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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

LPA is a chemorepellent for B16 melanoma cells: action through the cAMP-elevating LPA5 receptor.

Directory of Open Access Journals (Sweden)

Maikel Jongsma

Full Text Available Lysophosphatidic acid (LPA, a lipid mediator enriched in serum, stimulates cell migration, proliferation and other functions in many cell types. LPA acts on six known G protein-coupled receptors, termed LPA(1-6, showing both overlapping and distinct signaling properties. Here we show that, unexpectedly, LPA and serum almost completely inhibit the transwell migration of B16 melanoma cells, with alkyl-LPA(18:1 being 10-fold more potent than acyl-LPA(18:1. The anti-migratory response to LPA is highly polarized and dependent on protein kinase A (PKA but not Rho kinase activity; it is associated with a rapid increase in intracellular cAMP levels and PIP3 depletion from the plasma membrane. B16 cells express LPA(2, LPA(5 and LPA(6 receptors. We show that LPA-induced chemorepulsion is mediated specifically by the alkyl-LPA-preferring LPA(5 receptor (GPR92, which raises intracellular cAMP via a noncanonical pathway. Our results define LPA(5 as an anti-migratory receptor and they implicate the cAMP-PKA pathway, along with reduced PIP3 signaling, as an effector of chemorepulsion in B16 melanoma cells.

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

Science.gov (United States)

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.

Ethanol extract of Lycoris radiata induces cell death in B16F10 melanoma via p38-mediated AP-1 activation.

Science.gov (United States)

Son, Minsik; Kim, Aeyung; Lee, Jaewoo; Park, Chul-Hong; Heo, Jin-Chul; Lee, Hyun-Jin; Lee, Sang-Han

2010-08-01

Some active alkaloids isolated from Lycoris, a bulbous perennial herb, was shown to possess various anti-tumor and anti-inflammatory activities. In this study, we evaluated the in vitro apoptotic effect of ethanol extract from Lycoris radiata (LRE) and further probed the underlying molecular mechanisms of LRE effects. The survival rate of B16F10 melanoma cells exposed to LRE was decreased in a dose-dependent manner, cell growth was retarded by arresting cell cycle at G1 phase and apoptotic appearance such as caspase-3 activation as well as DNA fragmentation was observed by LRE treatment. In addition, LRE induced p38 and c-Jun phosphorylation, followed by activation of transcription factor AP-1. Pretreatment with the p38 inhibitor (SB203580) blocked LRE-induced AP-1 transcriptional activity, and curcumin, AP-1 inhibitor, dramatically inhibited LRE-induced apoptosis in B16F10 melanoma cells. Our results collectively indicate that LRE-mediated apoptosis occurs through the activation of p38 and AP-1 pathway and potentially LRE exhibits anti-cancer activity against B16F10 melanoma cells.

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

Science.gov (United States)

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.

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

Directory of Open Access Journals (Sweden)

Panpen Diawpanich

2008-07-01

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

Wogonin suppresses melanoma cell B16-F10 invasion and migration by inhibiting Ras-medicated pathways.

Directory of Open Access Journals (Sweden)

Kai Zhao

Full Text Available The patients diagnosed with melanoma have a bad prognosis for early regional invasion and distant metastases. Wogonin (5,7-dihydroxy-8-methoxyflavone is one of the active components of flavonoids that extracts from Scutellariae radix. Several previous studies reported that wogonin possesses antitumor effect against leukemia, gastrointestinal cancer and breast cancer. In this study, we used melanoma cell B16-F10 to further investigate the anti-invasive and anti-migratory activity of wogonin. Our date showed that wogonin caused suppression of cell migration, adhesion, invasion and actin remodeling by inhibiting the expression of matrix metalloproteinase-2 and Rac1 in vitro. Wogonin also reduced the number of the tumor nodules on the whole surface of the lung in vivo. Furthermore, the examination of mechanism revealed that wogonin inhibited Extracellular Regulated protein Kinases and Protein Kinase B pathways, which are both medicated by Ras. Insulin-like growth factor-1-induced or tumor necrosis factor-α-induced invasion was also inhibited by wogonin. Therefore, the inhibitory mechanism of melanoma cell invasion by wogonin might be elucidated.

B16F1 melanoma cells upregulate melanin synthesis after photodynamic therapy

International Nuclear Information System (INIS)

Moder, A.; Gassner, F.; Krammer, B.; Thalhamer, J.; Hammerl, P.

2003-01-01

Full text: The success of photodynamic therapy (PDT) of melanotic tumors is severely limited by insufficient penetration of light into deeper tissue layers. In this study, we analyzed the effect of PDT on the melanin production of the melanoma cell line B16F1. In vitro, these cells produce only little melanin. However, after PDT we found a dramatic elevation in intracellular melanin. Melanin production increased with, both, the concentration of the sensitizing agent and the light dose, and was found to continue for several hours after cell death. PDT-induced melanin synthesis was not prevented by the addition of cycloheximide or actinomycin D prior to irradiation, indicating that de-novo protein synthesis and transcriptional activity are not required for this effect. We also analyzed tyrosinase activity, a key enzyme in melanin biosynthesis, in PDT-treated B16 cells. Tyrosinase activity was found in PDT-treated as well as untreated cells. Cell fractionation experiments showed that tyrosinase was present in the cytosolic as well as the melanosomal fractions of, both, PDT-treated (melanin-high) as well as untreated (melanin-low) cells. These data indicate that PDT-induced production of melanin is not controlled at the transcriptional or translational level and that tyrosinase is not likely an essential regulator in this process. (author)

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

Energy Technology Data Exchange (ETDEWEB)

Souza, Lucas E.B., E-mail: lucasebsouza@usp.br [Department of Clinical Medicine, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Hemotherapy Center of Ribeirão Preto, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Almeida, Danilo C., E-mail: gudaalmeida@gmail.com [Department of Medicine – Nephrology, Laboratory of Clinical and Experimental Immunology, Federal University of São Paulo, São Paulo, SP (Brazil); Yaochite, Juliana N.U., E-mail: ueda.juliana@gmail.com [Department of Biochemistry and Immunology, Basic and Applied Immunology Program, School of Medicine of Ribeirão Preto, University of São Paulo (Brazil); Covas, Dimas T., E-mail: dimas@fmrp.usp.br [Department of Clinical Medicine, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Hemotherapy Center of Ribeirão Preto, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Fontes, Aparecida M., E-mail: aparecidamfontes@usp.br [Department of Genetics, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil)

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{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-MSCs enhanced

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

Radiation Changes the Metabolic Profiling of Melanoma Cell Line B16.

Directory of Open Access Journals (Sweden)

Lige Wu

Full Text Available Radiation therapy can be an effective way to kill cancer cells using ionizing radiation, but some tumors are resistant to radiation therapy and the underlying mechanism still remains elusive. It is therefore necessary to establish an appropriate working model to study and monitor radiation-mediated cancer therapy. In response to cellular stress, the metabolome is the integrated profiling of changes in all metabolites in cells, which can be used to investigate radiation tolerance mechanisms and identify targets for cancer radiation sensibilization. In this study, using 1H nuclear magnetic resonance for untargeted metabolic profiling in radiation-tolerant mouse melanoma cell line B16, we comprehensively investigated changes in metabolites and metabolic network in B16 cells in response to radiation. Principal component analysis and partial least squares discriminant analysis indicated the difference in cellular metabolites between the untreated cells and X-ray radiated cells. In radiated cells, the content of alanine, glutamate, glycine and choline was increased, while the content of leucine, lactate, creatine and creatine phosphate was decreased. Enrichment analysis of metabolic pathway showed that the changes in metabolites were related to multiple metabolic pathways including the metabolism of glycine, arginine, taurine, glycolysis, and gluconeogenesis. Taken together, with cellular metabolome study followed by bioinformatic analysis to profile specific metabolic pathways in response to radiation, we deepened our understanding of radiation-resistant mechanisms and radiation sensibilization in cancer, which may further provide a theoretical and practical basis for personalized cancer therapy.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

Benzodiazepines have high-affinity binding sites and induce melanogenesis in B16/C3 melanoma cells.

OpenAIRE

Matthew, E; Laskin, J D; Zimmerman, E A; Weinstein, I B; Hsu, K C; Engelhardt, D L

1981-01-01

We found that two markers of differentiation, tyrosinase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) activity and melanin synthesis, are induced by diazepam in B16/C3 mouse melanoma cells. We also demonstrated high-affinity binding sites for [3H]diazepam in these cells by radioreceptor assay, and we visualized binding to the cell surface by fluorescence microscopy with a benzodiazepine analog conjugated to a fluorescein-labeled protein. Our studies also showed tha...

Radiation sensitivity of B-16 melanoma

International Nuclear Information System (INIS)

Griem, M.L.; Malkinson, F.D.; Kalis, J.B.; Shefner, A.

1984-01-01

A model has been developed for radiation studies of melanoma. Β-16 melanoma (NCI), carried by subcutaneous implant in C57BL/6NCr mice was implanted instramuscularly into the right rear leg of female B6C3F1 mice. Test mice were inoculated with 1 x 10/sup 5/, 5 x 10/sup 5/, and 1 x 10/sup 6/ tumor cells to determine an appropriate tumor challenge for a reproducible and suitable median survival time. A challenge inoculum of 5 x 10/sup 5/ tumor cells was subsequently chosen as the standard tumor dose for test animals used in subsequent radiation dose response studies. Tumor-bearing test animals were treated with 500, 1000, 1500, or 2000 rads of 250 kV x-rays either 4 days or 14 days after tumor implantation. Only the tumorbearing leg of the test mouse was exposed during irradiation; the animal was otherwise protected by lead shielding. The median survival time of tumorbearing unirradiated mice was 24.4 days. Radiation on day 4 postinoculation was more effective than radiation administered on day 14. Median survival time for the 4 radiation dose groups given x-rays on day 4 were 31.0, 38.2, 59.0, and 60.0 days with progressive increases in radiation dose. Median survival times for mice irradiated on day 14 were 26.8, 31.8, 34.8, and 49.2 days as the radiation doses increased. This mouse melanoma model can be used in combined modality studies

Anti-Melanogenic Property of Geoditin A in Murine B16 Melanoma Cells

Directory of Open Access Journals (Sweden)

Chun-Tao Che

2012-02-01

Full Text Available Geoditin A, an isomalabaricane triterpene isolated from marine sponge Geodia japonica, has been demonstrated to induce apoptosis in leukemia HL60 cells and human colon HT29 cancer cells through an oxidative stress, a process also interfering with normal melanogenesis in pigment cells. Treatment of murine melanoma B16 cells with geoditin A decreased expression of melanogenic proteins and cell melanogenesis which was aggravated with adenylate cyclase inhibitor SQ22536, indicating melanogenic inhibition was mediated through a cAMP-dependent signaling pathway. Immunofluorescence microscopy and glycosylation studies revealed abnormal glycosylation patterns of melanogenic proteins (tyrosinase and tyrosinase-related protein 1, and a co-localization of tyrosinase with calnexin (CNX and lysosome-associated membrane protein 1 (LAMP-1, implicating a post-translational modification in the ER and a degradation of tyrosinase in the lysosome. Taken together, potent anti-melanogenic property and the relatively low cytotoxicity of geoditin A have demonstrated its therapeutic potential as a skin lightening agent.

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

Science.gov (United States)

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.

Antimelanogenic, Antioxidant and Antiproliferative Effects of Antrodia camphorata Fruiting Bodies on B16-F0 Melanoma Cells.

Directory of Open Access Journals (Sweden)

Jyh-Jye Wang

Full Text Available Antrodia camphorata is a fungus that is endemic to Taiwan, and its fruiting body has been used as a folk medicine for the prevention or treatment of diverse diseases. The present study is aimed at investigating the antimelanogenesis and antioxidation effect of the ethanolic extract of Antrodia camphorata fruiting body (EE-AC, as well as its antiproliferation effects in B16-F0 melanoma cells. Regarding antimelanogenic effects, EE-AC had effective cupric ions reducing capacity and expressed more potent inhibitory effect than kojic acid on mushroom tyrosinase activity. Moreover, EE-AC significantly inhibited cellular tyrosinase activity and the melanin content in B16-F0 cells at 12.5 μg/mL concentration without cell toxicities. Regarding antioxidant effects, EE-AC exhibited potent DPPH radical- and SOD-like-scavenging activities. Regarding antiproliferative effects, EE-AC exhibited a selective cytotoxic effect and markedly inhibited the migration ability of B16-F0 cells. EE-AC increased the population of B16-F0 cells at sub-G1 phase of the cell cycle. EE-AC also caused the increase of early apoptotic cells and chromatin condensation, which indicated the apoptotic effects in B16-F0 cells. We demonstrated that EE-AC possessed antimelanogenic, antioxidant and anti-skin cancer actions. The results would contribute to the development and application of cosmetics, healthy food and pharmaceuticals.

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

Science.gov (United States)

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).

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

Science.gov (United States)

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.

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

Directory of Open Access Journals (Sweden)

Taek-In Oh

2017-02-01

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

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

OpenAIRE

Valles, Soraya L; Benlloch, Mar?a; Rodriguez, Mar?a L; Mena, Salvador; Pellicer, Jos? A; Asensi, Miguel; Obrador, Elena; Estrela, Jos? M

2013-01-01

[EN] Background: 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. Methods: 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 f...

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

BFD-22 a new potential inhibitor of BRAF inhibits the metastasis of B16F10 melanoma cells and simultaneously increased the tumor immunogenicity

Energy Technology Data Exchange (ETDEWEB)

Ferreira, Adilson Kleber, E-mail: ferreira-kleber@usp.br [Laboratory of Tumor Immunology, University of São Paulo, São Paulo, SP (Brazil); Department of Medical Oncology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, Groningen (Netherlands); Pasqualoto, Kerly Fernanda Mesquita [Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, SP (Brazil); Kruyt, Frank A.E. [Department of Medical Oncology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, Groningen (Netherlands); Palace-Berl, Fanny [Laboratory of Drug Design and Development, University of São Paulo, São Paulo, SP (Brazil); Azevedo, Ricardo Alexandre [Laboratory of Tumor Immunology, University of São Paulo, São Paulo, SP (Brazil); Turra, Kely Medeiros [Laboratory of Cytopathology, Department of Clinical Chemistry and Toxicology, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP (Brazil); Rodrigues, Cecilia Pessoa; Ferreira, Ana Carolina Franco; Salomón, Maria Alejandra Clavijo [Laboratory of Tumor Immunology, University of São Paulo, São Paulo, SP (Brazil); Sá, Paulo Luiz de [Laboratory of Genetics, Butantan Institute, São Paulo, SP (Brazil); Farias, Camyla Fernandes; Figueiredo, Carlos Rogerio [Experimental Oncology Section, The Federal University of São Paulo, São Paulo, SP (Brazil); and others

2016-03-15

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 invasion 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. • BFD

Control of differentiation of melanoma cells

International Nuclear Information System (INIS)

Eguchi, Goro

1980-01-01

To develop the method to induce the appearance of differentiation in amelanotic melanoma, experimental control of differentiation in B-16 melanoma cells of mice was discussed. Human melanoma cells and yellow melanin pigment cells useful for a fundamental study of radiotherapy for cancer were cultured and were differentiated into some lines. Melanotic B-16 cells and amelanotic B-16 cells were irradiated with thermal neutron (neutron: 2.7 x 10 12 , γ-dose: 32.3 rad) after they were cultured in culture solution containing 10 γ/ml of 10 B-dopa for 13 hours. A fine structure 5 hours after the irradiation in one of 5 experimental cases showed aggregated disintegration of melanin pigment particles, markedly deformed and fragmentized nucleus, and structural changes in cell membrane. (Tsunoda, M.)

Calreticulin Fragment 39-272 Promotes B16 Melanoma Malignancy through Myeloid-Derived Suppressor Cells In Vivo

Directory of Open Access Journals (Sweden)

Xiao-Yan He

2017-10-01

Full Text Available Calreticulin (CRT, a multifunctional Ca2+-binding glycoprotein mainly located in the endoplasmic reticulum, is a tumor-associated antigen that has been shown to play protective roles in angiogenesis suppression and anti-tumor immunity. We previously reported that soluble CRT (sCRT was functionally similar to heat shock proteins or damage-associated molecular patterns in terms of ability to activate myeloid cells and elicit strong inflammatory cytokine production. In the present study, B16 melanoma cell lines expressing recombinant CRT fragment 39-272 (sCRT/39-272 in secreted form (B16-CRT, or recombinant enhanced green fluorescence protein (rEGFP (B16-EGFP, were constructed for investigation on the roles of sCRT in tumor development. When s.c. inoculated into C57BL/6 mice, the B16-CRT cells were significantly more aggressive (in terms of solid tumor growth rate than B16-EGFP controls in a TLR4- and myeloid-derived suppressor cells (MDSC-dependent manner. The B16-CRT-bearing mice showed increased Gr1+ MDSC infiltration in tumor tissues, accelerated proliferation of CD11b+Ly6G+Ly6Clow (G-MDSC precursors in bone marrow, and higher percentages of G-MDSCs in spleen and blood, which was mirrored by decreased percentage of dendritic cells (DC in periphery. In in vitro studies, recombinant sCRT/39-272 was able to promote migration and survival of tumor-derived MDSCs via interaction with TLR4, inhibit MDSC differentiation into DC, and also elicit expression of inflammatory proteins S100A8 and S100A9 which are essential for functional maturation and chemotactic migration of MDSCs. Our data provide solid evidence for CRT as a double-edged sword in tumor development.

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

Science.gov (United States)

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.

Cell cycle regulation by the Wee1 Inhibitor PD0166285, Pyrido [2,3-d] pyimidine, in the B16 mouse melanoma cell line

International Nuclear Information System (INIS)

Hashimoto, Osamu; Shinkawa, Masako; Torimura, Takuji; Nakamura, Toru; Selvendiran, Karuppaiyah; Sakamoto, Masaharu; Koga, Hironori; Ueno, Takato; Sata, Michio

2006-01-01

Wee1 kinase plays a critical role in maintaining G2 arrest through its inhibitory phosphorylation of cdc2. In previous reports, a pyridopyrimidine molecule PD0166285 was identified to inhibit Wee1 activity at nanomolar concentrations. This G2 checkpoint abrogation by PD0166285 was demonstrated to kill cancer cells, there at a toxic highest dose of 0.5 μM in some cell lines for exposure periods of no longer than 6 hours. The deregulated cell cycle progression may have ultimately damaged the cancer cells. We herein report one of the mechanism by which PD0166285 leads to cell death in the B16 mouse melanoma cell line. Tumor cell proliferation was determined by counting cell numbers. Cell cycle distribution was determined by flow cytometry. Morphogenesis analysis such as microtubule stabilization, Wee1 distribution, and cyclin B location were observed by immunofluorescence confocal microscopy. An immunoblot analysis of cdc2-Tyr15, cyclin D, E, p16, 21, 27, and Rb. A real-time PCR of the mRNA of cyclin D were completed. In our experiment, B16 cells also dramatically abrogated the G2 checkpoint and were found to arrest in the early G1 phase by treatment with 0.5 μM for 4 hours observed by flow cytometry. Cyclin D mRNA decreased within 4 hours observed by Real-time PCR. Rb was dephosphrylated for 24 hours. However, B16 cells did not undergo cell death after 0.5 μM treatment for 24 hours. Immnofluoscence microscopy showed that the cells become round and small in the morphogenesis. More interesting phenomena were that microtubule stabilization was blocked, and Wee1 distribution was restricted after treatment for 4 hours. We analyzed the effect of Wee1 inhibitor PD0166285 described first by Wang in the G2 transition in the B16 melanoma cell line. The inhibitor PD0166285 abrogated G2/M checkpoint inducing early cell division. Moreover, we found that the treatment of cells with the inhibitor is related to microtubule stabilization and decrease in cyclin D transcription

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

Directory of Open Access Journals (Sweden)

Ramesh Kumar Santhanam

2016-05-01

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

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Directory of Open Access Journals (Sweden)

Ching-Gong Lin

2009-09-01

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

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

Science.gov (United States)

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.

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

International Nuclear Information System (INIS)

Maeda, Masayo; Murakami, Manabu; Takegami, Tsutomu; Ota, Takahide

2008-01-01

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 commonly 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

Cytosolic DNA Sensor Upregulation Accompanies DNA Electrotransfer in B16.F10 Melanoma Cells

Directory of Open Access Journals (Sweden)

Katarina Znidar

2016-01-01

Full Text Available In several preclinical tumor models, antitumor effects occur after intratumoral electroporation, also known as electrotransfer, of plasmid DNA devoid of a therapeutic gene. In mouse melanomas, these effects are preceded by significant elevation of several proinflammatory cytokines. These observations implicate the binding and activation of intracellular DNA-specific pattern recognition receptors or DNA sensors in response to DNA electrotransfer. In tumors, IFNβ mRNA and protein levels significantly increased. The mRNAs of several DNA sensors were detected, and DAI, DDX60, and p204 tended to be upregulated. These effects were accompanied with reduced tumor growth and increased tumor necrosis. In B16.F10 cells in culture, IFNβ mRNA and protein levels were significantly upregulated. The mRNAs for several DNA sensors were present in these cells; DNA-dependent activator of interferon regulatory factor (DAI, DEAD (Asp-Glu-Ala-Asp box polypeptide 60 (DDX60, and p204 were significantly upregulated while DDX60 protein levels were coordinately upregulated. Upregulation of DNA sensors in tumors could be masked by the lower transfection efficiency compared to in vitro or to dilution by other tumor cell types. Mirroring the observation of tumor necrosis, cells underwent a significant DNA concentration-dependent decrease in proliferation and survival. Taken together, these results indicate that DNA electrotransfer may cause the upregulation of several intracellular DNA sensors in B16.F10 cells, inducing effects in vitro and potentially in vivo.

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

Science.gov (United States)

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 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.

