. These processes are implicated in the pathogenesis of Alzheimer's disease (AD. Although plasma Aβ has been investigated thoroughly, the presence and distribution of Aβ in human RBCs are still unclear. In this study, we quantitated Aβ40 and Aβ42 in human RBCs with ELISA assays, and provided evidence that significant amounts of Aβ could be detected in RBCs and that the RBC Aβ levels increased with aging. The RBC Aβ levels increased with aging. On the other hand, providing an antioxidant supplement (astaxanthin, a polar carotenoid to humans was found to decrease RBC Aβ as well as oxidative stress marker levels. These results suggest that plasma Aβ40 and Aβ42 bind to RBCs (possibly with aging, implying a pathogenic role of RBC Aβ. Moreover, the data indicate that RBC Aβ40 and Aβ42 may constitute biomarkers of AD. As a preventive strategy, therapeutic application of astaxanthin as an Aβ-lowering agent in RBCs could be considered as a possible anti-dementia agent. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN42483402.

  2. [Targeted therapies for melanoma].

    Science.gov (United States)

    Leiter, U; Meier, F; Garbe, C

    2014-07-01

    Since the discovery of activating mutations in the BRAF oncogene and also stimulation of immune mediated antitumor response in melanoma, there has been remarkable progress in the development of targeted therapies for unresectable and metastatic melanoma. This article addresses the latest developments of BRAF/MEK/ERK pathway signaling. In addition, the development of drugs to attack alternative mutations in melanoma, such as NRAS and KIT is described. Strategies for the management of BRAF inhibitor resistance, such as with combination therapy, are outlined. Antitumor immune therapies with monoclonal antibodies such as ipilimumab which acts by promoting T-cell activation or antibody blockade of programmed death-1 (PD-1) led to a long term response in metastatic melanoma. Results of latest clinical studies including the toxicity profile are described. Due to selective kinase inhibitors and immune checkpoint blockade, the therapy of unresectable metastatic melanoma has greatly improved and long-term survival of patients with metastatic melanoma seems a real possibility.

  3. Decoding Melanoma Metastasis

    Energy Technology Data Exchange (ETDEWEB)

    Damsky, William E. Jr. [Department of Dermatology, Yale School of Medicine, New Haven, Connecticut (United States); Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont (United States); Rosenbaum, Lara E.; Bosenberg, Marcus, E-mail: Marcus.Bosenberg@yale.edu [Department of Dermatology, Yale School of Medicine, New Haven, Connecticut (United States)

    2010-12-30

    Metastasis accounts for the vast majority of morbidity and mortality associated with melanoma. Evidence suggests melanoma has a predilection for metastasis to particular organs. Experimental analyses have begun to shed light on the mechanisms regulating melanoma metastasis and organ specificity, but these analyses are complicated by observations of metastatic dormancy and dissemination of melanocytes that are not yet fully malignant. Additionally, tumor extrinsic factors in the microenvironment, both at the site of the primary tumor and the site of metastasis, play important roles in mediating the metastatic process. As metastasis research moves forward, paradigms explaining melanoma metastasis as a step-wise process must also reflect the temporal complexity and heterogeneity in progression of this disease. Genetic drivers of melanoma as well as extrinsic regulators of disease spread, particularly those that mediate metastasis to specific organs, must also be incorporated into newer models of melanoma metastasis.

  4. The Danish Melanoma Database

    DEFF Research Database (Denmark)

    Hölmich, Lisbet Rosenkrantz; Klausen, Siri; Spaun, Eva;

    2016-01-01

    AIM OF DATABASE: The aim of the database is to monitor and improve the treatment and survival of melanoma patients. STUDY POPULATION: All Danish patients with cutaneous melanoma and in situ melanomas must be registered in the Danish Melanoma Database (DMD). In 2014, 2,525 patients with invasive...... melanoma and 780 with in situ tumors were registered. The coverage is currently 93% compared with the Danish Pathology Register. MAIN VARIABLES: The main variables include demographic, clinical, and pathological characteristics, including Breslow's tumor thickness, ± ulceration, mitoses, and tumor...... quality register. The coverage is high, and the performance in the five Danish regions is quite similar due to strong adherence to guidelines provided by the Danish Melanoma Group. The list of monitored indicators is constantly expanding, and annual quality reports are issued. Several important scientific...

  5. Genetics of familial melanoma

    DEFF Research Database (Denmark)

    Aoude, Lauren G; Wadt, Karin A W; Pritchard, Antonia L

    2015-01-01

    Twenty years ago, the first familial melanoma susceptibility gene, CDKN2A, was identified. Two years later, another high-penetrance gene, CDK4, was found to be responsible for melanoma development in some families. Progress in identifying new familial melanoma genes was subsequently slow; however......, with the advent of next-generation sequencing, a small number of new high-penetrance genes have recently been uncovered. This approach has identified the lineage-specific oncogene MITF as a susceptibility gene both in melanoma families and in the general population, as well as the discovery of telomere...... maintenance as a key pathway underlying melanoma predisposition. Given these rapid recent advances, this approach seems likely to continue to pay dividends. Here, we review the currently known familial melanoma genes, providing evidence that most additionally confer risk to other cancers, indicating...

  6. Decoding Melanoma Metastasis

    Directory of Open Access Journals (Sweden)

    Marcus Bosenberg

    2010-12-01

    Full Text Available Metastasis accounts for the vast majority of morbidity and mortality associated with melanoma. Evidence suggests melanoma has a predilection for metastasis to particular organs. Experimental analyses have begun to shed light on the mechanisms regulating melanoma metastasis and organ specificity, but these analyses are complicated by observations of metastatic dormancy and dissemination of melanocytes that are not yet fully malignant. Additionally, tumor extrinsic factors in the microenvironment, both at the site of the primary tumor and the site of metastasis, play important roles in mediating the metastatic process. As metastasis research moves forward, paradigms explaining melanoma metastasis as a step-wise process must also reflect the temporal complexity and heterogeneity in progression of this disease. Genetic drivers of melanoma as well as extrinsic regulators of disease spread, particularly those that mediate metastasis to specific organs, must also be incorporated into newer models of melanoma metastasis.

  7. Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders

    OpenAIRE

    2009-01-01

    Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells, such as postnatal fibroblasts to iPS cells, which resemble adherent embryonic stem cells. Here we report derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for disease modeling. Multiple human iPS ...

  8. Microdialysis in the femoral head of the minipig and in a blood cloth of human blood

    DEFF Research Database (Denmark)

    Bøgehøj, Morten Foged; Emmeluth, Claus; Overgaard, Søren

    2011-01-01

    Introduction Microdialysis can detect ischemia in soft tissue. In a previous study, we have shown the development of ischemia in the femoral head removed from patients undergoing total hip replacement. That study also raised some methodological questions that this study tries to answer: what...... is happening in the dead space around the catheter in the drill canal, and is there an equilibrium period after the insertion of the catheter? Material and methods In an ex-vivo study using 5 syringes with 5 mL human blood, a microdialysis catheter was inserted and microdialysis was performed over 3 h....... In an in-vivo study, a drill hole was made in the proximal part of the femur in 6 mature Göttingen minipigs and microdialysis was performed over 3 h. The pigs were kept normoventilated during the experiment. Results The ex-vivo microdialysis results showed that lactate kept a steady level and glucose...

  9. Metallic copper nanoparticles induce apoptosis in a human skin melanoma A-375 cell line

    Science.gov (United States)

    Chakraborty, Ruchira; Basu, Tarakdas

    2017-03-01

    In two earlier communications (Chatterjee et al 2012 Nanotechnology 23 085103, Chatterjee et al 2014 Nanotechnology 25 135101), we reported the development of a simple and unique method of synthesizing highly stable metallic copper nanoparticles (Cu NPs) with high antibacterial activity. Here we report on the cytotoxic potency of the NPs against cancer cells. The value of the IC50 dose of the Cu NPs against human skin cancer cell A-375 was found to be 1.71 μg ml‑1 only, which was much less than values reported so far, and this concentration had no cytotoxic effect on normal white blood cells. The NPs caused (i) lowering of cell membrane rigidity, (ii) DNA degradation, (iii) chromosomal condensation, (iv) cell cycle arrest in the G2/M phase, (v) depolarization of the mitochondrial membrane and (vi) apoptosis of cells. Cellular apoptosis occurred in the caspase-9-mediated intrinsic pathway. This study revealed that our Cu NPs had high anticancer properties by killing tumor cells through the apoptotic pathway. Since this particle has high antibacterial activity, our Cu NPs might be developed in future as a dual action drug—anticancer as well as antibacterial.

  10. PCR amplification of Bartonella koehlerae from human blood and enrichment blood cultures

    Directory of Open Access Journals (Sweden)

    Breitschwerdt Edward B

    2010-08-01

    Full Text Available Abstract Background Cats appear to be the primary reservoir host for Bartonella koehlerae, an alpha Proteobacteria that is most likely transmitted among cat populations by fleas (Ctenocephalides felis. Bartonella koehlerae has caused endocarditis in a dog and in one human patient from Israel, but other clinically relevant reports involving this bacterium are lacking. Despite publication of numerous, worldwide epidemiological studies designed to determine the prevalence of Bartonella spp. bacteremia in cats, B. koehlerae has never been isolated using conventional blood agar plates. To date, successful isolation of B. koehlerae from cats and from the one human endocarditis patient has consistently required the use of chocolate agar plates. Results In this study, Bartonella koehlerae bacteremia was documented in eight immunocompetent patients by PCR amplification and DNA sequencing, either prior to or after enrichment blood culture using Bartonella alpha Proteobacteria growth medium. Presenting symptoms most often included fatigue, insomnia, joint pain, headache, memory loss, and muscle pain. Four patients were also infected with Bartonella vinsonii subsp. berkhoffii genotype II. After molecular documentation of B. koehlerae infection in these patients, a serological test was developed and serum samples were tested retrospectively. Bartonella koehlerae antibodies were not detected (titers B. koehlerae antibody titers of 1:64 or greater. Conclusions Although biased by a study population consisting of individuals with extensive arthropod and animal exposure, the results of this study suggest that B. koehlerae bacteremia is more common in immunocompetent people than has been previously suspected. Future studies should more thoroughly define modes of transmission and risk factors for acquiring infection with B. koehlerae. In addition, studies are needed to determine if B. koehlerae is a cause or cofactor in the development of arthritis, peripheral

