Sample records for b16f10 melanoma cells

  1. Phosphodiesterase 4 regulates the migration of B16-F10 melanoma cells.

    PubMed

    Watanabe, Yoshihiro; Murata, Taku; Shimizu, Kasumi; Morita, Hiroshi; Inui, Madoka; Tagawa, Toshiro

    2012-08-01

    Phosphodiesterases (PDEs) are important regulators of signal transduction processes. Eleven PDE gene families (PDE1-11) have been identified and several PDE isoforms are selectively expressed in various cell types. PDE4 family members specifically hydrolyze cyclic AMP (cAMP). Four genes (PDE4A-D) are known to encode PDE4 enzymes, with additional diversity generated by the use of alternative mRNA splicing and the use of different promoters. While PDE4 selective inhibitors show therapeutic potential for treating major diseases such as asthma and chronic obstructive pulmonary disease, little is known concerning the role of PDE4 in malignant melanoma. In this study, we examined the role of PDE4 in mouse B16-F10 melanoma cells. In these cells, PDE4 activity was found to be ∼60% of total PDE activity. RT-PCR detected only PDE4B and PDE4D mRNA. Cell growth was inhibited by the cAMP analog, 8-bromo-cAMP, but not by the specific PDE4 inhibitors, rolipram and denbufylline, which increased intracellular cAMP concentrations. Finally, migration of the B16-F10 cells was inhibited by the PDE4 inhibitors and 8-bromo-cAMP, while migration was increased by a protein kinase A (PKA) inhibitor, PKI(14-22), and was not affected by 8-pCPT-2'-O-Me-cAMP, which is an analog of exchange protein activated by cAMP (Epac). The inhibitory effect of rolipram on migration was reversed by PKI(14-22). Based on these results, PDE4 appears to play an important role in the migration of B16-F10 cells, and therefore may be a novel target for the treatment of malignant melanoma.

  2. Jolkinolide B induces apoptosis and inhibits tumor growth in mouse melanoma B16F10 cells by altering glycolysis.

    PubMed

    Gao, Caixia; Yan, Xinyan; Wang, Bo; Yu, Lina; Han, Jichun; Li, Defang; Zheng, Qiusheng

    2016-10-31

    Most cancer cells preferentially rely on glycolysis to produce the energy (adenosine triphosphate, ATP) for growth and proliferation. Emerging evidence demonstrates that the apoptosis in cancer cells could be closely associated with the inhibition of glycolysis. In this study, we have found that jolkinolide B (JB), a bioactive diterpenoid extracted from the root of Euphorbia fischeriana Steud, induced tumor cells apoptosis and decreased the production of ATP and lactic acid in mouse melanoma B16F10 cells. Furthermore, we found that JB downregulated the mRNA expression of glucose transporter genes (Glut1, Glut3 and Glut4) and glycolysis-related kinase genes (Hk2 and Ldha) in B16F10 cells. Moreover, treatment with JB upregulated the mRNA expression of pro-apoptosis genes (Bax), downregulated the mRNA expression of anti-apoptosis genes (Bcl-2, Caspase-3 and Caspase-9), decreased the potential of mitochondrial membrane and increased reactive oxygen species (ROS) levels in B16F10 cells. Finally, intragastric administration of JB suppressed tumor growth and induced tumor apoptosis in mouse xenograft model of murine melanoma B16F10 cells. Taken together, these results suggest that JB could induce apoptosis through the mitochondrial pathway and inhibit tumor growth. The inhibition of glycolysis could play a crucial role in the induction of apoptosis in JB-treated B16F10 cells.

  3. Jolkinolide B induces apoptosis and inhibits tumor growth in mouse melanoma B16F10 cells by altering glycolysis

    PubMed Central

    Gao, Caixia; Yan, Xinyan; Wang, Bo; Yu, Lina; Han, Jichun; Li, Defang; Zheng, Qiusheng

    2016-01-01

    Most cancer cells preferentially rely on glycolysis to produce the energy (adenosine triphosphate, ATP) for growth and proliferation. Emerging evidence demonstrates that the apoptosis in cancer cells could be closely associated with the inhibition of glycolysis. In this study, we have found that jolkinolide B (JB), a bioactive diterpenoid extracted from the root of Euphorbia fischeriana Steud, induced tumor cells apoptosis and decreased the production of ATP and lactic acid in mouse melanoma B16F10 cells. Furthermore, we found that JB downregulated the mRNA expression of glucose transporter genes (Glut1, Glut3 and Glut4) and glycolysis-related kinase genes (Hk2 and Ldha) in B16F10 cells. Moreover, treatment with JB upregulated the mRNA expression of pro-apoptosis genes (Bax), downregulated the mRNA expression of anti-apoptosis genes (Bcl-2, Caspase-3 and Caspase-9), decreased the potential of mitochondrial membrane and increased reactive oxygen species (ROS) levels in B16F10 cells. Finally, intragastric administration of JB suppressed tumor growth and induced tumor apoptosis in mouse xenograft model of murine melanoma B16F10 cells. Taken together, these results suggest that JB could induce apoptosis through the mitochondrial pathway and inhibit tumor growth. The inhibition of glycolysis could play a crucial role in the induction of apoptosis in JB-treated B16F10 cells. PMID:27796318

  4. Melanogenesis inhibits respiration in B16-F10 melanoma cells whereas enhances mitochondrial cell content

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Meira, Willian Vanderlei; Heinrich, Tassiele Andréa; Cadena, Silvia Maria Suter Correia

    Melanoma is a rare and aggressive skin tumor; the survival of patients diagnosed late is fairly low. This high mortality rate is due to the characteristics of the cells that allow them to be resistant to radiotherapy and conventional chemotherapy, besides of being able to evade the immune system. Melanin, the pigment responsible for skin, hair and eye color, seems to be involved in this resistance. The main function of melanin is to protect the cells against ultraviolet (UV) light by absorbing this radiation and reactive oxygen species (ROS) scavenging. But this pigment may have also a role as photosensitizer,more » because when it is irradiated with UVA light (320-400 nm), the generation of ROS was detected. Besides, the melanogenesis stimulation on B16-F10 cells resulted in cell cycle arrest, induction of a quiescent state, change in the expression of several proteins and alterations on ADP/ATP ratio. The present study aimed to investigate the influence of melanogenesis stimulation in mitochondrial function of B16-F10 melanoma cells. Therefore, we analyzed cells respiration, mitochondrial membrane potential (Δψ{sub m}) and mitochondria mass in B16-F10 melanoma cells stimulated with 0.4 mM L-tyrosine and 10 mM NH{sub 4}Cl. Our results showed that the induction of melanin synthesis was able to reduce significantly the oxygen consumption after 48 h of stimulation, without changes of mitochondrial membrane potential when compared to non-stimulated cells. Despite of respiration inhibition, the mitochondria mass was higher in cells with melanogenesis stimulation. We suggest that the stimulation in the melanin synthesis might be promoting the inhibition of electrons transport chain by some intermediate compound from the synthesis of the pigment and this effect could contribute to explain the entry in the quiescent state. - Highlights: • Melanoma pigmentation alters mitochondrial respiration. • Induction of melanin synthesis by 48 h do not change mitochondrial

  5. Nanobiotechnological Nanocapsules Containing Polyhemoglobin-Tyrosinase: Effects on Murine B16F10 Melanoma Cell Proliferation and Attachment

    PubMed Central

    Wang, Yun; Chang, Thomas M. S.

    2012-01-01

    We have reported previously that daily intravenous infusions of a soluble nanobiotechnological complex, polyhemoglobin-tyrosinase [polyHb-Tyr], can suppress the growth of murine B16F10 melanoma in a mouse model. In order to avoid the need for daily intravenous injections, we have now extended this further as follows. We have prepared two types of biodegradable nanocapsules containing [polyHb-Tyr]. One type is to increase the circulation time and decrease the frequency of injection and is based on polyethyleneglycol-polylactic acid (PEG-PLA) nanocapsules containing [polyHb-Tyr]. The other type is to allow for intratumoural or local injection and is based on polylactic acid (PLA) nanocapsules containing [polyHb-Tyr]. Cell culture studies show that it can inhibit the proliferation of murine B16F10 melanoma cells in the “proliferation model”. It can also inhibit the attachment of murine B16F10 melanoma cells in the “attachment model.” This could be due to the action of tyrosinase on the depletion of tyrosine or the toxic effect of tyrosine metabolites. The other component, polyhemoglobin (polyHb), plays a smaller role in nanocapsules containing [polyHb-Tyr], and this is most likely by its depletion of nitric oxide needed for melanoma cell growth. PMID:23209910

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-10-01

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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Beninati, Simone, E-mail: beninati@bio.uniroma2.it; Oliverio, Serafina; Cordella, Martina

    2014-08-08

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

  9. Anti-cancer effects of CME-1, a novel polysaccharide, purified from the mycelia of Cordyceps sinensis against B16-F10 melanoma cells.

    PubMed

    Jayakumar, Thanasekaran; Chiu, Chong-Chi; Wang, Shwu-Huey; Chou, Duen-Suey; Huang, Yung-Kai; Sheu, Joen-Rong

    2014-01-01

    Matrix metalloproteinases (MMPs) play important roles in the invasion and migration of cancer cells. In melanoma, several signaling pathways are constitutively activated. Among these, the mitogen-activated protein kinase (MAPKs) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Therefore, the inhibition of MAPK signaling might be a crucial role for the treatment of melanoma cancer. We examined the anticancer effect of CME-1, a novel water-soluble polysaccharide fraction, isolated from Cordyceps sinensis mycelia on B16-F10 melanoma cells. B16-F10 cells were exposed to different concentrations of CME-1 (250, 500 and 800 μg/ml) for 24 h in 5% CO² incubator at 37°C. Western blot analysis was performed to detect the expression of MMP-1, p-p38 MAPK, p-ERK1/2, and IkB-α in B16-F10 cells. Cell migration test was performed by wound healing migration assay. CME-1 suppresses cell migration in a concentration-dependent manner. Western blotting analysis revealed that CME-1 led to the reduction on the expression levels of MMP-1 and down regulated the expression of phosphorylated extracellular signal-regulated kinase (ERK1/2 and p38 mitogen-activated protein kinase (p38 MAPK). CME-1 restored the IkB-degradation in B16F10 cells. These results indicate that CME-1 inhibited MMP-1 expressions in B16F10 melanoma cells through either NF-kB or ERK/p38 MAPK down regulation thereby inhibiting B16F10 cell migration. Therefore, we proposed that CME-1 might be developed as a therapeutic potential candidate for the treatment of cancer metastasis.

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

    PubMed

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

    2015-01-05

    Invasion and metastasis are the major causes of treatment failure in patients with cancer. Hinokitiol, a natural bioactive compound found in Chamacyparis taiwanensis, has been used in hair tonics, cosmetics, and food as an antimicrobial agent. In this study, we investigated the effects and possible mechanisms of action of hinokitiol on migration by the metastatic melanoma cell line, B16-F10, in which matrix metalloproteinase-1 (MMP-1) is found to be highly- expressed. Treatment with hinokitiol revealed a concentration-dependent inhibition of migration of B16-F10 melanoma cells. Hinokitiol appeared to achieve this effect by reducing the expression of MMP-1 and by suppressing the phosphorylation of mitogen- activated protein kinase (MAPK) signaling molecules such as extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK and c-Jun N-terminal kinases (JNK). On the other hand, hinokitiol treatment reversed IκB-α degradation and inhibited the phosphorylation of p65 nuclear factor kappa B (NF-κB) and cJun in B16-F10 cells. In addition, hinokitiol suppressed the translocation of p65 NF-κB from the cytosol to the nucleus, suggesting reduced NF-κB activation. Consistent with these in vitro findings, our in vivo study demonstrated that hinokitiol treatment significantly reduced the total number of mouse lung metastatic nodules and improved histological alterations in B16-F10 injected C57BL/6 mice. These findings suggest that treatment of B16-F10 cells with hinokitiol significantly inhibits metastasis, possibly by blocking MMP-1 activation, MAPK signaling pathways and inhibition of the transcription factors, NF-κB and c-Jun, involved in cancer cell migration. These results may accelerate the development of novel therapeutic agents for the treatment of malignant cancers. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Proteomic study reveals a co-occurrence of gallic acid-induced apoptosis and glycolysis in B16F10 melanoma cells.

    PubMed

    Liu, Cheng; Lin, Jen-Jie; Yang, Zih-Yan; Tsai, Chi-Chu; Hsu, Jue-Liang; Wu, Yu-Jen

    2014-12-03

    Gallic acid (GA) has long been associated with a wide range of biological activities. In this study, its antitumor effect against B16F10 melanoma cells was demonstrated by MTT assay, cell migration assay, wound-healing assay, and flow cytometric analysis. GA with a concentration >200 μM shows apoptotic activity toward B16F10 cells. According to Western blotting data, overexpressions of cleaved forms of caspase-9, caspase-3, and PARP-1 and pro-apoptotic Bax and Bad, accompanied by underexpressed anti-apoptotic Bcl-2 and Bcl-xL indicate that GA induces B16F10 cell apoptosis via mitochondrial pathway. The 2-DE based comparative proteomics was further employed in B16F10 cells with and without GA treatment for a large-scale protein expression profiling. A total of 41 differential protein spots were quantified, and their identities were characterized using LC-MS/MS analysis and database matching. In addition to some regulated proteins that were associated with apoptosis, interestingly, some identified proteins involved in glycolysis such as glucokinase, α-enolase, aldolase, pyruvate kinase, and GAPDH were simultaneously up-regulated, which reveals that the GA-induced cellular apoptosis in B16 melanoma cells is associated with metabolic glycolysis.

  12. Differential Effects of Methoxylated p-Coumaric Acids on Melanoma in B16/F10 Cells

    PubMed Central

    Yoon, Hoon Seok; Lee, Nam-Ho; Hyun, Chang-Gu; Shin, Dong-Bum

    2015-01-01

    As an approach to search for chemopreventive agents, we tested p-coumaric acid, 3-methoxy-p-coumaric acid (ferulic acid), and 3,5-dimethoxy-p-coumaric acid (sinapic acid) in B16/F10 melanoma cells. Intracellular melanin contents were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and cytotoxicity of the compounds were examined by lactate dehydrogenase (LDH) release. p-Coumaric acid showed inhibitory effect on melanogenesis, but ferulic acid increased melanin content, and sinapic acid had almost no effect on melanogenesis. Treatment with ferulic acid resulted in a 2 to 3 fold elevation in the production of melanin. Correlatively, cell viability decreased in a dose-dependent manner when treated with ferulic acid. However, ferulic acid did not affect the LDH release from the cells. Treatment with sinapic acid resulted in a 50~60% elevation in the release of LDH when treated with a 200 μg/mL concentration and showed neither cytostasis nor increase of melanin synthesis in a dose-dependent manner. Taken together, p-coumaric acid inhibits melanogenesis, ferulic acid induces melanogenesis, and sinapic acid exerts cytotoxic effects in B16/F10 murine melanoma cells. The results indicate that the addition of methoxy groups to p-coumaric acid shows the melanogenic or cytotoxic effects in melanoma cells compared to the original compound. Therefore, this study suggests the possibility that methoxylated p-coumaric acid, ferulic acid can be used as a chemopreventive agent. PMID:25866753

  13. Cytotoxicity of the coagulant Moringa oleifera lectin (cMoL) to B16-F10 melanoma cells.

    PubMed

    de Andrade Luz, Luciana; Rossato, Franco Aparecido; Costa, Rute Alves Pereira E; Napoleão, Thiago Henrique; Paiva, Patrícia Maria Guedes; Coelho, Luana Cassandra Breitenbach Barroso

    2017-10-01

    Moringa oleifera seeds are used in alternative medicine to treat inflammation, tumors and bacterial and protozoan infections, for example. The seeds contain lectins, which are carbohydrate-binding proteins with several biological properties including cytotoxicity to cancer cells. In this work, we examined the cytotoxicity of the coagulant M. oleifera lectin (cMoL) on B16-F10 murine melanoma cells. cMoL cytotoxic effects were evaluated through trypan blue assay and flow cytometry analysis. Mitochondrial superoxide levels and activation of caspases 3, 8 and 9 were measured. cMoL (1.5-16μM) reduced viability and caused cell death of B16-F10 cells with an IC 50 of 9.72μM. Flow cytometry analysis indicated induction of necrosis and suggested the presence of cells in late apoptosis. Specificity for tumor cells was observed since death of normal human fibroblasts (GN) was not higher than 20% in treatments with cMoL from 1.5 to 16μM. Microscopy images revealed rounded shape and reduction of volume in B16-F10 cells treated with cMoL. cMoL increased mitochondrial ROS production and promoted caspases 3, 8 and 9 activation in B16-F10 cells, indicating the activation of apoptosis-related pathway. In conclusion, this study demonstrates that cMoL is cytotoxic to B16-F10 cells, which stimulates more investigation on the anticancer potential of this lectin. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Casticin impairs cell migration and invasion of mouse melanoma B16F10 cells via PI3K/AKT and NF-κB signaling pathways.

    PubMed

    Shih, Yung-Luen; Chou, Hsiao-Min; Chou, Hsiu-Chen; Lu, Hsu-Feng; Chu, Yung-Lin; Shang, Hung-Sheng; Chung, Jing-Gung

    2017-09-01

    Casticin, a polymethoxyflavone, is one of the major active components obtained from Fructus viticis, which have been shown to have anticancer activities including induce cell apoptosis in human cancer cells. The aim of this study was to investigate the molecular mechanisms by which casticin inhibits cell migration and invasion of mouse melanoma B16F10 cells. Cell viability was examined by MTT assay and the results indicated that casticin decreased the total percentages of viable cells in dose-dependent manners. Casticin affected cell migration and invasion in B16F10 cells were examined by wound healing mobility assay and Boyden chamber migration and invasion assay and results indicated that casticin inhibited cell migration and invasion in dose-dependent manners. Western blotting was used to examine the protein expression of B16F10 cells after exposed to casticin and the results showed that casticin decreased the expressions of MMP-9, MMP-2, MMP-1, FAK, 14-3-3, GRB2, Akt, NF-κB p65, SOS-1, p-EGFR, p-JNK 1/2, uPA, and Rho A in B16F10 cells. Furthermore, cDNA microarray assay was used to show that casticin affected associated gene expression of cell migration and invasion and the results indicated that casticin affected some of the gene expression such as increased SCN1B (cell adhesion molecule 1) and TIMP2 (TIMP metallopeptidase inhibitor 2) and decreased NDUFS4 (NADH dehydrogenase (ubiquinone) Fe-S protein4), VEGFA (vascular endothelial growth factor A), and DDIT3 (DNA-damage-inducible transcript 3) which associated cell migration and invasion in B16F10 cells. Based on those observations, we suggest that casticin could be used as a novel anticancer metastasis of melanoma cancer in the future. © 2017 Wiley Periodicals, Inc.

  15. In vitro evaluation of low-intensity light radiation on murine melanoma (B16F10) cells.

    PubMed

    Peidaee, P; Almansour, N M; Pirogova, E

    2016-03-01

    Changes in the energy state of biomolecules induced by electromagnetic radiation lead to changes in biological functions of irradiated biomolecules. Using the RRM approach, it was computationally predicted that far-infrared light irradiation in the range of 3500-6000 nm affects biological activity of proto-oncogene proteins. This in vitro study evaluates quantitatively and qualitatively the effects of selected far-infrared exposures in the computationally determined wavelengths on mouse melanoma B16F10 cells and Chinese hamster ovarian (CHO) cells by MTT (thiazolyl blue tetrazolium bromide) cell proliferation assay and confocal laser-scanning microscopy (CLSM). This paper also presents the findings obtained from irradiating B16F10 and CHO cells by the selected wavelengths in visible and near-infrared range. The MTT results show that far-infrared wavelength irradiation induces detrimental effect on cellular viability of B16F10 cells, while that of normal CHO cells is not affected considerably. Moreover, CLSM images demonstrate visible cellular detachment of cancer cells. The observed effects support the hypothesis that far-infrared light irradiation within the computationally determined wavelength range induces biological effect on cancer cells. From irradiation of selected visible and near-infrared wavelengths, no visible changes were detected in cellular viability of either normal or cancer cells.

  16. Imatinib mesylate induction of ROS-dependent apoptosis in melanoma B16F0 cells.

    PubMed

    Chang, Shao-Ping; Shen, Shing-Chuan; Lee, Woan-Rouh; Yang, Ling-Ling; Chen, Yen-Chou

    2011-06-01

    Imatinib mesylate (STI571), a protein tyrosine kinase inhibitor, was shown to reduce the viability of several cancer cell lines via apoptosis induction; however, the role of reactive oxygen species (ROS) in STI571-induced melanoma cell apoptosis is still undefined. In this study, we investigated the contribution of ROS to STI571-induced apoptosis in melanoma B16F0 cells, and the apoptotic mechanism elicited by STI571 was illustrated. Using an in vitro cell culture system, the effects of STI571 on ROS production, cell cycle progression, caspase activation, and mitochondrial functions were examined via Western blotting, a flow cytometric analysis, an enzyme activity assay, and a DNA integrity assay. In pharmacological studies, the ROS scavenger, N-acetyl cysteine (NAC), the NADPH oxidase inhibitor, dipheylene iodide (DPI), and mitogen-activated protein kinase (MAPK) inhibitors (PD98059, SP600125, and SB203580) were applied to investigate the mechanism. STI571 reduced the viability of melanoma cells B16F0, but not human skin fibroblasts WS1, via apoptosis induction. Besides, apoptosis induced by STI571 was inhibited by the addition of NAC and DPI, and an increase in the intracellular peroxide level by STI571 was identified in melanoma B16F0 cells. Activation of caspases 3 and 9 enzyme activities accompanied by disrupting the mitochondria membrane potential in according with stimulating JNK and p38 protein phosphorylation was identified in STI571-treated B16F0 cells. STI571-mediated a ROS-dependent apoptosis potentiated by JNK inhibitor SP600125 was first identified in melanoma B16F0 cells. Our results support the idea that ROS-dependent apoptosis in STI571-treated melanoma cells B16F0. The combination of a JNK inhibitor with STI571 for treating melanomas is suggested for further in vivo studies. Copyright © 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  17. Phenothiazinium dyes in association with diode red laser against B16F10 melanoma cells: in vitro study

    NASA Astrophysics Data System (ADS)

    Miranda, Anderson F.; Santos, Gustavo M. P.; de Oliveira, Susana C. P. S.; Monteiro, Juliana S. C.; Sampaio, Fernando J. P.; Gomes Júnior, Rafael Araújo; Brugnera, Aldo; Gesteira, Maria F. M.; Zanin, Fátima A. A.; Pinheiro, Antônio Luiz B.; Vannier-Santos, Marcos A.

    2014-02-01

    In Brazil solar incidence is high and continuous throughout the year. Body exposure to sunlight may be a key point in the rates of individuals affected by melanoma and other types of skin cancer in many countries. Brazil already occupies the 15th place in the ranking of melanoma cases and the limitations presented by drugs used in the therapy of this cancer, new approaches are being used in an attempt to decrease the mortality of this malignancy. The aim of this study was to evaluate the effects of phenothiazinium dyes (PD) associated with laser light on murine melanoma (B16F10) in vitro by measuring cell growth using colorimetric assay before and after photodynamic therapy. We used a diode laser (λ660nm, 2.4 J/cm2, 40 mW, 60 s, CW) associated with PD at 12.5 μg/mL, time pre-irradiation of 30 minutes). The following groups were tested: control (LF-), PD (L-F+), Laser (L+F-), Laser + PD (L+F+). The results showed a significant reduction in cell growth in the group treated by the photodynamic therapy compared to the control at 24 and 48 h (p < 0.001). Were showing at 30 min PD has a dose-dependent response on B16F10 cells, but at 24 h did not demonstrated this response.

  18. Intravenous administration of bone marrow-derived multipotent mesenchymal stromal cells enhances the recruitment of CD11b{sup +} myeloid cells to the lungs and facilitates B16-F10 melanoma colonization

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Souza, Lucas E.B., E-mail: lucasebsouza@usp.br; Hemotherapy Center of Ribeirão Preto, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP; Almeida, Danilo C., E-mail: gudaalmeida@gmail.com

    The discovery that the regenerative properties of bone marrow multipotent mesenchymal stromal cells (BM-MSCs) could collaterally favor neoplastic progression has led to a great interest in the function of these cells in tumors. However, the effect of BM-MSCs on colonization, a rate-limiting step of the metastatic cascade, is unknown. In this study, we investigated the effect of BM-MSCs on metastatic outgrowth of B16-F10 melanoma cells. In in vitro experiments, direct co-culture assays demonstrated that BM-MSCs stimulated the proliferation of B16-F10 cells in a dose-dependent manner. For in vivo experiments, luciferase-expressing B16-F10 cells were injected through tail vein and mice weremore » subsequently treated with four systemic injections of BM-MSCs. In vivo bioluminescent imaging during 16 days demonstrated that BM-MSCs enhanced the colonization of lungs by B16-F10 cells, which correlated with a 2-fold increase in the number of metastatic foci. Flow cytometry analysis of lungs demonstrated that although mice harboring B16-F10 metastases displayed more endothelial cells, CD4 T and CD8 T lymphocytes in the lungs in comparison to metastases-free mice, BM-MSCs did not alter the number of these cells. Interestingly, BM-MSCs inoculation resulted in a 2-fold increase in the number of CD11b{sup +} myeloid cells in the lungs of melanoma-bearing animals, a cell population previously described to organize “premetastatic niches” in experimental models. These findings indicate that BM-MSCs provide support to B16-F10 cells to overcome the constraints that limit metastatic outgrowth and that these effects might involve the interplay between BM-MSCs, CD11b{sup +} myeloid cells and tumor cells. - Highlights: • BM-MSCs enhanced B16-F10 proliferation in a dose-dependent manner in vitro. • BM-MSCs facilitated lung colonization by B16-F10 melanoma cells. • BM-MSCs administration did not alter the number of endothelial cells and T lymphocytes in the lungs. • BM

  19. The biochemical characterization, stabilization studies and the antiproliferative effect of bromelain against B16F10 murine melanoma cells.

    PubMed

    São Paulo Barretto Miranda, Íngara Keisle; Fontes Suzart Miranda, Anderson; Souza, Fernanda Vidigal Duarte; Vannier-Santos, Marcos André; Pirovani, Carlos Priminho; Pepe, Iuri Muniz; Rodowanski, Ivanoé João; Ferreira, Katiúcia Tícila de Souza Eduvirgens; Mendes Souza Vaz, Luciano; de Assis, Sandra Aparecida

    2017-06-01

    The current study aims to extract bromelain from different parts (stem, crown, peels, pulp and leaves) of Ananas comosus var. comosus AGB 772; to determine of optimum pH and temperature; to test bromelain stability in disodium EDTA and sodium benzoate, and to investigate its pharmacological activity on B16F10 murine melanoma cells in vitro. The highest enzymatic activity was found in bromelain extracted from the pulp and peel. The optimum bromelain pH among all studied pineapple parts was 6.0. The optimum temperature was above 50 °C in all bromelain extracts. The fluorescence analysis confirmed the stability of bromelain in the presence of EDTA and sodium benzoate. Bromelain was pharmacologically active against B16F10 melanoma cells and it was possible verifying approximately 100% inhibition of tumor cell proliferation in vitro. Since bromelain activity was found in different parts of pineapple plants, pineapple residues from the food industry may be used for bromelain extraction.

  20. Combination of amino acids reduces pigmentation in B16F0 melanoma cells.

    PubMed

    Ishikawa, Masago; Kawase, Ichiro; Ishii, Fumio

    2007-04-01

    Amino acids, the building blocks of proteins, play significant roles in numerous physiological events in mammals. As the effects of amino acids on melanogenesis have yet to be demonstrated, the present study was conducted to identify whether amino acids, in particular alanine, glycine, isoleucine and leucine, influence melanogenesis in B16F0 melanoma cells. Glycine and L-isoleucine, but not D-isoleucine, reduced melanogenesis in a concentration-dependent manner without any morphological changes in B16F0 melanoma cells. L-Alanine and L-leucine, but not D-alanine and D-leucine, also reduced melanogenesis without any morphological changes in B16F0 melanoma cells. However these amino acids did not show a concentration-dependency. Combination of L-alanine and the other amino acids, particularly 4 amino acids combination, had an additive effect on the inhibition of melanogenesis compared with single treatment of L-alanine. None of the amino acids affected the activity of tyrosinase, a key enzyme in melanogenesis. These results suggest that L-alanine, glycine, L-isoleucine and L-leucine, but not the D-form amino acids, have a hypopigmenting effect in B16F0 melanoma cells, and that these effects are not due to the inhibition of tyrosinase activity. Combination of these 4 amino acids had the additive effect on hypopigmentation that was as similar as that of kojic acid.

  1. Biochemical basis of 4-hydroxyanisole induced cell toxicity towards B16-F0 melanoma cells.

    PubMed

    Moridani, Majid Y

    2006-11-18

    In the current work we investigated for the first time the biochemical basis of 4-hydroxyanisole (4-HA) induced toxicity in B16-F0 melanoma cells. It was found that dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased 4-HA induced toxicity towards B16-F0 cells whereas dithiothreitol, a thiol containing agent, and ascorbic acid (AA), a reducing agent, largely prevented 4-HA toxicity. TEMPOL and pyrogallol, free radical scavengers, did not significantly prevent 4-HA toxicity towards B16-F0 cells. GSH>AA>NADH prevented the o-quinone formation when 4-HA was metabolized by tyrosinase/O(2). 4-HA metabolism by horseradish peroxidase/H(2)O(2) was prevented more effectively by AA than NADH>GSH. We therefore concluded that quinone formation was the major pathway for 4-HA induced toxicity in B16-F0 melanoma cells whereas free radical formation played a negligible role in the 4-HA induced toxicity.

  2. Intravenous administration of bone marrow-derived multipotent mesenchymal stromal cells enhances the recruitment of CD11b(+) myeloid cells to the lungs and facilitates B16-F10 melanoma colonization.

    PubMed

    Souza, Lucas E B; Almeida, Danilo C; Yaochite, Juliana N U; Covas, Dimas T; Fontes, Aparecida M

    2016-07-15

    The discovery that the regenerative properties of bone marrow multipotent mesenchymal stromal cells (BM-MSCs) could collaterally favor neoplastic progression has led to a great interest in the function of these cells in tumors. However, the effect of BM-MSCs on colonization, a rate-limiting step of the metastatic cascade, is unknown. In this study, we investigated the effect of BM-MSCs on metastatic outgrowth of B16-F10 melanoma cells. In in vitro experiments, direct co-culture assays demonstrated that BM-MSCs stimulated the proliferation of B16-F10 cells in a dose-dependent manner. For in vivo experiments, luciferase-expressing B16-F10 cells were injected through tail vein and mice were subsequently treated with four systemic injections of BM-MSCs. In vivo bioluminescent imaging during 16 days demonstrated that BM-MSCs enhanced the colonization of lungs by B16-F10 cells, which correlated with a 2-fold increase in the number of metastatic foci. Flow cytometry analysis of lungs demonstrated that although mice harboring B16-F10 metastases displayed more endothelial cells, CD4 T and CD8 T lymphocytes in the lungs in comparison to metastases-free mice, BM-MSCs did not alter the number of these cells. Interestingly, BM-MSCs inoculation resulted in a 2-fold increase in the number of CD11b(+) myeloid cells in the lungs of melanoma-bearing animals, a cell population previously described to organize "premetastatic niches" in experimental models. These findings indicate that BM-MSCs provide support to B16-F10 cells to overcome the constraints that limit metastatic outgrowth and that these effects might involve the interplay between BM-MSCs, CD11b(+) myeloid cells and tumor cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Interactions of B16F10 melanoma cells aggregated on a cellulose substrate.

    PubMed

    Hindié, M; Vayssade, M; Dufresne, M; Quéant, S; Warocquier-Clérout, R; Legeay, G; Vigneron, P; Olivier, V; Duval, J-L; Nagel, M-D

    2006-09-01

    There is evidence that the shape of cells and their contact with a matrix direct the growth and the differentiation of both normal and cancer cells. Cells in 3D culture resemble the in vivo situation more closely than do those in conventional 2D cultures. We have studied the interactions and functions of B16F10 mouse melanoma cells, which spread and grow well on tissue culture polystyrene (tPS), when they were made to aggregate on cellulose-coated Petri dishes (CEL). This aggregation of melanoma cells on CEL was Ca2+ dependent and mediated by N-cadherins. The levels of N-cadherin and beta-catenin transcripts in cells cultured on CEL and tPS were similar, but those on CEL contained less beta-catenin protein. Immunoprecipitation and immunostaining showed that both N-cadherins and beta-catenins were present at the membranes of cells on CEL. Cells proliferated significantly more slowly after 48 h on CEL and the cellulose coating caused most of them to arrest in G1. We also compared the melanin contents and tyrosinase activity of cells on CEL and controls grown on tPS. Melanogenesis was induced in cells aggregated on CEL. A cellulose substrate thus appears to be an outstanding tool for studying cell-cell interactions and cell functions in 3D cultures.

  4. Isoliquiritigenin-Induced Differentiation in Mouse Melanoma B16F0 Cell Line

    PubMed Central

    Chen, Xiaoyu; Zhang, Bo; Yuan, Xuan; Yang, Fan; Liu, Jinglei; Zhao, Hong; Liu, Liangliang; Wang, Yanming; Wang, Zhenhua; Zheng, Qiusheng

    2012-01-01

    The chemotherapeutical treatment is very limited for malignant melanoma, a highly lethal disease occurs globally. Natural products derived from traditional Chinese medicine licorice are attractive in quest new treatments due to their anti-tumor activities. A new dietary flavonoid isoliquiritigenin (ISL) were thus investigated to indentify its anti-melanoma activities on mouse melanoma B16F0 cells in present study. Using biochemical and free radical biological experiments in vitro, we identified the pro-differentiated profiles of ISL and evaluated the role of reactive oxygen species (ROS) during B16F0 cell differentiation. The data showed a strong dose-response relationship between ISL exposure and the characteristics of B16F0 differentiation in terms of morphology changes and melanogenesis. The accumulated intercellular ROS during exposure are necessary to support ISL-induced differentiation, which was proven by additional redox modulators. It was confirmed further by the relative activities of enzymes and genes modulated melanogenesis in ISL-treatments with or without ROS modulators. The tumorigenicity of ISL-treated cells was limited significantly by using the colony formation assay in vitro and an animal model assay in vivo respectively. Our research demonstrated that isoliquiritigenin is a differentiation-inducing agent, and its mechanisms involve ROS accumulation facilitating melanogenesis. PMID:23304254

  5. Isoliquiritigenin-induced differentiation in mouse melanoma B16F0 cell line.

    PubMed

    Chen, Xiaoyu; Zhang, Bo; Yuan, Xuan; Yang, Fan; Liu, Jinglei; Zhao, Hong; Liu, Liangliang; Wang, Yanming; Wang, Zhenhua; Zheng, Qiusheng

    2012-01-01

    The chemotherapeutical treatment is very limited for malignant melanoma, a highly lethal disease occurs globally. Natural products derived from traditional Chinese medicine licorice are attractive in quest new treatments due to their anti-tumor activities. A new dietary flavonoid isoliquiritigenin (ISL) were thus investigated to indentify its anti-melanoma activities on mouse melanoma B16F0 cells in present study. Using biochemical and free radical biological experiments in vitro, we identified the pro-differentiated profiles of ISL and evaluated the role of reactive oxygen species (ROS) during B16F0 cell differentiation. The data showed a strong dose-response relationship between ISL exposure and the characteristics of B16F0 differentiation in terms of morphology changes and melanogenesis. The accumulated intercellular ROS during exposure are necessary to support ISL-induced differentiation, which was proven by additional redox modulators. It was confirmed further by the relative activities of enzymes and genes modulated melanogenesis in ISL-treatments with or without ROS modulators. The tumorigenicity of ISL-treated cells was limited significantly by using the colony formation assay in vitro and an animal model assay in vivo respectively. Our research demonstrated that isoliquiritigenin is a differentiation-inducing agent, and its mechanisms involve ROS accumulation facilitating melanogenesis.

  6. Anthocyanin determination in blueberry extracts from various cultivars and their antiproliferative and apoptotic properties in B16-F10 metastatic murine melanoma cells.

    PubMed

    Bunea, Andrea; Rugină, Dumitriţa; Sconţa, Zoriţa; Pop, Raluca M; Pintea, Adela; Socaciu, Carmen; Tăbăran, Flaviu; Grootaert, Charlotte; Struijs, Karin; VanCamp, John

    2013-11-01

    Blueberry consumption is associated with health benefits contributing to a reduced risk for cardiovascular disease, diabetes and cancer. The aim of this study was to determine the anthocyanin profile of blueberry extracts and to evaluate their effects on B16-F10 metastatic melanoma murine cells. Seven blueberry cultivars cultivated in Romania were used. The blueberry extracts were purified over an Amberlite XAD-7 resin and a Sephadex LH-20 column, in order to obtain the anthocyanin rich fractions (ARF). The antioxidant activity of the ARF of all cultivars was evaluated by ABTS, CUPRAC and ORAC assays. High performance liquid chromatography followed by electrospray ionization mass spectrometry (HPLC-ESI-MS) was used to identify and quantify individual anthocyanins. The anthocyanin content of tested cultivars ranged from 101.88 to 195.01 mg malvidin-3-glucoside/100g fresh weight. The anthocyanin rich-fraction obtained from cultivar Torro (ARF-T) was shown to have the highest anthocyanin content and antioxidant activity, and inhibited B16-F10 melanoma murine cells proliferation at concentrations higher than 500 μg/ml. In addition, ARF-T stimulated apoptosis and increased total LDH activity in metastatic B16-F10 melanoma murine cells. These results indicate that the anthocyanins from blueberry cultivar could be used as a chemopreventive or adjuvant treatment for metastasis control. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. The Cytotoxicity of Dacarbazine Potentiated by Sea Cucumber Saponin in Resistant B16F10 Melanoma Cells through Apoptosis Induction

    PubMed Central

    Baharara, Javad; Amini, Elaheh; Nikdel, Najme; Salek-Abdollahi, Farzaneh

    2016-01-01

    Background: Malignant melanoma is a highly aggressive malignant melanocytic neoplasm which resists against the most conventional therapies. Sea cucumber as one of marine organisms contains bioactive compounds such as polysaccharide, terpenoid and other metabolites which have anti-cancer, anti-tumor, anti-inflammatory and antioxidant properties. The present study was designed to investigate the anticancer potential of saponin extracted from sea cucumber Holothuria leucospilata alone and in combination with dacarbazine on B16F10 melanoma cell line. Methods: The B16F10 cell line was treated with different concentrations of saponin (0, 4, 8, 12, 16, 20 μg/ml), dacarbazine (0, 1200, 1400, 1600, 18000, 1200, 1400, 1600, 2000 μg/ml) and co-administration of saponin-dacarbazine (1200 da+8 sp, 1200 da+4 sp) for 24 and 48 hr and the cytotoxic effect was examined by MTT, DAPI, acridine orange/propodium iodide, flow cytometry and caspase colorimetric assay. Results: The results exhibited that sea cucumber saponin, dacarbazine, and co-administration of saponin-dacarbazine inhibited the proliferation of melanoma cells in a dose and time dependent manner with IC50 values of 10, 1400 and 4+1200 μg/ml, respectively. Morphological observation of DAPI and acridine orange/propodium iodide staining documented typical characteristics of apoptotic cell death. Flow cytometry assay indicated accumulation of IC50 treated cells in sub-G1 peak. Additionally, saponin extracted induced intrinsic apoptosis via up-regulation of caspase-3 and caspase-9. Conclusion: These results revealed that the saponin extracted from sea cucumber as a natural anti-cancer compound may be a new treatment modality for metastatic melanoma and the application of sea cucumber saponin in combination with dacarbazine demonstrated the strongest anti-cancer activity as compared with the drug alone. PMID:27563423

  8. The Cytotoxicity of Dacarbazine Potentiated by Sea Cucumber Saponin in Resistant B16F10 Melanoma Cells through Apoptosis Induction.

    PubMed

    Baharara, Javad; Amini, Elaheh; Nikdel, Najme; Salek-Abdollahi, Farzaneh

    2016-01-01

    Malignant melanoma is a highly aggressive malignant melanocytic neoplasm which resists against the most conventional therapies. Sea cucumber as one of marine organisms contains bioactive compounds such as polysaccharide, terpenoid and other metabolites which have anti-cancer, anti-tumor, anti-inflammatory and antioxidant properties. The present study was designed to investigate the anticancer potential of saponin extracted from sea cucumber Holothuria leucospilata alone and in combination with dacarbazine on B16F10 melanoma cell line. The B16F10 cell line was treated with different concentrations of saponin (0, 4, 8, 12, 16, 20 μg/ml), dacarbazine (0, 1200, 1400, 1600, 18000, 1200, 1400, 1600, 2000 μg/ml) and co-administration of saponin-dacarbazine (1200 da+8 sp, 1200 da+4 sp) for 24 and 48 hr and the cytotoxic effect was examined by MTT, DAPI, acridine orange/propodium iodide, flow cytometry and caspase colorimetric assay. The results exhibited that sea cucumber saponin, dacarbazine, and co-administration of saponin-dacarbazine inhibited the proliferation of melanoma cells in a dose and time dependent manner with IC50 values of 10, 1400 and 4+1200 μg/ml, respectively. Morphological observation of DAPI and acridine orange/propodium iodide staining documented typical characteristics of apoptotic cell death. Flow cytometry assay indicated accumulation of IC50 treated cells in sub-G1 peak. Additionally, saponin extracted induced intrinsic apoptosis via up-regulation of caspase-3 and caspase-9. These results revealed that the saponin extracted from sea cucumber as a natural anti-cancer compound may be a new treatment modality for metastatic melanoma and the application of sea cucumber saponin in combination with dacarbazine demonstrated the strongest anti-cancer activity as compared with the drug alone.

  9. Alternol induces an S-phase arrest of melanoma B16F0 cells.

    PubMed

    Liu, Liangliang; Zhang, Bo; Yuan, Xuan; Wang, Penglong; Sun, Xiling; Zheng, Qiusheng

    2014-03-01

    Alternol is a novel compound purified from the fermentation products of a microorganism in the yew tree bark. This study looks at the effects of alternol on the proliferation and cell cycle distribution of mouse melanoma cells. The inhibition of cell proliferation and changes in cell cycle distribution were analysed by sulforhodamine B and flow cytometry assays, respectively. mRNA expression of cyclin A, cyclin-dependent kinase 2 (CDK2), proliferating cell nuclear antigen (PCNA) and CDK inhibitor1A (p21) were measured by real-time reverse transcription PCR (RT-PCR). The protein levels of cyclin A, CDK2 and PCNA were analysed by Western blot analysis. p21 was measured by ELISA. Alternol treatment caused a significant decrease in the proliferation rate of B16F0 and B16F10 cells, which were significantly arrested in S phase, but this treatment had less effect on normal human embryonic kidney 293T cells. The mechanism by which alternol inhibits B16F0 proliferation in vitro may be associated with the inhibition of CDK2 and PCNA, and the activation of p21. © 2013 International Federation for Cell Biology.

  10. Stress hormones promote growth of B16-F10 melanoma metastases: an interleukin 6- and glutathione-dependent mechanism.

    PubMed

    Valles, Soraya L; Benlloch, María; Rodriguez, María L; Mena, Salvador; Pellicer, José A; Asensi, Miguel; Obrador, Elena; Estrela, José M

    2013-03-22

    Interleukin (IL)-6 (mainly of tumor origin) activates glutathione (GSH) release from hepatocytes and its interorgan transport to B16-F10 melanoma metastatic foci. We studied if this capacity to overproduce IL-6 is regulated by cancer cell-independent mechanisms. Murine B16-F10 melanoma cells were cultured, transfected with red fluorescent protein, injected i.v. into syngenic C57BL/6J mice to generate lung and liver metastases, and isolated from metastatic foci using high-performance cell sorting. Stress hormones and IL-6 levels were measured by ELISA, and CRH expression in the brain by in situ hybridization. DNA binding activity of NF-κB, CREB, AP-1, and NF-IL-6 was measured using specific transcription factor assay kits. IL-6 expression was measured by RT-PCR, and silencing was achieved by transfection of anti-IL-6 small interfering RNA. GSH was determined by HPLC. Cell death analysis was distinguished using fluorescence microscopy, TUNEL labeling, and flow cytometry techniques. Statistical analyses were performed using Student's t test. Plasma levels of stress-related hormones (adrenocorticotropin hormone, corticosterone, and noradrenaline) increased, following a circadian pattern and as compared to non-tumor controls, in mice bearing B16-F10 lung or liver metastases. Corticosterone and noradrenaline, at pathophysiological levels, increased expression and secretion of IL-6 in B16-F10 cells in vitro. Corticosterone- and noradrenaline-induced transcriptional up-regulation of IL-6 gene involves changes in the DNA binding activity of nuclear factor-κB, cAMP response element-binding protein, activator protein-1, and nuclear factor for IL-6. In vivo inoculation of B16-F10 cells transfected with anti-IL-6-siRNA, treatment with a glucocorticoid receptor blocker (RU-486) or with a β-adrenoceptor blocker (propranolol), increased hepatic GSH whereas decreased plasma IL-6 levels and metastatic growth. Corticosterone, but not NORA, also induced apoptotic cell death in

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

    PubMed

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

    2015-02-01

    Poor therapeutic results have been reported for treatment of malignant melanoma; therefore in this study, we have investigated inhibitory capacity of vitexin-2''-O-rhamnoside as well as the extract from which it was isolated, i.e. the ethyl acetate extract obtained from the leaves of Crataegus azarolus, on mouse melanoma (B16F10) proliferation. Cell viability was determined using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475nm. Ethyl acetate extract and vitexin-2''-O-rhamnoside exhibited significant anti-proliferative activity against B16F10 melanoma cells after incubation for 48hours with IC50s of 50μg/mL and 20μM, respectively. Furthermore, these two compounds have the ability to reduce the melanin content by inhibiting the tyrosinase activity of B16F10 cells. Thus, further investigations are merited to ascertain their potential application in treating hyperpigmentation disorders. Copyright © 2014. Published by Elsevier Masson SAS.

  12. Improving anticancer efficacy of (–)-epigallocatechin-3-gallate gold nanoparticles in murine B16F10 melanoma cells

    PubMed Central

    Chen, Cheng-Cheung; Hsieh, Dar-Shih; Huang, Kao-Jean; Chan, Yi-Lin; Hong, Po-Da; Yeh, Ming-Kung; Wu, Chang-Jer

    2014-01-01

    (–)-Epigallocatechin-3-gallate (EGCG), the major bioactive constituent in green tea, has been reported to effectively inhibit the formation and development of tumors. To maximize the effectiveness of EGCG, we attached it to nanogold particles (EGCG-pNG) in various ratios to examine in vitro cytotoxicity and in vivo anti-cancer activity. EGCG-pNG showed improved anti-cancer efficacy in B16F10 murine melanoma cells; the cytotoxic effect in the melanoma cells treated with EGCG-pNG was 4.91 times higher than those treated with EGCG. The enhancement is achieved through mitochondrial pathway-mediated apoptosis as determined by annexin V assay, JC-10 staining, and caspase-3, -8, -9 activity assay. Moreover, EGCG-pNG was 1.66 times more potent than EGCG for inhibition of tumor growth in a murine melanoma model. In the hemolysis assay, the pNG surface conjugated with EGCG is most likely the key factor that contributes to the decreased release of hemoglobin from human red blood cells. PMID:24855338

  13. Melanogenesis effect of Cordyceps militaris culture broth on the melanin formation of B16F0 melanoma cells.

    PubMed

    Cha, Jae-Young; Yang, Hyun-Ju; Park, Mi-Yeon; Choi, Seung-Tae; Moon, Hyung-In; Cho, Young-Su

    2011-10-13

    The effect of Cordyceps militaris culture broth (CMB) on melanogenesis in B16F0 melanoma cells was evaluated by measurement of the melanin concentration after 3 days of incubation. The B16F0 melanoma cells were treated with various concentrations of CMB 10-100 μg/mL and arbutin of 200 μM. Phenolic content and antioxidant activity of CMB were also measured. Phenolic content of CMB was 3.28 mg/g. The DPPH radical scavenging and ferric ion donating activities were 79.64% and 0.16, respectively. The melanin concentration and cell viability of melanoma cells by arbutin treatment decreased to 43% and 91% of the control, respectively. The CMB treatment showed a significant inhibitory effect of melanin production by 29%, 50%, and 56% at 50, 80, and 100 μg/mL concentration treatment, respectively, while over 90% of cells were viable. The CMB treatment at 50, 80, and 100 μg/mL concentrations in cultivation decreased extracellular melanin release induced by 3-isobutyl-1-methylxanthine (IBMX) treatment by 19%, 38%, and 48%, respectively. The CMB showed inhibitory activity against intracellular tyrosinase extracted from melanoma cells, while it had no inhibition on the activity of mushroom tyrosinase. The cellular glutathione contents were enhanced by CMB treatment in a concentration-dependent manner. These results suggested that CMB suppressed cellular tyrosinase activity and total melanin content in cultured B16F0 melanoma cells without any significant effects on cell proliferation and it might be candidate anti-melanogenic agent.

  14. Sulfasalazine, an inhibitor of the cystine-glutamate antiporter, reduces DNA damage repair and enhances radiosensitivity in murine B16F10 melanoma

    PubMed Central

    Shibata, Yuki; Shimizu, Takuto; Yoshioka, Chie; Maruo, Takuya

    2018-01-01

    The sodium-independent cystine-glutamate antiporter plays an important role in extracellular cystine uptake. It comprises the transmembrane protein, xCT and its chaperone, CD98. Because glutathione is only weakly cell membrane permeable, cellular uptake of its precursor, cystine, is known to be a key step in glutathione synthesis. Moreover, it has been reported that xCT expression affects the progression of tumors and their resistance to therapy. Sulfasalazine is an inhibitor of xCT that is known to increase cellular oxidative stress, giving it anti-tumor potential. Here, we describe a radio-sensitizing effect of sulfasalazine using a B16F10 melanoma model. Sulfasalazine decreased glutathione concentrations and resistance to H2O2 in B16F10 melanoma cells, but not in mouse embryonic fibroblasts. It synergistically enhanced the cyto-killing effect of X-irradiation in B16F10 cells. It inhibited cellular DNA damage repair and prolonged cell cycle arrest after X-irradiation. Furthermore, in an in vivo transplanted melanoma model, sulfasalazine decreased intratumoral glutathione content, leading to enhanced susceptibility to radiation therapy. These results suggest the possibility of using SAS to augment the treatment of radio-resistant cancers. PMID:29649284

  15. Vernolide-A, a sesquiterpene lactone from Vernonia cinerea, induces apoptosis in B16F-10 melanoma cells by modulating p53 and caspase-3 gene expressions and regulating NF-κB-mediated bcl-2 activation.

    PubMed

    Pratheeshkumar, Poyil; Kuttan, Girija

    2011-07-01

    In this study, we investigated the effect of vernolide-A on the induction of apoptosis as well as its regulatory effect on the activation of transcription factors in B16F-10 melanoma cells. Treatment of B16F-10 cells with nontoxic concentrations of vernolide-A showed the presence of apoptotic bodies and induced DNA fragmentation in a dose-dependent manner. Cell-cycle analysis and TUNEL assays also confirmed the observation. The proapoptotic genes, p53, Bax, caspase-9, and caspase-3, were upregulated in vernolide-A-treated cells, whereas the antiapoptotic gene, Bcl-2, was downregulated. vernolide-A treatment also showed a downregulation of cyclin D1 expression and upregulated p21 and p27 gene expression in B16F-10 melanoma cells. The study also reveals that vernolide-A treatment could alter the production and expression of proinflammatory cytokines and could inhibit the activation and nuclear translocation of p65, p50, and c-Rel subunits of nuclear factor-κB and other transcription factors, such as c-fos, activated transcription factor-2, and cyclic adenosine monophosphate response-element-binding protein in B16F-10 melanoma cells. These results suggest that vernolide-A induces apoptosis via activation of p53-induced, caspase-3-mediated proapoptotic signaling and suppression of NF-κB-induced, bcl-2-mediated survival signaling.

  16. Curcumin does not switch melanin synthesis towards pheomelanin in B16F10 cells.

    PubMed

    Wolnicka-Glubisz, Agnieszka; Nogal, Katarzyna; Żądło, Andrzej; Płonka, Przemysław M

    2015-01-01

    Melanin, the basic skin pigment present also in the majority of melanomas, has a huge impact on the efficiency of photodynamic, radio- or chemotherapies of melanoma. Moreover, the melanoma cells produce more melanin than normal melanocytes in adjacent skin do. Thus, attention has been paid to natural agents that are safe and effective in suppression of melanogenesis. B16F10 cells were studied by electron paramagnetic resonance (EPR) spectroscopy. The cells were cultured for 24-72 h in RPMI or DMEM with or without curcumin. The results confirmed that curcumin has no significant effect on B16F10 cells viability at concentrations of 1-10 µM. Curcumin at concentration of 10 µM significantly inhibited their proliferation and stimulated differentiation. We have not stimulated melanogenesis hormonally but we found a strong increase in melanogenesis in DMEM, containing more L-Tyr, as compared to RPMI. The EPR studies revealed that the effect of curcumin on melanogenesis in RPMI-incubated cells was not significant, and only in DMEM was curcumin able to inhibit melanogenesis. The effect of curcumin was only quantitative, as it did not switch eumelanogenesis towards pheomelanogenesis under any conditions. Interestingly, we observed elevation of production of hydrogen peroxide in DMEM-incubated cells, in parallel to the facilitation of melanogenesis. Curcumin significantly but transiently intensified the already pronounced generation of H2O2 in DMEM. We conclude that the quantitative effect of curcumin on melanogenesis in melanoma is intricate. It depends on the basic melanogenetic efficiency of the cells, and can be observed only in strongly pigmented cells. Qualitatively, curcumin does not switch melanogenesis towards pheomelanogenesis, either in strongly, or in weakly melanized melanoma cells.

  17. Inhibition of metastatic potential of B16-F10 melanoma cell line in vivo and in vitro by biflorin.

    PubMed

    Andrade Carvalho, Adriana; da Costa, Patrícia Marçal; Da Silva Souza, Luciana Gregório; Lemos, Telma Leda G; Alves, Ana Paula Negreiros Nunes; Pessoa, Cláudia; de Moraes, Manoel Odorico

    2013-08-14

    The aim of this study was to determine the antimetastatic potential of biflorin using in vivo and in vitro approaches. Biflorin was isolated from Capraria biflora collected in Fortaleza, Ceará, Brazil. Adhesion, migration and invasion assays were performed to avail of the antimetastatic potential of this quinone. Experimental metastasis was performed to avail of the antimetastatic potential of bilflorin using in vivo assay. Treatment with biflorin (25 and 50mg/kg/day) was shown to be effective in reducing B16-F10 melanoma metastasis in C57BL/6 mice. The administration of biflorin at 25mg/kg/day intraperitoneally inhibited the formation of metastases by about 57% compared to untreated control animals. When the animals were treated with 50mg/kg/day intraperitoneally, there was a 71% decrease in the number of lung metastases. Morphological assays showed the presence of hemosiderin and erythrocytes in the lung parenchyma, indicating the occurrence of hemorrhage, probably a side effect of biflorin. Biflorin at non-toxic concentrations (0.5, 1.0 and 1.5g/mL) was tested directly on B16-F10 cells in vitro, and it inhibited cell adhesion to type I collagen and cell motility using the wound-healing assay. These data suggest that biflorin has a promising antimetastatic potential, as shown by its anti-adhesion, anti-migration and anti-invasion properties against a metastatic melanoma cell line. However, further studies are essential to elucidate its mechanism of action. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Expression of Foxp3, CD25 and IL-2 in the B16F10 cancer cell line and melanoma is correlated with tumor growth in mice

    PubMed Central

    MIRANDA-HERNÁNDEZ, D.F.; FRANCO-MOLINA, M.A.; MENDOZA-GAMBOA, E.; ZAPATA-BENAVIDES, P.; SIERRA-RIVERA, C.A.; CORONADO-CERDA, E.E.; ROSAS-TARACO, A.G.; TAMÉZ-GUERRA, R.S.; RODRÍGUEZ-PADILLA, C.

    2013-01-01

    The forkhead box P3 (Foxp3) transcription factor is one of the most studied markers used to identify CD4+CD25+ regulatory T cells (Tregs), and has been identified as a key regulator in the development and function of Tregs. Foxp3 expression has been reported in a variety of solid human tumors, including melanoma. The aims of the present study were to analyze Foxp3 expression in B16F10 melanoma cells in vitro, to determine whether this expression was affected during tumor growth in a murine melanoma model and to correlate Foxp3 expression with CD25 expression, interleukin (IL)-2 production and tumor weight. Foxp3 expression was analyzed with quantitative (q)PCR, flow cytometry and confocal microscopy. CD25 expression was analyzed by flow cytometry, and cytokine production was measured by ELISA [IL-2, interferon (IFN)-γ, transforming growth factor (TGF)-β and IL-10] and flow cytometry (IL-2, IFN-γ, IL-4 and IL-5). Foxp3 and CD25 expression was detected in the B16F10 cells in culture and in the intratumoral B16F10 cells. An increase in Foxp3 and CD25 expression was observed in a time-dependent manner during tumor growth at 7, 14 and 21 days. The production of the IL-2, IL-10, IFN-γ and TGF-β cytokines was observed in the B16F10 cells and also detected in the tumoral microenvironment during tumor growth (7, 14 and 21 days). An increase in IL-2 and IL-10 production was observed, whereas IFN-γ production decreased in a time-dependent manner. The production of tumor necrosis factor (TNF)-α was not observed in culture, but was detected during tumor growth, whereas the production of IL-4 and IL-5 was not detected. These data showed a positive correlation between the expression of Foxp3, CD25 and IL-2 and tumor weight in murine melanoma. From these data, it may be suggested that Foxp3 participates in melanoma growth, the modulation of the IL-2, IFN-γ and TNF-α cytokines and CD25 expression, and that it also plays a possible role in immunosuppression. PMID:24179494

  19. Activation of neurokinin-1 receptor by substance P inhibits melanogenesis in B16-F10 melanoma cells.

    PubMed

    Ping, Fengfeng; Shang, Jing; Zhou, Jia; Song, Jing; Zhang, Luyong

    2012-12-01

    Skin pigmentation plays a number of valuable roles and its regulation is a complex process that is controlled by different factors. Substance P (SP) regulates many biological functions, including neurogenic inflammation, pain, and stress. However, to date, the regulatory role of SP in the control of melanogenesis has not been elucidated. The present study was designed to investigate the effects of SP on melanogenesis and to elucidate its underlying mechanism(s). After treatment for 48 h in mouse B16-F10 melanoma cells, SP (1 and 10nM) significantly down-regulated tyrosinase activity and melanin content. Importantly, western blot analysis revealed the presence of neurokinin-1 receptor (NK-1 R) in B16-F10 cells and the activation of it after SP treatment. It was also found that preincubation with NK-1 receptor antagonist Spantide I could partially reversed SP-induced down-regulations of tyrosinase activity, melanin content and the expression of tyrosinase and tyrosinase-related protein 1. Furthermore, SP could remarkably inhibit the expressions of microphtalmia-associated transcription factor (MITF) and p-p38 MAPK and stimulated p-p70 S6K1. These effects could also be partially reversed by the pretreatment with Spantide I. These results collectively suggested that SP inhibited melanogenesis in B16-F10 cells, which might be attributed to the fact that SP induces the activation of NK-1 receptor, stimulates the phosphorylation of p70 S6K1 and inhibits that of p38 MAPK, decreases the tyrosinase and tyrosinase-related protein 1 expression through MITF, finally resulting in the suppression of melanogenesis. These observations in vitro indicated that the regulation of the SP/NK-1 receptor system might be a useful novel management for skin pigmentation. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2016-08-01

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

  1. Characterization of thimet oligopeptidase and neurolysin activities in B16F10-Nex2 tumor cells and their involvement in angiogenesis and tumor growth.

    PubMed

    Paschoalin, Thaysa; Carmona, Adriana K; Rodrigues, Elaine G; Oliveira, Vitor; Monteiro, Hugo P; Juliano, Maria A; Juliano, Luiz; Travassos, Luiz R

    2007-07-09

    Angiogenesis is a fundamental process that allows tumor growth by providing nutrients and oxygen to the tumor cells. Beyond the oxygen diffusion limit from a capillary blood vessel, tumor cells become apoptotic. Angiogenesis results from a balance of pro- and anti-angiogenic stimuli. Endogenous inhibitors regulate enzyme activities that promote angiogenesis. Tumor cells may express pro-angiogenic factors and hydrolytic enzymes but also kinin-degrading oligopeptidases which have been investigated. Angiogenesis induced by B16F10-Nex2 melanoma cells was studied in a co-culture with HUVEC on Matrigel. A stimulating effect on angiogenesis was observed in the presence of B16F10-Nex2 lysate and plasma membrane. In contrast, the B16F10-Nex2 culture supernatant inhibited angiogenesis in a dose-dependent manner. This effect was abolished by the endo-oligopeptidase inhibitor, JA-2. Thimet oligopeptidase (TOP) and neurolysin activities were then investigated in B16F10-Nex2 melanoma cells aiming at gene sequencing, enzyme distribution and activity, influence on tumor development, substrate specificity, hydrolytic products and susceptibility to inhibitors. Fluorescence resonance energy transfer (FRET) peptides as well as neurotensin and bradykinin were used as substrates. The hydrolytic activities in B16F10-Nex2 culture supernatant were totally inhibited by o-phenanthrolin, JA-2 and partially by Pro-Ile. Leupeptin, PMSF, E-64, Z-Pro-Prolinal and captopril failed to inhibit these hydrolytic activities. Genes encoding M3A enzymes in melanoma cells were cloned and sequenced being highly similar to mouse genes. A decreased proliferation of B16F10-Nex2 cells was observed in vitro with specific inhibitors of these oligopeptidases. Active rTOP but not the inactive protein inhibited melanoma cell development in vivo increasing significantly the survival of mice challenged with the tumor cells. On Matrigel, rTOP inhibited the bradykinin - induced angiogenesis. A possible regulation of

  2. Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cunha, Elizabeth S.; Kawahara, Rebeca; Kadowaki, Marina K.

    Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cellmore » cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis. -- Highlights: Black-Right-Pointing-Pointer Melanogenesis stimulation by L-tyrosine+NH{sub 4}Cl in B16-F10 melanoma cells increases ROS levels. Black-Right-Pointing-Pointer Melanogenesis inhibits cell proliferation, and induced cell cycle arrest in the G1 phase. Black-Right-Pointing-Pointer Proteomic analysis showed alterations in proteins of the cell cycle and glucose metabolism. Black-Right-Pointing-Pointer RT-qPCR analysis confirmed alterations of metabolic targets after melanogenesis stimulation.« less

  3. Antitumor Effects In Vitro and In Vivo and Mechanisms of Protection against Melanoma B16F10-Nex2 Cells By Fastuosain, a Cysteine Proteinase from Bromelia fastuosa1

    PubMed Central

    Guimarães-Ferreira, Carla A; Rodrigues, Elaine G; Mortara, Renato A; Cabral, Hamilton; Serrano, Fabiana A; Ribeiro-dos-Santos, Ricardo; Travassos, Luiz R

    2007-01-01

    In the present work, the antitumor effect of fastuosain, a cysteine proteinase from Bromelia fastuosa, was investigated. In the intravenous model of lung colonization in C57Bl/6 mice, fastuosain and bromelain injected intraperitoneally were protective, and very few nodules of B16F10-Nex2 melanoma cells were detected. Tumor cells treated with fastuosain showed reduced expression of CD44 and decreased invasion through Matrigel, lost their cytoplasmic extensions and substrate adherence, and became round and detached, forming strongly bound cell clusters in suspension. Peritoneal cells recruited and activated by fastuosain treatment (mainly monocytic cells and lymphocytes) migrated to the lung, where pulmonary melanoma metastases grew. Adoptive transference of peritoneal cells recruited by fastuosain had no protective effect against lung metastases in recipient mice. Treatment of green fluorescent protein-chimeric animals with fastuosain did not change the number of cells that migrated to the lung, compared to PBS-injected control mice, but the number of positive major histocompatibility complex class II cells increased with fastuosain treatment. Murine antibodies against fastuosain, bromelain, and cathepsins B and L cross-reacted in ELISA and recognized surface and cytoplasmic components expressed on B16F10-Nex2 cells. Anti-fastuosain antibodies were cytotoxic/lytic to B16F10-Nex2 cells. Antitumor effects of fastuosain involve mainly the direct effect of the enzyme and elicitation of protective antibodies. PMID:17898868

  4. Higher cell stiffness indicating lower metastatic potential in B16 melanoma cell variants and in (-)-epigallocatechin gallate-treated cells.

    PubMed

    Watanabe, Tatsuro; Kuramochi, Hiromi; Takahashi, Atsushi; Imai, Kazue; Katsuta, Naoko; Nakayama, Tomonobu; Fujiki, Hirota; Suganuma, Masami

    2012-05-01

    To understand how nanomechanical stiffness affects metastatic potential, we studied the relationship between cell migration, a characteristic of metastasis, and cell stiffness using atomic force microscopy (AFM), which can measure stiffness (elasticity) of individual living cells. Migration and cell stiffness of three metastatic B16 melanoma variants (B16-F10, B16-BL6, and B16-F1 cells), and also effects of (-)-epigallocatechin gallate (EGCG), were studied using Transwell assay and AFM. Migration of B16-F10 and B16-BL6 cells was 3 and 2 times higher than that of B16-F1 cells in Transwell assay, and cell stiffness determined by AFM was also different among the three variants, although they have similar morphologies and the same growth rates: Means of Young's modulus were 350.8 ± 4.8 Pa for B16-F10 cells, 661.9 ± 16.5 Pa for B16-BL6 cells, and 727.2 ± 13.0 Pa for B16-F1 cells. AFM measurements revealed that highly motile B16-F10 cells have low cell stiffness, and low motile and metastatic B16-F1 cells have high cell stiffness: Nanomechanical stiffness is inversely correlated with migration potential. Treatment of highly motile B16-F10 cells with EGCG increased cell stiffness 2-fold and inhibited migration of the cells. Our study with AFM clearly demonstrates that cell stiffness is a reliable quantitative indicator of migration potential, and very likely metastatic potential, even in morphologically similar cells. And increased cell stiffness may be a key nanomechanical feature in inhibition of metastasis.

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

    PubMed

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

    2012-04-01

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

  6. Comparative Depigmentation Effects of Resveratrol and Its Two Methyl Analogues in α-Melanocyte Stimulating Hormone-Triggered B16/F10 Murine Melanoma Cells

    PubMed Central

    Yoon, Hoon-Seok; Hyun, Chang-Gu; Lee, Nam-Ho; Park, Sung-Soo; Shin, Dong-Bum

    2016-01-01

    Previous research showed that resveratrol (trans-3,4′,5-trihydroxystilbene) and pinostilbene (trans-3-methoxy-4′,5-dihydroxystilbene) were able to inhibit tyrosinase directly; however, anti-melanogenic effects of pterostilbene (trans-3,5-dimethoxy-4′-hydroxystilbene) and resveratrol trimethyl ether (RTE) have not been compared. To investigate the hypopigmentation effects of pterostilbene and RTE, melanin contents and intracellular tyrosinase activity were determined by western blot analysis. Firstly, pterostilbene showed the inhibitory effects on α-melanocyte stimulating hormone (MSH)-induced melanin synthesis stronger than RTE, resveratrol, and arbutin. Pterostilbene inhibited melanin biosynthesis in a dose-dependent manner in α-MSH-stimulated B16/F10 murine melanoma cells. Specifically, melanin content and intracellular tyrosinase activity were inhibited by 63% and 58%, respectively, in response to treatment with 10 μM of pterostilbene. The results of western blot analysis indicated that pterostilbene induced downregulation of tyrosinase protein expression and suppression of α-MSH-stimulated melan-A protein expression stronger than RTE or resveratrol. Based on these results, our study suggests that pterostilbene can induce hypopigmentation effects more effectively than resveratrol and RTE, and it functions via downregulation of protein expression associated with hyperpigmentation in α-MSH-triggered B16/F10 murine melanoma cells. PMID:27390733

  7. Characterization of thimet oligopeptidase and neurolysin activities in B16F10-Nex2 tumor cells and their involvement in angiogenesis and tumor growth

    PubMed Central

    Paschoalin, Thaysa; Carmona, Adriana K; Rodrigues, Elaine G; Oliveira, Vitor; Monteiro, Hugo P; Juliano, Maria A; Juliano, Luiz; Travassos, Luiz R

    2007-01-01

    Background Angiogenesis is a fundamental process that allows tumor growth by providing nutrients and oxygen to the tumor cells. Beyond the oxygen diffusion limit from a capillary blood vessel, tumor cells become apoptotic. Angiogenesis results from a balance of pro- and anti-angiogenic stimuli. Endogenous inhibitors regulate enzyme activities that promote angiogenesis. Tumor cells may express pro-angiogenic factors and hydrolytic enzymes but also kinin-degrading oligopeptidases which have been investigated. Results Angiogenesis induced by B16F10-Nex2 melanoma cells was studied in a co-culture with HUVEC on Matrigel. A stimulating effect on angiogenesis was observed in the presence of B16F10-Nex2 lysate and plasma membrane. In contrast, the B16F10-Nex2 culture supernatant inhibited angiogenesis in a dose-dependent manner. This effect was abolished by the endo-oligopeptidase inhibitor, JA-2. Thimet oligopeptidase (TOP) and neurolysin activities were then investigated in B16F10-Nex2 melanoma cells aiming at gene sequencing, enzyme distribution and activity, influence on tumor development, substrate specificity, hydrolytic products and susceptibility to inhibitors. Fluorescence resonance energy transfer (FRET) peptides as well as neurotensin and bradykinin were used as substrates. The hydrolytic activities in B16F10-Nex2 culture supernatant were totally inhibited by o-phenanthrolin, JA-2 and partially by Pro-Ile. Leupeptin, PMSF, E-64, Z-Pro-Prolinal and captopril failed to inhibit these hydrolytic activities. Genes encoding M3A enzymes in melanoma cells were cloned and sequenced being highly similar to mouse genes. A decreased proliferation of B16F10-Nex2 cells was observed in vitro with specific inhibitors of these oligopeptidases. Active rTOP but not the inactive protein inhibited melanoma cell development in vivo increasing significantly the survival of mice challenged with the tumor cells. On Matrigel, rTOP inhibited the bradykinin – induced angiogenesis. A

  8. In vitro and in vivo anticancer effects of Lithospermum erythrorhizon extract on B16F10 murine melanoma.

    PubMed

    Rajasekar, Seetharaman; Park, Da Jung; Park, Cheol; Park, Sejin; Park, Young Hoon; Kim, Sun Tae; Choi, Yung Hyun; Choi, Young Whan

    2012-11-21

    Lithospermum erythrorhizon has long been used in traditional Asian medicine for the treatment of diseases including skin cancer. In this study, hexane extract from the roots of Lithospermum erythrorhizon (LEH) was chemically characterized and its anticancer activity was tested against the most aggressive form of skin cancer. The in vitro anticancer studies viz. cell growth, cell cycle and apoptosis, and the expression of tumor regulating proteins were analyzed against B16F10 melanoma cells. In addition, C57BL/6 mice models were used to evaluate the in vivo anticancer potential of LEH. Mice were intraperitoneally injected with LEH at doses of 0.1 and 10mg/kg every 3 days. The tumor inhibition ratio was determined after 21 days of treatment and the histopathological analyses of the tumor tissues were compared. Further, LEH was purified and its active compounds were structurally elucidated and identified by NMR spectra and quantified by HPLC analyses. LEH effectively inhibits the growth of melanoma cells with an IC(50) of 2.73μg/ml. Cell cycle analysis revealed that LEH increased the percentage of cells in sub-G1 phase by dose dependent manner. LEH exhibited down regulation of anti-apoptotic Bcl-2 family proteins and up regulation of apoptotic Bax protein expression. Importantly, LEH induced cleavage of poly (ADP-ribose) polymerase (PARP) and activated the caspase cascade (caspase 3) with this cleavage mediating the apoptosis of B16F10 cells. LEH treatment at a dose of 10mg/kg for 21 days in experimental mice implanted with tumors resulted in significant reduction of the tumor growth (43%) and weight (36%). Histopathology analysis of LEH treated tumor tissues showed evidence of increased necrotic cells in a concentration dependent manner. Meanwhile, five naphthoquinone compounds [Shikonin (1); Deoxyshikonin (2); β-Hydroxyisovalerylshikonin (3); Acetylshikonin (4) and Isobutyrylshikonin (5)] were purified from LEH and responsible for its anticancer activity. LEH

  9. gamma-Glutamyl transpeptidase overexpression increases metastatic growth of B16 melanoma cells in the mouse liver.

    PubMed

    Obrador, Elena; Carretero, Julian; Ortega, Angel; Medina, Ignacio; Rodilla, Vicente; Pellicer, José A; Estrela, José M

    2002-01-01

    B16 melanoma (B16M) cells with high glutathione (GSH) content show rapid proliferation in vitro and high metastatic activity in the liver in vivo. gamma-Glutamyl transpeptidase (GGT)-mediated extracellular GSH cleavage and intracellular GSH synthesis were studied in vitro in B16M cells with high (F10) and low (F1) metastatic potential. GGT activity was modified by transfection with the human GGT gene (B16MF1/Tet-GGT cells) or by acivicin-induced inhibition. B16MF1/Tet-GGT and B16MF10 cells exhibited higher GSH content (35 +/- 6 and 40 +/- 5 nmol/10(6) cells, respectively) and GGT activity (89 +/- 9 and 37 +/- 7 mU/10(6) cells, respectively) as compared (P <.05) with B16MF1 cells (10 +/- 3 nmol GSH and 4 mU GGT/10(6) cells). Metastasis (number of foci/100 mm(3) of liver) increased in B16MF1 cells pretreated with GSH ester ( approximately 3-fold, P <.01), and decreased in B16MF1/Tet-GGT and B16MF10 cells pretreated with the GSH synthesis inhibitor L-buthionine (S,R)-sulphoximine ( approximately 5-fold and 2-fold, respectively, P <.01). Liver, kidney, brain, lung, and erythrocyte GSH content in B16MF1/Tet-GGT- or B16MF10-bearing mice decreased as compared with B16MF1- and non-tumor-bearing mice. Organic anion transporting polypeptide 1-independent sinusoidal GSH efflux from hepatocytes increased in B16MF1/Tet-GGT- or B16MF10-bearing mice ( approximately 2-fold, P <.01) as compared with non-tumor-bearing mice. Our results indicate that tumor GGT activity and an intertissue flow of GSH can regulate GSH content of melanoma cells and their metastatic growth in the liver.

  10. Subcellular localization and photodynamic activity of Photodithazine (glucosamine salt of chlorin e6) in murine melanoma B16-F10: an in vitro and in vivo study

    NASA Astrophysics Data System (ADS)

    Ono, Bruno Andrade; Pires, Layla; Nogueira, Marcelo Saito; Kurachi, Cristina; Pratavieira, Sebastião.

    2018-02-01

    Photodynamic therapy (PDT) is already a good option for the clinical treatment of several lesions, including mainly nonmelanoma skin cancers. However, cutaneous melanoma treatment remains a challenge when using PDT. One of the reasons for its reduced efficacy is the high pigmentation of melanoma cells. The object of our study is to evaluate the feasibility of the Photodithazine as a photosensitizer for melanoma. Photodithazine is already used in some malignant tumors with satisfactory results and has significant absorption band around 660 nm where the absorption of melanin is low. In this study, we measured the subcellular localization and photodynamic activity of Photodithazine (PDZ) in murine melanoma B16-F10 cell culture. Additionally, a PDT procedure was applied in an animal melanoma model. This first result demonstrates that Photodithazine is more localized at mitochondria in B16F10 cell culture and the cell viability is reduced to less than 90% using 1 µg/mL (PDZ) and 2 J/cm2. We also noticed a rapid PDZ (less than one hour) accumulation in a murine melanoma model. The treatment of melanoma resulted in 20 % more animal survival after one session of PDT compared with the control group. More studies are required to evaluate the cytotoxic effects of Photodithazine at human melanoma.

  11. Possible reduction of hepatoma formation by Smmu 7721 cells in SCID mice and metastasis formation by B16F10 melanoma cells in C57BL/6 mice by Agaricus blazei murill extract.

    PubMed

    Wu, Ming-Fang; Lu, Hsu-Feng; Hsu, Yu-Ming; Tang, Ming-Chu; Chen, Hsueh-Chin; Lee, Ching-Sung; Yang, Yi-Yuan; Yeh, Ming-Yang; Chung, Hsiung-Kwang; Huang, Yi-Ping; Wu, Chih-Chung; Chung, Jing-Gung

    2011-01-01

    Agaricus blazei Murill extract (ABM) has been reported to possess antitumor effects. In this study, the role of ABM in tumor growth and metastasis in vivo was evaluated in experimental Smmu 7721 hepatoma cells in severe combined immunodeficiency (SCID) mice and B16F10 melanoma cells lung metastasis in C57BL/6 mice. For the tumor growth model, the size of the liver tumor mass was about 10 mm to 20 mm in the control group. In comparison with the control group, the tumor mass seem to grow slowly with ABM treatment, especially at the high dose. For the tumor metastasis model, after a six-week treatment, the survival rates of B6 mice were 0%, 30%, 10% and 50% for control group, low, median and high concentration ABM treatment groups, respectively. The survival rate showed that pretreatment of C57BL/6 (B6) mice with ABM lengthened their lifespan after tumor cell inoculation, which supports the notion that ABM successfully reduced lung metastasis formation by B16F10 melanoma cells. The treatment effect was dependent on the concentration of ABM for tumor growth and metastasis in these models.

  12. Atmospheric-pressure plasma jet characterization and applications on melanoma cancer treatment (B/16-F10)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mashayekh, Shahriar; Rajaee, Hajar; Hassan, Zuhir M.

    2015-09-15

    A new approach in medicine is the use of cold plasma for various applications such as sterilization blood coagulation and cancer cell treatment. In this paper, a pin-to-hole plasma jet for biological applications has been designed and manufactured and characterized. The characterization includes power consumption via Lissajous method, thermal behavior of atmospheric-pressure plasma jet by using Infra-red camera as a novel method and using Speicair software to determine vibrational and transitional temperatures, and optical emission spectroscopy to determine the generated species. Treatment of Melanoma cancer cells (B16/F10) was also implemented, and tetrazolium salt dye (MTT assay) and flow cytometry weremore » used to evaluate viability. Effect of ultraviolet photons on cancerous cells was also observed using an MgF{sub 2} crystal with MTT assay. Finally, in-vivo studies on C57 type mice were also done in order to have a better understanding of the effects in real conditions.« less

  13. The lactoferricin B-derived peptide, LfB17-34, induces melanogenesis in B16F10 cells.

    PubMed

    Huang, Hsiu-Chin; Lin, Hsuan; Huang, Min-Chuan

    2017-03-01

    Lactoferricin B (LfcinB), a peptide of bovine lactoferrin (LfB), exhibits multiple biological functions, including antimicrobial, antiviral, antioxidant and immunomodulatory activities. However, the role of LfcinB-related peptides in melanogenesis remains unclear. In this study, a set of five LfcinB-related peptides was examined. We found that LfB17‑34, an 18-mer LfcinB-derived peptide, increased melanogenesis in B16F10 melanoma cells without significantly affecting cell viability. LfB17‑34 increased in vitro tyrosinase activity and melanin content in a dose-dependent manner. The results of RT-qPCR and western blot analyses showed that LfB17‑34 increased the mRNA and protein expression of tyrosinase and tyrosinase-related protein 1 (Trp1). Moreover, LfB17‑34 inhibited the phosphorylation of MAPK/Erk, but not p38 and Akt, and constitutively active MEK was able to reverse the LfB17-34-enhanced pigmentation, melanin content, and tyrosinase activity, suggesting a role of Erk signaling in the process of LfB17‑34-mediated pigmentation. Taken together, these results suggest that LfB17‑34 induces melanogenesis in B16F10 cells primarily through increased tyrosinase expression and activity and that LfB17‑34 could be further developed for the treatment of hypopigmentation disorders.

  14. The lactoferricin B-derived peptide, LfB17-34, induces melanogenesis in B16F10 cells

    PubMed Central

    Huang, Hsiu-Chin; Lin, Hsuan; Huang, Min-Chuan

    2017-01-01

    Lactoferricin B (LfcinB), a peptide of bovine lactoferrin (LfB), exhibits multiple biological functions, including antimicrobial, antiviral, antioxidant and immuno-modulatory activities. However, the role of LfcinB-related peptides in melanogenesis remains unclear. In this study, a set of five LfcinB-related peptides was examined. We found that LfB17-34, an 18-mer LfcinB-derived peptide, increased melanogenesis in B16F10 melanoma cells without significantly affecting cell viability. LfB17-34 increased in vitro tyrosinase activity and melanin content in a dose-dependent manner. The results of RT-qPCR and western blot analyses showed that LfB17-34 increased the mRNA and protein expression of tyrosinase and tyrosinase-related protein 1 (Trp1). Moreover, LfB17-34 inhibited the phosphorylation of MAPK/Erk, but not p38 and Akt, and constitutively active MEK was able to reverse the LfB17-34-enhanced pigmentation, melanin content, and tyrosinase activity, suggesting a role of Erk signaling in the process of LfB17-34-mediated pigmentation. Taken together, these results suggest that LfB17-34 induces melanogenesis in B16F10 cells primarily through increased tyrosinase expression and activity and that LfB17-34 could be further developed for the treatment of hypopigmentation disorders. PMID:28204812

  15. Tangeretin triggers melanogenesis through the activation of melanogenic signaling proteins and sustained extracellular signal- regulated kinase in B16/F10 murine melanoma cells.

    PubMed

    Yoon, Hoon Seok; Ko, Hee-Chul; Kim, Sang Suk; Park, Kyung Jin; An, Hyun Joo; Choi, Young Hun; Kim, Se-Jae; Lee, Nam-Ho; Hyun, Chang-Gu

    2015-03-01

    In order to test the effectiveness of tangeretin at ameliorating melanoma and melanoma-associated depigmentation, western blotting was used to assess the melanin content of treated melanoma cells. Tangeretin, a 4',5,6,7,8-pentamethoxyflavone, was found to trigger intracellular melanin production in a concentration-dependent manner in B16/F10 murine melanoma cells. Melanin content increased 1.74-fold in response to treatment with 25 μM of tangeretin, compared to that in non-treated cells. Examination of melanogenic protein expression showed that tyrosinase, tyrosinase-related protein (TRP)-1, and extracellular signal-regulated kinase (ERK) 1/2 levels increased in a dose-dependent manner. Furthermore, the expression of cyclic adenosine monophosphate response element binding protein (CREB) and microphthalmia transcription factor (MITF) was increased by tangeretin in 1 h and 4 h, respectively. Tangeretin- upregulated melanogenesis was suppressed by ERK 1/2 inhibitor and not by ERK1 inhibitor. These results suggest that tangeretin has therapeutic potential for melanoma and melanoma-associated depigmentation because it can induce hyperpigmentation through the activation of melanogenic signaling proteins and initiation of sustained ERK2 expression.

  16. Inhibition of ERK1/2 or AKT Activity Equally Enhances Radiation Sensitization in B16F10 Cells

    PubMed Central

    Kalal, Bhuvanesh Sukhlal; Fathima, Faraz; Pai, Vinitha Ramanath; Sanjeev, Ganesh; Krishna, Chilakapati Murali; Upadhya, Dinesh

    2018-01-01

    Background The aim of the study was to evaluate the radiation sensitizing ability of ERK1/2, PI3K-AKT and JNK inhibitors in highly radiation resistant and metastatic B16F10 cells which carry wild-type Ras and Braf. Methods Mouse melanoma cell line B16F10 was exposed to 1.0, 2.0 and 3.0 Gy of electron beam radiation. Phosphorylated ERK1/2, AKT and JNK levels were estimated by ELISA. Cells were exposed to 2.0 and 3.0 Gy of radiation with or without prior pharmacological inhibition of ERK1/2, AKT as well as JNK pathways. Cell death induced by radiation as well as upon inhibition of these pathways was measured by TUNEL assay using flow cytometry. Results Exposure of B16F10 cells to 1.0, 2.0 and 3.0 Gy of electron beam irradiation triggered an increase in all the three phosphorylated proteins compared to sham-treated and control groups. B16F10 cells pre-treated with either ERK1/2 or AKT inhibitors equally enhanced radiation-induced cell death at 2.0 as well as 3.0 Gy (P < 0.001), while inhibition of JNK pathway increased radiation-induced cell death to a lesser extent. Interestingly combined inhibition of ERK1/2 or AKT pathways did not show additional cell death compared to individual ERK1/2 or AKT inhibition. This indicates that ERK1/2 or AKT mediates radiation resistance through common downstream molecules in B16F10 cells. Conclusions Even without activating mutations in Ras or Braf genes, ERK1/2 and AKT play a critical role in B16F10 cell survival upon radiation exposure and possibly act through common downstream effector/s. PMID:29581812

  17. Inhibition of ERK1/2 or AKT Activity Equally Enhances Radiation Sensitization in B16F10 Cells.

    PubMed

    Kalal, Bhuvanesh Sukhlal; Fathima, Faraz; Pai, Vinitha Ramanath; Sanjeev, Ganesh; Krishna, Chilakapati Murali; Upadhya, Dinesh

    2018-02-01

    The aim of the study was to evaluate the radiation sensitizing ability of ERK1/2, PI3K-AKT and JNK inhibitors in highly radiation resistant and metastatic B16F10 cells which carry wild-type Ras and Braf . Mouse melanoma cell line B16F10 was exposed to 1.0, 2.0 and 3.0 Gy of electron beam radiation. Phosphorylated ERK1/2, AKT and JNK levels were estimated by ELISA. Cells were exposed to 2.0 and 3.0 Gy of radiation with or without prior pharmacological inhibition of ERK1/2, AKT as well as JNK pathways. Cell death induced by radiation as well as upon inhibition of these pathways was measured by TUNEL assay using flow cytometry. Exposure of B16F10 cells to 1.0, 2.0 and 3.0 Gy of electron beam irradiation triggered an increase in all the three phosphorylated proteins compared to sham-treated and control groups. B16F10 cells pre-treated with either ERK1/2 or AKT inhibitors equally enhanced radiation-induced cell death at 2.0 as well as 3.0 Gy (P < 0.001), while inhibition of JNK pathway increased radiation-induced cell death to a lesser extent. Interestingly combined inhibition of ERK1/2 or AKT pathways did not show additional cell death compared to individual ERK1/2 or AKT inhibition. This indicates that ERK1/2 or AKT mediates radiation resistance through common downstream molecules in B16F10 cells. Even without activating mutations in Ras or Braf genes, ERK1/2 and AKT play a critical role in B16F10 cell survival upon radiation exposure and possibly act through common downstream effector/s.

  18. Inhibitory Effect of Dried Pomegranate Concentration Powder on Melanogenesis in B16F10 Melanoma Cells; Involvement of p38 and PKA Signaling Pathways

    PubMed Central

    Kang, Su Jin; Choi, Beom Rak; Lee, Eun Kyoung; Kim, Seung Hee; Yi, Hae Yeon; Park, Hye Rim; Song, Chang Hyun; Lee, Young Joon; Ku, Sae Kwang

    2015-01-01

    Plants rich in antioxidant substances may be useful for preventing skin aging. Pomegranates, containing flavonoids and other polyphenolic compounds, are widely consumed due to their beneficial properties. We examined the underlying mechanisms of dried pomegranate concentrate powder (PCP) on melanin synthesis in B16F10 melanoma cells. The antioxidant effects of PCP were determined by measuring free radical scavenging capacity and transcript levels of antioxidant enzymes. To explore the inhibitory effects of PCP on melanin synthesis, we measured tyrosinase activity and melanin content in α-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 cells. In addition, the levels of tyrosinase-related protein-1 (TRP-1), TRP-2, tyrosinase, and microphthalmia-associated transcription factor (MITF) expression were determined by Western blotting. Changes in the phosphorylation status of protein kinase A (PKA), cAMP response element-binding protein (CREB), mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K), serine/threonine kinase Akt, and glycogen kinase 3β (GSK3β) were also examined. The free radical scavenging activity of PCP increased in a dose-dependent manner. In PCP-treated B16F10 cells, transcript levels of glutathione peroxidase-1 (GPx-1) were increased compared with α-MSH-stimulated cells. In addition, PCP led to the down-regulation of phospho-p38, phospho-PKA, phospho-CREB, phospho-GSK3β, MITF, and TRP-1 compared with α-MSH-stimulated B16F10 cells. We believe this effect may be associated with PCP activity, which leads to the inhibition of melanin production and tyrosinase activity. These results suggest that PCP decreases tyrosinase activity and melanin production via inactivation of the p38 and PKA signaling pathways, and subsequently decreases phosphorylation of CREB, MITF, and melanogenic enzymes. These observations provided new insights on the molecular mechanisms of the skin-whitening property of PCP. PMID:26473849

  19. Whitening and anti-wrinkle activities of ferulic acid isolated from Tetragonia tetragonioides in B16F10 melanoma and CCD-986sk fibroblast cells.

    PubMed

    Park, Hye-Jin; Cho, Jun-Hyo; Hong, Shin-Hyub; Kim, Dong-Hee; Jung, Hee-Young; Kang, In-Kyu; Cho, Young-Je

    2018-01-01

    Ferulic acid isolated from Tetragonia tetragonioides was tested for its whitening effect on the B16F10 mouse melanoma cell line and its anti-wrinkle activity on the CCD-986sk human dermal fibroblast cell line. Ferulic acid, one of the primary phenolic compounds that can be isolated from T. tetragonioides, has been reported to show potential as a functional food, for its whitening effect and anti-wrinkle activity. To measure its whitening and anti-wrinkle activities, cells were treated with ferulic acid isolated from T. tetragonioides at concentrations between 5 and 20 μM. Ferulic acid showed no cytotoxicity at concentrations up to 20 μM. Ferulic acid inhibited melanin synthesis, tyrosinase expression, and microphthalmia transcription factor expression in B16F10 cells stimulated with α-melanocyte stimulating hormone. Ferulic acid induced procollagen synthesis, hyaluronic acid synthesis, tissue inhibitor of metalloproteinase synthesis, and inhibited matrix metalloproteinase (MMP)-1 and MMP-9 expression in CCD-986sk cells stimulated with UV-B. On the basis of these results, we conclude that ferulic acid isolated from T. tetragonioides shows potential for use as a functional food, with whitening and anti-wrinkle activities.

  20. Identification of genes differentially expressed in B16 murine melanoma sublines with different metastatic potentials.

    PubMed

    Ishiguro, T; Nakajima, M; Naito, M; Muto, T; Tsuruo, T

    1996-02-15

    B16-F10 and B16-BL6 are B16 mouse melanoma sublines that preferentially metastasize to the lung following i.v. and s.c. injections, respectively. To study molecular mechanisms underlying the different metastatic behaviors exhibited by the B16 melanoma sublines, we performed differential hybridization of the genes transcribed in these cells and compared their expression levels. We isolated four genes that were highly expressed in B16-F10 cells but not in B16-BL6 cells: TI-225 (polyubiquitin), TI-229 (pyruvate kinase), TI-241 (LRF-1 homologue), and TI-227 (novel gene). Triosephosphate isomerase, 10-formyltetrahydrofolate dehydrogenase, tyrosinase-related protein 2, cytochrome c oxidase, ATP synthetase alpha subunit, RNA helicase, and ribosomal protein (L37, J1, acidic phosphoprotein), however, showed higher expression in B16-BL6 cells than in B16-F10 cells. Among these clones, transfection of TI-241 into the low metastatic clone F1 converted the parental cells from low- into high-metastatic cells. TI-241 may regulate the expression of various genes as a transcription factor in the complex process of metastasis.

  1. cRGD-installed docetaxel-loaded mertansine prodrug micelles: redox-triggered ratiometric dual drug release and targeted synergistic treatment of B16F10 melanoma

    NASA Astrophysics Data System (ADS)

    Zhong, Ping; Qiu, Min; Zhang, Jian; Sun, Huanli; Cheng, Ru; Deng, Chao; Meng, Fenghua; Zhong, Zhiyuan

    2017-07-01

    Combinatorial chemotherapy, which has emerged as a promising treatment modality for intractable cancers, is challenged by a lack of tumor-targeting, robust and ratiometric dual drug release systems. Here, docetaxel-loaded cRGD peptide-decorated redox-activable micellar mertansine prodrug (DTX-cRGD-MMP) was developed for targeted and synergistic treatment of B16F10 melanoma-bearing C57BL/6 mice. DTX-cRGD-MMP exhibited a small size of ca. 49 nm, high DTX and DM1 loading, low drug leakage under physiological conditions, with rapid release of both DTX and DM1 under a cytoplasmic reductive environment. Notably, MTT and flow cytometry assays showed that DTX-cRGD-MMP brought about a synergistic antitumor effect to B16F10 cancer cells, with a combination index of 0.37 and an IC50 over 3- and 13-fold lower than cRGD-MMP (w/o DTX) and DTX-cRGD-Ms (w/o DM1) controls, respectively. In vivo studies revealed that DTX-cRGD-MMP had a long circulation time and a markedly improved accumulation in the B16F10 tumor compared with the non-targeting DTX-MMP control (9.15 versus 3.13% ID/g at 12 h post-injection). Interestingly, mice treated with DTX-cRGD-MMP showed almost complete growth inhibition of B16F10 melanoma, with tumor inhibition efficacy following an order of DTX-cRGD-MMP > DTX-MMP (w/o cRGD) > cRGD-MMP (w/o DTX) > DTX-cRGD-Ms (w/o DM1) > free DTX. Consequently, DTX-cRGD-MMP significantly improved the survival rates of B16F10 melanoma-bearing mice. Importantly, DTX-cRGD-MMP caused little adverse effects as revealed by mice body weights and histological analyses. The combination of two mitotic inhibitors, DTX and DM1, appears to be an interesting approach for effective cancer therapy.

  2. [6]-Shogaol inhibits melanogenesis in B16 mouse melanoma cells through activation of the ERK pathway

    PubMed Central

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

    2013-01-01

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

  3. [6]-Shogaol inhibits melanogenesis in B16 mouse melanoma cells through activation of the ERK pathway.

    PubMed

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

    2013-02-01

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

  4. Feruloylserotonin inhibits hydrogen peroxide-induced melanogenesis and apoptosis in B16F10 and SK-Mel-2 melanoma cells.

    PubMed

    Cho, Hyejoung; Kim, Okjoon; Lee, Younghee; Kang, Li-Jung; Nguyen, Cam Ngoc; Ishihara, Atsushi; Kim, Hye-Eun

    2017-09-30

    Feruloylserotonin (FS) is a major bioactive component of safflower seeds, with documented strong antibacterial, anti-inflammatory, and free radical scavenging activities. Reactive oxygen species (ROS) can strongly induce melanogenesis and cell apoptosis. The present study aimed to investigate the ability of FS in preventing hydrogen peroxide (H 2 O 2 )-induced melanogenesis and cell apoptosis. Melanogenesis and apoptotic cell death were induced by transient exposure to H 2 O 2 in B16F10 and SK-Mel-2 melanoma cells. FS significantly inhibited melanogenesis and cell death in both cell lines. FS inhibited H 2 O 2 -induced melanin production by down-regulating CREB/MITF/TYR signaling via inhibited intracellular cAMP accumulation. Additionally, FS induced extracellular regulated kinase activation, which led to the degradation of MITF and consequently decreased TYR expression and melanin production in H 2 O 2 -stimulated cells. Furthermore, FS inhibited H 2 O 2 -induced apoptotic cell death by maintaining mitochondrial membrane potential. Therefore, FS might have potential use for cosmetic whitening and as a therapeutic agent for hyperpigmentation disorder. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Effect of bis(bilato)-1,2-cyclohexanediammineplatinum(II) complexes on lung metastasis of B16-F10 melanoma cells in mice.