Let-7b-mediated suppression of basigin expression and metastasis in mouse melanoma cells

International Nuclear Information System (INIS)

Fu, Tzu-Yen; Chang, Chia-Che; Lin, Chun-Ting; Lai, Cong-Hao; Peng, Shao-Yu; Ko, Yi-Ju; Tang, Pin-Chi

2011-01-01

Basigin (Bsg), also called extracellular matrix metalloproteinase inducer (EMMPRIN), is highly expressed on the surface of tumor cells and stimulates adjacent fibroblasts or tumor cells to produce matrix metalloproteinases (mmps). It has been shown that Bsg plays an important role in growth, development, cell differentiation, and tumor progression. MicroRNAs (miRNAs) are a class of short endogenous non-protein coding RNAs of 20-25 nucleotides (nt) that function as post-transcriptional regulators of gene expression by base-pairing to their target mRNAs and thereby mediate cleavage of target mRNAs or translational repression. In this study, let-7b, one of the let-7 family members, was investigated for its effect on the growth and invasiveness of the mouse melanoma cell line B16-F10. We have shown that let-7b can suppress the expression of Bsg in B16-F10 cells and also provided evidence that this suppression could result in the indirect suppression of mmp-9. The ability of B16-F10 cells transfected with let-7b to invade or migrate was significantly reduced. In addition, let-7b transfected B16-F10 cells displayed an inhibition of both cellular proliferation and colony formation. Furthermore, it was shown that the overexpression of let-7b in B16-F10 cells could reduce lung metastasis. Taken together, the present study identifies let-7b as a tumor suppressor that represses cancer cell proliferation and migration as well as tumor metastasis in mouse melanoma cells.

Let-7b-mediated suppression of basigin expression and metastasis in mouse melanoma cells

Energy Technology Data Exchange (ETDEWEB)

Fu, Tzu-Yen [Department of Animal Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Chang, Chia-Che [Institute of Biomedical Sciences, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Graduate Institute of Basic Medical Science, China Medical University, 91 Hsueh Shih Road, Taichung 40402, Taiwan (China); Lin, Chun-Ting [Department of Animal Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Lai, Cong-Hao [Institute of Biomedical Sciences, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Department of Life Sciences, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Peng, Shao-Yu; Ko, Yi-Ju [Department of Animal Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Tang, Pin-Chi, E-mail: pctang@dragon.nchu.edu.tw [Department of Animal Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China)

2011-02-15

Basigin (Bsg), also called extracellular matrix metalloproteinase inducer (EMMPRIN), is highly expressed on the surface of tumor cells and stimulates adjacent fibroblasts or tumor cells to produce matrix metalloproteinases (mmps). It has been shown that Bsg plays an important role in growth, development, cell differentiation, and tumor progression. MicroRNAs (miRNAs) are a class of short endogenous non-protein coding RNAs of 20-25 nucleotides (nt) that function as post-transcriptional regulators of gene expression by base-pairing to their target mRNAs and thereby mediate cleavage of target mRNAs or translational repression. In this study, let-7b, one of the let-7 family members, was investigated for its effect on the growth and invasiveness of the mouse melanoma cell line B16-F10. We have shown that let-7b can suppress the expression of Bsg in B16-F10 cells and also provided evidence that this suppression could result in the indirect suppression of mmp-9. The ability of B16-F10 cells transfected with let-7b to invade or migrate was significantly reduced. In addition, let-7b transfected B16-F10 cells displayed an inhibition of both cellular proliferation and colony formation. Furthermore, it was shown that the overexpression of let-7b in B16-F10 cells could reduce lung metastasis. Taken together, the present study identifies let-7b as a tumor suppressor that represses cancer cell proliferation and migration as well as tumor metastasis in mouse melanoma cells.

Gene transfer-applied BNCT (g-BNCT) for amelanotic melanoma in brain. Further upregulation of 10B uptake by cell modulation

International Nuclear Information System (INIS)

Iwakura, M.; Tamaki, N.; Hiratsuka, J.

2000-01-01

Our success in eradicating melanoma by single BNCT with BPA led to the next urgent theme, i.e. application of such BNCT for currently uncurable melanoma metastasis in brain. In order to establish 10 B-BPA-BNCT for melanoma in brain, we have investigated the pharmacokinetics of BPA which is most critical factor for successful BNCT, in melanotic and amelanotic and further tyrosinase gene-transfected amelanotic melanoma proliferating in brain having blood-brain-barrier, as compared to melanoma proliferating in skin. We have established three implanted models for melanoma in brain: 1) A1059 cells, amelanotic melanoma, 2) B16B15b cells, melanotic melanoma cells, highly metastatic to brain, and 3) TA1059 cells, with active melanogenesis induced by tyrosinase gene transfection. We would like to report the results of comparative analysis of the BPA uptake ability in these melanoma cells in both brain and skin. Based on these findings, we are further investigating to enhance 10 B-BPA uptake by not only g-BNCT but also by additional melanogenesis upregulating cell modulation. (author)

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

Neem leaf glycoprotein optimizes effector and regulatory functions within tumor microenvironment to intervene therapeutically the growth of B16 melanoma in C57BL/6 mice

Directory of Open Access Journals (Sweden)

Subhasis Barik

2015-01-01

Full Text Available Therapy with neem leaf glycoprotein (NLGP inhibits murine B16-melanoma in vivo and improves survivability. Studies on tumor-microenvironment (TME from NLGP treated mice (NLGP-TME suggests that anti-tumor effect is directly associated with enhanced CD8+T cell activity, dominance of type 1 cytokines/chemokine network with downregulation of suppressive cellular functions. NLGP-TME educated CD8+T cells showed higher perforin and granzymeB expression with greater in vitro cytotoxicity against B16 melanoma. These CD8+T cells showed proportionally lower FasR expression, denotes prevention from activation induced cell death by NLGP. Accumulated evidences strongly suggest NLGP influenced normalized TME allows CD8+T cells to perform optimally to inhibit melanoma growth.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Energy Technology Data Exchange (ETDEWEB)

Cunha, Elizabeth S.; Kawahara, Rebeca [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil); Kadowaki, Marina K. [Universidade Estadual do Oeste do Parana, Cascavel, PR (Brazil); Amstalden, Hudson G.; Noleto, Guilhermina R.; Cadena, Silvia Maria S.C.; Winnischofer, Sheila M.B. [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil); Martinez, Glaucia R., E-mail: grmartinez@ufpr.br [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil)

2012-09-10

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 cell 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.

A potent inhibitor of SIK2, 3, 3', 7-trihydroxy-4'-methoxyflavon (4'-O-methylfisetin, promotes melanogenesis in B16F10 melanoma cells.

Directory of Open Access Journals (Sweden)

Ayako Kumagai

Full Text Available Flavonoids, which are plant polyphenols, are now widely used in supplements and cosmetics. Here, we report that 4'-methylflavonoids are potent inducers of melanogenesis in B16F10 melanoma cells and in mice. We recently identified salt inducible kinase 2 (SIK2 as an inhibitor of melanogenesis via the suppression of the cAMP-response element binding protein (CREB-specific coactivator 1 (TORC1. Using an in vitro kinase assay targeting SIK2, we identified fisetin as a candidate inhibitor, possibly being capable of promoting melanogenesis. However, fisetin neither inhibited the CREB-inhibitory activity of SIK2 nor promoted melanogenesis in B16F10 melanoma cells. Conversely, mono-methyl-flavonoids, such as diosmetin (4'-O-metlylluteolin, efficiently inhibited SIK2 and promoted melanogenesis in this cell line. The cAMP-CREB system is impaired in A(y/a mice and these mice have yellow hair as a result of pheomelanogenesis, while Sik2(+/-; A(y/a mice also have yellow hair, but activate eumelanogenesis when they are exposed to CREB stimulators. Feeding Sik2(+/-; A(y/a mice with diets supplemented with fisetin resulted in their hair color changing to brown, and metabolite analysis suggested the presence of mono-methylfisetin in their feces. Thus, we decided to synthesize 4'-O-methylfisetin (4'MF and found that 4'MF strongly induced melanogenesis in B16F10 melanoma cells, which was accompanied by the nuclear translocation of TORC1, and the 4'-O-methylfisetin-induced melanogenic programs were inhibited by the overexpression of dominant negative TORC1. In conclusion, compounds that modulate SIK2 cascades are helpful to regulate melanogenesis via TORC1 without affecting cAMP levels, and the combined analysis of Sik2(+/- mice and metabolites from these mice is an effective strategy to identify beneficial compounds to regulate CREB activity in vivo.

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

Directory of Open Access Journals (Sweden)

Carla A. Guimarães-Ferreira

2007-09-01

Full Text Available 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 C57BI/6 mice, fastuosain and bromelain injected intraperitoneally were protective, 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, 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 PBSinjected control mice, but the number of positive major histocompatibility complex class II cells increased with fastuosain treatment. Murine antibodies against fastuosain, bromelain, cathepsins B and L crossreacted 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.

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

Science.gov (United States)

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 ( 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.

B16 melanoma tumor growth is delayed in mice in an age-dependent manner

Directory of Open Access Journals (Sweden)

Christina Pettan-Brewer

2012-08-01

Full Text Available A major risk factor for cancer is increasing age, which suggests that syngeneic tumor implants in old mice would grow more rapidly. However, various reports have suggested that old mice are not as permissive to implanted tumor cells as young mice. In order to determine and characterize the age-related response to B16 melanoma, we implanted 5×105 tumor cells into 8, 16, 24, and 32-month-old male C57BL/6 (B6 and C57BL/6×BALB/c F1 (CB6 F1 mice subcutaneously in the inguinal and axillary spaces, or intradermally in the lateral flank. Results showed decreased tumor volume with increasing age, which varied according to mouse genetic background and the implanted site. The B6 strain showed robust tumor growth at 8 months of age at the inguinal implantation site, with an average tumor volume of 1341.25 mm3. The 16, 24, and 32-month age groups showed a decrease in tumor growth with tumor volumes of 563.69, 481.02, and 264.55 mm3, respectively (p≤0.001. The axillary implantation site was less permissive in 8-month-old B6 mice with an average tumor volume of 761.52 mm3. The 24- and 32-month age groups showed a similar decrease in tumor growth with tumor volumes of 440 and 178.19 mm3, respectively (p≤0.01. The CB6F1 strain was not as tumor permissive at 8 months of age as B6 mice with average tumor volumes of 446.96 and 426.91 mm3 for the inguinal and axillary sites, respectively. There was a decrease in tumor growth at 24 months of age at both inguinal and axillary sites with an average tumor volume of 271.02 and 249.12 mm3, respectively (p≤0.05. The strain dependence was not apparent in 8-month-old mice injected intradermally with B16 melanoma cells, with average tumor volumes of 736.82 and 842.85 mm3 for B6 and CB6 F1, respectively. However, a strain difference was seen in 32-month-old B6 mice with an average decrease in tumor volume of 250.83 mm3 (p≤0.01. In contrast, tumor growth significantly decreased earlier in CB6 F1 mice with average

Syngeneic B16F10 Melanoma Causes Cachexia and Impaired Skeletal Muscle Strength and Locomotor Activity in Mice

Directory of Open Access Journals (Sweden)

Fabrício A. Voltarelli

2017-09-01

Full Text Available Muscle wasting has been emerging as one of the principal components of cancer cachexia, leading to progressive impairment of work capacity. Despite early stages melanomas rarely promotes weight loss, the appearance of metastatic and/or solid tumor melanoma can leads to cachexia development. Here, we investigated the B16F10 tumor-induced cachexia and its contribution to muscle strength and locomotor-like activity impairment. C57BL/6 mice were subcutaneously injected with 5 × 104 B16F10 melanoma cells or PBS as a Sham negative control. Tumor growth was monitored during a period of 28 days. Compared to Sham mice, tumor group depicts a loss of skeletal muscle, as well as significantly reduced muscle grip strength and epididymal fat mass. This data are in agreement with mild to severe catabolic host response promoted by elevated serum tumor necrosis factor-alpha (TNF-α, interleukin-6 (IL-6 and lactate dehydrogenase (LDH activity. Tumor implantation has also compromised general locomotor activity and decreased exploratory behavior. Likewise, muscle loss, and elevated inflammatory interleukin were associated to muscle strength loss and locomotor activity impairment. In conclusion, our data demonstrated that subcutaneous B16F10 melanoma tumor-driven catabolic state in response to a pro-inflammatory environment that is associated with impaired skeletal muscle strength and decreased locomotor activity in tumor-bearing mice.

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

Science.gov (United States)

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.

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.

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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

The antitumor activity of a doxorubicin loaded, iRGD-modified sterically-stabilized liposome on B16-F10 melanoma cells: in vitro and in vivo evaluation

Directory of Open Access Journals (Sweden)

Yu KF

2013-07-01

Full Text Available Ke-Fu Yu,1 Wei-Qiang Zhang,1 Li-Min Luo,1 Ping Song,1 Dan Li,1 Ruo Du,1 Wei Ren,1 Dan Huang,1 Wan-Liang Lu,1,2 Xuan Zhang,1 Qiang Zhang1,2 1Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing, People’s Republic of China; 2State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, People’s Republic of China Abstract: Considering the fact that iRGD (tumor-homing peptide demonstrates tumor-targeting and tumor-penetrating activity, and that B16-F10 (murine melanoma cells overexpress both αv integrin receptor and neuropilin-1 (NRP-1, the purpose of this study was to prepare a novel doxorubicin (DOX-loaded, iRGD-modified, sterically-stabilized liposome (SSL (iRGD-SSL-DOX in order to evaluate its antitumor activity on B16-F10 melanoma cells in vitro and in vivo. The iRGD-SSL-DOX was prepared using a thin-film hydration method. The characteristics of iRGD-SSL-DOX were evaluated. The in vitro leakage of DOX from iRGD-SSL-DOX was tested. The in vitro tumor-targeting and tumor-penetrating characteristics of iRGD-modified liposomes on B16-F10 cells were investigated. The in vivo tumor-targeting and tumor-penetrating activities of iRGD-modified liposomes were performed in B16-F10 tumor-bearing nude mice. The antitumor effect of iRGD-SSL-DOX was evaluated in B16-F10 tumor-bearing C57BL/6 mice in vivo. The average particle size of the iRGD-SSL-DOX was found to be 91 nm with a polydispersity index (PDI of 0.16. The entrapment efficiency of iRGD-SSL-DOX was 98.36%. The leakage of DOX from iRGD-SSL-DOX at the 24-hour time point was only 7.5%. The results obtained from the in vitro flow cytometry and confocal microscopy, as well as in vivo biodistribution and confocal immunofluorescence microscopy experiments, indicate that the tumor-targeting and tumor-penetrating activity of the iRGD-modified SSL was higher than that of unmodified SSL. In vivo antitumor activity

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

Directory of Open Access Journals (Sweden)

Guillaume Sarrabayrouse

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

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

Science.gov (United States)

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.

Antiproliferative and proapoptotic actions of okra pectin on B16F10 melanoma cells

NARCIS (Netherlands)

Vayssade, M.; Sengkhamparn, N.; Verhoef, R.P.; Delaigue, C.; Goundiam, O.; Vigneron, P.; Voragen, A.G.J.; Schols, H.A.; Nagel, M.D.

2010-01-01

The proliferation and apoptosis of metastatic melanoma cells are often abnormal. We have evaluated the action of a pectic rhamnogalacturonan obtained by hot buffer extraction of okra pods (okra RG-I) on melanoma cell growth and survival in vitro. We added okra RG-I containing an almost pure RG-I

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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.

Science.gov (United States)

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.

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

Science.gov (United States)

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 (Pcellular uptake. The Nos-Ag 2+ nanocrystals and Red-Br-Nos-Ag 2+ nanocrystals exhibited an IC 50 of 16.6μM and 6.5μM, significantly (Pcellular 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 caspase activation. Preliminary investigations substantiated that Nos-Ag 2+ nanocrystals and Red-Br-Nos-Ag 2+ nanocrystals must be further explored and utilized for the delivery of noscapinoids to melanoma cancer cells. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

Expansion of CD16-Negative Natural Killer Cells in the Peripheral Blood of Patients with Metastatic Melanoma

Directory of Open Access Journals (Sweden)

Shernan G. Holtan

2011-01-01

Full Text Available Altered natural killer (NK cell function is a component of the global immune dysregulation that occurs in advanced malignancies. Another condition associated with altered NK homeostasis is normal pregnancy, where robust infiltration with CD16− CD9+ NK cells can be identified in decidual tissues, along with a concomitant expansion of CD16− NK cells in the maternal peripheral blood. In metastatic melanoma, we identified a similar expansion of peripheral blood CD16− NK cells (median 7.4% in 41 patients with melanoma compared with 3.0% in 29 controls, P<.001. A subset of NK cells in melanoma patients also expresses CD9, which is characteristically expressed only on NK cells within the female reproductive tract. Expansion of CD16− NK cells was associated with elevated plasma transforming growth factor-beta (TGF-β levels (median 20 ng/ml, Spearman's ρ=0.81,P=.015. These findings suggest the possibility of exploring anti-TGF-β therapy to restore NK function in melanoma.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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

7-Piperazinethylchrysin inhibits melanoma cell proliferation by ...

African Journals Online (AJOL)

In B16F10 and A375 cells, treatment with PEC caused the inhibition ... Conclusion: PEC inhibited melanoma cell proliferation, apparently by blocking the cell cycle at G0/G1 .... all statistical analyses. .... Financial support from the Department of.

Vascular endothelial growth factor regulates melanoma cell adhesion and growth in the bone marrow microenvironment via tumor cyclooxygenase-2

Directory of Open Access Journals (Sweden)

Crende Olatz

2011-08-01

Full Text Available Abstract Background Human melanoma frequently colonizes bone marrow (BM since its earliest stage of systemic dissemination, prior to clinical metastasis occurrence. However, how melanoma cell adhesion and proliferation mechanisms are regulated within bone marrow stromal cell (BMSC microenvironment remain unclear. Consistent with the prometastatic role of inflammatory and angiogenic factors, several studies have reported elevated levels of cyclooxygenase-2 (COX-2 in melanoma although its pathogenic role in bone marrow melanoma metastasis is unknown. Methods Herein we analyzed the effect of cyclooxygenase-2 (COX-2 inhibitor celecoxib in a model of generalized BM dissemination of left cardiac ventricle-injected B16 melanoma (B16M cells into healthy and bacterial endotoxin lipopolysaccharide (LPS-pretreated mice to induce inflammation. In addition, B16M and human A375 melanoma (A375M cells were exposed to conditioned media from basal and LPS-treated primary cultured murine and human BMSCs, and the contribution of COX-2 to the adhesion and proliferation of melanoma cells was also studied. Results Mice given one single intravenous injection of LPS 6 hour prior to cancer cells significantly increased B16M metastasis in BM compared to untreated mice; however, administration of oral celecoxib reduced BM metastasis incidence and volume in healthy mice, and almost completely abrogated LPS-dependent melanoma metastases. In vitro, untreated and LPS-treated murine and human BMSC-conditioned medium (CM increased VCAM-1-dependent BMSC adherence and proliferation of B16M and A375M cells, respectively, as compared to basal medium-treated melanoma cells. Addition of celecoxib to both B16M and A375M cells abolished adhesion and proliferation increments induced by BMSC-CM. TNFα and VEGF secretion increased in the supernatant of LPS-treated BMSCs; however, anti-VEGF neutralizing antibodies added to B16M and A375M cells prior to LPS-treated BMSC-CM resulted in a

C5 Extract Induces Apoptosis in B16F10 Murine Melanoma Cells ...

African Journals Online (AJOL)

Purpose: To investigate the anti-cancer activities of C5 extract (C5E), a new herbal preparation from Korea, on B16F10 cells. Methods: The anti-proliferative effects of C5E were assessed by culturing B16F10 cells in the presence or absence of C5E. Cell cycle progression was analyzed by PI staining using flow cytometry.

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

Directory of Open Access Journals (Sweden)

Amy E Gilbert

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

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

Science.gov (United States)

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

Thymoquinone suppresses metastasis of melanoma cells by inhibition of NLRP3 inflammasome

Energy Technology Data Exchange (ETDEWEB)

Ahmad, Israr; Muneer, Kashiff M.; Tamimi, Iman A.; Chang, Michelle E.; Ata, Muhammad O. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, AL (United States); Yusuf, Nabiha, E-mail: nabiha@uab.edu [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, AL (United States); Veteran Affairs Medical Center, Birmingham, University of Alabama at Birmingham, AL (United States); Comprehensive Cancer Center, University of Alabama at Birmingham, AL (United States)

2013-07-01

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

Boron neutron capture therapy induces cell cycle arrest and DNA fragmentation in murine melanoma cells

Energy Technology Data Exchange (ETDEWEB)

Faiao-Flores, F. [Biochemical and Biophysical Laboratory, Butantan Institute, 1500 Vital Brasil Avenue, Sao Paulo (Brazil)] [Faculty of Medicine, University of Sao Paulo, 455 Doutor Arnaldo Avenue, Sao Paulo (Brazil); Coelho, P.R.P. [Institute for Nuclear and Energy Research, 2242 Lineu Prestes Avenue, Sao Paulo (Brazil); Arruda-Neto, J. [Physics Institute, University of Sao Paulo, 187 Matao Street, Sao Paulo (Brazil)] [FESP, Sao Paulo Engineering School, 5520 Nove de Julho Avenue, Sao Paulo (Brazil); Maria, Durvanei A., E-mail: durvaneiaugusto@yahoo.br [Biochemical and Biophysical Laboratory, Butantan Institute, 1500 Vital Brasil Avenue, Sao Paulo (Brazil)

2011-12-15

The melanoma is a highly lethal skin tumor, with a high incidence. Boron Neutron Capture Therapy (BNCT) is a radiotherapy which combines Boron with thermal neutrons, constituting a binary system. B16F10 melanoma and L929 fibroblasts were treated with Boronophenylalanine and irradiated with thermal neutron flux. The electric potential of mitochondrial membrane, cyclin D1 and caspase-3 markers were analyzed. BNCT induced a cell death increase and cyclin D1 amount decreased only in B16F10 melanoma. Besides, there was not caspase-3 phosphorylation.