  11. Copper(II) complexes encapsulated in human red blood cells.

    Science.gov (United States)

    Bonomo, R P; De Flora, A; Rizzarelli, E; Santoro, A M; Tabbí, G; Tonetti, M

    1995-09-01

    Copper(II) complexes were encapsulated in human red blood cells in order to test their possible use as antioxidant drugs by virtue of their labile character. ESR spectroscopy was used to verify whether encapsulation in red blood cells leads to the modification of such complexes. With copper(II) complexes bound to dipeptides or tripeptides, an interaction with hemoglobin was found to be present, the hemoglobin having a strong coordinative site formed by four nitrogen donor atoms. Instead, with copper(II) complexes with TAD or PheANN3, which have the greatest stability. ESR spectra always showed the original species. Only the copper(II) complex with GHL gave rise to a complicated behavior, which contained signals from iron(III) species probably coming from oxidative processes. Encapsulation of all copper(II) complexes in erythrocytes caused a slight oxidative stress, compared to the unloaded and to the native cells. However, no significant differences were observed in the major metabolic properties (GSH, glycolytic rate, hexose monophosphate shunt, Ca(2+)-ATPase) of erythrocytes loaded with different copper(II) complexes, with the exception of methemoglobin levels, which were markedly increased in the case of [Cu(GHL)H-1] compared to [Cu(TAD)]. This latter finding suggests that methemoglobin formation can be affected by the type of complex used for encapsulation, depending on the direct interaction of the copper(II) complex with hemoglobin.

  12. Human Umbilical Cord Blood Cell Transplantation in Neuroregenerative Strategies

    Directory of Open Access Journals (Sweden)

    Luisa R. Galieva

    2017-09-01

    Full Text Available At present there is no effective treatment of pathologies associated with the death of neurons and glial cells which take place as a result of physical trauma or ischemic lesions of the nervous system. Thus, researchers have high hopes for a treatment based on the use of stem cells (SC, which are potentially able to replace dead cells and synthesize neurotrophic factors and other molecules that stimulate neuroregeneration. We are often faced with ethical issues when selecting a source of SC. In addition to precluding these, human umbilical cord blood (hUCB presents a number of advantages when compared with other sources of SC. In this review, we consider the key characteristics of hUCB, the results of various studies focused on the treatment of neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, ischemic (stroke and traumatic injuries of the nervous system and the molecular mechanisms of hUCB-derived mononuclear and stem cells.

  13. Generation of induced pluripotent stem cells from human cord blood.

    Science.gov (United States)

    Haase, Alexandra; Olmer, Ruth; Schwanke, Kristin; Wunderlich, Stephanie; Merkert, Sylvia; Hess, Christian; Zweigerdt, Robert; Gruh, Ina; Meyer, Johann; Wagner, Stefan; Maier, Lars S; Han, Dong Wook; Glage, Silke; Miller, Konstantin; Fischer, Philipp; Schöler, Hans R; Martin, Ulrich

    2009-10-02

    Induced pluripotent stem cells (iPSCs) may represent an ideal cell source for future regenerative therapies. A critical issue concerning the clinical use of patient-specific iPSCs is the accumulation of mutations in somatic (stem) cells over an organism's lifetime. Acquired somatic mutations are passed onto iPSCs during reprogramming and may be associated with loss of cellular functions and cancer formation. Here we report the generation of human iPSCs from cord blood (CB) as a juvenescent cell source. CBiPSCs show characteristics typical of embryonic stem cells and can be differentiated into derivatives of all three germ layers, including functional cardiomyocytes. For future therapeutic production of autologous and allogeneic iPSC derivatives, CB could be routinely harvested for public and commercial CB banks without any donor risk. CB could readily become available for pediatric patients and, in particular, for newborns with genetic diseases or congenital malformations.

  14. Manipulation on human red blood cells with femtosecond optical tweezers

    Institute of Scientific and Technical Information of China (English)

    Ming Zhou; Haifeng Yang; Jianke Di; Enlan Zhao

    2008-01-01

    Different types of femtosecond optical tweezers have become a powerful tool in the modern biological field. However, how to control the irregular targets, including biological cells, using femtosecond optical tweezers remains to be explored. In this study, human red blood cells (hRBCs) are manipulated with femtosecond optical tweezers, and their states under different laser powers are investigated. The results indicate that optical potential traps only can capture the edge of hRBCs under the laser power from 1.4 to 2.8 mW, while it can make hRBCs turn over with the laser power more than 2.8 roW. It is suggested that femtosecond optical tweezers could not only manipulate biological cells, but also subtly control its states by adjusting the laser power.

  15. Regulation of the skeletal muscle blood flow in humans

    DEFF Research Database (Denmark)

    Mortensen, Stefan; Saltin, Bengt

    2014-01-01

    In humans, skeletal muscle blood flow is regulated by an interaction between several locally formed vasodilators including nitric oxide (NO) and prostaglandins. In plasma, ATP is a potent vasodilator that stimulates the formation of NO and prostaglandins and very importantly can offset local...... sympathetic vasoconstriction. ATP is released into plasma from erythrocytes and endothelial cells and the plasma concentration increases in both the feeding artery and the vein draining the contracting skeletal muscle. Adenosine also stimulates the formation of NO and prostaglandins, but the plasma adenosine...... and endothelial cells. In the interstitium, both ATP and adenosine stimulate the formation of NO and prostaglandins, but ATP has also been suggested to induce vasoconstriction and stimulate afferent nerves that signal to increase sympathetic nerve activity. Adenosine has been shown to contribute to exercise...

  16. The Danish Melanoma Database

    DEFF Research Database (Denmark)

    Hölmich, Lisbet Rosenkrantz; Klausen, Siri; Spaun, Eva

    2016-01-01

    AIM OF DATABASE: The aim of the database is to monitor and improve the treatment and survival of melanoma patients. STUDY POPULATION: All Danish patients with cutaneous melanoma and in situ melanomas must be registered in the Danish Melanoma Database (DMD). In 2014, 2,525 patients with invasive......, nature, and treatment hereof is registered. In case of death, the cause and date are included. Currently, all data are entered manually; however, data catchment from the existing registries is planned to be included shortly. DESCRIPTIVE DATA: The DMD is an old research database, but new as a clinical...

  17. Primary leptomeningeal melanoma.

    Science.gov (United States)

    Xie, Zhao-Yu; Hsieh, Kevin Li-Chun; Tsang, Yuk-Ming; Cheung, Wing-Keung; Hsieh, Chen-Hsi

    2014-06-01

    Primary melanoma of the central nervous system is a rare melanocytic tumor typically located in the leptomeninges. We report a 57-year-old woman with an intracranial leptomeningeal melanoma who presented with myoclonic seizures. Brain CT scan and MRI revealed a hemorrhagic intracranial tumor. The tumor was completely removed and leptomeningeal melanoma was proven pathologically. Follow-up imaging studies up to 19 months showed no recurrence of the disease. Here we present radiological, gross, and pathological images of leptomeningeal melanoma, discuss its characteristics, and review the relevant literature.

  18. Melanoma of the female urethra

    Directory of Open Access Journals (Sweden)

    Juan A Ramos

    2011-01-01

    Full Text Available Melanoma is a malignant tumor that can affect any area of the anatomical economy. Its appearance in the female urethra is extremely rare, with approximately 121 cases in indexed literature since 1966. The subject to be described is an 86-year-old woman who seeks assessment for intermittent macroscopic hematuria with blood clots of 3 months progression. On physical examination, there are no suspicious lesions detected on the surface of the skin. On external genital examination, it is observed a friable lesion at the level of the urethral meatus, with heterogeneous digitations, dark brown to black, and irregular polycyclic borders. No inguinal adenomegalies were palpated. Cystourethroscopy and biopsy of the lesion confirm the diagnosis. Melanoma of the female urethra is an extremely infrequent pathology. Due to lack of published case reports and the absence of prospective randomized trials on treatment outcomes, treatment must be directed using the same anatomical and surgical criteria for female urethral tumors, adding also the concepts of treatment of mucosal melanoma, even though its prognosis is different from the before mentioned.

  19. Circulating tumor cells in melanoma patients.

    Directory of Open Access Journals (Sweden)

    Gary A Clawson

    Full Text Available Circulating tumor cells (CTCs are of recognized importance for diagnosis and prognosis of cancer patients. With melanoma, most studies do not show any clear relationship between CTC levels and stage of disease. Here, CTCs were enriched (∼400X from blood of melanoma patients using a simple centrifugation device (OncoQuick, and 4 melanocyte target RNAs (TYR, MLANA, MITF, and MIF were quantified using QPCR. Approximately one-third of melanoma patients had elevated MIF and MLANA transcripts (p<0.0001 and p<0.001, respectively compared with healthy controls. In contrast, healthy controls had uniformly higher levels of TYR and MITF than melanoma patients (p<0.0001. There was a marked shift of leukocytes into the CTC-enriched fractions (a 430% increase in RNA recovery, p<0.001, and no relationship between CTC levels and stage of disease was found. CTCs were captured on microfabricated filters and cultured. Captured melanoma CTCs were large cells, and consisted of 2 subpopulations, based on immunoreactivity. One subpopulation (∼50% stained for both pan-cytokeratin (KRT markers and the common leukocyte marker CD-45, whereas the second subpopulation stained for only KRT. Since similar cells are described in many cancers, we also examined blood from colorectal and pancreatic cancer patients. We observed analogous results, with most captured CTCs staining for both CD-45/KRT markers (and for the monocyte differentiation marker CD-14. Our results suggest that immature melanocyte-related cells (expressing TYR and MITF RNA may circulate in healthy controls, although they are not readily detectable without considerable enrichment. Further, as early-stage melanomas develop, immature melanocyte migration into the blood is somehow curtailed, whereas a significant proportion of patients develop elevated CTC levels (based on MIF and MLANA RNAs. The nature of the captured CTCs is consistent with literature describing leukocyte/macrophage-tumor cell fusion hybrids

  20. Conditioned Media from Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Inhibits Melanogenesis by Promoting Proteasomal Degradation of MITF.