    PubMed

    Maeda, M; Suga, T; Takasuka, N; Hoshi, A; Sasaki, T

    1990-12-03

    New platinum(II) complexes, bis(bilato)-1,2-cyclohexanediammineplatinum(II) which were lipophilic and water-miscible, were tested for antitumor activity against lung nodules from intravenously injected B16-F10 melanoma cells in C57BL/6 mice by intravenous administration of the complexes in water suspension form. Among them, DACHP(litho)2 and DACHP(urso)2 had high antitumor activity but others had no activity. The antitumor activity of DACHP(urso)2 was increased significantly by injecting it three times; T/C was over 280% with 100-day survivors of 3 of 6 mice tested. Large amounts of total platinum were found in lung and liver tissues by atomic absorption spectroscopy after single intravenous injection of DACHP(urso)2 suspension in ICR mice.

  6. Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells.

    PubMed

    Park, Sun Joo; Kim, Yong Tae; Jeon, You Jin

    2012-04-01

    Reactive oxygen species (ROS) generation is linked to dynamic actin cytoskeleton reorganization, which is involved in tumor cell motility and metastasis. Thus, inhibition of ROS generation and actin polymerization in tumor cells may represent an effective anticancer strategy. However, the molecular basis of this signaling pathway is currently unknown. Here, we show that the Ecklonia cava-derived antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Steady-state intracellular ROS levels were higher in malignant B16F10 cells than in parental, nonmetastatic B16F0 cells. Elevation of ROS by H(2)O(2) treatment increased migration and invasion ability of B16F0 cells to level similar to that of B16F10 cells, suggesting that intracellular ROS signaling mediates the prometastatic properties of B16 mouse melanoma cells. ROS levels and the cell migration and invasion ability of B16 melanoma cells correlated with Rac1 activation and WAVE2 expression. Overexpression of dominant negative Rac1 and depletion of WAVE2 by siRNA suppressed H(2)O(2)-induced cell invasion of B16F0 and B16F10 cells. Similarly, dieckol attenuates the ROS-mediated Rac1 activation and WAVE2 expression, resulting in decreased migration and invasion of B16 melanoma cells. In addition, we found that dieckol decreases association between WAVE2 and NADPH oxidase subunit p47(phox). Therefore, this finding suggests that WAVE2 acts to couple intracellular Rac1/ROS signaling to the invasive migration of B16 melanoma cells, which is inhibited by dieckol.

  7. Antioxidant Dieckol Downregulates the Rac1/ROS Signaling Pathway and Inhibits Wiskott-Aldrich Syndrome Protein (WASP)-Family Verprolin-Homologous Protein 2 (WAVE2)-Mediated Invasive Migration of B16 Mouse Melanoma Cells

    PubMed Central

    Park, Sun Joo; Kim, Yong Tae; Jeon, You Jin

    2012-01-01

    Reactive oxygen species (ROS) generation is linked to dynamic actin cytoskeleton reorganization, which is involved in tumor cell motility and metastasis. Thus, inhibition of ROS generation and actin polymerization in tumor cells may represent an effective anticancer strategy. However, the molecular basis of this signaling pathway is currently unknown. Here, we show that the Ecklonia cava-derived antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Steady-state intracellular ROS levels were higher in malignant B16F10 cells than in parental, nonmetastatic B16F0 cells. Elevation of ROS by H2O2 treatment increased migration and invasion ability of B16F0 cells to level similar to that of B16F10 cells, suggesting that intracellular ROS signaling mediates the prometastatic properties of B16 mouse melanoma cells. ROS levels and the cell migration and invasion ability of B16 melanoma cells correlated with Rac1 activation and WAVE2 expression. Overexpression of dominant negative Rac1 and depletion of WAVE2 by siRNA suppressed H2O2-induced cell invasion of B16F0 and B16F10 cells. Similarly, dieckol attenuates the ROS-mediated Rac1 activation and WAVE2 expression, resulting in decreased migration and invasion of B16 melanoma cells. In addition, we found that dieckol decreases association between WAVE2 and NADPH oxidase subunit p47phox. Therefore, this finding suggests that WAVE2 acts to couple intracellular Rac1/ROS signaling to the invasive migration of B16 melanoma cells, which is inhibited by dieckol. PMID:22441674

  8. Oligoesculin fraction induces anti-tumor effects and promotes immune responses on B16-F10 mice melanoma.

    PubMed

    Mokdad Bzeouich, Imen; Mustapha, Nadia; Sassi, Aicha; Ghedira, Kamel; Ghoul, Mohamed; Chebil, Latifa; Luis, José; Chekir-Ghedira, Leila

    2016-08-01

    Laccase was used to enzymatically polymerize esculin. Oligoesculin fraction was obtained after ultrafiltration through a 5-kDa membrane. Several studies have been carried out to prove the effectiveness of natural substances such as immunomodulators to promote the anti-cancer activity in situ. The purpose of our report was to explore whether the anti-tumor potential of the oligoesculin fraction in vitro and in vivo is linked to its immunological mechanisms in melanoma-bearing mice. We revealed that oligoesculin fraction reduced B16-F10 proliferation and migration in vitro in a dose-related manner. Moreover, melanin synthesis and tyrosinase activity were inhibited in these melanoma cells in a concentration-dependent way. The anti-tumor potential of oligoesculin fraction was also assessed in vivo. Our results showed that intraperitoneal administration of oligoesculin fraction, at 50 mg/kg body weight (b.w.) for 21 days, reduced tumor size and weight with percentages of inhibition of 94 and 87 %, respectively. Oligoesculin fraction was effective in promoting lysosomal activity and nitric oxide (NO) production by peritoneal macrophages in tumor-implanted mice. In addition, the activities of natural killer (NK), cytotoxic T lymphocytes, and macrophages were significantly enhanced by oligoesculin fraction. These findings suggested that this polymer with its anti-tumor and immunomodulatory properties could be used for the treatment of melanoma.

  9. Effective adoptive transfer of haploidentical tumor-specific T cells in B16-melanoma bearing mice.

    PubMed

    Cui, Nai-peng; Xie, Shao-jian; Han, Jin-sheng; Ma, Zhen-feng; Chen, Bao-ping; Cai, Jian-hui

    2012-03-01

    Adoptive transfer of allogeneic tumor-specific T cells often results in severe graft-versus-host disease (GVHD). Here, we sought to maximize graft-versus-tumor and minimize GVHD by using haploidentical T cells in pre-irradiated B16-melanoma bearing mice. C57BL/6 mice bearing B16-melanoma tumors were irradiated with 0, 5, or 7 Gy total body irradiation (TBI), or 7 Gy TBI plus bone marrow transplantation. Tumor areas were measured every 3 days to assess the influence of irradiation treatment on tumor regression. B16-melanoma bearing mice were irradiated with 7 Gy TBI; sera and spleens were harvested at days 1, 3, 5, 7, 9, 11, and 13 after irradiation. White blood cell levels were measured and transforming growth factor β1 (TGF-b1) and interleukin 10 (IL-10) levels in serum were detected using enzyme-linked immunosorbent assay (ELISA) kits. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry were performed to test TGF-b1, IL-10 and Foxp3 mRNA levels and the proportion of CD4+CD25+Foxp3+ T-regulatory cells (Tregs) in spleens. B16-melanoma bearing C57BL/6 mice were irradiated with 7 Gy TBI followed by syngeneic (Syn1/Syn2) or haploidentical (Hap1/Hap2), dendritic cell-induced cytotoxic T lymphocytes (DC-CTLs) treatment, tumor areas and system GVHD were observed every 3 days. Mice were killed 21 days after the DC-CTLs adoptive transfer; histologic analyses of eyes, skin, liver, lungs, and intestine were then performed. Irradiation with 7 Gy TBI on the B16-melanoma-bearing mice did not influence tumor regression compared to the control group; however, it down-regulated the proportion of Tregs in spleens and the TGF-b1 and IL-10 levels in sera and spleens, suggesting inhibition of autoimmunity and intervention of tumor microenvironment. Adoptive transfer of haploidentical DC-CTLs significantly inhibited B16-melanoma growth. GVHD assessment and histology analysis showed no significant difference among the groups. Adoptive transfer of

  10. (2E,5E)-2,5-Bis(3-hydroxy-4-methoxybenzylidene) cyclopentanone Exerts Anti-Melanogenesis and Anti-Wrinkle Activities in B16F10 Melanoma and Hs27 Fibroblast Cells.

    PubMed

    Jung, Hee Jin; Lee, A Kyoung; Park, Yeo Jin; Lee, Sanggwon; Kang, Dongwan; Jung, Young Suk; Chung, Hae Young; Moon, Hyung Ryong

    2018-06-11

    Ultraviolet (UV) radiation exposure is the primary cause of extrinsic skin aging, which results in skin hyperpigmentation and wrinkling. In this study, we investigated the whitening effect of (2 E ,5 E )-2,5-bis(3-hydroxy-4-methoxybenzylidene)cyclopentanone (BHCP) on B16F10 melanoma and its anti-wrinkle activity on Hs27 fibroblasts cells. BHCP was found to potently inhibit tyrosinase, with 50% inhibition concentration (IC 50 ) values of 1.10 µM and 8.18 µM for monophenolase (l-tyrosine) and diphenolase (l-DOPA), and the enzyme kinetics study revealed that BHCP is a competitive-type tyrosinase inhibitor. Furthermore, BHCP significantly inhibited melanin content and cellular tyrosinase activity, and downregulated the levels of microphthalmia-associated transcription factor (MITF), phosphorylated levels of cAMP response element-binding (CREB) protein, and tyrosinase in α-melanocyte stimulating hormone (α-MSH)-induced B16F10 melanoma cells. Moreover, BHCP inhibited the phosphorylation of p65 and expression of matrix metalloproteinases (MMP-1, MMP-9, MMP-12, and MMP-13) in Hs27 fibroblasts stimulated with UV radiation. Therefore, our results demonstrate that BHCP may be a good candidate for the development of therapeutic agents for diseases associated with hyperpigmentation and wrinkling.

  11. A review of exosome separation techniques and characterization of B16-F10 mouse melanoma exosomes with AF4-UV-MALS-DLS-TEM.

    PubMed

    Petersen, Kevin E; Manangon, Eliana; Hood, Joshua L; Wickline, Samuel A; Fernandez, Diego P; Johnson, William P; Gale, Bruce K

    2014-12-01

    Exosomes participate in cancer metastasis, but studying them presents unique challenges as a result of their small size and purification difficulties. Asymmetrical field flow fractionation with in-line ultraviolet absorbance, dynamic light scattering, and multi-angle light scattering was applied to the size separation and characterization of non-labeled B16-F10 exosomes from an aggressive mouse melanoma cell culture line. Fractions were collected and further analyzed using batch mode dynamic light scattering, transmission electron microscopy and compared with known size standards. Fractogram peak positions and computed radii show good agreement between samples and across fractions. Ultraviolet absorbance fractograms in combination with transmission electron micrographs were able to resolve subtle heterogeneity of vesicle retention times between separate batches of B16-F10 exosomes collected several weeks apart. Further, asymmetrical field flow fractionation also effectively separated B16-F10 exosomes into vesicle subpopulations by size. Overall, the flow field flow fractionation instrument combined with multiple detectors was able to rapidly characterize and separate exosomes to a degree not previously demonstrated. These approaches have the potential to facilitate a greater understanding of exosome function by subtype, as well as ultimately allow for "label-free" isolation of large scale clinical exosomes for the purpose of developing future exosome-based diagnostics and therapeutics.

  12. A review of exosome separation techniques and characterization of B16-F10 mouse melanoma exosomes with AF4-UV-MALS-DLS-TEM

    PubMed Central

    Manangon, Eliana; Hood, Joshua L.; Wickline, Samuel A.; Fernandez, Diego P.; Johnson, William P.; Gale, Bruce K.

    2015-01-01

    Exosomes participate in cancer metastasis, but studying them presents unique challenges as a result of their small size and purification difficulties. Asymmetrical field flow fractionation with in-line ultraviolet absorbance, dynamic light scattering, and multi-angle light scattering was applied to the size separation and characterization of non-labeled B16-F10 exosomes from an aggressive mouse melanoma cell culture line. Fractions were collected and further analyzed using batch mode dynamic light scattering, transmission electron microscopy and compared with known size standards. Fractogram peak positions and computed radii show good agreement between samples and across fractions. Ultraviolet absorbance fractograms in combination with transmission electron micrographs were able to resolve subtle heterogeneity of vesicle retention times between separate batches of B16-F10 exosomes collected several weeks apart. Further, asymmetrical field flow fractionation also effectively separated B16-F10 exosomes into vesicle subpopulations by size. Overall, the flow field flow fractionation instrument combined with multiple detectors was able to rapidly characterize and separate exosomes to a degree not previously demonstrated. These approaches have the potential to facilitate a greater understanding of exosome function by subtype, as well as ultimately allow for “label-free” isolation of large scale clinical exosomes for the purpose of developing future exosome-based diagnostics and therapeutics. PMID:25084738

  13. Medicinal facilities to B16F10 melanoma cells for distant metastasis control with a supramolecular complex by DEAE-dextran-MMA copolymer/paclitaxel.

    PubMed

    Eshita, Yuki; Ji, Rui-Cheng; Onishi, Masayasu; Kobayashi, Takashi; Mizuno, Masaaki; Yoshida, Jun; Kubota, Naoji; Onishi, Yasuhiko

    2015-02-01

    The resistance of cancer cells to chemotherapeutic drugs (MDR) is a major problem to be solved. A supramolecular DEAE-dextran-MMA copolymer (DDMC)/paclitaxel (PTX) complex was obtained by using PTX as the guest and DDMC as the host having 50-300 nm in diameter. The drug resistance of B16F10 melanoma cells to paclitaxel was observed, but there is no drug resistance of melanoma cells to the DDMC/PTX complex in vitro. The cell death rate was determined using Michaelis-Menten kinetics, as the DDMC/PTX complex promoted allosteric supramolecular reaction to tubulin. The DDMC/PTX complex showed a very superior anti-cancer activity to paclitaxel alone in vivo. The median survival time (MST) of the saline, PTX, DDMC/PTX4 (particle size, 50 nm), and DDMC/PTX5 (particle size, 290 nm) groups were 120 h (T/C, 1.0), 176 h (T/C, 1.46), 328 h (T/C, 2.73), and 280 h (T/C, 2.33), respectively. The supramolecular DDMC/PTX complex showed the twofold effectiveness of PTX alone (p < 0.036). Histochemical analysis indicated that the administration of DDMC/PTX complex decreased distant metastasis and increased the survival of mice. A mouse of DDMC/PTX4 group in vivo was almost curing after small dermatorrhagia owing to its anti-angiogenesis, and it will be the hemorrhagic necrotic symptom of tumor by the release of "tumor necrosis factor alpha (TNF-α)" cytokine. As the result, the medicinal action of the DDMC/PTX complex will suppress the tumor-associated action of M2 macrophages and will control the metastasis of cancer cells.

  14. Treatment of metastatic melanoma B16F10 by the flavonoids tangeretin, rutin, and diosmin.

    PubMed

    Martínez Conesa, Cristina; Vicente Ortega, Vicente; Yáñez Gascón, M Josefa; Alcaraz Baños, Miguel; Canteras Jordana, Manuel; Benavente-García, Obdulio; Castillo, Julián

    2005-08-24

    Melanoma is one of the most frequently metastasizing malignant neoplasias. This study examines an experimental model of pulmonary metastasis and the B16F10 cell subline, highly metastatic in the lung. Antimetastatic effects of the flavonoids tangeretin, rutin, and diosmin were analyzed, and at the same time an analysis of the metastatic activity of ethanol was performed, considered to be necessary because it is used as a vehicle for administering the flavonoids. Lentini's model, which complements the macroscopic evaluation of nodule numbers by using a stereoscopic microscope and image analysis at the microscopic level, was used. The greatest reduction in the number of metastatic nodules (52%) was obtained with diosmin; similarly, the percentages of implantation, growth index, and invasion index (79.40, 67.44, and 45.23%, respectively), were all compared with those of the ethanol group, considered to be an effective control group. Rutin- and tangeretin-treated groups also showed reductions of the same index compared with the ethanol group. It would seem that structural factors would better explain these results and the antimetastatic activity of each flavonoid and the respective metabolites.

  15. Exosomes derived from B16F0 melanoma cells alter the transcriptome of cytotoxic T cells that impacts mitochondrial respiration.

    PubMed

    Bland, Cassidy L; Byrne-Hoffman, Christina N; Fernandez, Audry; Rellick, Stephanie L; Deng, Wentao; Klinke, David J

    2018-03-01

    While recent clinical studies demonstrate the promise of cancer immunotherapy, a barrier for broadening the clinical benefit is identifying how tumors locally suppress cytotoxic immunity. As an emerging mode of intercellular communication, exosomes secreted by malignant cells can deliver a complex payload of coding and noncoding RNA to cells within the tumor microenvironment. Here, we quantified the RNA payload within tumor-derived exosomes and the resulting dynamic transcriptomic response to cytotoxic T cells upon exosome delivery to better understand how tumor-derived exosomes can alter immune cell function. Exosomes derived from B16F0 melanoma cells were enriched for a subset of coding and noncoding RNAs that did not reflect the abundance in the parental cell. Upon exosome delivery, RNAseq revealed the dynamic changes in the transcriptome of CTLL2 cytotoxic T cells. In analyzing transiently coexpressed gene clusters, pathway enrichment suggested that the B16F0 exosomal payload altered mitochondrial respiration, which was confirmed independently, and upregulated genes associated with the Notch signaling pathway. Interestingly, exosomal miRNA appeared to have no systematic effect on downregulating target mRNA levels. Gene expression data are available in the GEO database under the accession SuperSeries number GSE102951. © 2018 Federation of European Biochemical Societies.

  16. Synthesis of dihydroresveratrol glycosides and evaluation of their activity against melanogenesis in B16F0 melanoma cells.

    PubMed

    Oode, Chisato; Shimada, Wataru; Izutsu, Yukiko; Yokota, Mariko; Iwadate, Takehiro; Nihei, Ken-ichi

    2014-11-24

    Dihydroresveratrol glucoside 1 isolated from Camellia oleifera and its xyloside derivative 2 were synthesized for the first time in 5 steps from TBS-protected aldehyde 4. Natural product 1 is a potent melanogenesis inhibitor in B16F0 melanoma cells (approximately 40 fold more potent than kojic acid). In contrast, the synthetic product 2 stimulates melanogenesis, suggesting that a single hydroxymethyl group in the glycoside substituent of dihydroresveratrols is responsible for inhibition or activation of melanogenesis. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  17. Synthesis and preclinical characterization of [18F]FPBZA: a novel PET probe for melanoma.

    PubMed

    Wu, Shih-Yen; Huang, Shih-Pin; Lo, Yen-Chen; Liu, Ren-Shyan; Wang, Shyh-Jen; Lin, Wuu-Jyh; Shen, Chih-Chieh; Wang, Hsin-Ell

    2014-01-01

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

  18. Triterpenoids Amplify Anti-Tumoral Effects of Mistletoe Extracts on Murine B16.F10 Melanoma In Vivo

    PubMed Central

    Strüh, Christian M.; Jäger, Sebastian; Kersten, Astrid; Schempp, Christoph M.; Scheffler, Armin; Martin, Stefan F.

    2013-01-01

    Purpose Mistletoe extracts are often used in complementary cancer therapy although the efficacy of that therapy is controversially discussed. Approved mistletoe extracts contain mainly water soluble compounds of the mistletoe plant, i.e. mistletoe lectins. However, mistletoe also contains water-insoluble triterpenoids (mainly oleanolic acid) that have anti-tumorigenic effects. To overcome their loss in watery extracts we have solubilized mistletoe triterpenoids with cyclodextrins, thus making them available for in vivo cancer experiments. Experimental design B16.F10 subcutaneous melanoma bearing C57BL/6 mice were treated with new mistletoe extracts containing both water soluble compounds and solubilized triterpenoids. Tumor growth and survival was monitored. In addition, histological examinations of the tumor material and tumor surrounding tissue were performed. Results Addition of solubilized triterpenoids increased the anti-tumor effects of the mistletoe extracts, resulting in reduced tumor growth and prolonged survival of the mice. Histological examination of the treated tumors showed mainly tumor necrosis and some apoptotic cells with active caspase-3 and TUNEL staining. A significant decrease of CD31-positive tumor blood vessels was observed after treatment with solubilized triterpenoids and different mistletoe extracts. Conclusion We conclude that the addition of solubilized mistletoe triterpenoids to conventional mistletoe extracts improves the efficacy of mistletoe treatment and may represent a novel treatment option for malignant melanoma. PMID:23614029

  19. Ethanol inhibits B16-BL6 melanoma metastasis and cell phenotypes associated with metastasis.

    PubMed

    Kushiro, Kyoko; Núñez, Nomelí P

    2012-01-01

    Every year, approximately 68,000 new cases of malignant melanoma are diagnosed in the US. Ethanol consumption inhibits metastasis of melanoma in mice, but the mechanism is not well understood. C57BL/6J ob/+ mice, given either water or 20% ethanol, were injected intravenously with B16-BL6 melanoma cells to determine pulmonary metastasis. The effects of ethanol on cell phenotypes and markers of the epithelial-to-mesenchymal transition were determined in cell culture. In mice, ethanol consumption inhibited experimental pulmonary metastasis. This inhibition was associated with decreased body weight, and levels of systemic leptin, and insulin. In cell culture, ethanol inhibited B16-BL6 cell motility, invasion, and anchorage-independent growth. Additionally, ethanol reduced Snai1 expression and increased E-cadherin expression. Lastly, ethanol increased the expression of Kiss1 metastasis-suppressor and the metastasis suppressor Nm23/nucleoside diphosphate kinase. In both animal and in cell culture conditions, ethanol inhibited the metastatic ability of B16-BL6 melanoma cells.

  20. Effect of vernolide-A, a sesquiterpene lactone from Vernonia cinerea L., on cell-mediated immune response in B16F-10 metastatic melanoma-bearing mice.

    PubMed

    Pratheeshkumar, P; Kuttan, Girija

    2011-09-01

    One of the major reasons for the rapid progression of cancers is the ability of tumor cells to escape from the immune surveillance mechanism of the body. Modulation of immune responses is highly relevant in tumor cell destruction. Effect of vernolide-A on the cell-mediated immune (CMI) response in metastatic condition was studied using C57BL/6 mice model. Administration of vernolide-A enhanced natural killer (NK) cell activity, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent complement-mediated cytotoxicity (ACC) and the activity was observed in treated group much earlier compared with the metastatic tumor-bearing control. Administration of vernolide-A significantly enhanced the production of interleukin (IL)-2 and interferon-gamma (IFN-γ) in metastatic tumor-bearing animals. In addition, vernolide-A significantly down-regulated the serum levels of proinflammatory cytokines such as IL-1β, IL-6, tumor necrosis factor-alpha (TNF-α), and granulocyte-macrophage colony-stimulating factor (GM-CSF) during metastasis. All these results demonstrate that vernolide-A could enhance the immune response against metastatic progression of B16F-10 melanoma cells in mice.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2009-08-01

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

  3. Caloric restriction induces heat shock response and inhibits B16F10 cell tumorigenesis both in vitro and in vivo.

    PubMed

    Novelle, Marta G; Davis, Ashley; Price, Nathan L; Ali, Ahmed; Fürer-Galvan, Stefanie; Zhang, Yongqing; Becker, Kevin; Bernier, Michel; de Cabo, Rafael

    2015-04-01

    Caloric restriction (CR) without malnutrition is one of the most consistent strategies for increasing mean and maximal lifespan and delaying the onset of age-associated diseases. Stress resistance is a common trait of many long-lived mutants and life-extending interventions, including CR. Indeed, better protection against heat shock and other genotoxic insults have helped explain the pro-survival properties of CR. In this study, both in vitro and in vivo responses to heat shock were investigated using two different models of CR. Murine B16F10 melanoma cells treated with serum from CR-fed rats showed lower proliferation, increased tolerance to heat shock and enhanced HSP-70 expression, compared to serum from ad libitum-fed animals. Similar effects were observed in B16F10 cells implanted subcutaneously in male C57BL/6 mice subjected to CR. Microarray analysis identified a number of genes and pathways whose expression profile were similar in both models. These results suggest that the use of an in vitro model could be a good alternative to study the mechanisms by which CR exerts its anti-tumorigenic effects.

  4. Caloric restriction induces heat shock response and inhibits B16F10 cell tumorigenesis both in vitro and in vivo

    PubMed Central

    Novelle, Marta G.; Davis, Ashley; Price, Nathan L.; Ali, Ahmed; Fürer-Galvan, Stefanie; Zhang, Yongqing; Becker, Kevin; Bernier, Michel; de Cabo, Rafael

    2015-01-01

    Caloric restriction (CR) without malnutrition is one of the most consistent strategies for increasing mean and maximal lifespan and delaying the onset of age-associated diseases. Stress resistance is a common trait of many long-lived mutants and life-extending interventions, including CR. Indeed, better protection against heat shock and other genotoxic insults have helped explain the pro-survival properties of CR. In this study, both in vitro and in vivo responses to heat shock were investigated using two different models of CR. Murine B16F10 melanoma cells treated with serum from CR-fed rats showed lower proliferation, increased tolerance to heat shock and enhanced HSP-70 expression, compared to serum from ad libitum-fed animals. Similar effects were observed in B16F10 cells implanted subcutaneously in male C57BL/6 mice subjected to CR. Microarray analysis identified a number of genes and pathways whose expression profile were similar in both models. These results suggest that the use of an in vitro model could be a good alternative to study the mechanisms by which CR exerts its anti-tumorigenic effects. PMID:25948793

  5. Genomic alterations in spontaneous and carcinogen-induced murine melanoma cell lines.

    PubMed

    Melnikova, Vladislava O; Bolshakov, Svetlana V; Walker, Christopher; Ananthaswamy, Honnavara N

    2004-03-25

    We have conducted an analysis of genetic alterations in spontaneous murine melanoma cell line B16F0 and its two metastatic clones, B16F1 and B16F10 and the carcinogen-induced murine melanoma cell lines CM519, CM3205, and K1735. We found that unlike human melanomas, the murine melanoma cell lines did not have activating mutations in the Braf oncogene at exon 11 or 15. However, there were distinct patterns of alterations in the ras, Ink4a/Arf, and p53 genes in the two melanoma groups. In the spontaneous B16 melanoma cell lines, expression of p16Ink4a and p19Arf tumor suppressor proteins was lost as a consequence of a large deletion spanning Ink4a/Arf exons 1alpha, 1beta, and 2. In contrast, the carcinogen-induced melanoma cell lines expressed p16Ink4a but had inactivating mutations in either p19Arf (K1735) or p53 (CM519 and CM3205). Inactivation of p19Arf or p53 in carcinogen-induced melanomas was accompanied by constitutive activation of mitogen-activated protein kinases (MAPKs) and/or mutation-associated activation of N-ras. These results indicate that genetic alterations in p16Ink4a/p19Arf, p53 and ras-MAPK pathways can cooperate in the development of murine melanoma.

  6. Differentiation-inducing and anti-proliferative activities of isoliquiritigenin and all-trans-retinoic acid on B16F0 melanoma cells: Mechanisms profiling by RNA-seq.

    PubMed

    Chen, Xiaoyu; Yang, Ming; Hao, Wenjin; Han, Jichun; Ma, Jun; Wang, Caixia; Sun, Shiguo; Zheng, Qiusheng

    2016-10-30

    Melanoma is a cancer that arises from melanocytes, specialized pigmented cells that are found predominantly in the skin. The incidence of malignant melanoma has significantly increased over the last decade. With the development of therapy, the survival rate of some kind of cancer has been improved greatly. But the treatment of melanoma remains unsatisfactory. Much of melanoma's resistance to traditional chemotherapy is believed to arise intrinsically, by virtue of potent growth and cell survival-promoting genetic alteration. Therefore, significant attention has recently been focused on differentiation therapy, as well as differentiation inducer compounds. In previous study, we found isoliquiritigenin (ISL), a natural product extracted from licorice, could induce B16F0 melanoma cell differentiation. Here we investigated the transcriptional response of melanoma differentiation process induced by ISL and all-trans-retinoic acid (RA). Results showed that 390 genes involves in 201 biochemical pathways were differentially expressed in ISL treatment and 304 genes in 193 pathways in RA treatment. Differential expressed genes (DGEs, fold-change (FC)≥10) with the function of anti-proliferative and differentiation inducing indicated a loss of grade malignancy characteristic. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated glutathione metabolism, glycolysis/gluconeogenesis and pentose phosphate pathway were the top three relative pathway perturbed by ISL, and mitogen-activated protein kinase (MAPK) signaling pathway was the most important pathway in RA treatment. In the analysis of hierarchical clustering of DEGs, we discovered 72 DEGs involved in the process of drug action. We thought Cited1, Tgm2, Xaf1, Cd59a, Fbxo2, Adh7 may have critical role in the differentiation of melanoma. The evidence displayed herein confirms the critical role of reactive oxygen species (ROS) in melanoma pathobiology and provides evidence for future targets in the

  7. Inhibitory Effects of Adlay Extract on Melanin Production and Cellular Oxygen Stress in B16F10 Melanoma Cells

    PubMed Central

    Huang, Huey-Chun; Hsieh, Wan-Yu; Niu, Yu-Lin; Chang, Tsong-Min

    2014-01-01

    The aim of this study was to determine the effects of adlay extract on melanin production and the antioxidant characteristics of the extract. The seeds were extracted by the supercritical fluid CO2 extraction (SFE) method. The effect of adlay extract on melanin production was evaluated using mushroom tyrosinase activity assay, intracellular tyrosinase activity, antioxidant properties and melanin content. Those assays were performed spectrophotometrically. In addition, the expression of melanogenesis-related proteins was determined by western blotting. The results revealed that the adlay extract suppressed intracellular tyrosinase activity and decreased the amount of melanin in B16F10 cells. The adlay extract decreased the expression of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase related protein-1 (TRP-1) and tyrosinase related protein-2 (TRP-2). The extract also exhibited antioxidant characteristics such as free radical scavenging capacity and reducing power. It effectively decreased intracellular reactive oxygen species (ROS) levels in B16F10 cells. We concluded that the adlay extract inhibits melanin production by down-regulation of MITF, tyrosinase, TRP-1 and TRP-2. The antioxidant properties of the extract may also contribute to the inhibition of melanogenesis. The adlay extract can therefore be applied as an inhibitor of melanogenesis and could also act as a natural antioxidant in skin care products. PMID:25244016

  8. Inhibition of melanogenesis by β-caryophyllene from lime mint essential oil in mouse B16 melanoma cells.

    PubMed

    Yang, C-H; Huang, Y-C; Tsai, M-L; Cheng, C-Y; Liu, L-L; Yen, Y-W; Chen, W-L

    2015-10-01

    Volatile essential oils of mint species are used for cosmetics and in skin care products. In this study, we evaluated the main chemical components of the lime mint and the anti-melanogenic properties of its main components. The essential oil was analysed by gas chromatography-mass spectrometry (GC/MS). The anti-melanogenic effects of mint essential oil and β-caryophyllene were investigated in B16F10 murine melanoma cells. The main components of lime mint essential oil were found to be D-limonene (41.10%), D-carvone (8.58%), δ-selinene (6.73%) and β-caryophyllene (6.24%). The lime mint essential oil reduced melanin production in a dose-dependent manner in murine B16F10 cells. β-Caryophyllene, one of the main compounds in lime mint essential oil, could reduce melanogenesis by down-regulating the expression of MITF, TRP-1, TRP-2 and tyrosinase, resulting in a decrease in melanin content decrease. These results reveal that lime mint essential oil and β-caryophyllene are considered to be valuable as potential skin-whitening agents. © 2015 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  9. Antitumoral effect of IL-12 gene transfected via liposomes into B16F0 cells.

    PubMed

    Speroni, Lucía; Gasparri, Julieta; de los A Bustuoabad, Victoria; Chiaramoni, Nadia S; Smagur, Andrzej; Szala, Stanisław; Taira, María C; del V Alonso, Silvia

    2009-01-01

    Murine melanoma B16F0 cells were transfected with SA:DPPC:DOPE (2:1:1 molar ratio) liposomes associated with a plasmid encoding murine IL-12. Stearylamine, a cationic lipid, showed a greater transfection efficiency compared to DOTAP-containing liposomes. The lipid:DNA ratio was 2:1 (w/w). Control groups were mock transfected or transfected with an empty plasmid (pNeo). pNeo or IL-12 transfected cells and controls were inoculated intradermically into the dorsal region of the foot or the lateral flank of C57BL6 mice. Results showed that IL-12 expression had a marked effect on in vivo growth of B16 melanoma tumors developed in both anatomic sites, significantly retarding their growth and prolonging host survival.

  10. Inhibitory components from the buds of clove (Syzygium aromaticum) on melanin formation in B16 melanoma cells.

    PubMed

    Arung, Enos Tangke; Matsubara, Eri; Kusuma, Irawan Wijaya; Sukaton, Edi; Shimizu, Kuniyoshi; Kondo, Ryuichiro

    2011-03-01

    In the course to find a new whitening agent, we evaluated the methanol extract from bud of clove (Syzygium aromaticum) on melanin formation in B16 melanoma cells. Eugenol and eugenol acetate were isolated as the active compounds and showed melanin inhibition of 60% and 40% in B16 melanoma cell with less cytotoxicity at the concentration of 100 and 200 μg/mL, respectively. Furthermore, an essential oil prepared from the bud of clove, which contain eugenol and eugenol acetate as dominant components, showed melanin inhibition of 50% and 80% in B16 melanoma cells at the concentration of 100 and 200 μg/mL, respectively. Copyright © 2010 Elsevier B.V. All rights reserved.

  11. Characterization in vitro and in vivo of progressively adriamycin-resistant B16-BL6 mouse melanoma cells.

    PubMed

    Ganapathi, R; Grabowski, D; Schmidt, H; Bell, D; Melia, M

    1987-07-01

    Adriamycin (ADR)-resistant sublines of B16-BL6 mouse melanoma selected by exposure to increasing concentrations of ADR were characterized in vitro for growth properties and in vivo for tumorigenicity and pulmonary metastases. The progressively resistant sublines adapted to grow in the presence of 0.025, 0.05, 0.1, and 0.25 microgram/ml ADR in monolayer culture were found to be 5-, 10-, 20-, and 40-fold ADR-resistant, respectively, compared to the parental sensitive cells, using a soft-agar colony assay and continuous ADR treatment for 7 days. The doubling time in monolayer culture of the parent sensitive and progressively ADR-resistant sublines of B16-BL6 melanoma cells was approximately 16-18 h. Although the colony-forming efficiency in soft agar of parental sensitive cells was only 0.5-4%, the 5-, 10-, 20-, and 40-fold ADR-resistant sublines had colony-forming efficiencies of 15, 20, 30, and 77%, respectively. Tumorigenicity in C57BL/6 mice of progressively ADR-resistant sublines was similar to parental sensitive cells following s.c. and i.p. implantation of 10(5)-10(6) tumor cells. Experimental pulmonary metastases were significantly lower in ADR-resistant sublines with progressive resistance. Additionally, unlike the parental sensitive and 5-fold ADR-resistant B16-BL6 cells, the 10-, 20-, and 40-fold ADR-resistant sublines were spontaneously nonmetastatic. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical detection of P-glycoprotein revealed the presence of a Mr 170,000 plasma membrane glycoprotein in the 40-fold ADR-resistant subline and its counterpart maintained for 1 year in ADR-free medium. Results from this study suggest that progressively ADR-resistant B16-BL6 mouse melanoma cells selected in vitro demonstrate a marked increase in colony formation in soft agar and a decrease in the ability to produce pulmonary metastases, without alterations in tumorigenicity.

  12. MR angiogenesis imaging with Robo4- vs. αVβ3-targeted nanoparticles in a B16/F10 mouse melanoma model

    PubMed Central

    Boles, Kent S.; Schmieder, Anne H.; Koch, Alexander W.; Carano, Richard A. D.; Wu, Yan; Caruthers, Shelton D.; Tong, Raymond K.; Stawicki, Scott; Hu, Grace; Scott, Michael J.; Zhang, Huiying; Reynolds, Benton A.; Wickline, Samuel A.; Lanza, Gregory M.

    2010-01-01

    The primary objective of this study was to utilize MR molecular imaging to compare the 3-dimensional spatial distribution of Robo4 and αVβ3-integrin as biosignatures of angiogenesis, in a rapidly growing, syngeneic tumor. B16-F10 melanoma-bearing mice were imaged with magnetic resonance (MR; 3.0 T) 11 d postimplantation before and after intravenous administration of either Robo4- or αVβ3-targeted paramagnetic nanoparticles. The percentage of MR signal-enhanced voxels throughout the tumor volume was low and increased in animals receiving αVβ3- and Robo4-targeted nanoparticles. Neovascular signal enhancement was predominantly associated with the tumor periphery (i.e., outer 50% of volume). Microscopic examination of tumors coexposed to the Robo4- and αVβ3-targeted nanoparticles corroborated the MR angiogenesis mapping results and further revealed that Robo4 expression generally colocalized with αVβ3-integrin. Robo4- and αVβ3-targeted nanoparticles were compared to irrelevant or nontargeted control groups in all modalities. These results suggest that αVβ3-integrin and Robo4 are useful biomarkers for noninvasive MR molecular imaging in syngeneic mouse tumors, but αVβ3-integrin expression was more detectable by MR at 3.0 T than Robo4. Noninvasive, neovascular assessments of the MR signal of Robo4, particularly combined with αVβ3-integrin expression, may help define tumor character prior to and following cancer therapy.—Boles, K. S., Schmieder, A. H., Koch, A. W., Carano, R. A. D., Wu, Y., Caruthers, S. D., Tong, R. K., Stawicki, S., Hu, G., Scott, M. J., Zhang, H., Reynolds, B. A., Wickline, S. A., and Lanza, G. M. MR angiogenesis imaging with Robo4- vs. αVβ3-targeted nanoparticles in a B16/F10 mouse melanoma model. PMID:20585027

  13. Vitamin C down-regulates VEGF production in B16F10 murine melanoma cells via the suppression of p42/44 MAPK activation.

    PubMed

    Kim, Ha Na; Kim, Hyemin; Kong, Joo Myung; Bae, Seyeon; Kim, Yong Sung; Lee, Naeun; Cho, Byung Joo; Lee, Seung Koo; Kim, Hang-Rae; Hwang, Young-il; Kang, Jae Seung; Lee, Wang Jae

    2011-03-01

    It is known that vitamin C induces apoptosis in several kinds of tumor cells, but its effect on the regulation of the angiogenic process of tumors is not completely studied. Vascular endothelial growth factor (VEGF) is the most well-known angiogenic factor, and it has a potent function as a stimulator of endothelial survival, migration, as well as vascular permeability. Therefore, we have investigated whether vitamin C can regulate the angiogenic process through the modulation of VEGF production from B16F10 melanoma cells. VEGF mRNA expression and VEGF production at protein levels were suppressed by vitamin C. In addition, we found that vitamin C suppressed the expression of cyclooxygenase (COX)-2 and that decreased VEGF production by vitamin C was also restored by the administration of prostaglandin E2 which is a product of COX-2. These results suggest that vitamin C suppresses VEGF expression via the regulation of COX-2 expression. Mitogen-activated protein kinases are generally known as key mediators in the signaling pathway for VEGF production. In the presence of vitamin C, the activation of p42/44 MAPK was completely inhibited. Taken together, our data suggest that vitamin C can down-regulate VEGF production via the modulation of COX-2 expression and that p42/44 MAPK acts as an important signaling mediator in this process. Copyright © 2010 Wiley-Liss, Inc.

  14. Ssanghwa-tang, an oriental herbal cocktail, exerts anti-melanogenic activity by suppression of the p38 MAPK and PKA signaling pathways in B16F10 cells

    PubMed Central

    2013-01-01

    Background Ssanghwa-tang (SHT) is a widely used medication for the treatment of fatigue, pain, inflammation, hypothermia, erectile dysfunction, cancer, and osteoporosis in Asia, however, role of SHT on the melanin synthesis has not been checked previously. Thus, the present study was designed to determine the effect of SHT on α-melanocyte stimulating hormone (α-MSH)-induced melanogensis and its mechanisms of action in murine B16F10 melanoma cells. Method Cellular melanin content and tyrosinase activity in murine B16F10 melanoma cells were determined after α-MSH stimulation with or without pre-treatment of SHT at the concentration of 250 and 500 μg/ml. Expression level of tyrosinase, tyrosinase-related protein 1 (TRP-1), TRP-2, microphthalmia-associated transcription factor (MITF), and activation of c-AMP-dependent protein kinase (PKA), c-AMP-related element binding protein (CREB), and mitogen-activated protein kinases (MAPKs) were examined by Western blot analysis. Results SHT significantly inhibited α-MSH-induced melanin synthesis and tyrosinase activity, and also decreased α-MSH-induced expression of MITF, tyrosinase, and TRP-1. In addition, SHT remarkably suppressed tyrosinase, CRE, and MITF luciferase reporter activity in a resting state as well as in α-MSH-stimulating condition. Phosphorylation of p38 MAPK by α-MSH stimulation was efficiently blocked by SHT pre-treatment. Moreover, SHT as an herbal cocktail showed synergistic anti-melanogenic effect compared with that of each single constituent herb. Conclusion SHT efficiently inhibited c-AMP-induced melanin synthesis in B16F10 cells via suppression of PKA and p38 MAPK signaling pathways and subsequently decreased the level of CREB phosphorylation, MITF, and melanogenic enzymes. These results indicate that SHT may be useful as herbal medicine for treating hyperpigmentation and cosmetics as a skin-whitening agent. PMID:23981281

  15. Silencing of diphthamide synthesis 3 (Dph3) reduces metastasis of murine melanoma.

    PubMed

    Wang, Lei; Shi, Yu; Ju, Peijun; Liu, Rui; Yeo, Siok Ping; Xia, Yinyan; Owlanj, Hamed; Feng, Zhiwei

    2012-01-01

    Melanoma is the most dangerous skin cancer due to its highly metastatic potential and resistance to chemotherapy. Currently, there is no effective treatment for melanoma once it is progressed to metastatic stage. Therefore, further study to elucidate the molecular mechanism underlying the metastasis of melanoma cells is urgently required for the improvement of melanoma treatment. In the present study, we found that diphthamide synthesis 3 (Dph3) is involved in the metastasis of B16F10 murine melanoma cells by insertional mutagenesis. We demonstrated that Dph3 disruption impairs the migration of B16F10 murine melanoma cells. The requirement of Dph3 in the migration of melanoma cells was further confirmed by gene silencing with siRNA in vitro. In corresponding to this result, overexpression of Dph3 significantly promoted the migratory ability of B16F10 and B16F0 melanoma cells. Moreover, down regulation of Dph3 expression in B16F10 melanoma cells strikingly inhibits their cellular invasion and metastasis in vivo. Finally, we found that Dph3 promotes melanoma migration and invasion through the AKT signaling pathway. To conclude, our findings suggest a novel mechanism underlying the metastasis of melanoma cells which might serve as a new intervention target for the treatment of melanoma.

  16. Inhibition of pulmonary metastasis of melanoma b16fo cells in C57BL/6 mice by a nutrient mixture consisting of ascorbic Acid, lysine, proline, arginine, and green tea extract.

    PubMed

    Roomi, M Waheed; Roomi, Nusrath; Ivanov, Vadim; Kalinovsky, Tatiana; Niedzwiecki, Aleksandra; Rath, Matthias

    2006-01-01

    The authors investigated the effect of a nutrient mixture (NM) on lung metastasis by B16F0 melanoma cells in C57BL/6 female mice. Mice were divided into equal groups (1 to 6) and injected via tail vein with B16F0 cells (groups 1 to 4), B16FO cells pretreated with NM (group 5), or saline (group 6). Groups 1, 3, 4, 5, and 6 were fed the control diet and group 2 the 0.5% NM supplemented diet. Groups 3 and 4 received NM intraperitoneally (IP) and intravenously (IV), respectively. Two weeks later, pulmonary metastatic colonies were counted. Pulmonary colonization was reduced by 63% in mice supplemented with NM diet, by 86% in mice receiving NM by IP and IV injections, and completely inhibited in mice injected with melanoma cells pretreated with NM. These results show that NM is effective in inhibiting the metastasis of B16FO melanoma cells.

  17. Prunus mume extract exerts antioxidant activities and suppressive effect of melanogenesis under the stimulation by alpha-melanocyte stimulating hormone in B16-F10 melanoma cells.

    PubMed

    Pi, KyungBae; Lee, KiBeom

    2017-10-01

    In the current study, we examined the antioxidant and skin-whitening properties of Prunus mume extract (PME). The ability of PME to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals was investigated in vitro. At a concentration of 1000 μg/mL, PME neutralized >45% free radical activity. Cell viability assessment with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that at concentrations <1500 μg/mL, PME does not exert cytotoxic effects on murine B16 melanoma (B16) cells. Morphological analysis disclosed that melanin production is inhibited in B16 cells treated with 250 nM α-melanocyte-stimulating hormone (α-MSH) and PME. We conclude that fruit extracts of P. mume exert a skin-whitening effect by inhibiting melanin production via regulation of melanogenesis-associated protein expression in melanocytes.

  18. Silencing of Diphthamide Synthesis 3 (Dph3) Reduces Metastasis of Murine Melanoma

    PubMed Central

    Wang, Lei; Shi, Yu; Ju, Peijun; Liu, Rui; Yeo, Siok Ping; Xia, Yinyan; Owlanj, Hamed; Feng, Zhiwei

    2012-01-01

    Melanoma is the most dangerous skin cancer due to its highly metastatic potential and resistance to chemotherapy. Currently, there is no effective treatment for melanoma once it is progressed to metastatic stage. Therefore, further study to elucidate the molecular mechanism underlying the metastasis of melanoma cells is urgently required for the improvement of melanoma treatment. In the present study, we found that diphthamide synthesis 3 (Dph3) is involved in the metastasis of B16F10 murine melanoma cells by insertional mutagenesis. We demonstrated that Dph3 disruption impairs the migration of B16F10 murine melanoma cells. The requirement of Dph3 in the migration of melanoma cells was further confirmed by gene silencing with siRNA in vitro. In corresponding to this result, overexpression of Dph3 significantly promoted the migratory ability of B16F10 and B16F0 melanoma cells. Moreover, down regulation of Dph3 expression in B16F10 melanoma cells strikingly inhibits their cellular invasion and metastasis in vivo. Finally, we found that Dph3 promotes melanoma migration and invasion through the AKT signaling pathway. To conclude, our findings suggest a novel mechanism underlying the metastasis of melanoma cells which might serve as a new intervention target for the treatment of melanoma. PMID:23185508

  19. Pim-3 enhances melanoma cell migration and invasion by promoting STAT3 phosphorylation.

    PubMed

    Liu, Jing; Qu, Xinyu; Shao, Liwei; Hu, Yuan; Yu, Xin; Lan, Peixiang; Guo, Qie; Han, Qiuju; Zhang, Jian; Zhang, Cai

    2018-03-04

    Melanoma is the deadliest form of commonly encountered skin cancer, and has fast propagating and highly invasive characteristics. Pim-3, a highly expressed oncogene in melanoma, is a highly conserved serine/threonine kinase with various biological activities, such as proliferation-accelerating and anti-apoptosis effects on cancer progression. However, whether Pim-3 regulates melanoma metastasis has not been determined. Here, we constructed a Pim-3-silencing short hairpin RNA (sh-Pim-3), a TLR7-stimulating ssRNA and a dual-function vector containing a sh-Pim-3 and a ssRNA, and transfected them into the B16F10 melanoma cell line to investigate the effects of Pim-3 on migration and invasion in melanoma. We found that sh-Pim-3 inhibited B16F10 cell migration and invasion in vitro. In a tumor-bearing mouse model, sh-Pim-3 significantly downregulated pulmonary metastasis of B16F10 melanoma cell in vivo. Mechanistically, sh-Pim-3 inhibited metastasis by regulating the expression of genes related to epithelial-mesenchymal transition (EMT). Further study revealed that by promoting the phosphorylation of STAT3 (signal transducer and activator of transcription 3), Pim-3 induced the expression of Slug, Snail, and ZEB1, which enhanced EMT-related changes and induced melanoma migration and invasion. Our study suggests that Pim-3 is a potential effective target for melanoma therapy.

  20. Noscapinoids bearing silver nanocrystals augmented drug delivery, cytotoxicity, apoptosis and cellular uptake in B16F1, mouse melanoma skin cancer cells.

    PubMed

    Soni, Naina; Jyoti, Kiran; Jain, Upendra Kumar; Katyal, Anju; Chandra, Ramesh; Madan, Jitender

    2017-06-01

    Noscapine (Nos) and reduced brominated analogue of noscapine (Red-Br-Nos) prevent cellular proliferation and induce apoptosis in cancer cells either alone or in combination with other chemotherapeutic drugs. However, owing to poor physicochemical properties, Nos and Red-Br-Nos have demonstrated their anticancer activity at higher and multiple doses. Therefore, in present investigation, silver nanocrystals of noscapinoids (Nos-Ag 2+ nanocrystals and Red-Br-Nos-Ag 2+ nanocrystals) were customized to augment drug delivery, cytotoxicity, apoptosis and cellular uptake in B16F1 mouse melanoma cancer cells. Nos-Ag 2+ nanocrystals and Red-Br-Nos-Ag 2+ nanocrystals were prepared separately by precipitation method. The mean particle size of Nos-Ag 2+ nanocrystals was measured to be 25.33±3.52nm, insignificantly (P>0.05) different from 27.43±4.51nm of Red-Br-Nos-Ag 2+ nanocrystals. Furthermore, zeta-potential of Nos-Ag 2+ nanocrystals was determined to be -25.3±3.11mV significantly (P<0.05) different from -15.2±3.33mV of Red-Br-Nos-Ag 2+ nanocrystals. The shape of tailored nanocrystals was slightly spherical and or irregular in shape. The architecture of Nos-Ag 2+ nanocrystals and Red-Br-Nos-Ag 2+ nanocrystals was crystalline in nature. FT-IR spectroscopy evinced the successful interaction of Ag 2+ nanocrystals with Nos and Red-Br-Nos, respectively. The superior therapeutic efficacy of tailored nanocrystals was measured in terms of enhanced cytotoxicity, apoptosis and cellular uptake. The Nos-Ag 2+ nanocrystals and Red-Br-Nos-Ag 2+ nanocrystals exhibited an IC 50 of 16.6μM and 6.5μM, significantly (P<0.05) lower than 38.5μM of Nos and 10.3μM of Red-Br-Nos, respectively. Finally, cellular morphological alterations in B16F1 cells upon internalization of Nos-Ag 2+ nanocrystals and Red-Br-Nos-Ag 2+ nanocrystals provided the evidences for accumulation within membrane-bound cytoplasmic vacuoles and in enlarged lysosomes and thus triggered mitochondria mediated apoptosis via

  1. Proteomic analyses for profiling regulated proteins/enzymes by Fucus vesiculosus fucoidan in B16 melanoma cells: A combination of enzyme kinetics functional study.

    PubMed

    Wang, Zhi-Jiang; Zheng, Li; Yang, Jun-Mo; Kang, Yani; Park, Yong-Doo

    2018-06-01

    Fucoidans are complex sulfated polysaccharides that have a wide range of biological activities. Previously, we reported the various effects of Fucus vesiculosus fucoidan on tyrosinase and B16 melanoma cells. In this study, to identify fucoidan-targeted proteins in B16 melanoma cells, we performed a proteomics study and integrated enzyme kinetics. We detected 19 candidate proteins dysregulated by fucoidan treatment. Among the probed proteins, the enzyme kinetics of two candidate enzymes, namely lactate dehydrogenase (LDH) as an upregulated protein and superoxide dismutase (SOD) as a downregulated enzyme, were determined. The enzyme kinetics results showed that Fucus vesiculosus fucoidan significantly inhibited LDH catalytic function while it did not affect SOD activity even at a high dose, while only slightly decreased activity (up to 10%) at a low dose. Based on our previous and present observations, fucoidan could inhibit B16 melanoma cells growth via regulating proteins/enzymes expression levels such as LDH and SOD known as cell survival biomarkers. Interestingly, both expression level and enzyme catalytic activity of LDH were regulated by fucoidan, which could directly induce the apoptotic effect on B16 melanoma cells along with SOD downregulation. This study highlights how combining proteomics with enzyme kinetics can yield valuable insights into fucoidan targets. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Induction of cell-mediated immunity against B16-BL6 melanoma in mice vaccinated with cells modified by hydrostatic pressure and chemical crosslinking.

    PubMed

    Eisenthal, A; Ramakrishna, V; Skornick, Y; Shinitzky, M

    1993-05-01

    In the preceding paper we have demonstrated an increase in presentation of both major histocompatibility complex antigens (MHC) and a tumor-associated antigen of the weakly immunogenic B16 melanoma by a straight-forward technique. The method consists in modulating the tumor cell membrane by hydrostatic pressure and simultaneous chemical crosslinking of the cell-surface proteins. In B16-BL6 melanoma, the induced antigenic modulation was found to persist for over 48 h, which permitted the evaluation of the ability of modified B16-BL6 cells to induce immunity against unmodified B16-BL6 cells. In the present study, we have shown that a significant systemic immunity was induced only in mice that were immunized with modified B16-BL6 melanoma cells, whereas immunization with unmodified B16-BL6 cells had only a marginal effect when compared to the results in control sham-immunized mice. The induced immunity was specific since a single immunization affected the growth of B16-BL6 tumors but had no effect on MCA 106, an antigenically unrelated tumor. The addition of interleukin-2 to the immunization regimen had no effect on the antitumor responses induced by the modified B16-BL6 cells. The cell-mediated immunity conferred by immunization with treated B16-BL6 cells was confirmed in experiments in vitro where splenocytes from immunized mice could be sensitized to proliferate by the presence of B16-BL6 cells. In addition, the altered antigenicity of these melanoma cells appeared to correlate with their increased susceptibility to specific effectors. Thus, 51Cr-labeled B16-BL6 target cells, modified by pressure and crosslinking, in comparison to control labeled target cells, were lysed in much greater numbers by effectors such as lymphokine-activated killer cells and allogeneic cytotoxic lymphocytes (anti-H-2b), while such cells remained resistant to lysis by natural killer cells. Our findings indicate that the physical and chemical modifications of the tumor cells that are

  3. Selective 2-( sup 18 F)fluorodopa uptake for melanogenesis in murine metastatic melanomas

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ishiwata, K.; Kubota, K.; Kubota, R.

    The relationship between 3,4-dihydroxy-2-({sup 18}F)fluoro-L-phenylalanine (2-({sup 18}F)FDOPA) uptake and melanogenesis was studied using mice bearing two B16 melanomas: B16-F1 has a higher melanin synthesis ability and a slower growing rate than the higher metastatic B16-F10. A significantly higher 2-({sup 18}F)FDOPA uptake by B16-F1 than by B16-F10 and a reverse relationship for the uptake of ({sup 14}C) 2-deoxy-2-fluoro-D-glucose and ({sup 3}H)thymidine were observed 1 hr postinjection. F1-to-F10 ratios of both the 2-({sup 18}F)FDOPA uptake and the acid-insoluble radioactivity increased to about 5 at 6 hr, which paralleled the melanin content. FM3A mammary carcinoma showed a 2-({sup 18}F)FDOPA uptake similar to themore » B16-F10 but without the acid-insoluble radioactivity. With D,L-DOPA loading, a 55% decreased uptake by FM3A 1 hr postinjection was significantly greater than the 20% reduction in both melanomas. O-Methylated 2-({sup 18}F)FDOPA was a predominant acid-soluble metabolite in all tumors. Whole-body autoradiography discriminated the two melanomas clearly. 2-({sup 18}F)FDOPA may be a promising tracer for the selective imaging of melanogenesis.« less

  4. Cytotoxicity of citral against melanoma cells: The involvement of oxidative stress generation and cell growth protein reduction.

    PubMed

    Sanches, Larissa Juliani; Marinello, Poliana Camila; Panis, Carolina; Fagundes, Tatiane Renata; Morgado-Díaz, José Andrés; de-Freitas-Junior, Julio Cesar Madureira; Cecchini, Rubens; Cecchini, Alessandra Lourenço; Luiz, Rodrigo Cabral

    2017-03-01

    Citral is a natural compound that has shown cytotoxic and antiproliferative effects on breast and hematopoietic cancer cells; however, there are few studies on melanoma cells. Oxidative stress is known to be involved in all stages of melanoma development and is able to modulate intracellular pathways related to cellular proliferation and death. In this study, we hypothesize that citral exerts its cytotoxic effect on melanoma cells by the modulation of cellular oxidative status and/or intracellular signaling. To test this hypothesis, we investigated the antiproliferative and cytotoxic effects of citral on B16F10 murine melanoma cells evaluating its effects on cellular oxidative stress, DNA damage, cell death, and important signaling pathways, as these pathways, namely, extracellular signal-regulated kinases 1/2 (ERK1/2), AKT, and phosphatidylinositol-3 kinase, are involved in cell proliferation and differentiation. The p53 and nuclear factor kappa B were also investigated due to their ability to respond to intracellular stress. We observed that citral exerted antiproliferative and cytotoxic effects in B16F10; induced oxidative stress, DNA lesions, and p53 nuclear translocation; and reduced nitric oxide levels and nuclear factor kappa B, ERK1/2, and AKT. To investigate citral specificity, we used non-neoplastic human and murine cells, HaCaT (human skin keratinocytes) and NIH-3T3 cells (murine fibroblasts), and observed that although citral effects were not specific for cancer cells, non-neoplastic cells were more resistant to citral than B16F10. These findings highlight the potential clinical utility of citral in melanoma, with a mechanism of action involving the oxidative stress generation, nitric oxide depletion, and interference in signaling pathways related to cell proliferation.

  5. E-cadherin determines Caveolin-1 tumor suppression or metastasis enhancing function in melanoma cells

    PubMed Central

    Lobos-González, L; Aguilar, L; Diaz, J; Diaz, N; Urra, H; Torres, V; Silva, V; Fitzpatrick, C; Lladser, A; Hoek, K.S.; Leyton, L; Quest, AFG

    2013-01-01

    SUMMARY The role of caveolin-1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E-cadherin in CAV1-dependent tumor suppression. Here we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E-cadherin, to unravel how CAV1 affects tumor growth and metastasis, and to assess how co-expression of E-cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10(cav-1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E-cadherin expression in B16F10(E-cad) cells reduces subcutaneous tumor formation, and lung metastasis when intravenously injected. Importantly, co-expression of CAV1 and E-cadherin in B16F10(cav1/E-cad) cells abolishes tumor formation, lung metastasis, increased Rac-1 activity and cell migration observed with B16F10(cav-1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac-1 activation in these cells. PMID:23470013

  6. Effects of genistein, a soybean-derived isoflavone, on proliferation and differentiation of B16-BL6 mouse melanoma cells.

    PubMed

    Yan, C H; Chen, X G; Li, Y; Han, R

    1999-01-01

    Genistein, a soybean-derived isoflavone, may contribute to the lower cancer incidence in South Asian countries. In this study, the effects and molecular mechanisms of genistein on growth and differentiation of B16-BL6 mouse melanoma cells were investigated. Genistein suppressed the growth of these melanoma cells. The IC50 value is 15.5 microM. On the other hand, genistein induced the changes of cell shape and cytoskeletal network. The cytoskeletal filaments were induced to form a bundle along the direction of elongation of the cells. Moreover, tyrosine phosphorylation levels of cytoskeleton-associated proteins decreased after the cells were exposed to 20 or 30 microM of genistein for 3 days. All these morphological and molecular changes were accompanied by appearance of the differentiated phenotypes. Genistein induced the increase of cellular melanin content, enhancement of tyrosinase activity, and decrease of colonization potentials in soft agar in a time-dependent and dose-dependent manner. The effective concentration was no more than 10 microM after 3 days' exposure. The tumorigenic potentials of B16-BL6 cells in C57BL/6 mouse also decreased after exposure to 20 or 30 microM of genistein for 3 days. When expressions of tumor-related genes were investigated in the differentiation-induced cells, the content of P53 dramatically increased while that of c-Myc protein decreased. Therefore, due to its ability to induce cellular and molecular changes, genistein suppressed the growth and induced differentiated phenotypes in B16-BL6 melanoma cells.

  7. Mechanism of the melanogenesis stimulation activity of (-)-cubebin in murine B16 melanoma cells.

    PubMed

    Hirata, Noriko; Naruto, Shunsuke; Ohguchi, Kenji; Akao, Yukihiro; Nozawa, Yoshinori; Iinuma, Munekazu; Matsuda, Hideaki

    2007-07-15

    (-)-Cubebin showed a melanogenesis stimulation activity in a concentration-dependent manner in murine B16 melanoma cells without any significant effects on cell proliferation. Tyrosinase activity was increased at 24-72 h after addition of cubebin to B16 cells, and then intracellular melanin amount was increased at 48-96 h after the treatment. The expression levels of tyrosinase were time-dependently enhanced after the treatment with cubebin. At the same time, the expression levels of tyrosinase mRNA were also increased after addition of cubebin. Furthermore Western blot analysis revealed that cubebin elevated the level of phosphorylation of p38 mitogen-activated protein kinase (MAPK). SB203580, a selective inhibitor of p38 MAPK, completely blocked cubebin-induced expression of tyrosinase mRNA in B16 cells. These results suggested that cubebin increased melanogenesis in B16 cells through the enhancement of tyrosinase expression mediated by activation of p38 MAPK.

  8. Differential biological effects of dehydroepiandrosterone (DHEA) between mouse (B16F10) and human melanoma (BLM) cell lines.

    PubMed

    Joshi, Kumud; Hassan, Sherif S; Ramaraj, Pandurangan

    2017-01-01

    Dehydroepiandrosterone (DHEA) is a weak androgen and had been shown to have anti-cancer, anti-adipogenic and anti-inflammatory effects on mouse and other rodent models, but not on humans, suggesting a systemic level difference between mouse and human. Our previous study on DHEA biological functions involving a variety of cell lines, suggested that the functional differences between mouse and human existed even at the cellular level. Hence, using mouse and human melanoma cell models, in-vitro effects of DHEA on cell growth, mechanism of cell death and mechanism of DHEA action were studied. Results indicated a differential biological effects of DHEA between mouse and human melanoma cell lines. These in-vitro studies also suggested that the differential biological effects observed between these two cell lines could be due to the difference in the way DHEA was processed or metabolized inside the cell.

  9. Inhibitory Effects of (2'R)-2',3'-dihydro-2'-(1-hydroxy-1-methylethyl)-2,6'-bibenzofuran-6,4'-diol on Mushroom Tyrosinase and Melanogenesis in B16-F10 Melanoma Cells.

    PubMed

    Zhu, Jing-Jie; Yan, Gui-Rui; Xu, Zhi-Jian; Hu, Xiao; Wang, Gai-Hong; Wang, Ting; Zhu, Wei-Liang; Hou, Ai-Jun; Wang, He-Yao

    2015-07-01

    (2'R)-2',3'-Dihydro-2'-(1-hydroxy-1-methylethyl)-2,6'-bibenzofuran-6,4'-diol (DHMB) is a natural compound extracted from Morus notabilis. It was found that DHMB acts as a competitive inhibitor against mushroom tyrosinase with a Ki value of 14.77 μM. Docking results further indicated that it could form strong interactions with one copper ion with a distance of 2.7 Å, suggesting the mechanism of inhibition might be due to chelating copper ions in the active site. Furthermore, melanin production in B16-F10 murine melanoma cells was significantly inhibited by DHMB in a concentration-dependent manner without cytotoxicity. The results of western blotting also showed that DHMB decreased 3-isobuty-1-methxlzanthine-induced mature tyrosinase expression. Taken together, these findings indicated that DHMB may be a new promising pigmentation-altering agent for agriculture, cosmetic, and therapeutic applications. Copyright © 2015 John Wiley & Sons, Ltd.

  10. The kin17 Protein in Murine Melanoma Cells

    PubMed Central

    Ramos, Anelise C.; Gaspar, Vanessa P.; Kelmer, Sabrina M. G.; Sellani, Tarciso A.; Batista, Ana G. U.; De Lima Neto, Quirino A.; Rodrigues, Elaine G.; Fernandez, Maria A.

    2015-01-01

    kin17 has been described as a protein involved in the processes of DNA replication initiation, DNA recombination, and DNA repair. kin17 has been studied as a potential molecular marker of breast cancer. This work reports the detection and localization of this protein in the murine melanoma cell line B16F10-Nex2 and in two derived subclones with different metastatic potential, B16-8HR and B16-10CR. Nuclear and chromatin-associated protein fractions were analyzed, and kin17 was detected in all fractions, with an elevated concentration observed in the chromatin-associated fraction of the clone with low metastatic potential, suggesting that the kin17 expression level could be a marker of melanoma. PMID:26610484

  11. Norartocarpetin from a folk medicine Artocarpus communis plays a melanogenesis inhibitor without cytotoxicity in B16F10 cell and skin irritation in mice

    PubMed Central

    2013-01-01

    Background Many natural products used in preventive medicine have also been developed as cosmeceutical ingredients in skin care products, such as Scutellaria baicalensis and Gardenia jasminoides. Norartocarpetin is one of the antioxidant and antityrosinase activity compound in Artocarpus communis; however, the cytotoxicity, skin irritation and antimelanogenesis mechanisms of norartocarpetin have not been investigated yet. Methods In the present study, cell viability in vitro and skin irritation in vivo are used to determine the safety of norartocarpetin. The melanogenesis inhibition of norartocarpetin was determined by cellular melanin content and tyrosinase in B16F10 melanoma cell. Moreover, we examined the related-melanogenesis protein by western blot analysis for elucidating the antimelanogenesis mechanism of norartocarpin. Results The result of the present study demonstrated that norartocarpetin not only present non-cytotoxic in B16F10 and human fibroblast cells but also non-skin irritation in mice. Moreover, our result also first found that norartocarpetin downregulated phospho-cAMP response element-binding (phospho-CREB) and microphthalmia-associated transcription factor (MITF) expression, which in turn decreased both synthesis of tyrosinases (TRP-1 and TRP-2) and cellular melanin content. This process is dependent on norartocarpetin phosphorylation by mitogen-activated protein kinases such as phospho-JNK and phospho-p38, and it results in decreased melanogenesis. Conclusion The present study suggests that norartocarpetin could be used as a whitening agent in medicine and/or cosmetic industry and need further clinical study. PMID:24325567

  12. Extracellular acidification by lactic acid suppresses glucose deprivation-induced cell death and autophagy in B16 melanoma cells.

    PubMed

    Matsuo, Taisuke; Sadzuka, Yasuyuki

    2018-02-19

    In solid tumors, cancer cells survive and proliferate under conditions of microenvironment stress such as poor nutrients and hypoxia due to inadequate vascularization. These stress conditions in turn activate autophagy, which is important for cancer cell survival. However, autophagy has a contrary effect of inducing cell death in cancer cells cultured in vitro under conditions of glucose deprivation. In this study, we hypothesized that supplementation of lactic acid serves as a means of cell survival under glucose-deprived conditions. At neutral pH, cell death of B16 murine melanoma cells by autophagy under glucose-deprived conditions was observed. However, supplementation of lactic acid suppressed cell death and autophagy in B16 melanoma cells when cultured in glucose-deprived conditions. Sodium lactate, which does not change extracellular pH, did not inhibit cell death, while HCl-adjusted acidic pH suppressed cell death under glucose-deprived conditions. These results suggested that an acidic pH is crucial for cell survival under glucose-deprived conditions. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Melanogenesis stimulation in murine B16 melanoma cells by Kava (Piper methysticum) rhizome extract and kavalactones.

    PubMed

    Matsuda, Hideaki; Hirata, Noriko; Kawaguchi, Yoshiko; Naruto, Shunsuke; Takata, Takanobu; Oyama, Masayoshi; Iinuma, Munekazu; Kubo, Michinori

    2006-04-01

    Melanogenesis stimulation activity of aqueous ethanolic extracts obtained from several different parts of five Piper species, namely Piper longum, P. kadsura, P. methysticum, P. betle, and P. cubeba, were examined by using cultured murine B16 melanoma cells. Among them, the extract of P. methysticum rhizome (Kava) showed potent stimulatory effect on melanogenesis as well as P. nigrum leaf extract. Activity-guided fractionation of Kava extract led to the isolation of two active kavalactones, yangonin (2) and 7,8-epoxyyangonin (5), along with three inactive kavalactones, 5,6-dehydrokawain (1), (+)-kawain (3) and (+)-methysticin (4), and a glucosylsterol, daucosterin (6). 7,8-Epoxyyangonin (5) showed a significant stimulatory effect on melanogenesis in B16 melanoma cells. Yangonin (2) exhibited a weak melanogenesis stimulation activity.

  14. Calcitonin gene-related peptide cooperates with substance P to inhibit melanogenesis and induces apoptosis of B16F10 cells.

    PubMed

    Zhou, Jia; Feng, Jun-Yi; Wang, Qian; Shang, Jing

    2015-07-01

    Skin is the largest organ in human body and works as biologically active barrier to provide critical preservation of body homeostasis. The skin is highly innervated by a plenitude of nerve fiber subpopulations, each carrying one or more neuronal mediators. Melanocyte itself also intimately contact with nerve fibers to form 'synaptic-like structure' and its functions may be directly regulated by the mediators contained in terminals of intra-epidermal nerve fibers. Clinical and biochemical studies have suggested that calcitonin gene-related peptide (CGRP) is involved in vitiligo skin. The present study was designed to investigate the effect of CGRP on epidermal melanocytes. After treatment with CGRP ranging from 0 to 500 ng/mL for 48 h, tyrosinase activity and melanogenesis were with little changes compared to treatment with medium only in B16F10 cells. Treatment with 500 ng/mL of CGRP cooperates with substance P (SP) (0.1-10 nM) to decrease tyrosinase activity and decrease melanin biosynthesis in B16F10 cells in a concentration-dependent manner. Furthermore, CGRP (8-37) antagonizes the synergistic effect of CGRP. The effect of CGRP on the cell apoptosis was examined. Treatments with 0-500 ng/mL of CGRP for 24 h, the expression levels of cleaved caspase-3, total caspase-3, cleaved caspase-9 and total caspase-9 were increased in a concentration-dependent manner. And 500 ng/mL of CGRP induced B16F10 cell apoptosis showed by TUNEL assay. In addition, Bax expression was up-regulated and Bcl-2 down-regulated in response to CGRP treatment. Hence, the Bax/Bcl-2 ratio was significantly increased. These in vitro observations indicate the pro-apoptotic impact of CGRP on B16F10 cell. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Meng; Long, Chaoqin; Yang, Guilan

    2016-03-11

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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ahmad, Israr; Muneer, Kashiff M.; Tamimi, Iman A.

    2013-07-01

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

  17. Mast cells promote melanoma colonization of lungs.

    PubMed

    Öhrvik, Helena; Grujic, Mirjana; Waern, Ida; Gustafson, Ann-Marie; Ernst, Nancy; Roers, Axel; Hartmann, Karin; Pejler, Gunnar

    2016-10-18

    Mast cells have been implicated in malignant processes, mainly through clinical correlative studies and by experiments performed using animals lacking mast cells due to defective c-kit signaling. However, mast cell-deficient mouse models based on c-kit defects have recently been questioned for their relevance. Here we addressed the effect of mast cells in a tumor setting by using transgenic Mcpt5-Cre+ R-DTA+ mice, in which the deficiency of mast cells is independent of c-kit defects. Melanoma cells (B16.F10) were administered either subcutaneously or intravenously into Mcpt5-Cre+ R-DTA+ mice or Mcpt5-Cre- R-DTA+ littermate controls, followed by the assessment of formed tumors. In the subcutaneous model, mast cells were abundant in the tumor stroma of control mice but were absent in Mcpt5-Cre+ R-DTA+ mice. However, the absence of mast cells did not affect tumor size. In contrast, after intravenous administration of B16.F10 cells, melanoma colonization of the lungs was markedly reduced in Mcpt5-Cre+ R-DTA+ vs. Mcpt5-Cre- R-DTA+ animals. Decreased melanoma colonization of the lungs in Mcpt5-Cre+ R-DTA+ animals was accompanied by increased inflammatory cell recruitment into the bronchoalveolar lavage fluid, suggesting that mast cells suppress inflammation in this setting. Further, qPCR analysis revealed significant alterations in the expression of Twist and E-cadherin in lungs of Mcpt5-Cre+ R-DTA+ vs. control Mcpt5-Cre- R-DTA+ animals, suggesting an impact of mast cells on epithelial-mesenchymal transition. In conclusion, this study reveals that mast cells promote melanoma colonization of the lung.

  18. CTLA-4 blockade plus adoptive T cell transfer promotes optimal melanoma immunity in mice

    PubMed Central

    Mahvi, David A.; Meyers, Justin V.; Tatar, Andrew J.; Contreras, Amanda; Suresh, M.; Leverson, Glen E.; Sen, Siddhartha; Cho, Clifford S.

    2014-01-01

    Immunotherapeutic approaches to the treatment of advanced melanoma have relied on strategies that augment the responsiveness of endogenous tumor-specific T cell populations (e.g., CTLA-4 blockade-mediated checkpoint inhibition) or introduce exogenously-prepared tumor-specific T cell populations (e.g., adoptive cell transfer). Although both approaches have shown considerable promise, response rates to these therapies remain suboptimal. We hypothesized that a combinatorial approach to immunotherapy using both CTLA-4 blockade and non-lymphodepletional adoptive cell transfer could offer additive therapeutic benefit. C57BL/6 mice were inoculated with syngeneic B16F10 melanoma tumors transfected to express low levels of the lymphocytic choriomeningitis virus peptide GP33 (B16GP33), and treated with no immunotherapy, CTLA-4 blockade, adoptive cell transfer, or combination immunotherapy of CTLA-4 blockade with adoptive cell transfer. Combination immunotherapy resulted in optimal control of B16GP33 melanoma tumors. Combination immunotherapy promoted a stronger local immune response reflected by enhanced tumor-infiltrating lymphocyte populations, as well as a stronger systemic immune responses reflected by more potent tumor antigen-specific T cell activity in splenocytes. In addition, whereas both CTLA-4 blockade and combination immunotherapy were able to promote long-term immunity against B16GP33 tumors, only combination immunotherapy was capable of promoting immunity against parental B16F10 tumors as well. Our findings suggest that a combinatorial approach using CTLA-4 blockade with non-lymphodepletional adoptive cell transfer may promote additive endogenous and exogenous T cell activities that enable greater therapeutic efficacy in the treatment of melanoma. PMID:25658614

  19. Anti-tumor angiogenesis effect of aminopeptidase inhibitor bestatin against B16-BL6 melanoma cells orthotopically implanted into syngeneic mice.

    PubMed

    Aozuka, Yasushi; Koizumi, Keiichi; Saitoh, Yurika; Ueda, Yasuji; Sakurai, Hiroaki; Saiki, Ikuo

    2004-12-08

    We investigated the effect of bestatin, an inhibitor of aminopeptidase N (APN)/CD13 and aminopeptidase B, on the angiogenesis induced by B16-BL6 melanoma cells. Oral administration of bestatin (100-200 mg/kg/day) was found to significantly inhibit the melanoma cell-induced angiogenesis in a mouse dorsal air sac assay. Additionally, anti-APN/CD13 mAb (WM15), which neutralizes the aminopeptidase activity in tumor cells, as well as bestatin inhibited the tube-like formation of human umbilical vein endothelial cells (HUVECs) in vitro. Furthermore, the intraperitoneal administration of bestatin (50-100 mg/kg/day) after the orthotopic implantation of B16-BL6 melanoma cells into mice reduced the number of vessels oriented towards the established primary tumor mass on the dorsal side of mice. These findings suggest that bestatin is an active anti-angiogenic agent that may inhibit tumor angiogenesis in vivo and tube-like formation of endothelial cells in vitro through its inhibition of APN/CD13 activity.

  20. Anti-tumor effect of Coriolus versicolor methanol extract against mouse B16 melanoma cells: in vitro and in vivo study.

    PubMed

    Harhaji, Lj; Mijatović, S; Maksimović-Ivanić, D; Stojanović, I; Momcilović, M; Maksimović, V; Tufegdzić, S; Marjanović, Z; Mostarica-Stojković, M; Vucinić, Z; Stosić-Grujicić, S

    2008-05-01

    Numerous studies have shown immunostimulatory and anti-tumor effects of water and standardized aqueous ethanol extracts derived from the medicinal mushroom, Coriolus versicolor, but the biological activity of methanol extracts has not been examined so far. In the present study we investigated the anti-tumor effect of C. versicolor methanol extract (which contains terpenoids and polyphenols) on B16 mouse melanoma cells both in vitro and in vivo. In vitro treatment of the cells with the methanol extract (25-1600 microg/ml) reduced melanoma cell viability in a dose-dependent manner. Furthermore, in the presence of the methanol extract (200 microg/ml, concentration IC(50)) the proliferation of B16 cells was arrested in the G(0)/G(1) phase of the cell cycle, followed by both apoptotic and secondary necrotic cell death. In vivo methanol extract treatment (i.p. 50 mg/kg, for 14 days) inhibited tumor growth in C57BL/6 mice inoculated with syngeneic B16 tumor cells. Moreover, peritoneal macrophages collected 21 days after tumor implantation from methanol extract-treated animals exerted stronger tumoristatic activity ex vivo than macrophages from control melanoma-bearing mice. Taken together, our results demonstrate that C. versicolor methanol extract exerts pronounced anti-melanoma activity, both directly through antiproliferative and cytotoxic effects on tumor cells and indirectly through promotion of macrophage anti-tumor activity.

  1. Tumor cytotoxicity by endothelial cells. Impairment of the mitochondrial system for glutathione uptake in mouse B16 melanoma cells that survive after in vitro interaction with the hepatic sinusoidal endothelium.

    PubMed

    Ortega, Angel L; Carretero, Julian; Obrador, Elena; Gambini, Juan; Asensi, Miguel; Rodilla, Vicente; Estrela, José M

    2003-04-18

    High GSH content associates with high metastatic activity in B16-F10 melanoma cells cultured to low density (LD B16M). GSH homeostasis was investigated in LD B16M cells that survive after adhesion to the hepatic sinusoidal endothelium (HSE). Invasive B16M (iB16M) cells were isolated using anti-Met-72 monoclonal antibodies and flow cytometry-coupled cell sorting. HSE-derived NO and H(2)O(2) caused GSH depletion and a decrease in gamma-glutamylcysteine synthetase activity in iB16M cells. Overexpression of gamma-glutamylcysteine synthetase heavy and light subunits led to a rapid recovery of cytosolic GSH, whereas mitochondrial GSH (mtGSH) further decreased during the first 18 h of culture. NO and H(2)O(2) damaged the mitochondrial system for GSH uptake (rates in iB16M were approximately 75% lower than in LD B16M cells). iB16M cells also showed a decreased activity of mitochondrial complexes II, III, and IV, less O(2) consumption, lower ATP levels, higher O(2) and H(2)O(2) production, and lower mitochondrial membrane potential. In vitro growing iB16M cells maintained high viability (>98%) and repaired HSE-induced mitochondrial damages within 48 h. However, iB16M cells with low mtGSH levels were highly susceptible to TNF-alpha-induced oxidative stress and death. Therefore depletion of mtGSH levels may represent a critical target to challenge survival of invasive cancer cells.

  2. Essential oil of Tridax procumbens L induces apoptosis and suppresses angiogenesis and lung metastasis of the B16F-10 cell line in C57BL/6 mice.

    PubMed

    Manjamalai, A; Kumar, M J Mahesh; Grace, V M Berlin

    2012-01-01

    To determine the effect of essential oil obtained from a traditionally used medicinal plant Tridax procumbens L, on lung metastasis developed by B16F-10 melanoma cells in C57BL/6 mice. Parameters studied were toxicity, lung tumor nodule count, histopathological features, tumor directed capillary vessel formation, apoptosis and expression levels of P53 and caspase-3 proteins. In vitro the MTT assay showed cytotoxicity was found to be high as 70.2% of cancer cell death within 24 hrs for 50 μg. In vivo oil treatment significantly inhibited tumor nodule formation by 71.7% when compared with untreated mice. Formation of tumor directed new blood vessels was also found to be inhibited to about 39.5%. TUNEL assays also demonstrated a significant increase in the number of apoptotic positive cells after the treatment. P53 and caspase-3 expression was also found to be greater in the essential oil treated group than the normal and cancer group. The present investigation showed significant effects of the essential oil of Tridax procumbens L in preventing lung metastasis by B16F-10 cell line in C57BL/6 mice. Its specific preventive effect on tumor directed angiogenesis and inducing effect on apoptosis warrant further studies at the molecular level to validate the significance of Tridax procumbens L for anticancer therapy.

  3. [Combined effect of cisplatin and caffeine on murine B16-BL6 melanoma cells].

    PubMed

    Yasutake, H; Tsuchiya, H; Sugihara, M; Tomita, K; Ueda, Y; Tanaka, M; Sasaki, T

    1989-05-01

    Combined effect of cisplatin and caffeine on murine B16-BL6 melanoma cells was studied. Synergistic inhibition of the cell growth was observed when caffeine (2 mM) was added continuously after one hour exposure of cisplatin. On the other hand, when caffeine was added before one hour exposure of cisplatin or one hour simultaneous exposure with cisplatin, synergistic effect was not shown. In the analysis of DNA histogram obtained from flow cytometry, S and G2/M accumulation was observed by the treatment of cisplatin and that accumulation was reduced by the combination of cisplatin and caffeine. From this findings, it was suggested that caffeine would inhibit DNA repair process. Furthermore, according to morphological studies with hematoxylin-eosin stain and Fontana-Masson stain, the addition of caffeine alone resulted in mild swelling of melanoma cells and the decrease of nuclear-cytoplasmic ratio. The combination of cisplatin and caffeine caused marked swelling of melanoma cells and remarkable increase of dendrite-like processes. Melanogenesis was also enhanced by the addition of these two drugs. Many matured melanosomes, increases of mitochondria, Golgi's apparatus and endoplasmic reticula were observed by the use of electron microscope. These findings implied that the combination of cisplatin and caffeine induced a differentiation of murine melanoma cells.

  4. [Study of migration and distribution of bone marrow cells transplanted animals with B16 melanoma ].

    PubMed

    Poveshchenko, A F; Solovieva, A O; Zubareva, K E; Strunkin, D N; Gricyk, O B; Poveshchenko, O V; Shurlygina, A V; Konenkov, V I

    2017-01-01

    Purpose. Reveal features migration and distribution of syngeneic bone marrow cells (BMC) and subpopulations (MSC) after transplantation into the recipient carrier B16 melanoma bodies. Methods. We used mouse male and female C57BL/6 mice. Induction of Tumor Growth: B16 melanoma cells implanted subcutaneously into right hind paw of female C57BL/6 mice at a dose of 2.5 x 105 cells / mouse. migration study in vivo distribution and BMC and MSC was performed using genetic markers - Y-chromosome specific sequence line male C57Bl/6 syngeneic intravenous transplantation in females using the polymerase chain reaction (PCR) in real time on Authorized Termal Cycler - Light Cycler 480 II / 96 (Roche). Introduction suspension of unseparated bone marrow cells, mesenchymal stem cells from donor to recipient male mice (syngeneic recipient female C57BL/6), followed by isolation of recipients of organs was performed at regular intervals, then of organ recipients isolated DNA. Results. It was shown that bone marrow cells positive for Y-chromosome in migrate lymphoid (lymph nodes, spleen, bone marrow) or in non-lymphoid organs (liver, heart, brain, skin) syngeneic recipients. In addition to the migration of cells from the bone marrow to other organs, there is a way back migration of cells from the circulation to the bone marrow. B16 melanoma stimulates the migration of transplanted MSCs and BMC in bone marrow. It is found that tumor growth enhanced migration of transplanted bone marrow cells, including populations of MSCs in the bone marrow. In the early stages of tumor formation MSC migration activity higher than the BMC. In the later stages of tumor formation undivided population of bone marrow cells migrate to the intense swelling compared with a population of MSCs. Conclusion. The possibility of using bone marrow MSCs for targeted therapy of tumor diseases, because migration of MSCs in tumor tissue can be used to effectively deliver anticancer drugs.

  5. Determination of cell cycle phases in live B16 melanoma cells using IRMS.

    PubMed

    Bedolla, Diana E; Kenig, Saša; Mitri, Elisa; Ferraris, Paolo; Marcello, Alessandro; Grenci, Gianluca; Vaccari, Lisa

    2013-07-21

    The knowledge of cell cycle phase distribution is of paramount importance for understanding cellular behaviour under normal and stressed growth conditions. This task is usually assessed using Flow Cytometry (FC) or immunohistochemistry. Here we report on the use of FTIR microspectroscopy in Microfluidic Devices (MD-IRMS) as an alternative technique for studying cell cycle distribution in live cells. Asynchronous, S- and G0-synchronized B16 mouse melanoma cells were studied by running parallel experiments based on MD-IRMS and FC using Propidium Iodide (PI) staining. MD-IRMS experiments have been done using silicon-modified BaF2 devices, where the thin silicon layer prevents BaF2 dissolution without affecting the transparency of the material and therefore enabling a better assessment of the Phosphate I (PhI) and II (PhII) bands. Hierarchical Cluster Analysis (HCA) of cellular microspectra in the 1300-1000 cm(-1) region pointed out a distribution of cells among clusters, which is in good agreement with FC results among G0/G1, S and G2/M phases. The differentiation is mostly driven by the intensity of PhI and PhII bands. In particular, PhI almost doubles from the G0/G1 to G2/M phase, in agreement with the trend followed by nucleic acids during cellular progression. MD-IRMS is then proposed as a powerful method for the in situ determination of the cell cycle stage of an individual cell, without any labelling or staining, which gives the advantage of possibly monitoring specific cellular responses to several types of stimuli by clearly separating the spectral signatures related to the cellular response from those of cells that are normally progressing.

  6. Effect of combination of taurine and azelaic acid on antimelanogenesis in murine melanoma cells

    PubMed Central

    2010-01-01

    Background Pigmentation in human skin is an important defense mechanism against sunlight or oxidative stress. Despite the protective role of melanin, abnormal hyperpigmentation such as freckles and chloasma sometimes can be serious aesthetic problems. Because of these effects of hyperpigmentation, people have considered the effect of depigmentation. Azelaic acid (AZ) is a saturated dicarboxylic acid found naturally in wheat, rye, and barley. Previously, we showed that AZ inhibited melanogenesis. In this study, we investigated the antimelanogenic activity of combination of AZ and taurine (Tau) in B16F10 mouse melanoma cells. Methods The mouse melanoma cell line B16F10 was used in the study. We measured melanin contents and tyrosinase activity. To gain the change of protein expression, we carried out western blotting. Results We investigated that AZ combined with taurine (Tau) show more inhibitory effects in melanocytes than the treatment of AZ alone. AZ combined with Tau inhibited the melanin production and tyrosinase activity of B16F10 melanoma cells without significant cytotoxicity. Also inhibitory effects after treatment with these combined chemical are stronger than AZ alone on melanogenesis. Conclusions These findings indicate that AZ with Tau might play an important role in the regulation of melanin formation and be useful as effective ingredients in antimelanogesis. PMID:20804622

  7. Effect of combination of taurine and azelaic acid on antimelanogenesis in murine melanoma cells.

    PubMed

    Yu, Ji Sun; Kim, An Keun

    2010-08-24

    Pigmentation in human skin is an important defense mechanism against sunlight or oxidative stress. Despite the protective role of melanin, abnormal hyperpigmentation such as freckles and chloasma sometimes can be serious aesthetic problems. Because of these effects of hyperpigmentation, people have considered the effect of depigmentation. Azelaic acid (AZ) is a saturated dicarboxylic acid found naturally in wheat, rye, and barley. Previously, we showed that AZ inhibited melanogenesis. In this study, we investigated the antimelanogenic activity of combination of AZ and taurine (Tau) in B16F10 mouse melanoma cells. The mouse melanoma cell line B16F10 was used in the study. We measured melanin contents and tyrosinase activity. To gain the change of protein expression, we carried out western blotting. We investigated that AZ combined with taurine (Tau) show more inhibitory effects in melanocytes than the treatment of AZ alone. AZ combined with Tau inhibited the melanin production and tyrosinase activity of B16F10 melanoma cells without significant cytotoxicity. Also inhibitory effects after treatment with these combined chemical are stronger than AZ alone on melanogenesis. These findings indicate that AZ with Tau might play an important role in the regulation of melanin formation and be useful as effective ingredients in antimelanogesis.

  8. The effect of Taurolidine on adherent and floating subpopulations of melanoma cells.

    PubMed

    Shrayer, D P; Lukoff, H; King, T; Calabresi, P

    2003-04-01

    The annual incidence of malignant melanoma is estimated at 10-12 per 100000 inhabitants in countries of Central Europe and the US, with more recent estimates showing a dramatic upward trend. Taurolidine (Carter/Wallace, Cranberry, NJ) is a novel, potentially effective, antitumor chemotherapeutic agent. We hypothesized that Taurolidine could inhibit the growth, induce apoptosis, affect the cell cycle and change morphology of melanoma cells. We expected this process to be different in adherent and floating subpopulations that may be reflective of solid tumors and their metastases. Analysis of MNT-1 human and B16F10 murine melanoma cells showed that at 72 h the IC(50) of Taurolidine was 25.4+/-3.3 microM for MNT-1 human melanoma cells and 30.9+/-3.6 microM for B16F10 murine melanoma cells. Taurolidine induced DNA fragmentation of melanoma cells in a dose-dependent manner. Taurolidine (75 and 100 microM) induced 52-97% Annexin-V binding (apoptosis), respectively. Evaluation of cell cycle after 72 h exposure to Taurolidine (0-100 microM) revealed that the percentage of melanoma cells in S phase increased from 27 to 40% in the adherent subpopulation and from 33 to 49% in the floating subpopulation. Phase contrast microscopy revealed a marked swelling of melanoma cells and decreasing cell numbers in adherent subpopulation starting at 24 h with 25 microM Taurolidine. Shrinkage of cells dominated at 75-100 microM Taurolidine. Using Cytospin assay in the floating population, we observed swelling of melanoma cells induced by 25-100 micro Taurolidine and appearance of giant (multinuclear) forms resulting from exposure to 75-100 micro Taurolidine. Some floating cells with normal morphology were observed with low concentrations of Taurolidine (0-25 microM). These data show that effects of Taurolidine may be different in adherent and floating subpopulations of melanoma cells. More importantly, floating subpopulations that may contain some viable melanoma cells, may be reflective

  9. Antioxidative and Anti-Melanogenic Activities of Bamboo Stems (Phyllostachys nigra variety henosis) via PKA/CREB-Mediated MITF Downregulation in B16F10 Melanoma Cells.

    PubMed

    Choi, Moon-Hee; Jo, Han-Gyo; Yang, Ji Hye; Ki, Sung Hwan; Shin, Hyun-Jae

    2018-01-30

    Phyllostachys nigra var. henosis, a domestic bamboo species, has been attracting much attention; its bioactive compounds (especially in the leaf) show antioxidant, anti-inflammatory, and anti-obesity activities. Little information is available on the antioxidative and anti-melanogenetic activities of the bioactive compounds in bamboo stems. The anti-melanogenic and antioxidative activities of the EtOAc fraction (PN3) of a P. nigra stem extract were investigated in a cell-free system and in B16F10 melanoma cells. PN3 consisted of a mixture of flavonoids, such as catechin, chlorogenic acid, caffeic acid, and p -coumaric acid. The antioxidant activity (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)), and hydroxyl radical scavenging) was evaluated, as well as the inhibition of reactive oxygen species (ROS) produced by the Fenton reaction. PN3 showed in vitro tyrosinase inhibition activity with the half maximal inbihitory concentration (IC 50 ) values of 240 μg/mL, and in vivo cytotoxic concentration ranges > 100 μg/mL. The protein expression levels and mRNA transcription levels of TYR , TRP-1 , and MITF were decreased in a dose-dependent manner by the treatment with PN3. PN3 interfered with the phosphorylation of intracellular protein kinase A (PKA)/cAMP response element-binding protein (CREB), demonstrating potent anti-melanogenic effects. PN3 could inhibit PKA/CREB and the subsequent degradation of microphthalmia-associated transcription factor (MITF), resulting in the suppression of melanogenic enzymes and melanin production, probably because of the presence of flavonoid compounds. These properties make it a candidate as an additive to whitening cosmetics.

  10. Antioxidative and Anti-Melanogenic Activities of Bamboo Stems (Phyllostachys nigra variety henosis) via PKA/CREB-Mediated MITF Downregulation in B16F10 Melanoma Cells

    PubMed Central

    Choi, Moon-Hee; Jo, Han-Gyo; Yang, Ji Hye; Ki, Sung Hwan

    2018-01-01

    Phyllostachys nigra var. henosis, a domestic bamboo species, has been attracting much attention; its bioactive compounds (especially in the leaf) show antioxidant, anti-inflammatory, and anti-obesity activities. Little information is available on the antioxidative and anti-melanogenetic activities of the bioactive compounds in bamboo stems. The anti-melanogenic and antioxidative activities of the EtOAc fraction (PN3) of a P. nigra stem extract were investigated in a cell-free system and in B16F10 melanoma cells. PN3 consisted of a mixture of flavonoids, such as catechin, chlorogenic acid, caffeic acid, and p-coumaric acid. The antioxidant activity (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)), and hydroxyl radical scavenging) was evaluated, as well as the inhibition of reactive oxygen species (ROS) produced by the Fenton reaction. PN3 showed in vitro tyrosinase inhibition activity with the half maximal inbihitory concentration (IC50) values of 240 μg/mL, and in vivo cytotoxic concentration ranges > 100 μg/mL. The protein expression levels and mRNA transcription levels of TYR, TRP-1, and MITF were decreased in a dose-dependent manner by the treatment with PN3. PN3 interfered with the phosphorylation of intracellular protein kinase A (PKA)/cAMP response element-binding protein (CREB), demonstrating potent anti-melanogenic effects. PN3 could inhibit PKA/CREB and the subsequent degradation of microphthalmia-associated transcription factor (MITF), resulting in the suppression of melanogenic enzymes and melanin production, probably because of the presence of flavonoid compounds. These properties make it a candidate as an additive to whitening cosmetics. PMID:29385729

  11. Melanoma-targeted delivery system (part 2): Synthesis, radioiodination and biological evaluation in B16F0 bearing mice.

    PubMed

    El Aissi, Radhia; Miladi, Imen; Chezal, Jean-Michel; Chavignon, Olivier; Miot-Noirault, Elisabeth; Moreau, Emmanuel

    2016-09-14

    Here we report the synthesis and radiolabelling with iodine-125 of a melanoma-selective prodrug (17a*) and its parent drug IUdR. The in vivo and ex vivo biodistributions of [(125)I](17a*) and [(125)I]IUdR were evaluated in a model of melanoma B16F0-bearing mice. The pharmacokinetic profile of [(125)I](17a*) suggests rapid release of the active drug [(125)I]IUdR after i.v. administration of [(125)I](17a*). Preliminary metabolism studies in dedicated compartments (i.e. blood, urine and tumour) yielded results consistent with this hypothesis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  12. In vitro anticancer evaluation of 5-fluorouracil lipid nanoparticles using B16F10 melanoma cell lines

    NASA Astrophysics Data System (ADS)

    Shenoy, Vikram S.; Gude, Rajiv P.; Murthy, Rayasa S. Ramachandra

    2013-05-01

    The present study is aimed to investigate the formulation and in vitro anticancer activities of solid lipid nanoparticles (SLNs) of 5-fluorouracil (5-FU) prepared using glyceryl monostearate (GMS) and cetyl palmitate (CP) by hot homogenization method. The lipids were selected based on the partition coefficient of 5-FU in lipids. The lipid nanoparticles were optimized for process and formulation parameters. The optimized nanoparticles were characterized for their zeta potential, morphology, release kinetics, and anticancer activity. Higher entrapments were achieved using a combination of emulsifiers. The zeta potential of the optimized CP and GMS SLN formulation were -8.26 and -9.35 mV, respectively. Both the optimized formulations were spherical. The in vitro release studies of SLNs of both the lipid carriers followed Peppas-Korsenmeyer equation when carried out at pH 3.5 and 7.4. The chemosensitivity assay carried out in B16F10 cell lines revealed that CP SLNs had better cytotoxicity than 5-FU solution and GMS SLNs at 48 h of incubation. Subtoxic concentration of 5-FU-loaded CP SLNs (0.12 μg/mL) possessed comparable antimigrational activity, colony inhibition activity, and cytopathic as that of 5-FU solution effects. The results indicated that encapsulating 5-FU in CP would be a promising delivery system for delivering 5-FU.