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

Science.gov (United States)

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.

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

Energy Technology Data Exchange (ETDEWEB)

Ryu, Min Sook [Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, 164, World cul-ro, Yeongtong-gu, Suwon, Gyeonggi-do 443-380 (Korea, Republic of); Woo, Min-Yeong [Department of Microbiology, Ajou University School of Medicine, 164, World cul-ro, Yeongtong-gu, Suwon, Gyeonggi-do 443-380 (Korea, Republic of); Department of Biomedical Sciences, The Graduate School, Ajou University (Korea, Republic of); Kwon, Daeho [Department of Microbiology, Kwandong University College of Medicine, Gangneung, Gangwon-do 210-701 (Korea, Republic of); Hong, Allen E. [Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, 164, World cul-ro, Yeongtong-gu, Suwon, Gyeonggi-do 443-380 (Korea, Republic of); Song, Kye Yong [Department of Pathology, Chung-Ang University College of Medicine, Dongjak-gu, Seoul 156-756 (Korea, Republic of); Park, Sun [Department of Microbiology, Ajou University School of Medicine, 164, World cul-ro, Yeongtong-gu, Suwon, Gyeonggi-do 443-380 (Korea, Republic of); Lim, In Kyoung [Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, 164, World cul-ro, Yeongtong-gu, Suwon, Gyeonggi-do 443-380 (Korea, Republic of)

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 the 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.

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

Science.gov (United States)

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.

Inhibitory effect of fentanyl citrate on the release of endothlin-1 induced by bradykinin in melanoma cells.

Science.gov (United States)

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.

The kin17 Protein in Murine Melanoma Cells

Directory of Open Access Journals (Sweden)

Anelise C. Ramos

2015-11-01

Full Text Available 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.

Efficient treatment of pigmented B16 melanoma using photosensitized long-circulating magnetofullerenosomes

International Nuclear Information System (INIS)

Babinec, Peter; Babincova, Melania; Sourivong, Paul; Leszczynska, Danuta

2005-01-01

Magnetic targeting was used for melanoma treatment with magnetofullerenosomes. After their intravenous administration, a permanent magnet was attached to the surface of B16 tumors in C57 mice for 24 h, followed by irradiation with an infrared laser pulse and subsequent illumination with a 776 nm diode laser for conventional photodynamic treatment. Tumor response was substantially better than that obtained with either treatment alone

PRL-3 siRNA inhibits the metastasis of B16-BL6 mouse melanoma cells in vitro and in vivo.

Science.gov (United States)

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.

Growth Inhibition of Re-Challenge B16 Melanoma Transplant by Conjugates of Melanogenesis Substrate and Magnetite Nanoparticles as the Basis for Developing Melanoma-Targeted Chemo-Thermo-Immunotherapy

Directory of Open Access Journals (Sweden)

Tomoaki Takada

2009-01-01

Full Text Available Melanogenesis substrate, N-propionyl-cysteaminylphenol (NPrCAP, is selectively incorporated into melanoma cells and inhibits their growth by producing cytotoxic free radicals. Magnetite nanoparticles also disintegrate cancer cells and generate heat shock protein (HSP upon exposure to an alternating magnetic field (AMF. This study tested if a chemo-thermo-immunotherapy (CTI therapy strategy can be developed for better management of melanoma by conjugating NPrCAP on the surface of magnetite nanoparticles (NPrCAP/M. We examined the feasibility of this approach in B16 mouse melanoma and evaluated the impact of exposure temperature, frequency, and interval on the inhibition of re-challenged melanoma growth. The therapeutic protocol against the primary transplanted tumor with or without AMF exposure once a day every other day for a total of three treatments not only inhibited the growth of the primary transplant but also prevented the growth of the secondary, re-challenge transplant. The heat-generated therapeutic effect was more significant at a temperature of 43∘C than either 41∘C or 46∘C. NPrCAP/M with AMF exposure, instead of control magnetite alone or without AMF exposure, resulted in the most significant growth inhibition of the re-challenge tumor and increased the life span of the mice. HSP70 production was greatest at 43∘C compared to that with 41∘C or 46∘C. CD+T cells were infiltrated at the site of the re-challenge melanoma transplant.

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

International Nuclear Information System (INIS)

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

2005-01-01

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 α-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 ≤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

The use of Zymosan A and bacteria anchored to tumor cells for effective cancer immunotherapy: B16-F10 murine melanoma model

Czech Academy of Sciences Publication Activity Database

Waldmannová, E.; Caisová, V.; Fáberová, J.; Sváčková, P.; Kovářová, M.; Sváčková, D.; Kumžáková, Z.; Jačková, A.; Vácová, N.; Nedbalová, P.; Horká, Marie; Kopecký, Jan; Ženka, J.

2016-01-01

Roč. 39, OCT (2016), s. 295-306 ISSN 1567-5769 Institutional support: RVO:68081715 ; RVO:60077344 Keywords : melanoma B16-F10, * cancer immunotherapy * frustrated phagocytosis Subject RIV: CB - Analytical Chemistry, Separation; EC - Immunology (BC-A) Impact factor: 2.956, year: 2016

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

Science.gov (United States)

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

2011-02-25

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

miR-193b Regulates Mcl-1 in Melanoma.

Science.gov (United States)

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.

Apoptosis of murine melanoma B16-BL6 cells induced by quercetin targeting mitochondria, inhibiting expression of PKC-alpha and translocating PKC-delta.

Science.gov (United States)

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

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

Science.gov (United States)

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.

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

Directory of Open Access Journals (Sweden)

Su Jin Kang

2015-10-01

Full Text Available 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.

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

Science.gov (United States)

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

Therapy of established B16-F10 melanoma tumors by a single vaccination of CTL/T helper peptides in VacciMax®

Directory of Open Access Journals (Sweden)

Korets-Smith Ella

2007-04-01

Full Text Available Abstract Background Melanoma tumors are known to express antigens that usually induce weak immune responses of short duration. Expression of both tumor-associated antigens p53 and TRP2 by melanoma cells raises the possibility of simultaneously targeting more than one antigen in a therapeutic vaccine. In this report, we show that VacciMax® (VM, a novel liposome-based vaccine delivery platform, can increase the immunogenicity of melanoma associated antigens, resulting in tumor elimination. Methods C57BL/6 mice bearing B16-F10 melanoma tumors were vaccinated subcutaneously 6 days post tumor implantation with a mixture of synthetic peptides (modified p53: 232–240, TRP-2: 181–188 and PADRE and CpG. Tumor growth was monitored and antigen-specific splenocyte responses were assayed by ELISPOT. Results Vaccine formulated in VM increased the number of both TRP2- and p53-specific IFN-γ producing splenocytes following a single vaccination. Vaccine formulated without VM resulted only in enhanced IFN-γ producing splenocytes to one CTL epitopes (TRP2:180–188, suggesting that VM overcomes antigen dominance and enhances immunogenicity of multiple epitopes. Vaccination of mice bearing 6-day old B16-F10 tumors with both TRP2 and p53-peptides formulated in VM successfully eradicated tumors in all mice. A control vaccine which contained all ingredients except liposomes resulted in eradication of tumors in no more than 20% of mice. Conclusion A single administration of VM is capable of inducing an effective CTL response to multiple tumor-associated antigens. The responses generated were able to reject 6-day old B16-F10 tumors.

Melanoma stem cells in experimental melanoma are killed by radioimmunotherapy

International Nuclear Information System (INIS)

Jandl, Thomas; Revskaya, Ekaterina; Jiang, Zewei; Harris, Matthew; Dorokhova, Olena; Tsukrov, Dina; Casadevall, Arturo; Dadachova, Ekaterina

2013-01-01

Introduction: In spite of recently approved B-RAF inhibitors and immunomodulating antibodies, metastatic melanoma has poor prognosis and novel treatments are needed. Melanoma stem cells (MSC) have been implicated in the resistance of this tumor to chemotherapy. Recently we demonstrated in a Phase I clinical trial in patients with metastatic melanoma that radioimmunotherapy (RIT) with 188-Rhenium( 188 Re)-6D2 antibody to melanin was a safe and effective modality. Here we investigated the interaction of MSC with RIT as a possible mechanism for RIT efficacy. Methods: Mice bearing A2058 melanoma xenografts were treated with either 1.5 mCi 188 Re-6D2 antibody, saline, unlabeled 6D2 antibody or 188 Re-labeled non-specific IgM. Results: On Day 28 post-treatment the tumor size in the RIT group was 4-times less than in controls (P < 0.001). The tumors were analyzed by immunohistochemistry and FACS for two MSC markers — chemoresistance mediator ABCB5 and H3K4 demethylase JARID1B. There were no significant differences between RIT and control groups in percentage of ABCB5 or JARID1B-positive cells in the tumor population. Our results demonstrate that unlike chemotherapy, which kills tumor cells but leaves behind MSC leading to recurrence, RIT kills MSC at the same rate as the rest of tumor cells. Conclusions: These results have two main implications for melanoma treatment and possibly other cancers. First, the susceptibility of ABCB5 + and JARID1B + cells to RIT in melanoma might be indicative of their susceptibility to antibody-targeted radiation in other cancers where they are present as well. Second, specifically targeting cancer stem cells with radiolabeled antibodies to ABCB5 or JARID1B might help to completely eradicate cancer stem cells in various cancers

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

Therapeutic T cells induce tumor-directed chemotaxis of innate immune cells through tumor-specific secretion of chemokines and stimulation of B16BL6 melanoma to secrete chemokines

Directory of Open Access Journals (Sweden)

Fox Bernard A

2007-11-01

Full Text Available Abstract Background The mechanisms by which tumor-specific T cells induce regression of established metastases are not fully characterized. In using the poorly immunogenic B16BL6-D5 (D5 melanoma model we reported that T cell-mediated tumor regression can occur independently of perforin, IFN-γ or the combination of both. Characterization of regressing pulmonary metastases identified macrophages as a major component of the cells infiltrating the tumor after adoptive transfer of effector T cells. This led us to hypothesize that macrophages played a central role in tumor regression following T-cell transfer. Here, we sought to determine the factors responsible for the infiltration of macrophages at the tumor site. Methods These studies used the poorly immunogenic D5 melanoma model. Tumor-specific effector T cells, generated from tumor vaccine-draining lymph nodes (TVDLN, were used for adoptive immunotherapy and in vitro analysis of chemokine expression. Cellular infiltrates into pulmonary metastases were determined by immunohistochemistry. Chemokine expression by the D5 melanoma following co-culture with T cells, IFN-γ or TNF-α was determined by RT-PCR and ELISA. Functional activity of chemokines was confirmed using a macrophage migration assay. T cell activation of macrophages to release nitric oxide (NO was determined using GRIES reagent. Results We observed that tumor-specific T cells with a type 1 cytokine profile also expressed message for and secreted RANTES, MIP-1α and MIP-1β following stimulation with specific tumor. Unexpectedly, D5 melanoma cells cultured with IFN-γ or TNF-α, two type 1 cytokines expressed by therapeutic T cells, secreted Keratinocyte Chemoattractant (KC, MCP-1, IP-10 and RANTES and expressed mRNA for MIG. The chemokines released by T cells and cytokine-stimulated tumor cells were functional and induced migration of the DJ2PM macrophage cell line. Additionally, tumor-specific stimulation of wt or perforin

The histone deacetylase inhibitor butyrate inhibits melanoma cell invasion of Matrigel.

Science.gov (United States)

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.

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

Science.gov (United States)

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

Phorbol 12-myristate 13-acetate enhances nm23 gene expression in murine melanocytes but not in syngeneic B16-BL6 melanoma variants.

Science.gov (United States)

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.

Melanoma cells show a heterogeneous range of sensitivity to ionizing radiation and are radiosensitized by inhibition of B-RAF with PLX-4032

International Nuclear Information System (INIS)

Sambade, Maria J.; Peters, Eldon C.; Thomas, Nancy E.; Kaufmann, William K.; Kimple, Randall J.; Shields, Janiel M.

2011-01-01

Purpose: To assess the relative radiosensitivities of a large collection of melanoma cell lines and to determine whether pharmacologic inhibition of mutant B-RAF with PLX-4032 can radiosensitize B-Raf+ melanoma cells. Materials and methods: A large collection of melanoma cell lines (n = 37) were treated with 0-8 Gy IR and clonogenic survival assays used to generate survival curves to rank relative radiosensitivities among the cell lines. The ability of a B-RAF inhibitor, PLX-4032, to radiosensitize highly radioresistant B-Raf+ cells was also assessed by clonogenic cell survival and spheroid invasion assays and the effects of treatment on the cell cycle assessed by FACS. Results: Melanoma cell lines displayed a very large, heterogeneous range of SF2 values (1.002-0.053) with a mean of 0.51. Cell lines with surviving fractions of 0.29 or less at SF2 and SF4 were observed at a high frequency of 18.9% and 70.2%, respectively. Treatment of B-Raf+ cells with the B-RAF inhibitor PLX-4032 in combination with radiation provided enhanced inhibition of both colony formation and invasion, and radiosensitized cells through an increase in G 1 arrest. Conclusions: Our data suggest that melanomas are not uniformly radioresistant with a significant subset displaying inherent radiosensitivity. Pharmacologic inhibition of B-RAF with PLX-4032 effectively radiosensitized B-Raf+ melanoma cells suggesting that this combination approach could provide improved radiotherapeutic response in B-Raf+ melanoma patients.

In vivo immunologic selection of class I major histocompatibility complex gene deletion variants from the B16-BL6 melanoma.

Science.gov (United States)

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.

Sticky siRNAs targeting survivin and cyclin B1 exert an antitumoral effect on melanoma subcutaneous xenografts and lung metastases

International Nuclear Information System (INIS)

Kedinger, Valerie; Erbacher, Patrick; Bolcato-Bellemin, Anne-Laure; Meulle, Aline; Zounib, Omar; Bonnet, Marie-Elise; Gossart, Jean-Baptiste; Benoit, Elodie; Messmer, Melanie; Shankaranarayanan, Pattabhiraman; Behr, Jean-Paul

2013-01-01

Melanoma represents one of the most aggressive and therapeutically challenging malignancies as it often gives rise to metastases and develops resistance to classical chemotherapeutic agents. Although diverse therapies have been generated, no major improvement of the patient prognosis has been noticed. One promising alternative to the conventional therapeutic approaches currently available is the inactivation of proteins essential for survival and/or progression of melanomas by means of RNA interference. Survivin and cyclin B1, both involved in cell survival and proliferation and frequently deregulated in human cancers, are good candidate target genes for siRNA mediated therapeutics. We used our newly developed sticky siRNA-based technology delivered with linear polyethyleneimine (PEI) to inhibit the expression of survivin and cyclin B1 both in vitro and in vivo, and addressed the effect of this inhibition on B16-F10 murine melanoma tumor development. We confirm that survivin and cyclin B1 downregulation through a RNA interference mechanism induces a blockage of the cell cycle as well as impaired proliferation of B16-F10 cells in vitro. Most importantly, PEI-mediated systemic delivery of sticky siRNAs against survivin and cyclin B1 efficiently blocks growth of established subcutaneaous B16-F10 tumors as well as formation and dissemination of melanoma lung metastases. In addition, we highlight that inhibition of survivin expression increases the effect of doxorubicin on lung B16-F10 metastasis growth inhibition. PEI-mediated delivery of sticky siRNAs targeting genes involved in tumor progression such as survivin and cyclin B1, either alone or in combination with chemotherapeutic drugs, represents a promising strategy for melanoma treatment

In vitro radiobiological evaluation of selective killing effects of 10B1-paraboronophenylalanine.HCl in the thermal neutron capture therapy of malignant melanoma cells

International Nuclear Information System (INIS)

Ichihashi, M.; Ueda, M.; Hayashibe, K.; Hatta, S.; Tsuji, M.; Mishima, Y.; Fukuda, H.; Kobayashi, T.; Kanda, K.

1985-01-01

In order to clarify the specific affinity of 10 B 1 -p-boronophenylalanine.HCl ( 10 B 1 -BPA) to melanoma cells, the killing effects of 10 B 1 -BPA in the thermal neutron capture treatment on both cultured melanotic and amelanotic melanoma cells were compared with those on non-melanoma cells, such as Alexander cells, HeLa cells and normal human fibroblasts. Cells in the plateau phase cultured in the usual medium for 4-7 days were incubated with the medium containing 50 μg/ml 10 B 1 -BPA for 20 hours until 2 hours before thermal neutron irradiation. After thermal neutron irradiation, the number of colonies consisting of more than 50 cells was counted to obtain the dose-survival curves. The melanotic cells pre-incubated with 10 B 1 -BPA had more enhanced killing sensitivity to thermal neutron irradiation than amelanotic melanoma cells pre-incubated similarly with 10 B 1 -BPA. 10 B 1 -BPA pre-incubation had no enhanced killing effects on Alexander cells, but had slightly enhanced killing effects on HeLa cells. These results indicate that 10 B 1 -BPA could be incorporated by a specific uptake mechanism of melanoma cells and accumulated within melanotic melanoma cells and that 10 B 1 -BPA at present could be the best chemical for the thermal neutron capture therapy of human malignant melanoma. (Namekawa, K.)

Increased projection of MHC and tumor antigens in murine B16-BL6 melanoma induced by hydrostatic pressure and chemical crosslinking.

Science.gov (United States)

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.

PPARalpha/gamma expression and activity in mouse and human melanocytes and melanoma cells.

Science.gov (United States)

Eastham, Linda L; Mills, Caroline N; Niles, Richard M

2008-06-01

We examined the expression of PPARs and the effects of PPARalpha and PPARgamma agonists on growth of mouse and human melanocytes and melanoma cells. PPARalpha,beta, and PPARgamma mRNA qualitative expression in melan-a mouse melanocytes, B16 mouse melanoma, human melanocytes, and A375 and SK-mel28 human melanoma cells was determined by RT-PCR, while quantitative PPARalpha mRNA levels were determined by QuantiGene assay. PPARalpha and PPARgamma protein was assessed by Western blotting. The effect of natural and synthetic PPAR ligands on cell growth was determined by either hemocytometer counting or crystal violet assay. PPAR transcriptional activity was determined by a PPRE-reporter gene assay, while knockdown of PPARalpha expression was achieved by transient transfection of siRNA. Both mouse and human melanoma cells produced more PPARalpha and PPARgamma protein compared to melanocytes. PPARalpha mRNA levels were elevated in human melanoma cells, but not in mouse melanoma cells relative to melanocytes. Silencing of PPARalpha in human melanoma cells did not alter cell proliferation or morphology. PPARgamma-selective agonists inhibited the growth of both mouse and human melanoma cells, while PPARalpha-selective agonists had limited effects. Increased expression of PPARalpha in melanoma relative to melanocytes may be a common occurrence, however its biologic significance remains to be determined. PPARgamma agonists may be useful for arresting the growth of some melanomas.

Antitumor effect of iRGD-modified liposomes containing conjugated linoleic acid–paclitaxel (CLA-PTX on B16-F10 melanoma

Directory of Open Access Journals (Sweden)

Du R

2014-06-01

Full Text Available Ruo Du,1 Ting Zhong,1 Wei-Qiang Zhang,1 Ping Song,1 Wen-Ding Song,1 Yang Zhao,1 Chao-Wang,1 Yi-Qun Tang,3 Xuan Zhang,1,2 Qiang Zhang1,2 1Department of Pharmaceutics, 2State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 3Department of Clinical Pharmacy, China Pharmaceutical University, Nanjing, People’s Republic of China Abstract: In the present study, we prepared a novel delivery system of iRGD (CRGDK/RGPD/EC-modified sterically stabilized liposomes (SSLs containing conjugated linoleic acid–paclitaxel (CLA-PTX. The anti-tumor effect of iRGD-SSL-CLA-PTX was investigated on B16-F10 melanoma in vitro and in vivo. The in vitro targeting effect of iRGD-modified SSLs was investigated in a real-time confocal microscopic analysis experiment. An endocytosis-inhibition assay was used to evaluate the endocytosis pathways of the iRGD-modified SSLs. In addition, the in vitro cellular uptake and in vitro cytotoxicity of iRGD-SSL-CLA-PTX were evaluated in B16-F10 melanoma cells. In vivo biodistribution and in vivo antitumor effects of iRGD-SSL-CLA-PTX were investigated in B16-F10 tumor-bearing mice. The induction of apoptosis by iRGD-SSL-CLA-PTX was evaluated in tumor-tissue sections. Real-time confocal microscopic analysis results indicated that the iRGD-modified SSLs internalized into B16-F10 cells faster than SSLs. The identified endocytosis pathway of iRGD-modified SSLs indicated that energy- and lipid raft-mediated endocytosis played a key role in the liposomes’ cellular uptake. The results of the cellular uptake experiment indicated that the increased cellular uptake of CLA-PTX in the iRGD-SSL-CLA-PTX-treated group was 1.9-, 2.4-, or 2.1-fold compared with that in the CLA-PTX group after a 2-, 4-, or 6-hour incubation, respectively. In the biodistribution test, the CLA-PTX level in tumor tissues from iRGD-SSL-CLA-PTX-treated mice at 1 hour (1.84±0.17 µg/g and 4 hours (1.17±0

Paullinia cupana Mart var. sorbilis, guaraná, reduces cell proliferation and increases apoptosis of B16/F10 melanoma lung metastases in mice

Directory of Open Access Journals (Sweden)

H. Fukumasu

2008-04-01

Full Text Available We showed that guaraná (Paullinia cupana Mart var. sorbilis had a chemopreventive effect on mouse hepatocarcinogenesis and reduced diethylnitrosamine-induced DNA damage. In the present experiment, we evaluated the effects of guaraná in an experimental metastasis model. Cultured B16/F10 melanoma cells (5 x 10(5 cells/animal were injected into the tail vein of mice on the 7th day of guaraná treatment (2.0 mg P. cupana/g body weight, per gavage and the animals were treated with guaraná daily up to 14 days until euthanasia (total treatment time: 21 days. Lung sections were obtained for morphometric analysis, apoptotic bodies were counted to calculate the apoptotic index and proliferating cell nuclear antigen-positive cells were counted to determine the proliferation index. Guaraná-treated (GUA animals presented a 68.6% reduction in tumor burden area compared to control (CO animals which were not treated with guaraná (CO: 0.84 ± 0.26, N = 6; GUA: 0.27 ± 0.24, N = 6; P = 0.0043, a 57.9% reduction in tumor proliferation index (CO: 23.75 ± 20.54, N = 6; GUA: 9.99 ± 3.93, N = 6; P = 0.026 and a 4.85-fold increase in apoptotic index (CO: 66.95 ± 22.95, N = 6; GUA: 324.37 ± 266.74 AB/mm², N = 6; P = 0.0152. In this mouse model, guaraná treatment decreased proliferation and increased apoptosis of tumor cells, consequently reducing the tumor burden area. We are currently investigating the molecular pathways of the effects of guaraná in cultured melanoma cells, regarding principally the cell cycle inhibitors and cyclins.