    Directory of Open Access Journals (Sweden)

    Eun Sung Kim

    Full Text Available Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs secrete various beneficial molecules, which have anti-apoptotic activity and cell proliferation. However, the effect of hUCB-MSCs in melanogenesis is largely unclear. In this study, we show that conditioned media (CM derived from hUCB-MSCs inhibit melanogenesis by regulating microphthalmia-associated transcription factor (MITF expression via the ERK signalling pathway. Treatment of hUCB-MSC-CM strongly inhibited the alpha-melanocyte stimulating hormone-induced hyperpigmentation in melanoma cells as well as melanocytes. Treatment of hUCB-MSC-CM induced ERK1/2 activation in melanocytes. In addition, inhibition of ERK1/2 suppressed the anti-pigmentation activity of the hUCB-MSC-CM in melanocytes and in vitro artificial skin models. We also found that the expression of MITF was appreciably diminished while expression of phosphorylated MITF, which leads to its proteasomal degradation, was increased in cells treated with hUCB-MSC-CM. These results suggested that hUCB-MSC-CM significantly suppresses melanin synthesis via MITF degradation by the ERK pathway activation.

  1. In Vitro Study on Lethal Effect of Human Choroidal Melanoma OCM-1 Cell Line by Repeating-70℃Freeze Thawing%In Vitro Study on Lethal Effect of Human Choroidal Melanoma OCM-1 Cell Line by Repeating-70℃ Freeze Thawing

    Institute of Scientific and Technical Information of China (English)

    Bin Liu; Yongping Li; Bo Zhang; Wenxin Zhang

    2007-01-01

    Objective: To investigate the effects of repeating -70℃ freeze thawing on human choroidal melanoma cell line OCM-1.Methods: OCM-1 cells were frozen by repeating -70℃ freeze thawing with various durations and frequencies. Then the inhibit rate of cells was examined by MTT essay.The cell viability was measured by monoclonal formation assay. We also used the HE staining, immunohistochemistry staining and the laser-scanning confocal microscopy (LSCM) to investigate the morphological changes of the cells.Results: The growth of OCM-1 cells was inhibited by repeating -70℃ freeze thawing in time-dependent and frequency-dependent manners (P < 0.01). Different morphous including necrosis and apoptosis of the cells could be observed after -70℃ freeze thawing by the LSCM.Conclusion: Repeating -70℃ freeze thawing can not only kill cells directly and induce considerable cells to apoptosis, but also inhibit the growth of the survivals. The kill and wound ratio of the cells disposal with different times and frequencies present variance. And the distinction when treated with different frequencies during the same time is much more significant than different times with the same frequency, which guide clinical workers to choose repeating cryotherapy with short term method instead of single cryotherapy with long term in choroidal melanoma treatment.

  2. A new treatment for human malignant melanoma targeting L-type amino acid transporter 1 (LAT1): A pilot study in a canine model

    Energy Technology Data Exchange (ETDEWEB)

    Fukumoto, Shinya; Hanazono, Kiwamu [Veterinary Internal Medicine, Department of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501 (Japan); Fu, Dah-Renn; Endo, Yoshifumi; Kadosawa, Tsuyoshi [Veterinary Oncology, Department of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501 (Japan); Iwano, Hidetomo [Veterinary Biochemistry, Department of Basic Veterinary Medicine, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501 (Japan); Uchide, Tsuyoshi, E-mail: uchide@rakuno.ac.jp [Veterinary Internal Medicine, Department of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501 (Japan)

    2013-09-13

    Highlights: •LAT1 is highly expressed in tumors but at low levels in normal tissues. •We examine LAT1 expression and function in malignant melanoma (MM). •LAT1 expression in MM tissues and cell lines is higher than those in normal tissues. •LAT1 selective inhibitors inhibit amino acid uptake and cell growth in MM cells. •New chemotherapeutic protocols including LAT1 inhibitors are effective for treatment. -- Abstract: L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities such as cellular growth, proliferation and maintenance. This amino acid transporter recently has received attention because of its preferential and up-regulated expression in a variety of human tumors in contrast to its limited distribution and low-level expression in normal tissues. In this study, we explored the feasibility of using LAT1 inhibitor as a new therapeutic agent for human malignant melanomas (MM) using canine spontaneous MM as a model for human MM. A comparative study of LAT expression was performed in 48 normal tissues, 25 MM tissues and five cell lines established from MM. The study observed LAT1 mRNA levels from MM tissues and cell lines that were significantly (P < 0.01) higher than in normal tissues. Additionally, MM with distant metastasis showed a higher expression than those without distant metastasis. Functional analysis of LAT1 was performed on one of the five cell lines, CMeC-1. [{sup 3}H]L-Leucine uptake and cellular growth activities in CMeC-1 were inhibited in a dose-dependent manner by selective LAT1 inhibitors (2-amino-2-norbornane-carboxylic acid, BCH and melphalan, LPM). Inhibitory growth activities of various conventional anti-cancer drugs, including carboplatin, cyclophosphamide, dacarbazine, doxorubicin, mitoxantrone, nimustine, vinblastine and vincristine, were significantly (P < 0.05) enhanced by combination use with BCH or LPM

  3. Ex vivo expansion of human peripheral blood progenitors.

    Science.gov (United States)

    Chabannon, C; Herrera-Rodriguez, D; Bardin, F; Mouren, M; Novakovitch, G; Blaise, D; Maraninchi, D; Mannoni, P

    1995-01-01

    Culture of human hematopoietic progenitors on a large scale could lead to several clinical applications within the near future, including the production of differentiated and functional cells, the increase in the number of early progenitors, especially stem cells, with such use as gene transfer, or the improvement of grafts used to limit the hematological toxicity associated with high-dose chemotherapy. In this case, one can still distinguish different objectives: improvement of grafts that contain low numbers of progenitors because of prior chemotherapies or because of marrow involvement for example, and qualitative changes in the graft content that would allow to envision the disappearance, or the further reduction, in the duration of absolute neutropenia that follows delivery of high dose chemotherapy ("nadir rescue"), despite substitution of mobilized blood cells to marrow cells and the in vivo use of hematopoietic growth factors. Additional advantages may be related to tumor purging in autologous expanded cells, and to the change in the ratio between hematopoietic progenitors and immunocompetent cells in allogeneic expanded populations. Therefore it appears that in vitro expansion currently raises two types of questions: the first ones are related to the definition of clinical or biological endpoints to be achieved, the second ones are related to "bioengineering", and deal with the efficiency and safety of progenitor cell cultures to be used for clinical applications. We here present preliminary results preparing future pilot clinical studies with ex vivo cultured human hematopoietic cells.

  4. Human red blood cells deformed under thermal fluid flow.

    Science.gov (United States)

    Foo, Ji-Jinn; Chan, Vincent; Feng, Zhi-Qin; Liu, Kuo-Kang

    2006-03-01

    The flow-induced mechanical deformation of a human red blood cell (RBC) during thermal transition between room temperature and 42.0 degrees C is interrogated by laser tweezer experiments. Based on the experimental geometry of the deformed RBC, the surface stresses are determined with the aid of computational fluid dynamics simulation. It is found that the RBC is more deformable while heating through 37.0 degrees C to 42.0 degrees C, especially at a higher flow velocity due to a thermal-fluid effect. More importantly, the degree of RBC deformation is irreversible and becomes softer, and finally reaches a plateau (at a uniform flow velocity U > 60 microm s(-1)) after the heat treatment, which is similar to a strain-hardening dominated process. In addition, computational simulated stress is found to be dependent on the progression of thermotropic phase transition. Overall, the current study provides new insights into the highly coupled temperature and hydrodynamic effects on the biomechanical properties of human erythrocyte in a model hydrodynamic flow system.

  5. Phase I study of GC1008 (fresolimumab: a human anti-transforming growth factor-beta (TGFβ monoclonal antibody in patients with advanced malignant melanoma or renal cell carcinoma.

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    John C Morris

    Full Text Available BACKGROUND: In advanced cancers, transforming growth factor-beta (TGFβ promotes tumor growth and metastases and suppresses host antitumor immunity. GC1008 is a human anti-TGFβ monoclonal antibody that neutralizes all isoforms of TGFβ. Here, the safety and activity of GC1008 was evaluated in patients with advanced malignant melanoma and renal cell carcinoma. METHODS: In this multi-center phase I trial, cohorts of patients with previously treated malignant melanoma or renal cell carcinoma received intravenous GC1008 at 0.1, 0.3, 1, 3, 10, or 15 mg/kg on days 0, 28, 42, and 56. Patients achieving at least stable disease were eligible to receive Extended Treatment consisting of 4 doses of GC1008 every 2 weeks for up to 2 additional courses. Pharmacokinetic and exploratory biomarker assessments were performed. RESULTS: Twenty-nine patients, 28 with malignant melanoma and 1 with renal cell carcinoma, were enrolled and treated, 22 in the dose-escalation part and 7 in a safety cohort expansion. No dose-limiting toxicity was observed, and the maximum dose, 15 mg/kg, was determined to be safe. The development of reversible cutaneous keratoacanthomas/squamous-cell carcinomas (4 patients and hyperkeratosis was the major adverse event observed. One malignant melanoma patient achieved a partial response, and six had stable disease with a median progression-free survival of 24 weeks for these 7 patients (range, 16.4-44.4 weeks. CONCLUSIONS: GC1008 had no dose-limiting toxicity up to 15 mg/kg. In patients with advanced malignant melanoma and renal cell carcinoma, multiple doses of GC1008 demonstrated acceptable safety and preliminary evidence of antitumor activity, warranting further studies of single agent and combination treatments. TRIAL REGISTRATION: Clinicaltrials.gov NCT00356460.