  13. Influence of anaesthetics on tumour-cell kill and repopulation in B16 melanoma treated with melphalan.

    PubMed Central

    Peacock, J. H.; Stephens, T. C.

    1978-01-01

    The influence of anaesthetics on the in vivo response of B16 melanoma to melphalan was studied using an in vitro cell-survival assay. Three anaesthetics were used, Saffan (Althesin) Sagatal (Nembutal) and Hypnorm. When Saffan was administered to tumour-bearing animals before melphalan there was a significant increase in tumour-cell kill. This effect was not observed with Sagatal or Hypnorm. Maximum increase in tumour-cell kill was achieved when Saffan was administered about 1 h before melphalan, and was dependent on Saffan dose. Clonogenic tumour-cell repopulation after melphalan was rapid (TD - 1 day) and the rate was similar from 2 levels of cell kill. When Saffan was combined with melphalan the repopulation rate was the same as with melphalan alone, and the increased cell kill was reflected in increased growth delay. The in vitro response of B16 melanoma cells to melphalan was unaltered by pretreatment with, or simultaneous exposure to Saffan. The results suggest that the mechanism of the enhanced cell kill in vivo is probably due to an indirect systemic effect, rather than a direct effect on the tumour cells. PMID:743490

  14. Differentiation- and apoptosis-inducing activities by pentacyclic triterpenes on a mouse melanoma cell line.

    PubMed

    Hata, Keishi; Hori, Kazuyuki; Takahashi, Saori

    2002-05-01

    In a study to investigate the relationship between the chemical structure and the differentiation-inducing activity of pentacyclic triterpenes, several lupane, oleanane, and ursane triterpenes were prepared and their effects on B16 2F2 melanoma cell differentiation and growth were examined. Eleven lupane triterpenes used in this study acted on the melanoma cells as a melanogen, but no induction of melanogenesis of B16 2F2 cells by oleanane and ursane was detected. The differences at C-17 of the lupane series and acetylation of the OH group at C-3 did not markedly influence their activities. However, the ED(50) value for up-regulation of melanin biosynthesis was markedly decreased by the oxidation of the OH group at C-3 of lupeol (1). Betulinic acid (11), its methyl ester (12), lup-28-al-20(29)-ene-3beta-ol (9), and lup-28-al-20(29)-en-3-one (10) inhibited B16 2F2 cell proliferation by induction of apoptosis. These findings suggested that the carbonyl group at C-17 might be essential for the apoptotic effects of these compounds on B16 2F2 cells.

  15. B16-BL6 melanoma cells release inhibitory factor(s) of active pump activity in isolated lymph vessels.

    PubMed

    Nakaya, K; Mizuno, R; Ohhashi, T

    2001-12-01

    We investigated whether supernatant cultured with melanoma cell lines B16-BL6 and K1735 or the Lewis lung carcinoma cell line (LLC) can regulate lymphatic pump activity with bioassay preparations isolated from murine iliac lymph vessels. B16-BL6 and LLC supernatants caused significant dilation of lymph microvessels with cessation of pump activity. B16-BL6 supernatant produced dose-related cessation of lymphatic pump activity. There was no significant tachyphylaxis in the supernatant-mediated inhibitory response of lymphatic pump activity. Pretreatment with 3 x 10(-5) M N(omega)-nitro-L-arginine methyl ester (L-NAME) or 10(-7) M or 10(-6) M glibenclamide and 5 x 10(-4) M 5-hydroxydecanoic acid caused significant reduction of supernatant-mediated inhibitory responses. Simultaneous treatment with 10(-3) M L-arginine and 3 x 10(-5) M L-NAME significantly lessened L-NAME-induced inhibition of the supernatant-mediated response, suggesting that endogenous nitric oxide (NO) plays important roles in supernatant-mediated inhibitory responses. Chemical treatment dialyzed substances of <1,000 molecular weight (MW), producing complete reduction of the supernatant-mediated response. In contrast, pretreatment with heating or digestion with protease had no significant effect on supernatant-mediated response. These findings suggest that B16-BL6 cells may release nonpeptide substance(s) of <1,000 MW, resulting in significant cessation of lymphatic pump activity via production and release of endogenous NO and activation of mitochondrial ATP-sensitive K(+) channels.

  16. Aqueous fraction from Cuscuta japonica seed suppresses melanin synthesis through inhibition of the p38 mitogen-activated protein kinase signaling pathway in B16F10 cells.

    PubMed

    Jang, Ji Yeon; Kim, Ha Neui; Kim, Yu Ri; Choi, Yung Hyun; Kim, Byung Woo; Shin, Hwa Kyoung; Choi, Byung Tae

    2012-05-07

    Semen cuscutae has been used traditionally to treat pimples and alleviate freckles and melasma in Korea. The present study aimed to investigate the inhibitory effect of Cuscuta japonica Choisy seeds on alpha-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis. The aqueous fraction from Semen cuscutae (AFSC) was used to determine anti-melanogenic effects by examination of cellular melanin contents, tyrosinase activity assay, cAMP assay and Western blot analysis for melanin synthesis-related signaling proteins in B16F10 mouse melanoma cells. AFSC markedly inhibited α-MSH-induced melanin synthesis and tyrosinase activity, and also decreased α-MSH-induced expression of microphthalmia-associated transcription factor (MITF) and tyrosinase-related proteins (TRPs). Moreover, AFSC significantly decreased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK) signaling through the down-regulation of α-MSH-induced cAMP. Furthermore, we confirmed that the specific inhibitor of p38 MAPK (SB203580)-mediated suppressed melanin synthesis and tyrosinase activity was further attenuated by AFSC. AFSC also further decreased SB203580-mediated suppression of MITF and TRP expression. These results indicate that AFSC inhibits p38 MAPK phosphorylation with suppressed cAMP levels and subsequently down-regulate MITF and TRP expression, which results in a marked reduction of melanin synthesis and tyrosinase activity in α-MSH-stimulated B16F10 cells. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  17. Rac-WAVE2 signaling is involved in the invasive and metastatic phenotypes of murine melanoma cells.

    PubMed

    Kurisu, Shusaku; Suetsugu, Shiro; Yamazaki, Daisuke; Yamaguchi, Hideki; Takenawa, Tadaomi

    2005-02-17

    WAVEs (WASP-family verprolin-homologous proteins) regulate the actin cytoskeleton through activation of Arp2/3 complex. As cell motility is regulated by actin cytoskeleton rearrangement and is required for tumor invasion and metastasis, blocking actin polymerization may be an effective strategy to prevent tumor dissemination. We show that WAVEs, especially WAVE2, are essential for invasion and metastasis of melanoma cells. Malignant B16F10 mouse melanoma cells expressed more WAVE1 and WAVE2 proteins and showed higher Rac activity than B16 parental cells, which are neither invasive nor metastatic. The effect of WAVE2 silencing by RNA interference (RNAi) on the highly invasive nature of B16F10 cells was more dramatic than that of WAVE1 RNAi. Membrane ruffling, cell motility, invasion into the extracellular matrix, and pulmonary metastasis of B16F10 cells were suppressed by WAVE2 RNAi. WAVE2 RNAi also had a profound effect on invasion induced by a constitutively active form of Rac (RacCA). In addition, ectopic expression of both RacCA and WAVE2 in B16 cells resulted in further increase in the invasiveness than that observed in B16 cells expressing only RacCA. Thus, WAVE2 acts as the primary effector downstream of Rac to achieve invasion and metastasis, suggesting that suppression of WAVE2 activity holds a promise for preventing cancer invasion and metastasis.

  18. The therapeutic potential of human adipose-derived mesenchymal stem cells producing CXCL10 in a mouse melanoma lung metastasis model.

    PubMed

    Mirzaei, Hamed; Salehi, Hossein; Oskuee, Reza Kazemi; Mohammadpour, Ali; Mirzaei, Hamid Reza; Sharifi, Mohammad Reza; Salarinia, Reza; Darani, Hossein Yousofi; Mokhtari, Mojgan; Masoudifar, Aria; Sahebkar, Amirhossein; Salehi, Rasoul; Jaafari, Mahmoud Reza

    2018-04-10

    Interferon γ-induced protein 10 kDa (IP-10) is a potent chemoattractant and has been suggested to enhance antitumor activity and mediate tumor regression through multiple mechanisms of action. Multiple lines of evidence have indicated that genetically-modified adult stem cells represent a potential source for cell-based cancer therapy. In the current study, we assessed therapeutic potential of human adipose derived mesenchymal stem cells (hADSC) genetically-modified to express IP-10 for the treatment of lung metastasis in an immunocompetent mouse model of metastatic melanoma. A Piggybac vector encoding IP-10 was employed to transfect hADSC ex vivo. Expression and bioactivity of the transgenic protein from hADSCs expressing IP-10 were confirmed prior to in vivo studies. Our results indicated that hADSCs expressing IP-10 could inhibit the growth of B16F10 melanoma cells and significantly prolonged survival. Immunohistochemistry analysis, TUNEL assay and western blot analysis indicated that hADSCs expressing IP-10 inhibited tumor cell growth, hindered tumor infiltration of Tregs, restricted angiogenesis and significantly prolonged survival. In conclusion, our results demonstrated that targeting metastatic tumor sites by hADSC expressing IP-10 could reduce melanoma tumor growth and lung metastasis. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Genistein suppresses adhesion-induced protein tyrosine phosphorylation and invasion of B16-BL6 melanoma cells.

    PubMed

    Yan, C; Han, R

    1998-07-03

    Protein tyrosine phosphorylation occurs as one of the earlier events in cancer cell-extracellular matrix (ECM) interaction. With immunoblot analysis and immunofluorescence microscopy, genistein was found to suppress the tyrosine phosphorylation of proteins located at the cell periphery, including a 125 kDa protein, when B16-BL6 melanoma cells attached to and interacted with ECM. When accompanied by the suppression of adhesion-induced protein tyrosine phosphorylation, the invasive potential of B16-BL6 cells through reconstituted basement membrane was decreased significantly. However, neither adhesive capability nor cell growth was significantly affected by genistein. Therefore, the interruption of cancer cell-ECM interaction by suppression of protein tyrosine phosphorylation may contribute to invasion prevention of genistein.

  20. Abnormally banded chromosomal regions in doxorubicin-resistant B16-BL6 murine melanoma cells.

    PubMed

    Slovak, M L; Hoeltge, G A; Ganapathi, R

    1986-08-01

    B16-BL6 murine melanoma cells were selected for cytogenetic evaluation during the stepwise development of increasing resistance in vitro to the antitumor antibiotic, doxorubicin (DOX). Karyotypic studies demonstrated extensive heteroploidy with both numerical and structural abnormalities which were not present in the parental DOX-sensitive B16-BL6 cells. Trypsin-Giemsa banding revealed the presence of several marker chromosomes containing abnormally banding regions (ABRs) in the 44-fold B16-BL6 DOX-resistant subline. These ABRs appeared to be more homogeneously staining at the higher DOX concentrations. Length measurements (ABR index) in seven banded metaphases indicated a direct correlation with increasing DOX concentration. When the DOX-resistant cells were grown in drug-free medium for 1 yr, the drug-resistant phenotype gradually declined in parallel with the level of resistance and the ABR index. DOX-induced cytogenetic damage examined by sister chromatid exchange methodology in parental B16-BL6 cells indicated a linear sister chromatid exchange:DOX dose-response relationship. However, after continuous treatment of parental B16-BL6 cells with DOX (0.01 microgram/ml) for 30 days, sister chromatid exchange scores were found to return to base-line values. The B16-BL6 resistant cells demonstrated a cross-resistant phenotype with N-trifluoroacetyladriamycin-14-valerate, actinomycin D, and the Vinca alkaloids but not with 1-beta-D-arabinofuranosylcytosine. The results suggest that ABR-containing chromosomes in DOX-resistant sublines may represent cytogenetic alterations of specific amplified genes involved in the expression of DOX resistance. Further studies are required to identify and define the possible gene products and to correlate their relationship to the cytotoxic action of doxorubicin.

  1. Mugil cephalus roe oil obtained by supercritical fluid extraction affects the lipid profile and viability in cancer HeLa and B16F10 cells.

    PubMed

    Rosa, A; Piras, A; Nieddu, M; Putzu, D; Cesare Marincola, F; Falchi, A M

    2016-09-14

    We explored the changes in viability and lipid profile occurring in cancer cells, murine melanoma cells (B16F10 cells) and human cervical carcinoma cells (HeLa cells), when exposed to 24 h-treatments with an n-3 PUFA-rich oil obtained by supercritical extraction with CO2 from Mugil cephalus processed roe (bottarga). The composition of the major lipid classes of bottarga oil was determined by the (13)C NMR technique. Reversed-phase HPLC with DAD/ELSD detection was performed to analyze cells' total fatty acid profile and the levels of phospholipids, total/free cholesterol, triacylglycerols, and cholesteryl esters. Cell-based fluorescent measurements of intracellular membranes and lipid droplets were performed on bottarga oil-treated cells using the Nile red staining technique. The treatments of cancer cells with bottarga oil reduced the viability and affected the fatty acid profile, with a significant n-3 PUFA increase in treated cells. Mullet roe oil uptake modulated the cancer cell lipid composition, inducing a remarkable incorporation of health beneficial n-3 PUFA in the polar and neutral lipid fractions. Bottarga oil treatment influenced the synthesis of intracellular membranes and accumulation of cytoplasmic lipid droplets in cancer cells.

  2. Inhibitory effects of 1-O-methyl-fructofuranose from Schisandra chinensis fruit on melanogenesis in B16F0 melanoma cells.

    PubMed

    Oh, Eun Young; Jang, Ji Yeon; Choi, Yung Hyun; Choi, Young Whan; Choi, Byung Tae

    2010-10-28

    1-O-methyl-fructofuranose (1-O-MFF) from the fruit of Schisandra chinensis is a traditional Korean medicinal herb that has a variety of beneficial properties. The effect of purified 1-O-MFF on melanogenesis including the activation of related signaling pathways was investigated. The inhibitory activities of 1-O-MFF were examined by melanin synthesis, tyrosinase activity assay, Western blot and flow cytometric analyses in B16F0 mouse melanoma cells. 1-O-MFF significantly inhibited both melanin synthesis and tyrosinase activity in a concentration-dependent manner, and reduced the expression of melanogenic proteins including microphthalmia-associated transcription factor (MITF), tyrosinase and tyrosinase-related protein-1. 1-O-MFF phosphorylated and activated melanogenesis inhibitory proteins such as mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and Akt. Flow cytometry confirmed that 1-O-MFF phosphorylated ERK and Akt proteins and recovered partially phosphorylated forms in cells treated with the MEK/ERK inhibitor compound PD98059 and the phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor compound LY294002. The suppressive effects of 1-O-MFF on melanogenesis may involve down-regulation of MITF and its downstream signal pathway via the activation of MEK/ERK or PI3K/Akt. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  3. Therapeutic efficacy of interleukin-2 activated killer cells against adriamycin resistant mouse B16-BL6 melanoma.

    PubMed

    Gautam, S C; Chikkala, N F; Lewis, I; Grabowski, D R; Finke, J H; Ganapathi, R

    1992-01-01

    Development of multidrug-resistance (MDR) remains a major cause of failure in the treatment of cancer with chemotherapeutic agents. In our efforts to explore alternative treatment regimens for multidrug-resistant tumors we have examined the sensitivity of MDR tumor cell lines to lymphokine activated killer (LAK) cells. Adriamycin (ADM) resistant B16-BL6 melanoma, L1210 and P388 leukemic cell lines were tested for sensitivity to lysis by LAK cells in vitro. While ADM-resistant B16-BL6 and L1210 sublines were found to exhibit at least 2-fold greater susceptibility to lysis by LAK cells, sensitivity of ADM-resistant P388 cell was similar to that of parental cells. Since ADM-resistant B16-BL6 cells were efficiently lysed by LAK cells in vitro, the efficacy of therapy with LAK cells against the ADM-resistant B16-BL6 subline in vivo was evaluated. Compared to mice bearing parental B16-BL6 tumor cells, the adoptive transfer of LAK cells and rIL2 significantly reduced formation of experimental metastases (P less than 0.009) and extended median survival time (P less than 0.001) of mice bearing ADM-resistant B16-BL6 tumor cells. Results suggest that immunotherapy with LAK cells and rIL2 may be a useful modality in the treatment of cancers with the MDR phenotype.

  4. In vitro and in vivo antitumor effects of 50 to 100-KDa components from B16 melanoma culture supernatant.

    PubMed

    Qin, Ying-Song; Zhang, X U; Zhang, Xiang-Yu

    2015-07-01

    The development of immunological therapies for melanoma has been of considerable concern in recent years. Whole tumor cell lysates have been used to develop antitumor vaccines, but the effective components of the lysates have not been identified. In the present study, protein elements were purified from the B16 supernatant to analyze the in vitro chemotaxis towards mouse spleen lymphocytes using a Boyden chamber. Prior to establishing a B16 melanoma model, C57BL/6 mice were vaccinated with these proteins, and melanoma growth, tumor appearance time and behavioral changes were observed. Next, the cytotoxicity and subsets of the tumor infiltrating lymphocytes, and the histological characteristics of the melanoma were analyzed. The isolated purified fragments of B16 melanoma culture supernatant had strong antitumor effects. The possible antitumor mechanism was delineated, and was identified to possibly be through the activation of cluster of differentiation 8-positive T cells and the promotion of B16 cell differentiation. These methods will provide a novel insight into understanding antitumor immunological mechanisms and provide a potential avenue for immunotherapy.

  5. Growth inhibition and differentiation of murine melanoma B16-BL6 cells caused by the combination of cisplatin and caffeine.

    PubMed

    Tsuchiya, H; Tomita, K; Yasutake, H; Ueda, Y; Tanaka, M; Sasaki, T

    1989-12-01

    We preliminarily investigated the combined effects of cisplatin and caffeine on murine melanoma B16-BL6 cells in vitro. When caffeine was added before or simultaneously with cisplatin, there was little growth inhibition. The addition of 2.0 mM caffeine after 1 h of exposure to cisplatin inhibited growth and induced cell differentiation. This treatment resulted in fewer cells, and the numbers of melanosomes and mitochondria and the amount of Golgi's complex and endoplasmic reticulum were increased. DNA histograms obtained by flow cytometry showed that cells treated with cisplatin alone accumulated in the G2/M phase, with a partial G2 block. The addition of 2.0 mM caffeine after 1 h of treatment with cisplatin reduced this block. Caffeine caused murine melanoma B16-BL6 cells treated with cisplatin to differentiate, and this inhibited growth.

  6. Anticancer effects of morin-7-sulphate sodium, a flavonoid derivative, in mouse melanoma cells.

    PubMed

    Li, Hua-Wen; Zou, Tang-Bin; Jia, Qing; Xia, En-Qin; Cao, Wen-Jun; Liu, Wen; He, Tai-Ping; Wang, Qin

    2016-12-01

    Increasing evidence supports the anticancer effects of morin in vitro and in vivo. However, the role of morin-7-sulphate sodium (NaMoS), a water-soluble flavonoid derivative synthesized from morin remains unclear. The present study investigated the tumor suppression by NaMoS in mouse melanoma cells. We synthesized the flavonoid derivative morin-7-sulphate sodium according to the method described for quercetin-sulphate derivative, and further isolated, purified and identified the compound. Cell proliferation in vitro was assessed using a CCK-8 assay. The wound healing assay was performed to evaluate cell motility, and flow cytometry was used to detect cellular apoptosis. Protein levels of vimentin, matrix metalloproteinase 9 (MMP9), phosphorylation of Akt1/2/3 (p-Akt1/2/3), extracellular signal-regulated kinase 1/2 (p-ERK1/2) and Caspase3 in B16F10 cells were detected by immunohistochemistry and Western blot. The results suggest that cell proliferation was markedly decreased in NaMoS-treated groups (1, 10, 25, 50, 100, 500, 1000μM) in a dose-dependent manner compared with the Control group and the IC 50 was 221.67μM at 48h. NaMoS at 200μM concentration significantly inhibited the invasion and promoted apoptosis of B16F10 cells. Moreover, protein level of Caspase3 increased significantly in B16F10 cells treated by NaMoS. Immunohistochemistry and Western blot further confirmed that NaMoS decreased the expression of vimentin, MMP9, p-Akt1/2/3 and p-ERK1/2 in B16F10 cells. This study provides robust evidence that NaMoS, a water-soluble flavonoid, manifests anticancer properties and may act as a signal transduction inhibitor in melanoma cells. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  7. Antiproliferative and Apoptotic Potential of Cyanidin-Based Anthocyanins on Melanoma Cells.

    PubMed

    Rugină, Dumitriţa; Hanganu, Daniela; Diaconeasa, Zoriţa; Tăbăran, Flaviu; Coman, Cristina; Leopold, Loredana; Bunea, Andrea; Pintea, Adela

    2017-04-30

    Elderberries are known for their high anthocyanins content, which have been shown to possess anti-proliferative and anti-cancer effects. Anthocyanins enriched extract (AEE) was obtained from elderberries and was characterized by LC/DAD/ESI-MS analysis. Five cyanidin-based anthocyanins were identified, among which Cy-3- O -samb was the major compound (51%). The total anthocyanins content of AEE was 495 mg Cy-3- O -samb/100 g FW. AEE inhibited proliferation of metastatic B16-F10 murine melanoma cells, in a concentration-dependent manner, with an IC50 of 264.3 μg/mL. LDH (lactate dehydrogenase), as a marker of membrane integrity, increased 74% in B16-F10 cells treated with 250 μg/mL AEE, compared to control. It was observed that apoptosis is the mechanism of melanoma cell death after AEE treatment, confirmed morphologically by acridine orange/ethidium bromide double staining and TUNEL analysis. These results indicate that elderberry-derived anthocyanins might be utilized in future applications as topical adjuvant in skin cancer therapy.

  8. Antiproliferative and Apoptotic Potential of Cyanidin-Based Anthocyanins on Melanoma Cells

    PubMed Central

    Rugină, Dumitriţa; Hanganu, Daniela; Diaconeasa, Zoriţa; Tăbăran, Flaviu; Coman, Cristina; Leopold, Loredana; Bunea, Andrea; Pintea, Adela

    2017-01-01

    Elderberries are known for their high anthocyanins content, which have been shown to possess anti-proliferative and anti-cancer effects. Anthocyanins enriched extract (AEE) was obtained from elderberries and was characterized by LC/DAD/ESI-MS analysis. Five cyanidin-based anthocyanins were identified, among which Cy-3-O-samb was the major compound (51%). The total anthocyanins content of AEE was 495 mg Cy-3-O-samb/100 g FW. AEE inhibited proliferation of metastatic B16-F10 murine melanoma cells, in a concentration-dependent manner, with an IC50 of 264.3 μg/mL. LDH (lactate dehydrogenase), as a marker of membrane integrity, increased 74% in B16-F10 cells treated with 250 μg/mL AEE, compared to control. It was observed that apoptosis is the mechanism of melanoma cell death after AEE treatment, confirmed morphologically by acridine orange/ethidium bromide double staining and TUNEL analysis. These results indicate that elderberry-derived anthocyanins might be utilized in future applications as topical adjuvant in skin cancer therapy. PMID:28468289

  9. Antitumoral, antioxidant, and antimelanogenesis potencies of Hawthorn, a potential natural agent in the treatment of melanoma.

    PubMed

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

    2016-06-01

    The lack of an efficient agent that does not have the disadvantage of low activity (kojic acid), high cytotoxicity, and mutagenicity (hydroquinone), poor skin penetration (arbutin), or low stability in formulation (glabridin) led us to continue our research on new antipigmentation/skin-lightening agents. Therefore, research of natural products that can modulate the metabolism of pigmentation is of great interest. Otherwise, malignant melanoma is one of the most aggressive forms of skin cancer, with high metastatic potential, and currently, there is no effective chemotherapy against invasive melanoma. Therefore, it is necessary to develop new drugs with potent activity and weak side effects against melanoma. The in-vitro anticancer effect of hawthorn was analyzed against B16F10 melanoma cells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effect of isolated compounds from hawthorn on melanogenesis in B16F10 melanoma cells was investigated by measuring the amounts of melanin and tyrosinase spectrophotometrically at 475 nm. Balb/c mice models inoculated with B16F10 mouse tumor cells were used to evaluate the in-vivo antitumoral potential of hawthorn by assessing its effect on the growth of transplanted tumors. The antioxidant potential of tested samples was evaluated in B16F10 and primary human keratinocyte cells using a cellular antioxidant activity assay. Hawthorn tested samples inhibited effectively the growth of melanoma cells in vitro. Furthermore, it appears that tested samples from hawthorn reduced melanogenesis by inhibiting the tyrosinase activity of B16F10 cells in a dose-dependent manner. In-vivo studies showed that hawthorn total oligomer flavonoids extract treatment at a dose of 150 mg/kg body weight for 21 days in implanted tumor mice resulted in significant inhibition of the tumor growth volume and weight. In addition, tested samples showed significant cellular antioxidant capacity against the reactive oxygen species

  10. Efficient glycoengineering of GM3 on melanoma cell and monoclonal antibody-mediated selective killing of the glycoengineered cancer cell

    PubMed Central

    Wang, Qianli; Zhang, Junping; Guo, Zhongwu

    2007-01-01

    To verify the principal of a new immunotherapeutic strategy for cancer, a monoclonal antibody 2H3 against N-phenylacetyl GM3, an unnatural form of the tumor-associated antigen GM3, was prepared and employed to demonstrate that murine melanoma cell B16F0 could be effectively glycoengineered by N-phenylacetyl-d-mannosamine to express N-phenylacetyl GM3 and that 2H3 was highly cytotoxic to the glycoengineered B16F0 cell in the presence of complements. It was further demonstrated that B16F0 cell could be glycoengineered 4-5 times more effectively than 3T3 A31 cell, a normal murine embryo fibroblast cell, and that the antibody and complement mediated cytotoxicity was at least 200 times more potent to the glycoengineered B16F0 cell than to the N-phenylacetyl-d-mannosamine-treated 3T3 A31 cell. These results show the promise for developing useful melanoma immunotherapies based on vaccination against N-phenylacetyl GM3 followed by treatment with N-phenylacetyl-d-mannosamine. PMID:17892942

  11. Efficient glycoengineering of GM3 on melanoma cell and monoclonal antibody-mediated selective killing of the glycoengineered cancer cell.

    PubMed

    Wang, Qianli; Zhang, Junping; Guo, Zhongwu

    2007-12-15

    To verify the principal of a new immunotherapeutic strategy for cancer, a monoclonal antibody 2H3 against N-phenylacetyl GM3, an unnatural form of the tumor-associated antigen GM3, was prepared and employed to demonstrate that murine melanoma cell B16F0 could be effectively glycoengineered by N-phenylacetyl-d-mannosamine to express N-phenylacetyl GM3 and that 2H3 was highly cytotoxic to the glycoengineered B16F0 cell in the presence of complements. It was further demonstrated that B16F0 cell could be glycoengineered 4-5 times more effectively than 3T3 A31 cell, a normal murine embryo fibroblast cell, and that the antibody and complement mediated cytotoxicity was at least 200 times more potent to the glycoengineered B16F0 cell than to the N-phenylacetyl-d-mannosamine-treated 3T3 A31 cell. These results show the promise for developing useful melanoma immunotherapies based on vaccination against N-phenylacetyl GM3 followed by treatment with N-phenylacetyl-d-mannosamine.

  12. Alcohol consumption suppresses metastasis of B16-BL6 melanoma in mice.

    PubMed

    Meadows, G G; Elstad, C A; Blank, S E; Gallucci, R M; Pfister, L J

    1993-03-01

    Female C57BL/6 mice were fed a defined, pelleted diet and given 10% w/v or 20% w/v ethanol in their drinking water. Natural killer (NK) cell cytolytic activity was compared between water-drinking and ethanol-consuming mice and in mice that were also treated with polyinosinic-polycytidylic acid (poly I:C) to augment NK cell activity or with anti-NK1.1 antibody to decrease activity. NK cell cytolytic activity was not altered in mice given 10% ethanol, but was decreased in mice given 20% ethanol compared to water-drinking mice. Poly I:C treatment increased and anti-NK1.1 antibody treatment decreased NK cell activity in both water-drinking and 20% ethanol-consuming mice. Experimental and spontaneous metastases of B16-BL6 melanoma were evaluated as a function of the duration of ethanol consumption before tumor inoculation and as a function of altered NK cell activity. Experimental metastasis was inhibited after 4 and also after 6.5 weeks of ethanol exposure. Poly I:C treatment inhibited tumor lung colonization irrespective of ethanol consumption. Anti-NK1.1 antibody treatment increased metastasis, although to a lesser degree in mice consuming 10% ethanol. Spontaneous metastasis was inhibited in mice consuming 10% ethanol for 4 weeks, and in mice consuming 20% ethanol for 1 and 4 weeks before melanoma inoculation.

  13. Evaluation of chromosomal aberrations induced by 188Re-dendrimer nanosystem on B16f1 melanoma cells.

    PubMed

    Tassano, Marcos; Oddone, Natalia; Fernández, Marcelo; Porcal, Williams; García, María Fernanda; Martínez-López, Wilner; Benech, Juan Claudio; Cabral, Pablo

    2018-06-19

    To study the rhenium-188 labeling of polyamidoamine (PAMAM) generation 4 (G4) dendrimer and its evaluation on biodistribution and chromosomal aberrations in melanoma cells induced by ionizing radiation as potential treatment agent. Dendrimers were first conjugated with Suc-HYNIC (succinimidyl 6-hydrazinopyridine-3-carboxylic acid hydrochloride). Dendrimer-HYNIC was then incubated with 188 ReO 4 - . Biodistribution was performed administrating 188 Re-dendrimer to normal (NM) or melanoma-bearing mice (MBM). Chromosome aberration test was conducted in order to measure treatment capacity of 188 Re-dendrimer in melanoma cells. Radiolabeling yield of dendrimer was approx. 70%. Biodistribution studies in NM showed blood clearance with hepatic and renal depuration. MBM showed a similar pattern of biodistribution with tumor uptake of 6% of injected dose. Aberrant metaphases quantified in control cells were 7%, increasing to 29.5% in cells treated with 15μCi (0.555 MBq) of 188 Re-dendrimer for 24 h. 188 Re-dendrimer can produce double-stranded breaks in DNA induced by ionizing radiation in melanoma cells in vitro.

  14. Modulation of radiochemoimmunotherapy-induced B16 melanoma cell death by the pan-caspase inhibitor zVAD-fmk induces anti-tumor immunity in a HMGB1-, nucleotide- and T-cell-dependent manner

    PubMed Central

    Werthmöller, N; Frey, B; Wunderlich, R; Fietkau, R; Gaipl, U S

    2015-01-01

    One prerequisite that radiotherapy (RT) and chemotherapy (CT) result in anti-tumor immune responses is triggering of immunogenic cell death forms such as necroptosis. The latter is inducible by inhibition of apoptosis with the pan-caspase inhibitor zVAD-fmk. The design of multimodal therapies that overcome melanoma's resistance to apoptosis is a big challenge of oncoimmunology. As hints exist that immune stimulation by hyperthermia (HT) augments the efficacy of melanoma therapies and that tumors can be sensitized for RT with zVAD-fmk, we asked whether combinations of RT with dacarbazine (DTIC) and/or HT induce immunogenic melanoma cell death and how this is especially influenced by zVAD-fmk. Necroptosis was inducible in poorly immunogenic B16-F10 melanoma cells and zVAD-fmk generally increased melanoma cell necrosis concomitantly with the release of HMGB1. Supernatants (SNs) of melanoma cells whose cell death was modulated with zVAD-fmk induced an upregulation of the activation markers CD86 and MHCII on macrophages. The same was seen on dendritic cells (DCs), but only when zVAD-fmk was added to multimodal tumor treatments including DTIC. DCs of MyD88 KO mice and DCs incubated with SNs containing apyrase did not increase the expression of these activation markers on their surface. The in vivo experiments revealed that zVAD-fmk decreases the tumor growth significantly and results in a significantly reduced tumor infiltration of Tregs when added to multimodal treatment of the tumor with RT, DTIC and HT. Further, a significantly increased DC and CD8+ T-cell infiltration into the tumor and in the draining lymph nodes was induced, as well as an increased expression of IFNγ by CD8+ T cells. However, zVAD-fmk did not further reduce tumor growth in MyD88 KO mice, mice treated with apyrase or RAG KO mice. We conclude that HMGB1, nucleotides and CD8+ T cells mediate zVAD-fmk induced anti-melanoma immune reactions in multimodal therapy settings. PMID:25973681

  15. Ascorbate supplementation inhibits growth and metastasis of B16FO melanoma and 4T1 breast cancer cells in vitamin C-deficient mice.

    PubMed

    Cha, John; Roomi, M Waheed; Ivanov, Vadim; Kalinovsky, Tatiana; Niedzwiecki, Aleksandra; Rath, Matthias

    2013-01-01

    Degradation of the extracellular matrix (ECM) plays a critical role in the formation of tumors and metastasis and has been found to correlate with the aggressiveness of tumor growth and invasiveness of cancer. Ascorbic acid, which is known to be essential for the structural integrity of the intercellular matrix, is not produced by humans and must be obtained from the diet. Cancer patients have been shown to have very low reserves of ascorbic acid. Our main objective was to determine the effect of ascorbate supplementation on metastasis, tumor growth and tumor immunohistochemistry in mice unable to synthesize ascorbic acid [gulonolactone oxidase (gulo) knockout (KO)] when challenged with B16FO melanoma or 4T1 breast cancer cells. Gulo KO female mice 36-38 weeks of age were deprived of or maintained on ascorbate in food and water for 4 weeks prior to and 2 weeks post intraperitoneal (IP) injection of 5x105 B16FO murine melanoma cells or to injection of 5x105 4T1 breast cancer cells into the mammary pad of mice. Ascorbate-supplemented gulo KO mice injected with B16FO melanoma cells demonstrated significant reduction (by 71%, p=0.005) in tumor metastasis compared to gulo KO mice on the control diet. The mean tumor weight in ascorbate supplemented mice injected with 4T1 cells was reduced by 28% compared to tumor weight in scorbutic mice. Scorbutic tumors demonstrated large dark cores, associated with increased necrotic areas and breaches to the tumor surface, apoptosis and matrix metalloproteinase-9 (MMP-9), and weak, disorganized or missing collagen I tumor capsule. In contrast, the ascorbate-supplemented group tumors had smaller fainter colored cores and confined areas of necrosis/apoptosis with no breaches from the core to the outside of the tumor and a robust collagen I tumor capsule. In both studies, ascorbate supplementation of gulo KO mice resulted in profoundly decreased serum inflammatory cytokine interleukin (IL)-6 (99% decrease, p=0.01 in the B16F0

  16. Induction of autocrine factor inhibiting cell motility from murine B16-BL6 melanoma cells by alpha-melanocyte stimulating hormone.

    PubMed

    Murata, J; Ayukawa, K; Ogasawara, M; Watanabe, H; Saiki, I

    1999-03-15

    We have previously reported that neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) successfully inhibited Matrigel invasion and haptotactic migration of B16-BL6 melanoma cells towards both fibronectin and laminin without affecting their growth. In the present study, we investigated the inhibitory mechanism of tumor cell motility by alpha-MSH. Alpha-MSH significantly blocked the autocrine motility factor (AMF)-enhanced cell motility. However, alpha-MSH did neither prevent the secretion of AMF from B16-BL6 cells nor alter the expression level of AMF receptor (gp78). On the other hand, alpha-MSH induced the secretion of the motility inhibitory factor(s) from B16-BL6 cells in a concentration- and time-dependent manner. The induction of the motility inhibitor(s) was proportional to increasing levels of intracellular cAMP induced by alpha-MSH as well as forskolin, and the activity was abolished by an adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA). The motility-inhibiting activity in conditioned medium (CM) from alpha-MSH-treated B16-BL6 cells was found to have a m.w. below 3 kDa after fractionation. This activity was abolished by boiling but insensitive to trypsin. The treatment of tumor cells with cycloheximide reduced the activity in alpha-MSH-stimulated CM. Our results suggest that alpha-MSH inhibited the motility of B16-BL6 cells through induction of autocrine factor(s).

  17. Growth Inhibition and Differentiation of Murine Melanoma B16‐BL6 Cells Caused by the Combination of Cisplatin and Caffeine

    PubMed Central

    Tomita, Katsuro; Yasutake, Hidetoshi; Ueda, Yoshimichi; Tanaka, Motohiro; Sasaki, Takuma

    1989-01-01

    We preliminarily investigated the combined effects of cisplatin and caffeine on murine melanoma B16‐BL6 cells in vitro. When caffeine was added before or simultaneously with cisplatin, there was little growth inhibition. The addition of 2.0 mM caffeine after 1 h of exposure to cisplatin inhibited growth and induced cell differentiation. This treatment resulted in fewer cells, and the numbers of melanosomes and mitochondria and the amount of Golgi's complex and endoplasmic reticulum were increased. DNA histograms obtained by flow cytometry showed that cells treated with cisplatin alone accumulated in the G2/M phase, with a partial G2 block. The addition of 2.0 mM caffeine after 1 h of treatment with cisplatin reduced this block. Caffeine caused murine melanoma B16‐BL6 cells treated with cisplatin to differentiate, and this inhibited growth. PMID:2516852

  18. Engineered fusokine GIFT4 licenses the ability of B cells to trigger a tumoricidal T cell response

    PubMed Central

    Deng, Jiusheng; Yuan, Shala; Pennati, Andrea; Murphy, Jordan; Wu, Jian Hui; Lawson, David; Galipeau, Jacques

    2014-01-01

    Engineered chimeric cytokines can generate gain-of-function activity in immune cells. Here we report potent antitumor activity for a novel fusion cytokine generated by N-terminal coupling of GM-CSF to IL-4, generating a fusokine termed GIFT4. B cells treated with GIFT4 clustered GM-CSF and IL-4 receptors on the cell surface and displayed a pan-STAT hyperphosphorylation associated with acquisition of a distinct phenotype and function described to date. In C57BL/6J mice, administration of GIFT4 expanded endogenous B cells and suppressed the growth of B16F0 melanoma cells. Further, B16F0 melanoma cells engineered to secrete GIFT4 were rejected immunologically in a B cell-dependent manner. This effect was abolished when GIFT4-expressing B16F0 cells were implanted in B cell-deficient mice, confirming a B cell-dependent antitumor effect. Human GIFT4-licensed B cells primed cytotoxic T cells and specifically killed melanoma cells in vitro and in vivo. Taken together, our results demonstrated that GIFT4 could mediate expansion of B cells with potent antigen-specific effector function. GIFT4 may offer a novel immunotherapeutic tool and define a previously unrecognized potential for B cells in melanoma immunotherapy. PMID:24938765

  19. Immunobiologic effects of cytokine gene transfer of the B16-BL6 melanoma.

    PubMed

    Strome, S E; Krauss, J C; Cameron, M J; Forslund, K; Shu, S; Chang, A E

    1993-12-01

    The genetic modification of tumors offers an approach to modulate the host immune response to relatively weak native tumor antigens. We examined the immunobiologic effects of various cytokine genes transferred into the poorly immunogenic B16-BL6 murine melanoma. Retroviral expression vectors containing cDNAs for interleukin 2, interleukin 4, interferon gamma, or a neomycin-resistant control were electroporated into a B16-BL6 tumor clone. Selected transfected clones were examined for in vitro cytokine secretion and in vivo tumorigenicity. When cells from individual clones were injected intradermally into syngeneic mice, the interleukin 4-secreting clone grew significantly slower than did the neomycin-resistant transfected control, while the growth of the interleukin 2- and interferon gamma-expressing clones was not affected. Despite minimal cytokine secretion by interferon gamma-transfected cells, these cells expressed upregulated major histocompatibility class I antigen and were more susceptible to lysis by allosensitized cytotoxic T lymphocytes compared with parental or neomycin-resistant transfected tumor targets. We observed diverse immunobiologic effects associated with cytokine gene transfer into the B16-BL6 melanoma. Interleukin 4 transfection of tumor resulted in decreased in vivo tumorigenicity that may be related to a host immune response. Further studies to evaluate the host T-cell response to these gene-modified tumors are being investigated.

  20. Potential ability of hot water adzuki (Vigna angularis) extracts to inhibit the adhesion, invasion, and metastasis of murine B16 melanoma cells.

    PubMed

    Itoh, Tomohiro; Umekawa, Hayato; Furuichi, Yukio

    2005-03-01

    The 40% ethanol eluent of the fraction of hot-water extract from adzuki beans (EtEx.40) adsorbed onto DIAION HP-20 resin has many biological activities, for example, antioxidant, antitumorigenesis, and intestinal alpha-glucosidase suppressing activities. This study examined the inhibitory effect of EtEx.40 on experimental lung metastasis and the invasion of B16-BL6 melanoma cells. EtEx.40 was found significantly to reduce the number of tumor colonies. It also inhibited the adhesion and migration of B16-BL6 melanoma cells into extracellular matrix components and their invasion into reconstituted basement membrane (matrigel) without affecting cell proliferation in vitro. These in vivo data suggest that EtEx.40 possesses a strong antimetastatic ability, which might be a lead compound in functional food development.

  1. Melanogenesis stimulation in murine B16 melanoma cells by Piper nigrum leaf extract and its lignan constituents.

    PubMed

    Matsuda, Hideaki; Kawaguchi, Yoshiko; Yamazaki, Miho; Hirata, Noriko; Naruto, Shunsuke; Asanuma, Yusuke; Kaihatsu, Takayuki; Kubo, Michinori

    2004-10-01

    A methanolic extract from the leaves of Piper nigrum L. showed a significant stimulatory effect on melanogenesis in cultured murine B16 melanoma cells. Activity-guided fractionation of the methanolic extract led to the isolation of two known lignans, (-)-cubebin (1) and (-)-3,4-dimethoxy-3,4-desmethylenedioxycubebin (2), together with a new lignan, (-)-3-desmethoxycubebinin (3). Among these lignans, 1 and 2 showed a significant stimulatory activity of melanogenesis without any significant effects on cell proliferation.

  2. Noninvasive and label-free detection of circulating melanoma cells by in vivo photoacoustic flow cytometry

    NASA Astrophysics Data System (ADS)

    Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Wei, Xunbin

    2015-03-01

    Melanoma is a malignant tumor of melanocytes. Circulating melanoma cell has high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC). PAFC is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. In our research, we developed in vitro experiments to prove the ability of PAFC system of detecting PA signals from melanoma cells. For in vivo experiments, we constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells B16F10 with subcutaneous injection. PA signals were detected in the blood vessels of mouse ears in vivo. By counting circulating melanoma cells termly, we obtained the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation. Our PAFC system is an efficient tool to monitor melanoma metastases, cancer recurrence and therapeutic efficacy.

  3. Electroporation transiently decreases GJB2 (connexin 26) expression in B16/BL6 melanoma cell line.

    PubMed

    Rangel, Marcelo Monte Mór; Chaible, Lucas Martins; Nagamine, Marcia Kazumi; Mennecier, Gregory; Cogliati, Bruno; de Oliveira, Krishna Duro; Fukumasu, Heidge; Sinhorini, Idércio Luiz; Mir, Lluis Maria; Dagli, Maria Lúcia Zaidan

    2015-02-01

    Connexins are proteins that form gap junctions. Perturbations in the cell membrane reportedly promote changes in the expression profile of connexins. Electroporation promotes destabilization by applying electrical pulses, and this procedure is used in electrochemotherapy and gene therapy, among others. This in vitro work aimed to study the interference of electroporation on the expression profile of GJB2 (Cx26 gene) and Connexin 26 in melanoma cell line B16/BL6. The techniques of immunocytochemistry, Western blot, and real-time PCR were used. After electroporation, cells showed a transient decrease in GJB2 mRNA. The immunostaining of Cx26 showed no noticeable change after electroporation at different time points. However, Western blot showed a significant reduction in Cx26 30 min after electroporation. Our results showed that electroporation interferes transiently in the expression of Connexin 26 in melanoma and are consistent with the idea that electroporation is a process of intense stress that promotes cell homeostatic imbalance and results in disruption of cell physiological processes such as transcription and translation.

  4. Engineered fusokine GIFT4 licenses the ability of B cells to trigger a tumoricidal T-cell response.

    PubMed

    Deng, Jiusheng; Yuan, Shala; Pennati, Andrea; Murphy, Jordan; Wu, Jian Hui; Lawson, David; Galipeau, Jacques

    2014-08-01

    Engineered chimeric cytokines can generate gain-of-function activity in immune cells. Here, we report potent antitumor activity for a novel fusion cytokine generated by N-terminal coupling of GM-CSF to IL4, generating a fusokine termed GIFT4. B cells treated with GIFT4 clustered GM-CSF and IL4 receptors on the cell surface and displayed a pan-STAT hyperphosphorylation associated with acquisition of a distinct phenotype and function described to date. In C57BL/6J mice, administration of GIFT4 expanded endogenous B cells and suppressed the growth of B16F0 melanoma cells. Furthermore, B16F0 melanoma cells engineered to secrete GIFT4 were rejected immunologically in a B-cell-dependent manner. This effect was abolished when GIFT4-expressing B16F0 cells were implanted in B-cell-deficient mice, confirming a B-cell-dependent antitumor effect. Human GIFT4-licensed B cells primed cytotoxic T cells and specifically killed melanoma cells in vitro and in vivo. Taken together, our results demonstrated that GIFT4 could mediate expansion of B cells with potent antigen-specific effector function. GIFT4 may offer a novel immunotherapeutic tool and define a previously unrecognized potential for B cells in melanoma immunotherapy. ©2014 American Association for Cancer Research.

  5. Biochemical Characterization of Ferulic Acid and Caffeic Acid Which Effectively Inhibit Melanin Synthesis via Different Mechanisms in B16 Melanoma Cells.

    PubMed

    Maruyama, Hiroko; Kawakami, Fumitaka; Lwin, Thet-Thet; Imai, Motoki; Shamsa, Fazel

    2018-01-01

    In this study, we examined the inhibitory effects of ferulic acid and caffeic acid on melanin production using a murine B16 melanoma cell line. The mechanisms by which the two acids inhibit melanin production were investigated by evaluating their effects on the activity of tyrosinase, which is involved is the first step of melanin biosynthesis. Ferulic acid showed no toxicity against the melanoma cells at any dose, whereas caffeic acid exerted cellular toxicity at concentrations higher than 0.35 mM. Both ferulic and caffeic acids effectively inhibited melanin production in the B16 melanoma cells. Ferulic acid reduced tyrosinase activity by directly binding to the enzyme, whereas no binding was observed between caffeic acid and tyrosinase. Both ferulic acid and caffeic acid inhibited casein kinase 2 (CK2)-induced phosphorylation of tyrosinase in a dose-dependent manner in vitro. Ferulic acid was found to be a more effective inhibitor of melanin production than caffeic acid; this difference in the inhibitory efficacy between the two substances could be attributable to the difference in their tyrosine-binding activity. Our analysis revealed that both substances also inhibited the CK2-mediated phosphorylation of tyrosinase.

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

    PubMed Central

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

    2014-01-01

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

  7. Properties of kojic acid and curcumin: Assay on cell B16-F1

    NASA Astrophysics Data System (ADS)

    Sugiharto, Ariff, Arbakariya; Ahmad, Syahida; Hamid, Muhajir

    2016-03-01

    Ultra violet (UV) exposure and oxidative stress are casually linked to skin disorders. They can increase melanin synthesis, proliferation of melanocytes, and hyperpigmentation. It is possible that antioxidants or inhibitors may have a beneficial effect on skin health to reduce hyperpigmentation. In the last few years, a huge number of natural herbal extracts have been tested to reduce hyperpigmentation. The objective of this study was to determine and to compare of kojic acid and curcumin properties to viability cell B16-F1. In this study, our data showed that the viability of cell B16-F1 was 63.91% for kojic acid and 64.12% for curcumin at concentration 100 µg/ml. Further investigation assay of antioxidant activities, indicated that IC50 for kojic acid is 63.8 µg/ml and curcumin is 16.05 µg/ml. Based on the data, kojic acid and curcumin have potential antioxidant properties to reduce hyperpigmentation with low toxicity effect in cell B16-F1.

  8. Effect of inhibition of polyamine biosynthesis by DL-alpha-difluoromethylornithine on the growth and melanogenesis of B16 melanoma in vitro and in vivo.

    PubMed

    Sunkara, P S; Chang, C C; Prakash, N J; Lachmann, P J

    1985-09-01

    The objective of the present study was to investigate the effect of polyamine depletion by alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase, on the growth and differentiation of B16 melanoma cells grown in culture and also as solid tumors in mice. Polyamine depletion by DFMO (2.5 mM) resulted in a complete inhibition of cell growth in culture and a 90% inhibition of viability of melanoma cells as determined by clonogenic assay at the end of 7 days after DFMO treatment. These results indicate that polyamine depletion induced by DFMO is cytotoxic to B16 melanoma cells in culture. Furthermore a 2- to 5-fold increase in tyrosinase activity and 10-fold accumulation of melanine were observed in polyamine depleted cells compared to control cultures. These effects of DFMO could easily be reversed by the addition of putrescine simultaneously with DFMO. Administration of different doses of DFMO in drinking water to B16 melanoma tumor bearing mice also resulted in an increase in tyrosinase activity and a dose dependent inhibition (86-90%) of tumor growth. Although one cannot rule out the possibility of induction of differentiated phenotype as a result of antiproliferative activity of DFMO, the data presented indicate that the unique sensitivity of melanoma to DFMO may be due to a combination of cell growth inhibition and concomitant induction of differentiation upon polyamine depletion. The results of the present study indicate that polyamines play an important role in growth and differentiation of melanoma and also provide an example of inhibition of tumor cell growth by induction of cellular differentiation.

  9. Inhibitory effects of Morinda citrifolia extract and its constituents on melanogenesis in murine B16 melanoma cells.

    PubMed

    Masuda, Megumi; Itoh, Kimihisa; Murata, Kazuya; Naruto, Shunsuke; Uwaya, Akemi; Isami, Fumiyuki; Matsuda, Hideaki

    2012-01-01

    The objective of this study was to examine the effects of Morinda citrifolia (noni) extract and its constituents on α-melanocyte stimulating hormone (α-MSH)-stimulated melanogenesis in cultured murine B16 melanoma cells (B16 cells). A 50% ethanolic extract of noni seeds (MCS-ext) showed significant inhibition of melanogenesis with no effect on cell proliferation. MCS-ext was more active than noni leaf and fruit flesh extracts. Activity guided fractionation of MCS-ext led to the isolation of two lignans, 3,3'-bisdemethylpinoresinol (1) and americanin A (2), as active constituents. To elucidate the mechanism of melanogenesis inhibition by the lignans, α-MSH-stimulated B16 cells were treated with 1 (5 μM) and 2 (200 μM). Time-dependent increases of intracellular melanin content and tyrosinase activity, during 24 to 72 h, were inhibited significantly by treatment with the lignans. The activity of 1 was greater than that of 2. Western blot analysis suggested that the lignans inhibited melanogenesis by down regulation of the levels of phosphorylation of p38 mitogen-activated protein kinase, resulting in suppression of tyrosinase expression.

  10. Genetically fluorescent melanoma bone and organ metastasis models.

    PubMed

    Yang, M; Jiang, P; An, Z; Baranov, E; Li, L; Hasegawa, S; Al-Tuwaijri, M; Chishima, T; Shimada, H; Moossa, A R; Hoffman, R M

    1999-11-01

    We report here the establishment and metastatic properties of bright, highly stable, green fluorescent protein (GFP) expression transductants of the B16 mouse malignant melanoma cell line and the LOX human melanoma line. The highly fluorescent malignant melanoma cell lines allowed the visualization of skeletal and multiorgan metastases after i.v. injection of B16 cells in C57BL/6 mice and intradermal injection of LOX cells in nude mice. The melanoma cell lines were transduced with the pLEIN expression retroviral vector containing the GFP and neomycin resistance genes. Stable B16F0 and LOX clones expressing high levels of GFP were selected stepwise in vitro in levels of G418 of up to 800 microg/ml. Extensive bone and bone marrow metastases of B16F0 were visualized by GFP expression when the animals were sacrificed 3 weeks after cell implantation. Metastases for both cell lines were visualized in many organs, including the brain, lung, pleural membrane, liver, kidney, adrenal gland, lymph nodes, skeleton, muscle, and skin by GFP fluorescence. This is the first observation of experimental skeletal metastases of melanoma, which was made possible by GFP expression. These models should facilitate future studies of the mechanism and therapy of bone and multiorgan metastasis of melanoma.

  11. Comparison of the in vitro and in vivo effects of retinoids either alone or in combination with cisplatin and 5-fluorouracil on tumor development and metastasis of melanoma.

    PubMed

    Liu, Xin; Chan, Sui Yung; Ho, Paul Chi-Lui

    2008-12-01

    Retinoids have previously been reported to inhibit proliferation of melanoma cell lines in vitro. However, the relative antimetastatic efficacy of various retinoids on melanoma in vivo is unknown. Therefore, we investigated the effects of different retinoids on the invasion and metastasis of murine melanoma B16-F10 cells in vitro and in vivo. Based on the findings, the antitumor effects of a selected retinoid either alone or in combination with cisplatin were also investigated in a preclinical mouse melanoma model. Cell proliferation and invasion analyses of murine melanoma B16-F10 cells were assessed in the presence of different retinoids, either alone or in combination with cisplatin (CDDP) or 5-fluorouracil (5-FU). Experimental lung metastasis assay was performed in this study to investigate the antimetastatic efficacy of retinoids. Additionally, a mouse melanoma model was used to assess the antitumor efficacy of a selected retinoid in combination with cisplatin. Retinoids showed significant antiproliferation and anti-invasion effects on murine melanoma B16-F10 cells. Pretreatment with retinoids increased the sensitivity to CDDP but not to 5-FU in in-vitro. Moreover, the number of metastatic colonies formed in the lungs of mice injected intravenously with B16-F10 cells was significantly reduced by injecting the respective retinoid once a day for 10 days. Treatment with a combination of cisplatin and 13-cis-retinoic acid resulted in a significant reduction in primary tumor size and the number of lung metastatic nodules in melanoma-bearing mice. These results suggest that retinoids not only exhibit antimetastatic effect, but also enhance the antitumor activity of cisplatin in vivo.

  12. A novel synthetic oleanane triterpenoid suppresses adhesion, migration and invasion of highly metastatic melanoma cells by modulating gelatinase signaling axis

    PubMed Central

    Sinha, Dona; Dutta, Kaustav; Ganguly, Kirat K.; Biswas, Jaydip; Bishayee, Anupam

    2014-01-01

    Background A methyl derivative natural triterpenoid amooranin (methyl-25-hydroxy-3-oxoolean-12-en-28-oate, AMR-Me) has been found to possess antiproliferative, proapoptotic and anti-inflammatory effects against established tumor cells. Large-scale synthesis of pure AMR-Me has eliminated the need of the natural phytochemical for further development of AMR-Me as an anticancer drug. Metastatic melanoma is a fatal form of cutaneous malignancy with poor prognosis and limited therapeutic options. It was hypothesized that antitumor pharmacological effect of AMR-Me could be linked to AMR-Me-mediated suppression of the metastatic potential of B16F10 murine melanoma. Methods AMR-Me was assessed for its antimetastatic efficacy by cell adhesion, migration and invasion assays in B16F10 cells. The signaling crosstalk was explored by gelatin zymography, Western blot, ELISA and immunocytochemistry. Results The results elicited that AMR-Me was successful in restricting the adhesion, migration and invasion of highly metastatic cells. The antimetastatic potential of this compound may be attributed to the reduced expression of membrane type 1 metalloproteinase (MT1-MMP) and matrix metalloproteinases (MMP-2 and MMP-9). AMR-Me was found to downregulate vascular endothelial growth factor (VEGF)/prosphorylated forms of focal adhesion kinase (pFAK397)/Jun N-terminus kinase (pJNK)/extracellular signal-regulated kinase (pERK). This, in turn, inhibited transcription factor nuclear factor-κB (NF-κB) and transactivation of MMPs. Moreover the activation of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) might have influenced the downmodulation of MT1-MMP, MMP-2 and MMP-9. Conclusion AMR-Me suppresses the activity of MT1-MMP, MMP-2 and MMP-9 by downregulation of VEGF/pFAK397/pJNK/pERK/NF-κB and activation of TIMP-1 and TIMP-2 in metastatic melanoma cell line, B16F10. General significance AMR-Me has the potential as an effective anticancer drug for metastatic melanoma which is a

  13. Relationship between 4-hydroxyanisole toxicity and dopa oxidase activity for three melanoma cell lines.

    PubMed

    Rodriguez-Vicente, J; Vicente-Ortega, V; Canteras-Jordana, M; Calderon-Rubiales, F

    1997-10-01

    We studied the response of mouse B16F10 and SK-MEL-28 and SK-MEL-1 human melanoma cell lines to treatment with 4-hydroxyanisole (4-HA), and attempted to relate the response to the dopa oxidase levels and the morphological characteristics of each cell line. Clear dose-response curves were observed after 24 h of treatment in each cell line, the 4-HA being more toxic to the B16F10 cells, with an ID50 value of 215 microM. This was much lower than that observed for the SK-MEL-28 and SK-MEL-1 cell lines (ID50 of 5.98 mM and 7.17 mM, respectively). There was a direct relationship between toxicity levels and dopa oxidase activity, since the highest specific activity was obtained for B16F10 (15.9 mU), while lower activity was registered for SK-MEL-28 (4.59 mU) and SK-MEL-1 (1.24 mU), which also showed lower 4-HA toxicity. Morphologically, we observed the typical characteristics of cellular injury, with swelling and dilation of the internal membranes and organelles, an increased number of vacuoles, and an increased number of abnormal multilamellar melanosomes or thick clumps of irregularly distributed melanin. On the other hand, we observed that the two cell lines with the lowest dopa oxidase activity contained more mature fully melanized melanosomes than B16F10, pointing to possible alterations in the melanosome transference mechanism and lower enzymatic activity in the mature melanosomes of these two human cell lines.

  14. Azelaic acid was sensitizing effect in the chemotherapeutic treatment of several melanoma cell lines.

    PubMed

    Rodriguez-Vicente, J; Vicente-Ortega, V; Canteras-Jordana

    1996-12-01

    Chemotherapy for melanoma results in low response and must be reinforced with sensitizer compounds. We believed that azelaic acid (AZA) could modulate melanomas' resistance to antineoplastics. Therefore we tried to compare in vitro treatment with antineoplastics alone versus AZA treatment followed by antineoplastics. We carried out MTT assays to evaluate the cytotoxicity of melphalan, lomustine (CCNU), fotemustine, and 4-Hydroxyanisole (4-HA) on three melanoma lines (B16F10, SK-MEL-28, and SK-MEL-1), and the modulating effect of pretreatment with AZA (1 mM). AZA showed a dose-dependent antineoplastic activity on the three lines. Melphalan was the most active drug followed by CCNU, fotemustine, and 4-HA. The most sensitive line was B16F10 and the least sensitive was SK-meL-1. Previous treatment with AZA of B16F10 reinforced the effect of melphalan (2.5 times), CCNU (10 times), and fotemustine (14 times); whereas for SK-MEL-28 and SK-MEL-1, only the cytotoxicity of CCNU and fotemustine increased. An antagonist effect was produced by 4-HA on all three lines. We concluded that AZA enhances in vitro cytotoxicity of CCNU and fotemustine.

  15. Synthesis of (S)-(+)-decursin and its analogues as potent inhibitors of melanin formation in B16 murine melanoma cells.

    PubMed

    Lee, Kyeong; Lee, Jee-Hyun; Boovanahalli, Shanthaveerappa K; Choi, Yongseok; Choo, Soo-Jin; Yoo, Ick-dong; Kim, Dong Hee; Yun, Mi Young; Lee, Gye Won; Song, Gyu-Yong

    2010-12-01

    We report the synthesis of a novel series of highly potent melanin inhibitors which were obtained through structural modification of an anticancer compound S-(+)-decursinol. The in vitro inhibitory potencies of the newly synthesized compounds were evaluated against α-MSH induced melanin production in B16 murine melanoma cells. Among the compounds evaluated, compounds 2, 3, 6b, 7a, 7b, 8a and 8b emerged as highly potent inhibitors of melanin production. Besides, these compounds demonstrated significantly low cytotoxicity. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

  16. Isoprenoid-substituted flavonoids from wood of Artocarpus heterophyllus on B16 melanoma cells: cytotoxicity and structural criteria.

    PubMed

    Arung, Enos Tangke; Yoshikawa, Keisuke; Shimizu, Kuniyoshi; Kondo, Ryuichiro

    2010-03-01

    As a result of cytotoxicity-guided fractionation, nine flavonoids, artocarpin (1), cudraflavone C (2), 6-prenylapigenin (3), kuwanon C (4), norartocarpin (5), albanin A (6), cudraflavone B (7), brosimone I (8) and artocarpanone (9) were identified from the methanol extract of the wood of Artocarpus heterophyllus, known commonly as Nangka in Indonesia. A structure-activity investigation of the effect of these isolated compounds (1-9) and structurally related compounds on B16 melanoma cells indicated that isoprenoid moiety substitutions in flavonoids enhance their cytotoxicity, and that the position of attachment and the number of isoprenoid-substituent moieties per molecule influence flavonoid cytotoxicity. 2009 Elsevier B.V. All rights reserved.

  17. Benzofuroxan derivatives N-Br and N-I induce intrinsic apoptosis in melanoma cells by regulating AKT/BIM signaling and display anti metastatic activity in vivo.

    PubMed

    Farias, C F; Massaoka, M H; Girola, N; Azevedo, R A; Ferreira, A K; Jorge, S D; Tavares, L C; Figueiredo, C R; Travassos, L R

    2015-10-27

    Malignant melanoma is an aggressive type of skin cancer, and despite recent advances in treatment, the survival rate of the metastatic form remains low. Nifuroxazide analogues are drugs based on the substitution of the nitrofuran group by benzofuroxan, in view of the pharmacophore similarity of the nitro group, improving bioavailability, with higher intrinsic activity and less toxicity. Benzofuroxan activity involves the intracellular production of free-radical species. In the present work, we evaluated the antitumor effects of different benzofuroxan derivatives in a murine melanoma model. B16F10-Nex2 melanoma cells were used to investigate the antitumor effects of Benzofuroxan derivatives in vitro and in a syngeneic melanoma model in C57Bl/6 mice. Cytotoxicity, morphological changes and reactive oxygen species (ROS) were assessed by a diphenyltetrasolium reagent, optical and fluorescence microscopy, respectively. Annexin-V binding and mitochondrial integrity were analyzed by flow cytometry. Western blotting and colorimetry identified cell signaling proteins. Benzofuroxan N-Br and N-I derivatives were active against murine and human tumor cell lines, exerting significant protection against metastatic melanoma in a syngeneic model. N-Br and N-I induce apoptosis in melanoma cells, evidenced by specific morphological changes, DNA condensation and degradation, and phosphatidylserine translocation in the plasma membrane. The intrinsic mitochondrial pathway in B16F10-Nex2 cells is suggested owing to reduced outer membrane potential in mitochondria, followed by caspase -9, -3 activation and cleavage of PARP. The cytotoxicity of N-Br and N-I in B16F10-Nex2 cells is mediated by the generation of ROS, inhibited by pre-incubation of the cells with N-acetylcysteine (NAC). The induction of ROS by N-Br and N-I resulted in the inhibition of AKT activation, an important molecule related to tumor cell survival, followed by upregulation of BIM. We conclude that N-Br and N-I are

  18. Monitoring the Systemic Human Memory B Cell Compartment of Melanoma Patients for Anti-Tumor IgG Antibodies

    PubMed Central

    Gilbert, Amy E.; Karagiannis, Panagiotis; Dodev, Tihomir; Koers, Alexander; Lacy, Katie; Josephs, Debra H.; Takhar, Pooja; Geh, Jenny L. C.; Healy, Ciaran; Harries, Mark; Acland, Katharine M.; Rudman, Sarah M.; Beavil, Rebecca L.; Blower, Philip J.; Beavil, Andrew J.; Gould, Hannah J.; Spicer, James; Nestle, Frank O.; Karagiannis, Sophia N.

    2011-01-01

    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. PMID:21559411

  19. Evaluation of a novel Arg-Gly-Asp-conjugated α-melanocyte stimulating hormone hybrid peptide for potential melanoma therapy.

    PubMed

    Yang, Jianquan; Guo, Haixun; Gallazzi, Fabio; Berwick, Marianne; Padilla, R Steven; Miao, Yubin

    2009-08-19

    The purpose of this study was to determine whether Arg-Gly-Asp (RGD)-conjugated α-melanocyte stimulating hormone (α-MSH) hybrid peptide could be employed to target melanocortin-1 (MC1) receptor for potential melanoma therapy. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys(3,4,10), DPhe(7), Arg(11)]α-MSH(3-13) {(Arg(11))CCMSH} to generate RGD-Lys-(Arg(11))CCMSH hybrid peptide. The MC1 receptor binding affinity of RGD-Lys-(Arg(11))CCMSH was determined in B16/F1 melanoma cells. The internalization and efflux, melanoma targeting and pharmacokinetic properties and single photon emission computed tomography/CT (SPECT/CT) imaging of (99m)Tc-RGD-Lys-(Arg(11))CCMSH were determined in B16/F1 melanoma cells and melanoma-bearing C57 mice. Clonogenic cytotoxic effect of RGD-Lys-(Arg(11))CCMSH was examined in B16/F1 melanoma cells. RGD-Lys-(Arg(11))CCMSH displayed 2.1 nM MC1 receptor binding affinity. (99m)Tc-RGD-Lys-(Arg(11))CCMSH showed rapid internalization and extended retention in B16/F1 cells. The cellular uptake of (99m)Tc-RGD-Lys-(Arg(11))CCMSH was MC1 receptor-mediated. (99m)Tc-RGD-Lys-(Arg(11))CCMSH exhibited high tumor uptake (14.83 ± 2.94% ID/g 2 h postinjection) and prolonged tumor retention (7.59 ± 2.04% ID/g 24 h postinjection) in B16/F1 melanoma-bearing mice. Nontarget organ uptakes were generally low except for the kidneys. Whole-body clearance of (99m)Tc-RGD-Lys-(Arg(11))CCMSH was rapid, with approximately 62% of the injected radioactivity cleared through the urinary system by 2 h postinjection. Flank melanoma tumors were clearly imaged by small animal SPECT/CT using (99m)Tc-RGD-Lys-(Arg(11))CCMSH as an imaging probe 2 h postinjection. Single treatment (3 h incubation) with 100 nM of RGD-Lys-(Arg(11))CCMSH significantly (p < 0.05) decreased the clonogenic survival of B16/F1 cells by 65% compared to the untreated control cells. Favorable melanoma targeting property of (99m)Tc-RGD-Lys-(Arg(11))CCMSH and remarkable cytotoxic effect of RGD

  20. Combination Immunotherapy of B16 Melanoma Using Anti–Cytotoxic T Lymphocyte–Associated Antigen 4 (Ctla-4) and Granulocyte/Macrophage Colony-Stimulating Factor (Gm-Csf)-Producing Vaccines Induces Rejection of Subcutaneous and Metastatic Tumors Accompanied by Autoimmune Depigmentation

    PubMed Central

    van Elsas, Andrea; Hurwitz, Arthur A.; Allison, James P.

    1999-01-01

    We examined the effectiveness of cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) blockade, alone or in combination with a granulocyte/macrophage colony-stimulating factor (GM-CSF)–expressing tumor cell vaccine, on rejection of the highly tumorigenic, poorly immunogenic murine melanoma B16-BL6. Recently established tumors could be eradicated in 80% (68/85) of the cases using combination treatment, whereas each treatment by itself showed little or no effect. Tumor rejection was dependent on CD8+ and NK1.1+ cells but occurred irrespective of the presence of CD4+ T cells. Mice surviving a primary challenge rejected a secondary challenge with B16-BL6 or the parental B16-F0 line. The same treatment regimen was found to be therapeutically effective against outgrowth of preestablished B16-F10 lung metastases, inducing long-term survival. Of all mice surviving B16-BL6 or B16-F10 tumors after combination treatment, 56% (38/68) developed depigmentation, starting at the site of vaccination or challenge and in most cases progressing to distant locations. Depigmentation was found to occur in CD4-depleted mice, strongly suggesting that the effect was mediated by CTLs. This study shows that CTLA-4 blockade provides a powerful tool to enhance T cell activation and memory against a poorly immunogenic spontaneous murine tumor and that this may involve recruitment of autoreactive T cells. PMID:10430624

  1. Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation.

    PubMed

    van Elsas, A; Hurwitz, A A; Allison, J P

    1999-08-02

    We examined the effectiveness of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade, alone or in combination with a granulocyte/macrophage colony-stimulating factor (GM-CSF)-expressing tumor cell vaccine, on rejection of the highly tumorigenic, poorly immunogenic murine melanoma B16-BL6. Recently established tumors could be eradicated in 80% (68/85) of the cases using combination treatment, whereas each treatment by itself showed little or no effect. Tumor rejection was dependent on CD8(+) and NK1.1(+) cells but occurred irrespective of the presence of CD4(+) T cells. Mice surviving a primary challenge rejected a secondary challenge with B16-BL6 or the parental B16-F0 line. The same treatment regimen was found to be therapeutically effective against outgrowth of preestablished B16-F10 lung metastases, inducing long-term survival. Of all mice surviving B16-BL6 or B16-F10 tumors after combination treatment, 56% (38/68) developed depigmentation, starting at the site of vaccination or challenge and in most cases progressing to distant locations. Depigmentation was found to occur in CD4-depleted mice, strongly suggesting that the effect was mediated by CTLs. This study shows that CTLA-4 blockade provides a powerful tool to enhance T cell activation and memory against a poorly immunogenic spontaneous murine tumor and that this may involve recruitment of autoreactive T cells.

  2. Anti-melanogenic activity of schaftoside in Rhizoma Arisaematis by increasing autophagy in B16F1 cells.

    PubMed

    Kim, Pan Soo; Shin, Ji Hyun; Jo, Doo Sin; Shin, Dong Woon; Choi, Dong-Hwa; Kim, Woo Jung; Park, Kyuhee; Kim, Jin Kyu; Joo, Chul Gue; Lee, Jong Suk; Choi, Yongmun; Shin, Yong Won; Shin, Joong Jin; Jeon, Hong Bae; Seo, Jin-Ho; Cho, Dong-Hyung

    2018-06-08

    Skin pigmentation involves multiple processes, including melanin synthesis, transport, and melanosome release. Melanin content determines skin color and protects against UV radiation-induced damage. Autophagy is a cooperative process between autophagosomes and lysosomes that degrades cellular components and organelles. In the present study, B16F1 cells were treated with Rhizoma Arisaematis extract (RA) and assessed for pigmentation and autophagy regulation. RA treatment suppressed the α-MSH-stimulated increase of melanogenesis and down-regulated the expression of tyrosinase and TRP1 proteins in B16F1 cells. In addition, autophagy was activated in RA-treated cells. Inhibition of autophagy reduced the anti-melanogenic activity of RA in α-MSH-treated B16F1 cells. We identified schaftoside as an effector molecule by LC-MS analysis of RA. Consistently, treatment of schaftoside showed anti-melanogenic effect and induced autophagy activation in B16F1 cells. Inhibition of autophagy by 3 MA treatment reduced the anti-melanogenic effect of the schaftoside and recovered expression level of melanogenesis regulators in α-MSH-treated B16F1 cells. Taken together, our results suggest that schaftoside from RA inhibits skin pigmentation through modulation of autophagy. Copyright © 2018. Published by Elsevier Inc.

  3. Neural cell adhesion molecule potentiates invasion and metastasis of melanoma cells through CAMP-dependent protein kinase and phosphatidylinositol 3-kinase pathways.

    PubMed

    Shi, Yu; Liu, Rui; Zhang, Si; Xia, Yin-Yan; Yang, Hai-Jie; Guo, Ke; Zeng, Qi; Feng, Zhi-Wei

    2011-04-01

    Neural cell adhesion molecule (NCAM) has been implicated in tumor metastasis yet its function in melanoma progression remains unclear. Here, we demonstrate that stably silencing NCAM expression in mouse melanoma B16F0 cells perturbs their cellular invasion and metastatic dissemination in vivo. The pro-invasive function of NCAM is exerted via dual mechanisms involving both cAMP-dependent protein kinase (PKA) and phosphatidylinositol 3-kinase (PI3K) pathways. Pharmacologic inhibition of PKA and PI3K leads to impaired cellular invasion. In contrast, forced expression of constitutively activated Akt, the major downstream target of PI3K, restores the defective cellular invasiveness of NCAM knock-down (KD) B16F0 cells. Furthermore, attenuation of either PKA or Akt activity in NCAM KD cells is shown to affect their common downstream target, transcription factor cAMP response element binding protein (CREB), which in turn down-regulates mRNA expression of matrix metalloproteinase-2 (MMP-2), thus contributes to impaired cellular invasion and metastasis of melanoma cells. Together, these findings indicate that NCAM potentiates cellular invasion and metastasis of melanoma cells through stimulation of PKA and PI3K signaling pathways thus suggesting the potential implication of anti-NCAM strategy in melanoma treatment. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Viwithan, a Standardized Withania somnifera Root Extract Induces Apoptosis in Murine Melanoma Cells.

    PubMed

    Sudeep, H V; Gouthamchandra, K; Venkatesh, B J; Prasad, K Shyam

    2018-01-01

    Withania somnifera is an Indian medicinal herb known for the multipotential ability to cure various therapeutic ailments as described in the ayurvedic system of medicine. In the present study, we have evaluated the antiproliferative activity of a standardized W. somnifera root extract (Viwithan) against different human and murine cancer cell lines. The cytotoxicity of Viwithan was determined using thiazolyl blue tetrazolium blue assay and crystal violet staining. The apoptotic changes in B16F1 cells following treatment with Viwithan were observed by acridine orange/ethidium bromide (AO/EB) staining and DNA fragmentation assay. The binding affinity of withanolides in Viwithan with antiapoptotic proteins B-cell lymphoma 2, B-cell lymphoma-extra large, and myeloid cell leukemia 1 (MCL-1) were studied using in silico approach. The half maximal inhibitory concentration (IC50) values of Viwithan against liver hepatocellular carcinoma, Henrietta Lacks cervical carcinoma cells, human colorectal carcinoma cell line, and Ehrlich ascites carcinoma cells were 1830, 968, 2715, and 633 μg/ml, respectively. Interestingly, Viwithan was highly effective against B16F1 cells with an IC50 value of 220 μg/ml after 24 h treatment. The morphological alterations of apoptotic cell death were clearly observed in the AO/EB-stained cells after treatment with Viwithan. Viwithan induced late apoptotic changes in treated B16F1 cells as evident by the ladder formation of fragmented DNA in a time-dependent manner. The findings of molecular docking showed that withanolides present in Viwithan have a more binding affinity with the antiapoptotic proteins, particularly MCL-1. We have reported for the first time that Viwithan with 5% withanolides has a potent cytotoxic effect, particularly against B16F1 murine melanoma cells among the different cancer cell lines tested. The present study reports for the first time that Viwithan, a standardized 5% Withania somnifera root extract, has potent

  5. Promotion or suppression of experimental metastasis of B16 melanoma cells after oral administration of lapachol

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Maeda, Masayo; Murakami, Manabu; Takegami, Tsutomu

    Lapachol [2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone] is a vitamin K antagonist with antitumor activity. The effect of lapachol on the experimental metastasis of murine B16BL6 melanoma cells was examined. A single oral administration of a high toxic dose of lapachol (80-100 mg/kg) 6 h before iv injection of tumor cells drastically promoted metastasis. This promotion of metastasis was also observed in T-cell-deficient mice and NK-suppressed mice. In vitro treatment of B16BL6 cells with lapachol promoted metastasis only slightly, indicating that lapachol promotes metastasis primarily by affecting host factors other than T cells and NK cells. A single oral administration of warfarin, the most commonlymore » used vitamin K antagonist, 6 h before iv injection of tumor cells also drastically promoted the metastasis of B16BL6 cells. The promotion of metastasis by lapachol and warfarin was almost completely suppressed by preadministration of vitamin K3, indicating that the promotion of metastasis by lapachol was derived from vitamin K antagonism. Six hours after oral administration of lapachol or warfarin, the protein C level was reduced maximally, without elongation of prothrombin time. These observations suggest that a high toxic dose of lapachol promotes metastasis by inducing a hypercoagulable state as a result of vitamin K-dependent pathway inhibition. On the other hand, serial oral administration of low non-toxic doses of lapachol (5-20 mg/kg) weakly but significantly suppressed metastasis by an unknown mechanism, suggesting the possible use of lapachol as an anti-metastatic agent.« less

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

    PubMed

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

    2016-03-01

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

  7. Inhibition of the metastatic spread and growth of B16-BL6 murine melanoma by a synthetic matrix metalloproteinase inhibitor.

    PubMed

    Chirivi, R G; Garofalo, A; Crimmin, M J; Bawden, L J; Stoppacciaro, A; Brown, P D; Giavazzi, R

    1994-08-01

    The synthetic matrix metalloproteinase inhibitor batimastat was tested for its ability to inhibit growth and metastatic spread of the B16-BL6 murine melanoma in syngeneic C57BL/6N mice. Intraperitoneal administration of batimastat resulted in a significant inhibition in the number of lung colonies produced by B16-BL6 cells injected i.v. The effect of batimastat on spontaneous metastases was examined in mice inoculated in the hind footpad with B16-BL6 melanoma. The primary tumor was removed surgically after 26-28 days. Batimastat was administered twice a day from day 14 to day 28 (pre-surgery) or from day 26 to day 44 (post-surgery). With both protocols, the median number of lung metastases was not significantly affected, but there was a significant reduction in the weight of the metastases. Finally, the effect of batimastat was examined on s.c. growth of B16-BL6 melanoma. Batimastat administered daily, starting at day of tumor transplantation, resulted in a significant growth delay, whereas treatment starting at advanced stage tumor only reduced tumor growth marginally. Our results indicate that a matrix metalloproteinase inhibitor can not only prevent the colonization of secondary organs by B16-BL6 cells but also limit the growth of solid tumors.

  8. Effects of dipotassium-trioxohydroxytetrafluorotriborate, K2[B3O3F4OH], on cell viability and gene expression of common human cancer drug targets in a melanoma cell line.

    PubMed

    Pojskic, Lejla; Haveric, Sanin; Lojo-Kadric, Naida; Hadzic, Maida; Haveric, Anja; Galic, Zoran; Galic, Borivoj; Vullo, Daniela; Supuran, Claudiu T; Milos, Mladen

    2016-12-01

    Recently it was found that dipotassium-trioxohydroxytetrafluorotriborate, K2(B3O3F4OH), is a potent and highly specific inhibitor of precancerous cell processes. We conducted gene expression profiling of human melanoma cells before and after treatment with two concentrations (0.1 and 1 mM) of this boron inorganic derivative in order to assess its effects on deregulation of genes associated with tumor pathways. Parallel trypan blue exclusion assay was performed to assess the cytotoxicity effects of this chemical. Treatment with K2(B3O3F4OH) induced a significant decrease of cell viability in melanoma cellline at both tested concentrations. Furthermore, these treatments caused deregulation of more than 30 genes known as common anti-tumor drug targets. IGF-1 and hTERT were found to be significantly downregulated and this result may imply potential use of K2(B3O3F4OH) as an inhibitor or human telomerase and insulin-like growth factor 1, both of which are associated with various tumor pathways.

  9. Nanodiamonds coupled with 5,7-dimethoxycoumarin, a plant bioactive metabolite, interfere with the mitotic process in B16F10 cells altering the actin organization.

    PubMed

    Gismondi, Angelo; Nanni, Valentina; Reina, Giacomo; Orlanducci, Silvia; Terranova, Maria Letizia; Canini, Antonella

    2016-01-01

    For the first time, we coupled reduced detonation nanodiamonds (NDs) with a plant secondary metabolite, citropten (5,7-dimethoxycoumarin), and demonstrated how this complex was able to reduce B16F10 tumor cell growth more effectively than treatment with the pure molecule. These results encouraged us to find out the specific mechanism underlying this phenomenon. Internalization kinetics and quantification of citropten in cells after treatment with its pure or ND-conjugated form were measured, and it was revealed that the coupling between NDs and citropten was essential for the biological properties of the complex. We showed that the adduct was not able to induce apoptosis, senescence, or differentiation, but it determined cell cycle arrest, morphological changes, and alteration of mRNA levels of the cytoskeletal-related genes. The identification of metaphasic nuclei and irregular disposition of β-actin in the cell cytoplasm supported the hypothesis that citropten conjugated with NDs showed antimitotic properties in B16F10 cells. This work can be considered a pioneering piece of research that could promote and support the biomedical use of plant drug-functionalized NDs in cancer therapy.

  10. Nanodiamonds coupled with 5,7-dimethoxycoumarin, a plant bioactive metabolite, interfere with the mitotic process in B16F10 cells altering the actin organization

    PubMed Central

    Gismondi, Angelo; Nanni, Valentina; Reina, Giacomo; Orlanducci, Silvia; Terranova, Maria Letizia; Canini, Antonella

    2016-01-01

    For the first time, we coupled reduced detonation nanodiamonds (NDs) with a plant secondary metabolite, citropten (5,7-dimethoxycoumarin), and demonstrated how this complex was able to reduce B16F10 tumor cell growth more effectively than treatment with the pure molecule. These results encouraged us to find out the specific mechanism underlying this phenomenon. Internalization kinetics and quantification of citropten in cells after treatment with its pure or ND-conjugated form were measured, and it was revealed that the coupling between NDs and citropten was essential for the biological properties of the complex. We showed that the adduct was not able to induce apoptosis, senescence, or differentiation, but it determined cell cycle arrest, morphological changes, and alteration of mRNA levels of the cytoskeletal-related genes. The identification of metaphasic nuclei and irregular disposition of β-actin in the cell cytoplasm supported the hypothesis that citropten conjugated with NDs showed antimitotic properties in B16F10 cells. This work can be considered a pioneering piece of research that could promote and support the biomedical use of plant drug-functionalized NDs in cancer therapy. PMID:26893562

  11. Metastatic melanoma imaging using a novel Tc-99m-labeled lactam-cyclized alpha-MSH peptide.

    PubMed

    Liu, Liqin; Xu, Jingli; Yang, Jianquan; Feng, Changjian; Miao, Yubin

    2017-11-15

    The purpose of this study was to determine the metastatic melanoma imaging property of 99m Tc(EDDA)-HYNIC-Aoc-Nle-CycMSH hex {hydrazinonicotinamide-8-aminooctanoic acid-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH 2 }. HYNIC-Aoc-Nle-CycMSH hex was synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The IC 50 value of HYNIC-Aoc-Nle-CycMSH hex was 0.78 ± 0.13 nM for B16/F10 melanoma cells. 99m Tc(EDDA)-HYNIC-Aoc-Nle-CycMSH hex displayed significantly higher uptake (14.26 ± 2.74 and 10.45 ± 2.31% ID/g) in B16/F10 metastatic melanoma-bearing lung than that in normal lung (0.90 ± 0.15 and 0.53 ± 0.14% ID/g) at 2 and 4 h post-injection, respectively. B16/F10 pulmonary metastatic melanoma lesions were clearly visualized by SPECT/CT using 99m Tc(EDDA)-HYNIC-Aoc-Nle-CycMSH hex as an imaging probe at 2 h post-injection, underscoring its potential as an imaging probe for metastatic melanoma detection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Blocking FGF2 with a new specific monoclonal antibody impairs angiogenesis and experimental metastatic melanoma, suggesting a potential role in adjuvant settings.

    PubMed

    de Aguiar, Rodrigo Barbosa; Parise, Carolina Bellini; Souza, Carolina Rosal Teixeira; Braggion, Camila; Quintilio, Wagner; Moro, Ana Maria; Navarro Marques, Fabio Luiz; Buchpiguel, Carlos Alberto; Chammas, Roger; de Moraes, Jane Zveiter

    2016-02-28

    Compelling evidence suggests that fibroblast growth factor 2 (FGF2), overexpressed in melanomas, plays an important role in tumor growth, angiogenesis and metastasis. In this study, we evaluated the therapeutic use of a new anti-FGF2 monoclonal antibody (mAb), 3F12E7, using for that the B16-F10 melanoma model. The FGF2 neutralizing effect of this antibody was certified by in vitro assays, which allowed the further track of its possible in vivo application. 3F12E7 mAb could be retained in B16-F10 tumors, as shown by antibody low-pH elution and nuclear medicine studies, and also led to reduction in number and size of metastatic foci in lungs, when treatment starts one day after intravenous injection of B16-F10 cells. Such data were accompanied by decreased CD34(+) tumor vascular density and impaired subcutaneous tumor outgrowth. Treatments starting one week after melanoma cell intravenous injection did not reduce tumor burden, remaining the therapeutic effectiveness restricted to early-adopted regimens. Altogether, the presented anti-FGF2 3F12E7 mAb stands as a promising agent to treat metastatic melanoma tumors in adjuvant settings. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  13. Downregulation of adaptor protein MyD88 compromises the angiogenic potential of B16 murine melanoma

    PubMed Central

    Araya, Paula; Nuñez, Nicolás Gonzalo; Mena, Hebe Agustina; Bocco, José Luis; Negrotto, Soledad; Maccioni, Mariana

    2017-01-01

    The mechanisms that link inflammatory responses to cancer development remain a subject of intense investigation, emphasizing the need to better understand the cellular and molecular pathways that create a tumor promoting microenvironment. The myeloid differentiation primary response protein MyD88 acts as a main adaptor molecule for the signaling cascades initiated from Toll-like receptors (TLRs) and the interleukin 1 receptor (IL-1R). MyD88 has been shown to contribute to tumorigenesis in many inflammation-associated cancer models. In this study, we sought to better define the role of MyD88 in neoplastic cells using a murine melanoma model. Herein, we have demonstrated that MyD88 expression is required to maintain the angiogenic switch that supports B16 melanoma growth. By knocking down MyD88 we reduced TLR-mediated NF-κB activation with no evident effects over cell proliferation and survival. In addition, MyD88 downregulation was associated with a decrease of HIF1α levels and its target gene VEGF, in correlation with an impaired capability to induce capillary sprouting and tube formation of endothelial cells. Melanomas developed from cells lacking MyD88 showed an enhanced secretion of chemoattractant ligands such as CCL2, CXCL10 and CXCL1 and have an improved infiltration of macrophages to the tumor site. Our results imply that cell-autonomous signaling through MyD88 is required to sustain tumor growth and underscore its function as an important positive modulator of tumor angiogenesis. PMID:28662055

  14. Label-free detection of circulating melanoma cells by in vivo photoacoustic flow cytometry

    NASA Astrophysics Data System (ADS)

    Wang, Xiaoling; Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Gao, Wenyuan; Tang, Shuo; Wei, Xunbin

    2016-03-01

    Melanoma is a malignant tumor of melanocytes. Melanoma cells have high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC), which is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. We have developed in vitro experiments to prove the ability of PAFC system of detecting photoacoustic signals from melanoma cells. For in vivo experiments, we have constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells, B16F10 with subcutaneous injection. PA signals are detected in the blood vessels of mouse ears in vivo. The raw signal detected from target cells often contains some noise caused by electronic devices, such as background noise and thermal noise. We choose the Wavelet denoising method to effectively distinguish the target signal from background noise. Processing in time domain and frequency domain would be combined to analyze the signal after denoising. This algorithm contains time domain filter and frequency transformation. The frequency spectrum image of the signal contains distinctive features that can be used to analyze the property of target cells or particles. The processing methods have a great potential for analyzing signals accurately and rapidly. By counting circulating melanoma cells termly, we obtain the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation.

  15. Efficacy of in vitro sensitized cells generated by in vivo priming with OK-432 for adoptive immunotherapy of the poorly immunogenic B16-Bl6 melanoma.

    PubMed

    Mukai, S; Kato, H; Kimura, S; Asai, K; Kawahito, Y; Inoue, M; Yamamura, Y; Sano, H; Sugino, S; Shu, S; Kondo, M

    1996-02-01

    We investigated the efficacy of the streptococcal preparation OK-432 as an adjuvant for in vivo priming in induction of sensitized cells for adoptive immunotherapy of the poorly immunogenic B16-BL6 (BL6) melanoma. C57BL/6 (B6) mice were immunized subcutaneously (s.c.) with 3 x 10(6) viable BL6 tumor cells admixed with various doses of OK-432 ranging from 1 to 100 micrograms in the foot-pad. Draining popliteal lymph nodes (LNs) were harvested 7 days after immunization and LN cells were further sensitized with irradiated tumor cells in the presence of 60-300 IU/ml of IL-2 for 11 days. These in vitro sensitized (IVS) cells (2 x 10(6)) were transferred intravenously (i.v.) to B6 mice bearing 4-day pulmonary metastases established by i.v. injection of 2-4 x 10(5) viable BL6 cells. The mice were also received intraperitoneally (i.p.) 4 x 10(4) IU/day of IL-2 for 4 days after adoptive transfer. Transfer of IVS cells from mice immunized by s.c. injection of tumor cells admixed with 10 micrograms of OK-432 significantly reduced the numbers of BL6 pulmonary metastases compared with that of control IVS' cells without the administration of OK-432 (P = 0.003). These effective IVS cells also significantly prolonged the survival of treated animals (P = 0.003). Functional IVS cells required in vitro stimulation with tumor cells. However, addition of OK-432 in the vaccine resulted in no enhancement of in vitro cytotoxicity and no characteristic change of phenotype of IVS cells. These results suggest that in vivo priming of OK-432 facilitates the sensitization of tumor-reactive T-cells. The procedure of in vivo priming with OK-432 may be beneficial in the adoptive immunotherapy of melanoma.

  16. Quercetin inhibits the invasion and mobility of murine melanoma B16-BL6 cells through inducing apoptosis via decreasing Bcl-2 expression.

    PubMed

    Zhang, X; Xu, Q; Saiki, I

    2000-01-01

    Quercetin has been known to have anti-tumor and anti-oxidation activities. In the present study, we have investigated its in vitro anti-metastatic activity. Quercetin inhibited the invasion and mobility of murine melanoma B16-BL6 cells in a dose-dependent manner but did not affect their adhesion to either laminin, fibronectin, or type VI collagen. Moreover, quercetin significantly inhibited the proliferation of B16-BL6 cells only in the case of time incubation longer than 48 h. Quercetin dose-dependently decreased the cell rates in S and G2-M phases of cell cycle. The effect of quercetin to cause a remarkable apoptosis of B16-BL6 cells was also demonstrated by flow cytometric assay as well as DNA fragmentation with a typical 180-bp ladder band in agarose electrophoresis and a quantitative analysis. Furthermore, quercetin markedly inhibited the expression of anti-apoptotic protein Bcl-2 but hardly influenced Bcl-XL. These results suggest that the inhibition of quercetin on invasiveness and migration of B16-BL6 cells are closely associated with the arrest of cell cycle as well as the induction of apoptosis by decreasing the Bcl-2 expression.

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

    PubMed

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

    2016-06-01

    DMAKO-05((S)-1-((5E,8E)-5,8-bis(hydroxyimino)-1,4-dimethoxy-5,8-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 3-methylbutanoate) is a novel oxime derivative of shikonin, the major component extracted from Chinese herb Lithospermun erythrorhizon. Here, we report that DMAKO-05 had an antitumor activity against mouse melanoma cell line B16F0. Our studies indicated that DMAKO-05 not only inhibited B16F0 proliferation and migration but also led to cell cycle arrest at G1 phase and cell apoptosis, in which DMAKO-05 triggered mitochondrial-mediated apoptosis signal including caspase-9/3 and PARP. In response to DMAKO-05 treatment, the Akt-mediated survival signals were remarkably attenuated in B16F0 cells. Collectively, DMAKO-05 has a strong cytotoxicity in B16F0 cells via inhibiting Akt activation, inducing G1 arrest, and promoting B16F0 cell apoptosis. DMAKO-05 might serve as a potential candidate lead compound for melanoma. © 2016 John Wiley & Sons A/S.

  18. Inhibition of B16-BL6 melanoma growth in mice by methionine-enkephalin.

    PubMed

    Murgo, A J

    1985-08-01

    The antitumor effect of methionine-enkephalin [( Met]enkephalin) was demonstrated in C57BL/6J mice inoculated with B16-BL6 melanoma cells. Local subcutaneous tumor growth was inhibited with a 50-micrograms dose daily for 7 or 14 days. The antitumor effect of [Met]enkephalin was inhibited by the administration of the opioid receptor antagonist naloxone. Naloxone alone had no significant effect on tumor growth.

  19. Viwithan, a Standardized Withania somnifera Root Extract Induces Apoptosis in Murine Melanoma Cells

    PubMed Central

    Sudeep, H.V.; Gouthamchandra, K.; Venkatesh, B. J.; Prasad, K. Shyam

    2017-01-01

    Background: Withania somnifera is an Indian medicinal herb known for the multipotential ability to cure various therapeutic ailments as described in the ayurvedic system of medicine. Objective: In the present study, we have evaluated the antiproliferative activity of a standardized W. somnifera root extract (Viwithan) against different human and murine cancer cell lines. Materials and Methods: The cytotoxicity of Viwithan was determined using thiazolyl blue tetrazolium blue assay and crystal violet staining. The apoptotic changes in B16F1 cells following treatment with Viwithan were observed by acridine orange/ethidium bromide (AO/EB) staining and DNA fragmentation assay. The binding affinity of withanolides in Viwithan with antiapoptotic proteins B-cell lymphoma 2, B-cell lymphoma-extra large, and myeloid cell leukemia 1 (MCL-1) were studied using in silico approach. Results: The half maximal inhibitory concentration (IC50) values of Viwithan against liver hepatocellular carcinoma, Henrietta Lacks cervical carcinoma cells, human colorectal carcinoma cell line, and Ehrlich ascites carcinoma cells were 1830, 968, 2715, and 633 μg/ml, respectively. Interestingly, Viwithan was highly effective against B16F1 cells with an IC50 value of 220 μg/ml after 24 h treatment. The morphological alterations of apoptotic cell death were clearly observed in the AO/EB-stained cells after treatment with Viwithan. Viwithan induced late apoptotic changes in treated B16F1 cells as evident by the ladder formation of fragmented DNA in a time-dependent manner. The findings of molecular docking showed that withanolides present in Viwithan have a more binding affinity with the antiapoptotic proteins, particularly MCL-1. Conclusion: We have reported for the first time that Viwithan with 5% withanolides has a potent cytotoxic effect, particularly against B16F1 murine melanoma cells among the different cancer cell lines tested. SUMMARY The present study reports for the first time that

  20. Preclinical evaluation of melanocortin-1 receptor (MC1-R) specific 68Ga- and 44Sc-labeled DOTA-NAPamide in melanoma imaging.