Ror2-Src signaling in metastasis of mouse melanoma cells is inhibited by NRAGE.

Science.gov (United States)

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.

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.

Science.gov (United States)

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.

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

Directory of Open Access Journals (Sweden)

Harish Chandra Pal

Full Text Available Malignant melanoma is responsible for approximately 75% of skin cancer-related deaths. BRAF plays an important role in regulating the mitogen-activated protein kinase (MAPK signaling cascade in melanoma with activating mutations in the serine/threonine kinase BRAF occurring in 60-70% of malignant melanomas. The BRAF-MEK-ERK (MAPK pathway is a key regulator of melanoma cell invasion. In addition, activation of NFκB via the MAPK pathway is regulated through MEK-induced activation of IKK. These pathways are potential targets for prevention and treatment of melanoma. In this study, we investigated the effect of fisetin, a phytochemical present in fruits and vegetables, on melanoma cell invasion and epithelial-mesenchymal transition, and delineated the underlying molecular mechanism. Treatment of multiple human malignant melanoma cell lines with fisetin (5-20 µM resulted in inhibition of cell invasion. BRAF mutated melanoma cells were more sensitive to fisetin treatment, and this was associated with a decrease in the phosphorylation of MEK1/2 and ERK1/2. In addition, fisetin inhibited the activation of IKK leading to a reduction in the activation of the NFκB signaling pathway. Treatment of cells with an inhibitor of MEK1/2 (PD98059 or of NFκB (caffeic acid phenethyl ester also reduced melanoma cell invasion. Furthermore, treatment of fisetin promoted mesenchymal to epithelial transition in melanoma cells, which was associated with a decrease in mesenchymal markers (N-cadherin, vimentin, snail and fibronectin and an increase in epithelial markers (E-cadherin and desmoglein. Employing three dimensional skin equivalents consisting of A375 cells admixed with normal human keratinocytes embedded onto a collagen-constricted fibroblast matrix, we found that treatment of fisetin reduced the invasive potential of melanoma cells into the dermis and increased the expression of E-cadherin with a concomitant decrease in vimentin. These results indicate that

Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

Science.gov (United States)

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

Cryptotanshinone has diverse effects on cell cycle events in melanoma cell lines with different metastatic capacity

OpenAIRE

Punchard, Neville

2011-01-01

Background and Purpose: Cryptotanshinone (CTs) is a major active component of Salvia miltiorrhiza, which is often used as Chinese herbal medicine in cancer therapy. Here, we systematically assessed the anti-tumor effect of CTs on two melanoma cell lines with low/high metastatic capacity (B16/B16BL6). Experimental Approach: MTT and LDH assays were used to evaluate cell growth and cytotoxicity. We assessed the effect of CTs on cell apoptosis or proliferation by Annexin V, TUNEL or BrdU assay. C...

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.

Science.gov (United States)

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.

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

Science.gov (United States)

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.

Immunomodulation of murine B16 melanoma metastasis: thymosin, thymectomy and irradiation

International Nuclear Information System (INIS)

Naylor, P.H.; Bhakoo, H.S.; Rosen, F.; Paolini, N.S.; Goldstein, A.L.

1983-01-01

Thymosin, a product of the endocrine system, was used to further define the effects of immunomodulation of metastasis. Adult thymectomized C57BL/6 mice, 4 wk post-irradiation (400 R) had a decrease in the number of pulmonary metastases (compared to controls) following tail vein injection of 5 X 10 4 B16 melanoma cells. Thymosin fraction 5 (fr. 5) administration (200 μg/mouse, 3 times weekly beginning 2 days post-thymectomy) returned the number of metastases to the nonthymectomized values. Thymosin treatment of sham-operated, sham-operated irradiated, or thymectomized nonirradiated mice did not significantly elevate the number of metastases compared to the respective controls. Variant tumors which have an increase in metastasis following thymectomy and irradiation were also used. Thymosin administration reversed the effects of thymectomy in such variants, resulting in a decrease in metastasis. Metastases in thymosin-treated control mice were not significantly altered. A role for the thymus in metastasis via an endocrine product (thymosin) is suggested by these studies. Since thymosin did not increase metastasis in intact mice with tumors, further clinical trials with thymosin in cancer patients are not counterindicated by the results. These experiments confirm that thymosin fr. 5 is an important probe of the immunoendocrine events involved in tumor growth and metastasis. (Auth.)

Tumour volume response, initial cell kill and cellular repopulation in B16 melanoma treated with cyclophosphamide and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea.

Science.gov (United States)

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

Engagement of αIIbβ3 (GPIIb/IIIa) with ανβ3 Integrin Mediates Interaction of Melanoma Cells with Platelets

Science.gov (United States)

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

Radiosensitizing effect of RHOB protein in melanoma cells

International Nuclear Information System (INIS)

Notcovich, C.; Grissi, C.; Sánchez Crespo, R.; Delgado, D.C.; Molinari, B.; Ibañez, I.L.; Durán, H.

2015-01-01

Melanoma cells are highly resistant to chemo or radiotherapy. DNA damage agents such as ionizing radiation induce apoptosis involving RhoB protein. In a great variety of tumors the levels of this protein decrease along tumor progression. RhoB is considered a tumor suppressor gene due to its antiproliferative and proapoptotic effect. Considering the aforementioned, the aim of this study was to characterize the radiobiological response of different human melanoma cell lines, and to evaluate the possible correlation between RhoB expression and radiosensitivity. The human melanoma cell lines A375, MELJ and SB2 were gamma-irradiated ( 137 Cs). Survival curves were obtained by clonogenic assay and fitted to the Linear-Quadratic (LQ) model. Radiosensitivity was evaluated by surviving fraction at 2 Gy (SF2). Results showed that MELJ was significantly more radioresistant (SF2=0.71) than A375 and SB2 (0.29 and 0.21 respectively. Expression levels of RhoB, evaluated by western blot, increased in all lines vs. non-irradiated control. SB2, the most radiosensitive cells, showed a greater induction (p<0.05) of RhoB. Finally, to study whether RhoB has a radiosensitizing effect, these cell lines were stably transfected with a wild type RhoB construction, a constitutively active RhoB mutant V14, or with the empty plasmid as control. For all cell lines higher expression level of this protein was found in RhoB or V14 transfected cells (p<0.05). Sensitization was evaluated by SF2. Significant radiosensitization was demonstrated in clones derived from A375 and SB2 ((p<0.05), while for MELJ cells, radio-sensitization was only found in clones overexpressing V14. In conclusion, the increase of RhoB in melanoma cell lines, either by radiation or transfection has a radiosensitizing effect. Thus, we propose RhoB modulation as a potential therapeutic tool to improve the radiation response of radioresistant melanoma. (authors)

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

Science.gov (United States)

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.

Nonlethal Levels of Zeaxanthin Inhibit Cell Migration, Invasion, and Secretion of MMP-2 via NF-κB Pathway in Cultured Human Uveal Melanoma Cells

Directory of Open Access Journals (Sweden)

Ming-Chao Bi

2016-01-01

Full Text Available Zeaxanthin at nonlethal dosages (3–10 μM significantly inhibited the cell migration of cultured uveal melanoma cells (C918 cell line as determined by wound healing assay and Boyden chamber assay. Matrigel invasion assay showed that cell invasion of uveal melanoma cells could be significantly inhibited by zeaxanthin. Secretion of MMP-2 by melanoma cells was significantly inhibited by zeaxanthin in a dose-dependent manner as measured by ELISA kit. Zeaxanthin also significantly inhibited the NF-κB levels in nuclear extracts of the UM cells, which is the upstream of the MMP-2 secretion. These results suggest that zeaxanthin might be a potentially therapeutic approach in the prevention of metastasis in uveal melanoma.

Z-100, an immunomodulatory extract of Mycobacterium tuberculosis strain Aoyama B, prevents spontaneous lymphatic metastasis of B16-BL6 melanoma.

Science.gov (United States)

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.

Innate immunity based cancer immunotherapy: B16-F10 murine melanoma model

Czech Academy of Sciences Publication Activity Database

Caisová, V.; Vieru, A.; Kumžáková, Z.; Glaserová, S.; Husniková, H.; Vácová, N.; Krejčová, G.; Paďouková, L.; Jochmanová, I.; Wolf, K. I.; Chmelař, J.; Kopecký, Jan; Ženka, J.

2016-01-01

Roč. 16, č. 1 (2016), č. článku 940. ISSN 1471-2407 Institutional support: RVO:60077344 Keywords : cancer immunotherapy * innate immunity * melanoma * neutrophils * resiquimod * mannan * phagocytosis Subject RIV: EC - Immunology Impact factor: 3.288, year: 2016

Germline RAD51B truncating mutation in a family with cutaneous melanoma

DEFF Research Database (Denmark)

Wadt, Karin A W; Aoude, Lauren G; Golmard, Lisa

2015-01-01

Known melanoma predisposition genes only account for around 40% of high-density melanoma families. Other rare mutations are likely to play a role in melanoma predisposition. RAD51B plays an important role in DNA repair through homologous recombination, and inactivation of RAD51B has been implicated...... in tumorigenesis. Thus RAD51B is a good candidate melanoma susceptibility gene, and previously, a germline splicing mutation in RAD51B has been identified in a family with early-onset breast cancer. In order to find genetic variants associated with melanoma predisposition, whole-exome sequencing was carried out...... on blood samples from a three-case cutaneous melanoma family. We identified a novel germline RAD51B nonsense mutation, and we demonstrate reduced expression of RAD51B in melanoma cells indicating inactivation of RAD51B. This is only the second report of a germline truncating RAD51B mutation. While...

Blockade of A2b Adenosine Receptor Reduces Tumor Growth and Immune Suppression Mediated by Myeloid-Derived Suppressor Cells in a Mouse Model of Melanoma

Directory of Open Access Journals (Sweden)

Raffaella Iannone

2013-12-01

Full Text Available The A2b receptor (A2bR belongs to the adenosine receptor family. Emerging evidence suggest that A2bR is implicated in tumor progression in some murine tumor models, but the therapeutic potential of targeting A2bR in melanoma has not been examined. This study first shows that melanoma-bearing mice treated with Bay 60-6583, a selective A2bR agonist, had increased melanoma growth. This effect was associated with higher levels of immune regulatory mediators interleukin-10 (IL-10 and monocyte chemoattractant protein 1 (MCP-1 and accumulation of tumor-associated CD11b positive Gr1 positive cells (CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs. Depletion of CD11b+Gr1+ cells completely reversed the protumor activity of Bay 60-6583. Conversely, pharmacological blockade of A2bR with PSB1115 reversed immune suppression in the tumor microenvironment, leading to a significant melanoma growth delay. PSB1115 treatment reduced both levels of IL-10 and MCP-1 and CD11b+Gr1+ cell number in melanoma lesions. These effects were associated with higher frequency of tumor-infiltrating CD8 positive (CD8+ T cells and natural killer T (NKT cells and increased levels of T helper 1 (Th1-like cytokines. Adoptive transfer of CD11b+Gr1+ cells abrogated the antitumor activity of PSB1115. These data suggest that the antitumor activity of PSB1115 relies on its ability to lower accumulation of tumor-infiltrating MDSCs and restore an efficient antitumor T cell response. The antitumor effect of PSB1115 was not observed in melanoma-bearing nude mice. Furthermore, PSB1115 enhanced the antitumor efficacy of dacarbazine. These data indicate that A2bR antagonists such as PSB1115 should be investigated as adjuvants in the treatment of melanoma.

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.

Science.gov (United States)

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.

Combined SEP and anti-PD-L1 antibody produces a synergistic antitumor effect in B16-F10 melanoma-bearing mice.

Science.gov (United States)

Hu, Zhengping; Ye, Liang; Xing, Yingying; Hu, Jinhang; Xi, Tao

2018-01-09

The increased PD-L1 induces poorer prognosis in melanoma. The treatment with PD-1/PD-L1 antibodies have a low response rate. The combination immunotherapies are the encouraging drug development strategy to receive maximal therapeutic benefit. In this study, we investigated the enhanced antitumor and immunomodulatory activity of combined SEP and αPD-L1 in B16-F10 melanoma-bearing mice. The results shown that combined SEP and αPD-L1 presented significant synergistic antitumor effects, increased the frequency of CD8 + and CD4 + T cells in spleen and tumor, cytotoxic activity of CTL in spleen, and IL-2 and IFN-γ levels in splenocytes and tumor. The combination treatment also produced synergistic increase in P-ERK1/2 level in spleen. Immunohistochemistry shown that SEP induced the PD-L1 expression in melanoma tissue possibly by promoting IFN-γ excretion, which led to the synergistic anti-tumor effects of aPD-L1 and SEP. Furthermore, in the purified T lymphocyte from the naive mice, the combination of SEP and αPD-L1 had more potent than SEP or αPD-L1 in promoting T lymphocyte proliferation and cytokines secretion including IL-2 and IFN-γ, at least partially by activating MEK/ERK pathway. Our study provides the scientific basis for a clinical trial that would involve combination of anti-PD-L1 mAb and SEP for sustained melanoma control.

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

Science.gov (United States)

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

Clinical significance of the molecular detection of melanoma cells circulating in the peripheral blood in melanoma patients.

Science.gov (United States)

Konstantopoulos, K; Psatha, M; Kalotychou, V; Frangia, N; Ioannovits, I; Meletis, I; Loukopoulos, D

2001-06-01

Blood circulating melanoma cells may be important for the spread of the disease. The current methods are not sensitive in detecting micro metastases. Tyrosinase mRNA can be detected in peripheral blood by a molecular test. As tyrosinase is expressed only in melanocytes and melanocytes normally do not circulate in the blood, the test may prove reliable in detecting circulating melanoma cells. we used a reverse-transcription polymerase chain reaction (RT-PCR) detecting tyrosinase mRNA in the blood. A prospective investigation in melanoma patients undergoing surgery was conducted; follow-up duration was 12 months. University Department Laboratory and Melanoma Clinic of a Tertiary Hospital. a total of 27 Greek patients with a diagnosis of malignant melanoma at different stages of the disease; 12 months follow-up after surgery. Samples form 12 healthy volunteers and 13 patients with chronic myelogenous leukemia served as controls. none. none. We detected mRNA tyrosinase in the peripheral blood in 16 out of 27 melanoma patients studied. No tyrosinase mRNA was detected in any of the 25 samples from the controls. Two of the 16 positive cases developed a metastasis within the next 12 months following testing. The other 14 positive cases remain metastasis free for this period, as also did the test negative cases. Detection of blood circulating melanoma cells by a RT-PCR technique, may be helpful in defining melanoma patients who are at risk for the spread of the disease.

Structure-activity relationship of prenyl-substituted polyphenols from Artocarpus heterophyllus as inhibitors of melanin biosynthesis in cultured melanoma cells.

Science.gov (United States)

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.

Augmented growth inhibition of B16-BL6 melanoma by combined treatment with a selective matrix metalloproteinase inhibitor, MMI-166, and cytotoxic agents.

Science.gov (United States)

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 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.

Ultra-structural cell distribution of the melanoma marker iodobenzamide: improved potentiality of SIMS imaging in life sciences

Directory of Open Access Journals (Sweden)

Papon Janine

2004-04-01

Full Text Available Abstract Background Analytical imaging by secondary ion mass spectrometry (SIMS provides images representative of the distribution of a specific ion within a sample surface. For the last fifteen years, concerted collaborative research to design a new ion microprobe with high technical standards in both mass and lateral resolution as well as in sensitivity has led to the CAMECA NanoSims 50, recently introduced onto the market. This instrument has decisive capabilities, which allow biological applications of SIMS microscopy at a level previously inaccessible. Its potential is illustrated here by the demonstration of the specific affinity of a melanoma marker for melanin. This finding is of great importance for the diagnosis and/or treatment of malignant melanoma, a tumour whose worldwide incidence is continuously growing. Methods The characteristics of the instrument are briefly described and an example of application is given. This example deals with the intracellular localization of an iodo-benzamide used as a diagnostic tool for the scintigraphic detection of melanic cells (e.g. metastasis of malignant melanoma. B16 melanoma cells were injected intravenously to C57BL6/J1/co mice. Multiple B16 melanoma colonies developed in the lungs of treated animals within three weeks. Iodobenzamide was injected intravenously in tumour bearing mice six hours before sacrifice. Small pieces of lung were prepared for SIMS analysis. Results Mouse lung B16 melanoma colonies were observed with high lateral resolution. Cyanide ions gave "histological" images of the cell, representative of the distribution of C and N containing molecules (e.g. proteins, nucleic acids, melanin, etc. while phosphorus ions are mainly produced by nucleic acids. Iodine was detected only in melanosomes, confirming the specific affinity of the drug for melanin. No drug was found in normal lung tissue. Conclusion This study demonstrates the potential of SIMS microscopy, which allows the

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

Science.gov (United States)

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 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. PMID:21645404

Novel (1E,3E,5E-1,6-bis(Substituted phenylhexa-1,3,5-triene Analogs Inhibit Melanogenesis in B16F10 Cells and Zebrafish

Directory of Open Access Journals (Sweden)

Jisun Oh

2018-04-01

Full Text Available The present study aimed to evaluate the anti-melanogenic activity of 1,6-diphenyl-1,3,5-hexatriene and its derivatives in B16F10 murine melanoma cells and zebrafish embryos. Twenty five (1E,3E,5E-1,6-bis(substituted phenylhexa-1,3,5-triene analogs were synthesized and their non-cytotoxic effects were predictively analyzed using three-dimensional quantitative structure-activity relationship approach. Inhibitory activities of these synthetic compounds against melanin synthesis were determined by evaluating melanin content and melanogenic regulatory enzyme expression in B16F10 cells. The anti-melanogenic activity was verified by observing body pigmentation in zebrafishes treated with these compounds. Compound #2, #4, and #6 effectively decreased melanogenesis induced by α-melanocyte-stimulating hormone. In particular, compound #2 remarkably lowered the mRNA and protein expression levels of microphthalmia-associated transcription factor (MITF, tyrosinase (TYR, tyrosinase-related protein 1 (TYRP1, and TYRP2 in B16F10 cells and substantially reduced skin pigmentation in the developed larvae of zebrafish. These findings suggest that compound #2 may be used as an anti-melanogenic agent for cosmetic purpose.

Bioconjugation of laminin peptide YIGSR with poly(styrene co-maleic acid) increases its antimetastatic effect on lung metastasis of B16-BL6 melanoma cells.

Science.gov (United States)

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.

miR-125b induces cellular senescence in malignant melanoma

DEFF Research Database (Denmark)

Nyholm, Anne Marie; Lerche, Catharina M; Manfé, Valentina

2014-01-01

transfected melanoma cell line Mel-Juso and then investigated the effect of the presence of a stable overexpression of miR-125b on growth by western blotting, flow cytometry and β-galactosidase staining. The tumourogenicity of the transfected cells was tested using a murine model and the tumours were further...... examined with in-situ-hybridization. RESULTS: In primary human tumours and in lymph node metastases increased expression of miR-125b was found in single, large tumour cells with abundant cytoplasm. A stable overexpression of miR-125b in human melanoma cell line Mel-Juso resulted in a G0/G1 cell cycle block...... and emergence of large cells expressing senescence markers: senescence-associated beta-galactosidase, p21, p27 and p53. Mel-Juso cells overexpressing miR-125b were tumourigenic in mice, but the tumours exhibited higher level of cell senescence and decreased expression of proliferation markers, cyclin D1 and Ki...

[Imiquimod combined with dendritic cell vaccine decreases Treg proportion and enhances anti-tumor responses in mice bearing melanoma].

Science.gov (United States)

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.

Gene silencing of beta-catenin in melanoma cells retards their growth but promotes the formation of pulmonary metastasis in mice.

Science.gov (United States)

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.

Suppression of adhesion-induced protein tyrosine phosphorylation decreases invasive and metastatic potentials of B16-BL6 melanoma cells by protein tyrosine kinase inhibitor genistein.

Science.gov (United States)

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.

Fucoidan from Sargassum sp. and Fucus vesiculosus reduces cell viability of lung carcinoma and melanoma cells in vitro and activates natural killer cells in mice in vivo

DEFF Research Database (Denmark)

Ale, Marcel Tutor; Maruyama, Hiroko; Tamauchi, Hidekazu

2011-01-01

Fucoidan is known to exhibit crucial biological activities, including anti-tumor activity. In this study, we examined the influence of crude fucoidan extracted from Sargassum sp. (MTA) and Fucus vesiculosus (SIG) on Lewis lung carcinoma cells (LCC) and melanoma B16 cells (MC). In vitro studies we...

Gelsolin functions as a metastasis suppressor in B16-BL6 mouse melanoma cells and requirement of the carboxyl-terminus for its effect.

Science.gov (United States)

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.

NKT cells act through third party bone marrow-derived cells to suppress NK cell activity in the liver and exacerbate hepatic melanoma metastases.