  6. Interferon alpha-induced reduction in the values of myeloid-derived suppressor cells in melanoma patients

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    Stanojević Ivan

    2015-01-01

    Full Text Available Background/Aim. Interaction between tumor cells and host’s immunoregulatory cells in creation of microenvironment that supports tumor progression is the focus of numerous investigations in recent years. Myeloid-derived suppressor cells (MDSCs are heterogeneous population of immature dendritic cells, macrophages and granulocytes. In cancer patients, these cells accumulate in tumor microenvironment, tumor-draining lymph nodes, peripheral blood and the liver and their numbers correlate with the stage of the disease and the metastatic disease. The aim of the study was to investigate the effect of interferon alpha on MDSCs percentage in peripheral blood of melanoma patients. Methods. The interferon treated melanoma patients were given subcutaneously interferon alpha, in optimal dose, for a period of at least 6 months before the analysis. Blood samples were collected from the melanoma patients (n = 91 and the age/sex matched healthy controls (n = 8. The following anti-human monoclonal antibodies were used for immunostaining: anti-CD15-FITC, anti-CD33-PE, anti-CD45-ECD, anti-HLA-DR PE/Cy5, anti-CD14-FITC, anti-CD16-PE and anti-CD11b-PE. Results. Comparison of myeloid-derived suppressor cells values in the stage 2 melanoma patients with and without interferon alpha therapy did not show a significant difference. When we compared the MDSCs values in the patients within stage 3 melanoma, we found a significant difference in granulocytic subset values between the interferon alpha-treated and the untreated group. Comparison of values of all suppressor cells populations between the interferon alpha-treated patients and healthy controls showed a significant increase in suppressor cells percentage in the melanoma patients. The granulocytic and total MDSCs values were significantly lower in the interferon alpha treated melanoma patients with progression in comparison with untreated patients with stable disease. Conclusion. We confirmed that interferon alpha

  7. DNA damage protecting and free radical scavenging properties of mycosporine-2-glycine from the Dead Sea cyanobacterium in A375 human melanoma cell lines.

    Science.gov (United States)

    Cheewinthamrongrod, Vipaporn; Kageyama, Hakuto; Palaga, Tanapat; Takabe, Teruhiro; Waditee-Sirisattha, Rungaroon

    2016-11-01

    Mycosporine-like amino acids (MAAs) are a group of natural sunscreen compounds that possess highly photoprotective properties. The most commonly found MAAs in marine organisms is shinorine, porphyra-334, and mycosporine-glycine. However, the halophilic species accumulate mycosporine-2-glycine (M2G) as the major MAA. In this study, we have investigated the protective effect of M2G against oxidative stress. In vitro radical scavenging activity revealed that M2G exhibited a significant inhibition with scavenging concentration (SC) 50 value of 22±1.4μM. In vivo analysis using the human melanoma A375 and a control cell line (NHSF) showed that M2G at low concentration (i.e. micromolar range) protected the cells against the oxidative stress (H2O2)-induced cell death. Comet assay to assess total DNA strand breaks demonstrated that M2G was not genotoxic and protected against the DNA damage by H2O2 treatment at the same level as ascorbic acid. To our knowledge, this is the first evidence demonstrating potential protective role of the natural sunscreen compound M2G against oxidative stress-induced DNA damage in human cell lines. The potent antioxidant activity combined with DNA protection ability of M2G may support its endorsement as a potential natural sunscreen with antioxidant property. These findings provide important clues for possible use of M2G in pharmaceutical and biotechnological applications.

  8. Triggering Apoptotic Death of Human Malignant Melanoma A375.S2 Cells by Bufalin: Involvement of Caspase Cascade-Dependent and Independent Mitochondrial Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Yu-Ping Hsiao

    2012-01-01

    Full Text Available Bufalin was obtained from the skin and parotid venom glands of toad and has been shown to induce cytotoxic effects in various types of cancer cell lines, but there is no report to show that whether bufalin affects human skin cancer cells. The aim of this investigation was to study the effects of bufalin on human malignant melanoma A375.S2 cells and to elucidate possible mechanisms involved in induction of apoptosis. A375.S2 cells were treated with different concentrations of bufalin for a specific time period and investigated for effects on apoptotic analyses. Our results indicated that cells after exposure to bufalin significantly decreased cell viability, and induced cell morphological changes and chromatin condensation in a concentration-dependent manner. Flow cytometric assays indicated that bufalin promoted ROS productions, loss of mitochondrial membrane potential (ΔΨm, intracellular Ca2+ release, and nitric oxide (NO formations in A375.S2 cells. Additionally, the apoptotic induction of bufalin on A375.S2 cells resulted from mitochondrial dysfunction-related responses (disruption of the ΔΨm and releases of cytochrome c, AIF, and Endo G, and activations of caspase-3, caspase-8 and caspase-9 expressions. Based on those observations, we suggest that bufalin-triggered apoptosis in A375.S2 cells is correlated with extrinsic- and mitochondria-mediated multiple signal pathways.

  9. Molecular biology of normal melanocytes and melanoma cells.

    Science.gov (United States)

    Bandarchi, Bizhan; Jabbari, Cyrus Aleksandre; Vedadi, Ali; Navab, Roya

    2013-08-01

    Malignant melanoma is one of the most aggressive malignancies in humans and is responsible for 60-80% of deaths from skin cancers. The 5-year survival of patients with metastatic malignant melanoma is about 14%. Its incidence has been increasing in the white population over the past two decades. The mechanisms leading to malignant transformation of melanocytes and melanocytic lesions are poorly understood. In developing malignant melanoma, there is a complex interaction of environmental and endogenous (genetic) factors, including: dysregulation of cell proliferation, programmed cell death (apoptosis) and cell-to-cell interactions. The understanding of genetic alterations in signalling pathways of primary and metastatic malignant melanoma and their interactions may lead to therapeutics modalities, including targeted therapies, particularly in advanced melanomas that have high mortality rates and are often resistant to chemotherapy and radiotherapy. Our knowledge regarding the molecular biology of malignant melanoma has been expanding. Even though several genes involved in melanocyte development may also be associated with melanoma cell development, it is still unclear how a normal melanocyte becomes a melanoma cell. This article reviews the molecular events and recent findings associated with malignant melanoma.

  10. Erythropoietic Potential of CD34+ Hematopoietic Stem Cells from Human Cord Blood and G-CSF-Mobilized Peripheral Blood

    Directory of Open Access Journals (Sweden)

    Honglian Jin

    2014-01-01

    Full Text Available Red blood cell (RBC supply for transfusion has been severely constrained by the limited availability of donor blood and the emergence of infection and contamination issues. Alternatively, hematopoietic stem cells (HSCs from human organs have been increasingly considered as safe and effective blood source. Several methods have been studied to obtain mature RBCs from CD34+ hematopoietic stem cells via in vitro culture. Among them, human cord blood (CB and granulocyte colony-stimulating factor-mobilized adult peripheral blood (mPB are common adult stem cells used for allogeneic transplantation. Our present study focuses on comparing CB- and mPB-derived stem cells in differentiation from CD34+ cells into mature RBCs. By using CD34+ cells from cord blood and G-CSF mobilized peripheral blood, we showed in vitro RBC generation of artificial red blood cells. Our results demonstrate that CB- and mPB-derived CD34+ hematopoietic stem cells have similar characteristics when cultured under the same conditions, but differ considerably with respect to expression levels of various genes and hemoglobin development. This study is the first to compare the characteristics of CB- and mPB-derived erythrocytes. The results support the idea that CB and mPB, despite some similarities, possess different erythropoietic potentials in in vitro culture systems.

  11. Raising the standard: changes to the Australian Code of Good Manufacturing Practice (cGMP) for human blood and blood components, human tissues and human cellular therapy products.

    Science.gov (United States)

    Wright, Craig; Velickovic, Zlatibor; Brown, Ross; Larsen, Stephen; Macpherson, Janet L; Gibson, John; Rasko, John E J

    2014-04-01

    In Australia, manufacture of blood, tissues and biologicals must comply with the federal laws and meet the requirements of the Therapeutic Goods Administration (TGA) Manufacturing Principles as outlined in the current Code of Good Manufacturing Practice (cGMP). The Therapeutic Goods Order (TGO) No. 88 was announced concurrently with the new cGMP, as a new standard for therapeutic goods. This order constitutes a minimum standard for human blood, tissues and cellular therapeutic goods aimed at minimising the risk of infectious disease transmission. The order sets out specific requirements relating to donor selection, donor testing and minimisation of infectious disease transmission from collection and manufacture of these products. The Therapeutic Goods Manufacturing Principles Determination No. 1 of 2013 references the human blood and blood components, human tissues and human cellular therapy products 2013 (2013 cGMP). The name change for the 2013 cGMP has allowed a broadening of the scope of products to include human cellular therapy products. It is difficult to directly compare versions of the code as deletion of some clauses has not changed the requirements to be met, as they are found elsewhere amongst the various guidelines provided. Many sections that were specific for blood and blood components are now less prescriptive and apply to a wider range of cellular therapies, but the general overall intent remains the same. Use of 'should' throughout the document instead of 'must' allows flexibility for alternative processes, but these systems will still require justification by relevant logical argument and validation data to be acceptable to TGA. The cGMP has seemingly evolved so that specific issues identified at audit over the last decade have now been formalised in the new version. There is a notable risk management approach applied to most areas that refer to process justification and decision making. These requirements commenced on 31 May 2013 and a 12 month

  12. Enhancement of HLA class II-restricted CD4+ T cell recognition of human melanoma cells following treatment with bryostatin-1.

    Science.gov (United States)

    Zhao, Dan; Amria, Shereen; Hossain, Azim; Sundaram, Kumaran; Komlosi, Peter; Nagarkatti, Mitzi; Haque, Azizul

    2011-01-01

    The majority of melanoma cells express detectable levels of HLA class II proteins, and an increased threshold of cell surface class II is crucial for the stimulation of CD4+ T cells. Bryostatin-1, a protein kinase C (PKC) activator, has been considered as a potent chemotherapeutic agent in a variety of in vitro tumor models. Little is known about the role of bryostatin-1 in HLA class II Ag presentation and immune activation in malignant tumors, especially in melanoma. In this study, we show that bryostatin-1 treatment enhances CD4+ T cell recognition of melanoma cells in the context of HLA class II molecules. We also show that bryostatin-1 treatment of melanoma cells increases class II protein levels by upregulating the class II transactivator (CIITA) gene. Flow cytometry and confocal microscopic analyses revealed that bryostatin-1 treatment upregulated the expression of costimulatory molecules (CD80 and CD86) in melanoma cells, which could prolong the interaction of immune cells and tumors. Bryostatin-1 also induced cellular differentiation in melanoma cells, and reduced tumorigenic factors such as pro-cathepsins and matrix-metalloproteinase-9. These data suggest that bryostatin-1 could be used as a chemo-immunotherapeutic agent for reducing tumorigenic potential of melanoma cells while enhancing CD4+ T cell recognition to prevent tumor recurrence. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. The impact of Ets-1 oncoprotein and human endoglin (CD105) on the recurrence of non-melanoma skin cancers.