    PubMed

    Nagy, Gábor; Dénes, Noémi; Kis, Adrienn; Szabó, Judit P; Berényi, Ervin; Garai, Ildikó; Bai, Péter; Hajdu, István; Szikra, Dezső; Trencsényi, György

    2017-08-30

    Alpha melanocyte stimulating hormone (α-MSH) enhances melanogenesis in melanoma malignum by binding to melanocortin-1 receptors (MC1-R). Earlier studies demonstrated that alpha-MSH analog NAPamide molecule specifically binds to MC1-R receptor. Radiolabeled NAPamide is a promising radiotracer for the non-invasive detection of melanin producing melanoma tumors by Positron Emission Tomography (PET). In this present study the MC1-R selectivity of the newly developed Sc-44-labeled DOTA-NAPamide was investigated in vitro and in vivo using melanoma tumors. DOTA-NAPamide was labeled with Ga-68 and Sc-44 radionuclides. The MC1-R specificity of Ga-68- and Sc-44-labeled DOTA-NAPamide was investigated in vitro and in vivo using MC1-R positive (B16-F10) and negative (A375) melanoma cell lines. For in vivo imaging studies B16-F10 and A375 tumor-bearing mice were injected with 44 Sc/ 68 Ga-DOTA-NAPamide (in blocking studies with α-MSH) and whole body PET/MRI scans were acquired. Radiotracer uptake was expressed in terms of standardized uptake values (SUVs). 44 Sc/ 68 Ga-labeled DOTA-NAPamide were produced with high specific activity (approx. 19 GBq/μmol) and with excellent radiochemical purity (99%<). MC1-R positive B16-F10 cells showed significantly (p≤0.01) higher in vitro radiotracer accumulation than that of receptor negative A375 melanoma cells. In animal experiments, also significantly (p≤0.01) higher Ga-68-DOTA-NAPamide (SUVmean: 0.38±0.02), and Sc-44-DOTA-NAPamide (SUVmean: 0.52±0.13) uptake was observed in subcutaneously growing B16-F10 tumors, than in receptor negative A375 tumors, where the SUVmean values of Ga-68-DOTA-NAPamide and Sc-44-DOTA-NAPamide were 0.04±0.01 and 0.07±0.01, respectively. Tumor-to-muscle (T/M SUVmean) ratios were approximately 15-fold higher in B16-F10 tumor-bearing mice, than that of A375 tumors, and this difference was also significant (p≤0.01) using both radiotracers after 60 min incubation time. Our newly synthesized 44 Sc

  1. Using adenovirus armed short hairpin RNA targeting transforming growth factor β1 inhibits melanoma growth and metastasis in an ex vivo animal model.

    PubMed

    Tai, Kuo-Feng; Wang, Chien-Hsing

    2013-12-01

    The transforming growth factor β (TGF-β) is the key molecule implicated in impaired immune function in human patients with malignant melanoma. TGF-β can promote tumor growth, invasion, and metastasis in advanced stages of melanoma. Blocking these tumor-promoting effects of TGF-β provides a potentially important therapeutic strategy for the treatment of melanoma. In this study, we used an adenovirus-based shRNA expression system and successfully constructed Ad/TGF-β1-RNA interference (RNAi) which mediated the RNAi for TGF-β1 gene silencing. We examined the effects of TGF-β1 protein knockdown by RNAi on the growth and metastasis of melanoma in C57BL/6 mice induced by the B16F0 cell line. The TGF-β1 hairpin oligonucleotide was cloned into adenoviral vector. The resulting recombinant adenoviruses infected murine melanoma cell line, B16F0, and designated as B16F0/TGF-β1-RNAi cells. The blank adenoviral vector also infected B16F0 cells and designed as B16F0/vector-control cells served as a control. TGF-β1 expression was reduced in B16F0/TGF-β1-RNAi cells compared with B16F0 cells and B16F0/vector-control cells. Three million wild-type B16F0 cells, B16F0/vector-control cells, and B16F0/TGF-β1-RNAi cells were injected subcutaneously into the right flanks of adult female syngeneic mice C57BL/6. The tumor sizes were 756.09 (65.35), 798.48 (78.77), and 203.55 (24.56) mm at the 14th day in the mice receiving B16F0 cells, B16F0/vector-control cells, and B16F0/TGFβ1-RNAi cells, respectively. The P value was less than 0.01 by 1-way analysis of variance. TGF-β1 knockdown in B16F0 cells enhanced the infiltration of CD4 and CD8 T cells in the tumor regions. C57BL/6 mice were evaluated for pulmonary metastasis after tail vein injection of 2 million B16F0 cells, B16F0/vector-control cells, and B16F0/TGF-β1-RNAi cells. The pulmonary metastasis also reduced significantly on days 14 day and 21 in mice injected with B16F0/TGF-β1-RNAi tumors. The blood vessel density of the

  2. Metastatic Melanoma Secreted IL-10 Down-Regulates CD1 Molecules on Dendritic Cells in Metastatic Tumor Lesions

    PubMed Central

    Gerlini, Gianni; Tun-Kyi, Adrian; Dudli, Christa; Burg, Günter; Pimpinelli, Nicola; Nestle, Frank O.

    2004-01-01

    CD1 molecules are expressed by antigen-presenting cells such as dendritic cells and mediate primary immune responses to lipids and glycolipids which have been shown to be expressed by various tumors. Glycolipids are expressed by melanoma cells but, despite their immunogenicity, no efficient spontaneous immune responses are elicited. As IL-10 has previously been shown to down-regulate CD1a on dendritic cells and is known to be expressed by various melanoma cell lines, we investigated if melanoma-derived IL-10 could down-regulate CD1 molecule expression on dendritic cells as a possible way to circumvent immune recognition. We found that CD1a, CD1b, CD1c, and CD1d were significantly down-regulated on dendritic cells in metastatic (n = 10) but not in primary melanoma lesions (n = 10). We further detected significantly higher IL-10 protein levels in metastatic than in primary melanomas. Moreover, supernatants from metastatic melanomas were significantly more effective in down-regulating CD1 molecules on dendritic cells than supernatants from primary melanoma cultures. This effect was blocked using a neutralizing IL-10 antibody in a dose dependent manner. Our findings suggest that metastatic but not primary melanomas can down-regulate CD1 molecules on infiltrating dendritic cells by secreting IL-10 which may represent a novel way to escape the immune response directed against the tumor. PMID:15579430

  3. A quantitative systems approach to identify paracrine mechanisms that locally suppress immune response to Interleukin-12 in the B16 melanoma model

    PubMed Central

    Kulkarni, Yogesh M.; Chambers, Emily; McGray, A. J. Robert; Ware, Jason S.; Bramson, Jonathan L.

    2012-01-01

    Interleukin-12 (IL12) enhances anti-tumor immunity when delivered to the tumor microenvironment. However, local immunoregulatory elements dampen the efficacy of IL12. The identity of these local mechanisms used by tumors to suppress immunosurveillance represents a key knowledge gap for improving tumor immunotherapy. From a systems perspective, local suppression of anti-tumor immunity is a closed-loop system - where system response is determined by an unknown combination of external inputs and local cellular cross-talk. Here, we recreated this closed-loop system in vitro and combined quantitative high content assays, in silico model-based inference, and a proteomic workflow to identify the biochemical cues responsible for immunosuppression. Following an induction period, the B16 melanoma cell model, a transplantable model for spontaneous malignant melanoma, inhibited the response of a T helper cell model to IL12. This paracrine effect was not explained by induction of apoptosis or creation of a cytokine sink, despite both mechanisms present within the co-culture assay. Tumor-derived Wnt-inducible signaling protein-1 (WISP-1) was identified to exert paracrine action on immune cells by inhibiting their response to IL12. Moreover, WISP-1 was expressed in vivo following intradermal challenge with B16F10 cells and was inferred to be expressed at the tumor periphery. Collectively, the data suggest that (1) biochemical cues associated with epithelial-to-mesenchymal transition can shape anti-tumor immunity through paracrine action and (2) remnants of the immunoselective pressure associated with evolution in cancer include both sculpting of tumor antigens and expression of proteins that proactively shape anti-tumor immunity. PMID:22777646

  4. Gallium-67-labeled lactam bridge-cyclized alpha-MSH peptides with enhanced melanoma uptake and reduced renal uptake.

    PubMed

    Guo, Haixun; Gallazzi, Fabio; Miao, Yubin

    2012-06-20

    The purpose of this study was to examine the melanoma targeting and pharmacokinetic properties of (67)Ga-DOTA-GGNle-CycMSHhex {(67)Ga-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and (67)Ga-NOTA-GGNle-CycMSHhex {(67)Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and compare with (67)Ga-DOTA-GlyGlu-CycMSH {(67)Ga-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]} we previously reported. DOTA-GGNle-CycMSHhex and NOTA-GGNle-CycMSHhex were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSHhex was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSHhex. The melanoma targeting and pharmacokinetic properties of (67)Ga-NOTA-GGNle-CycMSHhex and (67)Ga-DOTA-GGNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSHhex and DOTA-GGNle-CycMSHhex displayed comparable MC1 receptor binding affinities (1.6 vs 2.1 nM) in B16/F1 melanoma cells. Both (67)Ga-NOTA-GGNle-CycMSHhex and (67)Ga-DOTA-GGNle-CycMSHhex exhibited dramatically enhanced melanoma uptake and reduced renal uptake than (67)Ga-DOTA-GlyGlu-CycMSH in B16/F1 melanoma-bearing C57 mice. Furthermore, (67)Ga-NOTA-GGNle-CycMSHhex exhibited more favorable radiolabeling conditions (>85% radiolabeling yields started at 37 °C), as well as higher tumor/kidney uptake ratios than (67)Ga-DOTA-GGNle-CycMSHhex at 0.5, 2, and 24 h postinjection. High melanoma uptake coupled with low renal uptake highlighted the potential of (67)Ga-NOTA-GGNle-CycMSHhex for melanoma imaging and therapy.

  5. Gallium-67-Labeled Lactam Bridge-Cyclized Alpha-MSH Peptides with Enhanced Melanoma Uptake and Reduced Renal Uptake

    PubMed Central

    Guo, Haixun; Gallazzi, Fabio; Miao, Yubin

    2012-01-01

    The purpose of this study was to examine the melanoma targeting and pharmacokinetic properties of 67Ga-DOTA-GGNle-CycMSHhex {67Ga-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-Gly-Gly-Nle-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2} and 67Ga-NOTA-GGNle-CycMSHhex {67Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2} and compare with 67Ga-DOTA-GlyGlu-CycMSH {67Ga-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-dPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]} we previously reported. DOTA-GGNle-CycMSHhex and NOTA-GGNle-CycMSHhex were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSHhex was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSHhex. The melanoma targeting and pharmacokinetic properties of 67Ga-NOTA-GGNle-CycMSHhex and 67Ga-DOTA-GGNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSHhex and DOTA-GGNle-CycMSHhex displayed comparable MC1 receptor binding affinities (1.6 vs. 2.1 nM) in B16/F1 melanoma cells. Both 67Ga-NOTA-GGNle-CycMSHhex and 67Ga-DOTA-GGNle-CycMSHhex exhibited dramatically enhanced melanoma uptake and reduced renal uptake than 67Ga-DOTA-GlyGlu-CycMSH in B16/F1 melanoma-bearing C57 mice. Furthermore, 67Ga-NOTA-GGNle-CycMSHhexexhibited more favorable radiolabeling conditions (> 85% radiolabeling yields started at 37°C), as well as higher tumor/kidney uptake ratios than 67Ga-DOTA-GGNle-CycMSHhex at 0.5, 2 and 24 h post-injection. High melanoma uptake coupled with low renal uptake highlighted the potential of 67Ga-NOTA-GGNle-CycMSHhexfor melanoma imaging and therapy. PMID:22621181

  6. Cytotoxic Activity and Antiproliferative Effects of Crude Skin Secretion from Physalaemus nattereri (Anura: Leptodactylidae) on in vitro Melanoma Cells

    PubMed Central

    Cruz e Carvalho, Andréa; Prías Márquez, César Augusto; Azevedo, Ricardo Bentes; Joanitti, Graziella Anselmo; Pires Júnior, Osmindo Rodrigues; Fontes, Wagner; Castro, Mariana S.

    2015-01-01

    Anuran secretions are rich sources of bioactive molecules, including antimicrobial and antitumoral compounds. The aims of this study were to investigate the therapeutic potential of Physalaemus nattereri skin secretion against skin cancer cells, and to assess its cytotoxic action mechanisms on the murine melanoma cell line B16F10. Our results demonstrated that the crude secretion reduced the viability of B16F10 cells, causing changes in cell morphology (e.g., round shape and structure shrinkage), reduction in mitochondrial membrane potential, increase in phosphatidylserine exposure, and cell cycle arrest in S-phase. Together, these changes suggest that tumor cells die by apoptosis. This skin secretion was also subjected to chromatographic fractioning using RP-HPLC, and eluted fractions were assayed for antiproliferative and antibacterial activities. Three active fractions showed molecular mass components in a range compatible with peptides. Although the specific mechanisms causing the reduced cell viability and cytotoxicity after the treatment with crude secretion are still unknown, it may be considered that molecules, such as the peptides found in the secretion, are effective against B16F10 tumor cells. Considering the growing need for new anticancer drugs, data presented in this study strongly reinforce the validity of P. nattereri crude secretion as a rich source of new anticancer molecules. PMID:26457717

  7. Cytotoxic Activity and Antiproliferative Effects of Crude Skin Secretion from Physalaemus nattereri (Anura: Leptodactylidae) on in vitro Melanoma Cells.

    PubMed

    Cruz e Carvalho, Andréa; Márquez, César Augusto Prías; Azevedo, Ricardo Bentes; Joanitti, Graziella Anselmo; Pires Júnior, Osmindo Rodrigues; Fontes, Wagner; Castro, Mariana S

    2015-10-08

    Anuran secretions are rich sources of bioactive molecules, including antimicrobial and antitumoral compounds. The aims of this study were to investigate the therapeutic potential of Physalaemus nattereri skin secretion against skin cancer cells, and to assess its cytotoxic action mechanisms on the murine melanoma cell line B16F10. Our results demonstrated that the crude secretion reduced the viability of B16F10 cells, causing changes in cell morphology (e.g., round shape and structure shrinkage), reduction in mitochondrial membrane potential, increase in phosphatidylserine exposure, and cell cycle arrest in S-phase. Together, these changes suggest that tumor cells die by apoptosis. This skin secretion was also subjected to chromatographic fractioning using RP-HPLC, and eluted fractions were assayed for antiproliferative and antibacterial activities. Three active fractions showed molecular mass components in a range compatible with peptides. Although the specific mechanisms causing the reduced cell viability and cytotoxicity after the treatment with crude secretion are still unknown, it may be considered that molecules, such as the peptides found in the secretion, are effective against B16F10 tumor cells. Considering the growing need for new anticancer drugs, data presented in this study strongly reinforce the validity of P. nattereri crude secretion as a rich source of new anticancer molecules.

  8. In vivo UVA irradiation of mouse is more efficient in promoting pulmonary melanoma metastasis than in vitro

    PubMed Central

    2011-01-01

    Background We have previously shown in vitro that UVA increases the adhesiveness of mouse B16-F1 melanoma cells to endothelium. We have also shown in vivo that UVA exposure of C57BL/6 mice, i.v. injected with B16-F1 cells, increases formation of pulmonary colonies of melanoma. The aim of the present animal study was to confirm the previously observed in vivo UVA effect and to determine whether in vitro UVA-exposure of melanoma cells, prior the i.v. injection, will have an enhancing effect on the pulmonary colonization capacity of melanoma cells. As a second aim, UVA-derived immunosuppression was determined. Methods Mice were i.v. injected with B16-F1 cells into the tail vein and then immediately exposed to UVA. Alternatively, to study the effect of UVA-induced adhesiveness on the colonization capacity of B16-F1 melanoma, cells were in vitro exposed prior to i.v. injection. Fourteen days after injection, lungs were collected and the number of pulmonary nodules was determined under dissecting microscope. The UVA-derived immunosuppression was measured by standard contact hypersensitivity assay. Results and Discussion Obtained results have confirmed that mice, i.v. injected with B16-F1 cells and thereafter exposed to UVA, developed 4-times more of melanoma colonies in lungs as compared with the UVA non-exposed group (p < 0.01). The in vitro exposure of melanoma cells prior to their injection into mice, led only to induction of 1.5-times more of pulmonary tumor nodules, being however a statistically non-significant change. The obtained results postulate that the UVA-induced changes in the adhesive properties of melanoma cells do not alone account for the 4-fold increase in the pulmonary tumor formation. Instead, it suggests that some systemic effect in a mouse might be responsible for the increased metastasis formation. Indeed, UVA was found to induce moderate systemic immunosuppression, which effect might contribute to the UVA-induced melanoma metastasis in mice lungs

  9. Investigation of Hexadecylphosphocholine (miltefosine) usage in Pegylated liposomal doxorubicin as a synergistic ingredient: In vitro and in vivo evaluation in mice bearing C26 colon carcinoma and B16F0 melanoma.

    PubMed

    Teymouri, Manouchehr; Farzaneh, Hamidreza; Badiee, Ali; Golmohammadzadeh, Shiva; Sadri, Kayvan; Jaafari, Mahmoud Reza

    2015-12-01

    In this investigation, Hexadecylphosphocholine (HePC, miltefosine) was being used as a new ingredient in Pegylated liposomal doxorubicin (PLD) and different aspects of this integration such as its effect on doxorubicin (Dox) release and cell uptake, cytotoxicity of liposomes, in vivo distribution and half-life clearance time of Dox as well as median survival time were illustrated. The liposomal formulations were Pegylated liposomal doxorubicin containing 0, 0.5, 1, 2 and 4% mole ratios of HePC (HePC-PLD) and their respective Dox-free liposomes (HePC-PLs). The cells used were colon carcinoma (C26), adriamycin-resistant breast cancer (MCF-7-ADR), and B16F0 melanoma cell lines, of which C26 and B16F0 cells were exploited for tumoring in BALB/c and C57Bl/6 mice, respectively. In most cases, increase in miltefosine percentage resulted in physically liposomal instability, increased Dox delivery and toxicity and reduced blood half-life of Dox. Overall, HePC 4% -PLD and PLD differed significantly in many respects and it was considered too toxic to be injected at the same dose (15mg Dox/ kg) as PLD. Although HePC 2% -PLD could extend the median survival time marginally in comparison to PLD, the concept of HePC- containing liposomes merits further investigation. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Evaluation of two (125)I-radiolabeled acridine derivatives for Auger-electron radionuclide therapy of melanoma.

    PubMed

    Gardette, Maryline; Viallard, Claire; Paillas, Salomé; Guerquin-Kern, Jean-Luc; Papon, Janine; Moins, Nicole; Labarre, Pierre; Desbois, Nicolas; Wong-Wah-Chung, Pascal; Palle, Sabine; Wu, Ting-Di; Pouget, Jean-Pierre; Miot-Noirault, Elisabeth; Chezal, Jean-Michel; Degoul, Francoise

    2014-08-01

    We previously selected two melanin-targeting radioligands [(125)I]ICF01035 and [(125)I]ICF01040 for melanoma-targeted (125)I radionuclide therapy according to their pharmacological profile in mice bearing B16F0 tumors. Here we demonstrate in vitro that these compounds present different radiotoxicities in relation to melanin and acidic vesicle contents in B16F0, B16F0 PTU and A375 cell lines. ICF01035 is effectively observed in nuclei of achromic (A375) melanoma or in melanosomes of melanized melanoma (B16F0), while ICF01040 stays in cytoplasmic vesicles in both cells. [(125)I]ICF01035 induced a similar survival fraction (A50) in all cell lines and led to a significant decrease in S-phase cells in amelanotic cell lines. [(125)I]ICF01040 induced a higher A50 in B16 cell lines compared to [(125)I]ICF01035 ones. [(125)I]ICF01040 induced a G2/M blockade in both A375 and B16F0 PTU, associated with its presence in cytoplasmic acidic vesicles. These results suggest that the radiotoxicity of [(125)I]ICF01035 and [(125)I]ICF01040 are not exclusively reliant on DNA alterations compatible with γ rays but likely result from local dose deposition (Auger electrons) leading to toxic compound leaks from acidic vesicles. In vivo, [(125)I]ICF01035 significantly reduced the number of B16F0 lung colonies, enabling a significant increase in survival of the treated mice. Targeting melanosomes or acidic vesicles is thus an option for future melanoma therapy.

  11. In Vivo Imaging of Experimental Melanoma Tumors using the Novel Radiotracer 68Ga-NODAGA-Procainamide (PCA).

    PubMed

    Kertész, István; Vida, András; Nagy, Gábor; Emri, Miklós; Farkas, Antal; Kis, Adrienn; Angyal, János; Dénes, Noémi; Szabó, Judit P; Kovács, Tünde; Bai, Péter; Trencsényi, György

    2017-01-01

    The most aggressive form of skin cancer is the malignant melanoma. Because of its high metastatic potential the early detection of primary melanoma tumors and metastases using non-invasive PET imaging determines the outcome of the disease. Previous studies have already shown that benzamide derivatives, such as procainamide (PCA) specifically bind to melanin pigment. The aim of this study was to synthesize and investigate the melanin specificity of the novel 68 Ga-labeled NODAGA-PCA molecule in vitro and in vivo using PET techniques. Procainamide (PCA) was conjugated with NODAGA chelator and was labeled with Ga-68 ( 68 Ga-NODAGA-PCA). The melanin specificity of 68 Ga-NODAGA-PCA was tested in vitro , ex vivo and in vivo using melanotic B16-F10 and amelanotic Melur melanoma cell lines. By subcutaneous and intravenous injection of melanoma cells tumor-bearing mice were prepared, on which biodistribution studies and small animal PET/CT scans were performed for 68 Ga-NODAGA-PCA and 18 FDG tracers. 68 Ga-NODAGA-PCA was produced with high specific activity (14.9±3.9 GBq/µmol) and with excellent radiochemical purity (98%<), at all cases. In vitro experiments showed that 68 Ga-NODAGA-PCA uptake of B16-F10 cells was significantly ( p ≤0.01) higher than Melur cells. Ex vivo biodistribution and in vivo PET/CT studies using subcutaneous and metastatic tumor models showed significantly ( p ≤0.01) higher 68 Ga-NODAGA-PCA uptake in B16-F10 primary tumors and lung metastases in comparison with amelanotic Melur tumors. In experiments where 18 FDG and 68 Ga-NODAGA-PCA uptake of B16-F10 tumors was compared, we found that the tumor-to-muscle (T/M) and tumor-to-lung (T/L) ratios were significantly ( p ≤0.05 and p ≤0.01) higher using 68 Ga-NODAGA-PCA than the 18 FDG accumulation. Our novel radiotracer 68 Ga-NODAGA-PCA showed specific binding to the melanin producing experimental melanoma tumors. Therefore, 68 Ga-NODAGA-PCA is a suitable diagnostic radiotracer for the detection

  12. In Vivo Imaging of Experimental Melanoma Tumors using the Novel Radiotracer 68Ga-NODAGA-Procainamide (PCA)

    PubMed Central

    Kertész, István; Vida, András; Nagy, Gábor; Emri, Miklós; Farkas, Antal; Kis, Adrienn; Angyal, János; Dénes, Noémi; Szabó, Judit P.; Kovács, Tünde; Bai, Péter; Trencsényi, György

    2017-01-01

    Purpose: The most aggressive form of skin cancer is the malignant melanoma. Because of its high metastatic potential the early detection of primary melanoma tumors and metastases using non-invasive PET imaging determines the outcome of the disease. Previous studies have already shown that benzamide derivatives, such as procainamide (PCA) specifically bind to melanin pigment. The aim of this study was to synthesize and investigate the melanin specificity of the novel 68Ga-labeled NODAGA-PCA molecule in vitro and in vivo using PET techniques. Methods: Procainamide (PCA) was conjugated with NODAGA chelator and was labeled with Ga-68 (68Ga-NODAGA-PCA). The melanin specificity of 68Ga-NODAGA-PCA was tested in vitro, ex vivo and in vivo using melanotic B16-F10 and amelanotic Melur melanoma cell lines. By subcutaneous and intravenous injection of melanoma cells tumor-bearing mice were prepared, on which biodistribution studies and small animal PET/CT scans were performed for 68Ga-NODAGA-PCA and 18FDG tracers. Results: 68Ga-NODAGA-PCA was produced with high specific activity (14.9±3.9 GBq/µmol) and with excellent radiochemical purity (98%<), at all cases. In vitro experiments showed that 68Ga-NODAGA-PCA uptake of B16-F10 cells was significantly (p≤0.01) higher than Melur cells. Ex vivo biodistribution and in vivo PET/CT studies using subcutaneous and metastatic tumor models showed significantly (p≤0.01) higher 68Ga-NODAGA-PCA uptake in B16-F10 primary tumors and lung metastases in comparison with amelanotic Melur tumors. In experiments where 18FDG and 68Ga-NODAGA-PCA uptake of B16-F10 tumors was compared, we found that the tumor-to-muscle (T/M) and tumor-to-lung (T/L) ratios were significantly (p≤0.05 and p≤0.01) higher using 68Ga-NODAGA-PCA than the 18FDG accumulation. Conclusion: Our novel radiotracer 68Ga-NODAGA-PCA showed specific binding to the melanin producing experimental melanoma tumors. Therefore, 68Ga-NODAGA-PCA is a suitable diagnostic radiotracer for

  13. A multidisciplinary study using in vivo tumor models and microfluidic cell-on-chip approach to explore the cross-talk between cancer and immune cells.

    PubMed

    Mattei, Fabrizio; Schiavoni, Giovanna; De Ninno, Adele; Lucarini, Valeria; Sestili, Paola; Sistigu, Antonella; Fragale, Alessandra; Sanchez, Massimo; Spada, Massimo; Gerardino, Annamaria; Belardelli, Filippo; Businaro, Luca; Gabriele, Lucia

    2014-10-01

    A full elucidation of events occurring inside the cancer microenvironment is fundamental for the optimization of more effective therapies. In the present study, the cross-talk between cancer and immune cells was examined by employing mice deficient (KO) in interferon regulatory factor (IRF)-8, a transcription factor essential for induction of competent immune responses. The in vivo results showed that IRF-8 KO mice were highly permissive to B16.F10 melanoma growth and metastasis due to failure of their immune cells to exert proper immunosurveillance. These events were found to be dependent on soluble factors released by cells of the immune system capable of shaping the malignant phenotype of melanoma cells. An on-chip model was then generated to further explore the reciprocal interactions between the B16.F10 and immune cells. B16.F10 and immune cells were co-cultured in a microfluidic device composed of three culturing chambers suitably inter-connected by an array of microchannels; mutual interactions were then followed using time-lapse microscopy. It was observed that WT immune cells migrated through the microchannels towards the B16.F10 cells, establishing tight interactions that in turn limited tumor spread. In contrast, IRF-8 KO immune cells poorly interacted with the melanoma cells, resulting in a more invasive behavior of the B16.F10 cells. These results suggest that IRF-8 expression plays a key role in the cross-talk between melanoma and immune cells, and under-score the value of cell-on-chip approaches as useful in vitro tools to reconstruct complex in vivo microenvironments on a microscale level to explore cell interactions such as those occurring within a cancer immunoenvironment.

  14. Triflavin, an Arg‐Gly‐Asp‐containing Antiplatelet Peptide Inhibits Cell‐substratum Adhesion and Melanoma Cell‐induced Lung Colonization

    PubMed Central

    Sheu, Joen R.; Lin, Chao H.; Chung, Jih L.; Teng, Che M.

    1992-01-01

    Triflavin, an Arg‐Gly‐Asp (RGD) containing peptide purified from Trimeresurus flavoviridis snake venom, inhibits human platelet aggregation by blocking fibrinogen binding to fibrinogen receptors associated with glycoprotein Ilb/IIIa complex. In this study, we show that triflavin (1‐30 μg/mouse) inhibits B16F10 melanoma cell‐induced lung colonization in C57BL/6 mice in a dose‐dependent manner. In vitro, triflavin dose‐dependently inhibits adhesion of B16F10 melanoma cells to extracellular matrices (ECMs; i.e., fibronectin, fibrinogen, vitronectin, and collagen type I). Triflavin is approximately 600‐800 times more potent than GRGDS at inhibiting cell adhesion. In addition, triflavin dose‐dependently inhibits B16F10 cell‐induced platelet aggregation. These results imply that the inhibitory effect of triflavin on the adhesion of tumor cells to ECMs (e.g., fibronectin, vitronectin and collagen type I) and/or tumor cell‐induced platelet aggregation may be partially responsible for its antimetastatic activity in C57BL/6 mice. PMID:1399825

  15. Optimization of intracerebral tumour protection by active-specific immunization against murine melanoma B16/G3.12.

    PubMed

    Staib, L; Harel, W; Mitchell, M S

    2001-08-01

    Development of brain metastases despite extracerebral response to systemic immunotherapy is a common problem in melanoma patients. We have previously described a murine melanoma vaccine of interferon-gamma (IFNgamma)-treated, irradiated syngeneic B16/G3.12 and allogeneic (Cloudman) melanoma cells, plus the adjuvant DETOX, that is protective against subcutaneous (93%) or intracerebral (69%) syngeneic challenge. This study aimed to optimize this vaccine. Groups of nine or 10 mice were immunized five times in 5 weeks with: (i) complete vaccine +/- IFNgamma (VAC+, VAC-); (ii) syngeneic 2 x 106 G3.12 cells plus DETOX (Syn+D), (iii) 2 x 106 allogeneic Cloudman cells plus DETOX (Allo+D); (iv) VAC+ without DETOX (no DETOX); (v) DETOX alone (DETOX); or (vi) phosphate buffered saline (PBS). Mice were challenged subcutaneously with 104 viable G3.12 (or Cloudman cells) and after 35 days intracerebrally with 104 G3.12 cells. Expression of H-2 antigens (measured using fluorescence-activated cell sorting), splenocyte cytotoxicity (measured using 51Cr release) and median overall survival (OAS) were analysed using the log-rank test. VAC+, VAC- and G3.12 mice were equally protected from subcutaneous (s.c.) and intracerebral (i.c.) melanoma challenge (OAS 65 days for s.c., 30 days for i.c.). Protection was less (P < 0.05) in DETOX mice (48 days for s.c.), PBS mice (47 days for s.c., 21 days for i.c.) or no DETOX mice (51 days for s.c.). Allo+D mice showed s.c. (59 days) but not i.c. protection (20 days). IFNgamma incubation did not increase the effect in either the challenge cells or the vaccine cells (P > 0.05). Specific cytotoxicity was seen with G3.12 targets in VAC+ (27%) but not PBS (2%; P < 0.05) mice with equal NK (YAC-1) lysis (10% versus 7%; P< 0.05). Optimal protection against s.c./i.c. experimental murine melanoma was yielded by irradiated syngeneic cells plus DETOX. DETOX alone was not active. Upregulation of H-2 antigens with IFNgamma under these conditions does not

  16. Visualization and in vivo tracking of the exosomes of murine melanoma B16-BL6 cells in mice after intravenous injection.

    PubMed

    Takahashi, Yuki; Nishikawa, Makiya; Shinotsuka, Haruka; Matsui, Yuriko; Ohara, Saori; Imai, Takafumi; Takakura, Yoshinobu

    2013-05-20

    The development of exosomes as delivery vehicles requires understanding how and where exogenously administered exosomes are distributed in vivo. In the present study, we designed a fusion protein consisting of Gaussia luciferase and a truncated lactadherin, gLuc-lactadherin, and constructed a plasmid expressing the fusion protein. B16-BL6 murine melanoma cells were transfected with the plasmid, and exosomes released from the cells were collected by ultracentrifugation. Strong luciferase activity was detected in the fraction containing exosomes, indicating their efficient labeling with gLuc-lactadherin. Then, the labeled B16-BL6 exosomes were intravenously injected into mice, and their tissue distribution was evaluated. Pharmacokinetic analysis of the exosome blood concentration-time profile revealed that B16-BL6 exosomes disappeared very quickly from the blood circulation with a half-life of approximately 2min. Little luciferase activity was detected in the serum at 4h after exosome injection, suggesting rapid clearance of B16-BL6 exosomes in vivo. Moreover, sequential in vivo imaging revealed that the B16-BL6 exosome-derived signals distributed first to the liver and then to the lungs. These results indicate that gLuc-lactadherin labeling is useful for tracing exosomes in vivo and that B16-BL6 exosomes are rapidly cleared from the blood circulation after systemic administration. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Subcellular distribution of a new fluorinated, biocompatible, non-ionic telomeric carrier: a study in cultured B16 melanoma and rat skin fibroblasts.

    PubMed

    Chehade, F; Maurizis, J C; Pucci, B; Pavia, A A; Ollier, M; Veyre, A; Escaig, F; Jeanguillaume, C; Dennebouy, R; Slodzian, G; Hindié, E

    1996-05-01

    Tris-hydroxymethyl-amino-methane telomers bearing a fluorinated end have recently been proposed as potential drug carriers. Using ion microscopy, we have investigated the cell uptake and subcellular distribution of a perfluorinated telomere, called F-TAC, in two cell lines, malignant murine B16 melanoma and normal rat skin fibroblasts. Single layer cell cultures on gold plates were incubated with F-TAC at different concentrations. Ion microscopy using mass spectrometry enabled the detection of Fluorine 19 atoms entering into F-TAC constitution. This microanalytical study showed an elective cytoplasmic localization of the molecule, wherein the distribution is relatively homogeneous. Within same culture and incubation conditions, intercellular variations in F-TAC content were very low. In the malignant line, the intracellular concentration remains practically identical when increasing F-TAC concentration in the culture medium above 0.2 mg/ml, indicating that the uptake phenomenon is saturable. In conclusion, the F-TAC telomer easily crosses the plasma membrane, however, it has difficulties in crossing the nuclear membrane. It is likely that intracellular penetration is essentially due to rapid endocytosis of the telomer.

  18. Hedgehog signaling in the murine melanoma microenvironment.

    PubMed

    Geng, Ling; Cuneo, Kyle C; Cooper, Michael K; Wang, Hong; Sekhar, Konjeti; Fu, Allie; Hallahan, Dennis E

    2007-01-01

    The Hedgehog intercellular signaling pathway regulates cell proliferation and differentiation. This pathway has been implicated to play a role in the pathogenesis of cancer and in embryonic blood vessel development. In the current study, Hedgehog signaling in tumor related vasculature and microenvironment was examined using human umbilical vein endothelial cells and B16F0 (murine melanoma) tumors models. Use of exogenous Sonic hedgehog (Shh) peptide significantly increased BrdU incorporation in endothelial cells in vitro by a factor of 2 (P < 0.001). The Hedgehog pathway antagonist cyclopamine effectively reduced Shh-induced proliferation to control levels. To study Hedgehog signaling in vivo a hind limb tumor model with the B16F0 cell line was used. Treatment with 25 mg/kg cyclopamine significantly attenuated BrdU incorporation in tumor cells threefold (P < 0.001), in tumor related endothelial cells threefold (P = 0.004), and delayed tumor growth by 4 days. Immunohistochemistry revealed that the Hedgehog receptor Patched was localized to the tumor stroma and that B16F0 cells expressed Shh peptide. Furthermore, mouse embryonic fibroblasts required the presence of B16F0 cells to express Patched in a co-culture assay system. These studies indicate that Shh peptide produced by melanoma cells induces Patched expression in fibroblasts. To study tumor related angiogenesis a vascular window model was used to monitor tumor vascularity. Treatment with cyclopamine significantly attenuated vascular formation by a factor of 2.5 (P < 0.001) and altered vascular morphology. Furthermore, cyclopamine reduced tumor blood vessel permeability to FITC labeled dextran while having no effect on normal blood vessels. These studies suggest that Hedgehog signaling regulates melanoma related vascular formation and function.

  19. Uptake in melanoma cells of N-(2-diethylaminoethyl)-2-iodobenzamide (BZA2), an imaging agent for melanoma staging: relation to pigmentation.

    PubMed

    Mansard, Sandrine; Papon, Janine; Moreau, Marie-France; Miot-Noirault, Elisabeth; Labarre, Pierre; Bayle, Martine; Veyre, Annie; Madelmont, Jean-Claude; Moins, Nicole

    2005-07-01

    N-(2-diethylaminoethyl)-2-iodobenzamide (BZA(2)) has been singled out as the most efficacious melanoma scintigraphy imaging agent. Our work was designed to assess the mechanisms of the specific affinity of the radioiodinated iodobenzamide for melanoma tissue. We studied the cellular uptake and retention of [(125)I]-BZA(2) on various cell lines. In vitro, cellular [(125)I]-BZA(2) uptake was related to the pigmentation status of the cells: higher in pigmented melanoma cell lines (M4 Beu, IPC 227, B 16) than in a nonpigmented one (M3 Dau) and nonmelanoma cell lines (MCF 7 and L 929). Two mechanisms were assessed: binding of the tracer to melanin or to sigma receptors of melanoma cells. First, the uptake of [(125)I]-BZA(2) after melanogenesis stimulation by alpha-melanocyte-stimulating hormone and l-tyrosine increased in the B 16 melanoma cell line both in vitro and in vivo according to melanin concentration. Moreover, the binding of [(125)I]-BZA(2) to synthetic melanin was dependent on melanin concentration and could be saturated. Second, no competition was evidenced on M4 Beu cells between [(125)I]-BZA(2) and haloperidol, a sigma ligand, at concentrations < or =10(-6) M. We show that the specificity and sensibility of BZA(2) as a melanoma scintigraphic imaging agent are mostly due to interactions with melanic pigments.

  20. Tumor-suppressive effects of natural-type interferon-β through CXCL10 in melanoma

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kobayashi, Hikaru; Nobeyama, Yoshimasa, E-mail: nobederm@jikei.ac.jp; Nakagawa, Hidemi

    2015-08-21

    Introduction: Type 1 interferon is in widespread use as adjuvant therapy to inhibit melanoma progression. Considering the tumor-suppressive effects of local administration of interferon-β (IFN-β) on lymphatic metastasis, the present study was conducted to identify melanoma-suppressive molecules that are up-regulated by IFN-β treatment of lymphatic endothelial cells. Materials and methods: Lymphatic endothelial cells, fibroblasts, and melanoma cells were treated with natural-type IFN-β, and melanoma cells were treated with CXCL10. Genome-wide oligonucleotide microarray analysis was performed using lymphatic endothelial cells with or without IFN-β treatment. Quantitative real-time reverse transcription-PCR and an enzyme-linked immunosorbent assay were performed to examine CXCL10 expression. Amore » proliferation assay was performed to examine the effects of IFN-β and CXCL10 in melanoma cells. Results: Genome-wide microarray analyses detected CXCL10 as a gene encoding a secretory protein that was up-regulated by IFN-β in lymphatic endothelial cells. IFN-β treatment significantly induced CXCL10 in dermal lymphatic endothelial cells and melanoma cells that are highly sensitive to IFN-β. CXCL10 reduced melanoma cell proliferation in IFN-β-sensitive cells as well as resistant cells. Melanoma cells in which CXCL10 was knocked down were sensitive to IFN-β. CXCR3-B, which encodes the CXCL10 receptor, was up-regulated in melanoma cells with high sensitivity to IFN-β and down-regulated in melanoma cells with medium to low sensitivity. Conclusions: Our data suggest that IFN-β suppresses proliferation and metastasis from the local lymphatic system and melanoma cells via CXCL10. Down-regulation of CXCR3-B by IFN-β may be associated with resistance to IFN-β. - Highlights: • We search melanoma-suppressive molecules induced by IFN-β. • IFN-β induces a high amount of CXCL10 from lymphatic endothelial cells. • CXCL10 induction level in melanoma cells is

  1. The selective cytotoxicity of new triazene compounds to human melanoma cells.

    PubMed

    Sousa, Ana; Santos, Fábio; Gaspar, Maria Manuela; Calado, Susana; Pereira, João D; Mendes, Eduarda; Francisco, Ana Paula; Perry, Maria Jesus

    2017-08-01

    Metastatic melanoma still remains one the most difficult cancers to overcome. The aim of our research was the design of anti-tumour triazene compounds 3 for application to a melanoma-specific therapy. The strategy exploits the unique enzyme pathway of melanin biosynthesis for conversion of non-toxic prodrugs into toxic drugs in the melanoma cell. The compounds 3 were designed by coupling two active moieties, the alkylating triazenes and different tyrosinase substrates. All compounds 3 revealed to be chemically stable in isotonic phosphate buffer (PBS) at physiologic pH (t ½ ≥48h), and most of them showed to be slowly hydrolysed in human plasma (1.5≤t ½ (h)≤161). Compounds 3c-n revealed to be excellent tyrosinase substrates (0.74≤t ½ (min)≤6) with the best tyrosinase substrate 3l releasing MMT 45s after tyrosinase activation. Structure-activity relationship studies allowed the identification of the better structural features for enzyme affinity. Furthermore, the derivatives 3l and 3m showed cell selectivity with significant cytotoxic effects (IC 50 values of 46-65μM) against melanoma cell lines with tyrosinase overexpression MNT-1 and B16F10. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. In vitro and in vivo antitumor activity of the halogenated boroxine dipotassium-trioxohydroxytetrafluorotriborate (K2[B3O3F4OH]).

    PubMed

    Ivankovic, Sinisa; Stojkovic, Ranko; Galic, Zoran; Galic, Borivoj; Ostojic, Jelena; Marasovic, Maja; Milos, Mladen

    2015-06-01

    Dipotassium-trioxohydroxytetrafluorotriborate K2[B3O3F4OH] was listed as a promising new therapeutic for cancer diseases. For in vitro and in vivo investigation of its antitumor effects 4T1 mammary adenocarcinoma, B16F10 melanoma and squamous cell carcinoma SCCVII were used. The detailed in vitro investigation undoubtedly showed that K2[B3O3F4OH] affects the growth of cancer cells. The proliferation of cells depends on the concentration so that aqueous solution of K2[B3O3F4OH], the concentrations of 10(-4) M and less, does not affect cell growth, but the concentrations of 10(-3) M or more, significantly slows cells growth. B16F10 and SCCVII cells show higher sensitivity to the cytotoxic effects of K2[B3O3F4OH] compared to 4T1 cells. Under in vivo conditions, K2[B3O3F4OH] slows the growth of all three tumors tested compared to the control, and the inhibitory effect was most pronounced during the application of the substance. There is almost no difference if K2[B3O3F4OH] was applied intraperitoneally, intratumor, peroral or as ointment. Addition of 5-FU did not further increase the antitumor efficacy of K2[B3O3F4OH].

  3. Liposomes loaded with a STING pathway ligand, cyclic di-GMP, enhance cancer immunotherapy against metastatic melanoma.

    PubMed

    Nakamura, Takashi; Miyabe, Hiroko; Hyodo, Mamoru; Sato, Yusuke; Hayakawa, Yoshihiro; Harashima, Hideyoshi

    2015-10-28

    Malignant melanomas escape immunosurveillance via the loss/down-regulation of MHC-I expression. Natural killer (NK) cells have the potential to function as essential effector cells for eliminating melanomas. Cyclic di-GMP (c-di-GMP), a ligand of the stimulator of interferon genes (STING) signal pathway, can be thought of as a new class of adjuvant against cancer. However, it is yet to be tested, because technologies for delivering c-di-GMP to the cytosol are required. Herein, we report that c-di-GMP efficiently activates NK cells and induces antitumor effects against malignant melanomas when loaded in YSK05 lipid containing liposomes, by assisting in the efficient delivery of c-di-GMP to the cytosol. The intravenous administration of c-di-GMP encapsulated within YSK05-liposomes (c-di-GMP/YSK05-Lip) into mice efficiently induced the production of type I interferon (IFN) as well as the activation of NK cells, resulting in a significant antitumor effect in a lung metastasis mouse model using B16-F10. This antitumor effect was dominated by NK cells. The infiltration of NK cells was observed in the lungs with B16-F10 melanomas. These findings indicate that the c-di-GMP/YSK05-Lip induces MHC-I non-restricted antitumor immunity mediated by NK cells. Consequently, c-di-GMP/YSK05-Lip represents a potentially new adjuvant system for use in immunotherapy against malignant melanomas. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. 111In-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone peptide analogues for melanoma imaging.

    PubMed

    Miao, Yubin; Gallazzi, Fabio; Guo, Haixun; Quinn, Thomas P

    2008-02-01

    The purpose of this study was to examine the influence of the lactam bridge cyclization on melanoma targeting and biodistribution properties of the radiolabeled conjugates. Two novel lactam bridge-cyclized alpha-MSH peptide analogues, DOTA-CycMSH (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]) and DOTA-GlyGlu-CycMSH (DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]), were synthesized and radiolabeled with (111)In. The internalization and efflux of (111)In-labeled CycMSH peptides were examined in B16/F1 melanoma cells. The melanoma targeting properties, pharmacokinetics, and SPECT/CT imaging of (111)In-labeled CycMSH peptides were determined in B16/F1 melanoma-bearing C57 mice. Both (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH exhibited fast internalization and extended retention in B16/F1 cells. The tumor uptake values of (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH were 9.53+/-1.41% injected dose/gram (% ID/g) and 10.40+/-1.40% ID/g at 2 h postinjection, respectively. Flank melanoma tumors were clearly visualized with (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH by SPECT/CT images at 2 h postinjection. Whole-body clearance of the peptides was fast, with greater than 90% of the radioactivities cleared through urinary system by 2 h postinjection. There was low radioactivity (<0.8% ID/g) accumulated in blood and normal organs except kidneys at all time points investigated. Introduction of a negatively charged linker (-Gly-Glu-) into the peptide sequence decreased the renal uptake by 44% without affecting the tumor uptake at 4 h postinjection. High receptor-mediated melanoma uptakes coupled with fast whole-body clearance in B16/F1 melanoma-bearing C57 mice demonstrated the feasibility of using (111)In-labeled lactam bridge-cyclized alpha-MSH peptide analogues as a novel class of imaging probes for receptor-targeting melanoma imaging.

  5. CD133 Is Not Suitable Marker for Isolating Melanoma Stem Cells from D10 Cell Line.

    PubMed

    Rajabi Fomeshi, Motahareh; Ebrahimi, Marzieh; Mowla, Seyed Javad; Firouzi, Javad; Khosravani, Pardis

    2016-01-01

    Cutaneous melanoma is the most hazardous malignancy of skin cancer with a high mortality rate. It has been reported that cancer stem cells (CSCs) are responsible for malignancy in most of cancers including melanoma. The aim of this study is to compare two common methods for melanoma stem cell enriching; isolating based on the CD133 cell surface marker and spheroid cell culture. In this experimental study, melanoma stem cells were enriched by fluorescence activated cell sorting (FACS) based on the CD133 protein expression and spheroid culture of D10 melanoma cell line,. To determine stemness features, the mRNA expression analysis of ABCG2, c-MYC, NESTIN, OCT4-A and -B genes as well as colony and spheroid formation assays were utilized in unsorted CD133(+), CD133(-) and spheroid cells. Significant differences of the two experimental groups were compared using student's t tests and a two-tailed value of P<0.05 was statistically considered as a significant threshold. Our results demonstrated that spheroid cells had more colony and spheroid forming ability, rather than CD133(+) cells and the other groups. Moreover, melanospheres expressed higher mRNA expression level of ABCG2, c-MYC, NESTIN and OCT4-A com- pared to other groups (P<0.05). Although CD133(+) derived melanoma cells represented stemness fea- tures, our findings demonstrated that spheroid culture could be more effective meth- od to enrich melanoma stem cells.

  6. CD133 Is Not Suitable Marker for Isolating Melanoma Stem Cells from D10 Cell Line

    PubMed Central

    Rajabi Fomeshi, Motahareh; Ebrahimi, Marzieh; Mowla, Seyed Javad; Firouzi, Javad; Khosravani, Pardis

    2016-01-01

    Objective Cutaneous melanoma is the most hazardous malignancy of skin cancer with a high mortality rate. It has been reported that cancer stem cells (CSCs) are responsible for malignancy in most of cancers including melanoma. The aim of this study is to compare two common methods for melanoma stem cell enriching; isolating based on the CD133 cell surface marker and spheroid cell culture. Materials and Methods In this experimental study, melanoma stem cells were enriched by fluorescence activated cell sorting (FACS) based on the CD133 protein expression and spheroid culture of D10 melanoma cell line,. To determine stemness features, the mRNA expression analysis of ABCG2, c-MYC, NESTIN, OCT4-A and -B genes as well as colony and spheroid formation assays were utilized in unsorted CD133+, CD133- and spheroid cells. Significant differences of the two experimental groups were compared using student’s t tests and a two-tailed value of P<0.05 was statistically considered as a significant threshold. Results Our results demonstrated that spheroid cells had more colony and spheroid forming ability, rather than CD133+ cells and the other groups. Moreover, melanospheres expressed higher mRNA expression level of ABCG2, c-MYC, NESTIN and OCT4-A com- pared to other groups (P<0.05). Conclusion Although CD133+ derived melanoma cells represented stemness fea- tures, our findings demonstrated that spheroid culture could be more effective meth- od to enrich melanoma stem cells. PMID:27054115

  7. Growth inhibition of malignant melanoma by intermediate frequency alternating electric fields, and the underlying mechanisms.

    PubMed

    Chen, H; Liu, R; Liu, J; Tang, J

    2012-01-01

    This study investigated the antitumour effects of intermediate frequency alternating electric fields (IF-AEF) in a murine melanoma cell line (B16F10) and a mouse tumour model. IF-AEF was applied at 100 kHz. Proliferation of B16F10 cells in vitro was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. IF-AEF was applied in vivo to mice bearing B16F10 tumours. Terminal deoxy nucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay for apoptosis, and immunohistochemical detection of CD34 and vascular endothelial growth factor (VEGF), were performed. IF-AEF inhibited the proliferation of B16F10 cells in an electrical intensity and time-dependent manner. Treatment with IF-AEF for 7 days significantly inhibited the growth of tumours compared with untreated controls. IF-AEF induced apoptosis in vivo and reduced CD34-positive cell numbers; CD34 is a special marker of microvessel density. IF-AEF reduced microvessel density related to tumour growth and may serve as a therapeutic strategy for cancer treatment.

  8. The IL-6/sIL-6R treatment of a malignant melanoma cell line enhances susceptibility to TNF-{alpha}-induced apoptosis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wagley, Yadav; Yoo, Yung-Choon; Seo, Han Geuk

    2007-03-23

    Melanoma is an intractable tumor that has shown very impressive and promising response to local administration of high dose recombinant TNF-{alpha} in combination with IFN-{gamma} in clinical studies. In this study, we investigated the effect of IL-6/sIL-6R on TNF-{alpha}-resistant B16/F10.9 melanoma cells. A low dose of TNF-{alpha} or IL-6/sIL-6R had minimal affect on the cell growth. However, the highly active fusion protein of sIL-6R and IL-6 (IL6RIL6), covalently linked by a flexible peptide, sensitized TNF-{alpha}-resistant F10.9 melanoma cells to TNF-{alpha}-induced apoptosis. Stimulation of the cells with IL6RIL6 plus TNF-{alpha} resulted in both the activation of caspase-3 and the reduction ofmore » bcl-2 expression. Flow cytometry analysis showed that IL6RIL6-upregulated TNF-R55 and TNF-R75 expression, suggesting an increase in TNF-{alpha} responsiveness by IL6RIL6 resulting from the induction of TNF receptors. Moreover, exposure of F10.9 cells to neutralizing antibody to TNF-R55 significantly inhibited IL6RIL6/TNF-{alpha}-induced cytotoxicity. These results suggest that the IL6/sIL6R/gp130 system, which sensitizes TNF-{alpha}-resistant melanoma cells to TNF-{alpha}-induced apoptosis, may provide a new target for immunotherapy.« less

  9. Primary Tr1 cells from metastatic melanoma eliminate tumor-promoting macrophages through granzyme B- and perforin-dependent mechanisms.

    PubMed

    Yan, Hongxia; Zhang, Ping; Kong, Xue; Hou, Xianglian; Zhao, Li; Li, Tianhang; Yuan, Xiaozhou; Fu, Hongjun

    2017-04-01

    In malignant melanoma, tumor-associated macrophages play multiple roles in promoting tumor growth, such as inducing the transformation of melanocytes under ultraviolet irradiation, increasing angiogenesis in melanomas, and suppressing antitumor immunity. Because granzyme B- and perforin-expressing Tr1 cells could specifically eliminate antigen-presenting cells of myeloid origin, we examined whether Tr1 cells in melanoma could eliminate tumor-promoting macrophages and how the interaction between Tr1 cells and macrophages could affect the growth of melanoma cells. Tr1 cells were characterized by high interleukin 10 secretion and low Foxp3 expression and were enriched in the CD4 + CD49b + LAG-3 + T-cell fraction. Macrophages derived from peripheral blood monocytes in the presence of modified melanoma-conditioned media demonstrated tumor-promoting capacity, exemplified by improving the proliferation of cocultured A375 malignant melanoma cells. But when primary Tr1 cells were present in the macrophage-A375 coculture, the growth of A375 cells was abrogated. The conventional CD25 + Treg cells, however, were unable to inhibit macrophage-mediated increase in tumor cell growth. Further analyses showed that Tr1 cells did not directly eliminate A375 cells, but mediated the killing of tumor-promoting macrophages through the secretion of granzyme B and perforin. The tumor-infiltrating interleukin 10 + Foxp3 - CD4 + T cells expressed very low levels of granzyme B and perforin, possibly suggested the downregulation of Tr1 cytotoxic capacity in melanoma tumors. Together, these data demonstrated an antitumor function of Tr1 cells through the elimination of tumor-promoting macrophages, which was not shared by conventional Tregs.

  10. In-vitro and in-vivo inhibition of melanoma growth and metastasis by the drug combination of celecoxib and dacarbazine.

    PubMed

    Sadhu, Satya S; Wang, Shenggang; Averineni, Ranjith K; Seefeldt, Teresa; Yang, Yang; Guan, Xiangming

    2016-12-01

    Celecoxib has been found to be effective in cancer prevention and treatment. Its combination with other chemotherapeutic agents was reported to produce synergistic/additive effects on various cancers. Dacarbazine (DTIC) is one of the most commonly used drugs in the treatment of metastatic melanoma. This investigation aimed to determine the in-vitro and in-vivo effects of the drug combination of celecoxib and DTIC on melanoma growth and metastasis. Melanoma cells B16-F10 and SK-MEL-28, and female C57BL/6 mice were used for the study. Our in-vitro data showed that significant synergistic effects were obtained when celecoxib was used together with various concentrations of DTIC. A study with B16-F10 cells using flow cytometry analysis showed that the drug combination induced significantly more apoptosis than each drug used individually. Our in-vivo results showed that the drug combination was much more effective than each drug used alone for the inhibition of both melanoma growth and metastasis in the B16-F10+C57BL/6 mouse models. For melanoma growth, the median survival rates for phosphate-buffered saline (PBS) (control), celecoxib (30 mg/kg), DTIC-1 (10 mg/kg), DTIC-2 (positive control, 50 mg/kg), and the drug combination (DTIC 10 mg/kg+celecoxib 30 mg/kg) were 6, 6.5, 7.5, 7.5, and 9 days, respectively. For melanoma metastasis, the average number of metastatic tumors in murine lungs was 53.7±10.7, 31.8±18.6, 21.2±21.7, 7.0±9.0, and 0.8±2.0 for PBS, DTIC-1, celecoxib, the drug combination, and DTIC-2. Our results warrant further investigation of the combination as an effective treatment for melanoma patients.

  11. Zinc Oxide Nanoparticle-Poly I:C RNA Complexes: Implication as Therapeutics against Experimental Melanoma.

    PubMed

    Ramani, Meghana; Mudge, Miranda C; Morris, R Tyler; Zhang, Yuntao; Warcholek, Stanislaw A; Hurst, Miranda N; Riviere, Jim E; DeLong, Robert K

    2017-03-06

    There is current interest in harnessing the combined anticancer and immunological effect of nanoparticles (NPs) and RNA. Here, we evaluate the bioactivity of poly I:C (pIC) RNA, bound to anticancer zinc oxide NP (ZnO-NP) against melanoma. Direct RNA association to unfunctionalized ZnO-NP is shown by observing change in size, zeta potential, and absorption/fluorescence spectra upon complexation. RNA corona was visualized by transmission electron microscopy (TEM) for the first time. Binding constant (K b = 1.6-2.8 g -1 L) was determined by modified Stern-Volmer, absorption, and biological surface activity index analysis. The pIC-ZnO-NP complex increased cell death for both human (A375) and mouse (B16F10) cell lines and suppressed tumor cell growth in BALB/C-B16F10 mouse melanoma model. Ex vivo tumor analysis indicated significant molecular activity such as changes in the level of phosphoproteins JNK, Akt, and inflammation markers IL-6 and IFN-γ. High throughput proteomics analysis revealed zinc oxide and poly I:C-specific and combinational patterns that suggested possible utility as an anticancer and immunotherapeutic strategy against melanoma.

  12. A novel compound which sensitizes BRAF wild-type melanoma cells to vemurafenib in a TRIM16-dependent manner.

    PubMed

    Sutton, Selina K; Carter, Daniel R; Kim, Patrick; Tan, Owen; Arndt, Greg M; Zhang, Xu Dong; Baell, Jonathan; Noll, Benjamin D; Wang, Shudong; Kumar, Naresh; McArthur, Grant A; Cheung, Belamy B; Marshall, Glenn M

    2016-08-09

    There is an urgent need for better therapeutic options for advanced melanoma patients, particularly those without the BRAFV600E/K mutation. In melanoma cells, loss of TRIM16 expression is a marker of cell migration and metastasis, while the BRAF inhibitor, vemurafenib, induces melanoma cell growth arrest in a TRIM16-dependent manner. Here we identify a novel small molecule compound which sensitized BRAF wild-type melanoma cells to vemurafenib. High throughput, cell-based, chemical library screening identified a compound (C012) which significantly reduced melanoma cell viability, with limited toxicity for normal human fibroblasts. When combined with the BRAFV600E/K inhibitor, vemurafenib, C012 synergistically increased vemurafenib potency in 5 BRAFWT and 4 out of 5 BRAFV600E human melanoma cell lines (Combination Index: CI < 1), and, dramatically reduced colony forming ability. In addition, this drug combination was significantly anti-tumorigenic in vivo in a melanoma xenograft mouse model. The combination of vemurafenib and C012 markedly increased expression of TRIM16 protein, and knockdown of TRIM16 significantly reduced the growth inhibitory effects of the vemurafenib and C012 combination. These findings suggest that the combination of C012 and vemurafenib may have therapeutic potential for the treatment of melanoma, and, that reactivation of TRIM16 may be an effective strategy for patients with this disease.

  13. A novel compound which sensitizes BRAF wild-type melanoma cells to vemurafenib in a TRIM16-dependent manner

    PubMed Central

    Sutton, Selina K.; Carter, Daniel R.; Kim, Patrick; Tan, Owen; Arndt, Greg M.; Zhang, Xu Dong; Baell, Jonathan; Noll, Benjamin D.; Wang, Shudong; Kumar, Naresh; McArthur, Grant A.; Cheung, Belamy B.; Marshall, Glenn M.

    2016-01-01

    There is an urgent need for better therapeutic options for advanced melanoma patients, particularly those without the BRAFV600E/K mutation. In melanoma cells, loss of TRIM16 expression is a marker of cell migration and metastasis, while the BRAF inhibitor, vemurafenib, induces melanoma cell growth arrest in a TRIM16-dependent manner. Here we identify a novel small molecule compound which sensitized BRAF wild-type melanoma cells to vemurafenib. High throughput, cell-based, chemical library screening identified a compound (C012) which significantly reduced melanoma cell viability, with limited toxicity for normal human fibroblasts. When combined with the BRAFV600E/K inhibitor, vemurafenib, C012 synergistically increased vemurafenib potency in 5 BRAFWT and 4 out of 5 BRAFV600E human melanoma cell lines (Combination Index: CI < 1), and, dramatically reduced colony forming ability. In addition, this drug combination was significantly anti-tumorigenic in vivo in a melanoma xenograft mouse model. The combination of vemurafenib and C012 markedly increased expression of TRIM16 protein, and knockdown of TRIM16 significantly reduced the growth inhibitory effects of the vemurafenib and C012 combination. These findings suggest that the combination of C012 and vemurafenib may have therapeutic potential for the treatment of melanoma, and, that reactivation of TRIM16 may be an effective strategy for patients with this disease. PMID:27447557

  14. Triterpenoid saponins from Polaskia chichipe Backbg. and their inhibitory or promotional effects on the melanogenesis of B16 melanoma cells.

    PubMed

    Fujihara, Koji; Takahashi, Kunio; Koyama, Kiyotaka; Kinoshita, Kaoru

    2017-10-01

    Five new oleanane-type saponins 1-5 together with a known saponin 6 and a steroidal glycoside 7 were isolated from Polaskia chichipe Backbg., and their structures were determined from their 1D and 2D NMR and HRFABMS spectral data. The six isolated saponins 1-6 were tested for their effects on the melanogenesis of B16 melanoma 4A5 cells. Compound 1 exerted an inhibitory effect at 100 μM whereas compound 3 promoted melanogenesis at the same concentration, even though these two compounds contain the same aglycon structure. The dose-dependent activities of compounds 1 and 3 on melanin synthesis were investigated.

  15. Effect of MCM09, an active site-directed inhibitor of factor Xa, on B16-BL6 melanoma lung colonies in mice.

    PubMed

    Rossi, C; Hess, S; Eckl, R W; di Lena, A; Bruno, A; Thomas, O; Poggi, A

    2006-03-01

    Treatment with anticoagulant drugs has shown potential inhibitory effect on tumor invasion, although the relationship with clotting inhibition was not clear. The aim of our study was to evaluate the potential antitumor activity of MCM09, a newly developed, active site-directed, small molecule inhibitor of factor Xa (FXa) [WO0216312], and to relate the findings to anticlotting potency. MCM09 (0.1-10 mg kg(-1)) or heparin (H; 10 mg kg(-1)) was injected intravenously (i.v.), with 5 x 10(4) B16-BL6 melanoma cells, in C57BL/6 mice. Mice were killed after 18 days, to count lung colonies. Ex vivo anticoagulant activity was measured by activated partial thromboplastin time (APTT) on mouse plasma. MCM09, a selective inhibitor of FXa (IC-50 = 2.4 nm against human FXa), inhibited in a dose-dependent manner B16-BL6 melanoma lung colonies in mice. Mean lung metastasis number was 20.9 +/- 4.8 in controls (n = 10), 1.2 +/- 0.4 in mice treated with H, 10 mg kg(-1) i.v. (P < 0.01), 0.9 +/- 0.3, 9.2 +/- 2.2 and 15.5 +/- 2.6 in mice treated with MCM09, at 10 (P < 0.01), 1 (P < 0.05) and 0.1 mg kg(-1) i.v. (ns), respectively. MCM09 (10 mg kg(-1) i.v.) significantly prolonged APTT (57.1 +/- 10.2 s) 30 min after i.v. injection when compared with controls (25.3 +/- 1.6 s; P < 0.05). Lung colonies were 74.2-72.6% reduced by MCM09 (10 mg kg(-1)) given 60 or 120 min before cells, but not by MCM09 given 60 min thereafter, suggesting a direct cell interaction as a mechanism underlying antitumor activity.

  16. Melanogenesis stimulation in murine b16 melanoma cells by umberiferae plant extracts and their coumarin constituents.

    PubMed

    Matsuda, Hideaki; Hirata, Noriko; Kawaguchi, Yoshiko; Yamazaki, Miho; Naruto, Shunsuke; Shibano, Makio; Taniguchi, Masahiko; Baba, Kimiye; Kubo, Michinori

    2005-07-01

    Melanogenesis stimulation activities of seven ethanolic extracts obtained from Umbelliferae plants used as Chinese crude drugs, namely the roots of Angelica dahurica BENTH. et HOOK., A. biserrata SHEN et YUAN, Notopterygium incisum TING, Heracleum lanatum MICHX., and H. candicans WALL., and the fruits of Cinidium monnieri (L.) CUSSON and C. formosanum YABE, were examined by using cultured murine B16 melanoma cells. Among them, the extract (5, 25 microg/ml) of H. lanatum showed a potent stimulatory effect on melanogenesis with significant enhancement of cell proliferation in a dose-dependent manner. The melanogenesis stimulatory effects of sixteen coumarins (1-16) isolated from the seven Umbelliferae crude drugs were also examined. Among them, linear-furocoumarins [psoralen (1), xanthotoxin (2), bergapten (3), and isopimpinellin (4)] and angular-furocoumarin [sphondin (13)] exhibited potent melanogenesis stimulation activity. From the view point of structure-activity relationships, it may be assumed that a linear-furocoumarin ring having a hydrogen and/or methoxyl group at 5 and 8 positions such as 1, 2, 3 and 4 was preferable for the melanogenesis stimulation activity. The introduction of a prenyl group into the furocoumarin ring was disadvantageous. Coumarin derivatives having a simple coumarin ring were inactive.

  17. MicroRNA-193b represses cell proliferation and regulates cyclin D1 in melanoma.

    PubMed

    Chen, Jiamin; Feilotter, Harriet E; Paré, Geneviève C; Zhang, Xiao; Pemberton, Joshua G W; Garady, Cherif; Lai, Dulcie; Yang, Xiaolong; Tron, Victor A

    2010-05-01

    Cutaneous melanoma is an aggressive form of human skin cancer characterized by high metastatic potential and poor prognosis. To better understand the role of microRNAs (miRNAs) in melanoma, the expression of 470 miRNAs was profiled in tissue samples from benign nevi and metastatic melanomas. We identified 31 miRNAs that were differentially expressed (13 up-regulated and 18 down-regulated) in metastatic melanomas relative to benign nevi. Notably, miR-193b was significantly down-regulated in the melanoma tissues examined. To understand the role of miR-193b in melanoma, functional studies were undertaken. Overexpression of miR-193b in melanoma cell lines repressed cell proliferation. Gene expression profiling identified 314 genes down-regulated by overexpression of miR-193b in Malme-3M cells. Eighteen of these down-regulated genes, including cyclin D1 (CCND1), were also identified as putative miR-193b targets by TargetScan. Overexpression of miR-193b in Malme-3M cells down-regulated CCND1 mRNA and protein by > or = 50%. A luciferase reporter assay confirmed that miR-193b directly regulates CCND1 by binding to the 3'untranslated region of CCND1 mRNA. These studies indicate that miR-193b represses cell proliferation and regulates CCND1 expression and suggest that dysregulation of miR-193b may play an important role in melanoma development.

  18. MicroRNA-193b Represses Cell Proliferation and Regulates Cyclin D1 in Melanoma

    PubMed Central

    Chen, Jiamin; Feilotter, Harriet E.; Paré, Geneviève C.; Zhang, Xiao; Pemberton, Joshua G.W.; Garady, Cherif; Lai, Dulcie; Yang, Xiaolong; Tron, Victor A.

    2010-01-01

    Cutaneous melanoma is an aggressive form of human skin cancer characterized by high metastatic potential and poor prognosis. To better understand the role of microRNAs (miRNAs) in melanoma, the expression of 470 miRNAs was profiled in tissue samples from benign nevi and metastatic melanomas. We identified 31 miRNAs that were differentially expressed (13 up-regulated and 18 down-regulated) in metastatic melanomas relative to benign nevi. Notably, miR-193b was significantly down-regulated in the melanoma tissues examined. To understand the role of miR-193b in melanoma, functional studies were undertaken. Overexpression of miR-193b in melanoma cell lines repressed cell proliferation. Gene expression profiling identified 314 genes down-regulated by overexpression of miR-193b in Malme-3M cells. Eighteen of these down-regulated genes, including cyclin D1 (CCND1), were also identified as putative miR-193b targets by TargetScan. Overexpression of miR-193b in Malme-3M cells down-regulated CCND1 mRNA and protein by ≥50%. A luciferase reporter assay confirmed that miR-193b directly regulates CCND1 by binding to the 3′untranslated region of CCND1 mRNA. These studies indicate that miR-193b represses cell proliferation and regulates CCND1 expression and suggest that dysregulation of miR-193b may play an important role in melanoma development. PMID:20304954

  19. [The effect of Angelica sinensis on adhesion, invasion, migration and metastasis of melanoma cells].

    PubMed

    Gu, Qin; Xu, Jian-ya; Cheng, Luo-gen; Xia, Wei-jun

    2007-03-01

    To study the effect of Angelica sinensis on invasion, adhesion, migration and metastasis of B16-BL6 metastatic mouse melanoma cells and discuss its functional mechanism. The proliferation, adhesion, invasion and migration capacity of B16-BL6 metastatic cells was evaluated by MTT assay, adhesion assay and reconstituted basement membrane invasion and migration assay in vitro respectively. Mouse spontaneous melanoma model was used to study the effect of Angelica sinensis on metastasis in vivo. The extract of Angelica sinensis inhibited the proliferation of B16-BL6 metastatic cells and its migration capacity significantly. It regulated bidirectionally the adhesion of B16-BL6 metastatic cells to the basement component laminin while it had no effect on the invasion capacity. In the mouse spotaneous melanoma model, the lung metastatic nodes number and its volume were significantly decreased after continuously treated with the extract of Angelica sinensis at the concentration of 3.67 mg/kg. The extract of Angelica sinensis can inhibit the metastasis of of B16-BL6 metastatic mouse melanoma cells and its mechanism is maybe that Angelica sinensis can inhibit the B16-BL6 cells adhering to the ECM and reduce the migration of B16-BL6 cells.

  20. Antimetastatic effect of PSK, a protein-bound polysaccharide, against the B16-BL6 mouse melanoma.

    PubMed

    Matsunaga, K; Ohhara, M; Oguchi, Y; Iijima, H; Kobayashi, H

    1996-01-01

    We examined the effect of PSK, a protein-bound polysaccharide, upon in vivo metastasis and in vitro invasion of the B16-BL6 mouse melanoma cells. (1) PSK suppressed in vivo artificial and spontaneous lung metastases of B16-BL6 in C57BL/6 mice. (2) PSK in a dose-dependent fashion suppressed in vitro invasion and chemotaxis of the tumor cells using filters coated with a reconstituted basement membrane. (3) PSK had little effect on DNA synthesis in tumor cells in vitro, but suppressed tumor cell adhesion to, degradation of, and haptotaxis to components of the basement membrane. (4) PSK suppressed the binding of tumor cells to components of the basement membrane. These findings suggest that PSK may suppress metastasis through inhibition of tumor cell invasion and that this effect is the result of interactions between PSK and components of the basement membrane.

  1. Generation of T-cells reactive to the poorly immunogenic B16-BL6 melanoma with efficacy in the treatment of spontaneous metastases.

    PubMed

    Geiger, J D; Wagner, P D; Cameron, M J; Shu, S; Chang, A E

    1993-04-01

    The B16-BL6 (BL6) melanoma is a poorly immunogenic murine tumor that is highly invasive and spontaneously metastasizes from the primary site. Utilizing an established anti-CD3/interleukin-2 (IL-2) culture procedure, we have previously reported that lymph nodes (LNs) draining immunogenic murine sarcomas contained preeffector cells that could be activated to differentiate into therapeutic effector cells for adoptive immunotherapy. By contrast, LNs draining the poorly immunogenic BL6 melanoma were found not to be a reliable source of preeffector cells. Instead, sensitization of preeffector cells reactive to BL6 required the subcutaneous inoculation of tumor admixed with Corynebacterium parvum. LN cells draining these vaccination sites demonstrated therapeutic efficacy only after subsequent anti-CD3/IL-2 activation. The sensitization of preeffector cells was dependent on the presence of tumor antigen and an optimal dose of C. parvum (< or = 50 micrograms). Furthermore, kinetic analysis revealed that the preeffector response was transient after tumor vaccination. The therapeutic efficacy of anti-CD3/IL-2 activated LN cells was further evaluated in the treatment of spontaneous macroscopic BL6 visceral metastases. Spontaneous visceral metastases were induced in animals by inoculation with BL6 tumor in the footpad followed by amputation of the primary tumor 3 weeks later. The systemic transfer of 10(8) anti-CD3/IL-2 activated T-cells and the concomitant intraperitoneal administration of subtherapeutic doses of IL-2 1 week after amputation cured 50% of the animals and prolonged median survival time (MST) to > 140 days. All mice except one that received no treatment or was treated with IL-2 alone succumbed to visceral metastases with an MST of approximately 23 days. This study characterizes a model whereby the weak immune response to the BL6 melanoma can be positively or negatively modulated for the generation of antitumor reactive T-cells useful in adoptive immunotherapy.

  2. Inhibition of B16-BL6 melanoma lung colonies by semisynthetic sulfaminoheparosan sulfates from E. coli K5 polysaccharide.

    PubMed

    Poggi, Andreina; Rossi, Cosmo; Casella, Nicola; Bruno, Cristiana; Sturiale, Luisella; Dossi, Carla; Naggi, Annamaria

    2002-08-01

    Heparin (H), heparan sulfate (HS), and related glycosaminoglycans can inhibit cancer cell invasion, possibly due to their ability to interact with vascular growth factors, adhesion molecules, endoglycosidases, and signaling proteins, in addition to the well-known effects on the clotting system. We evaluated the antitumor activity of a series of semisynthetic sulfaminoheparosan sulfates (SAHSs) with different degree and distribution of sulfates, obtained by chemical modifications of the E. coli K5 polysaccharide, namely type A, B, and C compounds. B16-BL6 melanoma cells (10 5 cells/mouse) were injected intravenously (i.v.) in a lateral tail vein of C57BL6 mice at a dose of 0.5 mg/ mouse together with test compounds. Tumor lung nodules were significantly reduced as compared with controls only by H (95.5 +/- 1.0% inhibition), SAHS-2 (84.2 +/- 5.0% inhibition), and SAHS-4 (91.1 +/- 4.2% inhibition), among compounds tested. SAHS-2 and SAHS-4 are type B compounds, with a sulfate/carboxylate ratio similar to that of H. A typical mammalian HS showed only 54.8% inhibition. Supersulfated low-molecular-weight heparin and heparan sulfate (ssLMWH and ssLMWHS) showed an activity similar to that of unfractionated compounds. H and SAHS-4 inhibited dose dependently B16-BL6 lung colonies, with IC-50 values of 0.05 and 0.1 mg/mouse, respectively. The relationship with ex vivo anticoagulant potency was evaluated by activated partial thromboplastin time (aPTT) on mouse plasma at different time intervals after i.v. injection (0.1 to 0.5 mg/mouse) of the compound. H showed a dose-dependent anticoagulant activity lasting up to 2 hours, whereas SAHS-4 showed a potent anticoagulant effect only at a dose of 0.5 mg/mouse. Accordingly, H but not SAHS-4 consistently inhibited B16-BL6 lung colonies when given 1 hour before tumor cells. SAHS-4 derivatives, with different size and/or affinity depleted of AT binding sites, showed an inhibitory effect on B16-BL6 melanoma similar to that of SAHS-4

  3. Intravital third harmonic generation microscopy of collective melanoma cell invasion

    PubMed Central

    Weigelin, Bettina; Bakker, Gert-Jan; Friedl, Peter

    2012-01-01

    Cancer cell invasion is an adaptive process based on cell-intrinsic properties to migrate individually or collectively, and their adaptation to encountered tissue structure acting as barrier or providing guidance. Whereas molecular and physical mechanisms of cancer invasion are well-studied in 3D in vitro models, their topographic relevance, classification and validation toward interstitial tissue organization in vivo remain incomplete. Using combined intravital third and second harmonic generation (THG, SHG), and three-channel fluorescence microscopy in live tumors, we here map B16F10 melanoma invasion into the dermis with up to 600 µm penetration depth and reconstruct both invasion mode and tissue tracks to establish invasion routes and outcome. B16F10 cells preferentially develop adaptive invasion patterns along preformed tracks of complex, multi-interface topography, combining single-cell and collective migration modes, without immediate anatomic tissue remodeling or destruction. The data suggest that the dimensionality (1D, 2D, 3D) of tissue interfaces determines the microanatomy exploited by invading tumor cells, emphasizing non-destructive migration along microchannels coupled to contact guidance as key invasion mechanisms. THG imaging further detected the presence and interstitial dynamics of tumor-associated microparticles with submicron resolution, revealing tumor-imposed conditioning of the microenvironment. These topographic findings establish combined THG, SHG and fluorescence microscopy in intravital tumor biology and provide a template for rational in vitro model development and context-dependent molecular classification of invasion modes and routes. PMID:29607252

  4. [Effect of Spatholobus suberctus on adhesion, invasion, migration and metastasis of melanoma cells].

    PubMed

    Xu, Jian-Ya; Gu, Qin; Xia, Wei-Jun

    2010-10-01

    To study the effect of Spatholobus suberctus, a kind of Chinese Traditional Medicine which can dissolve the stasis by activating the blood circulation, on invasion, adhesion, migration and metastasis of B16-BL6 metastatic mouse melanoma cells and its mechanism. The proliferation, adhesion, invasion and migration capacity of B16-BL6 metastatic cells was evaluated by MTP assay, adhesion assay and reconstituted basement membrane invasion and migration assay in vitro respectively. Mouse spontaneous motility melanoma model was used to study the effect of Spatholobus suberctus on metastasis in vivo. At the highest innoxious concentration, the extracts of Spatholobus suberctus inhibited the adhesion and invasion capacity of B16-BL6 metastatic cells significantly. In the mouse spontaneous melanoma model, the lung metastatic nodes number and its volume were significantly decreased after continuously treated with the extracts of Spatholobus suberctu. The extracts of Spatholobus suberctu can inhibit the metastasis of of B16-BI6 metastatic mouse melanoma cells and its mechanism may be inhibiting the capability of B16-BL6 cells in adhering to the ECM and invading the basement membrane.

  5. TRPM5 mediates acidic extracellular pH signaling and TRPM5 inhibition reduces spontaneous metastasis in mouse B16-BL6 melanoma cells

    PubMed Central

    Maeda, Toyonobu; Suzuki, Atsuko; Koga, Kaori; Miyamoto, Chihiro; Maehata, Yojiro; Ozawa, Shigeyuki; Hata, Ryu-Ichiro; Nagashima, Yoji; Nabeshima, Kazuki; Miyazaki, Kaoru; Kato, Yasumasa

    2017-01-01

    Extracellular acidity is a hallmark of solid tumors and is associated with metastasis in the tumor microenvironment. Acidic extracellular pH (pHe) has been found to increase intracellular Ca2+ and matrix metalloproteinase-9 (MMP-9) expression by activating NF-κB in the mouse B16 melanoma model. The present study assessed whether TRPM5, an intracellular Ca2+-dependent monovalent cation channel, is associated with acidic pHe signaling and induction of MMP-9 expression in this mouse melanoma model. Treatment of B16 cells with Trpm5 siRNA reduced acidic pHe-induced MMP-9 expression. Enforced expression of Trpm5 increased the rate of acidic pHe-induced MMP-9 expression, as well as increasing experimental lung metastasis. This genetic manipulation did not alter the pHe critical for MMP-9 induction but simply amplified the percentage of inducible MMP-9 at each pHe. Treatment of tumor bearing mice with triphenylphosphine oxide (TPPO), an inhibitor of TRPM5, significantly reduced spontaneous lung metastasis. In silico analysis of clinical samples showed that high TRPM5 mRNA expression correlated with poor overall survival rate in patients with melanoma and gastric cancer but not in patients with cancers of the ovary, lung, breast, and rectum. These results showed that TRPM5 amplifies acidic pHe signaling and may be a promising target for preventing metastasis of some types of tumor. PMID:29108231

  6. TRPM5 mediates acidic extracellular pH signaling and TRPM5 inhibition reduces spontaneous metastasis in mouse B16-BL6 melanoma cells.

    PubMed

    Maeda, Toyonobu; Suzuki, Atsuko; Koga, Kaori; Miyamoto, Chihiro; Maehata, Yojiro; Ozawa, Shigeyuki; Hata, Ryu-Ichiro; Nagashima, Yoji; Nabeshima, Kazuki; Miyazaki, Kaoru; Kato, Yasumasa

    2017-10-03

    Extracellular acidity is a hallmark of solid tumors and is associated with metastasis in the tumor microenvironment. Acidic extracellular pH (pH e ) has been found to increase intracellular Ca 2+ and matrix metalloproteinase-9 (MMP-9) expression by activating NF-κB in the mouse B16 melanoma model. The present study assessed whether TRPM5, an intracellular Ca 2+ -dependent monovalent cation channel, is associated with acidic pH e signaling and induction of MMP-9 expression in this mouse melanoma model. Treatment of B16 cells with Trpm5 siRNA reduced acidic pH e -induced MMP-9 expression. Enforced expression of Trpm5 increased the rate of acidic pH e -induced MMP-9 expression, as well as increasing experimental lung metastasis. This genetic manipulation did not alter the pH e critical for MMP-9 induction but simply amplified the percentage of inducible MMP-9 at each pH e . Treatment of tumor bearing mice with triphenylphosphine oxide (TPPO), an inhibitor of TRPM5, significantly reduced spontaneous lung metastasis. In silico analysis of clinical samples showed that high TRPM5 mRNA expression correlated with poor overall survival rate in patients with melanoma and gastric cancer but not in patients with cancers of the ovary, lung, breast, and rectum. These results showed that TRPM5 amplifies acidic pH e signaling and may be a promising target for preventing metastasis of some types of tumor.

  7. Cinnamomum cassia Essential Oil Inhibits α-MSH-Induced Melanin Production and Oxidative Stress in Murine B16 Melanoma Cells

    PubMed Central

    Chou, Su-Tze; Chang, Wen-Lun; Chang, Chen-Tien; Hsu, Shih-Lan; Lin, Yu-Che; Shih, Ying

    2013-01-01

    Essential oils extracted from aromatic plants exhibit important biological activities and have become increasingly important for the development of aromatherapy for complementary and alternative medicine. The essential oil extracted from Cinnamomum cassia Presl (CC-EO) has various functional properties; however, little information is available regarding its anti-tyrosinase and anti-melanogenic activities. In this study, 16 compounds in the CC-EO have been identified; the major components of this oil are cis-2-methoxycinnamic acid (43.06%) and cinnamaldehyde (42.37%). CC-EO and cinnamaldehyde exhibited anti-tyrosinase activities; however, cis-2-methoxycinnamic acid did not demonstrate tyrosinase inhibitory activity. In murine B16 melanoma cells stimulated with α-melanocyte-stimulating hormone (α-MSH), CC-EO and cinnamaldehyde not only reduced the melanin content and tyrosinase activity of the cells but also down-regulated tyrosinase expression without exhibiting cytotoxicity. Moreover, CC-EO and cinnamaldehyde decreased thiobarbituric acid-reactive substance (TBARS) levels and restored glutathione (GSH) and catalase activity in the α-MSH-stimulated B16 cells. These results demonstrate that CC-EO and its major component, cinnamaldehyde, possess potent anti-tyrosinase and anti-melanogenic activities that are coupled with antioxidant properties. Therefore, CC-EO may be a good source of skin-whitening agents and may have potential as an antioxidant in the future development of complementary and alternative medicine-based aromatherapy. PMID:24051402

  8. 203Pb-Labeled Alpha-Melanocyte-Stimulating Hormone Peptide as an Imaging Probe for Melanoma Detection

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yubin, Miao; Figueroa, Said D.; Fisher, Darrell R.

    2008-05-01

    Abbreviations: a-MSH; alpha melanocyte stimulating hormone, DOTA; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, Re(Arg11)CCMSH; DOTA-[Cys3,4,10, D-Phe7, Arg11]a-MSH3-13, NDP; [Nle4,d-Phe7] a-MSH3-13. Abstract Peptide-targeted alpha therapy with 200 mCi of 212Pb-DOTA-Re(Arg11)CCMSH cured 45% of B16/F1 murine melanoma-bearing C57 mice in a 120-day study, highlighting its melanoma treatment potential. However, there is a need to develop an imaging surrogate for patient specific dosimetry and to monitor the tumor response to 212Pb-DOTA-Re(Arg11)CCMSH therapy. The purpose of this study was to evaluate the potential of 203Pb-DOTA-Re(Arg11)CCMSH as a matched-pair SPECT imaging agent for 212Pb-DOTA-Re(Arg11)CCMSH. Method: DOTA-Re(Arg11)CCMSH was labeled with 203Pb in 0.5 M NH4OAc buffer at pH 5.4. Themore » internalization and efflux of 203Pb-DOTA-Re(Arg11)CCMSH were determined in B16/F1 melanoma cells. The pharmacokinetics of 203Pb-DOTA-Re(Arg11)CCMSH were examined in B16/F1 melanoma-bearing C57 mice. A micro-SPECT/CT imaging study was performed with 203Pb-DOTA-Re(Arg11)CCMSH in a B16/F1 melanoma-bearing C57 mouse at 2 h post-injection. Results: 203Pb-DOTA-Re(Arg11)CCMSH was easily prepared in NH4OAc buffer and completely separated from the excess non-radiolabeled peptide by RP-HPLC. 203Pb-DOTA-Re(Arg11)CCMSH displayed fast internalization and extended retention in B16/F1 cells. Approximately 73% of 203Pb-DOTA-Re(Arg11)CCMSH activity internalized after a 20-min incubation at 25C. After incubating the cells in culture media for 20 min, 78% of internalized activity remained in the cells. 203Pb-DOTA-Re(Arg11)CCMSH exhibited similar biodistribution pattern with 212Pb-DOTA-Re(Arg11)CCMSH in B16/F1 melanoma-bearing mice. 203Pb-DOTA-Re(Arg11)CCMSH exhibited the peak tumor uptake of 12.00 +/- 3.20 %ID/g at 1 h post-injection. The tumor uptake gradually decreased to 3.43 +/- 1.12 %ID/g at 48 h post-injection. 203Pb-DOTA-Re(Arg11)CCMSH exhibited the peak tumor to

  9. The cryo-thermal therapy eradicated melanoma in mice by eliciting CD4+ T-cell-mediated antitumor memory immune response.

    PubMed

    He, Kun; Liu, Ping; Xu, Lisa X

    2017-03-23

    Tumor metastasis is a major concern in tumor therapy. In our previous studies, a novel tumor therapeutic modality of the cryo-thermal therapy has been presented, highlighting its effect on the suppression of distal metastasis and leading to long-term survival in 4T1 murine mammary carcinoma model. To demonstrate the therapeutic efficacy in other aggressive tumor models and further investigate the mechanism of long-term survival induced, in this study, spontaneous metastatic murine B16F10 melanoma model was used. The cryo-thermal therapy induced regression of implanted melanoma and prolonged long-term survival while inhibiting lung metastasis. It also promoted the activation of CD4 + CD25 - conventional T cells, while reduced the percentage of CD4 + CD25 + regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in the spleen, lung and blood. Furthermore, the cryo-thermal therapy enhanced the cytolytic function of CD8 + T cells and induced differentiation of CD8 + T cells into memory stem T cell (T SCM ), and differentiation of CD4 + T cells into dominant CD4-CTL, Th1 and Tfh subsets in the spleen for 90 days after the treatment. It was found that good therapeutic effect was mainly dependent on CD4 + T cells providing a durable memory antitumor immune response. At the same time, significant increase of serum IFN-γ was also observed to provide an ideal microenvironment of antitumor immunity. Further study showed that the rejection of re-challenge of B16F10 but not GL261 tumor in the treated mice in 45 or 60 days after the treatment, implied a strong systemic and melanoma-specific memory antitumor immunity induced by the treatment. Thus the cryo-thermal therapy would be considered as a new therapeutic strategy to prevent tumor recurrence and metastasis with potential clinical applications in the near future.

  10. Hydroalcoholic extract of Rhodiola rosea L. (Crassulaceae) and its hydrolysate inhibit melanogenesis in B16F0 cells by regulating the CREB/MITF/tyrosinase pathway.

    PubMed

    Chiang, Hsiu-Mei; Chien, Yin-Chih; Wu, Chieh-Hsi; Kuo, Yueh-Hsiung; Wu, Wan-Chen; Pan, Yu-Yun; Su, Yu-Han; Wen, Kuo-Ching

    2014-03-01

    We investigated the effects of an aqueous alcohol extract of Rhodiola rosea (R. rosea) and its hydrolysate on melanin synthesis and the mechanisms mediating the activity. The ratio of tyrosol to salidroside was 2.3 in hydroalcoholic extract, and 51.0 in hydrolysate. We found that R. rosea extract and its hydrolysate inhibited melanin synthesis and tyrosinase activity in mouse melanoma cells (B16F0 cells). R. rosea extract also inhibited gene and protein expression of melanocortin 1 receptor (MC1R) and inhibited c-AMP response element binding protein (CREB) phosphorylation, suppressed the activation of AKT and glycogen synthase kinase-3 beta (GSK3β), and inhibited the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase-related protein 1 (TRP-1). R. rosea hydrolysate inhibited the phosphorylation of CREB, the activation of AKT and GSK3β, and the expression of MITF and tyrosinase. Our results suggest that R. rosea extract is a novel tyrosinase inhibitor and that it exerts its effects by regulating the CREB/MITF/tyrosinase pathway in B16F0. Further in vivo studies are needed to determine the effectiveness of R. rosea extract as a skin whitening agent. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Quercetin inhibits the invasion of murine melanoma B16-BL6 cells by decreasing pro-MMP-9 via the PKC pathway.

    PubMed

    Zhang, Xian-Ming; Huang, Shao-Peng; Xu, Qiang

    2004-01-01

    On the basis of the inhibitory effect of quercetin on the invasion of melanoma B16-BL6 cells previously reported by us, the mechanisms of quercetin-mediated inhibition of invasion were further investigated in the present study. The ability of B16-BL6 cells to invade and migrate was evaluated in terms of the numbers of cells penetrating a reconstituted basement membrane in the Transwell coculture system. The relative levels and activities of matrix metalloproteinase-9 (MMP-9) and MMP-2 were determined by gelatin zymography and quantified using LabWorks 4.0 software. The quercetin-mediated inhibition of invasion was partially blocked by phorbol-12,13-dibutyrate (PDB), a PKC (protein kinase C) activator, and by doxorubicin, a PKC inhibitor. Only the proforms of MMP-9 (92 kDa) and MMP-2 (72 kDa) were detected by gelatin zymography. Quercetin dose-dependently decreased the gelatinolytic activity of pro-MMP-9. Doxorubicin also markedly reversed the quercetin-induced decrease. Quercetin showed a dose-dependent antagonism of increases in gelatinolytic activity of pro-MMP-9 induced by PDB and free fatty acid (another PKC activator). Together with the report that quercetin directly reduces PKC activity, the results reported here suggest that quercetin may inhibit the invasion of B16-BL6 cells by decreasing pro-MMP-9 via the PKC pathway.

  12. Cosmetic applications of glucitol-core containing gallotannins from a proprietary phenolic-enriched red maple (Acer rubrum) leaves extract: inhibition of melanogenesis via down-regulation of tyrosinase and melanogenic gene expression in B16F10 melanoma cells.

    PubMed

    Ma, Hang; Xu, Jialin; DaSilva, Nicholas A; Wang, Ling; Wei, Zhengxi; Guo, Liangran; Johnson, Shelby L; Lu, Wei; Xu, Jun; Gu, Qiong; Seeram, Navindra P

    2017-05-01

    The red maple (Acer rubrum) is a rich source of phenolic compounds which possess galloyl groups attached to different positions of a 1,5-anhydro-D-glucitol core. While these glucitol-core containing gallotannins (GCGs) have reported anti-oxidant and anti-glycative effects, they have not yet been evaluated for their cosmetic applications. Herein, the anti-tyrosinase and anti-melanogenic effects of a proprietary phenolic-enriched red maple leaves extract [Maplifa ™ ; contains ca. 45% ginnalin A (GA) along with other GCGs] were investigated using enzyme and cellular assays. The GCGs showed anti-tyrosinase activity with IC 50 values ranging from 101.4 to 1047.3 μM and their mechanism of tyrosinase inhibition (using GA as a representative GCG) was evaluated by chelating and computational/modeling studies. GA reduced melanin content in murine melanoma B16F10 cells by 79.1 and 56.7% (at non-toxic concentrations of 25 and 50 μM, respectively), and its mechanisms of anti-melanogenic effects were evaluated by using methods including fluorescent probe (DCF-DA), real-time PCR, and western blot experiments. These data indicated that GA was able to: (1) reduce the levels of reactive oxygen species, (2) down-regulate the expression of MITF, TYR, TRP-1, and TRP-2 gene levels in a time-dependent manner, and (3) significantly reduce protein expression of the TRP-2 gene. Therefore, the anti-melanogenic effects of red maple GCGs warrant further investigation of this proprietary natural product extract for potential cosmetic applications.

  13. Improved efficacy of therapeutic vaccination with viable human umbilical vein endothelial cells against murine melanoma by introduction of OK432 as adjuvant.

    PubMed

    Xu, Maolei; Xing, Yun; Zhou, Ling; Yang, Xue; Yao, Wenjun; Xiao, Wen; Ge, Chiyu; Ma, Yanjun; Yang, Jie; Wu, Jie; Cao, Rongyue; Li, Taiming; Liu, Jingjing

    2013-06-01

    Vaccination with xenogeneic or syngeneic endothelial cells targeting tumor angiogenesis is effective for inhibiting tumor growth. OK432, an effective adjuvant, was mixed with viable human umbilical vein endothelial cells (HUVECs) to prepare a novel HUVECs-OK432 vaccine, which could have an improved therapeutic efficacy. In this study, HUVECs-OK432 was administrated in mice by subcutaneous injection in a therapeutic procedure. The results showed that a stronger HUVEC-specific Abs and cytotoxic T lymphocyte immune response were elicited, which resulted in significant inhibition on the growth of B16F10 melanoma and remarkably prolonged survival of B16F10 melanoma-bearing mice compared with HUVECs. Besides, parallel results were obtained in vitro showing a stronger inhibition of HUVEC proliferation by immune sera of HUVECs-OK432 than that of HUVECs. Moreover, histochemistry and immunohistochemistry analysis showed that HUVECs-OK432 induced large areas of continuous necrosis within tumors and significantly reduced the vessel density, correlating well with the extent of tumor inhibition. Our present results suggest that OK432 could be employed as an effective adjuvant for HUVEC vaccines and therefore should be useful for adjuvant immunotherapy of cancer.

  14. Lentivirus-mediated bifunctional cell labeling for in vivo melanoma study

    PubMed Central

    Day, Chi-Ping; Carter, John; Bonomi, Carrie; Esposito, Dominic; Crise, Bruce; Ortiz-Conde, Betty; Hollingshead, Melinda; Merlino, Glenn

    2009-01-01

    SUMMARY Lentiviral vectors (LVs) are capable of labeling a broad spectrum of cell types, achieving stable expression of transgenes. However, for in vivo studies, the duration of marker gene expression has been highly variable. We have developed a series of LVs harboring different promoters for expressing reporter gene in mouse cells. Long-term culture and colony formation of several LV-labeled mouse melanoma cells showed that promoters derived from mammalian house-keeping genes, especially those encoding RNA polymerase II (Pol2) and ferritin (FerH), provided the highest consistency for reporter expression. For in vivo studies, primary B16BL6 mouse melanoma were infected with LVs whose luciferase-GFP fusion gene (Luc/GFP) was driven by either Pol2 or FerH promoters. When transplanted into syngeneic C57BL/6 mice, Luc/GFP-labeled B16BL6 mouse melanoma cells can be monitored by bioluminescence imaging in vivo, and GFP-positive cells can be isolated from the tumors by FACS. Pol2-Luc/GFP labeling, while lower in activity, was more sustainable than FerH-Luc/GFP labeling in B16BL6 over consecutive passages into mice. We conclude that Pol-2-Luc/GFP labeling allows long-term in vivo monitoring and tumor cell isolation in immunocompetent mouse melanoma models. SIGNIFICANCE In this study we have developed and identified lentiviral vectors that allow labeled mouse melanoma cells to maintain long-term and consistent expression of a bifunctional luciferase-GFP marker gene, even in syngeneic mice with an intact immune function. This cell-labeling system can be used to build immunocompetent mouse melanoma models that permit both tumor monitoring and FACS-based tumor cell isolation from tissues, greatly facilitating the in vivo study of melanoma. PMID:19175523

  15. ARMS depletion facilitates UV irradiation induced apoptotic cell death in melanoma.

    PubMed

    Liao, Yi-Hua; Hsu, Su-Ming; Huang, Pei-Hsin

    2007-12-15

    Tumor cells often aberrantly reexpress molecules that mediate proper embryonic development for advantageous growth or survival. Here, we report that ankyrin repeat-rich membrane spanning (ARMS), a transmembrane protein abundant in the developing and adult neural tissues, is overexpressed in melanoma, a tumor ontogenetically originating from neural crest. Immunohistochemical study of 79 melanocytic lesions showed significantly increased expression of ARMS in primary malignant melanomas (92.9%) and metastatic melanoma (60.0%) in comparison with benign nevocellular nevi (26.7%). To investigate the role of ARMS in melanoma formation, murine B16F0 melanoma cells with stable knockdown of ARMS were established by RNA interference. Down-regulation of ARMS resulted in significant inhibition of anchorage-independent growth in soft agar and restrictive growth of melanoma in severe combined immunodeficient mice. Importantly, depletion of ARMS facilitated UVB-induced apoptosis in melanoma cells through inactivation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK. Addition of MEK inhibitor PD98059 further sensitized ARMS-depleted melanoma cells to UVB-induced apoptosis, whereas constitutively active MEK rescued ARMS-depleted cells from apoptosis. We further showed that BRAF, a downstream signaling molecule of ARMS in ERK pathway, is not mutated as a constitutively active form in acral lentiginous melanoma; in contrast, BRAF(T1799A) mutation, which leads to constitutive activation of ERK signaling, was detected in 57.1% of superficial spreading melanoma. Our study suggests that overexpression of ARMS per se serves as one mechanism to promote melanoma formation by preventing stress-induced apoptotic death mediated by the MEK/ERK signaling pathway, especially in acral lentiginous melanoma, most of which does not harbor BRAF mutation.