Science.gov (United States)

Sadegh, Leila; Chen, Peter W; Brown, Joseph R; Han, Zhiqiang; Niederkorn, Jerry Y

2015-09-01

Uveal melanoma (UM) is the most common intraocular tumor in adults and liver metastasis is the leading cause of death in UM patients. We have previously shown that NKT cell-deficient mice develop significantly fewer liver metastases from intraocular melanomas than do wild-type (WT) mice. Here, we examine the interplay between liver NKT cells and NK cells in resistance to liver metastases from intraocular melanomas. NKT cell-deficient CD1d(-/-) mice and WT C57BL/6 mice treated with anti-CD1d antibody developed significantly fewer liver metastases than WT mice following either intraocular or intrasplenic injection of B16LS9 melanoma cells. The increased number of metastases in WT mice was associated with reduced liver NK cytotoxicity and decreased production of IFN-γ. However, liver NK cell-mediated cytotoxic activity was identical in non-tumor bearing NKT cell-deficient mice and WT mice, indicating that liver metastases were crucial for the suppression of liver NK cells. Depressed liver NK cytotoxicity in WT mice was associated with production of IL-10 by bone marrow-derived liver cells that were neither Kupffer cells nor myeloid-derived suppressor cells and by increased IL-10 receptor expression on liver NK cells. IL-10(-/-) mice had significantly fewer liver metastases than WT mice, but were not significantly different from NKT cell-deficient mice. Thus, development of melanoma liver metastases is associated with upregulation of IL-10 in the liver and an elevated expression of IL-10 receptor on liver NK cells. This impairment of liver NK activity is NKT cell-dependent and only occurs in hosts with melanoma liver metastases. © 2015 UICC.

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

Science.gov (United States)

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

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

Directory of Open Access Journals (Sweden)

Patricia Dillenburg-Pilla

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

Pro-inflammatory cytokine/chemokine production by reovirus treated melanoma cells is PKR/NF-κB mediated and supports innate and adaptive anti-tumour immune priming

Directory of Open Access Journals (Sweden)

Coffey Matt

2011-02-01

Full Text Available Abstract Background As well as inducing direct oncolysis, reovirus treatment of melanoma is associated with activation of innate and adaptive anti-tumour immune responses. Results Here we characterise the effects of conditioned media from reovirus-infected, dying human melanoma cells (reoTCM, in the absence of live virus, to address the immune bystander potential of reovirus therapy. In addition to RANTES, IL-8, MIP-1α and MIP-1β, reovirus-infected melanoma cells secreted eotaxin, IP-10 and the type 1 interferon IFN-β. To address the mechanisms responsible for the inflammatory composition of reoTCM, we show that IL-8 and IFN-β secretion by reovirus-infected melanoma cells was associated with activation of NF-κB and decreased by pre-treatment with small molecule inhibitors of NF-κB and PKR; specific siRNA-mediated knockdown further confirmed a role for PKR. This pro-inflammatory milieu induced a chemotactic response in isolated natural killer (NK cells, dendritic cells (DC and anti-melanoma cytotoxic T cells (CTL. Following culture in reoTCM, NK cells upregulated CD69 expression and acquired greater lytic potential against tumour targets. Furthermore, melanoma cell-loaded DC cultured in reoTCM were more effective at priming adaptive anti-tumour immunity. Conclusions These data demonstrate that the PKR- and NF-κB-dependent induction of pro-inflammatory molecules that accompanies reovirus-mediated killing can recruit and activate innate and adaptive effector cells, thus potentially altering the tumour microenvironment to support bystander immune-mediated therapy as well as direct viral oncolysis.

Inhibition of CSF-1R supports T-cell mediated melanoma therapy.

Directory of Open Access Journals (Sweden)

Marjolein Sluijter

Full Text Available Tumor associated macrophages (TAM can promote angiogenesis, invasiveness and immunosuppression. The cytokine CSF-1 (or M-CSF is an important factor of TAM recruitment and differentiation and several pharmacological agents targeting the CSF-1 receptor (CSF-1R have been developed to regulate TAM in solid cancers. We show that the kinase inhibitor PLX3397 strongly dampened the systemic and local accumulation of macrophages driven by B16F10 melanomas, without affecting Gr-1(+ myeloid derived suppressor cells. Removal of intratumoral macrophages was remarkably efficient and a modest, but statistically significant, delay in melanoma outgrowth was observed. Importantly, CSF-1R inhibition strongly enhanced tumor control by immunotherapy using tumor-specific CD8 T cells. Elevated IFNγ production by T cells was observed in mice treated with the combination of PLX3397 and immunotherapy. These results support the combined use of CSF-1R inhibition with CD8 T cell immunotherapy, especially for macrophage-stimulating tumors.

Cure of malignant melanoma by single thermal neutron capture treatment using melanoma-seeking compounds

International Nuclear Information System (INIS)

Mishima, Yutaka; Ichihashi, Masamitsu; Nakanishi, Takafumi

1985-01-01

Since not only malignant melanomas but also many kinds of human cancers, for example thyroid cancer and squamous cell carcinoma, synthesize their specific protein, much attention has been paid to the establishment of selective thermal neutron capture treatment of malignant melanoma as a prototype of such cancer cells. This paper presents 10 B chlorpromazine compounds and 10 B 1 -para-boronophenylalanine ( 10 B 1 -BPA) as tumor-seeking 10 B compounds which themselves possess selective affinity for the specific metabolic activity of the target cancer cells. An overview of the following studies on the effects of 10 B 1 -BPA in the thermal neutron capture treatment of melanoma is provided: 1) in vitro studies on specific enhanced melanoma cell killing effects of 10 B 1 -BPA; 2) in vivo studies on therapeutic effects of 10 B 1 -BPA using melanoma-bearing hamsters; and 3) preclinical therapeutic experiments using spontaneously occurring malignant melanoma in Duroc pig skin, including experiments in which melanoma was successfully cured. (Namekawa, K.)

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

International Nuclear Information System (INIS)

Nie, Yunzhong; Du, Leilei; Mou, Yongbin; Xu, Zhenjun; Weng, Leihua; Du, Youwei; Zhu, Yanan; Hou, Yayi; Wang, Tingting

2013-01-01

We previously found that the low frequency magnetic fields (LF-MF) inhibited gastric and lung cancer cell growth. We suppose that exposure to LF-MF may modulate immune function so as to inhibit tumor. We here investigated whether LF-MF can inhibit the proliferation and metastasis of melanoma and influence immune function. The effect of MF on the proliferation, cell cycle and ultrastracture of B16-F10 in vitro was detected by cell counting Kit-8 assay, flow cytometry, and transmission electron microscopy. Lung metastasis mice were prepared by injection of 2 × 10 5 B16-F10 melanoma cells into the tail vein in C57BL/6 mice. The mice were then exposed to an LF-MF (0.4 T, 7.5 Hz) for 43 days. Survival rate, tumor markers and the innate and adaptive immune parameters were measured. The growth of B16-F10 cells was inhibited after exposure to the LF-MF. The inhibition was related to induction of cell cycle arrest and decomposition of chromatins. Moreover, the LF-MF prolonged the mouse survival rate and inhibited the proliferation of B16-F10 in melanoma metastasis mice model. Furthermore, the LF-MF modulated the immune response via regulation of immune cells and cytokine production. In addition, the number of Treg cells was decreased in mice with the LF-MF exposure, while the numbers of T cells as well as dendritic cells were significantly increased. LF-MF inhibited the growth and metastasis of melanoma cancer cells and improved immune function of tumor-bearing mice. This suggests that the inhibition may be attributed to modulation of LF-MF on immune function and LF-MF may be a potential therapy for treatment of melanoma

Carbon ions of different linear energy transfer (LET values induce apoptosis & G2 cell cycle arrest in radio-resistant melanoma cells

Directory of Open Access Journals (Sweden)

Žakula Jelena

2016-01-01

Full Text Available Background & objectives: The main goal when treating malignancies with radiation is to deprive tumour cells of their reproductive potential. One approach is to induce tumour cell apoptosis. This study was conducted to evaluate the ability of carbon ions ( [12] C to induce apoptosis and cell cycle arrest in human HTB140 melanoma cells. Methods: In this in vitro study, human melanoma HTB140 cells were irradiated with the 62 MeV/n carbon ( [12] C ion beam, having two different linear energy transfer (LET values: 197 and 382 keV/μm. The dose range was 2 to 16 Gy. Cell viability was estimated by the sulforhodamine B assay seven days after irradiation. The cell cycle and apoptosis were evaluated 48 h after irradiation using flow cytometry. At the same time point, protein and gene expression of apoptotic regulators were estimated using the Western blot and q-PCR methods, respectively. Results: Cell viability experiments indicated strong anti-tumour effects of [12] C ions. The analysis of cell cycle showed that [12] C ions blocked HTB140 cells in G2 phase and induced the dose dependent increase of apoptosis. The maximum value of 21.8 per cent was attained after irradiation with LET of 197 keV/μm at the dose level of 16 Gy. Pro-apoptotic effects of [12] C ions were confirmed by changes of key apoptotic molecules: the p53, Bax, Bcl-2, poly ADP ribose polymerase (PARP as well as nuclear factor kappa B (NFκB. At the level of protein expression, the results indicated significant increases of p53, NFκB and Bax/Bcl-2 ratio and PARP cleavage. The Bax/Bcl-2 mRNA ratio was also increased, while no change was detected in the level of NFκB mRNA. Interpretation & conclusions: The present results indicated that anti-tumour effects of [12] C ions in human melanoma HTB140 cells were accomplished through induction of the mitochondrial apoptotic pathway as well as G2 arrest.

Phosphatase of Regenerating Liver-3 Promotes Motility and Metastasis of Mouse Melanoma Cells

Science.gov (United States)

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

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

Directory of Open Access Journals (Sweden)

Katherine Biron-Pain

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

Effects of taurolidine on radiosensitivity of murine melanoma cells and its mechanism

International Nuclear Information System (INIS)

Sun Baosheng; Liu Shixin; Wang Tiejun; Liu Linlin; Huang Guomin; Gong Shouliang

2008-01-01

Objective: To observe the effects of taurolidine on radiosensitivity of B16-F10 cells of murine melanoma via the enhancement of Bax and Bad proteins and induction of Bcl-2 protein. Methods: The apoptosis of B16-F10 cells was assessed after treated with 0, 10, 25, 50, 100 and 150 μmol·L -1 taurolidine, clone survival assay was used to detect the radiosensitivity of B16-F10 cells, and protein expressions were determined by Western blotting. Results: The apoptosis of 5% cells was induced in a dose-and time-dependent manner after B16-F10 cells were treated with 50 μmol·L -1 taurolidine. The survival rate decreased after treated with tautolidine in combination with 2 Gy X-irradiation with the increase of taurolidine concentration and doses of irradiation (P 0 and SER D q ) also increased with the increase of its concentration, there was significant difference between 50 μmol·L -1 taurolidine group and 10 μmol·L -1 taurolidine group (P<0.05); meantime, the level of proapototic protein Bax and Bad increased and the level of antiapoptotic protein Bcl-2 reduced. Conclusion: Taurolidine in combination with irradiation can enhance the radiosensitivity by the mediation of Bcl-2 family protein. (authors)

Pratol, an O-Methylated Flavone, Induces Melanogenesis in B16F10 Melanoma Cells via p-p38 and p-JNK Upregulation

Directory of Open Access Journals (Sweden)

You Chul Chung

2017-10-01

Full Text Available Tyrosinase is the rate-limiting enzyme critical for melanin synthesis. It controls pigmentation in the skin. Activation of tyrosinase is currently the most common approach in the development of tanning and haircare products. Pratol is a 7-hydroxy-4-methoxyflavone found in Trifolium pratense. In this study, we investigated the effects of pratol on melanogenesis. We also studied the mechanism of action of pratol in B16F10 mouse melanoma cells. The cells were treated with various concentrations (6.25, 12.5, 25, and 50 μM of pratol to observe its effects. The results showed that pratol significantly increased melanin content and tyrosinase activity in the cells without being cytotoxic. In addition, pratol strongly increased the expression of tyrosinase and tyrosinase-related protein-1 and 2 by enhancing the expression of microphthalmia-associated transcription factor. Furthermore, pratol stimulated melanogenesis via the phosphorylation of p38, c-Jun N-terminal kinases (JNK, and extracellular signal–regulated kinase (ERK. The findings from an assay searching for the inhibitor revealed that SB203580 (a specific p38 inhibitor or SP600125 (a p-JNK inhibitor attenuated pratol-induced cellular tyrosinase activity whereas PD98059 (an ERK inhibitor did not. Additionally, pratol interfered with the phosphorylation of p-AKT. We also found that pratol-induced melanogenesis was reversed by H89, which is a specific protein kinase A inhibitor. The results suggest that, owing to its multi-functional properties, pratol may be a potential tanning agent or a therapeutic agent for hair depigmentation in the cosmetic industry.

Natural compounds' activity against cancer stem-like or fast-cycling melanoma cells.

Directory of Open Access Journals (Sweden)

Malgorzata Sztiller-Sikorska

Full Text Available BACKGROUND: Accumulating evidence supports the concept that melanoma is highly heterogeneous and sustained by a small subpopulation of melanoma stem-like cells. Those cells are considered as responsible for tumor resistance to therapies. Moreover, melanoma cells are characterized by their high phenotypic plasticity. Consequently, both melanoma stem-like cells and their more differentiated progeny must be eradicated to achieve durable cure. By reevaluating compounds in heterogeneous melanoma populations, it might be possible to select compounds with activity not only against fast-cycling cells but also against cancer stem-like cells. Natural compounds were the focus of the present study. METHODS: We analyzed 120 compounds from The Natural Products Set II to identify compounds active against melanoma populations grown in an anchorage-independent manner and enriched with cells exerting self-renewing capacity. Cell viability, cell cycle arrest, apoptosis, gene expression, clonogenic survival and label-retention were analyzed. FINDINGS: Several compounds efficiently eradicated cells with clonogenic capacity and nanaomycin A, streptonigrin and toyocamycin were effective at 0.1 µM. Other anti-clonogenic but not highly cytotoxic compounds such as bryostatin 1, siomycin A, illudin M, michellamine B and pentoxifylline markedly reduced the frequency of ABCB5 (ATP-binding cassette, sub-family B, member 5-positive cells. On the contrary, treatment with maytansine and colchicine selected for cells expressing this transporter. Maytansine, streptonigrin, toyocamycin and colchicine, even if highly cytotoxic, left a small subpopulation of slow-dividing cells unaffected. Compounds selected in the present study differentially altered the expression of melanocyte/melanoma specific microphthalmia-associated transcription factor (MITF and proto-oncogene c-MYC. CONCLUSION: Selected anti-clonogenic compounds might be further investigated as potential adjuvants

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

Science.gov (United States)

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

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

International Nuclear Information System (INIS)

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.

2015-01-01

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

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.

Science.gov (United States)

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

The Standardized Extract of Juniperus communis Alleviates Hyperpigmentation in Vivo HRM-2 Hairless Mice and in Vitro Murine B16 Melanoma Cells.

Science.gov (United States)

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.

Expression of human papilloma virus type 16 E5 protein in amelanotic melanoma cells regulates endo-cellular pH and restores tyrosinase activity

Directory of Open Access Journals (Sweden)

Coccia Raffaella

2009-01-01

Full Text Available Abstract Background Melanin synthesis, the elective trait of melanocytes, is regulated by tyrosinase activity. In tyrosinase-positive amelanotic melanomas this rate limiting enzyme is inactive because of acidic endo-melanosomal pH. The E5 oncogene of the Human Papillomavirus Type 16 is a small transmembrane protein with a weak transforming activity and a role during the early steps of viral infections. E5 has been shown to interact with 16 kDa subunit C of the trans-membrane Vacuolar ATPase proton pump ultimately resulting in its functional suppressions. However, the cellular effects of such an interaction are still under debate. With this work we intended to explore whether the HPV16 E5 oncoprotein does indeed interact with the vacuolar ATPase proton pump once expressed in intact human cells and whether this interaction has functional consequences on cell metabolism and phenotype. Methods The expression of the HPV16-E5 oncoproteins was induced in two Tyrosinase-positive amelanotic melanomas (the cell lines FRM and M14 by a retroviral expression construct. Modulation of the intracellular pH was measured with Acridine orange and fluorescence microscopy. Expression of tyrosinase and its activity was followed by RT-PCR, Western Blot and enzyme assay. The anchorage-independence growth and the metabolic activity of E5 expressing cells were also monitored. Results We provide evidence that in the E5 expressing cells interaction between E5 and V-ATPase determines an increase of endo-cellular pH. The cellular alkalinisation in turn leads to the post-translational activation of tyrosinase, melanin synthesis and phenotype modulation. These effects are associated with an increased activation of tyrosine analogue anti-blastic drugs. Conclusion Once expressed within intact human cells the HPV16-E5 oncoprotein does actually interact with the vacuolar V-ATPase proton pump and this interaction induces a number of functional effects. In amelanotic melanomas these

Regulatory T cells and other lymphocyte subpopulations in patients with melanoma developing interferon-induced thyroiditis during high-dose interferon-α2b treatment.

Science.gov (United States)

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.

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

International Nuclear Information System (INIS)

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

2004-01-01

Our previous studies demonstrated that a synthetic peptide encompassing residues 185-203 of the noncollagenous (NC1) domain of the α3 chain of type IV collagen, named tumstatin, inhibits in vitro melanoma cell proliferation and migration. In the present study, B16F1 melanoma cells were stably transfected to overexpress the complete tumstatin domain (Tum 1-232) or its C-terminal part, encompassing residues 185-203 (Tum 183-232). Tumstatin domain overexpression inhibited B16F1 in vitro cell proliferation, anchorage-independent growth, and invasive properties. For studying the in vivo effect of overexpression, representative clones were subcutaneously injected into the left side of C57BL6 mice. In vivo tumor growth was decreased by -60% and -56%, respectively, with B16F1 cells overexpressing Tum 1-232 or Tum 183-232 compared to control cells. This inhibitory effect was associated with a decrease of in vivo cyclin D1 expression. We also demonstrated that the overexpression of Tum 1-232 or Tum 183-232 induced an in vivo down-regulation of proteolytic cascades involving matrix metalloproteinases (MMPs), especially the production or activation of MMP-2, MMP-9, MMP-13, as well as MMP-14. The plasminogen activation system was also altered in tumors with a decrease of urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) and a strong increase of plasminogen activator inhibitor-1 (PAI-1). Collectively, our results demonstrate that tumstatin or its C-terminal antitumor fragment, Tum 183-232, inhibits in vivo melanoma progression by triggering an intracellular transduction pathway, which involves a cyclic AMP (cAMP)-dependent mechanism

Downregulation of miR-125b in metastatic cutaneous malignant melanoma.

Science.gov (United States)

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.

Noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma.

Science.gov (United States)

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.

Effect of blocking TNF on IL-6 levels and metastasis in a B16-BL6 melanoma/mouse model.

Science.gov (United States)

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.

Imaging malignant melanoma with {sup 18}F-5-FPN

Energy Technology Data Exchange (ETDEWEB)

Feng, Hongyan; Xia, Xiaotian; Li, Chongjiao; Song, Yiling; Qin, Chunxia; Liu, Qingyao; Zhang, Yongxue; Lan, Xiaoli [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (China); Hubei Key Laboratory of Molecular Imaging (China)

2016-01-15

Radiolabelled benzamides are attractive candidates for targeting melanoma because they bind to melanin and exhibit high tumour uptake and retention. {sup 18}F-5-Fluoro-N-(2-[diethylamino]ethyl)picolinamide ({sup 18}F-5-FPN), a benzamide analogue, was prepared and its pharmacokinetics and binding affinity evaluated both in vitro and in vivo to assess its clinical potential in the diagnosis and staging of melanoma. {sup 18}F-5-FPN was prepared and purified. Its binding specificity was measured in vitro in two different melanoma cell lines, one pigmented (B16F10 cells) and one nonpigmented (A375m cells), and in vivo in mice xenografted with the same cell lines. Dynamic and static PET images using {sup 18}F-5-FPN were obtained in the tumour-bearing mice, and the static images were also compared with those acquired with {sup 18}F-FDG. PET imaging with {sup 18}F-5-FPN was also performed in B16F10 tumour-bearing mice with lung metastases. {sup 18}F-5-FPN was successfully prepared with radiochemical yields of 5 - 10 %. Binding of {sup 18}F-5-FPN to B16F10 cells was much higher than to A375m cells. On dynamic PET imaging B16F10 tumours were visible about 1 min after injection of the tracer, and the uptake gradually increased over time. {sup 18}F-5-FPN was rapidly excreted via the kidneys. B16F10 tumours were clearly visible on static images acquired 1 and 2 h after injection, with high uptake values of 24.34 ± 6.32 %ID/g and 16.63 ± 5.41 %ID/g, respectively, in the biodistribution study (five mice). However, there was no visible uptake by A375m tumours. {sup 18}F-5-FPN and {sup 18}F-FDG PET imaging were compared in B16F10 tumour xenografts, and the tumour-to-background ratio of {sup 18}F-5-FPN was ten times higher than that of {sup 18}F-FDG (35.22 ± 7.02 vs. 3.29 ± 0.53, five mice). {sup 18}F-5-FPN PET imaging also detected simulated lung metastases measuring 1 - 2 mm. {sup 18}F-5-FPN specifically targeted melanin in vitro and in vivo with high retention and affinity

Resveratrol prevents endothelial cells injury in high-dose interleukin-2 therapy against melanoma.