    Science.gov (United States)

    Chrisostomidis, Chrisostomos; Konofaos, Petros; Karypidis, Dimitrios; Lazaris, Andreas; Kostakis, Alkiviadis; Papadopoulos, Othon

    2015-09-01

    The aim of this study was to investigate if the expression of CD105 and Ets-1 was predictive of aggressive biologic behavior of non-melanoma skin cancers (NMSC) and to evaluate indicators of local recurrence. A total of 144 patients with NMSC were included in the current study. Surgical specimens were independently examined for diagnosis confirmation and immunohistochemical expression of Ets-1 and CD105 by two dermatopathologists. The most common tumor type was basal cell carcinoma (n = 76), followed by squamous cell carcinomas (SCC) (n = 65). The most common anatomic location was the head and neck area (n = 115). The follow-up was ˃ 2 years in all examined cases. A statistically significant correlation was found between tumor local recurrence and age (P = 0.03), Ets-1 expression (P ˂ 0.0001) and CD105 expression (P ˂ 0.0001). Our data confirm that both Ets-1 and CD105 show promise as prognostic markers for local recurrence of NMSC. However, this statement is made with caution, and additional studies, with larger populations, are necessary to examine the correlation between these two markers and local recurrence. A better understanding of the pathogenesis of local recurrence in primary NMSC may result in potential therapeutic interventions. © 2015 The International Society of Dermatology.

  14. Translational research in melanoma.

    Science.gov (United States)

    Ray, Madhury; Farma, Jeffrey M; Hsu, Cary

    2013-10-01

    Recent breakthroughs in the fundamental understanding of the cellular and molecular basis of melanoma have culminated in new therapies with unquestionable efficacy. Immunotherapy and targeted therapy strategies have completely transformed the contemporary management of advanced melanoma. The translational research behind these developments is discussed, with an emphasis on immune checkpoint blockade and inhibition of the mitogen-activated protein kinase signaling pathway.

  15. Bronchial malignant melanoma.

    Science.gov (United States)

    Weshler, Z; Sulkes, A; Kopolovitch, J; Leviatan, A; Shifrin, E

    1980-01-01

    We describe a case of malignant melanoma presenting initially as an endobronchial lesion located in the left main bronchus causing total atelectasis. This resolved with radiation therapy. Widespread metastases developed shortly thereafter. The differential diagnosis of primary and metastatic bronchial malignant melanoma is discussed. Other isolated case reports are reviewed.

  16. Dual receptor-targeting ⁹⁹mTc-labeled Arg-Gly-Asp-conjugated Alpha-Melanocyte stimulating hormone hybrid peptides for human melanoma imaging.

    Science.gov (United States)

    Xu, Jingli; Yang, Jianquan; Miao, Yubin

    2015-04-01

    The aim of this study was to examine whether the substitution of the Lys linker with the aminooctanoic acid (Aoc) and polyethylene glycol (PEG) linker could substantially decrease the non-specific renal uptake of (99m)Tc-labeled Arg-Gly-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) hybrid peptides. The RGD motif {Arg-Gly-Asp-DTyr-Asp} was coupled to [Cys(3,4,10), D-Phe(7), Arg(11)]α-MSH₃₋₁₃ via the Aoc or PEG₂ linker to generate RGD-Aoc-(Arg(11))CCMSH and RGD-PEG-(Arg(11))CCMSH. The biodistribution results of (99m)Tc-RGD-Aoc-(Arg(11))CCMSH and (99m)Tc-RGD-PEG₂-(Arg(11))CCMSH were examined in M21 human melanoma-xenografted nude mice. The substitution of Lys linker with Aoc and PEG₂ linker significantly reduced the renal uptake of (99m)Tc-RGD-Aoc-(Arg(11))CCMSH and (99m)Tc-RGD-PEG₂-(Arg(11))CCMSH by 58% and 63% at 2h post-injection. The renal uptake of (99m)Tc-RGD-Aoc-(Arg(11))CCMSH and (99m)Tc-RGD-PEG₂-(Arg(11))CCMSH was 27.93 ± 3.98 and 22.01 ± 9.89% ID/g at 2 h post-injection. (99m)Tc-RGD-Aoc-(Arg(11))CCMSH displayed higher tumor uptake than (99m)Tc-RGD-PEG₂-(Arg(11))CCMSH (2.35 ± 0.12 vs. 1.71 ± 0.25% ID/g at 2 h post-injection). The M21 human melanoma lesions could be clearly visualized by SPECT/CT using (99m)Tc-RGD-Aoc-(Arg(11))CCMSH as an imaging probe. The favorable effect of Aoc and PEG₂ linker in reducing the renal uptake provided a new insight into the design of novel dual receptor-targeting radiolabeled peptides. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Novel, high-yield red blood cell production methods from CD34-positive cells derived from human embryonic stem, yolk sac, fetal liver, cord blood, and peripheral blood.

    Science.gov (United States)

    Olivier, Emmanuel; Qiu, Caihong; Bouhassira, Eric E

    2012-08-01

    The current supply of red blood cells expressing rare blood groups is not sufficient to cover all the existing transfusion needs for chronically transfused patients, such as sickle cell disease homozygous carriers, because of alloimmunization. In vitro production of cultured red blood cells is slowly emerging as a possible complement to the existing collection-based red blood cell procurement system. The yield of cultured red blood cells can theoretically be maximized by amplifying the stem, progenitor, or precursor compartment. Here, we combined methods designed to expand these three compartments to optimize the yield of cultured red blood cells and found that exposing CD34(+) cells to a short pulse of cytokines favorable for erythroid differentiation prior to stem cell expansion followed by progenitor expansion produced the highest yield of erythroid cells. This novel serum-free red blood cell production protocol was efficient on CD34(+) cells derived from human embryonic stem cells, 6-8-week yolk sacs, 16-18-week fetal livers, cord blood, and peripheral blood. The yields of cells obtained with these new protocols were larger by an order of magnitude than the yields observed previously. Globin expression analysis by high-performance liquid chromatography revealed that these expansion protocols generally yielded red blood cells that expressed a globin profile similar to that expected for the developmental age of the CD34(+) cells.

  18. Familial malignant melanoma

    Energy Technology Data Exchange (ETDEWEB)

    Kopf, A.W.; Hellman, L.J.; Rogers, G.S.; Gross, D.F.; Rigel, D.S.; Friedman, R.J.; Levenstein, M.; Brown, J.; Golomb, F.M.; Roses, D.F.; Gumport, S.L.

    1986-10-10

    Characteristics associated with familial compared with nonfamilial malignant melanoma were assessed. These data were obtained from consecutive prospectively completed questionnaires on 1169 cases of cutaneous malignant melanoma. Of these, 69 patients indicated a positive family history for this cancer. Among the various clinical and histological variables compared, those that significantly correlated with the familial occurrence of malignant melanoma include younger age at first diagnosis, smaller diameter of the lesion, lower Clark level, decreased frequency of nonmelanoma skin cancer, and reduced prevalence of noncutaneous cancer. Increased awareness of malignant melanoma among family members could account for some of these observations. Identification of the familial variety of malignant melanoma has practical implications concerning early detection and prompt intervention.

  19. Synchronous anorectal melanoma

    Institute of Scientific and Technical Information of China (English)

    Drinko Balicevic; Karla Tomic; Miroslav Bekavac-Beslin; Igor Kovacevic; August Mijic; Mladen Belicza; Bozo Kruslin

    2006-01-01

    Anorectal melanoma is a very rare tumor with poor prognosis. Rectal bleeding is the most frequent symptom and surgical treatment ranges from local excision to radical abdominoperineal resection. We report a case of a 75-years-old male patient who presented with a history of recurrent rectal bleeding, and whose histopathological diagnosis was melanoma. Macroscopically, we found two distinct tumors in anorectal region, 0.5 cm and 1.5 cm from dentate line. The first one was pedunculated, on a thin stalk, measuring 1 cm in greatest diameter, and the second one was sessile and nodular measuring up to 2.8 cm in largest diameter. Microscopic examination and immunohistochemical analysis of both tumors confirmed the diagnosis of melanoma. This case represents multiple synchronous primary melanoma of the anorectal region, with a possibility that one of the lesions is primary melanoma and the second one is a satellite lesion.

  20. Interleukin-6 and melanoma

    DEFF Research Database (Denmark)

    Hoejberg, Lise; Bastholt, Lars; Schmidt, Henrik

    2012-01-01

    Interleukin-6 (IL-6) is a pleiotropic immunomodulatory cytokine produced by various types of cells, including melanoma cells. IL-6 plays a major role in the pathogenesis and development of malignancies. It promotes tumour growth by inhibition of apoptosis and induces tumour angiogenesis. IL-6...... is deregulated in many types of cancers, and increased serum concentration of IL-6 has been correlated with a worse prognosis in patients with different cancers, including melanoma. Several serum cytokines including IL-6 play an important role in the development and progression of melanoma; however, the specific...... biological functions of IL-6 in progression of melanoma are unknown. In this review, we present studies on cell cultures and mouse models and summarize published clinical studies on IL-6 and melanoma....

  1. Melanoma during pregnancy

    DEFF Research Database (Denmark)

    de Haan, Jorine; Lok, Christianne A; de Groot, Christianne J M

    2017-01-01

    The management of melanoma during pregnancy is challenging as maternal benefits and fetal risks need to be balanced. Here, we present an overview of the incidence, the demographic and clinical characteristics and the treatment modalities used. After analysis of obstetric, fetal and maternal outcome......, recommendations for clinical practice are provided. From the 'International Network on Cancer, Infertility and Pregnancy' database, pregnant patients with melanoma were identified and analysed. Sixty pregnancies were eligible for analysis. Fifty percent of the patients presented with advanced melanoma during...... pregnancy (14 stage III and 16 stage IV), and 27% were diagnosed with recurrent melanoma. Surgery was the main therapeutic strategy during pregnancy. Only four patients with advanced melanoma were treated during pregnancy with systemic therapy (n=1) or radiotherapy (n=3). Premature delivery was observed...