  16. Role of CD44 in lymphokine-activated killer cell-mediated killing of melanoma.

    PubMed

    Sun, Jingping; Law, Gabriela P; McKallip, Robert J

    2012-03-01

    In the current study, we examined the potential significance of CD44 expression on lymphokine-activated killer (LAK) cells in their interaction and killing of melanoma cells. Stimulation of splenocytes with IL-2 led to a significant increase in the expression of CD44 on T cells, NK cells, and NKT cells. Treatment of melanoma-bearing CD44 WT mice with IL-2 led to a significant reduction in the local tumor growth while treatment of melanoma-bearing CD44 KO mice with IL-2 was ineffective at controlling tumor growth. Furthermore, the ability of splenocytes from IL-2-treated CD44 KO mice to kill melanoma tumor targets was significantly reduced when compared to the anti-tumor activity of splenocytes from IL-2-treated CD44 WT mice. The importance of CD44 expression on the LAK cells was further confirmed by the observation that adoptively transferred CD44 WT LAK cells were significantly more effective than CD44 KO LAK cells at controlling tumor growth in vivo. Next, the significance of the increased expression of CD44 in tumor killing was examined and showed that following stimulation with IL-2, distinct populations of cells with low (CD44(lo)) or elevated (CD44(hi)) expression of CD44 are generated and that the CD44(hi) cells are responsible for killing of the melanoma cells. The reduced killing activity of the CD44 KO LAK cells did not result from reduced activation or expression of effector molecules but was due, at least in part, to a reduced ability to adhere to B16F10 tumor cells.

  17. IRF-8 Controls Melanoma Progression by Regulating the Cross Talk between Cancer and Immune Cells within the Tumor Microenvironment12

    PubMed Central

    Mattei, Fabrizio; Schiavoni, Giovanna; Sestili, Paola; Spadaro, Francesca; Fragale, Alessandra; Sistigu, Antonella; Lucarini, Valeria; Spada, Massimo; Sanchez, Massimo; Scala, Stefania; Battistini, Angela; Belardelli, Filippo; Gabriele, Lucia

    2012-01-01

    The transcription factor interferon regulatory factor-8 (IRF-8) is crucial for myeloid cell development and immune response and also acts as a tumor suppressor gene. Here, we analyzed the role of IRF-8 in the cross talk between melanoma cells and tumor-infiltrating leukocytes. B16-F10 melanoma cells transplanted into IRF-8-deficient (IRF-8-/-) mice grow more rapidly, leading to higher numbers of lung metastasis, with respect to control animals. These events correlated with reduced dendritic cell and T cell infiltration, accumulation of myeloid-derived suppressor cells and a chemokine/chemokine receptor expression profile within the tumor microenvironment supporting tumor growth, angiogenesis, and metastasis. Noticeably, primary tumors developing in IRF-8-/- mice displayed a clear-cut inhibition of IRF-8 expression in melanoma cells. Injection of the demethylating agent 5-aza-2′-deoxycytidine into melanoma-bearing IRF-8-/- animals induced intratumoral IRF-8 expression and resulted in the re-establishment of a chemokine/ chemokine receptor pattern favoring leukocyte infiltration and melanoma growth arrest. Importantly, intrinsic IRF-8 expression was progressively down-modulated during melanoma growth in mice and in human metastatic melanoma cells with respect to primary tumors. Lastly, IRF-8 expression in melanoma cells was directly modulated by soluble factors, among which interleukin-27 (IL-27), released by immune cells from tumor-bearing mice. Collectively, these results underscore a key role of IRF-8 in the cross talk between melanoma and immune cells, thus revealing its critical function within the tumor microenvironment in regulating melanoma progression and invasiveness. PMID:23308054

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

    PubMed

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

    2015-03-01

    4'-O-β-D-glucopyranosyl-quercetin-3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranoside (3C4'GQ), first isolated from Helminthostachys zeylanica root extract, was synthesized as a compound that stimulates intracellular melanogenesis. 3-O-methylquercetin (3MQ) and 3,4',7-O-trimethylquercetin (34'7TMQ) were synthesized as compounds that enhance extracellular melanin formation. The formation of dendrites and the expression of EBP50-PDZ interactor of 64 kDa (EPI64) relating to melanin transportation were investigated using B16 melanoma cells treated with 3C4'GQ, 3MQ, or 34'7TMQ in order to understand the mechanism underlying the observed activities. The influence of 3C4'GQ on the increase of intracellular melanin contents enhanced the expression of EPI64, exhibited no dendrite elongation activity, and inhibited melanin transportation. On the other hand, the increase of extracellular melanin content by 3MQ and 34'7TMQ inhibited the expression of EPI64 and formed elongated cells to stimulate melanin transportation. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

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

    PubMed Central

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

    2010-01-01

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

  20. Depigmenting effect of argan press-cake extract through the down-regulation of Mitf and melanogenic enzymes expression in B16 murine melanoma cells.

    PubMed

    Bourhim, Thouria; Villareal, Myra O; Gadhi, Chemseddoha; Hafidi, Abdellatif; Isoda, Hiroko

    2018-06-26

    Oil extraction from the kernels of Argania spinosa (L.) Skeels (Sapotaceae), an endemic tree of Morocco, produces argan press-cake (APC) used as a shampoo and to treat sprains, scabies, and for healing wounds. We have previously reported that argan oil has antimelanogenesis effect. Here, we determined if the by-product, APC, has melanogenesis regulatory effect using B16 murine melanoma cells. The effect of APC ethanol extract on cell proliferation and melanin content of B16 cells were measured, and to elucidate the mechanism involved, the expression level of melanogenic enzymes tyrosinase (TYR), dopachrome tautomerase (DCT), and tyrosinase-related protein 1 (TRP1) were determined and mRNA expression level of microphthalmia- associated transcription factor (Mitf) and Tyr genes were quantified. APC ethanol extract showed a significant melanin biosynthesis inhibitory effect on B16 cells in a time-dependent manner without cytotoxicity, which could be due to the decreased expression of TYR, TRP1, and DCT in a time-dependent manner. APC extract down regulated Mitf and Tyr. Decreased TRP1 and DCT levels could be due to post-translational modifications. These results suggest that APC extract may be used as a new source of natural whitening products and may be introduced as an important pharmacological agent for the treatment of hyperpigmentation disorders.

  1. Effect of DOTA position on melanoma targeting and pharmacokinetic properties of 111In-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone peptide.

    PubMed

    Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Prossnitz, Eric R; Sklar, Larry A; Miao, Yubin

    2009-11-01

    The purpose of this study was to examine the effect of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) position on melanoma targeting and pharmacokinetics of radiolabeled lactam bridge-cyclized alpha-melanocyte stimulating hormone (alpha-MSH) peptide. A novel lactam bridge-cyclized alpha-MSH peptide, Ac-GluGlu-CycMSH[DOTA] {Ac-Glu-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Lys(DOTA)]}, was synthesized using standard 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry. DOTA was directly attached to the alpha-amino group of Lys in the cyclic ring, while the N-terminus of the peptide was acetylated to generate Ac-GluGlu-CycMSH[DOTA]. The MC1 receptor binding affinity of Ac-GluGlu-CycMSH[DOTA] was determined in B16/F1 melanoma cells. Melanoma targeting and pharmacokinetic properties of Ac-GluGlu-CycMSH[DOTA]-111In were determined in B16/F1 melanoma-bearing C57 mice and compared to that of 111In-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp] (111In-DOTA-GlyGlu-CycMSH; DOTA was coupled to the N-terminus of the peptide). Ac-GluGlu-CycMSH[DOTA] displayed 0.6 nM MC1 receptor binding affinity in B16/F1 cells. Ac-GluGlu-CycMSH[DOTA]-111In was readily prepared with greater than 95% radiolabeling yield. Ac-GluGlu-CycMSH[DOTA]-111In exhibited high tumor uptake (11.42 +/- 2.20% ID/g 2 h postinjection) and prolonged tumor retention (9.42 +/- 2.41% ID/g 4 h postinjection) in B16/F1 melanoma-bearing C57 mice. The uptake values for nontarget organs were generally low (<1.3% ID/g) except for the kidneys 2, 4, and 24 h postinjection. DOTA position exhibited profound effect on melanoma targeting and pharmacokinetic properties of Ac-GluGlu-CycMSH[DOTA]-111In, providing a new insight into the design of lactam bridge-cyclized peptide for melanoma imaging and therapy.

  2. Comparative preclinical evaluation of 68Ga-NODAGA and 68Ga-HBED-CC conjugated procainamide in melanoma imaging.

    PubMed

    Trencsényi, György; Dénes, Noémi; Nagy, Gábor; Kis, Adrienn; Vida, András; Farkas, Flóra; Szabó, Judit P; Kovács, Tünde; Berényi, Ervin; Garai, Ildikó; Bai, Péter; Hunyadi, János; Kertész, István

    2017-05-30

    Malignant melanoma is the most aggressive form of skin cancer. The early detection of primary melanoma tumors and metastases using non-invasive PET imaging determines the outcome of this disease. Previous studies have shown that benzamide derivatives (e.g. procainamide) conjugated with PET radionuclides specifically bind to melanin pigment of melanoma tumors. 68 Ga chelating agents can have high influence on physiological properties of 68 Ga labeled bioactive molecules, as was experienced during the application of HBED-CC on PSMA ligand. The aim of this study was to assess this concept in the case of the melanin specific procaindamide (PCA) and to compare the melanin specificity of 68 Ga-labeled PCA using HBED-CC and NODAGA chelators under in vitro and in vivo conditions. Procainamide (PCA) was conjugated with HBED-CC and NODAGA chelators and was labeled with Ga-68. The melanin specificity of 68 Ga-HBED-CC-PCA and 68 Ga-NODAGA-PCA was investigated in vitro and in vivo using amelanotic (MELUR and A375) and melanin containing (B16-F10) melanoma cell lines. Tumor-bearing mice were prepared by subcutaneous injection of B16-F10, MELUR and A375 melanoma cells into C57BL/6 and SCID mice. 21±2days after tumor cell inoculation and 90min after intravenous injection of the 68 Ga-labelledlabeled radiopharmacons whole body PET/MRI scans were performed. 68 Ga-NODAGA-PCA and 68 Ga-HBED-CC-PCA were produced with excellent radiochemical purity (98%). In vitro experiments demonstrated that after 30 and 90min incubation time 68 Ga-NODAGA-PCA uptake of B16-F10 cells was significantly (p≤0.01) higher than the 68 Ga-HBED-CC-conjugated PCA accumulation in the same cell line. Furthermore, significant difference (p≤0.01 and 0.05) was found between the uptake of melanin negative and positive cell lines using 68 Ga-NODAGA-PCA and 68 Ga-HBED-CC-PCA. In vivo PET/MRI studies using tumor models revealed significantly (p≤0.01) higher 68 Ga-NODAGA-PCA uptake (SUVmean: 0.46±0.05, SUVmax: 1

  3. Single photon emission computed tomography/positron emission tomography imaging and targeted radionuclide therapy of melanoma: new multimodal fluorinated and iodinated radiotracers.

    PubMed

    Maisonial, Aurélie; Kuhnast, Bertrand; Papon, Janine; Boisgard, Raphaël; Bayle, Martine; Vidal, Aurélien; Auzeloux, Philippe; Rbah, Latifa; Bonnet-Duquennoy, Mathilde; Miot-Noirault, Elisabeth; Galmier, Marie-Josèphe; Borel, Michèle; Askienazy, Serge; Dollé, Frédéric; Tavitian, Bertrand; Madelmont, Jean-Claude; Moins, Nicole; Chezal, Jean-Michel

    2011-04-28

    This study reports a series of 14 new iodinated and fluorinated compounds offering both early imaging ((123)I, (124)I, (18)F) and systemic treatment ((131)I) of melanoma potentialities. The biodistribution of each (125)I-labeled tracer was evaluated in a model of melanoma B16F0-bearing mice, using in vivo serial γ scintigraphic imaging. Among this series, [(125)I]56 emerged as the most promising compound in terms of specific tumoral uptake and in vivo kinetic profile. To validate our multimodality concept, the radiosynthesis of [(18)F]56 was then optimized and this radiotracer has been successfully investigated for in vivo PET imaging of melanoma in B16F0- and B16F10-bearing mouse model. The therapeutic efficacy of [(131)I]56 was then evaluated in mice bearing subcutaneous B16F0 melanoma, and a significant slow down in tumoral growth was demonstrated. These data support further development of 56 for PET imaging ((18)F, (124)I) and targeted radionuclide therapy ((131)I) of melanoma using a single chemical structure.

  4. Vernonia cinerea Less. inhibits tumor cell invasion and pulmonary metastasis in C57BL/6 mice.

    PubMed

    Pratheeshkumar, Poyil; Kuttan, Girija

    2011-06-01

    The effect of Vernonia cinerea Less. extract on the inhibition of lung metastasis induced by B16F-10 melanoma cells was studied in C57BL/6 mice. V cinerea extract significantly (P < .001) inhibited lung tumor formation (78.8%) and significantly increased the life span (72.5%). Moreover, lung collagen hydroxyproline, uronic acid, and hexosamine and also serum sialic acid, γ-glutamyltransferase (GGT), and vascular endothelial growth factor (VEGF) levels were found to be significantly (P < .001) lower in treated animals compared with untreated controls. Histopathological analysis of the lung tissues also correlated with these findings. V cinerea treatment significantly inhibited the invasion of B16F-10 melanoma cells across the collagen matrix of the Boyden chamber. V cinerea also inhibited the migration of B16F-10 melanoma cells across a polycarbonate filter in vitro. It downregulated the production and expression of proinflammatory cytokines such as TNF (tumor necrosis factor)-α, IL (interleukin)-1β, IL-6, and GM-CSF (granulocyte monocyte colony-stimulating factor). V cinerea extract administration could suppress or downregulate the expression of matrix metalloproteinase (MMP)-2, MMP-9, lysyl oxidase, prolyl hydroxylase, K-ras, extracellular signal-regulated kinase (ERK)-1, ERK-2, and VEGF and also upregulate the expression of nm-23, tissue inhibitor of metalloproteinase (TIMP-1), and TIMP-2 in the lung tissue of metastasis-induced animals. It also inhibited the protein expression of MMP-2 and MMP-9 in gelatin zymographic analysis of B16F-10 cells. These results indicate that V cinerea could inhibit the metastatic progression of B16F-10 melanoma cells in C57BL/6 mice by regulating MMPs, VEGF, prolyl hydroxylase, lysyl oxidase, ERK-1, ERK-2, TIMPs, nm23, and proinflammatory cytokine gene expression in metastatic lung tissue.

  5. Gallium-67-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone peptide for primary and metastatic melanoma imaging.

    PubMed

    Guo, Haixun; Yang, Jianquan; Shenoy, Nalini; Miao, Yubin

    2009-12-01

    The purpose of this study was to examine the melanoma imaging properties of a novel 67Ga-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone (alpha-MSH) peptide. A lactam bridge-cyclized alpha-MSH peptide, DOTA-GlyGlu-CycMSH {DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]}, was synthesized and radiolabeled with 67Ga. The melanoma targeting and pharmacokinetic properties of 67Ga-DOTA-GlyGlu-CycMSH were determined in B16/F1 flank primary melanoma-bearing and B16/F10 pulmonary metastatic melanoma-bearing C57 mice. Flank primary melanoma and pulmonary metastatic melanoma imaging were performed by small animal single photon emission computed tomography (SPECT)/CT using 67Ga-DOTA-GlyGlu-CycMSH as an imaging probe. 67Ga-DOTA-GlyGlu-CycMSH was readily prepared with greater than 95% radiolabeling yield. 67Ga-DOTA-GlyGlu-CycMSH exhibited substantial tumor uptake (12.93 +/- 1.63%ID/g at 2 h postinjection) and prolonged tumor retention (5.02 +/- 1.35%ID/g at 24 h postinjection) in B16/F1 melanoma-bearing C57 mice. The uptake values for nontarget organs were generally low (<0.30%ID/g) except for the kidneys at 2, 4, and 24 h postinjection. 67Ga-DOTA-GlyGlu-CycMSH exhibited significantly (p < 0.05) higher uptakes (1.44 +/- 0.75%ID/g at 2 h postinjection and 1.49 +/- 0.69%ID/g at 4 h postinjection) in metastatic melanoma-bearing lung than those in normal lung (0.15 +/- 0.10%ID/g and 0.17 +/- 0.11%ID/g at 2 and 4 h postinjection, respectively). Both flank primary B16/F1 melanoma and B16/F10 pulmonary melanoma metastases were clearly visualized by SPECT/CT using 67Ga-DOTA-GlyGlu-CycMSH as an imaging probe 2 h postinjection. 67Ga-DOTA-GlyGlu-CycMSH exhibited favorable melanoma targeting and imaging properties, highlighting its potential as an effective imaging probe for early detection of primary and metastatic melanoma.

  6. Protein B61 as a new growth factor: expression of B61 and up-regulation of its receptor epithelial cell kinase during melanoma progression.

    PubMed

    Easty, D J; Guthrie, B A; Maung, K; Farr, C J; Lindberg, R A; Toso, R J; Herlyn, M; Bennett, D C

    1995-06-15

    Epithelial cell kinase (ECK) is a receptor protein tyrosine kinase, the role of which in melanoma biology is unclear. Here we studied the role of ECK during melanoma progression. ECK mRNA was overexpressed in virtually all melanoma lines tested, and levels were significantly higher in cell lines from distant metastases than primary melanomas; melanocytes were negative. Gene amplification was not detected in melanomas. Levels of ECK protein corresponded well with mRNA levels. B61 or LERK-1, recently identified as an ECK ligand, stimulated the growth of ECK-expressing melanoma cell lines, its first identified biological activity. Melanoma chemotaxis and chemoinvasion were not affected by B61. Growth of normal melanocytes was not affected. mRNA for B61 was detected in both melanoma cell lines and normal melanocytes. B61 was also identified by Western blotting and ECK binding activity with the use of a BIAcore binding assay in melanoma cell-conditioned media. These results suggest that B61 is an autocrine growth factor for melanomas but not normal melanocytes.

  7. A genome-wide shRNA screen identifies GAS1 as a novel melanoma metastasis suppressor gene.

    PubMed

    Gobeil, Stephane; Zhu, Xiaochun; Doillon, Charles J; Green, Michael R

    2008-11-01

    Metastasis suppressor genes inhibit one or more steps required for metastasis without affecting primary tumor formation. Due to the complexity of the metastatic process, the development of experimental approaches for identifying genes involved in metastasis prevention has been challenging. Here we describe a genome-wide RNAi screening strategy to identify candidate metastasis suppressor genes. Following expression in weakly metastatic B16-F0 mouse melanoma cells, shRNAs were selected based upon enhanced satellite colony formation in a three-dimensional cell culture system and confirmed in a mouse experimental metastasis assay. Using this approach we discovered 22 genes whose knockdown increased metastasis without affecting primary tumor growth. We focused on one of these genes, Gas1 (Growth arrest-specific 1), because we found that it was substantially down-regulated in highly metastatic B16-F10 melanoma cells, which contributed to the high metastatic potential of this mouse cell line. We further demonstrated that Gas1 has all the expected properties of a melanoma tumor suppressor including: suppression of metastasis in a spontaneous metastasis assay, promotion of apoptosis following dissemination of cells to secondary sites, and frequent down-regulation in human melanoma metastasis-derived cell lines and metastatic tumor samples. Thus, we developed a genome-wide shRNA screening strategy that enables the discovery of new metastasis suppressor genes.

  8. Identification of Lectins from Metastatic Cancer Cells through Magnetic Glyconanoparticles

    PubMed Central

    Kavunja, Herbert W.; Voss, Patricia G.

    2016-01-01

    Cancer cells can have characteristic carbohydrate binding properties. Previously, it was shown that a highly metastatic melanoma cell line B16F10 bound to galacto-side-functionalized nanoparticles much stronger than the corresponding less metastatic B16F1 cells. To better understand the carbohydrate binding properties of cancer cells, herein, we report the isolation and characterization of endogenous galactose binding proteins from B16F10 cells using magnetic glyconanoparticles. The galactose-coated magnetic glyconanoparticles could bind with lectins present in the cells and be isolated through magnet-mediated separation. Through Western blot and mass spectrometry, the arginine/serine rich splicing factor Sfrs1 was identified as a galactose-selective endogenous lectin, overexpressed in B16F10 cells, compared with B16F1 cells. In addition, galactin-3 was found in higher amounts in B16F10 cells. Finally, the glyconanoparticles exhibited a superior efficiency in lectin isolation, from both protein mixtures and live cells, than the corresponding more traditional microparticles functionalized with carbohydrates. Thus, the magnetic glyconanoparticles present a useful tool for discovery of endogenous lectins, as well as binding partners of lectins, without prior knowledge of protein identities. PMID:27110035

  9. Antitumoural effect of Synadenium grantii Hook f. (Euphorbiaceae) latex.

    PubMed

    de Oliveira, Thais Latansio; Munhoz, Antônio Carlos Mattar; Lemes, Bruna Mikulis; Minozzo, Bruno Rodrigo; Nepel, Angelita; Barison, Andersson; Fávero, Giovani Marino; Campagnoli, Eduardo Bauml; Beltrame, Flávio Luís

    2013-10-28

    Synadenium grantii Hook f. has traditionally been used to treat various neoplastic diseases in southern Brazil. Evaluation of the antitumoural potential of Synadenium grantii latex against B16F10 melanoma cell line using in vitro and in vivo models, as well as a phytochemical study of the latex. The in vitro antitumoural activity was performed using MTT and trypan blue assays with different latex concentrations (1.7 µg-7.0 µg/well and 1.22 mg-4.88 mg/well). Flow cytometry was used to determine the progression of the cell cycle. The in vivo activity was performed by subcutaneously injecting melanoma cells in the dorsum of C57BL6 mice, followed by treating the mice with a popular form of use of the latex (garrafada) administered orally. After sacrificing the animals, histological analysis of the organs was performed by hematoxylin-eosin staining. The phytochemical study of the latex was performed by NMR and chromatographic procedures and the extracts and isolated substances were evaluated by IR, 1D and 2D NMR analysis. The Synadenium grantii latex exhibited decreased cell viability of the melanoma line in a concentration and time-dependent manner, and also cell cycle arrest in the S-G2/M phase. The latex caused a 40% reduction in the volume of tumours of the mice with melanomas. Histological examination of the organs of these animals showed no differences between groups. The phytochemical investigation resulted in the isolation and identification of triterpene euphol and the steroid citrostadienol, which were tested against the strain of melanoma. Euphol showed no antitumoural activity, while the steroid citrostadienol showed reduced cytotoxic activity. The Synadenium grantii latex presented in vitro and in vivo cytotoxic effects with antitumoural activity against B16F10 melanoma cells. © 2013 Elsevier Ireland Ltd. All rights reserved.

  10. A novel role for autologous tumour cell vaccination in the immunotherapy of the poorly immunogenic B16-BL6 melanoma.

    PubMed

    Geiger, J D; Wagner, P D; Shu, S; Chang, A E

    1992-06-01

    The growth of immunogenic tumours stimulates the generation of tumour-sensitized, but not functional, pre-effector T cells in the draining lymph nodes. These pre-effector cells can mature into effector cells upon in-vitro stimulation with anti-CD3 and IL-2. In the current study, using a defined, poorly immunogenic tumour, B16-BL6 melanoma, the pre-effector cell response was not evident during progressive tumour growth but was elicited by vaccination with irradiated tumour cells admixed with Corynebacterium parvum. After anti-CD3/IL-2 activation, these cells were capable of mediating the regression of established pulmonary metastases. The efficacy of the vaccine depended on the doses of both tumour cells and the adjuvant. While higher numbers of tumour cells were more effective, an optimal dose (12.5 micrograms) of C. parvum was required. The dose of irradiation was not a critical factor. After vaccination, kinetic studies revealed that the pre-effector cell response was evident 4 days later and declined after 14 days. These observations illustrate the potential role of active immunization in the cellular therapy of cancer.

  11. Accumulation of cytolytic CD8{sup +} T cells in B16-melanoma and proliferation of mature T cells in TIS21-knockout mice after T cell receptor stimulation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ryu, Min Sook; Woo, Min-Yeong; Department of Biomedical Sciences, The Graduate School, Ajou University

    2014-10-01

    In vivo and in vitro effects of TIS21 gene on the mature T cell activation and antitumor activities were explored by employing MO5 melanoma orthograft and splenocytes isolated from the TIS21-knockout (KO) mice. Proliferation and survival of mature T cells were significantly increased in the KO than the wild type (WT) cells, indicating that TIS21 inhibits the rate of mature T cell proliferation and its survival. In MO5 melanoma orthograft model, the KO mice recruited much more CD8{sup +} T cells into the tumors at around day 14 after tumor cell injection along with reduced tumor volumes compared with themore » WT. The increased frequency of granzyme B{sup +} CD8{sup +} T cells in splenocytes of the KO mice compared with the WT may account for antitumor-immunity of TIS21 gene in the melanoma orthograft. In contrast, reduced frequencies of CD107a{sup +} CD8{sup +} T cells in the splenocytes of KO mice may affect the loss of CD8{sup +} T cell infiltration in the orthograft at around day 19. These results indicate that TIS21 exhibits antiproliferative and proapoptotic effects in mature T cells, and differentially affects the frequencies of granzyme B{sup +} CD8{sup +} T-cells and CD107a{sup +} CD8{sup +} T-cells, thus transiently regulating in vivo anti-tumor immunity. - Highlights: • Constitutive expression of TIS21 in splenocytes and upregulation by TCR stimulation. • Proliferation of mature T-cells in spleen of TIS21KO mice after TCR stimulation. • Inhibition of cell death in mature T-cells of TIS21KO mice compared with the wild type. • Inhibition of melanoma growth in TIS21KO mice and CD8{sup +} T cell infiltration in tumor. • Reduction of CD 107{sup +}CD8{sup +} T cells, but increased granzyme B{sup +} CD8{sup +} T cells in TIS21KO mice.« less

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-01-01

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

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

    PubMed

    Glud, Martin; Manfé, Valentina; Biskup, Edyta; Holst, Line; Dirksen, Anne Marie Ahlburg; Hastrup, Nina; Nielsen, Finn C; Drzewiecki, Krzysztof T; Gniadecki, Robert

    2011-06-01

    MicroRNAs (miRNAs) are small noncoding RNA molecules involved in gene regulation. Aberrant expression of miRNA has been associated with the development or progression of several diseases, including cancer. In a previous study, we found that the expression of miRNA-125b (miR-125b) was two-fold lower 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 of miR-125b induced typical senescent cell morphology, including increased cytoplasmatic/nucleus ratio and intensive cytoplasmatic β-galactosidase expression. In contrast, inhibition of miR-125b resulted in 30-35% decreased levels of spontaneous apoptosis. We propose that downregulation of miR-125b in an early cutaneous malignant melanoma can contribute to the increased metastatic capability of this tumor.

  15. Mitochondrial ATF2 translocation contributes to apoptosis induction and BRAF inhibitor resistance in melanoma through the interaction of Bim with VDAC1.

    PubMed

    Gao, Zongwei; Shang, Qingjuan; Liu, Zhaoyun; Deng, Chun; Guo, Chunbao

    2015-11-03

    The mitochondrial accumulation of ATF2 is involved in tumor suppressor activities via cytochrome c release in melanoma cells. However, the signaling pathways that connect mitochondrial ATF2 accumulation and cytochrome c release are not well documented. Several melanoma cell lines, B16F10, K1735M2, A375 and A375-R1, were treated with paclitaxel and vemurafenib to test the function of mitochondrial ATF2 and its connection to Bim and voltage-dependent anion channel 1 (VDAC1). Immunoprecipitation analysis was performed to investigate the functional interaction between the involved proteins. VDAC1 oligomerization was evaluated using an EGS-based crosslinking assay. The expression and migration of ATF2 to the mitochondria accounted for paclitaxel stimuli and acquired resistance to BRAF inhibitors. Mitochondrial ATF2 facilitated Bim stabilization through the inhibition of its degradation by the proteasome, thereby promoting cytochrome c release and inducing apoptosis in B16F10 and A375 cells. Studies using B16F10 and A375 cells genetically modified for ATF2 indicated that mitochondrial ATF2 was able to dissociate Bim from the Mcl-1/Bim complex to trigger VDAC1 oligomerization. Immunoprecipitation analysis revealed that Bim interacts with VDAC1, and this interaction was remarkably enhanced during apoptosis. These results reveal that mitochondrial ATF2 is associated with the induction of apoptosis and BRAF inhibitor resistance through Bim activation, which might suggest potential novel therapies for the targeted induction of apoptosis in melanoma therapy.

  16. Mitochondrial ATF2 translocation contributes to apoptosis induction and BRAF inhibitor resistance in melanoma through the interaction of Bim with VDAC1

    PubMed Central

    Deng, Chun; Guo, Chunbao

    2015-01-01

    Background The mitochondrial accumulation of ATF2 is involved in tumor suppressor activities via cytochrome c release in melanoma cells. However, the signaling pathways that connect mitochondrial ATF2 accumulation and cytochrome c release are not well documented. Methods Several melanoma cell lines, B16F10, K1735M2, A375 and A375-R1, were treated with paclitaxel and vemurafenib to test the function of mitochondrial ATF2 and its connection to Bim and voltage-dependent anion channel 1 (VDAC1). Immunoprecipitation analysis was performed to investigate the functional interaction between the involved proteins. VDAC1 oligomerization was evaluated using an EGS-based crosslinking assay. Results The expression and migration of ATF2 to the mitochondria accounted for paclitaxel stimuli and acquired resistance to BRAF inhibitors. Mitochondrial ATF2 facilitated Bim stabilization through the inhibition of its degradation by the proteasome, thereby promoting cytochrome c release and inducing apoptosis in B16F10 and A375 cells. Studies using B16F10 and A375 cells genetically modified for ATF2 indicated that mitochondrial ATF2 was able to dissociate Bim from the Mcl-1/Bim complex to trigger VDAC1 oligomerization. Immunoprecipitation analysis revealed that Bim interacts with VDAC1, and this interaction was remarkably enhanced during apoptosis. Conclusion These results reveal that mitochondrial ATF2 is associated with the induction of apoptosis and BRAF inhibitor resistance through Bim activation, which might suggest potential novel therapies for the targeted induction of apoptosis in melanoma therapy. PMID:26462148

  17. Inhibitory effect of fentanyl citrate on the release of endothlin-1 induced by bradykinin in melanoma cells.

    PubMed

    Andoh, Tsugunobu; Shinohara, Akira; Kuraishi, Yasushi

    2017-02-01

    Our previous study showed that the μ-opioid receptor agonist fentanyl citrate inhibits endothelin-1-and bradykinin-mediated pain responses in mice orthotopically inoculated with melanoma cells. We also demonstrated that bradykinin induces endothelin-1 secretion in melanoma cells. However, the analgesic mechanisms of fentanyl citrate remain unclear. Thus, the present study was conducted to determine whether fentanyl citrate affects bradykinin-induced endothelin-1 secretion in B16-BL6 melanoma cells. The amount of endothelin-1 in the culture medium was measured using an enzyme immunoassay. The expression of endothelin-1, kinin B 2 receptors, and μ-opioid receptors in B16-BL/6 melanoma cells was determined using immunocytochemistry. Fentanyl citrate inhibited bradykinin-induced endothelin-1 secretion. The inhibitory effect of fentanyl citrate on the secretion of endothelin-1 was attenuated by the μ-opioid receptor antagonist naloxone methiodide. The immunoreactivities of endothelin-1, kinin B 2 receptors, and μ-opioid receptors in B16-BL6 melanoma cells were observed. These results suggest that fentanyl citrate regulates bradykinin-induced endothelin-1 secretion through μ-opioid receptors in melanoma cells. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

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

    PubMed

    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. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. IGF-1 contributes to the expansion of melanoma-initiating cells through an epithelial-mesenchymal transition process.

    PubMed

    Le Coz, Vincent; Zhu, Chaobin; Devocelle, Aurore; Vazquez, Aimé; Boucheix, Claude; Azzi, Sandy; Gallerne, Cindy; Eid, Pierre; Lecourt, Séverine; Giron-Michel, Julien

    2016-12-13

    Melanoma is a particularly virulent human cancer, due to its resistance to conventional treatments and high frequency of metastasis. Melanomas contain a fraction of cells, the melanoma-initiating cells (MICs), responsible for tumor propagation and relapse. Identification of the molecular pathways supporting MICs is, therefore, vital for the development of targeted treatments. One factor produced by melanoma cells and their microenvironment, insulin-like growth factor-1 (IGF- 1), is linked to epithelial-mesenchymal transition (EMT) and stemness features in several cancers.We evaluated the effect of IGF-1 on the phenotype and chemoresistance of B16-F10 cells. IGF-1 inhibition in these cells prevented malignant cell proliferation, migration and invasion, and lung colony formation in immunodeficient mice. IGF-1 downregulation also markedly inhibited EMT, with low levels of ZEB1 and mesenchymal markers (N-cadherin, CD44, CD29, CD105) associated with high levels of E-cadherin and MITF, the major regulator of melanocyte differentiation. IGF-1 inhibition greatly reduced stemness features, including the expression of key stem markers (SOX2, Oct-3/4, CD24 and CD133), and the functional characteristics of MICs (melanosphere formation, aldehyde dehydrogenase activity, side population). These features were associated with a high degree of sensitivity to mitoxantrone treatment.In this study, we deciphered new connections between IGF-1 and stemness features and identified IGF-1 as instrumental for maintaining the MIC phenotype. The IGF1/IGF1-R nexus could be targeted for the development of more efficient anti-melanoma treatments. Blocking the IGF-1 pathway would improve the immune response, decrease the metastatic potential of tumor cells and sensitize melanoma cells to conventional treatments.

  20. Ajoene inhibits both primary tumor growth and metastasis of B16/BL6 melanoma cells in C57BL/6 mice.

    PubMed

    Taylor, Peter; Noriega, Raquel; Farah, Carla; Abad, María-Jesús; Arsenak, Miriam; Apitz, Rafael

    2006-08-08

    Ajoene is an organosulphur compound derived from garlic with important effects on several membrane-associated processes such as platelet aggregation, as well as being cytotoxic for tumor cell lines in vitro. In the present study, we investigated the effect of ajoene on different cell types in vitro, as well as its inhibitory effects on both primary tumors and metastasis in a mouse model. We found ajoene to inhibit tumor cell growth in vitro, but also to inhibit strongly metastasis to lung in the B16/BL6 melanoma tumor model in C57BL/6 mice. As far as we are aware, this is the first report of the anti-metastatic effect of ajoene. Ajoene also inhibited tumor-endothelial cell adhesion, as well as the in vivo TNF-alpha response to lipopolysaccharide. Possible mechanisms of its antitumoral activity are discussed in the light of these results.

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

    PubMed

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

    2011-02-01

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

  2. PRL-3 siRNA Inhibits the Metastasis of B16-BL6 Mouse Melanoma Cells In Vitro and In Vivo

    PubMed Central

    Qian, Feng; Li, Yu-Pei; Sheng, Xia; Zhang, Zi-Chao; Song, Ran; Dong, Wei; Cao, Shao-Xian; Hua, Zi-Chun; Xu, Qiang

    2007-01-01

    Phosphatase of regenerating liver-3 (PRL-3) has been proposed to promote the invasion of tumor cells to metastasis sites. However, the effect of PRL-3 on spontaneous metastasis has not been clearly demonstrated, and whether PRL-3 could become a new therapeutic target in malignant tumor is still unknown. In this study, we used PRL-3 siRNA as a molecular medicine to specifically reduce the expression of PRL-3 in B16-BL6 cells, a highly metastatic melanoma cell line. In vitro, PRL-3 siRNA significantly inhibited cell adhesion and migration, but had no effect on cell proliferation. In the spontaneous metastatic tumor model in vivo, PRL-3 siRNA treatment remarkably inhibited the proliferation of primary tumor, prevented tumor cells from invading the draining lymph nodes, and prolonged the life span of mice. Therefore, our results indicate that PRL-3 plays a critical role in promoting the whole process of spontaneous metastasis and tumor growth initiation, and that inhibiting PRL-3 will improve malignant tumor therapy. PMID:17592549

  3. PRL-3 siRNA inhibits the metastasis of B16-BL6 mouse melanoma cells in vitro and in vivo.

    PubMed

    Qian, Feng; Li, Yu-Pei; Sheng, Xia; Zhang, Zi-Chao; Song, Ran; Dong, Wei; Cao, Shao-Xian; Hua, Zi-Chun; Xu, Qiang

    2007-01-01

    Phosphatase of regenerating liver-3 (PRL-3) has been proposed to promote the invasion of tumor cells to metastasis sites. However, the effect of PRL-3 on spontaneous metastasis has not been clearly demonstrated, and whether PRL-3 could become a new therapeutic target in malignant tumor is still unknown. In this study, we used PRL-3 siRNA as a molecular medicine to specifically reduce the expression of PRL-3 in B16-BL6 cells, a highly metastatic melanoma cell line. In vitro, PRL-3 siRNA significantly inhibited cell adhesion and migration, but had no effect on cell proliferation. In the spontaneous metastatic tumor model in vivo, PRL-3 siRNA treatment remarkably inhibited the proliferation of primary tumor, prevented tumor cells from invading the draining lymph nodes, and prolonged the life span of mice. Therefore, our results indicate that PRL-3 plays a critical role in promoting the whole process of spontaneous metastasis and tumor growth initiation, and that inhibiting PRL-3 will improve malignant tumor therapy.

  4. [The role of gap junction communication in metastatic B16 melanoma in C57BL mice].

    PubMed

    Fëdorov, E S; Manikhas, G M; Petrishchëv, N N; Dubina, M V

    2006-01-01

    The study is concerned with the effects of non-specific blocking gap junction communication with oleamide as well as genesis and spreading of melanoma B16 metastases to the lung in mice C57B1. The blocking exerted no distinct influence on primary tumorigenesis but had a marked effect on metastatic spread. Oleamide treatment during tumor growth led to an increase in area covered by metastases. A correlation was established between metastatic frequency and dosage: 1 mg/kg was followed by an upsurge in frequency of secondary lung tumors while 10 mg/kg--by a drop.

  5. Antimetastatic potential of vernolide-A, a sesquiterpenoid from Vernonia cinerea L.

    PubMed

    Pratheeshkumar, P; Kuttan, Girija

    2012-01-01

    The inhibitory effect of vernolide-A (C(21)H(28)O(7)) on lung metastasis induced by B16F-10 melanoma cells was studied using C57BL/6 mice. Vernolide-A was administered in three different modalities such as simultaneously with tumor, prophylactic to tumor and after tumor development. Maximum inhibition in the metastasis was observed when vernolide-A was administered simultaneously with tumor. There was 89.39% inhibition of lung tumor nodule formation and 88.51% increase in the life span of metastatic tumor-bearing animals. Highly elevated levels of lung hydroxyproline, lung uronic acid, lung hexosamine, serum sialic acid, serum γ-glutamyl transpeptidase (GGT) and serum vascular endothelial growth factor (VEGF) in the metastatic control animals were found to be significantly lowered in the vernolide-A-treated animals. Histopathological analysis of lung tissues also correlated with these results. Vernolide-A administration downregulated the expression of matrix metalloproteinase-2 (MMP-2), MMP-9, extracellular-signal-regulated kinase-1 (ERK-1), ERK-2 and VEGF in the lung tissue of B16F-10 melanoma challenged animals. In the in vitro system, vernolide-A showed a significant inhibition of invasion of B16F-10 melanoma cells across the collagen matrix. Vernolide-A treatment also inhibited the migration of B16F-10 melanoma cells across a polycarbonate filter in vitro. Vernolide-A could inhibit MMP-2 and MMP-9 protein expression in gelatin zymographic analysis of B16F-10 cells. (3)H-thymidine proliferation assay showed that vernolide-A could inhibit the proliferation of B16F-10 melanoma cells in vitro. These results indicate that vernolide-A could inhibit the metastatic progression of B16F-10 melanoma cells in mice.

  6. Tumor cell cholesterol depletion and V-ATPase inhibition as an inhibitory mechanism to prevent cell migration and invasiveness in melanoma.

    PubMed

    Costa, Gildeíde Aparecida; de Souza, Sávio Bastos; da Silva Teixeira, Layz Ribeiro; Okorokov, Lev A; Arnholdt, Andrea Cristina Vetö; Okorokova-Façanha, Anna L; Façanha, Arnoldo Rocha

    2018-03-01

    V-ATPase interactions with cholesterol enriched membrane microdomains have been related to metastasis in a variety of cancers, but the underlying mechanism remains at its beginnings. It has recently been reported that the inhibition of this H + pump affects cholesterol mobilization to the plasma membrane. Inhibition of melanoma cell migration and invasiveness was assessed by wound healing and Transwell assays in murine cell lines (B16F10 and Melan-A). V-ATPase activity was measured in vitro by ATP hydrolysis and H + transport in membrane vesicles, and intact cell H + fluxes were measured by using a non-invasive Scanning Ion-selective Electrode Technique (SIET). Cholesterol depletion by 5mM MβCD was found to be inhibitory to the hydrolytic and H + pumping activities of the V-ATPase of melanoma cell lines, as well as to the migration and invasiveness capacities of these cells. Nearly the same effects were obtained using concanamycin A, a specific inhibitor of V-ATPase, which also promoted a decrease of the H + efflux in live cells at the same extent of MβCD. We found that cholesterol depletion significantly affects the V-ATPase activity and the initial metastatic processes following a profile similar to those observed in the presence of the V-ATPase specific inhibitor, concanamycin. The results shed new light on the functional role of the interactions between V-ATPases and cholesterol-enriched microdomains of cell membranes that contribute with malignant phenotypes in melanoma. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. The effect of low-level laser irradiation (In-Ga-Al-AsP - 660 nm) on melanoma in vitro and in vivo

    PubMed Central

    2009-01-01

    Background It has been speculated that the biostimulatory effect of Low Level Laser Therapy could cause undesirable enhancement of tumor growth in neoplastic diseases. The aim of the present study is to analyze the behavior of melanoma cells (B16F10) in vitro and the in vivo development of melanoma in mice after laser irradiation. Methods We performed a controlled in vitro study on B16F10 melanoma cells to investigate cell viability and cell cycle changes by the Tripan Blue, MTT and cell quest histogram tests at 24, 48 and 72 h post irradiation. The in vivo mouse model (male Balb C, n = 21) of melanoma was used to analyze tumor volume and histological characteristics. Laser irradiation was performed three times (once a day for three consecutive days) with a 660 nm 50 mW CW laser, beam spot size 2 mm2, irradiance 2.5 W/cm2 and irradiation times of 60s (dose 150 J/cm2) and 420s (dose 1050 J/cm2) respectively. Results There were no statistically significant differences between the in vitro groups, except for an increase in the hypodiploid melanoma cells (8.48 ± 1.40% and 4.26 ± 0.60%) at 72 h post-irradiation. This cancer-protective effect was not reproduced in the in vivo experiment where outcome measures for the 150 J/cm2 dose group were not significantly different from controls. For the 1050 J/cm2 dose group, there were significant increases in tumor volume, blood vessels and cell abnormalities compared to the other groups. Conclusion LLLT Irradiation should be avoided over melanomas as the combination of high irradiance (2.5 W/cm2) and high dose (1050 J/cm2) significantly increases melanoma tumor growth in vivo. PMID:19930543

  8. Rendomab B4, a monoclonal antibody that discriminates the human endothelin B receptor of melanoma cells and inhibits their migration

    PubMed Central

    Borrull, Aurélie; Allard, Bertrand; Wijkhuisen, Anne; Herbet, Amaury; Lamourette, Patricia; Birouk, Wided; Leiber, Denis; Tanfin, Zahra; Ducancel, Frédéric; Boquet, Didier; Couraud, Jean-Yves; Robin, Philippe

    2016-01-01

    ABSTRACT Metastatic melanoma is an aggressive cancer with a poor prognostic, and the design of new targeted drugs to treat melanoma is a therapeutic challenge. A promising approach is to produce monoclonal antibodies (mAbs) against the endothelin B receptor (ETB), which is known to be overexpressed in melanoma and to contribute to proliferation, migration and vasculogenic mimicry associated with invasiveness of this cancer. We previously described rendomab-B1, a mAb produced by DNA immunization. It is endowed with remarkable characteristics in term of affinity, specificity and antagonist properties against human ETB expressed by the endothelial cells, but, surprisingly, had poor affinity for ETB expressed by melanoma cells. This characteristic strongly suggested the existence of a tumor-specific ETB form. In the study reported here, we identified a new mAb, rendomab-B4, which, in contrast to rendomab-B1, binds ETB expressed on UACC-257, WM-266-4 and SLM8 melanoma cells. Moreover, after binding to UACC-257 cells, rendomab-B4 is internalized and colocalizes with the endosomal protein EEA-1. Interestingly, rendomab-B4, despite its inability to compete with endothelin binding, is able to inhibit phospholipase C pathway and migration induced by endothelin. By contrast, rendomab-B4 fails to decrease ERK1/2 phosphorylation induced by endothelin, suggesting a biased effect on ETB. These particular properties make rendomab-B4 an interesting tool to analyze ETB-structure/function and a promising starting point for the development of new immunological tools in the field of melanoma therapeutics. PMID:27390909

  9. Photodynamic therapy for melanoma: efficacy and immunologic effects

    NASA Astrophysics Data System (ADS)

    Avci, Pinar; Gupta, Gaurav K.; Kawakubo, Masayoshi; Hamblin, Michael R.

    2014-02-01

    Malignant melanoma is one of the fastest growing cancers and if it cannot be completely surgically removed the prognosis is bleak. Melanomas are known to be particularly resistant to both chemotherapy and radiotherapy. Various types of immunotherapy have however been investigated with mixed reports of success. Photodynamic therapy (PDT) has also been tested against melanoma, again with mixed effects as the melanin pigment is thought to act as both an optical shield and as an antioxidant. We have been investigating PDT against malignant melanoma in mouse models. We have compared B16F10 melanoma syngenic to C57BL/6 mice and S91 Cloudman melanoma syngenic to DBA2 mice. We have tested the hypothesis that S91 will respond better than B16 because of higher expression of immunocritical molecules such as MHC-1, tyrosinase, tyrosinase related protein-2 gp100, and intercellular adhesion molecule-1. Some of these molecules can act as tumor rejection antigens that can be recognized by antigen-specific cytotoxic CD8 T cells that have been stimulated by PDT. Moreover it is possible that DBA2 mice are intrinsically better able to mount an anti-tumor immune response than C57BL/6 mice. We are also studying intratumoral injection of photosensitzers such as benzoporphyrin monoacid ring A and comparing this route with the more usual route of intravenous administration.

  10. miR-193b Regulates Mcl-1 in Melanoma

    PubMed Central

    Chen, Jiamin; Zhang, Xiao; Lentz, Cindy; Abi-Daoud, Marie; Paré, Geneviève C.; Yang, Xiaolong; Feilotter, Harriet E.; Tron, Victor A.

    2011-01-01

    MicroRNAs play important roles in gene regulation, and their expression is frequently dysregulated in cancer cells. In a previous study, we reported that miR-193b represses cell proliferation and regulates cyclin D1 in melanoma cells, suggesting that miR-193b could act as a tumor suppressor. Herein, we demonstrate that miR-193b also down-regulates myeloid cell leukemia sequence 1 (Mcl-1) in melanoma cells. MicroRNA microarray profiling revealed that miR-193b is expressed at a significantly lower level in malignant melanoma than in benign nevi. Consistent with this, Mcl-1 is detected at a higher level in malignant melanoma than in benign nevi. In a survey of melanoma samples, the level of Mcl-1 is inversely correlated with the level of miR-193b. Overexpression of miR-193b in melanoma cells represses Mcl-1 expression. Previous studies showed that Mcl-1 knockdown cells are hypersensitive to ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-XL, and Bcl-w. Similarly, overexpression of miR-193b restores ABT-737 sensitivity to ABT-737–resistant cells. Furthermore, the effect of miR-193b on the expression of Mcl-1 seems to be mediated by direct interaction between miR-193b and seed and seedless pairing sequences in the 3′ untranslated region of Mcl-1 mRNA. Thus, this study provides evidence that miR-193b directly regulates Mcl-1 and that down-regulation of miR-193b in vivo could be an early event in melanoma progression. PMID:21893020

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

    PubMed

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

    2012-01-01

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

  12. Lubrol-RAFTs in melanoma cells: a molecular platform for tumor-promoting ephrin-B2-integrin-beta1 interaction.

    PubMed

    Meyer, Stefanie; Orsó, Evelyn; Schmitz, Gerd; Landthaler, Michael; Vogt, Thomas

    2007-07-01

    Ephrins control cell motility and matrix adhesion. These functions play a pivotal role in cancer progression, for example, in malignant melanomas. We have previously shown that the ephrin-B2-tumor-promoting action is partly mediated by integrin-beta1 interaction. However, the subcellular prerequisites for molecular interaction like molecular proximity and co-compartmentalization have not been elucidated yet. Specific cholesterol-rich microdomains, termed lipid rafts (RAFTs), are known to be essential for functional ephrin-B2 signalling and integrin-mediated effects. Therefore, we addressed the question whether RAFT co-compartmentalization of both molecules could provide the molecular platform for their tumor-promoting interaction. In this study, we show that overexpressed ephrin-B2 is not only compartmentalized to classical Triton X-100 RAFTs in B16 melanoma cells, but also to the recently defined Lubrol-RAFTs. Interestingly, in the melanoma cells investigated, integrin-beta1 is also preferentially detected in such Lubrol-RAFTs. Accordingly, the presence of ephrin-B2 and integrin-beta1 in RAFTs and their function in cell migration and matrix attachment are highly sensitive to RAFT disruption by cholesterol depletion. Confocal fluorescence microscopy analyses also support the concept of a close molecular proximity and functional interplay of ephrin-B2 and integrin-beta1 in the plasma membrane. We conclude that Lubrol-RAFTs probably represent the platform for tumor-promoting ephrin-B2-integrin-beta1 interaction, which could become an interesting target for future antitumoral therapies.

  13. The functional property of royal jelly 10-hydroxy-2-decenoic acid as a melanogenesis inhibitor.

    PubMed

    Peng, Chi-Chung; Sun, Hui-Tzu; Lin, I-Ping; Kuo, Ping-Chung; Li, Jen-Chieh

    2017-08-09

    It has been reported that royal jelly would reduce melanin synthesis and inhibit the expression of melanogensis related proteins and genes. In this study, we evaluate the anti-melanogenic and depigmenting activity of 10-hydroxy-2-decenoic acid (10-HDA) from royal jelly of Apis mellifera. In this study, we assesses the 10-HDA whitening activity in comparison with the changes in the intracellular tyrosinase activity, melanin content and melanin production related protein levles in B16F1 melanoma cells after treating with 10-HDA. Furthermore, the skin whitening effect was evaluated by applying a cream product containing with 0.5%, 1% and 2% of 10-HDA onto the skin of mice (C57BL/6 J) for 3 week to observe the effect of DL*-values. The results showed that 10-HDA inhibited the MITF protein expression (IC50 0.86 mM) in B16F1 melanoma cells. Western blot analysis revealed that 10-HDA inhibited the activity of tyrosinase and the expression of tyrosinase-related protein 1 (TRP-1), TRP-2, and microphthalmia-associated transcription factor (MITF) in B16F1 melanoma cells. In addition, the 10-HDA was applied on the skin of mice show significantly increased the average skin-whitening index (L value). The validation data indicated the potential of 10-HDA for use in suppressing skin pigmentation. The 10-HDA is proposed as a candidate to inhibit melanogenesis, thus it could be developed as cosmetics skin care products.

  14. Enhancement of radiosensitivity of melanoma cells by pegylated gold nanoparticles under irradiation of megavoltage electrons.

    PubMed

    Mousavi, Mehdi; Nedaei, Hassan Ali; Khoei, Samideh; Eynali, Samira; Khoshgard, Karim; Robatjazi, Mostafa; Iraji Rad, Rasoul

    2017-02-01

    Gold nanoparticles (GNP) have significant potential as radiosensitizer agents due to their distinctive properties. Several studies have shown that the surface modification of nanoparticles with methyl polyethylene glycol (mPEG) can increase their biocompatibility. However, the present study investigated the radiosensitization effects of mPEG-coated GNP (mPEG-GNP) in B16F10 murine melanoma cells under irradiation of 6 MeV Electron beam. The synthesized GNP were characterized by UV-Visible spectroscopy, dynamic light scattering, transmission electron microscopy, and zeta potential. Enhancement of radiosensitization was evaluated by the clonogenic assay at different radiation doses of megavoltage electron beams. It was observed that mPEG-GNP with a hydrodynamic size of approximately 50 nm are almost spherical and cellular uptake occurred at all concentrations. Both proliferation efficiency and survival fraction decreased with increasing mPEG-GNP concentration. Furthermore, significant GNP sensitization occurred with a maximum dose enhancement factor of 1.22 at a concentration of 30 μM. Pegylated-GNP are taken up by B16F10 cancer cells and cause radiosensitization in the presence of 6 MeV electrons. The radiosensitization effects of GNP may probably be due to biological processes. Therefore, the underlying biological mechanisms beyond the physical dose enhancement need to be further clarified.

  15. Impact of calcium ion on cytotoxic effect of the boroxine derivative, K2[B3O3F4OH].

    PubMed

    Ivankovic, Sinisa; Stojkovic, Ranko; Maksimovic, Milka; Galic, Borivoj; Milos, Mladen

    2016-01-01

    The effect of Ca 2+ ions on the cytotoxic ability of boron heterocyclic compound dipotassium-trioxohydroxytetrafluorotriborate (K 2 [B 3 O 3 F 4 OH]), on in vitro tumor cells (mammary adenocarcinoma 4T1, melanoma B16F10 and squamous cell carcinoma SCCVII) and non-tumoral fibroblast cells (mouse dermal L929 and hamster lung V79) was examined. At small concentrations of Ca 2+ ions (0.42 mM), K 2 [B 3 O 3 F 4 OH] (3.85 mM) has a very strong cytotoxic effect on all cancer cells tested (89.1, 85.6 and 84.6%) and significantly less effect on normal cells (19.5 and 24.2%), respectively. Applying larger concentrations of Ca 2+ ions (9.42-72.42 mM), at the same concentration of K 2 [B 3 O 3 F 4 OH], no significant cytotoxic effect was detected on cancer cells and normal cells investigated. The selective ability of K 2 [B 3 O 3 F 4 OH], in the medium with a low concentration of Ca 2+ ions has a strong cytotoxic effect on cancer cells and very weak effect in normal cells, opens up the possibility of its application in antitumor therapy.

  16. Solid Lipid Nanoparticles Carrying Temozolomide for Melanoma Treatment. Preliminary In Vitro and In Vivo Studies

    PubMed Central

    Ferrara, Benedetta; Biasibetti, Elena; Schiffer, Davide; Mellai, Marta; Annovazzi, Laura; Cangemi, Luigi; Muntoni, Elisabetta; Dianzani, Umberto

    2018-01-01

    Aim: To develop an innovative delivery system for temozolomide (TMZ) in solid lipid nanoparticles (SLN), which has been preliminarily investigated for the treatment of melanoma. Materials and Methods: SLN-TMZ was obtained through fatty acid coacervation. Its pharmacological effects were assessed and compared with free TMZ in in vitro and in vivo models of melanoma and glioblastoma. Results: Compared to the standard free TMZ, SLN-TMZ exerted larger effects, when cell proliferation of melanoma cells, and neoangiogeneis were evaluated. SLN-TMZ also inhibited growth and vascularization of B16-F10 melanoma in C57/BL6 mice, without apparent toxic effects. Conclusion: SLN could be a promising strategy for the delivery of TMZ, allowing an increased stability of the drug and thereby its employment in the treatment of aggressive malignacies. PMID:29364157

  17. Histopathology of normal skin and melanomas after nanosecond pulsed electric field treatment.

    PubMed

    Chen, Xinhua; James Swanson, R; Kolb, Juergen F; Nuccitelli, Richard; Schoenbach, Karl H

    2009-12-01

    Nanosecond pulsed electric fields (nsPEFs) can affect the intracellular structures of cells in vitro. This study shows the direct effects of nsPEFs on tumor growth, tumor volume, and histological characteristics of normal skin and B16-F10 melanoma in SKH-1 mice. A melanoma model was set up by injecting B16-F10 into female SKH-1 mice. After a 100-pulse treatment with an nsPEF (40-kV/cm field strength; 300-ns duration; 30-ns rise time; 2-Hz repetition rate), tumor growth and histology were studied using transillumination, light microscopy with hematoxylin and eosin stain and transmission electron microscopy. Melanin and iron within the melanoma tumor were also detected with specific stains. After nsPEF treatment, tumor development was inhibited with decreased volumes post-nsPEF treatment compared with control tumors (P<0.05). The nsPEF-treated tumor volume was reduced significantly compared with the control group (P<0.01). Hematoxylin and eosin stain and transmission electron microscopy showed morphological changes and nuclear shrinkage in the tumor. Fontana-Masson stain indicates that nsPEF can externalize the melanin. Iron stain suggested nsPEF caused slight hemorrhage in the treated tissue. Histology confirmed that repeated applications of nsPEF disrupted the vascular network. nsPEF treatment can significantly disrupt the vasculature, reduce subcutaneous murine melanoma development, and produce tumor cell contraction and nuclear shrinkage while concurrently, but not permanently, damaging peripheral healthy skin tissue in the treated area, which we attribute to the highly localized electric fields surrounding the needle electrodes.

  18. miR-193b Regulates Mcl-1 in Melanoma.

    PubMed

    Chen, Jiamin; Zhang, Xiao; Lentz, Cindy; Abi-Daoud, Marie; Paré, Geneviève C; Yang, Xiaolong; Feilotter, Harriet E; Tron, Victor A

    2011-11-01

    MicroRNAs play important roles in gene regulation, and their expression is frequently dysregulated in cancer cells. In a previous study, we reported that miR-193b represses cell proliferation and regulates cyclin D1 in melanoma cells, suggesting that miR-193b could act as a tumor suppressor. Herein, we demonstrate that miR-193b also down-regulates myeloid cell leukemia sequence 1 (Mcl-1) in melanoma cells. MicroRNA microarray profiling revealed that miR-193b is expressed at a significantly lower level in malignant melanoma than in benign nevi. Consistent with this, Mcl-1 is detected at a higher level in malignant melanoma than in benign nevi. In a survey of melanoma samples, the level of Mcl-1 is inversely correlated with the level of miR-193b. Overexpression of miR-193b in melanoma cells represses Mcl-1 expression. Previous studies showed that Mcl-1 knockdown cells are hypersensitive to ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-X(L), and Bcl-w. Similarly, overexpression of miR-193b restores ABT-737 sensitivity to ABT-737-resistant cells. Furthermore, the effect of miR-193b on the expression of Mcl-1 seems to be mediated by direct interaction between miR-193b and seed and seedless pairing sequences in the 3' untranslated region of Mcl-1 mRNA. Thus, this study provides evidence that miR-193b directly regulates Mcl-1 and that down-regulation of miR-193b in vivo could be an early event in melanoma progression. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  19. Apoptosis of murine melanoma B16-BL6 cells induced by quercetin targeting mitochondria, inhibiting expression of PKC-alpha and translocating PKC-delta.

    PubMed

    Zhang, Xian-Ming; Chen, Jia; Xia, Yu-Gui; Xu, Qiang

    2005-03-01

    In our previous study, quercetin was found to induce apoptosis of murine melanoma B16-BL6 cells. The cellular and molecular mechanism of quercetin-induced apoptosis was investigated in the present study. Nuclear morphology was determined by fluorescence microscopy. DNA fragmentation was analyzed by electrophoresis and quantified by the diphenylamine method. The transmembrane potential of mitochondria was measured by flow cytometry. Bcl-2, Bcl-X(L), PKC-alpha, PKC-beta, and PKC-delta were detected by Western blotting. Caspase activity was determined spectrophotometrically. Quercetin induced the condensation of nuclei of B16-BL6 cells in a dose-dependent pattern as visualized by Hoechst 33258 and propidium iodide dying. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, significantly enhanced apoptosis induced by quercetin, while doxorubicin, a PKC inhibitor, markedly decreased it. Both PMA and doxorubicin showed a consistent effect on the fragmentation of nuclear DNA caused by various dosages of quercetin. Quercetin dose-dependently led to loss of the mitochondrial membrane potential, which was also significantly reinforced or antagonized by PMA and doxorubicin, respectively. Moreover, PMA showed reinforcement, while doxorubicin showed significant antagonization, of the quercetin-mediated decrease in the expression of Bcl-2. Quercetin promoted caspase-3 activity in a dose-dependent manner, which was also regulated by PMA and doxorubicin with a pattern similar to that seen in their effect on apoptosis, mitochondrial membrane potential and Bcl-2 expression, but none of these were directly affected by PMA and doxorubicin. Free fatty acid and chlorpromazine, a PKC activator and inhibitor, respectively, did not interfere with these effects of quercetin. B16-BL6 cells expressed PKC-alpha, PKC-beta, and PKC-delta. Quercetin dose-dependently inhibited the expression of PKC-alpha but not that of PKC-beta and PKC-delta. Doxorubicin almost completely blocked the effect of

  20. The effect of ultraviolet radiation on choroidal melanocytes and melanoma cell lines: cell survival and matrix metalloproteinase production.

    PubMed

    Lai, Kenneth; Di Girolamo, Nick; Conway, Robert M; Jager, Martine J; Madigan, Michele C

    2007-05-01

    Ultraviolet radiation (UVR) can induce DNA damage and regulate the expression of factors important for tumour growth and metastasis, including matrix metalloproteinases (MMPs). Epidemiological studies suggest that chronic UVR exposure, especially during early adulthood, may be a risk factor in patients with choroidal melanoma. However, the effects of UV(R)-B on human choroidal melanocyte survival and growth are unknown. In this study, we investigated if UV(R)-B affected the in vitro survival, growth and MMP production of choroidal melanocytes and melanoma cells. Cultures of primary choroidal melanocytes and melanoma cell lines (OCM-1 and OCM-8) were exposed to UV(R)-B (0-30 mJ/cm(2)). The cell morphology and growth were examined, and cell viability was assessed using an MTT assay. Gelatin zymography was used to assess the enzymatic activity for MMP-2 and -9 in conditioned media following UV(R)-B treatment. UV(R)-B > or =20 mJ/cm(2) was cytotoxic for choroidal melanocytes. Cytotoxic doses of 5 to 10 mJ/cm(2) were found for OCM-8 and OCM-1 melanoma cell lines. Low levels of UV(R)-B (2.5 and 3.5 mJ/cm(2)) significantly reduced melanoma cell viability after 48 h, although melanocyte viability was not affected by doses of UV(R)-B <10 mJ/cm(2). Conditioned media from melanoma cells and melanocytes displayed pro-MMP-2 activity independent of UV(R)-B. Control and UV(R)-B-treated OCM-1 cells secreted active MMP-2 up to 72 h. Pro-MMP-9 activity was seen from 36 h for control and UV(R)-B-treated OCM-1 and OCM-8 cells. Melanocytes appeared more resistant to physiological doses of UV(R)-B than melanoma cells; the potential of melanocytes to initially survive DNA damage following UV(R)-B exposure may be relevant to the subsequent transformation of melanocytes to melanomas. Although UV(R)-B did not induce the production and/or activation of MMP-2 and -9 in melanocytes or melanoma cells, we are currently investigating whether DNA damage-response genes such as p53 and p21 can be

  1. Z-100, an immunomodulatory extract of Mycobacterium tuberculosis strain Aoyama B, prevents spontaneous lymphatic metastasis of B16-BL6 melanoma.

    PubMed

    Horii, Takayuki; Yoshinaga, Koji; Kobayashi, Nobuyoshi; Seto, Koichi; Orikawa, Yuki; Okamoto, Masahiro; Eta, Runa; Ohira, Yuta; Katsunuma, Kokichi; Hori, Yuko; Tanaka, Takao; Takei, Mineo

    2014-01-01

    Lymphatic metastasis is common in advanced-stage carcinoma and is associated with a poor prognosis. However, few effective treatments to inhibit it are available. Z-100 is an immunomodulatory extract of Mycobacterium tuberculosis strain Aoyama B that contains polysaccharides such as arabinomannan and mannan. Here, we investigated the inhibitory effect of Z-100 on spontaneous lymphatic metastasis. C57BL/6N mice injected subcutaneously with B16-BL6 melanoma cells in the right hind footpad were administered Z-100 subcutaneously in the right inguinal region on a daily basis. On day twenty-one after the injection, the right inguinal lymph nodes were excised, and the extent of metastasis, the number of immune cells, and the amount of granzyme B protein in the lymph nodes were examined. We also investigated the combined effect of Z-100 and irradiation in this model. Results showed that Z-100 reduced number of animals with metastasis, with respective metastasis rates of 85.7%, 42.9%, 7.1% and 0.0% in saline, 0.1 mg/kg Z-100, 1 mg/kg Z-100 and 10 mg/kg Z-100 group. Further, mice that had been given Z-100 were found to have more immune cells and granzyme B protein in the lymph nodes than control mice. The combination of low dose Z-100 and irradiation also inhibited spontaneous lymph node metastases. These findings suggest that Z-100 may be beneficial in preventing lymphatic metastasis by enhancing the immune response.

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

    PubMed Central

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

    2016-01-01

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

  3. Arrest of B16 Melanoma Cells in the Mouse Pulmonary Microcirculation Induces Endothelial Nitric Oxide Synthase-Dependent Nitric Oxide Release that Is Cytotoxic to the Tumor Cells

    PubMed Central

    Qiu, Hongming; Orr, F.William; Jensen, Derrek; Wang, Hui Helen; McIntosh, Alan R.; Hasinoff, Brian B.; Nance, Dwight M.; Pylypas, Susan; Qi, Ke; Song, Chun; Muschel, Ruth J.; Al-Mehdi, Abu-Bakr

    2003-01-01

    Metastatic cancer cells seed the lung via blood vessels. Because endothelial cells generate nitric oxide (NO) in response to shear stress, we postulated that the arrest of cancer cells in the pulmonary microcirculation causes the release of NO in the lung. After intravenous injection of B16F1 melanoma cells, pulmonary NO increased sevenfold throughout 20 minutes and approached basal levels by 4 hours. NO induction was blocked by NG-nitro-l-arginine methyl ester (L-NAME) and was not observed in endothelial nitric oxide synthase (eNOS)-deficient mice. NO production, visualized ex vivo with the fluorescent NO probe diaminofluorescein diacetate, increased rapidly at the site of tumor cell arrest, and continued to increase throughout 20 minutes. Arrested tumor cells underwent apoptosis with apoptotic counts more than threefold over baseline at 8 and 48 hours. Neither the NO signals nor increased apoptosis were seen in eNOS knockout mice or mice pretreated with L-NAME. At 48 hours, 83% of the arrested cells had cleared from the lungs of wild-type mice but only ∼55% of the cells cleared from eNOS-deficient or L-NAME pretreated mice. eNOS knockout and L-NAME-treated mice had twofold to fivefold more metastases than wild-type mice, measured by the number of surface nodules or by histomorphometry. We conclude that tumor cell arrest in the pulmonary microcirculation induces eNOS-dependent NO release by the endothelium adjacent to the arrested tumor cells and that NO is one factor that causes tumor cell apoptosis, clearance from the lung, and inhibition of metastasis. PMID:12547699

  4. Direct Melanoma Cell Contact Induces Stromal Cell Autocrine Prostaglandin E2-EP4 Receptor Signaling That Drives Tumor Growth, Angiogenesis, and Metastasis*

    PubMed Central

    Inada, Masaki; Takita, Morichika; Yokoyama, Satoshi; Watanabe, Kenta; Tominari, Tsukasa; Matsumoto, Chiho; Hirata, Michiko; Maru, Yoshiro; Maruyama, Takayuki; Sugimoto, Yukihiko; Narumiya, Shuh; Uematsu, Satoshi; Akira, Shizuo; Murphy, Gillian; Nagase, Hideaki; Miyaura, Chisato

    2015-01-01

    The stromal cells associated with tumors such as melanoma are significant determinants of tumor growth and metastasis. Using membrane-bound prostaglandin E synthase 1 (mPges1−/−) mice, we show that prostaglandin E2 (PGE2) production by host tissues is critical for B16 melanoma growth, angiogenesis, and metastasis to both bone and soft tissues. Concomitant studies in vitro showed that PGE2 production by fibroblasts is regulated by direct interaction with B16 cells. Autocrine activity of PGE2 further regulates the production of angiogenic factors by fibroblasts, which are key to the vascularization of both primary and metastatic tumor growth. Similarly, cell-cell interactions between B16 cells and host osteoblasts modulate mPGES-1 activity and PGE2 production by the osteoblasts. PGE2, in turn, acts to stimulate receptor activator of NF-κB ligand expression, leading to osteoclast differentiation and bone erosion. Using eicosanoid receptor antagonists, we show that PGE2 acts on osteoblasts and fibroblasts in the tumor microenvironment through the EP4 receptor. Metastatic tumor growth and vascularization in soft tissues was abrogated by an EP4 receptor antagonist. EP4-null Ptger4−/− mice do not support B16 melanoma growth. In vitro, an EP4 receptor antagonist modulated PGE2 effects on fibroblast production of angiogenic factors. Our data show that B16 melanoma cells directly influence host stromal cells to generate PGE2 signals governing neoangiogenesis and metastatic growth in bone via osteoclast erosive activity as well as angiogenesis in soft tissue tumors. PMID:26475855

  5. Immunoregulatory protein B7-H3 promotes growth and decreases sensitivity to therapy in metastatic melanoma cells.

    PubMed

    Flem-Karlsen, Karine; Tekle, Christina; Andersson, Yvonne; Flatmark, Kjersti; Fodstad, Øystein; Nunes-Xavier, Caroline E

    2017-09-01

    B7-H3 (CD276) belongs to the B7 family of immunoregulatory proteins and has been implicated in cancer progression and metastasis. In this study, we found that metastatic melanoma cells with knockdown expression of B7-H3 showed modest decrease in proliferation and glycolytic capacity and were more sensitive to dacarbazine (DTIC) chemotherapy and small-molecule inhibitors targeting MAP kinase (MAPK) and AKT/mTOR pathways: vemurafenib (PLX4032; BRAF inhibitor), binimetinib (MEK-162; MEK inhibitor), everolimus (RAD001; mTOR inhibitor), and triciribidine (API-2; AKT inhibitor). Similar effects were observed in melanoma cells in the presence of an inhibitory B7-H3 monoclonal antibody, while the opposite was seen in B7-H3-overexpressing cells. Further, combining B7-H3 inhibition with small-molecule inhibitors resulted in significantly increased antiproliferative effect in melanoma cells, as well as in BRAF V 600E mutated cell lines derived from patient biopsies. Our findings indicate that targeting B7-H3 may be a novel alternative to improve current therapy of metastatic melanoma. © 2017 The Authors Pigment Cell & Melonoma Research Published by John Wiley & Sons Ltd.

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

    PubMed Central

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

    2015-01-01

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

  7. Evaluation of (68)Ga- and (177)Lu-DOTA-PEG4-LLP2A for VLA-4-Targeted PET Imaging and Treatment of Metastatic Melanoma.

    PubMed

    Beaino, Wissam; Nedrow, Jessie R; Anderson, Carolyn J

    2015-06-01

    Malignant melanoma is a highly aggressive cancer, and the incidence of this disease is increasing worldwide at an alarming rate. Despite advances in the treatment of melanoma, patients with metastatic disease still have a poor prognosis and low survival rate. New strategies, including targeted radiotherapy, would provide options for patients who become resistant to therapies such as BRAF inhibitors. Very late antigen-4 (VLA-4) is expressed on melanoma tumor cells in higher levels in more aggressive and metastatic disease and may provide an ideal target for drug delivery and targeted radiotherapy. In this study, we evaluated (177)Lu- and (68)Ga-labeled DOTA-PEG4-LLP2A as a VLA-4-targeted radiotherapeutic with a companion PET agent for diagnosis and monitoring metastatic melanoma treatment. DOTA-PEG4-LLP2A was synthesized by solid-phase synthesis. The affinity of (177)Lu- and (68)Ga-labeled DOTA-PEG4-LLP2A to VLA-4 was determined in B16F10 melanoma cells by saturation binding and competitive binding assays, respectively. Biodistribution of the LLP2A conjugates was determined in C57BL/6 mice bearing B16F10 subcutaneous tumors, while PET/CT imaging was performed in subcutaneous and metastatic models. (177)Lu-DOTA-PEG4-LLP2A showed high affinity to VLA-4 with a Kd of 4.1 ± 1.5 nM and demonstrated significant accumulation in the B16F10 melanoma tumor after 4 h (31.5 ± 7.8%ID/g). The tumor/blood ratio of (177)Lu-DOTA-PEG4-LLP2A was highest at 24 h (185 ± 26). PET imaging of metastatic melanoma with (68)Ga-DOTA-PEG4-LLP2A showed high uptake in sites of metastases and correlated with bioluminescence imaging of the tumors. These data demonstrate that (177)Lu-DOTA-PEG4-LLP2A has potential as a targeted therapeutic for treating melanoma as well as other VLA-4-expressing tumors. In addition, (68)Ga-DOTA-PEG4-LLP2A is a readily translatable companion PET tracer for imaging of metastatic melanoma.

  8. Improving label-free detection of circulating melanoma cells by photoacoustic flow cytometry

    NASA Astrophysics Data System (ADS)

    Zhou, Huan; Wang, Qiyan; Pang, Kai; Zhou, Quanyu; Yang, Ping; He, Hao; Wei, Xunbin

    2018-02-01

    Melanoma is a kind of a malignant tumor of melanocytes with the properties of high mortality and high metastasis rate. The circulating melanoma cells with the high content of melanin can be detected by light absorption to diagnose and treat cancer at an early stage. Compared with conventional detection methods such as in vivo flow cytometry (IVFC) based on fluorescence, the in vivo photoacoustic flow cytometry (PAFC) utilizes melanin cells as biomarkers to collect the photoacoustic (PA) signals without toxic fluorescent dyes labeling in a non-invasive way. The information of target tumor cells is helpful for data analysis and cell counting. However, the raw signals in PAFC system contain numerous noises such as environmental noise, device noise and in vivo motion noise. Conventional denoising algorithms such as wavelet denoising (WD) method and means filter (MF) method are based on the local information to extract the data of clinical interest, which remove the subtle feature and leave many noises. To address the above questions, the nonlocal means (NLM) method based on nonlocal data has been proposed to suppress the noise in PA signals. Extensive experiments on in vivo PA signals from the mice with the injection of B16F10 cells in caudal vein have been conducted. All the results indicate that the NLM method has superior noise reduction performance and subtle information reservation.

  9. [18F]MEL050 as a melanin-targeted PET tracer: Fully automated radiosynthesis and comparison to 18F-FDG for the detection of pigmented melanoma in mice primary subcutaneous tumors and pulmonary metastases.

    PubMed

    Rizzo-Padoin, Nathalie; Chaussard, Michael; Vignal, Nicolas; Kotula, Ewa; Tsoupko-Sitnikov, Vadim; Vaz, Sofia; Hontonnou, Fortune; Liu, Wang-Qing; Poyet, Jean-Luc; Vidal, Michel; Merlet, Pascal; Hosten, Benoit; Sarda-Mantel, Laure

    2016-12-01

    Melanoma is a highly malignant cutaneous tumor of melanin-producing cells. MEL050 is a synthetic benzamide-derived molecule that specifically binds to melanin with high affinity. Our aim was to implement a fully automated radiosynthesis of [ 18 F]MEL050, using for the first time, the AllInOne™ synthesis module (Trasis), and to evaluate the potential of [ 18 F]MEL050 for the detection of pigmented melanoma in mice primary subcutaneous tumors and pulmonary metastases, and to compare it with that of [ 18 F]FDG. Automated radiosynthesis of [ 18 F]MEL050, including HPLC purification and formulation, were performed on an AllInOne™ synthesis module. [ 18 F]MEL050 was synthesized using a one-step bromine-for-fluorine nucleophilic heteroaromatic substitution. Melanoma models were induced by subcutaneous (primary tumor) or intravenous (pulmonary metastases) injection of B16-F10-luc2 cells in NMRI mice. The maximum percentage of [ 18 F]MEL050 Injected Dose per g of lung tissue (%ID/g Max) was determined on PET images, compared to [ 18 F]FDG and correlated to in vivo bioluminescence imaging. The automated radiosynthesis of [ 18 F]MEL050 required an overall radiosynthesis time of 48min, with a yield of 13-18% (not-decay corrected) and radiochemical purity higher than 99%. [ 18 F]MEL050 PET/CT images were concordant with bioluminescence imaging, showing increased radiotracer uptake in all primary subcutaneous tumors and pulmonary metastases of mice. PET quantification of radiotracers uptake in tumors and muscles demonstrated similar tumor-to-background ratio (TBR) with [ 18 F]MEL050 and [ 18 F]FDG in subcutaneous tumors and higher TBR with [ 18 F]MEL050 than with [ 18 F]FDG in pulmonary metastases. We successfully implemented the radiosynthesis of [ 18 F]MEL050 using the AllInOne™ module, including HPLC purification and formulation. In vivo PET/CT validation of [ 18 F]MEL050 was obtained in mouse models of pigmented melanoma, where higher [ 18 F]MEL050 uptake was observed

  10. 48 CFR 47.303-16 - F.o.b. designated air carrier's terminal, point of importation.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false F.o.b. designated air... Contracts 47.303-16 F.o.b. designated air carrier's terminal, point of importation. (a) Explanation of delivery term. F.o.b. designated air carrier's terminal, point of importation means free of expense to the...

  11. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Misu, Masayasu; Ouji, Yukiteru, E-mail: oujix@naramed-u.ac.jp; Kawai, Norikazu

    In spite of the strong expression of Wnt-10b in melanomas, its role in melanoma cells has not been elucidated. In the present study, the biological effects of Wnt-10b on murine B16F10 (B16) melanoma cells were investigated using conditioned medium from Wnt-10b-producing COS cells (Wnt-CM). After 2 days of culture in the presence of Wnt-CM, proliferation of B16 melanoma cells was inhibited, whereas tyrosinase activity was increased. An in vitro wound healing assay demonstrated that migration of melanoma cells to the wound area was inhibited with the addition of Wnt-CM. Furthermore, evaluation of cellular senescence revealed prominent induction of SA-β-gal-positive senescent cellsmore » in cultures with Wnt-CM. Finally, the growth of B16 melanoma cell aggregates in collagen 3D-gel cultures was markedly suppressed in the presence of Wnt-CM. These results suggest that Wnt-10b represses tumor cell properties, such as proliferation and migration of B16 melanoma cells, driving them toward a more differentiated state along a melanocyte lineage. - Highlights: • Wnt-10b inhibited proliferation and migration of melanoma cells. • Wnt-10b induced tyrosinase activity and senescence of melanoma cells. • Wnt-10b suppressed growth of cell aggregates in collagen 3D-gel cultures. • Wnt-10b represses tumor cell properties, driving them toward a more differentiated state along a melanocyte lineage.« less

  12. Climacostol reduces tumour progression in a mouse model of melanoma via the p53-dependent intrinsic apoptotic programme

    PubMed Central

    Perrotta, Cristiana; Buonanno, Federico; Zecchini, Silvia; Giavazzi, Alessio; Proietti Serafini, Francesca; Catalani, Elisabetta; Guerra, Laura; Belardinelli, Maria Cristina; Picchietti, Simona; Fausto, Anna Maria; Giorgi, Simone; Marcantoni, Enrico; Clementi, Emilio; Ortenzi, Claudio; Cervia, Davide

    2016-01-01

    Climacostol, a compound produced by the ciliated protozoan Climacostomum virens, displayed cytotoxic properties in vitro. This study demonstrates that it has anti-tumour potential. Climacostol caused a reduction of viability/proliferation of B16-F10 mouse melanoma cells, a rapidly occurring DNA damage, and induced the intrinsic apoptotic pathway characterised by the dissipation of the mitochondrial membrane potential, the translocation of Bax to the mitochondria, the release of Cytochrome c from the mitochondria, and the activation of Caspase 9-dependent cleavage of Caspase 3. The apoptotic mechanism of climacostol was found to rely on the up-regulation of p53 and its targets Noxa and Puma. In vivo analysis of B16-F10 allografts revealed a persistent inhibition of tumour growth rate when melanomas were treated with intra-tumoural injections of climacostol. In addition, it significantly improved the survival of transplanted mice, decreased tumour weight, induced a remarkable reduction of viable cells inside the tumour, activated apoptosis and up-regulated the p53 signalling network. Importantly, climacostol toxicity was more selective against tumour than non-tumour cells. The anti-tumour properties of climacostol and the molecular events associated with its action indicate that it is a powerful agent that may be considered for the design of pro-apoptotic drugs for melanoma therapy. PMID:27271364

  13. Design and evaluation of new Tc-99m-labeled lactam bridge-cyclized alpha-MSH peptides for melanoma imaging.

    PubMed

    Guo, Haixun; Gallazzi, Fabio; Miao, Yubin

    2013-04-01

    The purpose of this study was to examine the melanoma targeting and imaging properties of new (99m)Tc-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone (α-MSH) peptides using bifunctional chelating agents. MAG3-GGNle-CycMSH(hex), AcCG3-GGNle-CycMSH(hex), and HYNIC-GGNle-CycMSH(hex) peptides were synthesized, and their melanocortin-1 (MC1) receptor binding affinities were determined in B16/F1 melanoma cells. The biodistribution of (99m)Tc-MAG3-GGNle-CycMSH(hex), (99m)Tc-AcCG3-GGNle-CycMSH(hex), (99m)Tc(CO)3-HYNIC-GGNle-CycMSH(hex), and (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) were determined in B16/F1 melanoma-bearing C57 mice at 2 h postinjection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) were further examined because of its high melanoma uptake and fast urinary clearance. The IC50 values of MAG3-GGNle-CycMSH(hex), AcCG3-GGNle-CycMSH(hex), and HYNIC-GGNle-CycMSH(hex) were 1.0 ± 0.05, 1.2 ± 0.19, and 0.6 ± 0.04 nM in B16/F1 melanoma cells, respectively. Among these four (99m)Tc-peptides, (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) exhibited the highest melanoma uptake (14.14 ± 4.90% ID/g) and fastest urinary clearance (91.26 ± 1.96% ID) at 2 h postinjection. (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) showed high tumor to normal organ uptake ratios except for the kidneys. The tumor/kidney uptake ratios of (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) were 2.50 and 3.55 at 4 and 24 h postinjection. The melanoma lesions were clearly visualized by SPECT/CT using (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) as an imaging probe at 2 h postinjection. Overall, high melanoma uptake coupled with fast urinary clearance of (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) highlighted its potential for metastatic melanoma detection in the future.

  14. Design and Evaluation of New Tc-99m-Labeled Lactam Bridge-Cyclized Alpha-MSH Peptides for Melanoma Imaging

    PubMed Central

    Guo, Haixun; Gallazzi, Fabio; Miao, Yubin

    2013-01-01

    The purpose of this study was to examine the melanoma targeting and imaging properties of new 99mTc-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone (α-MSH) peptides using bifunctional chelating agents. MAG3-GGNle-CycMSHhex, AcCG3-GGNle-CycMSHhex and HYNIC-GGNle-CycMSHhex peptides were synthesized and their melanocortin-1 (MC1) receptor binding affinities were determined in B16/F1 melanoma cells. The biodistribution of 99mTc-MAG3-GGNle-CycMSHhex, 99mTc-AcCG3-GGNle-CycMSHhex, 99mTc(CO)3-HYNIC-GGNle-CycMSHhex and 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice at 2 h post-injection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex were further examined because of its high melanoma uptake and fast urinary clearance. The IC50 values of MAG3-GGNle-CycMSHhex, AcCG3-GGNle-CycMSHhex and HYNIC-GGNle-CycMSHhex were 1.0 ± 0.05, 1.2 ± 0.19 and 0.6 ± 0.04 nM in B16/F1 melanoma cells, respectively. Among these four 99mTc-peptides, 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex exhibited the highest melanoma uptake (14.14 ± 4.90% ID/g) and fastest urinary clearance (91.26 ± 1.96% ID) at 2 h post-injection. 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex showed high tumor to normal organ uptake ratios except for the kidneys. The tumor/kidney uptake ratios of 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex were 2.50 and 3.55 at 4 and 24 h post-injection. The melanoma lesions were clearly visualized by SPECT/CT using 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex as an imaging probe at 2 h post-injection. Overall, high melanoma uptake coupled with fast urinary clearance of 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex highlighted its potential for metastatic melanoma detection in the future. PMID:23418722

  15. [Preliminary study on molecular mechanism of curcumine anti-mouse melanoma].

    PubMed

    Gui, Fei; Ma, Wei-Feng; Cai, Shao-Hui; Li, Xiao-Kun; Tan, Yi; Zhou, Chun-Ling; Chen, Hong-Yuan

    2008-11-01

    To investigate the effects of curcumine on mouse B16 melanoma growth and possible mechanism of Bcl-2, P53 and glutathione in tumor cells. The inhibitory effect on growth of melanoma in vivo were examined by mice melanoma models transplanted B16 cells to C57BL/6J mice. MTT method was used to assay the contribution of curcumine to B16 cells in vitro. The apoptosis and expression of Bcl-2, P53 gene of B16 cells were analyzed by flow cytometry, and HPLC assay was used to detect the change of GSH in B16 melanom tissues of C57BL/6J mouse caused by curcumine. Curcumine had obvious inhibitory effect on the growth of mouse B16 melanoma in time and dose dependent manner and the gene expression of bcl-2 in B16 cells decreased after 24 hours supplied with curcumine, whereas P53 protein expression increased; Curcumine depressed the GSH quantity in melanoma tissues. The growth inhibitory effect of curcumine on mouse melanom is proved in vivo and in vitro respectively. Curcumine can induce some cells to apoptosis which may be relevant to downregulation of bcl-2 expression and upregulation of P53 expression as well as exhaustion of GSH in tumor organization.

  16. Radiation induces an antitumour immune response to mouse melanoma.

    PubMed

    Perez, Carmen A; Fu, Allie; Onishko, Halina; Hallahan, Dennis E; Geng, Ling

    2009-12-01

    Irradiation of cancer cells can cause immunogenic death. We used mouse models to determine whether irradiation of melanoma can enhance the host antitumour immune response and function as an effective vaccination strategy, and investigated the molecular mechanisms involved in this radiation-induced response. For in vivo studies, C57BL6/J mice and the B16F0 melanoma cell line were used in a lung metastasis model, intratumoural host immune activation assays, and tumour growth delay studies. In vitro studies included a dendritic cell (DC) phagocytosis assay, detection of cell surface exposure of the protein calreticulin (CRT), and small interfering RNA (siRNA)-mediated depletion of CRT cellular levels. Irradiation of cutaneous melanomas prior to their resection resulted in more than 20-fold reduction in lung metastases after systemic challenge with untreated melanoma cells. A syngeneic vaccine derived from irradiated melanoma cells also induced adaptive immune response markers in irradiated melanoma implants. Our data indicate a trend for radiation-induced increase in melanoma cell surface exposure of CRT, which is involved in the enhanced phagocytic activity of DC against irradiated melanoma cells (VIACUC). The present study suggests that neoadjuvant irradiation of cutaneous melanoma tumours prior to surgical resection can stimulate an endogenous anti-melanoma host immune response.

  17. The NAMPT/E2F2/SIRT1 axis promotes proliferation and inhibits p53-dependent apoptosis in human melanoma cells.

    PubMed

    Zhao, Hailong; Tang, Weiwei; Chen, Xiaowen; Wang, Siyu; Wang, Xianyan; Xu, Haiyan; Li, Lijuan

    2017-11-04

    Melanoma is the most common primary malignant neoplasm in adults, causing more deaths than any other skin cancer, necessitating the development of new target-based approaches. Current evidence suggests SIRT1, the mammalian nicotinamide adenine dinucleotide (NAD + )-dependent protein deacetylase, and nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting NAD + biosynthetic enzyme, together comprise a novel systemic regulatory network to play a pivotal role in cell proliferation and apoptosis. Nevertheless, how the regulation of this cofactor interfaces with signal transduction network remains poorly understood in melanoma. Here, we report NAMPT is highly expressed in melanomaassociated with poor overall survival in patients. Pharmacological and genetic inhibition of NAMPT decreased NAD + levels and melanoma cell proliferation capacity, and NAMPT knockdown induced apoptosis through the activity of the tumor suppressor p53. Next, we demonstrate NAMPT regulates the transcription factor E2F family member 2 (E2F2) in the apoptosis process. Downstream, E2F2 control the mRNA and protein levels of SIRT1. Finally, we find NAMPT mediates the apoptosis resistance of melanoma cells through NAMPT-E2F2-SIRT1 axis, more than NAD + -driven transcriptional program. Accordingly, our results demonstrated that NAMPT is a prognostic marker in melanoma, and the identificationofNAMPT-E2F2-SIRT1 pathway establishes another link between NAMPT and apoptosis events in melanoma, with therapeutic implications for this deadly cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Protective CD8 Memory T Cell Responses to Mouse Melanoma Are Generated in the Absence of CD4 T Cell Help

    PubMed Central

    Steinberg, Shannon M.; Zhang, Peisheng; Turk, Mary Jo

    2011-01-01

    Background We have previously demonstrated that temporary depletion of CD4 T cells in mice with progressive B16 melanoma, followed by surgical tumor excision, induces protective memory CD8 T cell responses to melanoma/melanocyte antigens. We also showed that persistence of these CD8 T cells is supported, in an antigen-dependent fashion, by concurrent autoimmune melanocyte destruction. Herein we explore the requirement of CD4 T cell help in priming and maintaining this protective CD8 T cell response to melanoma. Methodology and Principal Findings To induce melanoma/melanocyte antigen-specific CD8 T cells, B16 tumor bearing mice were depleted of regulatory T cells (Treg) by either temporary, or long-term continuous treatment with anti-CD4 (mAb clone GK1.5). Total depletion of CD4 T cells led to significant priming of IFN-γ-producing CD8 T cell responses to TRP-2 and gp100. Surprisingly, treatment with anti-CD25 (mAb clone PC61), to specifically deplete Treg cells while leaving help intact, was ineffective at priming CD8 T cells. Thirty to sixty days after primary tumors were surgically excised, mice completely lacking CD4 T cell help developed autoimmune vitiligo, and maintained antigen-specific memory CD8 T cell responses that were highly effective at producing cytokines (IFN-γ, TNF-α, and IL-2). Mice lacking total CD4 T cell help also mounted protection against re-challenge with B16 melanoma sixty days after primary tumor excision. Conclusions and Significance This work establishes that CD4 T cell help is dispensable for the generation of protective memory T cell responses to melanoma. Our findings support further use of CD4 T cell depletion therapy for inducing long-lived immunity to cancer. PMID:22046294

  19. Enzyme and Cancer Cell Selectivity of Nanoparticles: Inhibition of 3D Metastatic Phenotype and Experimental Melanoma by Zinc Oxide.

    PubMed

    DeLong, Robert K; Mitchell, Jennifer A; Morris, R Tyler; Comer, Jeffrey; Hurst, Miranda N; Ghosh, Kartik; Wanekaya, Adam; Mudge, Miranda; Schaeffer, Ashley; Washington, Laurie L; Risor-Marhanka, Azure; Thomas, Spencer; Marroquin, Shanna; Lekey, Amber; Smith, Joshua J; Garrad, Richard; Aryal, Santosh; Abdelhakiem, Mohamed; Glaspell, Garry P

    2017-02-01

    Biomedical applications for metal and metal oxide nanoparticles are rapidly increasing. Here their functional impact on two well-characterized model enzymes, Luciferase (Luc) or β-galactosidase (β-Gal) was quantitatively compared. Nickel oxide nanoparticle (NiO-NP) activated β-Gal (>400% control) and boron carbide nanoparticle (B4C-NP) inhibited Luc(<10% control), whereas zinc oxide (ZnO-NP) and cobalt oxide (Co3O4-NP) activated β-Gal to a lesser extent and magnesium oxide (MgO) moderately inhibited both enzymes. Melanoma specific killing was in the order; ZnO > B4C ≥ Cu > MgO > Co3O4 > Fe2O3 > NiO, ZnO-NP inhibiting B16F10 and A375 cells as well as ERK enzyme (>90%) and several other cancer-associated kinases (AKT, CREB, p70S6K). ZnO-NP or nanobelt (NB) serve as photoluminescence (PL) cell labels and inhibit 3-D multi-cellular tumor spheroid (MCTS) growth and were tested in a mouse melanoma model. These results demonstrate nanoparticle and enzyme specific biochemical activity and suggest their utility as new tools to explore the important model metastatic foci 3-D environment and their chemotherapeutic potential.

  20. The histone deacetylase inhibitor butyrate inhibits melanoma cell invasion of Matrigel.

    PubMed

    Kuwajima, Akiko; Iwashita, Jun; Murata, Jun; Abe, Tatsuya

    2007-01-01

    Histone deacetylase (HDAC) inhibitors have anticancer effects. Their effects on expression of cell adhesion molecules might be related to their effects on tumor cell invasion. Murine B16-BL6 cells were treated with the HDAC inhibitors, butyrate or trichostatin A. Melanoma cell invasion of the artificial basement membrane, Matrigel, was examined by Transwell chamber assay. Butyrate as well as trichostatin A inhibited the cell growth mainly by arresting the cell cycle. The cell invasion of Matrigel was inhibited by butyrate and trichostatin A. The butyrate treatment increased the cell-cell aggregation, although neither E-cadherin nor N-cadherin mRNA were up-regulated. Both mRNA expression and protein levels of the immunoglobulin superfamily cell adhesion molecules, Mel-CAM and L1-CAM, were increased in the butyrate-treated cells. The HDAC inhibitor butyrate blocked the B16-BL6 melanoma cell invasion of Matrigel, although it increased the expression of Mel-CAM and L1-CAM which are important to the metastatic potential.

  1. Photoacoustic detection of induced melanoma in vitro using a mouse model

    NASA Astrophysics Data System (ADS)

    Gupta, Sagar; Bhattacharya, Kiran; Newton, Jessica R.; Quinn, Thomas P.; Viator, John A.