Directory of Open Access Journals (Sweden)

Hongbing Guan

Full Text Available Immunotherapy with high-dose interleukin-2 (HDIL-2 is an effective treatment for patients with metastatic melanoma and renal cell carcinoma. However, it is accompanied by severe toxicity involving endothelial cell injury and induction of vascular leak syndrome (VLS. In this study, we found that resveratrol, a plant polyphenol with anti-inflammatory and anti-cancer properties, was able to prevent the endothelial cell injury and inhibit the development of VLS while improving the efficacy of HDIL-2 therapy in the killing of metastasized melanoma. Specifically, C57BL/6 mice were injected with B16F10 cells followed by resveratrol by gavage the next day and continued treatment with resveratrol once a day. On day 9, mice received HDIL-2. On day 12, mice were evaluated for VLS and tumor metastasis. We found that resveratrol significantly inhibited the development of VLS in lung and liver by protecting endothelial cell integrity and preventing endothelial cells from undergoing apoptosis. The metastasis and growth of the tumor in lung were significantly inhibited by HDIL-2 and HDIL-2 + resveratrol treatment. Notably, HDIL-2 + resveratrol co-treatment was more effective in inhibiting tumor metastasis and growth than HDIL-2 treatment alone. We also analyzed the immune status of Gr-1(+CD11b(+ myeloid-derived suppressor cells (MDSC and FoxP3(+CD4(+ regulatory T cells (Treg. We found that resveratrol induced expansion and suppressive function of MDSC which inhibited the development of VLS after adoptive transfer. However, resveratrol suppressed the HDIL-2-induced expansion of Treg cells. We also found that resveratrol enhanced the susceptibility of melanoma to the cytotoxicity of IL-2-activated killer cells, and induced the expression of the tumor suppressor gene FoxO1. Our results suggested the potential use of resveratrol in HDIL-2 treatment against melanoma. We also demonstrated, for the first time, that MDSC is the dominant suppressor cell than regulatory

Characterization and Inducing Melanoma Cell Apoptosis Activity of Mannosylerythritol Lipids-A Produced from Pseudozyma aphidis.

Directory of Open Access Journals (Sweden)

Linlin Fan

Full Text Available Mannosylerythritol lipids (MELs are natural glycolipid biosurfactants which have potential applications in the fields of food, cosmetic and medicine. In this study, MELs were produced from vegetable oil by Pseudozyma aphidis. Their structural data through LC/MS, GC/MS and NMR analysis revealed that MEL-A with two acetyls was the major compound and the identified homologs of MEL-A contained a length of C8 to C14 fatty acid chains. This glycolipid exhibited a surface tension of 27.69 mN/m at a critical micelle concentration (CMC, self-assembling into particles in the water solution. It was observed to induce cell growth-inhibition and apoptosis of B16 melanoma cells in a dose-dependent manner, as well as cause cell cycle arrest at the S phase. Further quantitative RT-PCR analysis and western blotting revealed an increasing tendency of both mRNA and protein expressions of Caspase-12, CHOP, GRP78 and Caspase-3, and a down-regulation of protein Bcl-2. Combined with the up regulation of signaling IRE1 and ATF6, it can be speculated that MEL-A-induced B16 melanoma cell apoptosis was associated with the endoplasmic reticulum stress (ERS.

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

Science.gov (United States)

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.

Loss of retrovirus production in JB/RH melanoma cells transfected with H-2Kb and TAP-1 genes.

Science.gov (United States)

Li, M; Xu, F; Muller, J; Huang, X; Hearing, V J; Gorelik, E

1999-01-20

JB/RH1 melanoma cells, as well as other melanomas of C57BL/6 mice (B16 and JB/MS), express a common melanoma-associated antigen (MAA) encoded by an ecotropic melanoma-associated retrovirus (MelARV). JB/RH1 cells do not express the H-2Kb molecules due to down-regulation of the H-2Kb and TAP-1 genes. When JB/RH1 cells were transfected with the H-2Kb and cotransfected with the TAP-1 gene, it resulted in the appearance of H-2Kb molecules and an increase in their immunogenicity, albeit they lost expression of retrovirus-encoded MAA recognized by MM2-9B6 mAb. Loss of MAA was found to result from a complete and stable elimination of ecotropic MelARV production in the H-2Kb/TAP-1-transfected JB/RH1 cells. Northern blot analysis showed no differences in ecotropic retroviral messages in MelARV-producing and -nonproducing melanoma cells, suggesting that loss of MelARV production was not due to down-regulation of MelARV transcription. Southern blot analysis revealed several rearrangements in the proviral DNA of H-2Kb-positive JB/RH1 melanoma cells. Sequence analysis of the ecotropic proviral DNA from these cells showed numerous nucleotide substitutions, some of which resulted in the appearance of a novel intraviral PstI restriction site and the loss of a HindIII restriction site in the pol region. PCR amplification of the proviral DNAs indicates that an ecotropic provirus found in the H-2Kb-positive cells is novel and does not preexist in the parental H-2Kb-negative melanoma cells. Conversely, the ecotropic provirus of the parental JB/RH1 cells was not amplifable from the H-2Kb-positive cells. Our data indicate that stable loss of retroviral production in the H-2Kb/TAP-1-transfected melanoma cells is probably due to the induction of recombination between a productive ecotropic MelARV and a defective nonecotropic provirus leading to the generation of a defective ecotropic provirus and the loss of MelARV production and expression of the retrovirus-encoded MAA. Copyright 1999

IGF-1 contributes to the expansion of melanoma-initiating cells through an epithelial-mesenchymal transition process.

Science.gov (United States)

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.

Engagement of αIIbβ3 (GPIIb/IIIa) with ανβ3 Integrin Mediates Interaction of Melanoma Cells with Platelets: A CONNECTION TO HEMATOGENOUS METASTASIS*

OpenAIRE

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.

2011-01-01

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 endothel...

Development of radioiodine-labeled 4-hydroxyphenylcysteamine for specific diagnosis of malignant melanoma

International Nuclear Information System (INIS)

Kobayashi, Masato; Nishii, Ryuichi; Shikano, Naoto; Flores, Leo G.; Mizutani, Asuka; Ogai, Kazuhiro; Sugama, Jyunko; Nagamachi, Shigeki; Kawai, Keiichi

2015-01-01

Introduction: A specific diagnosis for melanoma is strongly desired because malignant melanoma has poor prognosis. In a previous study, although radioiodine-125-labeled 4-hydroxyphenyl-L-cysteine ( 125 I-L-PC) was found to have good substrate affinity for tyrosinase enzyme in the melanin metabolic pathway, 123/131 I-L-PC had insufficient substrate affinity for tyrosinase to diagnose melanoma. In this study, we synthesized 4-hydroxyphenylcysteamine (4-PCA) and developed a novel radioiodine-125-labeled 4-hydroxyphenylcysteamine ( 125 I-PCA) to increase affinity for the melanin biosynthesis pathway. Methods: 4-PCA was separated with 2-hydroxyphenylcysteamine (2-PCA), which is an isomer of 4-PCA, and was examined using melting point, proton nuclear magnetic resonance, mass spectrometry and elemental analysis. 125 I-PCA was prepared using the chloramine-T method under no-carrier added conditions. We performed biodistribution experiments using B16 melanoma-bearing mice using 125 I-PCA, 125 I-L-PC, 125 I-α-methyl-L-tyrosine, 123 I-m-iodobenzylguanidine and 67 Ga-citrate. In vitro assay was performed with B16 melanoma cells, and affinity for tyrosinase, DNA polymerase and amino acid transport was evaluated using phenylthiourea, thymidine, ouabine and L-tyrosine inhibitor. In addition, partition coefficients of 125 I-PCA were evaluated. Results: In the synthesis of 4-PCA, analysis values did not differ between calculated and reported values, and 4-PCA was separated from 2-PCA at high purity. In biodistribution experiments, 125 I-PCA was accumulated and retained in B16 melanoma cells when compared with 125 I-L-PC. 125 I-PCA showed the highest values at 60 min after radiotracer injection in melanoma-to-muscle ratios, melanoma-to-blood ratios and melanoma-to-skin ratios. Accumulation of 125 I-PCA was significantly inhibited by phenylthiourea and thymidine. Partition coefficients of 125 I-PCA were lower than those of N-isopropyl-p-[ 123 I]iodoamphetamine and were not

The antimicrobial peptide nisin Z induces selective toxicity and apoptotic cell death in cultured melanoma cells.

Science.gov (United States)

Lewies, Angélique; Wentzel, Johannes Frederik; Miller, Hayley Christy; Du Plessis, Lissinda Hester

2018-01-01

Reprogramming of cellular metabolism is now considered one of the hallmarks of cancer. Most malignant cells present with altered energy metabolism which is associated with elevated reactive oxygen species (ROS) generation. This is also evident for melanoma, the leading cause of skin cancer related deaths. Altered mechanisms affecting mitochondrial bioenergetics pose attractive targets for novel anticancer therapies. Antimicrobial peptides have been shown to exhibit selective anticancer activities. In this study, the anti-melanoma potential of the antimicrobial peptide, nisin Z, was evaluated in vitro. Nisin Z was shown to induce selective toxicity in melanoma cells compared to non-malignant keratinocytes. Furthermore, nisin Z was shown to negatively affect the energy metabolism (glycolysis and mitochondrial respiration) of melanoma cells, increase reactive oxygen species generation and cause apoptosis. Results also indicate that nisin Z can decrease the invasion and proliferation of melanoma cells demonstrating its potential use against metastasis associated with melanoma. As nisin Z seems to place a considerable extra burden on the energy metabolism of melanoma cells, combination therapies with known anti-melanoma agents may be effective treatment options. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

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

Science.gov (United States)

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

Impact of sentinel lymphadenectomy on survival in a murine model of melanoma.

Science.gov (United States)

Rebhun, Robert B; Lazar, Alexander J F; Fidler, Isaiah J; Gershenwald, Jeffrey E

2008-01-01

Lymphatic mapping and sentinel lymph node biopsy-also termed sentinel lymphadenectomy (SL)-has become a standard of care for patients with primary invasive cutaneous melanoma. This technique has been shown to provide accurate information about the disease status of the regional lymph node basins at risk for metastasis, provide prognostic information, and provide durable regional lymph node control. The potential survival benefit afforded to patients undergoing SL is controversial. Central to this controversy is whether metastasis to regional lymph nodes occurs independent of or prior to widespread hematogenous dissemination. A related area of uncertainty is whether tumor cells residing within regional lymph nodes have increased metastatic potential. We have used a murine model of primary invasive cutaneous melanoma based on injection of B16-BL6 melanoma cells into the pinna to address two questions: (1) does SL plus wide excision of the primary tumor result in a survival advantage over wide excision alone; and (2) do melanoma cells growing within lymph nodes produce a higher incidence of hematogenous metastases than do cells growing at the primary tumor site? We found that SL significantly improved the survival of mice with small primary tumors. We found no difference in the incidence of lung metastases produced by B16-BL6 melanoma cells growing exclusively within regional lymph nodes and cells growing within the pinna.

The fibrinolytic system facilitates tumor cell migration across the blood-brain barrier in experimental melanoma brain metastasis

International Nuclear Information System (INIS)

Perides, George; Zhuge, Yuzheng; Lin, Tina; Stins, Monique F; Bronson, Roderick T; Wu, Julian K

2006-01-01

Patients with metastatic tumors to the brain have a very poor prognosis. Increased metastatic potential has been associated with the fibrinolytic system. We investigated the role of the fibrinolytic enzyme plasmin in tumor cell migration across brain endothelial cells and growth of brain metastases in an experimental metastatic melanoma model. Metastatic tumors to the brain were established by direct injection into the striatum or by intracarotid injection of B16F10 mouse melanoma cells in C57Bl mice. The role of plasminogen in the ability of human melanoma cells to cross a human blood-brain barrier model was studied on a transwell system. Wild type mice treated with the plasmin inhibitor epsilon-aminocaproic acid (EACA) and plg -/- mice developed smaller tumors and survived longer than untreated wild type mice. Tumors metastasized to the brain of wild type mice treated with EACA and plg -/- less efficiently than in untreated wild type mice. No difference was observed in the tumor growth in any of the three groups of mice. Human melanoma cells were able to cross the human blood-brain barrier model in a plasmin dependent manner. Plasmin facilitates the development of tumor metastasis to the brain. Inhibition of the fibrinolytic system could be considered as means to prevent tumor metastasis to the brain

Nestin is expressed in HMB-45 negative melanoma cells in dermal parts of nodular melanoma.

Science.gov (United States)

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.

The in-vitro and in-vivo inhibitory activity of biflorin in melanoma.

Science.gov (United States)

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.

Evaluation of radiation-induced genotoxicity on human melanoma cells (SK-MEL-37) by flow cytometry

International Nuclear Information System (INIS)

Bonfim, Leticia; Carvalho, Luma Ramirez de; Vieira, Daniel Perez

2017-01-01

Micronucleus assay is a test used to evaluate genotoxic damage in cells, which can be caused by various factors, like ionizing radiation. Interactions between radiation energies and DNA can cause breakage, leading to use chromosomal mutations or loss of genetic material, important events that could be induced in solid tumors to mitigate its expansion within human body. Melanoma has been described as a tumor with increased radio resistance. This work evaluated micronuclei percentages (%MN) in human melanoma cells (SK-MEL-37), irradiated by gamma radiation, with doses between 0 and 16Gy. Cell suspensions were irradiated in PBS by a "6"0Co source in doses between 0 and 16Gy, and incubated by 48h. Then cell membranes were lysed in the presence of SYTOX Green and EMA dyes, preserving nuclear membranes. Using this method, EMA-stained nuclei could be discriminated as those derived from dead cells, and SYTOX nuclei and micronuclei could be quantified. Micronuclei percentages were found to be proportional to dose, (R2 = 0.997). Only the highest dose (16Gy) could induce statistically significant increase of MN (p<0.0001), although cultures irradiated by 4, 8 and 16Gy showed significant increase of dead cell fractions. Calculation of the nuclei-to-beads ratio showed that 8 and 16Gy could reduce melanoma cell proliferation. Results showed that although cell death and loss of proliferative capacity could be observed on cultures irradiated at lower doses, genotoxic damage could be induced only on a higher dose. Resistance to radiation-induced genotoxicity could explain a relatively high radio resistance of melanoma tumors. (author)

Evaluation of radiation-induced genotoxicity on human melanoma cells (SK-MEL-37) by flow cytometry

Energy Technology Data Exchange (ETDEWEB)

Bonfim, Leticia; Carvalho, Luma Ramirez de; Vieira, Daniel Perez, E-mail: leticia.bonfim@ipen.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

2017-11-01

Micronucleus assay is a test used to evaluate genotoxic damage in cells, which can be caused by various factors, like ionizing radiation. Interactions between radiation energies and DNA can cause breakage, leading to use chromosomal mutations or loss of genetic material, important events that could be induced in solid tumors to mitigate its expansion within human body. Melanoma has been described as a tumor with increased radio resistance. This work evaluated micronuclei percentages (%MN) in human melanoma cells (SK-MEL-37), irradiated by gamma radiation, with doses between 0 and 16Gy. Cell suspensions were irradiated in PBS by a {sup 60}Co source in doses between 0 and 16Gy, and incubated by 48h. Then cell membranes were lysed in the presence of SYTOX Green and EMA dyes, preserving nuclear membranes. Using this method, EMA-stained nuclei could be discriminated as those derived from dead cells, and SYTOX nuclei and micronuclei could be quantified. Micronuclei percentages were found to be proportional to dose, (R2 = 0.997). Only the highest dose (16Gy) could induce statistically significant increase of MN (p<0.0001), although cultures irradiated by 4, 8 and 16Gy showed significant increase of dead cell fractions. Calculation of the nuclei-to-beads ratio showed that 8 and 16Gy could reduce melanoma cell proliferation. Results showed that although cell death and loss of proliferative capacity could be observed on cultures irradiated at lower doses, genotoxic damage could be induced only on a higher dose. Resistance to radiation-induced genotoxicity could explain a relatively high radio resistance of melanoma tumors. (author)

Diverse manifestations of tumorigenicity and immunogenicity displayed by the poorly immunogenic B16-BL6 melanoma transduced with cytokine genes.

Science.gov (United States)

Arca, M J; Krauss, J C; Strome, S E; Cameron, M J; Chang, A E

1996-05-01

We evaluated the in vivo response to the poorly immunogenic B16-BL6 (BL6) murine melanoma genetically altered to secrete interleukin-2 (IL-2), IL-4, interferon gamma (IFN gamma) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). Three parameters were evaluated: (1) tumorigenicity, (2) vaccination of naive animals, and (3) assessment of antitumor reactivity of T cells derived from tumor-draining lymph nodes (TDLN). Secretion of IL-2 abrogated the tumorigenicity of BL6, while IFN gamma and IL-4 partially reduced tumorigenicity, and GM-CSF had no effect. Protective immunity to wild-type tumor challenge could not be achieved by vaccination with irradiated cytokine-secreting tumors, although IL-2 and IL-4 secretion appeared to retard the growth of the challenge inoculum significantly. An alternative method to evaluate the immunogenicity of the cytokine-secreting tumors was to measure the ability of T cells obtained from TDLN to mediate regression of wild-type tumor in adoptive immunotherapy. Neither IL-2 nor IFN gamma secretion resulted in the induction of immune T cells. By contrast, GM-CSF and IL-4 secretion were found to induce immune T cells in the TDLN with GM-CSF being superior to IL-4. The combined secretion of GM-CSF and IL-4 did not lead to enhanced induction of immune T cells. GM-CSF secretion was found to upregulate B7-1 expression in TDLN, consistent with an increase in the population of antigen-presenting cells. These studies demonstrated that reduced tumorigenicity by cytokine secretion did not correlate with increased immunogenicity. With the cytokines examined, there was limited capability of developing protective immunity against the BL6 tumor. Nevertheless, GM-CSF and IL-4 secretion significantly enhanced T cell immune reactivity to the poorly immunogenic BL6 tumor.

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

DEFF Research Database (Denmark)

Adinolfi, Barbara; Carpi, Sara; Romanini, Antonella

2015-01-01

AIM: The current study was designed to characterize the anticancer effects of clotrimazole on human cutaneous melanoma cells. MATERIALS AND METHODS: The v-raf murine sarcoma viral oncogene homolog B1 V600E mutant melanoma cell line A375 was used as an in vitro model. Characterization tools includ...

Antitumor effect of cordycepin (3'-deoxyadenosine) on mouse melanoma and lung carcinoma cells involves adenosine A3 receptor stimulation.

Science.gov (United States)

Nakamura, Kazuki; Yoshikawa, Noriko; Yamaguchi, Yu; Kagota, Satomi; Shinozuka, Kazumasa; Kunitomo, Masaru

2006-01-01

An attempt was made to elucidate the molecular targetfor the antitumor effects of cordycepin (3'-deoxyadenosine) using non-selective and selective adenosine A1, A2a, A2b and A3 receptor agonists and antagonists. Although adenosine and 2'-deoxyadenosine (up to 100 microM) had no effect, cordycepin showed remarkable inhibitory effects on the growth curves of B16-BL6 mouse melanoma (IC50= 39 microM) and mouse Lewis lung carcinoma (IC50 = 48 microM) cell lines in vitro. Among the adenosine receptor agonists and antagonists used (up to 100 microM), only 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), a selective adenosine A3 receptor agonist, notably inhibited the growth of both mouse tumor cell lines (B16-BL6; IC50 = 5 microM, LLC; 14 microM). In addition, the tumor growth inhibitory effect of cordycepin was antagonized by 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191), a selective adenosine A3 receptor antagonist. These results suggest that cordycepin exerts inhibitory effects on the growth of mouse melanoma and lung carcinoma cells by stimulating adenosine A3 receptors on tumor cells.

LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

International Nuclear Information System (INIS)

Ghislin, Stephanie; Obino, Dorian; Middendorp, Sandrine; Boggetto, Nicole; Alcaide-Loridan, Catherine; Deshayes, Frederique

2012-01-01

Patients with metastatic melanoma have a poor median rate of survival. It is therefore necessary to increase our knowledge about melanoma cell dissemination which includes extravasation, where cancer cells cross the endothelial barrier. Extravasation is well understood during travelling of white blood cells, and involves integrins such as LFA-1 (composed of two chains, CD11a and CD18) expressed by T cells, while ICAM-1 is induced during inflammation by endothelial cells. Although melanoma cell lines cross endothelial cell barriers, they do not express LFA-1. We therefore hypothesized that melanoma-endothelial cell co-culture might induce the LFA-1/ICAM ligand/receptor couple during melanoma transmigration. A transwell approach has been used as well as blocking antibodies against CD11a, CD18 and ICAM-1. Data were analyzed with an epifluorescence microscope. Fluorescence intensity was quantified with the ImageJ software. We show here that HUVEC-conditioned medium induce cell-surface expression of LFA-1 on melanoma cell lines. Similarly melanoma-conditioned medium activates ICAM-1 expression in endothelial cells. Accordingly blocking antibodies of ICAM-1, CD11a or CD18 strongly decrease melanoma transmigration. We therefore demonstrate that melanoma cells can cross endothelial monolayers in vitro due to the induction of ICAM-1 and LFA-1 occurring during the co-culture of melanoma and endothelial cells. Our data further suggest a role of LFA-1 and ICAM-1 in the formation of melanoma cell clumps enhancing tumor cell transmigration. Melanoma-endothelial cell co-culture induces LFA-1 and ICAM-1 expression, thereby favoring in vitro melanoma trans-migration

LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

Directory of Open Access Journals (Sweden)

Ghislin Stephanie

2012-10-01

Full Text Available Abstract Background Patients with metastatic melanoma have a poor median rate of survival. It is therefore necessary to increase our knowledge about melanoma cell dissemination which includes extravasation, where cancer cells cross the endothelial barrier. Extravasation is well understood during travelling of white blood cells, and involves integrins such as LFA-1 (composed of two chains, CD11a and CD18 expressed by T cells, while ICAM-1 is induced during inflammation by endothelial cells. Although melanoma cell lines cross endothelial cell barriers, they do not express LFA-1. We therefore hypothesized that melanoma-endothelial cell co-culture might induce the LFA-1/ICAM ligand/receptor couple during melanoma transmigration. Methods A transwell approach has been used as well as blocking antibodies against CD11a, CD18 and ICAM-1. Data were analyzed with an epifluorescence microscope. Fluorescence intensity was quantified with the ImageJ software. Results We show here that HUVEC-conditioned medium induce cell-surface expression of LFA-1 on melanoma cell lines. Similarly melanoma-conditioned medium activates ICAM-1 expression in endothelial cells. Accordingly blocking antibodies of ICAM-1, CD11a or CD18 strongly decrease melanoma transmigration. We therefore demonstrate that melanoma cells can cross endothelial monolayers in vitro due to the induction of ICAM-1 and LFA-1 occurring during the co-culture of melanoma and endothelial cells. Our data further suggest a role of LFA-1 and ICAM-1 in the formation of melanoma cell clumps enhancing tumor cell transmigration. Conclusion Melanoma-endothelial cell co-culture induces LFA-1 and ICAM-1 expression, thereby favoring in vitro melanoma trans-migration.