  2. Biodistribution dosimetric study of radiopharmaceutical {sup 99mT}c Ixolaris in mice for melanoma diagnosis by molecular image and translational model for human beings; Estudo dosimetrico da biodistribuicao do radiofarmaco Ixolaris-{sup 99m}Tc em camundongos para diagnostico de melanoma atraves de imagem molecular e modelo translacional para humanos

    Energy Technology Data Exchange (ETDEWEB)

    Soriano, Sarah Canuto Silva

    2015-07-01

    The labeling of Ixolaris with {sup 99m}Tc was developed by Barboza et.al. (2013) aiming its use primarily in glioblastoma and after in melanoma diagnosis, a less common but very aggressive cancer and with high mortality rate. Preliminary tests on animals have proven its effectiveness of labeling but a dosimetric study to human clinical trials should be performed. This study aimed to: (1) determine the biokinetic model for the radiotracer {sup 99m}Tc-Ixolaris in mice by imaging dosimetry method; and (2) estimate the absorbed and effective dose resulting from the use of a new radiopharmaceutical for melanoma and metastases diagnosis in human beings, since a dosimetric study of new radiopharmaceuticals in animals is necessary to test them subsequently in humans and apply for registration in ANVISA. According to SPECT images, was found a latency period of 15 to 21 days for the development of lung metastasis in mice. Three C57BL6 mice, one control animal, and two animals with induced cell line B16-F10 murine melanoma were tested. The {sup 99m}Tc-Ixolaris radiopharmaceutical was administered intravenously in a caudal vein, and SPECT images were acquired 0.5 h, 1.5 h, 2.5 h, 3.5 h and 24 h post-administration for analysis and biodistribution quantification. The biokinetic model was determined and thus, obtained cumulative activity in order to estimate the absorbed dose in each organ. The mass and metabolic differences between mice and humans were considered and used to extrapolate the data acquired at different scales. Based on dose factors provided by the software MIRDOSE and Olinda (S factor), absorbed doses in irradiated target organs were calculated for the source organs, and finally the effective dose was estimated. The results indicate that for diagnostic exams conducted in human melanoma patients by administering approximately 25.7 MBq the estimated effective dose was 4.3 mSv. Comparing with effective doses obtained in other diagnostic techniques with {sup 99m

  3. Neurotropic melanoma with prominent melanization.

    Science.gov (United States)

    Barnhill, R L; Bolognia, J L

    1995-10-01

    Neurotropic melanoma has generally been described in the context of desmoplastic melanoma. The vast majority of melanomas displaying neurotropism contain relatively little or no melanin. Herein, we report an unusual case of neurotropic melanoma with prominent melanin content. The patient developed a tumor notable for pagetoid (superficial spreading) melanoma with partial regression and a deep component characterized by perineurial aggregates of melanophages and intraneural infiltration by melanoma cells. This case serves to alert dermatopathologists to the fact that the spectrum of neurotropic melanoma includes tumors with perineurial aggregates of pigment-containing cells.

  4. Melanoma: from mutations to medicine

    Science.gov (United States)

    Tsao, Hensin; Chin, Lynda; Garraway, Levi A.; Fisher, David E.

    2012-01-01

    Melanoma is often considered one of the most aggressive and treatment-resistant human cancers. It is a disease that, due to the presence of melanin pigment, was accurately diagnosed earlier than most other malignancies and that has been subjected to countless therapeutic strategies. Aside from early surgical resection, no therapeutic modality has been found to afford a high likelihood of curative outcome. However, discoveries reported in recent years have revealed a near avalanche of breakthroughs in the melanoma field—breakthroughs that span fundamental understanding of the molecular basis of the disease all the way to new therapeutic strategies that produce unquestionable clinical benefit. These discoveries have been born from the successful fruits of numerous researchers working in many—sometimes-related, although also distinct—biomedical disciplines. Discoveries of frequent mutations involving BRAF(V600E), developmental and oncogenic roles for the microphthalmia-associated transcription factor (MITF) pathway, clinical efficacy of BRAF-targeted small molecules, and emerging mechanisms underlying resistance to targeted therapeutics represent just a sample of the findings that have created a striking inflection in the quest for clinically meaningful progress in the melanoma field. PMID:22661227

  5. Melanocyte and Melanoma Cell Activation by Calprotectin

    Directory of Open Access Journals (Sweden)

    Stephanie H. Shirley

    2014-01-01

    Full Text Available Calprotectin, a heterodimer of S100A8 and S100A9, is a proinflammatory cytokine released from ultraviolet radiation-exposed keratinocytes. Calprotectin binds to Toll-like receptor 4, the receptor for advanced glycation end-products, and extracellular matrix metalloproteinase inducer on target cells to stimulate migration. Melanocytes and melanoma cells produce little if any calprotectin, but they do express receptors for the cytokine. Thus, keratinocyte-derived calprotectin has the potential to activate melanocytes and melanoma cells within the epidermis in a paracrine manner. We examined the ability of calprotectin to stimulate proliferation and migration in normal human melanocytes and melanoma cells in vitro. We first showed, by immunofluorescence and quantitative RT-PCR, that the melanocytic cells employed expressed a calprotectin receptor, the receptor for advanced end-products. We then demonstrated that calprotectin significantly enhanced proliferation, migration, and Matrigel invasion in both normal human melanocytes and melanoma cells. Thus, calprotectin is one of the numerous paracrine factors released by ultraviolet radiation-exposed keratinocytes that may promote melanomagenesis and is a potential target for melanoma prevention or therapy.

  6. Computational lipidology: predicting lipoprotein density profiles in human blood plasma.

    Directory of Open Access Journals (Sweden)

    Katrin Hübner

    2008-05-01

    Full Text Available Monitoring cholesterol levels is strongly recommended to identify patients at risk for myocardial infarction. However, clinical markers beyond "bad" and "good" cholesterol are needed to precisely predict individual lipid disorders. Our work contributes to this aim by bringing together experiment and theory. We developed a novel computer-based model of the human plasma lipoprotein metabolism in order to simulate the blood lipid levels in high resolution. Instead of focusing on a few conventionally used predefined lipoprotein density classes (LDL, HDL, we consider the entire protein and lipid composition spectrum of individual lipoprotein complexes. Subsequently, their distribution over density (which equals the lipoprotein profile is calculated. As our main results, we (i successfully reproduced clinically measured lipoprotein profiles of healthy subjects; (ii assigned lipoproteins to narrow density classes, named high-resolution density sub-fractions (hrDS, revealing heterogeneous lipoprotein distributions within the major lipoprotein classes; and (iii present model-based predictions of changes in the lipoprotein distribution elicited by disorders in underlying molecular processes. In its present state, the model offers a platform for many future applications aimed at understanding the reasons for inter-individual variability, identifying new sub-fractions of potential clinical relevance and a patient-oriented diagnosis of the potential molecular causes for individual dyslipidemia.

  7. Cerebral blood flow during static exercise in humans

    DEFF Research Database (Denmark)

    Rogers, H B; Schroeder, T; Secher, N H

    1990-01-01

    voluntary contraction (MVC) and utilized alternate legs. CBF (measured by the 133Xe clearance technique) was expressed by a noncompartmental flow index (ISI). Heart rate and mean arterial pressure increased from resting values of 73 (55-80) beats/min and 88 (74-104) mmHg to 106 (86-138) beats/min and 124......Cerebral blood flow (CBF) was determined in humans at rest and during four consecutive unilateral static contractions of the knee extensors. Each contraction was maintained for 3 min 15 s with the subjects in a semisupine position. The contractions corresponded to 8, 16, 24, and 32% of the maximal...... resistance increased from 1.5 (1.0-2.2) to 2.4 (1.4-3.0) mmHg. 100 g.min.ml-1 (P less than 0.025) at 32% of MVC. There was no difference in CBF between the two hemispheres at rest or during exercise. In contrast to dynamic leg exercise, static leg exercise is not associated with an increase in global CBF...

  8. In vitro characterization and inhibition of the CXCR4/CXCL12 chemokine axis in human uveal melanoma cell lines

    Directory of Open Access Journals (Sweden)

    Antecka Emilia

    2007-11-01

    Full Text Available Abstract Purpose The CXCR4/CXCL12 chemokine axis may play a critical role in guiding CXCR4+ circulating malignant cells to organ specific locations that actively secrete its ligand CXCL12 (SDF-1 such as bone, brain, liver, and lungs. We sought to characterize the presence of the CXCR4/CXCL12 axis in five uveal melanoma (UM cell lines in vitro. The ability of TN14003, a synthetic peptide inhibitor that targets the CXCR4 receptor complex, to inhibit this axis was also assessed. Methods Immunocytochemistry was performed against CXCR4 to confirm expression of this chemokine receptor in all five UM cell lines. Flow cytometry was preformed to evaluate CXCR4 cell surface expression on all five UM cell lines. A proliferation assay was also used to test effects TN14003 would have on cellular proliferation. Inhibition of cellular migration by specifically inhibiting the CXCR4/CXCL12 axis with TN14003 was also investigated. The binding efficacy of TN14003 to the CXCR4 receptor was assessed through flow cytometric methods. Results The CXCR4 receptor was present on all five UM cell lines. All five cell lines expressed different relative levels of surface CXCR4. TN14003 did not affect the proliferation of the five cell lines (p > 0.05. All cell lines migrated towards the chemokine CXCL12 at a level greater than the negative control (p Conclusion Interfering with the CXCR4/CXCL12 axis, using TN14003 was shown to effectively down regulate UM cell migration in vitro. Knowing that UM expresses the CXCR4 receptor, these CXCR4+ cells may be less likely to colonize distant organs that secrete the CXCL12 ligand, if treated with an inhibitor that binds CXCR4. Further studies should be pursued in order to test TN14003 efficacy in vivo.

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

    Directory of Open Access Journals (Sweden)

    Ming-Chao Bi

    2016-01-01

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

  10. Peginterferon Beta-1a Shows Antitumor Activity as a Single Agent and Enhances Efficacy of Standard of Care Cancer Therapeutics in Human Melanoma, Breast, Renal, and Colon Xenograft Models.