    2012-03-01

    Metastasis is a life threatening complex physiological phenomenon that involves the movement of cancer cells from one organ to another by means of blood and lymph. An understanding about metastasis is extremely important to device diagnostic systems to detect and monitor its spread within the body. For the first time we report rapid photoacoustic detection of the induced metastatic melanoma in mice in vitro using photoacoustic flowmetry. A new photoacoustic flow system is developed, that employs photoacoustic excitation coupled with an ultrasound transducer capable of determining the presence of individual, induced mouse melanoma cells (B16/F10) within the circulating system in vitro. Tumor was induced in mice by injecting mouse melanoma cells through tail vein into the C57BL/6 mice. A luciferase based in vivo bioluminescence imaging is performed to confirm the tumor load and multiple metastases in the tumor-induced mice. 1ml of blood obtained through cardiac puncture of the induced metastasized mice was treated to lyse the red blood cells (RBC) and enriched, leaving the induced melanoma in the peripheral blood mononuclear suspension (PBMC). A photoacoustic flowsystem coupled with an ultrasound transducer is used to detect the individual circulating metastatic melanoma cells from the enriched cell suspension.

  2. Direct Melanoma Cell Contact Induces Stromal Cell Autocrine Prostaglandin E2-EP4 Receptor Signaling That Drives Tumor Growth, Angiogenesis, and Metastasis.

    PubMed

    Inada, Masaki; Takita, Morichika; Yokoyama, Satoshi; Watanabe, Kenta; Tominari, Tsukasa; Matsumoto, Chiho; Hirata, Michiko; Maru, Yoshiro; Maruyama, Takayuki; Sugimoto, Yukihiko; Narumiya, Shuh; Uematsu, Satoshi; Akira, Shizuo; Murphy, Gillian; Nagase, Hideaki; Miyaura, Chisato

    2015-12-11

    The stromal cells associated with tumors such as melanoma are significant determinants of tumor growth and metastasis. Using membrane-bound prostaglandin E synthase 1 (mPges1(-/-)) mice, we show that prostaglandin E2 (PGE2) production by host tissues is critical for B16 melanoma growth, angiogenesis, and metastasis to both bone and soft tissues. Concomitant studies in vitro showed that PGE2 production by fibroblasts is regulated by direct interaction with B16 cells. Autocrine activity of PGE2 further regulates the production of angiogenic factors by fibroblasts, which are key to the vascularization of both primary and metastatic tumor growth. Similarly, cell-cell interactions between B16 cells and host osteoblasts modulate mPGES-1 activity and PGE2 production by the osteoblasts. PGE2, in turn, acts to stimulate receptor activator of NF-κB ligand expression, leading to osteoclast differentiation and bone erosion. Using eicosanoid receptor antagonists, we show that PGE2 acts on osteoblasts and fibroblasts in the tumor microenvironment through the EP4 receptor. Metastatic tumor growth and vascularization in soft tissues was abrogated by an EP4 receptor antagonist. EP4-null Ptger4(-/-) mice do not support B16 melanoma growth. In vitro, an EP4 receptor antagonist modulated PGE2 effects on fibroblast production of angiogenic factors. Our data show that B16 melanoma cells directly influence host stromal cells to generate PGE2 signals governing neoangiogenesis and metastatic growth in bone via osteoclast erosive activity as well as angiogenesis in soft tissue tumors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. microRNA-216b inhibits cell proliferation and migration in human melanoma by targeting FOXM1 in vitro and in vivo.

    PubMed

    Sun, Mengyao; Wang, Xiaopeng; Tu, Chen; Wang, Shuang; Qu, Jianqiang; Xiao, Shengxiang

    2017-12-01

    MicroRNAs (miRNAs) play an increasingly important role in cancer growth by coordinately suppressing genes that control cell migration, proliferation, and invasion. The above results can be achieved through the regulation of gene expression by miRNAs by suppressing translation or the direct sequence-specific degradation of the targeted mRNA. In the present study, we indicate that the expression of miR-216b could be effectively repressed both in human melanoma tissues through a comparison with primary melanoma and in human melanoma cell lines through a comparison with a normal human keratinocyte line. Moreover, miR-216b induced a clear decrease in melanoma cell proliferation and migration in vitro. Forkhead box M1 (FOXM1) was confirmed as a target gene of miR-216b, and the overexpression of miR-216b markedly repressed the luciferase activity of reporter plasmids containing the FOXM1 3'-UTR (untranslated region). Furthermore, miR-216b suppressed melanoma cell growth in nude mice in vivo, with the effects of miR-216b overexpression on melanoma cell growth and proliferation reversed by FOXM1 overexpression. The results demonstrated that miR-216b is a tumor suppressor in melanoma, identified the FOXM1 signaling pathway as a target of miR-216b action, and suggested a potential therapeutic role for miR-216b in melanoma. © 2017 International Federation for Cell Biology.

  4. Oncogenic B-Raf(V600E) abrogates the AKT/B-Raf/Mps1 interaction in melanoma cells.

    PubMed

    Zhang, Ling; Shi, Ruyi; He, Chanting; Cheng, Caixia; Song, Bin; Cui, Heyang; Zhang, Yanyan; Zhao, Zhiping; Bi, Yanghui; Yang, Xiaofeng; Miao, Xiaoping; Guo, Jiansheng; Chen, Xing; Wang, Jinfen; Li, Yaoping; Cheng, Xiaolong; Liu, Jing; Cui, Yongping

    2013-08-28

    Activating B-Raf mutations that deregulate the mitogen-activated protein kinase (MAPK) pathway commonly occur in cancer. Although B-Raf(V600E) induces increased Mps1 protein contributing to centrosome amplification and chromosome instability, the regulatory mechanisms of Mps1 in melanoma cells is not fully understood. Here, we report that Mps1/AKT and B-Raf(WT)/ERK signaling form an auto-regulatory negative feedback loop in melanoma cells; notably, oncogenic B-Raf(V600E) abrogates the negative feedback loop, contributing the aberrant Mps1 functions and tumorigenesis. Our findings raise the possibility that targeting the oncogenic B-Raf and Mps1, especially when used in combination could potentially provide great therapeutic opportunities for cancer treatment. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  5. Bioconjugation of laminin peptide YIGSR with poly(styrene co-maleic acid) increases its antimetastatic effect on lung metastasis of B16-BL6 melanoma cells.

    PubMed

    Mu, Y; Kamada, H; Kaneda, Y; Yamamoto, Y; Kodaira, H; Tsunoda, S; Tsutsumi, Y; Maeda, M; Kawasaki, K; Nomizu, M; Yamada, Y; Mayumi, T

    1999-02-05

    A comb-shaped polymeric modifier, SMA [poly(styrene comaleic anhydride)], which binds to plasma albumin in blood was used to modify the synthetic cell-adhesive laminin peptide YIGSR, and its inhibitory effect on experimental lung metastasis of B16-BL6 melanoma cells was examined. YIGSR was chemically conjugated with SMA via formation of an amide bond between the N-terminal amino group of YIGSR and the carboxyl anhydride of SMA. The antimetastatic effect of SMA-conjugated YIGSR was approximately 50-fold greater than that of native YIGSR. When injected intravenously, SMA-YIGSR showed a 10-fold longer plasma half-life than native YIGSR in vivo. In addition, SMA-YIGSR had the same binding affinity to plasma albumin as SMA, while native YIGSR did not bind to albumin. These findings suggested that the enhanced antimetastatic effect of SMA-YIGSR may be due to its prolonged plasma half-life by binding to plasma albumin, and that bioconjugation of in vivo unstable peptides with SMA may facilitate their therapeutic use. Copyright 1999 Academic Press.

  6. Role of β-catenin signaling in the anti-invasive effect of the omega-3 fatty acid DHA in human melanoma cells.

    PubMed

    Serini, Simona; Zinzi, Antonio; Ottes Vasconcelos, Renata; Fasano, Elena; Riillo, Maria Greca; Celleno, Leonardo; Trombino, Sonia; Cassano, Roberta; Calviello, Gabriella

    2016-11-01

    We previously found that docosahexaenoic acid (DHA), a dietary polyunsaturated fatty acid present at high level in fatty fish, inhibited cell growth and induced differentiation of melanoma cells in vitro by increasing nuclear β-catenin content. An anti-neoplastic role of nuclear β-catenin was suggested in melanoma, and related to the presence in the melanocyte lineage of the microphtalmia transcription factor (MITF), which interferes with the transcription of β-catenin/TCF/LEF pro-invasive target genes. In the present work we investigated if DHA could inhibit the invasive potential of melanoma cells, and if this effect could be related to DHA-induced alterations of the Wnt/β-catenin signaling, including changes in MITF expression. WM115 and WM266-4 human melanoma, and B16-F10 murine melanoma cell lines were used. Cell invasion was evaluated by Wound Healing and Matrigel transwell assays. Protein expression was analyzed by Western Blotting and β-catenin phosphorylation by immunoprecipitation. The role of MITF in the anti-invasive effect of DHA was analyzed by siRNA gene silencing. We found that DHA inhibited anchorage-independent cell growth, reduced their migration/invasion in vitro and down-regulated several Matrix Metalloproteinases (MMP: MMP-2, MT1-MMP and MMP-13), known to be involved in melanoma invasion. We related these effects to the β-catenin increased nuclear expression and PKA-dependent phosphorylation, as well as to the increased expression of MITF. The data obtained further support the potential role of dietary DHA as suppressor of melanoma progression to invasive malignancy through its ability to enhance MITF expression and PKA-dependent nuclear β-catenin phosphorylation. Copyright © 2016. Published by Elsevier Ireland Ltd.

  7. Phorbol 12-myristate 13-acetate enhances nm23 gene expression in murine melanocytes but not in syngeneic B16-BL6 melanoma variants.

    PubMed

    Huijzer, J C; McFarland, M; Niles, R M; Meadows, G G

    1996-03-01

    The nm23 gene has been described as a potential metastasis suppressor gene in certain rodent and human tumors. We previously demonstrated that tyrosine and phenylalanine restriction suppresses metastatic heterogeneity of B16-BL6 murine melanoma and selects for tumor variants with decreased metastatic potential. In this study, we investigated nm23 expression in the highly metastatic B16-BL6 (ND) melanoma, its nutritionally derived poorly metastatic (LT) variant, and the syngeneic non-tumorigenic Mel-ab melanocytes. No differences in nm23 expression were observed between ND and LT cells, and nm23 expression varied between different isolates. Previously, we showed that metastatic potential of 1-ND cells decreases and is not altered in 1-LT cells after prolonged in vitro cell passage; however, nm23 expression is equivalently increased by 2-fold. In 2-ND and 2-LT cells, expression of nm23 is not different at higher in vitro cell passage. Expression of nm23 decreased about 2-fold when phorbol 12-myristate 13-acetate (PMA) was removed from Mel-ab cells, which induces these cells to become quiescent. Although membrane-associated protein kinase C (PKC) activity decreased after prolonged PMA treatment in all cells, neither nm23 expression nor proliferation of ND and LT cells was affected by PMA. These data indicate that nm23 expression is related to proliferative activity rather than to the suppression of metastatic potential.

  8. In vivo, label-free, and noninvasive detection of melanoma metastasis by photoacoustic flow cytometry

    NASA Astrophysics Data System (ADS)

    Liu, Rongrong; Wang, Cheng; Hu, Cheng; Wang, Xueding; Wei, Xunbin

    2014-02-01

    Melanoma, a malignant tumor of melanocytes, is the most serious type of skin cancer in the world. It accounts for about 80% of deaths of all skin cancer. For cancer detection, circulating tumor cells (CTCs) serve as a marker for metastasis development, cancer recurrence, and therapeutic efficacy. Melanoma tumor cells have high content of melanin, which has high light absorption and can serve as endogenous biomarker for CTC detection without labeling. Here, we have developed an in vivo photoacoustic flow cytometry (PAFC) to monitor the metastatic process of melanoma cancer by counting CTCs of melanoma tumor bearing mice in vivo. To test in vivo PAFC's capability of detecting melanoma cancer, we have constructed a melanoma tumor model by subcutaneous inoculation of highly metastatic murine melanoma cancer cells, B16F10. In order to effectively distinguish the targeting PA signals from background noise, we have used the algorithm of Wavelet denoising method to reduce the background noise. The in vivo flow cytometry (IVFC) has shown a great potential for detecting circulating tumor cells quantitatively in the blood stream. Compared with fluorescence-based in vivo flow cytometry (IVFC), PAFC technique can be used for in vivo, label-free, and noninvasive detection of circulating tumor cells (CTCs).

  9. In vivo immunologic selection of class I major histocompatibility complex gene deletion variants from the B16-BL6 melanoma.

    PubMed

    Talmadge, J E; Talmadge, C B; Zbar, B; McEwen, R; Meeker, A K; Tribble, H

    1987-06-01

    The mechanism by which tumor allografts escape host immunologic attack was investigated. B16-BL6 cells (the bladder 6 subline of the B16 melanoma) (H-2b) were transfected with a gene (Dd) encoding an allogeneic class I major histocompatibility complex antigen. Clones that expressed Dd antigen were injected into the footpads of nonimmune syngeneic mice, syngeneic immune mice, and nude mice. Under conditions of immunologic selection a clone that contained multiple copies of the transfected gene formed variants that lacked the transfected gene. Primary tumors and pulmonary metastases of immunized mice and pulmonary metastases of nonimmunized mice had lost the Dd gene and, in most cases, all of the associated plasmid. In contrast, in immunodeficient nude mice, primary tumors and pulmonary metastases retained the Dd gene and the associated plasmid. Deletion of genes encoding cell surface antigens may be one of the mechanisms by which allogeneic tumors escape immunologic attack.

  10. Synthesis and characterization of a novel radioiodinated phenylacetamide and its homolog as theranostic agents for malignant melanoma.

    PubMed

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

    2016-01-01

    Melanin is an attractive target for the diagnosis and treatment of malignant melanoma. This study reports the preparation and biological characterizations of N-(2-(diethylamino)ethyl)-2-(3-(123/131)I-iodo-4- hydroxyphenyl)acetamide and N-(2-(diethylamino)ethyl)-3-(3-(123/131)I-iodo-4-hydroxyphenyl)propanamide (123/131)I-IHPA and 123/131I-IHPP) as novel melanin-specific theranostic agents. These two tracers were hydrophilic, exhibited good serum stability and high binding affinity to melanin. In vitro and in vivo studies revealed rapid, high and tenacious uptakes of both 131I-IHPA and 131I-IHPP in melanotic B16F0 cell line and in C57BL/6 mice bearing B16F0 melanoma, but not in amelanonic A375 cell line and tumors. Small-animal SPECT imaging also clearly delineate B16F0 melanoma since 1 h postinjection of 123I-IHPA and 123I-IHPP in tumor-bearing mice. Owing to the favorable biodistribution of 131I-IHPA and 131I-IHPP after intravenous administration, the estimated absorption dose was low in most normal organs and relatively high in melanotic tumor. The melanin-specific binding ability, sustained tumor retention, fast normal tissues clearance and acceptable projected human dosimetry supported that these two tracers are promising theranostic agents for melanin-positive melanoma.

  11. Photodynamic therapy mediated by acai oil (Euterpe oleracea Martius) in nanoemulsion: A potential treatment for melanoma.

    PubMed

    Monge-Fuentes, Victoria; Muehlmann, Luis Alexandre; Longo, João Paulo Figueiró; Silva, Jaqueline Rodrigues; Fascineli, Maria Luiza; de Souza, Paulo; Faria, Fernando; Degterev, Igor Anatolievich; Rodriguez, Anselmo; Carneiro, Fabiana Pirani; Lucci, Carolina Madeira; Escobar, Patricia; Amorim, Rivadávio Fernandes Batista; Azevedo, Ricardo Bentes

    2017-01-01

    Melanoma is the most aggressive and lethal form of skin cancer, responsible for >80% of deaths. Standard treatments for late-stage melanoma usually present poor results, leading to life-threatening side effects and low overall survival. Thus, it is necessary to rethink treatment strategies and design new tools for the treatment of this disease. On that ground, we hereby report the use of acai oil in nanoemulsion (NanoA) as a novel photosensitizer for photodynamic therapy (PDT) used to treat melanoma in in vitro and in vivo experimental models. NIH/3T3 normal cells and B16F10 melanoma cell lines were treated with PDT and presented 85% cell death for melanoma cells, while maintaining high viability in normal cells. Flow cytometry indicated that cell death occurred by late apoptosis/necrosis. Tumor bearing C57BL/6 mice treated five times with PDT using acai oil in nanoemulsion showed tumor volume reduction of 82% in comparison to control/tumor group. Necrotic tissue per tumor area reached its highest value in PDT-treated mice, supporting PDT efficacy. Overall, acai oil in nanoemulsion was an effective photosensitizer, representing a promising source of new photosensitizing molecules for PDT treatment of melanoma, a tumor with an inherent tendency to be refractory for this type of therapy. Copyright © 2016. Published by Elsevier B.V.

  12. Folate-conjugated immunoglobulin targets melanoma tumor cells for NK cell effector functions

    PubMed Central

    Skinner, Cassandra C.; McMichael, Elizabeth L.; Jaime-Ramirez, Alena C.; Abrams, Zachary B.; Lee, Robert J.; Carson, William E.

    2016-01-01

    The folate receptor (FR) is over-expressed on the vascular side of cancerous cells including those of the breast, ovaries, testes, and cervix. We hypothesized that a folate-conjugated immunoglobulin (F-IgG) would bind to the FR that is over-expressed on melanoma tumor cells to target these cells for lysis by natural killer (NK) cells. Folate receptor expression was confirmed in the Mel-39 (human melanoma) cell line by flow cytometry and immunoblot analysis, using KB (human oral epithelial) and F01 (human melanoma) as a positive and negative control, respectively. FR-positive and negative cell lines were treated with F-IgG or control immunoglobulin G (C-IgG) in the presence or absence of cytokines in order to determine NK cell ability to lyse FR-positive cell lines. NK cell activation was significantly upregulated and lysis of Mel 39 tumor cells enhanced following treatment with F-IgG, as compared to C-IgG at all effector:target (E:T) ratios (p<0.01). This trend was further enhanced by NK cell stimulation with the activating cytokine interleukin-12 (IL-12). NK cell production of cytokines such as interferon-gamma (IFN-γ), macrophage inflammatory protein 1 alpha (MIP-1α), and regulated on activation normal T-cell expressed and secreted (RANTES) were also significantly increased in response to co-stimulation with IL-12 stimulation and F-IgG-coated Mel 39 target cells, as compared to controls (p<0.01). In contrast, F-IgG did not bind to the FR-negative cell line F01 and had no significant effect on NK cell lysis or cytokine production. This research indicates the potential use of F-IgG for its ability to induce an immune response from NK cells against FR-positive melanoma tumor cells which can be further enhanced by the addition of cytokines. PMID:27035691

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

    PubMed Central

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

    2016-01-01

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

  14. Quantifying spontaneous metastasis in a syngeneic mouse melanoma model using real time PCR.

    PubMed

    Deng, Wentao; McLaughlin, Sarah L; Klinke, David J

    2017-08-07

    Modeling metastasis in vivo with animals is a priority for both revealing mechanisms of tumor dissemination and developing therapeutic methods. While conventional intravenous injection of tumor cells provides an efficient and consistent system for studying tumor cell extravasation and colonization, studying spontaneous metastasis derived from orthotopic tumor sites has the advantage of modeling more aspects of the metastatic cascade, but is challenging as it is difficult to detect small numbers of metastatic cells. In this work, we developed an approach for quantifying spontaneous metastasis in the syngeneic mouse B16 system using real time PCR. We first transduced B16 cells with lentivirus expressing firefly luciferase Luc2 gene for bioluminescence imaging. Next, we developed a real time quantitative PCR (qPCR) method for the detection of luciferase-expressing, metastatic tumor cells in mouse lungs and other organs. To illustrate the approach, we quantified lung metastasis in both spontaneous and experimental scenarios using B16F0 and B16F10 cells in C57BL/6Ncrl and NOD-Scid Gamma (NSG) mice. We tracked B16 melanoma metastasis with both bioluminescence imaging and qPCR, which were found to be self-consistent. Using this assay, we can quantitatively detect one Luc2 positive tumor cell out of 10 4 tissue cells, which corresponds to a metastatic burden of 1.8 × 10 4 metastatic cells per whole mouse lung. More importantly, the qPCR method was at least a factor of 10 more sensitive in detecting metastatic cell dissemination and should be combined with bioluminescence imaging as a high-resolution, end-point method for final metastatic cell quantitation. Given the rapid growth of primary tumors in many mouse models, assays with improved sensitivity can provide better insight into biological mechanisms that underpin tumor metastasis.

  15. The effects of different antineoplastic agents and of pretreatment by modulators on three melanoma lines.

    PubMed

    Rodriguez-Vicente, J; Vicente-Ortega, V; Canteras-Jordana, M

    1998-02-01

    The chemotherapy of melanoma patients must be improved because of the naturally poor response and acquired resistance of this disease. The authors used mouse (B16F10) and human (SK-MEL-28 and SK-MEL-1) melanoma lines for in vitro treatment with melphalan, lomustine, fotemustine, and 4-hydroxyanisole (4-HA) alone, combined and after pretreatment with buthionine sulfoximine (BSO), ethacrynic acid (EA), and azelaic acid (AZA). Melphalan was the most effective individual drug, followed by lomustine, fotemustine, and 4-HA. The simultaneous administration of two agents was disappointing, although some combinations slightly improved the response compared with the individual treatments. Pretreatment with BSO enhanced the cytotoxicity of melphalan and lomustine 10-fold in B16F10 and 7.5-fold in SK-MEL-28, increasing the toxicity of fotemustine in all 3 lines. EA potentiated lomustine and fotemustine 9-fold and melphalan 5-fold in B16F10 and SK-MEL-28. AZA increased the effectiveness of lomustine and fotemustine in B16F10 and to a lower degree in the two human lines. 4-HA was the poorest drug for sensitization; only B16F10 BSO followed by 4-HA treatment demonstrated increased toxicity, and all other combinations with 4-HA were negative or antagonistic. There was a strong relationship between dopa oxidase activity and the toxicity of 4-HA. B16F10 was the most sensitive to all treatments and SK-MEL-1 the most resistant. Melphalan was the most active individual drug and 4-HA the least. Combinations of two drugs did not result in improved activity compared with drugs administered alone. Pretreatment with modulator seems to be a potential method for enhancing some treatments.

  16. Therapeutic Efficacy of a {sup 188}Re-Labeled {alpha}-Melanocyte-Stimulating Hormone Peptide Analog in Murine and Human Melanoma-Bearing Mouse Models

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Miao, Yubin; Owen, Nellie K.; Fisher, Darrell R.

    2005-01-01

    The purpose of this study was to examine the therapeutic efficacy of {sup 188}Re-(Arg{sup 11})CCMSH in the B16/F1 murine melanoma and TXM13 human melanoma bearing mouse models. Method: (Arg11)CCMSH was synthesized and labeled with {sup 188}Re to form {sup 188}Re-(Agr{sup 11})CCMSH. B16/F1 melanoma tumor bearing mice were administrated with 200 Ci, 600 Ci and 2x400 Ci of {sup 188}Re-(Arg{sup 11})CCMSH via the tail vein, respectively. TXM13 melanoma tumor hearing mice were separately injected with 600 Ci, 2x400 Ci and 1000 Ci of 100Re-(Arg{sup 11})CCMSH through the tail vein. Two groups of 10 mice bearing either B16/F1 or TXM13 tumors weremore » injected with saline as untreated controls. Results: In contrast to the untreated control group, {sup 188}Re(Arg11)CCMSH yielded rapid and lasting therapeutic effects in the treatment groups with either B16/F1 or TXM13 tumors. The tumor growth rate was reduced and the survival rate was prolonged in the treatment groups. Treatment with 2x400 Ci of {sup 188}Re-Arg{sup 11}CCMSH significantly extended the mean life of B16/F1 tumor mice (p<0.05), while the mean life of TXm13 tumor mice was significantly prolonged after treatment with 600 Ci and 1000 Ci doses of {sup 188}Re-(Arg{sup 11})CCMSH (p<0.05 High-dose {sup 188}Re-(Arg{sup 11}))CCMSH produced no observed normal-tissue toxicity. Conclusions: The therapy study results revealed that {sup 188}Re-Arg11 CCMSH yielded significant therapeutic effects in both B16/F1 murine melanoma and TXM13 human melanoma bearing mouse models. {sup 188}Re-(Arg{sup 11})CCMSH appears to be a promising radiolabeled peptide for targeted radionuclide therapy of melanoma.« less

  17. Contribution of TMEM16F to pyroptotic cell death.

    PubMed

    Ousingsawat, Jiraporn; Wanitchakool, Podchanart; Schreiber, Rainer; Kunzelmann, Karl

    2018-02-20

    Pyroptosis is a highly inflammatory form of programmed cell death that is caused by infection with intracellular pathogens and activation of canonical or noncanonical inflammasomes. The purinergic receptor P2X 7 is activated by the noncanonical inflammasome and contributes essentially to pyroptotic cell death. The Ca 2+ activated phospholipid scramblase and ion channel TMEM16F has been shown earlier to control cellular effects downstream of purinergic P2X 7 receptors that ultimately lead to cell death. As pyroptotic cell death is accompanied by an increases in intracellular Ca 2+ , we asked whether TMEM16F is activated during pyroptosis. The N-terminal cleavage product of gasdermin D (GD-N) is an executioner of pyroptosis by forming large plasma membrane pores. Expression of GD-N enhanced basal Ca 2+ levels and induced cell death. We observed that GD-N induced cell death in HEK293 and HAP1 cells, which was depending on expression of endogenous TMEM16F. GD-N activated large whole cell currents that were suppressed by knockdown or inhibition of TMEM16F. The results suggest that whole cell currents induced by the pore forming domain of gasdermin-D, are at least in part due to activation of TMEM16F. Knockdown of other TMEM16 paralogues expressed in HAP1 cells suggest TMEM16F as a crucial element during pyroptosis and excluded a role of other TMEM16 proteins. Thus TMEM16F supports pyroptosis and other forms of inflammatory cell death such as ferroptosis. Its potent inhibition by tannic acid may be part of the anti-inflammatory effects of flavonoids.

  18. Endothelial cell differentiation into capillary-like structures in response to tumour cell conditioned medium: a modified chemotaxis chamber assay.

    PubMed

    Garrido, T; Riese, H H; Aracil, M; Pérez-Aranda, A

    1995-04-01

    We have developed a modified chemotaxis chamber assay in which bovine aortic endothelial (BAE) cells degrade Matrigel basement membrane and migrate and form capillary-like structures on type I collagen. This capillary formation occurs in the presence of conditioned media from highly metastatic tumour cell lines, such as B16F10 murine melanoma or MDA-MD-231 human breast adenocarcinoma, but not in the presence of conditioned medium (CM) from the less invasive B16F0 cell line. Replacement of tumour cell CM by 10 ng ml-1 basic fibroblast growth factor (bFGF) also results in capillary-like structure formation by BAE cells. An anti-bFGF antibody blocks this effect, showing that bFGF is one of the factors responsible for the angiogenic response induced by B16F10 CM in our assay. Addition of an anti-laminin antibody reduces significantly the formation of capillary-like structures, probably by blocking the attachment of BAE cells to laminin present in Matrigel. The anti-angiogenic compound suramin inhibits in a dose-dependent manner (complete inhibition with 100 microM suramin) the migration and differentiation of BAE cells on type I collagen in response to B16F10 CM. This assay represents a new model system to study tumour-induced angiogenesis in vitro.

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

    PubMed

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

    1998-03-01

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

  20. B-Raf inhibitor vemurafenib in combination with temozolomide and fotemustine in the killing response of malignant melanoma cells.

    PubMed

    Roos, Wynand P; Quiros, Steve; Krumm, Andrea; Merz, Stephanie; Switzeny, Olivier Jérôme; Christmann, Markus; Loquai, Carmen; Kaina, Bernd

    2014-12-30

    In the treatment of metastatic melanoma, a highly therapy-refractory cancer, alkylating agents are used and, for the subgroup of BRAFV600E cancers, the B-Raf inhibitor vemurafenib. Although vemurafenib is initially beneficial, development of drug resistance occurs leading to tumor relapse, which necessitates the requirement for combined or sequential therapy with other drugs, including genotoxic alkylating agents. This leads to the question whether vemurafenib and alkylating agents act synergistically and whether chronic vemurafenib treatment alters the melanoma cell response to alkylating agents. Here we show that a) BRAFV600E melanoma cells are killed by vemurafenib, driving apoptosis, b) BRAFV600E melanoma cells are neither more resistant nor sensitive to temozolomide/fotemustine than non-mutant cells, c) combined treatment with vemurafenib plus temozolomide or fotemustine has an additive effect on cell kill, d) acquired vemurafenib resistance of BRAFV600E melanoma cells does not affect MGMT, MSH2, MSH6, PMS2 and MLH1, nor does it affect the resistance to temozolomide and fotemustine, e) metastatic melanoma biopsies obtained from patients prior to and after vemurafenib treatment did not show a change in the MGMT promoter methylation status and MGMT expression level. The data suggest that consecutive treatment with vemurafenib and alkylating drugs is a reasonable strategy for metastatic melanoma treatment.

  1. The Cytolytic Amphipathic β(2,2)-Amino Acid LTX-401 Induces DAMP Release in Melanoma Cells and Causes Complete Regression of B16 Melanoma.

    PubMed

    Eike, Liv-Marie; Mauseth, Brynjar; Camilio, Ketil André; Rekdal, Øystein; Sveinbjørnsson, Baldur

    2016-01-01

    In the present study we examined the ability of the amino acid derivative LTX-401 to induce cell death in cancer cell lines, as well as the capacity to induce regression in a murine melanoma model. Mode of action studies in vitro revealed lytic cell death and release of danger-associated molecular pattern molecules, preceded by massive cytoplasmic vacuolization and compromised lysosomes in treated cells. The use of a murine melanoma model demonstrated that the majority of animals treated with intratumoural injections of LTX-401 showed complete and long-lasting remission. Taken together, these results demonstrate the potential of LTX-401 as an immunotherapeutic agent for the treatment of solid tumors.

  2. Intermittent hypoxia promotes melanoma lung metastasis via oxidative stress and inflammation responses in a mouse model of obstructive sleep apnea.

    PubMed

    Li, Lian; Ren, Fangyuan; Qi, Chao; Xu, Leiqian; Fang, Yinshan; Liang, Maoli; Feng, Jing; Chen, Baoyuan; Ning, Wen; Cao, Jie

    2018-02-12

    Recently, increased tumor incidence and cancer-related mortality have been reported among patients with obstructive sleep apnea (OSA). Intermittent hypoxia (IH), the hallmark feature of OSA, contributes to the metastasis of tumors. However, the molecular mechanisms by which tumor metastasis is accelerated by OSA-like IH remain to be elucidated. C57BL/6 J male mice were subjected to intravenous injection of B16F10 melanoma cells before receiving IH treatment. Then, the animals were randomly distributed into three groups (n = 8 each): normoxia (N) group, IH group, and antioxidant tempol group (IHT, exposed to IH after treatment with tempol). After the mice were sacrificed, the number and weight of lung metastatic colonies were assessed. The lung tissues with tumor metastasis were analyzed for markers of oxidative stress and inflammation and for HIF-1α using western blotting and real-time PCR (qRT-PCR). The level of reactive oxygen species (ROS) in B16F10 cell was also assessed after N, IH and IH with tempol treatments. Compared with normoxia, IH significantly increased the number and weight of mouse lung metastatic colonies. Treatment of B16F10 cells with IH significantly enhanced ROS generation. Lung tissues with tumor metastasis provided evidence of increased oxidative stress, as assessed by p22 phox and SOD mRNA levels and the NRF2 protein level, as well as increased inflammation, as assessed by TNF-α and IL-6 mRNA levels and the NF-κB P65 protein level. HIF-1α protein levels were increased in response to IH treatment. Tempol, an important antioxidant, ameliorated IH-induced melanoma lung metastasis in mice and reduced oxidative stress and inflammation responses. These results support the hypothesis that oxidative stress and inflammation responses play an important role in the pathogenesis of OSA-like IH-induced melanoma lung metastasis in mice. Antioxidant intervention provides a novel strategy for the prevention and treatment of cancer in OSA

  3. Increased projection of MHC and tumor antigens in murine B16-BL6 melanoma induced by hydrostatic pressure and chemical crosslinking.

    PubMed

    Ramakrishna, V; Eisenthal, A; Skornick, Y; Shinitzky, M

    1993-05-01

    The B16-BL6 melanoma, like most spontaneously arising tumors, is poorly immunogenic and expresses low levels of major histocompatibility complex (MHC) antigens. Treatment of cells of this tumor in vitro by hydrostatic pressure in the presence of adenosine 2',3'-dialdehyde (oxAdo), a membrane-impermeant crosslinker, caused elevated projection of MHC and a specific tumor antigen as demonstrated by flow-cytometric analysis. Maximum projection of both the MHC and the tumor antigens could be reached by application of 1200 atm for 15 min in the presence of 20 mM oxAdo. It is not yet clear whether this passive increase in availability of antigens on the cell surface originated from a dormant pool of antigens in the plasma membrane or from pressure-induced fusion of antigen-rich intracellular organelles (e.g. the endoplasmic reticulum). The immunogenic properties of the antigen-enriched B16-BL6 cells are described in the following paper.

  4. Regulatory T cell frequency in patients with melanoma with different disease stage and course, and modulating effects of high-dose interferon-alpha 2b treatment.

    PubMed

    Ascierto, Paolo A; Napolitano, Maria; Celentano, Egidio; Simeone, Ester; Gentilcore, Giusy; Daponte, Antonio; Capone, Mariaelena; Caracò, Corrado; Calemma, Rosa; Beneduce, Gerardo; Cerrone, Margherita; De Rosa, Vincenzo; Palmieri, Giuseppe; Castello, Giuseppe; Kirkwood, John M; Marincola, Francesco M; Mozzillo, Nicola

    2010-08-16

    High-dose interferon-alpha 2b (IFN-alpha 2b) is the only approved systemic therapy in the United States for the adjuvant treatment of melanoma. The study objective was to explore the immunomodulatory mechanism of action for IFN-alpha 2b by measuring serum regulatory T cell (Treg), serum transforming growth factor-beta (TGF-beta), interleukin (IL)-10, and autoantibody levels in patients with melanoma treated with the induction phase of the high-dose IFN-alpha 2b regimen. Patients with melanoma received IFN-alpha 2b administered intravenously (20 MU/m2 each day from day 1 to day 5 for 4 consecutive weeks). Serum Treg levels were measured as whole lymphocytes in CD4+ cells using flow cytometry while TGF-beta, IL-10, and autoantibody levels were measured using enzyme-linked immunosorbent assays. Twenty-two patients with melanoma received IFN-alpha 2b treatment and were evaluated for Treg levels. Before treatment, Treg levels were significantly higher in patients with melanoma when compared with data from 20 healthy subjects (P = 0.001; Mann-Whitney test). Although a trend for reduction of Treg levels following IFN-α 2b treatment was observed (average decrease 0.29% per week), statistical significance was not achieved. Subgroup analyses indicated higher baseline Treg levels for stage III versus IV disease (P = 0.082), early recurrence versus no recurrence (P = 0.017), deceased versus surviving patients (P = 0.021), and preoperative neoadjuvant versus postoperative adjuvant treatment groups (not significant). No significant effects were observed on the levels of TGF-beta, IL-10, and autoantibodies in patients with melanoma treated with IFN-alpha 2b. Patients with melanoma in this study showed increased basal levels of Treg that may be relevant to their disease and its progression. Treg levels shifted in patients with melanoma treated with IFN-alpha 2b, although no firm conclusions regarding the role of Tregs as a marker of treatment response or outcome can be made at

  5. The Cytolytic Amphipathic β(2,2)-Amino Acid LTX-401 Induces DAMP Release in Melanoma Cells and Causes Complete Regression of B16 Melanoma

    PubMed Central

    Eike, Liv-Marie; Mauseth, Brynjar; Camilio, Ketil André; Rekdal, Øystein; Sveinbjørnsson, Baldur

    2016-01-01

    In the present study we examined the ability of the amino acid derivative LTX-401 to induce cell death in cancer cell lines, as well as the capacity to induce regression in a murine melanoma model. Mode of action studies in vitro revealed lytic cell death and release of danger-associated molecular pattern molecules, preceded by massive cytoplasmic vacuolization and compromised lysosomes in treated cells. The use of a murine melanoma model demonstrated that the majority of animals treated with intratumoural injections of LTX-401 showed complete and long-lasting remission. Taken together, these results demonstrate the potential of LTX-401 as an immunotherapeutic agent for the treatment of solid tumors. PMID:26881822

  6. B-Raf inhibitor vemurafenib in combination with temozolomide and fotemustine in the killing response of malignant melanoma cells

    PubMed Central

    Krumm, Andrea; Merz, Stephanie; Switzeny, Olivier Jérôme; Christmann, Markus; Loquai, Carmen; Kaina, Bernd

    2014-01-01

    In the treatment of metastatic melanoma, a highly therapy-refractory cancer, alkylating agents are used and, for the subgroup of BRAFV600E cancers, the B-Raf inhibitor vemurafenib. Although vemurafenib is initially beneficial, development of drug resistance occurs leading to tumor relapse, which necessitates the requirement for combined or sequential therapy with other drugs, including genotoxic alkylating agents. This leads to the question whether vemurafenib and alkylating agents act synergistically and whether chronic vemurafenib treatment alters the melanoma cell response to alkylating agents. Here we show that a) BRAFV600E melanoma cells are killed by vemurafenib, driving apoptosis, b) BRAFV600E melanoma cells are neither more resistant nor sensitive to temozolomide/fotemustine than non-mutant cells, c) combined treatment with vemurafenib plus temozolomide or fotemustine has an additive effect on cell kill, d) acquired vemurafenib resistance of BRAFV600E melanoma cells does not affect MGMT, MSH2, MSH6, PMS2 and MLH1, nor does it affect the resistance to temozolomide and fotemustine, e) metastatic melanoma biopsies obtained from patients prior to and after vemurafenib treatment did not show a change in the MGMT promoter methylation status and MGMT expression level. The data suggest that consecutive treatment with vemurafenib and alkylating drugs is a reasonable strategy for metastatic melanoma treatment. PMID:25557167

  7. Application of genetically engineered Salmonella typhimurium for interferon-gamma-induced therapy against melanoma.

    PubMed

    Yoon, Wonsuck; Park, Yoo Chang; Kim, Jinseok; Chae, Yang Seok; Byeon, Jung Hye; Min, Sang-Hyun; Park, Sungha; Yoo, Young; Park, Yong Keun; Kim, Byeong Mo

    2017-01-01

    Salmonella have been experimentally used as anti-cancer agents, because they show selective growth in tumours. In this study, we genetically modified attenuated Salmonella typhimurium to express and secrete interferon-gamma (IFN-γ) as a tumouricidal agent to enhance the therapeutic efficacy of Salmonella. IFN-γ was fused to the N-terminal region (residues 1-160) of SipB (SipB160) for secretion from bacterial cells. Attenuated S. typhimurium expressing recombinant IFN-γ (S. typhimurium (IFN-γ)) invaded the melanoma cells and induced cytotoxicity. Subcutaneous administration of S. typhimurium (IFN-γ) also efficiently inhibited tumour growth and prolonged the survival of C57BL/6 mice bearing B16F10 melanoma compared with administration of phosphate-buffered saline (PBS), unmodified S. typhimurium or S. typhimurium expressing empty vector (S. typhimurium [Vec]) in a natural killer (NK) cell-dependent manner. Moreover, genetically modified Salmonella, including S. typhimurium (IFN-γ), showed little toxicity to normal tissues with no observable adverse effects. However, S. typhimurium (IFN-γ)-mediated tumour suppression was attributed to direct killing of tumour cells rather than to stable anti-tumour immunity. Collectively, these results suggest that tumour-targeted therapy using S. typhimurium (IFN-γ) has potential for melanoma treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Long non-coding RNA H19 promotes glucose metabolism and cell growth in malignant melanoma via miR-106a-5p/E2F3 axis.

    PubMed

    Luan, Wenkang; Zhou, Zhou; Ni, Xin; Xia, Yun; Wang, Jinlong; Yan, Yulan; Xu, Bin

    2018-03-01

    lncRNA H19 has been considered as an oncogenic lncRNA in many human tumours. In the present study, we identify the role and molecular mechanism of lncRNA H19 in melanoma. QRT-PCR was used to detect the expression of lncRNA H19 and E2F3 was detected in melanoma tissues. Cell counting kit-8 (CCK8), representative metabolites analysis was used to explore the biological function of lncRNA H19, miR-106a-5p and E2F3 in melanoma cells. Bioinformatics, luciferase reporter assays, MS2-RIP and RNA pull-down assay was used to demonstrate the molecular mechanism of lncRNA H19 in melanoma. We further test the function of lncRNA H19 in vivo though Xenograft tumour assay. We found that lncRNA H19 was increased in melanoma tissue, and lncRNA H19 was correlated with poor prognosis of melanoma patients. miR-106a-5p acts as a tumour suppressor in melanoma by targeting E2F3. E2F3 affects the melanoma cell glucose metabolism and growth. We also demonstrated that lncRNA H19 may function as the sponge of miR-106a-5p to up-regulate E2F3 expression, and consequently promote the glucose metabolism and growth of melanoma. This result elucidates a new mechanism for lncRNA H19 in melanoma development and provides a survival indicator and potential therapeutic target for melanoma patients.

  9. Ror2-Src signaling in metastasis of mouse melanoma cells is inhibited by NRAGE.

    PubMed

    Lai, Shan-Shan; Xue, Bin; Yang, Yang; Zhao, Li; Chu, Chao-Shun; Hao, Jia-Yin; Wen, Chuan-Jun

    2012-11-01

    The receptor tyrosine kinase (RTK) Ror2 plays important roles in developmental morphogenesis and mediates the filopodia formation in Wnt5a-induced cell migration. However, the function of Ror2 in noncanonical Wnt signaling resulting in cancer metastasis is largely unknown. Here, we show that Ror2 expression is higher in the highly metastatic murine B16-BL6 melanoma cells than in the low metastatic variant B16 cells. Overexpression of Ror2 increases the metastasis ability of B16 cells, and knockdown of Ror2 reduces the migration ability of B16-BL6 cells. Furthermore, the inhibition of Src kinase activity is critical for the Ror2-mediated cell migration upon Wnt5a treatment. The C-terminus of Ror2, which is deleted in brachydactyly type B (BDB), is essential for the mutual interaction with the SH1 domain of Src. Intriguingly, the Neurotrophin receptor-interacting MAGE homologue (NRAGE), which, as we previously reported, can remodel the cellular skeleton and inhibit cell-cell adhesion and metastasis of melanoma and pancreatic cancer, sharply blocks the interaction between Src and Ror2 and inhibits Ror2-mediated B16 cell migration by decreasing the activity of Src and focal adhesion kinase (FAK). Our data show that Ror2 is a potential factor in the tumorigenesis and metastasis in a Src-dependent manner that is negatively regulated by NRAGE. Copyright © 2012. Published by Elsevier Inc.

  10. A new O6-alkylguanine-DNA alkyltransferase inhibitor associated with a nitrosourea (cystemustine) validates a strategy of melanoma-targeted therapy in murine B16 and human-resistant M4Beu melanoma xenograft models.

    PubMed

    Rapp, Maryse; Maurizis, Jean C; Papon, Janine; Labarre, Pierre; Wu, Ting-Di; Croisy, Alain; Guerquin-Kern, Jean L; Madelmont, Jean C; Mounetou, Emmanuelle

    2008-07-01

    Chemoresistance to O(6)-alkylating agents is a major barrier to successful treatment of melanoma. It is mainly due to a DNA repair suicide protein, O(6)-alkylguanine-DNA alkyltransferase (AGT). Although AGT inactivation is a powerful clinical strategy for restoring tumor chemosensitivity, it was limited by increased toxicity to nontumoral cells resulting from a lack of tumor selectivity. Achieving enhanced chemosensitization via AGT inhibition preferably in the tumor should protect normal tissue. To this end, we have developed a strategy to target AGT inhibitors. In this study, we tested a new potential melanoma-directed AGT inhibitor [2-amino-6-(4-iodobenzyloxy)-9-[4-(diethylamino) ethylcarbamoylbenzyl] purine; IBgBZ] designed as a conjugate of O(6)-(4-iododbenzyl)guanine (IBg) as the AGT inactivator and a N,N-diethylaminoethylenebenzamido (BZ) moiety as the carrier to the malignant melanocytes. IBgBZ demonstrated AGT inactivation ability and potentiation of O(6)-alkylating agents (cystemustine, a chloroethylnitrosourea) in M4Beu highly chemoresistant human melanoma cells both in vitro and in tumor models. The biodisposition study on mice bearing B16 melanoma, the standard model for the evaluation of melanoma-directed agents, and the secondary ion mass spectrometry imaging confirmed the concentration of IBgBZ in the tumor and in particular in the intracytoplasmic melanosomes. These results validate the potential of IBgBZ as a new, more tumor-selective, AGT inhibitor in a strategy of melanoma-targeted therapy.

  11. Adoptive Cell Therapy of Melanoma with Cytokine-induced Killer Cells.

    PubMed

    Kim, Ji Sung; Kim, Yong Guk; Pyo, Minji; Lee, Hong Kyung; Hong, Jin Tae; Kim, Youngsoo; Han, Sang-Bae

    2015-04-01

    Melanoma is the most aggressive skin cancer and its incidence is gradually increasing worldwide. Patients with metastatic melanoma have a very poor prognosis (estimated 5-year survival rate of <16%). In the last few years, several drugs have been approved for malignant melanoma, such as tyrosine kinase inhibitors and immune checkpoint blockades. Although new therapeutic agents have improved progression-free and overall survival, their use is limited by drug resistance and drug-related toxicity. At the same time, adoptive cell therapy of metastatic melanoma with tumor-infiltrating lymphocytes has shown promising results in preclinical and clinical studies. In this review, we summarize the currently available drugs for treatment of malignant melanoma. In addition, we suggest cytokine-induced killer (CIK) cells as another candidate approach for adoptive cell therapy of melanoma. Our preclinical study and several previous studies have shown that CIK cells have potent anti-tumor activity against melanomas in vitro and in an in vivo human tumor xenograft model without any toxicity.

  12. Adoptive Cell Therapy of Melanoma with Cytokine-induced Killer Cells

    PubMed Central

    Kim, Ji Sung; Kim, Yong Guk; Pyo, Minji; Lee, Hong Kyung; Hong, Jin Tae; Kim, Youngsoo

    2015-01-01

    Melanoma is the most aggressive skin cancer and its incidence is gradually increasing worldwide. Patients with metastatic melanoma have a very poor prognosis (estimated 5-year survival rate of <16%). In the last few years, several drugs have been approved for malignant melanoma, such as tyrosine kinase inhibitors and immune checkpoint blockades. Although new therapeutic agents have improved progression-free and overall survival, their use is limited by drug resistance and drug-related toxicity. At the same time, adoptive cell therapy of metastatic melanoma with tumor-infiltrating lymphocytes has shown promising results in preclinical and clinical studies. In this review, we summarize the currently available drugs for treatment of malignant melanoma. In addition, we suggest cytokine-induced killer (CIK) cells as another candidate approach for adoptive cell therapy of melanoma. Our preclinical study and several previous studies have shown that CIK cells have potent anti-tumor activity against melanomas in vitro and in an in vivo human tumor xenograft model without any toxicity. PMID:25922594

  13. Nestin is expressed in HMB-45 negative melanoma cells in dermal parts of nodular melanoma.

    PubMed

    Kanoh, Maho; Amoh, Yasuyuki; Tanabe, Kenichi; Maejima, Hideki; Takasu, Hiroshi; Katsuoka, Kensei

    2010-06-01

    Nestin, a marker of neural stem cells, is expressed in the stem cells of the mouse hair follicle. The nestin-expressing hair follicle stem cells can differentiate into neurons, glia, keratocytes, smooth muscle cells and melanocytes in vitro. These pluripotent nestin-expressing stem cells are keratin 15 (K15)-negative, suggesting that they are in a relatively undifferentiated state. Recent studies suggest that the epithelial stem cells are important in tumorigenesis, and nestin expression is thought to be important in tumorigenesis. In the present study, we examined the expression of the hair follicle and neural stem cell marker nestin, as well as S-100 and HMB-45, in melanoma. Nestin immunoreactivity was observed in the HMB-45-negative melanoma cells in all five cases of amelanotic nodular melanomas. Moreover, nestin immunoreactivity was observed in the dermal parts in seven of 10 cases of melanotic nodular melanomas. Especially, nestin immunoreactivity was observed in the HMB-45-negative melanoma cells in the dermal parts of all 10 cases of HMB-45-negative amelanotic and melanotic nodular melanomas. On the other hand, nestin expression was negative in 10 of 12 cases of superficial spreading melanoma. These results suggest that nestin is an important marker of HMB-45-negative melanoma cells in the dermal parts of patients with nodular melanoma.

  14. A Subset of Host B-Lymphocytes Control Melanoma Metastasis Through a MCAM/MUC18-dependent Interaction: Evidence from Mice and Humans

    PubMed Central

    Staquicini, Fernanda I.; Tandle, Anita; Libutti, Steven K.; Sun, Jessica; Zigler, Maya; Bar-Eli, Menashe; Aliperti, Fabiana; Pérez, Elizabeth C.; Gershenwald, Jeffrey E.; Mariano, Mario; Pasqualini, Renata; Arap, Wadih; Lopes, José D.

    2008-01-01

    Host immunity affects tumor metastasis but the corresponding cellular and molecular mechanisms are not entirely clear. Here we show that a subset of B-lymphocytes (termed B-1 population) -- but not other lymphocytes -- have pro-metastatic effects on melanoma cells in vivo through a direct heterotypic cell-cell interaction. In the classic B16 mouse melanoma model, one mechanism underlying this phenomenon is a specific upregulation and subsequent homophilic interaction mediated by the cell surface glycoprotein MUC18 (also known as melanoma cell adhesion molecule; MCAM). Presence of B-1 lymphocytes in a panel of tumor samples from melanoma patients directly correlates with MUC18 expression in melanoma cells, indicating that the same protein interaction exists in humans. These results suggest a new but as yet unrecognized functional role for host B-1 lymphocytes in tumor metastasis and establish a biochemical basis for such observations. Our findings support the counterintuitive central hypothesis in which a primitive layer of the immune system actually contributes to tumor progression and metastasis in a mouse model and in melanoma patients. Given that monoclonal antibodies against MUC18 are in pre-clinical development but the reason for their anti-tumor activity is not well understood, these translational results are relevant in the setting of human melanoma, and perhaps of other cancers. PMID:18922915

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

    PubMed Central

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

    2014-01-01

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

  16. MicroRNA-9 up-regulates E-cadherin through inhibition of NF-κB1-Snail1 pathway in melanoma.

    PubMed

    Liu, Shujing; Kumar, Suresh M; Lu, Hezhe; Liu, Aihua; Yang, Ruifeng; Pushparajan, Anitha; Guo, Wei; Xu, Xiaowei

    2012-01-01

    MicroRNAs (miRNAs) are short non-coding RNAs that post-transcriptionally regulate gene expression. Hsa-miR-9 has been shown to have opposite functions in different tumour types; however, the underlying mechanism is unclear. Here we show that hsa-miR-9 is down-regulated in metastatic melanomas compared to primary melanomas. Overexpression of miR-9 in melanoma cells resulted in significantly decreased cell proliferation and migratory capacity with decreased F-actin polymerization and down-regulation of multiple GTPases involved in cytoskeleton remodelling. miR-9 overexpression induced significant down-regulation of Snail1 with a concomitant increase in E-cadherin expression. In contrast, knockdown of miR-9 increased Snail1 expression as well as melanoma cell proliferation and migration capacity. Mechanistically, miR-9 expression down-regulated NF-κB1 in melanoma and the effect was abolished by mutations in the putative miR-9 binding sites within the 3'-untranslated region (UTR) of NF-κB1. Anti-miR-9 miRNA inhibitor also increased the expression of NF-κB1. The effects of miR-9 on Snail1 expression and melanoma cell proliferation and migration were rescued by overexpression of NF-κB1 in these cells. Furthermore, miR-9 overexpression resulted in significantly decreased melanoma growth and metastasis in vivo. In summary, miR-9 inhibits melanoma proliferation and metastasis through down-regulation of the NF-κB1-Snail1 pathway. This study finds a new mechanism that miR-9 utilizes to decrease E-cadherin expression and inhibit melanoma progression. The results suggest that function of microRNAs is context and tumour type-specific. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  17. The plasma membrane Ca2+ pump PMCA4b inhibits the migratory and metastatic activity of BRAF mutant melanoma cells.

    PubMed

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

    2017-06-15

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

  18. Cordycepin (3'-deoxyadenosine) inhibits the growth of B16-BL6 mouse melanoma cells through the stimulation of adenosine A3 receptor followed by glycogen synthase kinase-3beta activation and cyclin D1 suppression.

    PubMed

    Yoshikawa, Noriko; Yamada, Shizuo; Takeuchi, Chihiro; Kagota, Satomi; Shinozuka, Kazumasa; Kunitomo, Masaru; Nakamura, Kazuki

    2008-06-01

    Cordyceps sinensis, a parasitic fungus on the larvae of Lepidoptera, has been used as a traditional Chinese medicine. We previously reported that the growth of B16-BL6 mouse melanoma (B16-BL6) cells was inhibited by cordycepin (3'-deoxyadenosine), an active ingredient of C. sinensis, and its effect was antagonized by MRS1191, a selective adenosine A3 receptor antagonist. In this study, the radioligand binding assay using [125I]-AB-MECA (a selective adenosine A3 receptor agonist) has shown that B16-BL6 cells express adenosine A3 receptors and that cordycepin binds to these receptors. We also confirmed the involvement of adenosine A3 receptors in the action of cordycepin using MRS1523 and MRS1220, specific adenosine A3 receptor antagonists. Next, indirubin, a glycogen synthase kinase-3beta (GSK-3beta) inhibitor, antagonized the growth suppression induced by cordycepin. Furthermore, the level of cyclin D1 protein in B16-BL6 cells was decreased by cordycepin using Western blot analysis. In conclusion, this study demonstrated that cordycepin inhibits the proliferation of B16-BL6 cells by stimulating adenosine A3 receptors followed by the Wnt signaling pathway, including GSK-3beta activation and cyclin D1 inhibition.

  19. Transforming Growth Factor-β2 is a Molecular Determinant for Site-specific Melanoma Metastasis in the Brain

    PubMed Central

    Zhang, Chenyu; Zhang, Fahao; Tsan, Rachel; Fidler, Isaiah J.

    2008-01-01

    Murine melanomas produce site-specific experimental brain metastases that reflect clinical reality. When injected into the internal carotid artery of mice, the K-1735 melanoma cells produce metastatic lesions only in the brain parenchyma, whereas the B16 melanoma cells and the somatic hybrid cells of the B16 x K-1735 melanoma cells produce metastatic lesions only in the leptomeninges and ventricles. In the present study, we identified TGF-β2, an isoform of the TGF-β family, as a molecular determinant of melanoma cell growth in the brain parenchyma. We found that the TGF-β2 mRNA was highly expressed by the K-1735 cells, whereas the B16 cells or any B16 x K-1735 somatic cell-cell fusion hybrids have low expression. Transfection of the TGF-β2 gene into B16 cells resulted in the production of microscopic metastatic lesions in the brain parenchyma, without a decrease in metastasis to the leptomeninges or ventricles. TGF-β2 knockdown in the K-1735 melanoma cells significantly reduced metastasis to the brain parenchyma but did not induce metastasis to the leptomeninges or ventricles. These data demonstrate that TGF-β2 expression by murine melanoma cells is necessary for the establishment and growth of metastases in the brain parenchyma. PMID:19141644

  20. Antitumor activity of monomethoxy poly(ethylene glycol)-poly (ε-caprolactone) micelle-encapsulated doxorubicin against mouse melanoma.

    PubMed

    Zheng, Lan; Gou, Maling; Zhou, Shengtao; Yi, Tao; Zhong, Qian; Li, Zhengyu; He, Xiang; Chen, Xiancheng; Zhou, Lina; Wei, Yuquan; Qian, Zhiyong; Zhao, Xia

    2011-06-01

    Doxorubicin (Dox) is one of the most commonly used and highly effective antineoplastic agents, but the clinical application of this broad spectrum drug is largely hampered by its poor stability and serious toxicity to normal tissues. Hence, it is essential to improve the therapeutic effect and decrease the systematic toxicity for the administration of doxorubicin. In our study, doxorubicin was incorporated into monomethoxy poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) micelle by a self-assembly method. The cytotoxicity and cellular uptake efficiency of Dox-loaded MPEG-PCL (Dox/MPEG-PCL) micelle against B16-F10 murine melanoma cells was examined by the methylthiazoltetrazolium (MTT) test and flow cytometry. The antitumor activity of Dox/MPEG-PCL was evaluated in C57BL/6 mice injected subcutaneously with B16-F10 cells. Toxicity was evaluated in tumor-free mice. Meanwhile, tumor proliferation, intratumoal angiogenesis and apoptotic cells were evaluated by PCNA, CD31 staining and TUNEL assay, respectively. Encapsulation of doxorubicin in MPEG-PCL micelle improved the cytotoxicity of doxorubicin and enhanced its cellular uptake on B16-F10 cell in vitro. Administration of Dox/MPEG-PCL micelle resulted in significant inhibition (75% maximum inhibition relative to controls) in the growth of B16-F10 tumor xenografts and prolonged the survival of the treated mice (P<0.05). These anti-tumor responses were associated with marked increase of tumor apoptosis and notable reduction of cell proliferation and intratumoral microvessel density (P<0.05). The system toxicity also decreased in the Dox/MPEG-PCL group compared with free doxorubicin group. Our data indicate that the encapsulation of doxorubicin in MPEG-PCL micelle improved the anti-tumor activity in vivo without conspicuous systemic toxic effects.

  1. Targeted radionuclide therapy of melanoma: anti-tumoural efficacy studies of a new 131I labelled potential agent.

    PubMed

    Bonnet-Duquennoy, Mathilde; Papon, Janine; Mishellany, Florence; Labarre, Pierre; Guerquin-Kern, Jean-Luc; Wu, Ting-Di; Gardette, Maryline; Maublant, Jean; Penault-Llorca, Frédérique; Miot-Noirault, Elisabeth; Cayre, Anne; Madelmont, Jean-Claude; Chezal, Jean-Michel; Moins, Nicole

    2009-08-01

    In recent years, there has been dramatic worldwide increase in incidence of malignant melanoma. Although localised disease is often curable by surgical excision, metastatic melanoma is inherently resistant to most treatments. In this context, targeted radionuclide therapy could be an efficient alternative. After pharmacomodulation study, we selected a quinoxaline derivative molecule (ICF01012) for its high, specific and long-lasting uptake in melanoma with rapid clearance from nontarget organs providing suitable dosimetry parameters for targeted radiotherapy. Aim of this study was to investigate, in vivo, efficacy of [(131)I]ICF01012 on nonmetastatic B16F0, metastatic B16Bl6 or human M4Beu melanoma tumours. First, colocalisation of ICF01012 with melanin by SIMS imaging was observed. Second, we showed that treatment drastically inhibited growth of B16F0, B16Bl6 and M4beu tumours whereas [(131)I]NaI or unlabelled ICF01012 treatment was without significant effect. Histological analysis and measure of PCNA proliferation marker expression showed that residual B16 tumour cells exhibit a significant loss of aggressiveness after treatment. This effect is associated with a lengthening of the treated-mice survival time. Moreover, with B16Bl6 model, 55% of the untreated mice had lung metastases whereas no metastasis was counted on treated group. Our data demonstrated a strong anti-tumoural effect of [(131)I]ICF01012 for radionuclide therapy on murine and human in vivo pigmented melanoma models, whatever their dissemination profiles and their melanin content be. Further studies will attempt to optimise therapy protocol by increasing the balance between the anti-tumoural effect and the safety on non-target organs.

  2. Pilot Study of 64Cu(I) for PET Imaging of Melanoma

    DOE PAGES

    Jiang, Lei; Tu, Yingfeng; Hu, Xiang; ...

    2017-05-31

    Currently, 64Cu(II) labeled tracers including 64CuCl 2 have been widely applied in the research of molecular imaging and therapy. Human copper transporter 1 (hCTR1) is the major high affinity copper influx transporter in mammalian cells, and specially responsible for the transportation of Cu(I) not Cu(II). Thus, we investigated the feasible application of 64Cu(I) for PET imaging. 64Cu(II) was reduced to 64Cu(I) with the existence of sodium L-ascorbate, DL-Dithiothreitol or cysteine. Cell uptake and efflux assay was investigated using B16F10 and A375 cell lines, respectively. Small animal PET and biodistribution studies were performed in both B16F10 and A375 tumor-bearing mice. Comparedmore » with 64Cu(II), 64Cu(I) exhibited higher cellular uptake by melanoma, which testified CTR1 specially influx of Cu(I). But, due to oxidation reaction in vivo, no significant difference between 64Cu(I) and 64Cu(II) was observed through PET images and biodistribution. In addition, radiation absorbed doses for major tissues of human were calculated based on the mouse biodistribution. Radiodosimetry calculations for 64/67Cu(I) and 64/67Cu(II) were similar, which suggested that although melanoma were with high radiation absorbed doses, high radioactivity accumulation by liver and kidney should be noticed for the further application. Thus, 64Cu(I) should be further studied to evaluate it as a PET imaging radiotracer.« less

  3. Pilot Study of 64Cu(I) for PET Imaging of Melanoma

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jiang, Lei; Tu, Yingfeng; Hu, Xiang

    Currently, 64Cu(II) labeled tracers including 64CuCl 2 have been widely applied in the research of molecular imaging and therapy. Human copper transporter 1 (hCTR1) is the major high affinity copper influx transporter in mammalian cells, and specially responsible for the transportation of Cu(I) not Cu(II). Thus, we investigated the feasible application of 64Cu(I) for PET imaging. 64Cu(II) was reduced to 64Cu(I) with the existence of sodium L-ascorbate, DL-Dithiothreitol or cysteine. Cell uptake and efflux assay was investigated using B16F10 and A375 cell lines, respectively. Small animal PET and biodistribution studies were performed in both B16F10 and A375 tumor-bearing mice. Comparedmore » with 64Cu(II), 64Cu(I) exhibited higher cellular uptake by melanoma, which testified CTR1 specially influx of Cu(I). But, due to oxidation reaction in vivo, no significant difference between 64Cu(I) and 64Cu(II) was observed through PET images and biodistribution. In addition, radiation absorbed doses for major tissues of human were calculated based on the mouse biodistribution. Radiodosimetry calculations for 64/67Cu(I) and 64/67Cu(II) were similar, which suggested that although melanoma were with high radiation absorbed doses, high radioactivity accumulation by liver and kidney should be noticed for the further application. Thus, 64Cu(I) should be further studied to evaluate it as a PET imaging radiotracer.« less

  4. The cathelicidin-BF Lys16 mutant Cbf-K16 selectively inhibits non-small cell lung cancer proliferation in vitro.

    PubMed

    Tian, Yuwei; Wang, Hui; Li, Bing; Ke, Mengyun; Wang, Jing; Dou, Jie; Zhou, Changlin

    2013-11-01

    The 30-amino acid antimicrobial peptide Cbf-K16 is a cathelicidin-BF (BF-30) Lys16 mutant derived from the snake venom of Bungarus fasciatus. Our previous study found that BF-30 selectively inhibited the proliferation of the metastatic melanoma cell line B16F10 in vitro and in vivo, but had a negligible effect on human lung cells. In the present study, it was demonstrated for the first time that Cbf-K16 selectively inhibits the proliferation of lung carcinoma cells in vitro, with low toxicity to normal cells. The half-maximal inhibitory concentrations (IC50) of Cbf-K16 against H460 human non-small cell lung carcinoma cells and mouse Lewis lung cancer cells were only 16.5 and 10.5 µM, respectively, which were much less compared to that of BF-30 (45 and 40.3 µM). Data using a transmission electron microscope (TEM) assay showed that, at 20 and 40 µM, Cbf-K16 induced the rupture of the cytoplasmic membrane, which was consistent with data obtained from lactate dehydrogenase (LDH) release assays. The LDH release increased from 17.8 to 52.9% as the duration and dosage of Cbf-K16 increased. Annexin V-fluorescein and propidium iodide staining assays indicated that there were no obvious apoptotic effects at the different dosages and times tested. In H460 cells, the rate of genomic DNA binding increased from 51.9 to 86.8% as the concentration of Cbf-K16 increased from 5 to 10 µM. These data indicate that Cbf-K16 selectively inhibits the proliferation of lung carcinoma cells via cytoplasmic membrane permeabilization and DNA binding, rather than apoptosis. Although Cbf-K16 displayed significant cytotoxic activity (40 µM) against tumor cells, in splenocytes no significant inhibitory effect was observed and hemolysis was only 5.6%. These results suggest that Cbf-K16 is a low-toxicity anti-lung cancer drug candidate.

  5. A Phosphoproteomic Comparison of B-RAFV600E and MKK1/2 Inhibitors in Melanoma Cells.

    PubMed

    Stuart, Scott A; Houel, Stephane; Lee, Thomas; Wang, Nan; Old, William M; Ahn, Natalie G

    2015-06-01

    Inhibitors of oncogenic B-RAF(V600E) and MKK1/2 have yielded remarkable responses in B-RAF(V600E)-positive melanoma patients. However, the efficacy of these inhibitors is limited by the inevitable onset of resistance. Despite the fact that these inhibitors target the same pathway, combination treatment with B-RAF(V600E) and MKK1/2 inhibitors has been shown to improve both response rates and progression-free survival in B-RAF(V600E) melanoma patients. To provide insight into the molecular nature of the combinatorial response, we used quantitative mass spectrometry to characterize the inhibitor-dependent phosphoproteome of human melanoma cells treated with the B-RAF(V600E) inhibitor PLX4032 (vemurafenib) or the MKK1/2 inhibitor AZD6244 (selumetinib). In three replicate experiments, we quantified changes at a total of 23,986 phosphosites on 4784 proteins. This included 1317 phosphosites that reproducibly decreased in response to at least one inhibitor. Phosphosites that responded to both inhibitors grouped into networks that included the nuclear pore complex, growth factor signaling, and transcriptional regulators. Although the majority of phosphosites were responsive to both inhibitors, we identified 16 sites that decreased only in response to PLX4032, suggesting rare instances where oncogenic B-RAF signaling occurs in an MKK1/2-independent manner. Only two phosphosites were identified that appeared to be uniquely responsive to AZD6244. When cells were treated with the combination of AZD6244 and PLX4032 at subsaturating concentrations (30 nm), responses at nearly all phosphosites were additive. We conclude that AZD6244 does not substantially widen the range of phosphosites inhibited by PLX4032 and that the benefit of the drug combination is best explained by their additive effects on suppressing ERK1/2 signaling. Comparison of our results to another recent ERK1/2 phosphoproteomics study revealed a surprising degree of variability in the sensitivity of phosphosites to

  6. Zinc phthalocyanines attached to gold nanorods for simultaneous hyperthermic and photodynamic therapies against melanoma in vitro.

    PubMed

    Freitas, L F; Hamblin, M R; Anzengruber, F; Perussi, J R; Ribeiro, A O; Martins, V C A; Plepis, A M G

    2017-08-01

    Studies indicate that hyperthermic therapy using gold nanorods and photodynamic activity with many photosensitizers can present a synergistic effect, and offer a great therapeutic potential, although more investigation needs to be performed before such approach could be implemented. We proposed to investigate the effect of the attachment of phthalocyanines on the surface of gold nanorods (well-characterized devices for hyperthermia generation) for the elimination of melanoma, one of the most important skin cancers due to its high lethality. Following the synthesis of nanorods through a seed-mediated method, the efficacy of photodynamic therapy (PDT) and hyperthermia was assessed separately. We chose to coat the nanorods with two tetracarboxylated zinc phthalocyanines - with or without methyl-glucamine groups. After the coating process, the phthalocyanines formed ionic complexes with the cetyltrimethylammonium bromide (CTAB) that was previously covering the nanoparticles. The nanorod-phthalocyanines complexes were analyzed by transmission electron microscopy (TEM), and their singlet oxygen and hydroxyl radical generation yields were assessed. Furthermore, they were tested in vitro with melanotic B16F10 and amelanotic B16G4F melanoma cells. The cells with nanoparticles were irradiated with laser (at 635nm), and the cell viability was assessed. The results indicate that the photodynamic properties of the phthalocyanines tested are enhanced when they are attached on the nanorods surface, and the combination of PDT and hyperthermia was able to eliminate over 90% of melanoma cells. This is a novel study because two tetracarboxylated phthalocyanines were used and because the same wavelength was irradiated to activate both the nanorods and the photosensitizers. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Biodistribution of modular nanotransporter carrying Auger electron emitter and targeted at melanoma cells in murine tumor model

    NASA Astrophysics Data System (ADS)

    Vorontsova, M. S.; Morozova, N. B.; Karmakova, T. A.; Rosenkranz, A. A.; Slastnikova, T. A.; Petriev, V. M.; Smoryzanova, O. A.; Tischenko, V. K.; Yakubovskaya, R. I.; Kaprin, A. D.; Sobolev, A. S.

    2017-09-01

    Recombinant modular nanotransporter containing α-melanocyte-stimulating hormone peptide sequence (MNT-MSH) as a ligand module was designed for nucleus-targeted delivery of cytotoxic agents into melanoma cells. MNT-MSH radiolabeled with Auger electron emitter (111In-NOTA-MNT-MSH) showed a high antitumor efficacy in mice bearing syngeneic melanoma after intratumoral (i.t.) injection. This study is aimed at evaluating the biodistribution of the radioconjugate in melanoma tumor model in vivo. 111In-NOTA-MNT-MSH was administered i.t. in C57Bl/6j mice bearing subcutaneously implanted B16-F1 murine melanoma cells, expressing high levels of MCR1. The tissue uptake of radioactivity was determined ex vivo by γ-counter measurements. The intravenous route of administration did not provide a desirable level of radioactivity accumulation in the tumor, possibly, due to a high uptake of the transporter in liver tissue. After i.t. administration 111In-NOTA-MNT-MSH provided a high local retention of radionuclide, ranged from 400 to 350 %ID/g within at least 48 hours post-injection. MNT containing Auger electron emitter and α-MSH peptide as vector ligand could be a promising basis for radiopharmaceutical preparations intended for melanoma treatment.

  8. Ethyl acetate extract from Panax ginseng C.A. Meyer and its main constituents inhibit α-melanocyte-stimulating hormone-induced melanogenesis by suppressing oxidative stress in B16 mouse melanoma cells.

    PubMed

    Jiang, Rui; Xu, Xiao-Hao; Wang, Ke; Yang, Xin-Zhao; Bi, Ying-Fei; Yan, Yao; Liu, Jian-Zeng; Chen, Xue-Nan; Wang, Zhen-Zhong; Guo, Xiao-Li; Zhao, Da-Qing; Sun, Li-Wei

    2017-08-17

    Hyperpigmentation disease involves darkening of the skin color due to melanin overproduction. Panax ginseng C.A. Meyer is a well-known traditional Chinese medicine and has a long history of use as a skin lightener to inhibit melanin formation in China, Korea and some other Asian countries. However, the constituents and the molecular mechanisms by which they affect melanogenesis are not fully clear. The purpose of this study was to identify the active ingredient in Panax ginseng C.A. Meyer extract that inhibits mushroom tyrosinase activity and to investigate the antioxidative capacity and molecular mechanisms of the effective extract on melanogenesis in B16 mouse melanoma cells. Aqueous extracts of Panax ginseng C.A. Meyer were successively fractionated with an equal volume of chloroform, ethyl acetate, and n-butyl alcohol to determine the effects by examining the activity of mushroom tyrosinase. The effective fraction was analyzed using HPLC and LC-MS. The antioxidative capacity and the inhibitory effects on melanin content, cell intracellular tyrosinase activity, and melanogenesis protein levels were determined in α-melanocyte-stimulating hormone (α-MSH)-treated B16 mouse melanoma cells. The ethyl acetate extract from Panax ginseng C.A. Meyer (PG-2) had the highest inhibiting effect on mushroom tyrosinase, mainly contained phenolic acids, including protocatechuic acid, vanillic acid, p-coumaric acid, salicylic acid, and caffeic acid, and exhibited apparent antioxidant activity in vitro. PG-2 and its main constituents significantly decreased melanin content, suppressed cellular tyrosinase activity, and reduced expression of tyrosinase protein to inhibit B16 cells melanogenesis induced by α-MSH, and no cytotoxic effects were observed. They also inhibited cellular reactive oxygen species (ROS) generation, increased superoxide dismutase (SOD) activity and glutathione (GSH) level in α-MSH-treated B16 cells effectively. And those activities of its main constituents

  9. Tumour volume response, initial cell kill and cellular repopulation in B16 melanoma treated with cyclophosphamide and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea.

    PubMed Central

    Stephens, T. C.; Peacock, J. H.

    1977-01-01

    The relationship between tumour volume response and cell kill in B16 melanoma following treatment in vivo with cyclophosphamide (CY) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) was investigated. Tumour volume response, expressed as growth delay, was estimated from measurements of tumour dimensions. Depression of in vitro colony-forming ability of cells from treated tumours was used as the measure of tumour cell kill. The relationship between these parameters was clearly different for the two agents studied. CY produced more growth delay (7.5 days) per decade of tumour cell kill than CCNU (2 to 3.5 days). The possibility that this was due to a technical artefact was rejected in favour of an alternative explanation that different rates of cellular repopulation in tumours treated with CY and CCNU might be responsible. Cellular repopulation was measured directly, by performing cell-survival assays at various times after treatment with doses of CY and CCNU which produced about 3 decades of cell kill. The rate of repopulation by clonogenic cells was much slower after treatment with CY than with CCNU, and this appears to account for the longer duration of the growth delay obtained with CY. PMID:921888

  10. Molecular Imaging and Radionuclide Therapy of Melanoma Targeting the Melanocortin 1 Receptor

    PubMed Central

    Zhang, Chengcheng; Lin, Kuo-Shyan; Bénard, François

    2017-01-01

    Melanoma is a deadly disease at late metastatic stage, and early diagnosis and accurate staging remain the key aspects for managing melanoma. The melanocortin 1 receptor (MC1 R) is overexpressed in primary and metastatic melanomas, and its endogenous ligand, the α-melanocyte-stimulating hormone (αMSH), has been extensively studied for the development of MC1 R-targeted molecular imaging and therapy of melanoma. Natural αMSH is not well suited for this purpose due to low stability in vivo. Unnatural amino acid substitutions substantially stabilized the peptide, while cyclization via lactam bridge and metal coordination further improved binding affinity and stability. In this study, we summarized the development and the in vitro and in vivo characteristics of the radiolabeled αMSH analogues, including 99mTc-, 111In-, 67 Ga-, or 125I-labeled αMSH analogues for imaging with single-photon emission computed tomography; 68Ga-, 64Cu-, or 18F-labeled αMSH analogues for imaging with positron emission tomography; and 188Re-, 177Lu-, 90Y-, or 212Pb-labeled αMSH analogues for radionuclide therapy. These radiolabeled αMSH analogues showed promising results with high tumor uptake and rapid normal tissue activity clearance in the preclinical model of B16F1 and B16F10 mouse melanomas. These results highlight the potential of using radiolabeled αMSH analogues in clinical applications for molecular imaging and radionuclide therapy of melanoma. PMID:29182034

  11. Amino Acid Signature in Human Melanoma Cell Lines from Different Disease Stages.

    PubMed

    Wasinger, Christine; Hofer, Alexandra; Spadiut, Oliver; Hohenegger, Martin

    2018-04-19

    Cancer cells rewire metabolism to sustain high proliferation rates. Beside glycolysis and glutaminolysis, amino acids substitute as energy source, feed fatty acid biosynthesis and represent part of the secretome of transformed cells, including melanoma. We have therefore investigated acetate, pyruvate and the amino acid composition of the secretome of human melanoma cells representing the early slow (WM35, WM278, WM793b and VM21) and metastatic fast (A375, 518a2, 6F and WM8) growth phase in order to identify possible signalling components within these profiles. Proliferation assays and a principle component analysis revealed a stringent difference between the fast and slow growing melanoma cells. Moreover, upon inhibition of the mevalonate pathway, glutamic acid and alanine were identified as the central difference in the conditional media. A supplementation of the media with glutamic acid and the combination with alanine significantly accelerated the proliferation, migration and invasion of early stage melanoma cells, but not metastatic cells. Finally, the inhibition of the mevalonate pathway abolished the growth advantage of the melanoma cells in a time dependent manner. Taken together, these data corroborate a stage specific response in growth and aggressiveness to extracellular glutamic acid and alanine, indicative for microenvironmental signalling of individual amino acids.

  12. Involvement of endothelin and ET(A) endothelin receptor in mechanical allodynia in mice given orthotopic melanoma inoculation.

    PubMed

    Fujita, Masahide; Andoh, Tsugunobu; Saiki, Ikuo; Kuraishi, Yasushi

    2008-02-01

    We investigated whether endothelin (ET) would be involved in skin cancer pain in mice. Orthotopic inoculation of B16-BL6 melanoma cells into the plantar region of the hind paw produced marked mechanical allodynia in C57BL/6 mice. Intraplantar injections of the ET(A)-receptor antagonist BQ-123 (0.3 - 3 nmol/site), but not the ET(B)-receptor antagonist BQ-788 (1 and 3 nmol/site), inhibited mechanical allodynia in mice with grown melanoma. In naive mice, an intraplantar injection of tumor extract (1 and 3 mg/site), which was prepared from the grown melanoma in the paw, produced mechanical allodynia, which was inhibited by BQ-123 and BQ-788 at doses of 3 and 10 nmol/site. An intraplantar injection of ET-1 (1 and 10 pmol/site) elicited licking behavior, which was increased in the melanoma-bearing hind paw. BQ-123 (3 and 10 nmol/site) inhibited licking induced by ET-1 (10 pmol/site). The level of mRNA of ET(A), but not ET(B), receptor, was significantly increased in the dorsal root ganglia on the inoculated side. Cultured B16-BL6 cells contained ET, and the melanoma mass increased the concentration of ET as it grew bigger. These results suggest that ET-1 and ET(A) receptor are at least partly involved in the induction of pain induced by melanoma cell inoculation.

  13. The functional property of egg yolk phosvitin as a melanogenesis inhibitor.

    PubMed

    Jung, Samooel; Kim, Dong Hee; Son, Jun Ho; Nam, Kichang; Ahn, Dong Uk; Jo, Cheorun

    2012-12-01

    Phosvitin is a phosphoglycoprotein present in egg yolk. More than half of the amino acids in phosvitin molecule are serine, of which >90% are phosphorylated. Therefore, phosvitin has a strong metal binding capability. The aim of this study was to investigate the effect of phosvitin on the inhibition of melanogenesis in melanoma cells. The results showed that phosvitin inhibited the activity of mushroom tyrosinase. Addition of phosvitin at a concentration of 50μg/ml, to B16F10 melanoma cells inhibited tyrosinase activity by approximately 42% and melanin synthesis by 17% compared to those in a control without phosvitin. Phosvitin inhibited the expression of tyrosinase, tyrosinase-related protein 1 (TRP-1), TRP-2, and microphthalmia-associated transcription factor (MITF) in B16F10 melanoma cells. In addition, phosvitin reduced the cellular cAMP concentration in B16F10 melanoma cells. These results indicate that phosvitin has the potential to be used as a melanogenesis inhibitor in the food and cosmetics industry. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. DW-F5: A novel formulation against malignant melanoma from Wrightia tinctoria

    PubMed Central

    Antony, Jayesh; Saikia, Minakshi; V, Vinod.; Nath, Lekshmi. R.; Katiki, Mohana Rao; Murty, M.S.R.; Paul, Anju; A, Shabna; Chandran, Harsha; Joseph, Sophia Margaret; S, Nishanth Kumar.; Panakkal, Elizabeth Jayex; V, Sriramya I.; V, Sridivya I.; Ran, Sophia; S, Sankar; Rajan, Easwary; Anto, Ruby John

    2015-01-01

    Wrightia tinctoria is a constituent of several ayurvedic preparations against skin disorders including psoriasis and herpes, though not yet has been explored for anticancer potential. Herein, for the first time, we report the significant anticancer properties of a semi-purified fraction, DW-F5, from the dichloromethane extract of W. tinctoria leaves against malignant melanoma. DW-F5 exhibited anti-melanoma activities, preventing metastasis and angiogenesis in NOD-SCID mice, while being non-toxic in vivo. The major pathways in melanoma signaling mediated through BRAF, WNT/β-catenin and Akt-NF-κB converging in MITF-M, the master regulator of melanomagenesis, were inhibited by DW-F5, leading to complete abolition of MITF-M. Purification of DW-F5 led to the isolation of two cytotoxic components, one being tryptanthrin and the other being an unidentified aliphatic fraction. The overall study predicts Wrightia tinctoria as a candidate plant to be further explored for anticancer properties and DW-F5 as a forthcoming drug formulation to be evaluated as a chemotherapeutic agent against malignant melanoma. PMID:26061820

  15. Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses.

    PubMed

    Schmitz, M; Graf, C; Gut, T; Sirena, D; Peter, I; Dummer, R; Greber, U F; Hemmi, S

    2006-06-01

    Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies.

  16. Dietary supplementation with secoisolariciresinol diglycoside (SDG) reduces experimental metastasis of melanoma cells in mice.

    PubMed

    Li, D; Yee, J A; Thompson, L U; Yan, L

    1999-07-19

    We investigated the effect of dietary supplementation with secoisolariciresinol diglycoside (SDG), a lignan precursor isolated from flaxseed, on experimental metastasis of B16BL6 murine melanoma cells in C57BL/6 mice. Four diets were compared: a basal diet (control group) and the basal diet supplemented with SDG at 73, 147 or 293 micromol/kg (equivalent to SDG provided in the 2.5, 5 or 10% flaxseed diet). Mice were fed the diet for 2 weeks before and after an intravenous injection of 0.6 x 10(5) tumor cells. At necropsy, the number and size of tumors that formed in the lungs were determined. The median number of tumors in the control group was 62, and those in the SDG-supplemented groups were 38, 36 and 29, respectively. The last was significantly different from the control (P < 0.01). Dietary supplementation with SDG at 73, 147 and 293 micromol/kg also decreased tumor size (tumor cross-sectional area and volume) in a dose-dependent manner compared with the control values. These results show that SDG reduced pulmonary metastasis of melanoma cells and inhibited the growth of metastatic tumors that formed in the lungs. It is concluded that dietary supplementation with SDG reduces experimental metastasis of melanoma cells in mice.

  17. Apoptosis through Bcl-2/Bax and Cleaved Caspase Up-Regulation in Melanoma Treated by Boron Neutron Capture Therapy

    PubMed Central

    Faião-Flores, Fernanda; Coelho, Paulo Rogério Pinto; Toledo Arruda-Neto, João Dias; Maria-Engler, Silvya Stuchi; Tiago, Manoela; Capelozzi, Vera Luiza; Giorgi, Ricardo Rodrigues; Maria, Durvanei Augusto

    2013-01-01

    Boron neutron capture therapy (BNCT) is a binary treatment involving selective accumulation of boron carriers in a tumor followed by irradiation with a thermal or epithermal neutron beam. The neutron capture reaction with a boron-10 nucleus yields high linear energy transfer (LET) particles, alpha and 7Li, with a range of 5 to 9 µm. These particles can only travel very short distances and release their damaging energy directly into the cells containing the boron compound. We aimed to evaluate proliferation, apoptosis and extracellular matrix (ECM) modifications of B16F10 melanoma and normal human melanocytes after BNCT. The amounts of soluble collagen and Hsp47, indicating collagen synthesis in the ECM, as well as the cellular markers of apoptosis, were investigated. BNCT decreased proliferation, altered the ECM by decreasing collagen synthesis and induced apoptosis by regulating Bcl-2/Bax in melanoma. Additionally, BNCT also increased the levels of TNF receptor and the cleaved caspases 3, 7, 8 and 9 in melanoma. These results suggest that multiple pathways related to cell death and cell cycle arrest are involved in the treatment of melanoma by BNCT. PMID:23527236

  18. Engagement of αIIbβ3 (GPIIb/IIIa) with ανβ3 Integrin Mediates Interaction of Melanoma Cells with Platelets

    PubMed Central

    Lonsdorf, Anke S.; Krämer, Björn F.; Fahrleitner, Manuela; Schönberger, Tanja; Gnerlich, Stephan; Ring, Sabine; Gehring, Sarah; Schneider, Stefan W.; Kruhlak, Michael J.; Meuth, Sven G.; Nieswandt, Bernhard; Gawaz, Meinrad; Enk, Alexander H.; Langer, Harald F.

    2012-01-01

    A mutual relationship exists between metastasizing tumor cells and components of the coagulation cascade. The exact mechanisms as to how platelets influence blood-borne metastasis, however, remain poorly understood. Here, we used murine B16 melanoma cells to observe functional aspects of how platelets contribute to the process of hematogenous metastasis. We found that platelets interfere with a distinct step of the metastasis cascade, as they promote adhesion of melanoma cells to the endothelium in vitro under shear conditions. Constitutively active platelet receptor GPIIb/IIIa (integrin αIIbβ3) expressed on Chinese hamster ovary cells promoted melanoma cell adhesion in the presence of fibrinogen, whereas blocking antibodies to aνβ3 integrin on melanoma cells or to GPIIb/IIIa significantly reduced melanoma cell adhesion to platelets. Furthermore, using intravital microscopy, we observed functional platelet-melanoma cell interactions, as platelet depletion resulted in significantly reduced melanoma cell adhesion to the injured vascular wall in vivo. Using a mouse model of hematogenous metastasis to the lung, we observed decreased metastasis of B16 melanoma cells to the lung by treatment with a mAb blocking the aν subunit of aνβ3 integrin. This effect was significantly reduced when platelets were depleted in vivo. Thus, the engagement of GPIIb/IIIa with aνβ3 integrin interaction mediates tumor cell-platelet interactions and highlights how this interaction is involved in hematogenous tumor metastasis. PMID:22102277

  19. Visualization of melanoma tumor with lectin-conjugated rare-earth doped fluoride nanocrystals

    PubMed Central

    Dumych, Tetiana; Lutsyk, Maxym; Banski, Mateusz; Yashchenko, Antonina; Sojka, Bartlomiej; Horbay, Rostyslav; Lutsyk, Alexander; Stoika, Rostyslav; Misiewicz, Jan; Podhorodecki, Artur; Bilyy, Rostyslav

    2014-01-01

    Aim To develop specific fluorescent markers for melanoma tumor visualization, which would provide high selectivity and reversible binding pattern, by the use of carbohydrate-recognizing proteins, lectins, combined with the physical ability for imaging deep in the living tissues by utilizing red and near infrared fluorescent properties of specific rare-earth doped nanocrystals (NC). Methods B10F16 melanoma cells were inoculated to C57BL/6 mice for inducing experimental melanoma tumor. Tumors were removed and analyzed by lectin-histochemistry using LABA, PFA, PNA, HPA, SNA, GNA, and NPL lectins and stained with hematoxylin and eosin. NPL lectin was conjugated to fluorescent NaGdF4:Eu3+-COOH nanoparticles (5 nm) via zero length cross-linking reaction, and the conjugates were purified from unbound substances and then used for further visualization of histological samples. Fluorescent microscopy was used to visualize NPL-NaGdF4:Eu3+ with the fluorescent emission at 600-720 nm range. Results NPL lectin selectively recognized regions of undifferentiated melanoblasts surrounding neoangiogenic foci inside melanoma tumor, PNA lectin recognized differentiated melanoblasts, and LCA and WGA were bound to tumor stroma regions. NPL-NaGdF4:Eu3+ conjugated NC were efficiently detecting newly formed regions of melanoma tumor, confirmed by fluorescent microscopy in visible and near infrared mode. These conjugates possessed high photostability and were compatible with convenient xylene-based mounting systems and preserved intensive fluorescent signal at samples storage for at least 6 months. Conclusion NPL lectin-NaGdF4:Eu3+ conjugated NC permitted distinct identification of contours of the melanoma tissue on histological sections using red excitation at 590-610 nm and near infrared emission of 700-720 nm. These data are of potential practical significance for development of glycans-conjugated nanoparticles to be used for in vivo visualization of melanoma tumor. PMID:24891277

  20. Nitric oxide-releasing nanoparticles: synthesis, characterization, and cytotoxicity to tumorigenic cells

    NASA Astrophysics Data System (ADS)

    Pelegrino, Milena T.; Silva, Letícia C.; Watashi, Carolina M.; Haddad, Paula S.; Rodrigues, Tiago; Seabra, Amedea B.

    2017-02-01

    Nitric oxide (NO) is involved in several biological processes, including toxicity against tumor cells. The aim of this study was to synthesize, characterize, and evaluate the cytotoxicity of NO-releasing chitosan nanoparticles. A thiol-containing molecule, mercaptosuccinic acid (MSA), was encapsulated (encapsulation efficiency of 99%) in chitosan/sodium tripolyphosphate nanoparticles (CS NPs). The obtained nanoparticles showed an average hydrodynamic size of 108.40 ± 0.96 nm and polydispersity index of 0.26 ± 0.01. MSA-CS NPs were nitrosated leading to S-nitroso-MSA-CS NPs, which act as NO donor. The cytotoxicity of CS NPs, MSA-CS NPs, and S-nitroso-MSA-CS NPs were evaluated in several tumor cells, including human hepatocellular carcinoma (HepG2), mouse melanoma (B16F10), and human chronic myeloid leukemia (K562) cell lines and Lucena-1, a vincristine-resistant K562 cell line. Both CS NPs and MSA-CS NPs did not cause toxic effects in these cells, whereas S-nitroso-MSA-CS NPs caused potent cytotoxic effects in all the tested tumor cell lines. The half-maximal inhibitory concentration values of S-nitroso-MSA-CS NPs were 19.7, 10.5, 22.8, and 27.8 μg·mL-1 for HepG2, B16F10, K562, and Lucena-1 cells, respectively. In contrast, S-nitroso-MSA-CS NPs exhibited lower cytotoxic to non-tumorigenic melanocytes (Melan-A) when compared with melanoma B16F10. Therefore, the results highlight the potential use of NO-releasing CS NPs in antitumor chemotherapy.

  1. Preparation and characterization of a novel Al(18)F-NOTA-BZA conjugate for melanin-targeted imaging of malignant melanoma.

    PubMed

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

    2016-08-15

    Melanin is an attractive target for the diagnosis and treatment of malignant melanoma. Previous studies have demonstrated the specific binding ability of benzamide moiety to melanin. In this study, we developed a novel (18)F-labeled NOTA-benzamide conjugate, Al(18)F-NOTA-BZA, which can be synthesized in 30min with a radiochemical yield of 20-35% and a radiochemical purity of >95%. Al(18)F-NOTA-BZA is highly hydrophilic (logP=-1.96) and shows good in vitro stability. Intravenous administration of Al(18)F-NOTA-BZA in two melanoma-bearing mouse models revealed highly specific uptake in B16F0 melanotic melanoma (6.67±0.91 and 1.50±0.26%ID/g at 15 and 120min p.i., respectively), but not in A375 amelanotic melanoma (0.87±0.21 and 0.24±0.09%ID/g at 15 and 120min p.i., respectively). The clearance from most normal tissues was fast. A microPET scan of Al(18)F-NOTA-BZA-injected mice also displayed high-contrast tumor images as compared with normal organs. Owing to the favorable in vivo distribution of Al(18)F-NOTA-BZA after intravenous administration, the estimated absorption dose was low in all normal organs and tissues. The melanin-specific binding ability, sustained tumor retention, fast normal tissues clearance and thelow projected human dosimetry supported that Al(18)F-NOTA-BZA is a very promising melanin-specific PET probe for melanin-positive melanoma. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Cu-64-Labeled Lactam Bridge-Cyclized α-MSH Peptides for PET Imaging of Melanoma

    PubMed Central

    Guo, Haixun; Miao, Yubin

    2012-01-01

    The purpose of this study was to examine and compare the melanoma targeting and imaging properties of 64Cu-NOTA-GGNle-CycMSHhex {64Cu-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and 64Cu-DOTA-GGNle-CycMSHhex {64Cu-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-GGNle-CycMSHhex}. Two lactam bridge-cyclized peptides, NOTA-GGNle-CycMSHhex and DOTA-GGNle-CycMSHhex, were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSHhex was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSHhex. The melanoma targeting and imaging properties of 64Cu-NOTA-GGNle-CycMSHhex and 64Cu-DOTA-GGNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSHhex and DOTA-GGNle-CycMSHhex displayed comparable MC1 receptor binding affinities (1.6 vs. 2.1 nM). The substitution of DOTA with NOTA dramatically increased the melanoma uptake and decreased the renal and liver uptake of 64Cu-NOTA-GGNle-CycMSHhex. The tumor uptake of 64Cu-NOTA-GGNle-CycMSHhex was between 12.39 ± 1.61 and 12.71 ± 2.68 % ID/g at 0.5, 2 and 4 h post-injection. The accumulation of 64Cu-NOTA-GGNle-CycMSHhex activity in normal organs was lower than 1.02 % ID/g except for the kidneys 2, 4 and 24 h post-injection. The tumor/liver uptake ratios of 64Cu-NOTA-GGNle-CycMSHhex were 17.96, 16.95 and 8.02, whereas the tumor/kidney uptake ratios of 64Cu-NOTA-GGNle-CycMSHhex were 2.52, 3.60 and 5.74 at 2, 4 and 24 h post-injection, respectively. Greater than 91% of the injected radioactivity cleared through the urinary system by 2 h post-injection. The substitution of DOTA with NOTA resulted in a dramatic increase in melanoma uptake and decrease in renal and liver uptake of 64Cu-NOTA-GGNle-CycMSHhex compared to 64Cu-DOTA-GGNle-CycMSHhex. High melanoma uptake coupled with low accumulation in non-target organs suggested 64Cu-NOTA-GGNle-CycMSHhex as a lead

  3. Preparation of coffee oil-algae oil-based nanoemulsions and the study of their inhibition effect on UVA-induced skin damage in mice and melanoma cell growth.