Enhanced therapeutic effect on murine melanoma and angiosarcoma cells by boron neutron capture therapy using a boronated metalloporphyrin

International Nuclear Information System (INIS)

Yamada, Yoshihiko; Ichihashi, Masamitsu; Kahl, S.B.; Toda, Ken-ichi.

1994-01-01

We have already achieved successful treatment of several human patients with malignant melanoma by boron neutron capture therapy (BNCT) using 10 B 1 -paraboronophenylalanine ( 10 B 1 -BPA·HCl). In this study we used a new compound, a manganese boronated protoporphyrin (Mn- 10 BOPP), and compared it to 10 B 1 -BPA·HCl with respect to uptake in murine melanoma and angiosarcoma cells as well as to their cell killing effect. 10 B uptake was measured in a new method, and the new compound was much more incorporated into both cells than 10 B 1 -BPA·HCl. Furthermore, melanoma and angiosarcoma cells preincubated with the new compound were 15 to 20 times more efficiently killed by BNCT than cells preincubated with 10 B 1 -BPA·HCl. (author)

Cloning and Characterization of the Genes Encoding the Murine Homologues of the Human Melanoma Antigens MART1 and gp100

Science.gov (United States)

Zhai, Yifan; Yang, James C.; Spiess, Paul; Nishimura, Michael I.; Overwijk, Willem W.; Roberts, Bruce; Restifo, Nicholas P.; Rosenberg, Steven A.

2008-01-01

The recent identification of genes encoding melanoma-associated antigens has opened new possibilities for the development of cancer vaccines designed to cause the rejection of established tumors. To develop a syngeneic animal model for evaluating antigen-specific vaccines in cancer therapy, the murine homologues of the human melanoma antigens MART1 and gp 100, which were specifically recognized by tumor-infiltrating lymphocytes from patients with melanoma, were cloned and sequenced from a murine B16 melanoma cDNA library. The open reading frames of murine MART1 and gp 100 encode proteins of 113- and 626-amino acids with 68.8 and 77% identity to the respective human proteins. Comparison of the DNA sequences of the murine MART1 genes, derived from normal melanocytes, the immortalized nontumorgenic melanocyte line Melan-a and the B16 melanoma, showed all to be identical. Northern and Western blot analyses confirmed that both genes encoded products that were melanocyte lineage proteins. Mice immunized with murine MART1 or gp 100 using recombinant vaccinia virus failed to produce any detectable T-cell responses or protective immunity against B16 melanoma. In contrast, immunization of mice with human gp 100 using recombinant adenoviruses elicited T cells specific for hgp100, but these T cells also cross reacted with B16 tumor in vitro and induced significant but weak protection against B16 challenge. Immunization with human and mouse gp100 together [adenovirus type 2 (Ad2)-hep100 plus recombinant vaccinia virus (rVV)-mgp100], or immunization with human gp100 (Ad2-hgp100) and boosting with heterologous vector (rVV-hgp100 or rVV-mgp100) or homologous vector (Ad2-hgp100), did not significantly enhance the protective response against B16 melanoma. These results may suggest that immunization with heterologous tumor antigen, rather than self, may be more effective as an immunotherapeutic reagent in designing antigen-specific cancer vaccines. PMID:9101410

Metastatic melanoma cells escape from immunosurveillance through the novel mechanism of releasing nitric oxide to induce dysfunction of immunocytes.

Science.gov (United States)

Zhang, X M; Xu, Q

2001-12-01

Nitric oxide (NO) is known to facilitate tumour metastasis through the promotion of angiogenesis, vascular dilation, platelet aggregation, etc. In the present study we explored its novel role in producing dysfunction of the host immune system in the metastasis of murine metastatic melanoma B16-BL6 cells. A significant reduction in the mixed lymphocyte reaction (MLR) was observed in the spleen cells from B16-BL6-bearing mice, but not in those from mice bearing the parent cell B16. When B16-BL6 cells were added in vitro to the MLR, a significant decrease was also found, even when they were co-cultured with the lymphocytes in two compartments of a Transwell chamber separated by an 8.0 microm filter. The supernatant from cultured B16-BL6 but not B16 cells, which had a greatly increased NO activity, significantly inhibited concanavalin A- and lipopolysaccharide-induced lymphocyte proliferation. A remarkably higher expression of inducible NO synthase (iNOS) was detected in B16-BL6 cells than in B16 cells. Nomega-Nitro-l-arginine (l-NNA), a NO synthase inhibitor and superoxide dismutase, significantly antagonized the above inhibition by B16-BL6 cells, while l-arginine, a NO precursor, and S-nitroso-N-acetyl-d,l-penicillamine, a NO donor, strengthened the inhibition. Furthermore, l-NNA significantly inhibited lung metastasis of B16-BL6 cells, while l-arginine tended to enhance the metastasis. The cytotoxicity of B16-BL6-specific T-cells was significantly decreased by pre-culture with B16-BL6 cells in a Transwell chamber or the culture supernatants of B16-BL6 cells, whereas l-iminoethyl-lysine, a selective inhibitor of iNOS, showed a significant recovery from the disease. These results suggest that NO released by metastatic tumour cells may impair the immune system, which facilitates the escape from immunosurveillance and metastasis of tumour cells.

Cell proliferation and expression of connexins differ in melanotic and amelanotic canine oral melanomas.

Science.gov (United States)

Teixeira, Tarso Felipe; Gentile, Luciana Boffoni; da Silva, Tereza Cristina; Mennecier, Gregory; Chaible, Lucas Martins; Cogliati, Bruno; Roman, Marco Antonio Leon; Gioso, Marco Antonio; Dagli, Maria Lucia Zaidan

2014-03-01

Melanoma is a malignant neoplasm occurring in several animal species, and is the most frequently found tumor in the oral cavity in dogs. Melanomas are classified into two types: melanotic and amelanotic. Prior research suggests that human amelanotic melanomas are more aggressive than their melanotic counterparts. This study evaluates the behavior of canine melanotic and amelanotic oral cavity melanomas and quantifies cell proliferation and the expression of connexins. Twenty-five melanomas (16 melanotic and 9 amelanotic) were collected from dogs during clinical procedures at the Veterinary Hospital of the School of Veterinary Medicine and Animal Science of the University of São Paulo, Brazil. After diagnosis, dogs were followed until death or euthanasia. Histopathology confirmed the gross melanotic or amelanotic characteristics and tumors were classified according to the WHO. HMB45 or Melan A immunostainings were performed to confirm the diagnosis of amelanotic melanomas. Cell proliferation was quantified both by counting mitotic figures and PCNA positive nuclei. Expressions of connexins 26 and 43 were evaluated by immunohistochemistry, qRT-PCR and Western blot. Dogs bearing amelanotic melanomas presented a shorter lifespan in comparison to those with melanotic melanomas. Cell proliferation was significantly higher in amelanotic melanomas. Expressions of Connexins 26 and 43 were significantly reduced in amelanotic melanomas. The results presented here suggest that oral cavity melanotic and amelanotic melanomas differ regarding their behavior, cell proliferation and connexin expression in dogs, indicating a higher aggressiveness of amelanotic variants.

Dihydromyricetin induces cell cycle arrest and apoptosis in melanoma SK-MEL-28 cells.

Science.gov (United States)

Zeng, Guofang; Liu, Jie; Chen, Hege; Liu, Bin; Zhang, Qingyu; Li, Mingyi; Zhu, Runzhi

2014-06-01

Dihydromyricetin (DHM) exhibits multiple pharmacological activities; however, the role of DHM in anti-melanoma activities and the underlying molecular mechanisms are unclear. The aim of the present study was to evaluate the effects of DHM on cell proliferation, cell cycle distribution and apoptosis in the human melanoma SK-MEL-28 cell line, and to explore the related mechanisms. The effect of DHM on cell proliferation was investigated by MTT assay, and cell cycle distribution was determined by flow cytometry. TUNEL assay was used to evaluate DHM-mediated apoptosis, and western blotting was applied to examine expression levels of p53, p21, Cdc25A, Cdc2, P-Cdc2, Bax, IKK-α, NF-κB p65, p38 and P-p38 proteins. The results revealed that DHM suppressed cell proliferation of SK-MEL-28 cells in a concentration- and time-dependent manner, and caused cell cycle arrest at the G1/S phase. DHM increased the production of p53 and p21 proteins and downregulated the production of Cdc25A, Cdc2 and P-Cdc2 proteins, which induced cell cycle arrest. Additionally, DHM significantly induced the apoptosis of SK-MEL-28 cells, and enhanced the expression levels of Bax proteins and decreased the protein levels of IKK-α, NF-κB (p65) and P-p38. The results suggest that DHM may be a novel and effective candidate agent to inhibit the growth of melanoma.

Differentiation of Human Malignant Melanoma Cells that Escape Apoptosis After Treatment with 9-Nitrocamptothecin In Vitro

Directory of Open Access Journals (Sweden)

Panayotis Pantazis

1999-08-01

Full Text Available After in-vitro exposure to 0.05 μmol/L 9-nitrocamptothecin (9NC for periods of time longer than 5 days, 65% to 80% of the human malignant melanoma SB1 B cells die by apoptosis, whereas the remaining cells are arrested at the G2-phase of the cell cycle. Upon discontinuation of exposure to 9NC the G2-arrested cells resume cell cycling or remain arrested depending on the duration of 9NC exposure. In contrast to cycling malignant cells, the cells irreversibly arrested at G2 exhibit features of normal-like cells, the melanocytes, as assessed by the appearance of dendrite-like structures; loss of proliferative activity; synthesis of the characteristic pigment, melanin; and, particularly, loss of tumorigenic ability after xenografting in immunodeficient mice. Further, the expression of the cyclin-dependent kinase inhibitor p16 is upregulated in the 9NC-treated, G1-arrested, but downregulated in density G1-arrested cells, whereas the reverse is observed in the expression of another cyclin-dependent kinase inhibitor, p21. These results suggest that malignant melanoma SB1B cells that escape 9NC-induced death by apoptosis undergo differentiation toward nonmalignant, normal-like cells.

Irradiated riboflavin diminishes the aggressiveness of melanoma in vitro and in vivo.

Directory of Open Access Journals (Sweden)

Daisy Machado

Full Text Available Melanoma is one of the most aggressive skin cancers due to its high capacity to metastasize. Treatment of metastatic melanomas is challenging for clinicians, as most therapeutic agents have failed to demonstrate improved survival. Thus, new candidates with antimetastatic activity are much needed. Riboavin (RF is a component of the vitamin B complex and a potent photosensitizer. Previously, our group showed that the RF photoproducts (iRF have potential as an antitumoral agent. Hence, we investigated the capacity of iRF on modulating melanoma B16F10 cells aggressiveness in vitro and in vivo. iRF decreases B16F10 cells survival by inhibiting mTOR as well as Src kinase. Moreover, melanoma cell migration was disrupted after treatment with iRF, mainly by inhibition of metalloproteinase (MMP activity and expression, and by increasing TIMP expression. Interestingly, we observed that the Hedgehog (HH pathway was inhibited by iRF. Two mediators of HH signaling, GLI1 and PTCH, were downregulated, while SUFU expression (an inhibitor of this cascade was enhanced. Furthermore, inhibition of HH pathway signaling by cyclopamine and Gant 61 potentiated the antiproliferative action of RF. Accordingly, when a HH ligand was applied, the effect of iRF was almost completely abrogated. Our findings indicate that Hedgehog pathway is involved on the modulation of melanoma cell aggressiveness by iRF. Moreover, iRF treatment decreased pulmonary tumor formation in a murine experimental metastasis model. Research to clarify the molecular action of flavins, in vivo, is currently in progress. Taken together, the present data provides evidence that riboflavin photoproducts may provide potential candidates for improving the efficiency of melanoma treatment.

Flavonoids apigenin and quercetin inhibit melanoma growth and metastatic potential.

Science.gov (United States)

Caltagirone, S; Rossi, C; Poggi, A; Ranelletti, F O; Natali, P G; Brunetti, M; Aiello, F B; Piantelli, M

2000-08-15

Flavonoids are a class of polyphenolic compounds widely distributed in the plant kingdom, which display a variety of biological activities, including chemoprevention and tumor growth inhibition. Our aim was to investigate the effects of several polyphenols on the growth and metastatic potential of B16-BL6 melanoma cells in vivo. Intraperitoneal administration of quercetin, apigenin, (-)-epigallocathechin-3-gallate (EGCG), resveratrol, and the anti-estrogen tamoxifen, at the time of i.m. injection of B16-BL6 cells into syngeneic mice, resulted in a significant, dose-dependent delay of tumor growth, without toxicity. The relative descending order of potency was EGCG > apigenin = quercetin = tamoxifen > resveratrol > control. Furthermore, polyphenols significantly potentiated the inhibitory effect of a non-toxic dose of cisplatin. When tested for the ability to inhibit lung colonization, quercetin, apigenin, and tamoxifen (but not EGCG or resveratrol) significantly decreased the number of B16-BL6 colonies in the lungs in a dose-dependent manner, with quercetin and apigenin being more effective than tamoxifen. Interestingly, quercetin, apigenin, and tamoxifen (but not EGCG or resveratrol) significantly decreased the invasion of B16-BL6 cells in vitro, with quercetin and apigenin being more effective than tamoxifen. This suggests that anti-invasive activity is one of the mechanisms underlying inhibition of lung colonization by quercetin and apigenin. In conclusion, quercetin and apigenin inhibit melanoma growth and invasive and metastatic potential; therefore, they may constitute a valuable tool in the combination therapy of metastatic melanoma. Copyright 2000 Wiley-Liss, Inc.

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

Directory of Open Access Journals (Sweden)

Visalini Muthusamy

2013-08-01

Full Text Available Ultraviolet (UV radiation activates cell signaling pathways in melanocytes. As a result of altered signaling pathways and UV-induced cellular damage, melanocytes can undergo oncogenesis and develop into melanomas. In this study, we investigated the effect of UV-radiation on p38 MAPK (mitogen-activated protein kinase, JNK and NFκB pathways to determine which plays a major role in stimulating TNFα secretion in human HEM (melanocytes and MM96L (melanoma cells. MM96L cells exhibited 3.5-fold higher p38 activity than HEM cells at 5 min following UVA + B radiation and 1.6-fold higher JNK activity at 15–30 min following UVB+A radiation, while NFκB was minimally activated in both cells. Irradiated HEM cells had the greatest fold of TNFα secretion (UVB: 109-fold, UVA + B: 103-fold & UVB+A: 130-fold when co-exposed to IL1α. The p38 inhibitor, SB202190, inhibited TNFα release by 93% from UVB-irradiated HEM cells. In the UVB-irradiated MM96L cells, both SB202190 and sulfasalazine (NFκB inhibitor inhibited TNFα release by 52%. Although, anisomycin was a p38 MAPK activator, it inhibited TNFα release in UV-irradiated cells. This suggests that UV-mediated TNFα release may occur via different p38 pathway intermediates compared to those stimulated by anisomycin. As such, further studies into the functional role p38 MAPK plays in regulating TNFα release in UV-irradiated melanocyte-derived cells are warranted.

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

Science.gov (United States)

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.

Mutation analysis of genes that control the G1/S cell cycle in melanoma: TP53, CDKN1A, CDKN2A, and CDKN2B

International Nuclear Information System (INIS)

Soto, José Luis; Cabrera, Carmen M; Serrano, Salvio; López-Nevot, Miguel Ángel

2005-01-01

The role of genes involved in the control of progression from the G1 to the S phase of the cell cycle in melanoma tumors in not fully known. The aim of our study was to analyse mutations in TP53, CDKN1A, CDKN2A, and CDKN2B genes in melanoma tumors and melanoma cell lines We analysed 39 primary and metastatic melanomas and 9 melanoma cell lines by single-stranded conformational polymorphism (SSCP). The single-stranded technique showed heterozygous defects in the TP53 gene in 8 of 39 (20.5%) melanoma tumors: three new single point mutations in intronic sequences (introns 1 and 2) and exon 10, and three new single nucleotide polymorphisms located in introns 1 and 2 (C to T transition at position 11701 in intron 1; C insertion at position 11818 in intron 2; and C insertion at position 11875 in intron 2). One melanoma tumor exhibited two heterozygous alterations in the CDKN2A exon 1 one of which was novel (stop codon, and missense mutation). No defects were found in the remaining genes. These results suggest that these genes are involved in melanoma tumorigenesis, although they may be not the major targets. Other suppressor genes that may be informative of the mechanism of tumorigenesis in skin melanomas should be studied

Photodynamic therapy for melanoma: efficacy and immunologic effects

Science.gov (United States)

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.

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

Science.gov (United States)

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%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 of melanoma tumors and metastases in vivo .

Inhibitory effect of rose hip (Rosa canina L.) on melanogenesis in mouse melanoma cells and on pigmentation in brown guinea pigs.

Science.gov (United States)

Fujii, Takashi; Ikeda, Katsumi; Saito, Morio

2011-01-01

The compounds present in rose hips exerting an inhibitory action against melanogenesis in B16 mouse melanoma cells were investigated by dividing an aqueous extract of rose hips (RE) into four fractions. The 50% ethanol eluate from a DIAION HP-20 column significantly reduced the production of melanin and was mainly composed of procyanidin glycosides. We also found that this 50% ethanol eluate reduced the intracellular tyrosinase activity and also had a direct inhibitory effect on tyrosinase obtained as a protein mixture from the melanoma cell lysate. We also investigated the effect of orally administering RE on skin pigmentation in brown guinea pigs, and found that the pigmentation was inhibited together with the tyrosinase activity in the skin. These data collectively suggest that proanthocyanidins from RE inhibited melanogenesis in mouse melanoma cells and guinea pig skin, and could be useful as a skin-whitening agent when taken orally.

Radiation of different human melanoma cell lines increased expression of RHOB. Level of this tumor suppressor gene in different cell lines

International Nuclear Information System (INIS)

Notcovich, C.; Molinari, B.; Duran, H.; Delgado González, D.; Sánchez Crespo, R.

2013-01-01

Previous results of our group show that a correlation exists between intrinsic radiosensitivity of human melanoma cells and cell death by apoptosis. RhoB is a small GTPase that regulates cytoskeletal organization. Besides, is related to the process of apoptosis in cells exposed to DNA damage as radiation. Also, RhoB levels decrease in a wide variety of tumors with the tumor stage, being considered a tumor suppressor gene due to its antiproliferative and proapoptotic effect. The aim of this study was to analyze the expression of RhoB in different human melanoma cell lines in relation to melanocytes, and evaluate the effect of gamma radiation on the expression of RhoB. We used the A375, SB2 and Meljcell lines, and the derived from melanocytes Pig1. It was found for all three tumor lines RhoB expression levels significantly lower than those of Pig1 (p <0.05), as assessed by semiquantitative RT-PCR . When tumor cells were irradiated to a dose of 2Gyinduction was observed at 3 hours RhoB irradiation. RhoB expression increased in all lines relative to non-irradiated control, showing a greater induction ( p< 0.05) for the more radiosensitive line SB2, consistent with apoptosis in response to radiation. The results allow for the first time in melanoma demonstrate that RhoB, as well as in other tumor types, has a lower expression in tumor cells than their normal counterparts. Moreover, induction in the expression of RhoB in irradiated cells may be associated with the process of radiation-induced apoptosis. The modulation of RhoB could be a new tool to sensitize radioresistant melanoma. (author)

Involvement of endothelin and ET(A) endothelin receptor in mechanical allodynia in mice given orthotopic melanoma inoculation.

Science.gov (United States)

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.

Pathway aberrations of murine melanoma cells observed in Paired-End diTag transcriptomes

Directory of Open Access Journals (Sweden)

Liu Edison

2007-06-01

Full Text Available Abstract Background Melanoma is the major cause of skin cancer deaths and melanoma incidence doubles every 10 to 20 years. However, little is known about melanoma pathway aberrations. Here we applied the robust Gene Identification Signature Paired End diTag (GIS-PET approach to investigate the melanoma transcriptome and characterize the global pathway aberrations. Methods GIS-PET technology directly links 5' mRNA signatures with their corresponding 3' signatures to generate, and then concatenate, PETs for efficient sequencing. We annotated PETs to pathways of KEGG database and compared the murine B16F1 melanoma transcriptome with three non-melanoma murine transcriptomes (Melan-a2 melanocytes, E14 embryonic stem cells, and E17.5 embryo. Gene expression levels as represented by PET counts were compared across melanoma and melanocyte libraries to identify the most significantly altered pathways and investigate the expression levels of crucial cancer genes. Results Melanin biosynthesis genes were solely expressed in the cells of melanocytic origin, indicating the feasibility of using the PET approach for transcriptome comparison. The most significantly altered pathways were metabolic pathways, including upregulated pathways: purine metabolism, aminophosphonate metabolism, tyrosine metabolism, selenoamino acid metabolism, galactose utilization, nitrobenzene degradation, and bisphenol A degradation; and downregulated pathways: oxidative phosphorylation, ATPase synthesis, TCA cycle, pyruvate metabolism, and glutathione metabolism. The downregulated pathways concurrently indicated a slowdown of mitochondrial activities. Mitochondrial permeability was also significantly altered, as indicated by transcriptional activation of ATP/ADP, citrate/malate, Mg++, fatty acid and amino acid transporters, and transcriptional repression of zinc and metal ion transporters. Upregulation of cell cycle progression, MAPK, and PI3K/Akt pathways were more limited to certain

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

Science.gov (United States)

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.