    Science.gov (United States)

    Boccia, Antonio; Virata, Cyrus; Lindner, Daniel; English, Nicki; Pathan, Nuzhat; Brickelmaier, Margot; Hu, Xiao; Gardner, Jennifer L; Peng, Liaomin; Wang, Xinzhong; Zhang, Xiamei; Yang, Lu; Perron, Keli; Yco, Grace; Kelly, Rebecca; Gamez, James; Scripps, Thomas; Bennett, Donald; Joseph, Ingrid B; Baker, Darren P

    2017-01-01

    Because of its tumor-suppressive effect, interferon-based therapy has been used for the treatment of melanoma. However, limited data are available regarding the antitumor effects of pegylated interferons, either alone or in combination with approved anticancer drugs. We report that treatment of human WM-266-4 melanoma cells with peginterferon beta-1a induced apoptotic markers. Additionally, peginterferon beta-1a significantly inhibited the growth of human SK-MEL-1, A-375, and WM-266-4 melanoma xenografts established in immunocompromised mice. Peginterferon beta-1a regressed large, established WM-266-4 xenografts in nude mice. Treatment of SK-MEL-1 tumor-bearing mice with a combination of peginterferon beta-1a and the MEK inhibitor PD325901 ((R)-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide) significantly improved tumor growth inhibition compared with either agent alone. Examination of the antitumor activity of peginterferon beta-1a in combination with approved anticancer drugs in breast and renal carcinomas revealed improved antitumor activity in these preclinical xenograft models, as did the combination of peginterferon beta-1a and bevacizumab in a colon carcinoma xenograft model.

  11. Targeting human dendritic cells via DEC-205 using PLGA nanoparticles leads to enhanced cross-presentation of a melanoma-associated antigen

    Directory of Open Access Journals (Sweden)

    Saluja SS

    2014-11-01

    Full Text Available Sandeep S Saluja,1 Douglas J Hanlon,1 Fiona A Sharp,2 Enping Hong,2 David Khalil,1 Eve Robinson,1 Robert Tigelaar,1 Tarek M Fahmy,2,3 Richard L Edelson1 1Department of Dermatology, Yale University School of Medicine, 2Department of Biomedical Engineering, Yale University, 3Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA Abstract: Targeting antigen to dendritic cells (DCs is a powerful and novel strategy for vaccination. Priming or loading DCs with antigen controls whether subsequent immunity will develop and hence whether effective vaccination can be achieved. The goal of our present work was to increase the potency of DC-based antitumor vaccines by overcoming inherent limitations associated with antigen stability and cross-presentation. Nanoparticles prepared from the biodegradable polymer poly(lactic-co-glycolic acid have been extensively used in clinical settings for drug delivery and are currently the subject of intensive investigation as antigen delivery vehicles for vaccine applications. Here we describe a nanoparticulate delivery system with the ability to simultaneously carry a high density of protein-based antigen while displaying a DC targeting ligand on its surface. Utilizing a targeting motif specific for the DC-associated surface ligand DEC-205, we show that targeted nanoparticles encapsulating a MART-127–35 peptide are both internalized and cross-presented with significantly higher efficiency than isotype control-coated nanoparticles in human cells. In addition, the DEC-205-labeled nanoparticles rapidly escape from the DC endosomal compartment and do not colocalize with markers of early (EEA-1 or late endosome/lysosome (LAMP-1. This indicates that encapsulated antigens delivered by nanoparticles may have direct access to the class I cytoplasmic major histocompatibility complex loading machinery, overcoming the need for “classical” cross-presentation and facilitating heightened DC

  12. Mapping blood flow directionality in the human brain.

    Science.gov (United States)

    Park, Sung-Hong; Do, Won-Joon; Choi, Seung Hong; Zhao, Tiejun; Bae, Kyongtae Ty

    2016-07-01

    Diffusion properties of tissue are often expressed on the basis of directional variance, i.e., diffusion tensor imaging. In comparison, common perfusion-weighted imaging such as arterial spin labeling yields perfusion in a scalar quantity. The purpose of this study was to test the feasibility of mapping cerebral blood flow directionality using alternate ascending/descending directional navigation (ALADDIN), a recently-developed arterial spin labeling technique with sensitivity to blood flow directions. ALADDIN was applied along 3 orthogonal directions to assess directional blood flow in a vector form and also along 6 equally-spaced directions to extract blood flow tensor matrix (P) based on a blood flow ellipsoid model. Tensor elements (eigenvalues, eigenvectors, etc) were calculated to investigate characteristics of the blood flow tensor, in comparison with time-of-flight MR angiogram. While the directions of the main eigenvectors were heterogeneous throughout the brain, regional clusters of blood flow directionality were reproducible across subjects. The technique could show heterogeneous blood flow directionality within and around brain tumor, which was different from that of the contralateral normal side. The proposed method is deemed to provide information of blood flow directionality, which has not been demonstrated before. The results warrant further studies to assess changes in the directionality map as a function of scan parameters, to understand the signal sources, to investigate the possibility of mapping local blood perfusion directionality, and to evaluate its usefulness for clinical diagnosis.

  13. Pro-apoptotic properties of (1,3)(1,4)-β-D-glucan from Avena sativa on human melanoma HTB-140 cells in vitro.

    Science.gov (United States)

    Parzonko, Andrzej; Makarewicz-Wujec, Magdalena; Jaszewska, Edyta; Harasym, Joanna; Kozłowska-Wojciechowska, Małgorzata

    2015-01-01

    In this study, the growth-inhibitory effect of polysaccharide (1,3)(1,4)-β-D-glucan from oat, Avena sativa L. grains was explored on the human skin melanoma HTB-140 cells in vitro. The oat β-D-glucan (OBG) exerted cytotoxic action on HTB-140 cells. After 24h of incubation, LD50 (concentration at which 50% of the cells were found dead) was obtained of 194.6 ± 9.8 μg/mL. The oat β-D-glucan caused a concentration-dependent increase of caspase-3/-7 activation and appearance of phosphatidylserine on the external surface of cellular membranes where it was bound to annexin V-FITC, demonstrating the induction of apoptosis. Intracellular ATP level decreased along with the mitochondrial potential, which suggested a mitochondrial pathway of apoptosis. A cell cycle analysis showed increase in the number of apoptotic cells, increase in the number of cells in G1 phase and decrease in the number of cells in G2/M. Although the detailed mechanism for the anti-tumor activity of the oat β-D-glucan still needs further investigation, this study provides preliminary insights into this direction along with perspectives of developing it as an anti-tumor agent.

  14. Absence of point mutation in the 12th codon of transformed c-Ha-rasl genes of human cancer of the breast, stomach, melanoma, and neuroblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Knyazev, P.G.; Schafer, R.; Willecke, K.V.; Seitz, I.F.

    1985-11-01

    In the authors' previous investigations, they established that the tumorous cell lines SK-BR-3 (breast cancer), LAN-1 (neuroblastoma), and a heterotransplant of malignant melanoma Jal contain transforming genes of Ha-ras type. Now, the authors report their results using restriction endonucleases of MspI and HpaII restriction to study nucleotide sequences 5'-CCGGC-3' and 3'GGCCG-5', which contain the 12th codon of GGC for the amino acid glycine in the normal allele of c-Ha-rasl in the three tumors listed above, in addition to human adenocarcinoma of the stomach (CaVSt) and normal cells corresponding to them. For hybridization of MspI/HpaII, fragments of chromosomal DNA isolated from cell lines SK-BR-3, and LAN-1, Ja-1 heterotransplant, and stomach adenocarcinoma CaVSt, the XmaI section of EJ oncogene, c-Ha-rasl (plasmid pEJ 6.6), labeled with /sup 32/P was used in down-translation reaction. Hybridization was performed in 3 x SSC buffer containing 5x Deinhardt's reagent and 10% dextran sulfate at 68/sup 0/C for 16-18 h. Washing of filters was conducted under rigid conditions. For autoradiography, Kodak XR-5 x-ray film in cartridges with reinforcing shields was used at -70/sup 0/C, exposure time of four to six days.

  15. Scanning electron microscopy of human and murine melanoma cells exposed to medium chain-length (C6-C12) dicarboxylic acids in tissue culture.

    Science.gov (United States)

    Breathnach, A S; Robins, E J; Bhasin, Y; Ethridge, L; Nazzaro-Porro, M; Passi, S; Picardo, M

    1987-07-01

    Human and murine (Harding-Passey and Cloudman) melanoma cells were exposed to various concentrations (1 x 10(-3) M-1 x 10(-1) M) of adipic (C6), azelaic (C9), and dodecanedioic (C12) acids for 1-6 hours in tissue culture, and the effects on shape and surface topography were examined by scanning electron microscopy. Effects, i.e., rounding up, concentration of microvilli, blebbing, and prominence of retraction fibrils were time and dose dependent, and for the same concentrations and exposure times, C12 had a greater effect than C9, and both a significantly greater effect than C6. These differential reactions to the three diacids parallel previously reported effects on cell kinetics and viability. The changes could be due to a prime effect on the cell membrane, or they might reflect phases of the cell cycle directed by action of the diacids on the nucleus; this latter seems unlikely. An effect on the cytoskeleton is possibly involved.

  16. Hybrids of Iron-Filled Multiwall Carbon Nanotubes and Anticancer Agents as Potential Magnetic Drug Delivery Systems: In Vitro Studies against Human Melanoma, Colon Carcinoma, and Colon Adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Sławomir Boncel

    2017-01-01

    Full Text Available Cell type, morphology, and functioning are key variables in the construction of efficient “drug-vehicle” hybrids in magnetic drug delivery. Iron-encapsulated multiwall carbon nanotubes (Fe@MWCNTs appear as promising candidates for theranostics due to in situ chemical catalytic vapor deposition (c-CVD synthesis, straightforward organic functionalization, and nanoneedle (1D behavior. Here, model hybrids were synthesized by exploring C-sp2 chemistry ((1+2-cycloaddition of nitrenes and amidation of the outer MWCNT walls combined with anticancer agents, that is, 5-fluorouracil (5FU, purpurin (Purp, and 1,8-naphthalimide DNA intercalators (NIDIs, via linkers. Analyses of the Fe@MWCNT vehicles by SEM, TEM, and Raman spectroscopy revealed their morphology while Mössbauer spectroscopy confirmed the presence of encapsulated ferromagnetic iron-based nanodomains. Cytotoxicity of the hybrids was studied using a 24 h MTS assay combined with the apoptosis and life cycle assays against human melanoma (Me45, colon carcinoma (HCT116+, and colon adenocarcinoma (Caco-2. The cells had different sensitivity to the vehicles themselves as well as to the hybrids. MWCNT-based covalent hybrids of 5FU and Purp emerged as the most promising systems against Me45 and HCT116+ cell lines with the highest in vitro cytotoxicity and proapoptotic activity. Furthermore, nanotubes bearing 4-nitro- and 4-(N-morpholinyl-1,8-naphthalimide DNA intercalators appear as a promising candidate for the treatment of Caco-2.