    PubMed

    Yang, Chu-Ching; Hung, Chi-Feng; Chen, Bing-Huei

    2017-01-01

    Coffee grounds, a waste by-product generated after making coffee, contains approximately 15% coffee oil which can be used as a raw material in cosmetics. Algae oil rich in docosahexaenoic acid (DHA) has been demonstrated to possess anticancer and anti-inflammation functions. The objectives of this study were to develop a gas chromatography-mass spectrometry (GC-MS) method for the determination of fatty acids in coffee oil and algae oil and prepare a nanoemulsion for studying its inhibition effect on ultraviolet A-induced skin damage in mice and growth of melanoma cells B16-F10. A total of 8 and 5 fatty acids were separated and quantified in coffee oil and algae oil by GC-MS, respectively, with linoleic acid (39.8%) dominating in the former and DHA (33.9%) in the latter. A nanoemulsion with a particle size of 30 nm, zeta potential -72.72 mV, and DHA encapsulation efficiency 100% was prepared by using coffee oil, algae oil, surfactant (20% Span 80 and 80% Tween 80), and deionized water. Differential scanning calorimetry (DSC) analysis revealed a high stability of nanoemulsion when heated up to 110°C at a pH 6, whereas no significant changes in particle size distribution and pH occurred over a 90-day storage period at 4°C. Animal experiments showed that a dose of 0.1% coffee oil-algae oil nanoemulsion was effective in mitigating trans-epidermal water loss, skin erythema, melanin formation, and subcutaneous blood flow. Cytotoxicity test implied effective inhibition of melanoma cell growth by nanoemulsion with an IC 50 value of 26.5 µg/mL and the cell cycle arrested at G2/M phase. A dose-dependent upregulation of p53, p21, cyclin B, and cyclin A expressions and downregulation of CDK1 and CDK2 occurred. Also, both Bax and cytochrome c expressions were upregulated and bcl-2 expression downregulated, accompanied by a rise in caspase-3, caspase-8, and caspase-9 activities for apoptosis execution. Collectively, the apoptosis pathway of melanoma cells B16-F10 may involve

  4. Preparation of coffee oil-algae oil-based nanoemulsions and the study of their inhibition effect on UVA-induced skin damage in mice and melanoma cell growth

    PubMed Central

    Chen, Bing-Huei

    2017-01-01

    Coffee grounds, a waste by-product generated after making coffee, contains approximately 15% coffee oil which can be used as a raw material in cosmetics. Algae oil rich in docosahexaenoic acid (DHA) has been demonstrated to possess anticancer and anti-inflammation functions. The objectives of this study were to develop a gas chromatography-mass spectrometry (GC-MS) method for the determination of fatty acids in coffee oil and algae oil and prepare a nanoemulsion for studying its inhibition effect on ultraviolet A-induced skin damage in mice and growth of melanoma cells B16-F10. A total of 8 and 5 fatty acids were separated and quantified in coffee oil and algae oil by GC-MS, respectively, with linoleic acid (39.8%) dominating in the former and DHA (33.9%) in the latter. A nanoemulsion with a particle size of 30 nm, zeta potential −72.72 mV, and DHA encapsulation efficiency 100% was prepared by using coffee oil, algae oil, surfactant (20% Span 80 and 80% Tween 80), and deionized water. Differential scanning calorimetry (DSC) analysis revealed a high stability of nanoemulsion when heated up to 110°C at a pH 6, whereas no significant changes in particle size distribution and pH occurred over a 90-day storage period at 4°C. Animal experiments showed that a dose of 0.1% coffee oil-algae oil nanoemulsion was effective in mitigating trans-epidermal water loss, skin erythema, melanin formation, and subcutaneous blood flow. Cytotoxicity test implied effective inhibition of melanoma cell growth by nanoemulsion with an IC50 value of 26.5 µg/mL and the cell cycle arrested at G2/M phase. A dose-dependent upregulation of p53, p21, cyclin B, and cyclin A expressions and downregulation of CDK1 and CDK2 occurred. Also, both Bax and cytochrome c expressions were upregulated and bcl-2 expression downregulated, accompanied by a rise in caspase-3, caspase-8, and caspase-9 activities for apoptosis execution. Collectively, the apoptosis pathway of melanoma cells B16-F10 may involve

  5. ABCB5 promotes melanoma metastasis through enhancing NF-κB p65 protein stability.

    PubMed

    Wang, Shenghao; Tang, Li; Lin, Junyu; Shen, Zhongliang; Yao, Yikun; Wang, Wei; Tao, Shuai; Gu, Chenjian; Ma, Jie; Xie, Youhua; Liu, Yanfeng

    2017-10-07

    Melanoma is the most aggressive type of skin cancer. Melanoma has an extremely poor prognosis because of its high potential for vascular invasion, metastasis and recurrence. The mechanism of melanoma metastasis is not well understood. ATP-binding cassette sub-family B member 5 (ABCB5) plays a key role in melanoma growth. However, it is uncertain what function ABCB5 may exert in melanoma metastasis. In this report, we for the first time demonstrate ABCB5 as a crucial factor that promotes melanoma metastasis. ABCB5 positive (ABCB5 + ) malignant melanoma initiating cells (MMICs) display a higher metastatic potential compared with ABCB5 negative (ABCB5 - ) melanoma subpopulation. Knockdown of ABCB5 expression reduces melanoma cell migration and invasion in vitro and melanoma pulmonary metastasis in tumor xenograft mice. ABCB5 and NF-κB p65 expression levels are positively correlated in both melanoma tissues and cell lines. Consequently, ABCB5 activates the NF-κB pathway by inhibiting p65 ubiquitination to enhance p65 protein stability. Our finding highlights ABCB5 as a novel pro-metastasis factor and provides a potential therapeutic target for melanoma. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. A New Concept of Enhancing Immuno-Chemotherapeutic Effects Against B16F10 Tumor via Systemic Administration by Taking Advantages of the Limitation of EPR Effect

    PubMed Central

    Yang, Yuting; Tai, Xiaowei; Shi, Kairong; Ruan, Shaobo; Qiu, Yue; Zhang, Zhirong; Xiang, Bing; He, Qin

    2016-01-01

    The enhanced permeability and retention (EPR) effect has been comfortably accepted, and extensively assumed as a keystone in the research on tumor-targeted drug delivery system. Due to the unsatisfied tumor-targeting efficiency of EPR effect being one conspicuous drawback, nanocarriers that merely relying on EPR effect are difficult to access the tumor tissue and consequently trigger efficient tumor therapy in clinic. In the present contribution, we break up the shackles of EPR effect on nanocarriers thanks to their universal distribution characteristic. We successfully design a paclitaxel (PTX) and alpha-galactosylceramide (αGC) co-loaded TH peptide (AGYLLGHINLHHLAHL(Aib)HHIL-Cys) -modified liposome (PTX/αGC-TH-Lip) and introduce a new concept of immuno-chemotherapy combination via accumulation of these liposomes at both spleen and tumor sites naturally and simultaneously. The PTX-initiated cytotoxicity attacks tumor cells at tumor sites, meanwhile, the αGC-triggered antitumor immune response emerges at spleen tissue. Different to the case that liposomes are loaded with sole drug, in this concept two therapeutic processes effectively reinforce each other, thereby elevating the tumor therapy efficiency significantly. The data demonstrates that the PTX/αGC-TH-Lip not only possess therapeutic effect against highly malignant B16F10 melanoma tumor, but also adjust the in vivo immune status and induce a more remarkable systemic antitumor immunity that could further suppress the growth of tumor at distant site. This work exhibits the capability of the PTX/αGC-TH-Lip in improving immune-chemotherapy against tumor after systemic administration. PMID:27698946

  7. Targeting protein kinase-b3 (akt3) signaling in melanoma.

    PubMed

    Madhunapantula, SubbaRao V; Robertson, Gavin P

    2017-03-01

    Deregulated Akt activity leading to apoptosis inhibition, enhanced proliferation and drug resistance has been shown to be responsible for 35-70% of advanced metastatic melanomas. Of the three isoforms, the majority of melanomas have elevated Akt3 expression and activity. Hence, potent inhibitors targeting Akt are urgently required, which is possible only if (a) the factors responsible for the failure of Akt inhibitors in clinical trials is known; and (b) the information pertaining to synergistically acting targeted therapeutics is available. Areas covered: This review provides a brief introduction of the PI3K-Akt signaling pathway and its role in melanoma development. In addition, the functional role of key Akt pathway members such as PRAS40, GSK3 kinases, WEE1 kinase in melanoma development are discussed together with strategies to modulate these targets. Efficacy and safety of Akt inhibitors is also discussed. Finally, the mechanism(s) through which Akt leads to drug resistance is discussed in this expert opinion review. Expert opinion: Even though Akt play key roles in melanoma tumor progression, cell survival and drug resistance, many gaps still exist that require further understanding of Akt functions, especially in the (a) metastatic spread; (b) circulating melanoma cells survival; and (c) melanoma stem cells growth.

  8. A Novel Inhibitor of STAT3 Activation Is Efficacious Against Established Central Nervous System Melanoma and Inhibits Regulatory T Cells

    PubMed Central

    Kong, Ling-Yuan; Abou-Ghazal, Mohamed K.; Wei, Jun; Chakraborty, Arup; Sun, Wei; Qiao, Wei; Fuller, Gregory N.; Fokt, Izabela; Grimm, Elizabeth A.; Schmittling, Robert J.; Archer, Gary E.; Sampson, John H.; Priebe, Waldemar; Heimberger, Amy B.

    2008-01-01

    Purpose Activation of STAT3 has been identified as a central mediator of melanoma growth and metastasis. We hypothesized that WP1066, a novel STAT3 blockade agent, has marked antitumor activity, even against the melanoma metastasis to brain, a site typically refractory to therapies. Experimental Design The antitumor activities and related mechanisms of WP1066 were investigated both in vitro on melanoma cell lines and in vivo on mice with subcutaneously syngeneic melanoma or with intracerebral melanoma tumors. Results WP1066 achieved an IC50 of 1.6 μM, 2.3 μM, and 1.5 μM against melanoma cell line A375, B16 and B16EGFRvIII, respectively. WP1066 suppressed the phosphorylation of JAK2 and STAT3 (Tyr705) in these cells. Tumor growth in mice with subcutaneously established syngeneic melanoma was markedly inhibited by WP1066 compared with that in controls. Long-term survival (>78 days) was observed in 80% of mice with established intracerebral syngeneic melanoma treated with 40 mg/kg of WP1066 in contrast to control mice who survived for a median of 15 days. Although WP1066 did not induce immunological memory or enhance humoral responses to EGFRvIII, this compound reduced the production of immunosuppressive cytokines and chemokines (TGF-β, RANTES, MCP-1, VEGF), markedly inhibited natural and inducible Treg proliferation, and significantly increased cytotoxic immune responses of T cells. Conclusions The antitumor cytotoxic effects of WP1066 and its ability to induce antitumor immune responses suggest that this compound has potential for the effective treatment of melanoma metastatic to brain. PMID:18794085

  9. Development of a (99m)Tc-labeled lactam bridge-cyclized alpha-MSH derivative peptide as a possible single photon imaging agent for melanoma tumors.

    PubMed

    Shamshirian, Danial; Erfani, Mostafa; Beiki, Davood; Fallahi, Babak; Shafiei, Mohammad

    2015-10-01

    Melanocortin-1 (MC1) receptor is an attractive melanoma-specific target which has been used for melanoma imaging and therapy. In this work, a new lactam bridge α-MSH analog was labeled with (99m)Tc via HYNIC and EDDA/tricine as coligands including gamma aminobutyric acid (GABA) as a three carbon chain spacer between HYNIC and the N-terminus of the cyclic peptide. Also, stability in human serum, receptor bound internalization, in vivo tumor uptake, and tissue biodistribution were thoroughly investigated. HYNIC-GABA-Nle-CycMSHhept was synthesized using a standard Fmoc strategy. Labeling was performed at 95 °C and analysis involved instant thin layer chromatography and high performance liquid chromatography methods. The receptor bound internalization rate was studied in MC1 receptor expressing B16/F10 cells. Biodistribution of radiopeptide was studied in nude mice bearing B16/F10 tumor. Labeling yield of >98 % (n = 3) was obtained corresponding to a specific activity of 81 MBq/nmol. Peptide conjugate showed efficient stability in the presence of human serum. The radioligand showed specific internalization into B16/F10 cells (12.45 ± 1.1 % at 4 h). In biodistribution studies, a receptor-specific uptake was observed in MC1 receptor-positive organs so that after 2 h the uptake in mouse tumor was 5.10 ± 0.08 % ID/g, while low accumulation in the kidney uptake was observed (4.58 ± 0.68 % ID/g at 2 h after injection). The obtained results show that the presented new designed labeled peptide conjugate may be a suitable candidate for diagnosis of malignant tumors.

  10. Low concentrations of Rhodamine-6G selectively destroy tumor cells and improve survival of melanoma transplanted mice.

    PubMed

    Kutushov, M; Gorelik, O

    2013-01-01

    Rhodamine-6G is a fluorescent dye binding to mitochondria, thus reducing the intact mitochondria number and inhibiting mitochondrial metabolic activity. Resultantly, the respiratory chain functioning becomes blocked, the cell "suffocated" and eventually destroyed. Unlike normal cells, malignant cells demonstrate a priori reduced mitochondrial numbers and aberrant metabolism. Therefore, a turning point might exist, when Rhodamine-induced loss of active mitochondria would selectively destroy malignant, but spare normal cells. Various malignant vs. non-malignant cell lines were cultured with Rhodamine-6G at different concentrations. In addition, C57Bl mice were implanted with B16-F10 melanoma and treated with Rhodamine-6G at different dosage/time regimens. Viability and proliferation of cultured tumor cells were time and dose-dependently inhibited, up to 90%, by Rhodamine-6G, with profound histological signs of cell death. By contrast, inhibition of normal control cell proliferation hardly exceeded 15-17%. Melanoma-transplanted mice receiving Rhodamine-6G demonstrated prolonged survival, improved clinical parameters, inhibited tumor growth and metastases count, compared to their untreated counterparts. Twice-a-week 10-6M Rhodamine-6G regimen yielded the most prominent results. We conclude that malignant, but not normal, cells are selectively destroyed by low doses of Rhodamine-6G. In vivo, such treatment selectively suppresses tumor progression and dissemination, thus improving prognosis. We suggest that selective anti-tumor properties of Rhodamine-6G are based on unique physiologic differences in energy metabolism between malignant and normal cells. If found clinically relevant, low concentrations of Rhodamine-6G might be useful for replacing, or backing up, more aggressive nonselective chemotherapeutic compounds.

  11. Electroporation driven delivery of both an IL-12 expressing plasmid and cisplatin synergizes to inhibit B16 melanoma tumor growth through an NK cell mediated tumor killing mechanism.

    PubMed

    Kim, Ha; Sin, Jeong-Im

    2012-11-01

    Combined therapy using chemotherapeutic drugs and immunotherapeutics offers some promise for treating patients with cancer. In this study, we evaluated whether cisplatin delivered by intratumoral (IT)-electroporation (EP) might enhance antitumor activity against established B16 melanoma and whether further addition of intramuscular (IM)-EP of IL-12 cDNA to IT-EP of cisplatin might augment antitumor therapeutic activity, with a focus on the underlining antitumor mechanism(s). When tumor (7 mm)-bearing animals were treated locally with cisplatin by IT-EP, they showed tumor growth inhibition significantly more than those without IT-EP. Moreover, IL-12 cDNA delivered by IM-EP was also able to inhibit tumor growth significantly more than control vector delivery. This tumor growth inhibition was mediated by NK cells, but not CD4+ T or CD8+ T cells, as determined by immune cell subset depletion and IFN-γ induction. Moreover, concurrent therapy using IT-EP of cisplatin plus IM-EP of IL-12 cDNA displayed antitumor therapeutic synergy. This therapeutic synergy appeared to be mediated by increased sensitivity of cisplatin-treated tumors to NK cell-mediated tumor killing. Taken together, these data support that cisplatin delivery by IT-EP plus IL-12 gene delivery by IM-EP are more effective at inducing antitumor therapeutic responses through increased sensitivity of cisplatin-treated tumors to NK cell-mediated tumor killing. This combined approach might have some implication for treating melanoma in patients.

  12. Melanoma exosomes deliver a complex biological payload that upregulates PTPN11 to suppress T lymphocyte function

    PubMed Central

    Wu, Yueting; Deng, Wentao; McGinley, Emily Chambers; Klinke, David J.

    2017-01-01

    Summary As exosomes are emerging as a new mode of intercellular communication, we hypothesized that the payload contained within exosomes is shaped by somatic evolution. To test this, we assayed the impact on primary CD8+ T cell function, a key mechanism for anti-tumor immunity, of exosomes derived from three melanoma-related cell lines. While morphologically similar, exosomes from each cell line were functionally different, as B16F0 exosomes dose-dependently suppressed T cell proliferation. In contrast, Cloudman S91 exosomes promoted T cell proliferation and Melan-A exosomes had a negligible effect on primary CD8+ T cells. Mechanistically, transcript profiling suggested that exosomal mRNA is enriched for full-length mRNAs that target immune-related pathways. Interestingly, B16F0 exosomes were unique in that they contained both protein and mRNA for Ptpn11, which inhibited T cell proliferation. Collectively, the results suggest that upregulation of PTPN11 by B16F0 exosomes to tumor infiltrating lymphocytes would bypass the extracellular control of the immune checkpoints. PMID:27930879

  13. Anti-tumor therapy with macroencapsulated endostatin producer cells

    PubMed Central

    2010-01-01

    Background Theracyte is a polytetrafluoroethylene membrane macroencapsulation system designed to induce neovascularization at the tissue interface, protecting the cells from host's immune rejection, thereby circumventing the problem of limited half-life and variation in circulating levels. Endostatin is a potent inhibitor of angiogenesis and tumor growth. Continuous delivery of endostatin improves the efficacy and potency of the antitumoral therapy. The purpose of this study was to determine whether recombinant fibroblasts expressing endostatin encapsulated in Theracyte immunoisolation devices can be used for delivery of this therapeutic protein for treatment of mice bearing B16F10 melanoma and Ehrlich tumors. Results Mice were inoculated subcutaneously with melanoma (B16F10 cells) or Ehrlich tumor cells at the foot pads. Treatment began when tumor thickness had reached 0.5 mm, by subcutaneous implantation of 107 recombinant encapsulated or non-encapsulated endostatin producer cells. Similar melanoma growth inhibition was obtained for mice treated with encapsulated or non-encapsulated endostatin-expressing cells. The treatment of mice bearing melanoma tumor with encapsulated endostatin-expressing cells was decreased by 50.0%, whereas a decrease of 56.7% in tumor thickness was obtained for mice treated with non-encapsulated cells. Treatment of Ehrlich tumor-bearing mice with non-encapsulated endostatin-expressing cells reduced tumor thickness by 52.4%, whereas lower tumor growth inhibition was obtained for mice treated with encapsulated endostatin-expressing cells: 24.2%. Encapsulated endostatin-secreting fibroblasts failed to survive until the end of the treatment. However, endostatin release from the devices to the surrounding tissues was confirmed by immunostaining. Decrease in vascular structures, functional vessels and extension of the vascular area were observed in melanoma microenvironments. Conclusions This study indicates that immunoisolation devices

  14. Anti-tumor therapy with macroencapsulated endostatin producer cells.

    PubMed

    Rodrigues, Danielle B; Chammas, Roger; Malavasi, Natália V; da Costa, Patrícia L N; Chura-Chambi, Rosa M; Balduino, Keli N; Morganti, Ligia

    2010-03-02

    Theracyte is a polytetrafluoroethylene membrane macroencapsulation system designed to induce neovascularization at the tissue interface, protecting the cells from host's immune rejection, thereby circumventing the problem of limited half-life and variation in circulating levels. Endostatin is a potent inhibitor of angiogenesis and tumor growth. Continuous delivery of endostatin improves the efficacy and potency of the antitumoral therapy. The purpose of this study was to determine whether recombinant fibroblasts expressing endostatin encapsulated in Theracyte immunoisolation devices can be used for delivery of this therapeutic protein for treatment of mice bearing B16F10 melanoma and Ehrlich tumors. Mice were inoculated subcutaneously with melanoma (B16F10 cells) or Ehrlich tumor cells at the foot pads. Treatment began when tumor thickness had reached 0.5 mm, by subcutaneous implantation of 107 recombinant encapsulated or non-encapsulated endostatin producer cells. Similar melanoma growth inhibition was obtained for mice treated with encapsulated or non-encapsulated endostatin-expressing cells. The treatment of mice bearing melanoma tumor with encapsulated endostatin-expressing cells was decreased by 50.0%, whereas a decrease of 56.7% in tumor thickness was obtained for mice treated with non-encapsulated cells. Treatment of Ehrlich tumor-bearing mice with non-encapsulated endostatin-expressing cells reduced tumor thickness by 52.4%, whereas lower tumor growth inhibition was obtained for mice treated with encapsulated endostatin-expressing cells: 24.2%. Encapsulated endostatin-secreting fibroblasts failed to survive until the end of the treatment. However, endostatin release from the devices to the surrounding tissues was confirmed by immunostaining. Decrease in vascular structures, functional vessels and extension of the vascular area were observed in melanoma microenvironments. This study indicates that immunoisolation devices containing endostatin

  15. Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells

    PubMed Central

    Si, Lingling; Yan, Xinyan; Hao, Wenjin; Ma, Xiaoyi; Ren, Huanhuan; Ren, Boxue; Li, Defang; Dong, Zhengping; Zheng, Qiusheng

    2018-01-01

    The aim of the present study was to determine the effects of Licochalcone D (LD) on the apoptosis and migration and invasion in human melanoma A375 cells. Cell proliferation was determined by sulforhodamine B assay. Apoptosis was assessed by Hoechst 33258 and Annexin V-FITC/PI staining and JC-1 assay. Total intracellular reactive oxygen species (ROS) was examined by DCFH-DA. Wound healing and Transwell assays were used to detect migration and invasion of the cells. The activities of matrix metalloproteinase (MMP-2 and MMP-9) were assessed via gelatin zymography. Tumor growth in vivo was evaluated in C57BL/6 mice. RT-PCR, qPCR, ELISA and western blot analysis were utilized to measure the mRNA and protein levels. Our results showed that LD inhibited the proliferation of A375 and SK-MEL-5 cells in a concentration-dependent manner. After treatment with LD, A375 cells displayed obvious apoptotic characteristics, and the number of apoptotic cells was significantly increased. Pro-apoptotic protein Bax, caspase-9 and caspase-3 were upregulated, while anti-apoptotic protein Bcl-2 was downregulated in the LD-treated cells. Meanwhile, LD induced the loss of mitochondrial membrane potential (ΔΨm) and increased the level of ROS. ROS production was inhibited by the co-treatment of LD and free radical scavenger N-acetyl-cysteine (NAC). Furthermore, LD also blocked A375 cell migration and invasion in vitro which was associated with the downregulation of MMP-9 and MMP-2. Finally, intragastric administration of LD suppressed tumor growth in the mouse xenograft model of murine melanoma B16F0 cells. These results suggest that LD may be a potential drug for human melanoma treatment by inhibiting proliferation, inducing apoptosis via the mitochondrial pathway and blocking cell migration and invasion. PMID:29565458

  16. Caffeic acid phenethyl ester suppresses melanoma tumor growth by inhibiting PI3K/AKT/XIAP pathway.

    PubMed

    Pramanik, Kartick C; Kudugunti, Shashi K; Fofaria, Neel M; Moridani, Majid Y; Srivastava, Sanjay K

    2013-09-01

    Melanoma is highly metastatic and resistant to chemotherapeutic drugs. Our previous studies have demonstrated that caffeic acid phenethyl ester (CAPE) suppresses the growth of melanoma cells and induces reactive oxygen species generation. However, the exact mechanism of the growth suppressive effects of CAPE was not clear. Here, we determined the potential mechanism of CAPE against melanoma in vivo and in vitro. Administration of 10 mg/kg/day CAPE substantially suppressed the growth of B16F0 tumor xenografts in C57BL/6 mice. Tumors from CAPE-treated mice showed reduced phosphorylation of phosphoinositide 3-kinase, AKT, mammalian target of rapamycin and protein level of X-linked inhibitor of apoptosis protein (XIAP) and enhanced the cleavage of caspase-3 and poly (ADP ribose) polymerase. In order to confirm the in vivo observations, melanoma cells were treated with CAPE. CAPE treatment suppressed the activating phosphorylation of phosphoinositide 3-kinase at Tyr 458, phosphoinositide-dependent kinase-1 at Ser 241, mammalian target of rapamycin at Ser 2448 and AKT at Ser 473 in B16F0 and SK-MEL-28 cells in a concentration and time-dependent study. Furthermore, the expression of XIAP, survivin and BCL-2 was downregulated by CAPE treatment in both cell lines. Significant apoptosis was observed by CAPE treatment as indicated by cleavage of caspase-3 and poly (ADP ribose) polymerase. AKT kinase activity was inhibited by CAPE in a concentration-dependent manner. CAPE treatment increased the nuclear translocation of XIAP, indicating increased apoptosis in melanoma cells. To confirm the involvement of reactive oxygen species in the inhibition of AKT/XIAP pathway, cells were treated with antioxidant N-acetyl-cysteine (NAC) prior to CAPE treatment. Our results indicate that NAC blocked CAPE-mediated AKT/XIAP inhibition and protected the cells from apoptosis. Because AKT regulates XIAP, their interaction was examined by immunoprecipitation studies. Our results show that CAPE

  17. Caffeic acid phenethyl ester suppresses melanoma tumor growth by inhibiting PI3K/AKT/XIAP pathway

    PubMed Central

    Srivastava, Sanjay K.

    2013-01-01

    Melanoma is highly metastatic and resistant to chemotherapeutic drugs. Our previous studies have demonstrated that caffeic acid phenethyl ester (CAPE) suppresses the growth of melanoma cells and induces reactive oxygen species generation. However, the exact mechanism of the growth suppressive effects of CAPE was not clear. Here, we determined the potential mechanism of CAPE against melanoma in vivo and in vitro. Administration of 10 mg/kg/day CAPE substantially suppressed the growth of B16F0 tumor xenografts in C57BL/6 mice. Tumors from CAPE-treated mice showed reduced phosphorylation of phosphoinositide 3-kinase, AKT, mammalian target of rapamycin and protein level of X-linked inhibitor of apoptosis protein (XIAP) and enhanced the cleavage of caspase-3 and poly (ADP ribose) polymerase. In order to confirm the in vivo observations, melanoma cells were treated with CAPE. CAPE treatment suppressed the activating phosphorylation of phosphoinositide 3-kinase at Tyr 458, phosphoinositide-dependent kinase-1 at Ser 241, mammalian target of rapamycin at Ser 2448 and AKT at Ser 473 in B16F0 and SK-MEL-28 cells in a concentration and time-dependent study. Furthermore, the expression of XIAP, survivin and BCL-2 was downregulated by CAPE treatment in both cell lines. Significant apoptosis was observed by CAPE treatment as indicated by cleavage of caspase-3 and poly (ADP ribose) polymerase. AKT kinase activity was inhibited by CAPE in a concentration-dependent manner. CAPE treatment increased the nuclear translocation of XIAP, indicating increased apoptosis in melanoma cells. To confirm the involvement of reactive oxygen species in the inhibition of AKT/XIAP pathway, cells were treated with antioxidant N-acetyl-cysteine (NAC) prior to CAPE treatment. Our results indicate that NAC blocked CAPE-mediated AKT/XIAP inhibition and protected the cells from apoptosis. Because AKT regulates XIAP, their interaction was examined by immunoprecipitation studies. Our results show that CAPE

  18. 64Cu-labeled alpha-melanocyte-stimulating hormone analog for microPET imaging of melanocortin 1 receptor expression.

    PubMed

    Cheng, Zhen; Xiong, Zhengming; Subbarayan, Murugesan; Chen, Xiaoyuan; Gambhir, Sanjiv Sam

    2007-01-01

    The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor (melanocortin type 1 receptor, or MC1R) plays an important role in the development and growth of melanoma cells. It was found that MC1R was overexpressed on most murine and human melanoma, making it a promising molecular target for melanoma imaging and therapy. Radiolabeled alpha-MSH peptide and its analogs that can specifically bind with MC1R have been extensively explored for developing novel agents for melanoma detection and radionuclide therapy. The goal of this study was to evaluate a 64Cu-labeled alpha-MSH analog, Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys(DOTA)-NH2 (DOTA-NAPamide), as a potential molecular probe for microPET imaging of melanoma and MC1R expression in melanoma xenografted mouse models. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated NAPamide was synthesized and radiolabeled with 64Cu (t1/2=12 h) in NH4OAc (0.1 M; pH 5.5) buffered solution for 60 min at 50 degrees C. Cell culture studies reveal rapid and high uptake and internalization of 64Cu-DOTA-NAPamide in B16F10 cells. Over 90% of receptor-bound tracer is internalized at 3 h incubation. A cellular retention study demonstrates that the receptor-bound 64Cu-DOTA-NAPamide is slowly released from the B16F10 cells into the medium; 66% of the radioactivity is still associated with the cells even after 3 h incubation. The biodistribution of 64Cu-DOTA-NAPamide was then investigated in C57BL/6 mice bearing subcutaneous murine B16F10 melanoma tumors with high capacity of MC1R and Fox Chase Scid mice bearing human A375M melanoma with a relatively low number of MC1R receptors. Tumor uptake values of 64Cu-DOTA-NAPamide are found to be 4.63 +/- 0.45% and 2.49 +/- 0.31% ID/g in B16F10 and A375M xenografted melanoma at 2 h postinjection (pi), respectively. The B16F10 tumor uptake at 2 h pi is further inhibited to 2.29 +/- 0.24% ID/g, while A375M tumor uptake at 2 h pi remains 2.20 +/- 0.41% ID/g with a coinjection of excess

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

    PubMed Central

    Jazirehi, Ali R.; Economou, James S.

    2012-01-01

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

  20. The modulation of Dicer regulates tumor immunogenicity in melanoma

    PubMed Central

    Hoffend, Nicholas C.; Magner, William J.; Tomasi, Thomas B.

    2016-01-01

    MicroRNAs (miRs) are small non-coding RNAs that regulate most cellular protein networks by targeting mRNAs for translational inhibition or degradation. Dicer, a type III endoribonuclease, is a critical component in microRNA biogenesis and is required for mature microRNA production. Abnormal Dicer expression occurs in numerous cancer types and correlates with poor patient prognosis. For example, increased Dicer expression in melanoma is associated with more aggressive tumors (higher tumor mitotic index and depth of invasion) and poor patient prognosis. However, the role that Dicer plays in melanoma development and immune evasion remains unclear. Here, we report on a newly discovered relationship between Dicer expression and tumor immunogenicity. To investigate Dicer's role in regulating melanoma immunogenicity, Dicer knockdown studies were performed. We found that B16F0-Dicer deficient cells exhibited decreased tumor growth compared to control cells and were capable of inducing anti-tumor immunity. The decrease in tumor growth was abrogated in immunodeficient NSG mice and was shown to be dependent upon CD8+ T cells. Dicer knockdown also induced a more responsive immune gene profile in melanoma cells. Further studies demonstrated that CD8+ T cells preferentially killed Dicer knockdown tumor cells compared to control cells. Taken together, we present evidence which links Dicer expression to tumor immunogenicity in melanoma. PMID:27356752

  1. The modulation of Dicer regulates tumor immunogenicity in melanoma.

    PubMed

    Hoffend, Nicholas C; Magner, William J; Tomasi, Thomas B

    2016-07-26

    MicroRNAs (miRs) are small non-coding RNAs that regulate most cellular protein networks by targeting mRNAs for translational inhibition or degradation. Dicer, a type III endoribonuclease, is a critical component in microRNA biogenesis and is required for mature microRNA production. Abnormal Dicer expression occurs in numerous cancer types and correlates with poor patient prognosis. For example, increased Dicer expression in melanoma is associated with more aggressive tumors (higher tumor mitotic index and depth of invasion) and poor patient prognosis. However, the role that Dicer plays in melanoma development and immune evasion remains unclear. Here, we report on a newly discovered relationship between Dicer expression and tumor immunogenicity. To investigate Dicer's role in regulating melanoma immunogenicity, Dicer knockdown studies were performed. We found that B16F0-Dicer deficient cells exhibited decreased tumor growth compared to control cells and were capable of inducing anti-tumor immunity. The decrease in tumor growth was abrogated in immunodeficient NSG mice and was shown to be dependent upon CD8+ T cells. Dicer knockdown also induced a more responsive immune gene profile in melanoma cells. Further studies demonstrated that CD8+ T cells preferentially killed Dicer knockdown tumor cells compared to control cells. Taken together, we present evidence which links Dicer expression to tumor immunogenicity in melanoma.

  2. Anti-melanoma vaccines engineered to simultaneously modulate cytokine priming and silence PD-L1 characterized using ex vivo myeloid-derived suppressor cells as a readout of therapeutic efficacy.

    PubMed

    Liechtenstein, Therese; Perez-Janices, Noemi; Blanco-Luquin, Idoia; Goyvaerts, Cleo; Schwarze, Julia; Dufait, Ines; Lanna, Alessio; Ridder, Mark De; Guerrero-Setas, David; Breckpot, Karine; Escors, David

    Efficacious antitumor vaccines strongly stimulate cancer-specific effector T cells and counteract the activity of tumor-infiltrating immunosuppressive cells. We hypothesised that combining cytokine expression with silencing programmed cell death ligand 1 (PD-L1) could potentiate anticancer immune responses of lentivector vaccines. Thus, we engineered a collection of lentivectors that simultaneously co-expressed an antigen, a PD-L1-silencing shRNA, and various T cell-polarising cytokines, including interferon γ (IFNγ), transforming growth factor β (TGFβ) or interleukins (IL12, IL15, IL23, IL17A, IL6, IL10, IL4). In a syngeneic B16F0 melanoma model and using tyrosinase related protein 1 (TRP1) as a vaccine antigen, we found that simultaneous delivery of IL12 and a PD-L1-silencing shRNA was the only combination that exhibited therapeutically relevant anti-melanoma activities. Mechanistically, we found that delivery of the PD-L1 silencing construct boosted T cell numbers, inhibited in vivo tumor growth and strongly cooperated with IL12 cytokine priming and antitumor activities. Finally, we tested the capacities of our vaccines to counteract tumor-infiltrating myeloid-derived suppressor cell (MDSC) activities ex vivo . Interestingly, the lentivector co-expressing IL12 and the PD-L1 silencing shRNA was the only one that counteracted MDSC suppressive activities, potentially underlying the observed anti-melanoma therapeutic benefit. We conclude that (1) evaluation of vaccines in healthy mice has no significant predictive value for the selection of anticancer treatments; (2) B16 cells expressing xenoantigens as a tumor model are of limited value; and (3) vaccines which inhibit the suppressive effect of MDSC on T cells in our ex vivo assay show promising and relevant antitumor activities.

  3. Anti-melanoma vaccines engineered to simultaneously modulate cytokine priming and silence PD-L1 characterized using ex vivo myeloid-derived suppressor cells as a readout of therapeutic efficacy

    PubMed Central

    Liechtenstein, Therese; Perez-Janices, Noemi; Blanco-Luquin, Idoia; Goyvaerts, Cleo; Schwarze, Julia; Dufait, Ines; Lanna, Alessio; Ridder, Mark De; Guerrero-Setas, David; Breckpot, Karine; Escors, David

    2014-01-01

    Efficacious antitumor vaccines strongly stimulate cancer-specific effector T cells and counteract the activity of tumor-infiltrating immunosuppressive cells. We hypothesised that combining cytokine expression with silencing programmed cell death ligand 1 (PD-L1) could potentiate anticancer immune responses of lentivector vaccines. Thus, we engineered a collection of lentivectors that simultaneously co-expressed an antigen, a PD-L1-silencing shRNA, and various T cell-polarising cytokines, including interferon γ (IFNγ), transforming growth factor β (TGFβ) or interleukins (IL12, IL15, IL23, IL17A, IL6, IL10, IL4). In a syngeneic B16F0 melanoma model and using tyrosinase related protein 1 (TRP1) as a vaccine antigen, we found that simultaneous delivery of IL12 and a PD-L1-silencing shRNA was the only combination that exhibited therapeutically relevant anti-melanoma activities. Mechanistically, we found that delivery of the PD-L1 silencing construct boosted T cell numbers, inhibited in vivo tumor growth and strongly cooperated with IL12 cytokine priming and antitumor activities. Finally, we tested the capacities of our vaccines to counteract tumor-infiltrating myeloid-derived suppressor cell (MDSC) activities ex vivo. Interestingly, the lentivector co-expressing IL12 and the PD-L1 silencing shRNA was the only one that counteracted MDSC suppressive activities, potentially underlying the observed anti-melanoma therapeutic benefit. We conclude that (1) evaluation of vaccines in healthy mice has no significant predictive value for the selection of anticancer treatments; (2) B16 cells expressing xenoantigens as a tumor model are of limited value; and (3) vaccines which inhibit the suppressive effect of MDSC on T cells in our ex vivo assay show promising and relevant antitumor activities. PMID:25954597

  4. Histological evaluation of intratumoral myxoma virus treatment in an immunocompetent mouse model of melanoma

    PubMed Central

    Doty, Rosalinda A; Liu, Jia; McFadden, Grant; Roy, Edward J; MacNeill, Amy L

    2013-01-01

    Two recombinant myxoma viruses (MYXV expressing a fluorescent protein [MYXV-Tred] and MYXV-Tred encoding murine interleukin-15 [MYXV-IL15]) were evaluated for therapeutic effects in an aggressive B16F10 melanoma model in immunocompetent mice. It was hypothesized that continuous expression of IL-15 within a tumor would recruit cytotoxic effector cells to induce an antitumor immune response and improve treatment efficacy. Weekly intratumoral injections were given to evaluate the effect of treatment on the median survival time of C57BL/6 mice bearing established B16F10 melanomas. Mice that received MYXV-Tred or MYXV-IL15 lived significantly longer than mice given treatment controls. Unexpectedly, the median survival time of MYXV-IL15-treated mice was similar to that of MYXV-treated mice. At 1, 2, and 4 days postinoculation, viral plaque assays detected replicating MYXV-Tred and MYXV-IL15 within treated tumors. At these time points in MYXV-IL15-treated tumors, IL-15 concentration, lymphocyte grades, and cluster of differentiation-3+ cell counts were significantly increased when compared to other treatment groups. However, viral titers, recombinant protein expression, and lymphocyte numbers within the tumors diminished rapidly at 7 days postinoculation. These data indicate that treatment with recombinant MYXV should be repeated at least every 4 days to maintain recombinant protein expression within a murine tumor. Additionally, neutrophilic inflammation was significantly increased in MYXV-Tred- and MYXV-IL15-treated tumors at early time points. It is speculated that neutrophilic inflammation induced by intratumoral replication of recombinant MXYV contributes to the antitumoral effect of MYXV treatment in this melanoma model. These findings support the inclusion of neutrophil chemotaxins in recombinant poxvirus oncolytic virotherapy. PMID:25866742

  5. Cytologic Features of Malignant Melanoma with Osteoclast-Like Giant Cells.

    PubMed

    Jiménez-Heffernan, José A; Adrados, Magdalena; Muñoz-Hernández, Patricia; Fernández-Rico, Paloma; Ballesteros-García, Ana I; Fraga, Javier

    2018-01-01

    Malignant melanoma showing numerous osteoclast-like giant cells (OGCs) is an uncommon morphologic phenomenon, rarely mentioned in the cytologic literature. The few reported cases seem to have an aggressive clinical behavior. Although most findings support monocyte/macrophage differentiation, the exact nature of OGCs is not clear. A 57-year-old woman presented with an inguinal lymphadenopathy. Sixteen years before, cutaneous malignant melanoma of the lower limb had been excised. Needle aspiration revealed abundant neoplastic single cells as well as numerous multinucleated OGCs. Occasional neoplastic giant cells were also present. Nuclei of OGCs were monomorphic with oval morphology and were smaller than those of melanoma cells. The immunophenotype of OGCs (S100-, HMB45-, Melan-A-, SOX10-, Ki67-, CD163-, BRAF-, CD68+, MiTF+, p16+) was the expected for reactive OGCs of monocyte/macrophage origin. The tumor has shown an aggressive behavior with further metastases to the axillary lymph nodes and oral cavity. Numerous OGCs are a rare and relevant finding in malignant melanoma. Their presence should not induce confusion with other tumors rich in osteoclastic cells. Since a relevant number of OGCs in melanoma may mean a more aggressive behavior, and patients may benefit from specific treatments, their presence should be mentioned in the pathologic report. © 2018 S. Karger AG, Basel.

  6. Evaluation of New Tc-99m-Labeled Arg-X-Asp-Conjugated Alpha-Melanocyte Stimulating Hormone Peptides for Melanoma Imaging

    PubMed Central

    Flook, Adam M.; Yang, Jianquan; Miao, Yubin

    2013-01-01

    The purpose of this study was to examine the melanoma targeting and imaging properties of two new 99mTc-labeled Arg-X-Asp-conjugated alpha-melanocyte stimulating hormone (α-MSH) peptides. RTD-Lys-(Arg11)CCMSH {c[Asp-Arg-Thr-Asp-DTyr]-Lys-Cys-Cys-Glu-His-DPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2} and RVD-Lys-(Arg11)CCMSH peptides were synthesized and their melanocortin-1 (MC1) receptor binding affinities were determined in B16/F1 melanoma cells. The biodistribution and melanoma imaging properties of 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The IC50 values of RTD-Lys-(Arg11)CCMSH and RVD-Lys-(Arg11)CCMSH were 0.7 ± 0.07 and 1.0 ± 0.3 nM in B16/F1 melanoma cells. Both 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH displayed high melanoma uptake. 99mTc-RTD-Lys-(Arg11)CCMSH exhibited the peak tumor uptake of 18.77 ± 5.13% ID/g at 2 h post-injection, whereas 99mTc-RVD-Lys-(Arg11)CCMSH reached the peak tumor uptake of 19.63 ± 4.68% ID/g at 4 h post-injection. Both 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH showed low accumulation in normal organs (<1.7% ID/g) except for the kidneys at 2 h post-injection. The renal uptake of 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH was 135.14 ± 23.62 and 94.01 ± 18.31% ID/g at 2 h post-injection, respectively. The melanoma lesions were clearly visualized by SPECT/CT using either 99mTc-RTD-Lys-(Arg11)CCMSH or 99mTc-RVD-Lys-(Arg11)CCMSH as an imaging probe at 2 h post-injection. Overall, the introduction of Thr or Val residue retained high melanoma uptake of 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH. However, high renal uptake of 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH need to be reduced to facilitate their future applications. PMID:23885640

  7. Evaluation of new Tc-99m-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone peptides for melanoma imaging.

    PubMed

    Flook, Adam M; Yang, Jianquan; Miao, Yubin

    2013-09-03

    The purpose of this study was to examine the melanoma targeting and imaging properties of two new (99m)Tc-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RTD-Lys-(Arg(11))CCMSH {c[Asp-Arg-Thr-Asp-DTyr]-Lys-Cys-Cys-Glu-His-DPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2} and RVD-Lys-(Arg(11))CCMSH peptides were synthesized, and their melanocortin-1 (MC1) receptor binding affinities were determined in B16/F1 melanoma cells. The biodistribution and melanoma imaging properties of (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The IC50 values of RTD-Lys-(Arg(11))CCMSH and RVD-Lys-(Arg(11))CCMSH were 0.7 ± 0.07 and 1.0 ± 0.3 nM in B16/F1 melanoma cells. Both (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH displayed high melanoma uptake. (99m)Tc-RTD-Lys-(Arg(11))CCMSH exhibited the highest tumor uptake of 18.77 ± 5.13% ID/g at 2 h postinjection, whereas (99m)Tc-RVD-Lys-(Arg(11))CCMSH reached the highest tumor uptake of 19.63 ± 4.68% ID/g at 4 h postinjection. Both (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH showed low accumulation in normal organs (<1.7% ID/g) except for the kidneys at 2 h postinjection. The renal uptake of (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH was 135.14 ± 23.62 and 94.01 ± 18.31% ID/g at 2 h postinjection, respectively. The melanoma lesions were clearly visualized by single-photon emission computed tomography (SPECT)/CT using either (99m)Tc-RTD-Lys-(Arg(11))CCMSH or (99m)Tc-RVD-Lys-(Arg(11))CCMSH as an imaging probe at 2 h postinjection. Overall, the introduction of Thr or Val residue retained high melanoma uptake of (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH. However, high renal uptake of (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH need to be reduced to facilitate their future applications.

  8. Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells.

    PubMed

    Si, Lingling; Yan, Xinyan; Hao, Wenjin; Ma, Xiaoyi; Ren, Huanhuan; Ren, Boxue; Li, Defang; Dong, Zhengping; Zheng, Qiusheng

    2018-05-01

    The aim of the present study was to determine the effects of Licochalcone D (LD) on the apoptosis and migration and invasion in human melanoma A375 cells. Cell proliferation was determined by sulforhodamine B assay. Apoptosis was assessed by Hoechst 33258 and Annexin V‑FITC/PI staining and JC‑1 assay. Total intracellular reactive oxygen species (ROS) was examined by DCFH‑DA. Wound healing and Transwell assays were used to detect migration and invasion of the cells. The activities of matrix metalloproteinase (MMP‑2 and MMP‑9) were assessed via gelatin zymography. Tumor growth in vivo was evaluated in C57BL/6 mice. RT‑PCR, qPCR, ELISA and western blot analysis were utilized to measure the mRNA and protein levels. Our results showed that LD inhibited the proliferation of A375 and SK‑MEL‑5 cells in a concentration‑dependent manner. After treatment with LD, A375 cells displayed obvious apoptotic characteristics, and the number of apoptotic cells was significantly increased. Pro‑apoptotic protein Bax, caspase‑9 and caspase‑3 were upregulated, while anti‑apoptotic protein Bcl‑2 was downregulated in the LD‑treated cells. Meanwhile, LD induced the loss of mitochondrial membrane potential (ΔΨm) and increased the level of ROS. ROS production was inhibited by the co‑treatment of LD and free radical scavenger N‑acetyl‑cysteine (NAC). Furthermore, LD also blocked A375 cell migration and invasion in vitro which was associated with the downregulation of MMP‑9 and MMP‑2. Finally, intragastric administration of LD suppressed tumor growth in the mouse xenograft model of murine melanoma B16F0 cells. These results suggest that LD may be a potential drug for human melanoma treatment by inhibiting proliferation, inducing apoptosis via the mitochondrial pathway and blocking cell migration and invasion.

  9. Combined blockade of Tim-3 and MEK inhibitor enhances the efficacy against melanoma

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Yang; Cai, Pengcheng; Wang, Ning

    Insights into the role of the mitogen-activated protein kinase (MAPK) pathway and immune checkpoints have led combined targeted therapy and immunotherapy to be a promising regimen. Trametinib, as a mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor, has demonstrated effectiveness in patients with advanced melanoma. T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3), an immune checkpoint molecule, participates in multiple negative regulation of antitumor immunity. We for the first time to our knowledge reported the combination of trametinib and anti-Tim-3 monoclonal antibody (mAb) in treating B16-F10 melanoma mice. We discovered that trametinib remarkably promoted apoptosis and inhibited cell proliferation while inhibition of MEKmore » improved the expression of Tim-3 and caused the decrease of CD8{sup +} T cells; to the contrary, anti-Tim-3 mAb enhanced antitumor immunity by stimulating CD8{sup +} T cells, thus the combined therapy produced potent antitumor effect cooperatively. Taken together, our study provides compelling evidence for combining trametinib and anti-Tim-3 mAb as a potential valuable regimen in treating melanoma. - Highlights: • The potent antitumor efficacy of combined trametinib and anti-Tim-3 mAb. • MEK inhibitor involves in up-regulation of Tim-3. • The combined medication provides new insights into melanoma treatment.« less

  10. A phase I trial of bortezomib and interferon-α-2b in metastatic melanoma.

    PubMed

    Markowitz, Joseph; Luedke, Eric A; Grignol, Valerie P; Hade, Erinn M; Paul, Bonnie K; Mundy-Bosse, Bethany L; Brooks, Taylor R; Dao, Thao-Vi; Kondalasula, Sri V; Lesinski, Gregory B; Olencki, Thomas; Kendra, Kari L; Carson, William E

    2014-01-01

    The possibility that cytokine administration could enhance the antitumor effects of proteasome inhibition was explored. It was found that coadministration of bortezomib and interferon-α (IFN-α) induced synergistic apoptosis in human melanoma cell lines and prolonged survival in a murine model of melanoma. A phase I study was conducted to determine the tolerability and the maximum tolerated dose of bortezomib when administered in combination with IFN-α-2b to patients with metastatic melanoma. Patients were treated on a 5-week cycle. In week 1 of cycle 1, patients received 5 million U/m(2) IFN-α subcutaneously thrice weekly. During weeks 2-4 of cycle 1, bortezomib was administered intravenously weekly along with IFN-α thrice weekly. There was a treatment break during week 5. After cycle 1, bortezomib was administered in combination with IFN-α. Bortezomib was administered in escalating doses (1.0, 1.3, or 1.6 mg/m) to cohorts of 3 patients. Sixteen patients were treated (8 women, 8 men; median age 59 y). Common grade 3 toxicities included fatigue (5), vomiting (3), and diarrhea (3). Grade 4 toxicities included fatigue (3) and lymphopenia (1). The maximum tolerated dose for bortezomib was 1.3 mg/m(2). One patient had a partial response, and 7 had stable disease. Progression-free survival was 2.5 months, and overall survival was 10.3 months. Bortezomib administration did not augment the ability of IFN-α to induce phosphorylation of STAT1 in circulating immune cells; however, it did lead to reduced plasma levels of proangiogenic cytokines. The combination of bortezomib and IFN-α can be safely administered to melanoma patients.

  11. Regulation of hematogenous tumor metastasis by acid sphingomyelinase

    PubMed Central

    Carpinteiro, Alexander; Becker, Katrin Anne; Japtok, Lukasz; Hessler, Gabriele; Keitsch, Simone; Požgajovà, Miroslava; Schmid, Kurt W; Adams, Constantin; Müller, Stefan; Kleuser, Burkhard; Edwards, Michael J; Grassmé, Heike; Helfrich, Iris; Gulbins, Erich

    2015-01-01

    Metastatic dissemination of cancer cells is the ultimate hallmark of malignancy and accounts for approximately 90% of human cancer deaths. We investigated the role of acid sphingomyelinase (Asm) in the hematogenous metastasis of melanoma cells. Intravenous injection of B16F10 melanoma cells into wild-type mice resulted in multiple lung metastases, while Asm-deficient mice (Smpd1−/− mice) were protected from pulmonary tumor spread. Transplanting wild-type platelets into Asm-deficient mice reinstated tumor metastasis. Likewise, Asm-deficient mice were protected from hematogenous MT/ret melanoma metastasis to the spleen in a mouse model of spontaneous tumor metastasis. Human and mouse melanoma cells triggered activation and release of platelet secretory Asm, in turn leading to ceramide formation, clustering, and activation of α5β1 integrins on melanoma cells finally leading to adhesion of the tumor cells. Clustering of integrins by applying purified Asm or C16 ceramide to B16F10 melanoma cells before intravenous injection restored trapping of tumor cells in the lung in Asm-deficient mice. This effect was revertable by arginine-glycine-aspartic acid peptides, which are known inhibitors of integrins, and by antibodies neutralizing β1 integrins. These findings indicate that melanoma cells employ platelet-derived Asm for adhesion and metastasis. PMID:25851537

  12. Structure-activity relationship of prenyl-substituted polyphenols from Artocarpus heterophyllus as inhibitors of melanin biosynthesis in cultured melanoma cells.

    PubMed

    Arung, Enos Tangke; Shimizu, Kuniyoshi; Kondo, Ryuichiro

    2007-09-01

    A series of prenylated, flavone-based polyphenols, compounds 1-8, were isolated from the wood of Artocarpus heterophyllus. These compounds, which have previously been shown not to inhibit tyrosinase activity, were found to be active inhibitors of the in vivo melanin biosynthesis in B16 melanoma cells, with little or no cytotoxicity. To clarify the structural requirement for inhibition, some structure-activity relationships were studied, in comparison with related compounds lacking prenyl side chains. Our experiments indicate that both prenyl and OH groups, as well as the type of substitution pattern, are crucial for the inhibition of melanin production in B16 melanoma cells.

  13. Cu-64-labeled lactam bridge-cyclized α-MSH peptides for PET imaging of melanoma.

    PubMed

    Guo, Haixun; Miao, Yubin

    2012-08-06

    The purpose of this study was to examine and compare the melanoma targeting and imaging properties of (64)Cu-NOTA-GGNle-CycMSH(hex) {(64)Cu-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and (64)Cu-DOTA-GGNle-CycMSH(hex) {(64)Cu-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-GGNle-CycMSH(hex)}. Two lactam bridge-cyclized peptides, NOTA-GGNle-CycMSH(hex) and DOTA-GGNle-CycMSH(hex), were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSH(hex) was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSH(hex). The melanoma targeting and imaging properties of (64)Cu-NOTA-GGNle-CycMSH(hex) and (64)Cu-DOTA-GGNle-CycMSH(hex) were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSH(hex) and DOTA-GGNle-CycMSH(hex) displayed comparable MC1 receptor binding affinities (1.6 vs 2.1 nM). The substitution of DOTA with NOTA dramatically increased the melanoma uptake and decreased the renal and liver uptake of (64)Cu-NOTA-GGNle-CycMSH(hex). The tumor uptake of (64)Cu-NOTA-GGNle-CycMSH(hex) was between 12.39 ± 1.61 and 12.71 ± 2.68% ID/g at 0.5, 2, and 4 h postinjection. The accumulation of (64)Cu-NOTA-GGNle-CycMSH(hex) activity in normal organs was lower than 1.02% ID/g except for the kidneys 2, 4, and 24 h postinjection. The tumor/liver uptake ratios of (64)Cu-NOTA-GGNle-CycMSHhex were 17.96, 16.95, and 8.02, whereas the tumor/kidney uptake ratios of (64)Cu-NOTA-GGNle-CycMSH(hex) were 2.52, 3.60, and 5.74 at 2, 4, and 24 h postinjection, respectively. Greater than 91% of the injected radioactivity cleared through the urinary system by 2 h postinjection. The substitution of DOTA with NOTA resulted in a dramatic increase in melanoma uptake and decrease in renal and liver uptake of (64)Cu-NOTA-GGNle-CycMSH(hex) as compared to (64)Cu-DOTA-GGNle-CycMSH(hex). High melanoma uptake coupled with low accumulation in nontarget

  14. Tumor cell-intrinsic PD-L1 promotes tumor-initiating cell generation and functions in melanoma and ovarian cancer

    PubMed Central

    Gupta, Harshita B; Clark, Curtis A; Yuan, Bin; Sareddy, Gangadhara; Pandeswara, Srilakshmi; Padron, Alvaro S; Hurez, Vincent; Conejo-Garcia, José; Vadlamudi, Ratna; Li, Rong; Curiel, Tyler J

    2016-01-01

    As tumor PD-L1 provides signals to anti-tumor PD-1+ T cells that blunt their functions, αPD-1 and αPD-L1 antibodies have been developed as anti-cancer immunotherapies based on interrupting this signaling axis. However, tumor cell-intrinsic PD-L1 signals also regulate immune-independent tumor cell proliferation and mTOR signals, among other important effects. Tumor-initiating cells (TICs) generate carcinomas, resist treatments and promote relapse. We show here that in murine B16 melanoma and ID8agg ovarian carcinoma cells, TICs express more PD-L1 versus non-TICs. Silencing PD-L1 in B16 and ID8agg cells by shRNA (‘PD-L1lo’) reduced TIC numbers, the canonical TIC genes nanog and pou5f1 (oct4), and functions as assessed by tumorosphere development, immune-dependent and immune-independent tumorigenesis, and serial transplantability in vivo. Strikingly, tumor PD-L1 sensitized TIC to interferon-γ and rapamycin in vitro. Cell-intrinsic PD-L1 similarly drove functional TIC generation, canonical TIC gene expression and sensitivity to interferon-γ and rapamycin in human ES2 ovarian cancer cells. Thus, tumor-intrinsic PD-L1 signals promote TIC generation and virulence, possibly by promoting canonical TIC gene expression, suggesting that PD-L1 has novel signaling effects on cancer pathogenesis and treatment responses. PMID:28798885

  15. Berberine suppressed epithelial mesenchymal transition through cross-talk regulation of PI3K/AKT and RARα/RARβ in melanoma cells.

    PubMed

    Kou, Yu; Li, Lei; Li, Hong; Tan, Yuhui; Li, Bin; Wang, Kun; Du, Biaoyan

    2016-10-14

    Berberine is a natural compound extracted from Coptidis rhizoma, and accumulating proof has shown its potent anti-tumor properties with diverse action on melanoma cells, including inhibiting cancer viability, blocking cell cycle and migration. However, the mechanisms of berberine have not been fully clarified. In this study, we identified that berberine reduced the migration and invasion capacities of B16 cells, and notably altered pluripotency of epithelial to mesenchymal transition associated factors. We found that berberine also downregulation the expression level of p-PI3K, p-AKT and retinoic acid receptor α (RARα) and upregulation the expression level of retinoic acid receptor β and γ (RARβ and RARγ). These effects of PI3 kinase inhibitor LY294002 treatment mimicked Berberine treatment except the expression level of RARγ. Moreover, Western blot analysis showed that the decreased PI3K and AKT phosphorylation, increased the epithelial maker E-cadherin, and upregulation level of RARβ while decreased the mesenchymal markers N-cadherin and downregulation level of RARα by incubation with LY294002 in mouse melanoma B16 cells. In conclusion, Our study reveal that berberine can reverse the epithelial to mesenchymal transition of mouse melanoma B16 cells and may be a useful adjuvant therapeutic agent in the treatment of melanoma through the PI3K/Akt pathway and inactivation PI3K/AKT could regulate RARα/RARβ expression. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  16. The in-vitro and in-vivo inhibitory activity of biflorin in melanoma.

    PubMed

    Vasconcellos, Marne C; Bezerra, Daniel P; Fonseca, Aluísio M; Araújo, Ana Jérsia; Pessoa, Cláudia; Lemos, Telma L G; Costa-Lotufo, Letícia V; de Moraes, Manoel Odorico; Montenegro, Raquel C

    2011-04-01

    Biflorin, an ortho-naphthoquinone, is an active compound found in the roots of Capraria biflora L. It has been reported that biflorin presents anticancer activity, inhibiting both tumor cell line growth in culture and tumor development in mice. The aim of this study was to examine the effectiveness of biflorin treatment using both in-vitro and in-vivo melanoma models. Biflorin displayed considerable cytotoxicity against all tested cell lines, with half maximal inhibitory concentration values ranging from 0.58 μg/ml in NCI H23 (human lung adenocarcinoma) to 14.61 μg/ml in MDA-MB-231 (human breast cancer) cell lines. In a second set of experiments using B16 melanoma cells as a model, biflorin reduced cell viability but did not cause significant increase in the number of nonviable cells. In addition, the DNA synthesis was significantly inhibited. Flow cytometry analysis showed that biflorin may lead to an apoptotic death in melanoma cells, inducing DNA fragmentation and mitochondria depolarization, without affecting membrane integrity. In B16 melanoma-bearing mice, administration of biflorin (25mg/day) for 10 days inhibited tumor growth, and also increased the mean survival rate from 33.3±0.9 days (control) to 44.5±3.4 days (treated). Our findings suggest that biflorin may be considered as a promising lead compound for designing new drugs to be used in the treatment of melanoma.

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

    PubMed Central

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

    2012-01-01

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

  18. NF-kappaB mediates mitogen-activated protein kinase pathway-dependent iNOS expression in human melanoma.

    PubMed

    Uffort, Deon G; Grimm, Elizabeth A; Ellerhorst, Julie A

    2009-01-01

    Tumor expression of inducible nitric oxide synthase (iNOS) predicts poor outcomes for melanoma patients. We have reported the regulation of melanoma iNOS by the mitogen-activated protein kinase (MAPK) pathway. In this study, we test the hypothesis that NF-kappaB mediates this regulation. Western blotting of melanoma cell lysates confirmed the constitutive expression of iNOS. Western blot detected baseline levels of activated nuclear extracellular signal-regulated kinase and NF-kappaB. Indirect immunofluorescence confirmed the presence of NF-kappaB p50 and p65 in melanoma cell nuclei, with p50 being more prevalent. Electrophoretic mobility shift assay demonstrated baseline NF-kappaB activity, the findings confirmed by supershift analysis. Treatment of melanoma cells with the MEK inhibitor U0126 decreased NF-kappaB binding to its DNA recognition sequence, implicating the MAPK pathway in NF-kappaB activation. Two specific NF-kappaB inhibitors suppressed iNOS expression, demonstrating regulation of iNOS by NF-kappaB. Several experiments indicated the presence of p50 homodimers, which lack a transactivation domain and rely on the transcriptional coactivator Bcl-3 to carry out this function. Bcl-3 was detected in melanoma cells and co-immunoprecipitated with p50. These data suggest that the constitutively activated melanoma MAPK pathway stimulates activation of NF-kappaB hetero- and homodimers, which, in turn, drive iNOS expression and support melanoma tumorigenesis.

  19. Atmospheric Retrievals of HAT-P-16b and WASP-11b/HAT-P-10b

    NASA Astrophysics Data System (ADS)

    McIntyre, Kathleen; Harrington, Joseph; Challener, Ryan; Lenius, Maria; Hartman, Joel D.; Bakos, Gaspar A.; Blecic, Jasmina; Cubillos, Patricio E.; Cameron, Andrew

    2018-01-01

    We report Bayesian atmospheric retrievals performed on the exoplanets HAT-P-16b and WASP-11b/HAT-P-10b. HAT-P-16b is a hot (equilibrium temperature 1626 ± 40 K, assuming zero Bond albedo and efficient energy redistribution), 4.19 ± 0.09 Jupiter-mass exoplanet orbiting an F8 star every 2.775960 ± 0.000003 days (Buchhave et al 2010). WASP-11b/HAT-P-10b is a cooler (1020 ± 17 K), 0.487 ± 0.018 Jupiter-mass exoplanet orbiting a K3 star every 3.7224747 ± 0.0000065 days (Bakos et al. 2009, co-discovered by West et al. 2008). We observed secondary eclipses of both planets using the 3.6 μm and 4.5 μm channels of the Spitzer Space Telescope's Infrared Array Camera (program ID 60003). We applied our Photometry for Orbits, Eclipses, and Transits (POET) code to produce normalized eclipse light curves, and our Bayesian Atmospheric Radiative Transfer (BART) code to constrain the temperature-pressure profiles and atmospheric molecular abundances of the two planets. Spitzer is operated by the Jet Propulsion Laboratory, California Institute of Technology, under a contract with NASA. This work was supported by NASA Planetary Atmospheres grant NNX12AI69G and NASA Astrophysics Data Analysis Program grant NNX13AF38G.

  20. Collaboration between tumor-specific CD4+ T cells and B cells in anti-cancer immunity.

    PubMed

    Guy, Thomas V; Terry, Alexandra M; Bolton, Holly A; Hancock, David G; Zhu, Erhua; Brink, Robert; McGuire, Helen M; Shklovskaya, Elena; Fazekas de St. Groth, Barbara

    2016-05-24

    The role of B cells and antibodies in anti-tumor immunity is controversial, with both positive and negative effects reported in animal models and clinical studies. We developed a murine B16.F10 melanoma model to study the effects of collaboration between tumor-specific CD4+ T cells and B cells on tumor control. By incorporating T cell receptor transgenic T cells and B cell receptor isotype switching B cells, we were able to track the responses of tumor-reactive T and B cells and the development of anti-tumor antibodies in vivo. In the presence of tumor-specific B cells, the number of tumor-reactive CD4+ T cells was reduced in lymphoid tissues and the tumor itself, and this correlated with poor tumor control. B cells had little effect on the Th1 bias of the CD4+ T cell response, and the number of induced FoxP3+ regulatory cells (iTregs) generated from within the original naive CD4+ T cell inoculum was unrelated to the degree of B cell expansion. In response to CD4+ T cell help, B cells produced a range of isotype-switched anti-tumor antibodies, principally IgG1, IgG2a/c and IgG2b. In the absence of CD4+ T cells, B cells responded to agonistic anti-CD40 administration by switching to production of IgG2a/c and, to a lesser extent, IgG1, IgG3, IgA and IgE, which reduced the number of lung metastases after i.v. tumor inoculation but had no effect on the growth of subcutaneous tumors.

  1. Melanoma exosomes deliver a complex biological payload that upregulates PTPN11 to suppress T lymphocyte function.

    PubMed

    Wu, Yueting; Deng, Wentao; McGinley, Emily Chambers; Klinke, David J

    2017-03-01

    As exosomes are emerging as a new mode of intercellular communication, we hypothesized that the payload contained within exosomes is shaped by somatic evolution. To test this, we assayed the impact on primary CD8+ T-cell function, a key mechanism for antitumor immunity, of exosomes derived from three melanoma-related cell lines. While morphologically similar, exosomes from each cell line were functionally different, as B16F0 exosomes dose-dependently suppressed T-cell proliferation. In contrast, Cloudman S91 exosomes promoted T-cell proliferation and Melan-A exosomes had a negligible effect on primary CD8+ T cells. Mechanistically, transcript profiling suggested that exosomal mRNA is enriched for full-length mRNAs that target immune-related pathways. Interestingly, B16F0 exosomes were unique in that they contained both protein and mRNA for PTPN11, which inhibited T-cell proliferation. Collectively, the results suggest that upregulation of PTPN11 by B16F0 exosomes to tumor infiltrating lymphocytes would bypass the extracellular control of the immune checkpoints. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Formaldehyde exposure impairs the function and differentiation of NK cells.

    PubMed

    Kim, Eun-Mi; Lee, Hwa-Youn; Lee, Eun-Hee; Lee, Ki-Mo; Park, Min; Ji, Kon-Young; Jang, Ji-Hun; Jeong, Yun-Hwa; Lee, Kwang-Ho; Yoon, Il-Joo; Kim, Su-Man; Jeong, Moon-Jin; Kim, Kwang Dong; Kang, Hyung-Sik

    2013-11-25

    We investigated the cytotoxic effects of formaldehyde (FA) on lymphocytes. FA-exposed mice showed a profound reduction not only in the number of natural killer (NK) cells but also in the expression of NK cell-specific receptors, but these mice did not exhibit decreases in the numbers of T or B lymphocytes. FA exposure also induced decreases in NK cytolytic activity and in the expression of NK cell-associated genes, such as IFN-γ, perforin and CD122. To determine the effect of FA on tumorigenicity, C57BL/6 mice were subcutaneously injected with B16F10 melanoma cells after FA exposure. The mass of the B16F10 tumor and the concentration of extravascular polymorphonuclear leukocytes were greater than those in unexposed tumor-bearing control mice. The number and cytolytic activity of NK cells were also reduced in B16F10 tumor-bearing mice exposed to FA. To determine how FA reduces the NK cell number, NK precursor (pNK) cells were treated with FA, and the differentiation status of the NK cells was analyzed. NK cell differentiation was impaired by FA treatment in a concentration-dependent manner. These findings indicate that FA exposure may promote tumor progression by impairing NK cell function and differentiation. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  3. [Imiquimod combined with dendritic cell vaccine decreases Treg proportion and enhances anti-tumor responses in mice bearing melanoma].

    PubMed

    Ren, Shurong; Wang, Qiubo; Zhang, Yanli; Lu, Cuixiu; Li, Ping; Li, Yumei

    2017-02-01

    Objective To investigate the therapeutic effect of Toll-like receptor 7 (TLR7) agonist imiquimod combined with dendritic cell (DC)-based tumor vaccine on melanoma in mice and the potential mechanism. Methods Melanoma-bearing mouse models were established by subcutanous injection of B16-OVA cells into C57BL/6 mice. DCs were isolated from mouse bone marrow and propagated in culture medium with recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant mouse interleukin-4 (rmIL-4). DC vaccine (OVA-DC) was prepared by overnight incubation of DCs added with chicken ovalbumin. C57BL/6 mice were separated into four groups which were treated with PBS, topical imiquimod application, OVA-DC intradermal injection and imiquimod plus OVA-DC, respectively. The tumor size was calculated by digital vernier caliper. Peripheral blood CD4 + FOXP3 + Tregs of the tumor-bearing mice was detected by flow cytometry. The cytotoxicity of splenic lymphocyte against B16-OVA was assessed in vitro by CCK-8 assay. Results Compared with the other three groups, B16-OVA-bearing mice treated with imiquimod plus DC vaccine had the smallest tumor volume. The percentage of CD4 + FOXP3 + Tregs decreased significantly in the combined treated mice. The combined treatment enhanced significantly cytotoxicity of splenic lymphocytes against B16-OVA cells. Conclusion Imiquimod combined with antigen-pulsed-DC vaccine could reduce CD4 + FOXP3 + Treg proportion and promote anti-tumor effect in mice with melanoma.

  4. Multifunctional particles for melanoma-targeted drug delivery.

    PubMed

    Wadajkar, Aniket S; Bhavsar, Zarna; Ko, Cheng-Yu; Koppolu, Bhanuprasanth; Cui, Weina; Tang, Liping; Nguyen, Kytai T

    2012-08-01

    New magnetic-based core-shell particles (MBCSPs) were developed to target skin cancer cells while delivering chemotherapeutic drugs in a controlled fashion. MBCSPs consist of a thermo-responsive shell of poly(N-isopropylacrylamide-acrylamide-allylamine) and a core of poly(lactic-co-glycolic acid) (PLGA) embedded with magnetite nanoparticles. To target melanoma cancer cells, MBCSPs were conjugated with Gly-Arg-Gly-Asp-Ser (GRGDS) peptides that specifically bind to the α(5)β(3) receptors of melanoma cells. MBCSPs consist of unique multifunctional and controlled drug delivery characteristics. Specially, they can provide dual drug release mechanisms (a sustained release of drugs through degradation of PLGA core and a controlled release in response to changes in temperature via thermo-responsive polymer shell), and dual targeting mechanisms (magnetic localization and receptor-mediated targeting). Results from in vitro studies indicate that GRGDS-conjugated MBCSPs have an average diameter of 296 nm and exhibit no cytotoxicity towards human dermal fibroblasts up to 500 μg ml(-1). Further, a sustained release of curcumin from the core and a temperature-dependent release of doxorubicin from the shell of MBCSPs were observed. The particles also produced a dark contrast signal in magnetic resonance imaging. Finally, the particles were accumulated at the tumor site in a B16F10 melanoma orthotopic mouse model, especially in the presence of a magnet. Results indicate great potential of MBCSPs as a platform technology to target, treat and monitor melanoma for targeted drug delivery to reduce side effects of chemotherapeutic reagents. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  5. Aurora kinase B inhibition reduces the proliferation of metastatic melanoma cells and enhances the response to chemotherapy.

    PubMed

    Porcelli, Letizia; Guida, Gabriella; Quatrale, Anna E; Cocco, Tiziana; Sidella, Letizia; Maida, Immacolata; Iacobazzi, Rosa M; Ferretta, Anna; Stolfa, Diana A; Strippoli, Sabino; Guida, Stefania; Tommasi, Stefania; Guida, Michele; Azzariti, Amalia

    2015-01-27

    The poor response to chemotherapy and the brief response to vemurafenib in metastatic melanoma patients, make the identification of new therapeutic approaches an urgent need. Interestingly the increased expression and activity of the Aurora kinase B during melanoma progression suggests it as a promising therapeutic target. The efficacy of the Aurora B kinase inhibitor barasertib-HQPA was evaluated in BRAF mutated cells, sensitive and made resistant to vemurafenib after chronic exposure to the drug, and in BRAF wild type cells. The drug effectiveness has been evaluated as cell growth inhibition, cell cycle progression and cell migration. In addition, cellular effectors of drug resistance and response were investigated. The characterization of the effectors responsible for the resistance to vemurafenib evidenced the increased expression of MITF or the activation of Erk1/2 and p-38 kinases in the newly established cell lines with a phenotype resistant to vemurafenib. The sensitivity of cells to barasertib-HQPA was irrespective of BRAF mutational status. Barasertib-HQPA induced the mitotic catastrophe, ultimately causing apoptosis and necrosis of cells, inhibited cell migration and strongly affected the glycolytic metabolism of cells inducing the release of lactate. In association i) with vemurafenib the gain in effectiveness was found only in BRAF(V600K) cells while ii) with nab-paclitaxel, the combination was more effective than each drug alone in all cells. These findings suggest barasertib as a new therapeutic agent and as enhancer of chemotherapy in metastatic melanoma treatment.

  6. Investigation of the Antimetastatic Effects of Agents that Inhibit Cell Adhesion or Protein Glycosylation

    PubMed Central

    Humphries, Martin J.; Matsumoto, Kazue; White, Sandra L.; Olden, Kenneth

    1987-01-01

    In this overview the authors describe their recent attempts to specifically interfere with the metastatic spread of B16-F10 melanoma cells. Using the experimental metastasis model system, inhibitory effects of (1) coinjection of cells with synthetic peptides derived from the glycoprotein fibronectin, which possess the ability to disrupt cell adhesion, and (2) treatment of cells with inhibitors of protein glycosylation and oligosaccharide processing have been examined. PMID:3295262

  7. In vitro cytotoxicity of Selol-loaded magnetic nanocapsules against neoplastic cell lines under AC magnetic field activation

    NASA Astrophysics Data System (ADS)

    Falqueiro, A. M.; Siqueira-Moura, M. P.; Jardim, D. R.; Primo, F. L.; Morais, P. C.; Mosiniewicz-Szablewska, E.; Suchocki, P.; Tedesco, A. C.

    2012-04-01

    The goals of this study are to evaluate invitro compatibility of magnetic nanomaterials and their therapeutic potential against cancer cells. Highly stable ionic magnetic fluid sample (maghemite, γ-Fe2O3) and Selol were incorporated into polymeric nanocapsules by nanoprecipitation method. The cytotoxic effect of Selol-loaded magnetic nanocapsules was assessed on murine melanoma (B16-F10) and oral squamous cell carcinoma (OSCC) cell lines following AC magnetic field application. The influence of different nanocapsules on cell viability was investigated by colorimetric MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. In the absence of AC magnetic field Selol-loaded magnetic nanocapsules, containing 100 µg/mL Selol plus 5 × 1012 particle/mL, showed antitumoral activity of about 50% on B16-F10 melanoma cells while OSCC carcinoma cells demonstrated drug resistance at all concentrations of Selol and magnetic fluid (range of 100-500 µg/mL Selol and 5 × 1012-2.5 × 1013 particle/mL). On the other hand, under AC applied fields (1 MHz and 40 Oe amplitude) B16-F10 cell viability was reduced down to 40.5% (±3.33) at the highest concentration of nanoencapsulated Selol. The major effect, however, was observed on OSCC cells since the cell viability drops down to about 33.3% (±0.38) under application of AC magnetic field. These findings clearly indicate that the Selol-loaded magnetic nanocapsules present different toxic effects on neoplastic cell lines. Further, the cytotoxic effect was maximized under AC magnetic field application on OSCC, which emphasizes the effectiveness of the magnetohyperthermia approach.

  8. Hinokitiol Exerts Anticancer Activity through Downregulation of MMPs 9/2 and Enhancement of Catalase and SOD Enzymes: In Vivo Augmentation of Lung Histoarchitecture.

    PubMed

    Huang, Chien-Hsun; Jayakumar, Thanasekaran; Chang, Chao-Chien; Fong, Tsorng-Harn; Lu, Shing-Hwa; Thomas, Philip Aloysius; Choy, Cheuk-Sing; Sheu, Joen-Rong

    2015-09-25

    Melanoma is extremely resistant to chemotherapy and the death rate is increasing hastily worldwide. Extracellular matrix promotes the migration and invasion of tumor cells through the production of matrix metalloproteinase (MMP)-2 and -9. Evidence has shown that natural dietary antioxidants are capable of inhibiting cancer cell growth. Our recent studies showed that hinokitiol, a natural bioactive compound, inhibited vascular smooth muscle cell proliferation and platelets aggregation. The present study is to investigate the anticancer efficacy of hinokitiol against B16-F10 melanoma cells via modulating tumor invasion factors MMPs, antioxidant enzymes in vitro. An in vivo mice model of histological investigation was performed to study the patterns of elastic and collagen fibers. Hinokitiol inhibited the expression and activity of MMPs-2 and -9 in B16-F10 melanoma cells, as measured by western blotting and gelatin zymography, respectively. An observed increase in protein expression of MMPs 2/9 in melanoma cells was significantly inhibited by hinokitiol. Notably, hinokitiol (1-5 μM) increased the activities of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) from the reduction in melanoma cells. Also, hinokitiol (2-10 µM) concentration dependently reduced in vitro Fenton reaction induced hydroxyl radical (OH·) formation. An in vivo study showed that hinokitiol treatment increased elastic fibers (EF), collagens dispersion, and improved alveolar alterations in the lungs of B16/F10 injected mice. Overall, our findings propose that hinokitiol may be a potent anticancer candidate through down regulation of MMPs 9/2, reduction of OH· production and enhancement of antioxidant enzymes SOD and CAT.

  9. Novel therapeutic mechanisms determine the effectiveness of lipid-core nanocapsules on melanoma models

    PubMed Central

    Drewes, Carine C; Fiel, Luana A; Bexiga, Celina G; Asbahr, Ana Carolina C; Uchiyama, Mayara K; Cogliati, Bruno; Araki, Koiti; Guterres, Sílvia S; Pohlmann, Adriana R; Farsky, Sandra P

    2016-01-01

    Melanoma is a severe metastatic skin cancer with poor prognosis and no effective treatment. Therefore, novel therapeutic approaches using nanotechnology have been proposed to improve therapeutic effectiveness. Lipid-core nanocapsules (LNCs), prepared with poly(ε-caprolactone), capric/caprylic triglyceride, and sorbitan monostearate and stabilized by polysorbate 80, are efficient as drug delivery systems. Here, we investigated the effects of acetyleugenol-loaded LNC (AcE-LNC) on human SK-Mel-28 melanoma cells and its therapeutic efficacies on melanoma induced by B16F10 in C57B6 mice. LNC and AcE-LNC had z-average diameters and zeta potential close to 210 nm and -10.0 mV, respectively. CytoViva® microscopy images showed that LNC and AcE-LNC penetrated into SK-Mel-28 cells, and remained in the cytoplasm. AcE-LNC in vitro treatment (18–90×109 particles/mL; 1 hour) induced late apoptosis and necrosis; LNC and AcE-LNC (3–18×109 particles/mL; 48 hours) treatments reduced cell proliferation and delayed the cell cycle. Elevated levels of nitric oxide were found in supernatant of LNC and AcE-LNC, which were not dependent on nitric oxide synthase expressions. Daily intraperitoneal or oral treatment (days 3–10 after tumor injection) with LNC or AcE-LNC (1×1012 particles/day), but not with AcE (50 mg/kg/day, same dose as AcE-LNC), reduced the volume of the tumor; nevertheless, intraperitoneal treatment caused toxicity. Oral LNC treatment was more efficient than AcE-LNC treatment. Moreover, oral treatment with nonencapsulated capric/caprylic triglyceride did not inhibit tumor development, implying that nanocapsule supramolecular structure is important to the therapeutic effects. Together, data herein presented highlight the relevance of the supramolecular structure of LNCs to toxicity on SK-Mel-28 cells and to the therapeutic efficacy on melanoma development in mice, conferring novel therapeutic mechanisms to LNC further than a drug delivery system. PMID:27099491

  10. Tks5-dependent formation of circumferential podosomes/invadopodia mediates cell-cell fusion.

    PubMed

    Oikawa, Tsukasa; Oyama, Masaaki; Kozuka-Hata, Hiroko; Uehara, Shunsuke; Udagawa, Nobuyuki; Saya, Hideyuki; Matsuo, Koichi

    2012-05-14

    Osteoclasts fuse to form multinucleated cells during osteoclastogenesis. This process is mediated by dynamic rearrangement of the plasma membrane and cytoskeleton, and it requires numerous factors, many of which have been identified. The underlying mechanism remains obscure, however. In this paper, we show that Tks5, a master regulator of invadopodia in cancer cells, is crucial for osteoclast fusion downstream of phosphoinositide 3-kinase and Src. Expression of Tks5 was induced during osteoclastogenesis, and prevention of this induction impaired both the formation of circumferential podosomes and osteoclast fusion without affecting cell differentiation. Tyrosine phosphorylation of Tks5 was attenuated in Src-/- osteoclasts, likely accounting for defects in podosome organization and multinucleation in these cells. Circumferential invadopodia formation in B16F0 melanoma cells was also accompanied by Tks5 phosphorylation. Co-culture of B16F0 cells with osteoclasts in an inflammatory milieu promoted the formation of melanoma-osteoclast hybrid cells. Our results thus reveal an unexpected link between circumferential podosome/invadopodium formation and cell-cell fusion in and beyond osteoclasts.

  11. Defective Cell Cycle Checkpoint Functions in Melanoma Are Associated with Altered Patterns of Gene Expression

    PubMed Central

    Kaufmann, William K.; Nevis, Kathleen R.; Qu, Pingping; Ibrahim, Joseph G.; Zhou, Tong; Zhou, Yingchun; Simpson, Dennis A.; Helms-Deaton, Jennifer; Cordeiro-Stone, Marila; Moore, Dominic T.; Thomas, Nancy E.; Hao, Honglin; Liu, Zhi; Shields, Janiel M.; Scott, Glynis A.; Sharpless, Norman E.

    2009-01-01

    Defects in DNA damage responses may underlie genetic instability and malignant progression in melanoma. Cultures of normal human melanocytes (NHMs) and melanoma lines were analyzed to determine whether global patterns of gene expression could predict the efficacy of DNA damage cell cycle checkpoints that arrest growth and suppress genetic instability. NHMs displayed effective G1 and G2 checkpoint responses to ionizing radiation-induced DNA damage. A majority of melanoma cell lines (11/16) displayed significant quantitative defects in one or both checkpoints. Melanomas with B-RAF mutations as a class displayed a significant defect in DNA damage G2 checkpoint function. In contrast the epithelial-like subtype of melanomas with wild-type N-RAS and B-RAF alleles displayed an effective G2 checkpoint but a significant defect in G1 checkpoint function. RNA expression profiling revealed that melanoma lines with defects in the DNA damage G1 checkpoint displayed reduced expression of p53 transcriptional targets, such as CDKN1A and DDB2, and enhanced expression of proliferation-associated genes, such as CDC7 and GEMININ. A Bayesian analysis tool was more accurate than significance analysis of microarrays for predicting checkpoint function using a leave-one-out method. The results suggest that defects in DNA damage checkpoints may be recognized in melanomas through analysis of gene expression. PMID:17597816

  12. Rb-Raf-1 interaction disruptor RRD-251 induces apoptosis in metastatic melanoma cells and synergizes with dacarbazine

    PubMed Central

    Singh, Sandeep; Davis, Rebecca; Alamanda, Vignesh; Pireddu, Roberta; Pernazza, Daniel; Sebti, Said; Lawrence, Nicholas; Chellappan, Srikumar

    2010-01-01

    Metastatic melanoma is an aggressive cancer with very low response rate against conventional chemotherapeutic agents such as dacarbazine (DTIC). Inhibitor of Rb-Raf-1 interaction (RRD-251) was tested against the melanoma cell lines SK-MEL-28, SK-MEL-5 and SK-MEL-2. RRD-251 was found to be a potent inhibitor of melanoma cell proliferation, irrespective of V600E B-Raf mutation status of the cell lines. In a SK-MEL-28 xenograft experiment, RRD-251 exerted a significant suppression of tumor growth compared to vehicle (p=0.003). Similar to in vitro effects, tumors from RRD-251 treated animals showed decreased Rb-Raf-1 interaction in vivo. Growth suppressive effects of RRD-251 were associated with induction of apoptosis as well as a G1 arrest, with an accompanying decrease in S-phase cells. RRD-251 inhibited Rb phosphorylation, and downregulated E2F1 protein levels in these cells. Real-time PCR analysis showed that RRD-251 caused downregulation of cell cycle regulatory genes thymidylate synthase (TS) and cdc6 as well as anti-apoptotic gene Mcl-1. Combinatorial treatment of RRD-251 and DTIC resulted in a significantly higher apoptosis in DTIC resistant cell lines SK-MEL-28 and SK-MEL-5, as revealed by increased Caspase-3 activity and PARP cleavage. Since aberrant Rb/E2F pathway is associated with melanoma progression and resistance to apoptosis, these results suggest that the Rb-Raf-1 inhibitor could be an effective agent for melanoma treatment, either alone or in combination with DTIC. PMID:21139044

  13. Rb-Raf-1 interaction disruptor RRD-251 induces apoptosis in metastatic melanoma cells and synergizes with dacarbazine.

    PubMed

    Singh, Sandeep; Davis, Rebecca; Alamanda, Vignesh; Pireddu, Roberta; Pernazza, Daniel; Sebti, Said; Lawrence, Nicholas; Chellappan, Srikumar

    2010-12-01

    Metastatic melanoma is an aggressive cancer with very low response rate against conventional chemotherapeutic agents such as dacarbazine (DTIC). Inhibitor of Rb-Raf-1 interaction RRD-251 was tested against the melanoma cell lines SK-MEL-28, SK-MEL-5, and SK-MEL-2. RRD-251 was found to be a potent inhibitor of melanoma cell proliferation, irrespective of V600E B-Raf mutation status of the cell lines. In a SK-MEL-28 xenograft experiment, RRD-251 exerted a significant suppression of tumor growth compared with vehicle (P = 0.003). Similar to in vitro effects, tumors from RRD-251-treated animals showed decreased Rb-Raf-1 interaction in vivo. Growth suppressive effects of RRD-251 were associated with induction of apoptosis as well as a G(1) arrest, with an accompanying decrease in S-phase cells. RRD-251 inhibited Rb phosphorylation and downregulated E2F1 protein levels in these cells. Real-time PCR analysis showed that RRD-251 caused downregulation of cell-cycle regulatory genes thymidylate synthase (TS) and cdc6 as well as the antiapoptotic gene Mcl-1. Combinatorial treatment of RRD-251 and DTIC resulted in a significantly higher apoptosis in DTIC resistant cell lines SK-MEL-28 and SK-MEL-5, as revealed by increased caspase-3 activity and PARP cleavage. Because aberrant Rb/E2F pathway is associated with melanoma progression and resistance to apoptosis, these results suggest that the Rb-Raf-1 inhibitor could be an effective agent for melanoma treatment, either alone or in combination with DTIC. ©2010 AACR.

  14. Sensitization of B16 tumor cells with a CXCR4 antagonist increases the efficacy of immunotherapy for established lung metastases

    PubMed Central

    Lee, Chih-hung; Kakinuma, Takashi; Wang, Julia; Zhang, Hong; Palmer, Douglas C.; Restifo, Nicholas P.; Hwang, Sam T.

    2008-01-01

    Expression of the chemokine receptor CXCR4 by tumor cells promotes metastasis, possibly by activating pro-survival signals that render cancer cells resistant to immune attack. Inhibition of CXCR4 with a peptide antagonist, T22, blocks metastatic implantation of CXCR4-transduced B16 (CXCR4-luc-B16) melanoma cells in lung, but not the outgrowth of established metastases, raising the question of how T22 can best be used in a clinical setting. Herein, whereas the treatment of CXCR4-luc-B16 cells in vitro with the CXCR4 ligand CXCL12 did not reduce killing induced by cisplatin or cyclophosphamide, CXCL12 markedly reduced Fas-dependent killing by gp100-specific (pmel-1) CD8+ T cells. T22 pretreatment restored sensitivity of CXCR4-luc-B16 cells to pmel-1 killing, even in the presence of CXCL12. Two immune-augmenting regimens were used in combination with T22 to treat experimental lung metastases. First, low-dose cyclophosphamide treatment (100 mg/kg) on day 5 in combination with T22 (days 4–7) yielded a ~70% reduction of B16 metastatic tumor burden in the lungs compared with cyclophosphamide treatment alone (P < 0.001). Furthermore, whereas anti–CTL antigen 4 (CTLA4) monoclonal antibody (mAb; or T22 treatment) alone had little effect on established B16 metastases, pretreatment with T22 (in combination with anti-CTLA4 mAb) resulted in a 50% reduction in lung tumor burden (P = 0.02). Thus, in vitro, CXCR4 antagonism with T22 renders B16 cells susceptible to killing by antigen-specific T cells. In vivo, T22 synergizes with cyclophosphamide or anti-CTLA4 mAb in the treatment of established lung metastases, suggesting a novel strategy for augmenting the efficacy of immunotherapy. PMID:17041104

  15. Gene silencing of beta-catenin in melanoma cells retards their growth but promotes the formation of pulmonary metastasis in mice.

    PubMed

    Takahashi, Yuki; Nishikawa, Makiya; Suehara, Tetsuya; Takiguchi, Naomi; Takakura, Yoshinobu

    2008-11-15

    Altered expression of beta-catenin, a key component of the Wnt signaling pathway, is involved in a variety of cancers because increased levels of beta-catenin protein are frequently associated with enhanced cellular proliferation. Although our previous study demonstrated that gene silencing of beta-catenin in melanoma B16-BL6 cells by plasmid DNA (pDNA) expressing short-hairpin RNA targeting the gene (pshbeta-catenin) markedly suppressed their growth in vivo, gene silencing of beta-catenin could promote tumor metastasis by the rearranging cell adhesion complex. In this study, we investigated how silencing of beta-catenin affects metastatic aspects of melanoma cells. Transfection of B16-BL6 cells with pshbeta-catenin significantly reduced the amount of cadherin protein, a cell adhesion molecule binding to beta-catenin, with little change in its mRNA level. Cadherin-derived fragments were detected in culture media of B16-BL6 cells transfected with pshbeta-catenin, suggesting that cadherin is shed from the cell surface when the expression of beta-catenin is reduced. The mobility of B16-BL6 cells transfected with pshbeta-catenin was greater than that of cells transfected with any of the control pDNAs. B16-BL6 cells stably transfected with pshbeta-catenin (B16/pshbeta-catenin) formed less or an equal number of tumor nodules in the lung than cells stably transfected with other plasmids when injected into mice via the tail vein. However, when subcutaneously inoculated, B16/pshbeta-catenin cells formed more nodules in the lung than the other stably transfected cells. These results raise concerns about the gene silencing of beta-catenin for inhibiting tumor growth, because it promotes tumor metastasis by reducing the amount of cadherin in tumor cells. (c) 2008 Wiley-Liss, Inc.

  16. Mutated PPP1R3B is recognized by T cells used to treat a melanoma patient who experienced a durable complete tumor regression

    PubMed Central

    Lu, Yong-Chen; Yao, Xin; Li, Yong F.; El-Gamil, Mona; Dudley, Mark E.; Yang, James C.; Almeida, Jorge R.; Douek, Daniel C.; Samuels, Yardena; Rosenberg, Steven A.; Robbins, Paul F.

    2013-01-01

    Adoptive cell therapy with tumor infiltrating lymphocytes (TILs) represents an effective treatment for patients with metastatic melanoma. However, most of the antigen targets recognized by effective melanoma reactive TILs remain elusive. In this study, patient 2369 experienced a complete response, including regressions of bulky liver tumor masses ongoing beyond seven years following adoptive TILs transfer. The screening of a cDNA library generated from the autologous melanoma cell line resulted in the isolation of a mutated PPP1R3B (protein phosphatase 1, regulatory (inhibitor) subunit 3B) gene product. The mutated PPP1R3B peptide represents the immunodominant epitope recognized by tumor reactive T cells in TIL 2369. Five years following adoptive transfer, peripheral blood T lymphocytes obtained from patient 2369 recognized the mutated PPP1R3B epitope. These results demonstrate that adoptive T cell therapy targeting a tumor-specific antigen can mediate long-term survival for a patient with metastatic melanoma. This study also provides an impetus to develop personalized immunotherapy targeting tumor-specific, mutated antigens. PMID:23690473

  17. Suppression of adhesion-induced protein tyrosine phosphorylation decreases invasive and metastatic potentials of B16-BL6 melanoma cells by protein tyrosine kinase inhibitor genistein.

    PubMed

    Yan, C; Han, R

    1997-01-01

    Protein tyrosine kinase (PTK) appears to be involved in the activation of signaling during cell attachment to and spreading on extracellular matrix (ECM) in the metastatic cascade. To verify the assumption that PTK inhibitors might impair ECM signaling and prevent cancer metastasis, the highly metastatic B16-BL6 mouse melanoma cells were exposed to the PTK inhibitor genistein for 3 days. The ability of the cells to invade through reconstituted basement membrane (Matrigel) and to establish experimental pulmonary metastatic foci in C57BL/6 mice decreased after genistein exposure. The genistein-treated cells were also prevented from attaching to Matrigel and spread extremely poorly on the ECM substratum. Immunoblot analysis showed that tyrosine phosphorylation of a 125-kD protein in response to cell spreading on Matrigel was suppressed in the genistein-treated cells. Adhesion-induced protein tyrosine phosphorylation represents the earlier and specific event in the activation of ECM signaling, so this result implied ECM signaling was impaired in the treated cells. With immunofluorescence microscopy, the adhesion-induced tyrosine phosphorylated proteins were located at the pericytoplasms of well-spread cells, but not at the periphery of poorly spread genistein-treated cells. Therefore, this paper suggests that genistein might impair ECM signaling and subsequently prevent cancer cells from spreading well and invading or establishing metastasis through the suppression of adhesion-induced protein tyrosine phosphorylation. PTKs and adhesion-induced protein tyrosine phosphorylation might play a role in the control of invasion and metastasis.

  18. Anti-melanoma activity of Forsythiae Fructus aqueous extract in mice involves regulation of glycerophospholipid metabolisms by UPLC/Q-TOF MS-based metabolomics study

    PubMed Central

    Bao, Jiaolin; Liu, Fang; Zhang, Chao; Wang, Kai; Jia, Xuejing; Wang, Xiaotong; Chen, Meiwan; Li, Peng; Su, Huanxing; Wang, Yitao; Wan, Jian-Bo; He, Chengwei

    2016-01-01

    Metabolomics is a comprehensive assessment of endogenous metabolites of a biological system in a holistic context. In this study, we evaluated the in vivo anti-melanoma activity of aqueous extract of Forsythiae Fructus (FAE) and globally explored the serum metabolome characteristics of B16-F10 melanoma-bearing mice. UPLC/Q-TOF MS combined with pattern recognition approaches were employed to examine the comprehensive metabolic signatures and differentiating metabolites. The results demonstrated that FAE exhibited remarkable antitumor activity against B16-F10 melanoma in C57BL/6 mice and restored the disturbed metabolic profile by tumor insult. We identified 17 metabolites which were correlated with the antitumor effect of FAE. Most of these metabolites are involved in glycerophospholipid metabolisms. Notably, several lysophosphatidylcholines (LysoPCs) significantly decreased in tumor model group, while FAE treatment restored the changes of these phospholipids to about normal condition. Moreover, we found that lysophosphatidylcholine acyltransferase 1 (LPCAT1) and autotaxin (ATX) were highly expressed in melanoma, and FAE markedly down-regulated their expression. These findings indicated that modulation of glycerophospholipid metabolisms may play a pivotal role in the growth of melanoma and the antitumor activity of FAE. Besides, our results suggested that serum LysoPCs could be potential biomarkers for the diagnosis and prognosis of melanoma and other malignant tumors. PMID:27991567

  19. Structural, spectroscopic and cytotoxicity studies of TbF3@CeF3 and TbF3@CeF3@SiO2 nanocrystals.

    PubMed

    Grzyb, Tomasz; Runowski, Marcin; Dąbrowska, Krystyna; Giersig, Michael; Lis, Stefan

    2013-01-01

    Terbium fluoride nanocrystals, covered by a shell, composed of cerium fluoride were synthesized by a co-precipitation method. Their complex structure was formed spontaneously during the synthesis. The surface of these core/shell nanocrystals was additionally modified by silica. The properties of TbF 3 @CeF 3 and TbF 3 @CeF 3 @SiO 2 nanocrystals, formed in this way, were investigated. Spectroscopic studies showed that the differences between these two groups of products resulted from the presence of the SiO 2 shell. X-ray diffraction patterns confirmed the trigonal crystal structure of TbF 3 @CeF 3 nanocrystals. High resolution transmission electron microscopy in connection with energy-dispersive X-ray spectroscopy showed a complex structure of the formed nanocrystals. Crystallized as small discs, 'the products', with an average diameter around 10 nm, showed an increase in the concentration of Tb 3+ ions from surface to the core of nanocrystals. In addition to photo-physical analyses, cytotoxicity studies were performed on HSkMEC (Human Skin Microvascular Endothelial Cells) and B16F0 mouse melanoma cancer cells. The cytotoxicity of the nanomaterials was neutral for the investigated cells with no toxic or antiproliferative effect in the cell cultures, either for normal or for cancer cells. This fact makes the obtained nanocrystals good candidates for biological applications and further modifications of the SiO 2 shell. .

  20. Structural, spectroscopic and cytotoxicity studies of TbF3@CeF3 and TbF3@CeF3@SiO2 nanocrystals

    NASA Astrophysics Data System (ADS)

    Grzyb, Tomasz; Runowski, Marcin; Dąbrowska, Krystyna; Giersig, Michael; Lis, Stefan

    2013-10-01

    Terbium fluoride nanocrystals, covered by a shell, composed of cerium fluoride were synthesized by a co-precipitation method. Their complex structure was formed spontaneously during the synthesis. The surface of these core/shell nanocrystals was additionally modified by silica. The properties of TbF3@CeF3 and TbF3@CeF3@SiO2 nanocrystals, formed in this way, were investigated. Spectroscopic studies showed that the differences between these two groups of products resulted from the presence of the SiO2 shell. X-ray diffraction patterns confirmed the trigonal crystal structure of TbF3@CeF3 nanocrystals. High resolution transmission electron microscopy in connection with energy-dispersive X-ray spectroscopy showed a complex structure of the formed nanocrystals. Crystallized as small discs, `the products', with an average diameter around 10 nm, showed an increase in the concentration of Tb3+ ions from surface to the core of nanocrystals. In addition to photo-physical analyses, cytotoxicity studies were performed on HSkMEC (Human Skin Microvascular Endothelial Cells) and B16F0 mouse melanoma cancer cells. The cytotoxicity of the nanomaterials was neutral for the investigated cells with no toxic or antiproliferative effect in the cell cultures, either for normal or for cancer cells. This fact makes the obtained nanocrystals good candidates for biological applications and further modifications of the SiO2 shell.