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

Directory of Open Access Journals (Sweden)

Manel B. Hammouda

2016-07-01

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

A phase I trial of bortezomib and interferon-α-2b in metastatic melanoma.

Science.gov (United States)

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.

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

Directory of Open Access Journals (Sweden)

Han-En Tsai

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

Dual Role of Host Par2 in a Murine Model of Spontaneous Metastatic B16 Melanoma

Czech Academy of Sciences Publication Activity Database

Olejár, Tomáš; Větvička, D.; Zadinová, M.; Poučková, P.; Kukal, J.; Ježek, Petr; Matěj, R.

2014-01-01

Roč. 34, č. 7 (2014), s. 3511-3515 ISSN 0250-7005 R&D Projects: GA ČR(CZ) GAP302/10/0346 Institutional support: RVO:67985823 Keywords : PAR2 * melanoma * metastasis * murine model Subject RIV: EA - Cell Biology Impact factor: 1.826, year: 2014

Microculture-based chemosensitivity testing: a feasibility study comparing freshly explanted human melanoma cells with human melanoma cell lines.

Science.gov (United States)

Marshall, E S; Finlay, G J; Matthews, J H; Shaw, J H; Nixon, J; Baguley, B C

1992-03-04

The culture of cancer cells has many applications in chemosensitivity testing and new drug development. Our goal was to adapt simple semiautomated microculture methods for testing the chemosensitivity of melanoma cells freshly recovered from patients' tumors. Cells were cultured on a substrate of agarose and exposed continuously to cytotoxic drugs, the effects of which were measured by determining the uptake of [3H]thymidine 4-7 days later. Immunocytochemical staining of cells cultured with 5-bromo-2'-deoxyuridine demonstrated that tumor cells were responsible for the measured thymidine incorporation. The effects of cytotoxic drugs were calculated as logarithmic 50% inhibitory concentrations and expressed as divergences from the mean in a log-mean graph. The inhibitory effects of amsacrine, etoposide, doxorubicin, cisplatin, mitomycin C, and fluorouracil were tested. Tumors differed widely in their sensitivity to these drugs, although sensitivity to the three topoisomerase-II-directed agents was highly correlated. Cells from two non-neoplastic hematopoietic progenitor cell lines (FT and 32D) showed chemosensitivity patterns distinct from those in the melanoma cells, indicating tissue selectivity. Two established melanoma cell lines, MM-96 and FME, were tested under the same conditions and showed sensitivity typical of at least some fresh specimens. These results support the validity of melanoma cell lines as models of freshly resected melanoma cells. If successfully applied to other tumor types, such semiautomated approaches could find wide application in routine hospital laboratories for the chemosensitivity testing of patients' tumor cells.

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs.

Science.gov (United States)

Timmons, Joshua J; Cohessy, Sean; Wong, Eric T

2016-05-24

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Inactivation of HTB63 human melanoma cells by irradiation with protons and gamma rays.

Science.gov (United States)

Ristic-Fira, Aleksandra; Petrovic, Ivan; Todorovic, Danijela; Koricanac, Lela; Vujèic, Miroslava; Demajo, Miroslav; Sabini, Gabriella; Cirrone, Pablo; Cuttone, Giacomo

2004-12-01

The effects of single irradiation with gamma rays and protons on HTB63 human melanoma cell growth were compared. The exponentially growing cells were irradiated with gamma rays or protons using doses ranging from 2-20 Gy. At 48 h of post-irradiation incubation under standard conditions, cell survival and induction of apoptotic cell death were examined. The best effect of the single irradiation with gamma rays was the reduction of cell growth by up to 26% (p=0.048, irradiation vs. control), obtained using the dose of 16 Gy. The same doses of proton irradiation, having energy at the target of 22.6 MeV, significantly inhibited melanoma cell growth. Doses of 12 and 16 Gy of protons provoked growth inhibition of 48.9% (p=0.003, irradiation vs. control) and 51.2% (p=0.012, irradiation vs. control) respectively. Irradiation with 12 and 16 Gy protons, compared to the effects of the same doses of gamma rays, significantly reduced melanoma cell growth (p=0.015 and p=0.028, protons vs. gamma rays, respectively). Estimated RBEs for growth inhibition of HTB63 cells ranged from 1.02 to 1.45. The electrophoretical analyses of DNA samples and flow cytometric evaluation have shown a low percentage of apoptotic cells after both types of irradiation. The better inhibitory effect achieved by protons in contrast to gamma rays, can be explained considering specific physical properties of protons, especially taking into account the highly localized energy deposition (high LET).

Natural CD8+25+ regulatory T cell-secreted exosomes capable of suppressing cytotoxic T lymphocyte-mediated immunity against B16 melanoma

International Nuclear Information System (INIS)

Xie, Yufeng; Zhang, Xueshu; Zhao, Tuo; Li, Wei; Xiang, Jim

2013-01-01

Highlights: •CD8 + 25 + regulatory T cells secrete tolerogenic exosomes. •CD8 + 25 + regulatory T cell-derived exosomes exhibit immunosuppressive effect. •CD8 + 25 + regulatory T cell-derived exosomes inhibit antitumor immunity. -- Abstract: Natural CD4 + 25 + and CD8 + 25 + regulatory T (Tr) cells have been shown to inhibit autoimmune diseases. Immune cells secrete exosomes (EXOs), which are crucial for immune regulation. However, immunomodulatory effect of natural Tr cell-secreted EXOs is unknown. In this study, we purified natural CD8 + 25 + Tr cells from C57BL/6 mouse naive CD8 + T cells, and in vitro amplified them with CD3/CD28 beads. EXOs (EXO Tr ) were purified from Tr cell’s culture supernatants by differential ultracentrifugation and analyzed by electron microscopy, Western blot and flow cytometry. Our data showed that EXO Tr had a “saucer” or round shape with 50–100 nm in diameter, contained EXO-associated markers LAMP-1 and CD9, and expressed natural Tr cell markers CD25 and GITR. To assess immunomodulatory effect, we i.v. immunized C57BL/6 mice with ovalbumin (OVA)-pulsed DCs (DC OVA ) plus Tr cells or EXO Tr , and then assessed OVA-specific CD8 + T cell responses using PE-H-2K b /OVA tetramer and FITC-anti-CD8 antibody staining by flow cytometry and antitumor immunity in immunized mice with challenge of OVA-expressing BL6–10 OVA melanoma cells. We demonstrated that DC OVA -stimulated CD8 + T cell responses and protective antitumor immunity significantly dropped from 2.52% to 1.08% and 1.81% (p OVA (p Tr , respectively. Our results indicate that natural CD8 + 25 + Tr cell-released EXOs, alike CD8 + 25 + Tr cells, can inhibit CD8 + T cell responses and antitumor immunity. Therefore, EXOs derived from natural CD4 + 25 + and CD8 + 25 + Tr cells may become an alternative for immunotherapy of autoimmune diseases

Quantifying rates of cell migration and cell proliferation in co-culture barrier assays reveals how skin and melanoma cells interact during melanoma spreading and invasion.

Science.gov (United States)

Haridas, Parvathi; Penington, Catherine J; McGovern, Jacqui A; McElwain, D L Sean; Simpson, Matthew J

2017-06-21

Malignant spreading involves the migration of cancer cells amongst other native cell types. For example, in vivo melanoma invasion involves individual melanoma cells migrating through native skin, which is composed of several distinct subpopulations of cells. Here, we aim to quantify how interactions between melanoma and fibroblast cells affect the collective spreading of a heterogeneous population of these cells in vitro. We perform a suite of circular barrier assays that includes: (i) monoculture assays with fibroblast cells; (ii) monoculture assays with SK-MEL-28 melanoma cells; and (iii) a series of co-culture assays initiated with three different ratios of SK-MEL-28 melanoma cells and fibroblast cells. Using immunostaining, detailed cell density histograms are constructed to illustrate how the two subpopulations of cells are spatially arranged within the spreading heterogeneous population. Calibrating the solution of a continuum partial differential equation to the experimental results from the monoculture assays allows us to estimate the cell diffusivity and the cell proliferation rate for the melanoma and the fibroblast cells, separately. Using the parameter estimates from the monoculture assays, we then make a prediction of the spatial spreading in the co-culture assays. Results show that the parameter estimates obtained from the monoculture assays lead to a reasonably accurate prediction of the spatial arrangement of the two subpopulations in the co-culture assays. Overall, the spatial pattern of spreading of the melanoma cells and the fibroblast cells is very similar in monoculture and co-culture conditions. Therefore, we find no clear evidence of any interactions other than cell-to-cell contact and crowding effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Role of β-catenin signaling in the anti-invasive effect of the omega-3 fatty acid DHA in human melanoma cells.

Science.gov (United States)

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.

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

Energy Technology Data Exchange (ETDEWEB)

Qin, J.-Z.; Xin, H. [Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University of Chicago Medical Center (United States); Nickoloff, B.J., E-mail: bnickol@lumc.edu [Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University of Chicago Medical Center (United States)

2010-05-28

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines.

Science.gov (United States)

Qin, J-Z; Xin, H; Nickoloff, B J

2010-05-28

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma. Copyright (c) 2010 Elsevier Inc. All rights reserved.

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

International Nuclear Information System (INIS)

Qin, J.-Z.; Xin, H.; Nickoloff, B.J.

2010-01-01

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.

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

Directory of Open Access Journals (Sweden)

Zih-Yun Wu

2016-03-01

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

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

Science.gov (United States)

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

Radiopharmaceuticals targeting melanoma

Energy Technology Data Exchange (ETDEWEB)

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

2008-02-15

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

Role of key-regulator genes in melanoma susceptibility and pathogenesis among patients from South Italy

International Nuclear Information System (INIS)

Casula, Milena; Sini, MariaCristina; Palomba, Grazia; The Italian Melanoma Intergroup; Palmieri, Giuseppe; Muggiano, Antonio; Cossu, Antonio; Budroni, Mario; Caracò, Corrado; Ascierto, Paolo A; Pagani, Elena; Stanganelli, Ignazio; Canzanella, Sergio

2009-01-01

Several genetic alterations have been demonstrated to contribute to the development and progression of melanoma. In this study, we further investigated the impact of key-regulator genes in susceptibility and pathogenesis of such a disease. A large series (N = 846) of sporadic and familial cases originating from South Italy was screened for germline mutations in p16 CDKN2A , BRCA2, and MC1R genes by DHPLC analysis and automated DNA sequencing. Paired primary melanomas and lymph node metastases from same patients (N = 35) as well as melanoma cell lines (N = 18) were analyzed for somatic mutations in NRAS, BRAF, and p16 CDKN2A genes. For melanoma susceptibility, investigations at germline level indicated that p16 CDKN2A was exclusively mutated in 16/545 (2.9%) non-Sardinian patients, whereas BRCA2 germline mutations were observed in 4/91 (4.4%) patients from North Sardinia only. Two MC1R germline variants, Arg151Cys and Asp294His, were significantly associated with melanoma in Sardinia. Regarding genetic events involved in melanoma pathogenesis at somatic level, mutually-exclusive mutations of NRAS and BRAF genes were observed at quite same rate (about two thirds) in cultured and in vivo melanomas (either primary or metastatic lesions). Conversely, p16 CDKN2A gene alterations were observed at increased rates moving from primary to metastatic melanomas and melanoma cell lines. Activation of the ERK gene product was demonstrated to be consistently induced by a combination of molecular alterations (NRAS/BRAF mutations and p16 CDKN2A silencing). Our findings further clarified that: a) mutation prevalence in melanoma susceptibility genes may vary within each specific geographical area; b) multiple molecular events are accumulating during melanomagenesis

Preventive effect of oral administration of 6-(methylsulfinyl)hexyl isothiocyanate derived from wasabi (Wasabia japonica Matsum) against pulmonary metastasis of B16-BL6 mouse melanoma cells.

Science.gov (United States)

Fuke, Yoko; Shinoda, Shoko; Nagata, Ikuko; Sawaki, Saeko; Murata, Mituyoshi; Ryoyama, Kazuo; Koizumi, Keiichi; Saiki, Ikuo; Nomura, Takahiro

2006-01-01

Effect of oral administration of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) or a 6-MITC-containing T-wasabi fraction from wasabi root (Wasabia japonica Matsum) to inhibit the macroscopic pulmonary metastasis was studied with a murine B16-BL6 melanoma model. Two administration routes, subcutaneous or intravenous, and two administration times, prior to or concomitant with tumor inoculation, of 6-MITC or T-wasabi against the metastatic foci formation in C57BL/6J mouse lungs were compared. The number of metastasized foci per lung in either subcutaneous or intravenous injection was significantly reduced by intake of 6-MITC or a T-wasabi fraction. The maximum reduction by a T-wasabi fraction reached to 82%. Fifty-six percent of foci formation was inhibited by a 2 week-prior administration of 6-MITC (200 microM), whereas only 27% inhibition was obtained by a concomitant administration with tumor inoculation. Neither 6-MITC nor T-wasabi at tested concentrations showed any toxic effects. Together with our previous results, a component of the Japanese pungent spice, wasabi appears to inhibit not only tumor cell growth but also tumor metastasis. Therefore, 6-MITC from wasabi is apparently a useful dietary candidate for controlling tumor progression.

Proteomic Analysis of Laser Microdissected Melanoma Cells from Skin Organ Cultures

Science.gov (United States)

Hood, Brian L.; Grahovac, Jelena; Flint, Melanie S.; Sun, Mai; Charro, Nuno; Becker, Dorothea; Wells, Alan; Conrads, Thomas P

2010-01-01

Gaining insights into the molecular events that govern the progression from melanoma in situ to advanced melanoma, and understanding how the local microenvironment at the melanoma site influences this progression, are two clinically pivotal aspects that to date are largely unexplored. In an effort to identify key regulators of the crosstalk between melanoma cells and the melanoma-skin microenvironment, primary and metastatic human melanoma cells were seeded into skin organ cultures (SOCs), and grown for two weeks. Melanoma cells were recovered from SOCs by laser microdissection and whole-cell tryptic digests analyzed by nanoflow liquid chromatography-tandem mass spectrometry with an LTQ-Orbitrap. The differential protein abundances were calculated by spectral counting, the results of which provides evidence that cell-matrix and cell-adhesion molecules that are upregulated in the presence of these melanoma cells recapitulate proteomic data obtained from comparative analysis of human biopsies of invasive melanoma and a tissue sample of adjacent, non-involved skin. This concordance demonstrates the value of SOCs for conducting proteomic investigations of the melanoma microenvironment. PMID:20459140

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Directory of Open Access Journals (Sweden)

Huey-Chun Huang

2014-09-01

Full Text Available 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.

The role of alpha-synuclein in melanin synthesis in melanoma and dopaminergic neuronal cells.

Directory of Open Access Journals (Sweden)

Tianhong Pan

Full Text Available The relatively high co-occurrence of Parkinson's disease (PD and melanoma has been established by a large number of epidemiological studies. However, a clear biological explanation for this finding is still lacking. Ultra-violet radiation (UVR-induced skin melanin synthesis is a defense mechanism against UVR-induced damage relevant to the initiation of melanoma, whereas, increased neuromelanin (NM, the melanin synthesized in dopaminergic neurons, may enhance the susceptibility to oxidative stress-induced neuronal injury relevant to PD. SNCA is a PD-causing gene coding for alpha-Synuclein (α-Syn that expresses not only in brain, but also in skin as well as in tumors, such as melanoma. The findings that α-Syn can interact with tyrosinase (TYR and inhibit tyrosine hydroxylase (TH, both of which are enzymes involved in the biosynthesis of melanin and dopamine (DA, led us to propose that α-Syn may participate in the regulation of melanin synthesis. In this study, by applying ultraviolet B (UVB light, a physiologically relevant stimulus of melanogenesis, we detected melanin synthesis in A375 and SK-MEL-28 melanoma cells and in SH-SY5Y and PC12 dopaminergic neuronal cells and determined effects of α-Syn on melanin synthesis. Our results showed that UVB light exposure increased melanin synthesis in all 4 cell lines. However, we found that α-Syn expression reduced UVB light-induced increase of melanin synthesis and that melanin content was lower when melanoma cells were expressed with α-Syn, indicating that α-Syn may have inhibitory effects on melanin synthesis in melanoma cells. Different from melanoma cells, the melanin content was higher in α-Syn-over-expressed dopaminergic neuronal SH-SY5Y and PC12 cells, cellular models of PD, than that in non-α-Syn-expressed control cells. We concluded that α-Syn could be one of the points responsible for the positive association between PD and melanoma via its differential roles in melanin synthesis in

lncRNA H19 predicts poor prognosis in patients with melanoma and regulates cell growth, invasion, migration and epithelial–mesenchymal transition in melanoma cells

Directory of Open Access Journals (Sweden)

Shi G

2018-06-01

Full Text Available Gaofeng Shi,1,2 Hu Li,2 Fengshan Gao,2 Qian Tan1 1Drum Tower Clinical Medical College of Nanjing Medical University, Nanjing, People’s Republic of China; 2Department of Plastic Surgery, the Affiliated Wuxi No 4 People’s Hospital of Jiangnan University, Wuxi, People’s Republic of China Introduction: Melanoma is a deadly malignancy and the poor prognosis of patients with advanced disease is relatively poor. Recent studies indicate that long non-coding RNAs are involved in the pathogenesis of malignant melanoma. This study aims to investigate the role of the long non-coding RNA H19 in melanoma and to explore the underlying molecular mechanisms. Materials and methods: The expression levels of H19 in clinical samples and melanoma cells were determined by quantitative real-time PCR. The cell growth and cell metastasis were assessed by Cell Counting Kit 8, cell invasion and wound healing assays. Cell apoptosis and cell cycle were determined by flow cytometry. Protein levels were determined by Western blotting assay. Results: H19 was highly expressed in melanoma tissues compared to normal adjacent skin tissues, and the tissue expression level of H19 from melanoma patients with metastasis was significantly higher than that from patients without distant metastasis. In addition, the high expression of H19 in melanoma tissues was associated with advanced tumor invasion and TNM stage, distal metastasis, lymph node metastasis and shorter overall survival in patients with melanoma. The in vitro functional assays showed that knockdown of H19 inhibited cell growth, invasion and migration and also induced cell apoptosis as well as G0/G1 arrest in melanoma cells. Further quantitative real-time PCR and Western blot experiments showed that knockdown of H19 differentially regulated the epithelial–mesenchymal transition (EMT-related gene expressions and reversed EMT in melanoma cell lines. Knockdown of H19 suppressed in vivo tumor growth and modulated the

Antitumor agent 25-epi Ritterostatin GN1N induces endoplasmic reticulum stress and autophagy mediated cell death in melanoma cells.

Science.gov (United States)

Riaz Ahmed, Kausar Begam; Kanduluru, Ananda Kumar; Feng, Li; Fuchs, Philip L; Huang, Peng

2017-05-01

Metastatic melanoma is the most aggressive of all skin cancers and is associated with poor prognosis owing to lack of effective treatments. 25-epi Ritterostatin GN1N is a novel antitumor agent with yet undefined mechanisms of action. We sought to delineate the antitumor mechanisms of 25-epi Ritterostatin GN1N in melanoma cells to determine the potential of this compound as a treatment for melanoma. Activation of the endoplasmic reticulum (ER) stress protein glucose-regulated protein 78 (GRP78) has been associated with increased melanoma progression, oncogenic signaling, drug resistance, and suppression of cell death. We found that 25-epi Ritterostatin GN1N induced cell death in melanoma cells at nanomolar concentrations, and this cell death was characterized by inhibition of GRP78 expression, increased expression of the ER stress marker CHOP, loss of mitochondrial membrane potential, and lipidation of the autophagy marker protein LC3B. Importantly, normal melanocytes exhibited limited sensitivity to 25-epi Ritterostatin GN1N. Subsequent in vivo results demonstrated that 25-epi Ritterostatin GN1N reduced melanoma growth in mouse tumor xenografts and did not affect body weight, suggesting minimal toxicity. In summary, our findings indicate that 25-epi Ritterostatin GN1N causes ER stress and massive autophagy, leading to collapse of mitochondrial membrane potential and cell death in melanoma cells, with minimal effects in normal melanocytes. Thus, 25-epi Ritterostatin GN1N is a promising anticancer agent that warrants further investigation.

Histopathology of normal skin and melanomas after nanosecond pulsed electric field treatment

Science.gov (United States)

Chen, Xinhua; Swanson, R. James; Kolb, Juergen F.; Nuccitelli, Richard; Schoenbach, Karl H.

2011-01-01

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 (Pelectric fields surrounding the needle electrodes. PMID:19730404

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

Science.gov (United States)

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.

Phenotypic stability of B16-BL6 melanoma exposed to low levels of tyrosine and phenylalanine.

Science.gov (United States)

Elstad, C A; Meadows, G G

1990-01-01

We previously demonstrated that tyrosine (Tyr) and phenylalanine (Phe) restriction suppresses metastatic heterogeneity of B16-BL6 (BL6) melanoma and selects for tumor variants with decreased metastatic potential. In this study, we investigate stability of this Tyr- and Phe-modulated tumor phenotype by sequentially transplanting BL6 in vivo into mice fed Low Tyr and Phe Diet. Metastatic potential of BL6 is suppressed after one subcutaneous passage. Suppression is unlikely to result from inhibition of tumor growth, since growth in vitro is significantly increased. The metastatic potential of the Tyr- and Phe-modulated tumor is unstable after in vivo passage, and lung colonizing ability is regenerated after ten in vivo passages. Conversely, the antimetastatic effect of Tyr and Phe restriction is stable after prolonged in vitro passage. The metastatic potential of tumors from mice fed Normal Diet is unstable after long-term in vitro culture. Sensitivity to adriamycin of BL6 from mice fed Low Tyr and Phe Diet is increased and is not altered by change in metastatic potential.

Comparative study of angio genesis radiopharmaceuticals for melanoma detection; Estudo comparativo de radiofarmacos para angiogenese na deteccao de melanoma

Energy Technology Data Exchange (ETDEWEB)