  17. Killing efficacy of a new silicon phthalocyanine in human melanoma cells treated with photodynamic therapy by early activation of mitochondrion-mediated apoptosis.

    Science.gov (United States)

    Barge, Jérôme; Decréau, Richard; Julliard, Michel; Hubaud, Jean-Claude; Sabatier, Anne-Sophie; Grob, Jean-Jacques; Verrando, Patrick

    2004-01-01

    Photodynamic therapy (PDT) is a promising therapeutic modality that utilizes a combination of a photosensitizer and visible light for the destruction of diseased tissues. Using human-pigmented melanoma cells, we examined the photokilling efficacy of new silicon-phthalocyanines (SiPc) that bore bulky axial substituents. The bis(cholesteryloxy) derivate (Chol-O-SiPc) displayed the best in vitro photokilling efficacy (LD(50) = 6-8 x 10(-9) M) and was seven to nine times more potent than chloro-aluminium Pc (ClAlPc), a known photosensitizer used as a reference. Although Chol-O-SiPc was half as potent as ClAlPc for promoting photo-oxidative membrane damage in a cell-free assay, early events of mitochondrion-mediated apoptosis upon PDT were triggered much faster, as demonstrated by kinetics studies examining cells with permeabilized mitochondrial membranes, cytochrome c release and caspase-9 activation. Inhibition of caspase-9 activity by a substrate analogue argued for its central role in the proapoptotic events leading to cell death by Chol-O-SiPc PDT. In addition, immunoblots showed that Bcl-2 antiapoptotic oncoprotein was not a primary target of Chol-O-SiPc in M3Dau cells treated with PDT. Conclusively, Chol-O-SiPc is a useful new photosensitizer with the property of triggering cell apoptosis mediated by mitochondria.

  18. Transient receptor potential canonical type 3 channels and blood pressure in humans

    DEFF Research Database (Denmark)

    Thilo, Florian; Baumunk, Daniel; Krause, Hans;

    2009-01-01

    There is evidence that transient receptor potential canonical type 3 (TRPC3) cation channels are involved in the regulation of blood pressure, but this has not been studied using human renal tissue. We tested the hypothesis that the expression of TRPC3 in human renal tissue is associated with blood...

  19. 3D Computer Simulations of Pulsatile Human Blood Flows in Vessels and in the Aortic Arch: Investigation of Non-Newtonian Characteristics of Human Blood

    CERN Document Server

    Sultanov, Renat A; Engelbrekt, Brent; Blankenbecler, Richard

    2008-01-01

    Methods of Computational Fluid Dynamics are applied to simulate pulsatile blood flow in human vessels and in the aortic arch. The non-Newtonian behaviour of the human blood is investigated in simple vessels of actual size. A detailed time-dependent mathematical convergence test has been carried out. The realistic pulsatile flow is used in all simulations. Results of computer simulations of the blood flow in vessels of two different geometries are presented. For pressure, strain rate and velocity component distributions we found significant disagreements between our results obtained with realistic non-Newtonian treatment of human blood and widely used method in literature: a simple Newtonian approximation. A significant increase of the strain rate and, as a result, wall sear stress distribution, is found in the region of the aortic arch. We consider this result as theoretical evidence that supports existing clinical observations and those models not using non-Newtonian treatment underestimate the risk of disru...

  20. 21 CFR 607.7 - Establishment registration and product listing of blood banks and other firms manufacturing human...

    Science.gov (United States)

    2010-04-01

    ... blood banks and other firms manufacturing human blood and blood products. 607.7 Section 607.7 Food and... Provisions § 607.7 Establishment registration and product listing of blood banks and other firms... permit any blood bank or similar establishment to ship blood products in interstate commerce. (b)...

  1. Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies

    OpenAIRE

    Meyer, Anke; Paroni, Federico; Günther, Kathrin; Dharmadhikari, Gitanjali; Ahrens, Wolfgang; Kelm, Sørge; Maedler, Kathrin

    2016-01-01

    Aims Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples. Methods The established PAXgeneTM blood collection method (Qiagen)...

  2. Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus.

    Science.gov (United States)

    Soares, Filipa A C; Pedersen, Roger A; Vallier, Ludovic

    2016-01-01

    This protocol describes the efficient isolation of peripheral blood mononuclear cells from circulating blood via density gradient centrifugation and subsequent generation of integration-free human induced pluripotent stem cells. Peripheral blood mononuclear cells are cultured for 9 days to allow expansion of the erythroblast population. The erythroblasts are then used to derive human induced pluripotent stem cells using Sendai viral vectors, each expressing one of the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc.

  3. Time-dependent histamine release from stored human blood products

    DEFF Research Database (Denmark)

    Nielsen, Hans Jørgen; Edvardsen, L; Vangsgaard, K

    1996-01-01

    Perioperative transfusion of whole blood has been shown to amplify trauma-induced immunosuppression, which could be attenuated by perioperative administration of histamine2 receptor antagonists. Supernatants from different blood products were, therefore, analysed for histamine content during...... storage. Whole blood (six units), plasma-reduced whole blood (six units), and plasma- and buffy coat-reduced (saline-adenine-glucose-mannitol) (SAGM) blood (six units) from unpaid healthy donors were stored in the blood bank for 35 days at 4 degrees C. Plasma histamine and total cell-bound histamine...... content at donation, and histamine concentration in samples drawn from the units on days 0, 2, 5, 9, 14, 21, 28 and 35 were analysed with an enzyme-linked immunosorbent assay. Median plasma histamine concentration was 4.8 (range 1.9-14.3) nmol/l (n = 18). Median total cell-bound histamine content was 417...

  4. Distribution and concentration of cyclosporin in human blood.

    OpenAIRE

    Atkinson, K.; Britton, K; Biggs, J.

    1984-01-01

    In patients receiving cyclosporin to minimise graft versus host disease after allogeneic bone marrow transplantation, whole blood cyclosporin concentration was roughly twice the serum concentration when blood was separated at 37 degrees C. In turn, blood separation at 37 degrees C resulted in a doubling of serum cyclosporin concentration compared with separation at room temperature. In vitro studies showed that the latter phenomenon was due to a temperature dependent partitioning of cyclospor...

  5. A Phase II Study of RO4929097 Gamma-Secretase Inhibitor in Metastatic Melanoma: SWOG 0933

    Science.gov (United States)

    Lee, Sylvia M.; Moon, James; Redman, Bruce G.; Chidiac, Tarek; Flaherty, Lawrence E.; Zha, Yuanyuan; Othus, Megan; Ribas, Antoni; Sondak, Vernon K.

    2014-01-01

    Background Aberrant Notch activation confers a proliferative advantage onto many human tumors, including melanoma. This phase II trial assessed the antitumor activity of RO4929097, a gamma-secretase inhibitor of Notch signaling, on the progression-free and overall survival of patients with advanced melanoma. Methods Chemotherapy-naïve patients with metastatic melanoma of cutaneous or unknown origin were treated with RO4929097 at a dose of 20 mg orally daily, 3 consecutive days per week. A two-step accrual design was used, with an interim analysis on the first 32 patients, and continuation of enrollment if ≥4/32 patients responded. Results Thirty-six patients from 23 institutions were enrolled; 32 patients were evaluable. RO4929097 was well-tolerated, and most toxicities were grade 1 or 2. The most common toxicities were nausea (53%), fatigue (41%), and anemia (22%). There was 1 confirmed partial response lasting 7 months, and 8 patients with stable disease lasting at least through week 12, with one of these continuing for 31 months. The 6-month PFS was 9% (95% CI: 2–22%), and 1-year OS was 50% (95% CI: 32–66%). Peripheral blood T cell assays showed no significant inhibition of IL-2 production, a surrogate pharmacodynamic marker of Notch inhibition, suggesting that the drug levels were insufficient to achieve Notch target inhibition. Conclusions RO4929097 showed minimal clinical activity against metastatic melanoma in this phase II trial, possibly due to inadequate exposure to therapeutic drug levels. While Notch inhibition remains a compelling target in melanoma, our results do not support further investigation of RO4929097 at this dose and schedule. PMID:25250858

  6. Time-dependent histamine release from stored human blood products

    DEFF Research Database (Denmark)

    Nielsen, Hans Jørgen; Edvardsen, L; Vangsgaard, K;

    1996-01-01

    storage. Whole blood (six units), plasma-reduced whole blood (six units), and plasma- and buffy coat-reduced (saline-adenine-glucose-mannitol) (SAGM) blood (six units) from unpaid healthy donors were stored in the blood bank for 35 days at 4 degrees C. Plasma histamine and total cell-bound histamine......Perioperative transfusion of whole blood has been shown to amplify trauma-induced immunosuppression, which could be attenuated by perioperative administration of histamine2 receptor antagonists. Supernatants from different blood products were, therefore, analysed for histamine content during.......0 (range 176.0-910.0) nmol/l in whole blood and 475.0 (range 360.0-1560.0) nmol/l in plasma-reduced whole blood, while it was undetectable in SAGM blood. Spontaneous histamine release increased in a time-dependent manner from a median of 6.7 (range 2.2-17.4) nmol/l at the time of storage to 175.0 (range 33...

  7. Changes in the human blood coagulating system during prolonged hypokinesia

    Science.gov (United States)

    Filatova, L. M.; Anashkin, O. D.

    1978-01-01

    Changes in the coagulating system of the blood were studied in six subjects during prolonged hypokinesia. Thrombogenic properties of the blood rose in all cases on the 8th day. These changes are explained by stress reaction due to unusual conditions for a healthy person. Changes in the blood coagulating system in the group subjected to physical exercise and without it ran a practically parallel course. Apparently physical exercise is insufficient to prevent such changes that appear in the coagulating system of the blood during prolonged hypokinesia.

  8. Stability of human chorionic gonadotropin and its alpha subunit in human blood.

    Science.gov (United States)