  1. The systematic study of the electroporation and electrofusion of B16-F1 and CHO cells in isotonic and hypotonic buffer.

    PubMed

    Usaj, Marko; Kanduser, Masa

    2012-09-01

    The fusogenic state of the cell membrane can be induced by external electric field. When two fusogenic membranes are in close contact, cell fusion takes place. An appropriate hypotonic treatment of cells before the application of electric pulses significantly improves electrofusion efficiency. How hypotonic treatment improves electrofusion is still not known in detail. Our results indicate that at given induced transmembrane potential electroporation was not affected by buffer osmolarity. In contrast to electroporation, cells' response to hypotonic treatment significantly affects their electrofusion. High fusion yield was observed when B16-F1 cells were used; this cell line in hypotonic buffer resulted in 41 ± 9 % yield, while in isotonic buffer 32 ± 11 % yield was observed. Based on our knowledge, these fusion yields determined in situ by dual-color fluorescence microscopy are among the highest in electrofusion research field. The use of hypotonic buffer was more crucial for electrofusion of CHO cells; the fusion yield increased from below 1 % in isotonic buffer to 10 ± 4 % in hypotonic buffer. Since the same degree of cell permeabilization was achieved in both buffers, these results indicate that hypotonic treatment significantly improves fusion yield. The effect could be attributed to improved physical contact of cell membranes or to enhanced fusogenic state of the cell membrane itself.

  2. Inferring the Impact of Regulatory Mechanisms that Underpin CD8+ T Cell Control of B16 Tumor Growth In vivo Using Mechanistic Models and Simulation.

    PubMed

    Klinke, David J; Wang, Qing

    2016-01-01

    A major barrier for broadening the efficacy of immunotherapies for cancer is identifying key mechanisms that limit the efficacy of tumor infiltrating lymphocytes. Yet, identifying these mechanisms using human samples and mouse models for cancer remains a challenge. While interactions between cancer and the immune system are dynamic and non-linear, identifying the relative roles that biological components play in regulating anti-tumor immunity commonly relies on human intuition alone, which can be limited by cognitive biases. To assist natural intuition, modeling and simulation play an emerging role in identifying therapeutic mechanisms. To illustrate the approach, we developed a multi-scale mechanistic model to describe the control of tumor growth by a primary response of CD8+ T cells against defined tumor antigens using the B16 C57Bl/6 mouse model for malignant melanoma. The mechanistic model was calibrated to data obtained following adenovirus-based immunization and validated to data obtained following adoptive transfer of transgenic CD8+ T cells. More importantly, we use simulation to test whether the postulated network topology, that is the modeled biological components and their associated interactions, is sufficient to capture the observed anti-tumor immune response. Given the available data, the simulation results also provided a statistical basis for quantifying the relative importance of different mechanisms that underpin CD8+ T cell control of B16F10 growth. By identifying conditions where the postulated network topology is incomplete, we illustrate how this approach can be used as part of an iterative design-build-test cycle to expand the predictive power of the model.

  3. Regulation of miR-21 expression in human melanoma via UV-ray-induced melanin pigmentation.

    PubMed

    Lin, Kuan-Yu; Chen, Chien-Min; Lu, Cheng-You; Cheng, Chun-Yuan; Wu, Yu-Hsin

    2017-08-01

    Excessive environmental ultraviolet (UV) radiation produces genetic mutations that can lead to skin cancer. This study was designed to assess the potential inhibitory activity of microRNA-21 (miR-21) on the UV irradiation-stimulated melanogenesis signal pathway in melanoma cells. The molecular mechanism of miR-21-induced inhibitory activity on UV-ray-stimulated melanogenesis-regulating proteins was examined in A375.S2 human melanoma and B16F10 mouse melanoma cells. UV irradiation for 30 min induced melanogenesis signal pathway by increasing melanin production and the number of A375.S2 cells. Similarly, UV radiation increased the expression of α-melanocyte-stimulating hormone (α-MSH) protein and decreased the melanogenesis-regulating signal, such as EGFR and Akt phosphorylation. Notably, miR-21 overexpression in UV-ray-stimulated A375.S2 cells decreased α-MSH expression and increased EGFR and Akt phosphorylation levels. Furthermore, miR-21 on UV-ray- induced melanogenesis was down-regulated by the Akt inhibitor and the EGFR inhibitor (Gefitinib). Results suggest that the suppressive activity of miR-21 on UV-ray-stimulated melanogenesis may involve the down-regulation of α-MSH and the activation in both of EGFR and Akt. © 2017 Wiley Periodicals, Inc.

  4. Introduction of an 8-aminooctanoic acid linker enhances uptake of 99mTc-labeled lactam bridge-cyclized α-MSH peptide in melanoma.

    PubMed

    Guo, Haixun; Miao, Yubin

    2014-12-01

    The purpose of this study was to examine the effects of amino acid, hydrocarbon, and polyethylene glycol (PEG) linkers on the melanoma targeting and imaging properties of (99m)Tc-labeled lactam bridge-cyclized HYNIC-linker-Nle-CycMSHhex (hydrazinonicotinamide-linker-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2) peptides. Four novel peptides (HYNIC-GGGNle-CycMSHhex, HYNIC-GSGNle-CycMSHhex, HYNIC-PEG2Nle-CycMSHhex, and HYNIC-AocNle-CycMSHhex) were designed and synthesized. The melanocortin-1 receptor binding affinities of the peptides were determined in B16/F1 melanoma cells. The biodistribution of (99m)Tc(ethylenediaminediacetic acid [EDDA])-HYNIC-GGGNle-CycMSHhex, (99m)Tc(EDDA)-HYNIC-GSGNle-CycMSHhex, (99m)Tc(EDDA)-HYNIC-PEG2Nle-CycMSHhex, and (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice at 2 h after injection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex were further examined because of its high melanoma uptake. The inhibitory concentrations of 50% (IC50) for HYNIC-GGGNle-CycMSHhex, HYNIC-GSGNle-CycMSHhex, HYNIC-PEG2Nle-CycMSHhex, and HYNIC-AocNle-CycMSHhex were 0.7 ± 0.1, 0.8 ± 0.09, 0.4 ± 0.08, and 0.3 ± 0.06 nM, respectively, in B16/F1 melanoma cells. Among these four (99m)Tc-labeled peptides, (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex displayed the highest melanoma uptake (22.3 ± 1.72 percentage injected dose/g) at 2 h after injection. (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex exhibited high tumor-to-normal-organ uptake ratios except for the kidneys. The tumor-to-kidney uptake ratios of (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex were 3.29, 3.63, and 6.78 at 2, 4, and 24 h, respectively, after injection. The melanoma lesions were clearly visualized by SPECT/CT using (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex as an imaging probe at 2 h after injection. High melanoma uptake and fast urinary clearance of (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex highlighted its

  5. Introduction of an 8-Aminooctanoic Acid Linker Enhances the melanoma uptake of Tc-99m-labeled Lactam Bridge-Cyclized Alpha-MSH Peptide

    PubMed Central

    Guo, Haixun; Miao, Yubin

    2015-01-01

    The purpose of this study was to examine the effects of amino acid, hydrocarbon and polyethylene glycol (PEG) linkers on melanoma targeting and imaging properties of 99mTc-labeled lactam bridge-cyclized HYNIC-linker-Nle-CycMSHhex {hydrazinonicotinamide-linker-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} peptides. Methods four novel peptides {HYNIC-GGGNle-CycMSHhex, HYNIC-GSGNle-CycMSHhex, HYNIC-PEG2Nle-CycMSHhex and HYNIC-AocNle-CycMSHhex} were designed and synthesized. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined in B16/F1 melanoma cells. The biodistribution of 99mTc(EDDA)-HYNIC-GGGNle-CycMSHhex, 99mTc(EDDA)-HYNIC-GSGNle-CycMSHhex, 99mTc(EDDA)-HYNIC-PEG2Nle-CycMSHhex and 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice at 2 h post-injection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex were further examined because of its high melanoma uptake. Results The IC50 values of HYNIC-GGGNle-CycMSHhex, HYNIC-GSGNle-CycMSHhex, HYNIC-PEG2Nle-CycMSHhex, and HYNIC-AocNle-CycMSHhex were 0.7 ± 0.1, 0.8 ± 0.09, 0.4 ± 0.08, and 0.3 ± 0.06 nM in B16/F1 melanoma cells, respectively. Among these four 99mTc-labeled peptides, 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex displayed the highest melanoma uptake (22.3 ± 1.72% ID/g) at 2 h post-injection. 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex exhibited high tumor to normal organ uptake ratios except for the kidneys. The tumor/kidney uptake ratios of 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex were 3.29, 3.63 and 6.78 at 2, 4 and 24 h post-injection. The melanoma lesions were clearly visualized by single photon emission computed tomography (SPECT)/CT using 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex as an imaging probe at 2 h post-injection. Conclusion High melanoma uptake and fast urinary clearance of 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex highlighted its potential for metastatic melanoma detection in the future

  6. Comparing the efficacy of photodynamic and sonodynamic therapy in non-melanoma and melanoma skin cancer.

    PubMed

    McEwan, Conor; Nesbitt, Heather; Nicholas, Dean; Kavanagh, Oisin N; McKenna, Kevin; Loan, Philip; Jack, Iain G; McHale, Anthony P; Callan, John F

    2016-07-01

    Sonodynamic therapy (SDT) involves the activation of a non-toxic sensitiser drug using low-intensity ultrasound to produce cytotoxic reactive oxygen species (ROS). Given the low tissue attenuation of ultrasound, SDT provides a significant benefit over the more established photodynamic therapy (PDT) as it enables activation of sensitisers at a greater depth within human tissue. In this manuscript, we compare the efficacy of aminolevulinic acid (ALA) mediated PDT and SDT in a squamous cell carcinoma (A431) cell line as well as the ability of these treatments to reduce the size of A431 ectopic tumours in mice. Similarly, the relative cytotoxic ability of Rose Bengal mediated PDT and SDT was investigated in a B16-melanoma cell line and also in a B16 ectopic tumour model. The results reveal no statistically significant difference in efficacy between ALA mediated PDT or SDT in the non-melanoma model while Rose Bengal mediated SDT was significantly more efficacious than PDT in the melanoma model. This difference in efficacy was, at least in part, attributed to the dark pigmentation of the melanoma cells that effectively filtered the excitation light preventing it from activating the sensitiser while the use of ultrasound circumvented this problem. These results suggest SDT may provide a better outcome than PDT when treating highly pigmented cancerous skin lesions. Copyright © 2016. Published by Elsevier Ltd.

  7. Development of polymeric nanopaclitaxel and comparison with free paclitaxel for effects on cell proliferation of MCF-7 and B16F0 carcinoma cells.

    PubMed

    Yadav, Deepak; Anwar, Mohammad Faiyaz; Garg, Veena; Kardam, Hemant; Beg, Mohd Nadeem; Suri, Suruchi; Gaur, Sikha; Asif, Mohd

    2014-01-01

    Paclitaxel is hydrophobic in nature and is recognized as a highly toxic anticancer drug, showing adverse effects in normal body sites. In this study, we developed a polymeric nano drug carrier for safe delivery of the paclitaxel to the cancer that releases the drug in a sustained manner and reduces side effects. N-isopropylacrylamide/ vinyl pyrrolidone (NIPAAm/VP) nanoparticles were synthesized by radical polymerization. Physico- chemical characterization of the polymeric nanoparticles was conducted using dynamic light scattering, transmission electron microscopy, scanning electron microscopy and nuclear magnetic resonance, which confirmed polymerization of formulated nanoparticles. Drug release was assessed using a spectrophotometer and cell viability assays were carried out on the MCF-7 breast cancer and B16F0 skin cancer cell lines. NIPAAm/ VP nanoparticles demonstrated a size distribution in the 65-108 nm range and surface charge measured -15.4 mV. SEM showed the nanoparticles to be spherical in shape with a slow drug release of ~70% in PBS at 38° over 96 h. Drug loaded nanoparticles were associated with increased viability of MCF-7 and B16F0 cells in comparison to free paclitaxel. Nano loaded paclitaxel shows high therapeutic efficiency by sustained release action for the longer period of time, i increasing its efficacy and biocompatibility for human cancer therapy. Therefore, paclitaxel loaded (NIPAAm/VP) nanoparticles may provide opportunities to expand delivery of the drug for clinical selection.

  8. Blue light inhibits proliferation of melanoma cells

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  9. Anticancer and Anti-Inflammatory Properties of Ganoderma lucidum Extract Effects on Melanoma and Triple-Negative Breast Cancer Treatment.

    PubMed

    Barbieri, Antonio; Quagliariello, Vincenzo; Del Vecchio, Vitale; Falco, Michela; Luciano, Antonio; Amruthraj, Nagoth Joseph; Nasti, Guglielmo; Ottaiano, Alessandro; Berretta, Massimiliano; Iaffaioli, Rosario Vincenzo; Arra, Claudio

    2017-02-28

    Among the most important traditional medicinal fungi, Ganoderma lucidum has been used as a therapeutic agent for the treatment of numerous diseases, including cancer, in Oriental countries. The aim of this study is to investigate the anti-inflammatory, anticancer and anti-metastatic activities of Ganoderma lucidum extracts in melanoma and triple-negative breast cancer cells. Ganoderma lucidum extracts were prepared by using common organic solvents; MDA-MB 231 and B16-F10 cell lines were adopted as cellular models for triple-negative breast cancer and melanoma and characterized for cell viability, wound-healing assay and measurement of cytokines secreted by cancer cells under pro-inflammatory conditions (incubation with lipopolysaccharide, LPS) and pretreatment with Ganoderma lucidum extract at different concentrations. Our study demonstrates, for the first time, how Ganoderma lucidum extracts can significantly inhibit the release of IL-8, IL-6, MMP-2 and MMP-9 in cancer cells under pro-inflammatory condition. Interestingly, Ganoderma lucidum extracts significantly also decrease the viability of both cancer cells in a time- and concentration-dependent manner, with abilities to reduce cell migration over time, which is correlated with a lower release of matrix metalloproteases. Taken together, these results indicate the possible use of Ganoderma lucidum extract for the therapeutic management of melanoma and human triple-negative breast cancer.

  10. Gelsolin functions as a metastasis suppressor in B16-BL6 mouse melanoma cells and requirement of the carboxyl-terminus for its effect.

    PubMed

    Fujita, H; Okada, F; Hamada , J; Hosokawa, M; Moriuchi, T; Koya, R C; Kuzumaki, N

    2001-09-01

    Gelsolin, an actin-binding protein, is implicated as a critical regulator in cell motility. In addition, we have reported that cellular levels of gelsolin are decreased in various tumor cells, and overexpression of gelsolin by gene transfer suppresses tumorigenicity. We sought to assess the effects of gelsolin overexpression on metastasis and to determine the importance of a carboxyl-terminus that confers Ca(2+) dependency on gelsolin for effects of its overexpression. Expression vectors with cDNA encoding either full-length wild-type or His321 mutant form, isolated from a flat revertant of Ras-transformed cells and a carboxyl-terminal truncate, C-del of gelsolin, were transfected into a highly metastatic murine melanoma cell line, B16-BL6. Expression of introduced cDNA in transfectants was confirmed using Western blotting, 2-dimensional gel electrophoresis and reverse transcription-polymerase chain reaction (RT-PCR). We characterized phenotypes of transfectants, such as growth rate, colony formation in soft agar, cell motility and metastasis formation in vivo. Transfectants expressing the wild-type, His321 mutant and C-del gelsolin exhibited reduced growth ability in soft agar. Although expression of integrin beta1 or alpha4 on the cell surface of transfectants was not changed, wild-type and His321 mutant gelsolin, except for C-del gelsolin, exhibited retardation of cell spreading, reduced chemotatic migration to fibronectin and suppressed lung colonization in spontaneous metastasis assay. Gelsolin may function as a metastasis suppressor as well as a tumor suppressor gene. The carboxyl-terminus of gelsolin is important for retardation of cell spreading, reduced chemotasis and metastasis suppression. Copyright 2001 Wiley-Liss, Inc.

  11. Host-Derived Pericytes and Sca-1+ Cells Predominate in the MART-1− Stroma Fraction of Experimentally Induced Melanoma

    PubMed Central

    Treviño-Villarreal, J. Humberto; Cotanche, Douglas A.; Sepúlveda, Rosalinda; Bortoni, Magda E.; Manneberg, Otto; Udagawa, Taturo

    2011-01-01

    Identification of cell types in tumor-associated stroma that are involved in the development of melanoma is hampered by their heterogeneity. The authors used flow cytometry and immunohistochemistry to demonstrate that anti–MART-1 antibodies can discriminate between melanoma and stroma cells. They investigated the cellular composition of the MART-1−, non-hematopoietic melanoma-associated stroma, finding it consisted mainly of Sca-1+ and CD146+ cells. These cell types were also observed in the skin and muscle adjacent to developing melanomas. The Sca-1+ cell population was observed distributed in the epidermis, hair follicle bulges, and tumor capsule. The CD146+ population was found distributed within the tumor, mainly associated with blood vessels in a perivascular location. In addition to a perivascular distribution, CD146+ cells expressed α-smooth muscle actin, lacked expression of endothelial markers CD31 and CD34, and were therefore identified as pericytes. Pericytes were found to be associated with CD31+ endothelial cells; however, some pericytes were also observed associated with CD31−, MART-1+ B16 melanoma cells that appeared to form blood vessel structures. Furthermore, the authors observed extensive nuclear expression of HIF-1α in melanoma and stroma cells, suggesting hypoxia is an important factor associated with the melanoma microenvironment and vascularization. The results suggest that pericytes and Sca-1+ stroma cells are important contributors to melanoma development. PMID:22147606

  12. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ferreira, Adilson Kleber, E-mail: ferreira-kleber@usp.br; Department of Medical Oncology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, Groningen; Pasqualoto, Kerly Fernanda Mesquita

    Benzofuroxan is an interesting ring system, which has shown a wide spectrum of biological responses against tumor cell lines. We investigated, herein, the antitumor effects of benzofuroxan derivatives (BFDs) in vitro and in a melanoma mouse model. Cytotoxic effects of twenty-two BFDs were determined by MTT assay. Effects of BFD-22 in apoptosis and cell proliferation were evaluated using Annexin V-FITC/PI and CFSE staining. In addition, the effects in the cell cycle were assessed. Flow cytometry, western blot, and fluorescence microscopy analysis were employed to investigate the apoptosis-related proteins and the BRAF signaling. Cell motility was also exploited through cell invasionmore » and migration assays. Molecular docking approach was performed in order to verify the BFD-22 binding mode into the ATP catalytic site of BRAF kinase. Moreover, the BFD-22 antitumor effects were evaluated in a melanoma murine model using B16F10. BFD-22 was identified as a potential hit against melanoma cells. BFD-22 induced apoptosis and inhibited cell proliferation of B16F10 cells. BFD-22 has suppressed, indeed, the migratory and invasive behavior of B16F10 cells. Cyclin D1 and CDK4 expression were reduced leading to cell cycle arrest at G0/G1 phase. Of note, phosphorylation of BRAF at Ser338 was strongly down-regulated by BFD-22 in B16F10 cells. The accommodation/orientation into the binding site of BRAF was similar of BAY43-9006 (co-crystallized inhibitor of BRAF, sorafenib). Importantly, BFD-22 presented in vivo antimetastatic effects and showed better therapeutic efficacy than sorafenib and taxol. BFD-22 can be considered as a new lead compound and, then, can be helpful for the designing of novel drug candidates to treat melanoma. - Highlights: • BFD-22 induces apoptosis effects of B16F10 cells by mitochondrial pathway. • BFD-22 provokes downstream in the MAPK/ERK kinase signaling cascade. • Molecular docking trials supported BRAF protein as potential target for BDF-22

  13. An Integrin-Targeting RGDK-Tagged Nanocarrier: Anticancer Efficacy of Loaded Curcumin.

    PubMed

    Das, Krishnendu; Nimushakavi, Sahithi; Chaudhuri, Arabinda; Das, Prasanta Kumar

    2017-05-22

    Herein we report the design and development of α 5 β 1 integrin-specific noncovalent RGDK-lipopeptide-functionalized single-walled carbon nanotubes (SWNTs) that selectively deliver the anticancer drug curcumin to tumor cells. RGDK tetrapeptide-tagged amphiphiles were synthesized that efficiently disperse SWNTs with a suspension stability index of >80 % in cell culture media. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)- and lactate dehydrogenase (LDH)-based cell viability assays in tumor (B16F10 melanoma) and noncancerous (NIH3T3 mouse fibroblast) cells revealed the non-cytotoxic nature of these RGDK-lipopeptide-SWNT conjugates. Cellular uptake experiments with monoclonal antibodies against α v β 3 , α v β 5 , and α 5 β 1 integrins showed that these SWNT nanovectors deliver their cargo (Cy3-labeled oligonucleotides, Cy3-oligo) to B16F10 cells selectively via α 5 β 1 integrin. Notably, the nanovectors failed to deliver the Cy3-oligo to NIH3T3 cells. The RGDK-SWNT is capable of delivering the anticancer drug curcumin to B16F10 cells more efficiently than NIH3T3 cells, leading to selective killing of B16F10 cells. Results of Annexin V binding based flow cytometry experiments are consistent with selective killing of tumor cells through the late apoptotic pathway. Biodistribution studies in melanoma (B16F10)-bearing C57BL/6J mice showed tumor-selective accumulation of curcumin intravenously administered via RGDK-lipopeptide-SWNT nanovectors. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Tumor cell-released TLR4 ligands stimulate Gr-1+CD11b+F4/80+ cells to induce apoptosis of activated T cells.

    PubMed

    Liu, Yan-Yan; Sun, Ling-Cong; Wei, Jing-Jing; Li, Dong; Yuan, Ye; Yan, Bin; Liang, Zhi-Hui; Zhu, Hui-Fen; Xu, Yong; Li, Bo; Song, Chuan-Wang; Liao, Sheng-Jun; Lei, Zhang; Zhang, Gui-Mei; Feng, Zuo-Hua

    2010-09-01

    Gr-1(+)CD11b(+)F4/80(+) cells play important roles in tumor development and have a negative effect on tumor immunotherapy. So far, the mechanisms underlying the regulation of their immunosuppressive phenotype by classical and alternative macrophage activation stimuli are not well elucidated. In this study, we found that molecules from necrotic tumor cells (NTC-Ms) stimulated Gr-1(+)CD11b(+)F4/80(+) cells to induce apoptosis of activated T cells but not nonstimulated T cells. The apoptosis-inducing capacity was determined by higher expression levels of arginase I and IL-10 relative to those of NO synthase 2 and IL-12 in Gr-1(+)CD11b(+)F4/80(+) cells, which were induced by NTC-Ms through TLR4 signaling. The apoptosis-inducing capacity of NTC-Ms-stimulated Gr-1(+)CD11b(+)F4/80(+) cells could be enhanced by IL-10. IFN-gamma may reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells only if their response to IFN-gamma was not attenuated. However, the potential of Gr-1(+)CD11b(+)F4/80(+) cells to express IL-12 in response to IFN-gamma could be attenuated by tumor, partially due to the existence of active STAT3 in Gr-1(+)CD11b(+)F4/80(+) cells and NTC-Ms from tumor. In this situation, IFN-gamma could not effectively reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells. Tumor immunotherapy with 4-1BBL/soluble programmed death-1 may significantly reduce, but not abolish the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells in local microenvironment. Blockade of TLR4 signaling could further reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and enhance the suppressive effect of 4-1BBL/soluble form of programmed death-1 on tumor growth. These findings indicate the relationship of distinct signaling pathways with apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and emphasize the importance of blocking TLR4 signaling to prevent the induction of T cell apoptosis by Gr-1(+)CD11b(+)F4/80(+) cells.

  15. MC1R and cAMP signaling inhibit cdc25B activity and delay cell cycle progression in melanoma cells

    PubMed Central

    Lyons, Jesse; Bastian, Boris C.; McCormick, Frank

    2013-01-01

    The melanocortin 1 receptor (MC1R) mediates the tanning response through induction of cAMP and downstream pigmentary enzymes. Diminished function alleles of MC1R are associated with decreased tanning and increased melanoma risk, which has been attributed to increased rates of mutation. We have found that MC1R or cAMP signaling also directly decreases proliferation in melanoma cell lines. MC1R overexpression, treatment with the MC1R ligand, or treatment with small-molecule activators of cAMP signaling causes delayed progression from G2 into mitosis. This delay is caused by phosphorylation and inhibition of cdc25B, a cyclin dependent kinase 1-activating phosphatase, and is rescued by expression of a cdc25B mutant that cannot be phosphorylated at the serine 323 residue. These results show that MC1R and cAMP signaling can directly inhibit melanoma growth through regulation of the G2/M checkpoint. PMID:23908401

  16. Phosphatase of Regenerating Liver-3 Promotes Motility and Metastasis of Mouse Melanoma Cells

    PubMed Central

    Wu, Xiaopeng; Zeng, Hu; Zhang, Xianming; Zhao, Ying; Sha, Haibo; Ge, Xiaomei; Zhang, Minyue; Gao, Xiang; Xu, Qiang

    2004-01-01

    Recent reports suggested that phosphatase of regenerating liver (PRL)-3 might be involved in colorectal carcinoma metastasis with an unknown mechanism. Here we demonstrated that PRL-3 expression was up-regulated in human liver carcinoma compared with normal liver. PRL-3 was also highly expressed in metastatic melanoma B16-BL6 cells but not in its lowly metastatic parental cell line, B16 cells. B16 cells transfected with PRL-3 cDNA displayed morphological transformation from epithelial-like shape to fibroblast-like shape. PRL-3-overexpressed cells showed much higher migratory ability, which could be reversed by specific anti-sense oligodeoxynucleotide and the phosphatase inhibitors sodium orthovanadate or potassium bisperoxo oxovanadate V. Meanwhile, the expression of the catalytically inactive PRL-3 mutations (D72A or C104S) significantly reduced the cell migratory capability. In addition, PRL-3 transfectants demonstrated altered extracellular matrix adhesive property and up-regulated integrin-mediated cell spreading efficiency. Furthermore, we confirmed that PRL-3 could facilitate lung and liver metastasis of B16 cells in an experimental metastasis model in mice, consistent with accelerated proliferation and growth rate both in vitro and in vivo. Together, these observations provide convincing evidence that PRL-3 truly plays a causal role in tumor metastasis. PMID:15161639

  17. PET Imaging of Very Late Antigen-4 in Melanoma: Comparison of 68Ga- and 64Cu-Labeled NODAGA and CB-TE1A1P-LLP2A Conjugates

    PubMed Central

    Beaino, Wissam; Anderson, Carolyn J.

    2014-01-01

    Melanoma is a malignant tumor derived from epidermal melanocytes, and it is known for its aggressiveness, therapeutic resistance, and predisposition for late metastasis. Very late antigen-4 (VLA-4; also called integrin α4β1) is a transmembrane noncovalent heterodimer overexpressed in melanoma tumors that plays an important role in tumor growth, angiogenesis, and metastasis by promoting adhesion and migration of cancer cells. In this study, we evaluated 2 conjugates of a high-affinity VLA-4 peptidomimetic ligand, LLP2A, for PET/CT imaging in a subcutaneous and metastatic melanoma tumor. Methods LLP2A was conjugated to 1,4,8,11-tetraazacyclotetradecane-1-(methane phosphonic acid)-8-(methane carboxylic acid) (CB-TE1A1P) and 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid (NODAGA) chelators for 68Ga and 64Cu labeling. The conjugates were synthesized by solid-phase peptide synthesis, purified by reversed-phase high-performance liquid chromatography, and verified by liquid chromatography mass spectrometry. Saturation and competitive binding assays with B16F10 melanoma cells determined the affinity of the compounds for VLA-4. The biodistributions of the LLP2A conjugates were evaluated in murine B16F10 subcutaneous tumor–bearing C57BL/6 mice. Melanoma metastasis was induced by intracardiac injection of B16F10 cells. PET/CT imaging was performed at 2, 4, and 24 h after injection for the 64Cu tracers and 1 h after injection for the 68Ga tracer. Results 64Cu-labeled CB-TE1A1P-PEG4-LLP2A and NODAGA-PEG4-LLP2A showed high affinity to VLA-4, with a comparable dissociation constant (0.28 vs. 0.23 nM) and receptor concentration (296 vs. 243 fmol/mg). The tumor uptake at 2 h after injection was comparable for the 2 probes, but 64Cu-CB-TE1A1P-PEG4-LLP2A trended toward higher uptake than 64Cu-NODAGA-PEG4-LLP2A (16.9 ± 2.2 vs. 13.4 ± 1.7 percentage injected dose per gram, P = 0.07). Tumor-to-muscle and tumor-to-blood ratios from biodistribution and PET/CT images

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matsuoka, Hiroshi; Department of Pharmacy, Nara Hospital, Kinki University School of Medicine, 1248-1 Ikoma, Nara 630-0293; Tsubaki, Masanobu

    2009-07-15

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

  19. Neural Cell Adhesion Molecule Potentiates the Growth of Murine Melanoma via β-Catenin Signaling by Association with Fibroblast Growth Factor Receptor and Glycogen Synthase Kinase-3β

    PubMed Central

    Liu, Rui; Shi, Yu; Yang, Hai Jie; Wang, Lei; Zhang, Si; Xia, Yin Yan; Wong, Jing Lin Jack; Feng, Zhi Wei

    2011-01-01

    The neural cell adhesion molecule (NCAM) was recently shown to be involved in the progression of various tumors with diverse effects. We previously demonstrated that NCAM potentiates the cellular invasion and metastasis of melanoma. Here we further report that the growth of melanoma is obviously retarded when the expression of NCAM is silenced. We found that the proliferation of murine B16F0 melanoma cells, their colony formation on soft agar, and growth of transplanted melanoma in vivo are clearly inhibited by the introduction of NCAM siRNA. Interestingly, change of NCAM expression level is shown to regulate the activity of Wnt signaling molecule, β-catenin, markedly. This novel machinery requires the function of FGF receptor and glycogen synthase kinase-3β but is independent of the Wnt receptors, MAPK-Erk and PI3K/Akt pathways. In addition, NCAM is found to form a functional complex with β-catenin, FGF receptor, and glycogen synthase kinase-3β. Moreover, up-regulation of NCAM140 and NCAM180 appears more potent than NCAM120 in activation of β-catenin, suggesting that the intracellular domain of NCAM is required for facilitating the β-catenin signaling. Furthermore, the melanoma cells also exhibit distinct differentiation phenotypes with the NCAM silencing. Our findings reveal a novel regulatory role of NCAM in the progression of melanoma that might serve as a new therapeutic target for the treatment of melanoma. PMID:21628472

  20. Neural cell adhesion molecule potentiates the growth of murine melanoma via β-catenin signaling by association with fibroblast growth factor receptor and glycogen synthase kinase-3β.

    PubMed

    Liu, Rui; Shi, Yu; Yang, Hai Jie; Wang, Lei; Zhang, Si; Xia, Yin Yan; Wong, Jing Lin Jack; Feng, Zhi Wei

    2011-07-22

    The neural cell adhesion molecule (NCAM) was recently shown to be involved in the progression of various tumors with diverse effects. We previously demonstrated that NCAM potentiates the cellular invasion and metastasis of melanoma. Here we further report that the growth of melanoma is obviously retarded when the expression of NCAM is silenced. We found that the proliferation of murine B16F0 melanoma cells, their colony formation on soft agar, and growth of transplanted melanoma in vivo are clearly inhibited by the introduction of NCAM siRNA. Interestingly, change of NCAM expression level is shown to regulate the activity of Wnt signaling molecule, β-catenin, markedly. This novel machinery requires the function of FGF receptor and glycogen synthase kinase-3β but is independent of the Wnt receptors, MAPK-Erk and PI3K/Akt pathways. In addition, NCAM is found to form a functional complex with β-catenin, FGF receptor, and glycogen synthase kinase-3β. Moreover, up-regulation of NCAM140 and NCAM180 appears more potent than NCAM120 in activation of β-catenin, suggesting that the intracellular domain of NCAM is required for facilitating the β-catenin signaling. Furthermore, the melanoma cells also exhibit distinct differentiation phenotypes with the NCAM silencing. Our findings reveal a novel regulatory role of NCAM in the progression of melanoma that might serve as a new therapeutic target for the treatment of melanoma.

  1. An electrochemical immunosensing method for detecting melanoma cells.

    PubMed

    Seenivasan, Rajesh; Maddodi, Nityanand; Setaluri, Vijaysaradhi; Gunasekaran, Sundaram

    2015-06-15

    An electrochemical immunosensing method was developed to detect melanoma cells based on the affinity between cell surface melanocortin 1 receptor (MC1R) antigen and anti-MC1R antibody (MC1R-Ab). The MC1R-Abs were immobilized in amino-functionalized silica nanoparticles (n-SiNPs)-polypyrrole (PPy) nanocomposite modified on working electrode surface of screen-printed electrode (SPE). Cyclic voltammetry was employed, with the help of redox mediator ([Fe(CN)6](3-)), to measure the change in anodic oxidation peak current arising due to the specific interaction between MC1R antigens and MC1R-Abs when the target melanoma cells are present in the sample. Various factors affecting the sensor performance, such as the amount of MC1R-Abs loaded, incubation time with the target melanoma cells, the presence of interfering non-melanoma cells, were tested and optimized over different expected melanoma cell loads in the range of 50-7500 cells/2.5 mL. The immunosensor is highly sensitive (20 cells/mL), specific, and reproducible, and the antibody-loaded electrode in ready-to-use stage is stable over two weeks. Thus, in conjunction with a microfluidic lab-on-a-chip device our electrochemical immunosensing approach may be suitable for highly sensitive, selective, and rapid detection of circulating tumor cells (CTCs) in blood samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Involvement of I-A-restricted B-B cell interaction in the polyclonal B cell differentiation induced by lipopolysaccharide

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Takahama, Y.; Ono, S.; Ishihara, K.

    1990-01-01

    The present study has examined a functional role of Ia molecules expressed on murine B cells in polyclonal B cell differentiation induced by lipopolysaccharide (LPS). Reverse, IgM PFC responses of unprimed B cells induced by LPS in the apparent absence of T cells and adherent accessory cells were markedly inhibited in a haplotype-specific manner by Fab monomer fragment of anti-class II (Ia) but not anti-class I MHC monoclonal antibody (mAb). However, the degree of inhibition of LPS responses of H-2-heterozygous F1 B cells expressing both parental I-A products by either one of anti-I-A mAb was at best half that ofmore » the parental B cells. Interestingly, when (B10 x B10.-BR)F1 (H-2b/k) B cells were fractionated into adherent and nonadherent populations by their ability to bind to parental B10 B cell monolayers, LPS responses of F1 B cells adherent to and nonadherent to the B10 B cell monolayers were selectively inhibited by anti-I-Ab and anti-I-Ak mAb, respectively. These results suggest that LPS-responsive F1 B cells comprise at least two separate populations with restriction specificity for only one of the parental I-A products expressed on B cells. In addition, it was demonstrated that the I-A-restriction specificity of LPS-responsive B cells is plastic and determined by H-2-genotype of bone marrow cells present during B cell ontogeny but not by that of radiation-resistant host elements. Namely, the LPS responses of B10-derived B cells from (B10 + B10.BR) (H-2b x H - 2k)F1 radiation bone marrow chimeras but not from B10 (H-2b x H-2k)F1 chimeras became sensitive to the inhibition of anti-I-Ak mAb in the presence of mitomycin C-treated I-Ak-positive B cells, supporting a notion of receptor-Ia molecules interactions rather than like-like interactions.« less

  3. Effects of amino acids on melanoma targeting and clearance properties of Tc-99m-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone peptides.

    PubMed

    Flook, Adam M; Yang, Jianquan; Miao, Yubin

    2013-11-14

    The purpose of this study was to examine the effects of amino acids on melanoma targeting and clearance properties of new (99m)Tc-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-Lys-(Arg(11))CCMSH {c[Arg-Ser-Asp-DTyr-Asp]-Lys-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RNleD-Lys-(Arg(11))CCMSH, RPheD-Lys-(Arg(11))CCMSH, and RdPheD-Lys-(Arg(11))CCMSH peptides were synthesized and evaluated for their melanocortin-1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of (99m)Tc-RSD-Lys-(Arg(11))CCMSH, (99m)Tc-RFD-Lys-(Arg(11))CCMSH, and (99m)Tc-RfD-Lys-(Arg(11))CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The substitution of Gly with Ser, Phe, and dPhe increased the MC1 receptor binding affinities of the peptides, whereas the substitution of Gly with Nle decreased the MC1 receptor binding affinity of the peptide. (99m)Tc-RSD-Lys-(Arg(11))CCMSH exhibited the highest melanoma uptake (18.01 ± 4.22% ID/g) and the lowest kidney and liver uptake among these (99m)Tc-peptides. The B16/F1 melanoma lesions could be clearly visualized by SPECT/CT using (99m)Tc-RSD-Lys-(Arg(11))CCMSH as an imaging probe. It is desirable to reduce the renal uptake of (99m)Tc-RSD-Lys-(Arg(11))CCMSH to facilitate its potential therapeutic application.

  4. Effects of Amino Acids on Melanoma Targeting and Clearance Properties of Tc-99m-Labeled Arg-X-Asp-Conjugated α-Melanocyte Stimulating Hormone Peptides

    PubMed Central

    Flook, Adam M.; Yang, Jianquan; Miao, Yubin

    2013-01-01

    The purpose of this study was to examine the effects of amino acids on melanoma targeting and clearance properties of new 99mTc-labeled Arg-X-Asp-conjugated alpha-melanocyte stimulating hormone (α-MSH) peptides. RSD-Lys-(Arg11)CCMSH {c[Arg-Ser-Asp-dTyr-Asp]-Lys-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RNleD-Lys-(Arg11)CCMSH, RPheD-Lys-(Arg11)CCMSH and RdPheD-Lys-(Arg11)CCMSH peptides were synthesized and evaluated for their melanocortin-1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of 99mTc-RSD-Lys-(Arg11)CCMSH, 99mTc-RFD-Lys-(Arg11)CCMSH and 99mTc-RfD-Lys-(Arg11)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The substitution of Gly with Ser, Phe and dPhe increased the MC1 receptor binding affinities of the peptides, whereas the substitution of Gly with Nle decreased the MC1 receptor binding affinity of the peptide. 99mTc-RSD-Lys-(Arg11)CCMSH exhibited the highest melanoma uptake (18.01 ± 4.22% ID/g) and the lowest kidney and liver uptake among these 99mTc-peptides. The B16/F1 melanoma lesions could be clearly visualized by SPECT/CT using 99mTc-RSD-Lys-(Arg11)CCMSH as an imaging probe. It is desirable to reduce the renal uptake of 99mTc-RSD-Lys-(Arg11)CCMSH to facilitate its potential therapeutic application. PMID:24131154

  5. MITF suppression improves the sensitivity of melanoma cells to a BRAF inhibitor.

    PubMed

    Aida, Satoshi; Sonobe, Yukiko; Tanimura, Hiromi; Oikawa, Nobuhiro; Yuhki, Munehiro; Sakamoto, Hiroshi; Mizuno, Takakazu

    2017-11-28

    Microphthalmia-associated transcription factor (MITF) is expressed in melanomas and has a critical role in melanocyte development and transformation. Because inhibition of MITF inhibits cell growth in melanoma, MITF is a potential therapeutic target molecule. Here, we report the identification of CH6868398, which has a novel chemical structure and suppresses MITF expression at the protein level in melanoma cells. CH6868398 showed cell growth inhibition activity against MITF-dependent melanoma cells both with and without BRAF mutation and also exhibited anti-tumor efficacy in a melanoma xenograft model. Because selective BRAF inhibitors are standard therapeutics for BRAF-mutated melanoma, we investigated the effect of CH6868398 with a BRAF inhibitor, PLX4720, on cell growth inhibition. The addition of CH6868398 enhanced the cell growth inhibition activity of PLX4720 in melanoma cell lines. Furthermore, combination of CH6868398 and PLX4720 efficiently suppressed MITF protein and enhanced cleavage of Caspase3 and poly (ADP-ribose) polymerase (PARP) in melanoma cell lines. These data support the therapeutic potential of CH6868398 as an anti-melanoma agent that reduces MITF protein levels in combination with BRAF inhibitors. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. RASSF6 exhibits promoter hypermethylation in metastatic melanoma and inhibits invasion in melanoma cells

    PubMed Central

    Mezzanotte, Jessica J; Hill, Victoria; Schmidt, M Lee; Shinawi, Thoraia; Tommasi, Stella; Krex, Dietmar; Schackert, Gabriele; Pfeifer, Gerd P; Latif, Farida; Clark, Geoffrey J

    2014-01-01

    Brain metastasis is a major contributor to cancer mortality, yet, the genetic changes underlying the development of this capacity remain poorly understood. RASSF proteins are a family of tumor suppressors that often suffer epigenetic inactivation during tumorigenesis. However, their epigenetic status in brain metastases has not been well characterized. We have examined the promoter methylation of the classical RASSF members (RASSF1A-RASSF6) in a panel of metastatic brain tumor samples. RASSF1A and RASSF2 have been shown to undergo promoter methylation at high frequency in primary lung and breast tumors and in brain metastases. Other members exhibited little or no methylation in these tumors. In examining melanoma metastases, however, we found that RASSF6 exhibits the highest frequency of inactivation in melanoma and in melanoma brain metastases. Most melanomas are driven by an activating mutation in B-Raf. Introduction of RASSF6 into a B-RafV600E-containing metastatic melanoma cell line inhibited its ability to invade through collagen and suppressed MAPK pathway activation and AKT. RASSF6 also appears to increase the association of mutant B-Raf and MST1, providing a potential mechanism by which RASSF6 is able to suppress MAPK activation. Thus, we have identified a novel potential role for RASSF6 in melanoma development. Promoter methylation leading to reduced expression of RASSF6 may play an important role in melanoma development and may contribute to brain metastases. PMID:25482183

  7. Augmented growth inhibition of B16-BL6 melanoma by combined treatment with a selective matrix metalloproteinase inhibitor, MMI-166, and cytotoxic agents.

    PubMed

    Hojo, Kanji; Maki, Hideo; Sawada, Takuko Yamada; Maekawa, Ryuji; Yoshioka, Takayuki

    2002-01-01

    MMI-166 is a selective matrix metalloproteinase (MMP) inhibitor. The purpose of this study was to evaluate the antitumor efficacy of the combined treatment of MMI-166 with paclitaxel or carboplatin. Mice bearing B16-BL6 melanoma were treated p.o. with MMI-166 from 1 day after tumor inoculation. The mice were administered i.v. with either paclitaxel or carboplatin at the maximum tolerated dose (MTD). MMI-166 monotherapy inhibited in vivo growth of the B16-BL6 tumor to an extent similar to that of paclitaxel or carboplatin monotherapy. When MMI-166 was combined with paclitaxel or carboplatin, the antitumor efficacy was significantly (p < 0.01) augmented in comparison with either MMI-166 or each cytotoxic agent alone. The hematotoxicity study demonstrated that daily treatment with MMI-166 did not affect the blood cell number in the mice and more importantly the combination of MMI-166 with paclitaxel did not augment the hematotoxicity caused by paclitaxel. An in vitro cytotoxicity study showed that MMI-166 itself has neither direct cytotoxicity against B16-BL6 tumor cells nor does it augment the cytotoxicity of paclitaxel or carboplatin. These results indicate that augmented antitumor activity of the combination treatment was not simply due to the augmentation of direct cytotoxic activity, but was rather an additive effect of the antitumor activities of different mechanisms. They suggest the effectiveness of a combination therapy of MMI-166 with paclitaxel or carboplatin in clinical therapy.

  8. Antitumor activity of a novel anti-vascular endothelial growth factor receptor-1 monoclonal antibody that does not interfere with ligand binding

    PubMed Central

    Tentori, Lucio; Scimeca, Manuel; Dorio, Annalisa S.; Atzori, Maria Grazia; Failla, Cristina M.; Morea, Veronica; Bonanno, Elena; D'Atri, Stefania; Lacal, Pedro M.

    2016-01-01

    Vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase transmembrane receptor that has also a soluble isoform containing most of the extracellular ligand binding domain (sVEGFR-1). VEGF-A binds to both VEGFR-2 and VEGFR-1, whereas placenta growth factor (PlGF) interacts exclusively with VEGFR-1. In this study we generated an anti-VEGFR-1 mAb (D16F7) by immunizing BALB/C mice with a peptide that we had previously reported to inhibit angiogenesis and endothelial cell migration induced by PlGF. D16F7 did not affect binding of VEGF-A or PlGF to VEGFR-1, thus allowing sVEGFR-1 to act as decoy receptor for these growth factors, but it hampered receptor homodimerization and activation. D16F7 inhibited both the chemotactic response of human endothelial, myelomonocytic and melanoma cells to VEGFR-1 ligands and vasculogenic mimicry by tumor cells. Moreover, D16F7 exerted in vivo antiangiogenic effects in a matrigel plug assay. Importantly, D16F7 inhibited tumor growth and was well tolerated by B6D2F1 mice injected with syngeneic B16F10 melanoma cells. The antitumor effect was associated with melanoma cell apoptosis, vascular abnormalities and decrease of both monocyte/macrophage infiltration and myeloid progenitor mobilization. For all the above, D16F7 may be exploited in the therapy of metastatic melanoma and other tumors or pathological conditions involving VEGFR-1 activation. PMID:27655684

  9. α2,6 sialylation associated with increased beta 1,6-branched N-oligosaccharides influences cellular adhesion and invasion.

    PubMed

    Ranjan, Amit; Kalraiya, Rajiv D

    2013-12-01

    Expression of β1,6-branched N-linked oligosaccharides have a definite association with invasion and metastasis of cancer cells. However, the mechanism by which these oligosaccharides regulate these processes is not well understood. Invasive variants of B16 murine melanoma, B16F10 (parent) and B16BL6 (highly invasive variant) cell lines have been used for these studies. We demonstrate that substitution of α2,6-linked sialic acids on multiantennary structures formed as a result of β1,6-branching modulate cellular adhesion on both extracellular matrix (ECM) and basement membrane (BM) components. Removal of α2,6 sialic acids either by enzymatic desialylation or by stably down-regulating the ST6Gal-I (enzyme that catalyses the addition of α2,6-linked sialic acids on N-linked oligosaccharides) by lentiviral driven shRNA decreased the adhesion on both ECM and BM components and invasion through reconstituted BM matrigel.

  10. Effects of the Amino Acid Linkers on the Melanoma-Targeting and Pharmacokinetic Properties of Indium-111-labeled Lactam Bridge-Cyclized α-MSH Peptides

    PubMed Central

    Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Miao, Yubin

    2011-01-01

    The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma targeting and pharmacokinetic properties of novel 111In-labeled lactam bridge-cyclized DOTA-[X]-CycMSHhex {1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2, X=GlyGlyNle, GlyGluNle or NleGlyGlu} peptides. Methods Three novel DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex and DOTA-NleGE-CycMSHhex peptides were designed and synthesized. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma targeting and pharmacokinetic properties of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. Results DOTA-GGNle-CycMSHhex and DOTA-GENle-CycMSHhex displayed 2.1 and 11.5 nM MC1 receptor binding affinities, whereas DOTA-NleGE-CycMSHhex showed 873.4 nM MC1 receptor binding affinity. The introduction of the -GlyGly- linker maintained high melanoma uptake while decreased the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex. The tumor uptake values of 111In-DOTA-GGNle-CycMSHhex were 19.05 ± 5.04 and 18.6 ± 3.56 % injected dose/gram (%ID/g) at 2 and 4 h post-injection. 111In-DOTA-GGNle-CycMSHhex exhibited 28, 32 and 42% less renal uptake values than 111In-DOTA-Nle-CycMSHhex we reported previously, and 61, 65 and 68% less liver uptake values than 111In-DOTA-Nle-CycMSHhex at 2, 4 and 24 h post-injection, respectively. Conclusion The amino acid linkers exhibited the profound effects on the melanoma targeting and pharmacokinetic properties of the 111In-labeled lactam bridge-cyclized α-MSH peptides. Introduction of the -GlyGly- linker maintained high melanoma uptake while reducing the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex, highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide for melanoma treatment when labeled with a therapeutic

  11. Dioscin augments HSV-tk-mediated suicide gene therapy for melanoma by promoting connexin-based intercellular communication

    PubMed Central

    Li, Bin; Wu, Yingya; Liu, Xijuan; Tan, Yuhui; Du, Biaoyan

    2017-01-01

    Suicide gene therapy is a promising strategy against melanoma. However, the low efficiency of the gene transfer technique can limit its application. Our preliminary data showed that dioscin, a glucoside saponin, could upregulate the expression of connexins Cx26 and Cx43, major components of gap junctions, in melanoma cells. We hypothesized that dioscin may increase the bystander effect of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) through increasing the formation of gap junctions. Further analysis showed that dioscin indeed could increase the gap junctional intercellular communication in B16 melanoma cells, resulting in more efficient GCV-induced bystander killing in B16tk cells. By contrast, overexpression of dominant negative Cx43 impaired the cell-cell communication of B16 cells and subsequently weakened the bystander effect of HSV-tk/GCV gene therapy. In vivo, combination treatment with dioscin and GCV of tumor-bearing mice with 30% positive B16tk cells and 70% wild-type B16 cells caused a significant reduction in tumor volume and weight compared to treatment with GCV or dioscin alone. Taken together, these results demonstrated that dioscin could augment the bystander effect of the HSV-tk/GCV system through increasing connexin-mediated gap junction coupling. PMID:27903977

  12. The Standardized Extract of Juniperus communis Alleviates Hyperpigmentation in Vivo HRM-2 Hairless Mice and in Vitro Murine B16 Melanoma Cells.

    PubMed

    Jegal, Jonghwan; Chung, Ki Wung; Chung, Hae Young; Jeong, Eun Ju; Yang, Min Hye

    2017-01-01

    In European folk medicine, the fruits of Juniperus communis are used in the treatment of skin-related disorders such as skin infection, itching, and psoriasis. Previously, we reported that the EtOAc fraction of J. communis (EAJC) contained tyrosinase inhibition properties in vitro non-cellular experiment. The aim of this study was to evaluate anti-melanogenic effect of standardized EAJC on a hyperpigmentation animal model. Therapeutic effects of EAJC toward skin hyperpigmentation were confirmed by both in vivo experiment and in vitro cell-based assay. Skin depigmenting effect was detected by topical treatment of EAJC for 11 d to HRM-2 melanin-possessing hairless mice. Histologic findings including significantly decreased melanin depositions could be observed in dorsal skin samples of EAJC-treated group. In addition, the EAJC (50 µg/mL) attenuated melanin production through down-regulation of tyrosinase activity and protein expression in B16 murine melanoma cells. According to the phytochemical analysis, EAJC was found to contain hypolaetin-7-O-β-D-xylopyranoside and isoscutellarein-7-O-β-D-xylopyranoside as main components. Hypolaetin-7-O-β-D-xylopyranoside was responsible for the skin-lightening effect of EAJC by reducing the number of melanocytes in dorsal skins of HRM-2 mice. The present study provided direct experimental evidence for skin-lightening effect of EAJC in UV-irradiated hairless mouse model. Therapeutic attempts with the J. communis might be useful in the management of skin pigmentation-related diseases.

  13. Evaluation of antitumoral and antimicrobial activity of Morinda lcitrifolia L. grown in Southeast Brazil.

    PubMed

    Candida, Thamyris; França, Jerônimo Pereira de; Chaves, Alba Lucilvânia Fonseca; Lopes, Fernanda Andrade Rodrigues; Gaiba, Silvana; Sacramento, Celio Kersul do; Ferreira, Lydia Masako; França, Lucimar Pereira de

    2014-01-01

    To evaluate the antitumor and antimicrobial activity of ethanolic extract of Morinda citrifolia L. fruit cultivated in southeastern Brazil. Preparation ethanolic extract of the fruit of Morinda citrifolia L. Culture of melanoma cells B16-F10 for treatment with ethanolic extract of Morinda citrifolia L. fruit to determine cell viability by MTT and determination temporal effect of ethanolic extract fruit on the cell growth B16-F10 for 8 days. Evaluation of antimicrobial activity of ethanolic extract fruit against Staphylococcus aureus and Escherichia coli by determination of Minimum Inhibitory Concentration (MIC). The ethanolic extract of Morinda citrifolia L. fruit (10mg/mL) decreased cellular activity and inhibited 45% the rate of cell proliferation of B16-F10 melanoma treated during period studied. The ethanolic extract of Morinda citrifolia L. fruit demonstrated antimicrobial activity inhibiting the growth of both microorganisms studied. Staphylococcus aureus was less resistant to ethanolic extract of Morinda citrifolia L. fruit than Escherichia coli, 1 mg/mL and 10 mg/mL, respectively. What these results indicate that the ethanolic extract of the fruit of Morinda citrifolia L. showed antitumor activity with inhibition of viability and growth of B16-F10 cells and also showed antibacterial activity as induced inhibition of growth of Staphylococcus aureus and Escherichia coli.

  14. Preventing the activation or cycling of the Rap1 GTPase alters adhesion and cytoskeletal dynamics and blocks metastatic melanoma cell extravasation into the lungs.

    PubMed

    Freeman, Spencer A; McLeod, Sarah J; Dukowski, Janet; Austin, Pamela; Lee, Crystal C Y; Millen-Martin, Brandie; Kubes, Paul; McCafferty, Donna-Marie; Gold, Michael R; Roskelley, Calvin D

    2010-06-01

    The Rap1 GTPase is a master regulator of cell adhesion, polarity, and migration. We show that both blocking Rap1 activation and expressing a constitutively active form of Rap1 reduced the ability of B16F1 melanoma cells to extravasate from the microvasculature and form metastatic lesions in the lungs. This correlated with a decreased ability of the tumor cells to undergo transendothelial migration (TEM) in vitro and form dynamic, F-actin-rich pseudopodia that penetrate capillary endothelial walls in vivo. Using multiple tumor cell lines, we show that the inability to form these membrane protrusions, which likely promote TEM and extravasation, can be explained by altered adhesion dynamics and impaired cell polarization that result when Rap1 activation or cycling is perturbed. Thus, targeting Rap1 could be a useful approach for reducing the metastatic dissemination of tumor cells that undergo active TEM. Copyright 2010 AACR.

  15. BRAF and MEK inhibitor therapy eliminates nestin expressing melanoma cells in human tumors.

    PubMed

    Doxie, Deon B; Greenplate, Allison R; Gandelman, Jocelyn S; Diggins, Kirsten E; Roe, Caroline E; Dahlman, Kimberly B; Sosman, Jeffrey A; Kelley, Mark C; Irish, Jonathan M

    2018-05-19

    Little is known about the in vivo impacts of targeted therapy on melanoma cell abundance and protein expression. Here, 21 antibodies were added to an established melanoma mass cytometry panel to measure 32 cellular features, distinguish malignant cells, and characterize dabrafenib and trametinib responses in BRAF V 600mut melanoma. Tumor cells were biopsied before neoadjuvant therapy and compared to cells surgically resected from the same site after 4 weeks of therapy. Approximately 50,000 cells per tumor were characterized by mass cytometry and computational tools t-SNE/viSNE, FlowSOM, and MEM. The resulting single cell view of melanoma treatment response revealed initially heterogeneous melanoma tumors were consistently cleared of Nestin expressing melanoma cells. Melanoma cells subsets that persisted to week 4 were heterogeneous but expressed SOX2 or SOX10 proteins and specifically lacked surface expression of MHC I proteins by MEM analysis. Traditional histology imaging of tissue microarrays from the same tumors confirmed mass cytometry results, including persistence of NES- SOX10+ S100β+ melanoma cells. This quantitative single cell view of melanoma treatment response revealed protein features of malignant cells that are not eliminated by targeted therapy. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  16. Non-Metastatic Cutaneous Melanoma Induces Chronodisruption in Central and Peripheral Circadian Clocks.

    PubMed

    de Assis, Leonardo Vinícius Monteiro; Moraes, Maria Nathália; Magalhães-Marques, Keila Karoline; Kinker, Gabriela Sarti; da Silveira Cruz-Machado, Sanseray; Castrucci, Ana Maria de Lauro

    2018-04-03

    The biological clock has received increasing interest due to its key role in regulating body homeostasis in a time-dependent manner. Cancer development and progression has been linked to a disrupted molecular clock; however, in melanoma, the role of the biological clock is largely unknown. We investigated the effects of the tumor on its micro- (TME) and macro-environments (TMaE) in a non-metastatic melanoma model. C57BL/6J mice were inoculated with murine B16-F10 melanoma cells and 2 weeks later the animals were euthanized every 6 h during 24 h. The presence of a localized tumor significantly impaired the biological clock of tumor-adjacent skin and affected the oscillatory expression of genes involved in light- and thermo-reception, proliferation, melanogenesis, and DNA repair. The expression of tumor molecular clock was significantly reduced compared to healthy skin but still displayed an oscillatory profile. We were able to cluster the affected genes using a human database and distinguish between primary melanoma and healthy skin. The molecular clocks of lungs and liver (common sites of metastasis), and the suprachiasmatic nucleus (SCN) were significantly affected by tumor presence, leading to chronodisruption in each organ. Taken altogether, the presence of non-metastatic melanoma significantly impairs the organism's biological clocks. We suggest that the clock alterations found in TME and TMaE could impact development, progression, and metastasis of melanoma; thus, making the molecular clock an interesting pharmacological target.

  17. Regulatory T cells and other lymphocyte subpopulations in patients with melanoma developing interferon-induced thyroiditis during high-dose interferon-α2b treatment.

    PubMed

    Soldevila, Berta; Alonso, Núria; Martínez-Arconada, Maria J; Granada, Maria L; Boada, Aram; Vallejos, Virginia; Fraile, Manuel; Fernández-Sanmartín, Marco A; Pujol-Borrell, Ricardo; Puig-Domingo, Manel; Sanmartí, Anna; Martínez-Cáceres, Eva M

    2013-04-01

    One of the side effects of interferon-alpha therapy is interferon-induced thyroiditis (IIT). The role of lymphocyte subpopulations in IIT melanoma patients remains to be defined. Our objective was to assess different peripheral blood lymphocyte subpopulations, mainly regulatory T cells (Tregs), in melanoma patients who developed IIT. From 30 melanoma patients receiving high-dose interferon (HDI)-alpha 2b (IFN-α2b) treatment, those who developed IIT (IIT patients) were selected and compared with patients who did not develop IIT (Co-MM) and healthy controls (Co-H). Peripheral blood mononuclear cells were obtained before treatment (BT), mid-treatment (MT), end of treatment (ET), 24 weeks post-treatment and at appearance of IIT (TT). Nine patients developed IIT (30%): four Hashimoto's thyroiditis and five destructive thyroiditis. An increase in Tregs was observed in both melanoma groups during HDI treatment. A decrease in CD3(+) , NKT lymphocyte subpopulations and Bcl2 expression on B cells was also observed in both groups. However, no changes were observed in the percentage of CD4(+) , CD8(+) , CD3(+) γδ(+) , CD19(+) , transitional B cells (CD24(high) CD38(high) CD19(+) CD27(-) ), natural killer (NK), invariant NKT (iNKT) lymphocytes and Th1/Th2 balance when BT was compared with ET. At TT, IIT patients had a higher Tregs percentage than Co-MM (P = 0·012) and Co-H (P = 0·004), a higher iNKT percentage than Co-MM (P = 0·011), a higher transitional B cells percentage than Co-H (P = 0·015), a lower CD3(+) percentage than Co-H (P = 0·001) and a lower Bcl2 expression on B cells than Co-H (P < 0·001). Our results point to the immunomodulatory effects of IFN-α on different lymphocyte subpopulations and a possible role of Tregs in melanoma patients who developed IIT. © 2012 Blackwell Publishing Ltd.

  18. PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines.

    PubMed

    Katona, Éva; Juhász, Tamás; Somogyi, Csilla Szűcs; Hajdú, Tibor; Szász, Csaba; Rácz, Kálmán; Kókai, Endre; Gergely, Pál; Zákány, Róza

    2016-03-01

    Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment.

  19. PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines

    PubMed Central

    KATONA, ÉVA; JUHÁSZ, TAMÁS; SOMOGYI, CSILLA SZŰCS; HAJDÚ, TIBOR; SZÁSZ, CSABA; RÁCZ, KÁLMÁN; KÓKAI, ENDRE; GERGELY, PÁL; ZÁKÁNY, RÓZA

    2016-01-01

    Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment. PMID:26717964

  20. Electrospun PBLG/PLA nanofiber membrane for constructing in vitro 3D model of melanoma.

    PubMed

    Wang, Yaping; Qian, Junmin; Liu, Ting; Xu, Weijun; Zhao, Na; Suo, Aili

    2017-07-01

    Though much progress in utilizing tissue engineering technology to investigate tumor development in vitro has been made, the effective management of human melanoma is still a challenge in clinic due to lack of suitable 3D culture systems. In this study, we prepared a poly(γ-benzyl-l-glutamate)/poly(lactic acid) (PBLG/PLA) nanofiber membrane by electrospinning and demonstrated its suitability as a matrix for 3D culture of melanoma cells in vitro. The electrospun PBLG/PLA nanofiber membrane displayed a smooth and uniform fibrous morphology and had a desirable water contact angle of 79.3±0.6°. The average diameter of PBLG/PLA nanofibers was 320.3±95.1nm that was less than that (516.2±163.3nm) of pure PLA nanofibers. The addition of PBLG into PLA decreased the cold crystallization peak of PLA fibers from 93 to 75°C. The in vitro biocompatibility of PBLG/PLA nanofiber membrane was evaluated with B16F10 cells using PLA nanofiber membrane as control. It was found that, compared to PLA nanofiber membrane, PBLG/PLA nanofiber membrane could better support cell viability and proliferation, as indicated by MTT assay and live-dead staining. SEM results revealed that PBLG/PLA rather than PLA nanofiber membrane promoted the generation of tumoroid-like structures. These findings clearly demonstrated that the electrospun PBLG/PLA nanofiber membrane could mimick the extracellular matrix of melanoma microenvironment and be a promising platform for 3D cell culture. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. 6-Thioguanine-loaded polymeric micelles deplete myeloid-derived suppressor cells and enhance the efficacy of T cell immunotherapy in tumor-bearing mice

    DOE PAGES

    Jeanbart, Laura; Kourtis, Iraklis C.; van der Vlies, André J.; ...

    2015-05-16

    Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that suppress effector T cell responses and can reduce the efficacy of cancer immunotherapies. We previously showed that ultra-small polymer nanoparticles efficiently drain to the lymphatics after intradermal injection and target antigen-presenting cells, including Ly6c hi Ly6g ₋monocytic MDSCs (Mo-MDSCs), in skin-draining lymph nodes (LNs) and spleen. Here, we developed ultra-small polymer micelles loaded with 6-thioguanine (MC-TG), a cytotoxic drug used in the treatment of myelogenous leukemia, with the aim of killing Mo-MDSCs in tumor-bearing mice and thus enhancing T cell-mediated anti-tumor responses. We found that 2 daysmore » post-injection in tumor-bearing mice (B16-F10 melanoma or E.G7-OVA thymoma), MC-TG depleted Mo-MDSCs in the spleen, Ly6c lo Ly6g + granulocytic MDSCs (G-MDSCs) in the draining LNs, and Gr1 int Mo-MDSCs in the tumor. In both tumor models, MC-TG decreased the numbers of circulating Mo- and G-MDSCs, as well as of Ly6c hi macrophages, for up to 7 days following a single administration. MDSC depletion was dose dependent and more effective with MC-TG than with equal doses of free TG. Finally, we tested whether this MDSC-depleting strategy might enhance cancer immunotherapies in the B16-F10 melanoma model. We found that MC-TG significantly improved the efficacy of adoptively transferred, OVA-specific CD8 + T cells in melanoma cells expressing OVA. Ultimately, these findings highlight the capacity of MC-TG in depleting MDSCs in the tumor microenvironment and show promise in promoting anti-tumor immunity when used in combination with T cell immunotherapies.« less

  2. 6-Thioguanine-loaded polymeric micelles deplete myeloid-derived suppressor cells and enhance the efficacy of T cell immunotherapy in tumor-bearing mice

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jeanbart, Laura; Kourtis, Iraklis C.; van der Vlies, André J.

    Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that suppress effector T cell responses and can reduce the efficacy of cancer immunotherapies. We previously showed that ultra-small polymer nanoparticles efficiently drain to the lymphatics after intradermal injection and target antigen-presenting cells, including Ly6c hi Ly6g ₋monocytic MDSCs (Mo-MDSCs), in skin-draining lymph nodes (LNs) and spleen. Here, we developed ultra-small polymer micelles loaded with 6-thioguanine (MC-TG), a cytotoxic drug used in the treatment of myelogenous leukemia, with the aim of killing Mo-MDSCs in tumor-bearing mice and thus enhancing T cell-mediated anti-tumor responses. We found that 2 daysmore » post-injection in tumor-bearing mice (B16-F10 melanoma or E.G7-OVA thymoma), MC-TG depleted Mo-MDSCs in the spleen, Ly6c lo Ly6g + granulocytic MDSCs (G-MDSCs) in the draining LNs, and Gr1 int Mo-MDSCs in the tumor. In both tumor models, MC-TG decreased the numbers of circulating Mo- and G-MDSCs, as well as of Ly6c hi macrophages, for up to 7 days following a single administration. MDSC depletion was dose dependent and more effective with MC-TG than with equal doses of free TG. Finally, we tested whether this MDSC-depleting strategy might enhance cancer immunotherapies in the B16-F10 melanoma model. We found that MC-TG significantly improved the efficacy of adoptively transferred, OVA-specific CD8 + T cells in melanoma cells expressing OVA. Ultimately, these findings highlight the capacity of MC-TG in depleting MDSCs in the tumor microenvironment and show promise in promoting anti-tumor immunity when used in combination with T cell immunotherapies.« less

  3. Downregulation of miR-125b in metastatic cutaneous malignant melanoma.

    PubMed

    Glud, Martin; Rossing, Maria; Hother, Christoffer; Holst, Line; Hastrup, Nina; Nielsen, Finn C; Gniadecki, Robert; Drzewiecki, Krzysztof T

    2010-12-01

    This study aimed to identify microRNA species involved in the earliest metastatic event in cutaneous malignant melanoma (MM). Samples from 28 patients with MM [stage T2 (tumor), M0 (distant metastasis)] were grouped by the presence of micrometastasis in the sentinel lymph nodes (N0/N1). Melanoma cells were harvested from primary, cutaneous MM tumors by laser-capture microdissection, and microRNA expression profiles were obtained by the microarray technique. Results were validated by quantitative reverse transcription PCR. We found that miR-125b was downregulated in the primary cutaneous melanomas that produced early metastases (T2, N1, M0) compared with the sentinel lymph node-negative (T2, N0, M0) melanomas. MiR-125b has earlier been found to be downregulated in other tumor types and in atypic naevi compared with the common acquired naevi. In conclusion, miR-125b may be involved in an early progression of cutaneous MM.

  4. Adenosine A(2B) receptor antagonist PSB603 suppresses tumor growth and metastasis by inhibiting induction of regulatory T cells.

    PubMed

    Kaji, Wakako; Tanaka, Satomi; Tsukimoto, Mitsutoshi; Kojima, Shuji

    2014-04-01

    Regulatory T cells (Treg) play a role in suppression of immune response, including anti-tumor immunity. We have recently reported that treatment of naïve CD4 T cells with adenosine A(2B) receptor antagonist PSB603 under Treg-skewing conditions inhibits expression of Foxp3, a marker of differentiation to Treg, without blocking IL-2 production or CD25 expression, which are activation markers, in CD4 T cells. We hypothesized that PSB603 suppresses cancer growth and metastasis by inhibiting induction of Treg, thereby facilitating anti-tumor immunity. In this study, we first examined the effect of PSB603 on tumor growth in B16 melanoma-bearing C57BL/6 mice. Administration of PSB603 significantly suppressed the increase of tumor volume as well as the increase of Treg population in these mice. The populations of CD4 and CD8 T cells were higher and splenic lymphocyte-mediated cytotoxicity towards B16 melanoma was significantly increased in PSB603-treated mice. We confirmed that PSB603 did not reduce the viability of B16 melanoma cells in vitro. Moreover, we also examined the effect of PSB603 on tumor metastasis in pulmonary metastasis model mice intravenously injected with B16 melanoma cells. The metastasis was also suppressed in PSB603-treated mice, in which the population of Treg was significantly lower. Overall, our results suggest that A(2B) receptor antagonist PSB603 enhances anti-tumor immunity by inhibiting differentiation to Treg, resulting in a delay of tumor growth and a suppression of metastasis.

  5. Noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma.

    PubMed

    Landen, Jaren W; Lang, Roland; McMahon, Steve J; Rusan, Nasser M; Yvon, Anne-Marie; Adams, Ashley W; Sorcinelli, Mia D; Campbell, Ross; Bonaccorsi, Paola; Ansel, John C; Archer, David R; Wadsworth, Patricia; Armstrong, Cheryl A; Joshi, Harish C

    2002-07-15

    Cellular microtubules, polymers of tubulin, alternate relentlessly between phases of growth and shortening. We now show that noscapine, a tubulin-binding agent, increases the time that cellular microtubules spend idle in a paused state. As a result, most mammalian cell types observed arrest in mitosis in the presence of noscapine. We demonstrate that noscapine-treated murine melanoma B16LS9 cells do not arrest in mitosis but rather become polyploid followed by cell death, whereas primary melanocytes reversibly arrest in mitosis and resume a normal cell cycle after noscapine removal. Furthermore, in a syngeneic murine model of established s.c. melanoma, noscapine treatment resulted in an 85% inhibition of tumor volume on day 17 when delivered by gavage compared with untreated animals (P melanoma progression by 83% on day 18 when delivered in drinking water (P melanoma cells through alterations in microtubule dynamics, with no detected toxicity to the host. Consequently, noscapine could be a valuable chemotherapeutic agent, alone or in combination, for the treatment of advanced melanoma.

  6. Downregulation of lncRNA H19 inhibits the migration and invasion of melanoma cells by inactivating the NF‑κB and PI3K/Akt signaling pathways.

    PubMed

    Liao, Zhichao; Zhao, Jun; Yang, Yun

    2018-05-01

    As the most aggressive type of skin cancer, melanoma seriously affects human health. Long noncoding (lncRNA) 19 has been demonstrated to be involved in the progression of a number of different types of human cancers. However, the involvement of lncRNA H19 in melanoma remains unknown. Therefore, the present study was performed to investigate the roles of H19 in the development and progression of melanoma. In the present study, 49 patients with melanoma were included. Expression of lncRNA H19 in tumor tissue, adjacent healthy tissue and various cell lines with different treatments was measured by reverse transcription‑quantitative polymerase chain reaction. The effects of H19 knockdown on melanoma cell proliferation, migration and invasion were detected by cell counting kit‑8, wound‑healing and transwell invasion assays, respectively. In addition, the effects of H19 knockdown on the expression of nuclear factor (NF)‑κB pathway‑associated proteins were investigated by western blotting. The results revealed that the expression level of H19 was significantly higher in tumor tissue than in the adjacent healthy tissue of 47 out of 49 patients. H19 knockdown significantly reduced the proliferation, migration and invasion ability of melanoma cells. H19 knockdown also inactivated the phosphoinositide 3‑kinase (PI3K)/protein kinase B (Akt) pathway, which in turn inhibited the activation of the NF‑κB signaling pathway. Thus, downregulation of lncRNA H19 may inhibit the migration and invasion of melanoma cells by inactivating the NF‑κB signaling pathway via the inactivation of the PI3K/Akt signaling pathway. The present study provided references for future studies on the pathogenesis of melanoma and the clinical treatment of this disease.

  7. Melanogenesis of murine melanoma cells induced by hesperetin, a Citrus hydrolysate-derived flavonoid.

    PubMed

    Huang, Yu-Chun; Liu, Kao-Chih; Chiou, Yi-Ling

    2012-03-01

    Melanogenesis is a complex process that modulates skin pigmentation to defend photodamage. Citrus is the most widely produced fruit crop in the world. People ingest various citrus fruits in their common diets. In the present study, the acid-hydrolyzed and un-hydrolyzed extracts of orange-type citrus fruits were subjected to analyze flavonoid compositions and assess their effects on melanin synthesis in murine B16-F10 melanoma cells. The acid-hydrolyzed extracts of Citrus sinensis, C. reticulata, and C. aurantium enhanced melanin production. Based on high-performance liquid chromatography (HPLC) analysis, the most abundant flavonoids that were found in citrus hydrolyzed extracts were hesperetin and naringenin. Hesperetin exhibited the most potent activity on melanin synthesis and induced tyrosinase and microphthalmia-associated transcription factor (MITF) expression. Moreover, hesperetin stimulated the activation of mitogen-activated protein kinases (MAPKs), phosphorylation of cAMP-responsive element binding protein (CREB) and glycogen synthase kinase-3β (GSK3β), and subsequently induced the accumulation of β-catenin. This study suggests that the citrus constituent hesperetin might have protective melanogenic potential as a cosmeceutical agent against skin photodamage. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Simulated Sunlight-Mediated Photodynamic Therapy for Melanoma Skin Cancer by Titanium-Dioxide-Nanoparticle-Gold-Nanocluster-Graphene Heterogeneous Nanocomposites.

    PubMed

    Cheng, Yan; Chang, Yun; Feng, Yanlin; Liu, Ning; Sun, Xiujuan; Feng, Yuqing; Li, Xi; Zhang, Haiyuan

    2017-05-01

    Simulated sunlight has promise as a light source able to alleviate the severe pain associated with patients during photodynamic therapy (PDT); however, low sunlight utilization efficiency of traditional photosensitizers dramatically limits its application. Titanium-dioxide-nanoparticle-gold-nanocluster-graphene (TAG) heterogeneous nanocomposites are designed to efficiently utilize simulated sunlight for melanoma skin cancer PDT. The narrow band gap in gold nanoclusters (Au NCs), and staggered energy bands between Au NCs, titanium dioxide nanoparticles (TiO 2 NPs), and graphene can result in efficient utilization of simulated sunlight and separation of electron-hole pairs, facilitating the production of abundant hydroxyl and superoxide radicals. Under irradiation of simulated sunlight, TAG nanocomposites can trigger a series of toxicological responses in mouse B16F1 melanoma cells, such as intracellular reactive oxygen species production, glutathione depletion, heme oxygenase-1 expression, and mitochondrial dysfunctions, resulting in severe cell death. Furthermore, intravenous or intratumoral administration of biocompatible TAG nanocomposites in B16F1-tumor-xenograft-bearing mice can significantly inhibit tumor growth and cause severe pathological tumor tissue changes. All of these results demonstrate prominent simulated sunlight-mediated PDT effects. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. The synthetic parasite-derived peptide GK1 increases survival in a preclinical mouse melanoma model.

    PubMed

    Pérez-Torres, Armando; Vera-Aguilera, Jesús; Hernaiz-Leonardo, Juan Carlos; Moreno-Aguilera, Eduardo; Monteverde-Suarez, Diego; Vera-Aguilera, Carlos; Estrada-Bárcenas, Daniel

    2013-11-01

    The therapeutic efficacy of a synthetic parasite-derived peptide GK1, an immune response booster, was evaluated in a mouse melanoma model. This melanoma model correlates with human stage IIb melanoma, which is treated with wide surgical excision; a parallel study employing a surgical treatment was carried out as an instructive goal. C57BL/6 mice were injected subcutaneously in the flank with 2×10(5) B16-F10 murine melanoma cells. When the tumors reached 20 mm3, mice were separated into two different groups; the GK1 group, treated weekly with peritumoral injections of GK1 (10 μg/100 μL of sterile saline solution) and the control group, treated weekly with an antiseptic peritumoral injection of 100 μL of sterile saline solution without further intervention. All mice were monitored daily for clinical appearance, tumor size, and survival. Surgical treatment was performed in parallel when the tumor size was 20 mm3 (group A), 500 mm3 (group B), and >500 mm3 (group C). The GK1 peptide effectively increased the mean survival time by 9.05 days, corresponding to an increase of 42.58%, and significantly delayed tumor growth from day 3 to 12 of treatment. In addition, tumor necrosis was significantly increased (p<0.05) in the treated mice. The overall survival rates obtained with surgical treatment at 6 months were 83.33% for group A, 40% for group B, and 0% for group C, with significant differences (p<0.05) among the groups. The GK1 peptide demonstrated therapeutic properties in a mouse melanoma model, as treatment resulted in a significant increase in the mean survival time of the treated animals (42.58%). The potential for GK1 to be used as a primary or adjuvant component of chemotherapeutic cocktails for the treatment of experimental and human cancers remains to be determined, and surgical removal remains a challenge for any new experimental treatment of melanoma in mouse models.

  10. Anticancer and Anti-Inflammatory Properties of Ganoderma lucidum Extract Effects on Melanoma and Triple-Negative Breast Cancer Treatment

    PubMed Central

    Barbieri, Antonio; Quagliariello, Vincenzo; Del Vecchio, Vitale; Falco, Michela; Luciano, Antonio; Amruthraj, Nagoth Joseph; Nasti, Guglielmo; Ottaiano, Alessandro; Berretta, Massimiliano; Iaffaioli, Rosario Vincenzo; Arra, Claudio

    2017-01-01

    Among the most important traditional medicinal fungi, Ganoderma lucidum has been used as a therapeutic agent for the treatment of numerous diseases, including cancer, in Oriental countries. The aim of this study is to investigate the anti-inflammatory, anticancer and anti-metastatic activities of Ganoderma lucidum extracts in melanoma and triple-negative breast cancer cells. Ganoderma lucidum extracts were prepared by using common organic solvents; MDA-MB 231 and B16-F10 cell lines were adopted as cellular models for triple-negative breast cancer and melanoma and characterized for cell viability, wound-healing assay and measurement of cytokines secreted by cancer cells under pro-inflammatory conditions (incubation with lipopolysaccharide, LPS) and pretreatment with Ganoderma lucidum extract at different concentrations. Our study demonstrates, for the first time, how Ganoderma lucidum extracts can significantly inhibit the release of IL-8, IL-6, MMP-2 and MMP-9 in cancer cells under pro-inflammatory condition. Interestingly, Ganoderma lucidum extracts significantly also decrease the viability of both cancer cells in a time- and concentration-dependent manner, with abilities to reduce cell migration over time, which is correlated with a lower release of matrix metalloproteases. Taken together, these results indicate the possible use of Ganoderma lucidum extract for the therapeutic management of melanoma and human triple-negative breast cancer. PMID:28264501

  11. Effect of blocking TNF on IL-6 levels and metastasis in a B16-BL6 melanoma/mouse model.

    PubMed

    Cubillos, S; Scallon, B; Feldmann, M; Taylor, P

    1997-01-01

    We studied the relationship between tumour necrosis factor (TNF) and interleukin 6 (IL-6) levels, and the metastatic process in C57BL/6 mice after intravenous inoculation of B16-BL6 melanoma cells. Bioactive TNF was not detectable in the sera of inoculated mice, but these animals did show higher TNF levels following intraperitoneal challenge with lipopolysaccharide (LPS) compared to control animals. Serum IL-6 levels were increased in inoculated animals. Injection of a hybrid molecule (p55-sf2) composed of the human p55 TNF receptor extracellular domain coupled to a human constant region backbone, decreased serum TNF (after LPS challenge) and IL-6 levels in inoculated animals. Lung metastases at 7-14 days were reduced, compared to human IgG-injected control animals, but this effect was lost at day 21 postinoculation. The results suggest that the reduction in the number of metastases may be related to the effect of blocking TNF activity.

  12. Quercetin-3-O-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside suppresses melanin synthesis by augmenting p38 MAPK and CREB signaling pathways and subsequent cAMP down-regulation in murine melanoma cells.

    PubMed

    Jung, Hyun Gug; Kim, Han Hyuk; Paul, Souren; Jang, Jae Yoon; Cho, Yong Hun; Kim, Hyeon Jeong; Yu, Jae Myo; Lee, Eun Su; An, Bong Jeun; Kang, Sun Chul; Bang, Byung Ho

    2015-11-01

    In this study, the effect of purified quercetin-3-O-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosid (QCGG) on melanogenesis was investigated. QCGG was isolated from the calyx of a traditional Korean medicinal herb, Persimmon (Diospyros kaki). The hypopigmentation effects of QCGG were determined by examination of cellular melanin contents, tyrosinase activity assay, cAMP assay, and Western blotting of α-MSH-stimulated B16F10 mouse melanoma cells. Our results showed that QCGG inhibited both melanin synthesis and tyrosinase activity in a concentration-dependent manner as well as significantly reduced the expression of melanogenic proteins such as microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1, tyrosinase-related protein-2, and tyrosinase. Moreover, QCGG inhibited intracellular cAMP levels, cAMP response element-binding protein (CREB), and p38 MAPK expression in α-MSH-stimulated B16F10 cells. Taken together, the suppressive effects of QCGG on melanogenesis may involve down-regulation of MITF and its downstream signaling pathway via phosphorylation of p38 MAPK and CREB along with reduced cAMP levels. These results indicate that QCGG reduced melanin synthesis by reducing expression of tyrosine and tyrosine-related proteins via extracellular signal-related protein kinase (ERK) activation, followed by down-regulation of CREB, p38, and MITF.

  13. Expression of Vitamin D-Activating Enzyme 1α-Hydroxylase (CYP27B1) Decreases during Melanoma Progression**

    PubMed Central

    Brożyna, Anna A.; Jóźwicki, Wojciech; Janjetovic, Zorica; Slominski, Andrzej T.

    2012-01-01

    Summary 1α-Hydroxylase (CYP27B1), the enzyme responsible for the synthesis of the biologically active form of vitamin D (1,25(OH)2D3), is expressed in the skin. To assess the correlation between progression of melanocytic tumors and CYP27B1, we analyzed its expression in 29 benign nevi, 75 primary cutaneous melanomas, 40 metastases, and 4 re-excision and 6 normal skin biopsies. Immunoreactivity for CYP27B1 was significantly lower in the vertical growth phase (VGP) and metastatic melanomas (0.6 and 0.5 arbitrary units [AU], respectively) in comparison with nevi and radial growth phase (RGP) tumors (1.2 and 1.1 AU, respectively); and expression was reduced in more advanced lesions (Clark levels III–V, Breslow thickness ≥2.1 mm; 0.8 and 0.7 AU, respectively). There was an inverse correlation between CYP27B1 and Ki-67 expression. Furthermore, CYP27B1 expression was reduced in primary melanomas that created metastases in comparison with non-metastasizing melanomas. Reduced CYP27B1 expression in RGP was related to shorter overall survival (810 vs 982 vs 1151 days in melanomas with absent, low, and high CYP27B1 immunoreactivity), and low CYP27B1 expression in RGP and VGP was related to shorter disease-free survival (114 vs 339 vs 737 days and 129 vs 307 vs 737 days, respectively, in melanomas with absent, low, and high CYP27B1). Also, CYP27B1 expression was inversely related to melanin in melanoma cells in vivo and melanoma cells cultured in vitro. Thus, reduction of CYP27B1 correlates with melanoma phenotype and behavior, and its lack affects the survival of melanoma patients, indicating a role in the pathogenesis and progression of this cancer. PMID:22995334

  14. Study of the nucleon radiative captures 8Li(n,γ)9Li, 9Be(p,γ)10B, 10Be(n,γ)11Be, 10B(p,γ)11C, and 16O(p,γ)17F at thermal and astrophysical energies

    NASA Astrophysics Data System (ADS)

    Dubovichenko, Sergey; Dzhazairov-Kakhramanov, Albert

    We have studied the neutron-capture reactions 8Li(n,γ)9Li and its role in the primordial nucleosynthesis. The n +8Li →9Li + γ reaction has a significant astrophysical interest because it includes one of the variants of chain of primordial nucleosynthesis processes of the Universe and thermonuclear reactions in type II supernovae. Furthermore, we consider the 9Be(p,γ)10B reaction in the astrophysical energy range in the modified potential cluster model (MPCM) with splitting of orbital states according to Young tableaux and, in some cases, with forbidden states (FS). The reaction 9Be(p,γ)10B plays an important role in primordial and stellar nucleosynthesis of light elements in the p shell. Hydrogen burning in second-generation stars occurs via the proton-proton (pp) chain and CNO cycle, with the 9Be(p,γ)10B reaction serving as an intermediate link between these cycles. Furthermore, the possibility of describing available experimental data for the total reaction cross-sections of neutron radiative capture on 10Be at thermal and astrophysical energies has been shown. This reaction is a part of one of the variants of the chain of primordial nucleosynthesis of the Universe due to which the elements with a mass of A > 11-12 may be formed. The results in the field of study of thermonuclear proton-capture reaction on 10B at ultralow, i.e., astrophysical energies will be presented further. The possibility of description of the experimental data for the astrophysical S-factor of the proton radiative capture on 16O to the ground state (GS) of 17F was considered in the frame of the MPCM with FS and classification of the states according to Young tableaux. It was shown that on the basis of the E1 transitions from the states of p16O scattering to the GS of 17F in the p16O channel generally succeed to explain the value of measured cross-sections at astrophysical energies.

  15. CDKN2B loss promotes progression from benign melanocytic nevus to melanoma

    PubMed Central

    McNeal, Andrew S.; Liu, Kevin; Nakhate, Vihang; Natale, Christopher A.; Duperret, Elizabeth K.; Capell, Brian C.; Dentchev, Tzvete; Berger, Shelley L.; Herlyn, Meenhard; Seykora, John T.; Ridky, Todd W.

    2015-01-01

    Deletion of the entire CDKN2B-CDKN2A gene cluster is among the most common genetic events in cancer. The tumor-promoting effects are generally attributed to loss of CDKN2A-encoded p16 and p14ARF tumor suppressors. The degree to which the associated CDKN2B-encoded p15 loss contributes to human tumorigenesis is unclear. Here we show that CDKN2B is highly upregulated in benign melanocytic nevi, contributes to maintaining nevus melanocytes in a growth-arrested premalignant state, and is commonly lost in melanoma. Using primary melanocytes isolated directly from freshly excised human nevi naturally expressing the common BRAF(V600E) activating mutation, nevi progressing to melanoma, and normal melanocytes engineered to inducibly express BRAF(V600E), we show that BRAF activation results in reversible, TGFβ-dependent, p15 induction that halts proliferation. Further, we engineer human skin grafts containing nevus-derived melanocytes to establish a new, architecturally faithful, in vivo melanoma model, and demonstrate that p15 loss promotes the transition from benign nevus to melanoma. PMID:26183406

  16. Prophylactic Dendritic Cell-Based Vaccines Efficiently Inhibit Metastases in Murine Metastatic Melanoma

    PubMed Central

    Sennikov, Sergey V.; Vlassov, Valentin V.; Zenkova, Marina A.

    2015-01-01

    Recent data on the application of dendritic cells (DCs) as anti-tumor vaccines has shown their great potential in therapy and prophylaxis of cancer. Here we report on a comparison of two treatment schemes with DCs that display the models of prophylactic and therapeutic vaccination using three different experimental tumor models: namely, Krebs-2 adenocarcinoma (primary tumor), melanoma (B16, metastatic tumor without a primary node) and Lewis lung carcinoma (LLC, metastatic tumor with a primary node). Dendritic cells generated from bone marrow-derived DC precursors and loaded with lysate of tumor cells or transfected with the complexes of total tumor RNA with cationic liposomes were used for vaccination. Lipofectamine 2000 and liposomes consisting of helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and cationic lipid 2D3 (1,26-Bis(1,2-de-O-tetradecyl-rac-glycerol)-7,11,16,20-tetraazahexacosan tetrahydrocloride) were used for RNA transfection. It was shown that DCs loaded with tumor lysate were ineffective in contrast to tumor-derived RNA. Therapeutic vaccination with DCs loaded by lipoplexes RNA/Lipofectamine 2000 was the most efficient for treatment of non-metastatic Krebs-2, where a 1.9-fold tumor growth retardation was observed. Single prophylactic vaccination with DCs loaded by lipoplexes RNA/2D3 was the most efficient to treat highly aggressive metastatic tumors LLC and B16, where 4.7- and 10-fold suppression of the number of lung metastases was observed, respectively. Antimetastatic effect of single prophylactic DC vaccination in metastatic melanoma model was accompanied by the reductions in the levels of Th2-specific cytokines however the change of the levels of Th1/Th2/Th17 master regulators was not found. Failure of double prophylactic vaccination is explained by Th17-response polarization associated with autoimmune and pro-inflammatory reactions. In the case of therapeutic DC vaccine the polarization of Th1-response was found nevertheless

  17. Prophylactic Dendritic Cell-Based Vaccines Efficiently Inhibit Metastases in Murine Metastatic Melanoma.

    PubMed

    Markov, Oleg V; Mironova, Nadezhda L; Sennikov, Sergey V; Vlassov, Valentin V; Zenkova, Marina A

    2015-01-01

    Recent data on the application of dendritic cells (DCs) as anti-tumor vaccines has shown their great potential in therapy and prophylaxis of cancer. Here we report on a comparison of two treatment schemes with DCs that display the models of prophylactic and therapeutic vaccination using three different experimental tumor models: namely, Krebs-2 adenocarcinoma (primary tumor), melanoma (B16, metastatic tumor without a primary node) and Lewis lung carcinoma (LLC, metastatic tumor with a primary node). Dendritic cells generated from bone marrow-derived DC precursors and loaded with lysate of tumor cells or transfected with the complexes of total tumor RNA with cationic liposomes were used for vaccination. Lipofectamine 2000 and liposomes consisting of helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and cationic lipid 2D3 (1,26-Bis(1,2-de-O-tetradecyl-rac-glycerol)-7,11,16,20-tetraazahexacosan tetrahydrocloride) were used for RNA transfection. It was shown that DCs loaded with tumor lysate were ineffective in contrast to tumor-derived RNA. Therapeutic vaccination with DCs loaded by lipoplexes RNA/Lipofectamine 2000 was the most efficient for treatment of non-metastatic Krebs-2, where a 1.9-fold tumor growth retardation was observed. Single prophylactic vaccination with DCs loaded by lipoplexes RNA/2D3 was the most efficient to treat highly aggressive metastatic tumors LLC and B16, where 4.7- and 10-fold suppression of the number of lung metastases was observed, respectively. Antimetastatic effect of single prophylactic DC vaccination in metastatic melanoma model was accompanied by the reductions in the levels of Th2-specific cytokines however the change of the levels of Th1/Th2/Th17 master regulators was not found. Failure of double prophylactic vaccination is explained by Th17-response polarization associated with autoimmune and pro-inflammatory reactions. In the case of therapeutic DC vaccine the polarization of Th1-response was found nevertheless

  18. Resistance to ursolic acid-induced apoptosis through involvement of melanogenesis and COX-2/PGE{sub 2} pathways in human M4Beu melanoma cancer cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hassan, Lama; Pinon, Aline; Limami, Youness

    Melanoma is one of the most aggressive forms of cancer with a continuously growing incidence worldwide and is usually resistant to chemotherapy agents, which is due in part to a strong resistance to apoptosis. Previously, we had showed that B16-F0 murine melanoma cells undergoing apoptosis are able to delay their own death induced by ursolic acid (UA), a natural pentacyclic triterpenoid compound. We had demonstrated that tyrosinase and TRP-1 up-regulation in apoptotic cells and the subsequent production of melanin were implicated in an apoptosis resistance mechanism. Several resistance mechanisms to apoptosis have been characterized in melanoma such as hyperactivation ofmore » DNA repair mechanisms, drug efflux systems, and reinforcement of survival signals (PI3K/Akt, NF-κB and Raf/MAPK pathways). Otherwise, other mechanisms of apoptosis resistance involving different proteins, such as cyclooxygenase-2 (COX-2), have been described in many cancer types. By using a strategy of specific inhibition of each ways, we suggested that there was an interaction between melanogenesis and COX-2/PGE{sub 2} pathway. This was characterized by analyzing the COX-2 expression and activity, the expression of tyrosinase and melanin production. Furthermore, we showed that anti-proliferative and proapoptotic effects of UA were mediated through modulation of multiple signaling pathways including Akt and ERK-1/2 proteins. Our study not only uncovers underlying molecular mechanisms of UA action in human melanoma cancer cells but also suggest its great potential as an adjuvant in treatment and cancer prevention.« less

  19. Novel alpha-MSH peptide analogs for melanoma targeting

    NASA Astrophysics Data System (ADS)

    Flook, Adam Michael

    Skin cancer is the one of the most diagnosed cancers in the United States with increasing incidence over the past two decades. There are three major forms of skin cancer but melanoma is the deadliest. It is estimated that 76,690 new diagnoses of melanoma and 9,480 deaths will occur in 2013. Melanoma accounts for approximately 1.6% of all cancer related deaths and is the 5 th leading diagnosed cancer in the United States. The mean survival rate of patients diagnosed with metastatic melanoma is six months, with five year survival rates of less than 5%. In this project, we describe the design and characterization of novel melanoma-targeting peptide analogs for use in diagnostic imaging of both primary and metastatic melanoma lesions. Novel alpha-MSH peptide conjugates were designed to target the melanocortin-1 receptor present and over-expressed on melanoma cells. These peptides were synthesized and their in-vitro melanocortin-1 receptor binding affinities were established in murine melanoma cells. Once binding affinities were determined, the peptides were radiolabeled with 99mTc utilizing a novel direct radiolabeling technique developed in our laboratory. The peptides were purified via reverse-phase high performance liquid chromatography and in-vivo melanoma targeting and pharmacokinetic properties were determined in B16/F1 melanoma-bearing female C57BL/6 mice. Biodistribution and SPECT/CT imaging studies were performed with the promising 99m Tc-labeled peptide conjugates. All alpha-MSH peptide conjugates tested showed low nanomolar binding affinity for the melanocortin-1 receptor. All peptides were readily radiolabeld with 99mTc with greater than 95% radiochemical purity. All 99mTc-labeled peptides displayed high specific in-vivo melanoma tumor uptake while maintaining low normal organ accumulation, and were excreted through the urinary system in a timely fashion. In addition, all tested 99mTc-labeld alpha-MSH peptides demonstrated clear visualization of in

  20. Increased apoptotic potential and dose-enhancing effect of gold nanoparticles in combination with single-dose clinical electron beams on tumor-bearing mice.

    PubMed

    Chang, Meng-Ya; Shiau, Ai-Li; Chen, Yu-Hung; Chang, Chih-Jui; Chen, Helen H-W; Wu, Chao-Liang

    2008-07-01

    High atomic number material, such as gold, may be used in conjunction with radiation to provide dose enhancement in tumors. In the current study, we investigated the dose-enhancing effect and apoptotic potential of gold nanoparticles in combination with single-dose clinical electron beams on B16F10 melanoma tumor-bearing mice. We revealed that the accumulation of gold nanoparticles was detected inside B16F10 culture cells after 18 h of incubation, and moreover, the gold nanoparticles were shown to be colocalized with endoplasmic reticulum and Golgi apparatus in cells. Furthermore, gold nanoparticles radiosensitized melanoma cells in the colony formation assay (P = 0.02). Using a B16F10 tumor-bearing mouse model, we further demonstrated that gold nanoparticles in conjunction with ionizing radiation significantly retarded tumor growth and prolonged survival compared to the radiation alone controls (P < 0.05). Importantly, an increase of apoptotic signals was detected inside tumors in the combined treatment group (P < 0.05). Knowing that radiation-induced apoptosis has been considered a determinant of tumor responses to radiation therapy, and the length of tumor regrowth delay correlated with the extent of apoptosis after single-dose radiotherapy, these results may suggest the clinical potential of gold nanoparticles in improving the outcome of melanoma radiotherapy.